research article downregulation of type ii diabetes...

Research ArticleDownregulation of Type II Diabetes Mellitusand Maturity Onset Diabetes of Young Pathways in HumanPancreatic Islets from Hyperglycemic Donors

Jalal Taneera Petter Storm and Leif Groop

Department of Clinical Sciences Diabetes amp Endocrinology Lund University Diabetes Center Skane University HospitalLund University 20502 Malmo Sweden

Correspondence should be addressed to Jalal Taneera jalaltaneeramedluse

Received 22 July 2014 Revised 22 September 2014 Accepted 25 September 2014 Published 14 October 2014

Academic Editor Daisuke Koya

Copyright copy 2014 Jalal Taneera et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Although several molecular pathways have been linked to type 2 diabetes (T2D) pathogenesis it is uncertain which pathwayhas the most implication on the disease Changes in the expression of an entire pathway might be more important for diseasepathogenesis than changes in the expression of individual genes To identify the molecular alterations in T2D DNA microarraysof human pancreatic islets from donors with hyperglycemia (119899 = 20) and normoglycemia (119899 = 58) were subjected to Gene SetEnrichment Analysis (GSEA) About 178KEGG pathways were investigated for gene expression changes between hyperglycemicdonors compared to normoglycemic Pathway enrichment analysis showed that type II diabetes mellitus (T2DM) and maturityonset diabetes of the young (MODY) pathways are downregulated in hyperglycemic donors while proteasome and spliceosomepathways are upregulated The mean centroid of gene expression of T2DM and MODY pathways was shown to be associatedpositively with insulin secretion and negatively with HbA1c level To conclude downregulation of T2DM and MODY pathways isinvolved in islet function andmight be involved in T2D Also the study demonstrates that gene expression profiles from pancreaticislets can reveal some of the biological processes related to regulation of glucose hemostats and diabetes pathogenesis

1 Introduction

T2D is a multifactorial disease characterized by increasedblood glucose level due to both a defect in insulin secretionfrom pancreatic beta-cells and impaired insulin action atthe target cells The disease is estimated to affect more than350 million people in 2020 worldwide (httpwwwidforgdiabetesatlas) and to contribute to other diseases such asatherosclerotic vascular disease blindness and kidney failure[1]

Several molecular pathways have been implicated in thedisease process insulin receptor signalling [2] carbohydratemetabolism [3] ER stress related pathway [4] cytokinesignalling [5] exocytosis [6] and oxidative phosphorylation[7 8] However it is unclear which of these or other pathwaysare disturbed in and might be responsible for T2D in itscommon form

DNA microarrays expression analysis enables scientiststo investigate the altered transcript levels in particular tissue

from individuals with specific diseases For example mRNAexpression profiles are generated from thousands of genesfrom samples of one of two classes such as cancer [9] Thedifferential expressed genes between classes can be rankedbased on their differentiation However the remaining chal-lenge is how to interpret a given list of genes into biologicalmechanism

Mootha et al have developed a statistical methodologycalled Gene Set Enrichment Analysis (GSEA) to definewhether a given gene set is significantly enriched in a list ofgenes ranked by their correlation with a phenotype of interest[8 10] GSEAhas been shown to have an increased capacity todetect modest but coordinated changes in prespecified set ofrelated genes GSEA has been successfully used to uncoveraltered metabolic pathways in several applications such ashuman diabetic muscle [8] comparingmousemodels of can-cer with human tumors using gene-expression profiling [11]lung cancer [10] characterization of acute megakaryoblastic

Hindawi Publishing CorporationJournal of Diabetes ResearchVolume 2014 Article ID 237535 7 pageshttpdxdoiorg1011552014237535

2 Journal of Diabetes Research

leukemia [12] and interaction between mRNA and miRNAin HIV-mediated neurodegeneration [13] and comparingwhole blood gene expression profiling from lean and obeseindividuals [14]

Here we employed GSEA to determine whether the178 selected KEGG pathways are altered between islet geneexpression from donors with normoglycemia and hyper-glycemia Pathway enrichment analysis showed that MODYand T2DM pathways are downregulated in hyperglycemicislets The mean centroid of gene expression of T2DM andMODY pathways was shown to be significantly associatedwith insulin secretion and HbA1c level which highlight thatthese pathways are involved in islet function

2 Materials and Methods

21 Human Pancreatic Islets Islets from cadaver donors(78 donors) were provided by the Nordic Islet Transplan-tation Program (wwwnordicisletsorg) Uppsala UniversityAll procedures were approved by the ethics committees atUppsala and Lund Universities Islets were obtained from 68nondiabetic donors (30 females 37 males age 59 plusmn 10 BMI259 plusmn 35 HbA1c 55 plusmn 11 and days of culture 35 plusmn 19)and 10 T2D donors (4 females 6 males age 607 plusmn 12 BMI281plusmn45 HbA1c 71plusmn12 and days of culture 2plusmn09) Purityof the islet preparations was assessed by dithizone staininginsulin content and contribution of exocrine and endocrinetissue as previously described [15] The islets were culturedin CMRL 1066 (ICN Biomedicals Costa Mesa CA USA)supplemented with 10mML HEPES 2mML l-glutamine50120583gmL gentamicin 025 120583gmL Fungizone (GIBCO BRLGaithersburg MD USA) 20120583gmL ciprofloxacin (BayerHealthcare Leverkusen Germany) and 10mML nicoti-namide at 37∘C (5 CO

2) prior to RNA preparation

22 Microarray Gene Expression in Human Pancreatic IsletsRNA was isolated with the AllPrep DNARNA Mini Kit(Qiagen Hilden Germany) RNA quality and concentrationwere measured using an Agilent 2100 bioanalyzer (Bio-Rad Hercules CA USA) and Nanodrop ND-1000 equip-ment (NanoDrop Technologies Wilmington DE USA)The microarrays (GeneChip Human Gene 10 ST) wereperformed using the Affymetrix standard protocol as previ-ously described [15] The array data were summarized andnormalized with robust multiarray analysis (RMA) methodusing the oligo package from BioConductor Also batchcorrection was done with COMBACT function from SVApackage from BioConductor All data areMIAME compliantand the raw data have been deposited in a MIAME database(GEO accession number GSE 50398 and GSE 50397)

23 Glucose-StimulatedInsulinSecretion Isletswerehand-pickedunder a stereomicroscope and preincubated for 30min at37∘C in Krebs Ringer bicarbonate (KRB) buffer (pH 74) con-taining (in mM) 120 NaCl 25 NaHCO

3 47 KCl 12 MgSO

4

25 CaCl2 12 KH

2PO 10 HEPES supplemented with 01

bovine serum albumin N-2 hydroxyethylpiperazine-N1015840-2-ethanesulfonic acid (10mmolL) and 1mmolL glucose Eachincubation vial contained 12 islets in 10mL KRB buffer

Table 1 Characteristics of human pancreatic donors

Normoglycemic Hyperglycemic119873 (malefemale) 58 (3424) 20 (119)Age (years) 609 plusmn 109 64 plusmn 89BMI 254 plusmn 29 285 plusmn 45HbA1c 54 plusmn 03 69 plusmn 10Purity 70 plusmn 16 63 plusmn 20Donors withdiabetes 0 10

Data represented as mean plusmn SD

solution and was treated with 95 O2-5 CO

2to obtain

constant pH and oxygenation After preincubation the bufferwas changed to a KRB buffer containing either 1mM (basalsecretion) or 167mM glucose (stimulated secretion) Theislets were then incubated for 1 h at 37∘C in ametabolic shaker(30 cycles per min) Immediately after incubation an aliquotof the medium was removed for analysis of insulin using aradioimmunoassay kit (Euro-Diagnostica Malmo Sweden)Insulin content in homogenized human islets was assessedby ELISA (Mercodia Uppsala Sweden) and values werenormalized to the total DNA in each sample as determinedby a fluorometric assay (Quant-iT PicoGreen InvitrogenMolecular Probes Stockholm Sweden)

24 Gene Set Enrichment Analysis (GSEA) The GSEA soft-ware tool (version 2013 wwwbroadinstituteorggsea) wasused to identify KEGG pathways (MSigDB version 40)that show an overrepresentation of up- or downregulatedgenes between donors with hyperglycemia (HbA1c gt 6119873 = 20) and normoglycemia (HbA1c lt 6 119873 = 30)Briefly an enrichment score was calculated for each geneset (ie KEGG pathway) by ranking each gene by theirexpression difference using Kolmogorov-Smirnov statisticcomputing a cumulative sum of each ranked in each geneset and recording the maximum deviation from zero as theenrichment score

25 Statistical Analysis Data are presented as means plusmn SDDifferences in expression levels were analyzed by Studentrsquos 119905-test or nonparametric Mann-Whitney tests Correlation testswere analyzed using nonparametric Spearmanrsquos tests Themean centroid represents the normalized gene expressionlevels of all genes from all individuals in the analysis with amean of 0 and a variance of 1 All statistical tests were per-formed using the Statistical Package for the Social Sciences(SPSS) version 190 software (SPSS Chicago IL USA)

3 Results

In this study we used human islet microarray expressiondata obtained from 78 donors The donors were subdividedinto 58 normoglycemic donors with HbA1c level lt6 and20 hyperglycemic donors with HbA1c level gt6 (Table 1)Normalized expression microarray data were subjected topathway analysis of GSEA algorithm using 178 Kyoto Ency-clopedia forGenes andGenomes (KEGG) pathways Pathway

Journal of Diabetes Research 3

Table 2 List of down- and upregulated pathways in hyperglycemic donors

Size NES NOM 119875 value FDR 119902 valueDownregulated KEGG pathwaysKEGG TYPE II DIABETES MELLITUS 45 minus1898 0 0077KEGG MATURITY ONSET DIABETES OF THE YOUNG 24 minus1812 0 0108KEGG OOCYTE MEIOSIS 108 minus1543 002 0485KEGG PROGESTERONE MEDIATED OOCYTE MATURATION 83 minus1453 004 070KEGG SNARE INTERACTIONS IN VESICULAR TRANSPORT 38 minus1583 005 0610Upregulated KEGG pathwaysKEGG PROTEASOME 44 2026 0005 0030KEGG SPLICEOSOME 125 1898 001 0075KEGG DNA REPLICATION 36 1621 004 0821KEGG PRIMARY IMMUNODEFICIENCY 35 1604 002 071KEGG CYTOKINE CYTOKINE RECEPTOR INTERACTION 251 1593 0003 0619KEGG GLYOXYLATE AND DICARBOXYLATE METABOLISM 16 1529 003 0614Ranking of the genes set was done using GSEA 2013 NES normalized enrichment score NOM nominal FDR false discovery rate

enrichments were evaluated by their normalized enrichmentscore (NES) nominal 119875 value and false discovery rates(FDR)

GSEA identified 4 pathways (119875 lt 005) which weredownregulated in the hyperglycemic islets compared tonormoglycemic islets (Table 2) At FDR lt 25 only twopathways (T2DM and MODY) were shown to be significant(Table 2 and Figures 1(a)-1(b)) On the other hand sixenriched pathways were upregulated in the hyperglycemicdonors (119875 lt 005) while at FDR lt 25 only two pathways(proteasome and spliceosome) were significant (Table 2 andFigures 1(c)-1(d))

Next we examined individual expression value of the 45genes in the T2DM and the 24 genes in MODY pathwayWe found that 14 out of the 45 genes of the T2DM pathway(31) and 9 out of the 24 genes ofMODY (375) contributedsignificantly to core enrichment whose expression was lowerin hyperglycemic than in normoglycemic donors (Figures2(a)-2(b)) Also expression of the 23 genes was signifi-cantly reduced in diabetic compared to nondiabetic donors(Table S1 in Supplementary Material available online athttpdxdoiorg1011552014237535) Insulin receptor sub-strate genes (IRS4) were shown to be lownot expressed inhuman pancreatic islets (Figure 2(a)) Glucokinase (GCK)solute carrier family 2 (facilitated glucose transporter)member 2 (SLC2A2) pancreatic and duodenal homeobox 1(PDX1) and v-maf musculoaponeurotic fibrosarcoma onco-gene homolog A (MAFA) overlapped between core enrich-ment of the two pathways Interestingly plotting the meancentroid of the 14 and 9 downregulated genes in T2DMand MODY pathways showed a positive correlation withinsulin secretion and negative correlation with an HbA1clevel (Figures 2(c)-2(d)) suggesting that these pathways areinvolved in regulation of insulin secretion and glycemicstatusMean centroid of the 31 and 55 upregulated genes in thecore enrichment of proteasome and spliceosome pathwaysshowed no correlation with insulin secretion but positivelycorrelated with HbA1c level (Figures 2(e)-2(f)) We also anal-ysed the real score of expression and differential expression of

genes in the core enrichment of proteasome and spliceosomepathways in normoglycemic versus hyperglycemic and innondiabetic versus diabetic donors (Tables S2 and S3)

4 Discussion

Although several pathways have been implicated in T2Dpathogenesis most of these studies were performed in non-pancreatic tissues In this study we used human pancreaticislets obtained from 78 donors Each donated pancreaticislet was systematically characterized by performing cDNAmicroarray in addition to measuring insulin response toglucose and glycemic status (HbA1c)

Our data presented additional evidence into the biologi-cal processes that differentially were regulated in pancreaticislets from normoglycemic and hyperglycemic donors Thedownregulated pathways (T2DM and MODY) in hyper-glycemic donors were due to decreased expression of severalprotein-encoded genes which indicate a reduction in proteinsynthesis in pancreatic islets Recently Del Rosario et alreported that regions in promoter of genes involved in T2DMand MODY pathways are more likely to be differentiallymethylated between diabetic and nondiabetic donors com-pared to other genes [16] In this study the islet expressionmean centroid of downregulated genes in T2DM andMODYpathways correlated positively with insulin secretion andnegatively with HbA1c level suggesting that appropriateexpression of these genes is required for sufficient insulinsecretion and glucose homeostasis

Notably most of the downregulated genes have beenimplicated in diabetes pathogenesis SLC2A2 (Glut2) isinvolved in 120573-cell function and insulin secretion [17] Micelacking SLC2A2 showed early diabetes and abnormal postna-tal pancreatic islet development [18] ABCC8 is a regulator ofATP-sensitive K(+) channels and insulin release A mutationin ABCC8 was observed in patients with hyperinsulinemichypoglycemia of infancy [19] and associated with T2D [20]KCNJ11 together with ABCC8 regulates transmembranepotential and thereby glucose-stimulated insulin secretion


0

000

025

050

00

2500 5000 7500 10000 12500 15000 17500 20000

minus06

minus025

minus05

minus04

minus03

minus02

minus01

Enric

hmen

t sco

re (E

S)

Enrichment profile

Ranking metric scores

Rank in ordered dataset

Zero cross at 9293

Rank

ed li

st m

etric

(Sig

nal2

Noi

se)

Hits

ldquoDIArdquo (positively correlated)

ldquoNONDIArdquo (negatively correlated)

Enrichment plot KEGG TYPE II DIABETES MELLITUS

(a)

000

025

050

minus025

00

minus06

minus07

minus05

minus04

minus03

minus02

minus01

Enric

hmen

t sco

re (E

S)0 2500 5000 7500 10000 12500 15000 17500 20000


Zero cross at 9293

Rank

ed li

st m

etric

(Sig

nal2

Noi

se)



Enrichment profile

Ranking metric scoresHits

KEGG MATURITY ONSET DIABETES OF THE YOUNGEnrichment plot

(b)

05

04

03

02

01

00

0 2500 5000 7500 10000 12500 15000 17500 20000

Enric

hmen

t sco

re (E

S)


000

025

050

minus025

Rank

ed li

st m

etric

(Sig

nal2

Noi

se)


Zero cross at 9293


Enrichment profile


Enrichment plot KEGG SPLICEOSOME

(c)

0 2500 5000 7500 10000 12500 15000 17500 20000


Enrichment profile


05

06

04

03

02

01

00

Enric

hmen

t sco

re (E

S)

000

025

050

minus025

Rank

ed li

st m

etric

(Sig

nal2

Noi

se)


Zero cross at 9293


Enrichment plot KEGG PROTEASOME

(d)

Figure 1 GSEA plot The analysis was performed against the KEGG database for differential enriched pathways between hyperglycemic andnormoglycemic islets Enrichment plots for the downregulated pathways are shown in graphs (a) and (b) and upregulated pathways are shownin graphs (b) and (c) The 119910-axis represents the value of the ranking metric the 119909-axis represents the rank for all genes Bottom plot of theranked list of all genes Top the enrichment score for the gene set as the analysis walks along the ranked list The score at the peak of the plotis the enrichment score (ES) for this gene set and those genes appearing before or at the peak are defined as core enrichment genes in thisgene set Lower levels of expression are represented in shades of blue and higher expression is represented in red


0100200300400

NormoglycemicHyperglycemic

500100015002000

Nor

mal

ized

mea

n ex

pres

sion

IRS4

IRS2

ABCC

8

CACN

A1A

CACN

A1C

CACN

A1D

GCK

KCNJ11

MAF

A

MAP

K10

PDX1

PIK3

R3

SLC2

A2

CACN

A1B

(a)

0

500

1000


600013000

Nor

mal

ized

mea

n ex

pres

sion

PAX6

IAPP

NEU

ROD1

NKX

2-2

NKX

6-1

GCK

MAF

A

PDX1

SLC2

A2

(b)

0

1

2

3

4

5

6

0 02 04 06 08 1Type II diabetes mellitus mean centroid

Insu

lin se

cret

ion

0

2

4

6

8

10

12

HbA

1c (

)

minus12 minus1 minus08 minus06 minus04 minus02

(c)

0

1

2

3

4

5

6

0 05 1Maturity onset diabetes of young mean centroid

Insu

lin se

cret

ion

024

6

810

12

HbA

1c (

)

minus05minus15 minus1

(d)

0

1

2

3

4

5

6

0 01 02 03Proteasome mean centroid

Insu

lin se

cret

ion

0

2

4

6

8

10

12

HbA

1c (

)

minus03 minus02 minus01

(e)

0

1

2

3

4

5

6

0 01 02 03 04Spliceosome mean centroid

Insu

lin re

leas

e

0

2

4

6

8

10

12

HbA

1c (

)


(f)

Figure 2 Genes differentially expressed in T2DMandMODYpathways Gene expression analysis of genes in the T2DMandMODYpathwayshowed that 14 out of the 45 genes of the T2DM (a) pathway (119875 lt 005) and 9 out of 24 genes of MODY (b) (119875 lt 005) have lower expressionin hyperglycaemic compared to normoglycemic donors (c) Correlation of mean centroid of the 14 downregulated genes in T2DM pathwayshowed positive correlation with insulin secretion (119877 = 033 119875 = 001) and negative correlation with HbA1c level (119877 = minus057 119875 = 000001)(d) Correlation of mean centroid of the 9 downregulated genes in MODY pathway showed positive correlation with insulin secretion (119877 =031 119875 = 001) and negative correlation with HbA1c level (119877 = minus059 119875 = 0000006) (e) Correlation of mean centroid of the 31 upregulatedgenes in proteasome pathway showed no correlation with insulin secretion (119877 = minus005 119875 = 07) and positive correlation with HbA1c level(119877 = 03 119875 = 003) (f) Correlation of mean centroid of the 55 upregulated genes in spliceosome pathway showed no correlation with insulinsecretion (119877 = 016 119875 = 023) and positive correlation with HbA1c level (119877 = 026 119875 = 006)

in pancreatic beta-cells A Glu23Lys polymorphism (E23K)has been associated with T2D and a modest impairmentin insulin secretion [21] In addition mutation in the genecauses a severe form of neonatal diabetes as well as matu-rity onset diabetes of the young type 11 (MODY11) [22]PDX1 is involved in the early development of the pancreasand plays a major role in glucose-dependent regulation of

insulin gene expression [23] Defects in this gene causedmaturity onset diabetes of the young type 4 (MODY4) [24]PAX6 point mutations in the PAX6 gene shown to disruptislet morphology and decreased numbers of 120573 120572 and PPcells [25] Also a mutation in the gene has been shownto cause early-onset diabetes [26] MAFA is required forislet beta-cell differentiation and activates the insulin and


glucagon promoters [27] MAFA functions as a downstreammediator of PAX6 in regulating the specification of insulinand glucagon expressing cells [28] NEUROD1 is reported toregulate expression of the insulin gene [29] andmutations inthis gene result in maturity onset diabetes of the young type6 (MODY6) [30] FOXA2 is involved in glucose homeostasisand regulates the expression of genes important for glucosesensing in pancreatic beta-cells and glucose homeostasis [31]Hence these data suggest that the downregulation of the 23genes in T2DMandMODYpathways is a causative for insulinsecretion impairment

Proteasomes are protein complexes with a main functionto regulate and degrade unnecessary or damaged proteinsby proteolysis while spliceosome is a complex molecularmachine assembled from snRNPs and protein complexesSplicing is a known process when spliceosome removesintrons from a transcribed pre-mRNA Both of the protea-some and the spliceosome pathways showed upregulationin hyperglycemic donors The findings are potentially veryimportant as there are several lines of evidence whichreported glucose or hyperglycemia to influence proteasomeand splicing Recent studies have shown that high glucoseand diabetes affect proteasome activity [32 33] The link ofhyperglycemia to proteasome raises several questions such ashow hyperglycemia can modulate proteasome targeting andactivity and whether this modulation occurs in a cell-specificmanner Hribal et al reported that chronic hyperglycemiaimpairs insulin secretion by affecting splicing in RIN 120573-cellline and human islet [34] Osmark et al reported pronouncedtissue-specific differences in the splicing of TCF7L2 withforms containing exons 4 and 15 being the most abundantin islets The incorporation of exon 4 in islets was shownto correlate positively with HbA1c levels [35] Also a shortTCF7L2mRNA variant in subcutaneous fat is associated withhyperglycemia and impaired insulin action in adipose tissue[36] Although these reports do not prove causality theysuggest an effect of plasma glucose levels on splicing

There is one confine in our study that must be acknowl-edgedThe study employedwhole pancreatic islet the relativecontribution of the transcriptional programs in specificcell types towards the observed gene expression differencescannot be clearly delineated However most of the downreg-ulated genes in the core enrichment of T2DM and MODYare more expressed in 120573-cell compared to alpha cells andexocrine cells as shown in recent published RNA-sequencingexpression data from sorted endocrine cells [37]

In conclusion the investigation of gene expression pro-files frompancreatic islets can illustrate some of the biologicalprocesses related to the regulation of glucose hemostats anddiabetes pathogenesis

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by grants from the SwedishResearchCouncil inclProjectGrant (LGDnr 251-2007-4037

collaborative Project Grant Dnr 521-2008-2974 strategicresearch area Grant (EXODIAB Dnr 2009-1039) and Lin-naeus Grant (LUDC) Dnr 349-2008-6589) Human pancre-atic islets were provided by the Nordic Network for ClinicalIslet Transplantation by the courtesy of O Korsgren UppsalaSweden supported by EXODIAB and grants from JDRF Theauthors thank SCIBLUGenomics at LundUniversity for theirhelp with microarrays preparation

References

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[2] X-F Huang and J-Z Chen ldquoObesity the PI3KAkt signalpathway and colon cancerrdquo Obesity Reviews vol 10 no 6 pp610ndash616 2009

[3] B R Henke and S M Sparks ldquoGlycogen phosphorylaseinhibitorsrdquo Mini-Reviews in Medicinal Chemistry vol 6 no 8pp 845ndash857 2006

[4] U Ozcan Q Cao E Yilmaz et al ldquoEndoplasmic reticulumstress links obesity insulin action and type 2 diabetesrdquo Sciencevol 306 no 5695 pp 457ndash461 2004

[5] P Trayhurn and I S Wood ldquoSignalling role of adipose tissueadipokines and inflammation in obesityrdquo Biochemical SocietyTransactions vol 33 no 5 pp 1078ndash1081 2005

[6] S A Andersson A H Olsson J L S Esguerra et al ldquoReducedinsulin secretion correlates with decreased expression of exo-cytotic genes in pancreatic islets from patients with type 2diabetesrdquoMolecular and Cellular Endocrinology vol 364 no 1-2 pp 36ndash45 2012

[7] A H Olsson B T Yang E Hall et al ldquoDecreased expressionof genes involved in oxidative phosphorylation in humanpancreatic islets from patients with type 2 diabetesrdquo EuropeanJournal of Endocrinology vol 165 no 4 pp 589ndash595 2011

[8] V K Mootha C M Lindgren K-F Eriksson et al ldquoPGC-1120572-responsive genes involved in oxidative phosphorylationare coordinately downregulated in human diabetesrdquo NatureGenetics vol 34 no 3 pp 267ndash273 2003

[9] T R Golub D K Slonim P Tamayo et al ldquoMolecular classi-fication of cancer class discovery and class prediction by geneexpression monitoringrdquo Science vol 286 no 5439 pp 531ndash5271999

[10] A Subramanian P Tamayo V K Mootha et al ldquoGene setenrichment analysis a knowledge-based approach for inter-preting genome-wide expression profilesrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 102 no 43 pp 15545ndash15550 2005

[11] A Sweet-Cordero S Mukherjee A Subramanian et al ldquoAnoncogenic KRAS2 expression signature identified by cross-species gene-expression analysisrdquo Nature Genetics vol 37 no1 pp 48ndash55 2005

[12] C Bourquin S Schreiber S Beck G Hartmann and S EndresldquoImmunotherapywith dendritic cells andCpGoligonucleotidescan be combined with chemotherapy without loss of efficacy inamousemodel of colon cancerrdquo International Journal of Cancervol 118 no 11 pp 2790ndash2795 2006

[13] L Zhou G M Pupo P Gupta et al ldquoA parallel genome-wide mRNA and microRNA profiling of the frontal cortex ofHIV patients with and without HIV-associated dementia shows


the role of axon guidance and downstream pathways in HIV-mediated neurodegenerationrdquo BMC Genomics vol 13 no 1article 677 2012

[14] V Skov S Knudsen M Olesen M L Hansen and L M Ras-mussen ldquoGlobal gene expression profiling displays a network ofdysregulated genes in non-atherosclerotic arterial tissue frompatients with type 2 diabetesrdquo Cardiovascular Diabetology vol11 article 15 2012

[15] J Taneera S Lang A Sharma et al ldquoA systems geneticsapproach identifies genes and pathways for type 2 diabetes inhuman isletsrdquo Cell Metabolism vol 16 no 1 pp 122ndash134 2012

[16] M C Del Rosario V Ossowski W C Knowler C Bogardus LJ Baier and R L Hanson ldquoPotential epigenetic dysregulationof genes associated with MODY and type 2 diabetes in humansexposed to a diabetic intrauterine environment an analysisof genome-wide DNA methylationrdquo Metabolism Clinical andExperimental vol 63 no 5 pp 654ndash660 2014

[17] I Barroso J Luan R P S Middelberg et al ldquoCandidate geneassociation study in type 2 diabetes indicates a role for genesinvolved in 120573-cell function as well as insulin actionrdquo PLoSBiology vol 1 no 1 p E20 2003

[18] M-T Gulllam E Hummler E Schaerer et al ldquoEarly diabetesand abnormal postnatal pancreatic islet development in micelacking Glut-2rdquoNature Genetics vol 17 no 3 pp 327ndash330 1997

[19] T L Hoffman E Blanco A Lane et al ldquoGlucose metabolismand insulin secretion in a patient with ABCC8 mutation andFanconi-Bickel syndrome caused by maternal isodisomy ofchromosome 3rdquo Clinical Genetics vol 71 no 6 pp 551ndash5572007

[20] J C Florez K A Jablonski S E Kahn et al ldquoType 2 diabetes-associated missense polymorphisms KCNJ11 E23K and ABCC8A1369S influence progression to diabetes and response tointerventions in the diabetes prevention programrdquo Diabetesvol 56 no 2 pp 531ndash536 2007

[21] A L Gloyn E R Pearson J F Antcliff et al ldquoActivatingmutations in the gene encoding the ATP-sensitive potassium-channel subunit Kir62 and permanent neonatal diabetesrdquo TheNew England Journal ofMedicine vol 350 no 18 pp 1838ndash18492004

[22] A Bonnefond J Philippe E Durand et al ldquoWhole-exomesequencing and high throughput genotyping identified KCNJ11as the thirteenth MODY generdquo PLoS ONE vol 7 no 6 ArticleID e37423 2012

[23] J Le Lay and R Stein ldquoInvolvement of PDX-1 in activation ofhuman insulin gene transcriptionrdquo Journal of Endocrinologyvol 188 no 2 pp 287ndash294 2006

[24] MM Sachdeva K C Claiborn C Khoo et al ldquoPdx1 (MODY4)regulates pancreatic beta cell susceptibility to ER stressrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 106 no 45 pp 19090ndash19095 2009

[25] A Hamasaki Y Yamada T Kurose et al ldquoAdult pancreaticislets require differential pax6 gene dosagerdquo Biochemical andBiophysical Research Communications vol 353 no 1 pp 40ndash462007

[26] M Nishi M Sasahara T Shono et al ldquoA case of novel de novopaired box gene 6 (PAX6) mutation with early-onset diabetesmellitus and aniridiardquoDiabetic Medicine vol 22 no 5 pp 641ndash644 2005

[27] Y Hang and R Stein ldquoMafA and MafB activity in pancreatic 120573cellsrdquo Trends in Endocrinology and Metabolism vol 22 no 9pp 364ndash373 2011

[28] I Artner B Blanchi J C Raum et al ldquoMafB is required forislet 120573 cell maturationrdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 104 no 10 pp 3853ndash3858 2007

[29] P Itkin-Ansari E Marcora I Geron et al ldquoNeuroD1 inthe endocrine pancreas localization and dual function as anactivator and repressorrdquo Developmental Dynamics vol 233 no3 pp 946ndash953 2005

[30] S Y Kristinsson E T Thorolfsdottir B Talseth et al ldquoMODYin Iceland is associated with mutations in HNF-1120572 and a novelmutation in NeuroD1rdquo Diabetologia vol 44 no 11 pp 2098ndash2103 2001

[31] H Wang B R Gauthier K A Hagenfeldt-Johansson MIezzi and C B Wollheim ldquoFoxa2 (HNF3120573) controls multiplegenes implicated in metabolism-secretion coupling of glucose-induced insulin releaserdquo Journal of Biological Chemistry vol277 no 20 pp 17564ndash17570 2002

[32] S Yadranji Aghdam Z Gurel A Ghaffarieh C M Sorensonand N Sheibani ldquoHigh glucose and diabetes modulate cellu-lar proteasome function implications in the pathogenesis ofdiabetes complicationsrdquo Biochemical and Biophysical ResearchCommunications vol 432 no 2 pp 339ndash344 2013

[33] H Liu S YuWXu and J Xu ldquoEnhancement of 26S proteasomefunctionality connects oxidative stress and vascular endothelialinflammatory response in diabetes mellitusrdquo ArteriosclerosisThrombosis and Vascular Biology vol 32 no 9 pp 2131ndash21402012

[34] M L Hribal L Perego S Lovari et al ldquoChronic hyperglycemiaimpairs insulin secretion by affecting insulin receptor expres-sion splicing and signaling in RIN beta cell line and humanislets of Langerhansrdquo The FASEB Journal vol 17 no 10 pp1340ndash1342 2003

[35] P Osmark O Hansson A Jonsson T Ronn L Groop andE Renstrom ldquoUnique splicing pattern of the TCF7L2 gene inhuman pancreatic isletsrdquo Diabetologia vol 52 no 5 pp 850ndash854 2009

[36] D Kaminska T Kuulasmaa S Venesmaa et al ldquoAdipose tissueTCF7L2 Splicing is regulated by weight loss and associates withglucose and fatty acid metabolismrdquo Diabetes vol 61 no 11 pp2807ndash2813 2012

[37] N C Bramswig L J Everett J Schug et al ldquoEpigenomicplasticity enables human pancreatic120572 to120573 cell reprogrammingrdquoJournal of Clinical Investigation vol 123 no 3 pp 1275ndash12842013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


leukemia [12] and interaction between mRNA and miRNAin HIV-mediated neurodegeneration [13] and comparingwhole blood gene expression profiling from lean and obeseindividuals [14]

Here we employed GSEA to determine whether the178 selected KEGG pathways are altered between islet geneexpression from donors with normoglycemia and hyper-glycemia Pathway enrichment analysis showed that MODYand T2DM pathways are downregulated in hyperglycemicislets The mean centroid of gene expression of T2DM andMODY pathways was shown to be significantly associatedwith insulin secretion and HbA1c level which highlight thatthese pathways are involved in islet function

2 Materials and Methods

21 Human Pancreatic Islets Islets from cadaver donors(78 donors) were provided by the Nordic Islet Transplan-tation Program (wwwnordicisletsorg) Uppsala UniversityAll procedures were approved by the ethics committees atUppsala and Lund Universities Islets were obtained from 68nondiabetic donors (30 females 37 males age 59 plusmn 10 BMI259 plusmn 35 HbA1c 55 plusmn 11 and days of culture 35 plusmn 19)and 10 T2D donors (4 females 6 males age 607 plusmn 12 BMI281plusmn45 HbA1c 71plusmn12 and days of culture 2plusmn09) Purityof the islet preparations was assessed by dithizone staininginsulin content and contribution of exocrine and endocrinetissue as previously described [15] The islets were culturedin CMRL 1066 (ICN Biomedicals Costa Mesa CA USA)supplemented with 10mML HEPES 2mML l-glutamine50120583gmL gentamicin 025 120583gmL Fungizone (GIBCO BRLGaithersburg MD USA) 20120583gmL ciprofloxacin (BayerHealthcare Leverkusen Germany) and 10mML nicoti-namide at 37∘C (5 CO

2) prior to RNA preparation

22 Microarray Gene Expression in Human Pancreatic IsletsRNA was isolated with the AllPrep DNARNA Mini Kit(Qiagen Hilden Germany) RNA quality and concentrationwere measured using an Agilent 2100 bioanalyzer (Bio-Rad Hercules CA USA) and Nanodrop ND-1000 equip-ment (NanoDrop Technologies Wilmington DE USA)The microarrays (GeneChip Human Gene 10 ST) wereperformed using the Affymetrix standard protocol as previ-ously described [15] The array data were summarized andnormalized with robust multiarray analysis (RMA) methodusing the oligo package from BioConductor Also batchcorrection was done with COMBACT function from SVApackage from BioConductor All data areMIAME compliantand the raw data have been deposited in a MIAME database(GEO accession number GSE 50398 and GSE 50397)

23 Glucose-StimulatedInsulinSecretion Isletswerehand-pickedunder a stereomicroscope and preincubated for 30min at37∘C in Krebs Ringer bicarbonate (KRB) buffer (pH 74) con-taining (in mM) 120 NaCl 25 NaHCO

3 47 KCl 12 MgSO

4

25 CaCl2 12 KH

2PO 10 HEPES supplemented with 01

bovine serum albumin N-2 hydroxyethylpiperazine-N1015840-2-ethanesulfonic acid (10mmolL) and 1mmolL glucose Eachincubation vial contained 12 islets in 10mL KRB buffer

Table 1 Characteristics of human pancreatic donors

Normoglycemic Hyperglycemic119873 (malefemale) 58 (3424) 20 (119)Age (years) 609 plusmn 109 64 plusmn 89BMI 254 plusmn 29 285 plusmn 45HbA1c 54 plusmn 03 69 plusmn 10Purity 70 plusmn 16 63 plusmn 20Donors withdiabetes 0 10

Data represented as mean plusmn SD

solution and was treated with 95 O2-5 CO

2to obtain

constant pH and oxygenation After preincubation the bufferwas changed to a KRB buffer containing either 1mM (basalsecretion) or 167mM glucose (stimulated secretion) Theislets were then incubated for 1 h at 37∘C in ametabolic shaker(30 cycles per min) Immediately after incubation an aliquotof the medium was removed for analysis of insulin using aradioimmunoassay kit (Euro-Diagnostica Malmo Sweden)Insulin content in homogenized human islets was assessedby ELISA (Mercodia Uppsala Sweden) and values werenormalized to the total DNA in each sample as determinedby a fluorometric assay (Quant-iT PicoGreen InvitrogenMolecular Probes Stockholm Sweden)

24 Gene Set Enrichment Analysis (GSEA) The GSEA soft-ware tool (version 2013 wwwbroadinstituteorggsea) wasused to identify KEGG pathways (MSigDB version 40)that show an overrepresentation of up- or downregulatedgenes between donors with hyperglycemia (HbA1c gt 6119873 = 20) and normoglycemia (HbA1c lt 6 119873 = 30)Briefly an enrichment score was calculated for each geneset (ie KEGG pathway) by ranking each gene by theirexpression difference using Kolmogorov-Smirnov statisticcomputing a cumulative sum of each ranked in each geneset and recording the maximum deviation from zero as theenrichment score

25 Statistical Analysis Data are presented as means plusmn SDDifferences in expression levels were analyzed by Studentrsquos 119905-test or nonparametric Mann-Whitney tests Correlation testswere analyzed using nonparametric Spearmanrsquos tests Themean centroid represents the normalized gene expressionlevels of all genes from all individuals in the analysis with amean of 0 and a variance of 1 All statistical tests were per-formed using the Statistical Package for the Social Sciences(SPSS) version 190 software (SPSS Chicago IL USA)

3 Results

In this study we used human islet microarray expressiondata obtained from 78 donors The donors were subdividedinto 58 normoglycemic donors with HbA1c level lt6 and20 hyperglycemic donors with HbA1c level gt6 (Table 1)Normalized expression microarray data were subjected topathway analysis of GSEA algorithm using 178 Kyoto Ency-clopedia forGenes andGenomes (KEGG) pathways Pathway








4 Discussion





0

000

025

050

00

2500 5000 7500 10000 12500 15000 17500 20000

minus06

minus025

minus05

minus04

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minus02

minus01

Enric

hmen

t sco

re (E

S)

Enrichment profile



Zero cross at 9293

Rank

ed li

st m

etric

(Sig

nal2

Noi

se)

Hits




(a)

000

025

050

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00

minus06

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minus04

minus03

minus02

minus01

Enric

hmen

t sco

re (E

S)0 2500 5000 7500 10000 12500 15000 17500 20000


Zero cross at 9293

Rank

ed li

st m

etric

(Sig

nal2

Noi

se)



Enrichment profile



(b)

05

04

03

02

01

00

0 2500 5000 7500 10000 12500 15000 17500 20000

Enric

hmen

t sco

re (E

S)


000

025

050

minus025

Rank

ed li

st m

etric

(Sig

nal2

Noi

se)


Zero cross at 9293


Enrichment profile



(c)

0 2500 5000 7500 10000 12500 15000 17500 20000


Enrichment profile


05

06

04

03

02

01

00

Enric

hmen

t sco

re (E

S)

000

025

050

minus025

Rank

ed li

st m

etric

(Sig

nal2

Noi

se)


Zero cross at 9293



(d)



0100200300400


500100015002000

Nor

mal

ized

mea

n ex

pres

sion

IRS4

IRS2

ABCC

8

CACN

A1A

CACN

A1C

CACN

A1D

GCK

KCNJ11

MAF

A

MAP

K10

PDX1

PIK3

R3

SLC2

A2

CACN

A1B

(a)

0

500

1000


600013000

Nor

mal

ized

mea

n ex

pres

sion

PAX6

IAPP

NEU

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NKX

2-2

NKX

6-1

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MAF

A

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A2

(b)

0

1

2

3

4

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6


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lin se

cret

ion

0

2

4

6

8

10

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1c (

)


(c)

0

1

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4

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cret

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024

6

810

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HbA

1c (

)


(d)

0

1

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cret

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0

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6

8

10

12

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)


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0

1

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0

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(f)











Acknowledgments



References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























4 Discussion





0

000

025

050

00

2500 5000 7500 10000 12500 15000 17500 20000

minus06

minus025

minus05

minus04

minus03

minus02

minus01

Enric

hmen

t sco

re (E

S)

Enrichment profile



Zero cross at 9293

Rank

ed li

st m

etric

(Sig

nal2

Noi

se)

Hits




(a)

000

025

050

minus025

00

minus06

minus07

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minus04

minus03

minus02

minus01

Enric

hmen

t sco

re (E

S)0 2500 5000 7500 10000 12500 15000 17500 20000


Zero cross at 9293

Rank

ed li

st m

etric

(Sig

nal2

Noi

se)



Enrichment profile



(b)

05

04

03

02

01

00

0 2500 5000 7500 10000 12500 15000 17500 20000

Enric

hmen

t sco

re (E

S)


000

025

050

minus025

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ed li

st m

etric

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nal2

Noi

se)


Zero cross at 9293


Enrichment profile



(c)

0 2500 5000 7500 10000 12500 15000 17500 20000


Enrichment profile


05

06

04

03

02

01

00

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hmen

t sco

re (E

S)

000

025

050

minus025

Rank

ed li

st m

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nal2

Noi

se)


Zero cross at 9293



(d)



0100200300400


500100015002000

Nor

mal

ized

mea

n ex

pres

sion

IRS4

IRS2

ABCC

8

CACN

A1A

CACN

A1C

CACN

A1D

GCK

KCNJ11

MAF

A

MAP

K10

PDX1

PIK3

R3

SLC2

A2

CACN

A1B

(a)

0

500

1000


600013000

Nor

mal

ized

mea

n ex

pres

sion

PAX6

IAPP

NEU

ROD1

NKX

2-2

NKX

6-1

GCK

MAF

A

PDX1

SLC2

A2

(b)

0

1

2

3

4

5

6


Insu

lin se

cret

ion

0

2

4

6

8

10

12

HbA

1c (

)


(c)

0

1

2

3

4

5

6


Insu

lin se

cret

ion

024

6

810

12

HbA

1c (

)


(d)

0

1

2

3

4

5

6


Insu

lin se

cret

ion

0

2

4

6

8

10

12

HbA

1c (

)


(e)

0

1

2

3

4

5

6


Insu

lin re

leas

e

0

2

4

6

8

10

12

HbA

1c (

)


(f)











Acknowledgments



References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

000

025

050

00

2500 5000 7500 10000 12500 15000 17500 20000

minus06

minus025

minus05

minus04

minus03

minus02

minus01

Enric

hmen

t sco

re (E

S)

Enrichment profile



Zero cross at 9293

Rank

ed li

st m

etric

(Sig

nal2

Noi

se)

Hits




(a)

000

025

050

minus025

00

minus06

minus07

minus05

minus04

minus03

minus02

minus01

Enric

hmen

t sco

re (E

S)0 2500 5000 7500 10000 12500 15000 17500 20000


Zero cross at 9293

Rank

ed li

st m

etric

(Sig

nal2

Noi

se)



Enrichment profile



(b)

05

04

03

02

01

00

0 2500 5000 7500 10000 12500 15000 17500 20000

Enric

hmen

t sco

re (E

S)


000

025

050

minus025

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ed li

st m

etric

(Sig

nal2

Noi

se)


Zero cross at 9293


Enrichment profile



(c)

0 2500 5000 7500 10000 12500 15000 17500 20000


Enrichment profile


05

06

04

03

02

01

00

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hmen

t sco

re (E

S)

000

025

050

minus025

Rank

ed li

st m

etric

(Sig

nal2

Noi

se)


Zero cross at 9293



(d)



0100200300400


500100015002000

Nor

mal

ized

mea

n ex

pres

sion

IRS4

IRS2

ABCC

8

CACN

A1A

CACN

A1C

CACN

A1D

GCK

KCNJ11

MAF

A

MAP

K10

PDX1

PIK3

R3

SLC2

A2

CACN

A1B

(a)

0

500

1000


600013000

Nor

mal

ized

mea

n ex

pres

sion

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IAPP

NEU

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MAF

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PDX1

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(b)

0

1

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6


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cret

ion

0

2

4

6

8

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1c (

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(c)

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024

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810

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HbA

1c (

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1

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5

6


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0

2

4

6

8

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1c (

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(e)

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4

5

6


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e

0

2

4

6

8

10

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HbA

1c (

)


(f)











Acknowledgments



References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0100200300400


500100015002000

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mal

ized

mea

n ex

pres

sion

IRS4

IRS2

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8

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CACN

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CACN

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(a)

0

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mal

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n ex

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sion

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0

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8

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HbA

1c (

)


(f)











Acknowledgments



References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























Acknowledgments



References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article downregulation of type ii diabetes...

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