Decreased TCF7L2 protein levels in type 2 diabetes mellitus correlate with downregulation of GIP- and GLP-1 receptors and impaired beta-cell function.

In this article, Figure 2F was incorrect. The correct panel is shown below. The authors sincerely apologise for this error.

Incretin-stimulated interaction between ß-cell Kv1.5 and Kvß2 channel proteins involves acetylation/deacetylation by CBP/SirT1.

The incretins, GIP (glucose-dependent insulinotropic polypeptide) and GLP-1 (glucagon-like peptide-1) are gastrointestinal hormones conferring a number of beneficial effects on ß-cell secretion, survival and proliferation. In a previous study, it was demonstrated that delayed rectifier channel protein Kv2.1 contributes to ß-cell apoptosis and that the prosurvival effects of incretins involve Kv2.1 PTMs (post-translational modifications), including phosphorylation and acetylation. Since Kv1.5 overexpression was also shown to stimulate ß-cell death, the present study was initiated in order to determine whether incretins modulate Kv1.5α-Kvß2 interaction via PTM and the mechanisms involved. GIP and GLP-1 reduced apoptosis in INS-1 ß-cells (clone 832/13) overexpressing Kv1.5, and RNAi (RNA interference)-mediated knockdown of endogenous Kv1.5 attenuated apoptotic ß-cell death. Both GIP and GLP-1 increased phosphorylation and acetylation of Kv1.5 and its Kvß2 protein subunit, leading to their enhanced interaction. Further studies demonstrated that CBP [CREB (cAMP-response-element-binding protein)-binding protein]/SirT1 mediated acetylation/deacetylation and interaction between Kvß2 and Kv1.5 in response to GIP or GLP-1. Incretin regulation of ß-cell function therefore involves the acetylation of multiple Kvα and Kvß subunits.

Quantitative micro positron emission tomography (PET) imaging for the in vivo determination of pancreatic islet graft survival.

Islet transplantation is an attractive approach for treating type-1 diabetes, but there is a massive loss of transplanted islets. It is currently only possible to estimate islet mass indirectly, through measurement of circulating C-peptide and insulin levels. This type of estimation, however, is not sufficiently sensitive or reproducible for follow-up of individuals who have undergone islet transplantation. Here we show that islet graft survival could be assessed for 1 month in diabetic NOD mice using 9-(4-[(18)F]-fluoro-3-hydroxymethylbutyl)guanine ([(18)F]FHBG)-positron emission tomography (PET) technology, the PET signal reflecting insulin secretory capacity of transplanted islets. Expression of the gene encoding viral interleukin-10 (vIL-10), was measurable in real time with PET scanning. Additionally, we addressed the clinical potential of this approach by visualizing transplanted islets in the liver, the preferred clinical transplantation site. We conclude that quantitative in vivo PET imaging is a valid method for facilitating the development of protocols for prolonging islet survival, with the potential for tracking human transplants.

Adipocyte expression of the glucose-dependent insulinotropic polypeptide receptor involves gene regulation by PPARγ and histone acetylation.

Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone that exerts insulinotropic and growth and survival effects on pancreatic ß-cells. Additionally, there is increasing evidence supporting an important role for GIP in the regulation of adipocyte metabolism. In the current study we examined the molecular mechanisms involved in the regulation of GIP receptor (GIPR) expression in 3T3-L1 cells. GIP acted synergistically with insulin to increase neutral lipid accumulation during progression of 3T3-L1 preadipocytes to the adipocyte phenotype. Both GIPR protein and mRNA expression increased during 3T3-L1 cell differentiation, and this increase was associated with upregulation of nuclear levels of sterol response element binding protein 1c (SREBP-1c) and peroxisome proliferator-activated receptor γ (PPARγ), as well as acetylation of histones H3/H4. The PPARγ receptor agonists LY171883 and rosiglitazone increased GIPR expression in differentiated 3T3-L1 adipocytes, whereas the antagonist GW9662 ablated expression. Additionally, both PPARγ and acetylated histones H3/H4 were shown to bind to a region of the GIPR promoter containing the peroxisome proliferator response element (PPRE). Knockdown of PPARγ in differentiated 3T3-L1 adipocytes, using RNA interference, reduced GIPR expression, supporting a functional regulatory role. Taken together, these studies show that GIP and insulin act in a synergistic manner on 3T3-L1 cell development and that adipocyte GIPR expression is upregulated through a mechanism involving interactions between PPARγ and a GIPR promoter region containing an acetylated histone region.

Decreased TCF7L2 protein levels in type 2 diabetes mellitus correlate with downregulation of GIP- and GLP-1 receptors and impaired beta-cell function.

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.

GIP increases human adipocyte LPL expression through CREB and TORC2-mediated trans-activation of the LPL gene.

GIP (glucose-dependent insulinotropic polypeptide) is a gastrointestinal hormone that regulates pancreatic islet function. Additionally, emerging evidence suggests an important physiological role for GIP in the regulation of adipocyte metabolism. In previous studies on the lipogenic effects of GIP, it was shown to increase adipocyte lipoprotein lipase (LPL) activity in both differentiated 3T3-L1 cells and human adipocytes through a pathway involving activation of protein kinase B (PKB)/Akt. In the current study, we examined the effects of GIP on LPL gene expression. GIP in the presence of insulin increased LPL gene expression in human adipocytes and LPL promoter activity in GIP receptor-expressing HEK-293 cells, and both effects were greatly reduced by the transcription inhibitor actinomycin D. Subsequent studies established that GIP increased phosphorylation of Serine 133 in cAMP-response element binding protein (CREB) and the nuclear localization of cAMP-responsive CREB coactivator 2 (TORC2) through a pathway involving phosphatidylinositol 3-kinase (PI3-K), PKB, and AMP-activated protein kinase (AMPK). However, in the presence of insulin, GIP failed to activate the cAMP/PKA pathway. Knockdown of CREB and TORC2 using RNA interference reduced LPL expression, supporting a functional regulatory role. GIP-induced phospho-CREB and TORC2 were shown to bind to a cAMP-response element (-II) site in the human LPL promoter and GIP increased protein-protein interactions of these two factors. The lipogenic effects of GIP in the presence of insulin are therefore at least partially mediated by upregulation of adipocyte LPL gene transcription through a pathway involving PI3-K/PKB/AMPK-dependent CREB/TORC2 activation.

Suppression of p38 MAPK and JNK via Akt-mediated inhibition of apoptosis signal-regulating kinase 1 constitutes a core component of the beta-cell pro-survival effects of glucose-dependent insulinotropic polypeptide.

Glucose-dependent insulinotropic polypeptide (GIP) potentiates glucose-stimulated insulin secretion, insulin biosynthesis, and beta-cell proliferation and survival. In previous studies GIP was shown to promote beta-cell survival by modulating the activity of multiple signaling modules and regulating gene transcription of pro- and anti-apoptotic bcl-2 family proteins. We have now evaluated the mechanisms by which GIP regulates the dynamic interactions between cytoplasmic bcl-2 family members and the mitochondria in INS-1 cells during apoptosis induced by treatment with staurosporine (STS), an activator of the mitochondria-mediated apoptotic pathway. STS induced translocation of bad and bimEL, activation of mitochondrial bax, release of mitochondrial cytochrome c, cleavage of caspase-3, and apoptosis. Each response was significantly diminished by GIP. Using selective enzyme inhibitors, overexpression of dominant-negative Akt, and Akt siRNA, it was demonstrated that GIP promoted beta-cell survival via Akt-dependent suppression of p38 MAPK and JNK and that combined inhibition was sufficient to explain the entire pro-survival responses to GIP during STS treatment. This signaling pathway also explained the pro-survival effects of GIP on INS-1 cells exposed to two other promoters of stress: thapsigargin (endoplasmic reticulum stress) and etoposide (genotoxic stress). Importantly, we discovered that GIP suppressed p38 MAPK and JNK via Akt-mediated changes in the phosphorylation state of the apoptosis signal-regulating kinase 1 in INS-1 cells and human islets, resulting in inhibition of its activity. Inhibition of apoptosis by GIP is therefore mediated via a key pathway involving Akt-dependent inhibition of apoptosis signal-regulating kinase 1, which subsequently prevents the pro-apoptotic actions of p38 MAPK and JNK.

Pancreatic beta-cell overexpression of the glucagon receptor gene results in enhanced beta-cell function and mass.

In addition to its primary role in regulating glucose production from the liver, glucagon has many other actions, reflected by the wide tissue distribution of the glucagon receptor (Gcgr). To investigate the role of glucagon in the regulation of insulin secretion and whole body glucose homeostasis in vivo, we generated mice overexpressing the Gcgr specifically on pancreatic beta-cells (RIP-Gcgr). In vivo and in vitro insulin secretion in response to glucagon and glucose was increased 1.7- to 3.9-fold in RIP-Gcgr mice compared with controls. Consistent with the observed increase in insulin release in response to glucagon and glucose, the glucose excursion resulting from both a glucagon challenge and intraperitoneal glucose tolerance test (IPGTT) was significantly reduced in RIP-Gcgr mice compared with controls. However, RIP-Gcgr mice display similar glucose responses to an insulin challenge. beta-Cell mass and pancreatic insulin content were also increased (20 and 50%, respectively) in RIP-Gcgr mice compared with controls. When fed a high-fat diet (HFD), both control and RIP-Gcgr mice developed similar degrees of obesity and insulin resistance. However, the severity of both fasting hyperglycemia and impaired glucose tolerance (IGT) were reduced in RIP-Gcgr mice compared with controls. Furthermore, the insulin response of RIP-Gcgr mice to an IPGTT was twice that of controls when fed the HFD. These data indicate that increased pancreatic beta-cell expression of the Gcgr increased insulin secretion, pancreatic insulin content, beta-cell mass, and, when mice were fed a HFD, partially protected against hyperglycemia and IGT.

Dipeptidyl peptidase IV inhibitors and diabetes therapy.

Current type 2 diabetes therapies are mainly targeted at stimulating pancreatic beta-cell secretion and reducing insulin resistance. A number of alternative therapies are currently being developed to take advantage of the actions of the incretin hormones Glucagon-Like Peptide-1 (GLP-1) and Glucose-dependent Insulinotropic Polypeptide (GIP). These hormones are released from the small intestine in response to nutrient ingestion and stimulate insulin secretion in a glucose-dependent manner. One approach to potentiating their actions is based on inhibiting dipeptidyl peptidase IV (DPP IV), the major enzyme responsible for degrading the incretins in vivo. DPP IV exhibits characteristics that have allowed the development of specific orally administered inhibitors with proven efficacy in improving glucose tolerance in animal models of diabetes. A number of clinical trials have demonstrated that DPP IV inhibitors are effective in improving glucose disposal and reducing hemoglobin A1c levels in type 2 diabetic patients and one inhibitor, sitagliptin, is now in therapeutic use, with others likely to receive FDA approval in the near future. Studies aimed at elucidating the mode of action of the inhibitors are still ongoing. Both enhancement of insulin secretion and reduction in glucagon secretion, resulting from the blockade of incretin degradation, are believed to play important roles in DPP IV inhibitor action. Preclinical studies indicate that increased levels of incretins improve beta-cell secretory function and exert effects on beta-cell mitogenesis and survival that can preserve beta-cell mass. Roles for other hormones, neuropeptides and cytokines in DPP IV inhibitor-medicated responses are also possible.

Incretin-based Therapies for Type 2 Diabetes.

There is a growing interest in developing therapeutic strategies for type 2 diabetes based on the actions of the hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). These hormones are the major incretins released from the intestine in response to nutrient ingestion, and they stimulate insulin secretion in a glucosedependent manner. Both peptides are degraded by the enzyme dipeptidyl peptidase-4 (DPP-4), thus terminating their actions. Studies in animal models of diabetes have shown that the incretins also exert a number of additional actions that improve glucose disposal. GLP-1 reduces food intake and gastric emptying, as well as inhibiting glucagon secretion. Injectable formulations of DPP-4-resistant GLP-1-related peptides (incretin mimetics) that are now in clinical use (exenatide) or undergoing trials (e.g. liraglutide) have been shown to reduce fasting and postprandial glucose and glycosylated hemoglobin (A1C) levels and induce weight loss. Oral administration of DPP-4 inhibitors potentiates the actions of incretins released during a meal. Clinical trials have demonstrated that DPP-4 inhibitors are weight-neutral drugs that also effectively reduce plasma glucose and A1C levels. One inhibitor, sitagliptin, is now available in Canada and the United States, and another, vildagliptin, has recently been approved by the European Union. Other inhibitors are under development. Preclinical studies indicate that treatment with incretin mimetics or DPP-4 inhibitors also preserves beta cell mass by exerting mitogenetic and prosurvival effects. It is not known whether similar effects occur in humans.

Type 2 diabetes--therapy with dipeptidyl peptidase IV inhibitors.

The sole application of an inhibitor of the dipeptidyl peptidase DP IV (also DP 4, CD26, DPP-IV or DPP-4) to a mammal subsequently leading to improved glucose tolerance marks a major breakthrough in metabolic research bearing the potential of a new revolutionary diabetes therapy. This was demonstrated in rat applying the specific DP IV inhibitor isoleucyl thiazolidine. It was published in 1996 for the first time that a specific DP IV inhibitor in a given dose was able to completely block glucagon-like peptide-1 (GLP-1) degradation in vivo resulting in improved insulin response accompanied, by accelerated peripheral glucose disposal. Later on, these results were confirmed by several research teams applying DP IV inhibitors intravenously or orally. Today, the DP IV inhibition for the treatment of metabolic disorders is a validated principle. Now, more than 10 years after the initial animal experiments, first DP IV inhibitors as investigational drugs are tested in phase 3 clinical trials.

Applications of dipeptidyl peptidase IV inhibitors in diabetes mellitus.

A number of alternative therapies for type 2 diabetes are currently under development that take advantage of the actions of the incretin hormones glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide on the pancreatic beta-cell. One such approach is based on the inhibition of dipeptidyl peptidase IV (DP IV), the major enzyme responsible for degrading the incretins in vivo. DP IV exhibits characteristics that have allowed the development of specific inhibitors with proven efficacy in improving glucose tolerance in animal models of diabetes and type 2 human diabetics. While enhancement of insulin secretion, resulting from blockade of incretin degradation, has been proposed to be the major mode of inhibitor action, there is also evidence that inhibition of gastric emptying, reduction in glucagon secretion and important effects on beta-cell differentiation, mitogenesis and survival, by the incretins and other DP IV-sensitive peptides, can potentially preserve beta-cell mass, and improve insulin secretory function and glucose handling in diabetics.

Long-term treatment with dipeptidyl peptidase IV inhibitor improves hepatic and peripheral insulin sensitivity in the VDF Zucker rat: a euglycemic-hyperinsulinemic clamp study.

Upon release into circulation, the potent insulin secretagogues glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are rapidly cleaved and inactivated by the enzyme dipeptidyl peptidase IV (DP IV). Long-term administration of specific DP IV inhibitors, so as to enhance circulating active GIP and GLP-1 levels, has been shown to improve glucose tolerance and beta-cell glucose responsiveness and to reduce hyperinsulinemia in the Vancouver diabetic fatty (VDF) rat model of type 2 diabetes. Using the VDF model, the current study was undertaken to examine the effects of long-term DP IV inhibitor treatment on insulin sensitivity. Euglycemic-hyperinsulinemic clamps were performed on two sets of conscious VDF rats treated with or without the DP IV inhibitor P32/98 (20 mg. kg(-1). day(-1) for 12 weeks). The protocol consisted of three sequential 90-min periods with insulin infusion rates of 0, 5, and 15 mU. kg(-1). min(-1) and included a constant infusion of [ (3)H]glucose for measure of hepatic and peripheral insulin sensitivity. Relative to untreated littermates, the treated animals showed a left shift in the sensitivity of hepatic glucose output to insulin (average reduction approximately 6 micro mol. kg(-1). min(-1)) and a marked gain in peripheral responsiveness to insulin, with glucose disposal rates increasing 105 and 216% in response to the two insulin steps (versus 2 and 46% in controls). These results provide the first demonstration of improved hepatic and peripheral insulin sensitivity after DP IV inhibitor therapy, and coupled with apparent improvements in beta-cell function, they offer strong support for the utility of these compounds in the treatment of diabetes.

Dipeptidyl peptidase IV inhibitor treatment stimulates beta-cell survival and islet neogenesis in streptozotocin-induced diabetic rats.

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.

Dipeptidyl peptidase IV-resistant [D-Ala(2)]glucose-dependent insulinotropic polypeptide (GIP) improves glucose tolerance in normal and obese diabetic rats.

The therapeutic potential of glucose-dependent insulinotropic polypeptide (GIP) for improving glycemic control has largely gone unstudied. A series of synthetic GIP peptides modified at the NH(2)-terminus were screened in vitro for resistance to dipeptidyl peptidase IV (DP IV) degradation and potency to stimulate cyclic AMP and affinity for the transfected rat GIP receptor. In vitro experiments indicated that [D-Ala(2)]GIP possessed the greatest resistance to enzymatic degradation, combined with minimal effects on efficacy at the receptor. Thus, [D-Ala(2)]GIP(1--42) was selected for further testing in the perfused rat pancreas and bioassay in conscious Wistar and Zucker rats. When injected subcutaneously in normal Wistar, Fa/?, or fa/fa Vancouver Diabetic Fatty (VDF) Zucker rats, both GIP and [D-Ala(2)]GIP significantly reduced glycemic excursions during a concurrent oral glucose tolerance test via stimulation of insulin release. The latter peptide displayed greater in vivo effectiveness, likely because of resistance to enzymatic degradation. Hence, despite reduced bioactivity in diabetic models at physiological concentrations, GIP and analogs with improved plasma stability still improve glucose tolerance when given in supraphysiological doses, and thus may prove useful in the treatment of diabetic states.

A novel pathway for regulation of glucose-dependent insulinotropic polypeptide (GIP) receptor expression in beta cells.

Glucose-dependent insulinotropic polypeptide (GIP) is secreted postprandially and acts in concert with glucose to stimulate insulin secretion from the pancreas. Here, we describe a novel pathway for the regulation of GIP receptor (GIPR) expression within clonal beta-cell lines, pancreatic islets, and in vivo. High (25 mM) glucose was able to significantly reduce GIPR mRNA levels in INS(832/13) cells after only 6 h. In contrast, palmitic acid (2 mM) and WY 14643 (100 microM) stimulated approximate doublings of GIPR expression in INS(832/13) cells under low (5.5 mM), but not high (25 mM), glucose conditions, suggesting that fat can regulate GIPR expression via PPARalpha in a glucose-dependent manner. Both MK-886, an antagonist of PPARalpha, and a dominant negative form of PPARalpha transfected into INS(832/13) cells caused a significant reduction in GIPR expression in low, but not high, glucose conditions. Finally, in hyperglycemic clamped rats, there was a 70% reduction in GIPR expression in the islets and a 71% reduction in GIP-stimulated insulin secretion from the perfused pancreas. Thus, evidence is presented that the GIPR is controlled at normoglycemia by the fatty acid load on the islet; however, when exposed to hyperglycemic conditions, the GIPR is down-regulated, which may contribute to the decreased responsiveness to GIP that is observed in type 2 diabetes.

Dipeptidyl peptidase IV inhibitors: how do they work as new antidiabetic agents?

A number of new approaches to diabetes therapy are currently undergoing clinical trials, including those involving stimulation of the pancreatic beta-cell with the gut-derived insulinotropic hormones (incretins), GIP and GLP-1. The current review focuses on an approach based on the inhibition of dipeptidyl peptidase IV (DP IV), the major enzyme responsible for degrading the incretins in vivo. The rationale for this approach was that blockade of incretin degradation would increase their physiological actions, including the stimulation of insulin secretion and inhibition of gastric emptying. It is now clear that both GIP and GLP-1 also have powerful effects on beta-cell differentation, mitogenesis and survival. By potentiating these pleiotropic actions of the incretins, DP IV inhibition can therefore preserve beta-cell mass and improve secretory function in diabetics.

Effect of glucagon-like peptide 1 (7-36 amide) on insulin-mediated glucose uptake in patients with type 1 diabetes.

OBJECTIVE: To examine the insulinomimetic insulin-independent effects of glucagon-like peptide (GLP)-1 on glucose uptake in type 1 diabetic patients. RESEARCH DESIGN AND METHODS: We used the hyperinsulinemic-euglycemic clamp (480 pmol. m(-2) x min(-1)) in paired randomized studies of six women and five men with type 1 diabetes. In the course of one of the paired studies, the subjects also received GLP-1 at a dose of 1.5 pmol. kg(-1) x min(-1). The patients were 41 +/- 3 years old with a BMI of 25 +/- 1 kg/m(2). The mean duration of diabetes was 23 +/- 3 years. RESULTS: Plasma glucose was allowed to fall from a fasting level of approximately 11 mmol/l to 5.3 mmol/l in each study and thereafter was held stable at that level. Plasma insulin levels during both studies were approximately 900 pmol/l. Plasma C-peptide levels did not change during the studies. In the GLP-1 study, plasma total GLP-1 levels were elevated from the fasting level of 31 +/- 3 to 150 +/- 17 pmol/l. Plasma glucagon levels fell from the fasting levels of approximately 14 pmol/l to 9 pmol/l during both paired studies. Hepatic glucose production was suppressed during the glucose clamps in all studies. Glucose uptake was not different between the two studies ( approximately 40 micromol. kg(-1) x min(-1)). CONCLUSIONS: GLP-1 does not augment insulin-mediated glucose uptake in lean type 1 diabetic patients.

Glucose-dependent insulinotropic polypeptide promotes beta-(INS-1) cell survival via cyclic adenosine monophosphate-mediated caspase-3 inhibition and regulation of p38 mitogen-activated protein kinase.

The incretin glucose-dependent insulinotropic polypeptide (GIP) is a major regulator of postprandial insulin secretion in mammals. Recent studies in our laboratory, and others have suggested that GIP is a potent stimulus for protein kinase activation, including the MAPK (ERK1/2) module. Based on these studies, we hypothesized that GIP could regulate cell fate and sought to examine the underlying mechanisms involved in GIP stimulation of cell survival. GIP potentiated glucose-induced beta-(INS-1)-cell growth to levels comparable with GH and GLP-1 while promoting cell survival in the face of serum and glucose-deprivation or treatment with wortmannin or streptozotocin. In the absence of GIP, 50% of cells died after 48 h of serum and glucose withdrawal, whereas 91 +/- 10% of cells remained viable in the presence of GIP [n = 3, P < 0.05; EC50 of 1.24 +/- 0.48 nm GIP (n = 4)]. Effects of GIP on cell survival and inhibition of caspase-3 were mimicked by forskolin, but pharmacological experiments excluded roles for MAPK kinase (Mek)1/2, phosphatidylinositol 3-kinase, protein kinase A, Epac, and Rap 1. Survival effects of GIP were ablated by the inhibitor SB202190, indicating a role for p38 MAPK. Furthermore, caspase-3 activity was also regulated by p38 MAPK, with a lesser role for Mek1/2, based on RNA interference studies. We propose that GIP is able to reverse caspase-3 activation via inhibition of long-term p38 MAPK phosphorylation in response to glucose deprivation (+/-wortmannin). Intriguingly, these findings contrasted with short-term phosphorylation of MKK3/6-->p38 MAPK-->ATF-2 by GIP. Thus, these data suggest that GIP is able to regulate INS-1 cell survival by dynamic control of p38 MAPK phosphorylation via cAMP signaling and lend further support to the notion that GIP regulation of MAPK signaling is critical for its regulation of cell fate.

In depth analysis of the N-terminal bioactive domain of gastric inhibitory polypeptide.

Gastric inhibitory polypeptide/glucose-dependent insulinotropic polypeptide (GIP) is an important gastrointestinal regulator of insulin release and glucose homeostasis following a meal. Strategies have been undertaken to delineate the bioactive domains of GIP with the intention of developing small molecular weight GIP mimetics. The molecular cloning of receptors for GIP and the related hormone GLP-1 (glucagon-like peptide-1) has allowed examination of the characteristics of incretin analogs in transfected cell models. The current report examines the N-terminal bioactive domain of GIP residing in residues 1-14 by alanine scanning mutagenesis and N-terminal substitution/modification. Further studies examined peptide chimeras of GIP and GLP-1 designed to localize bioactive determinants of the two hormones. The alanine scan of the GIP(1-14) sequence established that the peptide was extremely sensitive to structural perturbations. Only replacement of amino acids 2 and 13 with those found in glucagon failed to dramatically reduce receptor binding and activation. Of four GIP(1-14) peptides modified by the introduction of DP IV-resistant groups, a peptide with a reduced bond between Ala2 and Glu3 demonstrated improved receptor potency compared to native GIP(1-14). The peptide chimera studies supported recent results on the importance of a mid-region helix for bioactivity of GIP, and confirmed existence of two separable regions with independent intrinsic receptor binding and activation properties. Furthermore, peptide chimeras showed that binding of GLP-1 also involves both N- and C-terminal domains, but that it apparently contains only a single bioactive domain in its N-terminus. Together, these results should facilitate development of incretin based therapies using rational drug design for potential treatment of diabetes.