Regulation of wingless-type MMTV integration site family (WNT) signalling in pancreatic islets from wild-type and obese mice
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- Krützfeldt, J. & Stoffel, M. Diabetologia (2010) 53: 123. doi:10.1007/s00125-009-1578-2
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.
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.
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.
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 mice.
Keywordsβ-Catenin Microarray ob/ob Pancreatic islets Topgal Type 2 diabetes WNT4
Green fluorescent protein
Glycogen synthase kinase-3
Lymphoid enhancer factor
Pancreatic and duodenal homeobox 1
T cell factor
Wingless-type MMTV integration site
Among common type 2 diabetes risk genes, polymorphisms in the TCF7L2 gene provide the strongest disease association . The gene encodes for transcription factor 7-like-2, which interacts with β-catenin as a downstream effector of the WNT signalling pathway. Canonical WNT signalling involves binding of WNT molecules to cell-surface receptors called frizzled and to co-receptors, the lipoprotein receptor-related protein. Binding of WNT molecules leads to inhibition of glycogen synthase kinase-3 (GSK3β), which results in the stabilisation of cytosolic β-catenin. Subsequently, β-catenin translocates to the nucleus where it activates WNT target genes via binding to T cell factor (TCF)/lymphoid enhancer factor (LEF) promoter elements.
Polymorphisms in TCF7L2 are associated with impaired insulin secretion [2, 3] suggesting an important role of the WNT/β-catenin pathway in pancreatic islets. However, loss-of-function experiments in animal models have revealed conflicting results. Transgenic expression of an inhibitor of WNT signalling (Pdx1-tTa/TRE-axin) revealed a decrease of pancreatic beta cell mass by 50–60% and impaired glucose tolerance , but attenuation of WNT signalling in a different transgenic mouse model (Pdx1/Frz8CD) showed normal glucose tolerance and a relative increase in endocrine cell numbers compared with exocrine tissue . Furthermore, beta cell-specific deletion of β-catenin (Pdx1-Cre,Catnb lox/lox ) did not alter islet architecture or glucose tolerance at 6 or 12 weeks of age [6, 7], but deletion of β-catenin in pancreatic islets at later stages of development (RIP-Cre,Catnb lox/lox ) induced about 70% perinatal lethality, possibly because of hypoglycaemia .
Clearly, more research is needed to clarify whether WNT signalling is active in beta cells in vivo . Therefore, we studied activation of canonical WNT signalling in pancreatic islets from adult wild-type and obese mice using a reporter transgene (Topgal) and analysed regulation of the WNT/β-catenin signalling pathway at the molecular level using gene chip experiments.
All mice were housed as described previously . Principles of laboratory animal care (NIH, revised 1985) were followed. ob/ob mice (Lep −/−) were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Topgal mice were obtained 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 was 12.6 ± 0.7 (n = 36) vs 10.0 ± 0.2 mmol/l (n = 107, p < 0.05); and random 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.
Mouse beta cell line MIN6 cells were grown as described earlier . Adenoviruses were generated using the Rapid Adenovirus Production System (Viraquest, North Liberty, USA), employing the pVQ-CMV-K-Npa shuttle vector containing PCR-cloned murine Wnt4 cDNA.
Isolation of pancreatic islets and RT–PCR analysis
Pancreatic islets were isolated by collagenase digestion and differential centrifugation as previously described . Total RNA was extracted from double hand-picked islets using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and RT–PCR carried out for semiquantitative gene expression analysis as described earlier .
Oligonucleotide microarray analysis
Microarray analysis was performed as previously described using 10 μg total RNA from isolated islets per chip experiment . Duplicate arrays were performed for the ob/ob group and controls, respectively, and genes were considered differentially regulated if a significant p value was obtained according to the Affymetrix software (Santa Clara, CA, USA) in all of the four possible pair-wise comparisons.
Pancreases were perfused via the bile 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 containing 10% (wt/vol.) sucrose. Sections of 4 μm were mounted on slides and fixed for 2 min at room temperature in fixing solution. After washing, sections were stained overnight in the dark 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).
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 EDTA and 10% (vol./vol.) protease cocktail (Roche, Indianapolis, USA). Cells were lysed by one freeze/thaw cycle and subjected to ultracentrifugation (100,000 g, 90 min, 4°C). The resulting cytosolic fractions were separated by 10% (wt/vol.) SDS–PAGE and transferred onto nylon membranes. β-Catenin and GSK3β were visualised using monoclonal antibodies (anti-β-catenin [Sigma Aldrich, St Louis, USA], anti-GSK3β [BD Transduction Laboratories, Lexington, Kentucky, USA]), peroxidase-labelled second antibodies and enhanced chemiluminescence (PerkinElmer, Waltham, MA, USA). Anti-WNT4 polyclonal antibodies and recombinant WNT3A were purchased from R&D Systems (Minneapolis, USA).
Absence of activated WNT/β-catenin signalling in pancreatic islets in wild-type and obese mice in vivo
WNT4 is an abundant protein in pancreatic islets and upregulated in insulin-resistant mouse models
WNT4 is an inhibitor of the canonical WNT signalling pathway in pancreatic islets
The major finding of our study is the absence of activated canonical WNT signalling in adult pancreatic islets in vivo even in the presence of obesity and hyperinsulinaemia. This was surprising, since canonical wnt signalling can be activated by adipocyte-derived WNT signalling molecules  and high insulin levels . Importantly, our study identifies a potential mechanism for this discrepancy. In adult mice, WNT4 is an abundant WNT signalling molecule in pancreatic islets, and it is not detected in several other adult mouse tissues. In the context of the pancreatic islet, WNT4 is a specific inhibitor of canonical wnt signalling and robust upregulation of WNT4 was observed in pancreatic islets from two different insulin-resistant mouse models. Thus, WNT4 can provide a molecular link to prevent cross-talks between pancreatic islets and adipocyte-derived WNT signalling molecules (e.g. from adipocytes within the pancreas ) and between pancreatic islets and hyperinsulinaemia. Higher WNT4 expression in insulin-resistant mice might also induce non-canonical wnt signalling pathways, which, however, are diverse and poorly defined in mammalian tissues.
Previous studies reported different results on the impact of wnt/β-catenin signalling in beta cells. Deletion of β-catenin early in development (Pdx1-Cre,Catnb lox/lox ) did not show alterations in islet development and normal glucose tolerance later during adult life [6, 7]. In contrast, deletion of β-catenin in pancreatic islets at later stages of development (RIP-Cre,Catnb lox/lox ) induced about 70% perinatal lethality and the surviving mice showed only mild glucose intolerance later in adulthood . Beta cells might be specifically susceptible to loss of β-catenin around the time of birth, which is consistent with activation of endogenous canonical wnt signalling in the endocrine pancreas of newborn mice . However, β-catenin signalling in beta cells might be dispensable later in life since endogenous wnt signalling is not activated and possibly even actively suppressed. Genes that are downstream effectors of canonical WNT signalling such as TCF7L2 could impact pancreatic islets through β-catenin-independent aspects or at earlier stages of development.
We thank L. Castelo-Soccio and A.M.C. Brown (Weill Cornell Medical College, New York, USA) for technical advice. The work was supported by a grant to J. Krützfeldt from Deutsche Forschungsgemeinschaft (KR 2224/1-1).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.