Christopher H.S. McIntosh

The combined actions of glucose-dependent insulinotropic polypeptide (GIP) and truncated glucagon-like peptide-1 (tGLP-1) may fully account for the incretin effect. These hormones are released from the small intestine in response to oral glucose and stimulate insulin release. Recently, evidence has been provided demonstrating the degradation of GIP-(1-42) and GLP-1-(7-36)NH2 by the serum enzyme dipeptidyl peptidase IV (DPP IV) into the biologically inactive products GIP-(3-42) and GLP-1-(9-36)NH2. The objective of the current investigation was to develop a method to monitor the degradation of these hormones in vivo. Synthetic peptides were radiolabeled and purified by HPLC. Subsequent degradation of the peptides under various conditions was then monitored by further HPLC analysis. Incubation of [125I]GIP-(1-42) or [125I]GLP-1-(7-36)NH2 with Wistar rat serum or purified DPP IV resulted in the major N-terminal-truncated products [125I]GIP-(3-42) and [125I]GLP-1-(9-36)NH2. These products were significantly reduced when the specific DPP IV inhibitor diprotin A was included in the incubation mixture and were absent when serum from DPP IV-deficient rats was used. When the labeled peptides were infused into rats at hormone levels within the physiological range, over 50% was metabolized to the truncated forms within 2 min. These products were absent when the tracers were infused into DPP IV-deficient animals. It is concluded that DPP IV may be a primary inactivating enzyme of both GIP and tGLP-1 in vivo. As the N-terminal-truncated products of the DPP IV cleavage may not be distinguished from the biologically active hormone by currently employed assays, reports of circulating hormone levels should be reconsidered. The method described in this manuscript may be useful for investigating the durations of action of GIP and tGLP-1 in normal and pathophysiological conditions.

Recent studies into the physiology of the incretins glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) have added stimulation of beta-cell growth, differentiation, and cell survival to well-documented, potent insulinotropic effects. Unfortunately, the therapeutic potential of these hormones is limited by their rapid enzymatic inactivation in vivo by dipeptidyl peptidase IV (DP IV). Inhibition of DP IV, so as to enhance circulating incretin levels, has proved effective in the treatment of type 2 diabetes both in humans and in animal models, stimulating improvements in glucose tolerance, insulin sensitivity, and beta-cell function. We hypothesized that enhancement of the cytoprotective and beta-cell regenerative effects of GIP and GLP-1 might extend the therapeutic potential of DP IV inhibitors to include type 1 diabetes. For testing this hypothesis, male Wistar rats, exposed to a single dose of streptozotocin (STZ; 50 mg/kg), were treated twice daily with the DP IV inhibitor P32/98 for 7 weeks. Relative to STZ-injected controls, P32/98-treated animals displayed increased weight gain (230%) and nutrient intake, decreased fed blood glucose ( approximately 26 vs. approximately 20 mmol/l, respectively), and a return of plasma insulin values toward normal (0.07 vs. 0.12 nmol/l, respectively). Marked improvements in oral glucose tolerance, suggesting enhanced insulin secretory capacity, were corroborated by pancreas perfusion and insulin content measurements that revealed two- to eightfold increases in both secretory function and insulin content after 7 weeks of treatment. Immunohistochemical analyses of pancreatic sections showed marked increases in the number of small islets (+35%) and total beta-cells (+120%) and in the islet beta-cell fraction (12% control vs. 24% treated) in the treated animals, suggesting that DP IV inhibitor treatment enhanced islet neogenesis, beta-cell survival, and insulin biosynthesis. In vitro studies using a beta-(INS-1) cell line showed a dose-dependent prevention of STZ-induced apoptotic cell-death by both GIP and GLP-1, supporting a role for the incretins in eliciting the in vivo results. These novel findings provide evidence to support the potential utility of DP IV inhibitors in the treatment of type 1 and possibly late-stage type 2 diabetes.

Recent human genetics studies have revealed that common variants of the TCF7L2 (T-cell factor 7-like 2, formerly known as TCF4) gene are strongly associated with type 2 diabetes mellitus (T2DM). We have shown that TCF7L2 expression in the beta-cells is correlated with function and survival of the insulin-producing pancreatic beta-cell. In order to understand how variations in TCF7L2 influence diabetes progression, we investigated its mechanism of action in the beta-cell. We show robust differences in TCF7L2 expression between healthy controls and models of T2DM. While mRNA levels were approximately 2-fold increased in isolated islets from the diabetic db/db mouse, the Vancouver Diabetic Fatty (VDF) Zucker rat and the high fat/high sucrose diet-treated mouse compared with the non-diabetic controls, protein levels were decreased. A similar decrease was observed in pancreatic sections from patients with T2DM. In parallel, expression of the receptors for glucagon-like peptide 1 (GLP-1R) and glucose-dependent insulinotropic polypeptide (GIP-R) was decreased in islets from humans with T2DM as well as in isolated human islets treated with siRNA to TCF7L2 (siTCF7L2). Also, insulin secretion stimulated by glucose, GLP-1 and GIP, but not KCl or cyclic adenosine monophosphate (cAMP) was impaired in siTCF7L2-treated isolated human islets. Loss of TCF7L2 resulted in decreased GLP-1 and GIP-stimulated AKT phosphorylation, and AKT-mediated Foxo-1 phosphorylation and nuclear exclusion. Our findings suggest that beta-cell function and survival are regulated through an interplay between TCF7L2 and GLP-1R/GIP-R expression and signaling in T2DM.

The hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide (GLP)-1 act on the pancreas to potentiate glucose-induced insulin secretion (enteroinsular axis). These hormones (incretins) are rapidly hydrolyzed by the circulating enzyme dipeptidyl peptidase IV (DP IV) into biologically inactive NH2-terminally truncated fragments. This study describes the effect of inhibiting endogenous DP IV with a specific DP IV inhibitor, isoleucine thiazolidide (Ile-thiazolidide), on glucose tolerance and insulin secretion in the obese Zucker rat. In initial studies, the specificity of Ile-thiazolidide as an inhibitor of incretin degradation was determined using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. These results showed that inhibiting DP IV activity with Ile-thiazolidide blocked the formation of NH2-terminally truncated GIP and GLP-1. Oral administration of Ile-thiazolidide resulted in rapid inhibition of circulating DP IV levels by 65% in obese and lean Zucker rats. Suppression of DP IV levels enhanced insulin secretion in both phenotypes with the most dramatic effect occurring in obese animals (150% increase in integrated insulin response vs. 27% increase in lean animals). Ile-thiazolidide treatment improved glucose tolerance in both phenotypes and restored glucose tolerance to near-normal levels in obese animals. This was attributed to the glucose-lowering actions of increasing the circulating half-lives of the endogenously released incretins GIP and, particularly, GLP-1. This study suggests that drug manipulation of plasma incretin activity by inhibiting the enzyme DP IV is a valid therapeutic approach for lowering glucose levels in NIDDM and other disorders involving glucose intolerance.

The hormone glucose-dependent insulinotropic polypeptide (GIP) potently stimulates insulin secretion and promotes β-cell proliferation and cell survival. In the present study we identified Forkhead (Foxo1)-mediated suppression of the bax gene as a critical component of the effects of GIP on cell survival. Treatment of INS-1(832/13) β-cells with GIP resulted in concentration-dependent activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB)/Foxo1 signaling module. In parallel studies, GIP decreased bax promoter activity. Serial deletion analysis of the bax promoter demonstrated that the region -682 to -320, containing FHRE-II (5AAAACAAACA), was responsible for GIP-mediated effects. Foxo1 bound to FHRE-II in gel mobility shift assays, and Foxo1-FHRE-II interactions conferred GIP responsiveness to the bax promoter. INS-1 cells incubated under proapoptotic and glucolipotoxic conditions demonstrated increased nuclear localization of Foxo1 and bax promoter activity and decreased cytoplasmic phospho-PKB/Foxo1. GIP partially restored expression PKB/Foxo1 and bax promoter activity. Similar protective effects were found with dispersed islet cells from C57BL/6 mice, but not with those from GIP receptor knock-out (GIPR-/-) mice. GIP treatment reduced glucolipotoxicity-induced cell death in C57 BL/6 and Bax-/- islets, but not GIPR-/- mouse islets. Chronic treatment of Vancouver diabetic fatty Zucker rats with GIP resulted in down-regulation of Bax and up-regulation of Bcl-2 in pancreatic β-cells. The results show that PI3K/PKB/Foxo1 signaling mediates GIP suppression of bax gene expression and that this module is a key pathway by which GIP regulates β-cell apoptosis in vivo. The hormone glucose-dependent insulinotropic polypeptide (GIP) potently stimulates insulin secretion and promotes β-cell proliferation and cell survival. In the present study we identified Forkhead (Foxo1)-mediated suppression of the bax gene as a critical component of the effects of GIP on cell survival. Treatment of INS-1(832/13) β-cells with GIP resulted in concentration-dependent activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB)/Foxo1 signaling module. In parallel studies, GIP decreased bax promoter activity. Serial deletion analysis of the bax promoter demonstrated that the region -682 to -320, containing FHRE-II (5AAAACAAACA), was responsible for GIP-mediated effects. Foxo1 bound to FHRE-II in gel mobility shift assays, and Foxo1-FHRE-II interactions conferred GIP responsiveness to the bax promoter. INS-1 cells incubated under proapoptotic and glucolipotoxic conditions demonstrated increased nuclear localization of Foxo1 and bax promoter activity and decreased cytoplasmic phospho-PKB/Foxo1. GIP partially restored expression PKB/Foxo1 and bax promoter activity. Similar protective effects were found with dispersed islet cells from C57BL/6 mice, but not with those from GIP receptor knock-out (GIPR-/-) mice. GIP treatment reduced glucolipotoxicity-induced cell death in C57 BL/6 and Bax-/- islets, but not GIPR-/- mouse islets. Chronic treatment of Vancouver diabetic fatty Zucker rats with GIP resulted in down-regulation of Bax and up-regulation of Bcl-2 in pancreatic β-cells. The results show that PI3K/PKB/Foxo1 signaling mediates GIP suppression of bax gene expression and that this module is a key pathway by which GIP regulates β-cell apoptosis in vivo. The phosphatidylinositol 3-kinase (PI3K) 1The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; ANOVA, analysis of variance; Bax-/-, Bax knock-out; BSA, bovine serum albumin; FFA, free fatty acid; FHRE, Forkhead response element; Foxo1, Forkhead; GIP, glucose-dependent insulinotropic polypeptide; GIPR-/-, GIP receptor knock-out; PKB, protein kinase B; RFI, relative fluorescence intensity; TUNEL, terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling; VDF, Vancouver diabetic fatty. signaling pathway has emerged as a central route for regulating multiple cellular processes, including survival and proliferation of many cell types (1Brunet A. Datta S.R. Greenberg M.E. Curr. Opin. Neurobiol. 2001; 11: 297-305Crossref PubMed Scopus (1016) Google Scholar). The serine/threonine kinase protein kinase B (PKB; Akt) plays a major role in PI3K-mediated survival effects (2Franke T.F. Kaplan D.R. Cantley L.C. Cell. 1997; 88: 435-437Abstract Full Text Full Text PDF PubMed Scopus (1528) Google Scholar). Activated PKB can directly phosphorylate and, thereby, inactivate several components of the apoptotic machinery, including caspase 9 (3Cardone M.H. Roy N. Stennicke H.R. Salvesen G.S. Franke T.F. Stanbridge E. Frisch S. Reed J.C. Science. 1998; 282: 1318-1321Crossref PubMed Scopus (2735) Google Scholar), Bad (4Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4957) Google Scholar), glycogen synthase kinase-3β (5Pap M. Cooper G.M. J. Biol. Chem. 1998; 273: 19929-19932Abstract Full Text Full Text PDF PubMed Scopus (955) Google Scholar), and members of the Forkhead/winged helix (Forkhead; Foxo) family (6Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar). Recent studies have revealed that Foxo (FKHR) transcription factors (7Kaestner K.H. Knöchel W. Martinez D.E. Genes Dev. 2000; 14: 142-146PubMed Google Scholar) are important mediators of insulin and growth factor actions on gene expression downstream from PI3K and PKB (8Arden K.C. Biggs II, W.H. Arch. Biochem. Biophys. 2002; 403: 292-298Crossref PubMed Scopus (90) Google Scholar). Members of the Foxo subfamily share several structural features; in addition to the highly conserved central DNA binding Foxo domain (Fox box), Foxo members contain a C-terminal transactivation domain and three consensus phosphorylation sites for PKB located at Thr24, Ser256, and Ser316 (9Rena G. Woods Y.L. Prescott A.R. Peggie M. Unterman T.G. Williams M.R. Cohen P. EMBO J. 2002; 21: 2263-2271Crossref PubMed Scopus (201) Google Scholar). Foxo transcription factors regulate the expression of a wide variety of genes involved in the control of cell survival and apoptosis (6Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar, 8Arden K.C. Biggs II, W.H. Arch. Biochem. Biophys. 2002; 403: 292-298Crossref PubMed Scopus (90) Google Scholar), cell cycle progression (10Kops G.J. Medema R.H. Glassford J. Essers M.A. Dijkers P.F. Coffer P.J. Lam E.W. Burgering B.M. Mol. Cell. Biol. 2002; 22: 2025-2036Crossref PubMed Scopus (374) Google Scholar), protection against oxidative stress (11Nemoto S. Finkel T. Science. 2002; 295: 2450-2452Crossref PubMed Scopus (741) Google Scholar), and DNA repair (12Tran H. Brunet A. Grenier J.M. Datta S.R. Fornace Jr., A.J. DiStefano P.S. Chiang L.W. Greenberg M.E. Science. 2002; 296: 530-534Crossref PubMed Scopus (711) Google Scholar). Phosphorylation by PKB causes redistribution of Foxo1 from the nucleus to the cytoplasm, and the resulting decrease in nuclear Foxo1 has been proposed as the possible mechanism for the inhibition of Foxo1-mediated transcription (13Webster K.A. Circ. Res. 2004; 94: 856-859Crossref PubMed Scopus (31) Google Scholar). Regulation of insulin secretion involves the gut-derived peptide incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and the proglucagon gene products glucagon-like peptide-1(GLP-1)-(7–37) and GLP-1-(7–36)-amide (14Pederson R.A. Walsh J.H. Dockray G.J. Gut Peptides: Biochemistry and Physiology. Raven Press, Ltd., New York1994: 217-259Google Scholar, 15Kieffer T.J. Habener J.F. Endocr. Rev. 1999; 20: 876-913Crossref PubMed Google Scholar). Recently, anti-apoptotic functions of these hormones in pancreatic β-cells have been identified (16Farilla L. Hui H. Bertolotto C. Kang E. Bulotta A. Di Mario U. Perfetti R. Endocrinology. 2002; 143: 4397-4408Crossref PubMed Scopus (446) Google Scholar, 17Drucker D.J. Mol. Endocrinol. 2003; 17: 161-171Crossref PubMed Scopus Google Scholar, A. J. Endocrinol. 2002; PubMed Scopus Google Scholar, T. R.A. Endocrinology. 2003; PubMed Scopus Google Scholar) and, β-cell death is a major factor to of the that cell survival is GIP has been to PKB in INS-1 β-cells A. H. R. Mol. Endocrinol. 2001; PubMed Scopus Google Scholar), but the signaling pathway responsible the downstream have been In the present we demonstrated that GIP, the signaling nuclear Foxo1 with the Foxo1 response in INS-1 resulting in down-regulation of the bax gene glucolipotoxicity-induced we have that these are involved in of glucolipotoxicity-induced apoptosis in dispersed C57BL/6 mouse islet cells and Vancouver diabetic fatty Zucker rats in vivo. is the of of bax and the bax gene to the of cells were by C. were in with bovine and cells were were of Bax bax gene promoter was the and deletion were by with and for were the were and cells were at a of cells the was with of the bax of and the of control and Foxo1 and kinase and Foxo1 were by G. A. and T. G. Unterman were for to the the cells were with GIP for the in the to with and in of were the and with to for activity is as the activity of is involved in the phosphorylation of In studies in INS-1 cells were in containing and for with the were on cytoplasmic and nuclear and against and PKB and and Foxo1 and and and effects of PI3K on the phosphorylation of PKB cytoplasmic Foxo1 and nuclear Foxo1 were with GIP in the of the PI3K were to cells the as as GIP effects of PI3K on the phosphorylation of PKB cytoplasmic Foxo1 and nuclear Foxo1 INS-1 cells were with a of PI3K a of PI3K and in containing were for with of results from were was to and were relative to three in was with a bax promoter activity in INS-1 cells a pathway the PI3K/PKB/Foxo1 signaling module. In INS-1 cells were with a bax promoter and in containing BSA, and as The bax promoter are as the relative activity to the activity. of bax promoter activity. were with GIP for the of of GIP on bax promoter activity. INS-1 cells were incubated with of GIP for effects of PI3K on bax promoter activity. INS-1 cells were for with GIP in the of the PI3K were to cells a as as GIP effects of PI3K on bax promoter activity. INS-1 cells were with a bax promoter and PI3K a control INS-1 cells were and GIP was for effects of Foxo1 on bax promoter activity. INS-1 cells were with a bax promoter and Foxo1 kinase a control INS-1 cells were as for of INS-1 cells were with Foxo1 and were for with GIP, and was against The three in was with a and multiple of a in the bax Foxo1 binding of the bax promoter were to region by analysis of of the bax deletion and Foxo1 binding is INS-1 cells were with deletion and and were with GIP for The promoter are as the relative activity to the activity. Foxo1 to FHRE-II of the bax promoter. mobility shift was with nuclear and FHRE-II of consensus was as at to mobility shift of the was to binding GIP responsiveness of and FHRE-II The of and FHRE-II are of FHRE-II were to the for the in INS-1 cells were with and bax promoter FHRE-II and as for A. are of three in was with glucolipotoxicity-induced apoptosis INS-1 decreased bax promoter activity in response to treatment with GIP under glucolipotoxic INS-1 cells were with a bax promoter and and to of and for GIP was to and incubated for are as relative activity to activity. INS-1 cells were as and were against Bax and PKB Foxo1, and and Treatment with GIP under glucolipotoxic conditions resulted in decreased Bax protein expression increased PKB phosphorylation increased cytoplasmic Foxo1 phosphorylation and decreased nuclear Foxo1 were against Foxo1 and are of and three in was with glucolipotoxicity-induced apoptosis in dispersed mouse decreased bax expression in response to treatment with GIP under glucolipotoxic C57 BL/6 mouse islets, GIPR-/- islets, and Bax-/- were to of and for GIP was to and incubated for was from and transcription was to bax expression bax expression are as the control to expression dispersed mouse were as and were against Bax and PKB Foxo1 and Treatment with GIP under glucolipotoxic conditions resulted in decreased Bax protein expression increased PKB phosphorylation increased cytoplasmic Foxo1 phosphorylation and decreased nuclear Foxo1 are of and three in was with C57 FFA, C57 cytoplasmic and nuclear cells were at and and incubated on for were at in a and the was The nuclear was in and the were to studies on the of GIP on PKB and Foxo1 INS-1 cells cells were incubated in the of GIP at the in to of protein from were on a gel and of the was with PKB, Foxo1, and from were by of as a of the phosphorylation were the mobility shift for and FHRE-II were with the kinase and with of The was from a were incubated in a binding for Foxo1 BSA, and of with of the of the binding a to of was to the the assays, against Foxo1 was with nuclear for the addition of a were by on a gel in and at for were and for the at with and GIP receptor (GIPR-/-) Y. H. H. N. Y. A. S. M. A. H. T. T. J. Y. U. S. A. 1999; 96: PubMed Scopus Google Scholar) Bax knock-out Science. PubMed Scopus Google Scholar) were by of were by and dispersed to cells as by J. S.R. Mol. Endocrinol. 2001; PubMed Scopus Google Scholar). were in with bovine and of and cells dispersed mouse were incubated with a free fatty to containing GIP was to and incubated for of apoptotic cells was with the apoptosis Ltd., on the of by apoptotic analysis was a of the from the apoptotic cells to the of which are to the of were from a and to with control cells were with of serum the cells were for with GIP were in in for at and with and a were with and with The cells were a and were the GIP of GIP was to Zucker rats and in the to of the the rats were by of were at the of the and were and in for were in with the by the of on and the on from and control rats were to a for Bax and the were incubated with Bax and Bcl-2 and with and were with and a was in to DNA in apoptotic cells the apoptosis to the of was by analysis the of were a of were with those of and as relative fluorescence are as with the of in the to were the analysis and was with multiple the as in the to GIP a PKB/Foxo1 in INS-1 of INS-1 cells with GIP increased phosphorylation of PKB at but on PKB phosphorylation of PKB was by the of was by and was for GIP was concentration-dependent with of GIP treatment increased phosphorylation of Foxo1 at a with of phosphorylation of Foxo1 was by a in Foxo1 nuclear were from INS-1 cells and to with Foxo1 of GIP treatment the expression of nuclear Foxo1 was nuclear decrease was for and demonstrated a GIP with of The for nuclear of Foxo1 was that for phosphorylation of PKB and cytoplasmic Foxo1, that signaling by GIP are involved in the PI3K a of the GIP of PKB/Foxo1 phosphorylation of and by PI3K is for PKB activation D.R. M. P. N. Cohen P. EMBO J. Scopus Google Scholar), the PI3K and the PKB/Foxo1 signaling module was the of PI3K, and of PI3K decreased of PKB to GIP increased of Foxo1 in the and decreased of Foxo1 in the nucleus of PI3K in the PKB/Foxo1 signaling analysis was PI3K and of INS-1 cells decreased partially decreased in the cytoplasm, and increased nuclear in in the and in nuclear Foxo1 were reduced in the resulted in increased PKB phosphorylation with GIP of PKB cytoplasmic and nuclear Foxo1 was response to GIP treatment and In GIP was of a on PKB/Foxo1 in cells with PI3K to GIP, to in the results a role for PI3K in activation of the PKB/Foxo1 signaling GIP of the bax and the PI3K/PKB/Foxo1 bax Foxo1 binding to DNA transcription under conditions of growth factor that can to apoptosis (6Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar). to critical death genes involved in apoptosis as in the PI3K/PKB/Foxo1 signaling of the possible Foxo1 protein is a of cell is the bax of the bax gene is we the bax promoter the to of the bax gene promoter binding sites for Foxo1, in addition to nuclear factor and consensus the possible of Foxo1 in GIP of the bax promoter activity were Treatment of INS-1 cells with GIP for resulted in a decrease in bax promoter activity with control studies revealed of for GIP treatment of PI3K signaling with increased bax promoter activity and to GIP treatment Similar to the results with Foxo1, of resulted in of the effects of GIP on bax promoter of resulted in decreased bax promoter activity and with GIP treatment of of Foxo1, resulted in in bax promoter and GIP treatment decreased bax promoter activity to control of a kinase of Foxo1 Ser256, and increased bax promoter activity as with the and GIP was of activity the localization of Foxo1 was Foxo1 was found to from the nucleus to the in response to GIP the Foxo1 was in the nucleus results that the PI3K/PKB/Foxo1 module is a key component in the of bax promoter activity in the β-cell and that GIP activation of this pathway is involved in down-regulation of the bax gene the of Foxo1 from the of a Foxo1 in the bax involved in GIP responsiveness of the bax promoter was identified the and and and and and FHRE-II and to the consensus of growth protein the Foxo1 binding deletion from the of the to were The deletion from -682 to -320, containing GIP responsiveness deletion of a In gel assays, the of a was with the FHRE-II but not with the were to the of this the with the addition of a at to The addition of the nuclear and the FHRE-II and to the of a binding of Foxo1 to the of the FHRE-II in transactivation of the bax were the and the GIP responsiveness of was of three FHRE-II decreased resulting in decreased GIP responsiveness of these as with results that Foxo1 binding to FHRE-II is for transactivation of the bax of bax to of apoptosis resulting from is important of and treatment of β-cells with a of and free fatty has been to the diabetic M. W. R. 2002; PubMed Google Scholar). of the bax gene to the anti-apoptotic actions of GIP, INS-1 cells were incubated in a of and and and the of GIP was The addition of GIP on of apoptosis and in the of this of cells under glucolipotoxic conditions increased apoptosis in the of GIP the of apoptosis was reduced by bax promoter activity and protein expression were increased under glucolipotoxic with GIP treatment partially control and PKB and phosphorylation of Foxo1 in the cytoplasm, which decreased under glucolipotoxic were partially restored by GIP treatment and The expression of Foxo1 in the which increased under glucolipotoxic was partially restored we the of GIP on dispersed C57 BL/6 mouse under glucolipotoxic were GIP to those with INS-1 β-cells. Treatment with GIP reduced glucolipotoxicity-induced apoptotic cell death in dispersed C57 BL/6 mouse but not in those from GIPR-/- and in the of this GIPR-/- mouse to show to the with with C57BL/6 which from the of the anti-apoptotic actions of GIP In dispersed Bax-/- mouse to show increased to and GIP treatment reduced glucolipotoxicity-induced apoptotic cell death and bax and protein expression were increased in dispersed C57 BL/6 mouse to glucolipotoxic and GIP treatment partially restored those with and the of PKB and Foxo1 in the decreased under glucolipotoxic conditions and were partially restored by GIP nuclear Foxo1 was increased under glucolipotoxic conditions and partially restored by GIP the dispersed GIPR-/- mouse revealed to but responsiveness to GIP treatment Bax-/- mouse demonstrated to as with C57 BL/6 mouse islets, and GIP treatment restored protein expression to control results that GIP against glucolipotoxicity-induced apoptotic cell death partially down-regulation of bax gene transcription of Foxo1 from the of apoptosis on INS-1 cells and dispersed islet cells from GIPR-/-, and was the of were from a INS-1 were to those with control INS-1 islet cell were to those with control C57BL/6 islets. was with islet islet islet in a of GIP in of in treatment with GIP on β-cell apoptosis was with of peptide in Zucker of of pancreatic islet Bax protein expression was as under were with those of control and as from diabetic rats revealed increased Bax with of GIP treatment decreased Bax in from and of and In expression of the anti-apoptotic protein Bcl-2 were decreased in rats with of and GIP treatment increased to with from and with those from the revealed that the in Bcl-2 was to β-cells. in results a role for GIP in the of β-cell apoptosis the down-regulation of Bax and the up-regulation of In for the of DNA and by from rats of apoptosis of In from rats of in with GIP, islet cell apoptosis was reduced of has been proposed that GIP promotes cell survival at in by PI3K and downstream PKB G. R.A. 2004; Google Scholar). of PKB is to phosphorylate and, thereby, proapoptotic components of the cell death present the cytoplasm, including and caspase the nuclear of the pathway and role in cell survival have as been In the has been demonstrated that activation of the pathway by GIP results in phosphorylation of Foxo1, a of the Forkhead transcription factor to of Foxo1 in the from including the bax study the we the bax promoter and, the cell a promoter FHRE, FHRE-II was identified that is responsible for GIP-mediated effects. Foxo1 bound to FHRE-II of the bax and deletion of the FHRE-II region resulted in decreased activity as as GIP responsiveness in the FHRE-II region directly activity and GIP responsiveness of the promoter the of interactions for transactivation activity of the bax promoter was increased cells were with of Foxo1, a kinase of Foxo1, Recently, has that Foxo critical in cell and cell cycle progression (6Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. 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J. 2002; PubMed Scopus Google Scholar) and that in signaling in β-cells and insulin can for many of the with is the of β-cell Foxo1 activity In the studies, was that of the INS-1 β-cell dispersed C57 BL/6 mouse to of and to apoptosis by in nuclear Foxo1 and bax GIP treatment resulted in of glucolipotoxicity-induced apoptosis and in nuclear Foxo1 and bax expression in dispersed mouse and and and INS-1 β-cells and and The pathway by which GIP bax gene expression and glucolipotoxicity-induced apoptosis is and nuclear of Foxo1 The of bax gene expression in islet apoptosis was by the results with Bax-/- mouse islets. Bax-/- mouse to show increased to the of Bax in islet GIP treatment reduced glucolipotoxicity-induced apoptotic cell death Bax-/- mouse to in PKB and Foxo1 expression as with C57 BL/6 mouse islets, and GIP treatment partially restored protein expression effects of GIP on islet apoptosis and In study on the of GIP on apoptosis in INS-1 cells by serum and T. R.A. 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Science. 1998; PubMed Scopus Google Scholar). and anti-apoptotic family members can and that relative as a for the Cell. Full Text PDF PubMed Scopus Google Scholar). have found that GIP gene transcription protein C. and C. H. S. and this is to important pathway in the of β-cell that GIP the the and anti-apoptotic Bax and in the In phosphorylation of Foxo1, resulting in of the bax is to important pathway by which GIP regulates β-cell is apoptosis as the of islet β-cell death in resulting in relative of pancreatic β-cells. GIP of the key protein Bcl-2 is a major to the of β-cell and actions have for C. for INS-1 cells G. A. for PI3K T. G. Unterman for Foxo1 and for the GIPR-/- mice. with