Aims/hypothesis: TCF7L2 is a type 2 diabetes susceptibility gene and downstream effector of canonical wingless-type MMTV integration site family (WNT) signalling. However, it is unknown whether this pathway is active in adult pancreatic islets in vivo, and whether it is regulated in obesity. Methods: We analysed activation of endogenous WNT signalling in the endocrine pancreas from wild-type and obese mice (ob/ob) using a reporter transgene (Topgal). Regulation of WNT signalling was compared using gene chip experiments from isolated pancreatic islets. Activation of canonical WNT signalling in pancreatic islets and the mouse beta cell line MIN6 was measured using immunoblotting for cytosolic β-catenin. Results: Endogenous canonical WNT signalling was absent in the adult endocrine pancreas in both wild-type and obese mice. We identified WNT4 as an abundant WNT signalling molecule in adult pancreatic islets that is induced in two different insulin-resistant mouse models. Increased expression of WNT4 inhibited canonical WNT signalling in pancreatic islets and MIN6 cells. Conclusions/interpretation: Canonical WNT signalling is not active in adult beta cells in vivo. WNT4 provides a potential mechanism for suppression of canonical WNT signalling in obese mic

Krützfeldt, J.

Stoffel, M.

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ARTICLERegulation of wingless-type MMTV integration site family(WNT) signalling in pancreatic islets from wild-typeand obese miceJ. Krützfeldt & M. StoffelReceived: 7 July 2009 /Accepted: 15 September 2009 /Published online: 8 November 2009# Springer-Verlag 2009AbstractAims/hypothesis TCF7L2 is a type 2 diabetes susceptibilitygene and downstream effector of canonical wingless-typeMMTV integration site family (WNT) signalling. However, itis unknown whether this pathway is active in adult pancreaticislets in vivo, and whether it is regulated in obesity.Methods We analysed activation of endogenous WNTsignalling in the endocrine pancreas from wild-type andobese mice (ob/ob) using a reporter transgene (Topgal).Regulation of WNT signalling was compared using genechip experiments from isolated pancreatic islets. Activationof canonical WNT signalling in pancreatic islets and themouse beta cell line MIN6 was measured using immuno-blotting for cytosolic β-catenin.Results Endogenous canonical WNT signalling was absentin the adult endocrine pancreas in both wild-type and obesemice. We identified WNT4 as an abundant WNT signallingmolecule in adult pancreatic islets that is induced in twodifferent insulin-resistant mouse models. Increased expres-sion of WNT4 inhibited canonical WNT signalling inpancreatic islets and MIN6 cells.Conclusions/interpretation Canonical WNT signalling isnot active in adult beta cells in vivo. WNT4 provides apotential mechanism for suppression of canonical WNTsignalling in obese mice.Keywords β-Catenin .Microarray . ob/ob . Pancreaticislets . Topgal . Type 2 diabetes .WNT4AbbreviationsGFP Green fluorescent proteinGSK3β Glycogen synthase kinase-3LEF Lymphoid enhancer factorPDX1 Pancreatic and duodenal homeobox 1TCF T cell factorWNT Wingless-type MMTV integration siteIntroductionAmong common type 2 diabetes risk genes, polymorphismsin the TCF7L2 gene provide the strongest disease associ-ation [1]. The gene encodes for transcription factor 7-like-2,which interacts with β-catenin as a downstream effector ofthe WNT signalling pathway. Canonical WNT signallinginvolves binding of WNT molecules to cell-surface recep-tors called frizzled and to co-receptors, the lipoproteinreceptor-related protein. Binding of WNT molecules leadsto inhibition of glycogen synthase kinase-3 (GSK3β),which results in the stabilisation of cytosolic β-catenin.Subsequently, β-catenin translocates to the nucleus where itactivates WNT target genes via binding to T cell factor(TCF)/lymphoid enhancer factor (LEF) promoter elements.Polymorphisms in TCF7L2 are associated with impairedinsulin secretion [2, 3] suggesting an important role of theElectronic supplementary material The online version of this article(doi:10.1007/s00125-009-1578-2) contains supplementary material,which is available to authorised users.J. Krützfeldt :M. StoffelLaboratory of Metabolic Diseases, The Rockefeller University,New York, NY, USAPresent Address:J. Krützfeldt :M. Stoffel (*)ETH Zurich, Institute for Molecular Systems Biology,Laboratory of Metabolic Diseases,HPT E73, Wolfgang-Pauli Str. 16,CH-8093 Zurich, Switzerlande-mail: stoffel@imsb.biol.ethz.chDiabetologia (2010) 53:123–127DOI 10.1007/s00125-009-1578-2WNT/β-catenin pathway in pancreatic islets. However, loss-of-function experiments in animal models have revealedconflicting results. Transgenic expression of an inhibitor ofWNT signalling (Pdx1-tTa/TRE-axin) revealed a decrease ofpancreatic beta cell mass by 50–60% and impaired glucosetolerance [4], but attenuation of WNT signalling in a differenttransgenic mouse model (Pdx1/Frz8CD) showed normalglucose tolerance and a relative increase in endocrine cellnumbers compared with exocrine tissue [5]. Furthermore,beta cell-specific deletion of β-catenin (Pdx1-Cre,Catnblox/lox)did not alter islet architecture or glucose tolerance at 6 or12 weeks of age [6, 7], but deletion of β-catenin in pancreaticislets at later stages of development (RIP-Cre,Catnblox/lox)induced about 70% perinatal lethality, possibly because ofhypoglycaemia [8].Clearly, more research is needed to clarify whether WNTsignalling is active in beta cells in vivo [9]. Therefore, westudied activation of canonical WNT signalling in pancre-atic islets from adult wild-type and obese mice using areporter transgene (Topgal) and analysed regulation of theWNT/β-catenin signalling pathway at the molecular levelusing gene chip experiments.MethodsMice All mice were housed as described previously [10].Principles of laboratory animal care (NIH, revised 1985) werefollowed. ob/ob mice (Lep−/−) were purchased from JacksonLaboratories (Bar Harbor, ME, USA). Topgal mice wereobtained from E. Fuchs (Rockefeller University, New York,USA). For gene chip studies, age of mice was 9.7±0.2 (n=36)vs 8.6±0.1 weeks (n=117, p<0.05); random glucose was12.6±0.7 (n=36) vs 10.0±0.2 mmol/l (n=107, p<0.05); andrandom insulin was 4,669.1±1,567.8 (n=10) vs 68.8±10.3 pmol/l (n=10, p<0.05); all ob/ob vs control, respectively.Cell culture Mouse beta cell line MIN6 cells were grown asdescribed earlier [10]. Adenoviruses were generated usingthe Rapid Adenovirus Production System (Viraquest, NorthLiberty, USA), employing the pVQ-CMV-K-Npa shuttlevector containing PCR-cloned murine Wnt4 cDNA.Isolation of pancreatic islets and RT–PCR analysis Pancre-atic islets were isolated by collagenase digestion anddifferential centrifugation as previously described [10].Total RNA was extracted from double hand-picked isletsusing Trizol reagent (Invitrogen, Carlsbad, CA, USA) andRT–PCR carried out for semiquantitative gene expressionanalysis as described earlier [10].Oligonucleotide microarray analysis Microarray analysiswas performed as previously described using 10 μg totalRNA from isolated islets per chip experiment [11].Duplicate arrays were performed for the ob/ob group andcontrols, respectively, and genes were considered differen-tially regulated if a significant p value was obtainedaccording to the Affymetrix software (Santa Clara, CA,USA) in all of the four possible pair-wise comparisons.β-Galactosidase staining Pancreases were perfused via thebile duct with fixing solution (100 mmol/l NaPO4, pH 7.3,0.2% [vol./vol.] glutaraldehyde, 5 mmol/l EGTA, 2 mmol/l MgCl2) and immediately placed in ice-cold PBS contain-ing 10% (wt/vol.) sucrose. Sections of 4 μm were mountedon slides and fixed for 2 min at room temperature in fixingsolution. After washing, sections were stained overnight in thedark at 37°C in staining solution (1 mg/ml X-gal [5-bromo-4-chloro-3-indolyl-β-D-galactoside], 2.1 mg/ml K4Fe(CN)6·3H2O, 1.64 mg/ml K3Fe(CN)6, 2 mmol/l MgCl2,0.04% [wt/vol.] deoxycholate, 0.08% [vol./vol.] NP-40,100 mmol/l NaPO4, pH 7.3).Western blotting For detection of cytosolic β-catenin,MIN6 cells or pancreatic islets were harvested in TRIS-buffered saline (pH 7.5) containing 2 mmol/l dithiothreitol,2 mmol/l phenylmethylsulfonyl fluoride, 5 mmol/l EDTAand 10% (vol./vol.) protease cocktail (Roche, Indianapolis,USA). Cells were lysed by one freeze/thaw cycle andsubjected to ultracentrifugation (100,000g, 90 min, 4°C).The resulting cytosolic fractions were separated by 10%(wt/vol.) SDS–PAGE and transferred onto nylon mem-branes. β-Catenin and GSK3β were visualised usingmonoclonal antibodies (anti-β-catenin [Sigma Aldrich, StLouis, USA], anti-GSK3β [BD Transduction Laboratories,Lexington, Kentucky, USA]), peroxidase-labelled secondantibodies and enhanced chemiluminescence (PerkinElmer,Waltham, MA, USA). Anti-WNT4 polyclonal antibodiesand recombinant WNT3A were purchased from R&DSystems (Minneapolis, USA).ResultsAbsence of activated WNT/β-catenin signalling in pancreaticislets in wild-type and obese mice in vivo To detect canonicalwnt signalling, we used the transgenic reporter mousemodel Topgal, which harbours the LacZ gene under a TCF/LEF1-responsive promoter element. Endogenous activationof canonical wnt signalling in these mice results in β-galactosidase activity in all WNT-responsive cells. Immu-nohistochemistry demonstrated that marked β-galactosidaseactivity was detected in close proximity to islets withinsmall vessels of the pancreas (Fig. 1a). We crossed theTopgal transgene into mice that express green fluorescent124 Diabetologia (2010) 53:123–127protein (GFP) under the control of the pancreatic andduodenal homeobox 1 (PDX1) promoter (PDX-GFP) tolabel pancreatic beta cells and PDX1-positive duct cells.However, PDX1-positive cells in pancreatic islets andpancreatic ducts lack β-galactosidase activity (Fig. 1b, c).Lack of β-galactosidase activity in pancreatic islets was notcaused by technical constraints, as β-galactosidase wasreadily detectable in pancreatic islets from mutant Foxa2cexisexexduisexisexbexexveexexaDAPI DAPI DAPI DAPIdFig. 1 Activation of the WNT/β-catenin pathway in small pancreaticvessels, but not in the endocrine pancreas in vivo. Activation of theWNT/β-catenin pathway was visualised in cryosections from thepancreas of 2-month-old Topgal/wild-type (a), Topgal/PDX-GFP (b,c) or Topgal/ob/ob (d) mice by β-galactosidase staining (upperpanels). Lower panel shows fluorescence from the same sections withnuclear DAPI staining. Black arrow, β-galactosidase activity in asmall blood vessel. White arrow, PDX-positive duct cell with absentβ-galactosidase activity. du, pancreatic duct; ex, exocrine pancreas; is,islet; ve, blood vesselControl ob/ob–1.4-fold– 3.6-fold**– 2.4-fold**– 2.3-fold**+13.3-fold*Gapdh–RTMafaSlp4Glut2Wnt4+1.2-fold– 2.3-fold**– 2.6-fold**+2.2-fold**Gapdh– RTMafaGlut2Wnt4Control aP2-nSREBP-1cKidney50 kDa36 kDaWNT4β-CateninIsletsBrain LungLiver--WNT4β-CateninGSK3BControl 1Control 2ob/ob 1ob/ob 2a bc dFig. 2 Increased expression of Wnt4 in pancreatic islets from insulin-resistant mice. cDNA from isolated pancreatic islets from control miceor two different insulin-resistant mouse models (ob/ob, aP2-nSREBP-1c) was analysed using semiquantitative RT-PCR (a, b). Gapdh wasamplified to demonstrate equal amounts of cDNA. Quantitativemeasurements were obtained by densitometry and expressed as foldchange vs control. *p<0.05, **p<0.01 analysed by two-tailedstudent’s t test. −RT, without reverse transcriptase. c, d Amount ofWNT4 protein was measured by immunoblotting in whole cell lysates(50 μg protein or 150 islets, respectively), antibodies against β-catenin and GSK3β were used as loading controls. Expression ofWNT4 was compared between isolated pancreatic islets and differentadult mouse tissues in wild-type mice (c) and between pancreatic isletsisolated from control or ob/ob mice (d)Diabetologia (2010) 53:123–127 125mice [11] that have one inactivated Foxa2 allele by targetedinsertion of the LacZ gene (data not shown). Next, wecrossed the Topgal transgene into obese mice (ob/ob).Similarly to our observations in wild-type mice, activationof the WNT/β-catenin pathway was not detected inpancreatic islets from obese mice (Fig. 1d).WNT4 is an abundant protein in pancreatic islets andupregulated in insulin-resistant mouse models To investigatethe molecular basis for the absence of activated WNTsignalling in pancreatic islets in vivo we performed gene chipexperiments. We collected pancreatic islets from ob/ob miceand control mice (animal characteristics in the Methods). Wefound that 169 genes were consistently regulated, 105 geneswere increased and 64 genes were decreased (Electronicsupplementary material [ESM] Table 1). Importantly, weconfirmed downregulation of several genes in obese micethat are known to be decreased in beta cells duringhyperglycaemia, i.e. Glut2 (also known as Slc2a2), Mafa,Slp4 (also known as Sytl4) and G3pdh. Two members of theWNT signalling pathway, WNT4 and frizzled-related pro-tein, were upregulated in pancreatic islets from obese mice.We confirmed regulation of Wnt4 by semiquantitative RT–PCR (Fig. 2b). In addition, Wnt4 expression was alsoincreased in islets from a different insulin-resistant mousemodel, lipodystrophic mice that lack adipose tissue (aP2-nSREBP-1c [11]) (Fig. 2c). Western blot analysis of differentadult mouse tissues detected endogenous WNT4 protein onlyin pancreatic islets (Fig. 2b). Consistent with the regulationat the mRNA level, WNT4 protein is more abundant in isletsfrom obese mice compared with lean controls (Fig. 2d).Together, these data demonstrate that WNT4 protein isenriched in pancreatic islets and its expression is increased intwo different insulin-resistant mouse models.WNT4 is an inhibitor of the canonical WNT signallingpathway in pancreatic islets WNT members are notintrinsically canonical or non-canonical, but their signallingis dictated by the distinct set of receptors within a certaincell type [12]. To address the impact of WNT4 on theWNT/β-catenin pathway we overexpressed WNT4 inisolated pancreatic islets using an adenovirus system. Nostabilisation of β-catenin was noted when WNT4 levelswere increased in pancreatic islets (Fig. 3a). To characterisethe WNT/β-catenin pathway in beta cells in response toWNT4 in more detail we analysed MIN6 cells. Consistentwith the results obtained in pancreatic islets, adenoviralWNT4 did not activate the WNT/β-catenin pathway inMIN6 cells (Fig. 3b). In contrast, WNT4 efficientlyinhibited the stabilisation of cytosolic β-catenin that hadbeen induced by recombinant WNT3A. This inhibition wasspecific for the WNT receptor, since receptor-independentstabilisation of cytosolic β-catenin by the GSK3β inhibitorlithium chloride was not altered by Wnt4 (Fig. 3c). Theseresults demonstrate that WNT4 is an inhibitor of the WNT/β-catenin pathway in pancreatic islets and beta cells.DiscussionThe major finding of our study is the absence of activatedcanonical WNT signalling in adult pancreatic islets in vivoeven in the presence of obesity and hyperinsulinaemia. Thiswas surprising, since canonical wnt signalling can beactivated by adipocyte-derived WNT signalling molecules[13] and high insulin levels [14]. Importantly, our studyidentifies a potential mechanism for this discrepancy. Inadult mice, WNT4 is an abundant WNT signallingmolecule in pancreatic islets, and it is not detected inseveral other adult mouse tissues. In the context of thepancreatic islet, WNT4 is a specific inhibitor of canonicalwnt signalling and robust upregulation of WNT4 wasobserved in pancreatic islets from two different insulin-resistant mouse models. Thus, WNT4 can provide amolecular link to prevent cross-talks between pancreaticislets and adipocyte-derived WNT signalling molecules (e.g.from adipocytes within the pancreas [13]) and betweenpancreatic islets and hyperinsulinaemia. Higher WNT4expression in insulin-resistant mice might also inducenon-canonical wnt signalling pathways, which, however,are diverse and poorly defined in mammalian tissues.Ad-GFP:  +           +         – –Ad-WNT4: – – +         +β-CateninGSK3BAd-GFP: – + – +  Ad-WNT4: + – +  –Rec-WNT3A: ––+––+ ––+–++–++–++  ++– ++– ++– + Ad-GFP:       Ad-WNT4:     LiCl:     β-CateninGSK3Bb cβ-CateninGSK3BaFig. 3 WNT4 inhibits canonical WNT signalling in pancreatic isletsat the receptor level. Activation of the WNT/β-catenin pathway wasmeasured by immunoblotting of β-catenin in cytosolic fractions. aComparison of cytosolic β-catenin in isolated pancreatic islets afterinfection with control virus (Ad-GFP) or virus that overexpressesWNT4 (Ad-WNT4). b, c Effect of adenoviral WNT4 on activation ofthe WNT/β-catenin pathway in MIN6 cells by recombinant WNT3A(Rec-WNT3A, 30 ng/ml) (b) or lithium chloride (LiCl, 10 mmol/l) (c)126 Diabetologia (2010) 53:123–127Previous studies reported different results on the impactof wnt/β-catenin signalling in beta cells. Deletion of β-catenin early in development (Pdx1-Cre,Catnblox/lox) did notshow alterations in islet development and normal glucosetolerance later during adult life [6, 7]. In contrast, deletionof β-catenin in pancreatic islets at later stages of develop-ment (RIP-Cre,Catnblox/lox) induced about 70% perinatallethality and the surviving mice showed only mild glucoseintolerance later in adulthood [8]. Beta cells might bespecifically susceptible to loss of β-catenin around the timeof birth, which is consistent with activation of endogenouscanonical wnt signalling in the endocrine pancreas ofnewborn mice [6]. However, β-catenin signalling in betacells might be dispensable later in life since endogenouswnt signalling is not activated and possibly even activelysuppressed. Genes that are downstream effectors of canon-ical WNT signalling such as TCF7L2 could impactpancreatic islets through β-catenin-independent aspects orat earlier stages of development.Acknowledgements We thank L. Castelo-Soccio and A.M.C.Brown (Weill Cornell Medical College, New York, USA) for technicaladvice. The work was supported by a grant to J. Krützfeldt fromDeutsche Forschungsgemeinschaft (KR 2224/1-1).Duality of interest The authors declare that there is no duality ofinterest associated with this manuscript.References1. Pearson ER (2009) Translating TCF7L2: from gene to function.Diabetologia 52:1227–12302. 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Schinner S, Ulgen F, Papewalis C et al (2008) Regulation ofinsulin secretion, glucokinase gene transcription and beta cellproliferation by adipocyte-derived Wnt signalling molecules.Diabetologia 51:147–15414. Yi F, Sun J, Lim GE, Fantus IG, Brubaker PL, Jin T (2008) Crosstalk between the insulin and Wnt signalling pathways: evidencefrom intestinal endocrine L cells. Endocrinology 149:2341–2351Diabetologia (2010) 53:123–127 127

Regulation of wingless-type MMTV integration site family (WNT) signalling in pancreatic islets from wild-type and obese mice

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Regulation of wingless-type MMTV integration site family (WNT) signalling in pancreatic islets from wild-type and obese mice

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