254-OR: GLP-1(32-36) Peptide Promotes Diabetic Ischemia Recovery through Independent GLP-1R Pathway to Mediate Lipid Transport

The glucagon-like peptide 1 (GLP1) receptor is a member of Family B G protein-coupled receptors and represents an important drug target for type 2 diabetes. Despite recent solution of the structure of the amino-terminal domain of this receptor and that of several close family members, understanding of the molecular basis of natural ligand GLP1 binding to its intact receptor remains limited. The goal of this study was to explore spatial approximations between specific receptor residues within the carboxyl terminus of GLP1 and its receptor as normally docked. Therefore, we developed and characterized two high affinity, full-agonist photolabile GLP1 probes having sites for covalent attachment in positions 24 and 35. Both probes labeled the receptor specifically and saturably. Subsequent peptide mapping using chemical and proteinase cleavages of purified wild-type and mutant GLP1 receptor identified that the Arg(131)-Lys(136) segment at the juxtamembrane region of the receptor amino terminus contained the site of labeling for the position 24 probe, and the specific receptor residue labeled by this probe was identified as Glu(133) by radiochemical sequencing. Similarly, nearby residue Glu(125) within the same region of the receptor amino-terminal domain was identified as the site of labeling by the position 35 probe. These data represent the first direct demonstration of spatial approximation between GLP1 and its intact receptor as docked, providing two important constraints for the modeling of this interaction. This should expand our understanding of the molecular basis of natural agonist ligand binding to the GLP1 receptor and may be relevant to other family members.

Diabetes mellitus, defined as a fasting plasma glucose of ≥126 mg/dL or a glycosylated hemoglobin A1c level (HbA1c) of ≥6.5%, afflicts ≈12.9% of adults in the United States and nearly 285 million adults worldwide.1,2 Diabetes mellitus is a major risk factor for the development of cardiovascular disease, independently conferring a 2-fold excess risk of coronary heart disease and stroke.3 Macrovascular events in diabetes mellitus remain the leading cause of mortality, and the burden of cardiovascular disease attributable to diabetes mellitus has increased over the past decade.4 An increase in the prevalence of obesity has contributed to the rise in diabetes mellitus. Additionally, obesity independently increases the risk of cardiovascular disease in patients with diabetes mellitus.5 Although strict glycemic control unequivocally reduces the microvascular complications of diabetes mellitus, the macrovascular benefits of intensive therapy have been difficult to establish, with conflicting results from large clinical trials.6–9 Multifactorial strategies are recommended to reduce cardiovascular risk in diabetes mellitus through enhanced glycemic control, blood pressure reduction, lipid management, weight loss, and physical activity.10 Unfortunately, despite aggressive interventions for hyperglycemia, <50% of patients achieve standard HbA1c targets with conventional therapy.11 Polypharmacy is required to achieve glycemic control in the majority of patients within 3 years of diagnosis.12 Although combinations of drug classes can synergistically target multiple pathophysiological defects, novel therapies are required to manage diabetes mellitus and mitigate cardiovascular risks. Dipeptidyl-peptidase IV (DPP-IV) inhibitor and glucagon-like peptide-1 (GLP-1) receptor agonist incretin therapies were developed to complement conventional treatment options for diabetes mellitus. Despite promising initial reports of cardioprotective effects, DPP-IV inhibitors have failed to demonstrate improved cardiovascular outcomes in large clinical trials.13–15 Randomized studies to evaluate cardiovascular outcomes associated with GLP-1 receptor agonists are currently underway. This review presents …

An Albumin-Exendin-4 Conjugate Engages Central and Peripheral Circuits Regulating Murine Energy and Glucose Homeostasis

glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted from gut enteroendocrine L cells in response to nutrient ingestion that potentiates glucose-stimulated insulin secretion via direct actions on the islet β-cell GLP-1 receptor (GLP-1R) ([4][1]). Because of these properties, manipulation

In 1964, studies in just 2 subjects offered a simple, salient, and fundamental observation reported in 612 words: Glucose induces a greater insulin response when introduced through the gastrointestinal tract than when injected intravenously (the Figure, A).2 This finding built on studies dating to 1928 that injecting extracts of small intestine into animals lowered their glucose levels. Subsequently, this incretin effect was found to be mediated by glucagon-like peptide-1 (GLP1) and its action on pancreatic GLP1 receptors, in addition to contributions from glucose-dependent insulinotropic polypeptide.3,4 Moreover, the incretin response was found to be impaired in those with type 2 diabetes mellitus (T2D). We now know that the incretin axis also includes the enzyme dipeptidyl peptidase-IV (DPPIV), a serine protease that rapidly degrades GLP1 and other proteins.5 Ultimately, this arc of discovery led to new approved antidiabetic therapies: GLP1 analogs (exenatide, liraglutide) and DPPIV inhibitors (saxagliptin, sitagliptin, and, outside the United States, vildagliptin).4 For both classes of drugs, early preclinical experiments and smaller human studies suggest that targeting the incretin axis might address the elusive goal of an antidiabetic agent that improves cardiovascular disease.6,7 In the current issue of Circulation , Shah et al8 add this evolving story with their report that alogliptin, a DPPIV inhibitor in development, limits atherosclerosis and inflammation in 2 different mouse models. Given the increasing clinical use of approved incretin modulators, current large cardiovascular outcome trials with GLP1 agents and DPPIV inhibitors, and ongoing development of novel agents that target incretin signaling, further consideration of how the incretin axis might intersect the cardiovascular system is well warranted. Figure. A , The incretin effect. The well-documented phenomenon of oral glucose eliciting a higher insulin response than intravenous glucose at identical plasma levels of glucose is known as …

Insulin and glucagon are well-known peptide hormones that keep glucose levels within a healthy range in the body. But they are only part of a complex network that controls concentrations of this ubiquitous sugar in blood and tissues. Other molecules regulate glucose by controlling insulin secretion from the pancreas or protecting pancreatic β cells against stresses that lead to cellular dysfunction or cell death (1). One of these protective regulators is glucagon-like peptide 1 (GLP-1), a 30-amino-acid-long peptide produced in specialized epithelial cells of the intestine, called L cells, and also in the brain and other organs and tissues (2). GLP-1 belongs to a group of peptides that mediate the “incretin effect,” an endocrine response to glucose arising from food digestion in the intestines (2, 3). This response helps regulate food intake and the fate of dietary glucose. Specifically, GLP-1 is released from the intestinal cells when food is ingested and then binds to and activates the GLP-1 receptor (GLP-1R), a G protein–coupled receptor on many cell types, including β cells in which GLP-1R signaling stimulates insulin synthesis and secretion (3). Notably, the incretin effect stimulates insulin secretion from pancreatic β cells more strongly than exposure to glucose alone. An article published in the Journal of Biological Chemistry (4), recognized as a Classic here, added to our understanding of the incretin effect by showing that GLP-1R signaling protects β cells from cell death (Fig. 1). This finding was significant for preventing or managing type 2 diabetes, in which β-cell apoptosis occurs (5) and may contribute to insufficient pancreatic insulin production (6). Open in a separate window Figure 1. Li et al. (4) have shown that binding of the receptor agonist exendin-4 to GLP-1R on pancreatic β cells protects the cells from cellular injury and cytokine-induced apoptosis and thereby preserves glucose homeostasis in mice. Binding of GLP-1 to its cognate receptor on pancreatic β cells up-regulates intracellular cAMP levels, in turn reducing streptozotocin-induced β-cell death. Images of exendin-4 and GLP-1R (with GLP-1 bound) are from Ref. 7; image of cAMP is from Wikimedia, used under Creative Commons.

Dysfunction of glucagon-secreting α-cells participates in the progression of diabetes, and glucagon receptor (GCGR) antagonism is regarded as a novel strategy for diabetes therapy. GCGR antagonism upregulates glucagon and glucagon-like peptide 1 (GLP-1) secretion and, notably, promotes β-cell regeneration in diabetic mice. Here, we aimed to clarify the role of GLP-1 receptor (GLP-1R) activated by glucagon and/or GLP-1 in the GCGR antagonism–induced β-cell regeneration. We showed that in db/db mice and type 1 diabetic wild-type or Flox/cre mice, GCGR monoclonal antibody (mAb) improved glucose control, upregulated plasma insulin level, and increased β-cell area. Notably, blockage of systemic or pancreatic GLP-1R signaling by exendin 9-39 (Ex9) or Glp1r knockout diminished the above effects of GCGR mAb. Furthermore, glucagon-neutralizing antibody (nAb), which prevents activation of GLP-1R by glucagon, also attenuated the GCGR mAb–induced insulinotropic effect and β-cell regeneration. In cultured primary mouse islets isolated from normal mice and db/db mice, GCGR mAb action to increase insulin release and to upregulate β-cell–specific marker expression was reduced by a glucagon nAb, by the GLP-1R antagonist Ex9, or by a pancreas-specific Glp1r knockout. These findings suggest that activation of GLP-1R by glucagon participates in β-cell regeneration induced by GCGR antagonism in diabetic mice.Article HighlightsGlucagon receptor (GCGR) antagonism promotes β-cell regeneration in type 1 and type 2 diabetic mice and in euglycemic nonhuman primates. Glucagon and glucagon-like peptide 1 (GLP-1) can activate the GLP-1 receptor (GLP-1R), and their levels are upregulated following GCGR antagonism.We investigated whether GLP-1R activated by glucagon and/or GLP-1 contributed to β-cell regeneration induced by GCGR antagonism.We found that blockage of glucagon–GLP-1R signaling attenuated the GCGR monoclonal antibody–induced insulinotropic effect and β-cell regeneration in diabetic mice.Our study reveals a novel mechanism of β-cell regeneration and uncovers the communication between α-cells and β-cells in regulating β-cell mass.

We have previously shown that diabetes causes pericyte dysfunction, leading to loss of vascular integrity and vascular cognitive impairment and dementia (VCID). Glucagon-like peptide-1 (GLP-1) receptor agonists (GLP-1 RAs), used in managing type 2 diabetes mellitus, improve the cognitive function of diabetic individuals beyond glycaemic control, yet the mechanism is not fully understood. In the present study, we hypothesise that GLP-1 RAs improve VCID by preventing diabetes-induced pericyte dysfunction. Mice with streptozotocin-induced diabetes and non-diabetic control mice received either saline (NaCl 154 mmol/l) or exendin-4, a GLP-1 RA, through an osmotic pump over 28 days. Vascular integrity was assessed by measuring cerebrovascular neovascularisation indices (vascular density, tortuosity and branching density). Cognitive function was evaluated with Barnes maze and Morris water maze. Human brain microvascular pericytes (HBMPCs), were grown in high glucose (25 mmol/l) and sodium palmitate (200 μmol/l) to mimic diabetic conditions. HBMPCs were treated with/without exendin-4 and assessed for nitrative and oxidative stress, and angiogenic and blood-brain barrier functions. Diabetic mice treated with exendin-4 showed a significant reduction in all cerebral pathological neovascularisation indices and an improved blood-brain barrier (p<0.05). The vascular protective effects were accompanied by significant improvement in the learning and memory functions of diabetic mice compared with control mice (p<0.05). Our results showed that HBMPCs expressed the GLP-1 receptor. Diabetes increased GLP-1 receptor expression and receptor nitration in HBMPCs. Stimulation of HBMPCs with exendin-4 under diabetic conditions decreased diabetes-induced vascular inflammation and oxidative stress, and restored pericyte function (p<0.05). This study provides novel evidence that brain pericytes express the GLP-1 receptor, which is nitrated under diabetic conditions. GLP-1 receptor activation improves brain pericyte function resulting in restoration of vascular integrity and BBB functions in diabetes. Furthermore, the GLP-1 RA exendin-4 alleviates diabetes-induced cognitive impairment in mice. Restoration of pericyte function in diabetes represents a novel therapeutic target for diabetes-induced cerebrovascular microangiopathy and VCID.

Paying the Price for Eating Ice Cream: Is Excessive GLP-1 Signaling in the Brain the Culprit?

Glucagon-like peptide 2 (GLP-2) is an intestinotropic peptide that binds to GLP-2 receptor (GLP-2R), a class-B G protein-coupled receptor. The GLP-2R antagonist GLP-2(3-33) has relatively high partial agonistic activity, and there are as yet no ideal known potent GLP-2R antagonists. We therefore prepared several truncated forms of human GLP-2 and characterized them by binding and reporter assays to find antagonists more potent than GLP-2(3-33). We found that GLP-2(11-33) was the most potent orthosteric GLP-2R antagonist, with binding activity almost equal to those of GLP-2 and GLP-2(3-33) and weaker intrinsic agonistic activity than GLP-2(3-33). GLP-2(11-33) retained weak agonistic activity toward human, cynomolgus monkey, dog, and Syrian hamster GLP-2Rs. However, it had no agonistic activity toward rat GLP-2R. GLP-2(11-33) potentiated the agonistic activity of an ago-allosteric modulator of GLP-2R, compound 1 (N-[1-(2,5-dichlorothiophen-3-yl)-2-(phenylsulfanyl)ethylidene]hydroxylamine), synergistically toward human GLP-2R. In the case of rat GLP-2R, GLP-2(11-33) decreased the agonistic activity of compound 1, although GLP-2 and GLP-2(3-33) increased this activity additively. These findings suggest that the binding sites of the ago-allosteric modulator and GLP-2 overlap, at least in rat GLP-2R. GLP-2(11-33) is a novel, useful tool for analyzing the mode of action of agonists and ago-allosteric modulators of GLP-2R.

nonalcoholic fatty liver disease (NAFLD) encompasses a spectrum ranging from simple steatosis to steatohepatitis (NASH), increasing fibrosis and eventually, cirrhosis ([22][1]). Importantly, NASH accompanied by fibrosis and severe inflammation is the most relevant predictor for disease progression

Mucosal Adaptation to Enteral Nutrients is Dependent on the Physiologic Actions of Glucagon-Like Peptide-2 in Mice

It is increasingly clear that the gastrointestinal (GI) tract plays a major role in glucose regulation after meal ingestion. A prominent mechanism of GI influence over blood glucose is through the secretion of glucose-dependent insulinotropic polypeptide and glucagon-like peptide 1 (GLP-1), together termed incretins because of their actions to stimulate internal secretions (i.e., insulin). Indeed, common estimates are that 70% of β-cell release after meals is attributable to the incretins, and both of these peptides are necessary for normal glucose tolerance (1). Moreover, iv administration of GLP-1, and to a much lesser degree glucose-dependent insulinotropic polypeptide, enhances insulin secretion and improves glucose disposal in persons with type 2 diabetes. This has led to the development of two new classes of medication for diabetes, GLP-1 receptor agonists, which mimic the effects of endogenous GLP-1, and dipeptidyl peptidase IV (DPP-4) inhibitors, which prevent metabolism of the native peptide and extend its activity (2).

Glucagon like peptide 1 (GLP1) is an incretin hormone released from the enteroendocrine L-type cells of the lower gastrointestinal tract. The active isoforms of GLP1 are rapidly degraded (<2 min) by protease dipeptidyl peptidase-4 (DPP-4) after their release. Among its functions, GLP1 exerts a pivotal role in regulating glucose and lipid metabolism. In particular, GLP1 increases glucose stimulated insulin secretion, functional pancreatic β-cell mass and decreases glucagon secretion from pancreatic α-cells. GLP1 can also be a regulator of lipid and lipoprotein metabolism ameliorating diabetic dyslipidemia, liver steatosis, and promoting satiety. Interestingly, it has been found that GLP1 and GLP1 agonists can modulate the expression of different microRNAs (miRNAs), a ~22 nucleotides small non-coding RNAs, key modulators of protein expression. In particular, in pancreas, GLP1 increases the expression levels of miRNA-212 and miRNA-132, stimulating insulin secretion. Similarly, GLP1 decreases miRNA-338 levels, leading to an increase of pancreatic β-cell function, followed by an improvement of diabetic conditions. Moreover, GLP1 modulation of miRNAs expression in the liver regulates hepatic lipid storage. Indeed, GLP1 down-regulates miRNA-34a and miRNA-21 and up-regulates miRNA-200b and miRNA-200c expression in liver, reducing intra hepatic lipid accumulation and liver steatosis. Clinical and pre-clinical studies, discussed in this review, suggest that modulation of GLP1/miRNAs pathway may be a useful and innovative therapeutic strategy for prevention and treatment of metabolic disorders, such as diabetes mellitus and liver steatosis.

Glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and glucagon bind to related members of the same receptor superfamily and exert important effects on glucose homeostasis, insulin secretion, and energy regulation. The present study assessed the biological actions and therapeutic utility of novel GIP/glucagon/GLP-1 hybrid peptides. Nine novel peptides were synthesized and exhibited complete DPP-IV resistance and enhanced in vitro insulin secretion. The most promising peptide, [dA(2)]GLP-1/GcG, stimulated cAMP production in GIP, GLP-1, and glucagon receptor-transfected cells. Acute administration of [dA(2)]GLP-1/GcG in combination with glucose significantly lowered plasma glucose and increased plasma insulin in normal and obese diabetic (ob/ob) mice. Furthermore, [dA(2)]GLP-1/GcG elicited a protracted glucose-lowering and insulinotropic effect in high fat-fed mice. Twice daily administration of [dA(2)]GLP-1/GcG for 21 days decreased body weight and nonfasting plasma glucose and increased circulating plasma insulin concentrations in high fat-fed mice. Furthermore, [dA(2)]GLP-1/GcG significantly improved glucose tolerance and insulin sensitivity by day 21. Interestingly, locomotor activity was increased in [dA(2)]GLP-1/GcG mice, without appreciable changes in aspects of metabolic rate. Studies in knock-out mice confirmed the biological action of [dA(2)]GLP-1/GcG via multiple targets including GIP, GLP-1, and glucagon receptors. The data suggest significant promise for novel triple-acting hybrid peptides as therapeutic options for obesity and diabetes.