Effect of glucagon-like peptide-1 on insulin secretion

The insulinotropic actions of two forms of glucagon-like peptide 1 (GLP-1) containing 31 and 37 amino acid residues on perfused rat pancreas were compared with that of gastric inhibitory polypeptide (GIP), hitherto the most potent intestinal insulinotropic polypeptide known. The smaller form, C-terminally amidated GLP-1-(7-36), strongly enhanced insulin secretion stimulated by 11.1 mM D-glucose at a concentration as low as 0.1 nM. Comparable effects of GIP and GLP-1-(1-37) on insulin secretion were observed at concentrations of 1.0 nM and 10.0 nM, respectively. At the doses tested, neither GLP-1s nor GIP had any effect on insulin secretion induced by 3.3 mM D-glucose. At a concentration of 1.0 nM, GLP-1-(7-36 amide) also enhanced insulin secretion induced by 5 mM L-arginine whereas at concentrations of up to 10.0 nM, GLP-1-(1-37) did not. The results show that the smaller form of GLP-1 is more strongly insulinotropic than GIP. These findings suggest that the smaller GLP-1 may have a physiologically more important role as a modulator of insulin release.     

Role of glucagon-like peptide-1 in the pathogenesis and treatment of diabetes mellitus

Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted from enteroendocrine L cells in response to ingested nutrients. The first recognized and most important action of GLP-1 is the potentiation of glucose-stimulated insulin secretion in beta-cells, mediated by activation of its seven transmembrane domain G-protein-coupled receptor. In addition to its insulinotropic actions, GLP-1 exerts islet-trophic effects by stimulating replication and differentiation and by decreasing apoptosis of beta-cells. The GLP-1 receptor is expressed in a variety of other tissues important for carbohydrate metabolism, including pancreatic alpha-cells, hypothalamus and brainstem, and proximal intestinal tract. GLP-1 also appears to exert important actions in liver, muscle and fat. Thus, GLP-1 suppresses glucagon secretion, promotes satiety, delays gastric emptying and stimulates peripheral glucose uptake. The impaired GLP-1 secretion observed in type 2 diabetes suggests that GLP-1 plays a role in the pathogenesis of this disorder. Thus, because of its multiple actions, GLP-1 is an attractive therapeutic target for the treatment of type 2 diabetes, and major interest has resulted in the development of a variety of GLP-1 receptor agonists for this purpose. Ongoing clinical trials have shown promising results and the first analogs of GLP-1 are expected to be available in the near future.     

Free fatty acids administered into the colon promote the secretion of glucagon-like peptide-1 and insulin

We examined whether free fatty acids (FFAs) promote glucagon-like peptide-1 (GLP-1) secretion when administered into the intestinal tract. We found that an unsaturated long-chain FFA, alpha-linolenic acid (alpha-LA), resulted in increased plasma GLP-1 and insulin levels when administered into the colon. Such stimulatory effects were not apparent with either vehicle or a saturated middle-chain FFA, octanoic acid (OA). Concomitant with GLP-1 secretion, the administration of alpha-LA, but not vehicle or OA, also resulted in a significant increase in the population of pERK positive cells within the GLP-1 positive cells of the colonic mucosa. Moreover, colonic administration of alpha-LA into normal C3H/He mice caused a reduction in plasma glucose levels, as well as in type 2 diabetic model NSY mice. Our results indicate that the in vivo colonic administration of alpha-LA promotes secretion of incretin GLP-1 by activating the ERK pathway in L-cells and thereby enhances the secretion of insulin.     

利用GAP启动子在毕赤酵母中表达与纯化GLP-1类似物

胰高血糖素样肽-1(GLP-1)能提高II型糖尿病患者β胰腺细胞的胰岛素分泌量并能促进β胰腺细胞增殖,是潜在的糖尿病治疗药物。设计一种GLP-1类似物AGGH,即GLP-1(A2G)的二联体与人血清白蛋白(HSA)的N端连接,并在融合蛋白GGH前添加一个丙氨酸(A)。PCR获得融合基因aggh,将融合基因连接到p GAPZαA质粒中。在酵母中利用甘油醛三磷酸脱氢酶(GAP)启动子组成型表达外源蛋白AGGH。研究结果显示:筛选获得表达重组菌株,基因组PCR和western-blot验证正确;以葡萄糖为最优碳源培养下,表达量达到68 mg/L;5 L发酵罐中,发酵52 h蛋白产量最高达238 mg/L。蛋白经四步纯化后,获得纯度为95.8%的AGGH融合蛋白。与利用醇氧化酶1(AOX1)启动子表达的AGGH比较,发现两者产量和活性没有明显差异。但是,GAP启动子表达获得AGGH融合蛋白更加方便,且发酵时间减少了27.8%(20 h)。     

New insights concerning the glucose-dependent insulin secretagogue action of glucagon-like peptide-1 in pancreatic beta-cells.

The GLP-1 receptor is a Class B heptahelical G-protein-coupled receptor that stimulates cAMP production in pancreatic beta-cells. GLP-1 utilizes this receptor to activate two distinct classes of cAMP-binding proteins: protein kinase A (PKA) and the Epac family of cAMP-regulated guanine nucleotide exchange factors (cAMPGEFs). Actions of GLP-1 mediated by PKA and Epac include the recruitment and priming of secretory granules, thereby increasing the number of granules available for Ca(2+)-dependent exocytosis. Simultaneously, GLP-1 promotes Ca(2+) influx and mobilizes an intracellular source of Ca(2+). GLP-1 sensitizes intracellular Ca(2+) release channels (ryanodine and IP (3) receptors) to stimulatory effects of Ca(2+), thereby promoting Ca(2+)-induced Ca(2+) release (CICR). In the model presented here, CICR activates mitochondrial dehydrogenases, thereby upregulating glucose-dependent production of ATP. The resultant increase in cytosolic [ATP]/[ADP] concentration ratio leads to closure of ATP-sensitive K(+) channels (K-ATP), membrane depolarization, and influx of Ca(2+) through voltage-dependent Ca(2+) channels (VDCCs). Ca(2+) influx stimulates exocytosis of secretory granules by promoting their fusion with the plasma membrane. Under conditions where Ca(2+) release channels are sensitized by GLP-1, Ca(2+) influx also stimulates CICR, generating an additional round of ATP production and K-ATP channel closure. In the absence of glucose, no "fuel" is available to support ATP production, and GLP-1 fails to stimulate insulin secretion. This new "feed-forward" hypothesis of beta-cell stimulus-secretion coupling may provide a mechanistic explanation as to how GLP-1 exerts a beneficial blood glucose-lowering effect in type 2 diabetic subjects.     

Search for alpha-helical propensity in the receptor-bound conformation of glucagon-like peptide-1

To elucidate the receptor-bound conformation of glucagon-like peptide-1 (GLP-1), a series of conformationally constrained GLP-1 analogues were synthesized by introducing lactam bridges between Lys(i) and Glu(i)(+4) to form alpha-helices at various positions. The activity and affinity of these analogues to GLP-1 receptors suggested that the receptor-bound conformation comprises two alpha-helical segments between residues 11-21 and 23-34. It is notable that the N-terminal alpha-helix is extended to Thr(11), and that Gly(22) plays a pivotal role in arranging the two alpha-helices. Based on these findings, a highly potent bicyclic GLP-1 analogue was synthesized which is the most conformationally constrained GLP-1 analogue reported to date.     

The effect of glucagon-like peptide-1 in the management of diabetes mellitus: cellular and molecular mechanisms

Incretins, such as glucagon-like peptide-1 (GLP)-1, have been shown to elevate plasma insulin concentration. The purpose of this study is to investigate the cellular and molecular basis of the beneficial effects of GLP-1. Normal and diabetic male Wistar rats were treated with GLP-1 (50 ng/kg body weight) for 10 weeks. At the end of the experiment, pancreatic tissues were taken for immunohistochemistry, immunoelectron microscopy and real-time polymerase chain reaction studies. Samples of blood were retrieved from the animals for the measurement of enzymes and insulin. The results show that treatment of diabetic rats with GLP-1 caused significant (P?GLP-1 (10^?12–10^?6 M) induced significant (P?GLP-1-treated rats compared to controls. GLP-1 treatment induced significant (P?GLP-1-receptor genes in diabetic animals compared to controls. GLP-1 is present in pancreatic beta cells and significantly (P?GLP-1 is co-localized with insulin and seems to exert its beneficial effects by increasing cellular concentrations of endogenous antioxidant genes and other genes involved in the maintenance of pancreatic beta cell structure and function.     

Crystal structure of the ligand-bound glucagon-like peptide-1 receptor extracellular domain

The glucagon-like peptide-1 receptor (GLP-1R) belongs to Family B1 of the seven-transmembrane G protein-coupled receptors, and its natural agonist ligand is the peptide hormone glucagon-like peptide-1 (GLP-1). GLP-1 is involved in glucose homeostasis, and activation of GLP-1R in the plasma membrane of pancreatic beta-cells potentiates glucose-dependent insulin secretion. The N-terminal extracellular domain (nGLP-1R) is an important ligand binding domain that binds GLP-1 and the homologous peptide Exendin-4 with differential affinity. Exendin-4 has a C-terminal extension of nine amino acid residues known as the "Trp cage", which is absent in GLP-1. The Trp cage was believed to interact with nGLP-1R and thereby explain the superior affinity of Exendin-4. However, the molecular details that govern ligand binding and specificity of nGLP-1R remain undefined. Here we report the crystal structure of human nGLP-1R in complex with the antagonist Exendin-4(9-39) solved by the multiwavelength anomalous dispersion method to 2.2A resolution. The structure reveals that Exendin-4(9-39) is an amphipathic alpha-helix forming both hydrophobic and hydrophilic interactions with nGLP-1R. The Trp cage of Exendin-4 is not involved in binding to nGLP-1R. The hydrophobic binding site of nGLP-1R is defined by discontinuous segments including primarily a well defined alpha-helix in the N terminus of nGLP-1R and a loop between two antiparallel beta-strands. The structure provides for the first time detailed molecular insight into ligand binding of the human GLP-1 receptor, an established target for treatment of type 2 diabetes.     

The mechanism of glucagon-like peptide-1 participation in the osmotic homeostasis

We have found the physiological mechanism of intensification of the excessive fluid removal from the body under the action of glucagon-like peptide-1 and its analog exenatide. Under the water load in rats, exenatide significantly increased the clearance of lithium, reduced fluid reabsorption in the proximal tubule of the nephron and intensified reabsorption of sodium ions in the distal parts, which contributed to the formation of sodium-free water and faster recovery of osmotic homeostasis. Blocking this pathway with a selective antagonist of glucagon-like peptide-1 receptors slowed down the elimination of excessive water from the body.     

Reversal of obesity and insulin resistance by a non-peptidic glucagon-like peptide-1 receptor agonist in diet-induced obese mice

Background

Glucagon-like peptide-1 (GLP-1) is recognized as an important regulator of glucose homeostasis. Efforts to utilize GLP-1 mimetics in the treatment of diabetes have yielded clinical benefits. A major hurdle for an effective oral therapy has been the difficulty of finding a non-peptidic GLP-1 receptor (GLP-1R) agonist. While its oral bioavailability still poses significant challenges, Boc5, one of the first such compounds, has demonstrated the attainment of GLP-1R agonism in diabetic mice. The present work was to investigate whether subchronic Boc5 treatment can restore glycemic control and induce sustainable weight loss in diet-induced obese (DIO) mice, an animal model of human obesity and insulin resistance.

Methodology/Principal Findings

DIO mice were treated three times a week with Boc5 (0.3, 1 and 3 mg) for 12 weeks. Body weight, body mass index (BMI), food intake, fasting glucose, intraperitoneal glucose tolerance and insulin induced glucose clearance were monitored regularly throughout the treatment. Glucose-stimulated insulin secretion, β-cell mass, islet size, body composition, serum metabolic profiles, lipogenesis, lipolysis, adipose hypertrophy and lipid deposition in the liver and muscle were also measured after 12 weeks of dosing. Boc5 dose-dependently reduced body weight, BMI and food intake in DIO mice. These changes were associated with significant decreases in fat mass, adipocyte hypertrophy and peripheral tissue lipid accumulation. Boc5 treatment also restored glycemic control through marked improvement of insulin sensitivity and normalization of β-cell mass. Administration of Boc5 (3 mg) reduced basal but enhanced insulin-mediated glucose incorporation and noradrenaline-stimulated lipolysis in isolated adipocytes from obese mice. Furthermore, circulating leptin, adiponectin, triglyceride, total cholesterol, nonesterified fatty acid and high-density lipoprotein/low-density lipoprotein ratio were normalized to various extents by Boc5 treatment.

Conclusions/Significance

Boc5 may produce metabolic benefits via multiple synergistic mechanisms and may represent an attractive tool for therapeutic intervention of obesity and diabetes, by means of non-peptidic GLP-1R agonism.     

Characterization of high-affinity receptors for truncated glucagon-like peptide-1 in rat gastric glands

The truncated form of glucagon-like peptide-1 (TGLP-1, or proglucagon 78-108), secreted by the mammalian intestine, has potent pharmacological activities, stimulating insulin release and inhibiting gastric acid secretion. We have characterized high-affinity receptors for this peptide in rat isolated fundic glands. Scatchard analysis of binding studies using mono-125I-TGLP-1(7-36) amide as tracer showed a single class of binding site of Kd (4.4 +/- (SE) .08) x 10(-10) M, with a tissue concentration of 1.0 +/- 0.1 fmol sites/microgram DNA. Whole GLP-1 was approximately 700 times less potent in displacing tracer, while human GLP-2 and pancreatic glucagon produced no significant displacement at concentrations up to 10(-6) M. The data support a physiological role for TGLP-1 in the regulation of gastric acid secretion.     

Effects of glucagon-like peptide-1 on endothelial function in type 2 diabetes patients with stable coronary artery disease

GLP-1 stimulates insulin secretion, suppresses glucagon secretion, delays gastric emptying, and inhibits small bowel motility, all actions contributing to the anti-diabetogenic peptide effect. Endothelial dysfunction is strongly associated with insulin resistance and type 2 diabetes mellitus and may cause the angiopathy typifying this debilitating disease. Therefore, interventions affecting both endothelial dysfunction and insulin resistance may prove useful in improving survival in type 2 diabetes patients. We investigated GLP-1's effect on endothelial function and insulin sensitivity (S(I)) in two groups: 1) 12 type 2 diabetes patients with stable coronary artery disease and 2) 10 healthy subjects with normal endothelial function and S(I). Subjects underwent infusion of recombinant GLP-1 or saline in a random crossover study. Endothelial function was measured by postischemic FMD of brachial artery, using ultrasonography. S(I) [in (10(-4) dl.kg(-1).min(-1))/(muU/ml)] was measured by hyperinsulinemic isoglycemic clamp technique. In type 2 diabetic subjects, GLP-1 infusion significantly increased relative changes in brachial artery diameter from baseline FMD(%) (3.1 +/- 0.6 vs. 6.6 +/- 1.0%, P < 0.05), with no significant effects on S(I) (4.5 +/- 0.8 vs. 5.2 +/- 0.9, P = NS). In healthy subjects, GLP-1 infusion affected neither FMD(%) (11.9 +/- 0.9 vs. 10.3 +/- 1.0%, P = NS) nor S(I) (14.8 +/- 1.8 vs. 11.6 +/- 2.0, P = NS). We conclude that GLP-1 improves endothelial dysfunction but not insulin resistance in type 2 diabetic patients with coronary heart disease. This beneficial vascular effect of GLP-1 adds yet another salutary property of the peptide useful in diabetes treatment.     

New insights into the regulation of glucagon secretion by glucagon-like peptide-1.

Glucagon-like peptide-1 (GLP-1) is a potent incretin hormone currently under investigation for use as a novel therapeutic agent in the treatment of type 2 diabetes. One of several therapeutically important biological actions of GLP-1 in type 2 diabetic subjects is ability to induce strong suppression of glucagon secretion. The glucagonostatic action of GLP-1 results from its interaction with a specific G-protein coupled receptor resulting in the activation of adenylate cyclase and an increase in cAMP generation. In the pancreatic alpha-cell, cAMP, via activation of protein kinase A, interacts with a plethora of signal transduction processes including ion-channel activity and exocytosis of the glucagon-containing granules. In this short review, we will focus on recent advances in our understanding on the cellular mechanisms proposed to underlie the glucagonotropic action of GLP-1 and attempt to incorporate this knowledge into a working model for the control of glucagon secretion. Studies on the effects of GLP-1 on glucagon secretion are relevant to the pathogenesis of type 2 diabetes due to the likely contribution of hyperglucagonemia to impaired glucose tolerance in type 2 diabetes.     

Insulin contributes to fine-tuning of the pancreatic beta-cell response to glucagon-like peptide-1

Glucagon-like peptide-1 (GLP-1) stimulates insulin secretion from pancreatic β-cells in a glucose-dependent manner. However, factors other than glucose that regulate the β-cell response to GLP-1 remain poorly understood. In this study, we examined the possible involvement of insulin and receptor tyrosine kinase signaling in regulation of the GLP-1 responsiveness of β-cells. Pretreatment of β-cells with HNMPA, an insulin receptor inhibitor, and AG1478, an epidermal growth factor receptor inhibitor, further increased the cAMP level and Erk phosphorylation in the presence of exendin-4 (exe-4), a GLP-1 agonist. When β-cells were exposed to a high concentration of glucose (25 mM), which stimulates insulin secretion, exe-4-induced cAMP formation declined gradually as exposure time was increased. This decreased cAMP formation was not observed in the presence of HNMPA. HNMPA was able to further increase the exe-4-induced insulin secretion when β-cells were exposed to high glucose for 18 h. Treatment of β-cells with insulin significantly decreased exe-4-induced cAMP formation in a dose-dependent manner. Lowering the phospho-Akt level by HNMPA or LY294002, a PI3K inhibitor, further augmented exe-4-induced cAMP formation and Erk phosphorylation. These results suggest that insulin contributes to fine-tuning of the β-cell response to GLP-1.     

Small molecule ago-allosteric modulators of the human glucagon-like peptide-1 (hGLP-1) receptor

Following our previous publication describing the biological profiles, we herein describe the structure-activity relationships of a core set of quinoxalines as the hGLP-1 receptor agonists. The most potent and efficacious compounds are 6,7-dichloroquinoxalines bearing an alkyl sulfonyl group at the C-2 position and a secondary alkyl amino group at the C-3 position. These findings serve as a valuable starting point for the discovery of more drug-like small molecule agonists for the hGLP-1 receptor.     

Mechanisms mediating the diuretic and natriuretic actions of the incretin hormone glucagon-like peptide-1

Glucagon-like peptide-1 (GLP-1) is a gut incretin hormone considered a promising therapeutic agent for type 2 diabetes because it stimulates beta cell proliferation and insulin secretion in a glucose-dependent manner. Cumulative evidence supports a role for GLP-1 in modulating renal function; however, the mechanisms by which GLP-1 induces diuresis and natriuresis have not been completely established. This study aimed to define the cellular and molecular mechanisms mediating the renal effects of GLP-1. GLP-1 (1 μg·kg(-1)·min(-1)) was intravenously administered in rats for the period of 60 min. GLP-1-infused rats displayed increased urine flow, fractional excretion of sodium, potassium, and bicarbonate compared with those rats that received vehicle (1% BSA/saline). GLP-1-induced diuresis and natriuresis were also accompanied by increases in renal plasma flow and glomerular filtration rate. Real-time RT-PCR in microdissected rat nephron segments revealed that GLP-1 receptor-mRNA expression was restricted to glomerulus and proximal convoluted tubule. In rat renal proximal tubule, GLP-1 significantly reduced Na(+)/H(+) exchanger isoform 3 (NHE3)-mediated bicarbonate reabsorption via a protein kinase A (PKA)-dependent mechanism. Reduced proximal tubular bicarbonate flux rate was associated with a significant increase of NHE3 phosphorylation at the PKA consensus sites in microvillus membrane vesicles. Taken together, these data suggest that GLP-1 has diuretic and natriuretic effects that are mediated by changes in renal hemodynamics and by downregulation of NHE3 activity in the renal proximal tubule. Moreover, our findings support the view that GLP-1-based agents may have a potential therapeutic use not only as antidiabetic drugs but also in hypertension and other disorders of sodium retention.     

Chronic administration of ezetimibe increases active glucagon-like peptide-1 and improves glycemic control and pancreatic beta cell mass in a rat model of type 2 diabetes

Ezetimibe is a cholesterol-lowering agent targeting Niemann-Pick C1-like 1, an intestinal cholesterol transporter. Inhibition of intestinal cholesterol absorption with ezetimibe may ameliorate several metabolic disorders including hepatic steatosis and insulin resistance. In this study, we investigated whether chronic ezetimibe treatment improves glycemic control and pancreatic beta cell mass, and alters levels of glucagon-like peptide-1 (GLP-1), an incretin hormone involved in glucose homeostasis. Male LETO and OLETF rats were treated with vehicle or ezetimibe (10 mg kg⁻¹ day⁻¹) for 20 weeks via stomach gavage. OLETF rats were diabetic with hyperglycemia and significant decreases in pancreatic size and beta cell mass compared with LETO lean controls. Chronic treatment of OLETF rats with ezetimibe improved glycemic control during oral glucose tolerance test compared with OLETF controls. Moreover, ezetimibe treatment rescued the reduced pancreatic size and beta cell mass in OLETF rats. Interestingly, ezetimibe significantly decreased serum dipeptidyl peptidase-4 activity and increased serum active GLP-1 in OLETF rats without altering serum total GLP-1. These findings demonstrated that chronic administration of ezetimibe improves glycemic control and pancreatic beta cell mass, and increases serum active GLP-1 levels, suggesting possible involvement of GLP-1 in the ezetimibe-mediated beneficial effects on glycemic control.