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Salt intake and insulin sensitivity in healthy human volunteers

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Salt intake and insulin sensitivity in healthy human volunteers Powered By Docstoc
					                                                         Clinical Science (2007) 113, 141–148 (Printed in Great Britain) doi:10.1042/CS20060361         141




                                     Salt intake and insulin sensitivity in
                                               healthy human volunteers

Raymond R. TOWNSEND∗ , Shiv KAPOOR∗ and Christopher B. MCFADDEN†
∗
 Department of Medicine, University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104, U.S.A., and †Cooper
Medical Center, University of Medicine and Dentistry of New Jersey, One Cooper Plaza, Camden, NJ 08103, U.S.A.


A     B     S     T     R     A      C     T

          The literature on salt intake and insulin sensitivity presents a mixed picture, as some studies have
          shown an increase, whereas others have shown a decrease, in insulin action as sodium intake is
          enhanced. In some cases, this may relate to the study of salt intake in patients with co-morbidities
          such as hypertension or diabetes. In the present study, we selected healthy normotensive lean
          volunteers who underwent a euglycaemic clamp following 6 days of a low-salt diet (20 mmol
          sodium daily) and, subsequently, 6 days of a high-salt diet (200 mmol sodium daily). Our results
          show an increase in insulin-mediated glucose disposal during euglycaemic clamp conditions that
          was significantly higher following the high-salt diet compared with the low-salt diet (7.41 + 0.41
                                                                                                        −
          compared with 6.11 + 0.40 mg · kg−1 of body weight · min−1 respectively; P = 0.03). We measured
                                −
          calf blood flow before and during insulin infusion (no significant change after the two dietary salt
          interventions was detected) and plasma non-esterified fatty acids (also no significant differences
          were detected). We observed the expected increases in renin concentration and aldosterone
          activity in subjects on the low-salt diet, and also observed a significantly less increase in plasma
          noradrenaline concentration during euglycaemic insulin infusion following the high-salt compared
          with the low-salt diet. We propose that the 4–5-fold increase in serum aldosterone and the
          greater increase in plasma noradrenaline concentration following the low-salt intervention
          compared with the high-salt period may have contributed to the differences in insulin sensitivity
          following the adjustment in dietary sodium intake.




INTRODUCTION                                                        insulin-mediated glucose uptake is reduced (i.e. insulin
                                                                    resistance), more insulin secretion is usually required.
Each meal ingestion initiates an integrated series of                  The pathogenesis of insulin resistance is complex and
events, which facilitate digestion, delivery and storage of         only partially understood. We have shown previously [3]
nutrients in target tissues. The haemodynamic and meta-             a relationship between plasma renin activity and insulin-
bolic effects of insulin facilitate this process. The pivotal       stimulated glucose uptake measured during euglycaemic
role of insulin in nutrient storage stems from its ability          clamp conditions in normal volunteers. As plasma renin
to both stimulate cardiac output, thereby increasing                activity increased, insulin sensitivity fell [3], a finding
blood flow to [1] and transition through [2] the vascul-             also observed in hypertensive patients [4]. Alterations in
ature of insulin-sensitive target tissues such as skeletal          salt intake directly influence plasma renin activity, with
muscle, and to direct nutrient uptake (principally                  greater salt intake resulting in lower renin activity. Many
glucose and fatty acids). When the responsiveness of                of the studies over the last 2 decades, as summarized in
insulin-sensitive target tissues such as skeletal muscle to         Table 1, have pursued relationships between salt intake



Key words: calf blood flow, glucose disposal, insulin sensitivity, salt intake, sympathetic nervous system.
Abbreviations: BMI, body mass index; BP, blood pressure; CV, coefficient of variation; GCRC, General Clinical Research Center;
NEFA, non-esterified fatty acid; OGTT, oral glucose tolerance test; REE, resting energy expenditure.
Correspondence: Dr Raymond R. Townsend (email townsend@mail.med.upenn.edu).


                                                                                   C   The Authors Journal compilation   C   2007 Biochemical Society
142   R. R. Townsend, S. Kapoor and C. B. McFadden


      Table 1 Summary of studies on insulin sensitivity and sodium intake
      HOMA, homoeostatic model assessment; ↓, decrease; ↑, increase; →, no change.

                                                      Procedure used to assess       Insulin sensitivity response
      Author [reference]           Sample size (n)    insulin sensitivity            to salt administration       Comments

      Donovan et al. [5]             8∗               Euglycaemic clamp              ↓                          −
      Sharma et al. [6]             23∗               OGTT                           → or ↑                     Results differed by salt sensitivity of BP (→ = salt-resistant)
      Foo et al. [7]                18                Graded euglycaemic clamp       →                          −
      Facchini et al. [8]           19                3 h insulin-suppression test   →                          −
      Iwaoka et al. [9]             15                OGTT                           ↑                          All hypertensive
      Raji et al. [10]             426                HOMA                           → or ↑                     Depended on the nuances of renal blood flow responses to
                                                                                                                    angiotensin II (modulators compared with non-modulators)
      Melander et al. [29]          28                Euglycaemic clamp              ↓ or ↑                     Results varied by gender and salt sensitivity; salt-sensitive
                                                                                                                    subjects improved insulin sensitivity in higher salt intake;
                                                                                                                    salt-resistant subjects worsened
      Perry et al. [30]             15∗               Euglycaemic clamp              ↑                          −
          ∗ No   women included.




      and insulin sensitivity, with some studies suggesting salt                                 MATERIALS AND METHODS
      restriction increases insulin sensitivity [5], some finding a
      mixed result or no change in insulin sensitivity [6–8] and                                 All procedures were reviewed and approved by the Insti-
      other studies suggesting salt restriction reduces insulin                                  tutional Review Board of the University of Pennsylvania,
      sensitivity in hypertension [9,10]. Moreover, public                                       and all subjects provided written informed consent. The
      health initiatives that recommend reduced salt intake                                      protocol was also reviewed by the GCRC (General Clin-
      [11] often do not distinguish between those in whom                                        ical Research Center) Scientific Advisory Committee.
      clinical evidence implicates an increase in salt intake
      as injurious (such as those with hypertension or heart                                     Selection of subjects
      failure) and those without such co-morbidities where                                       Normotensive men and women, within 20 % of an ideal
      salt restriction could adversely affect parameters, such                                   body weight as defined by data from the Metropolitan
      as insulin action, without clear cardiovascular benefit                                     Life Insurance Company [14], of at least 18 years of age
      [12]. Thus information on the effects of salt restriction in                               were recruited by local advertisement and word of mouth.
      healthy individuals are needed to help keep public health                                  There was no ethnic exclusion. At enrolment, each subject
      policy on this issue well-informed.                                                        read and signed an informed consent form, and then
          In the present study, we measured insulin sensitivity                                  underwent a standard medical history and physical
      following 1 week of isocaloric nutrient intake on a high-                                  examination, 12-lead ECG and routine measures of
      salt diet compared with a low-salt diet. We examined                                       blood and urine chemistries, including urine toxicology
      three mechanisms that could potentially be responsible                                     for illegal substances. In addition, each subject also under-
      for changes induced by varying salt intake on insulin                                      went a standard 3-h OGTT (oral glucose tolerance test)
      sensitivity in healthy human subject volunteers, including                                 with 75 g of oral glucose [15] to exclude those with
      measures of (i) skeletal muscle blood flow, (ii) plasma                                     previously undiagnosed diabetes. Subjects with hyper-
      NEFA (non-esterified fatty acid) concentration, and                                         tension (> 140/> 90 mmHg) were excluded from the
      (iii) plasma catecholamine metabolism before and dur-                                      study. Subjects taking prescription medication for dia-
      ing hyperinsulinaemic euglycaemia. In addition, the                                        betes, thyroid disease, hypertension or sex-hormone
      effects of dietary salt manipulation on seated BP (blood                                   replacement were excluded. Subjects having laboratory
      pressure) and heart rate (on day 6 of the diet) and fasting                                values greater than twice the upper limit of normal were
      substrate oxidation, using indirect calorimetry during the                                 also excluded. Subjects with illegal substances present in
      clamp procedure, were determined. Given the growing                                        the urine would have been excluded; however, this did
      importance of the metabolic syndrome, whose pivotal                                        not occur in our screened subject population.
      finding is insulin resistance, as a risk for future cardio-
      vascular disease [13], we consider that the findings of                                     Study protocol
      studies such as this one would contribute to the body                                      An outline of the main study design is shown in Figure 1.
      of knowledge from which dietary recommendations are                                        Three euglycaemic clamp procedures were performed on
      drawn.                                                                                     each subject in the GCRC. The first clamp was performed


      C   The Authors Journal compilation       C    2007 Biochemical Society
                                                                                                          Salt intake and insulin sensitivity   143



                                                              for safety surveillance. One investigator measured seated
                                                              BPs on day 6 at 14.00 hours (obtained in duplicate and
                                                              averaged for a single systolic and diastolic value for each
                                                              subject) and also obtained a single measurement of the
                                                              heart rate of each subject. Urines samples (24 h) were
                                                              collected on days 6–7 for sodium and urine urea nitrogen
                                                              excretion.

                                                              Euglycaemic clamp procedure
                                                              The euglycaemic clamp and indirect calorimetry studies
                                                              were performed in the GCRC, as described previously
                                                              [3,16]. For the first euglycaemic clamp, subjects were
                                                              admitted to the GCRC between 17.00–18.00 hours on
                                                              the evening prior to the study. A 12-h urine collection
                                                              was obtained (18.00–06.00 hours) to estimate protein
                                                              oxidation from urea nitrogen excretion. During the 1-
                                                              week-long stay, 24-h urine samples were collected from
                                                              06.00 hours on the morning of day 6 to 06.00 hours on day
                                                              7 to determine sodium and urine urea nitrogen excretion.
                                                                 Table 2 shows the timing of the infusions, samples
Figure 1 Flow diagram detailing enrolment, study design,      and procedures during each euglycaemic clamp study. At
randomization and subject study completion                    06.30 hours on the day of the clamp study, an intravenous
Subj., subjects.                                              catheter was inserted into each arm. One catheter was
                                                              placed in the hand or wrist, and warmed to 55–60 ◦ C (to
after a single overnight fast and was incorporated into       ‘arterialize’ the blood [17]) from which all blood sampling
the protocol to familiarize the subject with the complex      was performed. The other catheter was placed in the
procedures undertaken during the clamp, as done by            opposite forearm and was used for all infusions. Subjects
others studying the effects of salt on insulin sensitivity    rested for approx. 30 min before any studies were
[7]. Moreover, this normal condition clamp allowed            initiated.
the unmanipulated insulin sensitivity of the subject             At 07.00 hours, a ‘blank’ blood sample for background
to be estimated. Women in this protocol underwent             deuterated glucose enrichment was obtained and was
each euglycaemic clamp within 10 days of the onset of         followed by a primed (4 mg/kg of body weight) and then
menses to minimize the effect cyclic hormonal changes         continuous (50 µg · kg−1 of body weight · min−1 ) infusion
have on insulin sensitivity. During the first admission        of [2 H6 ]glucose to determine the rate of appearance of
to the GCRC, each subject was interviewed by the              glucose as an estimate of hepatic glucose output.
GCRC dietician to ascertain caloric needs and to record          At 08.00 hours, samples for plasma renin and serum
food preferences to craft a palatable diet of 20 mmol         aldosterone concentrations were obtained to demonstrate
sodium/day. Subjects were re-admitted approx. 4 weeks         any physiological effects of the dietary salt manipulation.
later and randomized [using a pre-specified randomized         Plasma for adiponectin and resistin concentrations were
blocked (block = ten subjects) table generated by the         also collected at this time.
GCRC biostatistician and kept by the dietician] to               At 08.30 hours on the morning of clamp study, blood
receive the individualized 20 mmol sodium/day diet with       was sampled every 10 min to demonstrate stable whole-
either placebo tablets (ten per day; low-salt diet) or 1-g    blood glucose concentrations, by using the glucose oxi-
NaCl tablets (ten per day; high-salt diet). The tablets       dase method on a glucose analyser (YSI 2300; Yellow
were divided across three meals for the six consecutive       Springs). At the same time, expired air was collected
days. The diet was the same during each of the 1-             into an indirect calorimeter for 10 min via a canopy
week-long stays; the difference was limited to the NaCl       method to determine substrate oxidation. Expired air was
component administered as a supplement. If the subject        collected by a canopy system connected to a DeltaTrac
was randomized to the high-salt diet first, they underwent     II calorimeter (Sensormedic) for determination of CO2
a euglycaemic clamp on the morning of day 7 in the            production and O2 usage 30 min before insulin adminis-
GCRC (following six full days of the diet), and were re-      tration and during the last hour of the euglycaemic clamp
admitted approx. 4 weeks later. At that time, they received   study. Expired air was analysed at 60 s intervals for a total
the low-salt diet for 6 days, again undergoing the final       of 9–10 min, and was averaged for each measurement.
euglycaemic clamp on day 7 of that GCRC stay. Subjects           At 08.45 hours, calf blood flow was determined (five
were weighed daily. Vital signs were recorded at 10.00,       measurements; averaged for a single value) using supine
14.00 and 16.00 hours as per the usual GCRC protocol          calf strain-gauge plethysmography (Hokansen) [18].


                                                                          C   The Authors Journal compilation   C   2007 Biochemical Society
144   R. R. Townsend, S. Kapoor and C. B. McFadden


      Table 2 Time and events during the euglycaemic clamp study
                                                                       √
      The shading indicates an infusion during the time period. Each in a row indicates that a sample was drawn at that time. FFA, NEFAs; 15 % DEX, 15 % dextrose
      in water with 20 mmol/l KCl; ALDO, serum aldosterone concentration; NOREPI, plasma noradrenaline; Ind Cal, indirect calorimetry; QBf, calf blood flow measurement;
      ∗
        GLU, whole blood glucose (obtained at 10 min intervals from 07.00 to 09.00 hours, and sampled at 5 min intervals from 09.00 until 12.00 hours).




         At 08.55 hours and again at 09.00 hours, blood was                             Diagnostic). Aldosterone was determined by an RIA kit
      sampled to determine enrichment with [2 H6 ]glucose and                           (Diagnostic Products). The CVs (coefficients of variation)
      plasma NEFA concentrations.                                                       for both renin and aldosterone were < 5 and 7 % re-
         At 09.00 hours, samples for plasma noradrenaline and                           spectively. Plasma catecholamines were extracted using a
      insulin concentrations were drawn, which were repeated                            Bio-Rad Laboratories kit and were determined by HPLC
      periodically as shown in Table 2. At 09.00 hours, a                               using an EC 1640 detector (Bio-Rad Laboratories). The
      euglycaemic clamp was started [3,16]. A priming bolus                             CV for the catecholamines assay was < 8 %. All of the
      of normal human insulin (approx. 0.5 unit adjusted based                          assays were performed in the GCRC Core Laboratory.
      on body weight; Humulin-R; Eli Lilly) was followed by a                              Plasma insulin levels were determined by RIA
      constant infusion at a dose of 40 milli-units · m−2 · min−1                       (INCSTAR), plasma NEFAs were determined using a
      for 3 h (from 09.00 to 12.00 hours). Whole-blood glucose                          standard kit (Wako Bioproducts), plasma adiponectin
      concentrations were determined every 5 min, and a 15 %                            concentrations were determined by RIA, and plasma
      dextrose in 20 mmol/l KCl was administered at a rate suf-                         resistin concentrations were determined by ELISA.
      ficient to maintain whole-blood glucose concentrations at                          These assays were performed in the Diabetes and
      the average level observed between 08.30 and 09.00 hours                          Endocrine Research Center (University of Pennsylvania,
      (typically between 75 and 85 mg/dl; 4.2 and 4.7 mmol/l).                          Philadelphia, PA, U.S.A.).
         At 11.00 hours, the indirect calorimetry data were                                Indirect calorimetry data were analysed for carbohyd-
      repeated. At 11.15 hours, calf blood flow measurements                             rate and lipid substrate oxidation using formulae de-
      were repeated. From approx. 11.20 hours until insulin                             scribed previously [21].
      infusion was completed, the subject was left as undis-
      turbed as possible, so that the average glucose uptake                            Statistical analysis
      during the final 30 min of the 3-h insulin infusion repre-                         Values are expressed as means + S.E.M., and data were
                                                                                                                         −
      sented the target period.                                                         analysed using STATA v9.2. Differences in variables mea-
         Following 3 h of insulin infusion, all infusion solu-                          sured during euglycaemic clamps following the low-salt
      tions, except dextrose, were discontinued. Subjects ate a                         and high-salt diets were compared with a paired Student’s
      normal lunch, and the dextrose infusion tapered and dis-                          t test. Glucose uptake for each of the last three 10-min
      continued between 12.00 and 13.00 hours.                                          periods during insulin infusion was averaged for each
                                                                                        subject during each euglycaemic clamp and was used as
      Analyses and calculations                                                         their glucose uptake value for that dietary salt phase. A
      Urinary sodium was determined using a flame photo-                                 Mann–Whitney test was performed if the data failed an
      meter (IL-943; Instrumentation Laboratory). Glucose                               equal variance test. Two-tailed P values were used for the
      enrichment was determined using GC/MS [19] by Meta-                               Student’s t tests, with P values < 0.05 considered to be
      bolic Solutions, and the calculations for the rate of glucose                     statistically significant.
      appearance were performed using the Steele equation                                  We proposed a 15 % difference in glucose uptake as
      for steady state [20]. Active renin was determined                                potentially clinically significant, and determined that a
      using an immunoradiometric assay kit (Nichols Institute                           sample size of 20 subjects using an S.D. of 1.8 mg · kg−1 of


      C   The Authors Journal compilation   C   2007 Biochemical Society
                                                                                                                                       Salt intake and insulin sensitivity   145


Table 3 Demographics of the study subjects
Values are means + S.E.M.
                 −
                                              African American                        Caucasian

Characteristic           All subjects         Women               Men                 Women                    Men                    Asian men            Latino women

Subjects (n)             20                    4                   6                   3                        5                      1                    1
Age (years)               30 + 2
                              −                31 + 5
                                                   −               33 + 4
                                                                       −               26 + 4
                                                                                           −                    32 + 3
                                                                                                                    −                 28                   23
BMI (kg/m2 )             23.1 + 0.6
                              −               24.1 + 1.4
                                                   −              24.5 + 0.6
                                                                       −              23.1 + 0.2
                                                                                           −                   22.7 + 1.6
                                                                                                                    −                 19.1                 20.2
Glucose (mg/dl)           75 + 3
                              −                73 + 5
                                                   −               78 + 5
                                                                       −               70 + 6
                                                                                           −                    81 + 2
                                                                                                                    −                 63                   67


Table 4 Blood flow and indirect calorimetry before and during insulin infusion
Values are means + S.E.M. 1 kcal ≈ 4.184 J.
                 −
                                                            Low-salt diet                                            High-salt diet

Parameter                                                   Pre-insulin infusion     During insulin infusion         Pre-insulin infusion          During insulin infusion

Calf blood flow (cm3 · 100 g−1 of leg weight · min−1 )        2.3 + 0.2
                                                                 −                    2.8 + 0.3
                                                                                          −                           2.4 + 0.2
                                                                                                                          −                         2.8 + 0.2
                                                                                                                                                        −
REE (kcal · kg−1 of body weight · h−1 )                     22.7 + 1.2
                                                                 −                   24.2 + 0.9
                                                                                          −                          22.1 + 0.8
                                                                                                                          −                        23.1 + 0.8
                                                                                                                                                        −
Glucose oxidation (mg · kg−1 of body weight · min−1 )        0.8 + 0.3
                                                                 −                    2.4 + 0.4
                                                                                          −                           1.3 + 0.4
                                                                                                                          −                         3.2 + 0.5
                                                                                                                                                        −
Fat oxidation (mg · kg−1 of body weight · min−1 )            1.4 + 0.2
                                                                 −                    0.8 + 0.2
                                                                                          −                           1.1 + 0.2
                                                                                                                          −                         0.4 + 0.2
                                                                                                                                                        −


body weight · min−1 (based on our previous clamp studies                           although higher in subjects on the high-salt diet, was not
in normal volunteers [22]) would provide 80 % power to                             significantly different (P = 0.64) when compared with
detect a difference between the two interventions.                                 the low-salt dietary intervention.

                                                                                   Calorimetry and regional (calf) blood flow
RESULTS                                                                            Fasting substrate oxidation and calf blood flow on the
                                                                                   morning of the euglycaemic clamp before and following
The demographics of the study subjects are shown in                                2 h of euglycaemic insulin infusion are shown in Table 4.
Table 3. There were eight women and 12 men evaluated                               There were no significant differences in calf blood flow
in the protocol. Two subjects (both women) completed                               between the low-salt and high-salt diets before (P = 0.89)
only one of the two dietary phases. In both cases, this                            or during (P = 0.86) insulin infusion. Calf blood flow
was during the low-salt phase. One of the two subjects                             increased significantly during insulin infusion compared
required multiple attempts to re-start the blood sampling                          with the value obtained before insulin infusion began
line during the second euglycaemic clamp and declined                              (P < 0.03 for both low-salt and high-salt diets before com-
to return for the last intervention. The other subject                             pared with after insulin infusion on that particular diet).
moved out of the area shortly after completing the                                    Glucose oxidation was higher following the high-salt
second euglycaemic clamp.                                                          diet, but the results were not statistically significant
                                                                                   (P = 0.23). Similarly, lipid oxidation was lower following
BP, heart rate and weight changes on day                                           the high-salt diet compared with the low-salt diet, but not
6 after each dietary intervention                                                  significantly so (P = 0.27). REE (resting energy expendi-
Seated BPs at 14.00 hours on day 6 in subjects on the                              ture) was similar between the groups (P = 0.66), and the
low-salt diet were 111 + 2/65 + 2 mmHg, and seated BPs                             changes in REE and substrate oxidation during the eugly-
                       −       −
in the afternoon on day 6 in subjects on the high-salt                             caemic clamp were similar following the high-salt and
diet were 117 + 4/69 + 3 mmHg (P = 0.16 and P = 0.33                               low-salt diets. Difficulties with instrument calibration
               −      −
for changes in systolic and diastolic BPs respectively, in                         resulted in oxidation measurements only being obtained
subjects on the low-salt diet compared with the high-salt                          in 15 out of the 20 subjects.
diet). Seated heart rates in the afternoon on day 6 in
subjects on the low-salt and high-salt diets were 72 + 2                           Urine sodium, hormonal markers of
                                                      −
and 67 + 2 beats/min respectively (P = 0.10).
        −
                                                                                   sodium balance, rate of glucose
   The average weight on the morning of day 7 after                                appearance, cytokines and plasma NEFAs
6 days on the low-salt diet was 67.9 + 2.5 kg compared
                                      −                                            Urine sodium excretion, as per the protocol, averaged
with an average weight following 6 days on the high-salt                           23 + 4 and 194 + 12 mmol/day in subjects on the low-
                                                                                      −           −
diet of 69.6 + 2.4 kg. The average weight on day 7,
              −                                                                    salt and high-salt diets respectively. The low-salt diet


                                                                                                   C   The Authors Journal compilation       C   2007 Biochemical Society
146   R. R. Townsend, S. Kapoor and C. B. McFadden


      was associated with significant increases in renin (33 + 5
                                                              −
      compared with 16 + 2 µ-units/l in the high-salt diet;
                           −
      P < 0.001) and aldosterone (15.9 + 2.6 compared with
                                           −
      3.5 + 0.7 ng/dl in the high-salt diet; P < 0.03) concen-
          −
      trations. Simple pairwise correlations were performed
      using serum aldosterone concentrations in the low-salt
      and high-salt diets to compare the levels with the glucose
      uptake value for each subject. In the low-salt group, this
      pairwise correlation gave an r value of 0.36 (P = 0.17),
      whereas, in the high-salt group, an r value of − 0.02
      (P = 0.94) was obtained. Plasma adiponectin concentra-
      tions were 7.5 + 0.6 and 6.7 + 0.8 µg/ml in subjects on
                      −             −
      the low-salt and high-salt diets respectively (P = 0.42),
      and plasma resistin concentrations were 8.3 + 0.7 and
                                                       −
      6.8 + 0.7 ng/ml in subjects on the low-salt and high-
          −
      salt diets respectively (P = 0.14). The rate of glucose
      appearance was 1.9 + 0.01 and 2.1 + 0.9 mg · kg−1 of body
                          −              −
      weight · min−1 respectively, in subjects on the low-salt
      and high-salt diets (P = 0.13). Plasma NEFA concentra-
      tions were 0.51 + 0.22 and 0.43 + 0.15 mmol/l in subjects
                      −               −
      on the low-salt and high-salt diets respectively (P = 0.23).

      Euglycaemic clamp
      Blood glucose (Figure 2A) and insulin concentrations
      were similar during the clamps performed following the
      low-salt and high-salt diets. There were no differences in
      the insulin concentrations averaged over the last 30 min
      in the high-salt diet compared with the low-salt diet (P =
      0.82; Figure 2B). In the case of one subject (subject 3),
      the samples stored for the determination of the insulin
      concentrations (from all three clamp studies) were
      discarded accidentally before being assayed.
         Glucose uptake was significantly greater following the
      high-salt diet compared with the low-salt diet (average
      from 150–180 min was 7.41 + 0.41 compared with 6.11 +
                                   −                           −
      0.40 mg · kg−1 of body weight · min−1 respectively; P =
      0.03; Figure 2C). When the subjects were divided accord-
      ing to their BP response to the dietary intervention
      on day 6, half of the subjects were salt-sensitive (‘salt-
                                                                           Figure 2 Whole-blood glucose and insulin levels, and glu-
      sensitive’ was defined as an increase in BP of at least
                                                                           cose uptake during the euglycaemic clamp, in the low-salt
      3 mmHg). There were no significant differences in the
                                                                           ( ) and high-salt ( ) diets
      increase in the glucose uptake value in salt-resistant
                                                                           (A) Whole-blood glucose levels during the euglycaemic clamp before and after
      subjects compared with salt-sensitive subjects following
                                                                           insulin infusion during the diet interventions. (B) Plasma insulin levels during
      the high-salt and low-salt diets (results not shown).
                                                                           euglycaemic clamp conditions on the two diet interventions. (C) Glucose uptake
         Plasma noradrenaline concentrations were similar at
                                                                           during euglycaemic clamp conditions following the two diet interventions. Glucose
      the start of the euglycaemic clamp on day 7 of the low-salt
                                                                           uptake on the first GCRC admission (random salt intake) is shown by a broken
      and high-salt diets (198 + 18 and 173 + 13 pg/ml respect-
                               −             −                             line. M, glucose uptake.
      ively; P = 0.29). After 180 min of euglycaemic hyper-
      insulinaemia, plasma noradrenaline levels were signi-
      ficantly higher in the low-salt diet (238 + 25 pg/ml)
                                                     −                     noradrenaline and glucose uptake during the euglycaemic
      compared with the high-salt diet (181 + 19 pg/ml; P =
                                                 −                         clamps as follows. First, we calculated the slopes of the
      0.02). Assay problems were encountered when analysing                plots of hourly plasma noradrenaline concentrations over
      plasma catecholamines, such that there were only 17                  time during the two dietary salt conditions. Subsequently,
      paired samples available (0 and 180 min) for the low-salt            we calculated both the difference in slopes of the plasma
      group and 12 samples for the high-salt group. In addi-               noradrenaline concentration change over time after the
      tion, we performed simple pairwise correlations of                   two interventions and compared these slope differences


      C   The Authors Journal compilation   C   2007 Biochemical Society
                                                                                                           Salt intake and insulin sensitivity   147



with the differences in glucose uptake after the two            eight men, but was otherwise quite similar in design to
dietary salt conditions. This pairwise correlation gave an      the present study. We studied 20 subjects, both men and
r value of 0.46 (P = 0.13).                                     women, but, aside from the gender and numbers studied,
                                                                we have no additional explanation for the discordance in
                                                                the findings between the two investigations.
DISCUSSION                                                         Our results are also different from the findings of
                                                                Foo et al. [7]. Our biochemical (catecholamines, renin
The results of the present study have shown the anticip-        and aldosterone) changes were similar to those reported
ated stimulation of the renin–aldosterone axis by salt          by Foo et al. [7]; however, in their study, they observed
restriction, as well as a 21 % increase in insulin-stimulated   (unlike in the present study) an increase in calf blood
glucose uptake during euglycaemic clamp conditions              flow during the high-salt intervention, which did not
after 6 days of a high-salt diet compared with a low-salt       translate into greater glucose uptake. Foo et al. [7]
diet, which supports some findings of others [6,9,10] in         assessed insulin sensitivity using two doses of insulin
suggesting that restriction of salt intake reduces insulin      for 120 min at each dose, whereas we studied a single
sensitivity.                                                    insulin dose for 180 min. When we re-analysed our data
   Although we can only speculate on the mechanism(s)           at 120 min (see Figure 2C), we found that the differences
involved in this observation, we failed to show any sig-        in salt interventions were smaller at that point with a
nificant changes in skeletal muscle blood flow (before or         loss of statistical significance (P = 0.10). Moreover, the
during insulin infusion) or significant changes in plasma        differences in the serum aldosterone concentrations on
NEFA concentrations in subjects on the low-salt diet            the low-salt diet (mean, 15.9 ng/dl) compared with the
compared with the high-salt diet. In addition, there were       high-salt diet (mean, 3.5 ng/dl) in the present study were
no significant alterations in plasma adiponectin or resistin     approx. 4–5-fold higher in the low-salt diet compared
concentrations to assist in determining the mechanism           with approx. 2-fold in the study by Foo et al. [7].
behind the changes in insulin sensitivity. Our results are      Differences in the duration of insulin infusion and the
consistent, however, with a reduction in sympathetic            biochemical responses to salt deprivation may explain
neural activity in conjunction with a markedly lower            some of the differences between these two studies.
aldosterone concentration in the high-salt intervention,           The results of the present study are, to some extent,
which may have contributed to our observations of               in agreement with those of Melander et al. [29]. In their
insulin sensitivity in subjects on the two salt diets. These    study, a strong correlation between stimulation of the
data are consistent with our previous observations of           renin system (increased plasma renin activity and serum
an important role of the involuntary nervous system             aldosterone concentrations) and decreased insulin sensit-
in insulin sensitivity [23,24] and the known effects of         ivity was observed in the low-salt diet compared with the
aldosterone on insulin action [25,26].                          high-salt diet. No plasma catecholamine measurements
   Previous studies have shown that increases in salt           (only urine) were reported, so the present study differs
intake suppress sympathetic nervous system activity             in this respect. In their summary table, the difference in
[27]. In the present study, we observed that the plasma         glucose disposal, although higher in the high-salt intake,
noradrenaline concentration during insulin infusion             was not statistically significant (P = 0.10). It was only in
increased more during the low-salt diet compared with           the correlation of biochemical parameter changes with
the high-salt diet. Euglycaemic insulin infusions increase      insulin sensitivity that significant differences between the
plasma noradrenaline concentrations in a time-dependent         low-salt and high-salt diets were detected.
fashion [28]. Moreover, although not achieving statistical         The results of the present study are also similar in
significance (P = 0.10), the average heart rate in subjects      magnitude and direction to those reported by Perry et al.
on day 6 of the high-salt diet (66 + 2 beats/min) was lower
                                   −                            [30]. Their studies in normal volunteers demonstrated a
than the average heart rate in subjects on day 6 of the         15 % reduction in insulin sensitivity on a low-salt diet
low-salt diet (72 + 2 beats/min), consistent with reduced
                   −                                            (< 80 mmol/day) compared with an NaCl-supplemented
adrenergic activity during the high-salt intervention.          diet. In their study, a dose of insulin was used that
   Relatively recent work has shown that increased aldo-        achieved plasma concentrations approximately twice
sterone concentrations down-regulate insulin action in          those of the present study. They did not propose a mech-
target cells [26]. Interestingly, this in vitro study found a   anism to explain their results, since they were pursuing a
23 % reduction in glucose uptake when aldosterone was           role of angiotensin II on adipocyte metabolism (lipolysis
increased in the medium, similar to our 21 % difference         and glucose transport), but found no support for their
in glucose uptake during the high-salt diet compared            hypothesis that increases in angiotensin II were causative
with the low-salt diet [26].                                    in subjects on the low-salt diet.
   Our results do differ though from those of Donovan              Several limitations of our present study are important
et al. [5], who found that a high-salt diet decreased           to highlight. We studied only relatively young healthy
insulin-mediated glucose uptake. Their study enrolled           volunteers with a normal BMI (body mass index)


                                                                           C   The Authors Journal compilation   C   2007 Biochemical Society
148   R. R. Townsend, S. Kapoor and C. B. McFadden


      range, so our results are not generalizable to those                         10 Raji, A., Williams, G. H., Jeunemaitre, X. et al. (2001)
      with co-morbidities, such as diabetes or high BP, those                         Insulin resistance in hypertensives: effect of salt sensitivity,
                                                                                      renin status and sodium intake. J. Hypertens. 19, 99–105
      who are older than 45 years of age or obese subjects                         11 Food and Drug Administration (1984) Food labeling:
      (BMI > 27.5 kg/m2 ).                                                            declaration of sodium content of foods and label claims
                                                                                      for food on the basis of sodium content, final rule.
         In summary, we have observed a decrease in insulin-                          Fed. Register 49, 15.510–515.535
      mediated glucose uptake during euglycaemic clamp                             12 Controy, M. M. (2006) AMA targets sodium reduction as
      conditions in healthy volunteers given an isocaloric low-                       part of a plan to reduce cardiovascular disease
                                                                                      (http://www.medscape.com/viewarticle/536347).
      salt diet compared with a high-salt diet for 6 days. We did                  13 Reilly, M. P. and Rader, D. J. (2003) The metabolic
      not observe any significant changes in calf muscle blood                         syndrome: more than the sum of its parts? Circulation 108,
                                                                                      1546–1551
      flow or plasma NEFA concentrations, which might have                          14 Metropolitan Life Foundation (1983) Metropolitan Height
      explained this result. However, we did observe lower                            and Weight Tables, Stat. Bull. Metropol. Life Insur. Co.
                                                                                      64:21983
      sympathetic neural responses, as reflected by reduced                         15 National Diabetes Data Group. (1979) Classification and
      changes in plasma noradrenaline concentrations during                           diagnosis of diabetes mellitus and other categories of
      euglycaemic insulin infusion after the high-salt diet                           glucose intolerance. Diabetes 28, 1039–1057
                                                                                   16 Townsend, R. R. and DiPette, D. J. (1993) Pressor doses of
      compared with the low-salt diet. We also observed the                           angiotensin-II increase insulin-mediated glucose uptake in
      expected changes in serum aldosterone concentrations,                           normotensive man. Am. J. Physiol. 265, E362–E366
                                                                                   17 McGuire, E. A. H., Helderman, J. H., Tobin, J. D.,
      and propose that reductions in sympathetic neural activ-                        Andres, R. and Berman, M. (1976) Effects of arterial versus
      ation when insulin is infused after 1 week of salt loading                      venous sampling on analysis of glucose kinetics in man.
      in conjunction with a reduction in serum aldosterone                            J. Appl. Physiol. 41, 565–573
                                                                                   18 Hokanson, D. E., Sumner, D. S. and Strandness, Jr, D. E.
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                                                                                      Biomed. Eng. 22, 25–29
                                                                                   19 Guo, Z. K., Lee, W. N., Katz, J. and Bergner, A. E. (1992)
                                                                                      Quantitation of positional isomers of deuterium-labeled
      ACKNOWLEDGMENTS                                                                 glucose by gas chromatography/mass spectrometry.
                                                                                      Anal. Biochem. 204, 273–282
                                                                                   20 Steele, R., Wall, J., DeBodo, R. and Altszuler, N. (1956)
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      Health) grants M01-RR00040 and K24-DK-02684.                                    pool by the isotope dilution method. Am. J. Physiol. 187,
                                                                                      15–24
                                                                                   21 Jequier, E., Acheson, K. and Schutz, Y. (1987) Assessment
                                                                                      of energy expenditure and fuel utilization in man.
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                                                                           Received 13 December 2006/15 March 2007; accepted 10 April 2007
                                                                           Published as Immediate Publication 10 April 2007, doi:10.1042/CS20060361



      C   The Authors Journal compilation   C   2007 Biochemical Society

				
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Description: Special diet from the market to buy salt, if there is no use coarse salt. A pinch of salt bath each ankle and calf repeatedly in the massages, the intensity is appropriate to not cause pain. Each massage time is 15 to 20 minutes or so, massage the water with a high temperature soak a few minutes after the wash. If sitting with knee-deep soaking tub and massage your legs better.