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Diabetes genes identified by genome-wide association studies are regulated in mice by nutritional factors… Ho, Maggie M; Yoganathan, Piriya; Chu, Kwan Y; Karunakaran, Subashini; Johnson, James D; Clee, Susanne M Feb 25, 2013

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RESEARCH ARTICLE Open AccessDiabetes genes identified by genome-wideassociation studies are regulated in mice bynutritional factors in metabolically relevant tissuesand by glucose concentrations in isletsMaggie M Ho1, Piriya Yoganathan1, Kwan Yi Chu1, Subashini Karunakaran1, James D Johnson1,2and Susanne M Clee1*AbstractBackground: Genome-wide association studies (GWAS) have recently identified many new genetic variantsassociated with the development of type 2 diabetes. Many of these variants are in introns of known genes orbetween known genes, suggesting they affect the expression of these genes. The regulation of gene expression isoften tissue and context dependent, for example occurring in response to dietary changes, hormone levels, ormany other factors. Thus, to understand how these new genetic variants associated with diabetes risk may act, it isnecessary to understand the regulation of their cognate genes.Results: We identified fourteen type 2 diabetes-associated genes discovered by the first waves of GWAS for whichthere was little prior evidence of their potential role in diabetes (Adam30, Adamts9, Camk1d, Cdc123, Cdkal1,Cdkn2a, Cdkn2b, Ext2, Hhex, Ide, Jazf1, Lgr5, Thada and Tspan8). We examined their expression in metabolicallyrelevant tissues including liver, adipose tissue, brain, and hypothalamus obtained from mice under fasted, non-fasted and high fat diet-fed conditions. In addition, we examined their expression in pancreatic islets from thesemice cultured in low and high glucose. We found that the expression of Jazf1 was reduced by high fat feeding inliver, with similar tendencies in adipose tissue and the hypothalamus. Adamts9 expression was decreased in thehypothalamus of high fat fed mice. In contrast, the expression of Camk1d, Ext2, Jazf1 and Lgr5 were increased in thebrain of non-fasted animals compared to fasted mice. Most notably, the expression levels of most of the geneswere decreased in islets cultured in high glucose.Conclusions: These data provide insight into the metabolic regulation of these new type 2 diabetes genes that willbe important for determining how the GWAS variants affect gene expression and ultimately the development oftype 2 diabetes.Keywords: Type 2 diabetes, Gene expression, Genome-wide association, High fat diet, Feeding and fasting, Mice,Pancreatic islets, Glucotoxicity* Correspondence: susanne.clee@ubc.ca1Department of Cellular and Physiological Sciences, Life Sciences Institute,University of British Columbia, Vancouver, CanadaFull list of author information is available at the end of the article© 2013 Ho et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.Ho et al. BMC Genetics 2013, 14:10http://www.biomedcentral.com/1471-2156/14/10BackgroundNearly 350 million people world-wide are currentlyaffected by diabetes, and the number of people with type2 diabetes mellitus is increasing at an alarming rate [1].Type 2 diabetes results when the β-cells of the pancreasare no longer capable of producing sufficient insulin tomeet the body’s demands. Thus β-cell dysfunction is akey component of type 2 diabetes pathology. Althoughthe increased prevalence of obesity and resulting insulinresistance is contributing to the increased prevalence oftype 2 diabetes, many obese individuals are insulin re-sistant but do not develop diabetes [2]. Genetic factors,many of which have been proposed to affect β-cellfunction, play an important role in determining anindividual’s risk within this context [3-6]. In a smallnumber of individuals, type 2 diabetes is caused by raresingle gene mutations, but for most individuals type 2diabetes results from the combined effects of many com-mon single-nucleotide polymorphisms (SNPs), each ofwhich have a small effect on risk and likely interactwith each other and with environmental and lifestylefactors [7].Genome-wide association studies (GWAS) have re-cently revealed many novel SNPs associated with type 2diabetes. These include SNPs located in the regions nearTCF7L2, HHEX-IDE, EXT2, FTO, SLC30A8, IGF2BP2,CDKAL1, and CDKN2A-CDKN2B [8-13]. A secondphase of studies identified many additional variants,including those near JAZF1, TSPAN8-LGR5, THADA,ADAMTS9, NOTCH2-ADAM30, CDC123-CAMK1D, andKCNQ1 [14,15]. The two genes in which commonvariants were previously convincingly associated withtype 2 diabetes, PPARG and KCNJ11, were also identi-fied in these GWAS [12,16,17]. More recently, numerousother SNPs have been identified in additional GWASand meta-analyses [18].The association between variants in TCF7L2 and type2 diabetes, which was identified initially in pre-GWASstudies, has been almost universally replicated and hasthe largest effect on diabetes risk [19,20]. This transcrip-tion factor is known to play a role in WNT signallingand pancreatic development [20]. Variation in FTO hasbeen shown to influence risk of type 2 diabetes throughits effects on promoting obesity [8]. Variation withinIDE, the insulin degrading enzyme, has previously beenassociated with risk of type 2 diabetes in both humansand rats, although these findings were not consistentlyreplicated [21-26]. SLC30A8 is a zinc transporterexpressed in β-cells and is known to be involved in insu-lin granule formation and insulin secretion [27-29].IGF2BP2 encodes insulin like growth factor 2 mRNAbinding protein 2, which plays a role in RNA stabilityand localization and has been suggested to affect pancre-atic development [30]. NOTCH2 is a transcription factorimportant for pancreatic development [31] and theNotch pathway plays a role in adult beta-cell survival[32]. KCNQ1 is a voltage-gated potassium channel thathas been shown to affect insulin secretion [33]. However,the mechanisms by which the remainder of these genesaffect diabetes risk are largely unknown. Thus, wesought to obtain evidence of their potential role in meta-bolic disease.Most of the diabetes-associated SNPs were found innon-coding regions of the genome and are thus likely toaffect gene regulation. In order to understand how thesegenes affect type 2 diabetes and how the SNPs associatedwith diabetes affect gene expression, we need to firstunderstand the physiological processes that regulate theexpression of these genes. We examined the expressionpatterns of these potential new diabetes-susceptibilitygenes to determine which are expressed in tissues im-portant for the development of type 2 diabetes. Thismay also suggest the potential mechanism(s) by whichalterations in these genes affect diabetes risk (e.g. insulinsecretion versus insulin sensitivity). We also sought todetermine whether any of these genes are regulated byconditions known to alter the expression of metabolic-ally relevant genes. We examined the expression of thesegenes under fasting and non-fasting conditions (e.g. inresponse to insulin), which might be altered if they affectperipheral insulin sensitivity. Consumption of diets highin fats and sugars is associated with risk of developingtype 2 diabetes [34] and many genes that are critical forβ-cell function are regulated by glucose [35]. Thus, wealso compared their expression in fasted mice consum-ing a normal chow diet or a diet high in fat and sugar,and examined the expression of these genes in mousepancreatic islets cultured under low and high glucoseconcentrations. Here we show that most of the diabetes-associated genes are expressed in many metabolicallyrelevant tissues and the expression levels of several ofthese genes were decreased by high fat feeding or wereincreased in the fed state in the brain. In addition, wefound most of these genes are down-regulated byincreased glucose concentrations in mouse islets.ResultsTissue distribution of gene expressionHigh throughput gene expression profiling has pre-viously been performed to identify gene expressionpatterns across a wide variety of tissues [36], but suchmicroarray-based data must be complemented by accur-ate and quantitative analysis. Thus, we performed qPCRanalysis of the recently identified diabetes susceptibilitygenes across a panel of tissues (Table 1) to determinetheir relative expression levels in each tissue. Most ofthese genes were expressed in many metabolically rele-vant tissues including pancreatic islets, liver, white andHo et al. BMC Genetics 2013, 14:10 Page 2 of 12http://www.biomedcentral.com/1471-2156/14/10brown adipose tissue, skeletal muscle, and heart. Inaddition, most were expressed in the hypothalamus andin regions of the brain outside the hypothalamus. Few ofthese genes were robustly expressed in skeletal muscleor small intestine. We detected expression of all thegenes in pancreatic islets except Adam30, Cdkn2a, andLgr5. Together, these studies point to several metabolic-ally relevant tissues as potential key sites of action of thediabetes susceptibility genes identified by the GWAS.Regulation of GWAS diabetes genes by dietary status inthe liverTo determine whether these new type 2 diabetes suscep-tibility genes are regulated by nutritional manipulations,we examined whether there was a change in their ex-pression between tissues from fasted and non-fastedchow-fed mice, or between tissues from fasted chow andfasted high fat diet-fed mice. We used a common con-trol group (fasted, chow-fed mice) to which the non-fasted, chow-fed mice and the fasted, high fat diet-fedmice were compared. As the liver is a key metabolicorgan and a major target of insulin action, we firstexamined the expression of these genes in the liver(Figure 1). The expression of Jazf1 was decreased by ap-proximately 70% in mice fed a high fat diet compared tochow-fed controls. Similar differences were observed forAdamts9 (75% decrease) and Hhex (60% decrease).Whereas we could detect expression of the Arf isoformof Cdkn2a in chow-fed mice, its expression was detectedin only 2 samples from the high fat diet-fed mice. Incontrast to many classical transcriptional targets of insu-lin signalling, none of the genes examined in this studyhad significantly altered expression in the livers of fastedversus non-fasted mice. Again, while it was found inmost of the liver samples from the chow-fed fasted con-trol mice, the expression of Cdkn2a (Arf ) was detectedonly sporadically in the livers of non-fasted mice. WhileCdkn2b was expressed in all the samples, its expressiondid not differ between groups. No differences inTable 1 Relative expression patterns of GWAS diabetes genes in chow fed miceIsl Brain Hypo P-AT BAT Liver SkM Hrt St SI LI Lung Kid Spl Ov Ut ThyAdam30 NE Low NE Avg NE NE Avg Avg Avg NE NE NE NE Avg NE NE AvgAdamts9 Low Low Avg Avg Avg Avg NE Avg Avg NE Avg Avg Avg Avg High High AvgCamk1d Avg Avg Avg Avg Avg Avg Low Avg Avg NE High NE Avg Avg Avg Avg HighCdc123 Avg Avg Avg Avg Avg Avg Low Avg Avg NE Avg Low Avg Avg High Avg AvgCdkal1 Avg Avg Avg Avg Avg Avg Avg Avg Avg Avg Avg Low Avg Avg Avg Avg AvgCdkn2a (Arf ) Avg Avg Avg Avg Avg Avg Avg Avg Avg Avg Avg Avg Avg Avg Avg Avg AvgCdkn2a NE NE NE Avg Avg NE NE NE NE NE Avg NE Avg Avg NE NE LowCdkn2b High Avg Avg Avg Avg Avg NT Avg NT High NT NT Avg NT High Low LowExt2 Low Avg Avg Avg Avg Avg Low Avg Avg Avg Avg Low Avg Avg High Avg AvgHhex Low Avg Avg Avg Avg High Low Avg Low Low Avg Low Avg High Avg High AvgIde Avg Avg Avg Avg High Avg NT Avg NT Avg NT NT NT NT High Low LowJazf1 Avg Avg Avg Avg High NE NE Avg Low Low Avg Low Low Low Avg High AvgLgr5 NE High Avg Avg Low Avg Low Avg Avg NE Avg Low NE Avg High High AvgThada Avg Avg Avg Avg Avg Avg NE Avg Avg NE Avg Low Avg Avg High Avg AvgTspan8 Low Low Low Avg Avg Low Low Avg High NE High NE Avg High Avg High LowExpression levels are relative within a gene, but comparisons between genes cannot be made. Isl = islets, Hypo = hypothalamus, P-AT = parametrial white adiposetissue, BAT = brown adipose tissue, SkM = skeletal muscle (soleus),Hrt = heart, St = stomach, SI = small intestine (duodenum), LI = large intestine (distal), Kid =kidney, Spl = spleen, Ov = ovary, Ut = uterus, Thy = thymus. NE = not expressed, Ct values within 2 cycles of negative control; avg. = average expression (within 5-fold (2.3 cycles) of average expression across all tissues); high = >5 fold higher (Ct values >2.3 cycles lower) than average expression; low = >5 fold lower(Ct value >2.3 cycles higher) than average expression. NT = not tested.Adamts9 Hhex Jazf10., ChowFasted, HFDFasted, Chow****Fold ChangeFigure 1 Regulation of new diabetes genes by nutritionalstatus in the liver. Data are shown as fold-change relative to thoseobserved in the fasted chow-fed mice. The number of samples pergroup for the fasted chow-fed mice, fasted HFD-fed mice and non-fasted (fed) chow-fed mice, respectively are Adamts9 (7, 7, 4), Hhex(7, 5, 5), Jazf1 (7, 7, 8). P-values are shown for the indicated groupcompared to the fasted chow-fed controls. Data are shown as thefold change (2ΔΔCt) ± 2ΔΔCt±SE [87]. * P < 0.05, ** P < 0.01.Ho et al. BMC Genetics 2013, 14:10 Page 3 of 12http://www.biomedcentral.com/1471-2156/14/10expression were observed for Camk1d, Cdc123, Cdkal1,Ext2, Ide, Lgr5, Thada, or Tspan8 (not shown), al-though expression of Cdc123 was decreased ~35% inhigh fat diet-fed mice (P = 0.07). Thus, while the hepaticexpression of some of these new diabetes genes isregulated by chronic consumption of diets high in fatand sugar, we do not find evidence that they are acutelyregulated in response to feeding and fasting as might beexpected for key insulin-responsive genes.Regulation of GWAS diabetes genes by dietary status inadipose tissueAdipose tissue is a critical insulin-responsive tissue. Fatalso produces many factors, adipokines, that may con-tribute to whole body insulin sensitivity. We examinedwhether any of these diabetes susceptibility genes areregulated by nutritional status in adipose tissue (Figure 2).Cdkn2b and Thada expression levels were decreased ~50and 30%, respectively, in fasted chow versus high fatdiet-fed mice. Jazf1 expression may also be reduced(~45%, P = 0.06, not shown). In contrast Ide expres-sion levels were ~1.6 fold higher in high fat-fedmice. The expression of Adam30 was decreased over80% in fasted versus non-fasted mice, although thisdid not reach statistical significance (P = 0.06). Noneof the remaining genes expressed in adipose tissueshowed significant changes in expression for eithercondition (Figure 2), suggesting little involvement ofthese new diabetes genes in adipose tissue function.This is in contrast to many of the newly identifiedobesity genes [37].Regulation of GWAS diabetes genes by dietary status inthe brainRecent studies have shown important neuronal contributionsto the regulation of metabolism, insulin sensitivity and isletfunction, particularly within regions of the hypothalamus[38-41]. Thus, we examined the expression of the recentlyidentified type 2 diabetes susceptibility genes in the hypo-thalamus (Figure 3) and the remainder of the brain(Figure 4). In the hypothalamus, the expression ofAdamts9 and Camk1d were decreased by ~80%, in fastedchow versus fasted high fat diet-fed mice (Figure 3).Cdkn2b expression was similarly decreased, although thisdid not reach statistical significance (not shown). ThemRNA levels of Jazf1 and Thada were 60-70% lower inthe high fat diet-fed mice. Lgr5 also tended to bedecreased (not shown). In fasted versus non-fasted chow-fed mice, Cdkal1 expression was decreased by approxi-mately 50% (Figure 3). No significant differences werefound for Cdc123, the Arf isoform of Cdkn2a, Ext2, Hhex,Ide, Lgr5, or Tspan8.In the remainder of the brain outside the hypothal-amus none of the genes were significantly affected bythe consumption of the high fat diet, but strikingly manyof them were regulated by feeding/fasting status(Figure 4). Cdkn2b expression was increased ~3-fold byboth conditions, but neither reached statistical signifi-cance. Only four genes expressed in the brain, Cdc123,Cdkal1, Ide, and Thada, were not affected by either ex-perimental condition. Camk1d expression was increasedover 7-fold in non-fasted versus fasted chow-fed mice.Jazf1 and Lgr5 expression were increased 3.5 and 4.5-fold, respectively, in non-fasted mice. Ext2 and Hhexhad a 2 to 2.5-fold increase in expression in fasted ver-sus non-fasted mice. Tspan8 on the other hand, had asignificant 60% decrease in expression in non-fasted ver-sus fasted mice. Like in the liver, the expression of theArf isoform of Cdkn2a was low but detectable in bothchow-fed groups, but not in the mice consuming thehigh fat diet. Adam30 expression was only detected inthe brains of non-fasted chow-fed mice. Together, theseresults illustrate that the expression of several type 2 dia-betes susceptibility genes can be metabolically regulatedin the brain and point to the potential importance ofneuronal function in type 2 diabetes susceptibility.Regulation of GWAS diabetes genes by glucose inpancreatic isletsMany of the recently discovered type 2 diabetes geneshave been suggested to affect the development and/orfunction of pancreatic islets [6]. The function, growthand survival of β-cells can be regulated acutely andchronically by glucose [34]. Thus, we examined whetherthe new type 2 diabetes susceptibility genes are regulatedby overnight incubation in low (5 mM) or high (25 mM)Cdkn2b Ide Thada0123Fed, ChowFasted, HFDFasted, Chow***Fold ChangeFigure 2 Regulation of new diabetes genes by nutritionalstatus in adipose tissue. Data are shown as fold-change relative tothose observed in the fasted chow-fed mice (n = 7 for the fastedchow-fed group, 6 for the fasted HFD-fed mice and 8 for the fedchow-fed mice). P-values are for the comparison between theindicated group and the fasted, chow-fed control mice. Data areshown as the fold change (2ΔΔCt) ± 2ΔΔCt±SE [87]. * P < 0.05.Ho et al. BMC Genetics 2013, 14:10 Page 4 of 12http://www.biomedcentral.com/1471-2156/14/10glucose (Figure 5). Most genes were significantly ortended to be downregulated under conditions of highglucose. Cdkal1, Cdkn2a (Arf, P = 0.07), Ide, Jazf1,Camk1d, and Tspan8 (P = 0.06) expression levels weredecreased ~50-60%. Meanwhile, the expression ofCdkn2b, Hhex (P = 0.10), Cdc123, Adamts9 (P = 0.09),and Thada were reduced 30-40%. To ensure the isletsincubated in high glucose did not have globallydecreased expression, we examined the expression ofTxnip, which has been shown to be highly upregulatedby glucose [35] and found that its expression was stillsignificantly elevated in the islets cultured in high glu-cose (Figure 5). Mouse islets consist of β-cells andother cell types. Thus, the MIN6 β-cell line was alsoexamined. We found that all the genes were expressedin this cell line (not shown), although this does notpreclude that they also are expressed in other celltypes within the islet.Adamts9 Camk1d Cdkal1 Jazf1 Thada0., ChowFasted, HFDFasted, Chow*******Fold ChangeFigure 3 Regulation of new diabetes genes by nutritional status in the hypothalamus. Data are shown as fold-change relative to thoseobserved in the fasted chow-fed mice. The number of samples per group for the fasted chow-fed mice, fasted HFD-fed mice and non-fasted(fed) chow-fed mice, respectively are Adamts9 (7, 5, 4), Camk1d (7, 5, 5), Cdkal1 (10, 5, 9), Jazf1 (10, 8, 7), Thada (7, 5, 5). P-values are shown for theindicated group compared to the fasted chow-fed controls. Data are shown as the fold change (2ΔΔCt) ± 2ΔΔCt±SE [87]. * P < 0.05, ** P < 0.01.Adamts9Camk1dExt2HhexJazf1 Lgr5Tspan802468101214Fed, ChowFasted, HFDFasted, Chow*********Fold ChangeFigure 4 Regulation of new diabetes genes by nutritional status in the remainder of the brain. Data are shown as fold-change relative tothose observed in the fasted chow-fed mice. HFD = high fat diet. The number of samples per group for the fasted chow-fed mice, fasted HFD-fedmice and non-fasted (fed) chow-fed mice, respectively are (6, 5, 10) except for Adamts9 (6, 4, 10). P-values are shown for the indicated groupcompared to the fasted chow-fed controls. Data are shown as the fold change (2ΔΔCt) ± 2ΔΔCt±SE [87]. * P < 0.05, ** P < 0.01.Ho et al. BMC Genetics 2013, 14:10 Page 5 of 12http://www.biomedcentral.com/1471-2156/14/10DiscussionThe goal of the present study was to understandwhether metabolic factors affect the expression of thegenes recently implicated in the development of type 2diabetes for which there was little prior evidence of theirpotential role(s) in this disease. Although many add-itional SNPs have been identified in subsequent GWASand meta-analyses [18], we focussed these studies on thegenes identified in the first waves of GWAS, as thesehave been the subject of most follow-up studies to date.Specifically, we examined acute changes in expression ofthese genes in response to feeding and fasting andlonger term changes in the expression of these genes inresponse to a diet high in fat and sugar, recognized as acritical environmental risk factor for type 2 diabetes.It has been hypothesized that most of the new geneticvariants affect β-cell function, development or survivalbut not insulin sensitivity [6]. Consistent with this,we found all of the genes except Adam30 and Cdkn2awere expressed in pancreatic islets. These genes wereexpressed, however in the transformed β-cell line,MIN6. The expression of all the genes except Lgr5decreased following incubation of the islets in high glu-cose concentrations. It can thus be hypothesized thatthese genes may normally play a beneficial role in isletfunction, and a reduction in the expression of thesegenes could contribute to glucotoxic β-cell dysfunctionor survival. However, we also found evidence that mostof the genes could have potential roles in othermetabolically-relevant tissues. Genes affecting insulinsensitivity may be expected to be expressed in peripheralinsulin sensitive tissues, such as liver and adipose tissue,and be responsive to metabolic status. Consumption of ahigh fat diet was associated with a tendency for the ex-pression of several of these genes to be decreased. Simi-larly, many of the genes were regulated by feeding andfasting. Only the two splice isoforms of Cdkn2a had noevidence of metabolic regulation in any of the othertissues examined.Jazf1, also known as Tip27, encodes a transcriptionalrepressor of Nr2c2, an orphan nuclear receptor of thesteroid receptor family also known as TR4 and TAK1.Nr2c2 has been reported to modulate apoptosis [42,43]and its loss in mice is associated with reduced mito-chondrial function and increased oxidative stress, andconversely with reduced adipose tissue inflammation,hepatic steatosis and insulin resistance [44-46]. Jazf1-mediated alterations in Nr2c2 could thus affect both in-sulin sensitivity and β-cell function. Genetic variation inJazf1 has been variably associated with measures of insu-lin sensitivity and β-cell function [47-52], and our ex-pression data support roles for this gene in both. Jazf1was expressed in nearly all tissues examined and its ex-pression in islets was decreased following culture in highglucose-containing media. Consistent with a pathogenicrole in islets, it has recently been shown that JAZF1expression is reduced in individuals with type 2 dia-betes or hyperglycemia, and that JAZF1 expressionwas correlated with insulin secretion [53]. However, ourfindings suggest the reduced expression may be a conse-quence of their hyperglycemia, not the underlying cause.These data are consistent, however, with a role of Jazf1 inTxnipAdamts9Camk1dCdc123Cdkal1Cdkn2a (Arf)Cdkn2b Ext2Hhex IdeJazf1 Lgr5ThadaTspan80. Glucose5mM Glucose**********Fold ChangeFigure 5 Regulation of new diabetes genes by glucose levels in pancreatic islets. Data are shown as fold-change, (2ΔΔCt) ± 2ΔΔCt±SE [87],relative to those observed in the islets incubated in low (5 mM) glucose. Each group is the average of three replicates, each of which wascomprised of pooled islets from two mice. * P < 0.05, *** P < 0.001.Ho et al. BMC Genetics 2013, 14:10 Page 6 of 12http://www.biomedcentral.com/1471-2156/14/10further accelerating β-cell dysfunction once individualsdevelop hyperglycemia or perhaps impaired glucose toler-ance. Jazf1 expression in the liver and hypothalamus wasdecreased in mice fed the high fat diet, with the same ten-dency in adipose tissue. The GWAS SNPs may affect theexpression of Jazf1 in adipose tissue, suggesting that itsfunction in this tissue may be important for its role in type2 diabetes [54]. That we also observed changes in the ex-pression of Jazf1 in the brain and hypothalamus, suggestsfurther potentially important sites of action.Adamts9 is an anti-angiogenic factor known to beexpressed in vascular endothelial cells [55] and isimplicated in endoplasmic reticulum to Golgi transport[56]. Although some studies have found associations be-tween SNPs in this gene and various measures of insulinsensitivity or secretion [49,50], many others have not[47,48,52,57]. Microvascular structure affects both insu-lin secretion and sensitivity [58]. Adamts9 expressiontended to be downregulated by high glucose in islets andwas decreased in the hypothalamus and liver of high fatdiet-fed mice. These data suggest that this gene may playa role in the neural regulation of metabolism in additionto having effects, perhaps on insulin sensitivity, in theliver and also in islets that may be related to its role invascularization [59].Hhex encodes a homeobox transcription factor knownto be involved in pancreatic and liver development[60,61]. SNPs in HHEX have been associated withdecreased insulin secretion perhaps due to alterations invesicle docking [57,62-66]. Most studies have failed tofind associations between HHEX SNPs and insulin sensi-tivity [52,57,62-64,67], although associations with insulinclearance and hepatic insulin sensitivity have beenreported [52,64]. We found Hhex to be expressed in sev-eral tissues besides the pancreas, with evidence ofdecreased expression in the liver in response to high fatfeeding and increased expression in the brain in non-fasted mice. These findings provide potential support forroles of Hhex in metabolism outside the pancreas. TheGWAS SNPs associated with type 2 diabetes are locatedbetween Hhex and Ide. There was prior evidence for arole of Ide in type 2 diabetes [21,22,25,26]. We found Ideexpression to be increased by high fat feeding in adiposetissue and to be decreased by the incubation of islets inhigh glucose, consistent with a role of Ide in β-cell func-tion [63]. Combined, these data suggest potential rolesfor both Hhex and Ide in type 2 diabetes susceptibility.Little is known about the potential roles of Thada inmetabolic disease. It is a gene associated with a commonchromosomal breakpoint in thyroid cancers, that mayaffect cell death receptors [68]. We found widespreadexpression of Thada and noted its decreased expressionin islets following culture in high glucose. Thada expres-sion was also reduced in response to high fat feeding inboth adipose tissue and the hypothalamus. No evidencefor association of SNPs in this gene with insulin sensitiv-ity have been found [48,49,52,57], and while most stud-ies have not found associations with insulin secretion[47-49,57], an association with reduced insulin secretionin response to non-nutrient secretagogues and poten-tially β-cell mass has been reported [50]. Our data areconsistent with a primary role of this gene in the pan-creas in determining type 2 diabetes susceptibility, al-though indicate it may also have effects on theregulation of energy balance and metabolism.Although Adam30 (A disintegrin and matrix metalloprotease30) has been reported to be expressed only in the testis[69], we found evidence of expression in several metabol-ically relevant tissues including the brain, adipose tissue,heart and stomach. It was not expressed in islets. Weobserved a marked reduction of its expression in adiposetissue collected from non-fasting animals, providing a po-tential site of action whereby this gene may affect metab-olism and thus type 2 diabetes risk.In contrast to the above genes, the expression of othergenes were not widely altered aside from within pancre-atic islets, consistent with the primary mechanism bywhich they are associated with diabetes being throughalterations in islet biology. The mechanisms by whichSNPs at the CAMK1D-CDC123 locus affect diabetessusceptibility are unknown, and it is unclear which ofthese two genes is affected by the causative genetic vari-ation. Some, but not all, studies have found associationsof SNPs at this locus with insulin secretion, while noassociations with insulin sensitivity have been found[47-50,52,57]. There is evidence that genetic variationnear CAMK1D can affect its expression, at least inlymphocytes [54]. The expression of both genes wassimilarly reduced in islets cultured in high glucose,suggesting the possibility that they are under commonregulatory control in these cells. We found decreased ex-pression of Camk1d in the hypothalamus of high fat-fedmice and increased expression in other regions of thebrain in non-fasted mice, suggesting it may affect theneuronal control of metabolism or islet function. Incontrast, no substantial changes in Cdc123 expression inresponse to feeding and fasting or high fat diet con-sumption were observed. As the SNPs at this locus areprimarily associated with insulin secretion and the ex-pression of both genes in islets was altered, these datacannot distinguish which may be the causative gene.SNPs within CDKAL1 have been associated with insulin se-cretion and not insulin sensitivity [13,47,52,57,63,66,67,70].Cdkal1 is a tRNA modification enzyme. Specifically, this pro-tein is a methylthiotransferase that modifies tRNALys, stabiliz-ing interactions between the tRNA and mRNA, decreasingmisreading of its cognate codon [70]. Mice deficient in Cdkal1have impaired glucose tolerance and insulin secretion, andHo et al. BMC Genetics 2013, 14:10 Page 7 of 12http://www.biomedcentral.com/1471-2156/14/10evidence of β-cell ER stress [71,72]. We found that Cdkal1 ex-pression in pancreatic islets was decreased following incuba-tion in high glucose, which could contribute to β-celldysfunction in settings of hyperglycemia. Interestingly, we alsofound that Cdkal1 expression was reduced in the fed state inthe hypothalamus, suggesting it may have metabolic functionsin addition to those in insulin synthesis and secretion.The GWAS have identified SNPs at the Cdkn2a-Cdkn2blocus. The Cdkn2a (cyclin dependent kinase inhibitor 2a)gene has two alternative splice isoforms that encode dis-tinct proteins, Cdkn2a and Arf. The Arf isoform isgenerated by the use of an upstream alternative first exon.Both are involved in cell cycle control. We found expres-sion of the Arf isoform to be very low in chow-fed miceand not detectable in most tissues from high fat diet-fedmice. We found higher levels of expression of this gene inislets, and potential downregulation of its expression byhigh glucose concentrations. In contrast, the Cdkn2aisoform was found in a limited number of tissues and notin islets. These data suggest that Arf might be the relevantisoform of Cdkn2a, affecting diabetes by affecting β-cellmass. Given the loss of expression of this gene in high fatdiet-fed mice it is tempting to speculate that this may be amechanism affecting high fat diet-induced metabolic dys-function. The expression of Cdkn2b was decreased in adi-pose tissue of high fat-fed mice. As this gene encodes acell cycle inhibitor, this reduced expression may reflectincreased proliferation of adipocyte precursors or perhapsinfiltrating inflammatory cells. Interestingly, Cdkn2b ex-pression was also decreased in islets incubated in high glu-cose, consistent with a role in the regulation of β-cellmass and glucose-induced β-cell proliferation [73]. Thus,as with the Hhex-Ide locus, these studies cannot distin-guish whether Cdkn2a or Cdkn2b is the causative gene,and actually suggest a role for both in the development oftype 2 diabetes.Ext2 is a glycosyltranferase involved in the synthesis ofheparin sulphate, and mutations in this gene areassociated with abnormal bone growths (exostoses) [74].This gene may also be involved in neural development[75]. The association between SNPs in this gene andtype 2 diabetes has not been as well replicated[10,12,13,62]. We found increased expression of thisgene in brain, suggesting a possible site of action as towhere this gene could affect diabetes risk.Lgr5 is a seven transmembrane receptor and a mem-ber of the rhodopsin family [76]. It is a marker ofmitotically active intestinal stem cells and potentiatesWnt/β-catenin signalling [76,77]. This is the only genefor which we did not find a significant decrease in ex-pression in islets cultured in high glucose, although thiscertainly does not preclude it from having a role in pan-creatic and β-cell development. Its expression wasincreased in the brains of non-fasted mice, suggestinganother potential site of action through which it maymediate type 2 diabetes susceptibility.Tspan8, also known as Co-029, is a cell surface proteinimplicated in pancreatic, colon and liver tumors andtheir metastasis, possibly through interaction withintegrins [78]. Although some studies have foundassociations between SNPs in this gene and insulin sen-sitivity or secretion [47,57], others have not [48-50,52].Loss of this gene is associated with decreased bodyweight, although there were no detectable effects on glu-cose tolerance or insulin sensitivity [79]. In contrast tothat study which did not detect expression of this genein mouse pancreas [79], we found expression of thisgene in isolated pancreatic islets and suggested regula-tion of its expression by glucose. Tspan8 expression wassignificantly decreased in brains of fed compared tofasted chow-fed mice, suggesting it may also have a rolein the neural control of metabolism.In summary, we have identified nutritional regulationof many of the newly found type 2 diabetes-associatedgenes. As these studies were performed with a relativelysmall number of samples, it should be noted that smallerchanges in expression may also exist that we had insuffi-cient power to detect. These data provide support forthe involvement of these newly identified type 2 diabetessusceptibility genes in β-cell function and also suggestpotential roles for many of them in peripheral tissues,notably in the brain and hypothalamus, highlighting thepotential importance of neuronal regulation of metabol-ism and islet function to type 2 diabetes [38-41]. Ourstudy also highlights the tissue-specific regulation ofthese genes (changes in one or more tissues where thegene is expressed but not in all tissues), suggesting thatthe SNPs identified in the GWAS studies may need tobe examined in the appropriate tissues and under severalmetabolic contexts [37]. Indeed, recent studies aimed atidentifying genetic variants that affect gene expression(eQTLs) have found varying effects of these SNPs ongene expression in different tissues, particularly for SNPslocated within not between genes, and notably that theSNPs were more associated with expression of diabetes-associated genes in metabolically relevant tissues such asliver, adipose and muscle than in lymphocytes, which aresometimes used as a surrogate because they are easilyaccessible [80-82]. The abundant regulation of thesegenes by nutritional status found in our study alsosuggests there are likely gene-diet interactions involvingthese SNPs [83] that may be a complicating factor in fu-ture human studies to assess the functional implicationsof the associated SNPs.ConclusionsAs SNPs may affect the regulation of genes up to 1 Mbaway [84], future studies should examine the regulationHo et al. BMC Genetics 2013, 14:10 Page 8 of 12http://www.biomedcentral.com/1471-2156/14/10of other genes in the region of the associated SNPs toidentify other possible candidates. Future studies shouldalso examine the regulation of the remaining newlydiscovered type 2 diabetes-associated genes and theirneighbours. Studies to discover how the type 2 diabetes-associated GWAS SNPs affect the regulation of nearbygenes to promote diabetes will be important to realizethe value of the GWAS studies, however our findingssuggest that such studies will need to be carried out inthe appropriate tissues and under controlled environ-mental conditions.MethodsAnimalsFemale C57BL/6 J mice were housed in an environmen-tally controlled facility with 14 hour light cycles (7 am -9 pm) with unlimited water and were fed either a standardrodent chow (LabDiet 5010, Jamieson’s Pet FoodDistributors, Delta, BC, Canada) or a diet containing 60%calories from fat (primarily lard) and 20% calories fromsugar (sucrose and maltodextrin; D12492, Research Diets,New Brunswick, NJ) from weaning. Mice were sacrificedat 8 weeks of age by CO2 asphyxiation. Mice weresacrificed either after a physiological 4 hour fast (9 am -1 pm) or at 9 am without fasting, as indicated. Tissueswere rapidly collected and flash frozen in liquid nitrogen.All procedures were approved by the UBC Committee onAnimal Care and were performed according to CanadianCouncil on Animal Care guidelines.Pancreatic islet isolationIslets were isolated from chow-fed female C57BL/6 Jmice by collagenase digestion, using previously describedmodifications [32] of the filtration method reported bySalvalaggio et al. [85]. We handpicked islets into dishesof RPMI media (Invitrogen, Burlington, ON, Canada)containing either 5 mM glucose or 25 mM glucose. Al-though the mice were not fasted prior to islet isolation,the islets were incubated in these conditions overnightat 37°C and 5% CO2 prior to RNA isolation. Experimentswere performed in triplicate; each replicate comprised of50 islets from each of 2 mice, for a total of 100 islets perreplicate.Measurement of gene expressionRNA extraction and cDNA synthesis were performed asdescribed [37]. Primers were designed for all the genesfrom the first waves of type 2 diabetes GWAS for whichthere was no other evidence of their potential role intype 2 diabetes. Primers were designed to span an intronand to be located in exons common to all isoforms forany genes with alternatively spliced forms, except forCdkn2a which has two well characterized isoforms, Arfand Cdkn2a, for which specific primers were generated.The primer sequences used for each gene are providedin Additional file 1: Table S1. Because these studies wereperformed over a span of several months, and in con-junction with other studies [37], the sample numbersvaried between genes (e.g. due to the addition of newsamples or when samples were completely used).Gene expression was assessed by real-time quantitativereverse transcription PCR (qPCR) using SYBR Green I-based detection (PerfeCTa SYBR Green FastMix, QuantaBiosciences, Gaithersburg, MD), as previously described[37]. We confirmed that only a single product was amp-lified from all samples through melt-curve analysis inaddition to the direct visualization of PCR amplificationproducts on an agarose gel prior to real-time analysis.Gapdh was selected as the reference gene as its exp-ression was more consistent than β-actin (Actb),Cyclophilin (Ppib), Arbp, and 18S RNA across the differ-ent experimental conditions and tissues.Delta Ct (ΔCt) values were calculated by subtractingthe cycle threshold (Ct) value for each gene from the Ctvalue of the control gene amplified contemporaneously.Delta delta Ct (ΔΔCt) values for each sample werecalculated by subtracting the ΔCt of each sample fromthe average ΔCt of the fasted mice consuming chow(control) group or the islets incubated in 5 mM glucose.For each gene, negative controls included both a no re-verse transcriptase (no RT) and no template control(water).For the tissue distribution, genes were consideredexpressed if the Ct value was at least 2 cycles lower (i.e.higher expression) than the lowest value in the negativecontrols, which was typically undetected. To examinethe relative expression levels of each gene across thetissues in which it is expressed, we calculated the ave-rage Ct value for all the tissues that each gene is expressedin. Tissues with at least a 5-fold higher (2.3 cycles lower)expression than the average across tissues are shown asrelatively “high” expression of that gene, while tissueswhere expression was more than 5-fold lower (Ct values >2.3 cycles higher) than the average are indicated as hav-ing relatively “low” expression of that gene. For eachgene, these calculations permit the comparisons of thesame gene between tissues, however due to likely vari-ation in primer efficiency, comparisons cannot be madebetween genes.Statistical analysisThe study was comprised of three groups: two experi-mental groups (non-fasted chow-fed and fasted high fatdiet-fed) each compared to a single control group (fastedchow-fed). Changes in gene expression within liver, adi-pose tissue, brain and hypothalamus, were comparedusing non-parametric Mann–Whitney U-tests for eachof the experimental groups compared to the controlsHo et al. BMC Genetics 2013, 14:10 Page 9 of 12http://www.biomedcentral.com/1471-2156/14/10because of the small sample sizes [37]. This approachwas chosen over the Kruskal-Wallis test because thecomparison between the fasted high fat diet-fed andnon-fasted chow-fed mice was not meaningful, as bothfasting and diet conditions differed between these twogroups. Unadjusted P-values are presented [86]. Comparisonsbetween islets incubated in low and high glucose wereperformed by Student’s t-test. Statistics were performed on theΔΔCt values, prior to conversion to fold-change [37,87] usingPrism (GraphPad Software). Data are shown as fold change,calculated as 2ΔΔCt, with the upper and lower limits calculatedas 2ΔΔCt ± its standard error, respectively [87].Additional fileAdditional file 1: Table S1. Primer sequences.AbbreviationsCt: Cycle threshold; GWAS: Genome-wide association; HFD: High fat diet;qPCR: Quantitative reverse transcription real time PCR; SNP: Single nucleotidepolymorphism.Competing interestsThe authors declare they have no competing interests.Authors’ contributionsMH, PY, KC and SK performed the experiments. SC conceived of anddesigned the study. MH and SC analyzed and interpreted the data. Some ofthe experiments were performed in the laboratory of JJ. All authorscontributed to the writing of the manuscript and approved its final version.AcknowledgementsThe authors would like to thank Mr. Akiff Manji and Ms. Shadi Mahmoodi fortheir assistance with tissue collection. These studies were performed withfunding from the American Heart Association (0635234 N), CanadianDiabetes Association (OG-2-08-2600-SC), Heart and Stroke Foundation of BCand the Yukon and the Michael Smith Foundation for Health Research (CI-SCH-01423(07–01)). SMC is the Canada Research Chair in the Genetics ofObesity and Diabetes and is a Career Investigator of the Michael SmithFoundation for Health Research.Author details1Department of Cellular and Physiological Sciences, Life Sciences Institute,University of British Columbia, Vancouver, Canada. 2Department of Surgery,University of British Columbia, Vancouver, Canada.Received: 28 August 2012 Accepted: 21 February 2013Published: 25 February 2013References1. Danaei G, Finucane MM, Lu Y, Singh GM, Cowan MJ, Paciorek CJ, Lin JK,Farzadfar F, Khang YH, Stevens GA, et al: National, regional, and globaltrends in fasting plasma glucose and diabetes prevalence since,systematic analysis of health examination surveys and epidemiologicalstudies with 370 country-years and 2.7 million participants. Lancet 1980,378(9785):31–40.2. Forsen T, Eriksson J, Tuomilehto J, Reunanen A, Osmond C, Barker D: Thefetal and childhood growth of persons who develop type 2 diabetes.Ann Intern Med 2000, 133:176–182.3. 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BMC Bioinformatics 2006, 7:85.doi:10.1186/1471-2156-14-10Cite this article as: Ho et al.: Diabetes genes identified by genome-wideassociation studies are regulated in mice by nutritional factors inmetabolically relevant tissues and by glucose concentrations in islets.BMC Genetics 2013 14:10.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitHo et al. BMC Genetics 2013, 14:10 Page 12 of 12http://www.biomedcentral.com/1471-2156/14/10


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