Direct and indirect mechanisms regulating secretion of glucagon-like peptide-1 and glucagon-like peptide-2

The proglucagon-derived peptide family consists of three highly related peptides, glucagon and the glucagon-like peptides GLP-1 and GLP-2. Although the biological activity of glucagon as a counter-regulatory hormone has been known for almost a century, studies conducted over the past decade have now also elucidated important roles for GLP-1 as an antidiabetic hormone, and for GLP-2 as a stimulator of intestinal growth. In contrast to pancreatic glucagon, the GLPs are synthesized in the intestinal epithelial L cells, where they are subject to the influences of luminal nutrients, as well as to a variety of neuroendocrine inputs. In this review, we will focus on the complex integrative mechanisms that regulate the secretion of these peptides from L cells, including both direct and indirect regulation by ingested nutrients.     

Characterization of glucagon-like peptide-1 receptor beta-arrestin 2 interaction: a high-affinity receptor phenotype

To dissect the interaction between beta-arrestin ((beta)arr) and family B G protein-coupled receptors, we constructed fusion proteins between the glucagon-like peptide 1 receptor and (beta)arr2. The fusion constructs had an increase in apparent affinity selectively for glucagon, suggesting that (beta)arr2 interaction locks the receptor in a high-affinity conformation, which can be explored by some, but not all, ligands. The fusion constructs adopted a signaling phenotype governed by the tethered (beta)arr2 with an attenuated G protein-mediated cAMP signal and a higher maximal internalization compared with wild-type receptors. This distinct phenotype of the fusion proteins can not be mimicked by coexpressing wild-type receptor with (beta)arr2. However, when the wild-type receptor was coexpressed with both (beta)arr2 and G protein-coupled receptor kinase 5, a phenotype similar to that observed for the fusion constructs was observed. We conclude that the glucagon-like peptide 1 fusion construct mimics the natural interaction of the receptor with (beta)arr2 with respect to binding peptide ligands, G protein-mediated signaling and internalization, and that this distinct molecular phenotype is reminiscent of that which has previously been characterized for family A G protein-coupled receptors, suggesting similarities in the effect of (beta)arr interaction between family A and B receptors also at the molecular level.     

Design and synthesis of polyethylene glycol-modified biphenylsulfonyl-thiophene-carboxamidine inhibitors of the complement component C1s

Complement C1s protease inhibitors have potential utility in the treatment of diseases associated with activation of the classical complement pathway such as humorally mediated graft rejection, ischemia-reperfusion injury (IRI), vascular leak syndrome, and acute respiratory distress syndrome (ARDS). The utility of biphenylsulfonyl-thiophene-carboxamidine small-molecule C1s inhibitors are limited by their poor in vivo pharmacokinetic properties. Pegylation of a potent analog has provided compounds with good potency and good in vivo pharmacokinetic properties.     

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.     

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.     

Structural determinants for activity of glucagon-like peptide-2

Glucagon-like peptide-2 (GLP-2) is a 33 amino acid gastrointestinal hormone that regulates epithelial growth in the intestine. Dipeptidylpeptidase IV cleaves GLP-2 at the position 2 alanine, resulting in the inactivation of peptide activity. To understand the structural basis for GLP-2 action, we studied receptor binding and activation for 56 GLP-2 analogues with either position 2 substitutions or alanine replacements along the length of the peptide. The majority of position 2 substitutions exhibited normal to enhanced GLP-2 receptor (GLP-2R) binding; in contrast, position 2 substitutions were less well tolerated in studies of receptor activation as only Gly, Ile, Pro, alpha-aminobutyric acid, D-Ala, or nor-Val substitutions exhibited enhanced GLP-2R activation. In contrast, alanine replacement at positions 5,6,17, 20, 22, 23, 25, 26, 30, and 31 led to diminished GLP-2R binding. Position 2 substitutions containing Asp, Leu, Lys, Met, Phe, Trp, and Tyr, and Ala substitutions at positions 12 and 21 exhibited normal to enhanced GLP-2R binding but greater than 75% reduction in receptor activation. D-Ala(2), Pro(2) and Gly(2), Ala(16) exhibited significantly lower EC(50)s for receptor activation than the parent peptide (p < 0.01-0.001). Circular dichroism analysis indicated that the enhanced activity of these GLP-2 analogues was independent of the alpha-helical content of the peptide. These results indicate that single amino acid substitutions within GLP-2 can confer structural changes to the ligand-receptor interface, allowing the identification of residues important for GLP-2R binding and receptor activation.     

Neural regulation of glucagon-like peptide-1 secretion in pigs

Glucagon-like peptide (GLP)-1 is secreted rapidly from the intestine postprandially. We therefore investigated its possible neural regulation. With the use of isolated perfused porcine ileum, GLP-1 secretion was measured in response to electrical stimulation of the mixed, perivascular nerve supply and infusions of neuroactive agents alone and in combination with different blocking agents. Electrical nerve stimulation inhibited GLP-1 secretion, an effect abolished by phentolamine. Norepinephrine inhibited secretion, and phentolamine abolished this effect. GLP-1 secretion was stimulated by isoproterenol (abolished by propranolol). Acetylcholine stimulated GLP-1 secretion, and atropine blocked this effect. Dimethylphenylpiperazine stimulated GLP-1 secretion. In chloralose-anesthetized pigs, however, electrical stimulation of the vagal trunks at the level of the diaphragm had no effect on GLP-1 or GLP-2 and weak effects on glucose-dependent insulinotropic peptide and somatostatin secretion, although this elicited a marked atropine-resistant release of the neuropeptide vasoactive intestinal polypeptide to the portal circulation. Thus GLP-1 secretion is inhibited by the sympathetic nerves to the gut and may be stimulated by intrinsic cholinergic nerves, whereas the extrinsic vagal supply has no effect.     

Metformin effects on dipeptidylpeptidase IV degradation of glucagon-like peptide-1

There is current interest in the use of inhibitors of dipeptidyl peptidase IV (DP IV) as therapeutic agents to normalize glycemic excursions in type 2 diabetic patients. Data indicating that metformin increases the circulating amount of active glucagon-like peptide-1 (GLP-1) in obese nondiabetic subjects have recently been presented, and it was proposed that metformin might act as a DP IV inhibitor. This possibility has been investigated directly using a number of in vitro methods. Studies were performed on DP IV enzyme from three sources: 20% human serum, purified porcine kidney DP IV, and recombinant human DP IV. Inhibition of DP IV hydrolysis of the substrate Gly-Pro-pNA by metformin was examined spectrophotometrically. Effects of metformin on GLP-1([7-36NH2]) degradation were assessed by mass spectrometry. In addition, surface plasmon resonance was used to establish whether or not metformin had any effect on GLP-1([7-36NH2]) or GLP-1([9-36NH2]) interaction with immobilized porcine or human DP IV. Metformin failed to alter the kinetics of Gly-Pro-pNA hydrolysis or GLP-1 degradation tested according to established methods. Surface plasmon resonance recordings indicated that both GLP-1([7-36NH2]) and GLP-1([9-36NH2]) show micromolar affinity (K(D)) for DP IV, but neither interaction was influenced by metformin. The results conclusively indicate that metformin does not act directly on DP IV, therefore alternative explanations for the purported effect of metformin on circulating active GLP-1 concentrations must be considered.     

长效GLP-1受体激动剂的研发进展

胰高血糖素样肽-1(glucagon-like Peptide-1,GLP-1)是机体在葡萄糖等刺激下释放的一类肠促胰岛素。GLP-1具有促进葡萄糖依赖性胰岛素分泌,促进胰岛β细胞增殖并抑制其凋亡、抑制摄食、减慢胃排空和减轻体重等作用。GLP-1在体内极不稳定,易被二肽基肽酶-IV(dipeptidyl peptidase-IV,DPP-IV)降解,半衰期短。从墨西哥巨蜥蜴唾液中分离得到GLP-1的天然类似物Exendin-4与其有着相似的作用,且不易被DPP-IV降解。以GLP-1及Exendin-4为基础,研发长效GLP-1受体激动剂,已成为研发新型糖尿病治疗药物的热点之一。本文就长效GLP-1受体激动剂的研发现状予以综述。     

Synthesis and in vitro biological evaluation of new pyrimidines as glucagon-like peptide-1 receptor agonists

The therapeutic success of peptide glucagon-like peptide-1 (GLP-1) receptor agonists for the treatment of type 2 diabetes mellitus has inspired discovery efforts aimed at developing orally available small-molecule GLP-1 receptor agonists. In this study, two series of new pyrimidine derivatives were designed and synthesized using an efficient route, and were evaluated in terms of GLP-1 receptor agonist activity. In the first series, novel pyrimidines substituted at positions 2 and 4 with groups varying in size and electronic properties were synthesized in a good yield (78–90%). In the second series, the designed pyrimidine templates included both urea and Schiff base linkers, and these compounds were successfully produced with yields of 77–84%. In vitro experiments with cultured cells showed that compounds 3a and 10a (10^?15–10^?9 M) significantly increased insulin secretion compared to that of the control cells in both the absence and presence of 2.8 mM glucose; compound 8b only demonstrated significance in the absence of glucose. These findings represent a valuable starting point for the design and discovery of small-molecule GLP-1 receptor agonists that can be administered orally.     

Design of a long acting peptide functioning as both a glucagon-like peptide-1 receptor agonist and a glucagon receptor antagonist

The closely related peptides glucagon-like peptide (GLP-1) and glucagon have opposing effects on blood glucose. GLP-1 induces glucose-dependent insulin secretion in the pancreas, whereas glucagon stimulates gluconeogenesis and glycogenolysis in the liver. The identification of a hybrid peptide acting as both a GLP-1 agonist and a glucagon antagonist would provide a novel approach for the treatment of type 2 diabetes. Toward this end a series of hybrid peptides made up of glucagon and either GLP-1 or exendin-4, a GLP-1 agonist, was engineered. Several peptides that bind to both the GLP-1 and glucagon receptors were identified. The presence of glucagon sequence at the N terminus removed the dipeptidylpeptidase IV cleavage site and increased plasma stability compared with GLP-1. Targeted mutations were incorporated into the optimal dual-receptor binding peptide to identify a peptide with the highly novel property of functioning as both a GLP-1 receptor agonist and a glucagon receptor antagonist. To overcome the short half-life of this mutant peptide in vivo, while retaining dual GLP-1 agonist and glucagon antagonist activities, site-specific attachment of long chained polyethylene glycol (PEGylation) was pursued. PEGylation at the C terminus retained the in vitro activities of the peptide while dramatically prolonging the duration of action in vivo. Thus, we have generated a novel dual-acting peptide with potential for development as a therapeutic for type 2 diabetes.     

Effect of peripherally-injected glucagon-like peptide-1 on gastric mucosal blood flow

The aim of this study was to investigate the mechanisms involved in the effect of glucagon-like peptide-1 (GLP-1) on the decrease in gastric mucosal blood flow (GMBF) induced by intragastric ethanol.After preparation of the stomach for GMBF recording, a probe was placed to the gastric mucosa and basal GMBF recordings were obtained by a laser Doppler flowmeter after a 30-minute stabilization period. Following GLP-1 (1000 ng/kg; i.p.) injection, 1 ml of absolute ethanol was applied to the gastric chamber and GMBF was recorded continuously during a 30-minute period. GLP-1 (1000 ng/kg; i.p.) prevented the decrease in GMBF induced by ethanol. Nitric oxide (NO) synthase inhibitor L-NAME, (30 mg/kg; s.c.), calcitonine gene-related peptide (CGRP) receptor antagonist CGRP-(8–37) (10μg/kg; i.p.), and cyclooxygenase inhibitor indomethacin (5 mg/kg; i.p.) all inhibited the GMBF-improving effect of GLP-1.We concluded that, NO, CGRP and prostaglandins may be involved in the effect of peripherally-injected GLP-1 on GMBF reduction induced by intraluminal ethanol.     

Intracerebroventricular injection of glucagon-like peptide-1 changes lipid metabolism in chicks

Glucagon-like peptide-1 (GLP-1), derived from proglucagon, is thought to act as a negative regulator of energy homeostasis in mammals, since intracerebroventricular (ICV) injection of GLP-1 inhibits feeding behavior and enhances energy expenditure. The anorexigenic effect of GLP-1 is also observed in chicks, but whether brain GLP-1 enhances energy expenditure has not been investigated. The aim of the present study was to clarify the effect of ICV injection of GLP-1 on energy expenditure as well as metabolic changes in chicks. The injection of GLP-1 did not affect energy expenditure calculated from oxygen consumption and carbon dioxide production. On the other hand, the injection of GLP-1 significantly decreased respiratory quotient, suggesting that brain GLP-1 shifted the use of energy sources from carbohydrates to lipids. In support of this, ICV injection of GLP-1 increased plasma non-esterified fatty acid concentration while plasma glucose concentration was decreased. In conclusion, GLP-1 appears to act in the brain as a metabolic modulator rather than as a regulator of total energy expenditure in chicks.     

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.     

Synthesis of chalcone derivatives as potential anti-diabetic agents

Chalcones bearing electron donating or electron withdrawing substitutions were prepared and their glucose uptake activity was evaluated. Chalcone derivatives were synthesized in one step protocol with high purity and yield. Chalcones with chloro, bromo, iodo and hydroxy substitutions at position 2 on A-ring exhibited the highest activity with glucose medium concentration (210 to 236 mg/dl) compared to pioglitazone and rosiglitazone (230 and 263 mg/dl, respectively). Also chalcones with iodo substitution at position 3 on A-ring were comparably active (≤238 mg/dl). The structure-activity relationship of the tested chalcones was studied and the findings were supported statistically.     

Histological analysis of glucagon-like peptide-1 receptor expression in chicken pancreas

Glucagon-like peptide-1 (GLP-1) released from intestinal L cells in response to nutrient ingestion inhibits both gastrointestinal emptying and gastric acid secretion and promotes satiety. The main biological effect of GLP-1 is the stimulation of insulin secretion (thereby fulfilling the criterion for an incretin hormone) in order to reduce blood glucose levels in mammalian species. Chicken GLP-1 receptor (cGLP-1R) has also been identified in various tissues by gene expression analysis. Although certain effects of GLP-1 in mammals and birds are consistent, e.g., inhibition of food intake, whether GLP-1 has the same insulinotropic activity in chickens as in mammals is debated. Moreover, the expression of cGLP-1R in chicken pancreatic B cells has not been reported. The localization of cGLP-1R and its mRNA in pancreatic islets is studied by triple-immunofluorescence microscopy and in situ hybridization. Triple-immunofluorescence microscopy with antisera against cGLP-1R, somatostatin and insulin or glucagon revealed that cGLP-1R protein was exclusively localized in D cells producing somatostatin in chicken pancreatic islets. The D cells were localized in peripheral areas of the pancreatic islets and cGLP-1R mRNA was detected in the same areas, indicating that cGLP-1R mRNA was also expressed in D cells. This is the first report to demonstrate that cGLP-1R is expressed by D cells, not B cells as in mammals. Our study suggests that chicken GLP-1 performs its insulinotropic activity by a different mode of action from that of the mammalian hormone.     

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.     

Proteome analysis of recombinant Escherichia coli producing human glucagon-like peptide-1

The proteomic response of recombinant Escherichia coli producing human glucagon-like peptide-1 was analyzed by two-dimensional gel electrophoresis. Protein spots in two-dimensional gel could be identified by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and their expression profiles were compared with those of nonproducing cells. Thirty-five intracellular proteins exhibited differential expression levels between the production and control strains. These changes reflected physiological responses to heterologous peptide production in recombinant E. coli. Specifically, physiological changes included the down-regulation of proteins involved in the central carbon metabolism, biosynthesis of cellular building blocks and peptides, and up-regulation of cell protection proteins and some sugar transport proteins. This comprehensive analysis would provide useful information for understanding physiological alterations to heterologous peptide production and for designing efficient metabolic engineering strategies for the production of recombinant peptides in E. coli.     

The "cryptic" mechanism of action of glucagon-like peptide-2

Glucagon-like peptide-2 (GLP-2) is a peptide hormone with multiple beneficial effects on the intestine, including expansion of the mucosal surface area through stimulation of crypt cell proliferation, as well as enhancement of nutrient digestion and absorption. Recent advances in clinical trials involving GLP-2 necessitate elucidation of the exact signaling pathways by which GLP-2 acts. In particular, the GLP-2 receptor has been localized to several intestinal cell types that do not include the proliferating crypt cells, and the actions of GLP-2 have thus been linked to a complex network of indirect mediators that induce diverse signaling pathways. The intestinotropic actions of GLP-2 on the colon have been shown to be mediated through the actions of keratinocyte growth factor and insulin-like growth factor (IGF)-2, whereas small intestinal growth has been linked to IGF-1, IGF-2, and ErbB ligands, as well as the IGF-1 receptor and ErbB. The cellular source of these mediators remains unclear, but it likely includes the intestinal subepithelial myofibroblasts. Conversely, the anti-inflammatory and blood flow effects of GLP-2 are dependent on vasoactive intestinal polypeptide released from submucosal enteric neurons and nitric oxide, respectively. Finally, recent studies have suggested that GLP-2 not only modulates intestinal stem cell behavior but may also promote carcinogenesis in models of sporadic colon cancer. Further consideration of the molecular cross-talk and downstream signaling pathways mediating the intestinotropic effects of GLP-2 is clearly warranted.