GLP-1 expression in von Ebner's gland of diabetic rats

GLP-1, a peptidergic endocrine hormone, which associate with appetite control, glucose homeostasis and satiety. It might play an important role in the gustatory system. We tried to investigate the expression of GLP-1 in von Ebner's gland of diabetic and control rats, and the ultrastructure changes on von Ebner's gland of diabetes rats. GLP-1 expression in von Ebner's gland was evaluated by immunohistochemistry. The number of GLP-1 positive cells in diabetic rat von Ebner's gland was significantly higher than that in normal controls. Electron micrographs showed that a series of pathologic changes in von Ebner's gland of diabetes rats. The results suggest that GLP-1 have some effects within the gustatory systems, and elevated von Ebner's gland GLP-1 expression may be one cause of diabetic taste impairment.     

Divergent effects of GLP-1 analogs exendin-4 and exendin-9 on the expression of myosin heavy chain isoforms in C2C12 myotubes

Exendin 1-39 amide (Ex-4) and its truncated form exendin 9-39 amide (Ex-9) are peptides of non-mammalian nature, which act as an agonist and antagonist, respectively, of the glucagon-like peptide-1 (GLP-1) receptor in mammals. GLP-1 is an intestinal peptide that plays an important role in the regulation of glucose metabolism and glucose uptake in skeletal muscle; however, the effects of its two analogs (Ex-4 and Ex-9) on myofiber properties are still unclear. Here, we report the effects of Ex-4 and Ex-9 alone or in combination on the myosin heavy chain (MyHC) type composition and the glucose uptake capacity in differentiated C2C12 myotubes. Neither Ex-4 nor Ex-9 altered basal glucose uptake, whereas Ex-9 significantly increased insulin-stimulated glucose uptake, suggesting enhanced insulin sensitivity. The mRNA expression of MyHC I and 2A as well as the percentage of MyHC I protein was remarkably increased in Ex-9-treated myotubes. In contrast, Ex-4, alone or in combination with Ex-9, caused a significant reduction in MyHC 2A mRNA expression and the percentage of MyHC I protein. Consistent with the MyHC type switching peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α expression in myotubes was remarkably increased by Ex-9 yet was significantly inhibited by Ex-4. In addition, intracellular concentrations of free Ca²⁺ were increased in all treatment groups, but only Ex-9-treated myotubes showed higher calcineurin A protein content. Taken together, our data suggest that Ex-9 promotes oxidative differentiation in myotubes to improve cell insulin sensitivity, probably through calcineurin and PGC-1α mediated pathways.     

Comparative effects of the long-acting GLP-1 receptor ligands, liraglutide and exendin-4, on food intake and body weight suppression in rats

The glucagon-like-peptide-1 receptor (GLP-1R) agonists, liraglutide (Victoza) and the synthetic product of exendin-4 (Byetta), are approved for type II diabetes mellitus (T2DM) treatment and may be efficacious in obesity treatment as well, in part, due to the drugs' resistance to enzymatic degradation and prolonged half-life relative to endogenous GLP-1. To address the need to directly compare the food intake- and body weight-suppressive effects of these two GLP-1R ligands, acute and chronic dosing experiments were performed. Once-daily (q.d.) exendin-4 (0, 0.33, 1.5, and 3.0 μg/kg) and liraglutide (0, 50, 100, and 300 μg/kg, q.d.) both reduced the chow intake in nonobese rats in a dose-dependent fashion following either intraperitoneal (IP) or subcutaneous (SC) administration, whereas only liraglutide reduced 24 and 48 h body weight in nonobese, chow-maintained rats. Chow intake and body weight suppression by liraglutide were of greater magnitude and shorter latency following IP compared to SC delivery, whereas for exendin-4, the magnitude of intake-suppression was similar for IP and SC administration. The effects of chronic delivery (7 consecutive days; IP) of liraglutide (25 and 50 μg/kg; q.d.) and exendin-4 (3 μg/kg; q.d. and twice-daily (b.i.d.)) on food intake and body weight were also examined in diet-induced obese (DIO) rats. Liraglutide (50 μg/kg q.d.) and exendin-4 (3 μg/kg b.i.d.) were comparable in suppressing overall high fat/sucrose diet (HFS; 60% kcal from fat) intake. Both drugs regimens yielded marked weight loss over the 7-day period. The weight loss effect of liraglutide was achieved in the first 2 days and remained stable for the duration of the experiment; weight loss with exendin-4 appeared more linear over the 7-day period. In conclusion, administration of the GLP-1R ligands, exendin-4 (b.i.d.) and liraglutide (q.d.), lead to comparable and pronounced suppression of food intake and body weight in DIO rats, suggesting a potential role for these drugs as a clinical tool for obesity treatment.     

Expression and purification of exendin-4, a GLP-1 receptor agonist, in Escherichia coli

Exendin-4 is a 39 amino acid peptide isolated from salivary secretions of Gila monster (Heloderma suspectum). It shows 53% sequence similarity to glucagon-like peptide-1 (GLP-1), which is evaluated for the regulation of plasma glucose in type 2 diabetes. Exendin-4 is a potent and long-acting agonist of GLP-1 receptor. In the present study, the exendin-4 gene obtained by PCR with an enterokinase site at N-terminus and a termination codon at C-terminus was expressed in Escherichia coli strain BL21 (DE3) harboring pET32a(+). The fusion protein was purified by chromatography on Ni-NTA-agarose column. Recombinant exendin-4 was obtained by enterokinase cleavage of the fusion protein and subsequent purification. The yield of recombinant exendin-4 was 3.15mg/10g bacteria. The obtained recombinant exendin-4 shows glucose-lowering action in vivo.     

Brain activation following peripheral administration of the GLP-1 receptor agonist exendin-4

The aim of our study was to investigate the anorectic and brain stimulatory effects of various doses of exendin-4 (Ex-4) and to investigate the role of the vagus nerve in Ex-4-induced brain activation. A dose-related increase in c-fos mRNA expression was observed following Ex-4 administration (0.155-15.5 μg/kg). Doses of Ex-4 that caused anorexia without aversive effects (0.155, 0.775 μg/kg) induced c-fos expression in the hypothalamic arcuate and paraventricular (PVH; parvocellular) nuclei as well as in the limbic and brainstem structures. Doses of Ex-4 that caused aversion (1.55, 15.5 μg/kg) stimulated the same regions (in a more intense way) and additionally activated the magnocellular hypothalamic structures (supraoptic nucleus and PVH magnocellular). The brain c-fos pattern induced by Ex-4 showed both similarities and differences with that induced by refeeding. Subdiaphragmatic vagotomy significantly blunted the stimulation of c-fos mRNA expression induced by Ex-4 in the nodose ganglion, the medial part of nucleus of the solitary tract, and the parvocellular division of the PVH. Pretreatment with Ex-9-39 (330 μg/kg ip) impaired the neuronal activation evoked by Ex-4 in all brain regions and in the nodose ganglion. Effects of Ex-4 on hypothalamic-pituitary-adrenal axis activity were not altered by vagotomy. Results of this study demonstrate and relate the anorectic and brain stimulatory effects of aversive and nonaversive doses of Ex-4 and indicate that the activation of specific central regions induced by the peripheral administration of Ex-4 is, at least in part, dependent on the integrity of the vagus nerve.     

Differential structural properties of GLP-1 and exendin-4 determine their relative affinity for the GLP-1 receptor N-terminal extracellular domain

Glucagon-like peptide-1 (GLP-1) and exendin-4 (Ex4) are homologous peptides with established potential for treatment of type 2 diabetes. They bind and activate the pancreatic GLP-1 receptor (GLP-1R) with similar affinity and potency and thereby promote insulin secretion in a glucose-dependent manner. GLP-1R belongs to family B of the seven transmembrane G-protein coupled receptors. The N-terminal extracellular domain (nGLP-1R) is a ligand binding domain with differential affinity for Ex4 and GLP-1: low affinity for GLP-1 and high affinity for exendin-4. The superior affinity of nGLP-1R for Ex4 was previously explained by an additional interaction between nGLP-1R and the C-terminal Trp-cage of Ex4. In this study we have combined biophysical and pharmacological approaches thus relating structural properties of the ligands in solution to their relative binding affinity for nGLP-1R. We used both a tracer competition assay and ligand-induced thermal stabilization of nGLP-1R to measure the relative affinity of full length, truncated, and chimeric ligands for soluble refolded nGLP-1R. The ligands in solution and the conformational consequences of ligand binding to nGLP-1R were characterized by circular dichroism and fluorescence spectroscopy. We found a correlation between the helical content of the free ligands and their relative binding affinity for nGLP-1R, supporting the hypothesis that the ligands are helical at least in the segment that binds to nGLP-1R. The Trp-cage of Ex4 was not necessary to maintain a superior helicity of Ex4 compared to GLP-1. The results suggest that the differential affinity of nGLP-1R is explained almost entirely by divergent residues in the central part of the ligands: Leu10-Gly30 of Ex4 and Val16-Arg36 of GLP-1. In view of our results it appears that the Trp-cage plays only a minor role for the interaction between Ex4 and nGLP-1R and for the differential affinity of nGLP-1R for GLP-1 and Ex4.     

The GLP-1 analog exendin-4 modulates HSP72 expression and ERK1/2 activity in BTC6 mouse pancreatic cells

Lipotoxicity, an important factor in the pathogenesis of diabetes, leads to defective β-cell proliferation and increased apoptosis. Glucagon-like peptide-1 (GLP-1) analogs, which are used to treat type 2 diabetes, reduce endoplasmic reticulum stress and inflammation in pancreatic β-cells and improve their survival. However, their effects on the heat shock response (HSR) have not been elucidated yet. We investigated whether the GLP-1 analog exendin-4 exerts its protective effect by modulating the HSR and mitogen-activated protein kinases (MAPKs) in BTC-6 mouse pancreatic cells under palmitic acid (PA) stress. Expression patterns were analyzed using mass spectrometry, Western blotting, and qRT-PCR in the presence of 250 or 400 μM PA and 100 nM exendin-4. Additionally, we measured MAPK expression and phosphorylation using qRT-PCR and Western blotting, respectively. Upregulation of heat shock protein (HSP), notably HSP72, in the presence of PA, was attenuated by exendin-4. Despite the absence of global effects on the HSR system, exendin-4 attenuated the expression of other non-classical HSPs (GRP94, DNAJA1, and DNAJB6) in the presence of PA. Regarding MAPKs, only extracellular signal-regulated kinase (ERK) phosphorylation was highly increased by exendin-4 in both the presence and absence of PA. Furthermore, exendin-4 significantly alleviated PA-induced cell death, which was further confirmed with proteomics analysis where key cellular functions, including cellular growth, assembly, and organization, were improved by exendin-4 treatment. Thus, our results expand the protective role of GLP-1 analogs to include other cellular mechanisms involved in restoring normal β-cell homeostasis.     

Protective effects of GLP-1 analogues exendin-4 and GLP-1(9-36) amide against ischemia-reperfusion injury in rat heart

Glucagon-Like Peptide-1 (GLP-1) is an incretin peptide secreted from intestinal L-cells, whose potent plasma glucose-lowering action has prompted intense efforts to develop GLP-1 receptor-targeting drugs for treatment of diabetic hyperglycemia. More recently, GLP-1 and its analogues have been shown to exert cardiovascular effects in a number of experimental models. Here we tested exendin-4 (Exe-4), a peptide agonist at GLP-1 receptors, and GLP-1(9-36) amide, the primary endogenous metabolite of GLP-1 (both in the concentration range 0.03-3.0 nM), for their protective effects against ischemia-reperfusion injury (IRI) in an isolated rat heart preparation. When administered, the agents were only present for the first 15 min of a 120 min reperfusion period (postconditioning protocol). Exe-4, but not GLP-1(9-36) amide, showed a strong infarct-limiting action (from 33.2% +/-2.7% to 14.5% +/-2.2% of the ischemic area, p<0.05). This infarct size-limiting effect of Exe-4 was abolished by exendin(9-39) (Exe(9-39)), a GLP-1 receptor antagonist. In contrast, both Exe-4 and GLP-1(9-36) amide were able to augment left ventricular performance (left ventricular developed pressure and rate-pressure product) during the last 60 min of reperfusion. These effects were only partially antagonized by Exe(9-39). We suggest that Exe-4, in addition to being currently exploited in treatment of diabetes, may present a suitable candidate for postconditioning trials in clinical settings of IRI. The divergent agonist effects of Exe-4 and GLP-1(9-36), along with correspondingly divergent antagonistic efficacy of Exe(9-39), seem consistent with the presence of more than one type of GLP-1 receptor in this system.     

The GLP-1 agonist exendin-4 reduces food intake in nonhuman primates through changes in meal size

Exendin-4 (Ex4), a long-acting glucagon-like peptide-1 (GLP-1) receptor agonist, has been shown to reduce food intake and suppress gastric emptying in rodents and humans. In this study we investigated the effects of peripheral administration of Ex4 on food intake and meal patterns in adult male rhesus macaques. Rhesus macaques (n = 4) that had been trained to lever press for food pellets were injected intramuscularly 15 min before the start of their 6-h daily feeding period. Ex4 was given at doses of 0.10, 0.32, 0.56, 1.0, and 3.0 microg/kg. Ex4 suppressed food intake in a dose-dependent manner, with the 3.0 microg/kg dose completely preventing feeding during the 6-h period and the 0.10 microg/kg dose suppressing intake by 17%. Doses of 0.32, 0.56, 1.0, and 3.0 microg/kg caused significant reductions in cumulative intake at all six hourly time points. Ex4 inhibited food intake through a specific effect on meal size. Meal size was significantly reduced in a dose-dependent manner with significant reductions at the 0.32 and 1.0 microg/kg doses (P < 0.05). Day 2 and 3 intakes returned to baseline levels with no compensation for Ex4-induced feeding suppression. Administration of doses of 0.32 and 0.56 microg/kg Ex4 over 5 consecutive days led to sustained reductions in intake with no evidence of compensation. Again, these reductions were due to specific effects on meal size. These results demonstrate that activation of GLP-1 pathways has potent effects on the controls of meal size and overall food intake in a nonhuman primate model.     

Expression and purification of exendin-4 dimer in Escherichia coli and its interaction with GLP-1 receptor in vitro

Exendin-4 is a 39 amino acid peptide isolated from the Gila monster salivary gland. It is 53% homologous to GLP-1 and exhibits similar glucoregulatory activities. In this study, exendin-4 dimer (D-Ex4) was constructed, cloned into plasmid pET32a(+) and expressed in E. coli BL21(DE3). The fusion protein with His-tag at the N-terminus was purified with a Ni-NTA-agarose column. After proteolytic cleavage, D-Ex4 peptide with high purity was obtained by HPLC. The results obtained by chemical cross-linking showed that D-Ex4 maintained affinity to GLP-1 receptor.     

Oral self-nanoemulsifying formulation of GLP-1 agonist peptide exendin-4: development,characterization and permeability assesment on Caco-2 cell monolayer

Amino Acids - The objective of this study was to prepare a stable self-nanoemulsifying formulation of exendin-4, which is an antidiabetic peptide. As exendin-4 is commercially available only in...     

Screening of a phage display library of exendin-4 mutants with the extracellular domain of rat GLP-1 receptor

A phage display library of exendin-4 mutants was screened with the extracellular domain of rat glucagon-like peptide 1 receptor as the target. A novel variant of exendin-4 with higher affinity for the receptor fraction than that of the wild type was identified. The increased affinity was attributed to the substitution of Glu(16) by Val(16). Although the substitution probably caused an increased entropic cost to the helix region, the linker around Val(16) is more flexible resulting in the increase of affinity for the receptor.     

Glucagon-like peptide 1 (7-36) amide (GLP-1) and exendin-4 stimulate serotonin release in rat hypothalamus

Glucagon-like peptide 1 (7-36) amide (GLP-1) and exendin-4 are gastrointestinal hormones as well as neuropeptides involved in glucose homeostasis and feeding regulation, both peripherally and at the central nervous system (CNS), acting through the same GLP-1 receptor. Aminergic neurotransmitters play a role in the modulation of feeding in the hypothalamus and we have previously found that peripheral hormones and neuropeptides, which are known to modulate feeding in the central nervous system, are able to modify catecholamine and serotonin release in the hypothalamus. In the present paper we have evaluated the effects of GLP-1 and exendin-4 on dopamine, norepinephrine, and serotonin release from rat hypothalamic synaptosomes, in vitro. We found that glucagon-like peptide 1 (7-36) amide and exendin-4 did not modify either basal or depolarization-induced dopamine and norepinephrine release; on the other hand glucagon-like peptide 1 (7-36) amide and exendin-4 stimulated serotonin release, in a dose dependent manner. We can conclude that the central anorectic effects of GLP-1 agonists could be partially mediated by increased serotonin release in the hypothalamus, leaving the catecholamine release unaffected.     

Effects of Selenium and Exendin-4 on Glucagon-Like Peptide-1 Receptor, IRS-1, and Raf-1 in the Liver of Diabetic Rats

Selenium and exendin-4 exert antidiabetic effects by unknown mechanisms. Herein, we investigated their effects on the expression of glucagon-like peptide-1 receptor (GLP-1R), insulin receptor substrate-1 (IRS-1), and Raf-1 in the livers of rats with streptozotocin-induced diabetes. Diabetic rats were injected intraperitoneally with exendin-4 (0.03?μg/kg body weight) twice daily or treated with 5?ppm selenium as sodium selenite in drinking water for 4?weeks. Both selenium and exendin-4 reduced the hyperglycemia in diabetic rats. Induction of diabetes mellitus resulted in decreased level of GLP-1R and increased levels of IRS-1 and Raf-1 in the liver. Treatment of diabetic rats with selenium or exendin-4 resulted in increased level of GLP-1R and decreased levels of IRS-1 and Raf-1 in the liver, compared with the levels in diabetic rats. Therefore, the antidiabetic actions of selenium and exendin-4 involve their effects on GLP-1R, IRS-1, and Raf-1 levels in the liver.     

Local,exendin-(9-39)-insensitive,site of action of GLP-1 in canine ileum

Glucagon-like peptide-1 (GLP-1) modulates glucose levels following a meal, including by inhibition of gastric emptying and intestinal transport. Intra-arterial injection of GLP-1 into the gastric corpus, antrum, or pylorus of anesthetized dogs had no effect on the contractile activity of the resting or neurally activated stomach. GLP-1 injected intra-arterially inhibited intestinal segments when activated by enteric nerve stimulation but not by acetylcholine. Isolated ileum segments were perfused intra-arterially, instrumented with strain gauges to record circular muscle activity and with subserosal electrodes to stimulate enteric nerves. GLP-1 caused concentration-dependent inhibition of nerve-stimulated phasic but not tonic activity. This was absent during TTX-induced activity and partly prevented by N(G)-nitro-L-arginine. Exendin-(9-39), the GLP-1 antagonist, had no intrinsic activity and did not affect the actions of GLP-1. Capsaicin mimicked the effects of GLP-1 and may have reduced the effect of subsequent GLP-1. GLP-1 may mediate paracrine action on afferent nerves in the canine ileal mucosa using an unusual receptor.     

The importance of the nine-amino acid C-terminal sequence of exendin-4 for binding to the GLP-1 receptor and for biological activity

Exendin-4, a 39-amino acid (AA) peptide, is a long-acting agonist at the glucagon-like peptide-1 (GLP-1) receptor. Consequently, it may be preferable to GLP-1 as a long-term treatment for type 2 diabetes mellitus. Exendin-4 (Ex-4), unlike GLP-1, is not degraded by dipeptidyl peptidase IV (DPP IV), is less susceptible to degradation by neutral endopeptidase, and possesses a nine-AA C-terminal sequence absent from GLP-1. Here we examine the importance of these nine AAs for biological activity of Ex-4, a sequence of truncated Ex-4 analogs, and native GLP-1 and GLP-1 analogs to which all or parts of the C-terminal sequence have been added. We found that removing these AAs from Ex-4 to produce Ex (1-30) reduced the affinity for the GLP-1 receptor (GLP-1R) relative to Ex-4 (IC50: Ex-4, 3.22+/-0.9 nM; Ex (1-30), 32+/-5.8 nM) but made it comparable to that of GLP-1 (IC50: 44.9+/-3.2 nM). The addition of this nine-AA sequence to GLP-1 improved the affinity of both GLP-1 and the DPP IV resistant analog GLP-1 8-glycine for the GLP-1 receptor (IC50: GLP-1 Gly8 [GG], 220+/-23 nM; GLP-1 Gly8 Ex (31-39), 74+/-11 nM). Observations of the cAMP response in an insulinoma cell line show a similar trend for biological activity.     

早期糖尿病大鼠视网膜中血管生成素-1、血管内皮生长因子的表达

目的:研究血管生成素-1(angiopoietin-1,Ang-1)和血管内皮生长因子(vascular endothelial growth factor,VEGF)在早期糖尿病大鼠视网膜中的表达及其意义。方法:制备类似人类Ⅰ型糖尿病大鼠动物模型,取40只SD雄性大鼠分糖尿病组28只及正常对照组12只,糖尿病组28只SD雄性大鼠腹腔注射STZ65mg.Kg-1,建模成功后分别在1周、2周、3周、4周各取糖尿病组7只及正常对照组3只摘取大鼠眼球,应用免疫组化法检测Ang-1、VEGF在视网膜中的表达。结果:Ang-1、VEGF在正常大鼠视网膜的染色均为阴性,Ang-1在1、2、3周糖尿病大鼠视网膜内界膜、内核层及散在血管旁周细胞及Mǔller细胞中呈阳性表达,4周时呈阴性染色反应,Ang-1在糖尿病大鼠视网膜中表达1周阳性率约为39.5%,2周阳性率约为59.7%,3周阳性率约为78.9%,4周阳性率约为19.0%。VEGF在糖尿病大鼠视网膜中表达1周阳性率约为38.6%,2周阳性率约为54.3%,3周阳性率为78.9%,4周阳性率约为42%。结论:Ang-1、VEGF可能在糖尿病视网膜病变早期形成中起到重要...     

VIPergic innervation of diabetic pancreas in rats

Morphological, ultrastructural, and immunocytochemical studies of nerves containing vasoactive intestinal peptide (VIP) are described in diabetic pancreas of rats after streptozotocin treatment. The observations covered 48 hours and 6 months following streptozotocin treatment. At the ultrastructural level, degenerative changes were already observed in axons of nerve ganglia in the pancreas 48 hours (early stage) after streptozotocin treatment. These changes were hardly detected at the light microscopic level and VIP-like immunoreactivities were seen in nerves and fibers in such pancreas. The nerve ganglia were almost absent in the diabetic pancreas 6 months after streptozotocin treatment (late stage). Associated with the absence of nerve ganglia, nerves and fibers containing VIP-like immunoreactivities were also absent at this late stage. The findings indicated that the changes in the diabetic pancreas were neuropathic caused by metabolic disturbance after long standing diabetes and resulting absence of VIP-like immunoreactive nerves occurred. It was suggested from the present study that the changes in nerve ganglia began as damage at the ultrastructural level due to acute toxicity of the chemical at an early stage and then developed destruction and absence at a late stage.