exendin-(9-39) and glucagon-like-peptide-1-(7-36)amide

Topics: Animals; Brain; Feeding Behavior; Genes, fos; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Humans; Hyperglycemia; Hyperinsulinism; Neurons; Peptide Fragments; Protein Precursors; Rats; Rats, Zucker; Receptors, Glucagon; Venoms

Radiolabelled glucagon-like peptide 1 (GLP-1) receptor agonists have recently been shown to successfully image benign insulinomas in patients. Moreover, it was recently reported that antagonist tracers were superior to agonist tracers for somatostatin and gastrin-releasing peptide receptor targeting of tumours. The present preclinical study determines therefore the value of an established GLP-1 receptor antagonist for the in vitro visualization of GLP-1 receptor-expressing tissues in mice and humans.. Receptor autoradiography studies with (125)I-GLP-1(7-36)amide agonist or (125)I-Bolton-Hunter-exendin(9-39) antagonist radioligands were performed in mice pancreas and insulinomas as well as in human insulinomas; competition experiments were performed in the presence of increasing concentration of GLP-1(7-36)amide or exendin(9-39).. The antagonist (125)I-Bolton-Hunter-exendin(9-39) labels mouse pancreatic β-cells and mouse insulinomas, but it does not label human pancreatic β-cells and insulinomas. High affinity displacement (IC(50) approximately 2 nM) is observed in mouse β-cells and insulinomas with either the exendin(9-39) antagonist or GLP-1(7-36)amide agonist. For comparison, the agonist (125)I-GLP-1(7-36)amide intensively labels mouse pancreatic β-cells, mouse insulinoma and human insulinomas; high affinity displacement is observed for the GLP-1(7-36)amide in all tissues; however, a 5 and 20 times lower affinity is found for exendin(9-39) in the mouse and human tissues, respectively.. This study reports a species-dependent behaviour of the GLP-1 receptor antagonist exendin(9-39) that can optimally target GLP-1 receptors in mice but not in human tissue. Due to its overly low binding affinity, this antagonist is an inadequate targeting agent for human GLP-1 receptor-expressing tissues, as opposed to the GLP-1 receptor agonist, GLP-1(7-36)amide.

Topics: Animals; Gene Expression Regulation, Neoplastic; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Humans; Insulinoma; Isotope Labeling; Mice; Pancreas; Pancreatic Neoplasms; Peptide Fragments; Receptors, Glucagon

Glucagon-like peptide-1 (GLP-1) is a hormone predominately synthesized and secreted by intestinal L-cells. GLP-1 modulates multiple cellular functions and its receptor agonists are now used clinically for diabetic treatment. Interestingly, preclinical and clinical evidence suggests that GLP-1 agonists produce beneficial effects on dysfunctional hearts via acting on myocardial GLP-1 receptors. As the effects of GLP-1 on myocyte electrophysiology are largely unknown, this study was to assess if GLP-1 could affect the cardiac voltage-gated L-type Ca2+ current (I(Ca)).. The whole-cell patch clamp method was used to record I(Ca) and action potentials in enzymatically isolated cardiomyocytes from adult canine left ventricles.. Extracellular perfusion of GLP-1 (7-36 amide) at 5 nM increased I(Ca) by 23 ± 8% (p < 0.05, n = 7). Simultaneous bath perfusion of 5 nM GLP-1 plus 100 nM Exendin (9-39), a GLP-1 receptor antagonist, was unable to block the GLP-1-induced increase in I(Ca); however, the increase in I(Ca) was abolished if Exendin (9-39) was pre-applied 5 min prior to GLP-1 administration. Intracellular dialysis with a protein kinase A inhibitor also blocked the GLP-1-enhanced I(Ca). In addition, GLP-1 at 5 nM prolonged the durations of the action potentials by 128 ± 36 ms (p < 0.01) and 199 ± 76 ms (p < 0.05) at 50% and 90% repolarization (n = 6), respectively.. Our data demonstrate that GLP-1 enhances I(Ca) in canine cardiomyocytes. The enhancement of I(Ca) is likely via the cAMP-dependent protein kinase A mechanism and may contribute, at least partially, to the prolongation of the action potential duration.

Topics: Action Potentials; Animals; Calcium Channels, L-Type; Calcium Signaling; Cyclic AMP-Dependent Protein Kinases; Dogs; Enzyme Activation; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; In Vitro Techniques; Myocytes, Cardiac; Patch-Clamp Techniques; Peptide Fragments; Protein Kinase Inhibitors; Receptors, Glucagon; Time Factors

Several lines of evidence suggest that incretin-based therapies suppress the development of cardiovascular disease in type 2 diabetes. We investigated the possibility that glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) can prevent the development of atherosclerosis in Apoe (-/-) mice.. Apoe (-/-) mice (17 weeks old) were administered GLP-1(7-36)amide, GLP-1(9-36)amide, GIP(1-42) or GIP(3-42) for 4 weeks. Aortic atherosclerosis, oxidised LDL-induced foam cell formation and related gene expression in exudate peritoneal macrophages were determined.. Administration of GLP-1(7-36)amide or GIP(1-42) significantly suppressed atherosclerotic lesions and macrophage infiltration in the aortic wall, compared with vehicle controls. These effects were cancelled by co-infusion with specific antagonists for GLP-1 and GIP receptors, namely exendin(9-39) or Pro(3)(GIP). The anti-atherosclerotic effects of GLP-1(7-36)amide and GIP(1-42) were associated with significant decreases in foam cell formation and downregulation of CD36 and acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1) in macrophages. GLP-1 and GIP receptors were both detected in Apoe (-/-) mouse macrophages. Ex vivo incubation of macrophages with GLP-1(7-36)amide or GIP(1-42) for 48 h significantly suppressed foam cell formation. This effect was wholly abolished in macrophages pretreated with exendin(9-39) or (Pro(3))GIP, or with an adenylate cyclase inhibitor, MDL12,330A, and was mimicked by incubation with an adenylate cyclase activator, forskolin. The inactive forms, GLP-1(9-36)amide and GIP(3-42), had no effects on atherosclerosis and macrophage foam cell formation.. Our study is the first to demonstrate that active forms of GLP-1 and GIP exert anti-atherogenic effects by suppressing macrophage foam cell formation via their own receptors, followed by cAMP activation. Molecular mechanisms underlying these effects are associated with the downregulation of CD36 and ACAT-1 by incretins.

Topics: Acetyl-CoA C-Acetyltransferase; Animals; Apolipoproteins E; Atherosclerosis; Blotting, Western; CD36 Antigens; Cell Line; Cells, Cultured; Foam Cells; Gastric Inhibitory Polypeptide; Glucagon-Like Peptide 1; Humans; Incretins; Male; Mice; Mice, Knockout; Microscopy, Confocal; Peptide Fragments; Peptides; Real-Time Polymerase Chain Reaction

Increasing evidence from both clinical and experimental studies indicates that the insulin-releasing hormone, glucagon-like peptide-1 (GLP-1) may exert additional protective/reparative effects on the cardiovascular system. The aim of this study was to examine vasorelaxant effects of GLP-1(7-36)amide, three structurally-related peptides and a non-peptide GLP-1 agonist in rat aorta. Interestingly, all GLP-1 compounds, including the established GLP-1 receptor antagonist, exendin (9-39) caused concentration-dependent relaxation. Mechanistic studies employing hyperpolarising concentrations of potassium or glybenclamide revealed that these relaxant effects are mediated via specific activation of ATP-sensitive potassium channels. Further experiments using a specific membrane-permeable cyclic AMP (cAMP) antagonist, and demonstration of increased cAMP production in response to GLP-1 illustrated the critical importance of this pathway. These data significantly extend previous observations suggesting that GLP-1 may modulate vascular function, and indicate that this effect may be mediated by the GLP-1 receptor. However, further studies are required in order to establish whether GLP-1 related agents may confer additional cardiovascular benefits to diabetic patients.

Topics: Animals; Aorta, Thoracic; Cyclic AMP; DNA Primers; Gene Expression; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; In Vitro Techniques; KATP Channels; Male; Peptide Fragments; Rats; Rats, Sprague-Dawley; Receptors, Glucagon; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Vasodilation

Glucagon-like peptide-1 (GLP-1) (7-36) amide is a glucoregulatory hormone with insulinotropic and insulinomimetic actions. We determined whether the insulinomimetic effects of GLP-1 are mediated through its principal metabolite, GLP-1 (9-36) amide (GLP-1m).. Glucose turnover during two, 2-h, euglycemic clamps was measured in 12 lean and 12 obese (BMI <25 or >30 kg/m(2)) male and female subject volunteers with normal oral glucose tolerance test. Saline or GLP-1m were infused from 0 to 60 min in each study. Additionally, seven lean and six obese subjects underwent a third clamp in which the GLP-1 receptor (GLP-1R) antagonist, exendin (9-39) amide was infused from -60 to 60 min with GLP-1m from 0 to 60 min.. No glucose infusion was required in lean subjects to sustain euglycemia (glucose clamp) during saline or GLP-1m infusions. However, in obese subjects glucose infusion was necessary during GLP-1m infusion alone in order to compensate for a marked (>50%) inhibition of hepatic glucose production (HGP). Plasma insulin levels remained constant in lean subjects but rose significantly in obese subjects after termination of the peptide infusions. During GLP-1R blockade, infusion of glucose was immediately required upon starting GLP-1m infusions in all subjects due to a more dramatic reduction in HGP, as well as a delayed and modest insulinotropic response.. We conclude that GLP-1m potently inhibits HGP and is a weak insulinotropic agent. These properties are particularly apparent and pronounced in obese but only become apparent in lean subjects during GLP-1 (7-36) receptor blockade. These previously unrecognized antidiabetogenic actions of GLP-1m may have therapeutic usefulness.

Topics: Adult; Blood Glucose; Female; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucose Clamp Technique; Glucose Tolerance Test; Humans; Hypoglycemic Agents; Infusions, Parenteral; Insulin; Liver; Male; Middle Aged; Obesity; Peptide Fragments; Peptides; Receptors, Glucagon; Time Factors

Glucagon-like peptide-1 (GLP-1) receptor agonists have been reported to modulate gastrointestinal motility but the mechanism is essentially unknown. In the present studies, we investigated the potency and mechanism of action of GLP-1 receptor ligands on the isolated ileum of Suncus murinus, an insectivore used in anti-emetic research. Ileal segments were mounted in organ baths containing Kreb's solution. Cumulative concentration-response curves to GLP-1 (7-36) amide (0.1-300 nM) and exendin-4 (0.1-100 nM) were constructed in the absence and presence of exendin (9-39) amide (0.3-3 nM). GLP-1 (7-36) amide and exendin-4 induced concentration-dependent contractions yielding pEC50 values of 8.4+/-0.2 and 8.4+/-0.4, respectively. Exendin (9-39) antagonized the action of both agonists in a non-competitive reversible manner, with apparent pKB values of 9.5 and 9.7, respectively. Tetrodotoxin (1 microM), atropine (1 microM) and hexamethonium (500 microM) were used to determine the contractile mechanism of action of exendin-4. Tetrodotoxin and atropine significantly antagonized (P<0.01) the contractile action of exendin-4 (10 nM); hexamethonium (500 microM) had no action. These studies suggest that GLP-1 receptor agonists contract the ileum indirectly via postganglionic enteric neurones and an involvement of muscarinic receptors. These studies provide information relevant to the use of this species to estimate the therapeutic indexes of GLP-1 receptor agonists.

Topics: Animals; Exenatide; Glucagon-Like Peptide 1; Ileum; In Vitro Techniques; Male; Muscle Contraction; Peptide Fragments; Peptides; Shrews; Venoms

This study examined the biological effects of the GIP receptor antagonist, (Pro3)GIP and the GLP-1 receptor antagonist, exendin(9-39)amide.. Cyclic AMP production was assessed in Chinese hamster lung fibroblasts transfected with human GIP or GLP-1 receptors, respectively. In vitro insulin release studies were assessed in BRIN-BD11 cells while in vivo insulinotropic and glycaemic responses were measured in obese diabetic ( ob/ ob) mice.. In GIP receptor-transfected fibroblasts, (Pro(3))GIP or exendin(9-39)amide inhibited GIP-stimulated cyclic AMP production with maximal inhibition of 70.0+/-3.5% and 73.5+/-3.2% at 10(-6) mol/l, respectively. In GLP-1 receptor-transfected fibroblasts, exendin(9-39)amide inhibited GLP-1-stimulated cyclic AMP production with maximal inhibition of 60+/-0.7% at 10(-6) mol/l, whereas (Pro(3))GIP had no effect. (Pro(3))GIP specifically inhibited GIP-stimulated insulin release (86%; p<0.001) from clonal BRIN-BD11 cells, but had no effect on GLP-1-stimulated insulin release. In contrast, exendin(9-39)amide inhibited both GIP and GLP-1-stimulated insulin release (57% and 44%, respectively; p<0.001). Administration of (Pro(3))GIP, exendin(9-39)amide or a combination of both peptides (25 nmol/kg body weight, i.p.) to fasted (ob/ob) mice decreased the plasma insulin responses by 42%, 54% and 49%, respectively (p<0.01 to p<0.001). The hyperinsulinaemia of non-fasted (ob/ob) mice was decreased by 19%, 27% and 18% (p<0.05 to p<0.01) by injection of (Pro3)GIP, exendin(9-39)amide or combined peptides but accompanying changes of plasma glucose were small.. These data show that (Pro(3))GIP is a specific GIP receptor antagonist. Furthermore, feeding studies in one commonly used animal model of obesity and diabetes, (ob/ob) mice, suggest that GIP is the major physiological component of the enteroinsular axis, contributing approximately 80% to incretin-induced insulin release.

Topics: Animals; Cells, Cultured; Cricetinae; Cricetulus; Cyclic AMP; Diabetes Mellitus; Gastric Inhibitory Polypeptide; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Humans; Hyperinsulinism; Insulin; Insulin Secretion; Mice; Obesity; Peptide Fragments; Postprandial Period; Protein Precursors; Spectrometry, Mass, Electrospray Ionization

Activation of the hepatoportal glucose sensors by portal glucose infusion leads to increased glucose clearance and induction of hypoglycemia. Here, we investigated whether glucagon-like peptide-1 (GLP-1) could modulate the activity of these sensors. Mice were therefore infused with saline (S-mice) or glucose (P-mice) through the portal vein at a rate of 25 mg/kg. min. In P-mice, glucose clearance increased to 67.5 +/- 3.7 mg/kg. min as compared with 24.1 +/- 1.5 mg/kg. min in S-mice, and glycemia decreased from 5.0 +/- 0.1 to 3.3 +/- 0.1 mmol/l at the end of the 3-h infusion period. Coinfusion of GLP-1 with glucose into the portal vein at a rate of 5 pmol/kg. min (P-GLP-1 mice) did not increase the glucose clearance rate (57.4 +/- 5.0 ml/kg. min) and hypoglycemia (3.8 +/- 0.1 mmol/l) observed in P-mice. In contrast, coinfusion of glucose and the GLP-1 receptor antagonist exendin-(9-39) into the portal vein at a rate of 0.5 pmol/kg. min (P-Ex mice) reduced glucose clearance to 36.1 +/- 2.6 ml/kg. min and transiently increased glycemia to 9.2 +/- 0.3 mmol/l at 60 min of infusion before it returned to the fasting level (5.6 +/- 0.3 mmol/l) at 3 h. When glucose and exendin-(9-39) were infused through the portal and femoral veins, respectively, glucose clearance increased to 70.0 +/- 4.6 ml/kg. min and glycemia decreased to 3.1 +/- 0.1 mmol/l, indicating that exendin-(9-39) has an effect only when infused into the portal vein. Finally, portal vein infusion of glucose in GLP-1 receptor(-/-) mice failed to increase the glucose clearance rate (26.7 +/- 2.9 ml/kg. min). Glycemia increased to 8.5 +/- 0.5 mmol/l at 60 min and remained elevated until the end of the glucose infusion (8.2 +/- 0.4 mmol/l). Together, our data show that the GLP-1 receptor is part of the hepatoportal glucose sensor and that basal fasting levels of GLP-1 sufficiently activate the receptor to confer maximum glucose competence to the sensor. These data demonstrate an important extrapancreatic effect of GLP-1 in the control of glucose homeostasis.

Topics: Animals; Blood Glucose; Catheters, Indwelling; Enzyme-Linked Immunosorbent Assay; Fasting; Femoral Vein; Gastrointestinal Hormones; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Glucose; Hepatic Veins; Homeostasis; Infusions, Intravenous; Metabolic Clearance Rate; Mice; Mice, Inbred C57BL; Peptide Fragments; Portal System; Portal Vein; Protein Precursors; Tritium; Venoms

Glucagon-like peptide-1 (7-36)amide (tGLP-1), a representative humoral incretin, released into the portal circulation in response to a meal ingestion, exerts insulinotropic action through binding to the tGLP-1 receptor known to be a single molecular form thus far. We previously reported that the hepatic vagal nerve is receptive to intraportal tGLP-1, but not to non-insulinotropic full-length GLP-1-(1-37), through a mechanism mediated by specific receptor to the hormone. In the present study, we aimed to examine how modification of the receptor function alters this neural reception of tGLP-1, by using the specific agonist, exendin-4, and the specific antagonist, exendin (9-39)amide, of the receptor at doses known to exert their effects on the insulinotropic action of tGLP-1. Intraportal injection of 0.2 or 4.0 pmol tGLP-1, a periphysiological and pharmacological dose, respectively, facilitated significantly the afferent impulse discharge rate of the hepatic vagus in anesthetized rats, as reported previously. However, unexpectedly, intraportal injection of exendin-4 at a dose of 0.2 or 4.0 pmol, or of even 40.0 pmol, did not facilitate the afferents at all. Moreover, intraportal injection of exendin (9-39)amide at 100 times or more molar dose to that of tGLP-1, either 5 min before or 10 min after injection of 0.2 or 4.0 pmol tGLP-1, failed to modify the tGLP-1-induced facilitation of the afferents. The present results suggest that the neural reception of tGLP-1 involves a receptor mechanism distinct from that in the well-known humoral insulinotropic action.

Topics: Animals; Electrophysiology; Exenatide; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Injections, Intravenous; Liver; Male; Neurons, Afferent; Pancreas; Peptide Fragments; Peptides; Portal Vein; Rats; Rats, Wistar; Receptors, Glucagon; Vagus Nerve; Venoms

Glucagon-like peptide-1-(7---36) amide (GLP-1) is a potent incretin hormone secreted from distal gut. It stimulates basal and glucose-induced insulin secretion and proinsulin gene expression. The present study tested the hypothesis that GLP-1 may modulate insulin receptor binding. RINm5F rat insulinoma cells were incubated with GLP-1 (0.01-100 nM) for different periods (1 min-24 h). Insulin receptor binding was assessed by competitive ligand binding studies. In addition, we investigated the effect of GLP-1 on insulin receptor binding on monocytes isolated from type 1 and type 2 diabetes patients and healthy volunteers. In RINm5F cells, GLP-1 increased the capacity and affinity of insulin binding in a time- and concentration-dependent manner. The GLP-1 receptor agonist exendin-4 showed similar effects, whereas the receptor antagonist exendin-(9---39) amide inhibited the GLP-1-induced increase in insulin receptor binding. The GLP-1 effect was potentiated by the adenylyl cyclase activator forskolin and the stable cAMP analog Sp-5, 6-dichloro-1-beta-D-ribofuranosyl-benzimidazole-3', 5'-monophosphorothioate but was antagonized by the intracellular Ca(2+) chelator 1,2-bis(0-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM. Glucagon, gastric inhibitory peptide (GIP), and GIP-(1---30) did not affect insulin binding. In isolated monocytes, 24 h incubation with 100 nM GLP-1 significantly (P<0.05) increased the diminished number of high-capacity/low-affinity insulin binding sites per cell in type 1 diabetics (9,000+/-3,200 vs. 18,500+/-3,600) and in type 2 diabetics (15,700+/-2,100 vs. 28,900+/-1,800) compared with nondiabetic control subjects (25,100+/-2,700 vs. 26,200+/-4,200). Based on our previous experiments in IEC-6 cells and IM-9 lymphoblasts indicating that the low-affinity/high-capacity insulin binding sites may be more specific for proinsulin (Jehle, PM, Fussgaenger RD, Angelus NK, Jungwirth RJ, Saile B, and Lutz MP. Am J Physiol Endocrinol Metab 276: E262-E268, 1999 and Jehle, PM, Lutz MP, and Fussgaenger RD. Diabetologia 39: 421-432, 1996), we further investigated the effect of GLP-1 on proinsulin binding in RINm5F cells and monocytes. In both cell types, GLP-1 induced a significant increase in proinsulin binding. We conclude that, in RINm5F cells and in isolated human monocytes, GLP-1 specifically increases the number of high-capacity insulin binding sites that may be functional proinsulin receptors.

Topics: Adult; Animals; Exenatide; Female; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Humans; Insulin; Insulinoma; Male; Monocytes; Pancreatic Neoplasms; Peptide Fragments; Peptides; Proinsulin; Protein Precursors; Rats; Receptors, Glucagon; Tumor Cells, Cultured; Venoms

Central nervous system glucagon-like peptide-1-(7-36) amide (GLP-1) administration has been reported to acutely reduce food intake in the rat. We here report that repeated intracerebroventricular (i.c.v.) injection of GLP-1 or the GLP-1 receptor antagonist, exendin-(9-39), affects food intake and body weight. Daily i.c.v. injection of 3 nmol GLP-1 to schedule-fed rats for 6 days caused a reduction in food intake and a decrease in body weight of 16 +/- 5 g (P < 0.02 compared with saline-injected controls). Daily i.c.v. administration of 30 nmol exendin-(9-39) to schedule-fed rats for 3 days caused an increase in food intake and increased body weight by 7 +/- 2 g (P < 0.02 compared with saline-injected controls). Twice daily i.c.v. injections of 30 nmol exendin-(9-39) with 2.4 nmol neuropeptide Y to ad libitum-fed rats for 8 days increased food intake and increased body weight by 28 +/- 4 g compared with 14 +/- 3 g in neuropeptide Y-injected controls (P < 0.02). There was no evidence of tachyphylaxis in response to i.c.v. GLP-1 or exendin-(9-39). GLP-1 may thus be involved in the regulation of body weight in the rat.

Topics: Animals; Body Weight; Energy Intake; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Injections, Intraventricular; Male; Neurotransmitter Agents; Peptide Fragments; Rats; Rats, Wistar

Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) are potent insulinotropic peptides released from the small intestine. To examine their relative contribution to postprandial insulin release, a specific GIP antagonist (ANTGIP) and a GLP-1 antagonist, exendin-(9-39)-NH2, were infused into rats after an intragastric glucose meal. In control rats, plasma glucose and insulin levels rose gradually during the first 20 min and then decreased. Exendin-(9-39)-NH2 administration inhibited postprandial insulin secretion by 32% at 20 min and concomitantly increased plasma glucose concentrations. In contrast, ANTGIP treatment not only induced a 54% decrease in insulin secretion but also a 15% reduction in plasma glucose levels 20 min after the glucose meal. In vivo studies in rats demonstrated that glucose uptake in the upper small intestine was significantly inhibited by the ANTGIP, an effect that might account for the decrease in plasma glucose levels observed in ANTGIP-treated rats. When the two antagonists were administered to rats concomitantly, no potentiating effect on either insulin release or plasma glucose concentration was detected. Glucose meal-stimulated GLP-1 release was not affected by ANTGIP administration, whereas postprandial glucagon levels were diminished in rats receiving exendin-(9-39)-NH2. The results of these studies suggest that GIP and GLP-1 may share a common mechanism in stimulating pancreatic insulin release. Furthermore, the GIP receptor appears to play a role in facilitating glucose uptake in the small intestine.

Topics: Absorption; Animals; Blood Glucose; Dose-Response Relationship, Drug; Gastric Inhibitory Polypeptide; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Glucose; Insulin; Peptide Fragments; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptors, Gastrointestinal Hormone

In order to assess effects of glucagon-like peptide-1 (GLP-1) in the brain excluding the hypothalamus, the effects of GLP-1 (7-36) amide, a naturally produced active fragment, on the electroencephalogram and hippocampal single unit activities of anesthetized male Wistar rats were examined. I.c.v. injection of GLP-1 (7-36) amide decreased the hippocampal theta wave duration. Juxtacellular administration of GLP-1 (7-36) amide first increased and then decreased single unit activities recorded in the hippocampal CA1, which effects were antagonized by exendin (9-39) amide, a GLP-1 receptor antagonist, or 6-cyano-7-nitroquinoxaline-2,3-dione, a non-NMDA type glutamate receptor antagonist. These results indicate that GLP-1 receptors exist in the hippocampus and are involved in modulating hippocampal activity through an increase in the release of excitatory amino acid transmitters.

Topics: Animals; Aspartic Acid; Cerebral Cortex; Electroencephalography; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Glutamic Acid; Glutamine; Hippocampus; Injections, Intraventricular; Male; Neurons; Peptide Fragments; Peptides; Protein Precursors; Rats; Rats, Wistar; Receptors, Glucagon; Stimulation, Chemical; Ventromedial Hypothalamic Nucleus

cAMP is required for normal glucose-induced insulin release by pancreatic beta-cells. In a previous study, we showed that cAMP production in beta-cells depends on the expression of receptors for glucagon, glucagon-like peptide 1(7-36) amide [GLP-1(7-36) amide], and glucose-dependent insulinotropic polypeptide. Although the latter two peptides are thought to amplify meal-induced insulin release (incretin effect), the role of glucagon in the regulation of insulin release remains elusive. In the present study, we analyzed the interaction of glucagon with its own receptor and with the glucagon-like peptide 1 (GLP-1) receptor using purified rat beta-cells. Glucagon binding was partially displaced by 1 micromol/l des-His1-[Glu9]glucagon-amide, a glucagon receptor antagonist, and by 1 micromol/l GLP-1. Conversely, GLP-1 binding was competitively inhibited by high glucagon concentrations (Ki = 0.3 micromol/l). Glucagon-induced cAMP production in beta-cells was inhibited both by 1 micromol/l des-His1-[Glu9]glucagon-amide and exendin-(9-39)-amide, a specific GLP-1 receptor antagonist, whereas GLP-1-induced cAMP formation was suppressed only by exendin-(9-39)-amide. Finally, addition of 1 micromol/l exendin-(9-39)-amide to 20 mmol/l glucose-stimulated beta-cells did not antagonize the potentiating effect of 1 nmol/l glucagon, although it prevented 45% of glucagon potentiation when the peptide was administered at 10 nmol/l. Our data suggest that glucagon recognition via two distinct receptors allows pancreatic beta-cells to detect this peptide both when diluted in the systemic circulation and when concentrated as local signal in the islet interstitium.

Topics: Animals; Cells, Cultured; Cyclic AMP; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Insulin; Iodine Radioisotopes; Islets of Langerhans; Liver; Male; Peptide Fragments; Rats; Rats, Sprague-Dawley; Receptors, Glucagon

Glucagon-like peptide-1 (GLP-1) is the most potent endogenous insulin-stimulating hormone. In the present study the plasma stability and biological activity of a GLP-1 analog, [Ser]GLP-1(7-36)amide, in which the second N-terminal amino acid alanine was replaced by serine, was evaluated in vitro and in vivo. Incubation of GLP-1 with human or rat plasma resulted in degradation of native GLP-1(7-36)amide to GLP-1(9-36)amide, while [Ser]GLP-1(7-36)amide was not significantly degraded by plasma enzymes. Using glucose-responsive HIT-T15 cells, [Ser]GLP-1(7-36)amide showed strong insulinotropic activity, which was inhibited by the specific GLP-1 receptor antagonist exendin-4(9-39)amide. Simultaneous i.v. injection of [Ser]GLP-1(7-36)amide and glucose in rats induced a twofold higher increase in plasma insulin levels than unmodified GLP-1(7-36)amide with glucose and a fivefold higher increase than glucose alone. [Ser]GLP-1(7-36)amide induced a 1.5-fold higher increase in plasma insulin than GLP-1(7-36)amide when given 1 h before i.v. application of glucose. The insulinotropic effect of [Ser]GLP-1(7-36)amide was suppressed by i.v. application of exendin-4(9-39)amide. The present data demonstrate that replacement of the second N-terminal amino acid alanine by serine improves the plasma stability of GLP-1(7-36)amide. The insulinotropic action in vitro and in vivo was not impaired significantly by this modification.

Topics: Animals; Chromatography, High Pressure Liquid; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Glucose; Humans; Insulin; Insulin Secretion; Male; Peptide Fragments; Protein Precursors; Rats; Rats, Wistar; Receptors, Glucagon; Time Factors; Tumor Cells, Cultured

Topics: Animals; Avoidance Learning; Eating; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Neuropeptides; Peptide Fragments; Proto-Oncogene Proteins c-fos; Rats; Saccharin; Satiety Response; Taste

The GLP-1 structurally related peptides exendin-4 and exendin(9-39)amide were found to act, in rat liver and skeletal muscle, as agonist and antagonist, respectively, of the GLP-1(7-36)amide effects on glucose metabolism. Thus, like GLP-1(7-36)amide, exendin-4 increased glycogen synthase a activity and glucose incorporation into glycogen in both tissues and also stimulated exogenous D-glucose utilization and oxidation in muscle. These effects of GLP-1(7-36)amide and exendin-4 were inhibited by exendin(9-39)amide. Our findings provide further support to the proposed use of GLP-1, or exendin-4, as a tool in the treatment of diabetes mellitus. Thus, in addition to the well-known insulinotropic action of the peptides, they act both in liver and in muscle in a manner most suitable for restoration of glucose homeostasis, with emphasis on their positive effects upon glycogen synthesis in the two tissues and on the stimulation of exogenous glucose catabolism in muscle.

Topics: Animals; Cyclic AMP; Energy Metabolism; Enzyme Activation; Exenatide; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Glucose; Glycogen; Glycogen Synthase; Insulin; Liver; Muscle, Skeletal; Peptide Fragments; Peptides; Phosphorylase a; Rats; Rats, Wistar; Venoms

GLP-1-(7-36)-amide and exendin-4-(1-39) are glucagon-like peptide-1 (GLP-1) receptor agonists, whereas exendin-(9-39) is the only known antagonist. To analyze the transition from agonist to antagonist and to identify the amino acid residues involved in ligand activation of the GLP-1 receptor, we used exendin analogs with successive N-terminal truncations. Chinese hamster ovary cells stably transfected with the rat GLP-1 receptor were assayed for changes in intracellular cAMP caused by the test peptides in the absence or presence of half-maximal stimulatory doses of GLP-1. N-terminal truncation of a single amino acid reduced the agonist activity of the exendin peptide, whereas N-terminal truncation of 3-7 amino acids produced antagonists that were 4-10-fold more potent than exendin-(9-39). N-terminal truncation of GLP-1 by 2 amino acids resulted in weak agonist activity, but an 8-amino acid N-terminal truncation inactivated the peptide. Binding studies performed using 125I-labeled GLP-1 confirmed that all bioactive peptides specifically displaced tracer with high potency. In a set of exendin/GLP-1 chimeric peptides, substitution of GLP-1 sequences into exendin-(3-39) produced loss of antagonist activity with conversion to a weak agonist. The results show that receptor binding and activation occur in separate domains of exendin, but they are more closely coupled in GLP-1.

Topics: Amino Acid Sequence; Animals; Binding, Competitive; CHO Cells; Cricetinae; Cyclic AMP; Exenatide; Gastrointestinal Hormones; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Molecular Sequence Data; Peptide Fragments; Peptides; Rats; Receptors, Glucagon; Recombinant Fusion Proteins; Sequence Deletion; Structure-Activity Relationship; Transfection; Venoms

The sequence of glucagon-like peptide-1 (7-36) amide (GLP-1) is completely conserved in all mammalian species studied, implying that it plays a critical physiological role. We have shown that GLP-1 and its specific receptors are present in the hypothalamus. No physiological role for central GLP-1 has been established. We report here that intracerebroventricular (ICV) GLP-1 powerfully inhibits feeding in fasted rats. ICV injection of the specific GLP-1-receptor antagonist, exendin (9-39), blocked the inhibitory effect of GLP-1 on food intake. Exendin (9-39) alone had no influence on fast-induced feeding but more than doubled food intake in satiated rats, and augmented the feeding response to the appetite stimulant, neuropeptide Y. Induction of c-fos is a marker of neuronal activation. Following ICV GLP-1 injection, c-fos appeared exclusively in the paraventricular nucleus of the hypothalamus and central nucleus of the amygdala, and this was inhibited by prior administration of exendin (9-39). Both of these regions of the brain are of primary importance in the regulation of feeding. These findings suggest that central GLP-1 is a new physiological mediator of satiety.

Topics: Animals; Cerebral Ventricles; Eating; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Injections, Intraventricular; Male; Neuropeptide Y; Peptide Fragments; Proto-Oncogene Proteins c-fos; Rats; Satiation

Glucagon-like peptide 1 (GLP-1) is an insulinotropic hormone released after nutrient ingestion which is known to augment insulin secretion, inhibit glucagon release, and promote insulin-independent glucose disposition. To determine the overall effect of GLP-1 on glucose disposition after a meal we studied a group of healthy, conscious baboons before and after intragastric glucose administration during infusions of saline, and two treatments to eliminate the action of GLP-1: (a) exendin-[9-39] (Ex-9), a peptide receptor antagonist of GLP-1; or (b) an anti-GLP-1 mAb. Fasting concentrations of glucose were higher during infusion of Ex-9 than during saline (4.44 +/- 0.05 vs. 4.16 +/- 0.05 mM, P < 0.01), coincident with an elevation in the levels of circulating glucagon (96 +/- 10 vs. 59 +/- 3 ng/liter, P < 0.02). The postprandial glycemic excursions during administration of Ex-9 and mAb were greater than during the control studies (Ex-9 13.7 +/- 2.0 vs. saline 10.0 +/- 0.8 mM, P = 0.07; and mAb 13.6 +/- 1.2 vs. saline 10.6 +/- 0.9 mM, P = 0.044). The increments in insulin levels throughout the absorption of the glucose meal were not different for the experimental and control conditions, but the insulin response in the first 30 min after the glucose meal was diminished significantly during treatment with Ex-9 (Ex-9 761 +/- 139 vs. saline 1,089 +/- 166 pM, P = 0.044) and was delayed in three of the four animals given the neutralizing antibody (mAb 946 +/- 262 vs. saline 1,146 +/- 340 pM). Thus, elimination of the action of GLP-1 impaired the disposition of an intragastric glucose meal and this was at least partly attributable to diminished early insulin release. In addition to these postprandial effects, the concurrent elevation in fasting glucose and glucagon during GLP-1 antagonism suggests that GLP-1 may have a tonic inhibitory effect on glucagon output. These findings demonstrate the important role of GLP-1 in the assimilation of glucose absorbed from the gut.

Topics: Animals; Antibodies, Monoclonal; Blood Glucose; Fasting; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Glucose; Glucose Tolerance Test; Insulin; Male; Papio; Peptide Fragments; Receptors, Glucagon; Xylose

Glucagon is known to stimulate calcitonin secretion by thyroid C cells over a wide range of concentrations, raising the possibility of its interaction with several types of receptors. This study was designed to characterize receptors that mediate the effect of glucagon on a rat C cell line (CA-77). Binding studies, using radiolabeled [125I]glucagon and [125I]glucagon-like peptide-1-(7-36) amide ([125I]tGLP-1), to CA-77 plasma membranes demonstrated the presence of 1) a glucagon receptor with a dissociation constant (Kd) of 2.3 nM and relative potencies for structurally related peptides as follows: glucagon > oxyntomodulin > > tGLP-1; and 2) a tGLP-1 receptor with a Kd of 0.33 nM and relative potencies as follows: tGLP-1 > oxyntomodulin > glucagon. Glucagon stimulated calcitonin secretion from CA-77 cells in a dose-dependent manner over 4 orders of magnitude, with a maximal response of 312% over the basal value and an ED50 close to 50 nM. tGLP-1 induced a calcitonin release over 2 orders of magnitude, with a maximal response of 170% over the basal value and an ED50 close to 0.2 nM. Glucagon and tGLP-1 stimulated cAMP production in CA-77 cells to similar maximal levels over 4 and 2 orders of magnitude, respectively. The stimulation of cAMP production by glucagon at concentrations over 10 nM was suppressed by the tGLP-1 antagonist exendin-(9-39) amide, whereas the stimulation of calcitonin secretion was only partly abolished. Using a perifusion system of rat thyroid, glucagon and tGLP-1 stimulated calcitonin secretion in a calcium-dependent manner. It is concluded that glucagon and tGLP-1 receptors are expressed in the rat C cell line (CA-77) and in the normal rat thyroid. The effects of glucagon on calcitonin secretion observed at high concentrations are mediated in part through interaction with tGLP-1 receptors and via an additional non-cAMP-mediated mechanism.

Topics: Animals; Binding, Competitive; Calcitonin; Carcinoma, Medullary; Cyclic AMP; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Peptide Fragments; Perfusion; Rats; Rats, Wistar; Thyroid Gland; Thyroid Neoplasms; Tumor Cells, Cultured

This study was designed to determine the interactions of peptide exendin-(9-39) with the effect of glucagon-like peptide-1-(7-36) (GLP-1 (7-36)) amide and of exendin-4 on arterial blood pressure and heart rate in the rat. Both GLP-1 (7-36) amide and exendin-4 produced a dose-dependent increase in systolic, diastolic and mean arterial blood pressure, as well as in heart rate, although the effect of exendin-4 was more prolonged. These data indicate a longer functional half-life in vivo for exendin-4 as compared to GLP-1 (7-36) amide, which may have therapeutical applications. The antagonist effect of exendin-(9-39) on these cardiovascular parameters was also tested with 3000 ng of exendin-(9-39) intravenously administered 5 min before i.v. injection of 10 ng of either GLP-1 (7-36) amide or exendin-4. Under these experimental conditions the effect of the latter two peptides on arterial blood pressure and heart rate was blocked. By contrast, single administration of exendin-(9-39) did not modify cardiovascular parameters. These findings indicate that exendin-4 is an agonist and that exendin-(9-39) is an antagonist of the action of GLP-1 (7-36) amide on arterial blood pressure and heart rate. Therefore, the action of GLP-1 (7-36) amide on these parameters seems to be mediated through its own receptors.

Topics: Animals; Blood Pressure; Exenatide; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Heart Rate; Male; Peptide Fragments; Peptides; Rats; Rats, Sprague-Dawley; Venoms

Glucagon-like peptide 1 (7-37)/(7-36) amide (GLP-1) is derived from the intestinal proglucagon processing. It is considered an important insulin-releasing gut hormone. This study uses exendin (9-39) amide as a GLP-1 receptor antagonist to evaluate the contribution of GLP-1 to the incretin effect. Anesthetized rats were challenged by an intraduodenal glucose infusion to evaluate maximally occurring GLP-1 and gastric inhibitory polypeptide (GIP) plasma levels. Maximal immunoreactive (IR) GLP-1 plasma levels amounted to 10 pmol/l (IR-GIP 11 pmol/l). Exendin (9-39) amide abolished the insulin-stimulatory effect of 60 pmol of GLP-1 or of the GLP-1 agonist exendin-4 (0.5 nmol) injected as bolus, respectively. An intravenous bolus injection of 5.94 nmol of exendin (9-39) amide 3 min before enteral glucose infusion grossly reduced the total insulin secretory response (by 60%) and significantly increased circulating blood glucose levels (P < 0.05). In contrast, the GLP-1 antagonist left the insulin response after an intravenous glucose or glucose plus GIP (60 pmol) load unaltered. Our data support the concept that GLP-1 is an important incretin factor. Exendin (9-39) amide is a useful GLP-1 antagonist for in vivo studies.

Topics: Animals; Drug Interactions; Exenatide; Gastric Inhibitory Polypeptide; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Glucose; Insulin; Male; Peptide Fragments; Peptides; Protein Precursors; Radioimmunoassay; Rats; Rats, Wistar; Receptors, Glucagon; Venoms

Glucagon-like peptide-1 7-36 amide (GLP-1) has been postulated to be the primary hormonal mediator of the entero-insular axis but evidence has been indirect. The discovery of exendin (9-39), a GLP-1 receptor antagonist, allowed this to be further investigated. The IC50 for GLP-1 receptor binding, using RIN 5AH beta-cell membranes, was found to be 0.36 nmol/l for GLP-1 and 3.44 nmol/l for exendin (9-39). There was no competition by exendin (9-39) at binding sites for glucagon or related peptides. In the anaesthetized fasted rat, insulin release after four doses of GLP-1 (0.1, 0.2, 0.3, and 0.4 nmol/kg) was tested by a 2-min intravenous infusion. Exendin (9-39) (1.5, 3.0, and 4.5 nmol/kg) was administered with GLP-1 0.3 nmol/kg, or saline, and only the highest dose fully inhibited insulin release. Exendin (9-39) at 4.5 nmol/kg had no effect on glucose, arginine, vasoactive intestinal peptide or glucose-dependent insulinotropic peptide stimulated insulin secretion. Postprandial insulin release was studied in conditioned conscious rats after a standard meal. Exendin (9-39) (0.5 nmol/kg) considerably reduced postprandial insulin concentrations, for example by 48% at 15 min (431 +/- 21 pmol/l saline, 224 +/- 32 pmol/l exendin, P < 0.001). Thus, GLP-1 appears to play a major role in the entero-insular axis.

Topics: Anesthesia; Animals; Arginine; Cells, Cultured; Consciousness; Eating; Fasting; Gastric Inhibitory Polypeptide; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Glucose; Infusions, Intravenous; Insulin; Insulin Secretion; Male; Pancreas; Peptide Fragments; Peptides; Protein Precursors; Radioligand Assay; Rats; Rats, Wistar; Receptors, Glucagon; Vasoactive Intestinal Peptide

Glucagon-like peptide-I (GLP-I) is a potent insulinotropic peptide that mediates its actions at pancreatic B-cells via specific receptors. In the present study we stably expressed the rat B-cell GLP-I receptor in CHO cells and studied binding characteristics and receptor activation utilizing the naturally occurring receptor agonist GLP-I(7-36)-amide (GLP-I), the proglucagon-derived GLP-I-related peptide oxyntomodulin, the GLP-I receptor agonist exendin-4, and the specific antagonist exendin(9-39). The potencies to displace [125I]GLP-I from the receptor were GLP-I > exendin-4 > exendin(9-39) > oxyntomodulin, and to displace [125I]exendin-4 GLP-I = exendin-4 > exendin(9-39) > oxyntomodulin. cAMP production was stimulated equally by GLP-I and exendin-4. Oxyntomodulin was less potent to stimulate cAMP generation. Exendin(9-39) blocked the stimulatory action of GLP-I and exendin-4 on cAMP production, but not that of oxyntomodulin. This study shows that GLP-I and exendin-4 are potent agonists at the transfected rat B-cell GLP-I receptor whereas oxyntomodulin is only a weak GLP-I receptor agonist. Furthermore, exendin(9-39) is a potent GLP-I receptor antagonist. This peptide is a valuable tool to further study the physiological actions of GLP-I.

Topics: Animals; CHO Cells; Cricetinae; Evaluation Studies as Topic; Exenatide; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Lizards; Neurotransmitter Agents; Oxyntomodulin; Peptide Fragments; Peptides; Rats; Receptors, Cell Surface; Receptors, Glucagon; Venoms