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• W2102743706 abstract "The insulinotropic hormone GLP-1 (glucagon-like peptide-1) is a new therapeutic agent that preserves or restores pancreatic beta cell mass. We report that GLP-1 and its agonist, exendin-4 (Exd4), induce Wnt signaling in pancreatic beta cells, both isolated islets, and in INS-1 cells. Basal and GLP-1 agonist-induced proliferation of beta cells requires active Wnt signaling. Cyclin D1 and c-Myc, determinants of cell proliferation, are up-regulated by Exd4. Basal endogenous Wnt signaling activity depends on Wnt frizzled receptors and the protein kinases Akt and GSK3β but not cAMP-dependent protein kinase. In contrast, GLP-1 agonists enhance Wnt signaling via GLP-1 receptor-mediated activation of Akt and beta cell independent of GSK3β. Inhibition of Wnt signaling by small interfering RNAs to β-catenin or a dominant-negative TCF7L2 decreases both basal and Exd4-induced beta cell proliferation. Wnt signaling appears to mediate GLP-1-induced beta cell proliferation raising possibilities for novel treatments of diabetes. The insulinotropic hormone GLP-1 (glucagon-like peptide-1) is a new therapeutic agent that preserves or restores pancreatic beta cell mass. We report that GLP-1 and its agonist, exendin-4 (Exd4), induce Wnt signaling in pancreatic beta cells, both isolated islets, and in INS-1 cells. Basal and GLP-1 agonist-induced proliferation of beta cells requires active Wnt signaling. Cyclin D1 and c-Myc, determinants of cell proliferation, are up-regulated by Exd4. Basal endogenous Wnt signaling activity depends on Wnt frizzled receptors and the protein kinases Akt and GSK3β but not cAMP-dependent protein kinase. In contrast, GLP-1 agonists enhance Wnt signaling via GLP-1 receptor-mediated activation of Akt and beta cell independent of GSK3β. Inhibition of Wnt signaling by small interfering RNAs to β-catenin or a dominant-negative TCF7L2 decreases both basal and Exd4-induced beta cell proliferation. Wnt signaling appears to mediate GLP-1-induced beta cell proliferation raising possibilities for novel treatments of diabetes. The gut-derived insulinotropic hormone GLP-1 (glucagon-like peptide-1) and its long acting agonist exendin-4 (Exd4) are new agents for the treatment of diabetic patients (1Holst J.J. Expert Opin. Emerg. Drugs. 2004; 9: 155-166Crossref PubMed Scopus (95) Google Scholar). GLP-1 is a peptide hormone arising by its alternative enzymatic cleavage from proglucagon, the prohormonal precursor of GLP-1 (2Drucker D.J. Nauck M.A. Lancet. 2006; 368: 1696-1705Abstract Full Text Full Text PDF PubMed Scopus (2987) Google Scholar, 3Kieffer T.J. Habener J.F. Endocr. Rev. 1999; 20: 876-913Crossref PubMed Google Scholar). GLP-1 is released from the enteroendocrine cells of the gut in response to meals, stimulates glucose-dependent insulin secretion, and lowers blood glucose levels in type 2 diabetic subjects. Initial observations established that GLP-1 is a potent insulin secretagogue (4Mojsov S. Weir G.C. Habener J.F. J. Clin. Investig. 1987; 79: 616-619Crossref PubMed Scopus (681) Google Scholar). Subsequently, multiple anti-diabetogenic actions of GLP-1 were discovered, including the stimulation of the proliferation and the inhibition of the apoptosis of insulin-producing pancreatic beta cells (5Egan J.M. Bulotta A. Hui H. Perfetti R. Diabetes Metab. Res. Rev. 2003; 19: 115-123Crossref PubMed Scopus (185) Google Scholar, 6Li Y. Hansotia T. Yusta B. Ris F. Halban P.A. Drucker D.J. J. Biol. Chem. 2003; 278: 471-478Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar, 7List J.F. Habener J.F. Am. J. Physiol. 2004; 286: E875-E881Crossref PubMed Scopus (83) Google Scholar, 8Xu G. Stoffers D.A. Habener J.F. Bonner-Weir S. Diabetes. 1999; 48: 2270-2276Crossref PubMed Scopus (1073) Google Scholar). GLP-1 induces multiple signaling pathways intrinsic to beta cell function. Activation of the GLP-1 receptor (GLP-1R) by GLP-1 or Exd4 leads to the accumulation of cAMP and the activation of cAMP-dependent protein kinase A (PKA), 2The abbreviations used are:PKAcAMP-dependent protein kinasesiRNAsmall interfering RNACREcyclic AMP-response elementCREBCRE-binding proteinRTreverse transcriptionGAPDHglyceraldehyde-3-phosphate dehydrogenasePI3Kphosphatidylinositol 3-kinaseX-gal5-bromo-4-chloro-3-indolyl-β-d-galactopyranosideAPCadenomatous polyposis colidndominant-negativecaconstitutively activeChIPchromatin immunoprecipitationPBSphosphate-buffered salineBrdUrdbromodeoxyuridineEGFepidermal growth factorEGFREGF receptorLEFlymphocyte enhancer factorTCFT cell factorGTPγSguanosine 5′-3-O-(thio)triphosphateMAPKmitogen-activated protein kinaseERKextracellular signal-regulated kinaseMEKMAPK/ERK kinaseFzfrizzled. 2The abbreviations used are:PKAcAMP-dependent protein kinasesiRNAsmall interfering RNACREcyclic AMP-response elementCREBCRE-binding proteinRTreverse transcriptionGAPDHglyceraldehyde-3-phosphate dehydrogenasePI3Kphosphatidylinositol 3-kinaseX-gal5-bromo-4-chloro-3-indolyl-β-d-galactopyranosideAPCadenomatous polyposis colidndominant-negativecaconstitutively activeChIPchromatin immunoprecipitationPBSphosphate-buffered salineBrdUrdbromodeoxyuridineEGFepidermal growth factorEGFREGF receptorLEFlymphocyte enhancer factorTCFT cell factorGTPγSguanosine 5′-3-O-(thio)triphosphateMAPKmitogen-activated protein kinaseERKextracellular signal-regulated kinaseMEKMAPK/ERK kinaseFzfrizzled. mediated through the activation of adenylyl cyclase and the stimulatory G protein GαS. Recent studies determined that GLP-1 receptors activate several other second messengers, including mitogen-activated protein kinase (9Buteau J. Foisy S. Rhodes C.J. Carpenter L. Biden T.J. Prentki M. Diabetes. 2001; 50: 2237-2243Crossref PubMed Scopus (206) Google Scholar), phospholipase C (10Suzuki Y. Zhang H. Saito N. Kojima I. Urano T. Mogami H. J. Biol. Chem. 2006; 281: 28499-28507Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), intracellular Ca2+ (11Gomez E. Pritchard C. Herbert T.P. J. Biol. Chem. 2002; 277: 48146-48151Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), and phosphatidylinositol 3-kinase (12Hui H. Zhao X. Perfetti R. Diabetes Metab. Res. Rev. 2005; 21: 313-331Crossref PubMed Scopus (64) Google Scholar). The ability of the GLP-1 receptor to regulate such diverse responses appears to result from its promiscuous G protein coupling and its actions to mediate intracellular receptor crosstalk. For example, GLP-1 is reported to enhance beta cell proliferation via transactivation of the EGF receptor (EGFR) and its downstream effector PI3K (13Buteau J. Roduit R. Susini S. Prentki M. Diabetologia. 1999; 42: 856-864Crossref PubMed Scopus (354) Google Scholar). cAMP-dependent protein kinase small interfering RNA cyclic AMP-response element CRE-binding protein reverse transcription glyceraldehyde-3-phosphate dehydrogenase phosphatidylinositol 3-kinase 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside adenomatous polyposis coli dominant-negative constitutively active chromatin immunoprecipitation phosphate-buffered saline bromodeoxyuridine epidermal growth factor EGF receptor lymphocyte enhancer factor T cell factor guanosine 5′-3-O-(thio)triphosphate mitogen-activated protein kinase extracellular signal-regulated kinase MAPK/ERK kinase frizzled. cAMP-dependent protein kinase small interfering RNA cyclic AMP-response element CRE-binding protein reverse transcription glyceraldehyde-3-phosphate dehydrogenase phosphatidylinositol 3-kinase 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside adenomatous polyposis coli dominant-negative constitutively active chromatin immunoprecipitation phosphate-buffered saline bromodeoxyuridine epidermal growth factor EGF receptor lymphocyte enhancer factor T cell factor guanosine 5′-3-O-(thio)triphosphate mitogen-activated protein kinase extracellular signal-regulated kinase MAPK/ERK kinase frizzled. The canonical β-catenin-dependent Wnt signaling pathway is important in the modulation of cell proliferation, survival, migration and differentiation, and in organ development (14Gordon M.D. Nusse R. J. Biol. Chem. 2006; 281: 22429-22433Abstract Full Text Full Text PDF PubMed Scopus (1091) Google Scholar, 15Moon R.T. Sci. STKE 2005. 2005; : CM1Google Scholar, 16Moon R.T. Kohn A.D. De Ferrari G.V. Kaykas A. Nat. Rev. Genet. 2004; 5: 691-701Crossref PubMed Scopus (1559) Google Scholar, 17Stadeli R. Hoffmans R. Basler K. Curr. Biol. 2006; 16: R378-R385Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). It is also a regulator of stem cell fate determination and cancer (18Reya T. Clevers H. Nature. 2005; 434: 843-850Crossref PubMed Scopus (2964) Google Scholar, 19Willert K. Jones K.A. Genes Dev. 2006; 20: 1394-1404Crossref PubMed Scopus (501) Google Scholar). In the absence of Wnt ligand, cytoplasmic β-catenin is phosphorylated by GSK3β (glycogen synthase kinase 3β), within a protein complex containing axin and adenomatous polyposis coli (APC) protein. The phosphorylation of β-catenin by GSK3β results in its ubiquination and degradation (inactivation) (15Moon R.T. Sci. STKE 2005. 2005; : CM1Google Scholar, 20Kikuchi A. Kishida S. Yamamoto H. Exp. Mol. Med. 2006; 38: 1-10Crossref PubMed Scopus (165) Google Scholar). The binding of Wnt ligands to the frizzled receptors activates the intracellular protein, Dishevelled (Dvl), which inhibits APC-GSK3β-axin activity, leading to the accumulation of free cytosolic β-catenin. Subsequently, β-catenin translocates to the nucleus and forms a transcriptionally productive complex with members of the lymphocyte enhancer factor (LEF)/T cell factor (TCF) family of transcription factors, such as TCF7L2, to activate the expression of Wnt signaling target genes (19Willert K. Jones K.A. Genes Dev. 2006; 20: 1394-1404Crossref PubMed Scopus (501) Google Scholar). The role of Wnt signaling in pancreas development and functions is unclear. The expression of several Wnt and frizzled receptor genes has been detected in the developing pancreatic mesenchyme and epithelium. Misexpression of Wnt1 and Wnt5a in the early foregut results in agenesis or hypoplasia of the pancreas, respectively (21Heller R.S. Klein T. Ling Z. Heimberg H. Katoh M. Madsen O.D. Serup P. Gene Expr. 2003; 11: 141-147Crossref PubMed Scopus (76) Google Scholar). Deletion of β-catenin within the pancreatic epithelium results in either a loss (22Dessimoz J. Bonnard C. Huelsken J. Grapin-Botton A. Curr. Biol. 2005; 15: 1677-1683Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) or no change (23Wells J.M. Esni F. Boivin G.P. Aronow B.J. Stuart W. Combs C. Sklenka A. Leach S.D. Lowy A.M. BMC Dev. Biol. 2007; 7: 4Crossref PubMed Scopus (127) Google Scholar) in beta cell mass. Ectopic stabilization of β-catenin deregulates the normal mechanisms that control embryonic pancreas formation and postnatal organ growth (24Heiser P.W. Lau J. Taketo M.M. Herrera P.L. Hebrok M. Development (Camb.). 2006; 133: 2023-2032Crossref PubMed Scopus (189) Google Scholar). Based on a preliminary study of pancreatic islet gene expression profiling on microarrays, we recognized that GLP-1 and Exd4 induced the expression of genes in both the Wnt signaling pathway per se and target genes of the Wnt signaling pathway (Table S1). These genes are activated by the transcriptional complex of β-catenin and TCF/LEF. These microarray findings prompted us to examine in greater detail the regulation of Wnt signaling in pancreatic beta cells by GLP-1 agonists and the role of Wnt signaling in the GLP-1 agonist stimulation of beta cell proliferation. In this study, we examined the Exd4 activation of Wnt signaling in isolated islets and the INS-1 beta cell line using a β-catenin/TCF-activated reporter gene assay and found that GLP-1 and Exd4 enhance Wnt signaling. By using specific protein kinase inhibitors, dominant-negative isoforms of the kinases and of TCF7L2, and siRNA knockdown of β-catenin, we find that the basal Wnt signaling is dependent on endogenous Wnt ligands and requires active Akt and the inactivation of GSK3β. In marked contrast, GLP-1 and Exd4 activate Wnt signaling through the GLP-1 receptor coupled to the activation of cAMP-dependent protein kinase A (PKA), and the prosurvival kinase Akt/PKB, independent of GSK3β. Exd4-mediated activation of PKA phosphorylates β-catenin on Ser-675, a mechanism that appears to stabilize β-catenin and to enhance TCF7-L2 activation of gene expression in beta cells. We also provide evidence that active Wnt signaling stimulates the proliferation of beta cells as a dominant-negative TCF7L2 inhibits both basal and Exd4-stimulated INS-1 cell proliferation. Cell Culture and Transient Transfection—INS-1 cells (insulin-producing strain obtained from C. Wolheim, Geneva, Switzerland) were maintained in RPMI supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 μg/ml), and streptomycin (0.25 μg/ml) at 37 °C under 5% CO2 and at 95% humidity. Transfections were done with Lipofectamine 2000 (Invitrogen) according to the manufacturer's manual. For stable transfections, INS-1 cells were cotransfected with pcDNA3-myc-dnTCF7L2, encoding the dominant-negative mutant of TCF7L2 (dnTCF7L2), or a control empty vector and puromycin vector, containing a puromycin-selectable marker. Two days after their transfection, cells were selected for resistance to puromycin for 2 weeks. Puromycin-resistant clones were screened for dnTCF7L2 expression with a Myc monoclonal antibody against the Myc epitope after 2 weeks of selection, and positive clones were pooled. Plasmids—The TOPflash and FOPflash luciferase vectors were gifts from R. Moon, University of Washington. Dominant-negative form of the regulatory subunit of PKA was from G. S. McKnight, University of Washington. Dominant-negative form of the PI3K was from J. Du, Baylor College of Medicine. Dominant-negative forms of CREB were from Clontech. Isolated Mouse Pancreatic Islets—Mouse islets were isolated from the pancreata of TOPGAL mice transgenic for the LEF-lacZ Wnt signaling reporter (25Lacy P.E. Mt. Sinai J. Med. 1994; 61: 23-31PubMed Google Scholar). Freshly isolated islets were treated for 4 h with Exd4 with and without the addition of the PKA inhibitor H89 or the GLP-1R antagonist Exd-(9-39). β-Galactosidase activity was determined by incubation of the islets with X-gal for 6 h. For BrdUrd proliferation assay, islets were treated with 0.01% trypsin for 5 min while lightly disrupting them with a pipette. Islets were then resuspended in a mixture of media and/or retrovirus and then spun down at 2000 rpm for 60 min. Wnt Signaling Luciferase Reporter Assay (TOPflash)—INS-1 cells were plated into 24-well dishes 24 h before transfection with TOPflash (1 μg/well) or FOPflash (1 μg/well) using Lipofectamine 2000 (Invitrogen). Various concentrations of GLP-1 or Exd4 were then added to the culture medium 24 h following transfection for the indicated times. In studies in which inhibitors were used, LY294002 (10 μm), AG1478 (10 μm), H89 (10 μm), U0126 (10 μm), PD98059 (10 μm), SB203580 (1 μm), or Akt inhibitor IV (10 nm) were added concomitantly with Exd4. In studies in which wild-type, dominant-negative (dn), or constitutively active (ca) forms of kinase were used, dnPI3K, dnPKA, caPKA, dnGSK3β, caGSK3β, wild-type CREB, dnCREB, dnAkt, or caAkt (0.5 μg/well) was co-transfected with TOPflash. Luciferase activity in transfected cells was determined with a luciferase assay kit (Promega). Cyclic AMP-response Element Luciferase Reporter Assay—INS-1 cells were plated into 24-well dishes 24 h before transfection with cAMP-response element (CRE) luciferase (1 μg/well) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Various concentrations of GLP-1 or Exd4 were then added to the culture medium 24 h following transfection for the indicated time. dnTCF7L2 Retroviral Plasmids and Retroviral Infection of INS-1 Cells and Isolated Islets—A dnTCF7L2 fragment was subcloned into the BamHI and XhoI sites of retroviral vector pBMN (Orbigen). Retrovirus was prepared by transfecting phoenix cells with control (pBMN) or dnTCF7L2 retroviral vector (pBMN-dnTCF7L2). Virus-containing medium was collected 36 h after transfection and passed through a 0.45-μm syringe filter. Polybrene (hexadimethrine bromide; Sigma) was added to a final concentration of 8 μg/ml. This medium was then applied to subconfluent (50%) INS-1 cells (10-cm plates) or isolated islets for 36 h, and the infected cells were used in the subsequent cell proliferation assay. Wnt3A Conditioned Media—Wnt3A conditioned media and blank conditioned media were obtained from the ATCC. Wnt3A conditioned medium was prepared from TH L-M(TK-) cells transfected with a Wnt3A expression vector and selected in medium containing G418. Selected stable clones encode a secreted biologically active Wnt3A protein. Blank conditioned medium was collected from a parental cell line. Mfz8CRD-IgG Conditioned Media—Mfz8CRD-IgG was produced in 293 cells that were transiently transfected by the mfz8CRD-IgG cDNA (a kind gift from Dr. J. Nathans, The Johns Hopkins University) using Lipofectamine 2000. One day after transfection, cells were transferred to serum-free Dulbecco's modified Eagle's medium, and the conditioned medium was harvested after an additional 24 h. Control conditioned medium was obtained from untransfected 293 cells. Real Time RT-PCR—Real time RT-PCR was carried out by using the SYBR® Green QPCR kit (Stratagene). Briefly, INS-1 cells were treated with 10 nm Exd4 or PBS vehicle control for 4 h. Total RNA was reverse-transcribed to cDNA using Super-Script II reverse transcriptase (Invitrogen). PCR was performed to amplify cyclin D1 by using the following primers, forward 5′-TGTTTGAGACCTTCAACACC-3′ and reverse 5′-CCAGACAGCACTGTGTTGGC-3′, and to amplify Myc by using the following primers, forward 5′-CTGCTCTCCGTCCTATGTTG-3′ and reverse 5′-CCTGGATGATGATGTTCTTGATG-3′. For measurements of β-galactosidase and mRNA levels, real time quantitative PCR was used. Isolated islets prepared from TOPGAL mice were treated with 10 nm Exd4 or PBS vehicle control for 4 h. Total RNA was prepared from isolated islets of TOPGAL mice and reverse-transcribed to cDNA. Real time RT-PCR was performed to amplify the β-galactosidase encoding lacZ transcript by using the following primers, forward 5′-GTACGGCAGTTATCTGGAAG-3′ and reverse 5′-CATAACCACCACGCTCATCG-3′. Western Immunoblots—Membrane immunoblots were prepared from extracts of INS-1 cells and were interrogated with antisera to β-catenin as follows: total protein, the destabilizing GSK-3 phosphorylation sites (UpState catalog number 46-626), and the stabilizing PKA-mediated phosphorylation site, Ser-675, that stabilizes β-catenin (AnaSpec catalog number 29619). siRNA-mediated Knockdown of β-Catenin Expression—siRNAs against rat β-catenin (GenBank™ accession number NM_053357) were from Dharmacon (siRNA1 catalog number J100628-05, siRNA2 catalog number J-100628-06). 50 nm siRNAs were transfected into INS-1 cells using the Dharma reagent. siRNA-transfected cells were grown for 48 or 72 h, and aliquots of cells were harvested for Western immunoblot analysis and used for the TOPflash/FOPflash Wnt signaling reporter assay. Cell Proliferation Assay—The proliferation of dispersed isolated islets and INS-1 cells, stably transfected or transiently infected, was determined by measuring the incorporation of BrdUrd into newly synthesized DNA of proliferating cells. Cells were plated at 10,000/well or 20 islets/well in 96-well plates and treated with Exd4 or PBS overnight. Cells were pulse-labeled with BrdUrd 4 h before measurement. BrdUrd staining was measured by the DELFIA cell proliferation kit (PerkinElmer Life Sciences). Chromatin Immunoprecipitation (ChIP) Assays—For ChIP assays, INS-1 cells were treated with formaldehyde (1% final concentration) for 10 min at 37 °C. Cells were then pelleted for 4 min at 2000 rpm and resuspended in 200 μl of SDS lysis buffer. After sonication, cell supernatant was diluted 10-fold in ChIP dilution buffer. Anti-TCF7L2 (Santa Cruz Biotechnology) or β-catenin (Sigma) antibodies were added and incubated overnight at 4 °C. Afterward 60 μl of salmon sperm DNA/protein A-agarose slurry was added to the supernatant. After 1 h of incubation at 4 °C, the antibody-histone complex was collected by centrifugation. After elution and reverse cross-linking, DNA was recovered by phenol/chloroform extraction. DNAs were resuspended in 100 μl of TE buffer and analyzed by PCR using the following primers directed to cyclin D1 promoter, forward 5′-TGGAACTGCTTCTGGTGAAC-3′ and reverse 5′-CAGGAGAGGAAGTTGTTGGG-3′. GLP-1 and Exd4 Activate Wnt Signaling in INS-1 Cells and in Islets ex Vivo via the GLP-1 Receptor—Several of the following observations suggested to us that GLP-1 agonists may be involved in the activation of Wnt signaling in pancreatic beta cells. 1) GLP-1 agonists are known to enhance the proliferation (9Buteau J. Foisy S. Rhodes C.J. Carpenter L. Biden T.J. Prentki M. Diabetes. 2001; 50: 2237-2243Crossref PubMed Scopus (206) Google Scholar) and the neogenesis (8Xu G. Stoffers D.A. Habener J.F. Bonner-Weir S. Diabetes. 1999; 48: 2270-2276Crossref PubMed Scopus (1073) Google Scholar) of beta cells as well as the differentiation of both somatic (26Zalzman M. Anker-Kitai L. Efrat S. Diabetes. 2005; 54: 2568-2575Crossref PubMed Scopus (145) Google Scholar, 27Abraham E.J. Leech C.A. Lin J.C. Zulewski H. Habener J.F. Endocrinology. 2002; 143: 3152-3161Crossref PubMed Scopus (275) Google Scholar, 28Hisatomi Y. Okumura K. Nakamura K. Matsumoto S. Satoh A. Nagano K. Yamamoto T. Endo F. Hepatology. 2004; 39: 667-675Crossref PubMed Scopus (148) Google Scholar) and embryonic (29Bai L. Meredith G. Tuch B.E. J. Endocrinol. 2005; 186: 343-352Crossref PubMed Scopus (65) Google Scholar, 30Ku H.T. Zhang N. Kubo A. O'Connor R. Mao M. Keller G. Bromberg J.S. Stem Cells. 2004; 22: 1205-1217Crossref PubMed Scopus (108) Google Scholar, 31Lester L.B. Langeberg L.K. Scott J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14942-14947Crossref PubMed Scopus (167) Google Scholar, 32D'Amour K.A. Bang A.G. Eliazer S. Kelly O.G. Agulnick A.D. Smart N.G. Moorman M.A. Kroon E. Carpenter M.K. Baetge E.E. Nat. Biotechnol. 2006; 24: 1392-1401Crossref PubMed Scopus (1495) Google Scholar, 33Yue Z. Jiang T.X. Widelitz R.B. Chuong C.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 951-955Crossref PubMed Scopus (74) Google Scholar) stem cells into insulin-producing beta cells. 2) Components of the Wnt signaling pathway are known to be expressed in the pancreas during development, and experimentally induced genetic disruption of Wnt signaling impairs pancreas growth and functions (21Heller R.S. Klein T. Ling Z. Heimberg H. Katoh M. Madsen O.D. Serup P. Gene Expr. 2003; 11: 141-147Crossref PubMed Scopus (76) Google Scholar, 22Dessimoz J. Bonnard C. Huelsken J. Grapin-Botton A. Curr. Biol. 2005; 15: 1677-1683Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 24Heiser P.W. Lau J. Taketo M.M. Herrera P.L. Hebrok M. Development (Camb.). 2006; 133: 2023-2032Crossref PubMed Scopus (189) Google Scholar, 34Papadopoulou S. Edlund H. Diabetes. 2005; 54: 2844-2851Crossref PubMed Scopus (123) Google Scholar). Wnt signaling is an important pathway in the maintenance of the proliferation and the differentiation of embryonic and somatic stem cells (23Wells J.M. Esni F. Boivin G.P. Aronow B.J. Stuart W. Combs C. Sklenka A. Leach S.D. Lowy A.M. BMC Dev. Biol. 2007; 7: 4Crossref PubMed Scopus (127) Google Scholar, 35Feng Z. Srivastava A.S. Mishra R. Carrier E. Biochem. Biophys. Res. Commun. 2004; 324: 1333-1339Crossref PubMed Scopus (16) Google Scholar, 36Day T.F. Guo X. Garrett-Beal L. Yang Y. Dev. Cell. 2005; 8: 739-750Abstract Full Text Full Text PDF PubMed Scopus (1309) Google Scholar, 37Kleber M. Lee H.Y. Wurdak H. Buchstaller J. Riccomagno M.M. Ittner L.M. Suter U. Epstein D.J. Sommer L. J. Cell Biol. 2005; 169: 309-320Crossref PubMed Scopus (154) Google Scholar, 38Sato N. Meijer L. Skaltsounis L. Greengard P. Brivanlou A.H. Nat. Med. 2004; 10: 55-63Crossref PubMed Scopus (1709) Google Scholar, 39Teo R. Mohrlen F. Plickert G. Muller W.A. Frank U. Dev. Biol. 2006; 289: 91-99Crossref PubMed Scopus (68) Google Scholar). Collectively, these observations prompted us to examine the expression of genes expressed in Wnt signaling in beta cells using a focused Wnt signaling gene microarray (SuperArray, Invitrogen) and the clonal beta cell line INS-1 cells. Of the 118 probes represented on the Wnt signaling gene array, 37 were expressed above background in cultured INS-1 cells. Furthermore, we found that a 4-h exposure of the cells to Exd4 enhanced the expression of 14 of the genes, strongly suggesting that GLP-1 agonists activate components and target genes of the Wnt signaling pathway in INS-1 cells (supplemental Table S1). To investigate in more detail whether GLP-1 and Exd4 induce Wnt signaling in INS-1 cells, we used a Wnt signaling reporter assay (TOPflash/FOPflash) to measure Wnt signaling in INS-1 cells stimulated by Exd4. The TOPflash and FOPflash constructs contain the luciferase reporter either under the control of consensus TCF7L2-binding sites or mutated TCF7L2-binding sites, respectively. The ratio of TOPflash activity to FOPflash activity indicates the intensity of Wnt signaling. INS-1 cells were transfected with TOPflash or FOPflash, and 24 h later GLP-1 or derivatives thereof were added to the cell culture at the indicated doses. Luciferase activity was measured after 4 h of incubation (Fig. 1A) or the indicated times (Fig. 1B). The intensity of active Wnt signaling was determined by the TOPflash/FOPflash ratio. GLP-1-(7-36) and Exd4 activated Wnt signaling dose-dependently with maximum responses achieved at 5 and 1 nm, respectively (Fig. 1A). Rapidly after its secretion, intact GLP-1-(7-36) is proteolytically cleaved by the enzyme dipeptidyl peptidase IV, yielding the metabolite GLP-1-(9-36). Exendin-(9-39), a derivative of Exendin-4 (Exd4), is a specific and competitive antagonist of the GLP-1 receptor. These two inactive GLP-1R ligands did not activate Wnt signaling (TOPflash/FOPflash activity) at all concentrations tested (Fig. 1A). The activation of Wnt signaling by Exd4 occurred as early as 1 h and reached maximum levels after 4 h of exposure to the hormone (Fig. 1B). Furthermore, the Exd4 activation was antagonized by co-incubation with increasing amounts of the Exd4-(9-39) antagonist (Fig. 1A), indicating that the activation of Wnt signaling by GLP-1 or Exd4 occurs via the GLP-1 receptor. To further determine whether Exd4 induces Wnt signaling, mouse islets were isolated from a commonly used Wnt signaling reporter mouse model (TOPGAL mice) to measure the Wnt signaling stimulated by Exd4. TOPGAL mice are transgenic mice harboring a reporter gene TOPGAL, a β-galactosidase-encoding gene (lacZ) under the control of a regulatory sequence consisting of three consensus LEF/TCF-binding motifs upstream of a minimal c-fos promoter. This mouse provides an effective model for studying the Wnt signaling pathway (40DasGupta R. Fuchs E. Development (Camb.). 1999; 126: 4557-4568Crossref PubMed Google Scholar). Islets from TOPGAL mice were incubated with Exd4 or vehicle for 4 h and then treated with β-galactosidase substrate (X-gal) for an additional 4 h. The reaction products were examined under a phase contrast microscope. Islets treated with Exd4 exhibited a distinct blue color, representative of active Wnt signaling (Fig. 1C, upper panels). β-Galactosidase expression was inhibited by co-incubation with the GLP-1R antagonist Exd-(9-39) (Fig. 1C, lower panels). To quantitate the activation of lacZ by Exd4, we used real time PCR to measure levels of lacZ mRNA in islets treated with Exd4 or vehicle. lacZ mRNA increased by 2.6-fold in Exd4-treated TOPGAL islets, providing ex vivo evidence of Exd4-induced Wnt signaling activation in pancreatic islets (Fig. 1D). GLP-1 Interactions with the GLP-1R in INS-1 Cells and in Isolated Mouse Islets Are Coupled to the cAMP/PKA/CREB Signaling Pathway—Additional evidence that GLP-1 and Exd4 are acting on the known GLP-1R in INS-1 cells, and activate the cAMP/PKA/CREB signal transduction axis, was obtained by showing that both GLP-1-(7-36) and Exd4 agonists, and not the GLP-1-(9-36) and Exd-(9-39) antagonists, activated transcription from a luciferase reporter driven by a cAMP-response element (CRE) (Fig. 2A). The CRE-mediated transcription in response to GLP-1-(7-36) was attenuated by the PKA inhibitor H89 indicating that cAMP and active PKA are generated by GLP-1 activation of the GLP-1R in INS-1 cells (Fig. 2B). In addition, H89 abrogated Exd4-stimulated activation of β-galactosidase enzymatic activity and mRNA expression in islets isolated ex vivo from the TOPGAL Wnt signaling reporter mouse (Fig. 2, C and D). Insulin Does Not Activate Wnt Signaling in INS-1 Cells—Because GLP-1 agonists are insulin secretagogues, the effects of insulin on Wnt signaling were examined in INS-1 cells. Insulin (100 nm) had no effects on TOPFlash activities in conditions in which insulin readily stimulated the phosphorylation of insulin receptor substrate-1 (serine 1101), GSK3α (serine 21), and GSK3β (serine 9) (supplemental Fig. 2). We also found that there is no effect of insulin on GLP-1 stimulation of Wnt signaling in the INS-1 cells, as determined by the activation of the TopFlash reporter (supplemental Fig. 3). Wnt Ligands and Frizzled Receptors Mediate Basal Endogenous Wnt Signaling in INS-1 Cells—We observed that the TOPflash activity in INS-1 cells is 20-fold higher than FOPflash activity, indicating that these cells have substantial basal levels of Wnt signaling. To investigate whether the basal Wnt signaling in INS-1 cells is mediated by the canonical Wnt signaling pathway, we observed that the basal endogenous Wnt signaling is stimulated by the Wnt ligand Wnt3A in a d" @default.
• W2102743706 created "2016-06-24" @default.
• W2102743706 creator A5007905274 @default.
• W2102743706 creator A5034913449 @default.
• W2102743706 date "2008-03-01" @default.
• W2102743706 modified "2023-09-29" @default.
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