Christopher H.S. McIntosh

Improved glucose tolerance in Zucker fatty rats by oral administration of the dipeptidyl peptidase IV inhibitor isoleucine thiazolidide.

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 NHg-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 desorption7sol;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.

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.

An immunocytochemical investigation with monoclonal antibodies to somatostatin

SummaryFour monoclonal antibodies specific for somatostatin have been produced and characterized. These antibodies were used to assess the anatomical relationship of somatostatin-containing cells in the pancreas and gastrointestinal tract of man, baboon and rat with ten other peptide-containing endocrine cells. The peptides investigated were gastrin, cholecystokinin, motilin, secretin, neurotensin, gastric inhibitory polypeptide, gut-glucagon, pancreatic glucagon, pancreatic polypeptide and insulin.The only regions in which somatostatin cells were seen in close contact with another endocrine cell were in the pancreas and the gastric antrum. In the pancreas somatostatin cells were commonly seen in close contact with insulin, glucagon and pancreatic polypeptide cells and infrequent contact was demonstrable with the gastrin-immunoreactive cells in the antrum of both rat and man. In all other cases no evidence was obtained for a close anatomical relationship between somatostatin cells and the other enteroendocrine cells.

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.

Improved glucose tolerance in rats treated with the dipeptidyl peptidase IV (CD26) inhibitor Ile-thiazolidide.

The incretins glucose-dependent insulinotropic polypeptide (GIP1-42) and truncated forms of glucagon-like peptide-1 (GLP-1) are hormones released from the gut in response to ingested nutrients, which act on the pancreas to potentiate glucose-induced insulin secretion. These hormones are rapidly inactivated by the circulating enzyme dipeptidyl peptidase IV ([DPIV] CD26). This study describes the effect on glucose tolerance and insulin secretion of inhibiting endogenous DPIV in the rat using Ile-thiazolidide, a specific DPIV inhibitor. High-performance liquid chromatography (HPLC) analysis of plasma following in vivo administration of 125I-labeled peptides showed that inhibition of DPIV by about 70% prevented the degradation of 90.0% of injected 125I-GLP-17-36 after 5 minutes, while only 13.4% remained unhydrolyzed in rats not treated with the DPIV-inhibiting agent after only 2 minutes. Ile-thiazolidide treatment also increased the circulating half-life of intact GLP-17-36 released in response to intraduodenal (ID) glucose (as measured by N-terminal specific radioimmunoassay [RIA]). In addition, inhibition of DPIV in vivo resulted in an earlier increase and peak of plasma insulin and a more rapid clearance of blood glucose in response to ID glucose challenge. When considered with the HPLC data, these results suggest that the altered insulin profile is an incretin-mediated response. DPIV inhibition resulting in improved glucose tolerance may have therapeutic potential for the management of type 2 diabetes mellitus.

Glucose-dependent insulinotropic polypeptide (Gastric Inhibitory Polypeptide; GIP).

Glucose-dependent insulinotropic polypeptide (GIP; gastric inhibitory polypeptide) is a 42 amino acid hormone that is produced by enteroendocrine K-cells and released into the circulation in response to nutrient stimulation. Both GIP and glucagon-like peptide-1 (GLP-1) stimulate insulin secretion in a glucose-dependent manner and are thus classified as incretins. The structure of mammalian GIP is well conserved and both the N-terminus and central region of the molecule are important for biological activity. Following secretion, GIP is metabolized by the endoprotease dipeptidyl peptidase IV (DPP-IV). In addition to its insulinotropic activity, GIP exerts a number of additional actions including promotion of growth and survival of the pancreatic beta-cell and stimulation of adipogenesis. The brain, bone, cardiovascular system, and gastrointestinal tract are additional targets of GIP. The GIP receptor is a member of the B-family of G protein-coupled receptors and activation results in the stimulation of adenylyl cyclase and Ca(2+)-independent phospholipase A(2) and activation of protein kinase (PK) A and PKB. The Mek1/2-Erk1/2 and p38 MAP kinase signaling pathways are among the downstream pathways involved in the regulation of beta-cell function. GIP also increases expression of the anti-apoptotic Bcl-2 and decreases expression of the pro-apoptotic Bax, resulting in reduced beta-cell death. In adipose tissue, GIP interacts with insulin to increase lipoprotein lipase activity and lipogenesis. There is significant interest in potential clinical applications for GIP analogs and both agonists and antagonists have been developed for preclinical studies.

Dipeptidyl peptidase IV (DPIV/CD26) degradation of glucagon. Characterization of glucagon degradation products and DPIV-resistant analogs.

Over the past decade, numerous studies have been targeted at defining structure-activity relationships of glucagon. Recently, we have found that glucagon1–29 is hydrolyzed by dipeptidyl peptidase IV (DPIV) to produce glucagon3–29 and glucagon5–29; in human serum, [pyroglutamyl (pGlu)3]glucagon3–29 is formed from glucagon3–29, and this prevents further hydrolysis of glucagon by DPIV (H.-U. Demuth, K. Glund, U. Heiser, J. Pospisilik, S. Hinke, T. Hoffmann, F. Rosche, D. Schlenzig, M. Wermann, C. McIntosh, and R. Pederson, manuscript in preparation). In the current study, the biological activity of these peptides was examined in vitro. The amino-terminally truncated peptides all behaved as partial agonists in cyclic AMP stimulation assays, with Chinese hamster ovary K1 cells overexpressing the human glucagon receptor (potency: glucagon1–29 > [pGlu3]glu- cagon3–29 > glucagon3–29 > glucagon5–29 > [Glu9]glu- cagon2–29). In competition binding experiments, [pGlu3]glucagon3–29 and glucagon5–29 both demonstrated 5-fold lower affinity for the receptor than glucagon1–29, whereas glucagon3–29 exhibited 18-fold lower affinity. Of the peptides tested, only glucagon5–29 showed antagonist activity, and this was weak compared with the classical glucagon antagonist, [Glu9]glucagon2–29. Hence, DPIV hydrolysis of glucagon yields low affinity agonists of the glucagon receptor. As a corollary to evidence indicating that DPIV degrades glucagon (Demuth, et al., manuscript in preparation), DPIV-resistant analogs were synthesized. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry was used to assess DPIV resistance, and it allowed kinetic analysis of degradation. Of several analogs generated, only [d-Ser2] and [Gly2]glucagon retained high affinity binding and biological potency, similar to native glucagon in vitro. [d-Ser2]Glucagon exhibited enhanced hyperglycemic activity in a bioassay, whereas [Gly2]glucagon was not completely resistant to DPIV degradation.

Glucose-dependent insulinotropic polypeptide activates the Raf-Mek1/2-ERK1/2 module via a cyclic AMP/cAMP-dependent protein kinase/Rap1-mediated pathway.

The gastrointestinal hormone, glucose-dependent insulinotropic polypeptide (GIP), is one of the most important regulators of insulin secretion following ingestion of a meal. GIP stimulates insulin secretion from the pancreatic β-cell via its G protein-coupled receptor activation of adenylyl cyclase and other signal transduction pathways, but there is little known regarding subsequent protein kinase pathways that are activated. A screening technique was used to determine the relative abundance of 75 protein kinases in CHO-K1 cells expressing the GIP receptor and in two pancreatic β-cell lines (βTC-3 and INS-1 (832/13) cells). This information was used to identify kinases that are potentially regulated following GIP stimulation, with a focus on GIP regulation of the ERK1/2 MAPK pathway. In CHO-K1 cells, GIP induced phosphorylation of Raf-1 (Ser-259), Mek1/2 (Ser-217/Ser-221), ERK1/2 (Thr-202 and Tyr-204), and p90 RSK (Ser-380) in a concentration-dependent manner. Activation of ERK1/2 was maximal at 4 min and was cAMP-dependent protein kinase-dependent and protein kinase C-independent. Studies using a β-cell line (INS-1 clone 832/13) corroborated these findings, and it was also demonstrated that the ERK1/2 module could be activated by GIP in the absence of glucose. Finally, we have shown that GIP regulation of the ERK1/2 module is via Rap1 but does not involve Gβγ subunits nor Src tyrosine kinase, and we propose that cAMP-based regulation occurs via B-Raf in both CHO-K1 and β-cells. These results establish the importance of GIP in the cellular regulation of the ERK1/2 module and identify a role for cAMP in coupling its G protein-coupled receptors to ERK1/2 activity in pancreatic β-cells.

Activation of Lipoprotein Lipase by Glucose-dependent Insulinotropic Polypeptide in Adipocytes A ROLE FOR A PROTEIN KINASE B, LKB1, AND AMP-ACTIVATED PROTEIN KINASE CASCADE

Glucose-dependent insulinotropic polypeptide (GIP) has been mainly studied because of its glucose-dependent insulinotropic action and its ability to regulate β-cell proliferation and survival. Considerably less is known about the effects of GIP on fat metabolism, and the present study was directed at identifying the mechanisms underlying its stimulatory action on lipoprotein lipase (LPL). In differentiated 3T3-L1 adipocytes, GIP, in the presence of insulin, increased LPL activity and triglyceride accumulation through a pathway involving increased phosphorylation of protein kinase B (PKB) and reductions in phosphorylated LKB1 and AMP-activated protein kinase (AMPK). Knockdown of AMPK using RNA interference and application of the AMPK inhibitor, Compound C, supported this conclusion. In contrast, the other major incretin hormone, glucagon-like peptide-1, exhibited no significant effects on LPL activity or PKB, LKB1, or AMPK phosphorylation. Cultured subcutaneous human adipocytes showed similar responses to GIP but with greater sensitivity. Chronic elevation of circulating GIP levels in the Vancouver diabetic fatty Zucker rat in vivo resulted in increased LPL activity and elevated triglyceride accumulation in epidydimal fat tissue, combined with a modulation of PKB, LKB1, and AMPK phosphorylation similar to that observed in vitro. This appears to be the first demonstration of a GIP-stimulated signal transduction pathway involved in increasing fat storage in adipocytes.

Glucose-Dependent Insulinotropic Polypeptide-Mediated Up-Regulation of β-Cell Antiapoptotic Bcl-2 Gene Expression Is Coordinated by Cyclic AMP (cAMP) Response Element Binding Protein (CREB) and cAMP-Responsive CREB Coactivator 2

ABSTRACT The cyclic AMP (cAMP)/protein kinase A (PKA) cascade plays a central role in β-cell proliferation and apoptosis. Here, we show that the incretin hormone glucose-dependent insulinotropic polypeptide (GIP) stimulates expression of the antiapoptotic Bcl-2 gene in pancreatic β cells through a pathway involving AMP-activated protein kinase (AMPK), cAMP-responsive CREB coactivator 2 (TORC2), and cAMP response element binding protein (CREB). Stimulation of β-INS-1 (clone 832/13) cells with GIP resulted in increased Bcl-2 promoter activity. Analysis of the rat Bcl-2 promoter revealed two potential cAMP response elements, one of which (CRE-I [GTGACGTAC]) was shown, using mutagenesis and deletion analysis, to be functional. Subsequent studies established that GIP increased the nuclear localization of TORC2 and phosphorylation of CREB serine 133 through a pathway involving PKA activation and reduced AMPK phosphorylation. At the nuclear level, phospho-CREB and TORC2 were demonstrated to bind to CRE-I of the Bcl-2 promoter, and GIP treatment resulted in increases in their interaction. Furthermore, GIP-mediated cytoprotection was partially reversed by small interfering RNA-mediated reduction in BCL-2 or TORC2/CREB or by pharmacological activation of AMPK. The antiapoptotic effect of GIP in β cells is therefore partially mediated through a novel mode of transcriptional regulation of Bcl-2 involving cAMP/PKA/AMPK-dependent regulation of CREB/TORC2 activity.