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					                          Research Summaries Book
                               ENDO 2001
                        rd
               The 83 Annual Meeting of The Endocrine Society
                             June 20-23, 2001
                        Denver Convention Center
                             Press Room: Room A202
                           Phone Number: 303-228-8553
          Hours: Wednesday-Friday, 7:30am-5:00pm; Saturday, 8:00am-12 noon

                               HOW TO USE THIS BOOK
The ENDO 2001 Research Summaries Book has been designed to facilitate media coverage of the
83rd Annual Meeting of The Endocrine Society. The abstracts contained in this book have been
identified as those that would be of particular interest to journalists. They were selected from
among nearly 2,500 abstracts that will be presented at ENDO 2001.

Each abstract in this book is presented in its original scientific form, just as it appears in the
Program and Abstracts Book. A lay explanation of the research, which was prepared in
coordination with the research teams, follows each abstract. The lay versions include additional
background information on the area of research and, in many cases, the source of funding for the
research. The original abstracts appear on the left-hand pages and the lay explanations on the
facing right-hand pages.

The abstracts are divided into general topic areas, as denoted in the table of contents, which
follows this page. Hot Topics and Clinical Trials Symposia, the first section of this book, features
results of up-to-the-minute research and the latest clinical trials. At the end of the book, there are
indices of abstracts in numerical order, by author, date of presentation and by research funding.

The full ENDO 2001 program is available on The Endocrine Society Web page through the
On-line Science Planner at www.endo-society.org

                                      The Endocrine Society
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                       The Endocrine Society Public Affairs Contacts:
                Susan Koppi                                                Marisa Kessel
                Director, Public Affairs                                   Manager, Public Affairs
                Direct: 301-941-0252                                       Direct: 301-941-0255
        Email: skoppi@endo-society.org                            Email: mkessel@endo-society.org
   ENDO 2001 Embargo Policy


Unless otherwise noted, all abstracts presented at ENDO 2001
are embargoed until 6:00 a.m. (EST) on Wednesday, June 20,
  2001. The Endocrine Society reserves the right to lift the
embargo on specific abstracts that are selected for promotion
              prior to the start of ENDO 2001.




  For additional information, please contact The Endocrine
   Society’s Public Affairs department at 301-941-0255 or
                  mkessel@endo-society.org
Name: _____________________                 Organization: _____________
Email: _____________________                Phone: ___________________

                             2001 MEDIA SURVEY
Please take a few moments to complete the following survey regarding media services at
ENDO 2001. We appreciate hearing from you, and we will incorporate your feedback in
our future media plans.

1.     How did you hear about The Endocrine Society‘s Annual Meeting, ENDO 2001?

2.     What topics / presentations were of special interest to you at ENDO 2001?


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        Science Planner On-Line:
        Program and Abstract Book:
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        Other:

4.     Did you attend any of the press briefings during ENDO 2001? Why or why not?


5.     Were you able to arrange interviews with presenters?


6.     Was the press room adequately equipped (staff and resources) to facilitate your
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7.     Are you interested in future Endocrine Society meetings and announcements?


8.     How could we serve you better in the future?


Comments:


Thank you for taking time to fill out this survey. Please leave your completed form
                        with press room staff, or send to:

                       Marisa Kessel, Manager, Public Affairs
                               The Endocrine Society
                             4350 East West Highway
                                Bethesda, MD 20814
                                 PH: 301-941-0255
                                FAX: 301-941-0259
                         E-Mail: mkessel@endo-society.org
                             TABLE OF CONTENTS


        I.   Hot Topics/Clinical Trials Symposia
       II.   Aging (BS)
      III.   Diabetes (BS)
     IV.     Gene Expression (BS)
       V.    GnRH-Gonadotropins (BS)
     VI.     Growth Factors (general) (BS)
     VII.    GH-Prolactin-Regulation of Secrection (BS)
    VIII.    Hormones and Cancer (BS)
     IX.     Hormone Neoplasia (BS)
       X.    Hormones and Immune System (BS)
     XI.     HPA Axis (BS)
     XII.    IGFs (BS)
    XIII.    Intracellular Signalling (BS)
    XIV.     Neuroendocrinology (BS)
    XV.      Obesity (BS)
    XVI.     PTH-Calcium-Vitamin D-Bone (BS)
   XVII.     Reproduction-Gonadal Control (Female) (BS)
   XVIII.    Reproduction-Gonadal Control (Male) (BS)
    XIX.     Steroid Hormone Action (BS)
    XX.      Steroid Hormone Biosynthesis (BS)
    XXI.     Aging (CS)
   XXII.     Androgenic Disorders in women (CS)
   XXIII.    Autoimmune Endocrine Diseases (CS)
  XXIV.      Bone-Calcium-Parathyroid (CS)
   XXV.      Diabetes-Lipids-Metabolism (CS)
  XXVI.      Endocrine Genetics (CS)
  XXVII.     Glucocorticoids (CS)
 XXVIII.     Growth Hormone and IGFs (CS)
  XXIX.      Health Care Delivery (CS)
   XXX.      Hormones and Cancer (CS)
  XXXI.      Obesity (CS)
  XXXII.     Pediatric Endocrinology (CS)
 XXXIII.     Pituitary (CS)
 XXXIV.      Reproduction (Female) (CS)
 XXXV.       Reproduction (Male) Prostate (CS)
 XXXVI.      Thyroid (CS)
XXXVII.      Reproductive (Female) (CS)
XXXVIII.     Thyroid (MTP-Clinical Case)
 XXXIX.      Indices


BS = Basic Science
CS = Clinical Science
Clinical Trials Symposium
Thursday, June 21, 2001
9:30-11:00 a.m.
Ballroom 2
Denver Convention Center
Each year, the Clinical Trials Symposium includes announcements of
groundbreaking clinical trials that have never been presented at a
meeting. The ENDO 2001 Clinical Trial Symposium topics will
include diabetes, osteoporosis, muscle mass in elderly men, and
phytoestrogens.

Clinical Trial Symposium Presentations

   “Randomised, placebo-controlled clinical trial of recombinant
    human chorionic gonadotropin (hCG) on muscle mass, strength
    and physical activity in older men”
    Presented by Dr. Peter Liu, University of Sydney and Concord
    Hospital

   “Dietary soy containing phytoestrogens improves lipid profiles
    but does not activate coagulation in postmenopausal women”
    Presented by Dr. Helena Teede, Vascular Medicine Unit,
    Dandenong Hospital

   “Strict metabolic control with intensive insulin treatment reduces
    morbidity and mortality of intensive care dependent critical
    illness”
    Presented by Dr. Greet Van den Berghe, University Hospital
    Gasthuisberg University of Leuven

   “Maintenance of reduction in non-traumatic, non-vertebral
    fractures six months after discontinuation of LY333334
    [recombinant human parathyroid hormone (1-34), rhPTH(1-34)]
    use in postmenopausal women with osteoporosis”
    Presented by Dr. Robert Lindsay, Helen Hays Hospital
Hot Topics Symposium
Friday, June 22, 2001
9:30-11:00 a.m.
Ballroom 2
Denver Convention Center

The Hot Topics presentations focus on the most pivotal
developments in endocrinology. The ENDO 2001 Hot Topics
presentations will cover studies on the human genome as well as
novel hormones and their relationship to diabetes.

Hot Topics Symposium Presentations

   “The adipocyte-secreted protein ACRP30/ADIPOQ acts as a
    hepatic insulin sensitizer”
     Presented by Dr. Philipp Scherer, Albert Einstein College of
     Medicine

   “The human genome project and its implications for endocrine
    research”
     Presented by Dr. David Altshuler, Massachusetts General
     Hospital, Harvard Medical School

   “Resistin”
    Presented by Dr. Mitchell Lazar, University of Pennsylvania
    Medical School
                                                      HOT TOPICS/CLINICAL TRIALS
S20-1
STRICT METABOLIC CONTROL WITH INTENSIVE INSULIN TREATMENT REDUCES
MORBIDITY AND MORTALITY OF INTENSIVE CARE DEPENDENT CRITICAL ILLNESS
G Van den Berghe, F Weekers, C Verwaest, M Schetz, D Vlasselaers, P Ferdinande, P Lauwers, R
Bouillon. Intensive Care Medicine and Lab. Experiment. Medicine & Endocrinology, University of
Leuven, Leuven, Belgium

Hyperglycemia and insulin resistance are common in fed critically ill patients. There is no consensus about
the optimal level of metabolic control with insulin in such patients. In most ICUs, insulin is only
administered to prevent excessively elevated blood glucose levels.
We performed a prospective, randomized, controlled study to address this question. All mechanically
ventilated, adult patients admitted to our tertiary level ICU were included, after informed consent from the
closest family member. Only 5 patients participating in another study and 9 who were moribund or DNR
coded at ICU admission were excluded. At admission, patients were randomized to either strict
normalization of blood glucose levels (4.5-6.1 mmol/L) with continuously infused insulin (Actrapid HM)
during ICU stay or the current ICU policy, which comprises insulin when blood glucose exceeds 12
mmol/L in which case glycemia was clamped to 10-12 mmol/L. An interim safety analysis revealed a
difference in mortality and the study was ended for ethical reasons.
A total of 1548 patients were included, 765 in the intensive insulin schedule (IIS), 783 in the restrictive
insulin schedule (RIS). The 2 groups were comparable at inclusion for all factors influencing outcome
including age, gender, BMI, type of illness, severity scores (APII and TISS), secondary referral, history of
diabetes (13%) and malignancy. Analyses were made on intention to treat basis.
Preliminary results (>98% of files analyzed): IIS reduced ICU mortality by 43% (P=0.007) [death odds
ratio of 0.52 (0.33-0.81), P=0.004] and hospital mortality by 34% (P=0.01). Morbidity was reduced in the
IIS group: mean ICU stay by 22% (P=0.005), incidence of bacteremia and critical illness polyneuropathy to
half.
In conclusion, the data suggest that disturbances in glucose metabolism during critical illness are not
"adaptive and beneficial" since strict metabolic control with exogenous insulin substantially reduces
morbidity and mortality.

Clinical Science Symposium: Clinical Trials Symposium (9:30 AM-11:00 AM)

Presentation Date: Thursday, June 21, 2001; Time: 9:30 AM; Location: Ballroom 2




                                                   -1-
                                                       HOT TOPICS/CLINICAL TRIALS
S20-1
Lay explanation of abstract:

High blood glucose levels are common in intensive care-dependent critically ill patients. It is unknown
whether this is part of a beneficial adaptation to stress or a disturbance that should be treated. We postulated
the latter and performed a large prospective, randomized, controlled clinical study to test this hypothesis.

After informed consent from the closest family member, all mechanically ventilated, adult patients admitted to
our intensive care unit (ICU) were eligible for inclusion in the study, without selection. At ICU admission,
patients were randomly (by blinded envelopes) allocated to two treatment groups. In one group blood glucose
levels were strictly normalized (4.5-6.1 mmol/L) with continuously infused insulin (Actrapid HM), and these
patients were known to be on the intensive insulin schedule (IIS). The other group underwent the currently
applied strategy, which comprises insulin treatment only when blood glucose levels become excessively
elevated (>12 mmol/L), and these patients were controlled to a moderately elevated range (10-12 mmol/L),
known as the restrictive insulin schedule (RIS). An interim safety analysis revealed a difference in mortality
between the groups, and the study ended for ethical reasons.

A total of 1,548 patients were included, 765 in the IIS group, 783 in the RIS group. The two groups were
comparable at study start for all factors influencing the risk of death, including age, BMI, type and severity of
illness, whether or not they were referred from another ICU, history of diabetes, and malignancy.
IIS reduced ICU mortality by 43% (P=0.005) [63 deaths in the RIS group versus 35 in the IIS group; relative
risk of death in the IIS group, corrected for all baseline other predictors of ICU death, was 0.52 (0.33-0.82),
P=0.004] and hospital mortality was reduced by 34% (P=0.01).

Mortality reduction occurred exclusively in long-stay ICU patients and was due to prevention of lethal multiple
organ failure caused by severe infections. IIS indeed reduced the incidence of bloodstream infections, renal
failure, anemia, and critical illness polyneuropathy, as well as the need for dialysis, red cell transfusion,
prolonged mechanical ventilatory support, and intensive care. This translated to a substantially reduced use of
ICU resources.

The data suggest that disturbances in glucose metabolism during critical illness are not "adaptive and
beneficial" because strict metabolic control with insulin infusion substantially reduces morbidity and mortality.
Ever since the introduction of mechanical ventilation during the polio epidemics more than six decades ago,
few intensive care interventions, if any, have improved outcome to that extent. Preventing ultimately futile
intensive care with an inexpensive treatment reflects major progress in ICU medicine, because it prevents
human suffering while it prevents useless allocation of scarce ICU resources.

Additional background information:

Prolonged intensive care of such patients uses a large proportion (>60%) of the resources for this type of
acute medicine. Furthermore, prolonged critical illness represents substantial human suffering. Key
problems of prolonged critical illness are severe infections; renal failure; anemia; jaundice; a specific type
of polyneuropathy, in which extreme muscle weakness and paralysis perpetuate the need for mechanical
ventilation and intensive care. By correcting the ―diabetes of stress‖ with intensive insulin treatment,
several of the key problems of prolonged critical illness were prevented and mortality was substantially
reduced. This large study is the first to address this question in ICU patients. The results represent major
progress in ICU medicine as the beneficial effects were obtained with a simple and inexpensive treatment.

The research was supported by research grants from the Belgian Fund for Scientific Research (G. 0144.00
and G.3C05.95N to GVdB), the Research Council of the University of Leuven (OT 99/32 to GVdB), the
Belgian Foundation for Research in Congenital Heart Diseases (GVdB), and an unrestricted research grant
from Novo Nordisk, Denmark (GVdB). RB is holder of J.J. Servier Diabetes Research Chair.



                                                     -2-
                                                       HOT TOPICS/CLINICAL TRIALS

S20-2
MAINTENANCE OF REDUCTION IN NON-TRAUMATIC, NON-VERTEBRAL FRACTURES 6
MONTHS AFTER DISCONTINUATION OF LY333334 [RECOMBINANT HUMAN
PARATHYROID HORMONE (1-34), RHPTH(1-34)] USE IN POSTMENOPAUSAL WOMEN
WITH OSTEOPOROSIS
R Lindsay,1 WH Scheele,2 AD Clancy,2 B Mitlak.2 1Helen Hayes Hospital, West Haverstraw, New York;
2
  Eli Lilly and Company, Indianapolis, Indiana

In a controlled clinical trial, parathyroid hormone (1-34) (PTH), was shown to significantly reduce both
vertebral and non-traumatic, non-vertebral fractures by 65-69% and 53-54% respectively, in women with
one or more vertebral fractures (19 month median PTH exposure). PTH was associated with statistically
significant increases in bone mineral density in the lumbar spine (9.7-13.7%) and total hip (2.6-3.6%), and
total body bone mineral content (1.3-2.3%). The present study seeks to determine whether these fracture
effects persist when PTH is discontinued. Of the original study population, 77% or 1261 women
volunteered for the observation study; former placebo, n=413; former PTH20, n=436; former PTH40,
n=412. All women remained on calcium (1000 mg/day) and 400-1200 IU of vitamin D. Approximately
one-third of participants reported use of other forms of osteoporosis therapy after discontinuation of PTH
(36%, 31%, 29% for placebo, PTH20, PTH40, respectively). During the 6-month observation period, there
were no statistically significant changes in femoral neck or total body bone density. Seventeen women had
non-traumatic non-vertebral fractures (1.4%): placebo, n=10 (2.4%); PTH20, n=3 (0.7%); PTH40, n=4
(1.0%) (P=0.066). A Kaplan-Meier analysis of time from the original baseline to first non-traumatic non-
vertebral fracture demonstrated the persistence of a statistically significant PTH effect. From baseline of the
original study, 62 women reported fractures (4.9%): placebo, n=35 (8.5%); PTH20, n=14 (3.2%); PTH40,
n=13, 3.2% (P=0.001). This was statistically significant for each dose compared to placebo. In conclusion,
the positive effects of parathyroid hormone (1-34) in reducing the cumulative proportion of women with
non-traumatic, non-vertebral fractures compared to placebo continued to be significant 6 months after
discontinuation of treatment.

Clinical Science Symposium: Clinical Trials Symposium (9:30 AM-11:00 AM)

Presentation Date: Thursday, June 21, 2001; Time: 9:52 AM; Location: Ballroom 2




                                                     -3-
                                                      HOT TOPICS/CLINICAL TRIALS

S20-2
Lay explanation of abstract:

Parathyroid hormone is the main regulator of calcium and bone metabolism in the human body. Various
forms of parathyroid hormone (PTH), when given once daily, are known to stimulate formation of new
bone, increase bone density, and reduce the risk of bone fractures in postmenopausal women with the bone-
thinning disease osteoporosis. Until now, there has been little information about what happens when PTH
treatment is discontinued.

In this study, the investigators followed postmenopausal women with osteoporosis who had completed
approximately 19 months of treatment with recombinant human parathyroid hormone (1-34) [rhPTH (1-
34)] to determine if the reduced risk of fracture lasted over a six-month period following discontinuation of
the drug. Most patients did not take any other treatment for osteoporosis after treatment with rhPTH (1-34)
was finished. In comparison with women who took a placebo injection, those who had been treated with
rhPTH (1-34) continued to have lower risk of fracture during the 6 months of follow up. Continued follow-
up is needed to determine how long the apparent benefit of rhPTH (1-34) therapy lasts.

This study was funded by Eli Lilly and Company, which is developing this rhPTH (1-34) compound as the
proposed brand Fortéo™ (generic name: teriparatide injection, rDNA origin, Lilly).




                                                    -4-
                                                      HOT TOPICS/CLINICAL TRIALS

S20-3
RANDOMISED, PLACEBO-CONTROLLED CLINICAL TRIAL OF RECOMBINANT HUMAN
CHORIONIC GONADOTROPIN (HCG) ON MUSCLE MASS, STRENGTH AND PHYSICAL
ACTIVITY IN OLDER MEN
PY Liu, DJ Handelsman. Department of Andrology, ANZAC Research Institute, Concord Hospital and
University of Sydney, Sydney, NSW, 2139, Australia

Despite partial androgen deficiency, the safety and efficacy of androgen therapy in older men remains
controversial as controlled studies of testosterone (T) have given equivocal results. hCG can be
conveniently self-administered infrequently and increases not only circulating T but also estradiol and other
testicular steroids. We evaluated the efficacy and safety of 3 months treatment with subcutaneous
recombinant hCG (Ovidrel, Serono) on muscle mass, strength, mobility (gait & balance) and physical
activity in ambulant, community dwelling men >60 years old having partial androgen deficiency (T <= 15
nmol/L, twice). Forty eligible men (mean 67; range 60 - 85) were randomised to receive recombinant hCG
(5000 IU, 250 mg) or placebo by thrice weekly subcutaneous self-injection and were studied before,
monthly during and one month after treatment. All completed the study and treatment groups were well
matched for age, height and weight. hCG significantly increased body weight (~1 kg, p<0.05) and lean
body mass (~3 kg, P<0.001, bioimpedance) and decreased testis volume (~5 mL, p<0.05). Total and free
testosterone and estradiol were markedly (>= 2-fold, p<0.001) increased while LH, FSH, urea, total and
LDL cholesterol and triglycerides were significant decreased. There were no significand change in
hemoglobin, PSA, HDL cholesterol, osteocalcin, leptin or IGF-1. Shoulder and knee strength (peak torque)
as measured by isokinetic and isometric dynamometry were not significantly increased nor was physical
activity (accelerometry) or gait and balance (modified Guralnik and FICSIT performance batteries) altered.
Anthropometric measures of skinfold thickness (biceps, triceps, subscapular, suprailiac and midthigh) and
circumferences (midarm, waist, hip and midthigh), including the waist-hip ratio, did not significantly
change. We conclude that 3 months treatment with thrice weekly recombinant hCG demonstrates sustained
androgenic effects on hormones and muscle mass, acceptable cardiovascular and prostate safety markers
but had no effect on muscle strength or physical functioning.

Clinical Science Symposium: Clinical Trials Symposium (9:30 AM-11:00 AM)

Presentation Date: Thursday, June 21, 2001; Time: 10:15 AM; Location: Ballroom 2




                                                    -5-
                                                      HOT TOPICS/CLINICAL TRIALS

S20-3
Additional background information:

Modern, educated men have lost faith in traditional tonics that allegedly prolong youth and life. Now they
have become bedazzled by the misty allure of hormones, the contemporary embodiment of the fabled
fountain of youth. The modern doctor now plays the part of its monopolistic dispenser, under siege by
patients craving relief from aging, and encouraged by the pharmaceutical industry and franchised clinics.
Definitive answers, however, can be provided only by properly conducted clinical studies.

hCG is a hormone that stimulates the testis to produce more testosterone and related steroids. Unlike other
methods of testosterone supplementation, hCG may be self-administered three times a week as a relatively
painless injection under the skin. Overdosage is unlikely because any testosterone produced is intrinsically
limited by the steroidogenic capacity of the testis. hCG is therefore a novel form of testosterone
replacement that has not previously been studied.

This study was supported in part by the National Health and Medical Research Council of Australia
(Biomedical Postgraduate Medical Research Scholarship to Peter Y. Liu) and by Serono Australia (supply
of r-hCG and support for the study).




                                                    -6-
                                                      HOT TOPICS/CLINICAL TRIALS

S20-4
DIETARY SOY CONTAINING PHYTOESTROGENS IMPROVES LIPID PROFILES BUT DOES
NOT ACTIVATE COAGULATION IN POSTMENOPAUSAL WOMEN
HJ Teede, FS Dalais, D Kotsopoulos, E Malan, BP McGrath, R Peverill. Department of Medicine, Monash
University, Melbourne, Australia

Background- Estrogen therapy activates coagulation and increases thrombosis, potentially negating other
beneficial cardiovascular effects. The soybean is rich in isoflavone phytoestrogens, which are ligands for
estrogen receptors. Potentially, these isoflavones may mimic estrogenic effects on the hemostatic system.
To address the effect of phytoestrogens on coagulation and fibrinolysis, we measured circulating markers
of these processes, in a double blind, placebo-controlled trial.
Methods: 42 healthy postmenopausal women, 50-75 years, received either soy protein isolate (40g soy
protein, 118mg isoflavones) or casein placebo, with measurements at baseline and 3months.
Results: Baseline characteristics were similar in the two groups. In the soy group, compared to placebo,
urinary phytoestrogens increased, accompanied by an improvement in lipid profiles. Soy improved LDL
cholesterol (-0.71±0.16 vs 0.27±0.14mmol/L, p<0.05), the LDL to HDL ratio (-0.34±0.1 vs
0.18±0.09mmol/L, p<0.005) and triglycerides (-0.29±0.09 vs 0.01±0.1mmol/L, p<0.05). Soy did not
increase markers of coagulation (factor VIIc; 0.09±0.02 vs 0.06±0.02U/ml, prothrombin fragments 1+2;
0.14±0.04 vs 0.11±0.05nmol/L and soluble fibrin;-0.76±0.6 vs 0.44±0.6µg/mL), plasma fibrinolytic
inhibitory activity (PAI-1; -0.18±0.26 vs -0.21±0.21U/mL) or fibrinolysis (D-dimer; 9.9±8.7 vs -
4.3±5.8ng/mL) compared to placebo. Furthermore no effect of soy versus placebo was noted on von
Willebrand factor which is involved in platelet adhesion (vWAg; 0.07±0.7 vs 0.03±0.07U/ml).
Conclusions: In healthy postmenopausal women, dietary soy protein containing phytoestrogens favorably
effect lipid profiles. However soy protein with phytoestrogens does not appear to activate coagulation or
fibrinolysis, suggesting no biologically significant estrogenic effects on the hemostatic system and
supporting a potential role in cardioprotection.

Clinical Science Symposium: Clinical Trials Symposium (9:30 AM-11:00 AM)

Presentation Date: Thursday, June 21, 2001; Time: 10:37 AM; Location: Ballroom 2




                                                   -7-
                                                      HOT TOPICS/CLINICAL TRIALS

S20-4
Lay explanation of abstract:

Although estrogen in hormone replacement therapy in postmenopausal women improves cholesterol levels
and may protect against the build up of plaque in the arteries and subsequent heart disease, estrogen also
activates blood clotting and increases the risk of thrombosis. This adverse effect may potentially negate
other beneficial effects of estrogen on the cardiovascular system. The soybean is rich in phytoestrogens,
which have been shown to attach to estrogen receptors and which may act like estrogen in the body. We,
along with other investigators, have previously shown beneficial estrogen-like effects of phytoestrogens on
blood cholesterol levels. Potentially, these phytoestrogens may also mimic estrogen effects and increase the
risk of blood clotting. To address the effect of phytoestrogens on the hemostatic system, which controls
blood clotting, we measured circulating markers reflecting activation of the clotting system in a double
blind, placebo-controlled trial.

Forty-two healthy postmenopausal women, aged 50-75 years, received either dietary soy protein isolate
(40g soy protein, 118mg isoflavones) or milk protein placebo, with measurements at baseline and 3
months.

In the soy group, compared to placebo, urinary phytoestrogens increased, demonstrating that these products
were absorbed from the diet. This increase in phytoestrogen excretion was accompanied by an improvement
in blood cholesterol levels. Soy improved the problematic LDL (―bad‖) cholesterol levels and the LDL-to-
HDL ratio, as well as the blood fat or triglyceride level. However, soy with phytoestrogens, unlike estrogen,
did not appear to adversely increase the activation of the blood clotting system. Phytoestrogens caused no
increase in markers involved in blood clot formation or breakdown compared to placebo.

In healthy postmenopausal women, dietary soy protein containing phytoestrogens favorably affect lipid
profiles, similar to the effects observed with estrogen. However soy protein with phytoestrogens does not
appear to activate blood clotting, which suggests there are no biologically significant estrogenic effects on
the clotting system and supports a potential role in cardioprotection, compared with estrogen.

The study was funded by the National Heart Foundation of Australia.




                                                   -8-
                                                       HOT TOPICS/CLINICAL TRIALS

S38-1
RESISTIN
M.A. Lazar1,2. 1Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia,
PA, United States, 2Penn Diabetes Center, University of Pennsylvania, Philadelphia, PA, United States

Obesity is a major risk factor for insulin resistance and type 2 diabetes, but the mechanism underlying this
association is not well understood. PPAR gamma is a nuclear hormone receptor that is highly abundant in
adipose tissue and a major transcriptional regulator of adipose gene expression. Ligands for PPAR gamma
such as thiazolidinediones are effective insulin sensitizing agents. We hypothesized that these drugs down-
regulate the expression of a gene that is normally expressed in adipose tissue and contributes to insulin
resistance. A differential screen based on this hypothesis led to the identification of resistin, an adipose-
specific secreted protein whose expression is down-regulated by antidiabetic drugs. Increased serum
resistin levels are observed in mouse models of obesity and studies suggest that this contributes to insulin
resistance. Resistin antagonizes insulin action in responsive cell lines. Thus resistin is an adipocyte-derived
modulator of insulin sensitivity that may link obesity, diabetes, and the actions of antidiabetic drugs.

Symposium: Hot Topics Symposium (9:30 AM-11:00 AM)

Presentation Date: Friday, June 22, 2001; Time: 10:00 AM; Location: Ballroom 2




                                                     -9-
                                  HOT TOPICS/CLINICAL TRIALS

S38-1
RESISTIN



No lay abstract was provided.




                                - 10 -
                                                       HOT TOPICS/CLINICAL TRIALS

S38-2
THE ADIPOCYTE-SECRETED PROTEIN ACRP30/ADIPOQ ACTS AS A HEPATIC INSULIN
SENSITIZER
AH Berg,1 T Combatsiaris,1 X Du,2 M Brownlee,2,3 PE Scherer.1,3 1Cell Biology, Albert Einstein College of
Medicine, Bronx, New York; 2Medicine and Pathology, Albert Einstein College of Medicine, Bronx, New
York; 3Diabetes Research and Training Center, Albert Einstein College of Medicine, Bronx, New York

Acrp30 is a circulating protein synthesized in adipose tissue. A single injection of purified recombinant
Acrp30 in mice, leading to a 2-3-fold elevation in circulating Acrp30 levels, triggers a transient decrease in
basal glucose levels. Hyperglycemia in ob/ob, NOD and streptozotocin-treated mice is also transiently
abolished after similar treatment. The effect of Acrp30 on glucose is not associated with an increase in
insulin levels. However, in isolated hepatocytes, Acrp30 increases the ability of sub-physiologic levels of
insulin to suppress glucose production. We thus propose that Acrp30 is a potent insulin-sensitizer linking
adipose tissue and whole body glucose metabolism.

Symposium: Hot Topics Symposium (9:30 AM-11:00 AM)

Presentation Date: Friday, June 22, 2001; Time: 10:00 AM; Location: Ballroom 2




                                                    - 11 -
                                                       HOT TOPICS/CLINICAL TRIALS

S38-2
Lay explanation of abstract:

Acrp30 is a protein uniquely made in fat cells and released into the blood stream. Even though this protein
was cloned a number of years ago, the physiological function of this molecule has remained a mystery.
Acrp30 is a very complex molecule that is difficult to synthesize in sufficient quantities. We have recently
succeeded in doing this and administered the protein to mice. In response to even minor changes to serum
levels of Acrp30, glucose levels transiently drop.

In a number of diabetic mouse models, injection of Acrp30 leads to a temporary normalization of glucose
levels. In cell culture, we have been able to demonstrate that Acrp30 acts as an insulin sensitizer in liver
cells and leads to an inhibition of glucose production in these liver cells. Insulin-resistant humans tend to
have lower Acrp30 levels in their serum. Once we elucidate the mechanism by which Acrp30 manipulates
the ability of liver cells to release glucose, we will have an opportunity to identify new pharmacological
targets. The ability to sensitize the liver to insulin will be an effective way to normalize glucose levels in
diabetic patients.

Funding was from the National Institutes of Health NIDDK (1R01-DK55758) and the American Diabetes
Association.




                                                    - 12 -
                                                                                               AGING

OR24-1
PREVENTION OF OVARIECTOMY-INDUCED BONE LOSS WITH VITAMIN E THERAPY
SJ Wimalawansa, SS Surendran, C Thota. Internal Medicine, University Osteoporosis Program,, Medical
School Galveston, Galveston, Texas

Bone loss is associated with both oophorectomy and natural menopause. All drugs currently approved by
the FDA for prevention and treatment of osteoporosis have anti-osteoclastic bone resorption activities, and
are expensive. Drugs that are cost-effective and efficacious in promoting bone formation are unavailable at
present. Because free radicals of oxygen are known to accentuate bone resorption, and vitamin E (d-alpha-
tocopherol) is a well-established antioxidant, we examined the effects of this compound in the prevention
of bone loss in an ovariectomized (OVXed) rat model. Results of eight-week treatment in 32-week old
female rats with tocopherol were compared to those with standard estrogen treatment (n=12, each group),
the standard therapy for estrogen-deficiency-induced bone loss, or to those of an estrogen and tocopherol
combination treatment.
Ovariectomy induced a predictable loss of bone mineral density (BMD) when compared to sham-operated
control animals. This BMD loss was prevented by treatment with estrogen (estradiol cypionate, 50 |micro|g,
once per week) as well as by tocopherol (50 IU/ kg, once per week; equivalent of 400 IU/day orally for
humans), or by the combination treatment. Groups of rats treated with estrogen and tocopherol had a
significantly higher femur weight compared to vehicle-treated OVXed rats. There was no difference in
BMD or femur weights between tocopherol-treated OVXed animals or sham-operated vehicle-treated
animals. Biomarker data confirmed that both estrogen and tocopherol therapies decreased serum
osteocalcin and urinary deoxypyridinoline levels suggesting a decreased of bone turnover. More studies are
necessary to understand the mechanisms of action of tocopherol in prevention of bone loss. However, our
data suggest that d-alpha tocopherol (vitamin E) could be used as a highly cost-effective alternative
preventative therapy for estrogen deficiency-induced bone loss.

Basic Science Oral: Aging (Oral Session) (1:00 PM-2:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 1:00 PM; Location: A 108




                                                  - 13 -
                                                                                                 AGING

OR24-1
Lay explanation of abstract:

Weakness of bones (osteoporosis) often develops after removal of ovaries, after natural menopause in
women, and as part of aging among elderly men. Numerous studies have been conducted in order to
develop effective therapies for this condition. Although highly effective treatments are now available (and
approved by the Food and Drug administration), these medications are prohibitively expensive and some
are associated with significant adverse effects. Vitamin E (d1-alphatocopherol) is a frequently used over-
the-counter medication in North America for preventing cardiovascular and host of other diseases. Vitamin
E also is a well-established antioxidant. We therefore examined the effects of this compound in an animal
model of estrogen-deficient bone loss.

Results of eight-week treatments with vitamin E were compared to those with estrogen (the standard
therapy for postmenopausal bone loss) in a rat model of estrogen-deficiency-induced bone loss. Estrogen-
deficiency-induced by removal of ovaries led to a predictable loss of bone mineral density. This loss of
bone mineral was completely prevented by treatment with estrogen (as expected), as well as by Vitamin E.
Bone weight (another measure of improvement of calcium retention in bones) significantly increased in
Vitamin E-treated and estrogen-treated rats, in comparison with the controls. These data suggest that
Vitamin E, at appropriate doses, can be used as an effective alternative therapy for estrogen deficiency-
induced bone loss. If this proves to be the case in humans, this will be 10 times less expensive than any
other established, FDA-approved medication for this disorder. It is possible that this widely available, less
expensive therapy could be used as an efficacious, cost-effective alternative or as an adjunct to hormone
replacement therapy to offset postmenopausal bone loss.

Additional background information:

The cost of therapy with equivalent doses of Vitamin E for humans is about $4/month. This compares with
FDA-approved therapies for osteoporosis, which vary from $28 to $80 per month. Furthermore, Vitamin E
at these doses has virtually no adverse effects on the body, while all other FDA-approved medications for
osteoporosis have significant adverse effects, which require frequent monitoring. This certainly adds to
the cost of managing these patients. However, the appropriate dosing of Vitamin E for prevention and
treatment of osteoporosis in humans has yet to be established.




                                                   - 14 -
                                                                                               AGING

OR24-6
DOWN REGULATION OF PROTEOLYSIS CONTRIBUTES TO INCREASED MUSCLE MASS
AND STRENGTH FOLLOWING TESTOSTERONE ADMINISTRATION TO HEALTHY OLDER
MEN
TJ Marcell,1 J Jiang,1 AA Ferrando,2 M Sheffield-Moore,2 RR Wolfe,2 CR Bush,3 BA Luxon,3 RJ Urban.1
1
  Internal Medicine, University of Texas Medical Branch, Galveston, Texas; 2Surgery, University of Texas
Medical Branch, Galveston, Texas; 3HBC&G, University of Texas Medical Branch, Galveston, Texas

Aging in men is associated with decreased muscle mass and strength (sarcopenia) and reductions in
circulating testosterone (T) levels. T administration increases lean body mass (LBM) and strength in
hypogonadal men. This study assessed the benefit of physiologic T replacement on LBM given to healthy
older men. We administered a varied dose of testosterone enanthate (TE) to maintain nadir levels to seven
relatively hypogonadal older (> 60 yr; serum total T < 480 ng/dL) men for six-months. LBM and muscle
volume were determined by DEXA and MRI, strength by one-repetition maximum (1-RM), and protein
synthesis and net protein balance were assessed by stable isotope infusion. TE administration increased
total T levels and hematocrit into the normal range for young men. Total and leg LBM, leg muscle volume,
and leg 1-RM strength increased. Net muscle protein balance was also improved due to a decrease in net
protein breakdown. Gene expression that may have contributed to the decrease in protein breakdown was
assessed using CLONTECH Human cDNA Microarray. Of the 3600 genes that were screened
simultaneously by hybridizing with [32P], approximately 15% were expressed. Of interest was an increased
expression of fast-type sarcomeric structural genes, including fast myosin heavy chain (2.0x), myosin light
chain 3 (1.2x), and fast-troponin I (1.3x). As well, significant down-regulation of proteolytic genes were
observed, including the 26S proteosomal subunits (1.5x), ubiqitin-E2 conjugating enzyme (1.4x), calpain 1
(2.1x) and 94 (1.4x) subunits, and cadherin 3 (1.9x). Insulin-like growth factor (IGF) binding protein 1
expression was also decreased (2.0x), whereas IGFBP-5 was increased 1.7 fold. These data taken together
suggest that androgen effects on skeletal muscle protein breakdown may involve the down-regulation of
Ca2+-mediated proteolysis, and will direct future studies to investigate the genetic mechanisms of muscle
loss.

Clinical Science Oral: Aging (Oral Session) (1:00 PM-2:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 2:15 PM; Location: A 108




                                                  - 15 -
                                                                                                AGING

OR24-6
Lay explanation of abstract:

Normal healthy aging in men is associated with deleterious changes in physical function, including
decreased muscle mass and strength (sarcopenia) and reductions in circulating testosterone (T) levels. T
administration increases lean body mass (LBM) and strength in young healthy and hypogonadal men.

This study was undertaken to assess the benefit of administering T to healthy older men and learn whether
it helped improve LBM and strength. We administered intra-muscular injections of testosterone enanthate
(TE) every two weeks to increase basal T levels in seven healthy, community-dwelling, older (> 60 yr) men
who had low T levels (serum total T < 480 ng/dL), until their T levels matched those commonly found in
young men. Whole body and regional muscle mass and the muscle size (volume) were determined by dual
energy x-ray absorptiometry (DEXA) and MRI, respectively. Muscle strength was measured by one-
repetition maximum (1-RM), and protein synthesis and net protein balance were assessed by the infusion of
stably labeled proteins.

After six months of TE administration, circulating T levels and blood hematocrit were increased into the
normal range for young men. Total and leg LBM, leg muscle volume, and leg strength were also positively
increased. The net turnover of muscle proteins, protein balance, also improved because of less total protein
breakdown following TE treatment.

Molecular events that may have contributed to the decrease in protein breakdown and improved muscle
mass were assessed by screening muscle biopsy samples for 3,600 genes simultaneously using a human
cDNA microarray. Of particular interest was an increased expression of muscle structural genes, including
the fast-type myosin heavy chain, and a down-regulation of genes involved in protein breakdown.
Mitochondrial genes were also up-regulated, which might contribute to improved muscle energy
production. These data considered together suggest that androgen effects on skeletal muscle protein
breakdown may involve the down-regulation of Ca2+-mediated proteolysis and improved fat utilization
that leads to increased muscle mass. Future studies on age-related sarcopenia should continue to
investigate the genetic mechanisms of muscle loss.

We would like to gratefully acknowledge the grant support of the Claude D. Pepper Older American
Independence Center for Aging Research at the University of Texas Medical Branch (UTMB).




                                                  - 16 -
                                                                                                  AGING


P1-418
RELATIONSHIP BETWEEN PLASMA GROWTH HORMONE, ANTIOXIDANTS AND
OXIDATIVE DAMAGE IN PREMATURE AND DELAYED AGING MICE
HM Brown-Borg, SG Rakoczy, MA Romanick, MA Kennedy. Pharmacology, Physiology & Therapeutics,
University of North Dakota School of Medicine, Grand Forks, North Dakota

Plasma growth hormone deficiency and growth hormone (GH) excess are positively and negatively
correlated with longevity in rodents, respectively. One emerging explanation for the observed difference in
life span is the associated antioxidant status. Ames dwarf mic e live more than one year longer than wild
type mice and exhibit an enhanced antioxidative defense capacity. In contrast, mice that overexpress GH
live half as long and exhibit a depressed antioxidative system compared to wild type mice. This study was
conducted to evaluate levels of hydrogen peroxide (H2O2), the contribution of non-enzymatic antioxidants
and to assess oxidative damage in mice with altered GH status and life span. Tissues from Ames dwarf,
PEPCK-bGH transgenic mice and corresponding groups of wild type mice were examined for
mitochondrial H2O2 generation, glutathione (GSH), glutathione disulfide (GSSG), protein carbonyl content
(marker of oxidative protein damage) and 8-hydroxy-2-deoxyguanine (marker of oxidative DNA damage).
The le vels of H2O2 generated in liver mitochondria were significantly lower in dwarf mice compared to
wild type controls in the presence of different substrates. Liver glutathione levels were elevated 24, 53, and
24% in dwarf mice at 3, 12 and 24 months of age, respectively, when compared to wild type mice.

Basic Science Poster: Aging (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                   - 17 -
                                                                                                AGING


P1-418
Lay explanation of abstract:

A progressive decline in physiological systems occurs in all organisms. A popular theory that explains this
decline involves accumulation of damage to cell contents (DNA, proteins, lipids) by toxic chemicals
(oxidants) generated during normal cellular metabolisms. Delay of this decline is associated with extended
life span. There is previous evidence suggesting that different levels of growth hormone (a hormone
produced in the brain) in the blood may contribute to differences in life span in rodents. Mice that lack
growth hormone (Ames dwarf) exhibit both delayed aging (living more than a year longer than normal
mice) and enhanced defenses against cellular oxidants. In contrast, mice with high levels of serum growth
hormone (GH transgenics) live half as long as normal mice and exhibit a depressed oxidant defense system.

This study was conducted to evaluate levels of hydrogen peroxide (a toxic chemical produced by normal
cellular metabolism), glutathione (an antioxidant), and cellular damage in liver and brain tissues from 3
kinds of mice: mice lacking growth hormone, those with excess growth hormone, and those with normal
growth hormone levels.

Levels of hydrogen peroxide were significantly lower in long-living, growth-hormone-deficient mice. The
antioxidant, glutathione, was 24-53% higher in mice lacking growth hormone at 3, 12, and 24 months of
age. Cellular damage (to proteins) in the brain and liver tissues was also lower in these mice. Protein
damage in the liver and brain was increased in mice with high serum growth-hormone levels at 3 and 12
months of age. The results of these experiments suggest that growth-hormone-deficient dwarf mice dispose
of toxic metabolic chemicals better than either animals with normal or excess levels of growth hormone. A
greater ability to eliminate hydrogen peroxide results in less cellular damage and extended life span.

Additional background information:

We are working towards determining the mechanisms responsible for aging. We use an animal that lives
50% longer than normal age-matched sibling mice. These dwarf (growth hormone deficient) animals
appear to eliminate toxic byproducts of metabolism better than normal mice. Growth hormone appears to
have a role in this difference.

The American Federation of Aging Research and the National Institute on Aging funded this research.




                                                   - 18 -
                                                                                              AGING

P1-419
CHRONIC ESTROGEN EXPOSURE-INDUCED LOSS OF ESTROUS CYCLICITY IS
ACCOMPANIED BY CHANGES IN HYPOTHALAMIC MONOAMINES
SM MohanKumar, BS Kasturi, PS MohanKumar. Department of Veterinary Pathology, College of
Veterinary Medicine, Michigan State University, East Lansing, Michigan

Aging is marked by significant changes in estrous cyclicity. We hypothesized that alterations in
monoaminergic activity due to chronic exposure of the hypothalamus to estrogen could contribute to these
changes. To investigate this hypothesis, adult female rats (3-4 months of age) were either sham-implanted
(control) or implanted with estradiol-17ß slow-release pellets(0.02µg/day) (experimental) for 30 days
subcutaneously. Estrous cyclicity was observed periodically. At 30 days after implantation, about 20-25%
of the estradiol-implanted animals became acyclic and were in the state of constant estrus (CE). Groups of
Control and Experimental animals were sacrificed at 1200 h when they were in the state of proestrus
[Control (CPE) as well as experimental (EPE)] or CE [Experimental (ECE)]. The brains were removed
quickly, frozen, sectioned and a number of areas including the medial preoptic area (MPA), arcuate nucleus
(AN) and the suprachiasmatic nucleus (SCN) were microdissected using the Palkovits' microdissection
procedure. They were analyzed for norepinephrine (NE), dopamine (DA) and serotonin (5-HT)
concentrations (pg/µg protein) using HPLC-EC. In the MPA, NE concentrations decreased significantly in
ECE (13.8±1.7) rats compared to CPE (29.7±3.8) and EPE (20.3±3.8) rats.

Basic Science Poster: Aging (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                 - 19 -
                                                                                                AGING

P1-419
Lay explanation of abstract:

One of the most pronounced changes during aging is the end of reproductive or estrous cycles in almost all
species. Reproductive functions are controlled by a small area of the brain called the hypothalamus. In
rodents, reduction in neurochemical activity in the hypothalamus is believed to be one of the main reasons
for the loss of estrous cycles. However, the reason behind the reduction in brain neurochemicals is not
known. Female rats have short bursts of estrogen during their estrous cycles. Since they have short estrous
cycles, they are exposed to these short bursts of estrogen every 4-5 days. We hypothesized that this
estrogen could cause age-related changes in hypothalamic neurochemicals.

To test this, we implanted young female rats with slow-release estrogen pellets that released 20 ng of
estrogen per day for 30 days. This caused 20-25% of the young animals to stop having reproductive cycles.
When we examined specific areas of the hypothalamus that control reproduction, we found reduction in
neurochemical activity in these areas. In short, chronic exposure to low levels of estrogen was capable of
producing changes in estrous cycles and brain neurochemicals very similar to those seen during aging in
rodents. This shows that estrogen could be a major reason for the age-related end of cycles.

Additional background information:

Recent studies have provided evidence that estrogen is neuroprotective in the number of areas of the brain
using various animal modes. In contrast, this study provides evidence that apart from its neuroprotective
role in other areas of the brain, estrogen could alter neurochemical activity in the hypothalamus to disrupt
estrous cycles. Further, because estrogen is used in hormone replacement therapy in postmenopausal
women, the detrimental effects of estrogen on hypothalamic neurochemicals could play an important role in
modulating other hormones as well.

This research was funded by NIH grant AG 05980 from the National Institutes of Health.




                                                  - 20 -
                                                                                         DIABETES

OR12-4
GLUCOSE-DEPENDENT INSULINOTROPIC PEPTIDE IS A GROWTH AND ANTI-
APOPTOTIC FACTOR FOR BETA (INS-1) CELLS BY PLEIOTROPIC SIGNALING
D Hoersch,1 A Truemper,1 K Truemper,1 H Trusheim,1 R Arnold,1 B Goeke.2 1Endocrinology, Philipps-
University, Marburg, Germany; 2Gastroenterology, Ludwig-Maximailians University, Munich, Germany

Activation of the G-protein coupled receptor for glucose-dependent insulinotropic polypeptide (GIP)
facilitates insulin-release from pancreatic beta-cells. In the present study, we examined whether GIP also
acts as a growth and anti-apoptotic factor in the beta-cell line INS-1. Here, we show that GIP
synergistically with glucose between 2.5 mM and 15 mM induced cellular proliferation and inhibited
glucose- and serum starvation induced apoptosis by activation of pleiotropic signaling pathways. GIP
stimulated the signaling modules of protein kinase A (PKA)/cAMP regulatory element binder (CREB),
mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI 3-K)/protein kinase B
(PKB) in a glucose and dose-dependent manner. In contrast, janus kinase 2 and signal transducer and
activators of transcription 5/6 pathways were not stimulated by GIP. Activation of PI 3-K by GIP and
glucose was associated with insulin receptor substrate (IRS) isoforms IRS-2 and Gab-1 and PI 3-K
isoforms p85alpha, p110alpha, p110beta and p110gamma. Downstream of PI 3-K, GIP stimulated PKB and
PKBbeta isoforms and phosphorylated glycogen synthase kinase-3, forkhead transcription factor FKHR
and p70S6K. Signaling modules activated by GIP were dependent on glucose metabolism and calcium
influx and were tightly linked by multiple sites of activating and inhibiting cross-talks between pathways.
These data indicate that GIP functions synergistically with glucose as a pleiotropic mitogenic and anti-
apoptotic growth factor for insulin-producing beta-cells which may play a role for metabolic adaptations of
insulin-producing cells during type II diabetes.

Basic Science Oral: Diabetes II (1:00 PM-2:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 1:45 PM; Location: A 205




                                                  - 21 -
                                                                                              DIABETES

OR12-4

Lay explanation of abstract:

Type 2 Diabetes is characterized by peripheral insulin resistance, which means that the body needs more
insulin to keep the blood sugar at a normal range. Furthermore, there is a malfunction of the insulin-
producing beta cells of the pancreas. During the course of the disease, pancreatic beta cells increasingly
fail to compensate for the insulin-resistance by elevated insulin-secretion.

Our group therefore is interested in identifying and characterizing growth factors that are specific for beta
cells with the goal to improve the function and expand the mass of beta cells. Glucose-dependent
insulinotropic polypeptide (GIP) is a hormone secreted by cells in the gut. GIP senses glucose and fat load.
GIP acts on beta cells at specific receptors and facilitates insulin-secretion at elevated blood glucose-levels.
While this is a short-term effect and is lost in type 2 diabetes, we looked at putative long-term effects of
GIP on beta cells.

Our group found that GIP acts as a growth factor and also inhibits programmed cell death. Interestingly,
the receptor for GIP is able to activate several signal-transduction pathways in beta cells, which are also
tightly linked to each other. We hope that our results will lead to the development of drugs improving the
function and mass of beta cells during type 2 diabetes.

Additional background information:

Our group at the university of Marburg is small and mainly composed of a principal investigator working
with gifted and interested medical students. We perform most of our experiments using a cell-culture
model and apply a range of cell-biological and molecular biological techniques.

Our work has been supported in part by the Deutsche Forschungsgemeinschaft (DFG), the German
counterpart of the NIH.




                                                    - 22 -
                                                                                          DIABETES

P1-276

REGULATION OF PANCREATIC BETA CELL GROWTH AND SURVIVAL BY THE
SERINE/THREONINE PROTEIN KINASE AKT1
RL Tuttle,1 NS Gill,2 W Pugh,3 J Lee,3 B Koeberlein,4 EE Furth,2 KS Polonsky,3 A Naji,4 MJ Birnbaum.1
1
  HHMI/Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; 2Pathology & Laboratory
Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; 3Medicine, Washington University, St.
Louis, Missouri; 4Surgery, University of Pennsylvania, Philadelphia, Pennsylvania

Recently, the insulin signal transduction pathway has been implicated in both the regulation of hormone
secretion from the beta cell in mammals as well as the determination of cell and organ size in Drosophila
melanogaster. A known intermediary of many insulin-mediated events, the serine, threonine kinase Akt
(also known as PKB) has yet to be characterized with regards to beta cell regulation and growth. To address
these issues, we generated transgenic mice expressing, under the control of the rat insulin promoter, a form
of Akt1 (myr-Akt1) rendered constitutively active by amino-terminal myristoylation. These mice exhibited
a marked increase in islet mass caused by both an increase in islet number and an increase in islet size.
Interestingly, the transgene also caused an approximate doubling in beta cell diameter without major effects
on the other endocrine cells of the islet. These animals exhibited marked glucose tolerance while
maintaining peripheral insulin sensitivity. In addition, pancreata from the transgenic mice demonstrated a
typical insulin secretory response to a glucose infusion applied over time. Lastly, and perhaps most
interestingly, these animals were able to prevent low dose streptozotocin-induced diabetes mainly through
the inhibition of apoptosis. These studies implicate a novel role for Akt in mediating both beta cell growth
and survival.

Basic Science Poster: Diabetes I (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 23 -
                                                                                              DIABETES

P1-276
Lay explanation of abstract:

After a meal, the body utilizes some nutrients and stores others as fat, protein, and starch. Insulin regulates
this process by stimulating uptake of sugar into muscle and fat cells. Beta cells, located in the islets of
Langerhans of the pancreas, secrete insulin in a highly regulated process. Poor insulin production or poor
insulin response result in diabetes, for which there still is no cure. Most patients require lifelong treatment
with insulin or other medications.

Our study elucidates the role in the pancreatic beta cell of an important mediator of the insulin response, a
molecule known as Akt. The study also evaluates the potential therapeutic prospects of Akt. We generated
transgenic mice that express a constitutively active form of Akt exclusively in the pancreatic beta cells of
the islets. These animals exhibited a marked increase in islet mass caused by both an increase in islet
number and an increase in islet size. Interestingly, the size of the beta cell also increased significantly.
Given the need to generate sufficient human beta cells for transplantation, understanding the mechanism
through which Akt increases beta cell mass has potential ramifications for diabetic therapeutics.

Importantly, the beta cells of the transgenic mice were functional and had a typical insulin secretory
response to an infusion of glucose (form of sugar). Additionally, the insulin responsiveness of the
appropriate tissues was maintained in the transgenic mice. To further evaluate a role for Akt in preventing
diabetes, we challenged our transgenic mice with a drug that caused beta cell destruction and then diabetes.
The transgenic animals prevented cell loss and subsequent hyperglycemia; the control group of mice in the
litter went on to have hyperglycemia. Because a hallmark of diabetes is beta cell destruction, this study has
clinical importance. Overall, these studies implicate a novel role for Akt in mediating beta cell growth and
survival.

Additional background information:

Diabetes is an increasingly prevalent disease in the United States. One of its hallmarks is the destruction or
dysfunction of the insulin-producing cells of the pancreas, the beta cells. Recent research has focused on
the intracellular mediators of the insulin response. Interestingly, one of the major effectors of this response
is the protein kinase, Akt, which has yet to be characterized with regards to beta cell signaling,

Funds provided by the Howard Hughes Medical Institute and the National Institutes of Health allowed us to
study the role of Akt in beta




                                                    - 24 -
                                                                                            DIABETES

P1-296
IGF-I SUPPRESSES GLUCOSE-MEDIATED ACTIVATION OF CASPASES-3 AND -9 IN
NEURONS
GM Gehrke, JW Russell, EL Feldman. Neurology, University of Michigan, Ann Arbor, Michigan

High glucose initiates a caspase cascade leading to cellular apoptosis and, in the nervous system of diabetic
animals, neuronal loss in complications prone tissue. We have reported that glucose mediates a dose-
dependent apoptosis in 30-50% of human SH-SY5Y cells, a well established model of neuronal growth and
differentiation. The current study examines the activation sequence of specific caspases by glucose in an
effort to identify which caspases may serve as future therapeutic targets in the treatment of complications
prone tissues. The initiator caspases -8 and -9 activate the downstream effector caspases, including -3, -6,
and -7. Western blotting after glucose treatment of SH-SY5Y cells shows no activation of caspase-8. In
contrast, caspase-9 and caspase-3 cleavage, a measure of activity, is detected after 2 h in cells treated with
high glucose. Dominant negative (DN) caspase-8 and caspase-9 were transfected into SH-SY5Y cells.
Caspase-8 DN cells have similar cleavage patterns as untransfected cells. Caspase-9 DN cells exhibit little
caspase-9 cleavage and only 5.5% undergo glucose induced apoptosis. Yet under this same experimental
paradigm, caspase-3 cleavage was still present. This suggests that glucose mediated death is dependent on
caspase-9 activation and that caspase-3 activation is upstream of caspase-9 cleavage. Collectively our data
support the idea that glucose kills neurons in a caspase-8 independent, caspase-9 dependent manner. We are
currently examining the role of caspase-inhibition on glucose-mediated apoptosis in neurons. Supported by
NIH NS36778, NS38849, the Program for Understanding Neurological Diseases (PFUND) and the JDRF
Center for the Study of Complications in Diabetes.

Basic Science Poster: Diabetes I (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                   - 25 -
                                                                                             DIABETES

P1-296

Lay explanation of abstract:

Apoptosis is a process by which a cell commits suicide. Apoptosis occurs in normal development, but has
also been implicated in Alzheimer‘s disease and diabetes. Understanding how a cell undergoes apoptosis
when exposed to toxic substances is vital to understanding the progression of these diseases, as well as
identifying possible therapeutic interventions. One mechanism by which cells can undergo apoptosis
occurs through the activation of proteins called caspases. A cell under toxic stress can activate caspases,
which then cut and destroy other components of the cell leading to cell death.

Our laboratory has established that exposure of SH-SY5Y cells, a human neuronal cell line, to high levels
of glucose causes apoptosis. However, the specific caspases involved in this process are unknown.
Therefore, we wanted to determine which caspases were important for glucose-induced apoptosis in SH-
SY5Y cells, as well as the specific order in which these caspases were activated. Many scientists think that
caspase-8 is important in normal development and chemical-induced cell death. In contrast, caspase-9 is
reported to be important in stress and injury-induced cell death. When SH-SY5Y cells are exposed to high
levels of glucose causing cell death, caspase-3 and caspase-9 are activated, while caspase-8 is not.

Further experiments utilized dominant negative (DN) forms of caspase-8 and caspase-9. DN forms are
pieces of DNA that are placed inside cells and are able to interfere with the normal cellular activity of the
DNA being studied. In this case, DN forms of caspase-8 and caspase-9 were placed inside the SH-SY5Y
cells to interfere with the function of caspase-8 (caspase-8 DN cells) and caspase-9 (caspase-9 DN cells)
respectively. Under high glucose exposure, the caspase-8 DN cells showed the same level of caspase-8
activity when compared to normal SH-SY5Y cells that did not contain the caspase-8 DN form. The
caspase-9 DN cells when exposed to high levels of glucose also showed very little activation of caspase-9
and showed a decrease in cell death. However, the caspase-9 DN cells do reveal activation of caspase-3.
These data suggest that in these cells, caspase-3 is activated first, followed by activation of caspase-9,
which causes cells to die.

Research was funded under grant from NIH NS36778, NS38849, the Program for Understanding
Neurological Diseases (PFUND), and the JDRF Center for the Study for the Complications in Diabetes.




                                                    - 26 -
                                                                                          DIABETES

P1-352
UNCOUPLING OF OBESITY, DIABETES, AND HYPERCORTISOLISM IN POMC NULL
MUTANT MICE
BR Robinson,1,2 J Costa,3 S Bui,2 P Reed,2 U Hochgeschwender,2 MB Brennan.3 1Endocrinology, The
University of Oklahoma-HSC, Oklahoma City, Oklahoma; 2Developmental Biology, Oklahoma Medical
Research Foundation, Oklahoma City, Oklahoma; 3Eleanor Roosevelt Institute, Denver, Colorado

Obesity is commonly associated with insulin resistance, i.e. decreased response to a given dose of insulin.
This insulin resistance frequently precedes Type 2 diabetes. The mechanisms linking obesity and insulin
resistance are not known although studies on the mechanistic basis of obesity-induced insulin resistance
have revealed numerous potential links, making a single mechanism for explaining insulin insensitivity
unlikely.Obesity is also commonly associated with the presence of normal or elevated levels of
glucocorticoid. Glucocorticoid levels in most obese patients are at the higher end of the normal spectrum or
slightly elevated. Cortisol acts in an insulin-antagonistic fashion. Long-term elevation of cortisol limits
glucose utilization and stimulates glucose production. The majority of patients with Cushing's disease
develop insulin resistance, probably due to the high levels of cortisol.
We recently created a genetic mouse model of obesity, the POMC null mouse, which lacks all POMC-
encoded peptides and, as a consequence, lacks adrenal glands. Subsequently, POMC null mice have no
corticosterone and almost no epinephrine. While POMC null mutants are obese, they do not develop
diabetes. They have normal glucose and insulin levels in both the fed and fasted state, and they do not
develop insulin resistance and hyperglycemia over their lifespan. Further, they appear to be insulin
"hypersensitive", rather than insulin resistant, developing fatal hypoglycemia after insulin challenge.
POMC null mice are obese, yet they do not have hyperinsulinemia or type II diabetes. And POMC null
mice are obese despite lack of corticosterone. This mouse model shows that obesity, hypercortisolism and
diabetes are not mutually inclusive. At the same time, it offers the unique opportunity to dissect the
individual effects of corticosterone and obesity on glucose homeostasis.

Basic Science Poster: Diabetes II (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                   - 27 -
                                                                                            DIABETES

P1-352
Lay explanation of abstract:

Type 2 diabetes mellitus is a disease that affects approximately 6.6% of Americans and requires millions of
health care dollars per year. In type 2 diabetes the body becomes resistant to insulin, preventing tissues
from absorbing glucose from the blood stream. Also, type 2 diabetes is very often associated with obesity.
Obese type 2 diabetics have elevated levels of adrenal steroids (cortisol) in their blood.

It has been difficult to determine the exact role of obesity and body steroids in the development of diabetes
and the body's ability to use glucose correctly because these pathways are generally linked. Recently, we
have developed a mutant mouse model that is obese, not diabetic and has no blood steroids. Although this
does not prove that type 2 diabetes, cortisol levels and obesity are completely unrelated it does allow us to
focus on these aspects individually. We can now divide the development of this disease into separate
elements; that is, the complications of diabetes can be studied independently of the complications of
obesity. By comparing these mice to other mouse models of obesity and diabetes we may be able to
determine how these factors result in the development of type 2 diabetes in obesity.

Funding for this research comes from the Eleanor Roosevelt Institute, the Oklahoma Medical Research
Foundation, and the Oklahoma Center for Advancement of Science and Technology.




                                                   - 28 -
                                                                                          DIABETES

P1-368
RESCUE OF DIABETES DUE TO INTRAUTERINE GROWTH RETARDATION IN RATS
DA Stoffers,1 C Li,2 BM Desai,1 CA Stanley,2 RA Simmons.3 1Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania; 2Pediatrics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania;
3
  Neonatology, University of Pennsylvania, Philadelphia, Pennsylvania

Intrauterine growth retardation (IUGR) is a common complication of pregnancy and is associated with an
increased incidence of type 2 diabetes in adulthood. A rat model of IUGR, induced by bilateral uterine
artery ligation at 19 days of gestation, develops diabetes with progressive dysfunction in both insulin
secretion and insulin action. β-cell proliferation and PDX-1 mRNA levels of IUGR islets are significantly
lower than in controls, leading to a reduction in β-cell mass by 7 weeks of age. Exendin-4 (Ex-4) is a
glucagon-like-peptide-1 analog that stimulates β-cell neogenesis and proliferation. We hypothesized that
treatment with Ex-4 in the newborn period would increase β-cell mass in IUGR animals, improve glucose
tolerance, and increase glucose-stimulated insulin secretion. Sham-operated control (4 litters) and IUGR
rats (6 litters) were treated with Ex-4 (1 nmol/kg sc) or saline daily on postnatal days (PD) 0-6. Glucose
tolerance on PD10 was impaired in saline-treated IUGR rats compared to saline-treated controls (337±42
vs. 235.5±14 mg/dL at 60 min), and improved in Ex-4-treated IUGR rats (274±32.5mg/dL at 60 min).
Pancreatic minces were harvested on PD17, and perifusion studies were performed. Glucose concentration
in the perfusate was increased from 0 to 50 mM over 1 hour and secreted insulin was measured by RIA.
Basal insulin secretion was elevated in IUGR minces compared to control (2.2 vs. 0.5 ng/pancreas/min).
Ex-4 IUGR minces showed a reduced basal secretion rate (1.2 ng/pancreas/min) and ~40% increased
maximal secretion compared to saline-treated IUGR minces (7 vs. 5 ng/pancreas/min). At PD14, β cell
proliferation was reduced in IUGR rats compared to controls (4.9±0.5% versus 0.7±0.3% BrDU+ insulin+
cells). This was partially restored by Ex-4 treatment (2.9±0.5%). These results indicate that a period of
exposure to Ex-4 in the newborn period results in improved glucose tolerance and insulin secretion and
enhanced β cell proliferation. The implications of neonatal Ex-4 treatment in the IUGR rat for maintenance
of β cell mass and long-term protection from diabetes will be discussed.

Basic Science Poster: Diabetes II (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                   - 29 -
                                                                                             DIABETES

P1-368
Lay explanation of abstract:

Researchers have discovered that health problems in fetuses may lead to serious disease much later in life.
This research holds out the promise that improving the health of fetuses may have long-term health
benefits for many adults. Diabetes is a disease with roots in abnormal fetal growth and development. For
example, babies that are born too small often suffer from diabetes when they grow to adulthood.

One cause of such growth-retardation in the fetus is insufficient nutrient supply from the mother, which
researchers have speculated may cause permanent damage to the pancreas. A healthy pancreas is necessary
to produce insulin, a critical hormone that lets the body take nutrients from the bloodstream into cells of the
body. We have studied this process in the rat, in which fetal growth retardation can be induced by a simple
operation, during which the blood supply from the mother rat to the rat fetus is partially cut off. Growth-
retarded rat fetuses have a smaller pancreas that produces less insulin than normal.

We treated growth-retarded fetuses and normal fetuses with exendin-4 (Ex-4), a peptide hormone analog
that stimulates insulin secretion as well as growth of the insulin-producing cells of the pancreas. Our
hypothesis was that a brief treatment with Ex-4 right after birth could reverse some of the damage to the
pancreas in the growth-retarded fetuses. Six litters of growth-retarded rats were treated with Ex-4 on days
0-6 of life, and their pancreatic growth and function were followed at 2 weeks and 2 months of age. We
found that Ex-4 improved the function and growth of the pancreas, and that the effect appeared to be long
lasting. Additional studies will be required to fully uncover the degree and mechanisms of the observed
improvement in glucose tolerance. The results suggest that Ex-4 and other drugs may be used to treat
growth-retarded fetuses to possibly prevent or improve the effects of diabetes in adults.

These studies were supported by research grants from the Juvenile Diabetes Foundation and NIDDK (to
Doris Stoffers) and the American Diabetes Association and NIDDK (to Rebecca Simmons).




                                                    - 30 -
                                                                            GENE EXPRESSION

OR2-5
MECHANISM OF HISTONE H1-MEDIATED REPRESSION OF ESTROGEN RECEPTOR
TRANSCRIPTIONAL ACTIVITY
E Cheung, AS Zarifyan, WL Kraus. Department of Molecular Biology and Genetics, Cornell University,
Ithaca, New York

The estrogen receptor (ER) is a ligand-regulated transcriptional activator protein that belongs to the steroid
hormone family of nuclear receptors. We have previously shown that significant ligand-dependent
transcription by ER can only be observed when the estrogen-responsive DNA template is packaged into
chromatin containing the four core histones (i.e., H2A, H2B, H3, and H4). Thus, chromatin is an integral
part of ligand-regulated transcription by ER. In eukaryotic nuclei, chromatin also contains linker histones
(e.g., histone H1) that bind to the linker DNA between nucleosomes, leading to the formation of compact
higher order chromatin structures. Currently, the role of H1 and higher order chromatin structure in ER-
mediated transcriptional regulation is unclear. Thus, we have used a biochemical approach to examine a
potential role for H1 in ER transcriptional activity. More specifically, we have used an in vitro chromatin
assembly and transcription system that recreates ER-mediated transcription with chromatin templates.
Using purified recombinant ER alpha, as well as H1 purified from insect cells or calf thymus, we have
found that H1 is a potent inhibitor of ER-mediated transcription. In DNase I footprinting and nucleosome
disruption assays, we have found that H1 does not inhibit transcription by causing the displacement of ER
from its cognate DNA binding site, nor does it inhibit ER-mediated chromatin remodeling. Interestingly, in
single round transcription experiments, we observed that H1 inhibits ER activity by inhibiting transcription
initiation. However, H1 does not inhibit the stimulation of multiple rounds of transcription by ER (i.e.,
transcription reinitiation). Thus, H1-mediated repression of ER transcriptional activity is directed at the
initiation stage of transcription, perhaps by restricting preinitiation complex formation at the promoter.
Together, our findings suggest that histone H1, and possibly higher order chromatin structure, can have
specific effects on distinct aspects of ER-mediated transcription.

Basic Science Oral: Gene Expression I (1:00 PM-2:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 2:00 PM; Location: A 205




                                                    - 31 -
                                                                             GENE EXPRESSION

OR2-5
Lay explanation of abstract:

Estrogens are small molecules used to transmit signals from one part of the body to another. They play
important roles in the tissues in which they act, including the reproductive tract, nervous system, and bone.
Within us, estrogens are bound to proteins called estrogen receptors (ERs). ERs are activated by estrogens
and work in conjunction with other proteins, called coregulators, to turn genes on and off. Antiestrogens,
such as tamoxifen, are drugs that also bind to ERs. Antiestrogens block estrogen actions in the body and are
used as anti-tumor agents in diseases like breast cancer. In our work, we are interested in gaining a better
understanding of how estrogen and antiestrogens exert their gene regulatory effects through ERs and
coregulators. Because it is difficult to unravel molecular mechanisms that occur in the cell, we are studying
the regulation of estrogen receptor activity by estrogens and antiestrogens in a test tube (i.e., in vitro).

Specifically, we are using an in vitro system that accurately recreates the activity of estrogen, antiestrogens,
ERs, and coregulatory proteins on mini genes assembled with histone proteins into chromatin (chromatin
assembled DNA is the physiological state of genes inside of the cell). Using this biochemical assay, we
have found that a protein called histone H1 is a potent inhibitor of estrogen-mediated transcription.
Furthermore, it appears that this protein acts at a very specific point in the transcription process. In
summary, the results from these studies have provided us a better understanding of how estrogen actions
are regulated. The findings from these in vitro studies may lead to new ways to target ERs with drugs for
the treatment of breast cancer and other human diseases.

This research was funded by Burroughs Wellcome Fund and the NIH (# DK58110) to W. Lee Kraus and by
the Susan G. Komen Breast Cancer Foundation to Edwin Cheung.




                                                    - 32 -
                                                                          GENE EXPRESSION

OR13-2
TWO PROGESTERONE RECEPTOR (PR) ISOFORMS: DIFFERENTIAL GENE REGULATION
IN BREAST CANCER CELLS
JK Richer, BM Jacobsen, NG Manning, KB Horwitz. Department of Medicine, University of Colorado
Health Sciences Center, Denver, Colorado

Two isoforms of PR, PR-B and PR-A, exist naturally in tissues. PR-B are identical to PR-A but have 164
extra amino acids containing a strong activation function, AF3, at the N-terminal end. The two receptors
function differently in vitro, and breast cancers have widely divergent ratios of PR-A:PR-B. We now
demonstrate that the two PRs regulate different subset of genes in human breast cancer cells. To study
differential gene regulation by the two PRs, we utilize a model system in which PR-negative cells (T47D-
Y), were cloned from PR-positive wild-type T47Dco cells, then engineered to stably express either PR-B
(T47D-YB) or PR-A (T47D-YA) at equal levels to each other and to wild-type cells. Thus, PR-B vs PR-A
dependent transcription can be studied in a tightly controlled manner with identical cellular background and
basal levels of gene expression. YB, YA, and Y cells, were each treated with or without 10nM progesterone
for 6 hrs, in triplicate, time-separated, experiments. cDNAs derived from the 6 sets were hybridized to
AffymetrixTM microarray HFL6800 gene chips. Cluster analysis was performed using GeneSpring TM,
identifying patterns of statistically significant gene regulation. We have identified over 100 genes not
previously known to be PR regulated. Key genes were confirmed by RT-PCR, northern or western blotting.
66 genes are more than 2-fold upregulated uniquely by PR-B, 3 by PR-A, and 8 by both, clearly indicating
that the two PRs have entirely different functions. Other gene subsets are differentially downregulated.
Major regulated gene clusters are involved in steroid metabolism, cell adhesion, signal transduction,
calcium binding and transcriptional regulation. In sum, we can now distinguish genes that require AF3 and
are regulated uniquely by PR-B, from those that do not require AF-3 and are regulated by both PRs.
Furthermore, although PR-B are ~10-fold more active, we have identified a small number of genes
uniquely regulated by PR-A. Surprisingly, we also find a subset of genes regulated by PRs in a ligand-
independent manner. These studies have implications for discovery of progestins that activate only one PR
isoform, and for measurement of PRs in breast cancer.

Basic Science Oral: Gene Expression II (1:00 PM-2:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 1:15 PM; Location: C 207




                                                  - 33 -
                                                                           GENE EXPRESSION

OR13-2
Lay explanation of abstract:

Breast cancer is a disease of women, predominantly because of the impact that the ovarian hormones have
on growth and development of the breast. The two major ovarian hormones are estradiol and progesterone.
Presence of ―receptors‖ for estradiol and progesterone in breast cancers tells the oncologist whether a
particular tumor will respond to hormonal treatments and whether that tumor is likely to have a good
prognosis.

This study focuses on progesterone and progesterone receptors (PRs). In order to understand how
progesterone impacts the normal breast or breast cancers, one needs to understand which genes
progesterone turns on and off in the breast. Study of this has been difficult in the past, because
progesterone signals through two forms of PRs – called ―A‖ and ―B‖. We have developed breast cancer
cells that allow us to study the two PRs separately. These cells were used to screen 5,800 human genes for
their ability to be regulated by progesterone, through either the ―A‖ or ―B‖ receptors. We find, remarkably,
that the two PR receptors control different subsets of genes. These genes fall into interesting groups,
including ones involved in angiogenesis (development of blood vessels), in tumor metastasis, in cell growth
and apoptosis, and other functions.

Our first significant finding was that some breast cancers are ―A-rich‖ and others are ―B-rich‖. Our data
suggest that these two classes of breast cancers are likely to be biologically different. Our results could
lead to new methods to screen breast cancers for their ―A‖ vs. ―B‖ type. It is possible that one receptor
type is more harmful than the other. Our method could be used to fingerprint a breast cancer precisely for
―good‖ or ―bad‖ progesterone receptors. If this happens, the genes, not the receptors, will be measured as
markers of hormone response or prognosis.

Secondly, the genes we have discovered could be used to screen for new progesterone-like hormones,
which could specifically target one or the other PR receptor. For example, such hormones might, in theory,
suppress the activity only of the more harmful form of PR receptors. (For related reasons, such new
progestational hormones could also be useful for oral contraception or for hormone replacement therapy at
menopause.

This research was funded in part by the National Institutes of Health, the U.S. Army Medical Research
Command, the Komen Foundation, and the National Foundation for Cancer Research. The University of
Colorado has submitted a patent for use of these genes.




                                                   - 34 -
                                                                 GnRH-GONADOTROPINS

P3-473
NOVEL MECHANISMS FOR ENDOCRINE-DISRUPTING EFFECTS OF ENVIRONMENTAL
TOXICANTS: DIRECT EFFECTS ON GNRH NEURONS
AC Gore. Neurobiology, Mount Sinai School of Medicine, New York, New York

Environmental toxicants profoundly affect growth and developmental processes. However, the mechanisms
by which these toxicants exert these effects are not well-understood. It is likely that hypothalamic
gonadotropin-releasing hormone (GnRH) neurons, which integrate inputs and outputs to and from the
nervous and reproductive systems, mediate effects of environmental toxicants. Two organochlorine
pesticicides (methoxychlor and chlorpyrifos), and two PCB mixtures (Aroclor 1221 and Aroclor 1254)
were tes ted for their effects on GnRH gene expression in the hypothalamic GT1-7 cells, which have a
neuronal phenotype and synthesize and secrete GnRH. The toxicants chosen persist in New York City and
other urban environments and are relevant to human exposures.GT1-7 cells were treated with toxicants for
24 h, and GnRH gene expression and biosynthesis were quantified. In other experiments toxicants were co-
administered with the estrogen receptor antagonist, ICI 182,780 (ICI).
Both organochlorine pesticides had significant effects on GnRH gene expression. These effects were not
consistently blocked by ICI. Chlorpyrifos and methoxychlor also slightly stimulated peptide levels, and this
effect was blocked by ICI. These results indicate direct effects of organochlorine pesticides on GnRH
neurons, at a number of diverse biosynthetic levels.
For the PCBs, Aroclor 1221 was stimulatory to GnRH gene expression, particularly at post-transcriptional
levels. Aroclor 1221 stimulated an increase in GnRH peptide levels, suggesting a post-translational
regulation of GnRH biosynthesis as well. Aroclor 1254 was predominantly inhibitory to GnRH gene
expression, both at transcriptional and post-transcriptional levels, although at low doses, Aroclor 1254
stimulated GnRH mRNA by a post-transcriptional mechanism, and Aroclor 1254 did not alter GnRH
peptide levels. Effects of Aroclor 1221 on GnRH peptide levels were blocked by ICI, indicating that these
effects are mediated by the classical estrogen receptor alpha and/or beta. These results demonstrate a novel
mechanism for effects of environmental toxicants, directly on GnRH gene expression, and indicate a
hypothalamic level for endocrine disruption by these environmental toxicants.

Basic Science Poster: GnRH Neurons (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 35 -
                                                                    GnRH-GONADOTROPINS

P3-473
Lay explanation of abstract:

Endocrine-disrupting chemicals (EDCs) are toxic substances in the environment that can cause
developmental abnormalities. EDCs have been shown to have adverse effects on the reproductive system
and to cause neurological deficiencies.

We hypothesized that a group of cells in the brain that synthesize and release the hormone gonadotropin-
releasing hormone (GnRH) may mediate some of the neurological and reproductive effects of EDCs. The
cells producing GnRH are the key regulators of the reproductive endocrine system. While these cells play
an endocrine function, they are neurons, receiving and integrating inputs and outputs from other neurons in
the central nervous system. Thus, the neuroendocrine GnRH cells are a likely point of integration of signals
from EDCs, as their outputs affect the reproductive system as well as the brain. However, this has not
previously been explored.

We administered EDCs to a GnRH neuronal cell line, GT1 cells, in order to determine whether these
chemicals have direct effects on the GnRH system. We tested substances in recent use or that persist in the
environment. All substances are relevant to the urban New York City community. The choice of toxicants
was also intended to complement ongoing studies in the community that are measuring human exposures,
past and present, and designing interventions to reduce toxicant exposures.

Four toxicants were examined in our studies: two organochlorine pesticides (methoxychlor and
chlorpyrifos) and two PCB mixtures (Aroclor 1221 and Aroclor 1254) were administered to the GT1 cells
for 24 hours. Effects of the toxicants on GnRH gene expression and release were measured in order to
determine whether these toxicants had direct effects on these critical neuroendocrine cells.

We found that all four toxicants exerted potent effects on the GT1 cells. At high doses, they were
neurotoxic. At lower doses more subtle but significant effects were observed, including increases or
decreases in GnRH gene transcription, mRNA levels, and GnRH release, depending upon the dose and the
toxicant.

These data demonstrate for the first time that EDCs have direct effects on the GnRH system. The results
suggest that this may be an important and novel mechanism through which environmental toxicants disrupt
reproductive and neurological development. These results have implications for humans who have been
exposed to environmental toxicants and provide a model for testing effects of further substances such as
pesticides, industrial chemicals, and plastics, all of which may have endocrine-disrupting effects.

Additional background information:

In order to better understand the significance of the findings, it is important to understand a little about the
gonadotropin-releasing hormone (GnRH) neurons. These are a group of about 1000 neurons located in the
base of the brain (the hypothalamus) that synthesize and secrete the GnRH peptide. The GnRH molecule
regulates the rest of the reproductive axis, including the pituitary gland and the gonads (ovary in female,
testis in males). In the absence of GnRH, reproduction cannot occur, as is the case in the natural human
mutation Kallmann‘s syndrome, in which GnRH neurons do not develop normally. Because the GnRH
neurons can integrate information between the central nervous system and the reproductive endocrine axis,
we hypothesized that effects of endocrine-disrupting chemicals (EDCs), which exert their effects on both of
these biological systems, may be mediated at the level of the GnRH neuron. Therefore, the purpose of the
present experiments was to test effects of EDCs on the physiology and molecular biology of GnRH cells.

This research was funded by NIH Children‘s Environmental Health Center Grant (1 P50 ES09584-01) from
the NIEHS/EPA.



                                                    - 36 -
                                                                        GROWTH FACTORS

OR36-4
SYSTEMIC ADMINISTRATION OF IGF BINDING PROTEIN-4 (IGFBP-4) INCREASES BONE
FORMATION PARAMETERS IN MICE BY INCREASING IGF BIOAVAILABILITY VIA AN
IGFBP-4 PROTEASE DEPENDENT MECHANISM
N Miyakoshi, X Qin, Y Kasukawa, AK Srivastava, DJ Baylink, S Mohan. MDC, JL Pettis VA Medical
Center, Loma Linda, California

IGFBP-4, one of the most abundant IGFBPs produced by bone cells, is a potent inhibitor of IGF actions in
vitro. Accordingly, local administration of IGFBP-4 inhibited IGF-I-induced increase in bone formation
(BF) parameters in mice. Surprisingly systemic administration of IGFBP-4 at pharmacological doses
caused a significant increase in BF parameters. Based on the findings that IGFBP-4 administration caused a
shift in circulating IGF-I from the 150 kDa fraction which cannot cross the vascular endothelium into 50
kDa IGFBP-4+IGF complex and that mouse serum contains an IGF-dependent IGFBP-4 protease capable
of degrading IGFBP-4 to release IGFs, we proposed that the mechanism by which IGFBP-4 increases BF
may involve increased IGF bioavailability via proteolysis of IGFBP-4. To evaluate this hypothesis, we
produced wild-type (WT), protease-resistant (PR) and IGFBP-4 proteolytic fragments and determined their
effects on BF in mice using biochemical markers. PR IGFBP-4 was more potent than WT IGFBP-4 in
inhibiting IGF-I-induced mouse osteoblast cell proliferation in vitro and in inhibiting IGF-I-induced
increase in ALP activity in vivo. IGFBP-4 proteolytic fragments were inactive. Systemic administration of
WT but not PR IGFBP-4 increased serum osteocalcin, ALP and ALP in skeletal extracts in a dose-
dependent manner (40% ↑ at 1.25 nmol/mouse, p<0.05). Systemic administration of WT but not PR
IGFBP-4 increased free IGF-I levels (50% ↑ after 30 min treatment, P<0.05) in serum. If IGFBP-4
increases BF by a mechanism involving increased IGF-I availability in the circulation, we predict IGFBP-4
to be ineffective in mice deficient in circulating levels of IGF-I. Accordingly, IGF-I but not WT IGFBP-4
increased BF parameters in IGF-I midi mice which exhibit 70% reduced IGF-I levels compared to control
mice. Conclusions: 1) Systemic administration of IGFBP-4 increases BF parameters by increasing IGF
bioavailability via an IGFBP-4 protease dependent mechanism; and 2) We predict that physiological
regulation of IGF-I in the IGFBP-4+IGF complex may influence the levels of free IGF-I and, thereby, the
endocrine actions of IGF-I.

Basic Science Oral: Growth Factors (General) (11:00 AM-12:30 PM)

Presentation Date: Saturday, June 23, 2001; Time: 11:45 AM; Location: A 108




                                                 - 37 -
                                                                          GROWTH FACTORS

OR36-4

Lay explanation of abstract:

Osteoporosis is the most common bone disease that incurs large medical expenses. It is characterized by
thinning of the bones, which become fragile and break more easily. One out of every two white women will
have a osteoporotic fracture at some point in her lifetime. Osteoporosis occurs mainly because of : 1) lower
peak bone mass than normal at the end of sexual maturity; and 2) increased bone destruction rate that can
occur, for example, because of estrogen deficiency, without a corresponding increase in the formation of
new bone. Most available treatments for osteoporosis prevent bone loss by preventing bone destruction.
Because many osteoporotic patients who come for treatment have already lost a substantial amount of
bone, there is a need to develop treatments that increase bone mass by stimulating new bone formation. Our
basic research presented in this abstract explored the mechanism by which insulin-like growth factor
binding protein-4 (IGFBP-4) increases bone formation parameters in mice, when given systemically.

Insulin-like growth factors (IGFs) resemble insulin in structure and are very important in controlling the
amount of new bone formed both during development and during adulthood. IGFs are produced within
bone, where they act as local growth factors. In addition, IGFs circulate in blood in large amounts and
thereby act as endocrine hormones. The action of IGFs in blood is controlled by a set of proteins called
IGF binding proteins (IGFBPs) that bind IGFs and prevent them from being active. About 80% of IGFs in
the blood are present as a large complex by binding to IGFBP-3 and another protein (acid labile subunit,
ALS), which cannot go through the small pores of the blood vessel. The remaining 20% of IGFs are
present as small complex (e.g., IGFBP-4+IGF) that can freely move through the pores of the blood vessel
and thereby become available to bone and other tissues. In the present study, we administered IGFBP-4 to
mice so that more of the IGFs are bound to the smaller complex (IGFBP-4+IGF) and less is bound to the
larger complex (ALS+IGFBP-3+IGF).

There are two advantages with the smaller IGFBP-4+IGF complex. One, IGFBP-4 in this complex can be
broken down by proteases in the blood, thereby releasing IGFs to go through the pores of the blood vessel
to bone and other tissues to act as a growth factor. Second, the smaller IGFBP-4+IGF complex is able to
go through the pores of the blood vessel to bone where a protease can degrade IGFBP-4 to release IGF for
action. We confirmed this mechanism by using a form of IGFBP-4 that is resistant to degradation by
proteases in the blood.

Our basic research is significant. Based on our data, we predict that mechanisms that increase the level of
free IGF-I in serum could be used as therapy to increase bone formation and treat osteoporosis.

This work was supported by funds from the National Institutes of Health and the Veterans Administration.




                                                   - 38 -
                                                                         GROWTH FACTORS

OR36-6
MORPHOLOGIC RECAPITULATION OF HUMAN PITUITARY TUMORIGENESIS BY A
NOVEL TRANSFORMING ISOFORM OF FIBROBLAST GROWTH FACTOR RECEPTOR 4
SL Asa,1 L Zheng,2 S Ezzat.2 1Pathology, University Health Network, Toronto, Ontario, Canada;
2
  Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada

Fibroblast growth factors (FGFs) and altered FGF receptors (FGFRs) have been implicated in human
tumorigenesis. We have previously reported the identification of a novel pituitary tumor-derived N-
terminally truncated isoform of FGFR4 (ptd-FGFR4) that we isolated from primary human pituitaries. ptd-
FGFR4 transcription initiates in exon 5, resulting in an mRNA that encodes a polypeptide containing the
intact third Ig-like domain, a transmembrane domain, and an intact kinase region of FGFR4 but lacks a
signal peptide. ptd-FGFR4 is constitutively phosphorylated; it is transforming in vitro and in vivo. We now
demonstrate convincing evidence of a direct role for ptd-FGFR4 in pituitary tumorigenesis in transgenic
mice. Using a pituitary specific prolactin PRL promoter, we generated transgenic mice selectively
expressing ptd-FGFR4 in the pituitary. At 7 months, lactotrophs were numerous with intense cytoplasmic
FGFR4 expression. By the age of 11 months, 90% of the animals developed pituitary tumors that exhibited
disruption of the normal acinar architecture of the adenohypophysis and prominent vascularity. The tumor
cells contained cytoplasmic immunoreactivity for PRL as well as FGFR4. Other cell types of the
adenohypophysis were unaffected. Tumors were found equally in males and females, but the tumors were
significantly larger in females, with some exhibiting intracranial extension and invasion of brain. Males
consistently developed microadenomas that were found on microscopic examination. Age- and sex-
matched non-transgenic littermates had no pituitary pathology. Circulating PRL levels of transgenic
animals were higher than those of non-transgenic littermates and correlated with tumor size. These findings
demonstrate that a constitutively activated, cytoplasmic, truncated FGFR4 identified from primary human
pituitary tumors can morphologically recapitulate human pituitary tumorigenesis.

Basic Science Oral: Growth Factors (General) (11:00 AM-12:30 PM)

Presentation Date: Saturday, June 23, 2001; Time: 12:15 PM; Location: A 108




                                                  - 39 -
                                                                            GROWTH FACTORS

OR36-6

Lay explanation of abstract:

The pituitary gland is a pea-sized structure that lies under the brain, encased within the bone of the base of
the skull. It is the master gland that regulates the function of many hormone-producing glands. Tumors of
this gland can be seen on scans of the brain in up to 20% of adults. Some of these grow and function in a
manner that can have serious consequences on fertility, sexual function, heart function, and ultimately
longevity. The mechanisms that lead to the development of these tumors are largely unknown.

In this study, a specific defect in the processing of a gene responsible for a receptor that transmits growth
signals was identified in nearly 40% of human pituitary tumors. Re-constitution of this genetic defect in
living cells, immunologically tolerant mice, and mice genetically engineered to produce this defect only in
the pituitary resulted in tumorous cell growth. Perhaps the most remarkable aspect of this study is that it
was based on a genetic defect that is unique to this gland and whose re-capitulation in experimental models
faithfully reproduced the pathology of this gland as it is seen in patients.

The results of this study represent the blueprints required to devise more selective approaches to the
diagnosis and treatment of tumors of this gland. Moreover, this study provides a framework to characterize
generic defects in the various endocrine glands, which appear to behave distinctly from non-hormone
producing malignancies of the body.

This research was funded by the Medical Research Council of Canada/Canadian Institutes for Health
Research.




                                                    - 40 -
                                                                          GROWTH FACTORS

P3-380
EFFECTS OF RETINOIC ACID AND INSULIN-LIKE GROWTH FACTORS IN PRIMARY
HYPERPARATHYROIDISM
CK Wong, T Lai, CE Stewart, JR Farndon, JM Holly. Division of Surgery, Bristol Royal Infirmary, Bristol,
United Kingdom

Background: The precise cause of primary hyperparathyroidism (1HPT) is uncertain. The insulin-like
growth factor (IGF) system is multifunctional and ubiquitous in normal and neoplastic tissues. Retinoic
acid (RA), a derivative of vitamin A and a ligand for the IGF II receptor, is an important regulatory factor
in growth and differentiation of epithelial tissue and has been shown to inhibit cancer growth.Aims: To
assess the inhibitory actions of RA and its interactions with the IGF system in a 1HPT primary cell culture
model.
Methods: Fresh human parathyroid glands were obtained from 9 patients undergoing surgical treatment for
primary hyperparathyroidism. Glands were bisected for histology (frozen section) and immediate cell
culture. Tissue was dispersed by enzyme digestion and cells cultured in 24-well plates. Following adhesion,
the cells were transferred to serum free medium and dosed once with growth factors ± RA for 96hrs.
Proliferation was assessed by measuring tritiated thymidine incorporation.
Results: Compared with controls (n=5) IGF I and IGF II increased DNA synthesis by 55.9% ± 6.4 (sem)
and 51.1% ± 7.9 respectively (100ng/ml, P0.05) compared with wild-type IGF II.
Conclusion: These data implicate a novel antiproliferative role for RA in enhancing the pericellular
clearance of IGF II via the IGF II receptor thus preventing ligand activation of the IGF I receptor. This may
have broader implications for RA effects in other tumours.

Basic Science Poster: Growth Factors - 06 (General) (Poster Session) (11:00 AM-12:00 PM and 2:30 PM-
3:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                   - 41 -
                                                                            GROWTH FACTORS

P3-380
Lay explanation of abstract:

The parathyroid glands are four pea-sized glands behind the thyroid gland in the neck. These glands
produce a hormone (parathyroid hormone) that controls the serum calcium. Excess production of this
hormone causes excess calcium in the blood (hyperparathyroidism) that may cause osteoporosis, bone
fractures, and kidney stones. Imbalances of vitamins and growth factors have been suggested as causes of
this condition.

To date, surgery remains the only definitive treatment for hyperparathyroidism. This study presents novel
data on the mechanism and potential use of retinoic acid, a vitamin A derivative, as treatment for
hyperparathyroidism. We have proposed a mechanism: retinoic acid may modulate insulin-like growth
factor II availability. (This mechanism may also be important in cancer treatments.)

The insulin-like growth factor axis consists of growth factors I and II (IGF I and II) and corresponding
receptors I and II. Insulin-like growth factors are important growth factors in both normal and cancer
growth. Insulin-like growth factor receptor II (IGF receptor II) is a multi-functional receptor that binds
retinoic acid, mannose-6-phosphate, and IGF II for elimination, by transporting them to sites in the cell for
degradation. IGF receptor I binds both IGF I and IGF II to mediate their growth effects.

Our study evaluated the role of retinoic acid and insulin-like growth factors in primary
hyperparathyroidism. Parathyroid glands from hyperparathyroid patients undergoing surgery were used in
this study. Glands were dispersed and processed for cell culture experiments using retinoic acid and IGF I
and II. Nine glands from nine patients were included in this study. On its own, retinoic acid inhibited the
growth of parathyroid cells. IGF I and II stimulated the growth of parathyroid cells.

These data indicate a novel anti-proliferative role for retinoic acid in enhancing the clearance of IGF II
through the IGF receptor II, which prevents ligand activation of IGF receptor I. This may have broader
implications for retinoic acid effects in other tumors.

Further background information:

When retinoic acid was given in combination with IGF I and II, the growth response of IGF II was
completely abolished. To evaluate the role of IGF receptor II in mediating the actions of retinoic acid, we
used the IGF II analogues [Leu27]IGF II (10 to 20-fold reduced affinity for insulin-like growth factor
receptor I). The IGF II inhibitory effect of retinoic acid was enhanced in the presence of analogues
Leu27]IGF II compared with wild-type IGF II.


This project was completed at the Bristol Royal Infirmary, U.K. The project was sponsored by the Royal
College of Surgeons Research Fellowship, Mason‘s Medical Research Foundation, Hamamelis Trust.




                                                    - 42 -
                                                                         GROWTH FACTORS

P3-389
GROWTH HORMONE (GH) HAS POTENT ENDOTHELIUM-INDEPENDENT VASODILATING
EFFECTS IN VITRO
A Giustina,1 C Ceconi,2 A Cargnoni,2 F Manelli,1 R Lorusso,3 M Volterrani.2 1Internal Medicine,
University of Brescia, Brescia, Italy; 2Cardiology, S. Maugeri Foundation, Gussago, Italy; 3Cardiac
Surgery, Spedali Civili, Brescia, Italy

GH has been reported to exert acute beneficial effects in heart failure. The mechanisms of this action are
still unknown. We have hypothesised that GH may act via a reduction in peripheral vascular resistances.
The aim of our study was to evaluate these GH vasoactive properties and investigate the mechanisms
involved. Rabbit aortic strips were superfused in vitro with different vasoconstrictive agents (10-7M
noradrenaline N, 10-6 M PGF2 and 50mM KCl) in the presence of human rGH (0.1-5 mg/L). We observed
that: 1) GH potently counteracted the NA and PGF2-mediated contraction (maximal inhibition 36.6  3.5
and 57.2  9.1%, respectively); 2) GH activity was not dependent on endothelium since both NOS (10-4M
L-NAME) and COX (10-6M indomethacin) inhibitions did not reverse its effect; in addition the mechanical
removal of endothelium did not affect GH action; 3) GH depressed responsiveness of vascular smooth
muscle to KCl-induced contraction. In fact, in the presence of GH at 5 mg/L, we observed a shift to the
right of the KCl contraction curve (EC50 increased from 18.1  1.39 to 28.8  1.62; p<0.01); 4) GH
vasodilation did not depend on inhibition of calcium influx through L-type calcium channel; in fact, L-type
calcium blocker diltiazem (at 10-6M) did not affect GH activity. In conclusion, GH exerts potent
endothelium independent vasodilating action on rabbit aortic strips in vitro. We hypothesise that vascular
smooth muscle potassium channels may be involved in the GH-mediated vasodilatation.

Basic Science Poster: P3: Growth Factors - 06 (General) (Poster Session) (11:00 AM-12:00 PM and 2:30
PM-3:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 43 -
                                                                            GROWTH FACTORS

P3-389
Lay explanation of abstract:

We have made a new discovery: growth hormone (GH) has a potent vasodilating effect on rabbit aortic
strips in-vitro. Moreover, for the first time we have obtained some mechanistic insights into this effect,
which shows that this action is independent of endothelium and involves vascular smooth muscle relation
via potassium channels.

The aim of our study was to conduct an in-depth evaluation of the peripheral vasodilating properties of
growth hormone and the underlying mechanisms. In detail, we superinfused rabbit aortic strips with agents
that are known to induce potent vasoconstriction in this in vitro system. GH was able to reverse this
vasoconstriction independent of the presence of arterial endothelium.

The interesting part of this research is that it provides an explanation for the beneficial effects of GH in
patients with heart failure. Because this disease is one of the major causes of morbidity and mortality in the
western population and treatment for this disease is not yet very effective, our study suggests that there is a
role for GH as an adjunctive medical treatment of heart failure. Therefore, this basic study has potential
impact in the treatment of millions of patients with heart failure.

Additional background information:

Human studies have shown that acute prolonged infusion with recombinant human GH may have beneficial
effects in patients with heart failure, by decreasing pulmonary pressure and increasing cardiac output. To
allow a correct clinical use of growth hormone in patients with heart failure, it is crucial that we learn more
about the mechanism of this hormone action. In fact, both a direct inotropic action at the heart level and a
peripheral systemic vasodilating action have been hypothesized to be involved in the beneficial effect of
growth hormone in heart failure.

Research was funded by Fondazione Maugeri of Gussago, Italy and by Eli Lilly-Sesto Fiorentino, Italy.




                                                    - 44 -
                                                                            GROWTH FACTORS

P3-396
LEPTIN BONE MARROW CONCENTRATIONS DURING INITIAL CHEMOTHERAPY IN
CHILDREN SUFFERING FROM ACUTE LYMPHOBLASTIC LEUKEMIA (ALL)
H Wex,1 E Ponelis,1 U Mittler,1 R Dressendoerfer,2 K Mohnike,1 V Peter.1 1Pediatric Oncology, Otto-von-
Guericke University, Magdeburg, Germany; 2Diagnostic Systems Laboratories Inc., Sinsheim, Germany

Leptin is a secretory protein mainly produced by adipocytes. Leptin is not only critical for the regulation of
body weight but part of various endocrine axes, e.g. immune response, reproduction and hematopoesis.
Beside adipocytes other cells had been reported to secrete leptin (e.g. stroma cells). Leptin stimulates
normal myeloid and erythroid cell development. Hematopoetic progenitor cells express the leptin receptor,
implying a physiological role of leptin during expansion and differentiation of hematopoetic cells.
In order to investigate the role of leptin in childhood leukemia plasma levels of leptin were measured by
ELISA in 38 children at diagnosis of ALL. The tumour clones generally represents 50-90% of leucocytes.
At the time of diagnosis leptin concentrations were 0.92 ±0.79 ng/ml. After 33 days of chemotherapy
(Prednisone, Virncristin, Daunorubicin and Asparaginase) all children had reached hematological
remission. At day 33 leptin levels of 2.6 ±2.4 ng/ml were detected. In 32 of 38 patients an increase of
plasma leptin was found to the 2.8 fold of the initial levels (range: 0.14-3.39 at diagnosis, 0.65-11.62 at day
33; p < 0.01).
The underlying mechanism for the observed variability of plasma leptin levels are still unknown. On the
one hand, the presence of the tumour clone and the subsequent relative decrease of other cell types may
represent a direct cause for the low leptin levels at the time of diagnosis. On the other hand, indirect
regulatory effects such as the repression of leptin expression by cytokines secreted by the tumour clone
may play a role as well. Interestingly, glucocorticoids causes a 3 fold rise of leptin levels, which might
explain the 2.8 fold increase found after 33 days of chemotherapy.
In conclusion, we found changes in bone marrow plasma leptin concentrations in children with ALL before
and during chemotherapy. The potential usefulness of leptin as a prognostic indicator needs to be further
evaluated.

Basic Science Poster: Growth Factors - 06 (General) (Poster Session) (11:00 AM-12:00 PM and 2:30 PM-
3:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                    - 45 -
                                                                            GROWTH FACTORS

P3-396
Lay explanation of abstract:

The term ‗leptin‘ originates from the Greek word ‗leptos‘, which means ‗thin.‘ Leptin represents an
endogenous hormone that is a major regulator of body fat. During recent years, much information has been
collected to explain the role of leptin in weight regulation.

Leptin is predominantly produced by fat cells and released into the bloodstream. Leptin concentration
increases after food intake, which leads to decreasing appetite. In general, the basal level of leptin depends
on body mass. A high body mass is usually accompanied by an increase of body fat that leads to elevated
leptin levels compared to a person with normal body mass index.

Due to its regulatory ability to decrease appetite, the hormone leptin has generated much interest in the past
decade. In 1994, the gene encoding for leptin became a ‗celebrity gene‘ and physicians began to regard
obesity as a chronic disease. Physicians looked forward to treating obesity with a natural and safe weight-
reducing drug.

Meanwhile, current research has revealed that leptin is not only involved in fat regulation. Unexpectedly,
leptin receptors (discovered in 1998) were not found only in cells known to be involved in metabolic
functions, but also in capillaries, bone cells, brain, and cells of the immune system. Based on this tissue
distribution of leptin receptors, scientists suggested more complex functions for leptin. This new view on
leptin has been confirmed by numerous recent publications that prove that the hormone leptin is
functionally involved in diverse immune processes, in inflammatory processes, and in angiogenesis, bone
metabolism, and hematopoesis. Thus, leptin possesses proliferating effects on different cancer cells.

We found that cells from children suffering from leukemia express the receptor for leptin, which led us to
hypothesize that leptin present in the bloodstream of these children might function as ‗positive growth
factor‘ for the tumor cells. In this study the leptin concentration in blood plasma of these children was
measured by a commercially available enzyme kit.
All together, we analyzed 38 blood samples at the time of diagnosis of the leukemia, when usually 50-90%
of all white blood cells are tumor cells, and at day 33 of the chemotherapy, when the tumor cells have
widely disappeared and normal healthy blood cells started to come back. The leptin concentration at day 33
of chemotherapy was 2.8times the initial level.

The underlying mechanism for the observed changes in plasma leptin levels are still unknown. A plausible
explanation might be the enrichment of the tumor clone at the time of diagnosis, which leads subsequently
to a relative decrease of other cells that express and secret leptin.

In conclusion, we found changes in plasma leptin concentrations in children with leukemia before and
during chemotherapy. The increase of leptin is probably a consequence of a regeneration process of normal
blood and bone marrow cells. The potential usefulness of leptin as a prognostic indicator needs to be
further evaluated.

Funding was provided by W.A. Drenckmann Stiftung, Germany, and Magdeburger Förderkreis
krebskranker Kinder e.V., Germany.




                                                    - 46 -
                            GH-PROLACTIN-REGULATION OF SECRETION

P2-199

PLASMA GHRELIN LEVELS ARE INFLUENCED BY ACUTE AND CHRONIC FEEDING
STATES IN HUMANS
H Ariyasu,1 K Takaya,1 T Tagami,2 K Mori,1 Y Komatsu,1 Y Ogawa,1 K Hosoda,1 T Akamizu,1 T Usui,2
A Shimatsu,2 K Doi,1,3 H Hosoda,3 M Kojima,3 K Kangawa,3 K Nakao.1 1Department of Medicine and
Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, Japan; 2Clinical Research
Institute, Center for Endocrine and Metabolic Diseases, Kyoto National Hospital, Kyoto, Japan;
3
  Department of Biochemistry, National Cardiovascular Center Research Institute, Osaka, Japan

Ghrelin, an acylated peptide that was isolated as an endogenous ligand for the growth hormone (GH)
secretagogue receptor, strongly stimulates GH release in vivo and thus is likely to be involved in a novel
GH-regulating system. Although previous studies in rodents also suggest that ghrelin has actions involved
in energy homeostasis and that plasma ghrelin levels are influenced by feeding state, less information is
available about circulating ghrelin in humans. The aim of this study is to elucidate the source of circulating
ghrelin and the effects of feeding state on plasma ghrelin levels in humans. The plasma ghrelin
concentration in 61 normal subjects measured by a specific radioimmunoassay was 166.0 ± 10.1 fmol/ml.
Northern blot analysis of various human tissues identified ghrelin mRNA found most abundantly in the
stomach followed by the duodenum and jejunum. Plasma ghrelin levels in totally gastrectomized patients
were reduced to 35% of those in normal controls, suggesting that the stomach is a major source of
circulating ghrelin. Plasma ghrelin levels were increased by 31% after 12-h fasting and reduced by 22%
immediately after habitual feeding. In patients with anorexia nervosa, plasma ghrelin levels were markedly
elevated compared with those in sex- and age-matched controls (401.2 ± 58.4 vs. 192.8 ± 19.4 fmol/ml) and
were negatively correlated with body-mass indices. Two of severely affected patients, whose body-mass
indices were less than 11.0 kg/m2, showed more than 1,300 fmol/ml of plasma ghrelin levels. These
observations indicate that plasma ghrelin levels reflect both acute and chronic feeding states. In conclusion,
we have demonstrated that circulating ghrelin is derived from the stomach and its levels are influenced by
feeding state in humans, being consistent with the hypothesis that ghrelin is involved in energy
homeostasis.

Basic Science Poster: Growth Hormone-Prolactin: Regulation of Secretion II (11:00 AM-12:00 PM and
2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                   - 47 -
                            GH-PROLACTIN-REGULATION OF SECRETION

P2-199

Lay explanation of abstract:

Ghrelin is a newly discovered hormone found in the rat stomach. We examined the origin of ghrelin in
human blood and learned that the stomach contained ghrelin mRNA most abundantly and that blood
ghrelin levels were markedly reduced in gastrectomized patients (patients whose stomachs have been
removed or reduced surgically). These results indicate that ghrelin is mainly produced by the stomach and
secreted into the bloodstream in humans.

Because the stomach is a major source of blood ghrelin in humans, we examined the effects of fasting and
feeding on ghrelin levels. Using 10 healthy volunteers, we found that blood ghrelin levels are elevated by
31% after short-term (l2 hour) fasting and reduced by 22% after daily meals.

Moreover, we revealed that blood ghrelin levels are extremely high in female patients with anorexia
nervosa, a mental disease characterized by refusal to maintain a normal minimal body weight. Two
severely affected patients had seven times as much ghrelin in their blood as normal women. Blood ghrelin
levels were negatively correlated with body mass indexes. Those findings show that ghrelin is secreted
during both acute and chronic fasting states--short-term fasting and prolonged starvation. Previous studies
in rats showed that ghrelin stimulates appetite and induces accumulation of body fat. According to these
results, ghrelin may act protectively so a body can reduce energy loss during fasting and starvation.

In addition, we also found that ghrelin strongly induces growth hormone (GH) release in normal volunteers.
Synthetic ghrelin was injected intravenously in four male subjects. Blood samples were drawn and blood
GH levels were measured. Blood GH levels were markedly higher. With the highest dose of ghrelin (5.0
g/kg body weight), blood GH levels reached levels nearly 200 times as high as base levels. Thus, we
concluded that ghrelin is a very strong stimulator for GH release. It is noteworthy that GH also is known to
have important roles in lipid metabolism in adults. Ghrelin may regulate energy balance also through GH
secretion.

Overall, these findings mean that ghrelin may signal the central nervous system and control energy balance
in humans. Our findings here may contribute to discovering the mechanism for human obesity and
consequently to reduce many diseases related to obesity.

Additional background information:

Artificial compounds, named GH secretagogues (GHSs), which stimulate GH release, have been produced.
Ghrelin is a natural GHS in the human body. Although previous reports revealed that ghrelin stimulates GH
release and induces fat accumulation in rats, little is known about ghrelin in humans. This study showed
that ghrelin strongly acts to release GH and that it may be involved in human energy homeostasis.

This research was funded by research grants from the Japanese Ministry of Education, Science, and
Culture; the Japanese Ministry of Health and Welfare; and JSPS-RFTF
98L00801.




                                                   - 48 -
                           GH-PROLACTIN-REGULATION OF SECRETION

P2-215
INVERSE CHANGES IN PLASMA GROWTH HORMONE (GH) AND LEPTIN
CONCENTRATIONS IN POSTMENOPAUSAL WOMEN ARE NOT RELATED TO FAT LOSS
OR EXERCISE INTENSITY
KT Borer,1 M Sowers,2 GM Christman,3 JZ Kasa-Vubu.4 1Kinesiology, University of Michigan, Ann
Arbor, Michigan; 2Epidemiology, University of Michigan, Ann Arbor, Michigan; 3Obstetrics &
Gynecology, University of Michigan, Ann Arbor, Michigan; 4Pediatrics, University of Michigan, Ann
Arbor, Michigan

Leptin can stimulate GH secretion, and GH can inhibit leptin secretion, but it is not known whether the
effects are mediated by changes in body fat. We hypothesized that in postmenopausal women (1)
endurance training will lead to fat loss (2) in inverse proportion to exercise intensity,and (3) GH
concentration will increase and that of leptin decrease due to decreases in body fat.
Postmenopausal women (n=23) were stratified by age ( 57.8 yrs), weight (75.3 kg) and BMI (32kg/m2) and
assigned to intense exercise at 125% of ventilatory threshold, VT or moderate exercise (95% of VT. They
walked 4.8 km/day, five days/week,30 weeks. Body fat was assessed by DEXA, and GH 10 min)and leptin
(60 min) concentrations were measured, respectively by a chemiluminescent assay and RIA,between 18 h
and 08 h, before, and after 15 and 30 weeks of training. Characteristics of GH secretion was analyzed by
deconvolution. Results were: (1) Training led to fat loss (2) that was inversely proportional to training
intensity (r=-0.541, F=5.381, p=0.037); (3) changes in plasma leptin and plasma GH concentrations were
negatively correlated (r=-0.465, F=5.793, p=0.0254), but only plasma leptin (r=0.644, F=8.483,
p=0.013)and not GH change, was correlated with body fat change. Finally (4) neither training or its
intensity influenced GH secretory characteristics. Thus,endurance training reduces body fat level in
postmenopausal women and induces inverse changes in plasma GH and leptin concentrations that are not
mediated by body fat losses.
Support: U of M Clinical Research Partnership grant and NIH M01-RR-0042.

Basic Science Poster: Growth Hormone-Prolactin: Regulation of Secretion II (11:00 AM-12:00 PM and
2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                 - 49 -
                            GH-PROLACTIN-REGULATION OF SECRETION

P2-215
Lay explanation of abstract:

Currently, understanding of the mechanisms through which we partition nutrient energy between growth
(maintenance of lean body mass) and fat storage is incomplete. Growth hormone (GH) is the principal
endocrine controller of growth. GH increases synthesis of protein, maintains and enlarges lean body mass,
and diverts energy expenditure away from protein and toward fat. Its secretion increases when there is
energy shortage caused by either fasting or intense exercise.

Leptin is a relatively recently discovered hormone secreted by adipose tissue. It is believed to play an
important role in controlling the size of the adipose tissue mass by inhibiting appetite and stimulating
energy expenditure. Leptin is secreted by the adipose tissue in proportion to the size of total body fat mass.
Its secretion declines when there is a shortage of energy caused by either exercise or dieting. Thus, changes
in these two hormones are reciprocal when there is exercise-induced energy shortage. In addition, leptin
can stimulate GH secretion, and GH can stimulate leptin secretion.

Our study asked two questions:

        Are reciprocal changes in GH and leptin secretion mediated by changes in body fatness?
        Will the reciprocal changes in the secretion of two hormones be seen in overweight
         postmenopausal women subjected to energy expenditure in the form of walking?

We trained postmenopausal women for 30 weeks (3 miles, walking 5 times per day at either a slow or
intense pace). We measured GH in 10-minute blood samples and leptin in hourly blood samples at night
(between 8 p.m. and 8 a.m.), both before and after 15 and 30 weeks of training. We did analysis of GH
pulses by deconvolution. We also measured changes in body fat with DEXA (whole-body X-ray scan)
before and after training.

We found that:
    Reciprocal changes in GH and leptin secretion took place in postmenopausal women 15 weeks
       after training (changes in GH and leptin secretion between two measurement times were
       negatively correlated); and
    Slow walkers lost more fat than fast walkers, but only leptin and not GH change was correlated
       with body fat change.

Our results suggest that the secretion of leptin and GH are affected by exercise training in a reciprocally
interactive way, and that this interaction is not mediated by training-induced changes in body fat.
Additional research is needed for a better understanding of the role of leptin-GH interaction during
exercise.

This research was supported by NIH grant MO1-RR000042 and the University of Michigan School of
Medicine Clinical Research Partnership.




                                                    - 50 -
                                                                 HORMONES AND CANCER

OR4-5
EVALUATION OF LIGAND-DEPENDENT CHANGES IN ANDROGEN RECEPTOR
STRUCTURE BY PEPTIDE PROBES PROVIDES INSIGHT INTO THE MECHANISMS
UNDERLYING ANTI-ANDROGEN RESISTANCE
C Chang, DP McDonnell. Pharmacology and Cancer Biology, Duke University, Durham, North Carolina

Mutations in the androgen receptor (AR) are frequently found in relapsed prostate cancers, permitting anti-
androgens as well as estrogens and progestins to function as androgens. However, the mechanism by which
these mutations enable this shift in AR-pharmacology is still unknown. Resistance to anti-hormone therapy
arises also in estrogen receptor (ER)-positive breast cancers, where it is believed that alterations in cofactor
expression in the cells permit anti-estrogens like tamoxifen to function as agonists. In support of this
hypothesis we have shown that tamoxifen binding to ER allows the presentation of novel protein-protein
interaction surfaces on the receptor, enabling it to interact in an ectopic manner with transcriptional
coactivators. In this study we wanted to see if the same mechanisms would also apply with respect to anti-
androgen resistance. To explore this possibility, we used phage display to identify a series of LXXLL-
containing peptides that interact with the AF2 domain of AR. We found that although the binding of
peptides to wild-type AR was agonist dependent, these same peptides could also interact with the AR-
T877A variant, a mutant frequently found in anti-hormone refractory prostate cancers, in the presence of
either androgens or anti-androgens. This suggests that the agonist activity of anti-androgens and other
physiologically relevant ligands occurs because they, in the background of the mutation, allow AR-AF2 to
adopt an active conformation. Initially, this result seems to contradict other findings which suggest that
coactivator binding to AR-AF2 is not required for AR activity. In probing this further, we have determined
that the role of AR-AF2 appears to be to stabilize the overall structure of the receptor, allowing the amino
terminus to interact with appropriate coactivators. This contention is supported by the finding that over-
expression of the AF2-binding peptides does indeed block the interaction of the amino- and carboxyl-
terminal of AR, but does not attenuate AR transcriptional activity. Thus we believe that mutations in AR
which facilitate the formation of an AF2 pocket, have the potential to allow AR antagonists to manifest
agonist activity.

Basic Science Oral: Hormones & Cancer I (1:00 PM-2:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 2:00 PM; Location: A 106




                                                    - 51 -
                                                                HORMONES AND CANCER

OR4-5
Lay explanation of abstract:

About 180,000 new cases of prostate cancer will be diagnosed in the U.S. this year, and 32,000 deaths from
this disease will occur. These statistics make prostate cancer the second leading cause of cancer-related
death in men, after lung cancer. Exposure to androgens is a risk factor for prostate cancer because this class
of hormones, acting through a high-affinity receptor protein, can stimulate the growth of prostate cancer
cells. Not surprisingly, antiandrogens, compounds that bind to the receptor and block androgen access,
have become frontline therapies when managing prostate cancer. Although most patients initially respond
to this treatment, resistance to the existing classes of drugs usually arises within 2-3 years. The
mechanisms underlying this resistance are poorly understood, although it appears to be due in part to
mutations in the receptor which let antiandrogens be recognized by cells as androgens. We are interested in
defining the mechanism by which this alteration in androgen receptor (AR) pharmacology occurs, with a
view toward developing novel classes of antiandrogens that may provide a longer duration of response.

We have developed probes that can detect and distinguish between different AR-androgen and AR-
antiandrogen complexes. These probes were used to study the shape changes that occur in mutated ARs
upon binding different steroids. Using this approach, we made the surprising observation that some AR
mutants can adopt an ―active‖ form upon binding compounds other than those generally considered to be
androgens. For instance, flutamide, a widely used antiandrogen, inhibits the activity of the wild-type
androgen receptor, while it functions as an androgen on some mutant ARs. Not surprisingly, it has been
shown that some patients with progressing disease actually respond to withdrawal of the antiandrogen. The
failure of some other patients to respond to antiandrogen withdrawal may be because, as we have found,
deoxycorticosterone, a hormone produced in the adrenal gland, is recognized by some AR mutants as an
androgen. Indeed, when tested in vitro in prostate cancer cells bearing a mutated AR, deoxycorticosterone
was found to increase the synthesis of PSA, an established marker of androgen action. These observations
have important clinical implications and suggest that we should make it a priority to develop antiandrogens
whose biocharacter is not influenced by AR mutations.

This research was funded by NIH grant DK 48807 to DPM and a postdoctoral fellowship (DAMD 17-99-1-
9173) to C-Y C from U.S. Army Medical Research Acquisition Activity.




                                                   - 52 -
                                                              HORMONES AND CANCER

OR16-4

AP-1 BLOCKADE DELAYS THE ONSET OF TAMOXIFEN RESISTANCE IN A BREAST
CANCER MODEL
R. Schiff1, Y. Liu1, G. Ge1, Y. Zhang1, S.G. Hilsenbeck1, S.A. Fuqua1, P.H. Brown1 and C.K. Osborne1.
1
  Breast Center, Baylor College of Medicine, Houston, TX, United States

Acquired tamoxifen (Tam) resistance in breast cancer is a major clinical problem, but the underlying
mechanisms are largely unknown. We have recently shown in a xenograft model that the conversion of
breast tumors to a Tam-resistant (TamR) phenotype is due to the acquired ability of the tumor to be
stimulated by Tam. TamR in this model is associated with oxidative stress and increased JNK and AP-1
activities, events that we hypothesized could contribute to the onset of resistance. Recent data
demonstrating a significant increase in JNK and AP-1 activities in clinical TamR breast tumors supports
this hypothesis. To further investigate and confirm the role of AP-1 in the development of TamR, we
examined whether blocking AP-1 activity can affect or delay the emergence of the TamR phenotype. We
used human breast cancer MCF-7 Tet-Off-Tam67 cells, which upon discontinuation of doxycycline (Dox)
express a specific inhibitor of AP-1 (a dominant-negative c-Jun mutant, Tam67). Stably transfected
xenograft tumors were established in mice receiving supplemental estrogen and Dox to suppress expression
of the inhibitor. Estrogen pellets were then removed and the effects of Tam on tumor growth and the
emergence of resistance were determined. AP-1 blockade induced by expressing the c-Jun dominant-
negative during the Tam-sensitive/inhibited phase of tumor growth (44 days after the start of Tam) caused
reduction in tumor volume and significantly delayed tumor progression to resistance (p= 0.03). Median
time to develop resistance, defined as the time required for the tumor to reach 6 times its volume at the
beginning of Tam treatment, was prolonged by more than 50 days in the tumors in which AP-1 was
blocked, compared to control tumors. Strikingly, we also found that blocking AP-1 in tumors growing after
TamR had developed resulted in a rapid and marked decrease in tumor volume, with some tumors declining
to less than half their original volumes in less than 2 weeks. Thus, our results suggest that AP-1 may play
an important role in the development and/or growth of the TamR phenotype, and that agents that block AP-
1 activation might be useful in the clinic to delay or circumvent TamR.

Basic Science Oral Session: Hormones and Cancer (1:00 PM-2:30 PM)

Presentation Date: Thursday, June 21, 2001; Time 9:30 AM; Location Room C 205




                                                  - 53 -
                                                                HORMONES AND CANCER

OR16-4
Lay explanation of abstract:

Tamoxifen, a drug that blocks the action of the female hormone estrogen, is the most effective agent we
have for inhibiting the recurrence of many breast cancers after surgery or for treating them if they do recur.
Unfortunately, resistance to tamoxifen always develops eventually, and there is evidence that resistant
tumors are actually stimulated to grow by tamoxifen.

We have developed a model system in which human breast cancer cells are grown in mice – when tumors
begin to develop, tamoxifen causes their growth to stop, but after weeks or months, rapid growth resumes
in spite of continuing tamoxifen treatment. In studying this model, we have found that the onset of
tamoxifen-resistant growth is associated with increased activity of a particular transcription factor called
AP-1, which is responsible for inducing the synthesis of a number of proteins in the cell. An enzyme called
JNK, which can activate AP-1, is also increased in the tamoxifen-resistant tumor model. Furthermore, both
AP-1 and JNK are elevated in tamoxifen-resistant tumors from breast cancer patients.

But to prove directly that AP-1 is involved in tamoxifen resistance, we needed to show that inhibiting AP-1
would prevent or reverse the onset of resistant growth. We therefore took the same human breast cancer
cells used in our mouse model and inserted a gene that produces a specific inhibitor of AP-1. Furthermore
this gene is inducible, so that we can allow tumors to develop in the mice as usual and then, when
tamoxifen treatment starts, we can turn on the inserted gene to inhibit AP-1 activity. We found that
inhibiting AP-1 greatly prolongs the time before tamoxifen resistance appears. Even more striking, if we
let the tumors develop tamoxifen resistance and only then turn on the AP-1 inhibitor, the inhibitor still
stops the resistant tumor growth and there is a rapid decrease in tumor volume.

These results in our model system, along with the finding of elevated AP-1 in tamoxifen-resistant breast
cancers from patients, argue that AP-1 has an important role in developing resistant tumor growth during
tamoxifen treatment. Thus, our findings suggest that new agents to block AP-1, suitable for clinical use,
could be valuable in preventing or overcoming tamoxifen resistance in clinical breast cancer.

Funding source: SPORE in Breast Cancer, Dr. C Kent Osborne - P.I. This work was carried out in the new
Breast Center at Baylor College of Medicine, Houston; Dr. C Kent Osborne – Director.




                                                    - 54 -
                                                               HORMONES AND CANCER

P1-227

PROGESTERONE RECEPTORS A AND B LOCALISE IN DISCRETE NUCLEAR FOCI IN THE
NORMAL AND MALIGNANT ENDOMETRIUM
RL Arnett-Mansfield,1 A deFazio,1,2 PA Mote,1 CL Clarke.1 1Westmead Institute for Cancer Research,
University of Sydney at the Westmead Millennium Institute, Westmead, Australia; 2Department of
Gynaecological Oncology, Westmead Hospital, Westmead, Australia

The nuclear progesterone receptor (PR), which is expressed as two proteins (PRA and PRB) with different
functions, is frequently detected in uterine cancer and is an independent prognostic factor. Our previous
work has shown that, in endometrial cancer, there is a disruption of relative PR isoform expression from the
similar levels seen in normal endometrium, and that there is discrete punctate distribution of PRA and PRB
in the nuclei of endometrial cancers. Dual immunofluorescence and confocal microscopy were used in this
study to determine PRA and PRB distribution within the nucleus in the normal endometrium during the
menstrual cycle. PR was distributed either diffusely within the nucleus, or highly localised in discrete
subnuclear dots, depending on the menstrual cycle phase. In the proliferative phase, diffuse staining was
more common, with few cells containing nuclear dots, whereas in the secretory phase, there was a marked
increase in punctate staining. Both the numbers of cells with PR in nuclear dots, and the total numbers of
dots per nucleus, increased in the secretory phase. Individual analysis of PR isoforms showed that diffuse
and punctate patterns of distribution were noted for PRA and PRB, moreover that both isoforms were co-
located in the subnuclear dots. However, PRB was predominant in these nuclear dots, particularly in the
secretory phase. In summary, PR localisation within the nucleus of the endometrium alters with menstrual
cycle phase, from predominantly diffuse in the proliferative phase to associated with discrete subnuclear
dots in the secretory phase, during exposure to high circulating levels of progesterone. The nucleus is a
highly organised structure containing numerous specialised subnuclear structures including transcriptional
domains. The discrete subnuclear localisation of PR isoforms, particularly PRB, in the presence of
circulating progesterone suggests that altered intranuclear PR localisation is a consequence of ligand
binding and may be required for transcriptional activity in normal and malignant endometrium.

Basic Science Poster: Hormones & Cancer I (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 55 -
                                                               HORMONES AND CANCER

P1-227
Lay explanation of abstract:

Breast cancer is the most common cancer in women, and cancer of the uterus is the most common
gynecological cancer. The female hormones progesterone and estrogen are important for developing and
regulating the normal reproductive system. These hormones also are involved in breast and uterine cancer.

Progesterone acts through the progesterone receptor (PR). Presence of PR is a positive predictor to
hormonal response in advanced cancer disease, in which close to 86% of patients whose tumors contain PR
will respond to hormone therapy. Therefore the presence of PR is an important indicator of the likely
success of hormonal treatment of breast and uterine cancer.

We know that PR is expressed as two proteins, PRA and PRB, which have different functions. Our recent
work has shown that both FR proteins can be expressed in the same cell and that in the normal uterus, PRA
and PRB are seen in similar levels; there is a disruption, however, of PR protein levels expressed in uterine
cancer. Our preliminary data suggests that the two proteins are located in distinct areas of the nucleus,
which may be fundamentally important in the mechanism of their activity.

The aims of this study are to determine individual localization of the progesterone receptors. Sections of
normal human endometrium (membrane lining the uterus) from either the proliferative phase (days 5-14) or
secretory phase (days 15-28), of the menstrual cycle, were stained for the proteins PRA and PRB. In this
study, dual immunofluorescence was used to determine PRA and PRB distribution within the nucleus in the
normal endometrium during the menstrual cycle. We visualized this image with confocal microscopy. PR
was distributed either diffusely within the nucleus or highly localized in discrete dots, depending on the
menstrual cycle phase.

In the proliferative phase, when progesterone levels are low, diffuse staining was more common, with few
cells containing nuclear dots. In the secretory phase, however, in which high levels of progesterone
circulate, there was a marked increase in punctate staining. The discrete subnuclear localization of PR
protein in the presence of progesterone suggests that changes in PR localization are a consequence of
hormone binding to the receptor and may be required for cell activity in both normal and malignant
endometrial tissue. This project will help to provide a better understanding of the pathways involved in
progesterone responsiveness in breast and uterine cancers.

Rebecca Arnett-Mansfield is a scholar supported by the Australian Postgraduate Award from the
Commonwealth of Australia. The research is funded by the National Health and Medical Research Council
of Australia and the Westmead Millennium Foundation.




                                                   - 56 -
                                                               HORMONES AND CANCER

P1-241
INVOLVEMENT OF STEROID HORMONE RECEPTOR COACTIVATORS, E6-AP AND UBCS,
IN THE DEVELOPMENT OF BREAST TUMORS
X Gao,1 GM Clark,2 F Yan,1 Z Nawaz.1 1Molecular and Cellular Biology, Baylor College of Medicine,
Houston., Texas; 2Breast Center, Baylor College of Medicine, Houston, Texas

Steroid hormones, estrogen and progesterone, are known to play a major role in the development of breast
tumors by functioning through their cognate intracellular receptors, estrogen receptor (ER), and
progesterone receptor (PR), respectively. Coregulators of steroid hormone receptors are important
mediators of steroid receptors' function. Changes in the expression of these coactivators may contribute to
mammary gland tumorigenesis. Recently, our laboratory identified several ubiquitin pathway enzymes,
such as E6-associated protein ( E6-AP) and ubiquitin conjugating enzymes (UBCs), as coactivators of
steroid hormone receptors. Seperately, it was reported that E6-AP was overexpressed in a spontaneous
mouse model of mammary gland tumorigenesis. To study the expression profiles of E6-AP and UBCs in
human breast tumors, we examined 56 advanced stage human breast cancer biopsy samples. We found a
correlation between the expression of E6-AP and the expression of ER-alpha in these breast tumors using
Western blot analysis. The Spearman Rank Correlation Coefficient was 0.38 and the p value was 0.004,
indicating that this correlation was statistically significant. Furthermore, the expression of E6-AP also
correlated with that of UbcH7 (p=0.002), although the latter did not correlate with the expression of ER-
alpha ( p=-0.16). Our data provide the first evidence of a relationship between steroid hormone receptors
and their coactivators, E6-AP and UBCs, suggesting a possible role of these coactivators in mammary
gland tumorigenesis. It also indicates that E6-AP may be a potential target for breast cancer therapy.

Basic Science Poster: Hormones & Cancer I (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                   - 57 -
                                                                HORMONES AND CANCER

P1-241

Lay explanation of abstract:

Recently, significant progress has been made in the detection and treatment of breast cancers. However,
doctors feel helpless when endocrine therapy becomes ineffective for those patients who initially respond
well to the therapies. Therefore, it is critical to identify the molecular mechanisms associated with breast
cancer and with the development of endocrine-resistant tumors.

The steroid hormones estrogen and progesterone play a major role in the development of normal mammary
gland as well as in the development of breast tumors. The hormone molecules mediate their signaling
through intracellular receptors, called estrogen (ER) and progesterone (PR) receptors, respectively. The
functions of ER and PR are modulated by accessory proteins, called coregulators. The coregulators that
provide positive stimuli for receptors' function are coactivators, whereas those that provide negative stimuli
are corepressors. Changes in the expression of either coactivators or corepressors may contribute to the
development of breast tumors.

Our lab recently identified E6-associated protein (E6-AP) and ubiquitin conjugating enzymes (UBCs) as
co-activators of steroid hormone receptors. Separately, it was reported that E6-AP was over-expressed in a
spontaneous mouse model of breast tumors. To study the expression profiles of E6-AP and UBCs in human
breast tumors, we examined 56 advanced stage human breast cancer biopsy samples. We found a
correlation between the expression of E6-AP and the expression of ER in these breast tumor samples. The
Spearman Rank Correlation Coefficient was 0.38 and the p value was 0.004, showing that the correlation
was statistically significant. Our data provide the first evidence of a relationship between the expression
patterns of steroid hormone receptors and their coactivators, suggesting a possible role of E6-AP in
mammary gland tumor development. Hence, E6-AP may become the new targets for breast cancer therapy.


Additional background information:

Breast cancer is the most prevalent form of cancer (excluding skin cancer) among females in the United
States. About one in 10 women will have a form of breast cancer during her life. Though in recent years
significant progress has been made in detecting and treating the disease, the molecular basis of the disease
remains unclear. Endocrine therapy is the predominant treatment for patients with breast cancer, but these
therapies are ineffective for some patients and can become ineffective for some patients who initially
respond well to therapy. Therefore, it is critical to identify the molecular mechanisms associated with breast
cancer and with the development of endocrine-resistant tumors.

Cancer initially arises from abnormal expressions of certain key genes, which oversee vital cellular
processes. In the mammary gland, it is the task of estrogen (ER) and progesterone (PR) receptor proteins to
supervise the expression of these key and vital genes. ER and PR exert their regulatory control on a target
gene only in the presence of their cognate ligands, estrogen and progesterone.

The functions of ER and PR are complicated and finely tuned by accessory proteins, called coregulators.
Coregulators are divided into two groups, coactivators and corepressors. Coactivators provide positive
stimuli for nuclear receptor action, while corepressors provide negative stimuli for receptor functions. To
date, several coactivators have been cloned and identified. Recently, we identified novel coactivator
proteins, E6-AP and UBCs. These coactivator proteins fine-tuned the functions of ER and PR. The
expression of E6-AP could be used as a genetic marker for breast tumors.

This work is supported by grants from the Department of Defense and the National Institutes of Health.




                                                    - 58 -
                                                               HORMONES AND CANCER

P1-246

ANDROGEN RECEPTOR ACTIVATION INHIBITS THE GENOMIC EFFECT OF ESTRADIOL
IN MCF-7 CELLS. A MOLECULAR BASIS OF ITS ANTIPROLIFERATIVE ACTION
M Lanzino,1 F De Amicis,1 F Giordano,1 C Giordano,1 ML Panno,1 M Maggiolini,1 MJ McPhaul,2 S Ando.1
1
  Fac. of Pharmacy, University of Calabria, Rende, Italy; 2Int. Med., SWMed Center University of Texas,
Dallas, Texas

To investigate the link between androgens and human breast cancer, the hormone milieu present in pre-
postmenopausal women has been somehow translated in an in vitro model utilizing an hormone-dependent
breast cancer cell line MCF-7 exposed to DHEA, DHEAS, Androstenediol,T,DHT with or w/o E2.DHEAS
and Androstenediol stimulate growth of MCF-7 cell lines but reduce cell proliferation induced by E2 (1
nM). T and DHT (1-100 nM) instead inhibit MCF-7 cell proliferation independently of E2's presence. DHT
alone (100 nM) consistently inhibits MCF-7 cell proliferation by 50% of the basal rate and counteracts E2
proliferative action by 68%.
All this data well correlates with cell cycle analysis showing an enhanced number of cells in G0/G1 phase
after 6 days of DHT treatment.
Upon prolonged DHT exposure, Western blotting analysis shows a markedly increased AR content, while
immunohistochemistry indicates the AR translocation into the nucleus. So we assumed that the enhanced
activation of the AR might inhibit MCF-7 cell proliferation.
To better assess the role of the AR in inhibiting E2 action at genomic level, MCF-7 cells were transiently
cotransfected with the reporter plasmid XETL carrying luciferase sequence under the control of an ERE
and the full length AR or an AR carrying a mutation (C574) which abolishes its binding to DNA.
The overexpression of the AR markedly decreases the XETL expression upon E2 stimulation. The same
cells treated simultaneously with 100 nM of E2 and DHT show a more dramatic reduction of the luciferase
signal related to ERE transactivation, which is reversed by addition of OH-flutamide. This inhibitory effect
is no longer noticeable when MCF-7 cells are cotransfected with XETL and the mutant AR.
Taken together these data demonstrate that: 1)gonadal androgens are inhibitors of MCF-7 cell proliferation;
2) they antagonize the proliferative effects of E2 and this action appears to be strictly mediated by AR; 3)
the latter finding correlates with the inhibitory effects on E2 genomic action played by AR when it is
ectopically overexpressed in MCF-7 cells.

Basic Science Poster: Hormones & Cancer I (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 59 -
                                                                 HORMONES AND CANCER

P1-246
Lay explanation of abstract:

Hormones with androgenic properties are also present in women, where they are mainly secreted by the
adrenal gland and play an important physiological role. The in vitro model investigated in this study utilizes
an hormone-dependent breast cancer cell line exposed to androgens. Thus, it attempts to recreate the
hormonal ―milieu‖ features seen in pre- and post-menopausal states.

In this experiment, we were able to distinguish a double role of androgens. Some of them, precisely those
secreted by the adrenal gland, show opposed functional patterns: they inhibit breast tumor cell proliferation
in the presence of estradiol, the main steroid secreted by the ovary (e.g., in pre-menopausal condition) and
stimulate cell proliferation when estrogens are absent (e.g., in post-menopausal state).
Other powerful androgens, like testosterone and its conversion product (5-alpha-dihydrotestosterone),
inhibit cell proliferation in both conditions and thus seem to play a protective role in breast tumor
progression. It is well known that androgens exert their effects interacting with specific intracellular
proteins named androgen receptors. Thus, we have assumed that this activation may counteract the
proliferation of breast cancer cells.

To better assess the role of androgen receptor in breast cancer cells, we introduced into these cells DNA
codifying for the androgen receptor in order to transiently overexpress it and highlight its role in tumor cell
growth. In these circumstances, we have demonstrated that androgen receptor inhibits estrogen signal
promoting cell proliferation. What is new is that this study elucidates the molecular mechanism by which
the most powerful androgen (5alpha-dihydrotestosterone) and its own receptor may negatively interfere
with estrogen-promoting cell growth. These may yield potential therapies arising from the role of the most
powerful androgen in the breast cancer treatment.


Additional background information:

Up to now, the most promising tools in the hormonal treatment of breast cancer aimed to antagonize
estrogen action in promoting breast tumor growth. These tools are based on the use of anti-estrogen
molecules and aromatase inhibitors. The present study definitely suggests a candidate for an additional
pharmacological tool represented by powerful androgens as compounds that can counteract the estrogen-
induced tumor growth. These findings, which provide very attractive treatment potentialities, give rise to
the possibility of a clinical use of powerful androgens in women with breast cancer. To work effectively,
this pharmaceutical research also must yield new molecules able to activate androgen receptors in breast
cancer cells without causing other (virile) systemic effects.




                                                    - 60 -
                                                             HORMONES AND CANCER

P1-253

INFLAMMATION INDUCES CYCLOOXYGENASE-2 EXPRESSION IN PROSTATE LUMINAL
CELLS AND A HUMAN PROSTATIC INTRAEPITHELIAL NEOPLASIA CELL LINE
AC Levine,1 X Liu,1 S Yao,1 A Kirschenbaum.2 1Medicine (Endocrinology), Mount Sinai School of
Medicine, New York, New York; 2Urology, Mount Sinai School of Medicine, New York, New York

The human prostate gland is a common site of inflammation. Focal prostatic atrophy, which is associated
with chronic inflammation, is highly proliferative. Cyclooxygenase-2 (COX-2) is an inducible enzyme that
is over-expressed in cancer and inflammation. We previously demonstrated that both premalignant
(prostatic intraepithelial neoplasia or PIN) and malignant prostate cells express COX-2, whereas non-
malignant luminal epithelial cells do not express the enzyme. We hypothesized that inflammatory cytokines
induce the expression of COX-2 in luminal epithelial cells, leading to malignant transformation. Areas of
inflammation in non-cancerous prostates were immunostained for the expression of COX-2 and the anti-
death gene Bcl-2. We noted that luminal epithelial cells which were surrounded by T-lymphocytes were
induced to express both COX-2 and Bcl-2. We next tested the effects of two inflammatory cytokines,
transforming growth factor α (TGF-α) and interleukin-6 (IL-6) on COX-2 expression and PGE2 secretion
(a major COX-2 derived product) by a human PIN cell line. Northern blot analysis of COX-2 mRNA
expression levels demonstrated that TNF-alpha induced COX-2 mRNA in a time- and dose-dependent
fashion. COX-2 protein expression (Western Blot) also increased within 16h after TNF-α addition, with a
peak induction observed 24h after the addition of 1 ng/ml of TNF-α. Corresponding increases in PGE2
secretion were noted. IL-6 also induced COX-2 protein expression and PGE2 secretion in PIN cells. These
data demonstrate that non-cancerous prostate luminal epithelial cells are induced by surrounding
lymphocytes to express both COX-2 and the anti-death protein, Bcl-2. Inflammatory cytokines induce
COX-2 expression and PGE2 secretion by PIN cells. These data imply that the cytokine rich environment
in the inflammed prostate may promote cancer development and progression and that COX-2 inhibitors
may be useful in prostate cancer chemoprevention.

Basic Science Poster: Hormones & Cancer I (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                 - 61 -
                                                               HORMONES AND CANCER

P1-253

Lay explanation of abstract:

Human prostate samples (some normal, some cancerous) were stained to determine if they expressed an
enzyme called cyclooxygenase-2 (COX-2), an enzyme that is more abundant in inflamed, precancerous,
and cancerous cells of other organs. Indeed, we found that this enzyme was not expressed in normal
prostate secretory cells, but was expressed in precancerous and cancerous lesions. In addition, we found
that in areas of non-cancerous prostate where there was inflammation, the enzyme also was expressed.

We hypothesized that inflammation of the prostate causes more of this enzyme to be secreted by prostate
cells and that the enzyme, in turn, may help to initiate cancer and/or worsen cancer that already is in the
prostate. We demonstrated that the addition of substances produced by inflammatory cells (called
cytokines) does increase the expression of COX-2 in precancerous human prostate cells in the laboratory.
This information may have clinical relevance, because inhibitors of the COX-2 enzyme are FDA approved
and in clinical use for arthritis and may be useful also in the prevention or treatment of human prostate
cancer.

Funding for this project is from the T.J. Martell Foundation for Cancer, Leukemia and Aids Research.




                                                  - 62 -
                                                               HORMONES AND CANCER

P1-263
COMBINATION OF THE PURE SERM SCH 57068 WITH ESTRADIOL PROTECTS AGAINST
UTERINE AND BREAST STIMULATION WHILE PROVIDING BENEFICIAL EFFECTS ON
BONE AND LIPIDS
F Labrie, C Martel, M El-Alfy, S Gauthier, Y Merand, C Labrie. Oncology and Molecular Endocrinology
Laboratory, CHUL Research Center, Ste-Foy, Quebec, Canada

Despite the beneficial effects of hormone replacement therapy (HRT) on menopausal symptoms and the
role of estrogens in reducing bone loss and coronary heart disease, compliance to HRT is low. Women
decide not to take estrogens and stop treatment early because of the fear of breast and uterine cancer. A
new promising HRT is the combination of the pure SERM SCH57068 with estradiol (E 2). Ten- to 12-week
old female rats were ovariectomized (OVX) and treated orally once daily for 3 months with E2 (1 mg/kg),
SCH57068 (2.5 mg/kg) or E2 + SCH57068. Bone mineral density (BMD) of the lumbar spine (LS) was
10% lower in OVX control animals than in intact controls (p 2 to the SERM led to fat percentage values
below those observed in intact control animals. Serum cholesterol was increased from 2.01 ± 0.11 mmol/L
in intact animals to 2.46 ± 0.08 mmol/L in OVX controls. The administration of E 2 alone decreased serum
cholesterol levels to 1.37 ± 0.18 mmol/L (p 2 led to cholesterol levels significantly lower (0.87 ± 0.04 and
0.96 ±0.07 mmol/L, respectively) than those found in intact animals. A progressive inhibition of the
stimulatory effect of E2 (2 mg/kg) on endometrial epithelial height (EEH) and mammary gland was
observed following administration of increasing doses (0.01 to 10 mg/kg) of SCH57068. In fact, the
stimulatory effect of E2 on EEH was reversed by 83% at the 3 mg/kg dose of the SERM. The present data
show that the addition of SCH57068 to E2 blocks the stimulatory effect of estrogen on the mammary gland
and uterus, while on bone and lipids, SCH57068 exerts its own beneficial effects. A combined therapy with
SCH57068 and E2 could provide the benefits of E2 on vasomotor symptoms while the risks of breast and
uterine cancer would be eliminated by co-administration of the SERM.

Basic Science Poster: Hormones & Cancer I (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                   - 63 -
                                                                HORMONES AND CANCER

P1-263
Lay explanation of abstract:

Despite its beneficial effects in women at menopause, hormonal replacement therapy is not well accepted
by women because of fear of breast and uterine cancer. Among the women who accept treatment, 50% stop
treatment within a year.

The objective of this study is to develop a hormone replacement therapy with all the expected advantages,
but without the risk of breast and uterine cancer. In fact, this new approach would not only avoid the risk of
breast and uterine cancer, but would even prevent it. The approach combines a new 4 th generation SERM
(selective estrogen receptor modulator) SCH 57068 with an estrogen.

We know that the beneficial effect of estrogens on bone and lipids can be achieved by a new class of
compounds called SERMs. Such compounds, Raloxifene (Evista) being the first, do not, however, control
hot flashes, which is the primary reason why women consult their physician and use estrogens at
menopause.

Because we have developed the most potent SERM, SCH 57608, and it is the only SERM exerting a
complete and pure block on estrogen action in the mammary gland and uterus, we have used this compound
in the rat model. We have shown that the addition of SCH 57608 to estradiol does in fact block the
stimulatory effect of the estrogen on the mammary gland and uterus.

Moreover, at doses comparable to those used in women, results showed that while the mammary gland and
uterus were protected from the stimulatory effect of the estrogen by SCH 57608, the beneficial effects on
bone mineral density and cholesterol were maintained. Since SCH 57068 has very poor access to the brain,
estrogen should exert unhampered beneficial effects on hot flashes while all the peripheral beneficial
effects of hormone replacement therapy on bone and lipids would be achieved and breast and uterine
cancers would be prevented. Such a combination of SCH-068 and estradiol could well represent the ideal
response to the most important health concerns of women at menopause, namely osteoporosis, hot flashes,
and vascular disease, as well as breast and uterine cancer.

This research was funded by Endorecherche.




                                                   - 64 -
                                                              HORMONES AND CANCER

P2-630
PREGNANCY-RELATED SIGNALING PATHWAYS AS THERAPEUTIC TARGETS FOR
UTERINE LEIOMYOMA
K Cesen-Cummings,1 JA Copland,2 M Soloff,3 K Houston,4 CL Walker,4 BJ Davis.1 1Laboratory of
Women's Health, National Institute of Environmental Health Sciences, Research Triangle Park, North
Carolina; 2Endocrinology, University of Texas Medical Branch at Galveston, Galveston, Texas;
3
  Obstetrics/Gynecology, University of Texas Medical Branch at Galveston, Galveston, Texas;
4
  Carcinogenesis, University of Texas M.D. Anderson Cancer Center, Smithville, Texas

Uterine leiomyomas are the most common gynecological tumors in women, yet currently there is no
adequate medicinal therapy for this disease. Leiomyomas arise from myometrial smooth muscle cells, are
steroid hormone responsive, and share many biochemical and molecular characteristics associated with
myometrial cells of pregnancy and parturition. We questioned whether a phenotype characteristic of the
myometrium at parturition is also an attribute of leiomyomas that arise in the Eker rat. We found that E ker
rat leiomyomas and tumor-derived cells (ELT-3 cell line) express genes associated with parturition
including Cx43, COX-1, COX-2, and oxytocin receptors (OTR). Moreover, in the ELT-3 cells, 17β-
estradiol increases the expression of OTR, and oxytocin binding to OTR increases intracellular cytosolic
calcium demons trating that this signaling pathway is functional. Using the ELT-3 cell line, pregnancy-
related signaling pathways were investigated as potential targets for therapeutic intervention. COX-2-
mediated prostaglandin synthesis was evaluated as a possible the rapeutic avenue for decreasing leiomyoma
growth, however, prostaglandins, which are the final mediators of parturition, were found to stimulate,
rather than inhibit, proliferation of ELT-3 leiomyoma cells. Conversely, oxytocin (50 nM, 100 nM)
inhibited 17β-estra diol-induced growth of ELT-3 cells, indicating that signaling via the OTR was
antiproliferative in leiomyoma cells. These data suggest that signaling pathways mediated via OTR may be
useful therapeutic targets for uterine leiomyomas.

Basic Science Poster: Hormones & Cancer II (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 65 -
                                                               HORMONES AND CANCER

P2-630
Lay explanation of abstract:

Uterine leiomyomas or ―fibroids‖ are benign (noncancerous) tumors that are made up mostly of smooth
muscle cells in the uterus. Fibroids occur in as many as 70% of women in the United States. Fibroids cause
many health problems for women, including heavy bleeding and anemia, pelvic pain and pressure, frequent
urination, severe cramping, and infertility. Problems associated with fibroids are the most common reason
why women have their uterus removed (hysterectomy). Because of the negative physical, mental, and
economical effects associated with this major life-altering surgery, other treatment options for fibroids are
being studied.

The Eker rat is an animal that also develops uterine fibroids, and these animals can be used to study the
human disease. Indeed, the smooth muscle cells that make the uterine fibroid in both women and in the
Eker rat are similar to the smooth muscle cells of the uterus during pregnancy. We used cells (called ELT-3
cells) derived from an Eker tumor and treated them with hormones that are usually found in the uterus at
the time of labor to determine which hormones could make the tumor cells grow or stop growing. The
ELT-3 tumor cells grow in response to estrogen and the prostaglandins PGE 2 and PGF . The tumor cells,
however, do not grow when treated with oxytocin. Oxytocin treatment prevents the cells from growing in
the presence of estrogen. This suggests that oxytocin acting through the oxytocin receptor might be one
way to slow or stop the growth of fibroids in women.

Additional background information:

We do not yet fully understand why fibroids develop and grow. A genetic problem may be the cause, but
because this disease is so common among women, it is likely the environment plays a large role.
Environmental factors leading to fibroids may include chemical exposures, smoking, or diet. A major
focus of National Institute of Environmental Health Sciences (NIEHS) is to understand the role of these
environmental factors in the development of uterine fibroids. Our long-term goal is to prevent women from
developing fibroids, but first we must understand how and why fibroids grow. While these studies are
ongoing, we are investigating drugs for women who already have fibroids, in the hope that they can avoid
surgery.

This work is supported through NIEHS, NIH.




                                                   - 66 -
                                                               HORMONES AND CANCER

P3-663
COMPARISION OF THE ACTIVITY OF THIRD GENERATION SELECTIVE ESTROGEN
RECEPTOR MODULATORS (SERMS) IN A HUMAN ENDOMETRIAL CANCER CELL LINE
KS Bramlett, TP Burris. Gene Regulation, Bone, and Inflammation Research and Clinical Investigation, Eli
Lilly and Company, Indianapolis, Indiana

Selective estrogen receptor modulators (SERMs) are estrogen receptor (ER) ligands that function as
antagonists in some tissues, but have either partial or full agonist activity in others. Tamoxifen and
Raloxifene are two SERMs currently approved for clinical use. Tamoxifen is used for the treatment and
prevention of breast cancer; however, its uterotrophic activity precludes its use for other indications like
prevention of osteoporosis. Raloxifene, a SERM with an improved tissue selectivity profile, lacks the
uterotrophic activity of tamoxifen hence its use in the treatment and prevention of osteoporosis in post-
menopausal women. In this study, we examined the effects of several ER ligands including two
benzothiophenes (Arzoxifene (LY 353381), LY 117018), three triphenylethylenes (Droloxifene,
Clomiphene, Tamoxifen), and one tetrahydronapthylene in the human Ishikawa endometrial
adenocarcinoma cell line. The ability of these ER ligands to increase alkaline phosphatase (AP) activity, an
indicator of in vivo uterotrophic activity, was assessed. As expected 17-beta estradiol (E2) was a potent and
efficacious activator of AP activity and the level of maximal efficacy of other ER ligands is expressed in
terms of a percentage of the stimulation of AP by 1 micromolar E2. Tamoxifen stimulated AP activity to a
level 47% of that of E2. Droloxifene and Clomiphene also efficaciously stimulated AP activity (32% and
71%, respectively). The benzothiophene SERMs, such as Arzoxifene and the Raloxifene analog, LY
117018, displayed a superior profile exhibiting only 12% and 11% of E2 activity, respectively. The
tetrahydronapthylene enhanced AP activity to 18% of E2 control levels. Both the benzothiophene SERMs
were more potent antagonists of E2-stimulated AP activity than the tetrahydronapthylene. The IC50s for
Arzoxifene and LY117018 were 9.8 and 8.0 nM, respectively, while the tetrahydronapthylene was 20 nM.
Since one of the desirable attributes of SERMs is a tissue selectivity profile exhibiting minimal agonist
activity in uterine tissue, these data suggest that Arzoxifene displays a favorable profile.

Basic Science Poster: Hormones & Cancer III (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                   - 67 -
                                                                 HORMONES AND CANCER

P3-663
Lay explanation of abstract:

One of the molecules under development at Eli Lilly and Company is Arzoxifene, a selective estrogen
receptor modulator (SERM) currently in clinical trials for the treatment of metastatic breast cancer.

Preliminary safety data presented at the 2001 meeting of The Endocrine Society indicate that Arzoxifene,
unlike some other SERMS (for example, tamoxifen), can be administered without stimulating the
endometrium (lining of the uterus), which is associated with vaginal bleeding and endometrial cancer.

This finding, if confirmed in clinical trials, could have particular relevance for women with breast cancer
who have gone through menopause. Vaginal bleeding in the postmenopausal woman frequently requires
careful and potentially invasive evaluation to rule out endometrial cancer, notes Tom Burris, Ph.D., the
study‘s principal investigator and a research scientist at Eli Lilly and Company. A positive step forward in
the treatment of breast cancer would be to develop a therapeutic option that is safe and effective without
endometrial stimulation.

Additional background information:

This pre-clinical study used a very sensitive model of uterine stimulation known as the Ishikawa
endometrial cell line.

The Ishikawa endometrial cell line contains alkaline phosphatase, an enzyme whose expression is regulated
through the estrogen receptor; thus, it can serve as a pre-clinical model of estrogen receptor activation in
the endometrium. For instance, estradiol stimulates the endometrial estrogen receptor and results in an
increase in the synthesis (and activity) of alkaline phosphatase; this finding demonstrates that estrogen acts
as an estrogen receptor agonist in the endometrium. Conversely, an ideal SERM would induce less of an
increase in alkaline phosphatase synthesis (and activity) than estradiol; in this instance, such a finding
demonstrates that the SERM acts as an estrogen receptor antagonist in the endometrium.

In this study, three classes of SERMS (benzothiophenes, triphenylethylenes, and tetrahydronapthalene)
were tested in the Ishikawa model. Of the three classes of SERMS, the benzothiophenes (to which
Arzoxifene belongs) demonstrated the smallest increase in alkaline phosphatase activity relative to
estradiol, suggesting that the benzothiophenes should have very little agonist activity in uterine tissue; both
the triphenlyethylene and tetrahydronaphthalene agents demonstrated evidence of endometrial estrogen
receptor activation. The low IC50 of the benzothiophenes, relative to that of the tetrahydronaphthalene,
indicate that the benzothiophenes are very powerful inhibitors of estradiol-induced alkaline phosphatase
activity (i.e., potent estrogen receptor antagonists).

This study was conducted and funded by Eli Lilly and Company.




                                                    - 68 -
                                                                       HORMONE NEOPLASIA

OR50-4
THE ADHESION MOLECULE CEACAM1 IS SPECIFICALLY EXPRESSED IN PAPILLARY
STRUCTURES OF THYROID CARCINOMA
AM Bamberger,1 CM Bamberger,2 T Loning,1 C Wagener,3 SL Asa.4 1Institute of Pathology, University
Hospital Eppendorf, Hamburg, Germany; 2Medicine, University Hospital Eppendorf, Hamburg, Germany;
3
  Clinical Chemistry, University Hospital Eppendorf, Hamburg, Germany; 4Pathology, University Health
Network, Toronto, Ontario, Canada

The molecular mechanisms underlying the formation of benign and malignant thyroid tumors are largely
unknown. Furthermore, the molecular events leading to morphologically distinct thyroid tumor types are
not understood. Adhesion molecules, such as CEACAM1 (CD66a, BGP, C-CAM), play an important role
in maintaining normal tissue architecture and are, thus, often found to be dysregulated in neoplasia. The
purpose of the present study was to investigate the expression pattern of CEACAM1 in the normal human
thyroid gland, as well as in benign and malignant thyroid tumors. For this purpose, we performed
immunohistochemistry on 125 paraffin-embedded thyroid tissue samples (35 thyroids exhibiting nodular
hyperplasia, 40 papillary carcinomas, 35 follicular variant papillary carcinomas, 6 solid variant papillary
carcinomas, 5 follicular carcinomas, 4 insular carcinomas, of which 100 included nonnodular thyroid
tissue) using the 4D1/C2 anti-CEACAM1 monoclonal antibody, which specifically recognizes CEACAM1,
but not other members of the CEA family. In normal thyroid tissue, expression of CEACAM1 was present
in stromal blood vessels, but was absent from epithelial structures. The same was true for nodular
hyperplasia, follicular and solid variant papillary, follicular, and insular carcinomas. In contrast, most of the
papillary cracinomas displayed strong focal staining on the luminal border of the papillary epithelium. In
some follicular variant papillary carcinomas, microscopic areas of papillary architecture demonstrated
luminal staining. In summary, we demonstrate a highly specific expression pattern for CEACAM1 in the
human thyroid gland, with papillary structures showing strong expression and follicular structures beeing
CEACAM1-negative. CEACAM1 may, therefore, be implicated in the molecular morphogenesis of
papillary thyroid lesions.

Basic Science Oral: Hormone Neoplasia (1:30 PM-3:00 PM)

Presentation Date: Saturday, June 23, 2001; Time: 12:00 AM; Location: C 106




                                                     - 69 -
                                                                     HORMONE NEOPLASIA

OR50-4
Lay explanation of abstract:

The thyroid gland is a two-lobed structure in the middle of the neck that produces thyroid hormone, which
regulates the body‘s metabolic rate. This gland is frequently the site of tumor formation. Whether these
tumors are benign or malignant is a major question that has to be answered rigorously in order to apply
rational and appropriate treatment. A major diagnostic procedure in answering this question involves
examination of the appearance of cells aspirated from an identified thyroid nodule. Unfortunately, due to
the lack of objective criteria, this procedure carries a high rate of equivocal or indeterminate findings.

In this study, we examined the presence of a protein (CEACAM1) as a potential objective marker of
thyroid malignancy. This protein has thus far been shown to participate in the maintenance of normal tissue
architecture. In this study, we show that the presence of this protein in normal or non-malignant thyroid
disease is restricted to blood vessels and surrounding structures of the thyroid.In marked contrast, this
protein was widely distributed within the tongue-like protrusions characteristic of the most common type of
thyroid cancer papillary carcinoma.

The results of this study provide a potentially reliable and objective marker that can be applied to aspirated
cells from the thyroid to help confirm or refute the diagnosis of cancer in this gland.

This research was funded by the Medical Research Council of Canada/Canadian Institutes for Health
Research.




                                                    - 70 -
                                                                   HORMONE NEOPLASIA

OR50-5

REGULATION OF GENE EXPRESSION AND TRANSCRIPTION FACTORS IN MCF-7 CELLS
BY HUMAN CHORIONIC GONADOTROPIN
CV Rao,1 X Li,1 SK Manna,2 ZM Lei,1 BB Aggarwal.2 1Department of Ob/Gyn, University of Louisville
Health Sciences Center, Louisville, Kentucky; 2The University of Texas M. D. Anderson Cancer Center,
Houston, Texas

Human, rat and sow mammary glands and human breast cancer cell lines contain human chorionic
gonadotropin (hCG)/luteinizing hormone (LH) receptors. The activation of these receptors can exert anti-
proliferative and anti-invasive effects in MCF-7 cells. These and other studies support the concept that
completion of full term pregnancy at a young age confers lifetime protection against breast cancer in
women. To further advance our present understanding on anti breast cancer actions of hCG, MCF-7 cells
were treated with 50 ng/ml of highly purified hCG, and then analyzed for changes in gene expression using
Clontech's human cDNA expression arrays. The results demonstrated that treatment with hCG for 4 hrs
resulted in decreased expression of 11 genes and increased expression of one. After 24hrs of treatment, the
number of genes showing decreased expression reached 18, and the number of genes showing increased
expression reached six. The same genes were not involved in changes at both time points. The magnitude
of gene expression changes varied from modest to dramatic and came from eleven families, which included
transcription factors, oncogenes and tumor suppressors, extracellular matrix proteins, metabolism, etc.
However, the genes involved in metabolism by far outnumbered the other families after 24-hr treatment.
We next examined the effect of hCG on transcription factors, NF-kB and AP-1. Treatment of MCF-7 cells
with hCG resulted in an inhibition of TNF-alpha induced activation of NF-kB and AP-1. The inhibition of
NF-kB activation by hCG correlated with a decrease in the phosphorylation and degradation of IKB alpha.
These effects of hCG could be mimicked by treatment of cells with dibutyryl cAMP and inhibited by
treatment with adenyl cyclase inhibitor, dideoxyadenosine, suggesting the critical signaling role of cAMP.
In summary, our results suggest that the suppressive effects of hCG against breast cancer may also involve,
in part, modulation of gene expression through inhibition of activation of transcription factors via cAMP
signaling.

Basic Science Oral: Hormone Neoplasia (1:30 PM-3:00 PM)

Presentation Date: Saturday, June 23, 2001; Time: 2:30 PM; Location: C 106




                                                  - 71 -
                                                                    HORMONE NEOPLASIA

OR50-5

Lay explanation of abstract:

Studies from our laboratory, as well as others, have demonstrated that human and animal breast tissues and
human breast cell lines contain low levels of receptors that bind human chorionic gonadotropin (hCG) and
its structural and functional homolog, luteinizing hormone (LH). Activation of these receptors results in
anti-proliferative and anti-invasive actions in human breast cancer MCF-7 cells.

Our current ongoing basic science research study found further evidence that hCG works by inhibiting the
genes that are likely to be involved in promoting the carcinogenic process and increasing the expression of
genes that could be involved in inhibiting the carcinogenic process. These findings bring us one step closer
to potential hCG use in preventive treatment of young women who wish to delay their first pregnancy,
women who are at increased risk for breast cancer, or even those that have developed the disease. As a
therapeutic agent, hCG is inexpensive with tolerable physiological side effects, and is a better choice in
most cases when compared to alternatives.

Additional background information:

The incidence of breast cancer is the highest among all newly diagnosed cancers in women. The incidence
of breast cancer is increasing and currently is a leading cause of cancer-related deaths among American
women. Although numerous scientific breakthroughs have occurred, it is not completely clear what causes
this disease and how to prevent it. As in most other cancers, the etiology of breast cancer is probably
multifactorial and quite complex, and it may not be totally preventable. The current work with the
pregnancy hormone, hCG, as a protection against breast cancer represents a neglected but exciting and
potentially promising area in breast cancer research. Further research could lead to therapeutic use of hCG
in breast cancer prevention and/or treatment.




                                                  - 72 -
                                                                     HORMONE NEOPLASIA

OR50-6

CONDITIONAL EXPRESSION OF RET/PTC INDUCES A WEAK ONCOGENIC DRIVE IN
THYROID PCCL3 CELLS
E Puxeddu, J Wang, S Basu, JA Knauf, JA Fagin. Endocrinology and Metabolism, University of
Cincinnati, Cincinnati, Ohio

Chromosomal rearrangements linking the promoter/s and N-terminal domains of unrelated gene/s to the C-
terminus of RET result in the production of constitutively activated truncated forms of the receptor in
thyroid cells (RET/PTC). RET/PTC rearrangements are thought to be tumor-initiating events in the thyroid
as: 1) Radiation can directly induce RET/PTC rearrangements in vitro. 2) Chromosomal loci participating
in the RET/PTC1 rearrangement are juxtaposed during interphase in thyroid cells, providing a target for
simultaneous double strand DNA breaks leading to illegitimate recombination. 3) Overexpression of
RET/PTC isoforms in thyroid glands of transgenic mice results in tumors with a papillary histotype.
Despite this evidence, the early biological consequences of RET/PTC activation are not known. To explore
this, we generated clonal lines derived from well-differentiated rat thyroid PCCL3 cells with doxycyclin-
inducible expression of either RET/PTC1 or RET/PTC3. Expression of the chimeric genes was detectable
2h after doxycyclin, and was dose-dependent. As previously shown in other cell types, RET/PTC1 and 3
oligomerized, and displayed constitutive tyrosine kinase activity. Neither RET/PTC1 nor RET/PTC3
conferred cells with the ability to grow in the absence of TSH. Further analysis established that acute
RET/PTC1 or RET/PTC3 expression increased DNA synthesis, but this was associated with concomitant
induction of apoptosis, resulting in no significant net growth in the cell population. Acute expression of the
oncoproteins did, however, decrease TSH-mediated growth stimulation, and differentiated gene expression.
This was due to interference of TSH signaling by RET/PTC at multiple levels, through 1) decreased TSH-R
mRNA levels, 2) impairment of TSH and forskolin-induced adenylyl cyclase activity, and 3) decreased Br-
cAMP-induced DNA synthesis. As opposed to the early effects of RAS in thyroid cells, activation of
RET/PTC did not induce formation of micronuclei, suggesting that this oncoprotein may not predispose to
acquisition of large scale chromosomal abnormalities. We conclude that RET/PTC is a weak tumor
initiating event, and that secondary genetic or epigenetic changes are critical for clonal expansion.

Basic Science Oral: Hormone Neoplasia (1:30 PM-3:00 PM)

Presentation Date: Saturday, June 23, 2001; Time: 12:00 AM; Location: C 106




                                                   - 73 -
                                                                    HORMONE NEOPLASIA

OR50-6
Lay explanation of abstract:

The risk of developing thyroid cancer is increased substantially by exposure to radiation early in life. We
now know that radiation causes a specific chromosomal defect, a rearrangement of the RET gene, and that
this is the initiating event in developing most papillary thyroid carcinomas (PTC). However,
rearrangements of the RET gene also are seen in PTC from patients with no history of radiation exposure.

Several investigators have shown that when the expression of the RET/PTC oncogene is induced in thyroid
cells of transgenic mice, these mice develop lesions consistent with papillary thyroid carcinoma during
their natural life span. It is not known, however, what precise cellular events are involved after tumor
initiation by RET/PTC.

In this study, we initiated expression of the RET/PTC oncogene in thyroid cells in culture using a method
to activate the oncogene acutely by adding an antibiotic to the growth medium. Curiously, oncogenic
initiation by RET/PTC expression is not accompanied by a significant increase in cell growth. There is not
much cell growth, even though RET/PTC activation induces DNA synthesis. Simultaneously, however,
RET/PTC results in a stimulation of cell death. Concerted activation of both cellular growth and
programmed death result in a relatively stable cell population.

These findings indicate that the RET/PTC oncogene is a relatively weak oncogenic initiator. Additional
genetic or cellular abnormalities must take place for the clone to ultimately become transformed and
generate cancers. This might explain the high frequency of small nests of RET/PTC- positive papillary
carcinomas found in the glands of most adults, even those without thyroid cancer. These may simply
represent tumor clones initiated by RET/PTC activation, in which secondary progression steps have not yet
developed.




                                                   - 74 -
                                              HORMONES AND IMMUNE SYSTEM

OR51-1
ALTERED HYPOTHALAMIC-PITUITARY-ADRENAL (HPA) AXIS REGULATION IN TNFα-
DEFICIENT MICE
M Venihaki,1 G Kollias,2 KP Karalis.1 1Endocrinology, Children's Hospital, Boston, Massachusetts;
2
  Immunology, A. Fleming Biomedical Sciences Research Center, Varkiza, Greece

HPA axis activation following various stressors, including inflammation, is manifested by increased ACTH
and glucocorticoid secretion. TNFα, a potent proinflammatory cytokine, has been suggested to be an
important mediator of the stimulation of HPA axis in response to immune activation. TNFα is also
expressed in the adrenal gland and stimulates steroidogenesis. The aim of our study was to investigate the
impact of TNFα genetic deficiency in the stimulation of HPA axis by immune and non-immune stressors.
Thus, we used TNFα wild type (Tnfα+/+) and knockout mice (Tnfα-/-) either injected ip with 100µg LPS or
subjected to restraint stress for 15min. Activation of the HPA axis was evaluated by measurement of
plasma ACTH and corticosterone levels. Surprisingly, basal ACTH levels were significantly higher in
Tnfα-/- mice compared to their wild type littermates (Tnfα+/+ 35.33±5.13 vs Tnfα-/- 61±10 pg/mL), while
no difference was detected between their corresponding corticosterone levels (3.1±1.3 and 3.6±1.1µg/dL,
respectively). Following LPS injection or restraint stress, plasma ACTH levels were significantly elevated
and to the same extent (2-3x) in both Tnfα+/+ and Tnfα-/- mice. However, despite this similar elevation of
ACTH in both genotypes, corticosterone levels were significantly higher in Tnfα-/- compared to Tnfα+/+
mice following either of the two stressors (LPS: Tnfα+/+ 49.2±5.4 vs Tnfα-/- 71.2±6.2µg/dL; Restraint:
Tnfα+/+ 54.9±2.7 vs Tnfα-/- 67±3.9µg/dL). In summary, TNFα deficiency is associated with high basal
ACTH levels and enhanced stress-induced glucocorticoid release. Our findings suggest that endogenous
TNFα is a negative regulator of basal ACTH secretion and may act to restrain the adrenal glucocorticoid
response during stress. The tissue source of the amounts of TNFα that regulate the above basal and stress-
induced altered HPA axis activity remains to be identified.

Basic Science Oral: Hormones & Immune System (1:30 PM-3:00 PM)

Presentation Date: Saturday, June 23, 2001; Time: 1:30 PM; Location: A 109




                                                  - 75 -
                                                HORMONES AND IMMUNE SYSTEM

OR51-1
Lay explanation of abstract:

The hypothalamic-pituitary-adrenal (HPA) endocrine axis is one of the major hormonal systems affected by stress in
humans and rodents. Activation of the HPA axis by various stressors, including inflammation, causes increased
production of a brain hormone, pituitary ACTH. ACTH then reaches the adrenal gland and stimulates the release of
glucocorticoid. Increased secretion of glucocorticoid is one of the physiological reactions of the body to fight
inflammation, because this compound is one of the strongest anti-inflammatory agents in use.

During invasion by foreign organisms and with the inflammation that follows, several factors secreted from the
inflamed areas and from the immune system stimulate the HPA axis. Tumor necrosis factor alpha (TNF), which is
elevated in the blood of patients with a variety of inflammatory diseases, is one of these factors and has been found
to stimulate the HPA axis in humans. TNF works similarly in animals with experimental inflammation that mimics
the human diseases.

The aim of our study was to investigate the role of TNF deficiency, caused by deletion of the TNF gene, in the
stimulation of the HPA axis following inflammatory and non-inflammatory stressors. One example is restraint
stress, a psychological stress caused by immobilization. For this study, we used mice with or without TNF
deficiency. We either applied an acute systemic inflammation (by injection of 100 g of lipopolysaccharide; LPS),
which resembles human endotoxemia and septic shock, or 15 minutes of restraint stress. We evaluated activation of
the HPA axis by measuring blood levels of ACTH and glucocorticoid. Our results showed that during basal
conditions, TNF-deficient mice have higher levels of ACTH compared with normal mice. Following either of the
above- mentioned stressors, however, levels of ACTH were similarly elevated in both types of mice. Despite the
similar rise in blood ACTH level, TNF-deficient mice had significantly higher levels of glucocorticoid after
injection of LPS, but not after the restraint stress. This suggests a difference in the way the adrenal gland of TNF-
deficient mice responds to inflammatory stress and how it reacts to psychological stress.

Our results suggest that endogenous TNF may help to inhibit glucocorticoid production during inflammatory
stress. These findings, in addition to our ongoing studies, may contribute to the understanding of hormonal
responses in patients treated with TNF antagonists for such chronic inflammatory diseases as rheumatoid arthritis
and inflammatory bowel disease. Our studies also are providing useful insights for the design of new anti-
inflammatory agents.

This work was supported by an NIDDK grant RO1DK47977 to Dr. Katha Karatis and is part of a
collaboration with Dr. Kollias from Fleming Research Institute, Athens, Greece who created the TNF
mice.




                                                   - 76 -
                                                HORMONES AND IMMUNE SYSTEM

OR51-3

GENISTEIN INDUCES THYMIC ATROPHY THROUGH BOTH ER AND NON-ER MEDIATED
SIGNALING PATHWAYS
S Yellayi, A Naaz, T Sato, M Szewczykowski, P Cooke. Veterinary Biosciences, University of Illinois at
Urbana-Champaign, Urbana, Illinois

Genistein, a phytoestrogen present in soy, interacts with both estrogen receptor alpha (ER α) and ER[β] but
also has other functions. Estrogen has thymolytic effects, but the effects of phytoestrogens on thymus are
unclear. Here we examine d both estrogen receptor (ER) and non-ER mediated thymolytic effects of
genistein. Age-matched (13 weeks old) female mice (n = 5 for all groups) were ovariectomized and
beginning 7 days later injected (s/c) daily for 21 days with DMSO, E2 (125 and 300 ng/day) or genistein (5,
2, 0.5 and 0.2 mg/day); some animals were given E2 or genistein + ICI 182,780, the anti-estrogen (5
mg/week) to examine ER and non-ER mediated effects of genistein. Mice were killed and thymic, uterine,
vaginal and body weights determined. Thymic weight was normal in DMSO (control) mice. E2 induced a
50% reduction in thymic weight relative to controls, as previously published. Unexpectedly, there were
20%, 40%, 70% and 80% decreases in thymic weight at genistein doses of 0.2, 0.5, 2 and 5 mg/day,
respectively. Interestingly, ICI 182,780 totally blocked E2 effects on thymus, but blocked only 50%, 40%
and 16% of genistein effects at 5, 2 and 0.5 mg/day, respectively. These results indicate that at higher doses
(2 and 5 mg/day) genistein effects are mediated through both ER and non-ER pathways, but at the lower
dose (0.5 mg/day) the effects were mostly non-ER mediated. To insure that ICI 182,780 treatment inhibited
signaling through ER, we measured uterine and vaginal weights. I n both the E 2+ ICI and genistein + ICI
groups, uterine and vaginal weights were similar to controls. In conclusion these results show that genistein
produces thymic atrophy through both ER as well as non-ER mechanisms. In addition, these effects on the
th ymus were seen at doses (0.2 mg/day, equivalent to 8 mg/kg/day, based on a 25 g mice) comparable to
those in human infants drinking soy formula (6-11 mg/kg/day of total isoflavones). These results are the
first to show that genistein at doses similar to human infant exposure produces thymic atrophy in mice and
the effects at such low doses are primarily non-ER mediated possibly through inhibition of protein tyrosine
kinases and/or topoisomerase II.

Basic Science Oral: Hormones & Immune System (1:30 PM-3:00 PM)

Presentation Date: Saturday, June 23, 2001; Time: 2:00 PM; Location: A 109




                                                   - 77 -
                                               HORMONES AND IMMUNE SYSTEM

OR51-3

Lay explanation of abstract:

Although soy-based food has been associated with a variety of beneficial health effects, too much soy in a
diet can cause problems. The benefits of soy may involve the estrogenic effects of genistein, an isoflavone
present in soybeans and soy food products. However, the use of soy formula for newborns and infants has
led to exposure to high levels of both genistein and daidzein (another isoflavone present in soy) compared
with infants who drink only human or cow‘s milk. Infants drinking soy formula consume 6-11 mg of
isoflavones/kg/day, ten times more than adults eating a high soy diet. This high level of isoflavone
consumption by infants may be problematic, mostly because of the potential estrogenic effects of genistein.
Estrogens can have deleterious effects, and high doses can cause atrophy of the thymus gland, which is
needed early in life for development of normal immune functions.

In this study, we investigated the effects of the estrogenic soy isoflavone genistein on the thymus of
laboratory mice. Ovaries from female mice were removed, and beginning 7 days later, they were injected
daily for 21 days with estrogen (E2) or genistein (0.2-5 mg/day) or given no treatment (control). Some
animals were given E2 or genistein plus the anti-estrogen ICI 182,780 (5 mg/week) to determine if
genistein‘s effects were mediated through estrogen receptor (ER). Genistein at 0.2 mg/day caused a
decrease of approximately 20% in thymus gland (thymic) weight. In addition, higher doses of genistein
caused up to an 80% decrease in thymic weight. Furthermore, there were decreases of up to 90 % in
thymocyte numbers and also changes in the thymocyte subpopulations in genistein-treated animals.
Interestingly, ICI 182,780 totally blocked E2 effects on thymus, but only partially blocked the effects of
genistein, which indicates that genistein‘s effects are mediated through both ER and non-ER pathways.

This is the first report showing an in vivo effect of genistein on thymic atrophy. Genistein‘s effect as a
thymolytic substance can be seen in its ability to produce drastic decreases in thymic weight and thymocyte
number (greater than those caused by high doses of E2.) These effects were seen at doses as low as 0.2
mg/day, corresponding to 8 mg/kg. This is comparable to the level of exposure of human infants who
consume soy formula. Thus, soy formula, which accounts for 25% of formula sales in the U.S., may be
capable of inducing deleterious effects on the developing human infant thymus and immune system. This
topic needs to be investigated further.

This research was supported by the Illinois Council on Food and Agricultural Research (C-FAR).




                                                   - 78 -
                                               HORMONES AND IMMUNE SYSTEM

OR51-4

THE EFFECT OF GH ADMINISTRATION IN LPS-INDUCED SEPTIC SHOCK IS CRITICALLY
DEPENDENT ON TIMING OF TREATMENT
TK Hansen,1 S Thiel,2 F Dagnaes-Hansen,2 JO Jorgensen,1 A Flyvbjerg.1 1Medical Dept. M, Aarhus
University Hospital, Aarhus, Denmark; 2Dept. of Medical Microbiol. and Immunol., University of Aarhus,
Aarhus, Denmark

An excessive mortality from sepsis and multi-organ failure has recently been reported in critically ill
patients treated with high doses of growth hormone (GH). Septic shock is often induced by release of
lipopolysaccharides (LPS) from gram-negative bacteria, and the severity of the condition depends on the
degree of immune activation in the host.To address the influence of timing of GH administration on the
outcome in septic shock, we used a murine sepsis model. On day 0, 8 weeks old BALB/C mice were
injected with LPS (9.6 mg/kg ip.). Body weight (BW), food-, and water intake was recorded daily. The
mice were treated with GH (4 mg/kg/day), a GH-receptor antagonist (GHA), pegvisomant (1 mg/kg/day) or
placebo (Plc) using two daily sc. injections (n=12 in each group). In study I, the animals were treated from
day 0 through day 4, whereas in study II, treatment was initiated three days before the LPS injection
throughout day 4.
In both studies, the induction of septic shock was associated with a significant weight loss in all animals,
reaching a maximum on day 2. In study I, the GH treated animals were significantly less affected, and the
GHA treated significantly more affected, than the Plc treated animals on day 2 (weight loss from baseline;
GH: -5.0 ± 1.2 %; GHA: -15.8 ± 0.8 %; Plc: -12.5 ± 1.3 %; P < 0.0001). The GH group reached baseline
weight on day 3, while neither the GHA nor the Plc group had retrieved the loss by day 4. In study II, on
the other hand, the GHA group was significantly less affected than the GH and placebo group on day 2
(weight loss from baseline; GH: -18.5 ± 1.0 %; GHA: -13.6 ± 0.6 %; Plc: -17.1 ± 0.6 %, P < 0.0001), 4
mice died on day 2 (2 in the GH and 2 in the Plc group), and none of the groups had reached baseline
weights by day 4. In both studies, the changes in BW were reflected in food and water intake.
Our findings demonstrate, that the consequences of GH administration in septic shock are critically
dependent on the timing of treatment. It appears, that the detrimental effects of GH may depend on up-
regulation of inflammatory mediators, rather than acute metabolic actions of GH per se.

Basic Science Oral: Hormones & Immune System (1:30 PM-3:00 PM)

Presentation Date: Saturday, June 23, 2001; Time: 2:15 PM; Location: A 109




                                                  - 79 -
                                               HORMONES AND IMMUNE SYSTEM

OR51-4
Lay explanation of abstract:

Treatment of critically ill patients with growth hormone has been proposed as a way to prevent the
catabolic state and extensive muscle wasting typically seen in these patients. However, in a recent large-
scale, multi-center study of intensive care patients, a very surprising detrimental effect of growth hormone
treatment was observed. The excessive mortality among the growth-hormone-treated patients was almost
entirely due to sepsis or septic shock, which lead the investigators to conclude that a growth-hormone-
mediated change in the immune system occurred.

In this study, we used an animal (murine) model to further investigate the effects of treatment with growth
hormone or a novel growth hormone receptor antagonist in septic shock. When growth hormone treatment
and septic shock were initiated simultaneously, we found that growth hormone was clearly beneficial to the
animals. On the other hand, animals pre-treated with growth hormone were significantly more affected by
septic shock than animals treated with a growth-hormone-receptor antagonist.

Our findings demonstrate that the consequences of growth hormone treatment in septic shock are critically
dependent on the timing of treatment. It appears that the detrimental effects of growth hormone may
depend on up-regulation of one or more components of the immune system, rather than acute metabolic
actions of growth hormone as such. Characterization of these mechanisms and identification of possible
ways to inhibit them could benefit not only growth-hormone-treated patients, but also all patients exposed
to sepsis or septic shock. So far, the exact connection remains unclear, but we are currently investigating
the effects of growth hormone administration on a number of candidate immune factors.

The study was funded in part by The Danish Medical Research Council.




                                                   - 80 -
                                              HORMONES AND IMMUNE SYSTEM

P2-599
DEHYDROEPIANDROSTERONE (DHEA) AS A NOVEL THERAPEUTIC AGENT FOR ACUTE
HEPATIC INJURY
S Yoshida,1 A Nakajima,1 K Mukasa,1 M Omura,1 Y Nagashima,2 K Wada,3 H Sekihara.1 1The Third
Department of Internal Medicine, Yokohama City University, School of Medicine, Yokohama, Japan; 2The
Second Department of Pathology, Yokohama City University, School of Medicine, Yokohama, Japan; 3The
Department of Pharmacology, Osaka University, Graduate School of Dentistry, Osaka, Japan

Aim: DHEA, one of the predominant androgens secreted by the adrenal cortex, is a potential immunologic
regulator. In the present study, we examined the effect of DHEA on hepatic injury induced in mice by a
single injection of concanavalinA (ConA), on the induction of apoptosis in Hep-G2 cells and on the
production of inducible nitric oxide synthase (iNOS) in RAW264 murine macrophage cell line.
Methods: Mice were pretreated with DHEA and were injected i.v. with ConA(20mg/kg). Serum and liver
tissues were collected at 0, 8, 20 and 30hrs after ConA injection. The levels of TNF-α, cyclooxygenase
(COX)-2, iNOS, and macrophage migration inhibitory factor (MIF) mRNAs were measured in the liver
using relative quantitative RT-PCR. Apoptosis was detected in the tissue section by the TUNEL method
and by the anti-M30 antibody which recognizes the early stage of apoptosis. Hep-G2 cells were pretreated
with DHEA(10μM), then apoptosis induced with TNF-α and actinomycinD (ActD) was detected by
PI/Hoechst33342 staining. RAW264 cells treated with DHEA(10μM) after lipopolysaccharide (LPS,
10μg/ml) exposure were measured the levels of iNOS mRNA.
Results: The serum level of ALT on the DHEA-treated mice was significantly decreased and expressions of
inflammatory mediators, including TNF-α, iNOS were also dramatically decreased. The number of
apoptotic cells was much lower than that in control mouse liver. Immnohistochemical data showed that the
inflammatory infiltration of cells in the liver was markedly decreased in the DHEA-treated mice. In Hep-
G2 cells, fewer apoptotic cells were observed in the DHEA-treated cells than that in the controls. The
expression of iNOS on RAW264 cells compared to the DHEA-treated cells was decreased.
Conclusion: The present results suggest that DHEA can reduce inflammation in the liver via the inhibition
of expression of several and via the inhibition of apoptosis. Thus, DHEA may be a candidate for a novel
therapy for liver injury.

Basic Science Poster: Hormones & Immune System (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                 - 81 -
                                                HORMONES AND IMMUNE SYSTEM

P2-599

Lay explanation of abstract:

Most cases of hepatitis, including viral hepatitis and acute hepatitis, arise as an immunoreaction against
one‘s own body. The leading causes are inflammatory mediators from T-cells, immunocytes, or
macrophages. At the conclusion of these inflammatory reactions that become hepatitis disease, liver cell
death can occur and the patient‘s condition can worsen to cirrhosis of the liver or even liver cancer.

DHEA, one of the predominant androgens secreted by the adrenal cortex, is a potential immunologic
regulator. It is said that DHEA has anti-inflammatory effects. Thus, we examined the effect of DHEA on
patients with hepatitis. This study shows for the first time ever that DHEA could significantly control
hepatitis.

Additional background information:

DHEA is sold as a nutritional supplement in the United States. Controlling cases of hepatitis can help to
prevent the risk of liver cirrhosis or liver cancer. Thus, our finding that DHEA could significantly control
hepatitis may prove to be useful in protecting the public health.




                                                    - 82 -
                                               HORMONES AND IMMUNE SYSTEM

P2-601

ANABOLIC ACTIONS OF GROWTH HORMONE IN CATABOLIC STATES: ANALYSIS OF
DIFFERENTIAL GENE EXPRESSION IN RATS TREATED WITH GH AND LPS USING CDNA
MICRO ARRAYS
I Hoiden-Guthenberg,1 A Flores-Morales,1 G Norstedt,1 L Fryklund.2 1Molecular Medicine, Karolinska
Institute, Stockholm, Sweden; 2Endocrine Care, Pharmacia AB, Stockholm, Sweden

GH-treatment has previously been shown to exacerbate sepsis both in rats and humans. The present study
has investigated the possibility that GH interfered with the inflammatory reaction by activating genes
whose function are incompatible with the normal septic response. We have employed cDNA microarray
analysis and RNAse protection assays to identify genes that are differentially expressed in livers from
GH+LPS treated compared to LPS-treated rats. When using the micro-array approach, 110 genes were
identified and 52 of those encoded for proteins with known functions. Most genes were classified into four
different functional categories including intermediary metabolism, detoxification, signal transduction and
secreted products. Among the identified genes were GH-regulated genes described previously, such as
carbonic anhydrase III and LPS-regulated genes such as alpha-2-macroglobulin. The array results indicated
that GH altered the expression level of genes encoding for important metabolic enzymes such as L-type of
Pyruvate kinase, Pyruvate dehydrogenase kinase-4, enzymes within the fatty acid oxidation pathway and
Glutamine synthetase and dehydrogenase. The altered expression of these genes implies that GH may have
diverted amino acids from the liver and therefore disturbed the acute phase protein synthesis. The
expression of four different acute phase proteins was investigated and it was found that GH interfered with
the expression of these proteins both in rats treated with GH alone and in the presence of LPS. The
mechanism for these GH-mediated alterations was probably related to changes in signal transduction and
the array analysis and RNA:se protection assay showed that the expression of phosphatases PP2A, MKP1
and SOCS3, all known to be negative regulators of signalling was induced in GH + LPS-treated rats. From
these results, we can hypothesise that GH exacerbated the septic reaction by interfering with fuel
availability and protein synthesis in the liver and that the mechanisms behind the alterations involves
changes in flow of signals through MAPK and JAK-STAT pathways.

Basic Science Poster: Hormones & Immune System (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 83 -
                                                 HORMONES AND IMMUNE SYSTEM

P2-601
Lay explanation of abstract:

Our research provides new information about the actions of Growth Hormone (GH) in conjunction with
critical illness and sepsis. Most importantly, our results demonstrate the power of the newly developed
technique: cDNA microarray, in which the expression of a large number of genes is analyzed
simultaneously. This technique was used as a tool to dissect out anomalies that related to complex
physiological events.

GH is an anabolic hormone, promoting muscle build-up. The hormone is theoretically an ideal drug for
treating critically ill patients whose muscles are literally broken down. However, clinical studies have
shown that GH reduces the chance of survival.

In this study we describe several mechanisms that explain why GH causes patients‘ conditions to worsen.
We found that GH affected the ability of the liver to supply fuel, to carry out detoxification processes, and
to produce acute-phase reactants that are vital in supporting the body suffering from sepsis and trauma.
These hormone-related alterations involved changes in the flow of signals from the surface of cells to the
nucleus in three fundamental signalling pathways. Our results also may give insights into the general
mechanisms of how and why sepsis may progress into shock and death. An increased understanding of the
etiology of sepsis will of course benefit the development of appropriate intervention strategies. As yet, no
drugs are available that can ameliorate sepsis and septic shock.

The study used rats as research subjects. The rats were treated with GH for seven days followed by a single
injection of a bacterial endotoxin. The endotoxin itself induces a septic reaction in rats and the combination
of GH and endotoxin exacerbated the reaction. By extracting the genetic message from the livers of
hormone- and endotoxin-treated rats and comparing the specimens with those from rats treated with only
endotoxin, we used the cDNA-microarray technique to obtain a single real-time image of what was
occurring in the liver. This information has then been combined with results from other assays and
analyzed by computerized bioinformatics.

The immediate clinical implication of our study would be a recommendation that GH should not be used to
treat critically ill patients, a practice that has already been discontinued. Our study also demonstrates
plainly the dual function of GH as an anabolic endocrine agent and as a cytokine that affects the immune
system.

Additional background information:

Anyone can suddenly become critically ill, either as a result of trauma, complicated surgery, or severe
infections. Intensivists have been trying for years to find some way of alleviating the severity of the illness
as well as providing a faster route to recovery. Although great improvements in survival and disease have
been made through careful patient management and care, no drugs have yet been identified that could
accelerate the healing process and reduce the time needed for convalescence.

Dr Ingmarie Höiden-Guthenberg received a 2 year postdoctoral grant from Pharmacia AB in Sweden. As
well as funding from the Swedish Medical Research Foundation and by Lisa and Johan Grönbergs
foundation. The research has been funded by Pharmacia Corporation, by the Swedish Medical Research
foundation, and by Lisa and Johan Grönbergs foundation.




                                                    - 84 -
                                                                                            HPA AXIS

P3-194

INDIVIDUAL DIFFERENCES IN ANXIETY AND ADRENOCORTICAL RESPONSE TO
NOVELTY: CONTRIBUTION OF PARENTAL PHENOTYPE AND MATERNAL BEHAVIOR
DM Vazquez,1,2 M Kabbaj,2 MH Kabbaj,2 S Flagel,1 H Akil.2 1Pediatrics, University of Michigan, Ann
Arbor, Michigan; 2MHRI, University of Michigan, Ann Arbor, Michigan

Naive rats exposed to the mild stress of a novel environment may exhibit either high or low rates of
exploration. Based on this behavior animals are termed high (HR) or low responders (LR). The degree of
locomotor activity corresponds to the corticosterone stress response upon novelty (high in HR when
compared to LR). Studies have shown that early rearing environments influence subsequent adult behavior
and HPA axis responses of the offspring. However, developmental aspects have never been explored for
the naturally occurring HR/LR phenotype. We, thus hypothesized that individual difference in anxiety and
novelty seeking behavior result from interplay of family background and maternal factors. We have begun
to study four groups of animals resulting from the union of HR and LR parents: 1) HR:HR offspring (HR,
mother: HR, father), 2) HR:LR, 3) LR:HR, and 4) LR:LR. Maternal care was recorded from birth to day
14. Once adults, locomotion and corticosterone responses to novelty stress were measured. We observed
that, the LR mothers were active at licking and having passive contact during the 2nd week of life, a time
when these behaviors had significantly tapered off in HR mothers. In addition, there were differences in the
behavior of the dam as a function of whether the offspring were a product of a HR or an LR male union.
We also found that the HR:HR union: 1) results in a high proportion of adult progeny with high locomotor
activity (77 % HR animals, compared to 40-50% in other families), and 2) distinguishes best the stress
response to novelty.
Overall, these findings indicate that maternal behavior that had been previously shown to result in trans-
generational effects on stress and emotional responsiveness in the offspring is in fact associated with the
maternal HR/LR phenotype. A complex interplay of the genetic endowment of both parents, the maternal
behavior, and possibly the contribution of the offspring in eliciting that maternal behavior depending on its
family background is also suggested. These variables appear to all interact and result in differences in
emotional responsiveness in the offspring. Supported by NIMH42251.

Basic Science Poster: P3: H-P-A Axis II (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                   - 85 -
                                                                                             HPA AXIS

P3-194
Lay explanation of abstract:

Certain people are sensation seekers, while others may be timid, anxious and less willing to take risks.
These differences in emotional characteristics may result from genetic background, from rearing influences
during critical stages of development, or from life experiences. We wanted to learn more about the origins
of the differences in the emotional traits of individuals that occur naturally in many species, including rats
and humans.

Rats can be divided in two categories, based on their behavior in a new and therefore slightly stressful
environment: those that naturally explore a new environment intensely (‗less anxious‘ or ‗High
Responders‘) and those that may engage in very little exploration (‗highly anxious‘ or Low Responders‘).
The Low Responders are considered ‗highly anxious‘ because they score high on anxiety tests.
Interestingly, compared to these more timid animals, rats that take risks and have low anxiety have high
levels of the stress hormone, corticosterone, when exploring a new environment.

In this study, we selected 14 parent pairs of rats based on the ‗highly anxious‘/‘less anxious‘ classification
and studied the progeny that resulted from their union (112 subjects). We also measured the degree to
which each of the mother types took care of their young. We videotaped the maternal behavior daily for
two weeks and scored the time and frequency of nursing, licking, physical contact and mother‘s exploratory
activity. Our findings can be summarized as follows.

1) Parents that were ‗less anxious‘ had a greater number of offspring that were also ‗less anxious.‘
2) While mothers likely influence their offspring through their genes, they also affect them through their
   maternal behavior. The more sensation-seeking, ‗less anxious‘ mothers took good care of their young
   early in life, but tapered off on their care one week earlier than the more timid ‗highly anxious
   mothers.‘
3) Mothers also altered their behavior towards their pups as a function of the classification of the father.
   Thus, the ‗highly anxious‘ mothers caring for the offspring of a ‗less anxious‘ father nursed and licked
   them more than they nursed the offspring of a ‗highly anxious‘ father.
4) When the pups became adults, their hormonal response to stress was significantly affected by the
   classification of both their fathers and their mothers.

These findings are interesting because they underscore the interplay between genetic factors and early life
environment in shaping emotional styles and stress reactivity in adulthood. Perhaps the most newsworthy
aspect of this work is the finding that the pattern of care of mothers toward their young appears to be
determined by their own emotional characteristics. However, maternal behavior appears also to be changed
by characteristics of the young that were inherited from their father. We are pursuing the genetic and
neurobiological bases of these findings to learn more.

Additional background information:

Maternal behavior during early development can influence brain development. Meaney and co-workers
have shown that the initial 7 days of life are critical in producing altered stress responses when the animals
are checked as adults. Mothers who spend more time caring for their young produce offspring that are less
fearful with low stress hormone responses. They also have several biological alterations in key molecules
and key brain structures. The findings presented in this abstract suggest that the High Responder/Low
Responder dimension also is mediated in part by maternal behavior. The classification of the father also has
an effect on the offspring‘s classification, even though fathers are never in contact with pups, which points
to a genetic role in emotionality.




                                                    - 86 -
                                                                                                      IGFs

OR27-6

DIRECT LOCAL EFFECTS OF GH/TNF-α ON IGF-I/INSULIN SENSITIVITY IN SKELETAL
MUSCLE
CE Stewart, X He, JM Holly. Surgery, Uni. of Bristol, Bristol, United Kingdom

Type 2 diabetes/beta cell failure affects 140 million people worldwide. The diabetogenic effects of growth
hormone (GH) are well established, and GH hypersecretion has been implicated in the aetiology of insulin
resistance in catabolic conditions. Increasing evidence implicates other cytokines, e.g. TNFα, in these
insulin-resistant states. The net effects of increased circulating/local cytokine concentrations in diabetes
need to be clarified. Since skeletal muscle is a major target tissue of insulin action, we used murine C2 cell
lines and primary human myoblast cultures to investigate the relationship between local GH/TNFα and
IGF/insulin signalling. We have previously shown that TNFα blocks differentiation and induces apoptosis
in C2 cells, following suppression of IGF-II and IGFBP-5 secretion. We have since established that GH (20
ng/ml) does not alter differentiation or apoptosis relative to controls. IGF-I (100 ng/ml), which alone
caused a 25 % reduction in differentiation was not affected by a co-incubation with GH. Immunoblotting
demonstrated that while GH was without effect on IGFBP-5 secretion, it increased levels induced by IGF-I,
and enhanced IGF-induced MAP kinase activation, suggesting that GH may facilitate a pro-mitogenic role
of IGF-I in muscle. In our primary adult human myotubes, we have found that insulin promotes a dose
responsive glucose uptake compared with controls (142±15% (1µIU/ml), 198±35% (30 µIU/ml), p<0.01).
We have also found that TNFα (20 ng/ml), or GH (both of which are elevated in diabetes) suppress basal
glucose uptake compared with controls (to 63% and 26.9% vs 100 %, respectively). Insulin stimulated
glucose uptake (1 and 30 µIU/ml) was blocked by a pre-incubation with TNFα (98.5% and 87.3%,
respectively compared with TNFα alone), but still increased in the presence of GH (173.6% and 166.1%,
respectively compared with GH alone). These studies suggest that GH and TNFα may have differential
local effects on IGF/insulin-mediated actions in peripheral skeletal muscle. TNFα appears to be detrimental
to IGF/insulin stimulated survival, differentiation and glucose metabolism, whereas GH appears to be
facilitative to mitogenic/metabolic IGF/insulin mediated events.

Basic Science Oral: IGF Signaling (1:00 PM-2:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 2:15 PM; Location: Ballroom 4




                                                   - 87 -
                                                                                                       IGFs

OR27-6
Lay explanation of abstract:

The production and the release of the hormone insulin is particularly important in the control of circulating
blood sugar (glucose) levels, and thereby controls the body's metabolism. Insulin promotes the uptake of
glucose for use as energy in many tissues in the body, particularly in muscle. Because of this important
role, any abnormalities in insulin secretion or action may cause major clinical consequences, leading to the
syndrome Diabetes Mellitus, which affects 140 million people worldwide. If untreated, diabetes eventually
leads to coma and death.

Although direct effects of decreased insulin secretion are well recognized in diabetes, under certain
circumstances, the disease occurs not as a result of reduced insulin release, but as a consequence of reduced
actions. This condition can be associated with increased levels of other factors, such as growth hormone
(GH) and tumor necrosis factor-alpha (TNF), which modify insulin actions. The net effects of the
increased circulating levels of these factors and their influence in the development or progression of
diabetes need to be clarified.

Skeletal muscle is a major target tissue of insulin action. We therefore used skeletal muscle cells obtained
from commercial sources, or derived and grown from human skeletal muscle biopsies, to address whether
GH or TNF can alter the metabolic effects (glucose uptake) of insulin, or the growth, maturation, and
survival effects of its related protein, insulin-like growth factor-I (IGF).

We found that addition of TNF was capable of blocking maturation, of suppressing IGF production, and of
inducing the death of these cells. Additionally, it was capable of reducing baseline glucose uptake in the
muscle by 40 %, and of completely blocking the doubling in metabolism observed in the presence of
insulin. By contrast, while GH alone was without effect on muscle cell maturation or survival, we found
that it was capable of enhancing the growth-promoting activities of IGF. Like TNF, GH effectively
reduced the baseline glucose uptake of the cells (by 70 %). Insulin-stimulated glucose uptake still
increased, however, in the presence of GH.

These studies suggest that GH and TNF have contrasting local effects in skeletal muscle. TNF appears to
be detrimental to IGF or insulin-stimulated survival, maturation, or glucose metabolism, while GH appears
to assist in the growth promoting and metabolic events mediated by IGF or insulin, respectively. A clearer
understanding of the mechanisms involved may enable the development of new clinical interventions for
the treatment of a disease that is rapidly becoming a global epidemic.

Funding sources included the University of Bristol, Pharmacia, and the Association for International
Cancer Research (AICR).




                                                   - 88 -
                                                                                                      IGFs

P1-174

LOW IGF-1 SUPPRESSES VEGF-SURVIVAL SIGNALING IN RETINAL ENDOTHELIAL
CELLS: DIRECT CORRELATION WITH CLINICAL RETINOPATHY OF PREMATURITY
LE Smith,1 M Ju,1 C Perruzzi,2 E Engstrom,3 A Hard,3 J Lui,4 K Albertsson-Wikland,3 B Carlsson,5
A Niklasson,3 L Sjodell,3 D LeRoith,4 DL Senger,2 A Hellstrom.3 1Ophthalmology, Children's Hospital,
Harvard Medical School, Boston, Massachusetts; 2Pathology, Beth Israel Deaconess Medical Center,
Boston, Massachusetts; 3The Queen Silvia Children's Hospital, Goteborg, Sweden; 4Endocrine Branch,
NIH, Bethesda, Maryland; 5Research Centre for Endocrinology and Metabolism, University of Goteborg,
Goteborg, Sweden

Retinopathy of prematurity (ROP) is a blinding disease, initiated by lack of retinal vascular growth after
premature birth. We postulated that low serum IGF-1 levels in premature infants prevents normal retinal
vascular growth leading to ROP. In IGF-1-/- mice retinal vascular growth is retarded as seen in retinal flat
mount, despite the presence of vascular endothelial growth factor (VEGF), important to vessel
development. In retinal vascular endothelial cell culture, low levels of IGF-1 prevent VEGF-induced
activation of AKT, a kinase critical for endothelial cell survival. These data indicate that 50 ng/ml IGF-1,
which approximates a more normal physiological circulating concentration in newborns, acts together with
VEGF to activate AKT, and therefore supports endothelial cell survival in retina. By contrast, when IGF-1
is reduced to 10 ng/ml, comparable to the serum level present in premature infants likely to develop ROP,
VEGF activation is reduced 5-fold. Consequently, in such patients, lower than normal levels of IGF-1
likely translate into reduced AKT activation and reduced endothelial cell survival, despite the presence of a
constant level of VEGF. These results correlate with our clinical studies in premature infants and show that
ROP is associated with low IGF-1 levels. When IGF-1 is persistently low, vessels cease to grow, maturing
avascular retina becomes hypoxic and VEGF accumulates in the vitreous. As IGF-1 increases to a critical
level, retinal neovascularization is triggered. These data indicate that serum IGF-1 levels in premature
infants can predict which babies will develop retinopathy of prematurity and further suggests that early
restoration of insulin-like growth factor-1 in premature infants to normal levels could prevent this disease.

Basic Science Poster: IGFs I (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                   - 89 -
                                                                                                    IGFs

P1-174
Lay explanation of abstract:

Retinopathy of prematurity (ROP) is a major cause of childhood blindness. It is a retinal vascular disease
that occurs in premature infants, which starts with lack of normal growth of blood vessels in the eye. This
lack of growth happens after the baby leaves the normal environment of amniotic fluid in the mother's
uterus to emerge into a foreign world of air with high oxygen. It has been assumed that oxygen is the
major factor contributing to the development of ROP. Although oxygen is involved in the disease, it is not
necessarily the only factor contributing to the development of ROP. The environment in the womb clearly
provides some growth factors that are then lost when the baby enters the room air environment before its
time.

We examined the role of an important growth factor, insulin-like growth factor-1, or IGF-1, in this disease
process to determine whether this might be one of the critical factors lost after leaving the in utero
environment and to determine whether IGF-1 controls retinal blood vessel growth.

To determine if IGF-1 was critical to normal blood vessel growth, we first examined the retinal blood
vessel development in specially bred mice with the IGF-1 gene missing or "knocked out" to see if the loss
of the IGF-1 gene caused poor retinal blood vessel growth. The knockout mice had retinal blood vessels
that grew more slowly than those of normal mice, a pattern very similar to that of the premature babies with
ROP. We next asked the question: "How might loss of IGF-1 cause the abnormal blood vessel growth?"
We determined that IGF-1 controls an important chemical signaling pathway (VEGF activation of Akt) that
allows the survival of the cells, which make up the blood vessels. This finding explained how loss of IGF-1
could cause the disease, by preventing the normal survival of vascular endothelial cells.

Sometimes experiments in mice do not translate into what occurs in humans. To learn whether our
observations that low IGF-1 caused loss of blood vessel growth was also true in humans, we measured
IGF-1 levels in a group of premature infants to see if the babies who developed ROP had lower levels of
IGF-1 than babies who did not develop ROP. This was indeed the case. Babies without ROP (and normal
retinal blood vessel growth) had significantly higher levels of IGF-1 than those who developed the disease
(and had slow retinal blood vessel growth).

This work is important because it describes a possible cause of retinopathy of prematurity. The data suggest
that measurement of this growth factor could be used to predict which infants will develop the disease. In
addition, replacement of IGF-1 in premature infants might prevent the disease. Further clinical studies
based on these findings are in progress.

Additional background information:

We have been interested in the problem of ROP because the problem is increasing with the rise in
premature infants (some as small as 23 weeks gestation). When the disease was first described in the 1950's
it was clearly due to the use of high oxygen given to babies to allow them to survive. Oxygen use was then
curtailed and although the incidence of ROP fell, more infants died because their brains did not receive
enough oxygen. Oxygen is now given very carefully to premature infants, so that they receive just enough
to have a normal amount in their blood. Oxygen is no longer the major cause of the disease, yet more
infants are getting ROP because of their extreme premature status. We felt that there must be another
factor that was missing when the infants were born too soon and left the environment of the womb. This led
us to this course of research.. IGF-1 is a factor found in amniotic fluid that is no longer available after
premature birth.

Funding comes from the National Eye Institute (LEHS) and the V. Kann Rasmussen Foundation.




                                                   - 90 -
                                                                                                   IGFs

P1-198

COMPARISON OF PRIMARY SKELETAL MUSCLE CULTURES FROM PATIENTS WITH
AND WITHOUT CANCER
EJ Foulstone, JM Holly, CE Stewart. Dept. of Surgery, University of Bristol, Bristol, United Kingdom

Cachexia is associated with many chronic severe illnesses and is characterised by loss of lean body mass,
irrespective of nutritional intake. This muscle wasting significantly worsens patient prognosis. We
hypothesise that cachexia results from a shift in the balance of growth factors and cytokines that maintain
normal muscle, towards a more catabolic state. To explore this hypothesis we have developed a primary
adult human muscle cell model. Cells are cultured from biopsies of rectus muscle from patients undergoing
elective surgery, with (46, 33 male age 36-82) and without cancer (12, 4 male age 23-77). We have termed
these cultures malignant and benign respectively.The ability of the cultures to differentiate into multi-
nucleated myotubes was assessed. Creatine kinase activity at day 7 (D7) after placing into low serum media
(LSM) was measured. The isolated cultures are not homogenous populations of myoblasts and were stained
using an antibody to desmin, a muscle cell marker present in undifferentiated myoblasts and differentiated
myotubes. The percentage of myoblasts in the cell cultures at day 0 (D0) and the percentage of nuclei in
myotubes at D7 was calculated. Both benign and malignant cultures differentiated when placed into LSM.
In benign cultures the percentage of myoblasts at D0 decreased with patient age, at age 40 myoblasts were
59.5% of the cells, at 75 only 11%. However, the ability of the myoblasts to form myotubes was
maintained. This was not the case for malignant cultures.
Addition of 20ng/ml TNFα at D0 inhibited differentiation in both benign and malignant cultures. In benign
cultures addition of 30ng/ml IGF-I at D0 stimulated differentiation, 155.13%±28.2 over LSM alone.
However, out of 11 malignant cultures IGF-I stimulated differentiation in only 5. TNFα added
simultaneously with IGF-I blocked the IGF-I stimulated increase in differentiation.
We have started to explore the similarities and differences between the benign and malignant muscle cell
cultures. In subjects without malignancy the number of myoblasts with potential for muscle regeneration
decreases with age. In patients with cancer the age relationship is lost and cells may become resistant to
anabolic actions of IGF-I.

Basic Science Poster: IGFs I (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 91 -
                                                                                                       IGFs

P1-198
Lay explanation of abstract:

Many patients with long-term illnesses, including cancer, suffer from weight loss. In particular, they suffer
unwanted loss of muscle, in spite of nutritional intake. This loss of lean body mass is termed cachexia and
significantly decreases the chance of patient recovery and survival. In healthy individuals, there is a balance
in the muscle between cell growth and breakdown. Proteins called growth factors and cytokines maintain
this balance. We have suggested that in patients suffering from cachexia, this balance has been disturbed so
that more cell breakdown and not enough cell growth is occurring. To explore this theory, we have
extracted and grown (cultured) cells from muscle biopsies taken from adult patients undergoing surgery
both with (46, 33 male, age 36-82) and without (12, 4 male, age 23-77) cancer. We have termed these two
groups malignant and benign, respectively.

We were interested in looking at any differences in behavior between the two groups of cells. The cell
cultures are made up of muscle stem cells or myoblasts, which allow the muscle to repair and regenerate, as
well as cells of other tissue origin present in muscle. All the cells grow and divide in culture. The myoblasts
can be induced to differentiate, a terminal process in which the cells join together to form large multi-
nucleated tubes that make up normal muscle fibers. Differentiation can be measured chemically and
visually. We found that myoblasts in both benign and malignant cultures differentiated normally into tubes.
In the benign group the percentage of myoblasts in the cell cultures decreased with the age of the patient (in
the malignant cultures this age relationship was lost). However, their ability to differentiate was
unimpaired.

The positive muscle cell growth factor, insulin-like growth factor I (IGF-I), increased differentiation in all
of the benign cultures, but 5 out of 11 malignant cultures did not respond to IGF-I, which suggests that in
patients with cancer, muscle cells may become resistant to the actions of IGF-I. In response to tumor
necrosis factor alpha (TNF), a cytokine that has been implicated in cachexia, differentiation was inhibited
in all the cultures. TNF also blocked any increase in differentiation stimulated by IGF-I.
This study suggests that while muscle cells from patients with and without cancer generally behave
similarly, fundamental differences also exist. Understanding these differences will be important when
devising and implementing treatment.

This work was funded by the Association for International Cancer Research (AICR).




                                                    - 92 -
                                                                                                     IGFs

P1-203

VITAMIN E ATTENUATES THE DECLINE IN PLASMA IGF-I DURING THE ACUTE PHASE
RESPONSE TO ENDOTOXIN (LPS)
TH Elsasser,1 S Kahl,1 JL Sartin,2 E Connor,1 T Caperna.1 1Growth Biology Laboratory, USDA-ARS,
Beltsville, Maryland; 2Anatomy and Physiology, Auburn University College of Vet Med, Auburn, Alabama

Impaired growth and metabolic faltering are hallmark consequences of infection-driven acute phase
response (APR) with strong correlates to declines in plasma IGF-I concen-trations and loss of regulation of
IGF-I by growth hormone. It is recognized that significant changes in the intra-cellular oxidation-reduction
status occur during APR that, in conjunction with increased production of nitric oxide,peroxide, and
superoxide radicals, result in the formation of peroxynitrite and nitration in the form of nitrotyrosine (NT)
in the phenolic ring of tyrosine residues of proteins. The present study looked at the ability for vitamin E
pretreatment to affect nitration of liver proteins and changes in plasma levels of IGF-I as a function of
repeated challenge with 055:B5 E.coli LPS as a model for the APR. The nitration of JAK-2 kinase was
investigated further as a potential mechanism through which the regulation of IGF-I during APR could be
impaired. Female calves were challenged on two occasions 5 days apart with 3 mg/kg LPS (n=12) or saline
(n=4). Half the LPS calves were also treated daily for 5 days prior to each LPS challenge with vitamin E
(1000 IU/d, im). Six h after the second LPS challenge, liver tissue was obtained by biopsy and homo-
genized for extraction of intracellular proteins or fixed in Bouins solution for immunohistochemistry (IHC).
JAK-2 kinase was immunoprecipitated from the homogenate. Liver protein nitration was induced by LPS
as identified by western blot and IHC with anti-NT. JAK-2 was decreased 61% after repeated LPS
challenge and partially restored by Vitemin E; nitrated JAK-2 was identified in 3/12 LPS calves. IHC for
NT coupled with in situ hybridization for IGF-I mRNA suggested that the presence of intracellular NT-
proteins was correlated with an absence of IGF-I mRNA. Vitamin E decreased the formation of NT and the
duration of decreased plasma concentrations of IGF-I. The data support the hypothesis that some elements
of dysfunctional IGF-I regulation during the APR may be linked to an aberrant nitration of intracellular
protein and affected by pretreatment with vitamin E.

Basic Science Poster: IGFs I (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                   - 93 -
                                                                                                     IGFs

P1-203
Lay explanation of abstract:

Infection in young animals or people causes growth processes to be placed on hold while the immune
system assesses how severe the threat is and begins to respond. The slowdown in growth is most often
necessary to spare nutrients and make them available for other uses, such as function of the activated
immune system, fever, and synthesis of new proteins in the liver that help to stabilize the body during
infection stress. Sometimes the body overreacts to the stress, and the result is the improper formation of
proteins whose function is impaired. Many of these impaired proteins lose their function when certain
amino acids that make up the protein are chemically modified. This happens when nitrate is added to their
structure.

Our research showed that a protein important to the proper function of the growth-regulating hormone,
called "growth hormone" or JAK-2 kinase, is a target of such improper nitration. When this occurs, cells no
longer can be regulated properly by growth hormone. Vitamin E is praised for its ability to fight stress by
mopping up free radicals. The nitration of the proteins we described occurs in part because of the over-
production of some free radicals. Vitamin E treatment of endotoxin-challenged animals (a model that
causes the symptoms of a bacterial infection without the complications of actually infecting animals with a
live bacteria) resulted in a decreased production of nitrated proteins, and in maintenance of blood levels of
other hormones necessary to maintain normal metabolism and growth. The animals recovered sooner from
the effects of the endotoxin when they were pretreated with vitamin E.

Additional background information:

The actual experimental model was the growing beef animal, and the research was a part of the effort our
lab has undertaken: investigating nutritional methods to improve animal health, maintain a healthful food
product, avoid the use of antibiotics, and give farmers a science-based set of alternatives for animal
production that are effective, low cost, and easily introduced into farm practices. The timely use of
antioxidant compounds like vitamins may be beneficial to healthy animals.

This research was funded by the USDA in associated with CRIS# 1265-31000-013.




                                                   - 94 -
                                                                                                     IGFs

P2-262

IGFBP-3, INDEPENDENT OF IGF-1, IS A POTENT MITOGEN AND SURVIVAL FACTOR FOR
NORMAL BREAST EPITHELIAL CELLS
C McCaig, CM Perks, JM Holly. University Department of Surgery, Bristol Royal Infirmary, Bristol,
United Kingdom

We have demonstrated previously that IGFBP-3 (100ng/ml), in an IGF-independent manner, mildly
inhibited growth of Hs578T human breast cancer cells but markedly accentuated ceramide (C2)-induced
apoptosis. TGF-β can both stimulate or inhibit growth depending on cell type. In Hs578T cells, TGF-βcan
be growth inhibitory with an associated increase in IGFBP-3 levels. We aimed to look at the effects of non-
glycosylated (ng) IGFBP-3 on cell growth in the relatively normal breast epithelial cell line, MCF-10A, and
to investigate TGF-β effects on IGFBP-3 production, cell growth and survival. MCF-10A cells were a)
treated with added ngIGFBP-3 (100ng/ml) +/- a 1 hr pre-incubation with an IGF-I receptor antagonist
(100ng/ml), or b) treated with TGF-β (5ng/ml) alone for 3 days with either ngIGFBP-3 (50ng/ml)
administered daily or on the penultimate day, spiked with an apoptotic dose of C2 (25μM). Cell number
and death were determined by trypan blue counting and levels of endogeneous IGFBP-3 in conditioned
media were measured by radioimmuoassay. Added IGFBP-3 (100ng/ml) alone caused no cell death but
induced a significant increase in cell number (33.5%, p<0.01) and this mitogenic action of IGFBP-3 was
unaffected by the IGF-I receptor antagonist. TGF-β alone also had no effect on cell death but significantly
inhibited cell growth by 37.5% (p<0.005) which was accompanied by a 75% reduction in endogenous
levels of IGFBP-3. With adding exogenous IGFBP-3 over 3 days, the TGF-β-induced growth inhibition
was only 14.5%. Furthermore, adding C2 to cells pre-treated with TGF-β, dramatically accentuated cell
death (146%) relative to C2 alone. We have shown IGFBP-3 acts as a potent mitogen in MCF-10A cells.
TGF-β inhibited IGFBP-3 production and cell growth. This was counteracted if IGFBP-3 was replaced.
These actions of IGFBP-3 on cell growth are exerted in an IGF-independent manner. These data also
suggest that IGFBP-3 may act as a survival factor in this cell line. We have demonstrated that in contrast to
its actions on breast cancer cell lines, IGFBP-3 can act as a growth promoter and survival factor for MCF-
10A breast epithelial cells.

Basic Science Poster: P2: IGFs II (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                   - 95 -
                                                                                                        IGFs

P2-262

Lay explanation of abstract:

The insulin-like growth factors (IGFs) are soluble peptides that are key regulators of cell growth and death.
They are present throughout the body, bound to carrier proteins (IGFBPs) that tightly control the
availability and activity of the growth factors, but in addition have direct effects on cell function. The IGFs
and IGFBPs have been implicated in human breast cancer. We and others previously have demonstrated
that in cultures of human breast cancer cells the main carrier protein, IGFBP-3, acts as an enhancer of cell
death and a growth inhibitor.

We have now compared the direct actions of IGFBP-3 on a normal breast epithelial cell line with its effects
on cancer cells. In the normal cells, IGFBP-3 promoted cell growth and reduced cell death, independent of
its ability to bind to the growth factor. In addition, an inhibitor of epithelial cell growth increased the
secretion of IGFBP-3 from breast cancer cells but reduced its secretion from the normal cells.

The IGF-system is a key component in the regulation of normal tissue balance. The switch in action of
IGFBP-3 from an enhancer of growth and survival of normal breast epithelial cells, to an inhibitor of
growth and survival of cancer cells may represent a defense mechanism to restrict inappropriate growth.
Targeting interventions to enhance this natural tissue mechanism could provide a new strategy for
controlling breast cancer.

This work was funded by the AICR.




                                                    - 96 -
                                                       INTRACELLULAR SIGNALLING

OR53-4
PHOSPHATYDILINOSITOL 3 KINASE CONTROLS DISTINCT STEPS OF INSULIN
INTERNALIZATION AND INTRACELLULAR SORTING VIA PKC-ZETA AND AKT/PKB
F Fiory, F Oriente, S Santopietro, F Andreozzi, M Caruso, C Miele, G Condorelli, F Beguinot,
P Formisano. Dipartimento di Biologia e Patologia Cellulare e Molecolare, University of Napoli "Federico
II", Napoli, Italy

Insulin receptor (IR) tyrosine kinase activity is essential for insulin internalization. Following endocytosis,
insulin is then either degraded intracellularly or released, undegraded, into the extracellular medium
(retroendocytosis). However, the molecular mechanisms controlling IR endocytosis and further
intracellular sorting remain poorly defined. We have addressed this issue in L6 cells expressing human
insulin receptor (L6hIR). We found that insulin internalization was inhibited by about 50% in cells treated
with wortmannin (WM) or LY294002, which block phosphatydilinositol, 3 kinase (PI3K). Internalization
was not affected by bysindolylmaleimide and PD98059, which inhibit conventional protein kinase C (PKC)
isoforms and MEK/MAPK, respectively. WM and LY294002 inhibited insulin retroendocytosis by 70%
and insulin degradation by only 20%, thus impairing normal intracellular sorting. The effect of PI3K
inhibitors on insulin internalization was mimicked by transient transfection of either a dominant negative
PKCzeta mutant and PKCzeta antisense oligonucleotides. However, both insulin degradation and
retroendocytosis were reduced by 50% when PKCzeta was blocked. Conversely, overexpression of wild-
type PKCzeta in L6hIR cells increased by >2-fold insulin internalization, degradation and retroendocytosis.
At variance with PKCzeta, expression of wild-type and constitutively active Akt/PKB had no effect on
insulin internalization and increased by 2,5-fold insulin retroendocytosis with minor changes of insulin
degradation. Thus, PI3K controls different steps within insulin endocytic itinerary. PKCzeta appears to
mediate PI3K effect on insulin internalization while Akt/PKB directs intracellular sorting toward
retroendocytosis.

Basic Science Oral: Intracellular Signaling II (1:30 PM-3:00 PM)

Presentation Date: Saturday, June 23, 2001; Time: 2:15 PM; Location: A 108




                                                    - 97 -
                                                        INTRACELLULAR SIGNALLING
OR53-4
Lay explanation of abstract:

Insulin-resistance is present in many major diseases, such as diabetes mellitus, hypertension, and the
metabolic syndrome. Understanding how insulin works within the target tissues is crucial so that we can
identify effective strategies to prevent and/or cure abnormal insulin action. Many aspects of insulin‘s
mechanism of action have been uncovered in recent years, though many more still remain to be explored.

To reduce glycemic (glucose) levels in living organisms, insulin needs well-organized cellular structures.
For instance, insulin binds to specific proteins on the surface of target cells (insulin receptor IR), and
induces the activation of several enzymes, which leads to the regulation of glucose metabolism and that of
other nutrients. In addition, insulin-bound receptors are rapidly removed from the cell surface and
transported, within a system of vesicles, toward specific intracellular compartments. This process is also
known as endocytosis.

IR endocytosis is involved in clearing out insulin from circulation, and possibly in transferring insulin
signals from the surface to the interior of the cell. Thus, internalized insulin is then either degraded
intracellularly or recycled to the cell exterior, transported from one side to the other of the cell
(retroendocytosis). Impairment of insulin endocytosis has been described for both type 1 and type 2
diabetes mellitus. Nevertheless, the mechanisms regulating insulin and IR endocytosis have been only
partially assessed.

In this study, we investigated the molecular mechanisms by which insulin promotes its own endocytosis
and recycling in a model using rat muscle cell, named L6. These cells have been made more sensitive to
insulin by genetic engineering, so that they express many copies of the human IR. Interestingly, a unique
insulin-stimulated enzymatic activity (named phosphatydilinositol, 3, kinase -- PI3K) appears to control
both the endocytosis and the recycling of insulin, Following PI3K activation, however, two different
enzymes play specific roles in allowing either insulin endocytosis or recycling. In particular, a protein
named protein kinase C- (PKC) facilitates insulin internalization, while a different protein named protein
kinase B (PKB, also named Akt) is required mostly for insulin release outside of the cell. Selective
inhibition of PKC determines a reduction of insulin endocytosis, while blocking PKB/Akt leads to a
reduction of insulin retroendocytosis.

We believe that these findings will give further insights into understanding the mechanism of insulin action
on a molecular level. More work is needed to unravel the signals generated by the proteins that effectively
regulate insulin cellular traffic and contribute to the transmission of biological effects. This is an important
issue, because it will greatly help us to identify new potential therapies for diabetes mellitus and other
diseases characterized by defects in insulin action.

This work has been supported in part by grants from the European Community, the Italian Ministry of
University and Scientific Research, the Italian Association for Cancer Research (AIRC), and Telethon.




                                                    - 98 -
                                                                NEUROENDOCRINOLOGY

OR28-2
HIGH-INTENSITY EXERCISE ELICITS ACUTE ALTERATIONS OF GLUCOSE LEVELS AND
NEUROENDOCRINE SECRETION THAT VARY WITH CIRCADIAN PHASE OF EXERCISE
OM Buxton,1 AJ Scheen,2 M L'Hermite-Baleriaux,3 E Van Cauter.1,3 1Dept of Medicine, U Chicago,
Chicago, Illinois; 2Dept of Diabetes, Nutrition, Metabolic Disorders, ULG, Liege, Belgium; 3Dept of
Experimental Medicine, ULB, Brussels, Belgium
To examine the acute effects of high-intensity exercise on glucose metabolism and neuroendocrine
secretion at different circadian times, 5 groups of men 20-30 years of age (total n=40) participated in
studies involving no exercise (control) or exposure to morning, afternoon, evening, or nocturnal 1-hr
stairclimber exercise. Study conditions involved dim light exposure, constant glucose infusion, and
continuous bedrest (except during scheduled exercise or sleep) with iv sampling at 20-min intervals over 3
days. Circadian phase of exercise was estimated from melatonin profiles. Maximum changes (delta) in
glucose, growth hormone (GH), cortisol, and thyrotropin (TSH) levels were expressed relative to the mean
level during the hour preceding exercise (fig1) and were compared to changes at the same clocktime in the
controls. The main effect of EXERCISE was significant for all parameters (ANOVA,p<.001). The effects
of TIME of exercise (p≤.02) and EXERCISE*TIME (p≤.006) were significant for all except GH.
Decrements in glucose levels were minimal in the morning, afternoon, and evening (NS) but dramatic at
night (p<.001). Exercise induced significant GH increases at all circadian times (p≤.03). Increases in
cortisol were minimal in the morning (NS), modest during the afternoon (p<.04), but evening and nocturnal
exercise resulted in levels twofold higher than the circadian acrophase (p≤.01). Similarly, the variations of
TSH responses to exercise were minimal in the morning (NS), modest in the afternoon (p<.03), but
sustained and exceeding the circadian amplitude during evening and nocturnal exercise (p≤.001).
These findings indicate that the maximal neuroendocrine and glucose responses to high-intensity exercise
vary markedly with circadian phase.




Basic Science Oral: Neuroendocrinology (1:00 PM-2:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 1:15 PM; Location: C 207
                                                   - 99 -
                                                                NEUROENDOCRINOLOGY

OR28-2
Lay explanation of abstract:

Some times of day may be better than others for exercising. The body's response to exercise, including
changes in glucose and hormonal systems related to energy metabolism, may play a role in variations in
athletic performance during a day.

This study provides strong evidence for substantial changes in glucose metabolism and hormonal responses
to 1-hour, high-intensity exercise, depending on the time of day. Cortisol and thyrotropin fluctuate daily
with a 24-hr pattern partially controlled by the body's circadian clock. Growth hormone is modulated by
sleep and mealtime. Each of these hormones plays a role in energy metabolism.

Forty healthy men, between the ages of 20 and 30, were divided into five groups: a control group that did
no exercise, and groups that exercised vigorously for 1 hour on a stair-stepper in the morning, afternoon,
evening, or night. The circadian time of exercise was determined for each individual using the timing of
melatonin secretion, a marker of circadian "clock time." During all other times, the subjects were on
continuous bed-rest and constant glucose infusion to avoid interference by intermittent meals. Blood levels
of the "circadian hormones" melatonin, cortisol, and TSH and the levels of growth hormone and glucose
were compared to blood levels for the same time of day in the control subjects who remained on bed-rest.

Cortisol and thyrotropin were affected differently by exercise performed at different times of day.
Specifically, evening and night exercise caused large increases in the normal levels of these hormones.
Morning and afternoon exercise resulted in small increases. Decreases in glucose levels were minimal in
the morning, afternoon and evening, but dramatic at night. Levels of growth hormone increased
substantially with exercise at all times of day.

This broad study measured glucose levels and an array of hormones essential to understanding the complex
response to exercise. This study demonstrates variation in the effects of exercise at different times of day,
with circadian time precisely quantified, with a practical duration of exercise, and with an intensity (and
conditions) designed to elicit maximal metabolic effects.

Additional background information:

A high degree of temporal organization controls energy metabolism. This timing is seen in the distinct 24-
hour temporal patterns of secretion for each hormonal system. One hormone may be actively secreted in a
complex, pulsatile pattern while another may be in a quiescent period. These rhythmic patterns of hormonal
secretion are thought to provide internal temporal organization essential to the coordination of
physiological processes. Whether these major 24-hour variations in energy metabolism are involved in
differences in the peak metabolic response to exercise is unclear.

Many circadian rhythms, such as heart rate, oxygen consumption, and cardio-pulmonary function could
play a role in athletic performance. Several previous reviews have hypothesized that there may also be a
24-hour rhythm in athletic performance that may parallel the rhythm of the body's 24-hour circadian clock.
Physical exercise is associated with marked metabolic changes and can elicit a variety of neuroendocrine
responses. Although these metabolic and hormonal responses to morning exercise are well-documented,
few studies have examined the effects of exercise at other times of day.

This work was supported by grants from the Air Force Office of Scientific Research (F49620-96-1-0252 &
F49620-98-1-0028) and from the Department of Defense (Augmentation Award for Science and
Engineering Research Training F49620-96-1-0252). The University of Chicago Clinical Research Center
is supported by NIH grant RR-00055.




                                                  - 100 -
                                                               NEUROENDOCRINOLOGY

P2-138
PITUITARY LEPTIN GENE EXPRESSION IS INHIBITED BY NEONATAL
ANDROGENIZATION
BA Morash,1 E Ur,1 M Wilkinson.2,3 1Endocrinology, Dalhousie University, Halifax, Nova Scotia, Canada;
2
  Physiology & Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada; 3Obstetrics &
Gynaecology, Dalhousie University, Halifax, Nova Scotia, Canada

We have previously reported evidence of leptin gene expression in adult (Endocrinology 1999; 140: 5995)
and neonatal (Mol. Cell. Endocr.; In press) rat brain and pituitary gland. We have also shown that leptin
levels in female rat pituitary and cortex, but not hypothalamus, are developmentally regulated (Mol.Cell.
Endocr., In press). In rat adipocytes, a sexual dimorphism is apparent with respect to sex steroid-dependent
regulation of leptin gene expression. The leptin gene promoter includes an estrogen response element and
the hypothalamic-pituitary system contains estrogen and androgen receptors. We hypothesized that leptin
(ob) mRNA levels might be sexually dimorphic in rat brain and pituitary gland. Tissues (pituitary,
hypothalamus, cortex and subcutaneous fat) were collected on postnatal days (PD) 4, 14 and 22 from male,
female and androgenized female rats masculinized by a single dose of testosterone propionate (1mg; s.c.)
on PD 4. Leptin mRNA levels were evaluated using semi-quantitative RT-PCR analysis and previously
published primer sequences. Leptin mRNA levels were developmentally regulated in the pituitary and
cortex of male rats, paralleling the changes previously observed in female rats. In the pituitary, ob mRNA
levels were significantly higher during the early postnatal period and fell abruptly by PD 22 (~ 3-fold; p <
0.05). In the cortex, leptin expression was lowest at PD 4 and rose significantly by PD 14 (~ 4-fold; p <
0.05). By contrast, hypothalamic leptin mRNA levels were similar throughout PD 4-22. Gender
differences, most notably in the pituitary, were also observed. In the pituitary, ob mRNA levels were
significantly higher in female rats than in males at PD14 (2-fold; p < 0.01), but there were no sex
differences at PD 4 or PD 22. Androgenization of neonatal female rats profoundly reduced ob mRNA
levels at PD14 (3.5-fold; p < 0.01) and PD 22 (3-fold; p=0.05). We conclude that leptin gene expression is
developmentally regulated in the brain and pituitary and that pituitary leptin expression is sexually
dimorphic and sensitive to neonatal manipulation of sex steroid levels.

Basic Science Poster: Neuroendocrinology I (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 101 -
                                                                NEUROENDOCRINOLOGY

P2-138

Lay explanation of abstract:

Leptin is a newly discovered hormone, made by our fat cells and thought to play a role in the control of
body weight. At first, leptin was believed to suppress appetite and help burn fat. However, early clinical
trials have been disappointing in their attempts to use leptin to help overweight people lose weight.

We have provided a completely new perspective with our discovery that the brain and pituitary gland also
make leptin. In our most recent experiments, we have shown that the amount of leptin made in the pituitary
and brain of rats changes as the animal grows (i.e., from birth through puberty). We have also shown that
the male hormone testosterone can reduce leptin production in the pituitary of female animals. These
findings could have important implications in disorders such as polycystic ovary syndrome, which involve
overactivity of male hormone activity in women. Polycystic ovary syndrome is a relatively common and
often serious health concern among young women. Many of these patients are overweight, and some go on
to develop diabetes and heart disease. A better understanding of the regulation of leptin and fat may help
researchers find treatments to prevent polycystic ovary syndrome and its complications.

This work was funded by the Canadian Diabetes Association, the Dalhousie University Faculty of
Medicine, the Dalhousie University Internal Medicine Research Foundation, and the IWK Grace Research
Foundation.




                                                  - 102 -
                                                               NEUROENDOCRINOLOGY

P2-150
DECREASED SERUM LEPTIN LEVELS IN NONHUMAN PRIMATES TREATED WITH A
SELECTIVE (TYPE1) CORTICOTROPIN-RELEASING HORMONE RECEPTOR
ANTAGONIST
AR Ayala,1 M Gerald,2 S Bornstein,3 J Pushkas,2 KE Habib,1 G Cizza,1 S Lindell,2 K Pacak,3
D Ronsaville,1 KC Rice,4 GP Chrousos,3 JD Higley,2 PW Gold.1 1NIMH, National Institutes of Health
(NIH), Bethesda, Maryland; 2NIAAA, NIH, Poolesville, Maryland; 3PREB, NIH, Bethesda, Maryland;
4
  NIDDK, NIH, Bethesda, Maryland

The interactions between leptin, corticotropin-releasing hormone(CRH) and the hypothalamic-pituitary-
adrenal (HPA) axis are poorly understood, and are likely to be complex. Metabolic effects of leptin are
thought to be mediated through interactions with s everal orixigenic and anorexigenic neuropeptides
including CRH. To study this association, we sought to investigate the metabolic effects of the selective
nonpeptide CRH (type1) antagonist, Antalarmin,in nonhuman primates.
Antalarmin (A) or P lacebo (P) was administered (20 mg/kg orally) in a double-blind placebo-controlled
fashion to young adolescent rhesus monkeys (n=8) under basal conditions and during social separation, a
primate model of stress. Antalarmin was detected by chromatography (HPLC) in all animals receiving the
drug.
Plasma Leptin levels were lower during A administration as compared to P administration (p=0.052) in
both basal and stress conditions. (A group, mean= 0.688 ng/ml SD=0.167 vs. P group mean=0.938 ng/ml
SD=0.107). Leptin le vels during the washout (no drug) period were equal in both the antalarmin and
placeb o group (mean 0.856 vs.0.864 p= SD=0.04). Lower Leptin levels cannot be explained by differences
in either food consumption or weight gain, as daily behavioral observa tions revealed no significant
differences in feeding patterns and all subjects gained de velopmentally-expected weight during the study.
No significant differences in serum glucose, electrolytes and lipid profile between the groups were
observed.
This study suggests that pharmacological inhibition of CRH, a potent anorexic substance,with a selective
antagonist results in decreases of serum leptin, a peripheral satiety signal to the brain. This observation
confirms the existence of a bi-directional loop between CRH and the leptin system.

Basic Science Poster: Neuroendocrinology I (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                 - 103 -
                                                               NEUROENDOCRINOLOGY

P2-150
Lay explanation of abstract:

Leptin, a recently discovered hormone produced by fat cells, is known to regulate metabolism and body
weight by exerting its effects on key areas of the brain. Corticotropin-releasing hormone (CRH) is a protein
(hormone) produced mainly by the hypothalamus, a brain center that, among its various functions, regulates
metabolism and coordinates the hormonal response observed in stressful conditions. CRH is the main
stimulus for cortisol, a major stress-related hormone that coordinates the hypothalmic, pituitary, and
adrenal gland activation (―HPA‖ axis activation). Investigators believe that in conditions such as Major
Depression and Anorexia Nervosa, overactivation of the HPA system can result in metabolic problems that
include changes in body weight. Nonetheless, the mechanism involved in this process is not well
understood. The relationship between the CRH (stress system) and leptin may be part of the explanation.

To better understand this interaction , we used a CRH blocker (antagonist) and determined its effects on
blood leptin. Because of the experimental nature of the drug (not yet FDA approved), we used eight rhesus
monkeys to test our hypothesis. We administered the drug orally for 28 days and observed lower leptin
levels in monkeys that received the drug. This study suggests that pharmacological inhibition of CRH, a
potent anorexic substance with a selective antagonist, results in decreases of serum leptin, which is a
peripheral satiety signal to the brain. This observation confirms the existence of a bi-directional loop
between CRH and the leptin system.

This research was funded through the federal government, National Institutes of Health.




                                                  - 104 -
                                                                  NEUROENDOCRINOLOGY

P2-157
MONKEYS WITH HIGHER BASELINE CORTISOL LEVELS ACHIEVE LOWER SOCIAL
STATUS LATER IN LIFE
RR Dolney, MW Bridges, J Cavaretta, K Coleman, CJ Rogers, JL Cameron. Dept. of Psychiatry,
University of Pittsburgh, Pittsburgh, Pennsylvania

Previous studies have reported an inverse correlation between basal plasma cortisol levels and social status
in several primate species. There are at least two possible explanations for this finding; that having lower
social status is a stressful condition, which causes higher cortisol, or that animals with higher cortisol may
be more stress-sensitive, and thereby not as effective in social interactions needed to attain higher social
status. It has not been possible to differentiate between these possibilities in previous studies because
cortisol levels have been measured in animals after they have already been in established groups. In this
study, we measured baseline cortisol levels in monkeys living in single cages and then moved the monkeys
into social groups in order to test the hypothesis that monkeys with high baseline cortisol levels will
achieve lower social status when put into group conditions. We measured baseline cortisol levels in 25
adult female cynomolgus monkeys (Macaca fascicularis) living in single cages. Blood samples were
collected between 8:00-10:00 AM at five different times over the course of a year. Two years later,
monkeys were randomly assigned to groups (5-6 animals per group). We took daily focal samples to
measure dominance status. Dominance hierarchies were found to be linear in these groups. Correlations
between dominance status and mean cortisol (mean of 5 samples in the first year of study) were assessed by
dividing the animals in half, and comparing mean cortisol levels in the most dominant and least dominant
animals. We found that the animals who were least dominant had significantly higher baseline cortisol
levels prior to the grouping (p = 0.02) compared to those who achieved higher social status. These findings
suggest that animals with higher baseline cortisol levels achieve lower social rank later in life. It is possible,
however, that lifelong dominance status is attained very early in life and that even in this study where
animals had been maintained in single cages most of their life that dominance status developed prior to this
measurement of baseline cortisol.

Basic Science Poster: Neuroendocrinology I (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                    - 105 -
                                                                  NEUROENDOCRINOLOGY

P2-157
Lay explanation of abstract:

Previous studies have found that, in a number of different primate species, blood levels of cortisol are lower
in individuals with high-ranking social status than in individuals with low-ranking social status. While
acute release of cortisol in response to a specific stress is beneficial, chronic sustained cortisol release is
considered detrimental to health. Low-ranking primates have higher cortisol levels, which implies that they
are under a greater degree of chronic stress, and thus may suffer more stress-related disease, including
atherosclerosis and upper respiratory infections.

The cause and effect relationship between cortisol and social rank is still unclear. Low-ranking individuals
may have higher cortisol levels because their low social status leads to a greater amount of stress in their
lives (i.e., low status is stressful). Conversely, they may have had higher cortisol levels prior to being put in
a social condition and this physiological property could predict later social status (i.e., individuals with
higher cortisol levels become low-ranking). Our study was designed to differentiate between these two
possible explanations to learn the relationship between social status and circulating cortisol levels.

In our study, 25 female cynomolgus monkeys (an old world monkey), who had lived much of their lives in
single cages, were studied before and after being put into a group living situation. While they were still
living in single cages, they were trained to extend a leg to allow collection of a blood sample with a
minimum of stress. Cortisol levels were measured in a series of five blood samples collected in this
manner. Two years later, these females were moved into social group housing (4-6 monkeys/group). After
an adaptation period of several months, dominance status was determined by behavioral studies examining
displacement of animals by each other and the occurrence of threats and submissive gestures.

The results showed that animals with low-ranking status in the social groups had had higher baseline
cortisol levels two years earlier when they were housed alone. This finding supports the concept that certain
physiological variables may help identify the monkeys that will attain lower social status when placed in a
group environment. This finding adds to our increasing understanding that there are important links
between an individual's behavior and their health status.

Additional background information:

Cortisol, a hormone released by the adrenal glands, helps the body deal with stress. Major actions of
cortisol include mobilizing energy stores to deal with stressful situations and suppressing immune function,
such as an allergy attack, that impair the ability to cope with a stressor. Despite the beneficial actions of
acute cortisol release, there is substantial evidence that chronically elevated cortisol can lead to
consequences that are detrimental to health, including poor wound healing, an increased incidence of
infections and illnesses, and metabolic changes that can lead to obesity. Elevated cortisol levels are
considered an indicator of chronic stress. Other physiological indicators of chronic stress include higher
resting heart rate, increased heart rate responsiveness to stress, impaired reproductive function, impaired
glucose tolerance, and impaired immune function. Individuals with these indicators have more health
problems compared with individuals who don't have these indicators.

Our goal is to identify both physiological measures and behaviors that are linked to a higher likelihood of
developing stress-related diseases. Understanding these interactions will help clinicians identify people
who are susceptible to stress-related diseases. The ultimate goal is to develop therapies that will prevent
disease by targeting individuals who are particularly stress sensitive.




                                                    - 106 -
                                                               NEUROENDOCRINOLOGY

P2-162

PREMARIN AND TWO OF ITS COMPONENTS (DELTA 8,9-DIHYDROESTRONE SULFATE
AND ESTRONE SULFATE) BLOCK HOT FLUSHES IN A RAT MODEL
JM Funkhouser, I Merchenthaler. WHRI, Wyeth-Ayerst Research, Radnor, Pennsylvania

PremarinTM and the PremarinTM components, delta 8,9-dihydroestrone sulfate and estrone sulfate, were
tested in our rat hot flush model (Maturitas 8:67-76, 1986; Maturitas 30:307-316, 1998). The model relies
on the response (an increase in tail skin temperature) in the morphine addicted rat to naloxone, an opioid
receptor blocker. The elevated tail skin temperature response, or a flush, mimics in timing the human hot
flash response induced by menopause, ovariectomy or rapid drug withdrawal. The peak of the temperature
rise is 15 min after naloxone administration. In humans, the hot flash response can be suppressed by
treatment with PremarinTM or other estrogens. In an earlier study with the rat model (Maturitas 30:307-316,
1998) we demonstrated that ethynyl estradiol can suppress the tail skin temperature rise induced by
naloxone. In this study, PremarinTM or the PremarinTM components, delta 8,9-dehydroestrone sulfate or
estrone sulfate, were administered daily from days 6 to 13 after ovariectomy at concentrations of 0.03, 0.1,
0.3, 1.0, 3.0, and 10.0 mg/kg, orally. To make the animals morphine addicted, on days 8 and 10 after
ovariectomy, morphine pellets were subcutaneously implanted. On day 13, under Ketamine anesthesia, a
small thermistor, connected to a data acquisition system, was taped on the tail of the rats and naloxone was
injected subcutaneously to withdraw morphine effect. The tail skin temperature was monitored for one
hour. A two factor repeated measures, with one factor repeated, analysis of variance model was used to
analyze the change of tail skin temperature 15 min after naloxone injection. PremarinTM and estrone sulfate
suppressed the tail skin temperature rise at 3.0 and 10.0 mg/kg. Delta 8,9-dihydroestrone sulfate was more
active than both PremarinTM and estrone sulfate and suppressed the tail skin temperature rise at the lower
(1.0mg/kg) concentration as well. PremarinTM and Delta 8,9-dehydroestrone and estrone have been shown
in clinical studies to suppress hot flashes in women. The studies with the rat hot flush model indicate that
this animal model is mimicking the physiological response seen in humans and therefore, is likely a good
predictor of estrogen action.

Basic Science Poster: Neuroendocrinology I (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 107 -
                                                                 NEUROENDOCRINOLOGY

P2-162
Lay explanation of abstract:

The most common and characteristic symptom women experience during menopause (a period of life
during which women stop menstruating) is the hot flush (hot flash). This unpleasant event is associated
with sudden feeling of being overheated and profound sweating. Although the precise mechanism
underlying hot flushes is unknown, it is likely that a decline in estrogen secretion by the ovaries leads to
vasomotor instability that causes sudden vasodilatation, which results in hot flushes.

Estrogen replacement therapy (ERT) continues to be the most effective treatment of hot flushes since the
early 1900s. PremarinTM is the most commonly used estrogen for ERT in the United States and provides
other benefits, such as prevention of osteoporosis. The drug also is believed to reduce cardiovascular
diseases, decline in memory, and Alzheimer's disease.

In this study, we tested PremarinTM and the PremarinTM components, delta 8,9-dehydroestrone sulfate and
estrone sulfate, in our rat hot flush model (Maturitas 8:67-76, 1986) to learn whether we could identify
components with selective effect on hot flushes. The model measures an increase in tail skin temperature in
the morphine-addicted rat in response to naloxone, an opioid receptor blocker. The elevated tail skin
temperature response mimics in timing the human hot flush response induced by menopause (a period of
cessation of menstruation). PremarinTM or the PremarinTM components, delta 8,9-dehydroestrone sulfate or
estrone sulfate, were administered daily at different doses and tail skin temperature was measured at the
end of the treatment. Delta 8,9-dehydroestrone sulfate, as in human trials, was more active than both
PremarinTM and estrone sulfate and also suppressed tail skin temperature rise at a lower concentration.

The studies with the rat hot flush model indicate that this animal model is mimicking the physiological
response seen in humans and therefore, is likely a good predictor of estrogen action. Therefore, this animal
model could be of great importance for studying additional Premarin TM components, which may exhibit
tissue and cell selective actions.




                                                   - 108 -
                                                               NEUROENDOCRINOLOGY

P3-299
EFFECT OF CIGARETTE SMOKING ON β-ENDORPHIN AND δ-OPIATE RECEPTOR
Y Nakai,1 RY Osamura,2 Y Itho,2 N Sanno,3 Y Gomita.4 1Medical Technology, Kyoto University, Kyoto,
Japan; 2Pathology, Tokai University, Isehara, Japan; 3Neurosurgery, Nippon Medical School, Tokyo,
Japan; 4Pharmacology, Okayama University, Okayama, Japan

While cigarette smoking gives us mental pleasure, it induces psychiatric dependence. In order to clarify the
role of opioid peptides on the psychiatric dependence by smoking, the effect of chronic smoking on the
mRNA and peptide concentrations of β-endorphin, and also the mRNA concentrations of δ-opiate receptor
was examined in rat brain and pituitary gland. Ten adult male Wistar rats smoked fifteen cigarettes twice a
day for 7 days using the inhalation apparatus of Hamburg type-2. Ten control rats were set to the apparatus
without smoking. After this time the rats were rapidly decapitated. The brain and pituitary glands were
removed immediately and fixed quickly. Immunohistochemical localization of β-endorphin was studied in
hypothalamus and pituitary gland by avidin-biotin-complex method. The mRNA concentrations of β-
endorphin were measured by in situ hybridization using the synthetic oligonucleotide probes. The mRNA
concentrations of δ-opiate receptor were measured by in situ hybridization using the antisense riboprobes
prepared against the extracellular domain of the receptor. The pituitary cells expressing the δ-opiate
receptor were identified with mirror-section methods and serial section methods. The hypothalamus of the
rats under chronic smoking showed increased staining of β-endorphin immunopositive nerve fibers and
nerve cells that located close to portal vein. The mRNA concentrations of the δ-opiate receptor were
increased in the arcuate nucleus of the rat hypothalamus after chronic smoking. The mRNA concentrations
of β-endorphin and δ-opiate receptor were increased in the anterior pituitary of these rats after chronic
smoking. δ-Opiate receptors were expressed in the prolactin-producing cells, ACTH-producing cells and
GH-producing cells of the rat anterior pituitary gland. These results suggest that the increased function of
opioid peptide in the hypothalamus and pituitary under chronic smoking may play some role of the
psychiatric dependence by smoking (Supported by a grant from the Smoking Research Foundation).

Basic Science Poster: P3: Neuroendocrinology II (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 109 -
                                                               NEUROENDOCRINOLOGY

P3-299
Lay explanation of abstract:

While cigarette smoking gives us mental pleasure, it also induces psychiatric dependence. Opioid peptides,
endogenous opiate-like peptides such as enkephalins and endorphins, have been discovered in the brain,
pituitary, and other tissues. Later their receptors were also found in the brain and other tissues. Opioid
peptides and their receptors may play some role in alcohol dependence.

Recently, some scientists published work on the effect of naloxone, an opiate antagonist, on mood and/or
cognition after smoking. The results, however, were inconclusive. Until now, there has been no report on
the effect of chronic smoking on changes in opioid peptides and their receptors. Our study results suggest
that the increased function of opioid peptides in the brain in chronic smokers may play some role in
psychiatric dependence induced by smoking.

This research is supported by a grant from the Smoking Research Foundation.




                                                 - 110 -
                                                                 NEUROENDOCRINOLOGY

P3-306
BODY WEIGHT AND METABOLIC REGULATORY FACTORS ARE INFLUENCED BY
DIETARY SOY PHYTOESTROGENS
TD Lund,1,2 JP Porter,1 ED Lephart.1 1Neuroscience Center, Brigham Young University, Provo, Utah;
2
  Anatomy and Neurobiology, Colorado State University, Fort Collins, Colorado

Naturally occurring estrogen-like molecules in plants (Phytoestrogens), present via soy, in animal diets
exert many biological responses evoked by physiologic estrogens. This study characterized the effects of
dietary phytoestrogens on the expression of body, prostate and adipose tissue weights as well as arterial
blood pressure, and plasma insulin and leptin levels in adult Long-Evans rats. Using different experimental
protocols animals were fed either a phytoestrogen-rich diet (Phyto-600; that contained 600 micrograms of
phytoestrogens/gram of diet), which yielded similar plasma phytoestrogen levels to that of a human Asian
diet, or a phytoestrogen-low (Phyto-free) diet that yielded similar plasma phytoestrogen levels to that of a
human Western diet. Animals fed the Phyto-600 diet displayed decreased arterial blood pressure, body,
adipose tissue (males and females), and prostate weights compared to Phyto-free fed animals. In a subset of
animals at 80 days-old, one-half of the total number of male or female (random cycling) rats were: 1) kept
on their original diet (Phyto-600 or Phyto-free) or 2) changed to the alternate diet (i.e., Phyto-600 to Phyto-
free or Phyto-free to Phyto-600) for 40 days. Plasma insulin levels in males fed the Phyto-free diet were
significantly increased compared to males fed the Phyto-600 diet (lifelong) while the opposite was found in
females. Plasma insulin levels in females fed the Phyto-600 diet lifelong were significantly increased
compared to females fed the Phyto-free diet lifelong. No sex difference was found in Plasma leptin levels.
Plasma leptin levels in males and females fed the Phyto-free diet lifelong or short-term (i.e., Phyto-600 fed
animals changed to the Phyto-free diet) were significantly increased compared to males and females fed the
Phyto-600 diet lifelong or short-term (i.e., Phyto-free fed animals changed to the Phyto-600 diet). These
data suggest that consumption of phytoestrogens via a soy diet significantly:(1) decrease body, adipose
tissue, and prostate weights, (2) decrease arterial blood pressure, and (3) alter plasma insulin and leptin
levels during adulthood. Supported by, NSF grant, IBN-9507972 (EDL).

Basic Science Poster: P3: Neuroendocrinology II (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                   - 111 -
                                                                 NEUROENDOCRINOLOGY

P3-306
Lay explanation of abstract:

Phytoestrogens are naturally occurring estrogen-like molecules found in many plants (including soy). Soy
is used as the principal protein source in commercially available animal diets (and soy is frequently found
in human diets), and the phytoestrogens present in these diets through soy have been found to have
estrogen- or hormone-like effects. Thus, this study was conducted to determine the effects of consuming
phytoestrogens on body weight, prostate weight, and the accumulation of fat (adipose tissue weight).
Additionally, this study was conducted to determine whether phytoestrogen consumption would change
blood pressure, insulin levels (most often associated with diabetes, insulin regulates the metabolism of
carbohydrates, fats, and starches in the body), or leptin levels (produced mainly in adipose tissue, leptin is
known to inhibit food intake and increase energy expenditure) in adult Long-Evans rats.

In this study animals were fed either a phytoestrogen-rich diet (which could be thought of as similar to that
of a typical human ‗Asian‘ diet), or a phytoestrogen-low diet (which could be thought of as similar to that
of a typical human ‗Western‘ diet). Male and female animals fed the phytoestrogen-rich diet had decreased
blood pressure, decreased body weight and decreased body fat (adipose tissue) compared to phytoestrogen-
free fed animals. Additionally, males fed the phytoestrogen-rich diet had decreased prostate weight as
compared to males who were fed the phytoestrogen-free diet.

In another group of animals, at 80 days of age (in adulthood), half of the males and half of the females
either continued to be fed: 1) their original diet (phytoestrogen-rich or phytoestrogen-free) or 2) were
changed to the alternate diet for 40 days. Insulin levels in males fed the phytoestrogen-free diet were
significantly increased compared to males fed the phytoestrogen-rich diet lifelong, while the opposite effect
was found in females. Insulin levels in females fed the phytoestrogen-rich diet lifelong were significantly
increased compared to females fed the phytoestrogen-free diet lifelong. No sex difference was found in
leptin levels.

Leptin levels in male and female rats fed the phytoestrogen-free diet lifelong and males and females
initially fed the phytoestrogen-rich diet then changed to the phytoestrogen-free diet were significantly
increased compared with males and females fed the phytoestrogen-rich diet lifelong and males and females
initially fed the phytoestrogen-free diet and then changed to the phytoestrogen-rich diet. The findings of
this research suggest that eating a diet high in soy phytoestrogens significantly: 1) decreases body weight,
decreases body fat, and decreases prostate weight; 2) decreases blood pressure; and 3) alters insulin and
leptin levels during adulthood.

This work was supported, in part, by funds from The National Science Foundation (grant IBN-9507972)
and the BYU Dean‘s Fellowship in Neuroscience.




                                                   - 112 -
                                                                  NEUROENDOCRINOLOGY

P3-318
CEREBRAL GLUCOSE UPTAKE IN POSTMENOPAUSAL VOLUNTEERS BEFORE AND
AFTER TREATMENT WITH RALOXIFENE; AN FDG-PET PILOT STUDY
SJ Neele,1 R Evertz,1 DJ Veltman,2 AA Lammertsma,2 JC Netelenbos.1 1Endocrinology, Academic
Hospital Vrije Universiteit, Amsterdam, Netherlands; 2PET center, AZVU, Amsterdam, Netherlands

Background: Epidemiological and observational studies suggest favorable effects of estrogen replacement
therapy (ERT) on cognitive (especially verbal) performance, regional cerebral blood flow and the course of
Alzheimer Disease in postmenopausal women. However, ERT has drawbacks because of its peripheral side
effects. Raloxifene is a selective estrogen receptor modulator (SERM) with both estrogen agonistic and
antagonistic effects. Little is known about the effect of SERM's on cerebral functioning.
Aim: To assess whether raloxifene treatment affects cerebral glucose metabolism, as measured with [18F]-
2-fluoro-2-deoxy-D-glucose (FDG) PET, compared with placebo.
Methods: Twenty-six right-handed females, age 60-70 years, were treated with raloxifene 60 mg/day or
placebo according to a double-blind design during three months. FDG-PET scans were made at baseline
and after treatment. Statistical analysis was performed using SPM99 software (www.fil.ion.ucl.ac.uk).
Contrasts were computed for both main effects and group x time effects (threshold at p<0.001).
Results: Data were available from 22 women, 10 from the raloxifene group and 12 from the placebo group.
In the raloxifene group, relative glucose uptake was increased compared with placebo in L superior and
inferior parietal, L superior temporal, and R orbitofrontal as well as L inferior prefrontal cortices. A relative
decrease in glucose uptake compared with placebo was observed in L occipital and L medial temporal
cortices.
Conclusion: This pilot study shows that raloxifene treatment is associated with increased glucose uptake in
L-sided areas being associated with a number of cognitive functions, language in particular. Glucose uptake
was decreased in L occipital and medial temporal cortex, the latter finding corroborating functional MRI
data from a related study (Neele et al., JCEM 2001;86:1422-1424). Furthermore, these data are consistent
with earlier functional imaging data obtained in postmenopausal women treated with estrogen (Shaywitz et
al., 1999). Our results suggest that raloxifene may exert similar effects on CNS function as estrogen.

Basic Science Poster: P3: Neuroendocrinology II (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                    - 113 -
                                                                 NEUROENDOCRINOLOGY

P3-318
Lay explanation of abstract:

Hormone replacement therapy is often used by postmenopausal women for alleviation of climacteric
complaints. Currently, the effects of hormone replacement therapy on brain functioning have gained much
interest, because some studies have suggested favorable effects of estrogen therapy on memory and even on
the course of Alzheimer‗s disease. Because estrogen therapy may initiate unwanted side effects such as
breast cancer, alternatives are being developed to prevent these effects without losing the positive aspects
of estrogen treatment. Raloxifene is a compound that has been shown to have the same effect on bone
mineral density as estrogen but without the apparent effects on breast tissue. The drug‘s effects on breast
cancer and also on cardiovascular disease are currently under investigation in large clinical trials. Little is
known, however, about the effect of raloxifene on brain function.

To learn more about the effects of raloxifene treatment on the brain, we investigated how raloxifene would
affect glucose consumption in certain areas of the brain. Glucose is the most important source of energy for
brain cells. When glucose molecules are labeled with a radioactive compound it is possible to detect with a
brain scanner (PET-scan) where this labeled glucose is used in the brain, but also to estimate how much
glucose is used.

Twenty-six postmenopausal women between 60 and 70 years old participated in a 3-month-long study.
These women had to be free of hormone replacement therapy for at least 6 months, free of brain-
influencing medication, and free of apparent brain disease or familial history of brain disorders. During this
period, the women were assigned to receive a raloxifene or a placebo tablet each day. Before and after
treatment, brain scans were made in a resting condition.

With this experiment, we have shown for the first time that there are differences in glucose metabolism
between the women treated with raloxifene and those treated with placebo. Some areas of the brain showed
an increased glucose uptake, especially some areas on the left side of the brain that are associated with a
number of memory-related functions. Other areas showed a diminished uptake of glucose when compared
with the placebo-treated women. Our results resemble those found in another small study in which women
were treated with estrogens. It is therefore possible that raloxifene may exert the same effects on brain
glucose metabolism as estrogen. However, as with estrogen treatment, the effects of raloxifene on the brain
and especially on memory performance or brain diseases such as Alzheimer‘s disease, need to be clarified
in more detail in studies with many participants over a long period of time. Only then we will be able to
draw conclusions with regard to the proper implications for health care.




                                                   - 114 -
                                                                                            OBESITY

P1-381

OPPOSING ACTIONS OF IGF-1 AND TNFα ON HUMAN PREADIPOCYTE
DIFFERENTIATION AND CORTISOL METABOLISM
JW Tomlinson, I Bujalska, M Hewison, PM Stewart. Division of Medical Sciences, University of
Birmingham, Queen Elizabeth Hospital, Birmingham, United Kingdom

The incidence of obesity continues to rise yet the mechanisms that underpin its pathogenesis remain
unknown. Patients with cortisol excess develop central obesity however, circulating cortisol levels are
normal in simple obesity. We have postulated that within adipose tissue there is increased local cortisol
production due to enhanced expression of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). This
enzyme converts inactive cortisone to active cortisol. Both IGF-1 and TNFα regulate 11β-HSD1.
Clinically, TNFα levels are elevated in obesity and patients with GH/IGF-1 excess are lean. We therefore
examined the effect of IGF-1 and TNFα on human preadipocyte differentiation, and assessed whether their
actions are mediated via 11β-HSD1.Primary cultures of human preadipocytes from subcutaneous (sc) and
omental depots were used. Cultures were maintained in media known to promote preadipocyte
differentiation and treated with IGF-1 (1-100ng/ml) or TNFα (0.1-10ng/ml) for 14 days. Preadipocyte
differentiation was assessed by glycerol-3-phoshate dehydrogenase (G3PDH) activity and Oil Red O
(ORO) staining. 11β-HSD1 activity was assessed by specific enzyme assay.
IGF-1 treatment increased G3PDH activity in sc preadipocytes (eg. at 100ng/ml 150.0±15.9%, p
In primary cultures of human preadipocytes IGF-1 stimulates and TNFα inhibits differentiation. Divergent
regulation of 11β-HSD1 persists throughout differentiation and the altered generation of cortisol may serve
as an autocrine/paracrine neagtive feedback loop to counteract the direct action of growth factors and
cytokines.

Basic Science Poster: Obesity I: Leptin & Fat (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                 - 115 -
                                                                                               OBESITY

P1-381
Lay explanation of abstract:

Obesity is a major medical problem worldwide. In particular, central obesity (accumulation of fat around
the waist rather than hips) dramatically increases the risk of heart disease, high blood pressure, and
diabetes. Patients who have too much corticosteroid (cortisol), either because of medications taken or
because of an underlying disease, often develop central obesity.

We isolated fat cells from tissue taken from patients having elective abdominal surgery. These cells were
grown in media known to promote the accumulation of fat stores and treated with factors known to be
produced from mature fat cells (IGF-1 and TNF) for a period of 2 weeks. We then assessed the effect of
these factors on the amount of fat stored in these cells by measuring the activity of a specific enzyme,
which is involved in the synthesis of fat. We also stained the fat in these cells with a specific dye. We have
previously shown that fat cells also produce cortisol, and that cortisol increases fat stores. We therefore
also assessed the amount of cortisol that these cells can generate after treatment with IGF-1 and TNF.
Each experiment was performed between 3 to 6 times in triplicate.

IGF-1 increased the amount of fat stored, while TNF decreased it. Furthermore, in addition to directly
increasing fat stores, IGF-1 decreased the amount of cortisol produced by these cells. Conversely, TNF
decreased fat stores but increased cortisol generation. Therefore, IGF-1 has a direct action to promote the
formation of fat stores, but this is limited by decreasing the availability of cortisol to the cell. Similarly,
TNF has a direct action to decrease fat stores, but this is offset by increased cortisol generation. The
importance of this research is that it helps us to understand the relative contribution of factors such as IGF-
1, TNF, and cortisol in controlling the amount of fat stored in fat tissue. This may ultimately lead to the
development of new treatments for obesity.

This work was funded by the Medical Research Council (UK).




                                                   - 116 -
                                                                                             OBESITY


P3-102
ESTRADIOL-INDUCED ANOREXIA IS INDEPENDENT OF MCH AND LEPTIN
NA Tritos,1,2 G Lieberman,1,2 P Vezerides,1,2 E Maratos-Flier.1,2 1Joslin Diabetes Center, Boston,
Massachusetts; 2Harvard Medical School, Boston, Massachusetts

In male rodents estradiol (E2)induces anorexia and weight loss. Melanin concentrating hormone (MCH) is
an orexigenic neuropeptide that is downregulated by E2.To clarify the anorectic effects of E2 and elucidate
underlying mechanisms, we implanted male C57Bl mice with either E2(0.72 mg) pellets or placebo,
resulting in a 20 fold increase in serum E2 (P<0.0001). E2-implanted mice had a 68% decrease in food
intake (P<0.0001), a 11.2% decrease in body weight (P<0.0001) and a 28.2% decrease in body fat
(P=0.005).We speculated that a high fat diet would attenuate the anorectic response.In contrast, we found
that mice fed high fat (45%) diet showed a 99.7% decrease in food intake and a 90.6% reduction in serum
leptin (P<0.0001).62.5% of E2-implanted mice survived compared to 100% of placebo (P=0.05). Mortality
was observed only among E2-treated mice on high fat diet (P=0.05).E2-induced anorexia occurs
independently of leptin, as E2-implanted (0.72 mg) male ob/ob mice showed a 34.3% or 18.4% decrease in
food intake on the high or low fat diet, respectively (P<0.0001) and they lost 2.7% or 3.1% of body weight
on the high or low fat, respectively (P=0.09).16.6% of low fat and 14.3% of high fat, E2-treated mice
survived, compared with 100% of placebo (P=0.001).To evaluate the dose-response to E2, we implanted
C57Bl mice with low dose E2(0.18 mg)or placebo.E2-treated mice had a 66.5% or 15.6% decrease in food
intake on the high fat or low fat diet, respectively (P=0.0002) and a 9.2% or 4.0% decrease in body weight
on the high or low fat diet, respectively (P=0.02) with no mortality.There was no difference in water
intake,blood glucose and urine ketones.As others have speculated that MCH downregulation may mediate
E2-induced anorexia, we implanted MCH knockout and wild type mice with either E2(0.72 mg) or
placebo.E2-implanted mice had a 16.3% decrease in food intake and a 14.7% decrease in body weight
(P<0.0001).Both MCH knockout and wild type mice showed a similar decrease in body weight and food
intake (P=0.67).In conclusion, E2 treatment of male mice decreases food intake, body weight and serum
leptin.High fat fed C57Bl mice and ob/ob mice show a remarkable impairment in feeding. These effects are
independent of MCH and leptin.

Basic Science Poster: P3: Obesity II: Neuropeptides & Estrogen (11:00 AM-12:00 PM and 2:30 PM-3:30
PM)

Presentation Date: Friday, June 22, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 117 -
                                                                                              OBESITY

P3-102

Lay explanation of abstract:

Estradiol is known to inhibit feeding in male rodents, but the underlying mechanisms are not well
established. The current study sought to explore this observation and examine underlying mechanisms.

First, we examined the effect of two different doses of estradiol on feeding in male mice. Our work showed
that administration of a higher dose of estradiol (placed as a pellet under the skin) was associated with more
profound impairment in feeding and weight loss, which may lead to death. Second, estradiol-treated
animals fed a high-fat diet showed a more profound decrease in food intake, weight loss, and serum leptin
levels, which is contrary to the commonly observed stimulation of food intake and weight gain in mice fed
a high-fat diet. Third, we studied the effect of estradiol on feeding in genetically modified mice, including
ob/ob mice that lack leptin, and MCH knockout mice that lack MCH (also known as melanin-concentrating
hormone, an appetite-stimulating neuropeptide). We found that estradiol inhibited feeding in both ob/ob
mice and MCH knockout mice, indicating that neither leptin nor MCH have a role in mediating estradiol-
induced anorexia in male rodents. Fourth, we examined MCH and NPY (neuropeptide Y, another appetite-
stimulating neuropeptide) expression using in situ hybridization histochemistry in the hypothalamus, which
is a brain region involved in the regulation of appetite. This was accomplished using estradiol-treated (wild
type) mice. We found that the expression of both peptides increased in these mice, which indicates that
both MCH and NPY respond appropriately to the animals‘ decreased feeding and are not mediating the
estradiol-induced anorexia.

In conclusion, this study helps characterize estradiol-induced anorexia in male rodents, and attempts to
establish the underlying mechanisms, which still remain elusive. Further studies will be needed to clarify
the pathophysiologic mechanisms underlying estradiol-induced anorexia in male rodents.

Additional background information:

Estradiol inhibits feeding in male rodents. Our study sought to further describe this observation and
analyze underlying mechanisms.

This study was funded by a grant from the National Institute of
Diabetes and Digestive and Kidney Diseases (NIDDK) to Dr. E. Maratos-Flier.




                                                  - 118 -
                                                                                            OBESITY

P3-110
ESTROGEN RECEPTOR ALPHA (ERα) REGULATES THE EFFECT OF ESTROGEN ON
METABOLIC RATE (MR)
PA Heine,1 DR Jensen,3 JA Taylor,2 DB Lubahn,2 RH Eckel,3 PS Cooke.1 1Department of Veterinary
Biosciences, University of Illinois, Urbana, Illinois; 2Departments of Biochemistry and Child Health,
University of Missouri, Columbia, Missouri; 3Department of Medicine, University of Colorado Health
Sciences Center, Denver, Colorado

Estrogen has recently been shown to regulate a variety of target tissues in addition to its actions on the
female reproductive system. It is one of several hormones regulating adipose deposition and energy
balance. Reduced estrogen levels and a resultant increase in white adipose tissue (WAT) occur in
postmenopausal women as well as ovariectomized animals. Studies have shown that this increase in WAT
is not due to an increase in food consumption, but to a decrease in MR. With the development of the ERα
knockout (αERKO) mouse, we are now able to more closely examine the interaction of estrogen and MR.
We have previously shown that the αERKO mouse obese phenotype, seen in both males and females, does
not result from an increase in energy intake, but rather from a decrease in energy expenditure. The current
study corroborates these data while further showing that the difference in MR between αERKO and WT
mice is even greater at night, during nocturnal activity, than during the day. In the present study we have
compared energy expenditure differences between male sibling 17-month old αERKO and WT mice. WT
(n = 6) and αERKO (n = 6) mice underwent indirect calorimetry. Each mouse was housed individually in a
calorimetry unit for 3 consecutive days during which time data were collected. Compared to WT males,
αERKO males had significantly higher body weight (BW; 32.0±1.1 vs. 38.6 ±1.6 g, P=0.004), and
significantly lower energy intake (10.44±0.25 vs. 8.09±0.39 kcal/day, P<0.001), and MR (0.59±0.01 vs.
0.52±0.01 kcal/d/g BW, P=0.002). The difference in MR between the two genotypes was greatest at night,
when mice are most active (0.62±0.01 vs. 0.55 ±0.01 kcal/day/g BW, P=0.001). In conclusion, male mice
without ERα have a lower MR than WT male mice, especially during normal nocturnal activity. These
findings suggest that estrogen's maintenance of energy balance is likely controlled by signaling through
ERα. The current study also demonstrates that estrogen/ERα has a greater role in male physiology than
previously recognized.

Basic Science Poster: P3: Obesity II: Neuropeptides & Estrogen (11:00 AM-12:00 PM and 2:30 PM-3:30
PM)

Presentation Date: Friday, June 22, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 119 -
                                                                                                 OBESITY

P3-110
Lay explanation of abstract:

We have examined the relationship between the hormone estrogen, its receptor (ER), and obesity in both
males and females. Using mice that have been genetically altered so that they do not have ER (ER
knock outs; ERKO), we have found that fat increases when estrogen or estrogen signaling decreases. In
other words, if estrogen cannot act through ER, mice become obese. Interestingly, this is seen in both
male and female mice. Generally speaking, obesity develops in animals when they either eat too much or
exercise too little. Either of these possibilities will result in mice storing calories in the form of fat. ERKO
mice do not eat more food than animals with normal estrogen signaling, but they do have decreased energy
expenditure.

Therefore, obesity results because the ERKO mice have a lower energy expenditure, which appears to be
due to the loss of the action of estrogen through ER. Mice are nocturnal animals, meaning they are more
active at night than during the day. ERKO mice, however, are less active at night than their normal
counterparts.

This study is newsworthy for several reasons. It has not previously been shown that ER is important in
regulating activity. Also, although a relationship between estrogen and obesity in females has long been
recognized, this is the first demonstration that the receptor ER is important for regulating the development
of obesity in males. This study further is newsworthy because of the numerous health ramifications already
attributed to estrogen or estrogen supplementation. As this story continues to unfold, the findings linking
decreased levels of estrogen to the development of obesity are particularly likely to have an impact on the
daily lives of our aging population. As women and men age, circulating concentrations of hormones
change, commonly resulting in changes in overall health. Continued developments from our findings may
help explain why estrogen signaling is important for the overall health of people and animals.

Additional background information:

This research was conducted using six male ERKO mice paired with six normal mice. Mouse pairs were
17-month old siblings. Data were collected from the animals over a period of three consecutive days. Data
consisted of measuring the amount of food and water eaten, the weight of the animals, and detailed
measurements of the animals' energy expenditure. Statistical comparisons of the average data points from
each group showed that ERKO mice had a significantly higher body weight, but significantly lower food
consumption than normal mice. Additionally, energy expenditure of ERKO mice was significantly lower
than in normal mice. From this, we conclude that estrogen is regulating the development of obesity by
acting through ER.

This research was funded by Animal Health and Disease Research Funds from the University of Illinois.




                                                    - 120 -
                                                  PTH-CALCIUM-VITAMIN D-BONE

OR29-1
PARTIAL VITAMIN D ANTAGONISTS INHIBIT BREAST CANCER GROWTH IN VITRO AND
IN VIVO
J Barsony, C Segovis. LCBB, NIH/NIDDK, Bethesda, Maryland

Calcitriol has biphasic effect on cell growth; physiological doses stimulate cell proliferation while high
doses inhibit it. Calcemic effects hinders the use of calcitriol analogs for the treatment of breast and
prostate cancer. To inhibit the growth stimulatory effect, we synthesized partial calcitriol antagonists,
BCA11 and BCA21. They bind vitamin D receptors with high affinity. In MCF-7 cell extracts 1µM BCA11
acted as a weak agonist to increase 24-hydroxylase activity, while lower doses antagonized the effect of
calcitriol. Luciferase reporter assays gave similar results; BCA11 (0.1-100nM) and BCA21 (5-50nM)
inhibited calcitriol induced activity. In contrast, antiproliferative effects of BCA11 and calcitriol were
additive. BCA11 was 100x more effective than calcitriol to inhibit [3H]-thymidine incorporation in both
estrogen receptor positive (MCF-7, BR3, and T47D) and negative (BT20) breast cancer cells. BCA21 was
less effective than BCA11. More importantly, these antagonists inhibited the growth of human breast
cancer (MCF-7) in female athymic nude mice. Cells were injected into 7 weeks old mice. To generate
advanced stage tumors in the second experiment, cells were injected with Matrigel and estrogen pellets
were implanted. Four weeks after inoculation vehicle or hormones were injected every other day for 5
weeks. Tamoxifen (2mg/kg) decreased tumor volume 46% in the first experiment and 54% in the second.
BCA11 was most effective in 1.0µg/kg dose and decreased tumor volume 24% in the first experiment and
25% in the second. In addition, 5 tumors out of the 7 disappeared after BCA11 treatment in the first
experiment and 1 out of 17 in the second, while tumor number increased in the control groups. Addition of
Tamoxifen to BCA11 did not increase the effect, suggesting that they share a common mechanism of
action. BCA21 decreased tumor volume 17% and the agonist EB1089 (1µg/kg) was ineffective.
Histological analysis showed no abnormalities, and serum calcium, phosphorus, liver and renal functions
were within the normal range, indicating that our compounds have low toxicity. Our results show that a
selective vitamin D receptor antagonists can be used for breast cancer treatment, and suggest that BCA11
will be most useful for the treatment of estrogen resistant cancer.

Basic Science Oral: PTH-Calcium-Vitamin D-Bone (1:00 PM-2:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 1:00 PM; Location: A 106




                                                 - 121 -
                                                    PTH-CALCIUM-VITAMIN D-BONE

OR29-1

Lay explanation of abstract:

A team of NIDDK investigators led by Dr. Julia Barsony has developed new derivatives of vitamin D that
inhibit the growth of breast cancer cells in culture and in mice. These derivatives are based on calcitriol, the
hormonal form of vitamin D, which performs several important functions in the body. Because normal
concentrations of calcitriol stimulate growth in many cell types, including breast cancer cells, we modified
the calcitriol structure to make derivatives (BCA11 and BCA21) that counteract the hormone's ability to
stimulate cell growth. Current vitamin D derivatives have been able to mimic the way large doses of
calcitriol inhibit cancer growth, but these attempts were limited because of toxic levels of serum calcium
that resulted.

In our team's novel approach, these derivatives work by inhibiting vitamin D receptors inside the cells that
would normally bind calcitriol. In cell studies, our team found that BCA11 and BCA21 molecules bind
well to the vitamin D receptors, and they inhibit calcitriol's ability to bind to the receptor, regulate gene
transcription, and stimulate cell growth. As a result, the growth rate of breast cancer cells treated with low
concentrations of BCA11 was 50% of the growth rate of untreated cells. We also studied the anti-tumor
effects of these new substances in mice specially bred to tolerate the implantation of human breast cancer
cells. These studies on estrogen-responsive human breast cancer showed that BCA11 inhibited cancer
growth more than BCA21. The magnitude of this inhibitory effect was comparable to the effect of the
commonly used drug, Tamoxifen, which inhibits the ability of estrogen to stimulate growth. Both BCA11
and BCA21 prevented tumor growth without causing any discernible toxic side effects.

This study shows for the first time that inhibitors of vitamin D receptors can limit the growth of breast
cancer in cultured cells and in animals, and represents a new approach to potential drug development.
Because about 80% of breast cancers contain vitamin D receptors, while only 50% contain estrogen
receptors, targeting vitamin D receptors may make it possible to effectively treat more breast cancers. The
next step will be to further evaluate the effectiveness of these new substances in animals for the treatment
of breast cancers that do not respond to current hormonal treatments. We also plan to test other promising
substances to optimize the anticancer effect.

Additional background information:
This small group of researchers at the Laboratory of Cell Biochemistry and Biology, NIDDK, led by Dr.
Julia Barsony, Senior Investigator, has conducted studies on vitamin D actions for the past 10 years. We
developed derivatives of vitamin D to study the actions of the hormone and its intracellular receptor.
During these studies we searched for new derivatives that would antagonize the effects of calcitriol. To
make the new vitamin D receptor antagonists, the scientists made structural changes to vitamin D using the
principles developed to create estrogen receptor antagonists. Other NIDDK investigators contributed to this
work through NMR and mass spectrometry analyses of the compounds, and Vince Pozsgay of NICHD
assisted by advising on aspects of the chemical synthesis and purification. During the studies in cells, the
group discovered that these new compounds also inhibit the growth of various cancer cells in culture.
Because many drugs that work in cell culture are too toxic or ineffective in intact animals, a team led by
Colin Segovis, Chemist, scaled up the synthesis of these antagonists and tested them in mice with the
technical help of researchers at NCI Frederick and AniLytics Inc. Overall, more than 25 people have
contributed to this project.

The Intramural Research Program at NIDDK (National Institute of Diabetes, Digestive and
Kidney Diseases) supported these studies (under the protocol 1-Z01-DK60050-04).




                                                    - 122 -
                                                  PTH-CALCIUM-VITAMIN D-BONE

OR29-5
LOSS OF IMPRINTING ON THE MATERNAL GNAS1 ALLELE IN
PSEUDOHYPOPARATHYROIDISM 1B
SM Jan de Beur, Z Deng, J Cho, C Ding, MA Levine. Medicine and Pediatrics, Johns Hopkins University,
Baltimore, Maryland

Subjects with pseudohypoparathyroidism 1b (PHP 1b) have renal resistance to PTH and lack Albright's
hereditary osteodystrophy. By contrast to PHP 1a, levels of Gsα protein are not reduced, and the 13 exons
of GNAS1 that encode Gsα are normal. GNAS1 has 3 upstream alternative first exons (NESP55, XLαs, and
exon A) that are bidirectionally imprinted, and defective imprinting of maternal GNAS1 alleles has been
reported in PHP 1b. We analysed allelic expression and imprinting of GNAS1 in familial and sporadic PHP
1b. cDNA transcribed from mononuclear cell or transformed lymphoblast RNA was amplified with a
downstream primer in exon 6 and an upstream primer in either NESP55, XLαs, exon A, or exon 1. PCR
products were directly sequenced and allelic expression was determined using polymorphisms in exon 5 or
exon A. Exon 1 expression was biallelic in all subjects. Alternative exons showed normal uniallelic
expression in 16/16 unaffected PHP 1b family members, 2/2 normocalcemic PHP 1b gene carriers, and 4/4
PHP 1a patients. By contrast, exon A expression was biallelic in 6/6 familial and 5/5 sporadic PHP 1b
patients. XLαs and NESP55 expression was uniallelic in all but one PHP 1b subject, who had biallelic
expression of XLαs. Genomic DNA was digested with NgoMIV, a methylation-sensitive enzyme and
hybridized to an exon A probe. 13/13 PHP 1b patients showed biallelic digestion, indicating absence of
methylation at the NgoMIV restriction sites on the maternal and paternal alleles. By contrast, DNA from
normals, PHP 1a patients and normocalcemic PHP 1b gene carriers showed digestion of only one exon A
allele by NgoMIV. Direct sequence analysis of exon A after bisulfite treatment of genomic DNA confirmed
the lack of methylation at CpG islands on the maternal allele of PHP 1b patients. We conclude that
maternal imprinting of alternative GNAS1 exons is defective in PHP 1b. This defect may suppress
expression of Gsα from the maternal allele in imprinted tissues such as the kidney, thereby leading to
complete absence of Gsα protein and PTH resistance. The basis for the imprinting defect is currently under
study.

Basic Science Oral: PTH-Calcium-Vitamin D-Bone (1:00 PM-2:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 2:00 PM; Location: A 106




                                                 - 123 -
                                                   PTH-CALCIUM-VITAMIN D-BONE

OR29-5

Lay explanation of abstract:

Hypoparathyroidism results from failure of the parathyroid glands to make parathyroid hormone (PTH).
The parathyroid glands control calcium levels in the blood by acting on the skeleton, kidney, and intestine
to release or absorb calcium. When the parathyroid glands are damaged or malfunction, PTH is absent and
blood calcium levels fall. Pseudohypoparathyroidism (PHP) is a heterogeneous group of disorders
characterized by low blood calcium levels. In contrast to patients with hypoparathyroidism, these patients
have increased PTH levels and show resistance to the action of PTH. Patients with PHP are resistant to
PTH and other hormones because there is a defect in the protein known as a G protein (G 5) that transduces
the hormonal signal from the outside of the cell to the intracellular molecules that produce PTH‘s effects.

There are two distinct types of PHP, known as PHP 1a and PHP 1b. For purposes of discussing our study,
it is most important to know that patients with PHP 1a show mutations in the GNAS1 gene (the gene that
codes for G5) while patients with PHP 1b do not. The molecular defect that causes PHP 1b is not known.
(See below for more detailed background information.)

We sought to define the precise mechanism of PTH resistance in PHP 1b. Since no mutations in the GNAS1
gene have been identified in PHP 1b, we hypothesized that there may be aberrant imprinting that results in
altered expression of GNAS1 in patients with PHP 1b. We examined the methylation and expression of four
gene products encoded by GNAS1in blood from 13 patients with PHP 1b, 16 normal family members, and 4
PHP 1a subjects. The gene products were exon A, NESP55, XLs, and G5.

Aberrant imprinting was observed in one of the four genes encoded by GNAS1. Specifically, no
methylation of exon A was detected, whereas, in normal and PHP 1a individuals, the maternal gene was
methylated. Furthermore in PHP 1b patients, both the maternal and paternal genes were expressed. By
contrast, in normal and PHP 1a subjects only the paternal allele was expressed. In one PHP 1b patient, both
exon A and XLs were expressed from both the maternal and paternal gene suggesting that the imprinting
of the entire region may be controlled by a central mechanism. G5 was expressed from both the maternal
and paternal gene in all normal PHP 1a and PHP 1b subjects.

These results suggest that maternal imprinting of GNAS1 is defective in PHP 1b. This defect may suppress
expression of G5 from the maternal gene and lead to PTH resistance. Thus, defects in the GNAS1 gene
likely account for both PHP 1b and PHP 1a, although through distinct mechanisms. Mutations that disrupt
the structure and function of the GNAS1 gene account for the more global hormone resistance seen in PHP
1a, and the altered imprinting observed in PHP 1b likely accounts for the restricted PTH resistance. These
studies reveal a new mechanism of hormone resistance and may explain tissue-specific differences
observed in response to hormones.

Further background information:

In the type 1a variant of PHP, patients have reduced G5 (G5) protein in the blood owing to mutations in
the GNAS1 gene (the gene that codes for G5) located on chromosome 20. Patients with PHP 1a are
unresponsive to PTH and to several additional hormones whose receptors are coupled to G5. Patients with
PHP type 1a also manifest a different category of developmental and body defects, collectively called
A1bright‘s Hereditary Osteodystrophy (AHO). PHP type 1b is a clinically and biochemically distinct
disorder. Patients with PHP 1b lack features of AHO, have normal expression of G 5 protein in accessible
tissues, and manifest hormone resistance only to PTH. No mutations in the GNAS1 gene have been
identified in this disorder. Thus, the molecular defect is unknown.

This study was funded by the National Institutes of Health, National Institute of Diabetes, Digestive and
Kidney Disease.



                                                  - 124 -
                                                  PTH-CALCIUM-VITAMIN D-BONE

OR29-6
IDENTIFICATION AND CHARACTERIZATION OF THE GENE ENCODING THE
TUBEROINFUNDIBULAR PEPTIDE OF 39 RESIDUES (TIP39)
MR John, M Arai, KB Jonsson, H Jueppner. Endocrine Unit, Massachusetts General Hospital, Boston,
Massachusetts

The tuberoinfundibular peptide of 39 residues (TIP39) was recently isolated from bovine hypothalamus and
is a selective PTH2 receptor agonist (Nat Neuroscience 1999; 2:941-943). Subsequent in vitro studies
showed that the native peptide is also a potent antagonist on the PTH1 receptor. We have identified the
human (located on chromosome 19q13) and mouse genomic DNA sequence in the recently released
working draft sequences at NCBI. Subsequent alignment of two approximately 2 kb synthenic regions of
both genes along with sequences obtained from a 5' RACE product derived from human hypothalamic
cDNA, and RT-PCR products from murine brain poly-A+RNA, suggests that TIP39 contains a 61 amino
acid (aa) pro-sequence which is partially hydrophobic and may serve as the leader sequence. This sequence
is followed by the sequence encoding the secreted protein. Aa 1 to 43 of full-length TIP39 are encoded by
exon 1, while residues 44 to 100 are encoded by exon 2 which also contains the stop codon and
polyadenylation signal. Both coding exons of the TIP39 gene are GC-rich: human 74.3% vs. mouse 69.7%.
While the secreted human aa sequence is identical to the previously isolated bovine TIP39, mouse TIP39
differs by 4 aa. However, both peptides were equally potent in activating the human PTH2 receptor stably
expressed in LLCPK1 cells. A phylogenetic analysis of all known full-length prepro- or pro-peptides of the
TIP39, PTH and PTHrP families of ligands and human CRH as an outgroup was performed. It
demonstrated a distant relationship between TIP39, PTH and PTHrP, suggesting that they all may be
derived from a common ancestral precursor. In situ hybridization experiments using a murine TIP39 probe
(corresponding to aa 4 to 37 of secreted TIP39) and fresh frozen adult murine tissue sections showed
highest TIP39 mRNA expression stage-specifically in testicular seminiferous tubuli. In brain, specific
TIP39 expression was detected within focal areas of the substantia nigra, nucleus ruber, nucleus centralis
pontis and nucleus subparafascicularis thalami. We also detected TIP39 expression in kidney where it may
act as an antagonist at the PTH 1 receptor and thus may have a role in the regulation of calcium
homeostasis.

Basic Science Oral: PTH-Calcium-Vitamin D-Bone (1:00 PM-2:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 2:15 PM; Location: A 106




                                                 - 125 -
                                                   PTH-CALCIUM-VITAMIN D-BONE

OR29-6
Lay explanation of abstract:

Our study describes how we identified the genes encoding human and mouse tuberoinfundibular peptide of
39 residues (T1P39). This peptide was initially purified from bovine (cow) hypothalamus by Ted Usdin
(National Institutes of Health), and the amino acid sequence was published in Nature Neuroscience in 1999.

T1P39 is an interesting peptide since it appears to be the natural ligand for the parathyroid hormone type 2
receptor (PTH2R), which is found only in various areas of the brain, pancreas, placenta, and testis. It is
noteworthy that both T1P39 genes were identified through the use of widely available search tools to query
the recently released DNA sequences from the publicly funded human and mouse genome projects. Further
analysis of the genes was performed by standard laboratory techniques. We then performed an initial
characterization of the tissue-specific expression of T1P39.

The messenger RNA encoding T1P39 is expressed in several distinct areas of the brain, as well as in testis,
where its expression seems to be synchronized to different stages of spermatogenesis (the formation of
sperm). Nothing is known so far about the physiological role and importance of the T1P39-PTH2R system.
Identification of the genetic locus of T1P39 in the human and mouse genome should greatly facilitate
further studies of this hormone-receptor system. At the moment, we can only speculate that the system is
part of a neuro-endocrine signaling network, which may also be involved in sperm maturation. It is
conceivable that distinct genetic disorders (possibly including male infertility) are caused by mutations in
T1P39 or its receptor. Because we know the chromosomal location of the genes encoding these two
proteins, our research will be able to help genetic linkage studies with widely available polymorphic
microsatellite markers.

Additional background information:

Markus R. John, MD, Maya Arai, PhD and Kenneth B. Jonsson, MD, PhD are postdoctoral fellows at the
Endocrine Unit of the Massachusetts General Hospital (MGH), Boston. Harald Jueppner, M.D. is a
physician-scientist and principal investigator at MGH. He is a leader in the field of calcium metabolism and
has more than 20 years of experience in the discovery as well as functional and genetic characterization of
hormone-receptor systems.

This research was funded by the Deutsche Forschungsgemeinschaft (John) and the Swedish Foundation for
International Cooperation in Research and Higher Education (Jonsson).




                                                  - 126 -
                                                  PTH-CALCIUM-VITAMIN D-BONE

P1-496
REGULATION OF OSTEOPROTEGERIN GENE EXPRESSION AND PROTEIN PRODUCTION
BY ANDROGENS AND ANTI-ANDROGENS IN HUMAN OSTEOBLASTIC LINEAGE CELLS
LC Hofbauer,1 KC Hicok,2 D Chen,3 S Khosla.2 1Gastroenterology and Endocrinology, Philipps University,
Marburg, Germany; 2Endocrine Research Unit, Mayo Clinic, Rochester, Minnesota; 3Amgen , Inc,
Thousand Oaks, California

Both estrogen (E) and androgens have anti-resorptive effects on bone in vitro and in vivo, although recent
evidence indicates that, at physiological circulating levels, E is the dominant sex steroid regulating bone
resorption even in men. E and androgens suppress interleukin-6 production by osteoblastic cells and have
direct inhibitory effects on osteoclasts. However, while E has been shown to stimulate osteoprotegerin
(OPG) production by osteoblastic cells, the effects of androgens on OPG production have not been defined.
In this study, we assessed the regulation of OPG by androgens in two human osteoblastic lineage cells, the
hFOB/AR-6 (an immortalized fetal mature osteoblastic cell line which has been stably transfected with the
wild-type human androgen receptor), and MSC cells (normal undifferentiated adult stromal cells capable of
differentiating towards the mature osteoblastic phenotype). 5α-dihydrotestosterone (5α-DHT) dose-
dependently decreased OPG mRNA steady state levels and protein concentrations in hFOB/AR-6 cells by
up to 50% and 60%, respectively (P < 0.001). Inhibition of OPG mRNA levels and protein production by
5α-DHT was completely abrogated by the androgen receptor antagonist, hydroxyflutamide (OHF, 10 -6 M),
indicating that these effects are directly mediated by the androgen receptor. Of note, OHF at a
concentration of 10-7 M increased OPG mRNA levels and protein secretion by two- to three-fold (P <
0.005). 5α-DHT also dose-dependently decreased OPG protein secretion by 18% to 40% in the
untransformed MSC cells (P < 0.005). Interestingly, testosterone and dehydroepiandrosterone at
concentrations of 10-7 M also decreased OPG protein secretion by MSC cells by 46% and 43%,
respectively (P < 0.001). In conclusion, we demonstrate that androgens inhibit OPG mRNA levels and
protein secretion by osteoblastic cells. Since E has been shown to stimulate OPG production, the finding
that androgens decrease OPG levels may, in part, explain why they are less potent than E in decreasing
bone resorption, even in men.

Basic Science Poster: Vitamin D-Sex Steroids-PTH-Cartilage/Bone (11:00 AM-12:00 PM and 2:30 PM-
3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                 - 127 -
                                                   PTH-CALCIUM-VITAMIN D-BONE

P1-496

Lay explanation of abstract:

The female hormone, estrogen, and the male hormone, testosterone, have important skeletal effects. Recent
evidence from studies in humans, however, indicates that even in men, estrogen is the major sex hormone
that prevents bone tissue from breaking down. Previous studies by our group and others have shown that
estrogen increases the production of a protein called osteoprotegerin, which is an important and potent
inhibitor of bone breakdown. While both estrogen and testosterone have been shown to regulate the
production of other factors that inhibit bone breakdown in a similar way, possible effects of testosterone on
the bone-cell production of osteoprotegerin have not been studied.

In this study, we examined in detail the way that testosterone regulates bone-cell production of
osteoprotegerin in the laboratory. We found, in contrast to previous studies showing that estrogen increased
osteoprotegerin production by these cells, that testosterone had the opposite effect. In fact, testosterone
decreased osteoprotegerin production by bone cells. The effects depended on the dose of testosterone used.
In addition, the effects were prevented by a compound that opposes testosterone action. These results
indicate that the effects we observed were specific and mediated by the receptor molecule for testosterone
in these cells.

These findings provide a possible mechanism for the observation that in humans, estrogen appears to be a
much stronger inhibitor of bone breakdown than testosterone. We speculate that the difference in
osteoprotegerin production by bone cells caused by estrogen versus testosterone may be a key reason for
their differing effects on the skeleton.

Analogs of testosterone that result in increases in osteoprotegerin production could be developed that may
be able to increase the effectiveness of testosterone in stopping bone breakdown. These compounds may be
more effective than testosterone in preventing and treating osteoporosis in men.

This study was funded by the National Institute on Aging, National Institutes of Health.




                                                  - 128 -
                                                    PTH-CALCIUM-VITAMIN D-BONE

P1-497

A NON-STEROIDAL, SELECTIVE ANDROGEN RECEPTOR MODULATOR (SARM) WITH
EFFICACY ON BONE IN THE ORCHIECTOMIZED ADULT RAT
B Risek,1 JN Miner,1 G Evans,3 RT Turner,3 S Bain,2 V Shen,2 W Chang,1 RW Barnett,1 WT Schrader,1
J Rosen,1 A Negro-Vilar.1 1Ligand Pharmaceuticals, San Diego, California; 2SkeleTech, Inc., Bothell,
Washington; 3Orthopedic Research, Mayo Foundation, Rochester, Minnesota

Osteoporosis in men is a disease that is increasing in incidence and poses a serious health problem with an
increasingly elderly population. One approach to the treatment of osteoporosis in men is the development
of selective androgen receptor modulators (SARMs) that can stimulate formation of new bone with
substantially diminished proliferative activity in the prostate. In the present study we have used a skeletally
mature orchiectomized (ORX) male rat as an animal model of male hypogonadism for assessing the
efficacy of LGD2226, a non-steroidal, non-aromatizable, and non-5alpha-reducible SARM. The effects of
LGD2226 were assessed on bone turnover, bone mass and bone strength, and were compared with those
effects exerted on classic androgen-dependent targets, such as ventral prostate, seminal vesicle and levator
ani muscle. A substantial loss of bone density was observed in ORX animals, and this loss was prevented
by androgens. Biochemical markers of bone turnover revealed an early increase of bone resorption in
androgen-deficient rats, that was repressed in ORX rats treated with the oral SARM, LGD2226, during the
4-month treatment period. Differences in architectural properties and bone strength were detected by
histomorphometric and mechanical analyses, demonstrating beneficial effects of LGD2226 on bone quality
in androgen-deficient rats. Histomorphometric analysis of cortical bone revealed distinct anabolic activity
of LGD2226 in periosteal bone. LGD2226 was able to prevent bone loss and maintain bone quality in ORX
rats by stimulating bone formation, while also inhibiting bone turnover. In addition to bone, LGD2226
exerted anabolic activity on the levator ani muscle. Taken together, these results suggest that orally active,
non-steroidal SARMs may be useful therapeutics for both muscle and bone in elderly hypogonadal men
through their anabolic activities. Since SARMs both prevent bone loss, and also stimulate formation of new
bone they may have significant advantages relative to currently used anti-resorptive therapies.

Basic Science Poster: Vitamin D-Sex Steroids-PTH-Cartilage/Bone (11:00 AM-12:00 PM and 2:30 PM-
3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                   - 129 -
                                                   PTH-CALCIUM-VITAMIN D-BONE

P1-497
Lay explanation of abstract:

Osteoporosis, a disorder characterized by loss of bone strength and increases in fracture rates, is becoming
more prevalent. Osteoporosis poses a serious health problem to an aging population. To help prevent
osteoporosis, androgens build both bone and muscle mass. Unlike other agents, however, androgens also
can rebuild previously damaged bone. Current androgens have several problems that limit their use,
including an associated increased risk of prostate cancer and an inability to be taken orally. These problems
prevent the widespread use of androgens among elderly men.

We have approached the treatment of hypogonadism and sexual dysfunction as well as prevention and
treatment of osteoporosis in men by developing selective androgen receptor modulators (SARMs) that can
prevent and treat osteoporosis. SARMs also have the advantage of not stimulating prostate growth as
androgens do. Our study presents data for one of the novel molecules we have discovered, LGD2226, an
advanced candidate for clinical use.

We tested LGD2226, a non-steroidal SARM, for activity on the rate of bone turnover, bone mass, and bone
strength compared to its activity on the prostate and muscle. As expected in control animals, we observed a
substantial loss of bone density in animals deprived of androgens, a condition that mimics human
hypogonadism. Administering either androgens or LGD2226 prevented this loss. Similarly, bone turnover
increased in androgen-deficient animals, and this increase was prevented in animals treated with androgen
or with the SARM. We also demonstrated that LGD2226 increased bone strength when compared with a
control. Detailed analysis of the cortical bone, the dense outer portion of the bone, revealed significant
anabolic activity of LGD2226. This means that LGD2226 could increase the formation of new bone.
Further, we showed that LGD2226 prevented bone loss and enhanced bone quality in castrated rats by
stimulating bone formation and by inhibiting bone turnover, respectively.

LGD2226 also exerted anabolic activity in muscle. These agents have shown anabolic activity in
maintaining or restoring sexual function in animal models, an effect that might carry over to humans.
Overall, these results suggest that SARMs may be useful in enhancing both muscle and bone in elderly
men. Because SARMs prevent bone loss and also stimulate formation of new bone, they may have
significant advantages over the currently used anti-resorptive therapies.

This work was entirely supported by Ligand Pharmaceuticals.




                                                  - 130 -
                                                   PTH-CALCIUM-VITAMIN D-BONE

P1-503
BRAIN INJURY INDUCES PARATHYROID HORMONE-RELATED PROTEIN EXPRESSION
IN REACTIVE ASTROCYTES
JL Funk, CR Trout, H Wei, G Stafford, S Reichlin. Department of Medicine, University of Arizona,
Tucson, Arizona

Parathyroid hormone-related protein (PTHrP), a peptide whose function in brain is poorly understood, has
recently been shown to be expressed in immature, malignant, but not in normal astrocytes. To test the
hypothesis that PTHrP is induced in the reactive astrocytes of inflamed brain and thus acts as a mediator of
CNS inflammation, PTHrP expression was examined following cortical stab wound injury in rats, a classic
model of reactive gliosis, and in cerebral cortical rat astrocytes and glial-enriched mixed brain cells
exposed in vitro to the inflammatory cytokine, TNF-α. In normal brain and at the earliest time point
examined following cortical stab wound injury (day 1), neurons were the principal site of immunoreactive
PTHrP in the cerebral cortex. Over the subsequent 3 days, specific staining for PTHrP and for GFAP, a
marker of reactive astrocytes, was induced in astrocytes at the wound edge and in perivascular astrocytes,
reaching a maximum level of expression at the last time point examined (day 4). In astrocyte-enriched
cultures, the CNS cytokine, TNF-α, induced expression of both PTHrP and IL-6. IL-6 expression was also
induced by PTHrP(1-34). The stimulatory effects of PTHrP on IL-6 expression were additive to those of
TNF-α. Astrocyte proliferation in vitro was inhibited by PTHrP(1-34) and PTHrP(1-141), raising the
possibility that PTHrP, in addition to its stimulatory effect on IL-6 production during CNS inflammation,
functions to restrain the astrocytic response to brain injury. These studies provide evidence of regulated
PTHrP expression in the CNS, suggesting that PTHrP, in a manner analogous to its regulatory functions in
several non-CNS models of inflammation, may be an important mediator of the inflammatory response in
brain.

Basic Science Poster: Vitamin D-Sex Steroids-PTH-Cartilage/Bone (11:00 AM-12:00 PM and 2:30 PM-
3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 131 -
                                                    PTH-CALCIUM-VITAMIN D-BONE

P1-503

Lay explanation of abstract:

Our brain injury experiments have focused on the role of one particular agent, parathyroid hormone-related
protein (PTHrP). PTHrP is a peptide hormone that has already been administered safely to humans for the
treatment of non-neurologic diseases. However, in petri dish experiments, other laboratories have shown
that PTHrP is also capable of preventing nerve cell death. In our experiments, we asked: is PTHrP one of
the naturally protective agents that is produced by the ―glue‖ cells in the brain in response to injury?

Because the pattern of response to brain injury in humans is very similar to that in rodents, we used a
classic animal model of brain injury to answer this question. In this model, a small stab wound is made in
the brain. Outwardly, the animals do not show any obvious neurologic defects. However, close examination
of the brain within several days of injury shows that neurons in the wound track start to die and are
surrounded by activated ―glue‖ cells that are actively producing multiple beneficial or harmful agents. In
our studies, we demonstrated that PTHrP is indeed one of the natural agents produced by these activated
―glue‖ cells at the site of brain injury. Studies performed in petri dishes using ―glue‖ cells isolated from rat
brain also revealed additional pathways by which PTHrP could prevent brain injury, including its ability to
stimulate the production of other protective agents.

These results are exciting because they provide evidence that PTHrP, a hormone already given safely to
humans, might actually be one of the body‘s own protective agents for prevention of nerve cell damage.
Clinical trials are already being conducted to examine the ability of other naturally produced agents to
prevent nerve cell death in patients. Therefore, while additional pre-clinical studies must first be conducted,
these results suggest that PTHrP might also be a considered as a candidate drug for testing in patients with
brain injury to determine its ability to prevent permanent nerve deficiencies.

Additional background information:

Brain injury caused by trauma, infection, or stroke can result in permanent neurologic defects because of
damaged nerve cells in the brain. Recent progress in brain injury research has been achieved using stem
cells to replace these injured neurons. Another approach to treatment is to attempt to intervene at an earlier
time to prevent the loss of neurons from ever occurring. This approach is similar to what the body itself
does.

In response to many different types of brain injury, a common protective response is unleashed in the brain
at the site of injury to attempt to contain the damage. This response includes the ―activation‖ of local cells
to produce hormones or other agents that prevent nerve cell death. However, two types of agents produced
by these supporting ―glue‖ or glial cells can either help to maintain the health of neighboring nerve cells or
alternately, can contribute to their demise. The amount of ―damage control‖ that the brain is able to muster
is thus the result of the counterbalancing effects of these locally produced protective or harmful agents. By
identifying beneficial agents produced by glial cells in response to injury, researchers hope to harness the
body‘s own defense system so that these same agents can be administered to patients to prevent the loss of
nerve cells, and thus, prevent permanent nerve defects.

This research was funded by the National Institutes of Health.




                                                   - 132 -
                                                    PTH-CALCIUM-VITAMIN D-BONE

P1-508
LOCALIZATION OF THE GROWTH PLATE STEM-LIKE CELLS
V Abad,1 JL Meyers,1 M Weise,1 RI Gafni,1 KM Barnes,1 O Nilsson,2 JD Bacher,3 J Baron.1 1NICHD,
National Institutes of Health, Bethesda, Maryland; 2Dept of Woman and Child Health, Karolinska Institute,
Stockholm, Sweden; 3Office of Research Services, NIH, Bethesda, Maryland

Cell kinetic studies suggest that the growth plate contains a population of stem-like cells. One candidate for
the stem-like cell is the proliferative chondrocyte located at the top of the chondrocyte columns.
Alternatively, this topmost cell may have a limited ability to divide, and exhausted columns might be
gradually replaced by proliferation and differentiation of resting zone chondrocytes. Thus, the resting zone
chondrocyte is an alternative candidate for the stem-like cell. To distinguish these possiblities, we
surgically removed the proliferative and hypertrophic zones from the rabbit distal ulnar growth plate,
leaving only the resting zone. Within one week, a complete proliferative and hypertrophic zone often
regenerated.
The resting zone contains two types of cartilage: the reserve cartilage which lies closest to the proliferative
zone and contains flattened chondrocytes sometimes grouped in pairs and triplets and the epiphyseal
cartilage which lies farther from the proliferative zone and contains solitary round chondocytes.
Regeneration of a complete growth plate occurred only when the incision was made low in the resting
zone, leaving part of the reserve cartilage in place. Complete regeneration of the growth plate did not occur
when the incision was in the epiphyseal cartilage, thus removing all of the reserve cartilage. However, in
this situation, the lower edge of the remaining epiphyseal cartilage did undergo a partial transformation.
The solitary round chondrocytes were replaced by flat chondrocytes arranged in pairs and triplets, aligned
parallel to the long axis of the bone. Thus, the lower edge of the epiphyseal cartilage appeared to transform
into reserve cartilage. Because reserve chondrocytes can transform into proliferative chondrocytes, these
findings, taken together, suggest that chondrocytes in the epiphyseal cartilage, like those in the reserve
cartilage, may also have the potential to generate proliferative columns.
Our findings suggest that resting zone cartilage contains stem-like cells that give rise to clones of
proliferative chondrocytes.

Basic Science Poster: Vitamin D-Sex Steroids-PTH-Cartilage/Bone (11:00 AM-12:00 PM and 2:30 PM-
3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                   - 133 -
                                                    PTH-CALCIUM-VITAMIN D-BONE

P1-508
Lay explanation of abstract:

The growth plate, a thin layer of cartilage found near the ends of long bones, consists of three principal
layers: the proliferative zone, the hypertrophic zone, and the resting zone. The growth plate is responsible
for bone elongation during childhood. The proliferative zones and the hypertrophic zones each play an
important role in this growth process. The proliferative zone is the site of cell replication, while the
hypertrophic zone is the site of cell enlargement. The role of the resting zone, however, is unknown.

Some researchers have suggested that the resting zone might contain stem-like cells, which can generate
new groups of proliferative and hypertrophic cartilage cells. To test this hypothesis, we surgically removed
the proliferative and hypertrophic zones from a rabbit's distal ulnar growth plate, leaving only the resting
zone. Within one week, a complete proliferative and hypertrophic zone often regenerated. Our findings
suggest that resting zone cartilage contains stem-like cells that give rise to the proliferative and
hypertrophic cells.

Additional background information:

Previous studies suggest that growth plate stem-like cells may have a finite capacity to divide and that, as
this capacity is exhausted, childhood growth (in height) slows and eventually stops. Thus, understanding
the biology of these stem-like cells may provide important insights into childhood growth and growth
disorders.




                                                   - 134 -
                       REPRODUCTION-GONADAL CONTROL (FEMALE)
OR39-3
SUPPRESSIVE SUBTRACTIVE HYBRIDIZATION REVEALS UP-REGULATION OF THE
WNT SIGNALING ANTAGONIST, SECRETED FRIZZLED RELATED PROTEIN 4, IN PCOS
THECA CELLS
JR Wood,1 LK Christenson,1 JM McAllister,2 JF Strauss, III.1 1Center for Research on Reproduction &
Women's Health, University of Pennsylvania, Philadelphia, Pennsylvania; 2Department of Cellular and
Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania

Theca cell hypertrophy and excessive androgen production are hallmarks of polycystic ovary syndrome
(PCOS). Human theca cells from PCOS ovaries maintained in long-term culture display a stable
biochemical phenotype that includes increased androgen synthesis compared to normal theca cells. To
better understand which genes are involved in this PCOS theca cell phenotype, suppressive subtractive
hybridization (SSH) was carried out using poly (A)+ RNA collected from normal and PCOS theca cells.
Dot-blot analysis of the resulting SSH cDNA products demonstrated that 57 clones were up-regulated and
104 clones were down-regulated in PCOS compared to normal theca cells. Interestingly, the Wnt signaling
antagonist, secreted frizzled related protein 4 (sFRP4), was among the up-regulated clones in PCOS cells.
The Wnt signaling pathway, which brings about changes in cell proliferation, cell differentiation and cell
fate is activated by Wnt protein interaction with cognate Frizzled (Fz) receptors resulting in activation of
the β-catenin/TCF transcription complex. Quantitative RT-PCR analysis of 3 independent PCOS and 3
independent normal theca cell cultures confirmed a 4-fold greater abundance of sFRP4 transcripts in PCOS
theca cells. Furthermore, forskolin stimulation of normal and PCOS theca cells dramatically reduced sFRP4
transcripts. Quantitative RT-PCR also demonstrated equivalent expression of Wnt-4, Wnt-5a, Wnt-13 and
Fz1 in normal and PCOS theca cells. Taken together, these data suggest: 1) that the Wnt signaling pathway
is functional in human theca cells as reflected by expression of ligand, receptor, and ligand binding
inhibitor and 2) that increased expression of the ligand binding inhibitor sFRP4 in PCOS theca cells may
disrupt the normal Wnt signaling pathway, contributing to the PCOS theca cell phenotype. (Supported by
NIH grants U54HD34449, PL1HD06274, and T32HD07305).

Basic Science Oral: Hormonal Control of Follicular Development & Health (11:00 AM-12:30 PM)

Presentation Date: Saturday, June 23, 2001; Time: 11:30 AM; Location: A 207




                                                  - 135 -
                        REPRODUCTION-GONADAL CONTROL (FEMALE)
OR39-3
Lay explanation of abstract:

Polycystic ovary syndrome (PCOS) is a disorder that affects 5% to 10% of women of reproductive age.
PCOS is characterized by excess levels of male-type sex hormones (androgens) resulting in facial hair,
acne, and infertility. Women with PCOS often have other health problems, including an increased risk for
type II (late onset) diabetes and cancer of the uterus. Although there is increasing evidence that PCOS is a
genetic disorder, the exact genetic cause of PCOS is not known. Furthermore, the group of genes involved
in infertility and type II diabetes is not well defined. In the normal ovary, specific cells called theca cells
produce a limited amount of androgens. However, the theca cells in women with PCOS produce
abnormally high levels of androgen.

Because high androgen production causes many of the clinical symptoms of PCOS, we have used a
technique called suppressive subtractive hybridization to define genes that help cause high androgen
production in PCOS theca cells. In this technique, genes that are common to both the normal and PCOS
theca cells are ―subtracted out,‖ which lets researchers identify genes that are abnormally expressed in the
PCOS theca cells. One of the genes identified by this methodology was secreted frizzled related protein 4
(sFRP4), which interferes with a cell communication system called the Wnt signaling pathway. The Wnt
signaling pathway is activated by Wnt protein interaction with cell surface receptors called Frizzled (Fz).
This action leads to activation of genes in the cell nucleus, which in turn causes changes in cell
proliferation, differentiation, and cell fate. sFRP4 binds up Wnt and prevents it from working on the cell
surface receptors, effectively breaking up the Wnt signaling pathway.

Increased expression of sFRP4 in PCOS theca cells was confirmed by analysis of independent PCOS and
normal theca cell cultures by a second method called quantitative reverse transcriptase-polymerase chain
reaction (RT-PCR), which amplifies a gene transcript so that it can be detected and measured by a
fluorescent dye. Quantitative RT-PCR analysis also demonstrated that other components of the Wnt
signaling pathway, including multiple Wnt proteins and a Fz receptor are present in both PCOS and normal
theca cells.

These studies suggest that the Wnt pathway plays a role in the function of ovarian theca cells. The studies
also suggest the possibility that pathway disruption may contribute to symptoms of PCOS. Furthermore, a
better understanding of this disrupted pathway could bring about effective treatment targets for this
common disorder.

Additional background information:

Polycystic ovary syndrome (PCOS) has long been recognized as a common endocrine problem in women
of reproductive age. It is now known that this syndrome not only affects reproductive health (i.e., fertility)
but also impacts general health. Women with PCOS have an increased risk for type II diabetes and obesity
along with related health problems, including cardiovascular disease. While the symptoms of PCOS are
well defined, its etiology remains elusive, making treatment of the complex metabolic abnormalities
difficult. The Wnt signaling pathway was originally described by developmental biologists as important for
the proper organization of cells into tissues. More recently, it has become clear that the pathway is also
important in determining the fate of a cell; that is, does the cell grow, differentiate for a specific function,
or die. Our current research suggests that this pathway may also contribute to the abnormal function of
theca cells, and possibly other cells, in PCOS.

This research has been funded by three NIH grants U54HD34449, PL1HD06274, and T32HD07305.




                                                    - 136 -
                       REPRODUCTION-GONADAL CONTROL (FEMALE)

OR39-4
LEPTIN ATTENUATES FOLLICULAR APOPTOSIS AND ACCELERATES THE ONSET OF
PUBERTY IN IMMATURE RATS
R Sasson,1 B Almog,2 K Tajima,1 M Rubinstein,4 D Barkan,4 R Homburg,2 JB Lessing,2 A Gertler,3
A Amsterdam.1 1Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel;
2
  Department of Obstetrics and Gynecology, LIS Maternity Hospital, Tel Aviv Medical Center, Tel Aviv,
Israel; 3Faculty of Agriculture, Hebrew University, Rehovot, Israel; 4Department of Molecular Genetics,
Weizmann Institute of Science, Rehovot, Israel

Human and rat granulosa cells express receptors to leptin which synergies with glucocorticoid hormones in
stimulation of ovarian steroidogenesis. To examine whether leptin affects follicular development and
maturation, we injected recombinant ovine lepti n (300ng-10mg/animal) daily to immature 21 day-old
female rats. Non-treated rats reached puberty at 44.5 ±1.6 (n=9) days. In contrast, in leptin treated animals,
puberty was reached at 34.5 ±1.6 (n=9) days. Ovarian sections r e vealed hyperthyrophy of granulosa cells
in leptin treated animals. Moreover, the number of ovulations was 2-fold higher in the treated animals
compared to controls (3-4 ovulations versus 7-8 on the first three estrous cycles, P<0.001). Leptin
dramatically reduced incidence of follicular apoptosis measured by TUNEL, and was already evident after
7 days of leptin injection (12% of apoptosis in leptin treated group compare to 52% in controls, P<0.001).
Maximal protection against apoptosis was achieved at 1-3mg leptin/animal. To reveal whether modulation
of death and survival genes is involved in leptin attenuation of follicular apoptosis, we examined the
expression of the survival gene Bcl-2 and the death gene Bax in Western blots of ovarian homogenates.
There was a pronounced elevation in Bcl-2 expression during 7-14 days of leptin injection up to 16.3-fold
(P<0.001) compared to Bcl-2 expression in controls. Bax expression was elevated only 3.4 fold (P<0.001),
leading to an increase in the Bcl-2/Bax ratio of 4.7 fold (P<0.001). Expression of the tumor suppressor gene
p53 and the oncogene Mdm2 did not change significantly. Our data suggest that leptin may be involved in
accelerating follicular maturation by attenuating follicular atresia and increasing the ratio of Bcl-2/Bax.
Whether leptin could be considered in treatment of amenorrhea and unovulation should be further explored.

Basic Science Oral: Hormonal Control of Follicular Development & Health (11:00 AM-12:30 PM)

Presentation Date: Saturday, June 23, 2001; Time: 11:45 AM; Location: A 207




                                                   - 137 -
                        REPRODUCTION-GONADAL CONTROL (FEMALE)
OR39-4
Lay explanation of abstract:

Leptin is a recently identified hormone that is involved in controlling obesity. Secreted by fat cells, leptin
was discovered to be part of the regulation of sex steroid hormone production for such hormones as
estradiol and progesterone, in mammalian ovaries. Because infertility problems are often associated with
obesity or severe leanness (e.g., anorexia nervosa), the role of leptin in controlling puberty and the estrous
cycle in female rats was investigated in detail. Using a bacterial expression system we were able to
synthesize recombinant leptin that demonstrated high potency in rodents and sheep.

Rats 21-60 days of age were injected daily with moderate doses (0.3-10.0 g/animal) of leptin. Leptin
treatment advanced puberty dramatically. Whereas non-treated rats reached puberty at an average of
44.5±1.6 days, leptin-treated rats reached puberty at 34.5±1.6 days. Moreover, the number of ovulations
(number of matured eggs released to the fallopian tube and available for fertilization) was twice as high in
the treated animals as in the control animals. Specifically, the treated animals had 7-8 ovulations compared
with 3-4 ovulations for the control animals during the first three estrous cycles.

 These unexpected findings were correlated with a pronounced decrease in ovarian cell death, which is
characteristic of normal ovarian development. We discovered that the level of expression of Bcl-2, one of
the dominant genes associated with ovarian cell death, was much higher in leptin-treated animals than in
controls. The elevation of Bcl-2 protein in the ovary may be an important factor that leads to early puberty
and to increased ovulation, because it is known to act as a survival gene. Notably, no change was measured
in the animals‘ weight after puberty in the leptin-treated animals compared to controls.

Our results suggest a functional correlation between leptin administration and the age of onset of puberty,
as well as a correlation between leptin levels and the extent of ovulation. Because human leptin is already
prescribed as a drug to individuals who are deficient in this hormone, leptin should be further investigated
for possible treatment of late puberty and failure to ovulate (anovulation).

This research was partially supported by the Levin Center for applied research at the Weizmann Institute of
Science, the Hebrew University Faculty of Agriculture, Rehovot, Israel and the Lis Maternity Hospital, Tel
Aviv Medical Center.




                                                   - 138 -
                       REPRODUCTION-GONADAL CONTROL (FEMALE)

P2-356
PREGNANCY LOSS CAUSED BY ATRAZINE IN F344 RATS: EFFECTS ON SERUM
PROGESTERONE LEVELS
MG Narotsky, DS Best, SR Bielmeier, RL Cooper. Endocrinology Branch, US Environmental Protection
Agency, Research Triangle Park, North Carolina

Previously we reported that exposure to atrazine, a widely used herbicide, disrupted ovarian cycling in rats.
We also showed that atrazine can inhibit the ovulatory surge and pulsatile release of luteinizing hormone
(LH). More recently, we demonstrated that atrazine exposure on gestation days (GD) 6-10, roughly
coinciding with the LH-dependent period of pregnancy (GD 7-10), can cause pregnancy loss (i.e., full-litter
resorption) in rats. During the LH-dependent period, LH is required to maintain pregnancy by stimulating
luteal secretion of progesterone. As part of our efforts to confirm the hypothesis that atrazine-induced
pregnancy loss is LH mediated, we evaluated atrazine's effects on gestational progesterone levels. F344 rats
were treated by gavage with 0, 100, or 200 mg atrazine/kg/d on GD 6-10 (plug day = GD 0). Blood samples
were collected from the lateral tail vein once daily on GD 6-11. Serum progesterone levels were determined
by ELISA. Dams were allowed to deliver and their litters were examined postnatally. For females that did
not deliver, pregnancy loss was confirmed by staining uterine resorption sites with 10% ammonium sulfide.
All 7 control dams successfully maintained their pregnancies whereas 8 of 10 dams at 100 mg/kg and 6 of 9
dams at 200 mg/kg had pregnancy loss. Treated dams, particularly with pregnancy loss, had significantly
reduced serum progesterone levels on GD 11. For control, 100-mg/kg, and 200-mg/kg dams with live
litters, mean (± SE) GD-11 values were 170 ± 20, 98 ± 23, and 78 ± 13 ng/ml, respectively; whereas 100-
mg/kg and 200-mg/kg dams with pregnancy loss had respective values of 36 ± 6 and 64 ± 18 ng/ml.
Pregnancy loss was associated with progressively reduced progesterone levels resulting in marked
reductions by GD 11. Whereas all control values were >95 ng/ml, increasing numbers of dams with
pregnancy loss had moderately reduced values (<95 ng/ml) on GD 9-10, and markedly reduced values (<50
ng/ml) on GD 10-11. Although a direct ovarian effect remains possible, these findings are consistent with
reduced secretion of LH, and may reflect an LH-mediated mechanism. (This abstract does not necessarily
reflect EPA policy.)

Basic Science Poster: P2: Hormonal Control of Female Reproductive Tract I (11:00 AM-12:00 PM and
2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 139 -
                        REPRODUCTION-GONADAL CONTROL (FEMALE)
P2-356
Lay explanation of abstract:

Atrazine, a widely used herbicide, causes pregnancy loss in rats when administered during a critical period
in midpregnancy. In addition, rats with pregnancy loss have markedly reduced progesterone levels.
Progesterone, secreted by the ovary, is a hormone necessary for maintaining pregnancy. During this critical
period of pregnancy in the rat, luteinizing hormone (secreted by the pituitary gland) is required to maintain
ovarian secretion of progesterone. Thus, the reduction of progesterone levels, together with the timing of
exposure during this critical period, suggest that atrazine may disrupt pregnancy maintenance by interfering
with the secretion of luteinizing hormone by the pituitary gland.

Atrazine and similar pesticides are commonly used for weed control of corn, sorghum, sugarcane, citrus,
grapes, and other crops; these pesticides constitute the largest group of herbicides sold in the United States.
In areas of heavy use, these pesticides have been detected in sources of drinking water. The disruption of
pregnancy maintenance in rats, together with the potential for human exposure, heightens our concerns
about the potential health effects of atrazine and related pesticides.

In our study, rats received atrazine on gestation days 6 through 10. Blood samples were collected from a
tail vein once daily on days 6 through 11, and progesterone levels were measured. The rats were allowed to
deliver and their litters were examined. All seven control animals successfully maintained their pregnancies
whereas 14 of 19 atrazine-exposed animals had pregnancy loss. Animals with pregnancy loss had
progressively lower progesterone levels, which resulted in marked reductions by day 11. Although a direct
effect of atrazine on the ovary remains possible, these findings are consistent with reduced secretion of
luteinizing hormone and suggest an effect on the neuroendocrine control of the pituitary gland. Such a
mechanism of atrazine-induced pregnancy loss in the rat is not analogous to miscarriage in humans. It is
relevant, however, to mechanisms of human infertility. [This abstract does not necessarily reflect EPA
policy.]

Additional background information:

Atrazine and similar herbicides are commonly used for weed control of a variety of crops. These pesticides
have been demonstrated to cause early onset of mammary tumors in rats following long-term dietary
exposure. Research findings show that this effect is probably not because of a direct effect on the mammary
gland; instead, the effect is likely due to altered control of the secretion of luteinizing hormone and
prolactin, which in turn causes altered pituitary-ovarian function, which leads to an endocrine environment
that is conducive to mammary tumor growth.

We have previously reported that atrazine causes pregnancy loss in rats (Narotsky et al., 2001, Strain
comparisons of atrazine-induced pregnancy loss in the rat. Reproductive Toxicology 15:61-69).

Luteinizing hormone is needed for normal reproductive function in the male and for ovulation in females.
In rats, it is required during midgestation to maintain pregnancy. In humans, however, luteinizing hormone
is needed to maintain early pregnancy, before implantation of the embryo. The proposed mechanism of
atrazine-induced pregnancy loss in the rat is not analogous to miscarriage in humans but is relevant to
mechanisms of human infertility.

This research was funded by the U.S. Environmental Protection Agency.




                                                   - 140 -
                       REPRODUCTION-GONADAL CONTROL (FEMALE)

P2-360
ASSOCIATION OF ESTROGEN RECEPTOR-α GENE POLYMORPHISM WITH BONE
MINERAL DENSITY AND SYMPTOMS AT MENOPAUSE
EL Perez-Luque,1 LE Nava,1 F Sanchez-Marin,2 ME Fajardo,1 JM Malacara.1 1Inst Invest Medicas, U
Guanajuato, Leon, Mexico; 2Centr Invest Optica, Leon, Mexico

We selected 101 healthy non-related menopause women (mean age 52.4±0.5 years). We collected
anthropometric measurement weight and height to calculate body mass index (BMI), and menopause
symptoms: depression, sleep alterations, anxiety, hot flashes, empty nest syndrome, dispareunia and vaginal
dryness. FSH, LH, estradiol and estrone were measured in fasting blood samples. DNA was isolated from
peripheral blood leukocytes using Genomic DNA purification kit (Promega). To analyze the Pvu II and
Xba I RFPL in intron 1 of the estrogen receptor-alpha gene (ER-a), approximately 1.3 Kb fragments were
amplified by PCR using the oligonucleotide primers and PCR reaction program previously described. The
products of PCR were digested by the Pvu II and Xba I restriction endonuclease and separated on a 1%
agarose gel. PP and XX meaning the absence of restriction sites and pp or xx meaning the presence of sites
for both alleles. Bone mineral density (BMD) was measured by digital radiograph of the calcaneous bone
and digital x-ray sensor using a aluminum image for computerized correction of x-ray system. Mean values
BMI was 29.6± 5.2 Kg/m2 for FSH 26.7 (1.9-282) mIU/ml, for LH 29.5 (2.4-69) mIU/ml, for estradiol
14.3 (0.1-375) pg/ml and for estrone 7 (0.1-152.6) pg/ml. Genotype frequencies (according to Hardy-
Weinberg) for Pvu II were: PP= 0.10; Pp= 0.40; pp=0.50; for Xba I, XX=0.11; Xx=0.50; xx=0.39. No
differences between FSH, LH, estradiol, and estrone plasma levels were found among the Pvu II and Xba I
genotypes. Pvu II genotypes not found associated with BMD neither with symptoms at menopause.
However, significant associations were found among genotype XX with dispareunia (p = 0.012), vaginal
dryness (p = 0.011), and BMD (p = 0.003 when controlled for age and BMI). Combination of genotypes
Pvu II and Xba I, ppXX was found associated with higher BMD than other combinations (p = 0.004). BMD
correlated positively with BMI (p <0.001) and negatively with age (p<0.001). Conclusions: XX allele was
associated with dispareunia, vaginal dryness, and high BMD. The ppXX combined genotype was also
found associated with high BMD.(Supported by a CONACYT grant 458100-5-35276-M).

Basic Science Poster: P2: Hormonal Control of Female Reproductive Tract I (11:00 AM-12:00 PM and
2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                 - 141 -
                       REPRODUCTION-GONADAL CONTROL (FEMALE)
P2-360
Lay explanation of abstract:

At menopause, symptoms that result from lack of estrogen may not correlate well with the estrogen level
found in a woman's bloodstream. Therefore, we suspect that women have different hormone sensitivities.
Our study looked at polymorphism among estrogen receptors, which means that the receptors can have
different molecular forms. Each cell recognizes the hormone that influences it by means of a receptor.

We studied 101 healthy menopausal women to collect data about age, obesity, depression, symptoms of
sleep alterations, anxiety, hot flashes, empty nest syndrome, vaginal dryness, and painful intercourse. A
fasting blood sample was taken to measure the main hormones related to estrogen exposure: LH, FSH,
estradiol, and estrone. We isolated DNA from peripheral leukocytes. To analyze the estrogen-receptor
gene, a fragment was amplified, and the product was analyzed. Bone mineral density was measured using a
digital radiograph of the heel (calcaneous) bone. Higher bone mineral density was found in obese
individuals, and we suspect genetic factors may be associated with bone mineral density and with
symptoms of estrogen deficiency. Therefore, other factors may be considered when physicians recommend
hormone treatment for menopausal women.

We found that some types of estrogen receptors result in more frequent symptoms of menopause. The XX
allele is associated with vaginal dryness and discomfort during sexual activity. The combined ppXX
genotype was associated with higher bone mineral density. Those estrogen receptor genotypes, however,
were not associated with different LH, FSH, estrone, or estradiol serum levels. These finds are interesting
because they may provide an explanation for the different risks of women at menopause, even though they
apparently have a similar level of estrogen. The risks are related to clinical problems such as bone loss,
cardiovascular disease, emotional symptoms, and breast cancer. Further studies will let researchers identify
women with decreased estrogen sensitivity. Thus, hormone replacement therapy may become more
appropriate, because we will be able to identify woman who most need it.


This research was supported by CONACTY grant 458100-5-35276-M.




                                                  - 142 -
                       REPRODUCTION-GONADAL CONTROL (FEMALE)

P2-377
SUPPRESSION OF OOCYTE APOPTOSIS BY SPHINGOSINE-1-PHOSPHATE THERAPY IN
VIVO PRESERVES FERTILITY FOLLOWING RADIOTHERAPY
F Paris,2 GI Perez,1 RN Kolesnick,2 JL Tilly.1 1Vincent Center for Reproductive Biology, Massachusetts
General Hospital/Harvard Medical School, Boston, Massachusetts; 2Laboratory of Signal Transduction,
Memorial Sloan-Kettering Cancer Center, New York, New York

Premature ovarian failure and infertility are unfortunate side effects routinely observed in young girls and
reproductive age women treated for cancer. Using mice as a model, we have reported that oocytes undergo
apoptotic cell death as a pathological response to anticancer therapies (Nature Medicine 1997 3:1228).
Moreover, this oocyte death can be prevented, in vivo, by pretreating the mice with sphingosine-1-
phosphate (S1P), a ceramide antagonist, prior to initiating anticancer therapy (Nature Medicine 2000
6:1109). Herein we tested if oocytes protected in vivo remain competent for fertilization and
embryogenesis by monitoring pregnancy and live birth rates in irradiated female mice, without or with S1P
pretreatment. Young adult female mice (C57BL/6) were given intrabursal injections of vehicle (n=8) or a
maximally effective dose (0.2 mM final in the bursal cavity) of S1P (n=8) two hours before exposure to 0.1
Gy of ionizing radiation. Under these conditions, >80% of the immature oocytes are subsequently lost in
vehicle-pretreated mice, whereas no oocyte loss is observed in S1P-pretreated mice (Nature Medicine 2000
6:1109). Two months following irradiation, the mice were individually caged with young adult male mice,
and pregnancy and live birth rates were monitored in four separate mating trials. In vehicle-treated mice,
pregnancy and live birth rates were low, with a mean value of only 38% (SEM of 8%). By comparison,
pregnancy and live birth rates in mice protected by S1P were significantly (P<0.001) higher, with a mean
value of 88% (SEM of 4%), comparable to those of non-irradiated females. Importantly, S1P-protected
females gave birth to normal size litters, F1-generation neonates matured into adults without any obvious
abnormalities, and mating of these F1 animals yielded normal litters of F2-generation mice. We conclude
that in vivo preservation of ovarian function and fertility in females following cancer therapy can be
achieved using S1P-based therapies. (Supported by NIH R01-AG12279, R01-ES08430, R01-CA423852,
and Vincent Memorial Research Funds).

Basic Science Poster: P2: Hormonal Control of Female Reproductive Tract II (11:00 AM-12:00 PM and
2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 143 -
                       REPRODUCTION-GONADAL CONTROL (FEMALE)
P2-377
Lay explanation of abstract:

The quality of a cancer survivor‘s life after therapy has become a critical issue, in part because of the
extended years of life that recent treatments provide. One of the most worrisome side effects of
conventional cancer treatments is damage to the gonads. This problem is compounded in females because
the ovaries, unlike the testes, are incapable of germ cell renewal. As a consequence, the inappropriate
destruction of female germ cells (oocytes or ―eggs‖) after exposure to chemotherapeutic drugs and
radiation is irreparable, often leading to premature menopause and infertility. Considering recent estimates
that 1 in 52 human females between birth and age 39 (i.e., the pre-reproductive and reproductive years) will
be diagnosed with and presumably treated for cancer, new strategies to minimize or prevent gonadal
damage during treatment would have a profound impact.

We recently published a study in Nature Medicine (October 2000, volume 6, pages 1109-1114). We
demonstrated that pretreatment of young adult female mice with a naturally occurring lipid (referred to as
sphingosine-1-phosphate or S1P) before irradiation completely protected the ovarian germ cell population
from this pathological insult. By comparison, female mice pretreated with a placebo showed massive egg
depletion, a characteristic ovarian response to radiotherapy.

As an important follow-up to this work, the data presented in this abstract describe results from mating
trials performed with female mice, pretreated with placebo or S1P, following radiotherapy. As anticipated,
pregnancy and live birth rates in young adult female mice treated with placebo before radiotherapy were
reduced to less than 50% of normal rates, while S1P-protected females became pregnant and gave birth at
rates comparable to non-irradiated females (almost 90%).

Importantly, the offspring conceived from the ―protected‖ eggs develop into adults without discernible
defects and have normal fertility rates. These data collectively suggest that S1P therapy represents a
promising new technology that could help preserve ovarian function and fertility in female cancer patients.

This research was funded by the Vincent Memorial Research Funds and the National Institutes of Health.




                                                  - 144 -
                        REPRODUCTION-GONADAL CONTROL (FEMALE)

P2-389
THE ROLE OF LEPTIN IN FASTING AND OVARIAN STEROIDOGENEIS IN THE DOMESTIC
HEN
H Paczoska-Eliasiewicz,1 J Rzasa,1 M Proszkowiec,1 A Hrabia,1 A Sechman,1 A Gertler.2 1Dept. Animal
Physiology, University of Agriculture, Krakow, Poland; 2Institute of Biochemistry, Food Science and
Nutrition, The Hebrew University, Rehovot, Israel

To evaluate whether leptin affects fasting-related events in laying hens, 4 treatments were conducted: (1)
fed ad lib; (2) fed ad lib + leptin (3) fasted and (4) fasted + leptin. Animals were fasted for five days and
given two IP daily leptin injections (250 ug/kg bw). Fasting resulted in an arrest of egg lay, decrease in the
ovary weight and lowered plasma levels of LH, GH, P4, E2, TG, FFA and PL. The T4, rT3, total and HDL
cholesterol were increased, whereas FSH, T3 and glucose were not changed. Leptin treatment caused
further decrease in P4, TG, FFA and PL but attenuated the decrease in ovary weight, E2 and slowed down
the arrest of egg lay. Other parameters were not changed. In fed hens leptin decreased T4, FFA and PL,
increased HDL cholesterol and had no other effects. This results clearly show that in chickens leptin is
involved in the adaptation to starving. To better understand regulation of ovarian steroidogenesis the effect
of leptin (alone or with LH, GH or IGF-I) was tested in vitro in a culture of intact non-hierarchical follicles
(1-4, 4-6 and 6-8 mm) and in granulosa (F1G, F2G and F3G) and theca (F1T, F2T, F3T) layers of the three
largest preovulatory follicles. The culture was continued for 24 h in absence or presence of 10 ng leptin/ml.
Secretion of E2 was determined in small follicles and in theca layers while P4 was tested in granulosa cells.
Leptin alone had no effect in all three follicle types, whereas LH, GH and IGF-I increased E2 secretion.
This increase was further enhanced by leptin in LH and GH treatments. Leptin attenuated the IGF-I effect
in medium and large but not in small follicles. In F1G leptin decreased and LH increased the P4 secretion
and LH effect was attenuated by leptin. GH and IGF-I had no effect at all. No hormonal effects were noted
in F2G and F3G. GH and IGF-I increased the E2 secretion in F1T, F2T and F3T and LH in F3T only.
Leptin alone had no effect but attenuated the LH effect in F3T and IGF-I effects in F2T and F2T, whereas
the GH effect was not changed. Thus, leptin effect on streoidogenesis is dependent upon hormonal
combinations and developmental stage of the follicles.

Basic Science Poster: Hormonal Control of Female Reproductive Tract II (11:00 AM-12:00 PM and 2:30
PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                   - 145 -
                        REPRODUCTION-GONADAL CONTROL (FEMALE)
P2-389
Lay explanation of abstract:

Leptin, the adipose-derived hormone discovered in 1994 by Zang et al., acts in mammals as a signal
between peripheral lipid stores and the central nervous system. Plasma leptin levels correlate with fat stores
and respond to changes in energy balance. Increasing evidence shows that leptin plays a key role in
regulating food intake, energy expenditure, and whole-body energy homeostasis. Some have proposed that
leptin serves a primary role as an anti-obesity hormone, and that this role commonly is thwarted by leptin
resistance. It seems likely that leptin has evolved for a dominant functionacting as a mediator for
adaptation to starvation. Compelling evidence also indicates that leptin plays role in the control of
neuroendocrine function and is critical for normal reproduction. The decrease in leptin levels during fasting
as well as reduced levels of LH and gonadal steroids serves as an important component of the impaired
fertility that accompanies this state.

Chicken leptin was cloned by Taouis et al. in 1998 and recombinant chicken leptin was prepared in our lab.
The decrease in food intake after intracerebroventricular injection of leptin in broiler and layer chickens
suggests that in birds, leptin plays a similar role in regulating energy balance as it does in mammals. Its
importance for reproduction in chickens is unknown. In layer chickens, egg production declines with age
and is followed by natural molting, which improves egg production because the hen rejuvenates. During
molting, the reproductive organs regress and egg laying stops. In poultry husbandry, artificially induced
molting has been developed as a way to increase egg production efficiency and lengthen the total egg-
laying period of the hen in an economic way. Cessation of egg laying can be induced by several methods
but the most common method has been, and continues to be, fasting. Answering the question of whether
leptin affects events occurring in the chicken ovary during fasting is therefore of great practical importance.

In our study, the injections of recombinant leptin to fasting hens: 1) reduced regression of the ovary, 2)
delayed cessation of egg laying 3) influenced progesterone and estradiol plasma levels, 4) changed the
thyroid activity, and 5) affected lipid metabolism. Our results clearly show that in chickens, leptin serves as
a signal for adaptation to starvation. Use of recombinant chicken leptin has a potential to serve as a mean to
regulate physiological events related to egg laying.

This research was funded by the Commission for Scientific Research, Poland (for Dr. Paczoska),
and the Ministry of Agriculture, Poultry Science Division, Israel (for Prof. Gertler).




                                                   - 146 -
                           REPRODUCTION-GONADAL CONTROL (MALE)
P2-498
DEMONSTRATION OF PIASX-ALPHA EXPRESSION IN TESTES OF MEN WITH NORMAL
SPERMATOGENESIS AND ITS CHARACTERIZATION IN RODENTS
PN Schlegel,1,2 A Mielnik,2 PT Chan,1 PL Morris.2,3 1Urology, Weill Medical College of Cornell
University, New York, New York; 2Center for Biomedical Research, Population Council, NY, New York;
3
  The Rockefeller University, NY, New York

Limited data are available on gene expression during human spermatogenesis. In order to elucidate human
genes associated with spermatogenesis, differential display PCR (ddPCR) was applied to mRNA extracted
from testes of men with normal spermatogenesis and compared to tissues with Sertoli cell-only pattern.
Several transcripts were identified and confirmed as being expressed at high levels in tissues with normal
spermatogenesis but were absent in testes with Sertoli cell-only pattern. Full length cDNA cloning
demonstrated that one of these transcripts was PIASxa, a protein inhibitor of the JAK-STAT signalling
pathway. Northern blot analysis of mRNA isolated from multiple human organs demonstrated that PIASxa
expression is limited to the testis. Using a cDNA probe with high homology between rodent and human
sequences, analysis of cell-specific PIASxa expression was performed. Expression of PIASxa was
evaluated in rodent germ cells purified by centrifugal elutriation and compared with developmental studies
using mouse and rat testes. High levels of PIASxa expression were seen in spermatogonia, pachytene
spermatocytes, and round spermatids. No expression was seen in isolated Leydig or Sertoli cells. Increased
levels of PIASxa mRNA were concordant with spermatocytic development in day 20-30 mouse testes.
Together these data show that PIASxa is expressed in a testis-specific manner in humans and rodents with
expression restricted to spermatogonia, pachytene spermatocyte and round spermatids. These studies have
identified expression of PIASxa transcripts in human testes with normal spermatogenesis, with no
expression of PIASxa detectable in the testes of men with Sertoli cell-only. Ongoing studies are evaluating
the role of PIASxa in human spermatogenesis and infertility.

Basic Science Poster: Reproduction-Gonadal Control (Male) (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 147 -
                            REPRODUCTION-GONADAL CONTROL (MALE)
P2-498
Lay explanation of abstract:

Infertility affects about 10-15% of all married couples, and of those affected, 30-50% have a male factor
contributing to the condition. However, limited knowledge exists about the clinical causes of male
infertility. Although many cases of male infertility are thought to be related to genetic problems, little
biomedical investigation has focused on the genetic basis of human sperm development and production
(spermatogenesis). While studies in rodent models can provide some insight into human spermatogenesis,
not all observations in rodents can be translated to human conditions.

To better understand the genetic basis of male fertility, we applied new molecular research techniques to
the study of material biopsied from the testes of fertile men compared with those from men with different
infertility characteristics. Our research showed that human testes without germ cells (sperm precursors)
failed to express the PIASx-alpha (PIASx) gene, which codes for a factor unique to the testis. Although
the role of human PIASx is unknown, our studies characterized its deficiency in certain types of male
infertility. Based on our data, an important function for this newly identified PIASx factor during human
spermatogenesis is proposed. Understanding the role of PIASx may eventually lead to new treatment
strategies for male infertility.

Additional background information:

Clinical biopsy sample materials were obtained with appropriate informed consent. Our research identified
the absence of a unique testis gene called PIASx in patients with particular types of male infertility. Our
findings indicate that PIASx
Further studies in rodents also were performed, and these confirmed that PIASx is found only in
developing sperm and their precursors. As this protein has the molecular characteristics of a growth
inhibitor, studies will next evaluate its role in regulating sperm production and male fertility.

This research was funded in part by NIH / NICHD R01 HD29428 to PLM. Support for this study also was
provided by the Department of Urology, Weill Medical College to PNS.




                                                  - 148 -
                            REPRODUCTION-GONADAL CONTROL (MALE)

P2-502
TARGETED ANDROGEN RECEPTOR MUTATIONS IN MICE TO PROBE FUNCTIONS IN
MALE DEVELOPMENT
A Scheller,1 M Brogley,1 G Shetty,2 ML Meistrich,2 DM Robins.1 1Human Genetics, University of
Michigan Medical School, Ann Arbor, Michigan; 2Experimental Radiation Oncology, University of Texas
M. D. Anderson Cancer Center, Houston, Texas

Androgen receptor (AR) activated by bound testosterone promotes male differentiation during
embryogenesis, survival of male organs via antiapoptosis, and full virility at puberty. The AR roles in these
processes were addressed by exchanging the transactivation domains in the amino terminus unique to AR
with those from the related glucocorticoid receptor (GR) by homologous recombination. This mutant
receptor retains the AR DNA binding and ligand binding domains. Such a receptor (GAA) in transfection
activates some steroid-responsive promoters, but not those activated solely by androgens. In part, this is
because the AR amino terminus is known to interact directly with the rest of the receptor, and this feature is
required for many AR-specific functions. To examine if these observations in vitro are relevant and as
significant in the intact animal, targeted recombination on the single X chromosome in ES cells replaced
the AR first exon coding residues with the analogous region of GR. Chimeric mice resulting from the
fusion of these ES cells with blastocysts were not fully virilized: only 1 of 34 "males" was fertile. Chimeras
5-9 months old with mid to high ES cell contribution had smaller testes. The much less extensive
seminiferous tubules harbored fewer germ cells, most of which did not pass the spermatocyte stage.
Hyperplasias, apparently Leydig in orgin, filled in the peritubular spaces. Epididymal tubules were sparse
but enlarged with PAS+ filled lumina. When present, other glandular structures of the male urogenital tract
were small and irregular in shape. AR-directed functions in normal urogenital cells evidently failed to fully
maintain or supplant the mutant ES cells. This points to bidirectional intercellular communications in this
organ system. The failure to support complete spermatogenesis, and the expression level of GAA as well as
male-specific genes, currently are being examined. Future receptor mutants will include alterations
expected to be less severe, and will be used to detail amino terminal domains of AR responsible for subsets
of gene expression in male development.

Basic Science Poster: Reproduction-Gonadal Control (Male) (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                   - 149 -
                            REPRODUCTION-GONADAL CONTROL (MALE)
P2-502
Lay explanation of abstract:

In animals constructed by combining embryonic stem cells mutated at the androgen receptor gene with
cells from a normal embryo, the male sexual organs are smaller. Overall male fertility is largely lost. This
implies that adjacent cells talk to each other, becoming mutually dependent for long-term survival and
compensating poorly for losses of neighboring cells. This confirms similar fusions performed previously
between mutant mouse embryos or between cell types comprising certain tissues of mutant origin.
However, here we extend those methods by using embryonic stem cells that accept desired mutations
targeted to any gene of interest. These cells subsequently can contribute to any part or whole of an embryo.

We are studying steroid hormone receptors as we address the basic research questions of why steroids have
distinct effects on the body. To do this, we exchanged different parts from two representative steroid
receptors and currently are examining how these constructs behave in mice when normal protein effectively
is replaced with mutant protein. Because this work remains in progress, we are examining the effects on
male sexual development, as well as maintenance of male body parts and male hormonal pathways. There
are future implications from this study for male diseases arising under the influence of steroid hormones,
such as prostate cancer.

Steroid hormones govern diverse processes set up during development that are maintained throughout
adulthood. Male/female sexual structures and function, changes in females during pregnancy,
concentration of salts and metabolites in body fluids plus rapid stress responses all include the effects of
androgens/estrogens, progestins, or cortisols, respectively. The steroids are small, closely related
molecules with such slight differences in structure that any information that influences these bodily
processes must be conveyed through nuclear steroid receptor proteins. Steroid receptors binding the
steroids then bind to chromosomal DNA sequences to help increase or decrease the accumulation of RNA,
which in turn changes the levels of proteins and affects the roles for each of the steroids. The steroid
receptors have closely related structures in their tails, which includes the DNA binding domains and steroid
binding domains, but are completely different in their large head areas, which harbor distinct RNA
transcription activation domains. We have exchanged the heads between androgen receptor (AR) and
glucocorticoid receptor (GR) to see what parts of AR are required for the androgen-specific steroid
response.

A GR-head/AR-tail hybrid receptor was chosen as a targeted mutation in the androgen receptor gene on the
mouse X chromosome in embryonic stem cells. On a test DNA sequence activated by AR but not GR, the
prototype for this hybrid acted the same as GR, evidently because the AR tail interacts with parts of the AR
head, and will shut down a steroid receptor unless these head parts are present. When male ES cells with
only the GR-AR receptor were mixed with normal cells and combined into an embryo, the resulting mice
were mostly intersexed (a combination of the sexes), but with a male bias. Many male organs from the
male urogenital tract were reduced in size or missing, giving an indication of how sensitive to androgens
the tissues are and/or how much intercellular communication is occurring.

This research was supported by the NIH, with supplemental funding from the University of Michigan
Comprehensive Cancer Center and the Michigan Diabetes Research Training Consortium.




                                                  - 150 -
                                                          STEROID HORMONE ACTION
OR18-4
A NEW CLASS OF GLUCOCORTICOID RECEPTOR-DEPENDENT NON-STEROIDAL ANTI-
INFLAMMATORY AGENTS
JN Miner,1 J Hu,1 M Elgort,1 P Jacobsen,2 B Lane,2 G Carter,2 M Coghlan,2 M Nakane,2 C Lin,2 J Rosen,1
E Berger,1 S Tian,1 M Iskandar,1 C Tyree.1 1Ligand Pharmaceuticals, San Diego, California; 2Abbott
Laboratories, Abbott Park, Illinois

Glucocorticoids are widely used as powerful anti-inflammatory drugs, but their use is ultimately limited by
severe side effects. We and others have hypothesized that the strong anti-inflammatory activity of
glucocorticoids is associated with transcriptional repression of a large number of inflammatory cytokines,
while the side effects are associated with transcriptional activation of genes in basic metabolic pathways.
We have characterized a novel class of non-steroidal, anti-inflammatory glucocorticoid receptor ligands
termed selective glucocorticoid receptor modulators (SGRMs). We show that these compounds are capable
of steroid-like transcriptional repression of inflammatory genes but fail to fully activate gene expression.
To understand this activity, we have used both biochemical and cellular cofactor interaction assays to
compare SGRMs with steroidal glucocorticoids. We have demonstrated that only a subset of specific co-
regulators that bind in response to steroidal ligands can associate in the presence of these compounds. This
suggests that upon binding, these SGRMs change receptor structure in a manner different from steroidal
compounds. These selective cofactor interactions lead to unique tissue selectivity profiles and may provide
significant therapeutic benefit over steroidal glucocorticoid receptor ligands in the treatment of a wide
variety of inflammatory diseases.

Basic Science Oral: Steroid Hormone Action (1:00 PM-2:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 1:45 PM; Location: A 109




                                                  - 151 -
                                                          STEROID HORMONE ACTION
OR18-4
Lay explanation of abstract:

Corticosteroids (steroids) such as prednisone and dexamethasone provide excellent therapeutic benefits in
many inflammatory diseases. Unfortunately, currently used steroids cause several serious side effects that
ultimately limit their use. For example, corticosteroid use leads to an increased risk for osteoporosis and
fractures, hyperglycemia, hypertension, stunted growth in children, and several other major side effects.
Discovering new compounds that maintain the anti-inflammatory activity of steroids with minimal side
effects could yield significantly improved medications.

Steroids take effect through the glucocorticoid receptor (GR), a member of a family known as the nuclear
receptors. The GR becomes activated when it binds to a steroid or other ligand, which causes the GR to
regulate the transcription of many genes. Some of these genes are related to the anti-inflammatory effect of
activated GR, while others are associated with side effects. In order to separate these two activities, we
explored differences between the mechanism that governs regulation of genes involved in the anti-
inflammatory effect and the mechanism governing regulation of genes that cause some of the side effects.

We established tests that could determine whether one or both sets of genes are regulated. All tested
steroids activated the GR to regulate both sets of genes. We were able, however, to identify non-steroidal
compounds that bound to the GR and regulated the genes involved in inflammation, but that had little effect
on the genes involved in side effects. Additional in vitro studies confirmed this separation of activities.
Furthermore, in vivo studies presented in this and an accompanying report by Jacobean et al. demonstrate
the in vivo efficacy of this non-steroidal class of compound. This class of compound also showed reduced
effects on glucose homeostasis that could lead to diabetes after long-term steroid treatment. Thus, we feel
that we made an important contribution by identifying compounds with the efficacy of steroids but with
fewer side effects.

We then investigated the mechanism of action for these compounds. Recently, a variety of proteins
(cofactors) have been shown to bind to nuclear receptors. Cofactors involved in repressing the activity of a
receptor (corepressors) tend to be bound in the absence of ligand, while cofactors that are required for
transcriptional activation (coactivators) associate with the receptor after ligand is bound. We showed that a
distinct set of cofactors are bound to the receptor in the presence of steroids, but only a subset of those
cofactors are associated in the presence of our non-steroidal compounds. We hope that these differences
can be used to further refine these compounds, as well as to discover new compounds with improved
activity.

This research was supported by Ligand Pharmaceuticals and Abbott Laboratories.




                                                  - 152 -
                                                          STEROID HORMONE ACTION
OR31-6
THE ROLE OF THE C-TERMINAL EXTENSION FROM THE STEROID RECEPTOR DNA
BINDING DOMAIN IN DNA BINDING AFFINITY AND BINDING SITE PREFERENCE
VM Senkus Melvin,1 DP Edwards.1,2 1Molecular Biology Program, Univ. of Colo. HSC, Denver, Colorado;
2
  Pathology Dept., Univ. of Colo. HSC, Denver, Colorado

The DNA binding domains (DBD) of nuclear hormone receptors contain a conserved zinc finger core that
recognizes the major groove of hormone response elements (HREs). The DBDs of RXR heterodimer and
orphan receptors contain an additional C-terminal extension (CTE) that is structured (α-helix and extended
loop GRIP box, respectively) and makes minor groove DNA contacts important for binding affinity and
recognition of extended half-HREs. Available data suggests the steroid receptor DBDs lack a similar CTE.
However, using purified, recombinant steroid receptor DBDs that contain equivalent CTE regions, we
observed a substantially higher binding affinity (100-fold) of the estrogen receptor (ER) α and ERβ DBDs
for estrogen response elements (EREs) than the PR DBD for progesterone respone elements (PREs).
Additionally, the ERαDBD has a 3 fold higher affinity than ERβ DBD for full and half-site EREs.
Sequence alignments indicate that the ERα DBD contains an orphan receptor-like GRIP box in the CTE
region suggesting that ERα has a functional CTE accounting for the higher DNA binding affinity.
Swapping the CTE region between the two ER subtypes switched the DNA binding affinity difference
between the ER α and β DBDs for both full and half-EREs. Truncation of the CTEs substantially reduced
DNA binding affinity of ERα, ERβ, and PR DBDs for full-HREs. Truncation of the PR DBD also resulted
in reduction in binding affinity to half-PREs, while in contrast the truncated ER DBDs failed to bind half-
EREs entirely. We have previously shown that the chromatin, high mobility group-1/-2 proteins (HMG-1/-
2) enhance DNA binding affinity of ER and PR DBDs for their respective HREs. The PR DBD lacking a
CTE continued to respond to addition of HMG-1/-2 with enhanced DNA binding, while the ER DBDs
lacking a CTE did not. These results implicate participation of the steroid receptor CTE in DNA binding
affinity, binding site selection and interaction with HMG-1/-2. Additionally, the CTE does not appear to be
functionally equivalent among steroid receptors since it is critical for ER, but not PR DBDs to interact with
half-HREs and HMG-1/-2.

Basic Science Oral: Functional Analysis of Steroid Hormone Receptors (1:00 PM-2:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 2:15 PM; Location: A 207




                                                  - 153 -
                                                          STEROID HORMONE ACTION
OR31-6
Lay explanation of abstract:

Estrogen regulates the growth and maintenance of female reproductive tissues such as the breast and uterus,
by altering the genes expressed in these tissues. Normally, estrogen binds to a cellular protein called the
estrogen receptor (ER). This receptor becomes ―activated‖ when it is bound to estrogen and binds to
genomic (gene-containing) DNA to affect when and where a specific gene is expressed. Misregulation of
gene expression through this hormonal signal can result in unregulated growth, even cancer, because of the
important role estrogen and the estrogen receptor play in reproductive tissue growth.

Until recently, we believed that estrogen bound only one hormone receptor, ER, but a new estrogen
receptor has been identified, ER. ER binds to the estrogen hormone in a manner similar to ER. Many
research groups are interested in comparing the gene regulation by ER and ER. These studies may lead
to a better understanding of the role of estrogen in both normal and cancerous reproductive tissues.

Hormone receptors can be structurally divided into distinct ―domains.‖ One part of the receptor, the
hormone-binding domain, binds the hormone. The DNA- binding domain (DBD) binds to DNA. Although
these domains work together in the full-length receptors to regulate genes, they can be dissected to analyze
their different functions independently. We are currently comparing the DNA-binding characteristics of
ER and ER using only the DNA-binding domain (DBD) fragments to understand DNA binding strength
and specificity.

Our studies show that ER DBD binds to target DNA elements more strongly than ER DBD does. This
difference observed in the test tube can be magnified many times in the complex environment of the cell or
tissue, which could result in very different estrogen effects through these two ERs. We have further
dissected this difference in DNA-binding strength to a small subdomain of the DBD termed the CTE. By
molecularly ―swapping‖ this subdomain between ER and ER, we can change the DNA-binding strength.
This CTE domain also is essential for ERs binding to DNA sequences often found in genes that respond to
estrogen. Therefore, the CTE appears to play an important role in how the ERs recognize DNA. Our
molecular mapping of the DNA binding function could help to explain some functional differences
between these two forms of estrogen receptor, because DNA binding and direct gene regulation is tightly
linked for estrogen receptor function.

This research was funded by the National Institutes of Health.




                                                  - 154 -
                                                          STEROID HORMONE ACTION
P2-30
EFFECT OF NORMAL POLYMORPHIC GLUTAMINE REPEAT LENGTH IN THE HUMAN
ANDROGEN RECEPTOR (AR) ON TRANSCRIPTIONAL ACTIVITY AND AR PROTEIN
LEVEL
D Ding, L Xu, M Menon, GP Reddy, ER Barrack. Urology, Henry Ford Hospital & Health Sciences
Center, Detroit, Michigan

The human AR gene contains CAG and GGC repeats that are polymorphic in length, and encode glutamine
(Q) and glycine repeats, respectively, in the N-terminal transactivation domain of the AR. The goal of the
present study was to determine whether Q repeat lengths in the normal range affect AR function. We
constructed expression vectors of human AR with 14-25 CAGs and 16 GGCs (the most common GGC
repeat length). DU145 human prostate cancer cells, which do not express endogenous AR, were
cotransfected with AR plasmid and an androgen-responsive reporter plasmid (firefly luciferase under
control of the PSA gene promoter), in the presence of 10 nM R1881, an AR agonist. Cells were also
cotransfected with a Renilla luciferase expression plasmid to monitor transfection efficiency. Firefly and
Renilla luciferase expression was measured enzymatically, and AR protein was quantitated by immunoblot
analysis. AR transcriptional activity was expressed as units of firefly luciferase per unit amount of AR
protein, and AR protein level was expressed as units of AR protein per unit of Renilla luciferase. There was
an inverse relationship between these measures of AR activity and AR level, but it was not a direct function
of Q length. An AR with 17 CAGs had the highest transcriptional activity per unit of AR protein, yet
yielded the lowest level of AR protein per unit of transfection. AR/CAG20 and AR/CAG21, the most
common alleles, were at the other end of the scale (low activity, high protein). We reported previously that
men with aggressive prostate cancer had a higher than expected frequency of germline AR alleles with 17
CAGs (Hakimi et al, 1997). We suggest that an AR/CAG17 might play a unique role in the development of
aggressive disease, either by increasing AR activity or by lowering AR protein level. The effect of a higher
activity AR in prostate cancer is easily reconciled with the ability of AR to act as a positive transciptional
regulator. Alternatively, if AR also had a role as a negative regulator, then a lower AR protein level could
attenuate this block and allow cells to generate signals that increase tumor aggressiveness. (D.D. & L.X.
contributed equally. Supported by CA68645.).

Basic Science Poster: Steroid Hormone Action II (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                   - 155 -
                                                           STEROID HORMONE ACTION
P2-30
Lay explanation of abstract:

Many proteins (e.g., insulin) are identical in amino acid sequence among individuals in the general
population; such strict conservation of these sequences suggests that variation would not be tolerated
without a bad effect on function. In contrast, other proteins [e.g., glutathione S-transferase P1 (GSTP1) and
androgen receptor (AR)] differ in sequence among individuals. These variants are referred to as
polymorphisms, and the genes that encode them are allele variants.

Many polymorphisms affect protein function (e.g., GSTP1; work was published by Barnabas et al.),
whereas others have not been studied. The AR gene (located on the X chromosome) is extensively
polymorphic; alleles differ in CAG and GGC repeat lengths and encode AR proteins with different
glutamine and glycine repeat lengths. The biological relevance of this polymorphism is notable because
these repeats are located in the N-terminal domain, which is required for the transcriptional activity of the
AR.

Glutamine repeats (encoded by a CAG repeat) are present in many transcription factors, some of which
activate and others repress transcription, and a role of these repeats has been implicated in function.
Glutamine repeats can form beta sheets held together by hydrogen bonds, and may thus function as ‗polar
zippers‘ by bringing together transcription factors. Thus, it is conceivable that the length of the AR
glutamine repeat could affect transcription factor activity of the AR by affecting protein-protein
interactions. Androgen effects mediated by the AR could thereby differ depending on glutamine repeat
length. Given the important role of androgens in both men and women, AR polymorphism might help to
account for variations in the degree of androgenization in the population.

Our laboratory has been studying the relevance of this normal polymorphism of the AR glutamine and
glycine repeats, both by measuring allele frequencies in men with prostate cancer, and by measuring the
effects of glutamine and glycine repeat length on recombinant AR transcription factor activity and AR
protein levels in an experimental system. Because of the important role of androgen hormones in both men
and women, AR polymorphism studies may help explain variations in the extent of androgenization in the
population.

This research was funded by NCI grant CA68645.




                                                   - 156 -
                                                          STEROID HORMONE ACTION
P2-66
MINERALOCORTICOIDS INFLUENCE THE GROWTH AND DIFFERENTIATION OF
PLURIPOTENT HUMAN STEM CELLS
M Mirshahi,1 S Emadi,2 F Valamanesh,1 N Golestaneh,1 MC Le Bousse-Kerdiles,2 MK AGARWAL.3 1E
9912, Inserm, Paris, France; 2U 268, Inserm, Villejuif, France; 3UMR 8532, Cnrs, Villejuif, France

Mineralocorticoids are generally believed to regulate the amiloride-sensitive sodium channel (ASSC) in the
apical membrane of epithelial cells in appropriate target tissues. Their potential influence on other types of
cells and organs has remained largely unelucidated. We studied the mineralocorticoid receptor (MCR)-
mediated effect of steroid agonists and antagonists on pluripotent stem cells that were isolated either from
human peripheral blood or human bone marrow samples using established procedures. Megakaryoblasts
from the bone marrow, and peripheral CD34+ cells, exhibited both the MCR and the ASSC (alpha subunit
of the epithelial sodium channel or ENaC) in immunofluorescence developed in the presence of specific
polyclonal antibodies raised against these two proteins. Whereas the MCR was apparent as a
nucleocytoplasmic protein, the ASSC was primarily membrane-bound, as expected from studies with the
classical MCR-specific target cells. Using the primers 5'-AGGCTACCACAGTCTCCCTG-3' (sense) and
5'-GCAGTGTAAAATCTCC AGTC-3' (antisense), the RT-PCR analysis revealed the presence of message
for the MCR in CD41+ megakaryoblast and its subsequent progeny 164. The presence of the ASSC was
similarly confirmed with the aid of the primers 5'-CTGCCTTTATGGATGGT-3' (sense) and 5'-
GTTCAGCTCGAAGAAGA-3' (antisense). Sequence analysis of the residues 1-778 in the PCR products,
from CD41+ megakaryocyte and the leukemic cell line HEL derived from the CD34+ parent, revealed
almost 100% homology with the residues 2944-2525 of human kidney MCR gene. Similarly, the residues
1-495 of αENaC cDNA shared almost 100% homology with the residues 1197-1692 of the human kidney
ENaC gene. The growth of blood CD34+ cells was altered both by the natural steroid aldosterone and two
synthetic MCR-specific antagonists RU 26752 and ZK 91587. These steroids also induced apoptic cell
death in the HEL cell line. These and other studies reveal that the receptor- mediated action of
mineralocorticoids appears to influence the normal and abnormal growth and differentiation of pluripotent
stem cells, contrary to the contemporary notion that they are unique features of selected epithelial cells.

Basic Science Poster: Steroid Hormone Action II (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                   - 157 -
                                                            STEROID HORMONE ACTION

P2-66

Lay explanation of abstract:

Steroid hormones are produced by special glands in the body (gonads, adrenals, etc.) and are transported by
blood to different organs where they regulate a wide variety of tissue-specific functions. The adrenal gland
produces mineralocorticoids that control the sodium-water balance by binding to a cellular protein called
the mineralocorticoid receptor. This alters the synthesis of tissue-specific proteins that control entry of
sodium into the cell, such as the epithelial sodium channel protein present in the cell membrane. Water also
enters the cell to maintain physiological isotonicity. Regulation of the sodium channel by the
mineralocorticoid receptor is believed to be specific to the epithelial cells located where sodium is to be
absorbed from fluids (skin, intestine).

Two important problems have rarely been investigated: (1) the effect of mineralocorticoids on other types
of cells; and (2) the effect of those hormones on pluripotent stem cells, present in early fetal life. Stem cells
later differentiate into red blood cells, white blood cells that include lymphocytes, basophils, neutrophils,
monocytes, and certain cancer cells (leukemia).

We found that pluripotent stem cells contained both the mineralocorticoid receptor and the sodium channel.
The growth of some of those cells was altered by mineralocorticoids and synthetic drugs that inhibit their
action. The development of rat fetuses also was altered dramatically when they were deprived of natural
mineralocorticoid by synthetic drugs that block the receptor.

Additional background information:

These findings open possibilities for further work in endocrine research. Future studies may someday yield
important implications for human health, as follows: 1) both the receptor and the sodium channel may
contribute to the differentiation of stem cells, contrary to existing ideas, which say the effect would be
limited to the epithelial cell in the adult; 2) development from fetal to adult life may be critically regulated
by mineralocorticoids; 3) this hormone may be used for regulating growth of certain cancers (leukemia)
and used as a growth factor for the large-scale production of normal cells for human transfusion; and 4)
diseases resistant to other treatments may respond to mineralocorticoids.

This research was funded by Inserm and CNRS, Paris, France.




                                                    - 158 -
                                                STEROID HORMONE BIOSYNTHESIS
OR7-6
IMMUNOHISTOCHEMICAL LOCALIZATION OF THE STEROIDOGENIC ACUTE
REGULATORY (STAR) PROTEIN IN THE MOUSE BRAIN

SR King,1,2,3 T Ishii,5 SD Ginsberg,4 KL Parker,5 RG Smith,1,3 DJ Lamb.2,3 1Huffington Center on Aging,
Baylor College of Medicine, Houston, Texas; 2Scott Dept. of Urology, Baylor College of Medicine,
Houston, Texas; 3Dept. of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas;
4
  Depts. of Pediatrics and Neuroscience, Baylor College of Medicine, Houston, Texas; 5Depts. of Internal
Medicine and Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas

There is increasing evidence that steroids are synthesized de novo in the brain at pharmacologically
significant levels. If steroid production is an essential process in the brain, then it may be subject to trophic
regulation. The regulated step in steroidogenesis is the delivery of cholesterol from the outer mitochondrial
membrane to the inner, where P450scc converts it to the first steroid formed, pregnenolone. This step is
mediated by the Steroidogenic Acute Regulatory (StAR) protein, which is present in all tissues where
steroid synthesis is hormonally regulated. Mutations in StAR result in congenital lipoid adrenal
hyperplasia, a condition in which patients have a profound deficit in steroid production and can die from
adrenal steroid insufficiency.To examine whether StAR protein is present in brain tissues, we performed
immunohistochemistry using anti-StAR antisera from Dr. D.B. Hales (Endocrinology 141:4000, 2000) on
frozen free-floating 40-μm sections from mouse brains. Studies reveal specific and intense staining of
several neuronal populations, including neuronal cell bodies and processes of striatal medium spiny
neurons, raising the possibility that StAR participates in neurosteroid biosynthesis. Immunoreactivity was
also found in pontine neurons whereas only background staining was observed in the pons of day-old
StAR-deficient mice. The data indicate that StAR is present in specific brain regions in levels sufficient to
promote neurosteroidogenesis. Thus, these results suggest that neurosteroid synthesis may be a regulated
process. Future studies will assess the full range of StAR expression within the central nervous system.
(Supported by Gilson Longenbaugh Foundation and Alzheimer's Association NIRG-00-2250 (SDG), NIH
DK54028 (KLP); SRK was supported by NIH T32-DKO7763-01.).

Basic Science Oral: Steroid Hormone Biosynthesis I (1:00 PM-2:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 2:15 PM; Location: A 207




                                                    - 159 -
                                              STEROID HORMONE BIOSYNTHESIS
OR7-6
Lay explanation of abstract:

The Steroidogenic Acute Regulatory (StAR) protein has been known for several years to be associated with
production of steroids in peripheral tissues. We demonstrated that StAR protein is expressed in the brain,
confirming the hypothesis that steroids are synthesized in the brain by a similar mechanism.

All steroids in the body are made from cholesterol. Cholesterol is first converted to pregnenolone, which is
the precursor of all the various steroids. StAR mediates this rate-limiting step in steroid synthesis.
Naturally occurring mutations in the StAR gene that render StAR inactive cause congenital lipoid adrenal
hyperplasia, a potentially fatal condition in which patients do not make enough steroids and must therefore
be administered daily steroid injections. In addition, because the level of testosterone, which is required for
male development, is practically zero in these patients, males look like females.

To examine whether StAR protein is in the brain, we performed immunohistochemistry on mouse brain
sections and as a control, sections from genetically engineered mice that lack StAR. These experiments
revealed StAR protein expression in the brain, especially in pontine (in the pons) neurons of the brain stem
and striatal neurons.

Our new discovery, that StAR protein is present in the brain, is important because it indicates that
neurosteroids are produced by a StAR-dependent pathway and the production of these steroids appears to
be important enough to be regulated by the brain. The presence of StAR in the pons and the striatum
suggests that neurosteroids may be physiologically relevant for normal brain function. Future studies will
assess the full range of StAR expression, and therefore, neurosteroid production within the central nervous
system. Because StAR levels correlate with steroid synthesis in other tissues, future correlations with StAR
expression may show whether the progression or cause of certain neurological diseases and conditions
involves disruption of neurosteroid biosynthesis.

Currently, the study of the role of neurosteroids in behavior and other brain functions remains in its early
stages. Already laboratories are considering the clinical applications of neurosteroids, such as anesthetics
and anticonvulsants. This may represent an additional avenue of treatment for many neurological
disorders. Our finding adds urgency to researching the importance and function of these compounds in the
brain.

Additional background information:

Tissues in the body such as the testes, the ovary, and the adrenal gland produce steroids. Recent studies
suggest a function for steroids in the brain and that the brain can synthesize its own steroids
(―neurosteroids‖). Investigations of the various types of neurosteroids so far suggest that some possess
anesthetic and anticonvulsant properties and may be involved in alcohol tolerance, protection of neurons
from death, and behaviors associated with premenstrual syndrome (PMS) and postpartum depression.
Since the steroidogenic acute regulatory (StAR) protein mediates the rate-limiting step in steroid synthesis
in other steroidogenic tissues, we investigated whether StAR is expressed in the brain as well. While the
importance of neurosteroids is not clear, our discovery that StAR protein is present in the brain suggests
that steroid production is an important neurologic function.

These studies are supported by grants from the Gilson Longenbaugh Foundation and Alzheimer's
Association (NIRG-00-2250) to Dr. Stephen D. Ginsberg and from the NIH (DK54028) to Dr. Keith L.
Parker. Dr. Steven R. King is supported by a postdoctoral NIH training grant (T32-DKO7763-01).




                                                   - 160 -
                                              STEROID HORMONE BIOSYNTHESIS
OR41-4
ALTERED NEUROSTEROIDOGENESIS IN NP-C DISEASE CONTRIBUTES TO
NEURODEGENERATION
LD Griffin,2 CL Brown,1 SH Mellon.1 1Ob, Gyn & Repro. Sci., UCSF, San Francisco, California;
2
  Neurology, UCSF, San Francisco, California

Niemann-Pick type C disease, an autosomal recessive neurodegenerative disease is caused by mutations in
the lysosomal NPC1 protein. Lack of NPC1 protein results in accumulation of cholesterol esters, and
abnormal transport of cholesterol and other molecules in NPC1+ vesicles may be an important factor
leading to neurodegeneration. Among other functions of cholesterol, it is used for neurosteroid synthesis.
We have shown previously that certain neurosteroids act to promote axonal and dendritic growth in
neocortical neuronal cultures. Others have shown effects of the neurosteroid progesterone on myelin
formation, and of 3α5α-tetrahydroprogesterone (ALLO) on neurite growth and regression of hippocampal
neurons. Thus, altered neurosteroid synthesis may result in abnormal neuronal development. We used a
mouse model of NP-C to study the synthesis of neurosteroids in different brain regions, to determine if
there were alterations in specific neurosteroidogenic pathways. Our data indicate that adult NP-C mouse
brains contain substantially less pregnenolone and DHEA than do normal Balb/c mice. Allo synthesis from
progesterone is also decreased. At 9 wks, after the appearance of ataxia, we detected brain region-specific
decreases in 5αreductase and 3αHSD activities. The brains from these mice synthesize only 10% as much
ALLO as their age-matched littermate controls. Differences are also seen at birth and 5 weeks of age, prior
to the onset of symptoms. P450scc, 3ßHSD, and 5α-reductase expression are significantly reduced in the
brain regions in which they are normally expressed. There is also ectopic cellular expression, and altered
subcellular localization of these enzymes. Our data indicate region-specific changes in NP-C mouse brains,
suggesting that the glia and neurons that express those neurosteroidogenic enzymes have preferentially
degenerated, have failed to migrate to their correct location, and/or that loss of NPC1 results in diminished
neurosteroidogenic enzyme expression and activity. While it is still unknown how NPC1 defects lead to the
disease pathology, our data suggest that decreased synthesis of neurosteroids may be a factor leading to
neurodegeneration.

Basic Science Oral: Steroid Hormone Biosynthesis II (11:00 AM-12:30 PM)

Presentation Date: Saturday, June 23, 2001; Time: 11:45 AM; Location: C 207




                                                  - 161 -
                                              STEROID HORMONE BIOSYNTHESIS
OR41-4

Lay explanation of abstract:

Niemann-Pick type C (NP-C) disease is a genetic disease of childhood that causes progressive intellectual
and motor deterioration leading to incoordination, dementia, seizures, and death. It is caused by the
mutation of a protein called NPC1, which is important for normal cholesterol processing and for the
transport of cholesterol and other molecules within all cells of the body. Cholesterol is used for many
functions, including synthesis of steroids in the brain (called neurosteroids).

We have shown recently that neurosteroids, particularly DHEA and allopregnanolone (which is formed
from the hormone progesterone), affect growth and differentiation of neurons. Neurosteroids are
synthesized in the brain in a developmentally patterned, regional-specific pattern. We hypothesize that
neurosteroids are important in the developmental patterning of neuronal networks, and that they will
contribute to the overt clinical phenotype (genetic make-up) seen in human beings with NP-C, and in a
mouse model with NP-C disease. We used this mouse model to examine the production of neurosteroids
over time in this disease.

We studied the NP-C mice from birth through the end of their lifespan (about 70 days). We showed that the
brains of these mice have decreased concentrations of the neurosteroids pregnenolone and DHEA. Also, the
brains of these animals synthesize about 10% of the allopregnanolone synthesized by a normal mouse
brain. We further showed that the progressive loss of activity of several neurosteroidogenic enzymes, 5
alpha reductase, and 3 alpha hydroxysteroid dehydrogenase begins to occur as early as birth and well
before the onset of any symptoms of the disease.

Furthermore, we found that there is expression of 5 alpha reductase in cells that normally don‘t express this
enzyme, and that there are cells present in the outer layer of the cerebellum that normally aren‘t there.
These experiments indicate region-specific steroid changes in NP-C mouse brains. The results also suggest
that the neurons and their supporting cells, the glia, that express those steroids have degenerated and have
in some cases failed to migrate to their correct location. We speculate that the loss of this NPC1 protein in
NP-C leads to diminished neurosteroidogenic enzyme expression and activity. While we don‘t know how
NPC1 defects lead to the disease pathology, our data suggest that decreased synthesis of neurosteroids may
be a factor leading to neurodegeneration.

This research is supported by the National Neimann-Pick Foundation, Ara Parseghian Medical Research
Foundation, March of Dimes, and the National Institutes of Health.




                                                  - 162 -
                                                                                                  AGING
P1-436
PREDICTIVE DEMOGRAPHICS OF 110 CONSECUTIVE COMMUNITY-DWELLING
RESIDENTS WITH HIP FRACTURE
LA Fitzpatrick,1 J Loftus,2 M Bolander.2 1Department of Medicine, Mayo Clinic, Rochester, Minnesota;
2
  Department of Orthopedics, Mayo Clinic, Rochester, Minnesota

Hip fractures result in 10-20% excess mortality in 1 year and only 1/3 of patients regain their
independence. We gathered data from 112 consecutive community patients admitted to Mayo Medical
Center with a hip fracture (fx) to determine possible risk factors. Each morning the surgical register was
reviewed and cases confirmed by operative reports. Data were collected by chart review and bedside
interview. Two patients were excluded from analysis as their fxs were due to trauma and both were less
than 40 years of age. The average age was 70.8 ± 10.6 years (38 to 98), BMI was 26.28 ± 7.06, 29% were
male and 71% were female. 22 of 110 patients (20%) had a prior diagnosis of osteoporosis. At the time of
fx, 45% (49/110) of patients had known prior fractures (10% ankle, 15% prior hip, 2% pelvic, 25% Colles',
2% vertebral). Balance problems were identified by history in many patients: 25% reported
dementia/chronic brain syndrome, 11% had transient ischemic attacks, and only 2% complained of vertigo.
Gait instability, focal cortical degeneration syndrome and functional gait problems were each less than 1%.
Of 59 patients available for complete interview, less than 3% were chair or bed ridden, 3% were inactive,
28% were home ambulators, and 45% were community ambulators. 19% of patients were fully active and
brisk walkers. In conclusion, a large number (45%) of patients with hip fx had prior fx, especially Colles' fx
(25%). Few patients were bed ridden and vertigo was rare at the time of fx. Organic brain
syndrome/dementia was associated with fracture in one-quarter of the cases. These data provide
information for target areas for prevention and suggest that early intervention in patients who have had a
prior fracture may be important to prevent future hip fractures.

Clinical Science Poster: Aging (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                   - 163 -
                                                                                                     AGING
P1-436

Lay explanation of abstract:

Identifying risk factors for hip fracture is an important first step in targeting interventions that prevent
future fractures. In this study, every patient admitted to Mayo Medical Center with a hip fracture was
interviewed, and their charts were reviewed carefully to determine the specific risk factors associated with
hip fracture. The average age of patients with hip fracture was 71 years. Approximately one-third were
male and two-thirds were female. Only 20% of patients realized that they had a diagnosis of osteoporosis
at the time of their fracture. However, 45% of patients had had a previous fracture. Up to 25% had had
fractures of the wrist (Colles') and 15% had had a previous hip fracture. To determine if cognitive issues
were associated with hip fractures, the patient or a family member was interviewed. One-quarter of the
patients had dementia or chronic brain syndrome, and 11% had transient ischemic attacks.

Before their hip fracture, most patients were very active. Only 3% were chair- or bedridden and only
another 3% were inactive. Twenty percent were fully active and brisk walkers, while the others were able
to get around easily at home or in the community.

This study emphasizes the need to identify patients at high risk at the time of their first fracture and to
intervene with appropriate therapy. Most patients were active before the hip fracture, emphasizing the
importance of early intervention to prevent the marked disability that can occur after the event.

Additional background information:

The death rate associated with hip fractures in the elderly population is substantial. After hip fracture, the
all-cause mortality rate is 24% at 12 months. As the American population ages, substantial hospital
resources will be utilized to care for patients with hip fractures. In the United States, the lifetime risk of hip
fracture is 17.5% for women and 6.0% for men. With our aging baby-boomer population, we will see
substantial societal implications as fracture numbers are expected to double by the year 2040. Functional
recovery of patients is not common enough, and patients frequently have pronounced limitations. At the
end of one year, only one-half can walk unaided and even fewer can perform all activities of daily living
independently.




                                                    - 164 -
                                                                                                  AGING
P1-437
ANTI-MüLLERIAN HORMONE SERUM LEVELS: A PUTATIVE MARKER FOR OVARIAN
AGING
A de Vet,1 JS Laven,1 FH de Jong,2 AP Themmen,3 BC Fauser.1 1Division Reproductive Medicine,
Department Obstetrics and Gynecology, Erasmus University Medical Center Rotterdam, Rotterdam,
Netherlands; 2Internal Medicine, Erasmus University Medical Center Rotterdam, Rotterdam, Netherlands;
3
  Endocrinology and Reproduction, Erasmus University Medical Center Rotterdam, Rotterdam, Netherlands

Previous studies in mice indicate that anti-Müllerian hormone (AMH) is involved in the depletion of the
stock of primordial follicles, suggesting a role in ovarian aging. The current study was designed to
investigate whether AMH serum concentrations may be used as a marker for ovarian aging in 41 young (29
± 4 years) normo-ovulatory volunteers. In each woman early follicular AMH levels were assessed in two
cycles with a time interval of 2.6 ± 1.7 (SD) years. AMH concentrations are compared with other markers
associated with ovarian aging such as number of antral follicles as assessed by ultrasound, follicle-
stimulating hormone (FSH) and inhibin B serum concentrations. AMH levels were found to decrease
significantly over time (median 2.1 (0.1-7.4) mg/L during visit 1, vs 1.3 (0.0-5.0) mg/L during visit 2, P <
0.001), whereas antral follicle number, FSH and inhibin B levels did not change. During visit 1 and 2,
AMH levels correlated with age (r = -0.40, P = 0.01 and r = -0.57, P < 0.001) and number of antral follicles
(r = 0.66, P < 0.001 and r = 0.71, P < 0.001), to a lesser extend with FSH (r = -0.36, P = 0.02 and r = -0.36,
P = 0.02), but not with inhibin B. In conclusion, serum AMH levels decrease over time in young normo-
ovulatory women whereas other markers of ovarian aging did not change during this time interval.
Secondly, AMH concentrations correlate with age and FSH but most strongly with antral follicle number.
AMH concentrations may represent a novel marker for ovarian agin.

Clinical Science Poster: Aging (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                   - 165 -
                                                                                                    AGING

P1-437

Lay explanation of abstract:

Due to the widespread delay of childbearing, arising from emancipatory developments and facilitated by
easy access to contraceptives, the average female age at birth of her first child has dramatically risen in the
modern Western world. Because there is an age-related decline in fertility, the absolute number of couples
suffering from infertility has risen substantially. It is nowadays accepted that this age-related fertility
decline is the result of a decrease in the quantity as well as the quality of ovarian follicles. This so-called
―ovarian reserve‖ rather than chronological age determines a woman‘s individual reproductive potential.

Existing markers for ovarian aging, such as follicle-stimulating hormone (FSH) and inhibin B are
associated with the final depletion of ovarian follicles, and therefore, do not necessarily indicate diminished
ovarian reserve. Accurate assessment of ovarian reserve could be of use to counsel each individual patient
and to tailor infertility treatment to the needs and options of each patient. A new marker, called Anti-
Mullerian Hormone (AMH), for assessment of ovarian reserve has been identified.
AMH levels were assessed twice in 41 young women with normal regular menstrual cycles. The time
interval between the first and second AMH measurement varied from 1 up to 5 years. AMH levels were
compared to existing markers for ovarian aging, such as number of antral follicles (areas in the ovary where
eggs develop and are released and where hormones are produced) as assessed by ultrasound, FSH levels,
and inhibin B serum concentrations.

AMH levels were found to decrease significantly over time, while the number of follicles, FSH, and inhibin
B levels did not change. Secondly, AMH levels were higher when more ovarian follicles were seen during
the ultrasound investigation. Although to a lesser extent, a similar association was found between AMH
concentrations and age. Therefore, AMH seems a promising marker to assess the decreasing number of
follicles more commonly referred to as ovarian reserve.

This research was funded by the ―Stichting Voortplantingsgeneeskunde Rotterdam‖ Rotterdam, The
Netherlands.




                                                    - 166 -
                                                                                               AGING
P1-438
HIGHLY EFFICACIOUS AND COST-EFFECTIVE ALENDRONATE REGIMEN FOR
POSTMENOPAUSAL OSTEOPOROSIS: A FOUR-YEAR CLINICAL STUDY
SJ Wimalawansa. Internal Medicine, Robert Wood Johnson Medical School, New Brunswick, New Jersey

Bisphosphonate therapy is highly effective for osteoporosis. Difficuly in taking alendronate daily, and the
upper GI adverse effects are responsible for poor compliance. The primary aim of this study was to see
whether infrequent (i.e., once-a-week) of alendronate has the same efficacy as once-daily therapy on BMD.
The secondary aims were to assess adverse effects profile and whether this regimen improves the adherence
to therapy, in comparison with the daily regimen. A total of 180 patients (mean age 62 yrs) with BMD <2.0
SD in the lumbar spine were included in this study. The first group of patients (n=89) received alendronate
once a week (60 mg) for two years. At the end of two years, they were given the choice of continuing the
same dose of alendronate (60 mg once a week) or taking a single tablet of 40 mg of alendronate once a
week for further two-years (a total of four years). This group was compared with a group of patients (n=91)
who received the standard recommended dose of alendronate 10 mg per day for four-year duration. Bone
mineral density (BMD) was measured by DXA in the lumbar spine and in the total hip at the beginning and
then annually. Results on changes of both BMD and biochemical markers are remarkably similar in both
groups over the four years duration of the study. Patients on both regimens had equal and significant
suppression of biochemical markers; serum osteocalcin and urinary N-telopeptides. In this study, patient
compliance with alendronate, as accounted with pill counting was ~88% in the once a week regimen and
72% in the once daily regimen. Patients who discontinue the regime due to adverse GI effects during this
study are 8% for the once a week group and 14% for the once daily treatment groups, respectively.
Alendronate therapy significantly increase the bone mass both in the lumbar spine and the hip, decreased
markers of bone turnover, and generally well tolerated in both doses. However, the compliance and the
adverse effect profiles were better with the once a week regimen. This study gives an insight into a highly
effective, safe and economical way of administration of oral alendronate to achieve the expected BMD
outcome in patients with established osteoporosis.

Clinical Science Poster: P1: Aging (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                 - 167 -
                                                                                                AGING
P1-438

Lay explanation of abstract:

Osteoporosis is a common and debilitating condition that affects mostly postmenopausal women.
Although highly effective therapies are now available, adherence to treatment is poor due to undesirable
side effects and inconvenience of taking daily medications. Alendronate is one such efficacious FDA-
approved drug for osteoporosis. However, because of difficulties in complying with the daily routine of
taking the drug and the unpleasant upper gastrointestinal effects, patients show poor compliance with
alendronate therapy. The primary aim of this study was to see whether infrequent administration (i.e., once
a week) of alendronate has the same benefit as once daily administration of this drug in strengthening bone.
The secondary aims were to assess whether one could minimize the adverse effects and also whether this
new regimen for taking the medication will improve adherence to therapy.

A total of 180 patients were included in this study. The first group of patients (n=89) received alendronate
once a week (60 mg) for two years. At the end of two years, they were given the choice of continuing the
same dose of alendronate (60 mg once a week) or taking a single tablet of 40 mg of alendronate once a
week for an additional two years (a total follow-up period of four years). The second group received the
standard recommended dose of alendronate (n=91), 10 mg taken daily for four years. Bone mineral density,
a measure of the calcium content of the bone, was measured in the lumbar spine and in the total hip at the
beginning and then annually, and data were compared among these three groups.

Results on changes of both bone density and biochemical markers are remarkably similar in these groups
over the four years‘ duration of the study; this is true of the other variables indicating the response to
therapy. In this study, patient compliance with alendronate was 88% in the once-a-week regimen and 72%
in the once daily regimen. In the once-a-week treatment group, 8% of patients discontinued this therapy,
whereas 14% of patients discontinued in the once daily treatment group. In both treatment groups,
alendronate therapy significantly increased bone mass, decreased markers of bone turnover, and generally
was well tolerated. This study demonstrated for the first time that while improving the compliance and
adverse effects, once-a-week administered alendronate is a highly effective, safe, and economical way to
increase bone mass in patients with osteoporosis.

Additional background information:

Alendronate is sold under the trademark of FOSAMAX in North America. The company that makes this
medication, Merck Pharmaceuticals, recently announced a 70-mg tablet, which can now be taken as one
pill once a week.




                                                  - 168 -
                                                                                                AGING
P1-445
PERIMENOPAUSAL WOMEN HAVE HIGH FSH AND ACTIVIN, WITH FREQUENT
ANOVULATION DESPITE ULTRASOUND SIGNS OF FOLLICLE GROWTH
A Sakaris,1 H Eckholdt,2 G Neal-Perry,3 A Nussbaum,1 L Weingart,1 G Adel,1 B Isaac,1 N Santoro.1
1
  Ob/Gyn & Women's Health, Albert Einstein College of Medicine, New York, New York; 2Neurology,
Albert Einstein College of Medicine, New York, New York; 3Ob/Gyn, Beth Israel Med Ctr, New York,
New York

We compared daily serum hormones and serial ultrasound monitoring for one cycle in 13 perimenopausal
women (PERI; age> or =43) to 5 midreproductive aged controls (>19 but <34 yrs). As of this submission,
LH, FSH, estradiol (E2), progesterone (P; DELFIA), inhibin A (Inh A; DSL), and activin A (Act A;
Serotec) have been assayed. 10/13 PERI women ovulated; all ovulatory cycles were standardized to the LH
surge and compared using mixed models repeated measures analysis of variance.
Results: Act A was increased in PERI women compared to controls (mean = 0.8+/-0.1 [SEM] vs 0.2 +/-
0.05 ng/ml, p=0.001), and did not vary by cycle stage. Inh A followed a pattern similar to E2 with a
midcycle mean of 70+/-20(SEM)in PERI and 65+/-42 pg/ml in controls. Three anovulatory PERI women
had had variable estradiols but Inh A<47pg/ml, despite ultrasound presence of a dominant follicle >12mm.
Late follicular phase E2 was not significantly higher in PERI women (309+/-67pg/ml vs 263+/-42pg/ml;
p=0.4) and late luteal P trended lower in PERI women. FSH was consistently elevated in PERI women
(mean=10 mIU/ml vs 4mIU/ml; p=0.001), with an accentuation of the between group differences in the
early follicular and late luteal phases. Conclusions: 1. Perimenopausal women produce consistently more
FSH and Act A than younger, cycling women. 2. Decreased Inh A was not observed in this study, perhaps
due to the use of an assay (DSL) different from prior studies (Serotec). 3. In 3/13 perimenopausal women
studied, ovulation failed to occur despite clear-cut ultrasound evidence of follicle growth. 4. Inh A may be
a marker for adequacy of folliculogenesis in older women. (Supported by NIH AG12222 to N.).

Clinical Science Poster: P1: Aging (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 169 -
                                                                                                  AGING
P1-445

Lay explanation of abstract:

Why do women‘s cycles become less frequent and less predictable as they approach menopause? Our
group has examined carefully hormones and ultrasound images of the uterus and ovaries in women in their
late 40s. We performed blood testing every day during one menstrual cycle and checked the growth of the
ovaries every couple of days through an entire cycle. This information was compared to that of regularly
cycling, normal young women in their 20s and 30s.

We noted several interesting findings. First, several of the older women we studied had what appeared to be
normal cycles in that their menstrual period came exactly when it was expected, yet they did not ovulate in
that cycle and had fairly unusual ultrasound pictures of the ovary during the cycle of study. This is very
different from the cycles of younger women, because almost every single cycle in young, healthy women
with regular periods is an ovulatory cycle.

The hormones produced by the ovaries also differed between the older and younger women. The older
women tended overall to produce fewer hormones (except for estrogen), reflecting less healthy growth of
their follicles (microstructures in the ovary stored for future hormone production and egg release). There
was also a smaller group of follicles available for growth in their ovaries. These differences help to explain
the relative infertility that arises as women‘s ovaries age and helps point to possible ways to measure the
loss of eggs and follicles over time that is known to occur with aging in women.

Additional background information:

Our previous findings have shown that the ovary does not simply ‗poop out‘ with reproductive aging and
that a series of dynamic changes in hormones occurs. There may be wider swings in hormones during
perimenopause (the time period before menopause) in women, and this may be linked to problems with
irregular and unpredictable bleeding.

These studies are funded by a grant to Dr. Santoro from the NIH (AG12222) and the National Institute on
Aging. They are an extension of previous research that has helped to characterize the patterns of hormones
produced by women who are entering menopause.




                                                   - 170 -
                                          ANDROGENIC DISORDERS IN WOMEN
OR32-1
EFFECTS OF ANDROGEN ADMINISTRATION TO NORMAL FEMALES: COMPARISON
WITH SYMPTOMS OF POLYCYSTIC OVARIAN SYNDROME
AW Toorians, LJ Gooren, H Asscheman. Endocrinology, Vrije Universiteit Medical Center, Amsterdam,
Netherlands

Polycystic ovary syndrome (PCOS) is characterized by chronic anovulation, hyperandrogenism, and insulin
resistance. Cardiovascular risk profiles of women with PCOS show a shift into the male direction. Cross-
sectional studies do not allow to distinguish between the contribution of hyperandrogenism and of
hyperinsulinism to these cardiovascular risk profiles, which are in any case intertwined. Administration of
250mg testosterone-esters (T) i.m. every 14 days to female-to-male transsexuals allowed to monitor
prospectively the effects of androgen administration on shifts of cardiovascular risk factors. All these
females had, before T administration, regular cycles, and mean BMI, and were between 18-40 yrs. Our
subjects compare best with non-obese women with PCOS. One year of T treatment led to: 1) A significant
elevation of T levels with a suppression of LH. 2) To morphological changes of the ovaries identical to
those in PCOS. 3) Significant increases of BMI and of the visceral fat area while subcutaneous abdominal
and gluteofemoral fat decreased. 4) No effect on insulin sensitivity in clamp studies. 5) No effect on blood
pressure. 6) No effect on plasma cholesterol, LDL-cholesterol, VLDL-cholesterol, and free fatty acid, but a
significant decrease levels of HDL-cholesterol, and it subfractions HDL2, HDL3 with an increase in
hepatic lipase activity. Triglyceride levels did not change. 7) Before the start of T administration visceral
fat area (assessed by MRI) was an important determinant of plasminogen activator inhibitor type-1 (PAI-1)
levels, independently of insulin sensitivity and plasma levels of insulin and triglycerides. T treatment did
not change levels of PAI-1 and of antigen of tissue-type plasminogen activator (t-PA) but the relationship
between visceral fat and PAI-1 levels was no longer demonstrable. In subjects with PCOS PAI-1 levels are
correlated with BMI and fasting insulin levels. Conclusions: one year of exogenous T-induced
hyperandrogenism 1) does not induce hyperinsulinism / insulin resistance, which is a key symptom of
PCOS 2) induces an accumulation of visceral fat, without an effect on PAI-1 levels or t-PA. 3) has no effect
on blood pressure. 4) induces a lipid profile similar to that of women with PCOS.

Clinical Science Oral: Female Androgenic Disorders (1:00 PM-2:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 1:00 PM; Location: C 106




                                                  - 171 -
                                           ANDROGENIC DISORDERS IN WOMEN
OR32-1

Lay explanation of abstract:

Polycystic ovarian syndrome (PCOS) is a condition affecting 3-4 % of fertile women. The syndrome
derives its name from the cystic changes in the ovaries, which are usually enlarged. Main characteristics are
anovulation (no monthly production of an egg), menstrual irregularities, and an excess of male hormone,
often with hairiness and acne. These women are often obese, sometimes with a male type fat distribution
(fat mainly in the belly region), and a tendency to develop diabetes. They may have unfavorable lipid
profiles and other metabolic factors predisposing them to heart disease. PCOS is complex, and doctors find
it difficult to agree on the nature of the disease. Is the excess of androgens the core phenomenon, or is
excess of male hormone the result of other aspects of this disease? Thus, it is difficult to define the best
possible treatment. Men suffer more from heart disease than women, so the idea has emerged that excess of
male hormone in these women with PCOS is accountable for their unfavorable risk profile for heart
disease.

In our clinic each year approximately 20-30 transsexual healthy women undergo a sex change to male.
They receive treatment with male hormones. By closely monitoring the changes these non-diseased
transsexual women undergo after administration of male hormones, we were able to chart which PCOA
symptoms likely are explained by the excess of male hormone in women with PCOS.

Our findings: 1) the ovaries of the transsexual women show the same cystic changes and enlargement as
found in PCOS; 2) as expected, male hormones induce male patterns of hair growth and acne; 3) male
hormone induces a male type of fat distribution with more fat in the belly region; 4) some changes in lipid
profiles found in PCOS are from excess of male hormones but others do not change unfavorably; 5) no
evidence exists that male hormones increase the likelihood of developing diabetes; 6) no effect on blood
pressure was seen; and 7) no strong evidence exists that male hormones strongly accelerate the
development of heart disease.

In conclusion: 1) we have now a fair idea which features of PCOS are due to excess of male hormones 2)
the effects that male hormones have on the risks of developing heart disease were less serious than
traditionally feared. So there must be other explanations for the higher rate of heart disease in men.

Additional background information:

In our clinic, transsexuals receive treatment in an academic setting allowing unique scientific observations
of the role of male hormones in females and of female hormones in males. It is increasingly clear that the
terms ―male‖ and ―female‖ hormones are becoming obsolete. Men need some female hormones and women
need some male hormones for normal biological functioning. Lack of these small amounts of hormones in
each other strongly impairs quality of life. Caring for transsexuals in our clinic puts us at the forefront of
this research area.

It has been difficult to find funding for this type of research because it has been almost impossible to
convince funding agencies that changes observed in transsexuals are not exotic and can benefit other
groups of patients. The university has sparingly funded most of the research. Only one external research
grant has been received so far, from the Netherlands Organization for Scientific Research (NWO).




                                                   - 172 -
                                          ANDROGENIC DISORDERS IN WOMEN
OR32-4
GLOBAL FIBRINOLYTIC CAPACITY IS DECREASED IN POLYCYSTIC OVARY
SYNDROME
BO Yildiz,1 IC Haznedaroglu,2 S Kirazli,2 M Bayraktar.1 1Endocrinology, Hacettepe University Hospital,
Ankara, Turkey; 2Hematology, Hacettepe University Hospital, Ankara, Turkey

The polycystic ovary syndrome (PCOS) is associated with an increased incidence of cardiovascular disease
(CVD). Insulin resistance, hyperandrogenism and dyslipidemia are well-known cardiovascular risk factors
in PCOS. Impaired fibrinolysis could also contribute to the development of CVD in PCOS. Global
fibrinolytic capacity (GFC) is a recently developed method which is reflected by the amount of generated
D-dimer when the fibrinolysis of a freeze-dried fibrin clot is stopped by introducing aprotinin. It was
previously shown that GFC is sensitive to all the factors involved in the process of fibrinolysis. The aim of
this study was to evaluate whether women with PCOS have any alterations in the GFC, and other essential
hemostatic parameters. Thirty-one women with PCOS (mean age 22.6 y, mean BMI 23.6 kg/m2) and 11
age and BMI matched healthy controls participated. We measured GFC and triglycerides, total cholesterol,
HDL cholesterol, lipoprotein-a, prothrombin time, partial thromboplastin time, thrombin time, antithrombin
III, factor II, V, VII, X, fibrinogen, plasminogen, alpha2-antiplasmin, D-dimer. Glucose and insulin (at
baseline and during a 75g 2-h OGTT) were also measured and insulin resistance (IR) was assessed by
homeostatic model assessment (HOMA).GFC was found to be significantly lower in PCOS group
compared with control group (1.62±1.12 versus 3.54±2.01 μg/ml, p=0.005). The PCOS group had
significantly higher plasminogen activity (%) (92±16 versus 76±9, p<0.05). All the other coagulation and
fibrinolysis parameters were comparable between the two groups. The PCOS group had lower HDL-
cholesterol and higher IR values (53±10 versus 61±9 mg/dl, p<0.05; and 2.3±1.1 versus 1.2±0.3, p<0.05
respectively). All subjects had normal glucose tolerance. GFC was correlated with free testosterone
negatively and with HDL-cholesterol positively (r= -0.384, p<0.05; r= 0.421, p<0.05 respectively). There
was no correlation between GFC and IR values.
Our results suggest that women with PCOS have impaired fibrinolysis as reflected by the decreased GFC.
This impairment is not related to the insulin resistance and may increase the risk of CVD in PCOS.

Clinical Science Oral: Female Androgenic Disorders (1:00 PM-2:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 1:45 PM; Location: C 106




                                                  - 173 -
                                           ANDROGENIC DISORDERS IN WOMEN
OR32-4

Lay explanation of abstract:

Polycystic ovary syndrome (PCOS) is a common disease, affecting approximately five percent of women
of reproductive age. It is characterized by irregular or absent periods, excess hair growth on face and body,
acne, infertility, lipid abnormalities and, often, obesity. In addition, PCOS is frequently associated with
decreased sensitivity to insulin (insulin resistance).

There is significant evidence that women with PCOS are at risk for cardiovascular disease. Insulin
resistance, elevated levels of androgens, decreased levels of HDL or ―good‖ cholesterol, and elevated levels
of LDL or ―bad‖ cholesterol are well-known cardiovascular risk factors in PCOS.The coagulation system
that produces blood clots and fibrinolytic system that dissolves blood clots compete for the regulation of
blood clotting in the body. An increase in coagulation or a decrease in fibrinolysis results in thrombotic
tendency and contributes to the development of cardiovascular disease. Global fibrinolytic capacity is a
recently developed method to evaluate fibrinolysis. It is sensitive to all the factors involved in the process
of fibrinolysis.

Thirty-one non-obese PCOS patients (mean age 22 years) and eleven control women with normal ovarian
function who had similar demographic characteristics participated in this study. Coagulation and
fibrinolysis parameters and serum lipids were measured. Glucose and insulin levels (at baseline and during
a standard 2-hour oral glucose tolerance test) were also measured. Insulin resistance was measured based
on a mathematical modeling, called ―homeostatic model assessment‖. All the participants had normal
glucose tolerance. The women with PCOS had significantly higher insulin resistance values and lower
HDL-cholesterol levels than those of control group, as expected.

As a new and clinically important finding, global fibrinolytic capacity values were found to be significantly
lower in PCOS group than those in control group. Other coagulation and fibrinolysis parameters were
comparable between the two groups. Global fibrinolytic capacity was correlated with free testosterone
negatively and with HDL-cholesterol positively. Interestingly, there was no correlation between global
fibrinolytic capacity and insulin resistance values.

Our results suggest that women with PCOS have impaired fibrinolysis as reflected by the decreased global
fibrinolytic capacity. In theory, a decrease in fibrinolysis (reduced ability to dissolve blood clots) might be
expected to result in an increase in the deposition of fibrin and subsequent formation of thrombus. Thus,
impaired fibrinolysis (not related to insulin resistance) could be added to other cardiovascular risk factors
such as altered lipid metabolism, hormonal derangements, obesity, and insulin resistance in PCOS. Global
fibrinolytic capacity seems to be a simple and accurate marker of the process of fibrinolysis. Because of the
increased cardiovascular risk even in young and thin PCOS patients, early diagnosis, close and long-term
follow-up, and treatment of modifiable risk factors are essential in PCOS.

Additional background information:
Insulin is a hormone released from the pancreas after a meal and it allows the organs of the body to take up
energy in the form of glucose. In PCOS there is a resistance (decreased sensitivity) of cells in the body to
insulin, so the pancreas makes more insulin to try and compensate. The patency (state of being freely open
or exposed) of the space inside of blood vessels is vital for all organs and systems. This is especially true
for the vessels of the heart. Two systems compete for the regulation of blood clotting. One is coagulation
system that produces clots, the other is fibrinolytic system designed to dissolve blood clots. Increased
coagulation or decreased fibrinolysis (reduced ability to dissolve blood clots) results in thrombotic
tendency, blockage of blood flow in vessels that can lead to heart attack and stroke. Global fibrinolytic
capacity is a recently developed method that is sensitive to all the factors involved in the process of
fibrinolysis. The aim of this study was to evaluate whether women with PCOS have any alterations in the
global fibrinolytic capacity and other essential coagulation and fibrinolysis parameters, any of which could
contribute to the risk of cardiovascular disease in PCOS.




                                                   - 174 -
                                          ANDROGENIC DISORDERS IN WOMEN
P2-462
FLUTAMIDE EFFECTS ON HIRSUTISM, ACNE, MENSTRUAL CYCLICITY AND
OVULATION RESTORATION
P Falasca, M Poggi, A Stigliano, S Monti, F Sciarra, V Toscano. Dep Fisiopatologia Medica - II
Endocrinologia, Universita' La Sapienza, Roma, Italy

Menstrual disturbances are frequently associated to hirsutism and acne and may be related to chronic
anovulation. In the pathogenesis of chronic anovulation androgens per se may play an important role. Aim
of the present study is to evaluate the efficacy of flutamide on acne, hirsutism, menstrual disorders and
ovulation restoration. 96 patients with mean age of 26±7 yrs, were studied: 60 patients with hirsutism , 19
with acne and 17 with both symptoms. Menstrual disorders were present in 53% of the patients and
ovulation only in 36%. To all patients, after a written informed consent, 250 mg/die of flutamide
(Flutamide Ipsen) was administered for 1 year. Patients were evaluated after 6 and 12 months of treatment
and 3 months after flutamide withdrawal. Blood transaminases were checked after 15 day and then every
month. RESULTS (mean±SD): 24 patients (25%) dropped out the protocol, because of the no-show at the
scheduled monthly control. 8 patients (8.3%) dropped out the protocol, because of an impairment in liver
function. In the remaining patients transaminases remained unchanged during all period of treatment and 3
month after drug withdrawal. Hirsutism degree (Ferriman-Gallwey scale) was significantly reduced at 6
(8.9±2.4 vs 10.9±3.9, p<0.02) and 12 months (8.9±1.9, p<0.01), and remained unchanged 3 month after the
drug withdrawal (8.8±2.2, p<0.02). Acne degree (Lucky scale) improved significantly at 6 (1.7±1.2 vs
3.8±1.2, p<0.0001) and at 12 (1.4±0.5, p<0.0001) months and remained unchanged after the withdrawal
(2±1.2, p<0.01). Normal menstrual cycles were restored during treatment in 74% of the patients studied and
remained regular 3 month after the withdrawal in 84%. 84% of the patients showed ovulatory cycles during
treatment and in 83% cycles remained ovulatory 3 month after the withdrawal. In conclusion our data show
that a pure androgen receptor blocker is highly effective not only in the control of skin manifestations of
hyperandrogenism, but may also improve menstrual cyclicity and restore ovulation in the majority of the
patients, confirming the direct role of androgens in the impairment of ovarian function.

Clinical Science Poster: P2: Androgenic Disorders in Women (11:00 AM-12:00 PM and 2:30 PM-3:30
PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                 - 175 -
                                           ANDROGENIC DISORDERS IN WOMEN

P2-462

Lay explanation of abstract:

Hirsutism (hairiness) and acne represent the most important clinical manifestations of hyperandrogenism
(over-production of androgens) in young females. Menstrual disturbances are frequently associated with
these skin manifestations and may be related to the continuous absence of ovulatory cycles.

In the problem of chronic anovulation (lack of ovulation), androgens per se may play an important role, as
recently suggested by the studies performed in monkeys using testosterone or dihydrotestosterone
treatment. The aim of the present study is to evaluate the efficacy of flutamide, an androgen receptor
blocker, on acne, hirsutism, menstrual disorders, and ovulation restoration. Ninety-six patients with mean
age of 267 years were studied: 60 patients with hirsutism , 19 with acne and 17 complaining of both
symptoms. Menstrual disorders were present in 53% of the patients. Ovulation (documented by plasma
progesterone values during the second phase of the menstrual cycle higher than 6 ng/ml) occurred in only
36% of the patients. After giving written informed consent, all patients received 250 mg/die of flutamide
(Flutamide Ipsen) for one year. Patients were evaluated after 6 months and 12 months of treatment and 3
months after flutamide withdrawal. Blood transaminases were checked after 15 days and then every month.

Twenty-four patients (25%) dropped out the protocol because of they did not attend the scheduled monthly
control. Eight patients (8.3%) dropped out of the protocol because of an impairment in liver function. In
the remaining, patients liver function remained unchanged during the entire period of treatment and 3
month after drug withdrawal. Hirsutism degree (Ferriman-Gallwey scale) was significantly reduced at 6
(8.92.4 vs. 10.93.9, p<0.02; all results given as meanSD) and 12 months (8.91.9, p<0.01), and
remained unchanged 3 months after the drug withdrawal (8.82.2, p<0.02). Acne degree (Lucky scale)
improved significantly at 6 (1.71.2 vs 3.81.2, p<0.0001) and at 12 (1.40.5, p<0.0001) months and
remained unchanged after the withdrawal (2.1.2, p<0.01). Normal menstrual cycles were restored during
treatment in 74% of the patients studied, and these remained regular 3 months after the drug withdrawal in
84% of these patients. Eighty-four percent of the patients showed ovulatory cycles during treatment, and in
83% of these patients, cycles remained ovulatory 3 months after the flutamide withdrawal. In conclusion,
our data show that an androgen receptor blocker like flutamide, is highly effective not only in the control of
skin manifestations of hyperandrogenism but may also improve menstrual cycle regularity and restore
ovulation in the majority of the patients. These results confirm the direct, receptor-mediated role of
androgens in the impairment of ovarian function.

Additional background information:

Acne and increased hair growth often develop in young females together with menstrual cycle disturbances,
which may be mediated by an increased action of androgens at the ovarian level. Many young women are
unwilling to accept this esthetic problem, and ask the doctor for treatment. Most frequently they are treated
with birth control pills to stop ovarian function, but not to improve that function. Usually the treatment was
continued for a long time with metabolic consequences. It is not yet clear whether this unusual use for these
drugs plays a role in breast cancer development. For this reason, we are suggesting an alternative treatment
using a drug that will act at skin and ovarian levels without any blockage of ovarian function. This will lead
to a correction of the menstrual cycle disturbances, together with improvement of esthetic problems like
excess hair and acne.




                                                   - 176 -
                                           ANDROGENIC DISORDERS IN WOMEN
P2-463
POLYCYSTIC OVARIAN SYNDROME IN LEAN, YOUNG WOMEN: COMBINED
METFORMIN-FLUTAMIDE TREATMENT IS MORE EFFECTIVE THAN MONOTHERAPY
L Ibanez,1 C Valls,2 A Ferrer,1 F Rodriguez-Hierro,1 M Marcos,3 F de Zegher.4 1Endocrinology Unit,
Hospital Sant Joan de Deu, University of Barcelona, Barcelona, Spain; 2Hormonal Laboratory, Hospital
Sant Joan de Deu, University of Barcelona, Barcelona, Spain; 3Endocrinology Unit, Hospital de Terrassa,
Terrassa, Spain; 4Pediatrics, University of Leuven, Leuven, Belgium

The endocrine hallmarks of Polycystic Ovarian Syndrome (PCOS) in lean, young women are
hyperinsulinism, hyperandrogenism and dyslipidemia. Insulin-sensitizing and antiandrogen monotherapies
act through different pathways and result in a different spectrum of endocrine-metabolic effects.We
assessed whether combined metformin-flutamide (Met-Flu) treatment is more effective than either
monotherapy to correct endocrine-metabolic anomalies in lean, young women with PCOS (n=24; mean age
19 yr; BMI 21.9 Kg/m2). At inclusion, each woman had hirsutism, hyperinsulinism (oGTT; peak insulin
>150 µuU/mL), adrenal and/or ovarian hyperandrogenism (diagnosis by GnRH-agonist test) and
dyslipidemia. Women were randomly assigned to receive Flu (250 mg), Met (1275 mg) or Met-Flu once
daily. Clinical and hormonal assessments were performed before and 6 months after start of treatment.
Each treatment was well tolerated, and was followed by a decrease in hirsutism score and in serum
testosterone, and by a less atherogenic lipid profile. The fasting glucose to insulin ratio rose only after Met
and Met-Flu; the LDL/HDL ratio dropped more with Met than with Flu. Combined Met-Flu treatment
further decreased the LDL/HDL ratio and was superior to monotherapy in raising SHBG and reducing
triglycerides, androstenedione and DHEAS. Met and Met-Flu induced eumenorrhea and ovulation more
readily than Flu.
In conclusion, combined Met-Flu was found to be more effective than monotherapy, in particular to correct
dyslipidemia and hyperandrogenism. Low-dose Met-Flu may become an induction treatment of choice in
young women with PCOS.

Clinical Science Poster: Androgenic Disorders in Women (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                   - 177 -
                                            ANDROGENIC DISORDERS IN WOMEN
P2-463

Lay explanation of abstract:

Polycystic ovary syndrome (PCOS), which affects approximately 10% of women of reproductive age, is
characterized by elevated serum androgen levels (hyperandrogenism, due to increased ovarian and usually
also adrenal hypersecretion) and chronic failure to ovulate. This condition is the most common cause of
infertility in women. It is usually associated with hirsutism (excessive body hair growth following a male
pattern) and menstrual disturbances (oligomenorrhea, which means menstrual cycles of more than 45 days
duration, or amenorrhea, which means fewer than 5 cycles/year).

Hyperinsulinemia and insulin resistance and dyslipidemia are among the metabolic abnormalities
associated with PCOS, both in obese and non-obese women. Insulin resistance is defined as impaired
sensitivity to the effects of insulin on glucose utilization. Dyslipidemia is characterized by high levels of
serum total cholesterol, tryglicerides and low-density lipoprotein (LDL)-cholesterol, and decreased
concentrations of serum high-density lipoprotein (HDL)-cholesterol. Insulin resistance and dyslipidemia
are considered part of the metabolic ensemble of Syndrome X, which confers an increased risk for the
development of type 2 diabetes and cardiovascular disease.

A single therapy (monotherapy) with either antiandrogens or insulin sensitizers are among the most
commonly used therapeutic approaches in PCOS. Treatment with flutamide, a non-steroidal anti-androgen,
which prevents the binding of testosterone to its receptor, reduces hirsutism and reduces circulating levels
of androgens, triglycerides and LDL-cholesterol in adolescents and women with PCOS, but does not
restore normal menstrual cycles. In addition, it fails to increase HDL-cholesterol, known as good
cholesterol, or to decrease hyperinsulinemia; thus, it fails to affect two major risk factors for subsequent
cardiovascular disease. Insulin-sensitizing treatment with metformin is known to reduce the
hyperinsulinism, the hyperandrogenism, and the blood vessel risk of the lipid profile, and to restore normal
menstruation and ovulatory function, but it may be less effective in decreasing hirsutism.

Our study assessed whether combined low-dose flutamide+metformin treatment is more effective than
either antiandrogen or insulin-sensitizing monotherapy to correct endocrine-metabolic anomalies in 31 non-
obese, young women with PCOS [age, 19.2 yr; body mass index, 21.9 Kg/m2 (normal range = 18-25)]. The
patients were randomly assigned to receive flutamide (250 mg), metformin (1275 mg) or
flutamide+metformin, once daily, for 6 months.

All treatments were well tolerated; indices of liver and kidney function were stable throughout the study.
Dual treatment with flutamide+metformin matched or exceeded the efficacy of the most effective
monotherapy, as it virtually normalized hyperandrogenemia and dyslipidemia, indicating that
hyperinsulinism and hyperandrogenism each contribute to these endocrine-metabolic abnormalities.
Flutamide was accompanied by a decrease in circulating hemoglobin, while metformin monotherapy was
accompanied by an increase in circulating hemoglobin; during our co-treatment study the metformin effect
prevailed, and circulating hemoglobin increased.

Of the two monotherapies studied in this cohort, metformin seems to be more effective than flutamide, as
judged by safe near-normalization of ovulation rate and menstrual pattern, by increased hemoglobin, and
by correction of hyperinsulinism, dyslipidemia, and hyperandrogenemia. Accordingly, metformin is a
monotherapy of first choice, in particular if there is a concomitant risk or wish for pregnancy; continuous
metformin treatment during pregnancy in women with PCOS appears to safely reduce first-trimester
spontaneous abortion and does not appear to be harmful to the fetus (teratogenic).Through interactions that
remain to be fully uncovered, flutamide+metformin co-treatment was superior to either monotherapy in
normalizing lipid problems and in decreasing hyperandrogenism. Hence, anti-androgen and insulin-
sensitizing co-treatment may be regarded as first choice in the initial approach of PCOS, specifically if
there is no risk for pregnancy (because of potential flutamide teratogenicity) and if therapy aims at long-
term prevention of type 2 diabetes and cardiovascular disease related to insulin resistance and dyslipidemia.




                                                    - 178 -
                                           ANDROGENIC DISORDERS IN WOMEN
P2-468
ANDROGEN DEFICIENCY IN WOMEN WITH ANOREXIA NERVOSA
KK Miller, A Klibanski, SK Grinspoon. Neuroendocrine Unit, Massachusetts General Hospital, Boston

Androgen deficiency is associated with osteopenia and decreased lean body mass. Chronic undernutrition
causes dysfunction of the hypothalamic-pituitary-gonadal and adrenal axes, thereby affecting critical
sources of testosterone and its precursors in women. We hypothesized that amenorrheic women with
anorexia nervosa would have reduced serum testosterone levels compared to normals and that
hypoandrogenemia would be a determinant of bone density(BD)in this population. We therefore
determined serum testosterone levels, BD and body composition in 89 women with anorexia nervosa(AN)
not taking estrogen and 30 eumenorrheic normal-weight controls(C) of comparable age [25±1 (AN) vs.
24±1 (C) years]. Body composition and BD were measured using dual x-ray absorptiometry (Hologic
4500). Body mass index (BMI) was significantly lower in the women with AN compared with control
subjects (17.2±0.2 vs. 21.1±0.3 kg/m², p < 0.0001). Serum total testosterone levels were significantly lower
in the women with AN compared with controls (23.0±1.5 vs. 31.7±2.6 ng/dl, p = 0.004). In a stepwise
regression model, IGF-1 accounted for 31% of the variation in serum testosterone levels seen (r² = 0.31, p =
0.0009), and serum testosterone was significantly correlated with total body BD T score (r = 0.41, p =
0.05), lateral spine T score (r= 0.22, p = 0.05), total hip T score (r= 0.23, p = 0.05), body mass index (r=
0.27, p = 0.01), total body fat (r= 0.32, p = 0.02), age (r= - 0.32, p = 0.003), and number of months of prior
estrogen therapy (r = -0.26, p= 0.02). The association between serum testosterone and total body BD T
score remained significant even after controlling for BMI. However, the correlations between testosterone
and BD at other sites were not significant after controlling for BMI. Our data demonstrate decreased serum
testosterone in women with anorexia nervosa. Reduced IGF-1 and BMI may contribute to the
hypoandrogenemia seen in this population. The association between serum testosterone and bone mineral
density may in part reflect the effect of BMI on BD in this population. The pathophysiologic basis of
androgen deficiency and the effects of androgen replacement in women with anorexia nervosa remain to be
determined.

Clinical Science Poster: Androgenic Disorders in Women (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 179 -
                                          ANDROGENIC DISORDERS IN WOMEN
P2-468

Lay explanation of abstract:

Anorexia nervosa is an increasingly prevalent disorder in young women, one that is characterized by severe
low weight, absent menstrual periods, and osteoporosis. We hypothesized that because ovarian function is
not normal in these women, they would have low blood testosterone levels. Moreover, we hypothesized
that testosterone levels would be important determinants of bone density in these women.

We therefore measured blood testosterone levels and bone density in 89 women with anorexia nervosa, and
30 healthy women of comparable age with regular menstrual periods. Our results indicate that testosterone
levels are significantly lower in women with anorexia nervosa compared with healthy women of similar
age (23.0 vs. 31.7 ng/dl). Moreover, testosterone levels correlated with bone density at many skeletal sites.
Thus, our data suggest that low blood levels of testosterone might contribute to the low bone density
observed in women with anorexia nervosa. However, the importance of testosterone as a factor contributing
to low bone density has not been determined, and the effect of testosterone administration on bone density
is unknown.

Additional background information:

Anorexia nervosa is a chronic disorder of self-imposed caloric restriction that affects 1-2% of college-age
women. Severe osteoporosis is a common complication of this eating disorder. Low testosterone levels in
men are associated with osteoporosis, and testosterone administration results in an increase in bone density
in men. Women also produce testosterone, but their blood levels are about one-tenth those present in men.
The ovaries and adrenal glands, which are responsible for testosterone production in women, do not
function normally in women with anorexia nervosa. For example, they have absent menstrual periods
which reflect abnormal ovarian function, and their adrenal glands over-produce cortisol, a stress hormone.
It is not known whether women with anorexia nervosa have reduced testosterone levels compared with men
and whether this contributes to the osteoporosis observed in this population.

This study was funded by an NIH grant.




                                                  - 180 -
                                      AUTOIMMUNE ENDOCRINE DISORDERS
P3-208
SELENIUM SUBSTITUTION REDUCES THYROID PEROXIDASE AUTOANTIBODY
CONCENTRATION IN PATIENTS WITH AUTOIMMUNE THYROIDITIS
B Gasnier, R Gaertner, M Angstwurm, J Dietrich. Endocrinology, Medizinische Klinik University, Munich,
Germany

It has previously shown, that selenium deficiency may contribute to the development and the maintenance
of autoimmune thyroiditis. In addition, selenium dependent enzymes have several modulatory effects on
the immune system. We therefore performed a blinded, placebo controlled prospective study in female
patients (n=72, mean age 42y) with autoimmune thyroiditis and TPOAb and/or TgAb above 350 U/ml,
measured by an immuno-luminiszenz assay (Byk Sangtec). The primary endpoint of the study was the
change in the autoantibody concentration as a marker for the activity of the disease. Patients were
randomised into two age and antibody matched groups groups; 36 patients received 200 µg sodium selenite
per day for three months, 36 patients received placebo. All patients were substituted with L-thyroxine to
maintain TSH within the normal range. TPOAb and TgAb concentrations, thyroid hormone levels as well
as ultrasound of the thyroid were monitored. After 3 months, 9 patients in the selenium treated group had
completely normalized antibody titers in contrast to 2 patients in the placebo group (chiquadtrat test:
p=0.02). Ultrasound of the thyroid showed a normalised echogeneity in these patients. The mean TPOAb
concentrations decreased significantly to 51% (paired t-test: p=0.042) in the selenium group versus 90%
(p=0.73) in the placebo group. The TgAb concentrations were unchanged in both groups. A subgroup
analysis of those patients with TPOAb >1200 U/ml (n=8) revealed a mean 40% reduction in the selenium
treated patients compared to a 10% increase in the placebo group. The mean TSH, FT4 and FT3 levels
were unchanged in both groups during the study.
We conclude from this pilot study, that a selenium substitution with 200 µg sodium selenite may improve
the inflammatory activity in patients with autoimmune thyroiditis. Whether this effect is specific for
autoimmune thyroiditis or may also be effective in other organ specific autoimmune diseases has to be
investigated.

Clinical Science Poster: P3: Autoimmune Endocrine Diseases (11:00 AM-12:00 PM and 2:30 PM-3:30
PM)

Presentation Date: Friday, June 22, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                - 181 -
                                        AUTOIMMUNE ENDOCRINE DISORDERS
P3-208

Lay explanation of abstract:

Autoimmune thyroid diseases are very common. About 10 % of females and 2 % of males suffer from this
disorder. Until now, there was no possibility to prevent the onset of the disease; it was only possible to treat
them symptomatically by giving thyroid hormones, or in case of thyrotoxicosis, by blocking thyroid
hormone synthesis.

Selenium is a trace element, important for the function of several enzymes that modulate the immune
function. It has been shown previously, that in areas with low selenium intake, the prevalence of thyroid
autoimmune diseases is more common. Therefore, we investigated whether a supplementation of selenium
(200 µg/day) for three months in patients with thyroid autoimmune disease (Hashimotos´ thyroiditis) may
change the autoantibody (thyroid peroxidase antibodies) concentrations, which are reflecting the activity of
the disease. This selenium supplementation has no side effects, it even has been shown to prevent the
development of several cancers and to improve immune function (for review see: M.P. Rayman, The
importance of selenium to human health. Lancet Vol. 356, 2000, p. 233). In addition, the selenium
supplementation is very cheap.

We performed a placebo-controlled prospective study, including 72 age-matched females (mean age 42y)
with active autoimmune thyroiditis. Thirty-six of them received sodium selenite, 200 µg orally per day, and
36 received placebo. The patients were informed and agreed to participate in the study. We found that the
antibody concentrations significantly decreased to 51% in the treatment group, and 9 patients out of the
treatment group were completely normalized in antibody titers and thyroid ultrasound compared to only 2
in the placebo group. This clearly indicates that patients with autoimmune thyroid disease benefit from
selenium supplementation. We can suggest, especially during onset of the autoimmune thyroid diseases,
that a selenium supplementation prevents progression of the disease. We must further investigate whether
other, more severe autoimmune diseases can be influenced or even prevented. Selenium therapy would be
a new, cost-effective and safe concept of treatment.

Additional background information:

The trace element selenium is an essential nutrient of fundamental importance to human health. In the past
few years, several studies have shown a hitherto unsuspected role for this element. Selenium has a
component of selenoproteins, most of which have important enzymatic functions. Most selenoproteins have
an important role in the redox system, and their function is to scavenge free radicals. This function helps to
maintain membrane integrity, protects from protacyclin production, and reduces the propagation of further
oxidative damage to biomolecules such as lipids, lipoproteins, and DNA. (These biomolecules are
associated with increased risk of conditions such as atherosclerosis, cancer and also autoimmune disease.)

We were especially interested in the role of selenium in treatment of autoimmune thyroid diseases, because
it already has been shown that in endemic selenium deficiency, the prevalence of this disease is increased.
We succeeded in demonstrating in a clinical trial that in patients with active autoimmune thyroiditis,
supplementation with moderate, low-sodium selenite orally significantly decreases autoantibody
concentrations and disease activity. This beneficial effect of selenium has to be studied further in more
severe autoimmune diseases.

This study has not been funded by the government. We received free medication from: biosyn –
Arzneimittel GmbH, 70734 Fellbach, Germany.




                                                    - 182 -
                                      AUTOIMMUNE ENDOCRINE DISORDERS
P3-211
HIGH PREVALENCE OF AUTOIMMUNE THYROIDITIS IN PATIENTS WITH POLYCYSTIC
OVARY (PCO) SYNDROME
R Gaertner, N Mehlmauer. Endocrinology, Medizinische Klinik Innenstadt, University, Munich, Germany

Females are known to have a 5-10 fold higher incidence of organ specific autoimmune diseases including
autoimmune thyroiditis compared to males, suggesting a role of female hormones in the pathogenesis of the
disease. During the perimenopause, the incidence of newly developing autoimmune thyroiditis is about
twice as high as during normal reproductive life. The low progesterone levels and still elevated estrogen
levels may contribute to this increased autoimmune activity. As patients with PCO syndrome also have low
progesterone levels due to their anovulatory cycles, we now investigated in a prospective study the
incidence of autoimmune thyroiditis in caucasian patients with PCO syndrome. Over a period of 12 months
all patients with PCO syndrome (n=58), mean age 25y±8.4 were included in the study. PCO syndrome has
been documented by increased LH/FSH ratio >2, elevated plasma testosterone levels, hypertrichosis,
oligomenorrhea and polycystic ovaries shown by ultrasound. These patients were tested for TPOAb as well
as TgAb concentrations by a commercial test kits (Byk Sangtec). In addition TSH, FT4, FT3 was
determined and ultrasound of the thyroid was performed. For control, the incidence of postitive TPOAb and
TgAb concentrations (>100 U/ml) in 106 age matched patients was investigated. In the control patients,
10% had positive TPOAb and/or TgAb. In contrast, from the 58 patients with PCO syndrome, 25 (43.1%,
p=0.0001) had elevated TPOAb (mean 246±251U/ml and/or TgAb (mean 361±578 U/ml), 20 of them
hypoechogeneity in thyroid ultrasound. Eight patients were under L-thyroxine substitution and two patients
operated because of Graves disease. Free thyroid hormone levels as well as TSH was normal in all patients.
This prospective study clearly demonstrates a 4-fold higher prevalence of thyroid specific antibodies in
patients with documented PCO syndrome compared to controls, and 17% of them had clinical autoimmune
thyroiditis. This might be due to the impaired progesterone/estrogen levels compared to normal young
females and therefore a stimulation of T- and B-lymphocyte activity or due to different genetic background.

Clinical Science Poster: P3: Autoimmune Endocrine Diseases (11:00 AM-12:00 PM and 2:30 PM-3:30
PM)

Presentation Date: Friday, June 22, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                 - 183 -
                                       AUTOIMMUNE ENDOCRINE DISORDERS
P3-211

Lay explanation of abstract:

Females are known to have a 5-10-fold higher incidence of organ-specific autoimmune diseases including
autoimmune thyroiditis compared with males. This higher incidence suggests that female hormones play a
role in the pathogenesis of this disease. During perimenopause (time just before menopause), the incidence
of newly developing autoimmune thyroiditis is about twice as high as during normal reproductive life. The
low progesterone levels and still elevated estrogen levels during this period may contribute to this increased
autoimmune activity.

Because young females with PCO syndrome (polycystic ovary syndrome) also have low progesterone
levels due to their anovulatory (non-ovulating) cycles, we investigated in a prospective study the incidence
of autoimmune thyroiditis in Caucasian patients with PCO syndrome. Over a period of 12 months, all
patients with PCO syndrome (n=58), mean age 25+8.4y were included in the study. PCO syndrome has
been documented by an increased LH/FSH ratio >2, low progesterone and/or elevated plasma testosterone
levels, hypertrichosis, oligomenorrhea, and polycystic ovaries shown by ultrasound in most of the patients.
All patients were tested for TPOAb as well as TgAb concentrations by commercial test kits (Byk Sangtec).
In addition TSH, FT4, FT3 was determined and ultrasound of the thyroid was performed. For control, the
incidence of positive TPOAb and TgAb concentrations (>100 U/ml) in 106 age-matched patients was
investigated. In the control patients, 10% had positive TPOAb and/or TgAb. In contrast, among the 58
patients with PCO syndrome, 25 (43.1%, p=0.0001) had elevated TPOAb and/or TgAb, 20 of them
hypoechogeneity in thyroid ultrasound. Eight patients were under L-thyroxine substitution, and two
patients already had had operations because of Graves´ disease. Free thyroid hormone levels as well as
TSH was normal in all patients.

  This prospective study clearly demonstrates a 4-fold higher prevalence of autoimmune thyroiditis in
patients with documented PCO syndrome. This might be due to the impaired estrogen/progesterone levels
compared to normal young females and therefore a stimulation of T- and B-lymphocyte activity or due to
different genetic background. The clinical consequence should be that all patients with PCO syndrome
should be tested and followed up for their thyroid function. The result of the study also is of interest
because it gives further insight into the pathogenesis of autoimmune diseases. Estrogens, if not
counteracted by progesterones, seem to be responsible for the increased activity of the immune system.
Therefore, a patient‘s genetic disposition for developing autoimmune diseases might propagate the disease.

Additional background information:

The results of this study have two implications:
1) Clinical: PCO syndrome is a common disease; about 5% of all women are suffering from this disease.
We found that about half of these women also have autoimmune thyroiditis, so these patients also have to
be tested for thyroid function and vice versa. In addition, progesterone substitution in these patients might
be useful in preventing the disease.
2) Pathophysiological: It is known from in vitro studies that estrogens stimulate the immune system,
whereas progesterones decrease this activity. Our hypothesis therefore was, that females with a lack of
normal progesterone production but normal estrogens might develop a higher incidence of autoimmune
diseases. Because patients with PCO syndrome, which is characterized by low progesterone levels, have a
4-fold higher incidence of autoimmune thyroiditis, this seems to support our hypothesis. It also appears that
progesterones or derivatives of this hormone with a specific target to immune competent cells are a suitable
immunosuppressive agent.

This study has not been funded by the government or a company. All work was performed without external
financial support.




                                                   - 184 -
                                                   BONE-CALCIUM-PARATHYROID
OR42-1
THE MINIMAL SERUM LEVEL OF ESTRADIOL TO IMPROVE CATABOLIC BONE
METABOLISM IN PATIENTS WITH ANOREXIA NERVOSA
M Hotta,1 K Jibiki,2 R Okaji,2 E Odagiri,2 K Sato,1 K Takano.1 1Department of Medicine, Tokyo Women's
Medical University, Tokyo, Japan; 2Radioisotope Center, Tokyo Women's Medical University, Tokyo,
Japan

Estrogen deficiency is one of the risk factors for bone loss in patients with anorexia nervosa (AN). Serum
estradiol (E2) levels in severely emaciated AN patients are typically undetectable by conventional assay
methods. To investigate the relationship between serum E2 levels and bone metabolism, we measured
serum E2 levels using a highly sensitive radioimmnoassay (ESTR-US-CT, CIS International, Saclay,
France), bone formation marker (serum osteocalcin), and bone resorption marker (urinary excretion of C-
terminal telopeptides of collagen type Ι (CrossLaps) ) in 58 female AN patients. Serum E2 levels decreased
to less than 10 pg/mL in 29 of 58 AN patients and were undetectable in 5 patients (<1.36 pg/mL). Serum
E2 levels were positively correlated with BMI (r=0.775, p<0.0001). Urinary CrossLaps excretion was the
highest (775 ± 91 µg/mmol Cr) in patients whose serum level of E2 was below 1.36 pg/mL and was
significantly decreased (545 ± 59 µg/mmol Cr) in patients with serum E2 level ranging from 1.36 to 3
pg/mL (p=0.018). Urinary CrossLaps excretion was further decreased as serum E2 levels increased, and
reached those of age-matched healthy women (176 ± 25 µg/mmol Cr) in patients with a serum E2 level
between 7 and 9 pg/mL (206 ± 23 µg/mmol Cr). In 5 patients with the undetectable serum E2 level, urinary
CrossLaps excretion was studied again when their BMI increased. The bone resorption marker was
significantly decreased (p<0.0041) when serum E2 levels increased to detectable values, namely 2.3 to 3.0
pg/mL (2.67 ± 0.12 pg/mL). Serum osteocalcin levels in patients with serum E2 levels below 5 pg/mL were
decreased, and were raised to the normal range in patients with E2 levels of 5 to 11 pg/mL.
We have demonstrated that markedly increased bone resorption in severely emaciated AN patients is
significantly decreased when serum E2 levels increase from the undetectable level to values of 1.36 to 3
pg/mL, suggesting that catabolic bone metabolism in AN patients may be improved at very low
concentrations of serum E2.

Clinical Science Oral: Bone-Calcium-Parathyroid (11:00 AM-12:30 PM)

Presentation Date: Saturday, June 23, 2001; Time: 11:00 AM; Location: A 106




                                                 - 185 -
                                                   BONE-CALCIUM-PARATHYROID
OR42-1

Lay explanation of abstract:

Estrogen deficiency is one of the risk factors for bone loss in patients with anorexia nervosa (AN). The
influence of estrogen deficiency on bone metabolism is characterized by faster breakdown of bone tissue
(bone resorption). However, serum estradiol (E2) levels are too low in AN patients to be detected by
conventional assay methods in which the highest sensitivity is 10 pg/mL.

To investigate the relationship between serum E2 levels and bone metabolism, we measured serum E2
levels in 58 female AN patients. We used a highly sensitive radioimmunoassay (ESTR-US-CT, CIS
International, Saclay, France) for formation marker (serum osteocalcin) and bone resorption marker
(urinary excretion of C-terminal telopeptide of collagen type I (Cross Laps)

Serum E2 levels decreased to less than 10 pg/mL in 29 of 58 AN patients and were undetected in 5 patients
(<1.36 pg/mL). Serum E2 levels increased in proportion to body mass index (BMI) and correlated
positively with BMI (r=0.775, P<0.0001). Urinary excretion of bone resorption marker was highest in
patients with serum E2 levels ranging from 1.35 to 3 pg/mL (p=0.018). Bone resportion marker was further
decreased as serum E2 levels increased and reached those of age-matched healthy women, in patients with
a serum E2 level between 7 and 9 pg/mL.

We have demonstrated that bone resorption is very sensitive to extremely low concentrations of E2.
Previous reports indicated that serum E2 levels from 3.6 to 5 pg/mL would exert an effect on bone
resorption. We have indicated that the minimal serum E2 levels for normalization of bone resorption range
from 7 to 9 pg/mL. These findings should be clinically useful for estrogen replacement therapy in AN
patients.




                                                 - 186 -
                                                   BONE-CALCIUM-PARATHYROID
OR42-4
INCREASED BONE DENSITY FOLLOWING TWO YEARS OF GROWTH HORMONE(GH)
TREATMENT IN MEN WITH IDIOPATHIC OSTEOPOROSIS
P Gillberg,1 H Mallmin,2 M Petren-Mallmin,3 O Ljunggren,1 S Ljunghall,1 AG Nilsson.1 1Department of
Internal Medicine, University Hospital, Uppsala, Sweden; 2Department of Surgical Sciences, University
Hospital, Uppsala, Sweden; 3Department of Radiology, University Hospital, Uppsala, Sweden

GH has been suggested as an anabolic agent in the treatment of osteoporosis. We have investigated the
effects of continuous and intermittent GH treatment on bone turnover, bone size, bone mineral density
(BMD) and content (BMC) in 29 men, age 27-62 years, with idiopathic osteoporosis, defined as BMD in
lumbar spine (LS) or femoral neck (FN) <-2.5 SD of the mean BMD of a young reference population. The
patients were randomly assigned to treatment with recombinant human GH 0.4 µg daily (group A, n=14) or
0.8 µg/day for 14 days every three months (group B, n=15) for 24 months, with a follow-up period of 12
months. All patients received supplements with calcium and vit D3 daily during all 36 months. At baseline,
after 2 weeks, 1, 6, 12, 18 , 24, 30 and 36 months fasting morning urine and serum samples were obtained
for assay of IGF-I, bone markers and routine lab tests. Body composition, BMD and BMC were determined
by DXA at baseline and every 6 months. Radiograms of the right hand were obtained at baseline and after
24 months to evaluate changes in the cortical thickness (CT) of the second metacarpal bone. After two
years, there was an increase in BMD LS by 4.1% in group A and in total body (TB) by 2.6% in group A
and 2.7% in group B. BMC and lean body mass increased and fat mass decreased in both groups. After 36
months the BMD in LS and TB and BMC had increased by 4.6%, 3.7% and 4.0% respectively in group A
and by 6.6%, 3.2% and 3.3% in group B. The serum osteocalcin level was increased in both groups after
one month and remained so throughout the study. In group A, the urinary deoxypyridinoline excretion was
increased during the GH treatment period. In group B it was only increased after one month of treatment.
There were no significant changes in CT during the study. This study shows that 2 years of treatment with
GH in men with idiopathic osteoporosis results in an increase in BMD and BMC which is sustained and
possibly further enhanced for at least one year posttreatment.

Clinical Science Oral: Bone-Calcium-Parathyroid (11:00 AM-12:30 PM)

Presentation Date: Saturday, June 23, 2001; Time: 11:45 AM; Location: A 106




                                                 - 187 -
                                                     BONE-CALCIUM-PARATHYROID
OR42-4

Lay explanation of abstract:

In most cases, osteoporosis is caused by an increased activity in cells that are engaged in the breakdown of
calcified bone. Most treatments for this disease have been directed to decrease this activity. Growth
Hormone (GH), a hormone produced by the pituitary gland, has been suggested to be an anabolic agent in
the treatment of osteoporosis, not only to inhibit breakdown of calcified bone, but also to stimulate the
formation of new bone.

In this study we have investigated the effects of GH given every day to 14 men (Group A) and for two
weeks every three months to 15 men (Group B), age 27-62 years, with idiopathic osteoporosis for a period
of two years. The follow-up period was one year. All men received calcium and vitamin D in addition to
the GH treatment. The density (BMD) and bone mineral content (BMC) of the skeleton was measured by
DXA, a radiographic examination.

After three years, there was an increase in BMD in lumbar spine and in BMC in Group A by 4.6% and
4.0%, respectively, and in Group B by 6.6% and 3.3%, respectively. This study shows for the first time that
GH treatment for two years in men with idiopathic osteoporosis results in increased BMD and BMC.

Additional background information:

In females, most osteoporosis is caused by the estrogen deficiency that menopause induces. In men, the
main reasons for osteoporosis are glucocorticoid therapy, gastro-intestinal diseases, and alcoholism.
However, in about a third of all cases, no secondary cause for the disease is found, and the condition then is
termed primary or idiopathic osteoporosis. There have only been a few studies concerning the treatment of
this condition with Growth Hormone, and to our knowledge no study of this duration has been conducted
before.

This study was supported by Pharmacia & Upjohn.




                                                   - 188 -
                                                  BONE-CALCIUM-PARATHYROID
OR42-5
EFFECT OF LY333334 [RECOMBINANT HUMAN PARATHYROID HORMONE (1-34),
RHPTH(1-34)] ON BONE DENSITY WHEN GIVEN TO POSTMENOPAUSAL WOMEN
RECEIVING HORMONE REPLACEMENT THERAPY (HRT)
LG Ste-Marie,1 WH Scheele,2 S Jasqui,3 SL Schwartz,4 G Pohl,2 GA Gaich.2 1Ctre Recherche CHUM, Hop.
Saint-Luc, Montreal, Quebec, Canada; 2Eli Lilly and Company, Indianapolis, Indiana; 3Instituto Nacional
de la Nutricion Salvador Zubiran, Mexico D.F., Mexico; 4Diabetes & Glandular Research Center, San
Antonio, Texas

Parathyroid hormone, given once daily, activates new bone formation and increases bone mass in patients
with osteoporosis. The purpose of this study was to determine the effectiveness of 40µg of rhPTH(1-34)
(PTH40) when used in combination with ongoing or de novo HRT. A randomized, placebo-controlled
clinical trial was conducted in 5 countries. Postmenopausal women with osteopenia or osteoporosis were
stratified into 2 groups: current use of HRT for >1 yr or no use of HRT for >1 yr. Within these strata,
women were randomly assigned to either PTH40 (n=122) or placebo (n=125), given once daily by self-
injection (median exposure of 15 mos). All women received HRT. Bone mineral density (BMD) or content
(BMC) was measured by DXA. rhPTH(1-34)+HRT significantly increased BMD at the lumbar spine and
proximal femur compared to HRT alone, whether started simultaneously with, or added to, ongoing HRT.
In the rhPTH(1-34)+ de novo HRT group, there was also an increase in total body BMC and ultradistal
radius BMD. In conclusion, these results suggest that rhPTH(1-34) may be used effectively in combination
with HRT; however, the results do not establish that the combination would yield greater fracture risk
reduction than that achieved with rhPTH(1-34) alone.

Skeletal site (BMD     Ongoing HRT       PTH40 + ongoing         de novo HRT      PTH40 + de novo
g/cm2, BMC g)          (% change)        HRT (% change)          (% change)       HRT (% change)
Lumbar spine BMD       1.5               11.2 (P<0.001)          4.6              16.9 (P<0.001)
Total hip BMD          0.2               3.4 (P<0.001)           2.9              7.3 (P<0.001)
Femoral neck BMD       0.7               3.2 (P<0.001)           3.4              7.5 (P<0.001
Total body BMC         -0.7              3.1                     2.6              6.2 (P<0.001)
Radius - ultradistal
                       0.6               0.6                     1.8              4.2 (P<0.05)
BMD
Radius - 1/3 distal
                       -0.3              -0.7                    1.4              0.6
BMD


Clinical Science Oral: Bone-Calcium-Parathyroid (11:00 AM-12:30 PM)

Presentation Date: Saturday, June 23, 2001; Time: 12:00 PM; Location: A 106




                                                - 189 -
                                                   BONE-CALCIUM-PARATHYROID
OR42-5

Lay explanation of abstract:

Parathyroid hormone is the main regulator of calcium and bone metabolism in the human body. Various
forms of parathyroid hormone (PTH), when given once daily, are known to stimulate formation of new
bone, increase bone density, and reduce the risk of bone fractures in postmenopausal women having the
bone-thinning disease osteoporosis. In contrast, hormone replacement therapy (HRT) slows or stops bone
loss by reducing destruction of bone.

This study explored the effects of PTH and HRT in postmenopausal women with osteoporosis. The primary
goal was to determine if a specific form of PTH [rhPTH (1-34)} + HRT would increase bone density more
than either treatment alone. A second goal was to learn whether the two treatments could be given together
safely.

Patients given rhPTH (1-34) + HRT together had substantial increases in bone density at the hip and spine,
and no safety issues were identified. The authors concluded that rhPTH (1-34) + HRT can be used
effectively together to treat osteoporosis, but cautioned that the study did not establish that the two
treatments together would give greater protection from fractures than rhPTH 1-34 alone.

This study was funded by Eli Lilly and Company, which is developing this rhPTH (1-34) compound as the
proposed brand Fortéo™ (generic name: teriparatide injection, rDNA origin, Lilly).




                                                 - 190 -
                                                     BONE-CALCIUM-PARATHYROID
P1-464
RISEDRONATE PREVENTS FIRST VERTEBRAL FRACTURE IN POSTMENOPAUSAL
WOMEN
E Boling,1 M Hooper,2 I Barton.3 1Loma Linda University, Loma Linda, California; 2Concord Repatriation
Hospital, Concord, Australia; 3Procter & Gamble Pharmaceuticals, Staines, United Kingdom

It has been demonstrated that the presence of vertebral fractures is associated with increased risk for future
fracture. Consequently, prevention of the first vertebral fracture is an important component of osteoporosis
management. The effectiveness of risedronate (RIS) in reducing the risk of the first vertebral fracture in
postmenopausal women at risk for fracture was examined in 3 studies ranging in length from 1.5-3 years.
BMD-NA (North America) and BMD-MN (Multi-National) enrolled 1191 patients based on low lumbar
spine bone mineral density (T-score <-2), VERT-NA (North America) enrolled 2458 patients based on a
combination of prevalent vertebral fractures and low spinal BMD. Patients received RIS or placebo daily,
in addition to calcium (1000 mg/d), and < 500 IU/d vitamin D if levels were < 40 nmol/L. A total of 383
patients were enrolled without prevalent vertebral fracture and low spinal BMD (LS T-score < -2.5). Across
the studies, the mean age ranged from 63-71 years and the mean LS T-score was -3.2.
Risedronate significantly reduced the risk of the first vertebral fracture by 70% (p=0.03) in women without
prevalent vertebral fractures and low LS BMD (T-score < -2.5) in studies up to 3 years. Adjustments for
age or baseline LS T-score did not affect the reduction in risk.
To conclude, risedronate is effective in reducing the risk of the first vertebral fracture in postmenopausal
women at risk for vertebral fracture, an important goal in the management of osteoporosis.

Clinical Science Poster: Bone-Calcium-Parathyroid I (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 191 -
                                                      BONE-CALCIUM-PARATHYROID
P1-464

Lay explanation of abstract:

Studies have shown that people with osteoporosis who have backbone (vertebral) fractures are at an
increased risk for developing even more fractures in the future. Therefore, an important part of osteoporosis
management is preventing the first vertebral fracture.

The effectiveness of risedronate in reducing the risk of the first vertebral fracture in postmenopausal
women at risk for fracture was examined in three studies (BMD-MN, BMD-NA and VERT-NA) over a
1.5 - 3 year period. A total of 4,032 women, who either had vertebral fractures or were at a high risk of
developing fractures, were included in these three studies. This analysis looked at the 383 patients who did
not already have a vertebral fracture but were at risk for developing a fracture as determined by bone
mineral density testing. The test measures the difference in a patient‘s bone mass compared with peak bone
mass in a young adult of the same sex. The mean age across all studies ranged from 63 - 71 years. All of
the women in the study were given risedronate or placebo with a supplement of calcium and vitamin D (to
help absorb calcium), if needed. The studies found that women who took risedronate reduced the risk of
developing a first vertebral fracture by 70%, which is statistically significant compared to women who
received placebo.

In conclusion, by taking risedronate, postmenopausal women who are at risk of vertebral fractures can
possibly avoid them, which is an important goal in the management of osteoporosis.

Additional background information:

Risedronate is a drug of the bisphosphonate class and is sold under the trade name Actonel® in most
countries for the prevention and treatment of postmenopausal osteoporosis and of osteoporosis caused by
taking glucocorticoids (like prednisone). Risedronate is also effective in the treatment of Paget‘s disease of
bone. Risedronate works by inhibiting bone resorption, and was recently shown to prevent hip fractures
over a period of 3 years in women with osteoporosis, as reported in The New England Journal of Medicine
(McClung et al., N Engl J Med 2001;344;333-340).

Osteoporosis is a disease characterized by low bone mass and structural deterioration of bone, leading to
bone fragility and an increased likelihood of bone fracture, especially of the hip, spine (vertebral fractures),
and wrist. Postmenopausal osteoporosis affects millions of women worldwide, including 22 million
American women. Women can lose up to 25% of their bone mass in the first 5 years following menopause,
making their bones more likely to weaken and break. About 40% of women over age 50 will have an
osteoporosis-related fracture in their lifetimes. Vertebral fractures can cause severe back pain, loss of
height, a curved back and stooped posture. This can lead to disability, loss of independence, and increased
health care costs. Intervening with a bisphosphonate like risedronate helps reduce the risk of vertebral
fractures within one year. Furthermore, studies have shown that approximately 1 in 5 women experience a
subsequent fracture within one year of sustaining their first vertebral fracture.

This study was funded by Procter & Gamble Pharmaceuticals, Inc., Cincinnati, Ohio, and by Aventis
Pharma, Bridgewater, New Jersey.




                                                    - 192 -
                                                    BONE-CALCIUM-PARATHYROID
P1-470
BONE FORMATION AND BONE RESORPTION ARE INDEPENDENTLY MEDIATED IN LEAN
ANOREXIA NERVOSA
B Galusca, M Kadem, D Frerre, F Lang, C Mounier, B Estour. CHU, St Etienne, France

Osteoporosis occurs in lean anorexia nervosa (AN) as a result of a bone metabolism uncoupling, which
includes a decreased bone formation and an increased bone resorption. It is known that multiple hormonal
abnormalities impair the bone turnover in this disease. Conversely, the refeeding induces a recover of this
unbalance. There is no data proving a correlation between osteocalcine (BGP) and cross laps (CTX), in
AN. So, the hypothesis of an independent pathway for each process is possible. The aim of this study is to
evaluate the impact of hormonal and nutrition anomalies on bone markers regulation in AN.
Material and Method: In a group of 60 subjects with AN (DSM IV), mean BMI=14.1 kg/m2 (9 to 17.5), the
following parameters were determined: bone markers (serum intact BGP and CTX - mean of 6 points
circadian cycles), nutritional markers (BMI, bioelectric impedance measurements - fat and lean mass),
hormones (fT3, fT4, LH, FSH, SDHA, IGF1, metadrenaline, normetadrenaline, mean of GH and cortisol),
other bone metabolism parameters (PTH, Ca, vitamin D). A statistical analysis has been performed using a
correlation matrix (including all the parameters and their log values) and a step by step regression modeling
for BGP and CTX.
Results: There is no correlation between BGP and CTX in lean AN (r=0.13, p0.45, p<0.001). BGP values
showed a moderate correlation (r>0.45, p<0.001) with fT3 (r=0.58), IGF1 (0.63), mean cortisol (0.67) and
mean GH (r=0.53). CTX was also moderately correlated with BMI (r=0.5), fat mass (0.51),
normetadrenaline (r=0.48) and IGF1 (r=0.46). No other significant correlations have been found. In a step-
by-step regression model using 5 parameters, 66% of BGP variance was due to fT3, cortisol and IGF1, with
no effect of GH or FSH. In a model with 4 parameters, 40% of the CTX variance was determined by BMI.
Discussion and Conclusions: The lack of correlation between BGP and CTX in this group of lean AN was
explained by an independent mediation for each marker. So, CTX levels seem to be sensitive to nutritional
parameters like BMI. On the other hand the impact of fT3, cortisol, IGF1 on BGP levels proved the role of
these hormones in osteblastic receptor regulation. These results will be discussed before starting an
interventional trial on bone mass in lean AN.

Clinical Science Poster: Bone-Calcium-Parathyroid I (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 193 -
                                                   BONE-CALCIUM-PARATHYROID
P1-470

Lay explanation of abstract:


Osteoporosis is a frequent complication of anorexia nervosa (AN). It occurs at the same time because of
reduced bone formation and increased bone newborn. The effect of the hormonal and nutritional factors on
osteoporosis is poorly understood.

Thus, this study performed on 60 subjects with lean AN evaluated the influence of several hormonal and
nutritional parameters acting at the bone level on markers of the bone formation (osteocalcin) and bone
resorbtion (cross-laps). The results confirmed a prevalent hormonal effect (increased cortisol, decreased
IGF1 and fT3) on the reduction of bone formation. We have shown for the first time the role of
malnutrition in elevating the rate of bone newborn. Weight gain appears to be the best way to modify
levels of bone markers. Weight gain acts directly on bone resorbtion and indirectly on the bone formation
through the horomones cortisol and IGF1. However, weight gain among women with AN is often difficult,
so medical treatment might be beneficial and improve levels of these markers. Such treatment might
include anticortisolics, recombinant IGF, T3, and biphosphonates.


Additional background information:

This work has been published as : Anne Caillot-Augusseau, Marie-Hélène Lafage-Proust, Philippe
Margaillan, Nathalie Vergely, Sylvie Faure, Stephane Paillet, François Lang, Christian Alexandre, Bruno
Estour - Weight gain reverses bone turnover and restores circadian variation of bone newborn in anorexic
patients, Clinical Endocrinology (2000) 52, 113-121

This study was supported by a governmental research funding.




                                                 - 194 -
                                                   BONE-CALCIUM-PARATHYROID
P3-139
HIGH PHYTOESTROGEN INTAKE INCREASES BONE MINERAL DENSITY IN SOUTHERN
CHINESE WOMEN
J Mei, SS Yeung, AW Kung. Department of Medicine, The University of Hong Kong, Queen Mary
Hospital, Hong Kong, China

Animal studies demonstrated that phytoestrogen had protective effect against bone loss after ovariectomy.
However, data on dietary phytoestrogen intake as well as their relationship with bone mineral density
(BMD) in human are not available. 650 southern Chinese women, aged 19 to 86 years, were recruited to
determine their dietary phytoestrogen intakes by a food frequency questionnaire. BMDs at the lumbar spine
and hip region were measured using dual energy X-ray absorptiometry (DXA). The subjects were analyzed
according to tertiles of various phytoestrogen intakes. Among the postmenopausal women (n=357),
significant difference at L2-4 BMD (6.4%, p<0.05) was found between the highest and the lowest intake of
isoflavones after adjusting for age, height, weight, years since menopause, smoking, alcohol drinking, HRT
usage and daily calcium intake. The similar findings at femoral neck BMD (5.0%, p<0.05), trochanter
BMD (5.5%, p<0.05) were observed between the highest and the lowest intake of phytosterols. Women
with the highest intake of isoflavones had significantly lower serum PTH (19.24 ± 1.89 vs 27.23 ± 2.01
pg/mL, p<0.05), osteocalcin (4.88 ± 0.64 vs 6.67 ± 0.71 mg/L, p =0.07) and urinary N-telopeptides (NTx)
(34.28 ± 5.49 vs 51.02 ± 5.57 mmol BCE/mol Cr, p<0.05) when compared to those with the lowest intake
of isoflavones. Women with the higher intake of phytosterols also had lower ALP and osteocalcin values.
No association between dietary phytoestrogen and BMD was seen in premenopausal women (n=293). In
conclusion, habitual high dietary phytoestrogen intake is associated with higher BMD at both hip and spine
in postmenopausal women. The mechanism is probably through suppressing secondary
hyperparathyroidism and inhibiting high bone turnover associated with estrogen withdrawal.

Clinical Science Poster: P3: Bone-Calcium-Parathyroid II (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                 - 195 -
                                                   BONE-CALCIUM-PARATHYROID
P3-139

Lay explanation of abstract:

Phytoestrogens are a group of compounds found mainly in legumes. They are structurally similar to human
endogenous estrogen and possess weak estrogenic properties. These compounds, called phytoestrogens, are
found in several foods: soybeans and other beans, peas, lentils, soybean sprouts, podded plants, and soy
products such as tofu and soymilk. Only trace amounts of phytoestrogens are found in other plants.

This study investigated the phytoestrogen content in a typical Chinese diet and determined the link between
dietary phytoestrogen intake and bone mineral density (BMD) in postmenopausal Chinese women. A
higher level of bone mineral density correlates with stronger bones. A 10% reduction in BMD is associated
with a two-fold increase in bone fracture risk.

Among the 357 subjects we studied, the average phytoestrogen intake was 21mg/day. This figure is more
than seven times that of the Western population. We found that women with high daily phytoestrogen
intake had significantly higher BMD (+6.4%) at the lumbar spine compared to those with low daily
phytoestrogen intake. (High phytoestrogen intake was 60mg/day, the equivalent of about 2 pieces of
tofu/day or 50 g dry soybeans/day or 3 cups of soymilk/day. Low phytoestrogen intake was 3 mg/day,
equivalent to about 3 pieces of tofu/month, or 75 g dry soybeans/month or 3 cups of soymilk/month).

Our results suggest that phytoestrogen might exert a protective effect by suppressing secretion of some
bone regulatory hormones. This results in a reduction of bone loss associated with estrogen withdrawal in
postmenopausal women and helps prevent osteoporosis.

This research was funded by the Osteoporosis and Endocrinology Fund of the University of Hong Kong.




                                                 - 196 -
                                                   BONE-CALCIUM-PARATHYROID
P3-141
EFFECTS OF CONSUMPTION OF SOY CONTAINING ISOFLAVONES BY
POSTMENOPAUSAL WOMEN ON BIOCHEMICAL MARKERS OF BONE TURNOVER
LJW Lu,1 KE Anderson,1 JJ Grady,1 M Nagamani.2 1Preventive Medicine and Community Health, The
University of Texas Medical Branch, Galveston, Texas; 2Obstetrics and Gynecology, The University of
Texas Medical Branch, Galveston, Texas

Osteoporosis is a major health threat to millions of American women. Asian women consuming soy have
lower hip fracture rates than white women in the West. Soy isoflavones have weak estrogen-like activity
and may have bone-preserving effects. This study determined the effects of soy consumption on
biochemical markers of bone metabolism in 12 healthy postmenopausal women (49 to 66 yrs old, 1-13 yrs
postmenopausal) not on hormone replacement therapy. All women provided fasting blood samples 4 times
during 8 weeks of baseline observation period, 1 time at 2, 4, 10 and 16 weeks after soy feeding (total 4
times), and 1 time at 1-, 2- and 16-week after termination of soy feeding (total 3 times). During the soy
diet, women ingested a 36 oz-portion of soymilk daily that contained ~112 mg (range 76 to 161 mg) of
isoflavones. All the blood samples were analyzed for serum markers of bone turnover and PTH. Serum
osteocalcin and PTH (specific for the intact molecule) were measured by IRMA (Diagnostic Systems Lab.,
Webster, TX) and bone specific alkaline phosphatase and total deoxypyridinoline (bone resorption marker)
by enzyme-linked immunosorbent assays (Metra Biosystems, Mountain View, CA). Consumption of
soymilk containing >112 mg of isoflavones (aglycone units) induced time-dependent increases in bone
specific alkaline phosphatase by up to 17.9% (P = 0.002 by linear trend test and P=0.008 for comparison
between means of baseline and soy feeding periods), osteocalcin by up to 34% (P=0.064 by linear trend test
and P=0.006 for comparison between means of baseline and soy feeding periods), and deoxypyridinoline
by up to 21% (P = 0.045 by linear trend test and P=0.05 for comparison between means of baseline and soy
feeding periods). These stimulatory effects were reversed one week after termination of soymilk ingestion.
We observed significant within-subject variations in markers of bone metabolism. Soy feeding did not
affect PTH levels. These preliminary results suggest that soy may have bone-sparing effects. Supported by
USPHS CA65628, CA56273, CA45181, and NIH NCRR GCRC MO1 RR00073.

Clinical Science Poster: Bone-Calcium-Parathyroid II (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                 - 197 -
                                                    BONE-CALCIUM-PARATHYROID
P3-141

Lay explanation of abstract:

Osteoporosis is a major health threat to millions of American women. Asian women consuming soy have
lower hip fracture rates than white women in the West. Estrogen is known to be a critical player in
regulating bone strength, and plant estrogens may have similar effects. Soy isoflavones have weak
estrogen-like activity, and it has been previously suggested that these plant estrogens have bone-preserving
effects—thus explaining the low rate of hip fracture observed in Asian women consuming soy.

We studied 12 healthy postmenopausal women who were not on hormone replacement therapy to
determine whether soy consumption could alter bone metabolism. We measured the markers in blood that
may indicate the rate at which bone cells are replaced (turnover) in these women before, while, and after
they were on a soy diet containing ~112 mg (range 76 to 161 mg) of isoflavones for 16 weeks. We found
that soy consumption significantly increased the levels of markers for bone turnover in the blood of these
women, suggesting that the plant estrogens in soy may stimulate bone cell activities and may have bone-
sparing effects. More studies are needed to determine whether soy diet may reduce hip fracture rates in
American women.

Additional background information:

This research was conducted in the Department of Preventive Medicine and Community Health and the
General Clinical Research Center at the University of Texas Medical Branch, Galveston, TX. This
research was funded by the National Cancer Institute and the National Center for Research Resources -
General Clinical Research Centers Program of the National Institutes of Health, the American Institute for
Cancer Research, and the John Sealy Memorial Endowment. The research team includes Lee-Jane W. Lu,
Ph.D., Manubai Nagamani, M.D., Karl E. Anderson, M.D., and James J. Grady, Dr. P.H.




                                                  - 198 -
                                                   BONE-CALCIUM-PARATHYROID
P3-143
THE EFFECTS OF DIETARY SOY PROTEIN CONTAINING PHYTOESTROGENS ON LIPIDS
AND INDICES OF BONE TURNOVER IN POSTMENOPAUSAL WOMEN
F Dalais,1 HJ Teede,2 D Kotsopoulos,2 BP McGrath,2 PR Ebeling.3 1International Health and Development
Unit, Monash University, Alfred Hospital, Melbourne, Australia; 2Department of Medicine, Monash
University, Dandenong Hospital, Melbourne, Australia; 3Department of Endocrinolgy, Melbourne
University, Royal Melbounre Hospital, Melbourne, Australia

Background: It is well documented that estrogen replacement in postmenopausal women reduces bone
turnover, improves bone density and appears to reduce fracture risk. The soybean is rich in isoflavone
phytoestrogens, which are ligands for estrogen receptors. Potentially, these isoflavones may mimic
estrogenic effects on bone. To address the effect of phytoestrogens on bone turnover, we have measured
urinary bone turnover markers, in a double blind, placebo-controlled trial.Methods: 78 healthy
postmenopausal women were randomised to either dietary soy protein isolate (n=38) (40g soy protein,
118mg isoflavones) or casein placebo (n=40), with measurements at baseline and 3months. Results:
Urinary phytoestrogen excretion increased significantly in the soy group only. Lipid profiles improved with
a significant decrease in LDL cholesterol (-0.6±0.1 vs -0.29±0.09mmo/L, p<0.05), triglycerides
(-0.22±0.07 vs +0.01±0.05mmol/L, p<0.005) and LDL:HDL ratio (-0.32±0.1 vs 0.2±0.1, p<0.005) in the
soy group compared to placebo. No changes were noted in the indices of bone turnover in the soy
compared to the placebo group in either pyridinoline (-4.5±3.1 vs -0.8±3.1nmol/mmolCr, p=0.4) or
deoxypyridinoline (-1.5±1.1 vs -0.3±0.7nmol/mmolCr, p=0.4). Conclusion: In healthy postmenopausal
women, soy protein dietary supplementation containing phytoestrogens, does not appear to have estrogenic
effects on markers of bone resorption. Soy protein does have favorable effects on lipids, however these
differ from those observed with oral estrogen including a fall in triglycerides and no change in HDL. These
findings suggest that soy protein dietary supplementation containing phytoestrogens may not have
biologically significant estrogenic effects on bone turnover and that the lipid effects may be mediated
through non-estrogenic mechanisms.

Clinical Science Poster: P3: Bone-Calcium-Parathyroid II (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                 - 199 -
                                                   BONE-CALCIUM-PARATHYROID
P3-143

Lay explanation of abstract:

Estrogen in hormone replacement therapy in postmenopausal women reduces bone loss and improves bone
strength, leading to a reduction in fracture risk. The soybean is rich in phytoestrogens, which are plant-
based compounds that have been shown to attach to the estrogen receptor and may act like estrogen in the
body. We, along with other investigators, have previously shown beneficial, estrogen-like effects of
phytoestrogens on blood cholesterol levels. Potentially, these phytoestrogens may also mimic estrogen
effects on bone. To address the effect of phytoestrogens on bone in postmenopausal women, we completed
a double-blind, placebo-controlled trial.

This study involved 78 postmenopausal women, randomized to two groups, either dietary soy
supplementation (n=38) or milk protein placebo (n=40), for 3 months. Urine and blood samples were
collected at baseline and 3 months. Urinary phytoestrogen excretion was measured to reflect compliance.
Lipid profiles were completed including total plasma, LDL and HDL cholesterol, as well as triglycerides.
Urine chemical markers that reflect bone activity were used as markers of bone resorption.

Urinary phytoestrogen excretion increased in the soy group only. There was a significant decrease in blood
fat or triglyceride level and in the cholesterol LDL:HDL ratio in the soy group, compared to placebo.
Depite these observed lipid effects, soy with phytoestrogens did not significantly change bone activity.

We concluded that soy protein dietary supplementation containing phytoestrogens exerts favorable effects
on lipids. However, these findings suggest that soy protein dietary supplementation containing
phytoestrogens may not have biologically significant estrogenic effects on bone in postmenopausal women.

The study was funded by the National Heart Foundation of Australia.




                                                 - 200 -
                                                   BONE-CALCIUM-PARATHYROID
P3-144
RISEDRONATE THERAPY FOR 5 YEARS RESULTS IN SUSTAINED REDUCTION OF
VERTEBRAL FRACTURE RISK.
NB Watts,1 J Brown,2 D Hosking,3 S Adami,4 H Mulder,5 J Reginster,6 A Chines,7 C Kasibhatla.7 1Emory
University, Atlanta, Georgia; 2Le Centre hospitalier universitaire de Quebec, Montreal, Quebec, Canada;
3
  Nottingham City Hospital, Nottingham, United Kingdom; 4Clinicizzato di Valeggio, Valeggio, Italy;
5
  Research Center Good Clinical Practice, Rotterdam, Netherlands; 6University of Leige, Leige, Belgium;
7
  Procter & Gamble Pharmaceuticals, Mason, Ohio

Most trials of osteoporosis treatment that measure fractures are done as three-year double-blind studies.
Extensions usually enroll completed patients into open-label follow-up. Thus, it has not been possible to
determine long-term efficacy of bisphosphonate therapy. Two large 3-year fracture end-point trials with
risedronate (VERT-NA, VERT-MN) provided placebo-controlled two-year extensions permitting
evaluation of both efficacy and safety over 5 years. 130 placebo subjects and 135 who received risedronate
5 mg daily in VERT-MN continued their originally-assigned treatment in years 4 and 5. During the 2 year
extension, new vertebral fractures occurred in 22.3% of placebo-treated subjects and 11.1% of risedronate-
treated patients (p=0.011). Over 5 years, there was a 49% reduction in subjects with new vertebral
fractures.
In the VERT-NA extension, there were numerically fewer clinical fractures during years 4 and 5 with
risedronate (6/44, 13.6%) compared with placebo (9/42, 21.4%) (p=NS). Bone histology after 5 years of
risedronate treatment showed normal lamellar bone with no pathologic findings. Based on mineralizing
surface, bone turnover was reduced by 50%. We conclude that after 5 years of risedronate treatment the
early reduction in vertebral fractures is sustained, BMD is maintained or increases and bone turnover
remains appropriately reduced. Bone biopsies after 5 years of risedronate showed no histologic
abnormalities. In general, risedronate was well-tolerated over 5 years of treatment.

Clinical Science Poster: Bone-Calcium-Parathyroid II (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                 - 201 -
                                                      BONE-CALCIUM-PARATHYROID
P3-144

Lay explanation of abstract:

Osteoporosis affects millions of women, and the prevalence is increasing as our population ages. Because
it is a chronic condition, treatment may need to be continued for many years; however, few studies have
studied the long-term effects of osteoporosis treatments. Clinicians and women need to know what the
benefits and risks of long-term treatment may be.

We studied the long-term use of risedronate, a pyridinyl bisphosphonate that has been shown previously to
reduce the risk of vertebral and nonvertebral fractures in women with postmenopausal osteoporosis. The
earlier double-blind, placebo-controlled studies (studies VERT-MN and VERT-NA) lasted for 3 years and
enrolled women who had a history of vertebral fracture. Some women who completed these studies were
enrolled in extension studies in which they continued to receive risedronate 5 mg (135 women in VERT-
MN; 44 women in VERT-NA) or placebo (130 women in VERT-MN; 42 women in VERT-NA) for an
additional two years, while the double-blind was maintained.

During these two years, 22.3% of placebo-treated women experienced new vertebral fractures (as
determined by x-rays) in study VERT-MN, compared with 11.1% of risedronate-treated women; this
difference between groups was statistically significant. Over the entire 5 years of study, the risk of new
vertebral fracture was reduced by 49% in risedronate-treated women compared with placebo-treated
women, a difference that was highly statistically significant. Bone density continued to increase or was
maintained during the extension study. Similarly, there were numerically fewer clinical fractures (any bone
fractures that came to clinical attention) during years 4 and 5 with risedronate treatment (13.6%) than with
placebo (21.4%) in the VERT-NA study, although this difference between groups was not statistically
significant. Examination of bone samples from women in the VERT-NA study extension showed that bone
is normal with continuing risedronate use through 5 years. Risedronate continued to be well tolerated
during the additional two years of treatment, with no increased incidence of upper gastrointestinal tract side
effects compared with placebo.

These risedronate studies provide the first placebo-controlled data, collected over a five-year period, for
any bisphosphonate therapy. The studies showed that the fracture benefits observed in the first three years
are maintained with continuing treatment through five years.

Additional background information:
Risedronate is a drug of the bisphosphonate class and is sold under the trade name Actonel® in most
countries for the prevention and treatment of postmenopausal osteoporosis and of osteoporosis caused by
taking glucocorticoids (like prednisone). Risedronate is also effective in the treatment of Paget‘s disease of
bone. Risedronate works by inhibiting bone resorption, and was recently shown to prevent hip fractures
over 3 years in women with osteoporosis, as reported in The New England Journal of Medicine (McClung
et al., N Engl J Med 2001;344;333-340).

Osteoporosis is a disease characterized by low bone mass and structural deterioration of bone, leading to
bone fragility and an increased likelihood of bone fracture, especially of the hip, spine (vertebral fractures),
and wrist. Postmenopausal osteoporosis affects millions of women worldwide, including 22 million
American women. Women can lose up to 25% of their bone mass in the first 5 years following menopause,
making their bones more likely to become weak and fracture. About 40% of women over age 50 will
have an osteoporosis-related fracture in their lifetimes. Vertebral fractures can cause severe back pain, loss
of height, and a curved back and stooped posture. Intervening with a bisphosphonate such as risedronate
helps reduce the risk of vertebral fractures as rapidly as one year. Furthermore, studies have shown that
approximately 1 in 5 women experience a subsequent fracture within one year of sustaining their first
vertebral fracture.

This research was funded by Procter & Gamble Pharmaceuticals, Inc., Cincinnati, Ohio, and by Aventis
Pharma, Bridgewater, New Jersey.



                                                    - 202 -
                                                     BONE-CALCIUM-PARATHYROID
P3-145
TSE-424, A NOVEL TISSUE SELECTIVE ESTROGEN, REDUCES BIOCHEMICAL INDICES
OF BONE METABOLISM IN A DOSE RELATED FASHION
S Ronkin,1 E Baracat,2 L Roma,3 L Clarke,1 P Boudes,1 G Constantine,1 R Lindsay.4 1Wyeth-Ayerst
Research, Radnor, Pennsylvania; 2Escola Paulista de Medicina, Sao Paulo, Brazil; 3Wyeth-Whitehall
Laboratories, Sao Paulo, Brazil; 4Helen Hayes Hospital, West Haverstraw, New York

TSE-424 is a novel tissue selective estrogen with estrogen agonist effect on bone and estrogen antagonist
effect on breast and endometrium in preclinical models. This study is a prospective, sequentially
randomized, double-blind clinical trial evaluating 4 doses of TSE-424 on biochemical indices of bone
metabolism. Premarin|?circledR?| 0.625 mg/ MPA 2.5 mg is used as a positive control. All groups were
given calcium (600 mg/day). 360 healthy postmenopausal women (approximately 60 per arm) were
enrolled based upon elevated uNTX (> 30 nmol/mol Cr).

Results:There was a non-statistically significant decrease in markers of bone resorption in the placebo
group which was likely related to increased calcium intake. As expected, PMPA produced a marked
statistically significant fall in all markers. TSE-424 produced a dose related decrease in uNTX, uCTX,
sCTX and BSAP, with significant reductions at 10 and 20 mg/day (decreases in dPYR did not achieve
statistical significance, decrease in OC achieved statistical significance for the 10 mg dose). TSE-424, up to
20 mg daily, was safe and well-tolerated.

Conclusion:This controlled clinical trial demonstrates dose related reductions in bone remodeling with
TSE-424, as measured by markers of bone resorption and formation. TSE-424 behaves like an estrogen
agonist on the human skeleton.

Bone markers at 6 months (median % change from baseline)
                                                               PBO 2.5mg 5 mg 10 mg 20 mg PMPA
Urinary N-Telopeptide (uNTX)                                   -14.4 -16.0 -16.0 -25.0 -33.2* -56.3*
Urinary C-Telopeptide (uCTX)                                   -10.3 -3.0    -7.9 -25.4* -25.0* -63.6*
Serum C-Telopeptide (sCTX)                                     -8.5 -13.6 -14.2 -21.4* -30.9* -64.3*
Bone-specific Alkaline Phosphatase (BSAP)                      -7.2 -3.5     -2.3 -16.1* -12.8* -37.2*
*p-value vs. placebo <0.05


Clinical Science Poster: Bone-Calcium-Parathyroid II (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                   - 203 -
                                                       BONE-CALCIUM-PARATHYROID
P3-145

Lay version of abstract:

TSE-424 is a novel tissue selective estrogen that protects bone without stimulating breast or endometrium
(lining of the uterus) in preclinical models. This trial is a prospective, sequentially randomized, double-
blind, controlled clinical trial treating approximately 360 postmenopausal women for 6 months.

Low doses of TSE-424 produced dose-related reductions in markers of bone remodeling that were
statistically significant when compared to placebo, indicating that TSE-424 protects bone. TSE-424, up to
20 mg daily over 6 months, was safe and well-tolerated. TSE-424 behaves like an estrogen agonist on the
human skeleton.

Additional background information:

TSE-424 is a third generation selective estrogen receptor modulator that has been stringently designed to
provide beneficial effects of estrogen on bone and other tissues without stimulating endometrial or breast
tissue. This is the first report of data from phase II trials exploring the clinical effects of various doses of
TSE-424. Higher doses of TSE-424 are currently under study.

Biochemical indices of bone turnover are surrogate markers for bone loss. Reduction in these indices
indicates reduced bone turnover and protection against the bone loss associated with osteoporosis.

Phase III clinical trials will explore change in bone mineral density and reduction of fracture incidence.

This trial was funded by Wyeth-Ayerst Research.




                                                     - 204 -
                                                  DIABETES-LIPIDS-METABOLISM
OR20-1
ACUTE INHIBITION OF LIPOLYSIS IMPROVES INSULIN SENSITIVITY IN PATIENTS
WITH HIV LIPODYSTROPHY AND INSULIN RESISTANCE
C Hadigan,1 J Rabe,1 N Aliabadi,1 J Breu,2 S Grinspoon.1 1Neuroendocrine Unit, Massachusetts General
Hospital, Boston, Massachusetts; 2Clinical Research Center, Massachusetts Institute of Technology,
Cambridge, Massachusetts

HIV lipodystrophy is associated with significant fat redistribution, dyslipidemia and insulin resistance,
however, the mechanism of insulin resistance remains unknown in this large patient population. Patients
with HIV infection and lipodystrophy have elevated free fatty acids (FFA). We hypothesized that increased
FFA levels resulting from visceral obesity contribute to insulin resistance in HIV-infected patients with
lipodystrophy. In order to determine the role of FFA in insulin resistance among these patients, we
performed frequent sampled intravenous glucose tolerance tests (FSIGTT) following administration of
acipimox, a potent inhibitor of lipolysis (1000 mg in 2 divided doses) compared to placebo in 6 HIV-
infected men with lipodystrophy and hyperinsulinemia. Each patient completed 2 FSIGTT's separated by 3-
7 days and acipimox and placebo were administered in a double-blind randomized order. Patients were
44.5±2.3 y (mean±SEM), had a BMI of 27.2±1.1 kg/m2, a waist-to-hip ratio of 0.99±0.02, and the duration
of HIV was 7.6±1.3 y. All patients were on a protease inhibitor with a mean duration of 41.3±5.7 months.
Patients were markedly insulin resistant (insulin sensitivity [SI] 0.81±0.4 10 -4×min-1×µU/ml compared to
normative values of 7.56 10-4×min-1×µU/ml for healthy men). FFA levels were significantly reduced after
administration of acipimox compared to placebo (FFA area under the curve: acipimox 94.1±11.6 vs.
placebo 144.4±15.0 mmol/L/270 min, p=0.008). Acipimox resulted in a significant increase in insulin
sensitivity (SI 1.46±0.5 10-4×min-1×µU/ml, p=0.04). These data demonstrate that acute inhibition of
lipolysis and a reduction in FFA levels are associated with improved insulin sensitivity in patients with
HIV lipodystrophy and insulin resistance. Long-term strategies to reduce FFA concentrations may be
useful in the treatment of the metabolic disturbances associated with HIV lipodystrophy.

Clinical Science Oral: Diabetes-Lipids-Metabolism I (1:00 PM-2:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 1:00 PM; Location: Ballroom 1




                                                - 205 -
                                                     DIABETES-LIPIDS-METABOLISM
OR20-1

Lay explanation of abstract:

Researchers at Massachusetts General Hospital are investigating the use of Acipimox to understand the
metabolic complications of HIV lipodystrophy. Patients with HIV lipodystrophy experience significant
changes in body fat distribution, which often are accompanied by abnormalities in sugar and insulin
regulation and high cholesterol levels. These patients typically have high levels of free fatty acids, which
may interfere with insulin and sugar metabolism. Acipimox is a medication that blocks the production of
fatty acids and is used to lower cholesterol levels. It is not currently available in the US.

In this study, seven men with HIV infection and lipodystrophy received Acipimox and underwent testing of
insulin sensitivity. Results showed that short-term administration of Acipimox reduced levels of free fatty
acids and thereby enhanced the action of insulin in regulating blood sugar levels. Insulin sensitivity, a
measure of the ability to process sugar in response to insulin, was found to be significantly improved and
nearly doubled on the day patients received Acipimox compared to a placebo. This demonstrates that
elevated free fatty acids contribute to abnormal insulin metabolism in this patient population. Furthermore,
it suggests that long term administration of Acipimox may have a role in the treatment of insulin resistance
in this syndrome.

Additional background information:

HIV infected individuals frequently develop metabolic abnormalities that may be related to their HIV
medications, HIV virus, or a combination of factors. It is estimated that the majority of patients treated with
new potent antiretroviraI therapy are affected. This syndrome is called HIV lipodystrophy and is
characterized by increased levels of insulin, triglycerides, and cholesterol, which are known cardiovascular
disease risk factors. In most cases, there is also a disturbance in fat distribution, and patients may lose
subcutaneous fat in the face, arms and legs, and/or gain fat in the abdomen. This syndrome is of significant
concern for the HIV-positive population, because of an increased risk of cardiovascular disease and marked
changes in appearance.

This research was funded by the National Institutes of Health and the Clinical Nutrition Research Center at
Harvard.




                                                   - 206 -
                                                    DIABETES-LIPIDS-METABOLISM
P1-310
REDUCED PLASMA TESTOSTERONE IN MEN IS ASSOCIATED WITH SUPPRESSED
INSULIN RESPONSE TO ACUTE MYOCARDIAL INFARCTION
PJ Pugh,2 KM English,2 RD Jones,1 KS Channer,2 TH Jones.1 1Institute of Endocrinology, University of
Sheffield, Sheffield, United Kingdom; 2Department of Cardiology, Royal Hallamshire Hospital, Sheffield,
United Kingdom

Background: Low plasma testosterone levels have been associated with insulin resistance in men. Impaired
carbohydrate metabolism influences the outcome following acute myocardial infarction (AMI) and may be
improved by androgen replacement therapy. We examined the relation of androgens with circulating
insulin levels in non-diabetic men with AMI.Methods: 22 male patients presenting with AMI were
recruited. Blood was taken on admission (day 1) and on days 2, 3 and 6, between 0800 and 0900hours.
Levels of total testosterone, 17β-oestradiol and insulin were measured. Group comparisons were made
using student's t test. Correlations were performed using Pearson's test for parametric data and Spearman's
test for non-parametric data.
Results: Testosterone levels fell on day 2 from 12.0nM to 9.8nM (p=0.03), then rose to exceed baseline on
day 6. Insulin levels rose markedly from 26mU/L to 58m/L on day 2, 70mU/L on day 3 and 71mU/L on
day 6 (p=0.001).
Higher baseline testosterone was associated with a greater rise in insulin on days 2 (r=0.55, p=0.009) and 3
(r=0.49, p=0.02) and with insulin levels on days 2 (r=0.50, p=0.02) and 3 (r=0.59, p=0.004). Subjects in the
highest quartile of baseline testosterone had higher insulin levels on days 2 (106mU/L v 40mU/L, p=0.009),
3 (122mU/L v 51mU/L, p=0.003) and 6 (106mU/L v 59mU/L, p=0.02). The initial testosterone/oestradiol
ratio correlated with the rise (r=0.54, p=0.009) and level of insulin on day 3 (r=0.51, p=0.02). Subjects who
subsequently developed heart failure or left ventricular dysfunction (LVD) had lower baseline testosterone
levels (11.2nM v 14.8nM, p=0.04) and smaller initial rise in insulin (20mU/L v 73mU/L ,p=0.03).
Conclusion: We conclude from these data that lower initial testosterone levels, in non-diabetic men with
AMI, are associated with a suppressed insulin response and with the development of heart failure or LVD.
Higher androgen levels are associated with a better outcome, which may be linked to an effect on
carbohydrate metabolism.

Clinical Science Poster: Diabetes-Lipids-Metabolism I (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 207 -
                                                     DIABETES-LIPIDS-METABOLISM
P1-310

Lay explanation of abstract:

In a prospective study, we examined the link between sex hormones and insulin in men who had had heart
attacks. Recent evidence suggests that sex hormones may play a role in carbohydrate metabolism, which if
impaired can lead to a poorer recovery after a heart attack. The sex hormones may affect carbohydrate
metabolism through an effect on insulin levels. Carbohydrate metabolism is controlled by insulin and is
impaired in diabetic patients.

We studied 22 men admitted to the hospital with an acute heart attack. Blood was taken on four occasions
over 6 days. We measured levels of testosterone (male sex hormone), estradiol (female sex hormone), and
insulin in each blood sample. We found that levels of testosterone fell significantly in the first 24 hours.
Estradiol levels remained constant. Insulin levels rose markedly over the 6 days.

In patients with higher baseline testosterone levels, the rise in insulin was marked. Insulin level was much
reduced, however, in those with lower baseline testosterone levels. Estradiol had no apparent influence on
insulin levels. We also found that patients who developed complications from their heart attack had lower
testosterone levels and a smaller rise in insulin. This suggests that, in men, testosterone protects the heart
from damage during a heart attack. This effect may be the result of greater insulin production.

These findings are of particular interest for two reasons. First, they go against the conventional wisdom that
testosterone is bad for the heart. This notion has arisen from the excess cardiac death rate in men compared
with women, which appears to be an erroneous conclusion. Second, the importance of maintaining proper
"metabolic control" during a heart attack is gaining increasing recognition. Our significant finding is that
sex hormones may influence this control.

Additional background information:

Other research in this area from our department has included a trial of testosterone therapy for men with
stable angina, which improved significantly. We also measured other parameters in the present study,
which supported a protective role for testosterone in men with heart disease. These findings may lead to
trials of sex hormones as therapy for patients who have acute heart attacks.

This project was funded by the Sheffield Hospitals Charitable Trust, UK.




                                                   - 208 -
                                                   DIABETES-LIPIDS-METABOLISM
P1-319
SUSTAINED BENEFITS OF METFORMIN THERAPY IN HIV LIPODYSTROPHY
C Hadigan,1 J Rabe,1 B Davis,2 N Basgoz,2 S Grinspoon.1 1Neuroendocrine Unit, Massachusetts General
Hospital, Boston, Massachusetts; 2Infectious Disease Division, Massachusetts General Hospital, Boston,
Massachusetts

Insulin resistance, in association with lipodystrophy, is increasingly recognized among HIV-infected
patients. We conducted a double-blind randomized 3 month trial of metformin for HIV-infected patients
with lipodystrophy and insulin resistance and previously demonstrated improvement in insulin levels,
weight, waist circumference and diastolic blood pressure compared to placebo. In order to assess the
efficacy and safety of prolonged metformin therapy, eligible subjects who completed the randomized study
received a 6 month open-label metformin treatment extension. Nineteen of 25 potential patients were
eligible to receive open label metformin based on pre-established efficacy criteria. Patients completed a
standard 75 g oral glucose tolerance test (OGTT) at baseline and follow-up visits every 3 mo. In an analysis
of the overall effect of metformin at the end of treatment (n=19), metformin significantly reduced insulin
and glucose area under the curve (AUC) following OGTT (p<=0.04). Among patients initially randomized
to metformin (n=11) who received 9 mo of treatment, the mean change in insulin AUC was -3066±1845
µU/mL/120 min. Among patients initially randomized to placebo who received 6 mo of open-label
treatment (n=8), insulin AUC was reduced by 3403±2367 µU/mL/120 min. Statistically significant
reductions in BMI (p=0.002), waist circumference (p=0.003) and waist-to-hip ratio (p=0.02) were also seen
in response to metformin. The reduction in insulin AUC observed after 3 mo in patients initially
randomized to metformin was maintained during the 6 mo continuation. At the end of the study, there was
no statistical difference in insulin AUC response to metformin between patients regardless of initial
randomization. However, the reduction in waist circumference and BMI tended to be greater in patients
who received 9 vs. 6 mo of metformin (p=0.08). Metformin was well tolerated and no one discontinued
treatment due to side effects. There was no increase in lactic acid levels associated with metformin. These
data demonstrate a sustained benefit of metformin to reduce insulin resistance in patients with HIV
infection and lipodystrophy.

Clinical Science Poster: Diabetes-Lipids-Metabolism I (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 209 -
                                                     DIABETES-LIPIDS-METABOLISM
P1-319

Lay explanation of abstract:

Researchers at Massachusetts General Hospital recently completed a nine-month study of metformin for the
treatment of insulin resistance associated with HIV lipodystrophy syndrome. This syndrome is of
increasing concern for the HIV- positive population as patients experience significant changes in fat
distribution often accompanied by abnormalities in sugar and insulin regulation. This study demonstrated
that a course of nine months of metformin is a safe and effective treatment for insulin resistance associated
with HIV lipodystrophy. The benefits of metformin therapy included reduced insulin levels and decreased
weight and waist circumference. In this way, metformin treatment may help reduce the overall
cardiovascular disease risk profile for these patients.

The first three months of the study involved a double-blind, placebo-controlled trial of 25 patients. Results
of this portion of the study have been previously reported. Nineteen patients from this initial study received
open treatment with metformin for an additional six months. Nine months of metformin led to a greater
reduction in weight and waist circumference than six months of treatment. Significant reductions in insulin
levels persisted at the end of the study, regardless of the duration of treatment. There was no increase in
lactic acid levels, a rare potential toxic side effect of metformin. This study offers further evidence that
metformin therapy may provide long-term benefits for patients with insulin resistance and lipodystrophy.
The treatment also may potentially reduce the risk of cardiovascular disease and diabetes in these patients.

Additional background information:

HIV infected individuals frequently develop metabolic abnormalities that may be related to their HIV
medications, HIV virus, or a combination of factors. It is estimated that the majority of patients treated with
new potent antiretroviral therapy are affected. This syndrome is referred to as HIV lipodystrophy and is
characterized by increased levels of insulin, triglyceride and cholesterol, which are known cardiovascular
disease risk factors. In most cases, there is also a disturbance in fat distribution and patients may lose
subcutaneous fat in the face, arms and legs and/or gain fat in the abdomen. This syndrome is of significant
concern for the HIV-positive population due to the increased risk of cardiovascular disease and marked
changes in appearance.

This research was funded by the National Institutes of Health.




                                                   - 210 -
                                                   DIABETES-LIPIDS-METABOLISM
P1-321
EFFECT OF SWITCHING TO INSULIN PUMP THERAPY IN PATIENTS WITH TYPE 1
DIABETES AND SEVERE HYPOGLYCEMIA ASSOCIATED WITH MULTIPLE DAILY
INJECTIONS OF INSULIN
L Siegel, J Hey-Hadavi, K Herold, C Tuck, R Goland. Naomi Berrie Diabetes Center & Dept. of Medicine,
Columbia University, New York, New York

The relationship between strict metabolic control of diabetes and hypoglycemia is complex. Some studies,
including the DCCT, have shown that intensive glycemic control with multiple daily injection (MDI) of
insulin or continuous subcutaneous insulin infusion (CSII) is associated with increased hypoglycemia.
However, some recent reports have demonstrated a decrease in hypoglycemia with insulin pump therapy.
Despite intensive diabetes education, some patients experience significant hypoglycemia with MDI. We
hypothesized that switching from MDI to CSII in these patients would lead to a decrease in hypoglycemic
events because of the more predictable insulin action offered by CSII. We therefore evaluated the incidence
of hypoglycemia and level of metabolic control in 12 patients who were started on CSII because of severe,
recurrent hypoglycemia with MDI. The mean age of the patients was 32 yrs (range, 5 to 65 yrs); 6 were
male and 6 were female. The mean duration of type 1 diabetes was 15 yrs. At baseline, all patients reported
2-4 episodes of hypoglycemia per week including frequent nocturnal hypoglycemia. Severe hypoglycemia,
requiring assistance from another person, occurred 2-3 times per month in all patients; 4 patients had a
history of a hypoglycemic seizure. Patients received intensive insulin pump training, diabetes education,
and nutritional counseling. Within several weeks of initiating CSII, symptomatic hypoglycemia was
reduced, nocturnal hypoglycemia was eliminated in all patients, and there were no episodes of
hypoglycemia requiring assistance from another person or hypoglycemic seizures. Mean HbA1c at baseline
was 6.0% and fell to 5.9% after 3 months and 5.5% at 6 months of pump therapy.

Clinical Science Poster: Diabetes-Lipids-Metabolism I (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                 - 211 -
                                                    DIABETES-LIPIDS-METABOLISM
P1-321

Lay explanation of abstract:

The best approach to preventing the severe long-term complications of diabetes (blindness, kidney failure,
amputation) is to intensively control blood sugar toward normal levels. The major side effect from
intensive treatment with multiple daily injections of insulin is low blood sugar (hypoglycemia), which
could be serious and even life threatening.

We switched 12 patients whose treatment with insulin shots led to severe hypoglycemia to treatment with
an insulin pump. An insulin pump provides insulin in a more predictable manner than multiple daily
injections. These 12 patients had type 1 diabetes and ranged in age from 5-65 years. All patients reported 2-
4 episodes of hypoglycemia per week including frequent nighttime hypoglycemia. Severe hypoglycemia
requiring assistance from another person occurred 2-3 times a month. Four patients had a history of
hypoglycemic seizures. Within several weeks of insulin pump therapy initiation, hypoglycemia was
reduced in all patients. In addition, all patients' glucose control also improved after starting the pump.

These findings are important because many people associate insulin pump therapy with an increased risk
for hypoglycemia, and our findings suggest the opposite. Insulin pump therapy appears to be a major
advance for patients with type 1 diabetes.

Our findings are important clinically because improved treatments for type 1 diabetes are very much
needed. Insulin pump therapy is an excellent alternative for patients with type 1 diabetes, especially those
whose treatment is complicated by severe hypoglycemia. Insulin pump therapy can be used successfully in
children and adults. Our study showed that in just several weeks, hypoglycemia was reduced through
insulin pump therapy.

Additional background information:

An insulin pump is a new technologically advanced approach to treating diabetes. Many people with
diabetes struggle with high and low blood sugars, 5-6 injections of insulin a day, and strict eating
schedules. A pump can give them more flexibility and a better quality of life.

Due to advancements in technology, insulin pump therapy is a treatment option that is very successful and
adaptable to an active lifestyle. Insulin pumps are lightweight, easy to use, and are about the size of a
beeper. Patients do not have to take multiple insulin shots daily; instead they insert a small needle under
the skin once every 2 to 3 days. The pump delivers insulin 24 hours a day, according to a plan programmed
individually for each patient. It allows programmed changes in basal insulin delivery to compensate for
periods of high hypoglycemia risk, even during the night. An insulin pump provides the patient with
excellent blood glucose control and reduced hypoglycemia.

This study was funded by the Naomi Berrie Diabetes Center at Columbia Presbyterian, New York, NY.




                                                  - 212 -
                                                   DIABETES-LIPIDS-METABOLISM
P2-557
ALTERNATIVE TREATMENTS OF TYPE 2 DIABETES MELLITUS (T2DM)
G Arsenis,1,2 D Goettelman.1 1Endocrine/Medicine, VAMC, Bay Pines, Florida; 2Medicine, Univ of South
Florida, Tampa, Florida

The aim of this study is to try to reverse or arrest the progress of the disease in T2DM. We studied 51
patients with T2DM who failed treatment with large doses of oral hypoglycemics and/or insulin. Dietary
excesses in foods with high glycemic and insulinemic index were reduced for six months, i.e. animal
protein (AP) reduced from 2-3 times a day to once every other day and plain sugars were stopped.
Isocaloric amounts of vegetable protein were given in place of AP to maintain the same total daily protein
intake. Patients liked the new diet and the fact that they did not have to fast, take low calorie diets or
receive appetite suppressants. The metabolic profile (MP) [HbA1C, lipids, blood sugar(BS)] was
determined prior and at the end of the study. Results in 31 patients who followed the low AP diet showed
that they did not lose any weight and the MP improved to normal or close to normal values. Thus, HbA1C
decreased by 30% from 9.98±0.51 to 7.02±0.44%, p30%. Twenty patients did not improve MP because six
of them followed low calorie diet and lost weight, 5-100 lbs, but did not practice a low AP diet; the
remaining 14 subjects continued on AP, 2-3 times a day, but changed from red meat to fish and poultry. In
summary, the patients who followed the low AP diet achieved optimal control in BS, HbA1C, cholesterol
and close to optimal levels in LDL, TG and HDL. Improvement in the MP was accomplished by a low AP
diet alone, diet with reduced medication or diet with the same dose of medication. We conclude that the
reduction of AP and sugar can reverse the course of the disease or improve the MP by enhancing the effect
of the failing oral agents and/or insulin in T2DM.

Clinical Science Poster: Diabetes-Lipids-Metabolism II (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                 - 213 -
                                                    DIABETES-LIPIDS-METABOLISM
P2-557

Lay explanation of abstract:

The aim of this study is to reverse or arrest the progress of Type 2 diabetes mellitus (T2DM), and we found
benefits from the diet we studied. We studied 51 patients with T2DM who failed treatment on high dose of
sugar pills and/or insulin. Dietary excesses in foods that stimulate insulin secretion and raise blood sugar
(BS) were reduced for six months. Thus, animal protein (AP) reduced from 2-3 times a day to once every
other day and plain sugars were eliminated. AP was replaced with equal amounts of vegetable protein. The
caloric content remained the same to prevent weight loss. Patients liked the new diet because they did not
have to fast, adhere to low-calorie diets or take appetite suppressants.

Blood tests HbA1C, lipids, BS] were done before and at the end of the study. Results from 31 patients who
followed low AP diet showed no weight loss and abnormal results returned toward normal. The HbA1C
(measures the long-term control on BS) was decreased by 30% from 9.980.5 1 to 7.02±0.44%. p<0.000l.
Total cholesterol was reduced by 32% from 277.5020.32 to l86.946.75 mg/dL, p<0.004. Triglycerides
(Tg) dropped by 60%, from 611.56144.68 to 247.6248.22 mg/dL, p<0.03. Low-density density
lipoprotein (LDL) decreased by 35% from 180.60+10.18 to 116.406.64 mg/dL, p<0.05. High-density
lipoprotein (HDL) increased by 10% from 34.872.63 to 38.311.67 mg/dL, p<0.02.

Three patients reduced the insulin dose by 50%, and two discontinued insulin. Four patients had to stop the
sugar pills and six discontinued one or two of the cholesterol pills. Quality of life and compliance were
improved. Short-term cost was reduced by more than 30%. In 20 patients out of the 51, the results did not
improve, because six of them did not follow the low AP diet; instead they followed a low-calorie diet and
lost 5-100 lbs. The remaining 14 subjects failed to improve because they continued on the same amount of
AP, which was changed from red meat to fish and poultry.

In summary, patients who followed the low AP diet achieved optimal BS, HbA1C, cholesterol, LDL, Tg
and HDL control, while on diet alone or on combination of diet and reduced dose of medication. In
conclusion, the reduction in AP and sugar can reverse or improve the course of the disease by enhancing
the effect of sugar pills and/or insulin in the treatment of T2DM.




                                                  - 214 -
                                                    DIABETES-LIPIDS-METABOLISM
P2-559
INCREASED MORTALITY SEEN WITH HIGH DOSE SULFONYLUREAS OR
SULFONYLUREA-METFORMIN COMBINATION MAY REFLECT HYPOGLYCEMIA - A
META-ANALYSIS OF 11 LARGE TRIALS
R Innerfield. Epidemiology & Clinical Trials Branch, The National Diabetes Center, Olney, Maryland

Data from the Mayo Clinic suggests sulfonylureas are associated with enhanced cardiovascular mortality
following angioplasty procedures. In settings of randomized, controlled, clinical trials, low, fixed-doses of
sulfonylureas seem to improve or not change mortality measures whereas high, fixed of sulfonylureas
increase cardiovascular mortality. Initial randomization to very low-potential hypoglycemic metformin
monotherapy significantly improved cardiovascular mortality in the UKPDS. Initial randomization to very
high-potential hypoglycemic metformin + sulfonylurea combination therapy significantly increased
cardiovascular mortality in the UKPDS. US Clinical Trials submitted to FDA for metformin initial
regulatory approval showed significantly increased mortality of patients randomized to metformin on
combination metformin plus sulfonylureas. [Hypoglycemia was suggested to hve played a potentially
causal role]. Obervational data from Malmo show increased mortality in diabetic patients taking
combination metformin plus sulfonylureas. Data from the Bezafibrate Infarction Prevention Trial show an
increased cardiovascular mortality from patients on sulfonylureas alone or on combination metformin plus
sulfonylureas. Long-term pharmacologic interventions to very tightly control Type 2 Diabetes not only are
ineffective in reducing mortality, but may contribute to increased morbidity and mortality [except within
the context of acute mi and by insulin.] Patients with established coronary artery disease should therefore
probably not be on oral agents but well-controlled on insulin. Current ADA Treatment Guidelines for Type
2 glycemic control of target A1c at 7% and action point at 8% should be maintained and not tightened. The
NHLBI ACCORD trial -Prevention of Cardiovascular Disease in Diabetes Mellitus ("Action to Control
Cardiovascular Risk in Diabetes") - currently underway to test the "extremely tight control hypothesis"
should therefore be 2-tailed.

Clinical Science Poster: Diabetes-Lipids-Metabolism II (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 215 -
                                                      DIABETES-LIPIDS-METABOLISM
P2-559
Lay explanation of abstract:
Studies have shown reasonably conclusively that very good control of blood sugars over the long haul does
very little to improve one's chances of survival with type 2 diabetes. Because cliniciansespecially we
endocrinologistsare still reluctant to accept this notion, another trial is currently underway at the NIH
(National Heart Lung and Blood Institute) to see if even tighter control could make an impact on reducing
diabetic mortality.

An additional analysis reported at the current Endocrine Society meeting suggests that the strong
correlation between poor diabetic control and increased total or cardiovascular mortality may be explained
by common inflammatory hormones called cytokines. The analysis further suggests that either these
cytokines or the atherosclerotic process itself may secondarily induce 1) worsening glucose tolerance, 2)
new onset of diabetes, or 3) worsening of existing diabetic control. Nevertheless, one other possible
explanation of why tight control of blood sugars has so far failed to improve diabetic survival is that some
of the therapeutic strategies used to treat type 2 diabetes may be harmful, in and of themselves.

Thus, a meta-analysis of several randomized, controlled, long-term clinical trials was launched to provide
insight into this question. The analysis does seem to suggest increases in death when sulfonylureas (agents
very commonly used to control blood sugars) are taken either 1) singly at higher doses and compared with
lower doses or 2) combined with metformin and compared with metformin alone. Increases in death were
statistically and clinically significant. The increase in all-cause deaths was +1270 to +1750 excess deaths
per 10,000 treated patients over a 10-year period. Deaths from cardiovascular causes increased by +955 to
+1630 excess deaths per 10,000 treated patients over a 10-year period.).

The primary question at issue is whether any specific types of therapy employed in diabetes treatment
might make a difference, either favorably or unfavorably, upon overall and cardiovascular diabetic
mortality. One major class of anti-diabetic drugs still prominently employed today is the sulfonylureas.
This class increases insulin production while decreasing blood flow to already oxygen-deprived [heart]
muscle. These effects are a direct function of the sulfonylurea drug concentration as well as its major
mechanism of action. The question was asked, therefore, whether or not there may be an effect of drug
concentration upon overall diabetic survival. Indeed, this did appear to be the case in an analysis across
several large clinical trials. (Nevertheless, the administration of this class of drug at very high [and fixed]
levels in the most famous of these trials - the UGDP - was such that it might also have induced blood
sugars to drop to very low levels. Indeed, low blood sugars from this class of drugs has in point of fact
been shown elsewhere to have been associated with an increased diabetic mortality.)

The next question asked was whether data might exist that compares the treatment differences of a therapy
designed to implement significantly lower blood sugars, such as the early prescription of sulfonylurea
combination with metformin, versus a therapy designed to cause less significant hyperglycemia, such as
metformin single therapy. Again, this within-trial subgroup meta-analysis did appear to show that therapies
designed to maintain lower blood sugars were significantly more harmful than those that do not.

Several other recent studies have also shown increased cardiovascular mortality with sulfonylurea-
metformin combination or with sulfonylurea therapy alone (monotherapy) particularly in a group with
coronary artery disease. From this analysis, it would appear that:
1. High doses of sulfonylureas should be avoided, particularly in patients with coronary artery disease;
2. The combination of sulfonylureas with metformin should be avoided, particularly in patients with
    coronary artery disease;
3. Extremely tight control of diabetes in the (NHLBI) ACCORD trial should include advisement that a
    real possibility exists that severe adverse effects might ensue, and patients should be so advised in the
    informed consent process; and
4. The ADA should NOT tighten its recommended guidelines of a 7% HbA1c goal and an 8% HbA1c
    action point.

This research was privately funded.



                                                    - 216 -
                                                    DIABETES-LIPIDS-METABOLISM
P2-564
EFFECTS OF CHRONIC MODERATE ALCOHOL INTAKE ON FASTING INSULIN AND
GLUCOSE CONCENTRATIONS AND INSULIN SENSITIVITY IN POSTMENOPAUSAL
WOMEN: A CONTROLLED-DIETARY INTERVENTION STUDY
MJ Davies,1 DT Baer,1 JT Judd,1 E Brown,2 W Campbell,2 PR Taylor.2 1Beltsville Human Nutrition
Research Center, ARS-USDA, Beltsville, Maryland; 2Cancer Prevention Studies Branch, NCI-DHHS,
Rockville, Maryland

Reduced fasting insulin concentrations and improved insulin sensitivity are associated with moderate
alcohol intake in cross-sectional studies. These changes may be related to the improved lipid profile
associated with moderate alcohol intake and the cardioprotective effects of alcohol. To date, no controlled-
feeding studies have examined the effects of chronic alcohol consumption on fasting insulin and glucose
concentrations and insulin sensitivity. We addressed whether chronic consumption of low to moderate
amounts of alcohol influences glucose and insulin homeostasis in non-diabetic, postmenopausal women
who were not on hormone replacement therapy. Fifty-two women were assigned to consume either 15 g
alcohol/d (1 drink), 30 g alcohol/d (2 drinks), or an isocaloric placebo beverage (no alcohol) for 8-wk as
part of a controlled diet in a randomized crossover design. All foods and beverages were provided and
energy intake was adjusted to maintain constant body weight. Serum was collected from fasted volunteers
on three separate days at the end of each dietary period and then pooled. Glucose was measured
enzymatically (Sigma) and insulin with an ELISA (DSL). Fasting glucose and insulin concentrations were
used to assess insulin sensitivity via the quantitative insulin sensitivity check index (QUICKI). Fasting
glucose, fasting insulin (log-transformed), and QUICKI were compared among treatments with a mixed
model ANOVA. Consumption of 30 g alcohol/d compared to placebo significantly reduced fasting insulin
concentration (7.50 ± 0.96 vs. 9.10 ± 1.24 mU/L; p = 0.003) and increased QUICKI (0.367 ± 0.005 vs.
0.358 ± 0.006; p = 0.006). Fasting insulin concentration and QUICKI were not, however, different after
consumption of 15 g alcohol/d compared to placebo. Moreover, fasting glucose concentration was not
different across treatments. These data demonstrate that chronic consumption of two alcoholic drinks per
day reduces fasting insulin concentrations and improves insulin sensitivity in postmenopausal women.

Clinical Science Poster: Diabetes-Lipids-Metabolism II (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 217 -
                                                    DIABETES-LIPIDS-METABOLISM
P2-564

Lay explanation of abstract:

In a carefully controlled diet study with postmenopausal women, moderate alcohol consumption was
associated with changes in serum insulin and blood lipids, which could reduce risk of degenerative
diseases. Several studies, including this one, have shown that men and women who consume low-to-
moderate amounts of alcohol (1 to 2 drinks per day) have improved blood lipid profiles that may be
associated with reduced risk of cardiovascular disease, which in turn may lead to reduced risk of heart
disease and stroke.

In our study with postmenopausal women, in addition to improved blood lipid profiles, a reduction in
serum insulin concentrations was noted. Elevated insulin concentrations and reduced insulin sensitivity (the
ability of the body to respond to circulating insulin) are related to increased risk for adult-onset (Type 2)
diabetes, as well as cardiovascular disease.

There have been few clinical trials and no controlled diet studies showing the effects of moderate alcohol
consumption on fasting insulin concentrations and insulin sensitivity. Menopause is not only associated
with increased risk for cardiovascular disease, but also with increased risk for adult-onset diabetes.
Therefore, we investigated whether chronic consumption of low-to-moderate amounts of alcohol
influenced fasting glucose and insulin, and insulin sensitivity in non-diabetic, post-menopausal women who
were not on hormone replacement therapy.

As part of a controlled diet, 52 women consumed, in random order, one alcoholic drink per day, two
alcoholic drinks per day, or a non-alcoholic beverage each for 8 weeks. All foods and beverages were
provided by the Beltsville Human Nutrition Research Center. Blood was collected at the end of each
dietary period and used to measure glucose and insulin concentrations, which were then used to assess
insulin sensitivity using the quantitative insulin sensitivity check index. Consumption of two alcoholic
drinks per day compared with a non-alcoholic beverage significantly reduced fasting insulin concentration
and increased insulin sensitivity. However, one alcoholic drink daily compared with a non-alcoholic
beverage did not change fasting insulin concentration and insulin sensitivity. Moreover, fasting glucose
concentration was not affected by alcohol consumption.

This controlled diet study is the first to show that moderate alcohol consumption (2 drinks per day) reduces
fasting insulin concentrations and enhances insulin sensitivity in non-diabetic, postmenopausal women.
The mechanisms by which alcohol affects insulin concentration and sensitivity are unknown. These
findings may assist physicians in medically assessing their patients and in interpreting clinical data. These
data do not suggest that non-drinkers should consume alcohol to obtain these benefits, but the results may
provide a guide for individuals who consume alcohol in moderation. Consuming greater amounts of alcohol
is associated with reductions in or loss of the health benefits noted for moderate alcohol consumption.

This research was funded by the Agricultural Research Service, U.S. Department of Agriculture, and by the
National Cancer Institute, National Institutes of Health, Department of Health and Human Services.




                                                  - 218 -
                                                   DIABETES-LIPIDS-METABOLISM
P2-579
THE EFFECT OF SUPERVISED AND UNSUPERVISED LONG TERM EXERCISE ON BODY
COMPOSITION AND LIPID PROFILE IN TYPE 2 DIABETES
S Alam, CL Pentecost, M Stolinski, P Croos, PH Sonksen, AM Umpleby. Department of Medicine, St
Thomas' Hospital, London, United Kingdom

Objectives:Type 2 diabetes is characterised by insulin resistance and dyslipidemia. Previous studies have
shown that aerobic exercise for 12 weeks can lead to improved insulin sensitivity and an improved lipid
profile. The aim of this study was to compare the effects of supervised and unsupervised exercise in type 2
diabetics who had failed to show improved glycaemic control with diet and tablets for a period of 6 months.
Methods:18 poorly controlled, moderately obese type 2 diabetics were recruited. All patients were given an
initial training session and a personal exercise programme. Patients were then randomised into two groups,
a supervised exercise group (n=9) and a non-supervised exercise group (n=9) matched for age and BMI.
Patients in the supervised group exercised under supervision with a trainer 4 times /week at 70% of VO2
max for six months. Patients in the non-supervised group were asked to exercise 4 times /week at 70% of
VO2 max for the same period. Fasting blood samples were taken before and after 6 months of the exercise
programme.
Results: All results are presented as mean±SD. In the supervised group there was a significant decrease in
BMI (30.6±3.3, at 0 months to 29.7±3.1 kg/m2 at 6 months; p< 0.02), HbA1c (8.7±0.7 to 7.7± 0.7 %; p<
0.01), abdominal fat (39.2±1.2 to 36.1±0.2%; p< 0.03). HDL cholesterol was significantly increased from
0.99±0.2 to 1.2±0.2mmol/l; (p < 0.02).
Conclusions: Patients in the supervised exercise group showed a significant improvement in body
composition and lipid profile as compared to the unsupervised group over a six month period. This suggests
a need for supervision when exercise is used as part of the management of type 2 diabetics.

Clinical Science Poster: Diabetes-Lipids-Metabolism II (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                 - 219 -
                                                    DIABETES-LIPIDS-METABOLISM
P2-579

Lay explanation of abstract:

In a study to investigate the effect of a 6-month supervised exercise program in people with type 2 diabetes,
we have shown that regular exercise under the supervision of a personal trainer is a very effective form of
treatment. Blood glucose control was substantially improved. This improvement was as good as that seen
with current forms of drug therapy, but it provided a significant weight loss.

Patients with type 2 diabetes are at an increased risk of developing heart disease. This has been linked to
low levels of beneficial HDL (high-density lipoprotein) cholesterol, high levels of abdominal fat, and
reduced sensitivity of the body to insulin (known as insulin resistance). The HDL cholesterol helps remove
excess cholesterol from the blood so high levels are actually good for the heart.

In this study, we found that a supervised exercise program increased good (HDL) cholesterol. There was
also an improvement in the body‘s sensitivity to insulin and body fat around the abdomen was reduced.
This suggests that supervised exercise may also reduce the risk of heart disease.

Although patients with type 2 diabetes are often asked to exercise, they rarely do so. This study compares
simple advice about an exercise program against the same program supervised by a personal trainer. In this
study, funded by the British Heart Foundation, 18 moderately overweight people with poorly controlled
type 2 diabetes volunteered to participate in the study. Their baseline fitness was measured on a treadmill.
Patients were randomly split into two groups, supervised and unsupervised exercise programs. Patients in
the supervised group exercised under supervision with a personal trainer 4 times/week. Those in the
unsupervised group were asked to exercise on their own 4 times/week following an initial session with the
trainer. Patients in both groups, at their first interview, were given guidelines on what type and how much
exercise to do depending on personal preferences and measured fitness levels. Blood samples were taken at
the start and end of the 6-month exercise period. At the end of the study, the supervised group showed
improvements in fitness, body composition, glucose, and cholesterol levels.

This study clearly demonstrates the benefits of a supervised exercise program in type 2 diabetes and
suggests that this should be offered as part of a Diabetes Service.

This research was funded by the British Heart Foundation.




                                                  - 220 -
                                                                   ENDOCRINE GENETICS
P3-553
VARIATION AT THE CAPN10 (CALPAIN 10) GENE AND PATHOGENESIS OF POLYCYSTIC
OVARIAN SYNDROME: EXTENDED DATA DEMONSTRATES NO EVIDENCE FOR A
MAJOR ROLE IN EUROPID POPULATIONS
L Haddad,3 N Gharani,3 J Evans,2 S Franks,1 MI McCarthy.3 1Dept. of Reproductive Science and Medicine,
Imperial College, London, United Kingdom; 2Dept. of Diabetes and Vascular Medicine, University of
Exeter, Exeter, United Kingdom; 3Imperial College Genetics and Genomics Research Institute, Imperial
College, London, United Kingdom

Polycystic ovary syndrome (PCOS) is a common endocrine disorder affecting ~5-10% of women of
reproductive age. Apart from its typical manifestations PCOS encompasses a multitude of metabolic
disorders such as insulin resistance and dyslipidaemia and shows clear physiological and epidemiological
overlap with type 2 diabetes (T2D). The calpain 10 (CAPN10) gene was recently identified as a major T2D
gene in Mexican Americans (MA). We have applied family-based association methods to determine
whether the CAPN10 gene plays a wider role in insulin-resistant states. We studied 146 Europid parent-
offspring trios ascertained through symptomatic ultrasound-confirmed PCOS probands [median (IQR) age:
32.4 (25.6-39.2)y; BMI 24.0 (16.7-31.3)kgm-2]. A further 185 PCOS subjects were available for
intermediate trait analyses. Four CAPN10 variants (SNPs 43, 44, 19, 63) implicated in diabetes-
susceptibility were analysed using the transmission disequilibrium test (TDT). Homozygosity for the
common allele at SNP43 was most strongly associated with MA T2D but no association was evident in
PCOS (χ2=0.08). Furthermore we observed no excess transmission at SNP44 (χ2=0.78), SNP19 (χ2=0.59)
or SNP63 (χ2=4.51, nominal p=0.028 (NS after correction)). Multilocus TDT revealed reduced haplotype
diversity (only 5/16 possible haplotypes were present at >1.2%), but none showed excess transmission
(χ2=7.68, p=0.18) to affected offspring. Haplotype frequencies in all 331 PCOS subjects did not differ from
those in >800 Europid control chromosomes (p=0.57). Finally, intermediate trait analysis (for BMI, WHR,
lipids, fasting insulin/glucose) revealed no relationship with genotype in all 331 PCOS subjects. We
conclude that no association between CAPN10 variation and PCOS in European subjects was detected and
hence, that there is no evidence that CAPN10 plays a role in susceptibility to insulin-resistant states.

Clinical Science Poster: Endocrine Genetics (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                 - 221 -
                                                                     ENDOCRINE GENETICS

P3-553

Lay explanation of abstract:

Polycystic Ovary Syndrome (PCOS) is the name given to a condition in which women have ovaries that
contain many small cysts and have not developed properly. Associated with these polycystic ovaries is a
range of symptoms, which include irregular periods, or no periods at all, reduced fertility, unwanted facial
or body hair (hirsutism), acne, and occasionally loss of hair from the top of the head. Polycystic ovary
syndrome is a common disorder and affects about 5-10% of women of reproductive age. There is evidence
to show that some women with polycystic ovaries are more prone to weight gain. Polycystic ovary
syndrome can also make some women more susceptible to certain health problems in later life, such as an
increased risk of developing type 2 diabetes and heart disease. It is believed that a certain overlap exists
between polycystic ovary syndrome and type 2 diabetes and that similar mechanisms and genes may be
involved in development of both diseases.

Much effort has gone into the search for diabetes susceptibility genes. One such gene, called calpain 10 on
chromosome 2 has been shown to be involved in type 2 diabetes in Mexican-Americans as well as in other
European populations.

Our aim was to study these changes or variants in the calpain 10 gene to see if they play a role in polycystic
ovary syndrome as well. Our sample consisted of 331 women with PCOS, all of European descent; we also
had maternal and paternal DNA samples from 146 of these women. We looked for these calpain 10 variants
in our samples and examined the data to see if one or more of the variants was over-presented in our PCOS
women. We found no relationship between any of the calpain 10 variants and our PCOS samples. Hence,
we conclude that there is no evidence for the involvement of the calpain 10 gene in susceptibility to PCOS.

Additional background information:

Patients for this study have been from both fertility and endocrine clinics and were all of Caucasian descent
(mostly British/Irish). This study included 2 types of subjects, PCOS trios and PCOS cases. Trios are
symptomatic women with PCOS for whom DNA from both mother and father were available. Cases are
individual PCOS women with no parents or siblings.

This work has been funded by the Medical Research Council UK.




                                                   - 222 -
                                                                  ENDOCRINE GENETICS
P3-555
LINKAGE AND ASSOCIATION OF INSULIN RECEPTOR SUBSTRATE-1 VARIANTS WITH
POLYCYSTIC OVARY SYNDROME
L Haddad,1 K Vourvouhaki,1 N Gharani,1 S Franks,2 MI McCarthy.1 1Imperial College Genetics and
Genomics Research Institute, Imperial College, London W12 0NN, United Kingdom; 2Department of
Reproductive Science and Medicine, Imperial College, London W12 0NN, United Kingdom

Polycystic ovary syndrome (PCOS) is a heterogeneous disorder with associated endocrine and metabolic
features. Defects in both secretion and action of insulin are components of the PCOS phenotype and
contribute to the high prevalence of type 2 diabetes in women with PCOS. The molecular mechanisms
underlying insulin resistance in these conditions is unknown, though a post-receptor signalling defect has
been demonstrated in PCOS. The metabolic and mitogenic actions of insulin in target tissues are mediated
by phosphorylation of insulin receptor substrates (IRS) 1 and 2 leading to downstream activation of PI-3
kinase, MAP kinase and other signalling pathways. Associations between variants in the IRS1 gene
(Gly972Arg and Ala513Pro) and type 2 diabetes has been reported in some but not all studies. Our aim was
to examine the role of the IRS-1 missense mutations Gly972Arg and Ala513Pro in PCOS using family-
based and case-control association methods. Our study cohort consisted of 138 Europid parent-offspring
trios with ultrasound confirmed PCOS [median (IQR) age: 30.8 (22.5-39.7)y; BMI 24.3 (17.6-30.9)kgm-2].
We used PCR based assays and the transmission disequilibrium test (TDT) to look for preferential
transmission of alleles at both SNPs. There was no evidence of excess transmission of either allele for
Ala513Pro (χ2=1.08). However, for Gly972Pro, excess transmission of the wild-type Gly972 was evident
(χ2=10.16, p=0.0014). A further 158 PCOS cases and 141 controls were available for case-control
association analysis. The frequency of alleles at codons 513 and 972 did not differ significantly in cases
and controls (χ2=0.24 and 0.66 respectively). Finally, intermediate trait analysis for both codon 513 and
972 (for BMI, WHR, lipid profiles, fasting insulin/glucose) revealed no relationship with genotype in all
296 PCOS cases. Although these results are not entirely conclusive the highly significant TDT score
suggests a role for variation in the gene cannot be excluded and warrants further investigation.

Clinical Science Poster: Endocrine Genetics (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                 - 223 -
                                                                     ENDOCRINE GENETICS
P3-555

Lay explanation of abstract:

Polycystic ovary syndrome (PCOS) is the name given to a common condition that affects between 5% and
10% of women and probably constitutes the most common endocrine disorder in women of reproductive
age. Having PCOS significantly alters the quality of life of pre-menopausal women and is associated with
significant mortality and morbidity in later years. Associated with PCOS is a range of symptoms including
hirsutism (facial and body hair), acne, occasional loss of hair, as well as irregular periods or no periods at
all.

Some of the symptoms of PCOS are triggered by imbalances in various hormones that control the
menstrual cycle. One hormone, insulin, produced by the pancreas in response to glucose is thought to be
involved. Many women with PCOS have insulin resistance in which the body's tissues (except the liver and
the ovary) resist the effects of insulin, so that the body has to produce more insulin to compensate. This
hyperinsulinaemia and insulin resistance as well as abnormal lipid levels form part of the 'metabolic
syndrome' associated with PCOS. PCOS represents a major risk factor for type 2 diabetes. Prevalence of
type 2 diabetes in young obese women is between 11% and 38%. Women with PCOS have a 7-fold
increased risk development of type 2 diabetes in later life.

At the moment, our understanding of the precise mechanisms leading to the development of PCOS is
incomplete. Finding the genes responsible for this disease may provide the necessary clues for a better
understanding, and consequently treatment and prevention of this condition. Insulin exerts its action on
glucose in a step-wise fashion of binding and activating specific molecules, such as the insulin receptor
substrate 1 (IRS-1). Variation in the DNA sequence of the IRS-1 gene has been shown to play a role in
insulin resistance and type 2 diabetes. We chose to study 2 variants in the IRS-1 gene (these variants are
known as Gly972Arg and Ala513Pro) in our PCOS subjects. Using techniques like polymerase chain
reaction (PCR) analysis, we were able to show that one of the variants in the IRS-1 gene (Gly972Arg) may
contribute to the pathogenesis of PCOS. As they stand, our results are not entirely conclusive but given the
important role this gene may play in the insulin action cascade, further investigation is warranted.

Additional background information:

Polycystic ovary syndrome tends to run in families. It is known that the chance that any given person will
develop PCOS over their lifetime depends on a complex mix of factors, some genetic and some
environmental. One data-set used in this study consists of trios, in which samples are obtained from both
parents as well as the daughter with PCOS. This family configuration is very useful when looking at
transmission of genetic material from parents to affected offspring. Another type of data-set used is a case-
control data-set, in which genetic material from individual women with PCOS (cases, no other family
member available) is compared with DNA collected from women who do not have PCOS (controls).


This work was funded by the Medical Research Council, UK.




                                                   - 224 -
                                                                          GLUCOCORTICOIDS
OR21-1
SALIVARY CORTISOL/CORTISONE MEASUREMENTS: CUSHING'S SYNDROME AND
STATINS
H Raff,1 JW Findling,1 RJ Singh.2 1Endocrine Research Laboratory, St. Luke's Med Ctr, Milwaukee,
Wisconsin; 2Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota

The measurement of late-night salivary cortisol is emerging as a simple and accurate method to screen for
Cushing's syndrome. In the process of screening many patients for Cushing's syndrome, we discovered 2
patients with very elevated late-night salivary cortisol levels (>500 nmol/L; normal 7.0 nmol/L. Two had
elevated F/E ratios (12.0 and 18.0) and were also found to be on statin therapy. Although we are unaware
of a mechanism for statin therapy to inhibit the conversion of cortisol to cortisone in the salivary gland and
not the kidney epithelium, it may be responsible for falsely elevated salivary cortisol levels during
screening for Cushing's syndrome.

Clinical Science Oral: Cortisol: Too Much, Too Little (1:00 PM-2:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 1:00 PM; Location: Ballroom 4




                                                   - 225 -
                                                                          GLUCOCORTICOIDS
OR21-1

Lay explanation of abstract:

Cushing‘s syndrome is caused by increased secretion of cortisol from the adrenal glands. It can
masquerade as other diseases such as obesity, hypertension, and diabetes (metabolic syndrome X).
Therefore, the diagnosis of Cushing‘s syndrome is extremely important but quite challenging. We have
developed a simple saliva test to screen for Cushing‘s disease. Cortisol in the saliva reflects cortisol in the
blood, and cortisol is normally low at night. Therefore, patients can take samples of their own saliva at
home at bedtime using a special device, mail them to our laboratory, and we can screen them for Cushing‘s.

The current study investigated whether measuring another steroid in the saliva – cortisone – might be
helpful in evaluating certain enzyme defects that may also be present in the kidney.

We studied 14 patients with elevated salivary cortisol levels. Interestingly, two of them had very high
cortisol levels but they did not appear to have Cushing‘s syndrome. We then found that their cortisone
levels were lower than expected. The only thing these patients had in common was the use of statin drugs
to treat lipid disorders. When these drugs were discontinued, salivary cortisol and cortisone levels
normalized in one patient and improved in the other. We conclude that measuring salivary cortisol is an
excellent modality for screening for Cushing‘s syndrome, but that clinicians must be aware of other factors
that may elevate salivary cortisol.

Additional background information:

Hypertension, obesity, and diabetes are on the increase! It is extremely important that Cushing‘s syndrome
be eliminated as a cause of these common findings before treatment starts. Appropriate treatment of
Cushing‘s syndrome can reduce hypertension, obesity, and diabetes and save patients from prolonged and
unnecessary therapy. Appropriate treatment can also improve the diminished psychological status and
lower quality of life that often accompany Cushing‘s syndrome. This simple screening test using saliva
sampling by the patients at home is a simple approach to making the correct diagnosis.

The funding source was Aurora Health Care, Mayo Clinic, Rochester, Minn.




                                                   - 226 -
                                                  GROWTH HORMONES AND IGFS
OR44-3
INSULIN-LIKE GROWTH FACTOR I IS CRITICAL TO RETINOPATHY OF PREMATURITY
A Hellstrom,1,2 E Engstrom,2 B Carlsson,3 K Albertsson-Wikland,2 A Niklasson,2 A Hard,1 L Sjodell,1
L Smith.4 1Pediatric Ophthalmology, Clinical Neurosciences, Goteborg, Sweden; 2International Pediatric
Growth Research Center, Women and Children's Health, Goteborg, Sweden; 3Research Center fo
Endocrinology and Metabolism, Internal Medicine, Goteborg, Sweden; 4Ophthalmology, Children's
Hospital, Boston, Massachusetts

Background: Retinopathy of prematurity (ROP) is a major cause of blindness in children in the developed
world. Current treatment results in destruction of retina, and is only partially effective at reducing
blindness. Experimental studies have indicated that IGF-I plays an important role in the development of
retinopathy. Aim: To investigate the relationship between longitudinal serum levels of IGF-I and
retinopathy of prematurity.
Methods and patients: Children without obvious malformations and chromosomal aberrations, born at The
Queen Silvia Children's Hospital in Goteborg between December 1999 and September 2000 with a
gestational age (GA) at birth <32 weeks were invited to participate in the present study. Twenty-nine
children (median GA at birth 28 weeks, range 24-32 weeks) were included. Blood-samples were taken
once/week from birth to discharge from hospital. Total IGF-I was analyzed by a IGFBP-blocked RIA.
Examinations for retinopathy were routinely performed weekly according to a specific protocol.
Results: The mean duration of time from birth to IGF-1 reaching 30ng/ml was 19 days in infants who did
not develop ROP (n=19) compared to 58 days for those that developed ROP stage 2-4 (n=10), (P=0.0001).
The median IGF-1 level at the same gestational age was consistently lower in infants who developed ROP
than those who did not develop ROP with a difference at 34 weeks of 25 ng/ml (ROP 2-4) versus 43ng/ml
(without ROP) P=0.002. Maximum IGF-I in the period gestational age 30 to 36 weeks was significantly
lower among the children with ROP than the children without ROP (38ng/ml versus 52ng/ml, P=0.03).
Conclusion: The present study suggests that serum IGF-I is critical to retinopathy of prematurity as we
found abnormal serum IGF-I levels among the children who developed retinopathy of prematurity.

Clinical Science Oral: GH & IGF-I (Pediatrics) (11:00 AM-12:30 PM)

Presentation Date: Saturday, June 23, 2001; Time: 11:30 AM; Location: Ballroom 1




                                                - 227 -
                                                     GROWTH HORMONES AND IGFS
OR44-3

Lay explanation of abstract:

Retinopathy of prematurity (ROP) is a major cause of blindness in children in the developed world. In
infants who develop ROP, growth of blood vessels in the retina slows or ceases at birth, and this leaves a
maturing but avascular (without blood vessels) retina, known as a hypoxic peripheral retina. The limit of
the blood supply to the retina in the initial phase of ROP appears to determine the subsequent degree of
neovascularization, the late destructive stage of ROP, with the attendant risks of retinal detachment and
blindness. If it were possible to allow blood vessels to grow normally in all premature infants, as they do in
the uterus, the late damaging neovascular phase of ROP would not occur.

Current treatment of ROP results in destruction of retina, and is only partially effective at reducing
blindness. Several growth factors, however, have been proposed as agents to regulate blood vessel growth.
In animal studies, low levels of insulin-like growth factor-I (IGF-I) prevent normal retinal vascular growth.
We hypothesized that ROP may be associated with an inability of some premature infants to attain normal
serum levels of IGF-1, corresponding to those normally found in fetuses during pregnancy. IGF-1 is
associated with growth of fetal tissue, and it has been shown that IGF-1 levels after premature birth are
lower than gestational-age-matched fetal levels in the uterus. These IGF-1 levels may be lower in
premature infants because of loss of IGF-1 sources from placenta and from amniotic fluid, in which IGF-1
levels increase rapidly from the second to the third trimester. This time period, the second semester, is often
when pre-term children who are at risk of developing ROP are born. Furthermore, many premature infants
are undernourished, which further lowers IGF-1.

To test the hypothesis that a prolonged period of low IGF-1 levels after premature birth is associated with
lack of vascular growth followed by ROP, we prospectively measured IGF-1 plasma levels and examined
the retinas weekly after birth in twenty-nine premature infants born at gestational ages 24 to 31 weeks at
The Queen Silvia Children‘s Hospital, Göteborg, Sweden, between December 1999 to December 2000.

The mean duration of time from birth to IGF-1 reaching a ―critical level‖ of 30 ng/ml was 19 days in
infants who developed no ROP compared to 58 days for those that developed ROP, which confirmed the
hypothesis that prolonged low levels of IGF-1 were associated with ROP. Maximum IGF-1 was
significantly lower among the children with ROP (38 ng/ml) than the children without ROP (52 ng/ml).
Premature infants with higher IGF-1 levels had more normal vascular development and did not develop
ROP

This work has direct clinical implications for diagnosis and treatment of ROP. These findings suggest that
IGF-1 levels can be used to predict which babies will develop ROP. The differences in pattern of IGF-1
levels between patients that do and do not develop ROP suggest that administration of IGF-1 early after
birth may prevent this disease. Since ROP is correlated with other developmental problems, increasing
IGF-1 levels to the level of infants without ROP may also improve neurological development and somatic
growth.


This research was funded by the Göteborg Medical Society, the Frimurare-Barnhusdirektionen and the
Swedish Medical Research Council (#7905, 10863 and #13515).




                                                   - 228 -
                                                                HEALTH CARE DELIVERY
P3-684
MEETING THE NEEDS OF YOUNG WOMEN WITH PREMATURE OVARIAN FAILURE
NH Alzubaidi, 2 HL Chapin,2 KA Calis,1 VH Vanderhoof,2 LM Nelson.2 1Pharmacy, Clinical Center,
National Institutes of Health, Bethesda, Maryland; 2Gynecologic Endocrinology Unit, National Institute of
Child Health and Human Development, NIH, Bethesda, Maryland

Background: Two-thirds of women with spontaneous premature ovarian failure have osteopenia when they
present to a tertiary level medical research center (Anasti et al., Obstet Gynecol 1998;91:12). This is despite
the fact that 85% of them had been prescribed some form of estrogen replacement. Here we investigate
delay in diagnosis as a contributor to the development of osteopenia in these women.
Methods: We conducted a structured interview with 28 women with premature ovarian failure. The
interview was designed to elicit contributing factors that were under the patient's control and those factors
beyond the patient's control.
Results: Nearly all were college graduates (27/28, 96%) and 10 had graduate degrees (38%). More than
one-quarter (8/28, 29%) waited more than a year before they sought medical evaluation for the loss of
menstrual regularity. Most women [23/28 (82%)] did not search out medical information regarding
menstrual irregularity before contacting their health care provider. Nearly one-half of women considered
the ovary a fertility organ and not a source of hormones (13/28, 46%). Once patients saw a physician, in
nearly two thirds of the cases (18/28, 64%) more than a year elapsed before the diagnosis of premature
ovarian failure was established. Over one-half of women (15/28, 54%) saw at least three physicians before
diagnosis. Nearly two-thirds of women (18/28, 64%) were unsatisfied with the availability of information
about premature ovarian failure.
Conclusions: Our findings are preliminary and biased by recall and the fact that we are a tertiary center.
Nonetheless, these findings in well-educated women suggest that a strong need exists to further
characterize the factors contributing to delay in detecting ovarian insufficiency. There is an apparent need
to educate young women that the ovary functions as an important endocrine organ and that loss of
menstrual regularity may not only be a sign of impending ovarian insufficiency but also a risk factor for
osteoporosis.

Clinical Science Poster: Health Care Delivery (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                   - 229 -
                                                                HEALTH CARE DELIVERY
P3-684
Lay explanation of abstract:

A recent NIH study suggests that young women should consider the menstrual cycle as a vital sign of good
health. This is because having a regular menstrual cycle each month is a sign that the ovaries are
functioning normally. In young women, this is important because the ovaries play an important role in
building and maintaining strong bones. Young women can view the menstrual cycle as a vital sign
reflecting risk for developing osteoporosis. They should record a menstrual diary and seek medical advice
promptly should their cycles become irregular.

This study is important because it provides evidence that young women often experience a delay in the
diagnosis of premature ovarian failure, a condition that if not treated properly can lead to osteoporosis at a
young age. The investigators previously had found osteopenia, evidence suggesting weaker bones, in two-
thirds of women with spontaneous premature ovarian failure that they treat at the NIH Clinical Center.

The investigators conducted this study to determine if delay in diagnosis might be contributing to the bone
loss these young women suffer. They interviewed 28 women with premature ovarian failure to elicit
contributing factors that were under the patients‘ control and factors that were beyond the patients‘ control.
Nearly all patients were college graduates (96%), and 10 had graduate degrees (38%). Nearly 30% of the
women waited more than a year before they sought medical evaluation for the loss of menstrual regularity.
Most women (82%) did not search out medical information regarding menstrual irregularity before
contacting their health care provider. Nearly one-half of women (46%) considered the ovary a fertility
organ and not a source of hormones that help maintain good health. Once patients saw a physician, in
nearly two-thirds of the cases (64%) more than a year elapsed before the diagnosis of premature ovarian
failure was established. Over one-half of women (54%) saw at least three physicians before diagnosis.
Apparently there is a need to educate young women that the ovary functions as an important endocrine
organ and that loss of menstrual regularity may not only be a sign of impending ovarian insufficiency but
also a risk factor for osteoporosis.

Additional background information:
Ovarian failure affects 1% of women by age 40. These menopausal symptoms are related to estrogen
deficiency. Young women deficient in estrogen may also be at increased risk of developing heart disease
or osteoporosis at an earlier age.In the past, doctors referred to premature ovarian failure as ―premature
menopause.‖ This is because at the time it was believed that in this disorder, the ovary had stopped
working permanently and irreversibly. We now know this is not true. In some cases of premature ovarian
failure the ovary can work normally again even years after the diagnosis. Today most specialists who deal
with this condition prefer to use the term ―premature ovarian failure‖ rather than ―premature menopause.‖
Specialists believe this lets them communicate more accurately and clearly with young women who
develop this condition. We have seen women who have developed premature ovarian failure as early as age
14. One half of the women that we see here at the NIH Intramural Research Program develop the condition
before they reach their 32nd birthday. Normal menopause occurs because the ovary runs out of critical
units that are called primordial follicles. These are tiny microscopic structures in the ovary that are stored
for future hormone production and egg release. Women have all these tiny units already formed in their
ovaries when they are born and can‘t make any new ones. Each month, women normally use a small
portion of their store of primordial follicles. Once all these units have been completely used up, the ovary
can‘t make normal amounts of estrogen any longer. Normal menopause occurs because the ovary runs out
of these critical follicles. In other words, menopause happens because of ―follicle depletion.‖ However,
this situation is not true of many women with premature ovarian failure. Many women with premature
ovarian failure have ―follicle dysfunction.‖ This means there are still primordial follicles remaining in the
ovary, but something prevents them from working normally.

The National Institute of Child Health and Human Development Intramural Research Program funded this
study.




                                                   - 230 -
                                                              HEALTH CARE DELIVERY
P3-685
IMPROVED PERIOPERATIVE GLYCEMIC CONTROL BY AN ENDOCRINOLOGY TEAM
DOES NOT INCREASE COSTS IN PATIENTS WITH DIABETES
AC Vora,1 RC Polomano,2 V Eddinger,1 C Hollenbeak,1 T Girdharry,2 D Martin,2 RA Gabbay.1 1Div. of
Endocrinology, Penn State Milton S. Hershey Med Ctr, Hershey, Pennsylvania; 2Dept of Anesthesiology,
Penn State Milton S. Hershey Med Ctr, Hershey, Pennsylvania

Although patients with diabetes and Coronary Artery Disease (CAD) are more likely to require Coronary
Artery Bypass Graft surgery (CABG), their outcomes are worse than those without diabetes. Poor glycemic
control could underlie the increased risk of infections and improving glycemic control perioperatively, may
improve outcomes. The financial impact of clinical care resources needed to maintain strict glycemic
control has not been determined. Specifically, it is unclear whether interventions to optimize control are
associated with improvement in measures of institutional efficiency (length of hospital stay and associated
cost).Our Intensive Glycemic Control Protocol (IGCP), using an intravenous insulin regimen under the
supervision of the endocrine consultants, significantly improved glycemic control. Outcomes of CABG
surgery patients with diabetes receiving our IGC Protocol (n=107, year 2000) were compared to those
diabetics having CABG prior to the use of the Protocol (n=81, year 1999). An extensive cost analysis was
performed on data procured from our financial database, TrendStar, which incorporated the additional costs
from our protocol such as glucose monitoring every 2 hours, pharmacy expenditures and professional fees
for endocrine consultations.
Overall hospital costs were not affected by the intervention (control $21,442; IGCP $21,076). There was no
significant difference between length of stay from surgery to discharge (LOS) in the two groups (control,
5.98 days; IGCP, 5.48 days). Incidence of deep sternal wound infections (DSWI) was 4.94% in controls
and 4.67% with the IGCP.
We demonstrate that glycemic control can be improved by implementation of our clinical care guidelines
with no additional health care costs. Therefore, endocrinologist involvement was a revenue neutral service
that significantly improved the management of patients with diabetes undergoing major vascular surgery.
The decreasing trend for LOS and rate of postoperative DSW infections associated with strict glycemic
control is currently being evaluated further.

Clinical Science Poster: Health Care Delivery (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                 - 231 -
                                                               HEALTH CARE DELIVERY
P3-685
Lay explanation of abstract:

Eighty percent of all deaths in diabetic patients result from cardiovascular disease; about three-fourths of
these are a result of coronary artery disease. Patients with diabetes and coronary artery disease are more
likely to need coronary artery bypass graft surgery (CABG) than patients who don‘t have diabetes but
diabetic patients experience worse outcomes from the surgery. About 250,000 patients with diabetes have
CABG done each year in the US. The average length of stay in hospital after CABG is about 3-6 days
longer in diabetic patients. Furthermore, they have a 2-2.5 fold increased risk of developing major
infections compared to non-diabetic patients. Surgery in patients with diabetes is therefore associated with
poorer outcomes and increased costs.

Preliminary evidence suggests that controlling blood glucose levels during hospitalization could reduce
these complications. The financial impact of the clinical care resources needed to maintain strict glycemic
control has not been determined. Specifically, it is unclear whether interventions to optimize control are
associated with reduction in length of hospital stay and associated costs.

Our Intensive Glycemic Control Protocol (IGCP), using an intravenous insulin regimen under the
supervision of an endocrinologist, significantly improved glycemic control in patients undergoing CABG at
the Penn State-Hershey Medical Center. Outcomes of CABG surgery patients with diabetes who received
our IGC Protocol (n=107, year 2000) were compared to those diabetics having CABG before the use of the
Protocol (n=8l, year 1999). An extensive cost analysis of our protocol was performed, incorporating the
additional costs such as glucose monitoring every 2 hours, pharmacy expenditures, and professional fees
for endocrine consultations.

There was a trend towards a reduction in length of hospital stay and risk of infections in patients treated
with the IGC protocol, but this failed to reach statistical significance. Overall hospital costs were not
affected by the intervention (control $21,442, IGCP $21,076). We demonstrated that glycemic control can
be improved, with no additional health care costs, by implementing our clinical care guidelines.

Endocrinologist involvement proved to be a revenue-neutral service that significantly improved the
management of patients with diabetes who were undergoing major vascular surgery. The promising trends
of decreasing length of stay and decreasing rate of postoperative DSW infections associated with strict
glycemic control are being evaluated further as more patients enroll.

This study had no external funding sources.




                                                  - 232 -
                                                                HORMONES AND CANCER
P2-660
HORMONE DEPENDENT BREAST CANCER: INFLUENCE OF AN ENDURANCE EXERCISE
PROGRAM
C Peters, T Schulz, H Loetzerich, H Michna. Morphology and Tumor Research, German Sport University,
Cologne, Germany

Objective: In the context of prevention and rehabilitation of cancer sports is currently acknowledged, since
numerous independent studies indicate a beneficial influence of moderate exercise on the immune system
and psychological parameters. It is well established that there is a strong relationship between
psychological parameters and the immune system and it is well known, that stress may be immune-
suppressive which is documented by a decreased activity of natural killer cells.
Methods: We investigated the influence of a moderate exercise training on the immune system of 49 breast
cancer patients. Therefore, 24 women trained 5 times a week in a rehabilitation cure and 2 times a week for
further six months in an ambulatory group; the other 25 women served as controls. At the beginning of the
cure, after the cure and after 6 months blood samples were taken to determine immune functions, e.g.
phagocytosis of granulocytes and monocytes, natural killer cell activity and leukocyte subpopulations. In
parallel, psychological parameters were tested with questionaires (EZ, FPI-R).
Results: The moderate training showed positive effects on different immune functions: phagocytic activitiy
of granulocytes and monocytes as well as the natural killer cell cytotoxicity increased. In addition, in regard
to psychological effects moderate exercise training is able to enhance quality of life.
Conclusion: Our results demonstrate that moderate exercise training may restabilize reduced
immunological functions in female breast cancer patients as well as improve psychological behaviour and
mood. Therefore, moderate exercise training should be considered to be integrated part in the rehabilitation
program of breast cancer patients.

Clinical Science Poster: Hormones & Cancer (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                   - 233 -
                                                                HORMONES AND CANCER
P2-660

Lay explanation of abstract:

In industrialized nations, one out of every four people dies from cancer. Although medical technology has
improved during the last few years, we still need improved cancer prevention, therapy, and rehabilitation.
In the context of prevention and rehabilitation of breast cancer, participation in exercise and sports appears
promising. Several independent studies indicate that moderate exercise can stimulate the immune system
and improve psychological parameters. It is well established that there is a strict relationship between
psychological parameters and immune system function. Stress can be immune-suppressive, which has been
documented by a decreased activity of natural killer cells.

We therefore investigated the influence of moderate exercise training on the immune system of 49 breast
cancer patients. Twenty-four women trained five times a week in a rehabilitation program (cure). During
the following three months they trained in an ambulatory group three times a week. Another 25 women
served as a control group. At the beginning of the cure, right after the cure, and 6 months after the cure,
blood samples were taken to determine immune functions, such as the activity of special innate immune
functions like the phagocytosis of granulocytes and monocytes or the cytotoxicity of natural killer cells
which are known to play an important role in immunosurveillance against spontaneously arising tumors.
Simultaneously, psychological parameters were tested with the help of questionnaires (EZ, FPI-R).

Regular endurance training seemed to have an immune-stimulating effect on different cells of the immune
system: While phagocytic activity of granulocytes and monocytes as well as the cytotoxicity of natural
killer cells increased in the training group, the levels in the control groups remained the same after 6 weeks
of the cure. After another five months of training, the measured immune functions of the training group
increased and reached a healthy level, whereas the basic immune functions in the control group did not
increase. In addition, in regard to psychological effects moderate exercise, training is able to enhance
quality of life, which is important for personal psychosocial well being and future health.

Our results demonstrate that moderate exercise training may be able to restabilize reduced immunological
functions in female breast cancer patients as well as to stabilize psychological behavior. Therefore,
moderate exercise training should be considered as an integral part of the rehabilitation program of cancer
patients.

This study was supported by the Society for the Prevention of Cancer within Northrhine-Westfalia (NRW),
Germany and by Boehringer Mannheim, Germany.




                                                   - 234 -
                                                                HORMONES AND CANCER
P2-668
COGNITIVE PATTERNS IN TRANSSEXUALS TREATED WITH EXOGENOUS HORMONES
MT Prendiville,1 AB Wisniewski,2 TT Nguyen,1 AS Dobs.1 1Medicine, Johns Hopkins University,
Baltimore, Maryland; 2Pediatrics, Johns Hopkins University, Baltimore, Maryland

Gonadal hormones have been shown to have activational effects on cognitive functions in transsexuals. Our
group further supported activational effects of sex steroid hormones in populations of post menopausal
women receiving estrogen and testosterone hormone therapy. Previous work on female to male transsexual
(FM) populations indicates activational effects of testosterone on spatial cognitive functions. The aim of the
present study is to investigate the effects of super-physiological levels of estrogen on visuo-spatial and
perceptual speed cognitive functions of male to female (MF) transsexuals. We hypothesized that these
cognitive functions can be manipulated with large doses of exogenous estrogen. Thirteen MF (47 ± 6.5
years old) transsexual subjects receiving exogenous estrogen for at least 3 months had a total estrogen level
of 1053.07 ± 285.6 pg/dl and estradiol of 474.4 ± 134.9 pg/dl. With the exception of the visual memory
task, the MF group showed no significant differences from the eugonadal control men (56 ± 5.5 years old)
on all visuo-spatial and all perceptual speed tasks. However, MF subjects exhibited a significant decrease in
performance on a visual memory task (F(1,21) = 6.569, p = 0.018). This pattern of performance is similar
to that observed in previous work with post menopausal women receiving double the standard dose of
estrogen hormone replacement therapy (1.25 mg esterified estrogen) (Wisniewski, submitted). In summary,
super-physiological levels of estrogen did not cause MF transsexuals to exhibit enhanced patterns of
performance on perceptual speed cognitive tasks. In addition, there is some indication that elevated levels
of estrogen may have an adverse effect on visuo-spatial memory building.

Clinical Science Poster: Hormones & Cancer (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                   - 235 -
                                                                HORMONES AND CANCER
P2-668

Lay explanation of abstract:

The aim of this study was to investigate the effects of super-physiological levels of estrogen on visuo-
spatial and perceptual-speed cognitive functions of transsexuals who changed from male to female (MF).
Thirteen (47  6.5 years old) transsexual subjects receiving exogenous estrogen for at least 3 months
received higher than normal (super-physiological) levels of estrogen. With the exception of the visual
memory task, the MF group showed no significant differences from men in a control group (56  5.5 years
old) on all visuo-spatial and all perceptual speed tasks. The MF subjects exhibited a significant decrease in
performance on a visual memory task. This pattern of performance is similar to that observed in previous
work with post-menopausal women receiving twice the standard dose of estrogen hormone replacement
therapy. In summary, super-physiological levels of estrogen did not cause MF transsexuals to exhibit
enhanced patterns of performance on perceptual speed cognitive tasks.

Additional background information:

We had shown previously that sex hormones, such as testosterone, do affect the brain. The level of
testosterone determines which of the two hemispheres of the brain is dominant. This effect on hemisphere
dominance can translate to cognitive effects. For example, we have shown that testosterone can increase the
dominance of the right hemisphere, which in turn improves a person‘s spatial ability. We have studied
testosterone in both men and post-menopausal women.




                                                   - 236 -
                                                                                              OBESITY
P1-561
DIET-INDUCED LONG TERM CHANGES IN BODY FAT AND SERUM LEPTIN CAN BE
PREDICTED IN OBESE FEMALES WITH THE G-2548→A LEPTIN PROMOTER
POLYMORPHISM .
G.H. Scholz1, G. Flehmig1, M. Scholz3, D. Gutknecht1 and K. Huse2. 1Department of Internal Medicine III,
University of Leipzig, Leipzig, Germany, 2Department of Genome Analysis, Institute of Molecular
Biotechnology, Jena, Germany, 3Department of Nutrition, ETZ-M.Scholz, Leipzig, Germany

Leptin is involved in appetite and weight regulation in humans. A single nucleotide polymorphism in the
leptin promoter was found to be related to weight loss in moderate obese women [1] and to extreme obesity
[2]. The mutation destroys the restriction site for Cfo I. We have investigated the relationship between this
polymorphism, body fat and leptin changes during a 1000 kcal weight reducing program in obese females
by RFLP analysis of DNA. Body fat (body impedance analysis) and leptin levels were measured and
nutritional guidance was given every two weeks. Delta body fat and delta leptin were calculated at 2 and 10
weeks in a group of females (n=93, age: 45±12y, BMI: 32.8±5.9 kg/m2). Delta leptin levels at 10w differed
significantly (p=0.07) in the three genotypes (Cfo -/-:15.0±12.2, Cfo+/-:7.4±12.6, Cfo+/+:7.7±12.8 ng/ml as
did delta body fat (Cfo-/-:6.2±2.6, Cfo+/-:5.2±2.9, Cfo+/+:4.0±2.1 kg, p=0.007). Leptin at baseline and delta
leptin at 2w were analysed in a multiple linear regression model to calculate the predictive power on delta
leptin 10w.
In the Cfo-/- genotype (n=24) leptin 0w and delta leptin 2w are predictive (p<0.001 and p<0.002),
(R2adj.(%):89.4). In the Cfo+/- genotype (n=37) and in the Cfo+/+ genotype (n=32) delta leptin 2w remained
predictive (p=0.001) but leptin 0w lost its predictive power and the explained variances are reduced to
R2adj.(%):63.1 and 36.4.
The Cfo leptin promoter polymorphism seems to be related to the achieved fat changes. Females with the
Cfo-/- genotype may have better chances to reduce their body fat. Long term changes in leptin can be
predicted in the Cfo-/- genotype but less in the other genotypes, mainly because of a significant contribution
of the leptin level at baseline in the Cfo-/- genotype. This kind of prediction might help to improve weight
reducing programs by individualizing dietary recommendations. 1. Mammes O et al. (1998) Diabetes
47:487-489; 2. Li WD et al. (1999) Ann Hum Genet 63:227-234

Clinical Science Poster Session: Obesity (11:00 AM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                   - 237 -
                                                                                              OBESITY
P1-561
Lay explanation of abstract:

Programs for the reduction of body weight (body fat) are difficult to adapt to individual needs, because
many factors are involved in regulating appetite and physical activity. Our research should help to predict
the outcomes of a weight-loss program and help identify individuals who should receive special support.
Because leptin plays a key role in weight regulation, we analyzed the relationship between changes in body
fat and serum leptin levels during a 1000 kcal weight-reducing program and the genotypes of obese females
(n=93; age: 45±12years; BMI: 32.8±5.9 kg/m2).

The genotypes were characterized by a single nucleotide polymorphism (G to A) in one (AG) or both
alleles (AA, GG) of the leptin promoter, which participates in the regulation of leptin synthesis. DNA-
sequencing and restriction fragment-length analysis was used to identify the mutation. Body weight, body
fat (by bioelectric impedance analysis) and leptin (by radioimmunoassay) were measured initially, at 2
weeks, and at 10 weeks, respectively. Leptin is an integrating indicator for changes in body fat and energy
intake. Delta leptin levels at 10 weeks differed significantly (p=0.07) in the three genotypes
(AA:15.0±12.2, AG:7.4±12.6, GG:7.7±12.8 ng/ml) as did delta body fat (AA:6.2±2.6, AG:5.2±2.9,
GG:4.0±2.1 kg, p=0.007). Females homozygous for the A-allele may be more likely to reduce their amount
of body fat.

To predict the outcome of the dietary intervention we used a mathematical model that asks: How strong is
the influence of the initial leptin value/body fat content and the change of leptin/body fat after 2 weeks and
the change of leptin/body fat after 10 weeks? The achieved predictive value is named R² (maximum: 100).
We found that reduction of serum leptin and body fat values after 10 weeks depended on the genotype of an
individual. If a female with the DNA-mutation from G to A (AA-type) is characterized by both a high
initial leptin level or body fat content and successfully reduces leptin levels or body fat content within the
first 2 weeks, she will be able to continue the reduction within the 10-week period (R² for leptin: 89.4 and
fat: 44.1) without additional advice. In females homozygous for the G-allele, the reliability for this
prediction is much lower (R² for leptin: 36.4 and fat: 11.4). Practically, this means that AA-females with
only a small reduction of leptin levels or body fat content after 2 weeks and any GG-female (with no regard
to their 2 weeks results) are in need of additional diet/exercise advice in order to achieve their personal
goals.

Additional background information:

Weight loss and maintenance are difficult to achieve according to long-term studies worldwide. Life style
changes in the last century with lower physical activity levels and inappropriate high-energy (excess
calories) intake probably cause most weight gain. In 1994, leptin, a hormone involved in the regulation of
appetite, energy consumption and weight maintenance, was discovered. In 1995 a method to measure leptin
in human serum was available for our group. In 1995, we started an observational study measuring leptin
and other biochemical and morphometric parameters during a 1000 kcal weight-reducing program. In 1996
we published one of the first series of data showing that leptin and body fat dissociate during long-term
dietary intervention (Scholz, G.H. et al., Hormone and Metabolic Research, 28 (1996) 718-723). We
suggested that there are interesting differences in the response of body fat and leptin among individuals, as
well as the possibility that in some individuals, low leptin values during their diet may indicate relative
leptin deficiency. In recent years, we have focused on the differences in the kinetics of body fat and leptin,
respectively, in obese females during intentional weight loss. We investigated possible genetic factors,
which may explain our previous observations. The G-A polymorphism in the leptin promoter is one step in
this direction and allowed us to develop a genotype-based advisory program in an experimental setting.

The research program was funded by the ETZ-M.Scholz, Leipzig, and the University of Leipzig.




                                                   - 238 -
                                                                                           OBESITY
P1-566
REDUCTION OF WEIGHT AND WAIST CIRCUMFERENCE AND MODIFICATION OF
CORONARY RISK FACTORS WITH METFORMIN AND CARBOHYDRATE-MODIFIED DIET
IN NON-DIABETIC MEN WITH MIDLIFE WEIGHT GAIN AND HYPERINSULINEMIA
HR Mogul,1 W Lee,1 S Zhang,1 BI Weinstein,2 AL Southren,1 SJ Peterson.1 1Medicine, New York Medical
College, Valhalla, New York; 2Biochemistry, New York Medical College, Valhalla, New York

Introduction: The associations of age-related total and abdominal adiposity with coronary risk factors are
well established. Pharmacotherapy has not been specifically evaluated in the treatment of midlife weight
(wt) gain. The goal of this analysis was to evaluate metformin as an adjunct to carbohydrate-modulated diet
for reduction of wt and waist circumference (WC) and coronary risk modification in men with
hyperinsulinemia (AUC-insulin≥100μU/ml). We conducted a retrospective review of 16 consecutive non-
diabetic men (55.3±3.4 yrs, BMI 36.1±1.6kg/m2) with progressive midlife wt gain (despite attempted
intervention) and increased WC (47.0±2.0") seen in a wt management program. METHODS Patients were
treated with metformin (1500mg/day) and calorie/carbohydrate-reduced diet. Paired t-tests assessed
changes in wt, WC, blood pressure, lipids, insulin and HOMA-R in 14 patients who returned for ≥1 follow-
up visit. RESULTS Metformin produced significant wt decrements at 1, 3, 6 month and final visits
(respectively 10.8±2.1, 15.4±3.7, 20.4±3.9 and 21.2±3.5 lbs (p= .000, .002, .001, .000) with ≥5% wt loss in
11 of 14 patients at 3 mos. CONCLUSION Preliminary data in a distinct subset of non-diabetic,
hyperinsulinemic men suggests that metformin and carbohydrate-modulation produces wt loss and highly
desirable modification of abdominal adiposity, HOMA-R and other coronary risks.

                                                      Total-     HDL-
          Weight(se) Waist       SBP       DBP                                LDL-Chol TG         HOMA-
                                                      Chol       Chol
          lbs        inches      mmHg      mmHg                               mg/dL    mg/dL      R
                                                      mg/dL      mg/dL
                       47.9      142.7     85.7       217.3                   142.8     184.0
Baseline 253.0 (3.5)                                             40.5 (2.7)                       4.3 (0.6)
                       (0.9)     (4.5)     (2.5)      (11.7)                  (9.0)     (23.9)
Final
          231.8        43.5      128.4     74.0       190.2      40.1         115.9     122.0     2.3
visit
P-value   0.000        0.000     0.007     0.000      0.041      0.870        0.017     0.025     0.007


Clinical Science Poster: Aspects of Human Obesity (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                   - 239 -
                                                                                                OBESITY
P1-566
Lay explanation of abstract:

The study describes a novel obesity treatment in non-diabetic men with central obesity (increased fat
accumulation at the waistline) and increased insulin levels. Findings suggest that a diabetes drug,
metformin, in combination with diet modifications may be useful for long-term weight management in
people with insulin abnormalities. More than half of American adults are now considered overweight.
Overweight and obese people of all ages have an increased risk for developing diabetes, heart disease,
certain types of cancer, including breast, prostate and colon, and earlier death from all causes compared to
normal weight individuals. Although metformin has been used to treat obesity in non-diabetics in Europe
and has been shown to reduce the amount of food that people eat, it has not been studied as a primary
treatment for obesity or related coronary risk factors in the United States.

Our findings are newsworthy because very few effective weight reduction strategies are currently available
and recent studies show an alarming increase in the number of overweight and obese Americans. These
reported interventions could be effective for many overweight people who typically have undetected insulin
abnormalities and are at high risk for the development of heart disease. These strategies might have an
impact on many overweight Americans. The diet and medication programs were easily implemented,
required minimal physician contact, and induced permanent lifestyle changes in the study population.
Thus, when targeted to a population of overweight and obese men, the reported combination of diet and
medication could be both clinically effective and economical.

The study addressed an important endocrine abnormality, insulin resistance and hyperinsulinemia. In the
study subjects, obesity treatment was matched to a specific underlying metabolic defect, hyperinsulinemia.
Identification of hyperinsulinemia in 16 men with previously normal blood sugars was an important first
step in the research. This set the stage for the use of metformin, a drug known to produce weight loss in
diabetic and non-diabetic patients in European obesity studies.

Sixteen non-diabetic men (average age 55) with hyperinsulinemia (based on a standard test of sugar
tolerance) were treated with a calorie- and fat-restricted diet in which carbohydrates were reduced and
added sugars (or sweets) were eliminated and were given metformin 3-4 times a day. The study evaluated
response to treatment in consecutive patients who returned for at least one follow-up visit (14/16 who
started in this study). All the men had been unable to lose weight, despite conventional medical care that
included diet and participating in a regular exercise program (at least 3 time a week). Metformin produced
significant weight loss at 1-, 3-, 6-month, and final visits (respectively 10.8, 15.4, 20.4, and 21.2 lbs) with
11 of 14 men losing 5% or more of their body weight at 3 months. At the final visit, significant changes
were observed in the following risk factors for heart disease (as shown in the average baseline compared
with follow-up group measurements):
      Waist circumference ( 43 vs. 47 inches)
      Diastolic blood pressure (85.7 vs. 74.0)
      Total cholesterol (217.3 vs. 190.2 mg/dl )
      LDL cholesterol (142.8 vs. 115.9 mg/dl )
      Triglycerides (184.0 vs. 122.0 mg/dl)
      Fasting insulin (16.3 vs. 10.0 U/ml)
      HOMA-R – a measure of insulin resistance (4.3 vs. 2.3).

Preliminary data in a distinct subset of non-diabetic, hyperinsulinemic men (who had been unable to lose
weight previously despite exercise and conventional weight reduction) suggests that metformin and
carbohydrate-modulation produces weight loss and highly desirable modification of abdominal fat,
HOMA-R, and other coronary risks. Our findings provide hope for new weight loss and behavior
modifications among overweight adults.

The research was not funded.




                                                   - 240 -
                                                                                            OBESITY
P1-567
USE OF ROSIGLITAZONE IN TREATMENT OF HYPERINSULINEMIC OBESITY IN NON-
DIABETICS
RC Shoemaker,1 AR Cobitz.2 1McCready Hospital, Pokomoke, Maryland; 2GlaxoSmithKline, Collegeville,
Pennsylvania

Treatment of obesity in hyperinsulinemic (insulin-resistant) patients who do not have diabetes is difficult,
with sustained weight loss rarely obtained. A new approach involving restriction of amylose in the diet has
demonstrated weight loss of 0.5 lbs/week.Thiazolidinediones reduce insulin resistance and hence their use
in the treatment of hyperinsulinemic patients with obesity, but not diabetes, could present a new approach
to weight loss for these patients. A pilot study performed on 100 patients in 1998 showed that troglitazone
combined with an amylose-free diet improved weight loss in these patients without adverse effects and was
not associated with dyslipidema or edema. A repeat study combining rosiglitazone (RSG) with an amylose-
restricted diet is reported here.
Patients who presented for management of weight loss with a BMI >29 kg/m2, a fasting insulin level ≥10
ng/mL, and a fasting blood sugar ≤110 mg/dL were treated with RSG 4 mg bd for 12 weeks.
In the group compliant with diet (n=30), average weight loss was 1.5 lbs/week for women and 1.7 lbs/week
for men. Total cholesterol fell by 8.6%, LDL-cholesterol by 10.1%, and HDL-cholesterol increased by
4.6%. Average loss of hip circumference was 2.4 inches in women, and the average loss in waist
circumference was 1.6 inches in patients of both sexes. Average change in body composition in the
compliant group was 4.6% fat loss and 3.1 lb increase in lean body mass. 80% of compliant patients
reported a sensation of warmth without fever. Edema was not observed. Those non-compliant with diet
(n=10) gained 0.3 lbs/week, had a rise of total and LDL-cholesterol of 10%, and did not notice warmth.
20% of the non-compliant group developed edema. There were no adverse health effects noted in either
group.
In conclusion, the combination of RSG with an amylose-free diet in hyperinsulinemic, non-diabetic, obese
patients resulted in rapid weight loss and significant decreases in hip and waist circumference combined
with a favorable safety profile. The beneficial effects of RSG in a weight-loss program may be due to the
induction of transcription of genes coding for uncoupling protein as well as glucose transport proteins.
These effects may be blocked by dietary amylose.

Clinical Science Poster: Aspects of Human Obesity (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 241 -
                                                                                                   OBESITY
P1-567

Lay explanation of abstract:

Obesity has reached epidemic proportions in the US. Despite a public awareness of the importance of
exercise and calorie restriction, dieters seldom lose more than 30 pounds and rarely do those few successful
dieters keep the weight off long term. Some unsuccessful dieters have a hormone problem associated with
their obesity that is called ―insulin resistance.‖ These patients don‘t have diabetes, which brings low blood
sugar, but they have too much insulin in their bloodstream. The problem comes from the fact that their
insulin doesn‘t work properly to remove sugar from the blood and put it into liver and muscle cells, where
it would normally either be stored in glycogen or immediately burned for fuel.

High insulin levels are associated with other metabolic problems. The high-insulin patient converts sugar to
fat and stores it efficiently, often has high blood pressure, and often has high cholesterol and triglycerides.
High-insulin patients also are more likely to have heart disease and gout, among other illnesses.

A new group of FDA-approved medications used to help treatment of diabetes, called thiazolidinediones,
shows great promise in treating insulin resistance. These medicines, including rosiglitazone (Avandia), are
safe for patient use. Unlike the first FDA-approved medication in this class, Rezulin, they don‘t cause liver
problems. Unlike sulfonylurea medications or patient-administered insulin, they don‘t create the risk of
low-blood-sugar reactions. They work by activating a group of genes that help the body move sugar into
the cells more efficiently.

In this study, the authors used a special diet and rosiglitazone therapy to treat 40 of the most refractory,
obese patients attending a weight-loss clinic. These patients all had failed many attempts to lose weight and
keep it off. With explicit FDA- and IRB-approved informed consent, these patients eliminated a particular
starchy carbohydrate, amylose, from their diet. They ate 8 ounces of protein a day, and 3 servings each of
fresh fruit and vegetables that grow above the ground. They did not count calories, portion size was not
restricted, and fat grams were not counted.

The results were astounding. With the diet alone, the patients lost only one half pound per week. With the
diet and medication, women lost 1.5 pounds per week during the 12-week study, and men lost 1.7 pounds
per week. Women lost 2.4 inches around their hips and 1.6 inches off their waist. There were no adverse
events during the study.

This study will need to be verified in larger clinical trials at multiple sites, but if the results are confirmed,
rosiglitazone and the special no-amylose diet has the promise of defeating the increasing problem of
obesity by activating the body‘s own metabolic machinery to burn fat.

Additional background information:

This study was funded by GlaxoSmithKline.




                                                     - 242 -
                                                                                        OBESITY
P1-571
INCREASED RESPONSE OF THE HYPOTHALAMIC-PITUITARY-ADRENAL AXIS TO
NALOXONE IN OBESE HUMANS
EJ Nye, JE Grice, GI Hockings, CR Strakosch, DJ Torpy, MM Walters, GV Crosbie, RV Jackson. Dept. of
Medicine, University of Queensland (GPH), Brisbane, Australia

Obesity may be associated with HPA axis hyperactivity in humans. Ten obese subjects (mean wt 123.3 kg,
LBM 61.1 kg, BMI 43 kg/m2), 10 normal weight subjects (mean wt 84.3 kg, mean LBM 66.9 kg, mean
BMI 26.7 kg/m2) had 4 afternoon randomised, single-blinded tests: NAL (125 µg/kg iv), NAL-LBM (100
µg/kg LBM iv), AVP (0.0143 IU/kg iv) and NAL/AVP (125 µg/kg + 0.0143 IU/kg iv). We measured
plasma ACTH (RIA) and cortisol (HPLC). Responses (mean±SE) are shown in the table. No differences in
basal hormone levels were seen. In controls, NAL and AVP gave similar responses (all P=NS), whereas
NAL/AVP gave significantly higher peak and AUC responses (P<0.05 vs NAL or AVP alone). Obese
patients with NAL had higher ACTH responses than controls (AUC, P<0.05). Peak cortisols were not
different but obese subjects had greater cortisol AUC (P<0.05). With NAL-LBM, both groups received a
similar NAL dose (P=NS). ACTH peaks were not different but AUC had a considerably greater magnitude
in the obese group (P<0.1). Indeed, 4 obese subjects had ACTH AUCs that were much greater than (AUC
mean + 2SD) for controls. Cortisol responses in the NAL-LBM test were not different. There was no
difference in responses to AVP or NAL/AVP between the groups. Within the obese group, unlike controls,
NAL gave ACTH and cortisol responses that were not different from the combined NAL/AVP stimulus. In
conclusion, obese persons may have an enhanced opioid tone, or an increased hypothalamic and/or
pituitary sensitivity to endogenous CRH.

TEST              Normals - ACTH Normals - Cortisol Obese - ACTH Obese - Cortisol
NAL        Peak 7.8+/-1.1          362+/-43           13.7+/-3.1      456+/-24
           AUC 167.8+/-43.9        10360+/-1940       570.3+/-284.5 24011+/-2945
AVP        Peak 9.9+/-1.6          363+/-31           9.4+/-1.6       351+/-46
           AUC 143.0+/-37.4        8708+/-1662        166.0+/-34.8    12110+/-3453
NAL/AVP Peak 26.7+/-4.9            490+/-18           20.0+/-4.5      544+/-34
           AUC 811.4+/-174.3       24602+/-2095       650.2+/-152.6 30875|/-2961
NAL-LBM Peak 6.4+/-1.0             403+/-44           9.4+/-1.7       439+/-32
           AUC 135.0+/-39.4        14042+/-1882       296.5+/-60.7    18901+/-2663

Clinical Science Poster: Aspects of Human Obesity (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                               - 243 -
                                                                                                OBESITY
P1-571

Lay explanation of abstract:

We found evidence that one of the body's major stress hormone systems, the hypothalamic-pituitary-
adrenal axis, may respond excessively to stimulation in obese humans compared with normal weight
control subjects. Normally, the axis can be activated by a variety of stimuli, including everyday things
such as waking up or eating, more severe physical stresses such as injury or illness, and emotional stress.
Activation leads to a cascade of progressive hormone secretion, with the end product being release of the
hormone cortisol from the adrenal glands. The body needs a certain amount of cortisol to function
effectively, and its actions are widespread. However, in conditions where it is over-produced, it can have
cause problems, including high blood pressure; such metabolic disturbances as increased blood sugar and
insulin resistance; and an increase in fat mass, particularly central or abdominal fat. Such ill effects are seen
in individuals who have an over-production of cortisol as a result of a pituitary or adrenal tumor, for
example. These effects also are common among overweight people.

Unlike people with tumors, in whom cortisol secretion may be continually high, obese people are believed
to have relatively normal, unstimulated cortisol secretion. Our research suggests that, at least in some obese
people, cortisol can be over-produced in response to stimulation of the hypothalamic-pituitary-adrenal axis.
Furthermore, these results and those from previous studies lead us to believe that the cause of this defect
occurs at the first step in the chain of activation of the hypothalamic-pituitary-adrenal axis, the release of
the hormone CRH from the hypothalamus gland in the brain.

If our initial findings are confirmed by further studies, there may be significant implications for some
overweight people. Patients whose obesity is linked with over-activity of the hypothalamic-pituitary-
adrenal axis might feel relieved to learn of an identifiable and potentially treatable cause for their weight
problem.

Ten obese and 10 normal weight subjects underwent four tests with different intravenous medications,
designed to stimulate stress hormone release. Our significant findings came from two tests in particular.
Naloxone, which stimulates the first step in the cascade (CRH release), followed by the second step (ACTH
release) and the third step (cortisol release), caused significantly higher blood plasma levels of ACTH and
cortisol in obese subjects. However, another intravenous medication, AVP, which bypasses the first step
and directly stimulates ACTH and then cortisol, caused no difference in ACTH and cortisol levels between
the two groups. We concluded that obese subjects had increased CRH release when the entire axis was
stimulated by naloxone, as it would be in response to other stimuli of this axis.

Additional background information:

The Australasian Society for the Study of Obesity states that 40% of Australian adults are significantly
overweight. Many of these people are particularly unsuccessful at losing weight and then maintaining a
normal body weight. We believe that for some of them, there may be a link between their obesity and a
complex system of hormones secreted in the brain and the adrenal glands. Among their many functions,
these hormones are known to control or manage fat mass, appetite, behavior, and the body‘s responses to
stress. One particular hormone system is known as the hypothalamic-pituitary-adrenal axis, because the
major hormones released come from the hypothalamus in the brain, the pituitary gland and the adrenal
glands. The end product of this hormone system is the release of cortisol from the adrenals. In conditions
when it is over-produced, cortisol can have harmful effects. We have shown that cortisol is over-produced
in some overweight people because of a defect in secretion of the first hormone in the chain, CRH, which
comes from a part of the brain called the hypothalamus. Other hormones, such as leptin, whose
concentration is proportional to fat mass, and Neuropeptide Y, which controls food intake and energy
expenditure, also are linked in this complex system of hormones that may influence obesity.

Our research was funded by a grant from the National Health and Medical Research Council of Australia.




                                                    - 244 -
                                                                                            OBESITY
P1-573
LICORICE REDUCES BODY FAT MASS IN OBESE MEN AND WOMEN
MJ Mattarello,1 I Karbowiak,2 A Ermolao,1 CB De Palo,1 P Spinella,3 M Zaccaria,1 C Scaroni,1
D Nacamulli,1 D Armanini.1 1Medical and Surgical Sciences, University Padua, Padua, Italy; 2Clinical
Pathology, Piove di Sacco Hospital, Piove di Sacco Padua, Italy; 3Clinical and Experimental Medicine,
University Padua, Padua, Italy

Licorice (L) is used as mouth refresher or sweetener. The active principle, glycyrrhetinic acid (GE), is
responsible for the occurrence of side effects. The most common side effect is a hypokalemic hypertension
when high amounts of licorice preparations, containing a high amounts of GE, are consumed. This side
effect is due to inhibition of 11-hydroxysteroid dehydrogenase (11HSD), the enzyme that catalyzes the
conversion of cortisol to cortisone, thereby minimizing the binding of cortisol to mineralocorticoid
receptors. L is also able to reduce serum testosterone by blocking 17-HSD. We here report another effect of
L in reducing body fat mass. We studied 15 normal subjects (7 male age 22-26 yo and 8 female, age 21-26
yo) who consumed for two months 3.5 g a day of a commercial preparation of L containing 7.6% of GE.
Body fat mass (expressed as % of total body weight) was calculated by skin fold thickness and by
bioelectrical impedance analysis (BIA). Extracellular water (expressed as % of total body water) was
measured by BIA. Table: (*p<0.02 **p<0.001).L was able to reduce body fat mass and to suppress
aldosterone, without any change in blood pressure and in BMI. A possible explanation of this finding is that
the strong taste of L prevents subjects from tasting more food. It can not be excluded that licorice acts on
the metabolism of fat or on the 11-HSD at the level of fat cells.

                 male                  male                     female                              female
                 before                2 months after           before                              2 months after
body mass
                 24.32.1               24.3*plusmn*2.4**        20.2*plusmn*2.0                     20.1*plusmn*1.9
index BMI
extracellular
water % body     41.8*plusmn*2.9       47.0*plusmn*2.3*         48.2*plusmn*1.4                     49.3*plusmn*2.1*
water

body fat mass
                 12.0*plusmn*2.1       10.8*plusmn*2.9          24.9*plusmn*5.1                     22.1*plusmn*5.3
%body weigh

total
cholesterol                                                     169*plusmn*2*plusmn*2.49            169*plusmn*20
mg/dL
HDL
cholesterol                                                     55.8*plusmn*17                      58.4*plusmn*17
mg/dL
PRA ng/ml/h      3.9*plusmn*3.3        1.2*plusmn*1,7**         3.7*plusmn*2.3                      2.4*plusmn*2.4*
pl aldosterone
                 18.7*plusmn*8.1       5.9*plusmn*8.0**         21.6*plusmn*25                      5.6*plusmn*3.5**
ng/dL


Clinical Science Poster: Aspects of Human Obesity (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 245 -
                                                                                                OBESITY
P1-573

Lay explanation of abstract:

Licorice has been used for 5,000 years as a medical plant. The product is also used as sweetener and mouth
refresher. The active principle of licorice roots is glycyrrhizic acid and it is known that the compound can
produce also side effects like sodium and water retention and hypokalemia when consumed in large
amounts. Our research shows that licorice also is able to reduce body fat mass (this effect already had been
discovered in 400 B.C., when Theoprhastus reported that
the root can suppress the appetite).

We have studied 15 normal subjects (7 male and 8 female, 21-26 years old) who consumed 3.5 g a day for
two months, a commercial preparation of licorice (pure extract of licorice roots containing 7.6% of the
active principle without sugar and other additives). Body fat mass, expressed as % of total body weight)
was calculated by skin fold thickness and by bioelectrical impedance analysis (BIA). Intracellular water
was measured by BIA.

Licorice was able to reduce significantly body fat mass (1.5% of body weight in man and 2.5% in women)
and suppress plasma aldosterone, without any change in body mass index or in blood pressure. In all cases,
extracellular water significantly increased.

These subjects did not lose weight because licorice produced also water retention, as demonstrated by the
measurement of extracellular water. When specifically asked, all subjects noted a reduced appetite for food,
probably due to the sharp taste of licorice sticks. A possible additional explanation comes from previous
studies that demonstrated that licorice directly can affect metabolism of cholesterol and fat and that licorice
can affect 11-hydroxysteroid dehydrogenase, the enzyme responsible for the conversion of cortisol into
cortisone and cortisone to cortisol. This enzyme recently has been found in certain types of fat. These
findings suggest that licorice may be useful in treating obesity in conjunction with a diuretic or low sodium
diet to prevent water retention.

Additional background information:

Licorice is a medicinal plant that has been used for centuries, particularly as an appetite suppressant. It can
produce side effects when ingested in large amounts. Licorice affects the type II 11-beta hydroxysteroid
dehydrogenase and can block the inactivation of cortisol at the kidney level. It has also an effect on 17-
hydroxysteroid dehydrogenase and can reduce plasma testosterone, but this effect seems irrelevant in
healthy subjects whose testosterone levels are always in the normal range. Our study showed that licorice
can reduce fat mass, but further investigation is needed to fully explain the mechanism of action and the
value of its use in obese subjects.

Research was funded by MURST (Italian University funding source).




                                                    - 246 -
                                                                                           OBESITY
P1-576
ANDROGEN RECEPTOR GENE GGN REPEATS AND INDICES OF OBESITY IN OLDER
ADULTS
BM Koppanati,1 S Williams,2 DR Gustafson.1 1Nutrition and Food Sciences, Utah State University, Logan,
Utah; 2Genomics Core Facility, University of Utah, Salt Lake City, Utah

Polymorphic repeat regions in the androgen receptor (AR) gene may regulate the metabolism of androgens.
Androgens may play an important role in determining abdominal and total obesity in men and women. We
examined the association between the GGN repeat size in the AR gene and indices of obesity in healthy
older Caucasian adults. A cross-sectional study was conducted that included 99 men (51-93 years) and 115
women (51-92 years). Blood samples were collected, DNA was extracted from lymphocytes, and the GGN
repeat size of the AR gene was quantified using Perkin Elmer Applied Biosystems GeneScan and
Genotyper software. Anthropometric measurements included body weight, height, and waist and hip
circumferences. Waist was measured at the umbilicus, the iliac crest (wstili) and mid-way between the iliac
crest and the lowest rib. Waist to hip ratios (WHR) were calculated using these three waist circumferences.
Body mass index (BMI) was calculated as kg/m2. We evaluated relationships between the GGN repeat size
and all indices of obesity: waist circumferences, WHRs, waist to height ratios, and BMI, in men and
women separately. The most common GGN repeat size was 17 (range 7-21) in men and women and
accounted for 49% and 55% of all alleles, respectively. Our primary finding was that men with a GGN
repeat size of 17 had a significantly lower WHR (mean = 0.97 ± 0.04) measured at wstili than men who did
not have 17 GGN repeats (mean = 0.99 ± 0.05; p = 0.036). In women, no significant associations were
found between GGN repeat size and indices of obesity. These data show that rarer GGN alleles in men may
be more associated with obesity phenotypes. Sponsored by NRICGP/USDA Award Number 97-35-207-
4619 and the Utah Agricultural Experiment Station.

Clinical Science Poster: Aspects of Human Obesity (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                 - 247 -
                                                                                             OBESITY
P1-576

Lay explanation of abstract:

Researchers estimate that obesity leads to approximately 280,000 adult deaths in the United States each
year, but little is known about the causes of the disease. Overweight and obesity are occurring in epidemic
proportions in the United States and around the world, and a newly released study is helping researchers
understand how a gene related to male sex hormones may contribute to the disease. These latest findings
suggest the possibility that differences in sex hormone action might determine how men distribute their
body fat, a key factor because obesity that occurs around the waist (being ‗apple-shaped‘) is more
associated with disease.

In the study, researchers measured whether there was an association between differences in what is called
the androgen receptor gene, total obesity, and waist obesity in 214 men and women over 50 years of age.
They found that men who had the most common form of the androgen receptor gene had a significantly
lower waist-to-hip ratio than men who had an uncommon form of the gene. A waist-to-hip ratio is often
calculated to determine the relative amount of waist fat in a person.

The androgen receptor gene was not related to obesity in women. These results point to the role of genes
and sex hormones in determining obesity patterns in men.

Additional background information:

Androgens are male sex hormones and estrogens are female sex hormones, although men and women make
both types. Sex hormones are released into the bloodstream and travel to specific target cells on which they
have their effects. The actions of sex hormones can occur only after they have entered the cell and are
bound to a receptor. For example, the androgen receptor binds male sex hormones in the cells of men and
women.

Receptors may differ in function between people, depending on the DNA that a person inherits from their
parents. DNA strands are like necklaces made up of genes. DNA can be compared to a necklace of real
pearls, where the pearls are unique genes and their unique combinations determine every aspect and
function of the body.

More than 50% of U.S. adults are overweight, and nearly 25% are obese. Obesity increases risk for age-
related disease, such as cardiovascular diseases, diabetes, and certain cancers.

This study was funded by the U.S. Department of Agriculture, National Research Initiative Grant Program,
and the Utah State University Agricultural Experiment Station.




                                                  - 248 -
                                                       PEDIATRIC ENDOCRINOLOGY
P1-654
ENDOCRINE OUTCOME IN CHILDREN WITH MEDULLOBLASTOMA (PNET) TREATED
WITH LOW DOSE CRANIOSPINAL IRRADIATION (CSRT)
W Xu, A Janss, D Ng, A Kelly, R Packer, P Phillips, T Moshang. Pediatrics, Children's Hosp, Phila,
Pennsylvania

Young children with PNET treated with CSRT and chemotherapy have significant morbidity. In 1987-90,
low dose (18Gy) CSRT and chemotherapy were used to treat 10 children with PNET. The protocol was
discontinued because 3 died. This report compares the endocrine outcome in the 7 surviving patients
(Group LD) to a group (group HD) who were treated with standard CSRT (23-40 Gy) and chemotherapy.
Patients: Group LD (6 males and 1 female) was diagnosed with PNET at a mean age of 3.5 yrs. Of the 7
children, one was not GHD and not treated with GH. One was GHD and treated for one year with GH but
GH was discontinued after a stroke. Mean age at their last clinic visit was 15.2 yrs and some still on GH
therapy. Group HD was diagnosed and treated at mean age of 5.4 yrs and consisted of 11 children (5
males). All of the children in HD were treated with GH. The mean age of group HD at their last visit was
17.2 yrs. The dose of GH used in both groups was 0.3 mg/kg/week. Hypothyroidism, adrenal insufficiency,
pubertal delay and early puberty were treated. Results: There was no difference between the two groups for
mid-parental height (mean height SDS 0.12 in LD and SDS -0.16 in HD). For group LD, the near adult
heights achieved at their most recent clinic visit (mean SDS - 0.86 +/- 1.2), were statistically taller (p<
0.006) than adult heights achieved for group HD (SDS -2.4 +/-0.8). Moreover, the near adult heights for
patients in LD were not statistically different from mid-parental heights (p>0.09), unlike group HD whose
adults heights were significantly shorter than mid-parental heights (pConclusion: Low dose CSRT resulted
in a significantly better height outcome. Furthermore there was clearly less hypothyroidism, adrenal
insufficiency and hypogonadism. The mortality rate of 30% is similar to published survival data for PNET
children treated with high dose CSRT. These data suggest that a new trial using low dose CSRT in children
with PNET may be justified.

Clinical Science Poster: Pediatric Endocrinology I (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                 - 249 -
                                                        PEDIATRIC ENDOCRINOLOGY
P1-654

Lay explanation of abstract:

Medulloblastoma is a potentially lethal tumor treated with radiotherapy and chemotherapy. High doses of
radiation (like the ones used to treat these tumors), especially in young children, are associated with poor
growth and other endocrine abnormalities. In 1987-1990, a protocol using low-dose (LD) radiation and
chemotherapy was used to treat 10 children with medulloblastoma. The protocol was discontinued because
3 died. The 7 surviving patients were diagnosed at a mean age of 3.5 years (6 males and 1 female) similar
to a group treated with standard high-dose (HD) irradiation, diagnosed at a mean age of 5.4 years (5 males
and 6 females). Six of the seven patients in the LD group were growth hormone deficient and treated with
growth hormone (GH); in one the GH was discontinued after 1 year. All of the children in the high-dose
group were growth hormone deficient and treated with similar doses of GH. The children in the LD group
achieved an adult height statistically taller than the adult heights achieved by the children in treated with
HD irradiation. Moreover, the near-adult heights for the patients in the LD group were similar to their
parents‘ height, unlike the HD group, whose adult heights were significantly shorter than their parents‘
height. Of other endocrine conditions in the HD group, 10 had hypothyroidism, 2 had adrenal insufficiency,
2 had hypogonadism, and 1 had early puberty. In contrast, in the LD group, only 1 had hypothyroidism and
1 had early puberty.

Recent publications indicate that the long-term survival of children with medulloblastoma treated with
high-dose radiation is not different from the survival rate among our 10 children treated with low dose
radiation. In conclusion, these retrospective data, based upon small numbers, suggest that low-dose
radiation resulted in a survival rate similar to high-dose irradiation but with much better outcome in terms
of height and endocrine characteristics. These data suggest further that a low-dose radiation treatment
should be studied in a larger number of children with medulloblastoma.

Additional background information:

The investigators at the Children‘s Hospital of Philadelphia have had a longstanding interest in long-term
effects of cancer therapy, especially in children with brain tumors. The present study involved children
treated under a research protocol supported over 10 years ago. The retrospective analyses of the outcomes
of these children were not supported by extramural funds, except for support of the research Fellow, Dr.
Diva Ng: NIH training grant # 5T32DK07314.




                                                   - 250 -
                                                         PEDIATRIC ENDOCRINOLOGY
P3-46
SEX DIFFERENCES IN THE RELATIONSHIP OF FAT MASS TO BONE MINERAL CONTENT
IN CHILDREN AND ADOLESCENTS
AL Ackerman,1,2 JC Thornton,2 J Wang,2 RN Pierson,2 LS Levine,1 M Horlick.1,2 1Pediatrics, Columbia
University, New York, New York; 2Body Composition Unit, St. Luke's-Roosevelt Hospital Center, New
York, New York

Background: With an increase in the prevalence of childhood obesity, there has been a concern about the
long term adverse health sequelae. Given that childhood and adolescence are critical periods for bone
mineral acquisition, it is important to examine the effect of obesity on bone mineral content(BMC). In the
literature, there have been conflicting reports about the relationship between obesity and bone mass. Some
studies have reported lower than expected BMC in obese and overweight children while other studies have
not. (Goulding 2000, Weiler 2000, Ellis 2000)Objective: To examine the relationship of fat mass(FM) to
BMC.
Methods: The following were measured in 152 white girls (ages 5-18) and 192 white boys (ages 3-18):
Height (cm); weight (kg); BMC(g); non-bone fat free mass (nbFFM)(kg) and FM(kg) by dual energy X-ray
absorptiometry. The relationship of BMC to FM was analyzed by regression analysis including nbFFM,
height, Tanner stage, age and sex as independent variables. Pubertal status was established by the criteria of
Tanner.
Results: Log(FM) was a significant determinant of log(BMC) in girls only. In both boys and girls,
log(nbFFM ) was the most significant contributor to log(BMC). The effect of puberty on log(BMC) was
evident at Tanner stage 5 in boys and Tanner stage 4 and 5 in girls. This model explained 97% of the
variability in BMC in this population.
Conclusion: We found that FM was a significant determinant of BMC in girls only and this relationship
was independent of Tanner stage and height. However, the contribution of fat mass to BMC in girls is one
tenth that of nbFFM and non-existent in boys, suggesting that in two individuals of the same weight, the
one with greater fat mass will have compromised BMC. These results confirm that BMC is greater in late
puberty in both boys and girls but that the negative effect of obesity persists. The implication for a pediatric
population with a high incidence of obesity may be lower peak bone mineral mass.
Supported by NIDDK 37352.

Clinical Science Poster: P3: Pediatric Endocrinology II (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                    - 251 -
                                                         PEDIATRIC ENDOCRINOLOGY
P3-46

Lay explanation of abstract:

Osteoporosis is a major health problem in the aging population, particularly in women. The best way to
prevent osteoporosis in adulthood is to achieve higher bone mass during growth. Childhood and
adolescence is the only time that people gain bone. The recent epidemic of pediatric obesity makes it
important to examine the effects of fat on bone. We found that fat contributes to bone in girls, but not in
boys. Others have studied the effect of obesity on bone by examining obese children compared with normal
children and encountered conflicting results. Our study analyzed the relationship of fat to bone in healthy
children and adolescents with a range of weight from normal to obese.

The volunteers for this study were 152 white girls (ages 5-18 years) and 192 white boys (ages 3-18 years).
Height, weight, and stage of puberty were measured. Total body bone mass in grams, fat mass in
kilograms, and non-fat mass in kilograms were measured by dual energy x-ray absorptiometry (DXA) scan.
The greatest contributor to bone in both boys and girls was non-fat mass. The contribution of fat to bone in
girls was one-tenth that of non-fat mass and was the same at all stages of puberty. There was no
contribution of fat to bone in boys.

This observation is newsworthy and interesting for two reasons. First, the sex difference in the contribution
of fat to bone is of scientific interest in understanding the process of bone growth. Second, it implies that
there may be long-term bone health consequences for growing boys and girls who are obese. That is, if two
children of the same sex are the same weight, the one with more fat will have less bone. If a boy and girl
have the same amount of fat, the effect on bone gain will be worse for the boy than the girl.

Although osteoporosis has not been thought to be common among obese adults, most obese adults were not
obese children. However, we are now facing an unprecedented rate of obesity in children. The implications
for boys and girls who are obese during the period of bone growth may be lower bone mass and possible
osteoporosis. This may be especially important in boys. The relationship of fat to bone will be analyzed in
children from other ethnic backgrounds to see if the sex difference is present in all groups. Understanding
the sex difference in the influence of fat on bone may help in prevention of osteoporosis.

Additional background information:

This study was prompted by concerns about the effect of the current epidemic of pediatric obesity on bone
in growing children. For example, it has been reported that overweight girls have a higher than expected
rate of arm fractures. At least three studies evaluated bone mineral mass in obese compared to normal
children, and arrived at conflicting conclusions. We felt it was important to analyze the bone and fat results
we had obtained in a large group of healthy children.

The measurements analyzed in this study were obtained from participants in the Pediatric Body
Composition Project at St. Luke's-Roosevelt Hospital Center in New York City. The volunteers were 1200
healthy children and adolescents of African-American, Asian, Hispanic, and white backgrounds from the
New York metropolitan area. Multiple noninvasive and painless methods of body composition were
performed, including dual energy x-ray absorptiometry (DXA), which is the most common method for
measurement of bone mineral mass but also measures fat and non-fat mass.

There are ethnic differences in body composition and bone mass in children and adults. We chose to
analyze the information on the white participants initially, because this is the ethnic group with the greatest
fracture rate in obese girls, according to published evidence. This also is the group with the greatest
recognized rate of osteoporosis. We plan to perform analyses on other ethnic groups.




                                                   - 252 -
                                                      PEDIATRIC ENDOCRINOLOGY
P3-47
ABNORMAL BONE MINERAL ACCRUAL IN ADOLESCENT GIRLS WITH ANOREXIA
NERVOSA
LA Soyka, M Misra, K Miller, S Grinspoon, A Klibanski. Neuroendocrine Unit, Mass General Hospital,
Boston, Massachusetts

Osteopenia is common in adolescent girls with anorexia nervosa (AN) at a time of peak bone mass accrual.
We have reported decreased bone formation markers in such girls, primarily determined by nutritional
status. We hypothesized that reduced bone mineral accrual occurs in AN and would persist with chronic
undernutrition. Therefore, 38 teenage girls (19 AN and 19 controls) had assessment of BMD and body
composition by DEXA, bone age, and calcium status at baseline, 6 and 12 mo. Subjects were matched for
bone age (AN 15.1 +/- 0.4 yrs, control 15.0 +/- 0.4 yrs, mean +/- SEM) and age (AN 15.4 +/- 0.4 yrs,
control 14.6 +/- 0.4 yrs). Mean duration of AN was 14 mos, duration of amenorrhea was 21 mos, and 7
patients were pre-menarchal. At baseline, BMI, lean body mass and lumbar BMD were significantly
reduced in AN (AN 16.4 +/- 0.5 kg/m2 vs. control 22.0 +/- 0.5 kg/m2, p=< 0.0001; AN 33.6 +/- 1.0 kg vs.
control 37.4 +/- 1.0 kg, p=0.009; AN 0.89 +/- 0.02 g/cm2 vs. control 0.96 +/- 0.02 g/cm2,
p=0.04),respectively. At follow up, nutritional status in AN improved (BMI increase = 9.2 +/- 1.9% at 6
mo, 15.2 +/- 2.6% at 12 mo), with 4 recovered subjects at 6 mo and 8 recovered at 12 mo. However, lumbar
BMD at 6 mo (AN 0.88 +/- 0.03 g/cm2 vs. control 0.98 +/- 0.02 g/cm2, p=0.01) and 12 mo (AN 0.88 +/-
0.02 g/cm2 vs. control 0.98 +/- 0.03 g/cm2, p=0.008) remained significantly reduced in AN compared with
control, even in recovered subjects. This was due to significant increases in BMD in controls vs. no change
in AN at 6 mo (AN - 0.00 +/- 0.01 g/cm2 vs. control 0.02 +/- 0.01 g/cm2, p=0.02) and 12 mo (AN 0.00 +/-
0.01 g/cm2 vs. control 0.04 +/- 0.01 g/cm2, p=0.04). The most significant determinant of change in lumbar
BMD at 12 mo was change in lean body mass at 12 mo in both AN (r=0.62, p=0.008) and control (r=0.80,
p=0.0003). Dietary calcium deficiency was present in 42% of AN and 50% of control at baseline and
remained deficient in 33% or more of subjects at follow up. No correlation between calcium intake and
BMD was found. Therefore, 1)spinal osteopenia in adolescent girls with AN persists in spite of recovery,
2)poor bone mineral accrual occurs in AN, and 3) change in lean body mass is a major determinant of
change in lumbar BMD in both AN and healthy girls.

Clinical Science Poster: Pediatric Endocrinology II (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                 - 253 -
                                                        PEDIATRIC ENDOCRINOLOGY
P3-47

Lay explanation of abstract:

Thinning of the bones is common in teenage girls who have anorexia nervosa during the time when
maximum bone strength is being built. Thin bones are a health concern, because they have a higher risk of
breaking. We have previously shown that girls with anorexia nervosa have low levels of bone cell activity,
which depends on good nutrition.

In this study, we examined 19 teenage girls with anorexia and 19 healthy girls who underwent
measurement of bone thickness and body composition (proportions of fat and lean tissue) by dual energy x-
ray absorptiometry and assessment of skeletal age and calcium intake over a 12-month period. At the start
of the study, we found that the girls with anorexia nervosa had thinner bones at the lower spine than the
healthy girls. Although the girls with anorexia nervosa generally had increased their weight during the
period of this study, their bones remained thin at the 6-month and 12-month visits, compared with bones of
the healthy girls.

Even those girls with anorexia nervosa who recovered weight and resumed menstrual periods had
persistently thin bones. This was due to increases in bone thickness over time in healthy girls compared to
no changes in bone thickness in girls with anorexia nervosa. Change in the amount of lean body tissue was
thought to be the main factor that determined change in the bone thickness of the lower spine in girls with
anorexia nervosa and healthy girls at the 12-month visit.

Another finding of the study was that at least one third of the teenage girls studied were deficient in
calcium and vitamin D intake at all study visits during a time when their skeletal need for calcium was at its
highest. This data suggests that bones in girls with anorexia nervosa are thin because there is no increase in
bone over time compared with healthy girls who continue to increase bone thickness. Often teenage girls
with anorexia nervosa have persistently thin bones, even when they recover from anorexia. This is further
evidence that nutritional status has a major impact on bone thickness in teenage girls. Treatment efforts
should be directed at increasing bone formation in girls with anorexia nervosa.

This research was funded by the Genentech Foundation for Growth end Development and NIH grants RO1-
DK-52625 and MO1-RR-O1066.




                                                   - 254 -
                                                                                        PITUITARY
OR22-6
EFFECTS OF 12 MONTHS GH REPLACEMENT ON CARDIAC PERFORMANCE IN ADULT
GH DEFICIENCY (GHD): COMPARISON WITH UNTREATED GHD PATIENTS AND
HEALTHY CONTROLS
C Di Somma,1 M Pietrosante,1 A Cuocolo,2 A Giaccio,1 FW Rossi,1 G Aimaretti,3 G Tamburrano,4
G Coronel,5 M Salvatore,2 G Lombardi,1 A Colao.1 1Depts of Molecular and Clinical Endocrinology and
Oncology, "Federico II" University, Naples, Italy; 2Nuclear Medicine, "Federico II" University, Naples,
Italy; 3Internal Medicine Endocrinology Division, University, of Turin, Italy; 4Clinical Science
Endocrinology Section, University "La Sapienza", Rome, Italy; 5Medical Dept, Novo Nordisk, Rome, Italy

Cardiac performance by equilibrium radionuclide angiography was prospectively evaluated in 15 adult
GHD patients (age 18-76 yrs) treated with rGH (0.15-1 mg/day) for 12 months (group A), 15 adult GHD
patients (age 18-76 yrs) who refused GH replacement (group B) and 35 healthy subjects (age 18-73 yrs)
(group C). At study entry, GH after ARG+GHRH test, IGF-I levels were similar in group A and B, while
they were significantly lower in group C. A significant decrease of left ventricular (LV) ejection fraction
(LVEF) both at rest and at peak exercise, exercise-induced changes of LVEF (ΔEF), exercise duration and
capacity was found in group A and B as compared to group C. 36.6% of GHD patients had an impaired
LVEF at rest and 76.7% had an inadequate LVEF response at peak exercise. Patients of group A and B had
similar baseline hemodynamic parameters and exercise capacity and duration. After 12 months, IGF-I
levels normalized in all patients of group A and were significantly reduced in those of group B.

Clinical Science Oral: Pituitary I (1:00 PM-2:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 2:15 PM; Location: C 106




                                                 - 255 -
                                                                                        PITUITARY
OR22-6

Lay explanation of abstract:

This study is part of a very large protocol to investigate the role of pituitary hormones on heart structure
and cardiovascular function. Increased cardiovascular deaths have been reported among patients who have
hypopituitarism. Growth hormone (GH) deficiency has been called the major cause of cardiovascular death.

We found that patients with adult GH deficiency have several alterations of cardiovascular risk parameters
and of cardiac performance that can be improved by GH replacement therapy. An additional finding was
that patients with GH deficiency who refused GH treatment showed further impairment of cardiac
performance after 12 months. Lipid profile was also worse after 12 months. The results indicate the need
for GH replacement in GH-deficient adults.

Additional background information:

All patients in our study had hypopituitarism and GH deficiency, either acquired during childhood
(congenital GHD) or adulthood (pituitary surgery/irradiation). All patients had undergone surgery for
tumors of the hypothalamus-pituitary region or had a history of cranial trauma (prevalence of GH
deficiency greater than 30%).

We received grants from Health Ministry of Italy, National Council of Research, Campania Region. All
pharmaceutical companies producing GH and registered in Italy gave partial support to this study.




                                                  - 256 -
                                                                                         PITUITARY
OR34-5
ASSOCIATION BETWEEN ANDROGENS AND BONE DENSITY IN WOMEN WITH
HYPOPITUITARISM
KK Miller, BM K. Biller, E Arena, A Klibanski. Neuroendocrine Unit, Massachusetts General Hospital,
Boston, Massachusetts

Hypopituitarism is associated with osteopenia and reduced lean body mass (LBM). We have recently
shown that hypoandrogenemia is also associated with this disorder. Both LBM and androgens influence
bone mass. We hypothesized that androgens and LBM are important determinants of bone density (BD) in
hypopituitary women. Moreover, because IGF-1 may stimulate adrenal androgens, we investigated whether
serum androgens increase with GH administration and may contribute to the beneficial effects of GH
replacement. Women with adult-onset pituitary disease (N=16) and stimulated GH < 5 ng/ml underwent
BD and body composition testing by DXA. Subjects were randomized to GH at 12.5 mcg/kg/d (Genentech,
South San Francisco, CA) or placebo for 12 months in a double-blind protocol. Serum androgen levels
were obtained at 0, 1, 3, 6, 9 and 12 months. Univariate regression analysis revealed strong correlations
between androgen levels and BD [lateral spine and DHEAS (r=0.68, p=0.03), total hip and free testosterone
(T) (r=0.60, p=0.02), Ward´s triangle and DHEAS (r=0.68, p=0.004), Ward´s triangle and free T (r=0.54,
p=0.004), femoral neck and free T (r=0.52, p=0.04), femoral neck and DHEAS (r=0.51, p=0.04)]. LBM
strongly correlated with BD [total hip (r=0.80, p=0.0002), total body (r=0.78, r=0.0003), trochanter
(r=0.74, p=0.001), Ward´s triangle (r=0.56, p=0.02), femoral neck (r=0.53, p=0.04), and AP spine (r=0.52,
p=0.04)]. In stepwise regression models, DHEAS determined 47% of the variation in Ward´s triangle
(R²=0.47, p=0.004) and 46% of lateral spine BD (R²=0.46, p=0.03). LBM determined 64% of the variation
in total hip (R²=0.64, p=0.0002), 55% of trochanter (R²=0.55, p=0.001), 28% of femoral neck (R²=0.28,
p=0.04) and 27% of AP spine BD (R²=0.27, p=0.04). Serum androgens did not change in either group (GH
or placebo). In conclusion, androgen levels and LBM may be important determinants of BD in women with
hypopituitarism. GH did not result in an increase in androgen levels; it is therefore unlikely that androgens
mediate the beneficial effects of GH. It remains to be determined whether androgen replacement or an
increase in LBM will increase BD in this population.

Clinical Science Oral: Pituitary II (1:00 PM-2:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 2:00 PM; Location: Ballroom 1




                                                  - 257 -
                                                                                        PITUITARY
OR34-5

Lay explanation of abstract:

Women with impaired pituitary function, also known as hypopituitarism, are known to be at risk for
osteoporosis and to have low blood androgen levels. We hypothesized that androgens, such as testosterone
and DHEAS, and muscle mass would be important determinants of bone density in such women. We also
sought to determine whether growth hormone therapy results in increased androgen levels. We therefore
measured blood androgen levels, bone density, muscle, and fat mass in 16 women with hypopituitarism.

The women in the study were randomly assigned to receive growth hormone or placebo for 12 months, and
blood androgen levels were measured. We found that DHEAS, testosterone, and muscle mass were
important determinants of bone density in these women. DHEAS blood levels accounted for nearly 50% of
the differences in spinal bone density between individual women, whereas muscle mass accounted for over
60% of the differences in hip bone density observed. Blood androgen levels did not increase in women who
received growth hormone therapy compared with those who received placebo.

Therefore, androgens, such as testosterone and DHEAS, and muscle mass may be important for bone health
in women with hypopituitarism, who are known to be at risk for osteoporosis and bone fractures.


Additional background information:

The pituitary gland controls many of the hormones in the body, including the ovaries and adrenal glands.
Patients with impaired pituitary function have been shown to be at risk for osteoporosis and obesity. Low
levels of androgens, such as testosterone, have been shown to be associated with osteoporosis, increased
fat, and decreased muscle in men. We have shown that women with impaired pituitary function have low
blood androgen levels, including testosterone and DHEAS. It is not known whether low androgen levels
contribute to the osteoporosis observed in women with hypopituitarism, and further research would be
helpful.

This study was funded by the NIH and Genentech, Inc.




                                                 - 258 -
                                                                                        PITUITARY
OR46-5
LOW DOSE DHEA AFFECTS BEHAVIOR IN THE ANDROGEN-DEFICIENT WOMAN - A
PLACEBO-CONTROLLED TRIAL
G Johannsson,1 P Burman,2 L Wiren,1 B Eden Engstrom,2 A Nilsson,1 B Jonsson,3 BA Bengtsson,1
FA Karlsson.2 1Research Center for Endocrinology and Metabolism, Sahlgrenska University Hospital,
Goteborg, Sweden; 2Dept of Medicine, Inst of Medical Sciences, University Hospital, Uppsala, Sweden;
3
  Dept of Women's and Children's Health, Karolinska Institute, Stockholm, Sweden

Women with pituitary insufficiency have abnormally low androgens, due to lack of adrenal and ovarian
stimulation. We treated 38 women, age 25-65 years, with pituitary insufficiency with DHEA (30 mg daily,
n=9, 45 years of age) for 6 months in a randomized, placebo-controlled double-blind study, followed by 6
months open treatment. The administration of DHEA raised the serum levels of DHEAS into normal, and
androstendione and testosterone into subnormal age-related reference ranges, increased hematocrit and the
LDL/HDL-cholesterol ratio, and lowered HDL-cholesterol and Apo A-1. No significant changes occurred
compared to placebo in total cholesterol, insulin, IGF-1 or bone markers.Androgen effects on skin and/or
sexual hair were observed in no case after placebo and in 84% (32/38) of the women following 6 months
treatment with DHEA. These androgen effects were correlated to the serum levels of DHEAS. Partners
noted significant improvements of their spouses regarding alertness, stamina and the executive ability, and
the sexual relation tended to improve (p=0.06). Increased libido after the controlled part of the study was
reported by 50% of the women on 30 mg DHEA and by 0% on 20 mg DHEA. Following subsequent 6
months of open treatment with DHEA, 100% of those receiving 30 mg and 41% of the those receiving 20
mg, reported increased libido.
In conclusion, oral daily administration of a low dose of DHEA to adult women with androgen deficiency
due to pituitary insufficiency induces androgen effects in skin, sexual hair, and change in behavior, with
little adverse effects on metabolism.

Clinical Science Oral: Pituitary III (11:00 AM-12:30 PM)

Presentation Date: Saturday, June 23, 2001; Time: 12:00 PM; Location: Ballroom 2




                                                  - 259 -
                                                                                        PITUITARY
OR46-5

Lay explanation of abstract:

In normal women, the adrenal glands are the main source of ―male‖ hormones (androgens), hormones of
importance for physical strength and some aspects of mental energy. In women with pituitary diseases
(mostly benign tumors that are treated with surgery or radiation therapy), the adrenal glands are often
dormant. The adrenals don‘t act normally because the pituitary-derived hormone ACTH
(adrenocorticotrophic hormone), which normally stimulates the adrenal glands to secrete androgens, is at
low levels.

We carried out a 6-month, placebo-controlled study and examined effects of DHEA on metabolism and
behavior in 38 women with pituitary disease. The dose we used was low (aimed to restore the DHEA
levels in the blood into the normal range) and therefore unlikely to cause adverse effects. The results
showed that the women receiving DHEA experienced androgenic effects in skin and hair, and that their
spouses reported positive effects on mood and behavior, in particular on stamina, alertness, and decision-
making. The biochemical parameters moved toward normal.

Based on our findings, we think that all women with pituitary diseases leading to adrenal insufficiency
should be given an androgen supplement. Such treatment should be monitored by an endocrinologist.

Support for this research came from the Uppsala University Research Fund and Gothenburg University
Research Fund. No commercial/pharmaceutical support was received.




                                                  - 260 -
                                                            REPRODUCTION (FEMALE)
OR10-1
WOMEN WITH EXERCISE ASSOCIATED AMENORRHEA HAVE A PARADOXICAL
CHANGE IN METABOLIC PARAMETERS
SE Best,1 MN dela Cruz,1 AE Ambinder,1 RN Pierson,2 J Wang,2 CN Boozer,2 M Ferin,1 MP Warren.1
1
  Obstetrics and Gynecology and Medicine, Columbia College of Physicians and Surgeons, New York, New
York; 2St. Luke's-Roosevelt Hospital Center, New York, New York

The persistence of hypothalamic amenorrhea in exercise associated amenorrhea in individuals of normal
weight is poorly understood and is thought to be related to a restraining metabolic signal. We compared
women of normal weight with hypothalamic amenorrhea to normal exercising controls to determine if
differences in metabolic parameters could differentiate between the two groups. We studied 6 subjects with
amenorrhea and 16 normal controls. Months at present weight was significantly lower in the group with
amenorrhea (4.8±3.6 vs. 24.06±17.26; p 40K is a direct reflection of muscle which is metabolically active
tissue while fat is not. Thus this increase would be expected to be associated with an increased RMR. The
dichotomy in these findings suggests a physiological paradox preventing increases in metabolic rate. These
changes may be associated with mechanisms designed to prevent weight loss in this group and increase
caloric efficiency. This mechanism may account for the persistence of amenorrhea in this group and along
with the variation in weight suggest that fluctuations in weight may be disrupting normal mechanisms to
establish menstrual cyclicity.

Clinical Science Oral: Female Reproduction (1:00 PM-2:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 1:00 PM; Location: C 207




                                                 - 261 -
                                                              REPRODUCTION (FEMALE)

OR10-1
Lay explanation of abstract:

Hypothalamic amenorrhea is a condition in which menstrual periods stop too soon. It is associated with a
failure of normal signals from an area in the brain called the hypothalamus. This failure is thought to be due
to stress and/or insufficient energy (caloric) intake.

We studied women with this condition to determine whether a metabolic alteration could be found. The
women were of normal weight, and when compared to controls, they showed a trend toward lowering their
metabolic rate despite high muscle mass. Because muscle mass accounts for most of metabolic rate, this
suggests that these women have a metabolic adjustment that lets them increase caloric efficiency (i.e., use
fewer calories) despite high muscle mass. This group also showed fluctuations in weight, which suggested
yo-yo dieting.

This research was funded by the National Institutes of Health.




                                                   - 262 -
                                                             REPRODUCTION (FEMALE)

OR10-4
ENDOMETRIOTIC AND ADENOMYOTIC LESIONS DISPLAY THE SAME CYCLICAL
ESTROGEN(ER), PROGESTERONE(PR)AND PROGESTERONE ISOFORM B RECEPTOR
(PRB) EXPRESSION AS THE FUNCTIONAL UNIT OF BASAL (ZONE IV) ENDOMETRIUM
AND ARCHIMYOMETRIUM
G Leyendecker,1 M Herbertz,1 G Kunz,1 G Mall.2 1Obstetrics and Gynecology, Klinikum Darmstadt,
Darmstadt, Germany; 2Pathology, Klinikum Darmstadt, Darmstadt, Germany

We tested the hypothesis that both adenomyotic and endometriotic lesions are derived from basal
endometrium with surrounding myometrium resulting from metaplasia of the ectopic endometrial stroma.
Uteri with adenomyosis obtained by hysterectomy, excised endometriotic lesions and menstrual blood of
women with endometriosis and adenomyosis were used. All women had normal menstrual cycles and aged
32-49 years. ER, PR PRB immunohistochemistry was performed with the use of specific monoclonal
antibodies. In the outer myometrium, the archimyometrium, zones I-III of the endometrium the typical
pattern of ER and PR expression could be demonstrated with a constantly high expression in the outer
myometrium and a cyclically changing expression in the archimetrial components of the uterine wall. Basal
endometrium of zone IV displayed a specific cyclic pattern resulting in a different ER and PR expression
between the functionalis and the basalis in the secretory phase (SP). PRB expression was high in the
functionalis and basalis of the eutopic endometrium during the late proliferative and was absent in both
layers during the late SP. Endometriotic and adenomyotic lesions completely mimicked the cyclical pattern
of the zone IV basalis. In menstrual blood of patients with endometriosis endometrial fragments with weak
staining (functionalis) and strong staining (basalis) could be demonstrated. The peristromal muscular tissue
of endometriotic and adenomyotic lesions displayed the same cyclical pattern of ER and PR expression as
the archimyometrium. These data suggest that endometriosis and adenomyosis are derived from the basal
layer of the endometrium. We propose that dislocated basal endometrium has stem cell character capable of
resuming embryonic growth potential and resulting in the ectopic formation of all archimetrial components
such as epithelium, stroma and smooth muscle cells.

Clinical Science Oral: Female Reproduction (1:00 PM-2:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 1:45 PM; Location: C 207




                                                  - 263 -
                                                             REPRODUCTION (FEMALE)
OR10-4

Lay explanation of abstract:

Our data take research in endometriosis in a completely new direction. The data show, as have our previous
results, that endometriosis is primarily a disease of the uterus. The mechanism of the disease is probably
uterine auto-traumatization from uterine peristalsis (wave-like movement) and hyperperistalsis (and also by
iatrogenic traumatization, arising from medical treatment)). Fragments of basal endometrium (the lining of
the uterus) are detached and transported into the peritoneal cavity where they implant and grow. Basal
endometrial fragments re-activate embryonal growth potential in that all components of the primordial
uterus such as epithelium, stroma, and smooth muscle cells are formed in the ectopic site. Basal
endometrium and therefore endometriotic lesions respond differentially to progestins and other hormonal
treatment in comparison to the functional layer of the endometrium, from which endometriosis has been
believed to be derived up to now. This may explain why hormonal treatment of endometriosis is not very
effective. Future research in endometriosis should focus on the functional unit of basal endometrium and
the innermost layer of the myometrium, the archimyometrium. This layer controls directed sperm transport
by peristaltic activity. Infertile women with endometriosis display a hyperfunction of this layer, which
warrants further research.

Additional background information:

The uterus is a highly sophisticated organ. Not only does it provide the site of implantation, protect the
growth of the embryo/fetus, and provide its final expulsion, but the uterus also provides other fundamental
functions in the early process of reproduction, such as defense against inflammation and uterine peristalsis
for directed, rapid sperm transport into the fallopian tube where the egg has been released each month. In
infertile women with endometriosis, there is uterine hyperperistalsis that results in auto-traumatization of
the endometrial-archimyometrial unit. This leads not only to the detachment of fragments of basal
endometrium and its implantation in the pelvic area, but also to the infiltration of basal endometrium into
the myometrium resulting in adenomyosis (implantation of epithelial tissue), which in turn leads to
destruction of the myometrial wall and impedes directed sperm transport.

This study was unfunded clinical research.




                                                  - 264 -
                                                            REPRODUCTION (FEMALE)

OR10-5
PRENATAL ANDROGEN EXCESS IMPAIRS BLASTOCYST DEVELOPMENT IN ADULT
FEMALE RHESUS MONKEYS UNDERGOING GONADOTROPIN STIMULATION FOR IN
VITRO FERTILIZATION
DA Dumesic,1 RD Schramm,2 A Paprocki,2 E Peterson,2 R Zhou,2 DH Abbott.2 1OB/GYN, Mayo Clinic,
Rochester, Minnesota; 2Wisconsin Regional Primate Research Center, University of Wisconsin, Madison,
Wisconsin

Prenatally androgenized (PA) adult female rhesus monkeys exposed in utero to testosterone propionate
(TP) show luteinizing hormone (LH) hypersecretion and hyperandrogenism. To examine the effect of
prenatal androgen excess on oocyte meiotic and developmental competency, 5 early-treated (E) and 5 late-
treated (L) ovulatory PA females (15-35 days of TP therapy starting on gestational days 40-44 or 100-115,
respectively) and 5 control (C) females were studied. Females received recombinant (r) human (h) follicle-
stimulating hormone (FSH) injections (30 IU, IM daily) for 7-8 days. rh chorionic gonadotropin (CG; 1000
IU, IM) was given 1 day later and oocyte retrieval was performed 27 hours after rhCG. Serum FSH, LH,
estradiol (E2), progesterone (P4), androstenedione (A4), testosterone (T) and dihydrotestosterone (DHT)
levels were measured before rFSH treatment (basal), on the last day of rFSH injection and at oocyte
retrieval. Age (C 17.8+1.2, EPA 19.9+ 0.5, LPA 17.1+0.9 years), body weight (C 9.0+1.0, EPA 9.5+0.6,
LPA 8.9+0.7 kg) and basal FSH levels (C 1.8+0.4, EPA 1.8+0.3, LPA 1.7+0.1 ng/ml) were similar among
female groups. Basal LH levels, however, were greater in EPA (0.39+0.02) than LPA (0.22+0.06) or C
females (0.24+0.04 ng/ml; p+8, EPA 15+6, LPA 22+4) and the incidence of oocyte nuclear maturation (C
59+8, EPA 55+8, LPA 55+10 % metaphase II oocytes) and fertilization (C 60+11, EPA 65+18, LPA
77+9%) also were comparable among female groups. Nevertheless, only 8+6% of normally fertilized
oocytes developed to blastocysts in EPA compared to C (62+7%, p=0.02) or LPA (48+16%, p=0.04)
females. Early prenatal exposure of adult female rhesus monkeys to TP may therefore impair
developmental, but not meiotic, competency of in vivo matured oocytes, suggesting that a similar
phenomenon in human fetal development could reduce fecundity in women with androgen excess.
Supported by NIH grants RR14093 & RR13635.

Clinical Science Oral: Female Reproduction (1:00 PM-2:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 2:00 PM; Location: C 207




                                                 - 265 -
                                                              REPRODUCTION (FEMALE)
OR10-5

Lay explanation of abstract:

Our study shows that subtle abnormalities of hormone secretion from the pituitary of the mother have long-
term effects on early embryo development. Because we exposed our research monkeys to androgens (male
hormones) while they were still fetuses, they developed some abnormalities. These appear to be similar to
characteristics seen in women who have polycystic ovarian syndrome. Thus, we believe that our animal
model may eventually help address the question of why women with polycystic ovarian syndrome are at
risk for miscarriage. Because embryo development in humans is linked to infertility and pregnancy
loss, this study will be of interest to those interested in increasing fertility.

Additional background information:

About 15 years ago, several pregnant rhesus monkeys carrying female offspring were given testosterone
injections to determine the long-term programming effects of androgens in adulthood. As these offspring
reached adulthood, they exhibited abnormalities similar to those of women with polycystic ovary syndrome
(such as abnormalities of LH and insulin secretion).

All of these offspring monkeys underwent in vitro fertilization procedures in a manner similar to that
performed in women. This study abstract showed that embryo development from the eggs of these same
monkeys also was impaired, which raises the possibility that hormonal abnormalities in women have
profound effects on embryo development and possibly miscarriage.

This study was funded by the National Institutes of Health.




                                                  - 266 -
                                                             REPRODUCTION (FEMALE)


OR10-6
MATERNAL PLASMA CORTICOTROPIN-RELEASING HORMONE TRAJECTORIES VARY
DEPENDING ON THE ETIOLOGY OF PRETERM DELIVERY
S McGrath,1 M McLean,2 D Smith,1 A Bisits,1 W Giles,1 R Smith.1 1Mothers and Babies Research Centre,
John Hunter Hospital, Newcastle, Australia; 2Endocrinology, Westmead Hospital, Sydney, Australia

While second trimester maternal plasma corticotropin-releasing Hormone (CRH) concentrations are
elevated in women destined to deliver preterm, the positive predictive value of an individual test of CRH is
low. We have reanalysed data from acohort of women with multiple sampling to determine if the trajectory
of increase of placental CRH provided additional information over a single sample.In this cohort 2-4
samples assayed for CRH were available on 305 women, the general form of the exponential equation
y=aebt was fitted to the CRH data of each individual by the Gauss-Newton non-linear least squares method.
This generated parameters a (y-intercept) and b (rate of rise) in CRH for each woman. Nonparametric
statistical techniques were applied to compare the results for the group of women who delivered at term
with the group of women who delivered spontaneously preterm and the group who were delivered preterm
by induction or cesarian section because of obstetric indication.
The 3 clinically defined groups have significantly different parameters, a and b, which describe the CRH
trajectory. The group that delivered preterm because of obstetric indications had a similar mean y-intercept
but significantly greater mean rate of rise in CRH than the term group (0.3491 vs. 0.1788, 95% CI 0.2331-
0.4615 vs. 0.1394-0.2330). The group that delivered spontaneously preterm had a significantly greater
mean y-intercept than the group that delivered preterm because of obstetric indication (17.08 vs 1.83, 95%
CI 5.89-28.43 vs. 0.03-5.19) but a similar mean rate of rise to the group which delivered at term.
These data suggest that spontaneous preterm delivery is associated with an abnormal setting of the
production of CRH present from very early in pregnancy while women who experience an induced preterm
delivery are characterised by rapidly rising placental CRH. These data further suggest that clinical
abnormalities associated with preterm delivery by induction or cesarian section are associated with
abnormalities that lead to a rapidly increasing CRH. Trajectories for CRH provide information beyond that
obtained from a single sample.

Clinical Science Oral: Female Reproduction (1:00 PM-2:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 2:15 PM; Location: C 207




                                                  - 267 -
                                                            REPRODUCTION (FEMALE)
OR10-6

Lay explanation of abstract:

We followed the levels of a hormone (corticotrophin-releasing hormone) in the blood of a large group of
pregnant women. We discovered that a pattern of increase of the hormone predicted whether the woman
would deliver spontaneously before her due date (preterm) or would be induced to deliver prematurely by
her obstetrician.

Women who delivered spontaneously preterm started with high levels of the hormone which then increased
throughout pregnancy in the same pattern as levels in normal women, to a peak level at the time of birth.
Women whose obstetricians decided to deliver them preterm because of obstetric complications started
pregnancy with normal concentrations but levels of the hormone rose more rapidly than normal.

These different patterns of hormonal change during pregnancy suggest that different diseases during
pregnancy have " signatures" or typical patterns of change. These patterns in hormones can be recognized
and used to diagnose the problem and predict outcomes. The rapidly increasing levels of corticotrophin-
releasing hormone also suggest that these pregnancies are "stressed."




                                                 - 268 -
                                                               REPRODUCTION (FEMALE)


P2-404
SERUM LEPTIN LEVELS IN RURAL AND URBAN GIRLS AT PUBERTY
AL Valencia, JM Malacara, LE Nava, EL Perez-Luque. Inst Invest Medicas, Univ Guanajuato, Leon,
Mexico

There is strong evidence that leptin, participates in the regulation of the reproduction. We examined the
possible participation of leptin in pubertal development in girls under different nutritional, comparing
serum leptin levels in girls from urban and rural settings. A total of 265 healthy girls (134 living in a large
city, 126 living in a rural population with less than 1000 inhabitants) aged 8 to 15 yrs. The mean ages were
10.7 both for the rural and urban groups. Weight, height, four skinfold thickness and the Tanner pubertal
stage were registered. Body fat percentage (BFP) was calculated according to Westrate and Deurenberg.
Leptin, estradiol, FSH and LH were measured in a fasting blood sample.
Comparing both groups of study, by age, rural girls had lower stature at all age groups, however, weight
and BMI were not significantly different in most age groups. Comparing the groups by Tanner stage
development, stature, weight and BMI were not significantly different. BFP was higher in urban girls at
Tanner stage 2 (p=0.003). During development, leptin increased from 6.4 + 5.2 ng/dl in the prepubertal
group and 10.5 + 9.3 ng/dl in early puberty to 17.5 + 11.8 ng/dl in the postmenarcheal group. A stepwise
multiple regression procedure showed a significant association of leptin with BMI (r=.49, p<.000) and BFP
(r=0.35, p<.001) but not with the pubertal development. At the initiation of development (Tanner 2) leptin
levels were higher in the urban group with a significant difference(p=0.003). The age at Tanner stage 2 was
lower in the group or urban origin (9.4±1.2 vs 10.2±1.2, p=0.009). Using Mann-Whitney U test, estradiol
levels showed a marginal higher levels in the urban group in Tanner 5 (p= 0.079). Estradiol levels were
significantly correlated with leptin levels (r=0.17, p=0.007).
Conclusion. Our results show that serum leptin levels in girls with suboptimal nutritional conditions are
highly correlated with body fat but not with the pubertal development. Girls from the city have an earlier
onset of puberty. At that time, they also have higher BFP figures and higher serum leptin concentrations.
This finding is difficult to reconcile with the concept of a critical role of peripheral levels of leptin in the
onset of puberty.

Clinical Science Poster: P2: Female Reproduction (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                    - 269 -
                                                               REPRODUCTION (FEMALE)
P2-404

Lay explanation of abstract:

We studied pubertal development in girls with good and less-than-good (suboptimal) nutrition to learn
about leptin levels in the blood. The onset of puberty was earlier for urban girls, at 9.4 years as compared
with young women living in rural areas, who underwent puberty at 10.2 years. Previously, serum leptin
levels had not been correlated with pubertal development.

Such findings are of interest, however, because current concepts say that puberty is triggered when a
certain body composition is reached. Leptin, a recently discovered hormone produced in fat tissue, has been
proposed as a peripheral signal for the beginning of sexual maturation. The study of mechanisms that
regulate this process is interesting because girls who have an early or late sexual development may suffer
important consequences. To carry out this work, we studied 265 girls aged 8 to 15 years (135 living in a
city and 126 living rurally) and collected data on age, weight, height, and body fat percentage as calculated
by skinfold thickness.

Rural girls were not as tall as city girls. Studying the young women according to their pubertal
development, however, height and weight were similar for both groups. Leptin was highly associated with
the body mass index and with body fat percentage, but not with pubertal development. At the onset of
puberty (Tanner's stage 2), serum leptin levels and body fat percentage were significantly higher in rural
than in urban girls. This finding questions the proposed critical role of leptin in the initiation of puberty.

Additional background information:

The initiation of puberty occurs at earlier ages in recent times and in industrialized countries. Many
scientists propose that it results from a certain body composition. According to this view, the main
indicator--serum leptin levels--should trigger puberty. However, other signals must also be involved in this
process, because our findings showed that leptin levels did not serve as a trigger. Further work should
define a possible interaction of genetic, nutritional, and environmental factors.

This research was supported by CONACTY grant 458100-5-35276-M.




                                                   - 270 -
                                                            REPRODUCTION (FEMALE)


P2-410
CIRCULATING NITRIC OXIDE IS RELATED TO SEX STEROID MILIEU IN BOTH SEXES:
AN OVERVIEW
S Valenti, L Fazzuoli, L Bocca, D Cavallero, M Giusti. Department of Endocrinology and Metabolism,
University of Genova, Genova, Italy

Introduction: Circulating nitric oxide (NO)is mainly produced by the vascular endotelium, depending on
the sex steroid milieu. Our ongoing studies are focused on the evaluation of NO levels in males and females
in different clinical conditions.
Materials and Methods: 50 women aged 28-40 affected by pathological hyperprolactimaemia (HPRL)on
dopamine-agonist therapy, 10 women aged 18-29 affected by anorexia nervosa (AN) during pulsatile
LHRH therapy and 47 healthy controls aged 25-40 were studied troughout the menstrual cycle (days 7, 14,
21, 28). NO levels were assayed by means of the Greiss reaction and sex steroids by RIA. 10 males-to-
female transexuals (TRANS) aged 18-32 were also studied before and during treatment with the
antiandogen flutamide (750 mg/die p.o. for 12 month).
Results: 1)In healthy women NO fluctuates during the menstrual cycle been lowest during on day 21 and
showing a negative correlation with progesterone; 2)In HPRL NO levels are lower than healthy women at
any day of the cycle, but still negatively correlated to progesterone on day 21; 3) In AN the lowest NO
levels were seen and three-months pulsatile LHRH treatment was necessary to restore the physiological
pattern of NO secretion; 4)In TRANS anti-androgen treatment promptly increased NO levels, despite
modest increase in estradiol.
Conclusion: Our ongoing studies show that circulating NO is related to sex steroids milieu in both females
and males. In females NO appears to be produced under tonic stimulation by estradiol and episodically
inhibited by PG; on the other hand, in males NO seems to be under tonic inhibition by androgens and
promptly increased by pharmacologically induced rise in E2. A gender-related difference in the control of
NO secretion may thus exist.

Clinical Science Poster: Female Reproduction (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                 - 271 -
                                                             REPRODUCTION (FEMALE)
P2-410

Lay explanation of abstract:

Circulating nitric oxide (NO) is produced by the vascular endothelium and plays a major role in
cardiovascular protection. NO concentration is related to sex hormone concentration. NO is high in the
presence of estrogens and low in the presence of androgens. In women, NO level varies during the
menstrual cycle—higher during the follicular phase (leading up to ovulation) and lower during the luteal
phase (after ovulation). Our ongoing studies have shown that NO concentration is lower in women with
diseases characterized by menstrual problems (such as hyperprolactinemia and anorexia nervosa) than in
healthy women in a control group matched for age.

Ten patients affected by anorexia nervosa, in whom menstruation had been completely absent for at least
one year, underwent pulsatile treatment with luteinizing hormone-releasing hormone (LHRH) to restore
normal menstrual cycles. While 17-estradiol levels returned to normal after about one month of treatment,
normal NO concentrations were restored only after at least three months.

We also confirmed that NO concentration is lower in males than in females. To investigate the relationship
between NO and sex hormones in greater depth, we studied the effects of androgen blockade treatment on
10 male-to-female transsexuals who wanted to undergo surgery for gender reassignment. In these subjects,
treatment with the androgen receptor blocker flutamide markedly increased NO concentrations within one
month, and modestly increased 17-estradiol. We believe the regulation of NO secretion may be different
in the two genders. In females, NO seems to be produced by the stimulation of 17-estradiol and to be
episodically inhibited by progesterone. In males, however, NO appears to be constantly inhibited by
androgens, because this inhibition is promptly removed when a patient receives a drug treatment to block
androgens, as is the case with male-to-female transsexuals.

Considered in total, our data reinforce the concept of a gender difference in NO concentrations. NO is
higher in women than in men, and this difference is partly responsible for the better cardiovascular
protection of women who are of reproductive age when compared with age-matched men or post-
menopausal women. Women with menstrual disturbances are likely to have poorer cardiovascular
protection than healthy women, because of their lower NO levels.

These ongoing studies are supported by a grant from the University of Genova (Genoa) to Dr. Sandra
Valenti, under the supervision of Prof. Massimo Giusti.




                                                  - 272 -
                                                           REPRODUCTION (FEMALE)

P2-422
MENOPAUSAL HORMONE REPLACEMENT THERAPY (HRT) HAS DIVERSE
PROTHROMBOTIC EFFECTS
TA Worster,1 DH Ku,2 YS Arkel,2 KA Ault,2 DI Spratt.1,2 1Ob/Gyn, Maine Medical Center, Portland,
Maine; 2M.M.C. Research Institute, Maine Medical Center, Scarborough, Maine

HRT in postmenopausal women is associated with an increased risk of thromboembolic events. We
obtained initial data regarding the diversity of effects of HRT on coagulation using a broad panel of
recently developed laboratory assays. Prothrombotic pathways were assessed with Thrombus precursor
Protein (TpP), endogenous thrombin potential with and without activated protein C (ETP±APC), and
platelet activation (%CD62P positive platelets) and aggregation. The thrombolytic pathway was assessed
with thrombin activatable fibrinolysis inhibitor (TAFI). 12 postmenopausal women received conjugated
estrogens 0.625mg (CE 0.625) for 4w, then CE 1.25 for 4w, then CE 1.25+medroxyprogesterone 5mg for
2w (CE+MP). Assays were performed at baseline then at the end of each treatment phase. A positive effect
in a woman was defined as an increase in a parameter of ≥20% and to above the normal range. If an
increase in TpP was observed, samples were also analyzed for thrombin-antithrombin (TAT) and
prothrombin fragments 1+2 (PF1.2) as confirmatory measurements of fibrin generation. Results: Increases
in one or more parameters were observed in 6 of 12 women receiving CE and/or CE+MP. A marked
increase in TpP in four women (baseline vs. therapy: 0.75±0.58SE to 12.58±3.9 μg/mL) reflected increased
fibrin generation and was confirmed by increased TAT (1.22±0.31 to 19.26±5.49μg/L) and PF1.2
(1.63±0.34 to 4.36±2.06nmol/L). ETP+APC increased in 3 women (0.45±0.08 to 1.06±0.01OD/min)
reflecting acquired resistance to protein C. Platelet activation was increased in 4 women on CE and/or
CE+MP (18.0±3.5 to 43.7±13.3%) with platelet aggregation increased in only one woman. TAFI increased
in 2 women. Increases in different combinations of parameters of coagulation pathways were observed in
different women receiving different regimens of HRT. Increases were not greater with the higher dose of
CE. These data suggest that HRT effects on the coagulation cascade are common. Effects are diverse with
different effects occurring in different women. Additional research may be undertaken to further define
these effects and to determine which are most common and which may be related to clinical
thromboembolic events.

Clinical Science Poster: Female Reproduction (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                - 273 -
                                                             REPRODUCTION (FEMALE)
P2-422

Lay explanation of abstract:

Normal blood clotting in humans involves many elements and reactions. We know that hormone
replacement therapy (HRT) affects blood clotting in at least some postmenopausal women. These effects
result in increased risk for major blood clots (such as in the lungs) and possibly increased risk of heart
attack in some women who already have heart disease. However, we do not yet know exactly how
estrogen and/or progestins affect the clotting system or how to predict these effects in individual women.

In this study, we used recently developed laboratory techniques to evaluate the effects of HRT on several
reactions in the blood clotting process in 12 healthy postmenopausal women. Each woman was
sequentially given two doses of estrogen, then a progestin was added. Before therapy and after each
change in therapy, blood was collected for laboratory analysis of platelet activation and other pathways
leading to clot formation as well as the pathway leading to dissolution of the blood clot. In this way, we
could determine whether any effect of estrogen increased with higher doses and whether progestin also
affects blood clotting. We could also determine which part(s) of the clotting process is (are) affected.

Effects on the coagulation system were very common (occurring in half of the women). Rather than just
one effect, effects varied with changes in more than one element of the clotting process, as observed in 5 of
those 6 women. Different effects were observed in different women. Changes occurred in some women
with estrogen only and in some women with estrogen and progestin together. The most common effect was
enhancement of processes involved in forming a blood clot (rather than inhibition of processes that dissolve
a blood clot).

These results provide initial information upon which to base additional studies of effects of HRT on blood
clotting. These data do not yet tell us that these six women are more likely to form blood clots while taking
HRT. From a clinical standpoint, we can now focus on specific parts of the coagulation system and begin
to study whether some forms of HRT may be safer than other forms with respect to blood clotting. From a
physiological standpoint, we can now work on sorting out the precise mechanisms by which estrogens and
progestins affect blood clotting and how they vary in different women. Both avenues of information will
help refine our practices of prescribing HRT, particularly in women with increased risk for developing
major blood clots.

Funding was provided by the Maine Medical Center (M.M.C.) Medical Staff Research Committee and the
M.M.C. Research Institute (M.M.C.R.I.).




                                                  - 274 -
                                                             REPRODUCTION (FEMALE)
P2-427
METFORMIN REDUCES EARLY PREGNANCY LOSS IN POLYCYSTIC OVARY SYNDROME
DJ Jakubowicz,2 MJ Iuorno,1 S Jakubowicz,2 KA Roberts,1 JE Nestler.1 1Division of Endocrinology and
Metabolism, VCU/Medical College of Virginia, Richmond, Virginia; 2Hospital de Clinicas Caracas,
Central University of Venezuela, Caracas, Venezuela

Polycystic ovary syndrome (PCOS) is the most common form of female infertility in the Western world. In
addition to poor conception rates, pregnancy loss rates are high (30-50%) during the first trimester. We
hypothesized that hyperinsulinemic insulin reisistance contributes to early (first trimester) pregnancy loss
in PCOS, and that decreasing hyperinsulinemic insulin resistance with metformin during pregnancy would
reduce the rate of early pregnancy loss. We conducted a retrospective study of all women with polycystic
ovary syndrome who were seen in an academic endocrinology clinic within the past four and a half years
and who become pregnant during that time. 101 women received metformin during pregnancy (metformin
group) and 31 women did not (control group). The early pregnancy loss rate in the metformin group was
12.9% as compared with 41.9% in the control group (P=0.001). In the subset of women in each group with
a prior history of miscarriage, the early pregnancy loss rate was 15.7% in the metformin group (N=51) as
compared to 58.3% in the control group (N=12) (P=0.005). We conclude that metformin administration
during pregnancy reduces first trimester pregnancy loss in women with the polycystic ovary syndrome.

Clinical Science Poster: Female Reproduction (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 275 -
                                                             REPRODUCTION (FEMALE)
P2-427

Lay explanation of abstract:

Polycystic ovary syndrome is the most common form of infertility in the Western world. In fact, it is the
most common reason for female infertility in the United States. In addition to poor conception rates, many
women with polycystic ovary syndrome also will suffer early miscarriage during pregnancy. Pregnancy
loss during the first trimester of pregnancy is more common in women with this disease. Women with this
disorder are at three times the risk for early loss of pregnancy or spontaneous abortion as normal women.

This study was performed to determine whether treatment with the insulin-lowering drug metformin would
reduce the rate of early pregnancy loss in polycystic ovary syndrome. We examined the outcome of
pregnancy among 96 women with polycystic ovary syndrome. In this group 65 were treated with an
insulin-lowering drug called metformin throughout pregnancy, and 31 women did not receive metformin
during pregnancy. We compared the number of miscarriages in the treated group with those who were
untreated with metformin.

The retrospective trial took place over 4 years. We found that the women treated with metformin had a
miscarriage rate of 8.8%, while those not treated with metformin had a miscarriage rate of 42%. We also
found no incidence of fetal abnormality due to the drug among those treated with metformin. This study
suggests that metformin reduces early pregnancy loss in women with polycystic ovary syndrome, and that
it is safe for use in pregnancy.

This study was supported by the National Institutes of Health.




                                                  - 276 -
                                                                  REPRODUCTION (MALE)
OR11-3
DOSE-DEPENDENT EFFECTS OF TESTOSTERONE ON INSULIN SENSITIVITY,PLASMA
LIPIDS, APOLIPOPROTEINS, AND C-REACTIVE PROTEIN IN HEALTHY, YOUNG MEN
A Singh,1 S Hsia,1 P Alaupovic,2 I Sinha-Hikim,1 L Woodhouse,1 R Shen,1 S Bhasin.1 1Endocrinology,
Charles R Drew University, Los Angeles, California; 2Lipid and Lipoprotein Lab, Oklahoma Medical
Research Foundation, Oklahoma, Oklahoma

The effects of testosterone (T)on cardiovascular (CVD) risk in men are unknown. While supraphysiological
doses of T decrease plasma HDL-C, its effects on insulin sensitivity, inflammation markers, and
apolipoproteins remain poorly understood. We do not know if T's effects are dose-dependent, or its
anabolic effects occur at doses, not affecting the CVD risk factors. To determine the effects of graded doses
of T, 61 eugonadal men, 18-35 years, were randomized to one of the five groups to receive monthly
injection of GnRH agonist to suppress endogenous T secretion, and weekly injection of 25, 50, 125, 300
and 600 mg T enanthate for 20 weeks. Energy and protein were standardized. This regimen resulted in
nadir testosterone conc. of 253, 306, 542, 1345, and 2370 ng/dl at the 25, 50, 125, 300, 600 mg doses,
respectively. Plasma HDL-C (change +4.5± 3.20 mg/dl) and apo-A1 (change +9 ± 3 mg/dl. P=0.021)
increased at 25 mg dose and decreased in 600-mg group (change in HDL − 8 ± 2 mg/dl, P=0.0008; change
in apo-a1 − 16 ± 2 mg/dl, P=0.0001). There was no significant change in total cholesterol, LDL-C, VLDL,
triglycerides, apo-B, and apo- CIII levels at any dose. Insulin sensitivity index (Si), glucose disposal index
(Sg), and acute insulin response to glucose (AIRg), based on Bergman Minimal Model, did not change
significantly at any T dose nor did the levels of C-reactive protein in any group. Significant increments in
fat-free mass and muscle size were seen at T doses that did not affect cardiovascular risk factors.
Conclusion. Over a wide range of doses including those associated with significant gains in fat-free mass, T
administartion had no adverse effect on measures of insulin sensitivity, plasma lipids , apolipoproteins and
C-reactive protein. Only the highest dose of testosterone (600 mg) was associated with reduction in plasma
HDL cholesterol and apolipoprotein A1. Further studies are needed to determine whether T
supplementation affects the risk of atherosclerotic heart disease in older men with low T levels.

Clinical Science Oral: Male Reproduction (1:00 PM-2:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 1:30 PM; Location: C 106




                                                   - 277 -
                                                                   REPRODUCTION (MALE)
OR11-3

Lay explanation of abstract:

Testosterone is a hormone, essential for normal growth of bone, muscle, and appropriate sexual
characteristics in males. Many males lack testosterone because of various diseases, and these patients need
testosterone replacement. Testosterone in very high doses can decrease the good cholesterol (HDL-C),
which is believed to have a protective effect on the heart. The higher the HDL-C levels, the better
protection against heart attacks and strokes. The effect of different doses of testosterone on other risk
factors is unknown. These factors include insulin sensitivity, an inflammation marker called C-reactive
protein, and other cholesterols called apolipoproteins. The idea is to find the appropriate dose of
testosterone, with beneficial effects and without deleterious effects on the body. We wanted to find out
what kinds of effects the different doses of testosterone have on some of the risk factors for coronary artery
disease.

This study was conducted in 61 healthy men who had normal testosterone levels. They were divided into 5
groups. The subjects' own testosterone production was suppressed with a drug called GnRH, given as
monthly injections. Testosterone was given weekly by injections. Each group received 25, 50, 125, 300,
and 600 mg weekly injections, respectively, in groups 1 through 5 for 20 weeks. Calories and protein intake
were standardized during the study. Blood levels of testosterone achieved were 253, 306, 542, 1345, and
2370 nanograms/100 cc corresponding with the above dosage. Normal in the adult male is 300 to 1200
nanograms/100 cc.

We measured various good and bad cholesterols, collecting data on total cholesterol, triglycerides, LDL-
cholesterol, HDL-cholesterol and apolipoprotein A1 (together known as good cholesterols), apo-CIII and
apo-B. We also measured insulin sensitivity index, glucose disposal index, and acute insulin response to
glucose before and after treatment, in order to assess whether subjects were becoming insulin resistant.
Significant decrease in the HDL-C and apo-A1 was seen only with the 600-mg dose of testosterone; a slight
increase was seen with the 25-mg dose. There were no changes seen with the other cholesterols or
parameters at any other doses. Significant increase in muscle mass and strength was seen at various other
doses.

We concluded that over a wide range of testosterone doses, including those associated with a gain in
muscle mass, there were no adverse effects on the various cardiovascular factors, except with the 600-mg
dose, which was associated with a reduction in the good cholesterols HDL-C and apo-A1.

Additional background information:

More studies are needed to determine the effects of testosterone replacement and its relation to the risk of
atherosclerosis in an elderly population with low testosterone levels. The other important implication of
these findings is that men and athletes who abuse androgens to improve sports ability are putting
themselves at a higher risk for coronary artery disease and strokes.

This research was funded by the NIEA.




                                                   - 278 -
                                                                                           THYROID
OR35-2
TIME REQUIRED TO ACHIEVE AN ADEQUATE TSH LEVEL IN ORDER TO PERFORM
WHOLE BODY SCANS AND/OR ADMINISTER RADIOIODINE THERAPEUTIC DOSES IN
PATIENTS WITH WELL DIFFERENTIATED THYROID CARCINOMA
RG Sanchez,1 E Sosa,1 A Vazquez,1 L Martinez,1 I Hernandez,1 R Munoz,2 M Mercado.1
1
  Medicine/Endocrinology, Hospital de Especialidades, Centro Medico Nacional S. XXI, IMSS, Mexico
City, Mexico; 2Nuclear Medicine, Hospital de Especialidades, Centro Medico Nacional S. XXI, IMSS,
Mexico City, Mexico

In the follow up of patients with well differentiated thyroid carcinoma (WTC) a TSH > 30 uU/mL is
required in order to perform whole body scans (WBS), determine thyroglobulin levels and administer
therapeutic doses of radioactive iodine. The latter implies that the patient most spend a variable amount of
time in symptomatic hypothyroidism, which can be incapacitating and at least in theory, promote the
growth of cancer cells. Elderly patients and those with other comorbid conditions should benefit from
avoiding long periods of hypothyroidism. We undertook this study to evaluate what is the minimum time
required to achieve a TSH > 30 uU/mL in both, recently operated patients and patients already on thyroid
hormone suppressive therapy.
We studied 2 groups of patients. Group I (3 males, 18 females, mean age 46.3 years) consisted of 21
subjects who had been thyroidectomized in the past and were already on thyroid hormone (TH)
suppression; TH was discontinued and thyroid function tests were performed weekly for 4 weeks thereafter.
Group II (1 male, 9 females, mean age 44.3)consisted of 10 recently operated patients, who had never been
on TH, but who also underwent thyroid function testing on a weekly basis for 4 weeks after thyroidectomy.
At the end of the second week, 43% of patients in group I and 70% in group II had a TSH > 30 uU/mL. All
patients in group II and 86% of patients in group I, had achieved a TSH > 30 uU/mL by the end of the third
week. Free T4 levels significantly inversely correlated with TSH levels at all time points in both groups.
We conclude that the time required to achieve an adequate TSH level in order to perform a WBS or
administer a therapeutic dose of radioidine is less than traditionally accepted. We suggest that a TSH
should be obtained 2 weeks after thyroidectomy or after discontinuing TH since a significant proportion of
patients will then be ready for scanning and/or treatment.

Clinical Science Oral: Thyroid Cancer (1:00 PM-2:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 1:15 PM; Location: Ballroom 2




                                                  - 279 -
                                                                                               THYROID
OR35-2

Lay explanation of abstract:

Patients with thyroid cancer usually have a good prognosis as long as they receive appropriate treatment.
Treatment consists of a complete removal of the thyroid gland followed shortly afterward by administration
of radioactive iodine, which is intended to destroy residual thyroid tissue. Patients with this condition are
later-on started on thyroid hormone replacement therapy, which besides providing him or her with the
thyroid hormone they can no longer produce, helps to suppress the growth of any residual thyroid cancer
cells. The follow-up of these patients takes advantage of the fact that thyroid tissue avidly takes up iodine
(regardless if it is radioactive or not). Therefore, administration of radioactive iodine helps us visualize
areas of the body where residual thyroid cells are located. Furthermore, administering a larger amount of
radioactive iodine can destroy residual cancer cells. The problem is that in order to perform a radioactive
iodine scan and administer treatment doses of radioiodine, the patient needs to be off thyroid hormone
medication traditionally for 6 weeks. During this period, the patient is rendered thyroid hormone deficient,
which makes him or her feel ill, and in certain circumstances, this deficiency can be dangerous if there is
also a heart, lung, or neurological disorder.

We studied 31 patients who had undergone operations for thyroid cancer. Twenty-one had their surgery
one or two years before the study and were already taking thyroid hormone (group 1), and 10 had recently
had operations (group 2).

Our goal was to determine the minimum amount of time required for a patient to become sufficiently
hypothyroid (low in thyroid hormone) in order to perform scans and/or administer radioactive iodine. For
this purpose, we serially measured thyroid function weekly after the thyroid hormone had been
discontinued, until they showed frank hypothyroidism (thyroid hormone deficiency). At the end of the
third week, all patients in group 1 and 86% of patients in group 2 had reached a sufficient degree of thyroid
hormone deficiency in order to undergo thyroid scans and/or radioiodine treatment.

Our results contrast with the traditional practice of waiting 6 weeks after thyroid hormone withdrawal
before performing scans or treatments with radioiodine. It is important to reduce the time patients spend
being thyroid deficient, because this increases their quality of life and it may also decrease the costs of lost
labor (days a patient must spend away from work).

This research was funded locally.




                                                    - 280 -
                                                                                             THYROID
P1-598
THE CHANGE OF PULMONARY ARTERY PRESSURE AND SERUM VASCULAR
ENDOTHELIAL GROWTH FACTOR (VEGF) IN GRAVES' DISEASE BEFORE AND AFTER
TREATMENT
D Kim, J Seo, H Kim, C Choi, S Ihm, J Yoo, M Choi, H Yoo, S Park. Department of Medicine, Hallym
University, College of Medicine, Seoul, South Korea

Exertional symptoms, dyspnea, and impaired effort tolerance are common in patients with Graves' disease.
Proposed explanations include high output left heart failure, ineffective oxygen utilization, and respiratory
muscle weakness. In addition, cases of pulmonary hypertension in patients with Graves' disease have also
been reported. Recently, it has been demonstrated that vascular endothelial growth factor (VEGF) is
produced by thyroid follicular epithelial cells in response to stimulation of the TSH receptor. Secreted
VEGF stimulates Flt receptors on endothelial cells in a paracrine manner, leading to endothelial cell
proliferation. A significant increase in serum VEGF levels in Graves' disease patients has been observed
and the role of VEGF in pulmonary hypertension suggested. To evaluate the role of VEGF in Graves'
disease, we performed two-dimensional and Doppler echocardiographic examination (Hewlett Packard
Sonos 2500) to determine pulmonary artery pressure and measured serum VEGF using ELISA (R&D
systems) in 28 untreated Graves' disease patients before and after treatment (25 patients with
propylthiouracil and 3 with RAI) and 10 normal controls. Pulmonary artery (PA) pressure was increased in
Graves' patients compared to normal controls (mean 23.5 vs. 29.3 mmHg).
In conclusion, 44% of untreated Graves' disease showed pulmonary hypertension and pulmonary artery
pressure was decreased after treatment. The pathogenesis of pulmonary hypertension in Graves' disease
needs further studies.

Clinical Science Poster: Thyroid I (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Wednesday, June 20, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 281 -
                                                                                            THYROID
P1-598

Lay explanation of abstract:

Hyperthyroidism is a condition in which the thyroid gland is overactive. This condition develops when the
thyroid produces too much thyroid hormone. Graves‘ disease is most common cause of hyperthyroidism
and is believed to be caused by an antibody that stimulates the thyroid to produce too much thyroid
hormone. Symptoms of exertion, dyspnea, and impaired tolerance for exertion are common in patients with
Graves‘ disease. Proposed explanations include heart failure, ineffective oxygen utilization, and respiratory
muscle weakness.

We think that pulmonary hypertension is another possible cause. We studied 28 untreated Graves‘ disease
patients before and after treatment and measured the pressure of the pulmonary artery non-invasively using
Doppler sonography.

Our result showed that 44% of untreated Graves‘ disease patients showed pulmonary hypertension.
Pulmonary artery pressure decreased after treatment. The pulmonary artery pressure was related to the level
of TBII that is one of the immunological markers in the blood of Graves‘ disease patients. One patient had
severe heart failure symptoms because of Graves‘ disease. His respiratory difficulty was not well
controlled, despite improvement in the symptoms of heart failure. Thus, we think that other factors, such as
pulmonary hypertension, were involved in his respiratory difficulty.

We then studied the relationship between pulmonary hypertension and thyroid disease, especially Graves‘
disease. Our result clearly demonstrated that more than 40% of untreated Graves‘ disease patients had
pulmonary hypertension and that pulmonary artery pressure was decreased after treatment.




                                                  - 282 -
                                                                                          THYROID
P2-533
BENIGN THYROID NODULES DO NOT GROW
P Tannirandorn, SA Fish, JE Langer, SJ Mandel. Div. of Endocrinology and Ultrasound, Univ. of PA
School of Medicine, Philadelphia, Pennsylvania

The appropriate management of a thyroid nodule with benign cytology is uncertain. Many endocrinologists
recommend suppressive thyroid hormone therapy (T4 Rx) with the suggestion that it may prevent further
growth. This recommendation is based upon the assumption that benign nodules grow. However, there are
few data on the natural growth history of nodules. Using ultrasound (US), we evaluated the growth pattern
in nodules considered to be benign after fine needle aspiration.132 benign nodules from 93 patients (M 11;
F 82; mean age 52 years) had been previously aspirated with US guidance and were reimaged by US on
average 12 months later. Nodule size was recorded in 3 dimensions; increase or decrease in size was
defined if 2 dimensions had a >10% increase/decrease from baseline. No patient was on TSH suppressive
T4 Rx, although 13 were taking replacement T4 Rx with normal TSH values. 79 (60%) nodules remained
stable in size (mean diameter 17 mm to 17 mm, p NS), 45 (34%) decreased in size (mean diameter 18 to 12
mm, p< 0.05). There was no significant difference in the growth pattern of solid (89) vs. complex (43)
nodules. The 8 nodules that grew were reaspirated and cytology was again benign. At the time of this
report, a follow-up third US in 3 of these 8 patients documented that 2 nodules remained stable and one had
decreased in size.
US documents that the vast majority (94%) of nodules with benign cytology remain stable or decrease in
size when reassessed 1 year after FNA. Repeat cytology in the 6% that grew agreed with the initial benign
cytology. Overall, the lack of significant growth suggests that nodules have either reached their maximum
size or are growing very slowly and may require a longer surveillance period. Therefore, routine
administration of TSH suppressive T4 Rx to prevent growth is unwarranted.

Clinical Science Poster: Thyroid Cancer (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                 - 283 -
                                                                                          THYROID
P2-533

Lay explanation of abstract:

As people age, thyroid nodules become increasingly common. The best way to determine whether a nodule
is cancerous is to biopsy it. Fortunately, the majority of nodules (90%) are benign (non-cancerous).
However, physicians differ about the best way to treat a nodule after a biopsy has shown it is benign. Some
patients are given thyroid hormone to put the thyroid to ―rest,‖ with the thinking that the nodule won‘t
grow. We studied more than 100 patients who had undergone nodule biopsies that showed non-cancerous
cells. None were instructed to take thyroid hormones to ―shrink‖ nodules. On average, one year after their
biopsy, nodule size was remeasured by ultrasound. Only 6% of nodules grew; these were biopsied again
and found to be still non-cancerous. The majority of nodules (60%) remained stable in size or shrunk
(34%). Therefore, we concluded that at least in the United States, the routine use of thyroid hormone to
shrink nodules in patients with normal thyroid function is unnecessary, as only 6% grew.

This research was funded by the University of Pennsylvania School of Medicine.




                                                 - 284 -
                                                                                           THYROID
P3-157
A CASE OF RESISTANCE TO THYROID HORMONE (RTH) COMPLICATED WITH
PRIMARY HYPOTHYROIDISM: DIFFICULT CASE TO MANAGE
S Fukata,1 K Kuma,1 K Motomura,2 GA Brent,2 M Sugawara.2 1Kuma hospital, Kobe, Japan; 2West Los
Angeles VA Medical Center and UCLA School of Medicine, Los Angeles, California

Resistance to thyroid hormone (RTH) is characterized by elevated circulating thyroid hormones,
unsuppressed TSH levels and peripheral refractoriness to hormone action. RTH associated with
Hashimoto's thyroiditis is rare. We report a 41 year-old woman, who has RTH and Hashimoto's thyroiditis,
developed primary hypothyroidism. This case illustrates difficulties in evaluating thyroid status and its
management. She was born from parents with consanguine marriage. She had persistent elevation of serum
free thyroid hormone with normal or high serum TSH. Before seen by us, she received methimazol and
underwent subtotal thyroidectomy in 1993. When she was seen by us in 1998, she was on methimazole and
her thyroid gland was diffusely enlarged with an estimated volume of 62 ml (normal: <15) by ultrasound.
Thyroid function revealed normal serum free T4 and T3 levels with serum TSH level of greater than 100
U/ml (normal:0.40-4.50). Repeated thyroid function test 2 months after discontinuation of methimazol
showed normal serum TSH, slightly increased serum free thyroid hormone and positive anti-microsomal
antibody. Thyroid hormone  receptor analysis from lymphocytes showed a point mutation at the position
438 replacing arginine with histidine. She was observed without any medication and her goiter became
smaller. She visited 2 years later because of increasing goiter size of 63.9 ml. Her thyroid function showed
TSH of 72.97 U/ml, normal free thyroid hormone levels and strongly positive anti-microsomal antibody
titer. She was clinically hypothyoid and l-thyroxine treatment with 100 g daily ameliorated her
hypothyoid symptoms. This case presents the difficulty in detecting thyroid failure and establishing
euthyoid marker in the presence of RTH. We also speculate that RTH is present in some of patients with
Hashimoto's thyroiditis who present persistent subclinical hypothyroidism.

Clinical Science Poster: Pediatric Endocrinology II (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 285 -
                                                                                             THYROID
P3-157

Lay explanation of abstract:

An increase in thyroid hormone level in the blood generally indicates an overactive thyroid gland, or
hyperthyroidism. A decrease in thyroid hormone level in the blood suggests an inactive thyroid
glandhypothyroidism. Our human body recognizes excess or decreased thyroid hormone level in the
pituitary gland in the brain and tries to control thyroid gland activity by adjusting the secretion of thyroid
stimulating hormone (TSH). For instance, increased TSH secretion is seen in patients with hypothyroidism.
Recognition of excess or decreased thyroid hormone is accomplished through the thyroid hormone
receptor, a protein complex that binds thyroid hormone and begins thyroid hormone action. We use blood
levels of thyroid hormone and TSH to evaluate thyroid function, assuming that our hormone recognition
system is intact.

We are reporting on a patient who has a defect in recognizing thyroid hormone. Physicians were confused
about whether this patient had hyperthyroidism or hypothyroidism. We found a single mutation in the
thyroid hormone receptor gene, causing dysfunction of the receptor and the difficulty in interpreting thyroid
status. It is important for health care personnel to consider this type of receptor dysfunction whenever a
bizarre relationship between thyroid hormone level and thyroid stimulation hormone is found. If this type
of disorder is not recognized, patients may undergo unnecessary surgery or drug therapy.

We studied a 41 year-old woman, who initially exhibited high thyroid hormone levels in the blood. She
never showed normalization of both thyroid hormone and TSH levels after surgery or drug therapy for
possible hyperthyroidism. Then, she developed distinct symptoms of hypothyroidism; however, her
thyroid hormone levels in the blood were normal and thyroid stimulating hormone levels were high. We
speculated the possibility of the defect in the thyroid hormone recognition system, because thyroid
hormone levels in the blood did not reflect true thyroid status. DNA was extracted from lymphocytes and
the sequence of thyroid hormone  receptor gene was analyzed. A point mutation was observed at amino
acid position 438, where histidine was substituted for arginine.

This is a valuable case study, because mutation of the thyroid receptor involving only one amino acid
substitution can cause tremendous difficulty when evaluating thyroid status.

Funding for this research came from Kuma Hospital, Kobe, Japan and Veterans Affair Research Funds.




                                                   - 286 -
                                                                                            THYROID
P3-167
TSH IS A POOR TEST TO ASSESS THE SEVERITY OF TISSUE HYPOTHYROIDISM AS
COMPARED TO METABOLIC EVALUATION AT THE PERIPHERAL TARGET ORGANS
C Meier, P Trittibach, M Kunz, M Guglielmetti, B Muller, J Staub. Division of Endocrinology, Department
of Medicine, University Hospital, Basel, Switzerland

Background: While serum TSH is clearly the most sensitive test for early diagnosis of hypothyroidism, no
data are available for the evaluation of TSH as a measure for the degree of tissue hypothyroidism. As a
diagnostic tool, tissue markers provide quantitative measurements of the cellular action of thyroid
hormones at the peripheral target organs. The aim of the study was to compare basal TSH with peripheral
parameters of target tissues for the assessment of the severity of tissue hypothyroidism. Methods: 58
women with overt hypothyroidism (OH; TSH 50.5±3.4 mIU/L; age 56.3±1.5 yrs) and 153 women with
subclinical hypothyroidism (SCH; TSH 13.5±1.1 mIU/L; age 53.8±1.1 yrs) were investigated. In all
patients thyroid hormone levels and the following tissue markers were measured: ankle reflex time (ART),
circulating creatine kinase (CK), clinical scores (Billewicz, Zulewski), total cholesterol (TC) and LDL-
cholesterol levels (LDL-C). Severity of hypothyroidism was classified either by fT4 concentrations or by
metabolic manifestation (based on ART).
Results: In OH we found no correlation between tissue markers and plasma TSH levels (r=0.14 to 0.27,
p=ns), in contrast to the fT4 levels, which correlated well with all metabolic tests (r=-0.53 to -0.75,
p<0.0001). In SCH, no significant correlations between the different tissue markers and either TSH nor fT4
levels were found. By metabolic grading (by ART values), documenting progressive tissue hypothyroidism,
we could demonstrate a poor, non-significant increase of TSH in relation to the severity of tissue
hypothyroidism, but a clear effect on other peripheral tissue markers (scores, p=0.02; CK, p=0.001; TC,
p=0.01; LDL-C, p=0.02) and decreased thyroid hormones (fT4 and T3, p=0.001).
Conclusions: TSH is a poor test for the evaluation of tissue hypothyroidism and clinical scores compared to
the tests of peripheral hormone action. This may be explained by maximal stimulation of the pituitary
thyrotrops (top of the dose-response curve) or possibly by a different effect on thyroid hormone receptors at
the central or peripheral target tissues.

Clinical Science Poster: P3: Thyroid II (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 287 -
                                                                                               THYROID
P3-167

Lay explanation of abstract:

Primary hypothyroidism is a common endocrine disease affecting predominantly women over 40 years old.
In the overt stage, typical clinical signs and symptoms like weakness, fatigue, cold intolerance,
constipation, weight change, depression, and dry skin together with decreased thyroid hormone
concentrations lead to the diagnosis. The introduction of new assays for thyroid-stimulating hormone
(TSH) measurements and their widespread application as first-line test of thyroid function has led to early
recognition of the hypothyroid state.

Serum TSH is clearly the most sensitive routine test for screening and early diagnosis of hypothyroidism.
However, no data are available for the evaluation of TSH as a measure for the degree of hypothyroidism at
the tissue level. As a diagnostic tool, tissue markers provide quantitative measurements of the cellular
action of thyroid hormones at the peripheral target organs.

The aim of this study was to compare basal TSH with peripheral parameters of target tissues to assess the
severity of tissue hypothyroidism. We investigated 58 women with overt hypothyroidism and 153 women
with mild hypothyroidism with elevated TSH levels but normal thyroid hormone concentrations. In all
patients, thyroid hormone levels and the following tissue markers were measured: ankle reflex time,
circulating creatine kinase levels, clinical scores, total cholesterol, and LDL-cholesterol levels. Severity of
hypothyroidism was classified either biochemically by free T4 concentrations or by metabolic
manifestation (based on ankle reflex time).

In overt hypothyroidism, we found no correlation between tissue markers and plasma TSH levels in
contrast to free T4 levels, which correlated well with all metabolic tests. In mild hypothyroidism, no
significant correlations between the different tissue markers and either TSH or free T4 levels were found.
By metabolic grading (by the ankle reflex values), which documents progressive tissue hypothyroidism, we
could demonstrate no significant increase of TSH in relation to the severity of tissue hypothyroidism. There
was a clear effect on other peripheral tissue markers and decreased thyroid hormones .

We demonstrated that in overt hypothyroidism, serum TSH is a poor test to estimate the severity of tissue
hypothyroidism. This is in direct contrast to the generally accepted view that TSH is the best and most
reliable screening test for assessing routine thyroid function. This finding may be explained by maximal
stimulation of the pituitary cells secreting TSH (top of the dose-response curve) or possibly by a different
effect on thyroid hormone receptors at the central or peripheral target tissues.

This study was supported by grants from the Swiss Research Foundation and by the Thyroid Research Unit,
Division of Endocrinology of the University Hospital, Basel, Switzerland.




                                                    - 288 -
                                                           REPRODUCTIVE (FEMALE)

P2-397
EVIDENCE FOR AN ACTIVATED RENIN-ANGIOTENSIN SYSTEM BY HORMONE
REPLACEMENT THERAPY (HRT): ANGIOTENSINOGEN LEVELS ARE HIGHER IN
POSTMENOPAUSAL WOMEN WITH HRT
S Claudi-Boehm,1 B Winkelmann,2 KE Siegler,2 V Maier,3 S Schilling,3 BO Boehm.3 1Department of
Obstetrics and Gynaecology, University of Ulm, Ulm, Germany; 2Herzzentrum Ludwigshafen,
Ludwigshafen, Germany; 3Division of Endocrinologie and Diabetes, University of Ulm, Ulm, Germany

Advantages and disadvantages of HRT are still not clear. A positive modification of classical
cardiovascular risk factors was described. However, HRT may also increase risk in women with established
HRT. We have addressed the role of the RAS in HRT as an explanation for negative cardio-vascular
effects. Methods: Cross-sectional analysis of women with (N = 109) and without HRT (N = 796). The
cohort was matched for BMI and age. Angiotensinogen was determined by angiotensin I measurement after
angiotensinogen cleavage with human recombinant renin. Angiotensinogen-gene polymorphisms (ANG174
and ANG235) was determined by PCR. Results: Angiotensinogen level was significantly increased in
women with HRT (mean 2599 pmol/ml + SD 1447 vs. 1442 + SD 669; P< 0.001), whereas polymorphisms
of the angiotensinogen gene was not (ANG174: P=0.169, ANG235: P=0.242). A trend for an increased
diastolic level was seen with HRT (mean: 79,7 mmHg + SD 11.2 mmHg vs. 80.8 + SD 11.2; P=0.248).
Conclusions: HRT increased angiotensinogen levels significantly and therefore exerts a profound
stimulatory effect on the RAS in humans. However, the levels of diastolic blood pressure were non-
significantly higher in the group with HRT. This may indicate an estrogen-induced shift towards pressure
lowering peptides that may partially antagonize the increase of blood pressure induced by the
angiotensinogen increase.

Clinical Science Poster: Female Reproduction (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                - 289 -
                                                            REPRODUCTIVE (FEMALE)
P2-397
Lay explanation of abstract:

Hormone replacement therapy (HRT) has been used for a long time as a way to prevent atherosclerosis in
older women. However, the advantages of HRT in postmenopausal women are not clear. HRT use has been
linked with protection against classical cardiovascular risk factors, such an increase of high-density
lipoprotein concentration (i.e., an improvement of the lipid profile through the use of HRT), which may
protect against atherosclerosis. An ongoing and controversial discussion involves hormone replacement
therapy (HRT): Why does HRT provide some positive effects on various surrogate risk markers (i.e., lipid
profile) while it also appears to harm women with established coronary heart disease? On the negative
side, HRT also may directly activate the renin-angiotensin-aldosterone system (RAAS), and this activation
may explain the harmful effects of HRT in women with coronary heart disease. Activation of the RAAS is
a major determinant of progression of cardiovascular diseases and also has profound effects on
cardiovascular homeostasis.

We studied the role of HRT on the renin-angiotensin-aldosteron system with groups of postmenopausal
women taking (N=109) and not taking HRT (N=796). All women had a high-risk profile for cardiovascular
disease. The groups studied were matched for age and body mass index. In women taking HRT, the
hormone angiotensinogen (the precursor molecule for the generation of various vasoactive peptides) was
found to be significantly higher compared with the matched women in the control group. An association of
angiotensinogen levels with angiotensinogen gene polymorphisms could not be found in HRT users. There
was a trend for an increase of diastolic blood pressure in women taking HRT.

We showed that HRT increased the level of the precursor molecule angiotensinogen significantly, which
indicates a potentially harmful activation of the RAAS. The level of diastolic blood pressure rose but was
not significantly increased in HRT users, which indicated that HRT may have a double effect. Estrogens
modulate the RAAS, which causes an increase of RAAS activity. This activation may be harmful,
especially in women with established coronary heart disease. However, an estrogen-induced shift towards
pressure-lowering peptides was also found in HRT users. While activation of the RAAS can be risky in the
case of women with established cardiovascular disease, the risk may be counterbalanced somewhat by the
dual activity of estrogen on the RAAS.

Additional background information:
A large number of prospective trials have shown the beneficial effects of angiotensin-I converting enzyme
(ACE) inhibitor therapy in heart failure, post-myocardial infarction, and in preventing cardiovascular
disease. These data clearly indicate that the RAAS is a major determinant for the progression of
cardiovascular disease, which is the leading killer of women in Western countries. Negative cardiovascular
effects were found in the recent Heart and Estrogen/progestin Replacement Study (HERS). We suggested
that an increase in angiotensinogen, which is the precursor molecule for the various regulatory peptides,
may not only be influenced by HRT, but may also be controlled by a well-known gene polymorphism of
the angiotensinogen gene. This gene polymorphism recently has been found to control blood pressure,
especially diastolic blood pressure. Therefore, we thought that we could scan candidate genes to fine gene
markers that could help to explain the positive and negative clinical endpoints reported from primary and
secondary prevention trials with HRT. We showed that HRT has a two-way action on the RAAS and may
modulate both the vasoconstrictor and the vasodilator system. An ongoing genetic analysis of the study
cohort described will address genes that directly effect the generation of vasoactive peptides (see:
Winkelmann BR, März W, Boehm BO: Rationale and design of the LURIC study – a resource for
functional genomics, pharmacogenomics and long-term prognosis of cardiovascular disease.
Pharmacogenomics 2001;2: S7–S73).

Grant support came from the German Research Council (DFG).




                                                 - 290 -
                                                             REPRODUCTIVE (FEMALE)
P2-400
CORTISOL SECRETION IN WOMEN WITH FUNCTIONAL HYPOTHALAMIC
AMENORRHEA (FHA) TREATED WITH COGNITIVE BEHAVIOR THERAPY
SL Berga,1,2 TL Loucks,1 MD Marcus,2 M Krohn,1 JL Cameron.2 1Ob, Gyn, and Repro Sci, Univ Pittsburgh
Sch of Medicine and Magee-Womens Hospital, Pgh, Pennsylvania; 2Psychiatry, Univ Pittsburgh Sch of
Medicine, Pgh, Pennsylvania

FHA is a common and theoretically reversible form of anovulation due to reduced GnRH/LH drive.
Women with FHA also display other neuroendocrine secretory alterations, including increased cortisol and
decreased T3 and T4 levels. Behavioral concomitants of FHA include dysfunctional attitudes, mild
undernutrition, and/or increased energy expenditure. Our monkey model of FHA revealed a synergistic
impact of psychosocial stress and mild metabolic imbalance in the induction of FHA. Given these
considerations, we hypothesized that cognitive behavior therapy (CBT) targeted to ameliorate problematic
behaviors and attitudes would reverse FHA and that treatment would be accompanied by a decline in
cortisol secretion. 14 women with FHA of normal body weight, who met stringent inclusion and exclusion
criteria, including absence of eating disorders and psychiatric conditions, were randomized to 16
manualized sessions of CBT or observation (OBS) over 20 wks. Treated and observed women did not
differ in baseline characteristics, including BMI and age. Ovarian function was monitored before, during,
and after CBT or OBS with weekly levels of estradiol (E2 pg/mL), and progesterone (P nmol/L). Cortisol
patterns were monitored before and after CBT or OBS in 9 of 14 subjects by obtaining blood samples
Q15min x 24h. Of the 7 women treated, 6 fully recovered (E2 > 100 and/or P > 5) and 1 partially recovered
(E2 > 60 but P < 5). Of the 7 subjects observed, 1 fully recovered, 1 partially recovered, and 5 did not
recover. CBT yielded a higher rate of recovery (100%) than OBS (29%), χ 2 = 0.009. Nocturnal cortisol
decreased 14.7% ± 4.9 (SE) in treated (n=5) vs. 1.9%±4.7 in observed (n=4), Mann-Whitney, one-sided.
In conclusion, CBT restored ovarian function and reduced cortisol secretion. These findings demonstrate
that combined psychogenic and metabolic stress plays a causal role in FHA. These findings provide
additional evidence that a tailored behavioral intervention offers an efficacious treatment option that also
avoids the pitfalls of pharmacological modalities.

Clinical Science Poster: Female Reproduction (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Thursday, June 21, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 291 -
                                                                 REPRODUCTIVE (FEMALE)
P2-400

Lay explanation of abstract:

Our research team presents the first study that demonstrates that a psychological intervention can restore
fertility by reducing the activation of the hypothalamic-pituitary-adrenal axis. It also demonstrates that
multiple small stressors that seemingly would have minimal impact on reproductive competence can play a
major role in causing failure to ovulate (anovulation). Fortunately, by identifying and correcting the
stressors, fertility can be restored without resorting to expensive medications. Importantly, this study also
highlights the important links between the mind and the body.

This study demonstrates that a psychological intervention can help to improve endocrine measures of stress
(cortisol). Thus, this study shows the important contribution lifestyle factors play in modulating overall
health and reproductive health in particular. To reverse stress-induced anovulation, both mental and
metabolic sources of stress need to be identified and addressed.

The current standard of practice other than observation is to offer hormonal interventions such as ovulation
induction if fertility is desired and oral contraceptives if fertility is not desired. These solutions are costly,
and moreover, they can hide other endocrine disturbances. Because these endocrine disturbances pose a
risk to overall health, it is important to have a therapy that restores the endocrine system, including (but not
limited to) the reproductive system. Cognitive behavior therapy offers a holistic treatment that is cost
effective and easy to implement.

Additional background information:
The menstrual cycle involves a complex sequence of hormone signals that begin in the brain. A part of the
brain called the hypothalamus produces a releasing factor, GnRH, which stimulates the pituitary gland (the
master gland at the base of the brain) to secrete two hormones into the bloodstream, luteinizing hormone
(LH) and follicle-stimulating hormone (FSH). In turn, LH and FSH stimulate the ovaries to produce eggs.
If the stimulation is sufficient and the ovaries contain responsive, good -quality eggs, ovulation (release of
the egg) will result and fertilization becomes possible. There are many reasons for not ovulating
(anovulation), but the most common reason is a reduction in the brain message, GnRH. A prolonged
reduction in GnRH drive causes amenorrhea (absence of periods). This condition is called functional
hypothalamic amenorrhea (FHA; also called stress-induced anovulation) if the reduction in GnRH is not
due to a tumor or another organic cause. Most physicians don‘t know that FHA involves more than a
reduction in GnRH drive and resultant anovulation/amenorrhea. Women with FHA also typically have
increased cortisol levels and reduced thyroid hormone levels. These endocrine changes signal a metabolic
imbalance. There are other hormonal components of FHA as well. Because the role of the hypothalamus
is to maintain endocrine homeostasis (direct endocrine adaptations that allow survival), all of these
endocrine alterations reflect the body‘s coping response to commonplace challenges such as undernutrition,
excess energy expenditure, or psychological stress.

Unfortunately, the constellation of endocrine adaptations that help the body cope with psychological or
metabolic challenge in the short term are not good for overall health in the long term. A prolonged loss of
the ovarian hormones, estradiol and progesterone, can cause harmful effects. Likewise, sustained increases
in cortisol and prolonged decreases in thyroid can compromise overall health. Thus, replacing the missing
ovarian hormones is not enough. Some of the conditions linked to FHA include osteoporosis,
cardiovascular disease, depression, and cognitive impairment. If drugs are employed to induce ovulation,
then there are risks for the fetus as well, such as intrauterine growth restriction, prematurity, and
compromised neuropsychological development.

This study has been funded for eight years by the National Institute of Mental Health.




                                                     - 292 -
                                                   THYROID (MTP-CLINICAL CASE)

P3-152
THE HIGH FREQUENCY OF REPORTED NEWBORNS WITH CONGENITAL CENTRAL
HYPOTHYROIDISM DUE TO A TSHB 313ΔT MUTATION IS CAUSED BY A FOUNDER
EFFECT
H Brumm,1 H Biebermann,1 A Pfeufer,4 C Heinrichs,3 L Duprez,2 R Gessner,5 A Grueters.1 1Pediatric
Endocrinology, Charite CVK, Berlin, Germany; 2IRI Faculte de Medecine ULB, Campus Erasme, Brussels,
Belgium; 3Hopital Universitaire des Enfants Reine Fabiola, Brussels, Belgium; 4Kerckhoff Clinic, Max-
Planck-Gesellschaft, Frankfurt (Main), Germany; 5Laboratory Medicine and Pathobiochemistry, Charite
CVK, Berlin, Germany

The introduction of neonatal TSH-screening was an enormous acquirement for the detection of congenital
hypothyroidism. However, in very rare cases central hypothyroidism is caused by TSH-deficiency. These
patients are missed in TSH screening due to their low TSH levels. Within the last 10 years an increasing
number of patients was reported with autosomal recessive mutations in the gene for the β-subunit of TSH
(TSHB) as the molecular cause of the disease. Beside single cases of E12X, G29R and Q49X, the most
frequent TSHB mutation 313ΔT (C105V) has been described in 6 apparently unrelated families. This
frequent occurence of an identical mutation may be due to a founder effect or to a mutational hot spot at
this position. Therefore, it has been discussed whether a neonatal T4 screening in addition to the TSH
determination is advisable. To estimate the disease risk in the general population due to this mutation we
screened 500 unrelated individuals and did not detect any 313ΔT allele indicating an overall rather low
prevalence. In 4 affected families and additional 8 unaffected families haplotype analysis of the TSHB
region containing 5 single nucleotide polymorphism sites revealed the presence of 8 different haplotypes.
In all 8 parental lines of the 4 affected families 313ΔT mutations occurred exclusively on the same
haplotype which had a prevalence of 40 % in the population. Extending the haplotype by two flanking
microsatellites revealed little heterogeneity of the disease haplotypes. All these data suggest a
monophyletic origin of the recessive TSHB 313ΔT mutation and no significant population prevalence.
Cases seem to be clustered in certain geographic areas leading to a locally increased possibility of
heterozygous mating. The identification and genetic counselling of heterozygous carriers in affected
families seems to be warranted.

Clinical Science Poster: Thyroid II (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)

Presentation Date: Friday, June 22, 2001; Time: 11:00 AM; Location: Exhibit Hall




                                                  - 293 -
                                                     THYROID (MTP-CLINICAL CASE)

P3-152

Lay explanation of abstract:

In rare cases, newborns are affected by an inherited mutation causing a reduced function of their thyroid
gland (hypothyroidism), which then cannot produce thyroid hormones. Untreated, this condition results in
severe mental and physical retardation of the growing child. Fortunately, if detected in the first days of life,
the disease can be treated with doses of missing hormones in a daily oral dose and the child will have a
fully normal development.

To detect every affected child (1 out of 3,000 newborns), most developed countries have nationwide
screening programs. In Europe, this is done by measuring the level of a hormone important for thyroidal
development, thyroid-stimulating hormone (TSH). In case of a hypofunctioning thyroid gland (so-called
primary hypothyroidism), the TSH level is higher in order to stimulate the thyroid gland to produce thyroid
hormones, so TSH level is abnormally high. Therefore, an elevated TSH level in the newborn indicates a
hypofunction of the thyroid gland and the child is examined further.

In some very rare cases (approximately 1 out of 100,000 newborns), the defect is not situated in the thyroid
gland but the TSH itself (so-called secondary hypothyroidism). In this situation, no elevated TSH-levels
can be detected, although the children later on will also suffer from symptoms of thyroid hypofunction.
These cases can only be detected by measuring the products of thyroid synthesisthe thyroid hormones,
which are measured in the United States. This recently started a vivid discussion—Is it necessary to change
the method of newborn screening in Europe? Recently, many new cases of secondary hypothyroidism have
been observed. Strikingly, one definite mutation in the gene for TSH has appeared in different parts of the
world, in four German cities, in Belgium and in Brazil. The basic question is whether this alteration is
because of a mutational hot spot that indicates that many cases of secondary hypothyroidism will arise in
the future. These cases would not be caught with traditional European TSH screening. Yet an additional
screening for thyroid hormones inevitably would add costs to the public health system. If the problem is not
a genetic mutation, however, the increased incidence of secondary hypothyroidism may be because of
parental relationships among all families affected by this mutation, which would be a suitable explanation
for the observed new appearances. If that were true, a widespread screening would be unnecessary.

Our research, using molecular biological investigation with a method comparable to fatherhood analysis
clearly showed a parental relationship of all investigated families in Germany and Belgium with one
common founder about 3,700 years ago. Hence, no change in screening procedures is needed, because this
specific mutation did not arise in other populations and will not do so in the future. In conclusion, one does
not expect appearances of children affected with that kind of TSH abnormality in the future. Only families
at risk would need to employ further testing. These cases impressively demonstrate that genetic alterations,
which occurred by chance in one person many generations ago, can spread through the world, carried by
families who had no idea they were related to each other.

This study was supported by a scholarship given to H. Brumm by the German Society of Endocrinology.




                                                    - 294 -
INDEX OF ABSTRACTS
 Abstract #     Page #
   S20-1          1
   S20-2          3
   S20-3          5
   S20-4          7
   S38-1          9
   S38-2          11
   OR24-1         13
   OR24-6         15
   P1-418         17
   P1-419         19
   OR12-4         21
   P1-276         23
   P1-296         25
   P1-352         27
   P1-368         29
   OR2-5          31
   OR13-2         33
   P3-473         35
   OR36-4         37
   OR36-6         39
   P3-380         41
   P3-389         43
   P3-396         45
   P2-199         47
   P2-215         49
   OR4-5          51
   OR16-4         53
   P1-227         55

      - 295 -
Abstract #     Page #
  P1-241         57
  P1-246         59
  P1-253         61
  P1-263         63
  P2-630         65
  P3-663         67
  OR50-4         69
  OR50-5         71
  OR50-6         73
  OR51-1         75
  OR51-3         77
  OR51-4         79
  P2-599         81
  P2-601         83
  P3-194         85
  OR27-6         87
  P1-174         89
  P1-198         91
  P1-203         93
  P2-262         95
  OR53-4         97
  OR28-2         99
  P2-138         101
  P2-150         103
  P2-157         105
  P2-162         107
  P3-299         109
  P3-306         111
  P3-318         113
  P1-381         115



     - 296 -
Abstract #     Page #
  P3-102         117
  P3-110         119
  OR29-1         121
  OR29-5         123
  OR29-6         125
  P1-496         127
  P1-497         129
  P1-503         131
  P1-508         133
  OR39-3         135
  OR39-4         137
  P2-356         139
  P2-360         141
  P2-377         143
  P2-389         145
  P2-498         147
  P2-502         149
  OR18-4         151
  OR31-6         153
  P2-30          155
  P2-66          157
  OR7-6          159
  OR41-4         161
  P1-436         163
  P1-437         165
  P1-438         167
  P1-445         169
  OR32-1         171
  OR32-4         173
  P2-462         175



     - 297 -
Abstract #     Page #
  P2-463         177
  P2-468         179
  P3-208         181
  P3-211         183
  OR42-1         185
  OR42-4         187
  OR42-5         189
  P1-464         191
  P1-470         193
  P3-139         195
  P3-141         197
  P3-143         199
  P3-144         201
  P3-145         203
  OR20-1         205
  P1-310         207
  P1-319         209
  P1-321         211
  P2-557         213
  P2-559         215
  P2-564         217
  P2-579         219
  P3-553         221
  P3-555         223
  OR21-1         225
  OR44-3         227
  P3-684         229
  P3-685         231
  P2-660         233
  P2-668         235



     - 298 -
Abstract #     Page #
  P1-561         237
  P1-566         239
  P1-567         241
  P1-571         243
  P1-573         245
  P1-576         247
  P1-654         249
  P3-46          251
  P3-47          253
  OR22-6         255
  OR34-5         257
  OR46-5         259
  OR10-1         261
  OR10-4         263
  OR10-5         265
  OR10-6         267
  P2-404         269
  P2-410         271
  P2-422         273
  P2-427         275
  OR11-3         277
  OR35-2         279
  P1-598         281
  P2-533         283
  P3-157         285
  P3-167         287
  P2-397         289
  P2-400         291
  P3-152         293




     - 299 -
                         INDEX OF ABSTRACTS BY SUBJECT


                              I.        Hot Topics/Clincial Trials Symposia

                              Abstract #                       Page #

                                     S20-1                        1
                                     S20-2                        3
                                     S20-3                        5
                                     S20-4                        7
                                     S38-1                        9
                                     S38-2                        11



                              II.       Aging (BS)

                              Abstract #                       Page #

                                     OR24-1                       13
                                     OR24-6                       15
                                     P1-418                       17
                                     P1-419                       19



                              III.      Diabetes (BS)

                              Abstract #                       Page #

                                     OR12-4                       21
                                     P1-276                       23
                                     P1-296                       25
                                     P1-352                       27
                                     P1-368                       29



                              IV.       Gene Expression (BS)

                              Abstract #                       Page #

                                     OR2-5                        31
                                     OR13-2                       33



NOTE: BS = Basic Science
      CS = Clinical Science



                                              - 300 -
V.        GnRH-Gonadotropins (BS)

Abstract #                     Page #

       P3-473                      35



VI.       Growth Factors (general) (BS)

Abstract #                     Page #

       OR36-4                      37
       OR36-6                      39
       P3-380                      41
       P3-389                      43
       P3-396                      45



VII.      GH-Prolactin-Regulation of Secrection (BS)

Abstract #                     Page #

       P2-199                      47
       P2-215                      49



VIII.     Hormones and Cancer (BS)

Abstract #                     Page #

       OR4-5                       51
       OR16-4                      53
       P1-227                      55
       P1-241                      57
       P1-246                      59
       P1-253                      61
       P1-263                      63
       P2-630                      65
       P3-663                      67




                - 301 -
IX.       Hormone Neoplasia (BS)

Abstract #                      Page #

       OR50-4                        69
       OR50-5                        71
       OR50-6                        73



X.        Hormones and Immune System (BS)

Abstract #                      Page #

       OR51-1                        75
       OR51-3                        77
       OR51-4                        79
       P2-599                        81
       P2-601                        83



XI.       HPA Axis (BS)

Abstract #                      Page #

       P3-194                        85



XII.      IGFs (BS)

Abstract #                      Page #

       OR27-6                        87
       P1-174                        89
       P1-198                        91
       P1-203                        93
       P2-262                        95



XIII.     Intracellular Signalling (BS)

Abstract #                      Page #

       OR53-4                        97




                - 302 -
XIV. Neuroendocrinology (BS)

Abstract #               Page #

      OR28-2                   99
      P2-138                   101
      P2-150                   103
      P2-157                   105
      P2-162                   107
      P3-299                   109
      P3-306                   111
      P3-318                   113



XV.      Obesity (BS)

Abstract #               Page #

      P1-381                   115
      P3-102                   117
      P3-110                   119



XVI. PTH-Calcium-Vitamin D-Bone (BS)

Abstract #               Page #

      OR29-1                   121
      OR29-5                   123
      OR29-6                   125
      P1-496                   127
      P1-497                   129
      P1-503                   131
      P1-508                   133




               - 303 -
XVII. Reproduction-Gonadal Control (Female) (BS)

Abstract #                   Page #

      OR39-3                     135
      OR39-4                     137
      P2-356                     139
      P2-360                     141
      P2-377                     143
      P2-389                     145




XVIII. Reproduction-Gonadal Control (Male) (BS)

Abstract #                   Page #

      P2-498                     147
      P2-502                     149

XIX. Steroid Hormone Action (BS)

Abstract #                   Page #

      OR18-4                     151
      OR31-6                     153
      P2-30                      155
      P2-66                      157



XX.      Steroid Hormone Biosynthesis (BS)

Abstract #                   Page #

      OR7-6                      159
      OR41-4                     161




               - 304 -
XXI. Aging (CS)

Abstract #                Page #

    P1-436                    163
    P1-437                    165
    P1-438                    167
    P1-445                    169



XXII. Androgenic Disorders in Women (CS)

Abstract #                Page #

    OR32-1                    171
    OR32-4                    173
    P2-462                    175
    P2-463                    177
    P2-468                    179



XXIII. Autoimmune Endocrine Diseases (CS)

Abstract #                Page #

    P3-208                    181
    P3-211                    183



XXIV. Bone-Calcium-Parathyroid (CS)

Abstract #                Page #

    OR42-1                    185
    OR42-4                    187
    OR42-5                    189
    P1-464                    191
    P1-470                    193
    P3-139                    195
    P3-141                    197
    P3-143                    199
    P3-144                    201
    P3-145                    203




             - 305 -
 XXV. Diabetes-Lipids-Metabolism (CS)

 Abstract #                   Page #

     OR20-1                         205
     P1-310                         207
     P1-319                         209
     P1-321                         211
     P2-557                         213
     P2-559                         215
     P2-564                         217
     P2-579                         219



 XXVI. Endocrine Genetics (CS)

 Abstract #                   Page #

     P3-553                         221
     P3-555                         223



XXVII. Glucocorticoids (CS)

 Abstract #                   Page #

     OR21-1                         225



XXVIII. Growth Hormone and IGFs (CS)

 Abstract #                   Page #

     OR44-3                         227



XXIX.   Health Care Delivery (CS)

 Abstract #                   Page #

     P3-684                         229
     P3-685                         231




              - 306 -
XXX.      Hormones and Cancer (CS)

 Abstract #                  Page #

       P2-660                    233
       P2-668                    235



XXXI.     Obesity (CS)

 Abstract #                  Page #

       P1-561                    237
       P1-566                    239
       P1-567                    241
       P1-571                    243
       P1-573                    245
       P1-576                    247




XXXII. Pediatric Endocrinology (CS)

 Abstract #                  Page #

       P1-654                    249
       P3-46                     251
       P3-47                     253



XXXIII. Pituitary (CS)

 Abstract #                  Page #

       OR22-6                    255
       OR34-5                    257
       OR46-5                    259




                - 307 -
XXXIII. Reproduction (Female) (CS)

 Abstract #                 Page #

     OR10-1                     261
     OR10-4                     263
     OR10-5                     265
     OR10-6                     267
     P2-404                     269
     P2-410                     271
     P2-422                     273
     P2-427                     275



XXXV. Reproduction (Male) Prostate (CS)

 Abstract #                 Page #

     OR11-3                     277



XXXVI. Thyroid (CS)

 Abstract #                 Page #

     OR35-2                     279
     P1-598                     281
     P2-533                     283
     P3-157                     285
     P3-167                     287



XXXVII. Reproductive (Female) (CS)

 Abstract #                 Page #

     P2-397                     289
     P2-400                     291



XXXVIII. Thyroid (MTP-Clinical Case)

 Abstract #                 Page #

     P3-152                     293




              - 308 -
         INDEX OF ABSTRACTS BY DATE
                   WEDNESDAY, JUNE 20, 2001
OR2-5     P1-227                   P1-368     P1-503
OR4-5     P1-241                   P1-381     P1-508
OR7-6     P1-246                   P1-418     P1-561
OR10-1    P1-253                   P1-419     P1-566
OR10-4    P1-263                   P1-436     P1-567
OR10-5    P1-276                   P1-437     P1-571
OR10-6    P1-296                   P1-438     P1-573
OR11-3    P1-310                   P1-445     P1-576
P1-174    P1-319                   P1-464     P1-598
P1-198    P1-321                   P1-470     P1-654
P1-203    P1-352                   P1-497     PI-496

                   THURSDAY, JUNE 21, 2001

OR12-4    P2-162                   P2-422     P2-599
OR13-2    P2-199                   P2-427     P2-601
OR16-4    P2-215                   P2-462     P2-630
OR18-4    P2-262                   P2-463     P2-660
OR20-1    P2-356                   P2-468     P2-668
OR21-1    P2-360                   P2-498     S20-1
OR22-6    P2-377                   P2-502     S20-2
P2-30     P2-389                   P2-533     S20-3
P2-66     P2-397                   P2-557     S20-4
P2-138    P2-400                   P2-559
P2-150    P2-404                   P2-564
P2-157    P2-410                   P2-579

                     FRIDAY, JUNE 22, 2001

OR24-1    OR35-2                   P3-157     P3-473
OR24-6    P3-46                    P3-167     P3-553
OR27-6    P3-47                    P3-194     P3-555
OR28-2    P3-102                   P3-208     P3-663
OR29-1    P3-110                   P3-211     P3-684
OR29-5    P3-139                   P3-299     P3-685
OR29-6    P3-141                   P3-306     S38-1
OR31-6    P3-143                   P3-318     S38-2
OR32-1    P3-144                   P3-380
OR32-4    P3-145                   P3-389
OR34-5    P3-152                   P3-396

                   SATURDAY, JUNE 23, 2001
OR36-4    OR44-3                   OR53-4
OR36-6    OR46-5
OR39-3    OR50-4
OR39-4    OR50-5
OR41-4    OR50-6
OR42-1    OR51-1
OR42-4    OR51-3
OR42-5    OR51-4

                         - 309 -
INDEX OF ABSTRACTS BY AUTHOR
      Lead Author               Abstract #

      Abad, V                   P1-508
      Ackerman, AL              P3-46
      Alam, S                   P2-579
      Alzubaidi, NH             P3-684
      Ariyasu, H                P2-199
      Arnett-Mansfield, RL      P1-227
      Arsenis, G                P2-557
      Asa, SL                   OR36-6
      Ayala, AR                 P2-150
      Bamberger, AM             OR50-4
      Barsony J                 OR29-1
      Berg, AH                  S38-2
      Berga, SL                 P2-400
      Best, SE                  OR10-1
      Boling, E                 P1-464
      Borer, KT                 P2-215
      Bramlett, KS              P3-663
      Brown-Borg, HM            P1-418
      Brumm, H                  P3-152
      Buxton OM                 OR28-2
      Cesen-Cummings, K         P2-630
      Chang, C                  OR4-5
      Cheung, E                 OR2-5
      Claudi-Boehm, S           P2-397
      Dalais, F                 P3-143
      Davies, MJ                P2-564
      de Vet, A                 P1-437
      Di Somma, C               OR22-6
      Ding, D                   P2-30
      Dolney, RR                P2-157
      Dumesic, DA               OR10-5
      Elsasser, TH              P1-203
      Falasca, P                P2-462
      Fiory, F                  OR53-4
      Fitzpatrick, LA           P1-436
      Foulstone, EJ             P1-198

                      - 311 -
Lead Author               Abstract #

Fukata, S                 P3-157
Funk, JL                  P1-503
Funkhouser, JM            P2-162
Gaertner, R               P3-211
Galusca, B                P1-470
Gao, X                    P1-241
Gasnier, B                P3-208
Gehrke, GM                P1-296
Gillberg, P               OR42-4
Giustina, A               P3-389
Gore, AC                  P3-473
Griffin, LD               OR41-4
Haddad, L                 P3-553
Haddad, L                 P3-555
Hadigan, C                OR20-1
Hadigan, C                P1-319
Hansen, TK                OR51-4
Heine, PA                 P3-110
Hellstrom, A              OR44-3
Hoersch, D                OR12-4
Hofbauer, LC              P1-496
Hoiden-Guthenberg, I      P2-601
Hotta, M                  OR42-1
Ibanez, L                 P2-463
Innerfield, R             P2-559
Jakubowicz, DJ            P2-427
Jan de Beur, SM           OR29-5
Johannsson, G             OR46-5
John, MR                  OR29-6
Kim, D                    P1-598
King, SR                  OR7-6
Koppanati, BM             P1-576
Labrie, F                 P1-263
Lanzino, M                P1-246
Lazar, MA                 S38-1
Levine, AC                P1-253
Leyendecker, G            OR10-4



                - 312 -
Lead Author               Abstract #

Lindsay, R                S20-2
Liu, PY                   S20-3
Lu, LJW                   P3-141
Lund, TD                  P3-306
Marcell, TJ               OR24-6
Mattarello, MJ            P1-573
McCaig, C                 P2-262
McGrath, S                OR10-6
Mei, J                    P3-139
Meier, C                  P3-167
Miller, KK                OR34-5
Miller, KK                P2-468
Miner, JN                 OR18-4
Mirshahi, M               P2-66
Miyakoshi, N              OR36-4
Mogul, HR                 P1-566
MohanKumar, SM            P1-419
Morash, BA                P2-138
Nakai, Y                  P3-299
Narotsky, MG              P2-356
Neele, SJ                 P3-318
Nye, EJ                   P1-571
Paczoska-Eliasiewicz, H   P2-389
Paris, F                  P2-377
Perez-Luque, EL           P2-360
Peters, C                 P2-660
Prendiville, MT           P2-668
Pugh, PJ                  P1-310
Puxeddu, E                OR50-6
Raff, H                   OR21-1
Rao, CV                   OR50-5
Richer, JK                OR13-2
Risek, B                  P1-497
Robinson, BR              P1-352
Ronkin, S                 P3-145
Sakaris, A                P1-445
Sanchez, RG               OR35-2


               - 313 -
Lead Author              Abstract #
Sasson, R                OR39-4
Scheller, A              P2-502
Schiff, R                OR16-4
Schlegel, PN             P2-498
Scholz, GH               P1-561
Senkus Melvin, VM        OR31-6
Shoemaker, RC            P1-567
Siegel, L                P1-321
Singh, A                 OR11-3
Smith, LE                P1-174
Soyka, LA                P3-47
Ste-Marie, LG            OR42-5
Stewart, C               OR27-6
Stoffers, DA             P1-368
Tannirandorn, P          P2-533
Teede, HJ                S20-4
Tomlinson, JW            P1-381
Toorians, AW             OR32-1
Tritos, NA               P3-102
Tuttle, RL               P1-276
Valencia, AL             P2-404
Valenti, S               P2-410
Van den Berghe, G        S20-1
Vasquez, DM              P3-194
Venihaki, M              OR51-1
Vora, AC                 P3-685
Watts, NB                P3-144
Wex, H                   P3-396
Wimalawansa, SJ          OR24-1
Wimalawansa, SJ          P1-438
Wong, CK                 P3-380
Wood, JR                 OR39-3
Worster, TA              P2-422
Xu, W                    P1-654
Yellayi, S               OR51-3
Yildiz, BO               OR32-4
Yoshida, S               P2-599



               - 314 -
                             CORPORATE RESEARCH INDEX

Corporation/Foundation Name                                              Abstract #
Abbott Laboratories                                                           OR18-4
American Diabetes Association                                                 S38-2, P1-368
American Federation of Aging Research                                         P1-418
American Institute for Cancer Research                                        P3-141
Animal Health and Disease Research Funds, University of Illinois              P3-110
Ara Parseghian Medical Research Foundation                                    OR41-4
Association for International Cancer Research (AICR)                          OR27-6, P1-198, P2-262
Aurora Health Care, Mayo Clinic, Rochester, Minn                              OR21-1
Aventis Pharma, Bridgewater, NJ                                               P1-464, P3-144
Belgian Foundation for Research in Congenital Heart Diseases                  S20-1
Belgian Fund for Scientific Research                                          S20-1
biosyn – Arzneimittel GmbH, Germany                                           P3-208
Boehringer Mannheim, Germany                                                  P2-660
British Heart Foundation                                                      P2-579
Burroughs Wellcome Fund                                                       OR2-5
BYU Dean's Fellowship in Neuroscience                                         P3-306
Canadian Diabetes Association                                                 P2-138
Claude D. Pepper Older American Independence Center for Aging Research        OR24-6
Clinical Nutrition Research Center at Harvard.                                OR20-1
CNRS, Paris, France                                                           P2-66
CONACTY                                                                       P2-360, P2-404
Dalhousie University Internal Medicine Research Foundation                    P2-138
Danish Medical Research Council                                               OR51-4
Deutsche Forschungsgemeinschaft                                               OR29-6, OR12-4
Eleanor Roosevelt Institute                                                   P1-352
Eli Lilly and Company                                                         S20-2, P3-663, OR42-5
Eli Lilly-Sesto Fiorentino, Italy                                             P3-389
Endorecherche.                                                                P1-263
ETZ-M                                                                         P1-561
Fondazione Maugeri of Gussago, Italy                                          P3-389
Frimurare-Barnhusdirektionen                                                  OR44-3
Genentech Foundation for Growth and Development                               P3-47
Genentech, Inc.                                                               OR34-5
German Research Council                                                       P2-397
German Society of Endocrinology                                               P3-152
Gilson Longenbaugh Foundation and Alzheimer's Association                     OR7-6
Göteborg Medical Society                                                      OR44-3
Gothenburg University Research Fund                                           OR46-5
Hamamelis Trust                                                               P3-380
                                         - 315 -
Hebrew University Faculty of Agriculture                                     OR39-4
Howard Hughes Medical Institute                                              P1-276
Illinois Council on Food and Agricultural Research (C-FAR).                  OR51-3
Inserm                                                                       P2-66
Italian Association for Cancer Research (AIRC)                               OR53-4
Italian Ministry of University and Scientific Research                       OR53-4
IWK Grace Research Foundation.                                               P2-138
JDRF Center for the Study for the Complications in Diabetes                  P1-296
John Sealy Memorial Endowment                                                P3-141
Juvenile Diabetes Foundation                                                 P1-368
Kuma Hospital, Kobe, Japan                                                   P3-157
Levin Center for applied research at the Weizmann Institute of Science       OR39-4
Ligand Pharmaceuticals                                                       P1-497, OR18-4
Lis Maternity Hospital, Tel Aviv Medical Center.                             OR39-4
Lisa and Johan Grönbergs Foundation                                          P2-601
Magdeburger Förderkreis krebskranker Kinder e.V., Germany                    P3-396
Maine Medical Center Medical Staff Research Committee                        P2-422
Maine Medical Center Research Institute                                      P2-422
March of Dimes                                                               OR1-4
Mason‘s Medical Research Foundation                                          P3-380
Medical Research Council (UK)                                                P1-381, P3-553, P3-555
Medical Research Council of Canada/Canadian Institutes for Health Research   OR50-4, OR36-6
Merck Pharmaceuticals                                                        P1-438
Michigan Diabetes Research Training Consortium                               P2-502
MURST (Italian University funding source)                                    P1-573
Naomi Berrie Diabetes Center at Columbia Presbyterian, New York              P1-321
National Eye Institute (LEHS)                                                P1-174
National Foundation for Cancer Research                                      OR13-2
National Health and Medical Research Council of Australia                    P1-227, P1-571, S20-3
National Heart Foundation of Australia                                       S20-4, P3-143
National Neimann-Pick Foundation                                             OR41-4
National Research Initiative Grant Program                                   P1-576
National Science Foundation                                                  P3-306
Netherlands Organization for Scientific Research                             OR32-1
Novo Nordisk, Denmark                                                        S20-1
Oklahoma Center for Advancement of Science and Technology                    P1-352
Oklahoma Medical Research Foundation                                         P1-352
Osteoporosis and Endocrinology Fund of the University of Hong Kong           P3-139
Pharmacia                                                                    OR27-6
Pharmacia & Upjohn                                                           OR42-4
Pharmacia AB, Sweden                                                         P2-601
Procter & Gamble Pharmaceuticals, Cincinnati, OH                             P1-464, P3-144
Research Council of the University of Leuven                                 S20-1

                                           - 316 -
Royal College of Surgeons Research Fellowship                                   P3-380
Serono Australia                                                                S20-3
Sheffield Hospitals Charitable Trust, UK                                        P1-310
Smoking Research Foundation                                                     P3-299
Society for the Prevention of Cancer, Northrhine-Westfalia (NRW), Germany       P2-660
SPORE in Breast Cancer (Clinical Trial)                                         OR16-4
Stichting Voortplantingsgeneeskunde Rotterdam                                   P1-437
Susan G. Komen Breast Cancer Foundation                                         OR2-5, OR13-2
Swedish Foundation for International Cooperation in Research and Higher Educ.   OR29-6
Swedish Medical Research Council                                                OR44-3
Swedish Medical Research Foundation                                             P2-601
Swiss Research Foundation                                                       P3-167
T.J. Martell Foundation for Cancer, Leukemia and AIDS Research                  P1-253
Telethon                                                                        OR53-4
University Hospital, Basel, Switzerland                                         P3-167
University of Bristol                                                           OR27-6
University of Genova, Italy                                                     P2-410
University of Leipzig, Germany                                                  P1-561
University of Michigan Comprehensive Cancer Center                              P2-502
University of Michigan School of Medicine Clinical Research Partnership         P2-215
University of Pennsylvania School of Medicine                                   P2-533
Uppsala University Research Fund                                                OR46-5
Utah State University Agricultural Experiment Station                           P1-576
V. Kann Rasmussen Foundation                                                    P1-174
Vincent Memorial Research Funds                                                 P2-377
W.A. Drenckmann Stiftung, Germany                                               P3-396
Weill Medical College, Dept. of Urology                                         P2-498
Westmead Millennium Foundation                                                  P1-227
Wyeth-Ayerst Research                                                           P3-145




                                           - 317 -

				
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