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					Identification of Gene Expression
Changes in Skeletal Muscle from
Diabetic and Normal Rats Treated by
Oral Administration of Vanadyl Sulfate

G.R. Willsky1, L.-H. Chi1*, Y. Liang2, and D.C. Crans3
State University of New York at Buffalo Dept. of Biochemistry1 or
Division of Biostatistics, Dept. of Social and Preventive Medicine2 and
Colorado State University, Dept. of Chemistry3
*Lai-Har   Chi : lchi@buffalo.edu
       HISTORY of VANADIUM and DIABETES


 1822          Discovered in Sweden by Nils Sefstrom
 1875 Physiology studied in England by Priestly
 1899 Given by Lyonnet to treat diabetes in France
 1954-59       Lowers cholesterol (Curran and Lewis)
 1974          Inhibits the Na, K- ATPase (Cantley)
 1980 Inhibits glucose transport in fat cells (Karlish and
                Kleinzellor)
 1985 Oral administration alleviated symptoms of diabetes in
       rats with STZ induced diabetes (McNeill)
 1986          V dependent nitrogenase found in bacteria (Robson)
 1995          Clinical trials in humans with V salts (Boden, Cohen,
                         Kahn, Goldfine)
      RATIONALE for STUDYING the EFFECTS of VANADYL
    SULFATE on GENE EXPRESSION in NORMAL and DIABETIC
            RAT MUSCLE USING DNA MICROARRAY

●   Diabetes and the biological effects of metal complexes are
    complex phenomena involving many components.
    Therefore, DNA microarray analysis is an ideal technique
    to use to study this problem.

●   Muscle was chosen because it is the largest reservoir for
    insulin stimulated glucose transport in mammals.

●   The Affymetrix RGU34A rat chip system with
    approximately 8000 probe sets was chosen because it
    included all currently identified rat genes.

●   The two hypotheses were:
    1. Metal treatment of diabetic animals should restore
       gene expression patterns to normal.
    2. Diabetes and VS treatment of diabetes would have
       opposite effects on gene expression.
     EXPERIMENTAL GROUPS

Four Groups were analyzed, each with n=5
    Normal Untreated [N]
    Diabetic Untreated [D]
    Normal Treated [N/ VOSO4 (VS)]
    Diabetic Treated [D/ VOSO4 (VS)]

Two factors in the analysis:
      Treatment vs No Treatment (VS)
      Disease vs No Disease (Diabetes)
                     ANIMAL PROTOCOL
STZ injection    VS start                             Rat Sacrificed
                                                    Tissues Isolated
                                                                


DAY     -10         0          7         14         21           28
                            Four week treatment period         
                            Dose about 0.6 mmol V/kg/day
   1. Diabetes was induced by injection of streptozotocin in Male
      Wistar outbred rats.
   2. During Treatment: Blood Glucose 2 per week
                           Urine ketones 1 per week
                           Fluid drunk, body weight daily
   3. End Point Determinations:
          Serum Cholesterol, Triglyceride
          DNA microarray analysis using muscle RNA
      EFFECT of ORAL ADMINISTRATION of VOSO4 (VS) on
       BLOOD GLUCOSE in DIABETIC and NORMAL RATS

                                  600
          Blood glucose (mg/dL)
                                  500                                               N

                                  400                                               D

                                  300
                                            **         ***
                                                             *** ***                D/VS
                                                 ***
                                  200
                                                                 *** *** ***        N/VS

                                  100       *** *** *** *** *** *** *** ***
                                                                                  ** p<0.01, *** p<0.001
                                   0
                                              *** *** ***      ***     *** ***    compared to diabetic
                                        0              10              20    30
                                                                                     Data represents
                                                         Time (Day)                   mean ± SEM.


Oral administration of VS significantly lowers the elevated blood
glucose of diabetes, but only slightly lowers blood glucose of normals.
      EFFECT of ORAL ADMINISTRATION of VOSO4(VS) on
      SERUM CHOLESTEROL and TRIGLYCERIDE LEVELS
                   in SELECTED RATSa .

    Group                  n        Cholesterol     Triglyceride
                                     (mg/dl)           (mg/dl)

    Normal                 4         99 ± 5**        145 ± 55***
    Normal/VS              5        105 ± 22***       78 ± 6***
    Diabetic               3        194 ± 30        1569 ± 289
    Diabetic/VS            3         72 ± 2***       161 ± 18***


    ** p<0.01, *** p<0.001 compared to diabetic
aSamples from some of the rats used in the microarray experiments were
randomly selected for lipid analysis. Data is presented as the mean ± SEM .

Oral administration of VS lowers diabetic hyperlipidemia, does not
lower normal serum cholesterol but does lower normal serum
triglyceride.
      BIOLOGICAL CONCLUSIONS


        Oral administration of Vanadyl Sulfate
to diabetic rats lowers elevated blood glucose
and elevated serum cholesterol and
triglyceride.


       Oral administration of Vanadyl Sulfate
to normal rats does not lower blood glucose
or serum lipids enough to cause clinical
symptoms.
     ANALYSIS of GLOBAL GENE EXPRSSION in RAT
      MUSCLE USING AFFYMETRIX RGU34A CHIPS

                                                Hybridize onto
                 Isolate Total    Label cRNA      Rat Chip
                     RNA




1 Rat per Chip
                                 4 Groups x 5 Chips = 20 Chips
5 Chips per Group
           The ANATOMY of an AFFYMETRIX
                    PROBE SET


           Probe Set:
 (11-16 probe pairs representing   Perfect Match (PM) Probe
    various parts of the gene)     …AAGTCACGATGCATC…




  B   Probe Pair:                   MisMatch (MM) Probe
one PM and one MM probe            …AAGTCACCATGCATC…
             INTENSITY BAR GRAPH of PM and MM of 2
               PROBE SETS from the SAME GENE with
                 ABSENT CALL and PRESENT CALL



Absent Call (i.e. expression of probe set was absent)




Present Call (i.e. expression of probe set was present)




   M33648 = 3-hydroxy-3-methylglutaryl-CoA synthase
      USE of AFFYMETRIX PM and MM
                SYSTEM


        Use of both Perfect Match (PM) and
Mismatch (MM) to obtain one final signal
intensity data can lead to unnecessary loss of
probe set data.


       We used signal intensities determined
by the Affymetrix algorithms, which used MM
and PM data, for our current analysis.
         REPRESENTATIVE SIGNAL SCATTER PLOT: DIABETIC
             TREATED with VS COMPARED to NORMAL



Red dots - probe sets present
         (P) in both samples
Blue dots - probe sets present
         in only one of the two
         samples
Yellow dots - probe sets absent
         (A) or marginally
         detected (M) in either
         of these two samples




           This plot represents one of 25 matrices prepared to compare these groups. The
Y axis is the VS treated diabetic probe sets, while the X axis is the normal probe sets.
Probe sets absent or marginally detected in all chips were deleted from the analysis.
Approximately 5000 of the 8000 probe sets on the RGU34A chip were included in these
analyses.
       FALSE POSITIVE RATE (FPR) DECISION

         To determine the robustness of the array data, and to verify
 that n = five for each group was large enough, we determined the
 overall false positive rate using statistical methods outside of the
 Affymetrix Software system, modeling our approach on the array
 work of A. Reiner, D.Yekutieli and Y. Benjamini (2003)
 Bioinformatics 19: 368-375.

1. Decision threshold = FPR tolerance/# of probe sets tested
                         = 0.1/5055
                         = 0.0000197
2. Performed t-test for the mean signal intensity of each probe
    set in pair-wise comparison.
3. Found 1 probe set with a p-value < decision threshold
4. Therefore, false positive rate > 10%
AFFYMETRIX (MAS 5.0) DATA ANALYSIS
           Probe Signal Intensity Data


             Background Correction


      Scaled Signal with Target Intensity 2500
                          Probe sets absent or marginally detected in
                          all chips were deleted from the analysis.
         5 x 5 Matrices (25 comparisons)

           Signal Log2 Ratio obtained


      Identified Probe Sets with an Increase
or Decrease in at least 80% of the 25 comparisons
        PROBE SETS with DIFFERENTIAL GENE EXPRESSION in
        COMPARISON of NORMAL and DIABETIC RATS TREATED
             and UNTREATED with VANADYL SULFATE

                 A                  B                C                   D
              Diabetic       Diabetic treated Diabetic treated     Normal treated
             vs Normal          vs Diabetic     vs Normal            vs Normal

 Increase        110               52                  6                  35

 Decrease         14               127                 5                  1
    There were 46 probe sets, representing 33 genes, identified as showing opposite
changes when gene expression in Diabetic rats was compared to that in Normal rats
(column A) and when gene expression in Diabetic treated rats was compared to gene
expression in Diabetic untreated rats (column B). The 46 probe sets were 37% of the
124 probe sets whose expression was modified by Diabetes (column A)
    There were very few probe sets identified when gene expression in the Diabetic
treated rat was compared to the Normal Rat (column C), which shows that VS
treatment restored diabetes induced gene expression towards the normal pattern of
expression.
    VS treatment of N rats (column D) did not affect gene expression as much as VS
treatment of D rats (column B)
VS CORRECTS DIABETES INDUCED CHANGES in GENE EXPRESSION
       Accession Number            Probe Set Description
      UP REGULATED BY DIABETES
                                 Lipid Metabolism
       D00569                    2,4-dienoyl-CoA reductase
       M33648                    HMG-CoA synthase *
       U40001                    hormone-sensitive lipase
       A1237731                  lipoprotein lipase
       AI169612                  fatty acid binding protein 4
                                  Transport/Trafficking
       AB000507                   aquaporin 7
       AF072411                   fatty acid translocase/CD36
       L07736                     carnitine palmitoyltransferase I
       M11794                     metallothionein-2 and metallothionein-1*
       S76779                     apolipoprotein E liver

                                  Signal Transduction
       AA900476                   Cbp/p300-interacting transactivator
       U05014                     PHAS-I
       U12187                     ras-related protein (rad)
       X81193                     muscle LIM protein

                                  Other
       J03914/U86635              glutathione S-transferase subunits *

      DOWN REGULATED BY DIABETES
       J03190                  5-aminolevulinate synthase
   * genes whose product has been specifically shown to be vanadium related.
          GENES WHOSE EXPRESSION WAS TESTED
                 USING REAL TIME PCR
        Up-regulated by Diabetes
Vanadium Related Genes    glutathione S transferase - GSH-ST
                          HMGCoA synthetase -HMG CoA S
                          metallothionein 1 & 2

Lipid Metabolism           fatty acid translocase/CD36 - FATCD36
                           2,4-dienoyl CoA reductase -dienoyl CoA R

Signal Transduction        PHAS-1
                           ras-related protein - rad

Other                    carnitine palmitoyltransferase - CPT
                         creatine kinase - CK
                         myosin heavy chain - MHC
        Down-regulated by Diabetes
                         5-aminolevulinate synthase - ALS
         As seen on the next panel 2 comparisons for each of these 11 genes were
made. Only one comparison for CK did not confirm, which means that 4.5% of the
comparisons with Real time PCR confirmed the DNA Microarray data. This result is
consistent with our previous statistical determination of the False Positive Rate.
                                  HOW DO MICROARRAY RESULTS COMPARE
                                            to REAL TIME PCR?
                         Affy [n=5] vs Real Time PCR[n=6,3 affy samples& 3 non-affy samples]
       D vs N (Affy)                D vs N (Real Time PCR)                  D/VS vs D (Affy)            D/VS vs D (Real Time PCR)
                   10

                    8
                    6
Mean Fold Change




                    4

                    2
                    0

                    -2
                    -4
                    -6
                    -8
                   -10

                   -12
                                                                               PHAS-I
                                                                      ALS
                                                            HMGCoAS




                                                                                                                     RAD
                                                                                        DiECoAR



                                                                                                  FAT-CD36




                                                                                                                           CK
                                    MHC




                                                                                                             CPT-I
                         GSH-ST




                                          Metallothionein
                                                1&2




                                                                            Gene ID
          GENERAL TWO WAY ANOVA ANALYSIS
                   of ARRAY DATA
        Global ANOVA: Array and Treatment for all probe
           sets:
               Yijk =  + Ai + Tj + (AT)ij + ijk
                        i = 1…20
                        j = 1…4
                       k = 1…5055
                : overall mean
                : random error
               A : main effect of Array
               T : main effect of Treatment
           Result: p < 0.0001
               This means that there are significant
           differences in each array set in addition to the
           difference cause by treatment.
Array analysis done by Dr. Yulan Liang, Division of Biostatistics SUNY at Buffalo.
         TWO FACTORIAL DESIGNED ANOVA ANALYSIS OF ARRAY
         DATA to TEST BIOLOGICAL QUESTION: DOES TREATMENT
          ACT DIFFERENTLY in the NORMAL and DISEASE STATE?

                    Treatment (VS)
                     _        +              For the overall analysis p< 0.0001
                                             showing high interaction between
              _      N         N/VS          Disease and Treatment, i.e. VS
Disease (D)                                  treatment affects diabetic and
              +      D         D/VS          normal animals differently


                         No Treatment           D       N        p < 0.0001
      DISEASE            Treatment             D/VS    N/VS
                         No Disease              N      N/VS     p = 0.0013
      TREATMENT          Disease                 D      D/VS

  The above table shows that differences between treatment for the
  disease factor and differences between disease for the treatment
  factor are significant.
 Array analysis done by Dr. Yulan Liang, Division of Biostatistics, SUNY at Buffalo.
                  CONCLUSIONS
      Oral administration of Vanadyl Sulfate to
Diabetic rats lowers elevated blood glucose and
elevated serum cholesterol and triglyceride.
      Oral administration of Vanadyl Sulfate to
Diabetic rats restores the overall gene expression
pattern towards that seen in Normal rats.
      Gene expression in Vanadyl Sulfate treated
Diabetic rats compared to untreated Diabetic rats
results in opposite changes to that observed when
gene expression in Diabetic rats is compared to that in
Normal Rats for about 37% of the genes whose
expression is altered by Diabetes.
       Oral administration of Vanadyl Sulfate has
different effects on gene expression in Normal and
Diabetic rats as determined using both biological and
statistical analyses .
                     ACKNOWLEDGEMENTS
SUNY at Buffalo                       Roswell Park Cancer
                                      Institute (RPCI)
Willsky Laboratory                    Norma Nowak
Gail Willsky (gwillsky@buffalo.edu)   Lesleyann Hawthorne
Josephine Alfano
Michael Godzala
Peter Kaszynski                       Joslin Diabetes Center
La Tanya Austin                       C.Ronald Kahn
A. Jake Ocque                         Allison Goldfine
Rebecca Grayson                       M.- E. Patti
Rohit Gupta
Division of Biostatistics             Colorado State University
Yulan Liang                           Debbie Crans
Toxicology Research Center
                                            Funded by NIH
Paul Kostyniak

				
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posted:9/23/2011
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