glucagon-like-peptide-1-(7-36)amide and Insulinoma

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-(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

Glucagon-like peptide (7-36) amide (GLP-1) acutely inhibits food and water consumption in rats after intracerebroventricular (icv) administration. To assess the potential for desensitization of these effects, we investigated the effects of chronic icv administration of GLP-1 on food consumption and body weight in Sprague-Dawley (SD) rats and Zucker (fa/fa) obese rats. In vitro functional densensitization of the GLP-1 receptor was not observed after overnight exposure of Rin m5F insulinoma cells to GLP-1 at concentrations up to 10 nM. Administration of GLP-1 to SD rats (30 microg icv twice a day for 6 days) resulted in significant reductions in 24-hour food consumption each day (25 +/- 1%). Continuous icv infusion of GLP-1 for 7 and 14 days significantly inhibited cumulative food consumption and reduced body weight in SD rats. In the genetically obese Zucker rat, chronic dosing with GLP-1 (30 microg icv) once a day for 6 days caused significant reductions in food consumption each day and a reduction in body weight. These results indicate that the GLP-1 pathways in the central nervous system controlling food consumption do not desensitize after chronic exposure to GLP-1 and suggest that agonists of the central GLP-1 receptor may be effective agents for the treatment of obesity.

Topics: Animals; Body Weight; Eating; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Injections, Intraventricular; Insulinoma; Male; Neurotransmitter Agents; Obesity; Pancreatic Neoplasms; Peptide Fragments; Rats; Rats, Sprague-Dawley; Rats, Zucker; Receptors, Glucagon; Tumor Cells, Cultured

Central infusion of glucagon-like peptide-1-(7-36) amide (GLP-1) and intraperitoneal (i.p.) injection of lithium chloride (LiCl) produce similar patterns of c-Fos induction in the rat brain. These similarities led us to assess the hypothesis that neuronal activity caused by i.p. injection of LiCl involves activation of central GLP-1 pathways. We therefore determined if third-ventricular (i3vt) infusion of a GLP-1 receptor antagonist would block LiCl-induced c-Fos expression in the brainstem. Relative to rats pretreated with i3vt infusion of vehicle, pretreatment with the potent GLP-1 receptor antagonist, des His1 Glu9 exendin-4 (10.0 microgram), significantly attenuated LiCl-induced (76 mg/kg; i.p.) c-Fos expression in several brainstem regions, including the area postrema, the nucleus of the solitary tract, and the lateral parabrachial nucleus. While central infusion of des His1 Glu9 exendin-4 also blocked GLP-1-induced (10.0 microgram) anorexia and c-Fos expression, the antagonist produced no independent effects on food intake or c-Fos expression. These results suggest that LiCl-induced c-Fos expression in the rat brainstem is mediated, at least in part, by GLP-1 receptor signaling.

Topics: Animals; Brain Stem; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Guinea Pigs; Infusions, Parenteral; Injections, Intraperitoneal; Injections, Intraventricular; Insulin; Insulin Secretion; Insulinoma; Lithium Chloride; Male; Neurons; Peptide Fragments; Proto-Oncogene Proteins c-fos; Rats; Rats, Long-Evans; Receptors, Glucagon; Signal Transduction; Tumor Cells, Cultured

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is abundant in mammalian serum, but the source of the circulating enzyme is unknown. Pancreatic islets have been reported to contain and secrete GPI-PLD. In this report we examined the regulation of GPI-PLD secretion from beta TC3 cells, a mouse insulinoma cell line. In the absence of glucose, phorbol myristic acid (0.1 microM) stimulated insulin secretion by 2.5-fold and GPI-PLD secretion by 2-fold. Carbachol (5 microM), glucagon-like peptide I-(7-36) amide (0.1 microM), and isobutylmethylxanthine (0.1 mM) had no significant effect on insulin or GPI-PLD secretion in the absence of glucose. Glucose (16.7 mM) stimulated both GPI-PLD and insulin secretion from beta TC3 cells by 55% and 235%, respectively. In addition, glucose potentiated the secretagogue effect of isobutylmethylxanthine, phorbol myristic acid, and glucagon-like peptide I on both insulin and GPI-PLD secretion. By immunohistochemistry and confocal microscopy, beta TC3 cells contain both insulin and GPI-PLD, which generally colocalized intracellularly. However, GPI-PLD secretion differed from insulin secretion by a higher rate of basal release (2.8% vs. 0.23%/h), a lower magnitude of response to secretagogues, and a more prolonged period of increased secretion. These results demonstrate that beta TC3 cells secrete GPI-PLD in response to insulin secretagogues and suggest that GPI-PLD may be secreted via the regulated pathway in these cells.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Carbachol; Cycloheximide; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Glucose; Immunohistochemistry; Insulin; Insulin Secretion; Insulinoma; Islets of Langerhans; Kinetics; Mice; Pancreatic Neoplasms; Peptide Fragments; Phospholipase D; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

This study concerns whether the pancreatic beta cell expresses cell-surface ectopeptidases that are capable of proteolysis of peptide hormones and neuropeptides that modify glucose-dependent insulin release. These biochemical investigations of the RINm5F cell line found that these cells express ectopeptidases. We have characterized the limited endoproteolysis of GLP-1 (7-36) amide that occurs in the presence of RINm5F plasma membranes. The products and the sensitivity to specific peptidase inhibitors of the proteolysis is characteristic of neutral endopeptidase (NEP) 24.11. Vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), amylin, glucagon, glucose-dependent insulinotropic polypeptide (GIP), and exendin-4 also undergo proteolysis in the presence of RIN cell membranes. NEP 24.11-activity in RIN cell membranes was confirmed using a specific fluorogenic assay, by histochemistry, and by comparison with the recombinant enzyme with respect to the kinetics of proteolysis of GLP-1 (7-36) amide and of a fluorogenic substrate. Specific fluorogenic assays revealed the presence of aminopeptidase N and the absence of aminopeptidase A and of dipeptidylpeptidase IV.

Topics: Amino Acid Sequence; Animals; Cell Membrane; Gastrointestinal Hormones; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Hydrolysis; Insulinoma; Peptide Fragments; Peptide Hydrolases; Rats; Tumor Cells, Cultured

The glucagon-like peptide 1 (7-36) amide (GLP-1) receptor mediates the insulinotropic effects of the incretin hormone GLP-1. To elucidate the tissue-specific regulation of the GLP-1 receptor we screened a human genomic library with a human GLP-1 receptor cDNA. The gene spans 40 kb and consists of at least seven exons. The promoter contained no TATA- or CAAT-boxes, but several other putative cis-regulatory recognition sequences including three Sp1 binding sites. Transient transfections of GLP-1 receptor producing and non-producing cells with promoter/ reporter gene constructs revealed that the putative Sp1 binding sites and several other silencer and tissue specific elements are important for the activity. Therefore, 3000 bp upstream the GLP-1 receptor coding sequences comprise regulatory elements essential for the tissue- and cell-specific transcription of the gene.

Topics: Base Sequence; Cloning, Molecular; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Humans; Insulinoma; Molecular Sequence Data; Peptide Fragments; Promoter Regions, Genetic; Receptors, Glucagon; Sequence Analysis, DNA; Transcription, Genetic; Tumor Cells, Cultured

Topics: 1-Methyl-3-isobutylxanthine; Animals; Calcium; Cytoplasm; Fura-2; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Insulin; Insulinoma; Islets of Langerhans; Male; Neurotransmitter Agents; Peptide Fragments; Phosphodiesterase Inhibitors; Protein Precursors; Rats; Rats, Sprague-Dawley; Sincalide; Tumor Cells, Cultured

In the insulin-secreting beta-cell line beta TC3, stimulation with 11.2 mmol/l glucose caused a rise in the intracellular free Ca2+ concentration ([Ca2+]i) in only 18% of the tested cells. The number of glucose-responsive cells increased after pretreatment of the cells with glucagon-like peptide I (GLP-I)(7-36)amide and at 10(-11) mol/l; 84% of the cells responded to glucose with a rise in [Ca2+]i. GLP-I(7-36)amide induces a rapid increase in [Ca2+]i only in cells exposed to elevated glucose concentrations (> or = 5.6 mmol/l). The action of GLP-I(7-36)amide and forskolin involved a 10-fold increase in cytoplasmic cAMP concentration and was mediated by activation of protein kinase A. It was not associated with an effect on the membrane potential but required some (small) initial entry of Ca2+ through voltage-dependent L-type Ca2+ channels, which then produced a further increase in [Ca2+]i by mobilization from intracellular stores. The latter effect reflected Ca(2+)-induced Ca2+ release and was blocked by ryanodine. Similar increases in [Ca2+]i were also observed in voltage-clamped cells, although there was neither activation of a background (Ca(2+)-permeable) inward current nor enhancement of the voltage-dependent L-type Ca2+ current. These observations are consistent with GLP-I(7-36) amide inducing glucose sensitivity by promoting mobilization of Ca2+ from intracellular stores. We propose that this novel action of GLP-I(7-36)amide represents an important factor contributing to its insulinotropic action.

Topics: Animals; Calcium; Calcium Channels; Cell Line; Colforsin; Cyclic AMP; Cytoplasm; Dose-Response Relationship, Drug; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Glucose; Inositol Phosphates; Insulin; Insulin Secretion; Insulinoma; Islets of Langerhans; Kinetics; Mice; Mice, Transgenic; Pancreatic Neoplasms; Peptide Fragments; Ryanodine; Time Factors; Tumor Cells, Cultured

GLP-1 (glucagon-like peptide 1 (7-36) amide) plays an important role in the regulation of insulin secretion and proinsulin gene expression of pancreatic beta-cells. Patients with insulinoma tumors show uncontrolled insulin hypersecretion. This study demonstrates the molecular cloning of a cDNA for the GLP-1 receptor from a human insulinoma employing a lambda-gt11 cDNA library. The cloned cDNA encoded a seven transmembrane domain protein of 463 amino acids which showed high homology to the GLP-1 receptor in normal human pancreas. Four amino acid exchanges were found in comparison to a receptor sequence obtained from regular pancreatic islets. When transfected transiently into COS-7 or stably into fibroblast CHL cells a high affinity receptor was expressed which coupled to the adenylate cyclase with normal basal cAMP and increasing intracellular cAMP levels under GLP-1 stimulation. The receptor accepted GLP-1 and the non-mammalian agonist exendin-4 as high affinity ligands. In transfected COS-7 cells, GLP-1 did not influence intracellular calcium, whereas in the stably transfected fibroblasts GLP-1 transiently increased intracellular calcium to a small extent. The understanding of GLP-1 receptor regulation and signal transduction will aid in the discovery of compounds that act as agonists of the GLP-1 receptor for potential use in the treatment of diabetes and will facilitate the understanding of its expression under normal and pathophysiological conditions.

Topics: Amino Acid Sequence; Animals; Blotting, Northern; Calcium; Cell Line; Cloning, Molecular; Cyclic AMP; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Humans; Insulinoma; Molecular Sequence Data; Peptide Fragments; Rats; Receptors, Glucagon; Recombinant Proteins; RNA; Sequence Homology, Amino Acid; Signal Transduction; Transfection

125I-glucagon-like peptide 1(7-36)amide was covalently cross-linked to a specific binding protein in human insulinoma cell membranes. A single radiolabeled band at M(r) 63,000 was identified by SDS-PAGE after solubilization of the ligand-binding protein complex. The molecular weight of this apparent GLP-1 receptor in human endocrine pancreatic tissue was of identical size as the GLP-1 receptor on rat insulinoma-derived RINm5F cell membranes. The radiolabeled band was undetectable when 1 microM of unlabeled GLP-1(7-36)amide or of the GLP-1 antagonist exendin(9-39)amide was included in the binding assay. Utilizing isolated poly-A+ RNA from the human insulinoma and a 1,500 bp Eco-RI fragment of the cDNA coding for the rat GLP-1(7-36)amide receptor for Northern blot analysis, a main hybridization signal at about 7 kb was found by Northern blotting. Our data provide the first direct evidence of the existence of GLP-1 receptors in human endocrine pancreatic tissue.

Topics: Animals; Cell Membrane; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Humans; Insulinoma; Islets of Langerhans; Pancreatic Neoplasms; Peptide Fragments; Rats; Receptors, Cell Surface; Receptors, Glucagon; Tumor Cells, Cultured

To examine the structure-activity relationships in the insulinotropic activity of glucagon-like peptide-1(7-36) amide (GLP-1(7-36)amide), we synthesized 16 analogues, including eight which were designed by amino acid substitutions at positions 10 (Alal0), 15 (Serl5), 16 (Try16), 17 (Arg17), 18 (Lys18), 21 (Gly21), 27 (Lys27) and 31 (Asp31) of GLP-1(7-36)amide with an amino acid of GH-releasing factor possessing only slight insulinotropic activity, and three tentative antagonists including [Glu15]-GLP-1(8-36)amide. Their insulinotropic activities were assessed by rat pancreas perfusion experiments, and binding affinity to GLP-1 receptors and stimulation of cyclic AMP (cAMP) production were evaluated using cultured RINm5F cells. Insulinotropic activity was estimated as GLP-1(7-36)amide = Tyr16 > Lys18, Lys27 > Gly21 > Asp31 >> Ser15, Arg17 > Ala10 >> GRF > [Glu15]-GLP-1(8-36) amide. Displacement activity against 125I-labelled GLP-1(7-36)amide binding and stimulatory activity for cAMP production in RINm5F cells correlated well with their insulinotropic activity in perfused rat pancreases. These results demonstrate that (1) positions 10 (glycine), 15 (aspartic acid) and 17 (serine) in the amino acid sequence of GLP-1(7-36)amide, in addition to the N-terminal histidine, are essential for its insulinotropic activity through its binding to the receptor, (2) the amino acid sequences for the C-terminal half of GLP-1(7-36)amide also contribute to its binding to the receptor, although they are less important compared with those of the N-terminal half, and (3) [Glu15]-GLP-1(8-36)amide is not an antagonist of GLP-1(7-36)amide as opposed to des-His1 [Glu9]-glucagon amide which is a potent glucagon antagonist.

Topics: Animals; Cell Line; Cyclic AMP; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Insulin; Insulinoma; Male; Pancreas; Peptide Fragments; Perfusion; Rats; Rats, Wistar; Receptors, Cell Surface; Receptors, Glucagon; Stimulation, Chemical; Structure-Activity Relationship

We investigated the effect of amylin on the glucagon-like peptide-1(7-36)amide (GLP-1(7-36)amide) induced stimulation of cAMP production in RINm5F cells. Amylin and the structurally related calcitonin gene-related peptide (CGRP) inhibited the stimulatory effect of GLP-1(7-36)amide on cAMP generation while substance P was without effect. Amylin had no effect on the forskolin-induced cAMP-generation. These findings suggest that amylin alters the biological action of the incretin hormone GLP-1(7-36)amide. This could at least partly contribute to an amylin-induced impaired glucose tolerance which has been previously observed.

Topics: Adenylyl Cyclases; Amyloid; Animals; Cell Line; Colforsin; Cyclic AMP; Gastrointestinal Hormones; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Glucose Intolerance; Insulinoma; Islet Amyloid Polypeptide; Islets of Langerhans; Models, Biological; Peptide Fragments; Peptides; Rats; Substance P

Neuropeptide-Y (NPY) is a 36-amino acid peptide known to inhibit glucose-stimulated insulin secretion in various animal models in vitro and in vivo. NPY is thought to be one of the mediators of sympathetic action in the pancreas through nerve endings surrounding the islets, and it has recently been shown to be synthesized within the islets of Langerhans. To elucidate the potential role of NPY in the endocrine pancreas, we studied the expression and regulation of NPY secretion in a rat insulinoma cell line (INS-1). NPY mRNA and peptide are highly expressed and secreted by INS-1 cells. NPY levels were determined by a sensitive and specific two-site amplified enzyme-linked immunosorbent assay. Incubation of INS-1 cells with various glucose concentrations did not modify NPY secretion; however, stimulation of adenylate cyclase by forskolin induced a dose- and time-dependent increase in NPY release in the medium. The glucagon-like peptide-I-(7-36) amide (GLP-1), a known gluco-incretin in humans, induced at low concentration (10(-9) M) a similar expression of NPY mRNA and peptide secretion in INS-1 cells. On the other hand, the inhibition of cAMP accumulation by the alpha 2-adrenergic agonist clonidine decreased NPY secretion. In conclusion, 1) high levels of gene expression and secretion of NPY are found in a rat insulinoma cell line (INS-1). 2) Accumulation of cAMP induced by forskolin or a gluco-incretin (GLP-1) induces a further increase in NPY gene expression and release. 3) NPY secretion is not modulated by low or high glucose concentrations in the medium. 4) Induction of NPY, a known inhibitor of insulin secretion, may represent a novel counterregulatory mechanism of insulin secretion, limiting the stimulatory effect of GLP-1 on insulin secretion.

Topics: Adenylyl Cyclases; Animals; Cell Differentiation; Clonidine; Colforsin; Cyclic AMP; Enzyme Activation; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Neoplastic; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Insulin; Insulin Secretion; Insulinoma; Neuropeptide Y; Pancreatic Neoplasms; Peptide Fragments; Protein Kinase C; Rats; Receptors, Neuropeptide Y; Tumor Cells, Cultured

Exendin-4 purified from Heloderma suspectum venom shows structural relationship to the important incretin hormone glucagon-like peptide 1-(7-36)-amide (GLP-1). We demonstrate that exendin-4 and truncated exendin-(9-39)-amide specifically interact with the GLP-1 receptor on insulinoma-derived cells and on lung membranes. Exendin-4 displaced 125I-GLP-1, and unlabeled GLP-1 displaced 125I-exendin-4 from the binding site at rat insulinoma-derived RINm5F cells. Exendin-4 had, like GLP-1, a pronounced effect on intracellular cAMP generation, which was reduced by exendin-(9-39)-amide. When combined, GLP-1 and exendin-4 showed additive action on cAMP. They each competed with the radio-labeled version of the other peptide in cross-linking experiments. The apparent molecular mass of the respective ligand-binding protein complex was 63,000 Da. Exendin-(9-39)-amide abolished the cross-linking of both peptides. Exendin-4, like GLP-1, stimulated dose dependently the glucose-induced insulin secretion in isolated rat islets, and, in mouse insulinoma beta TC-1 cells, both peptides stimulated the proinsulin gene expression at the level of transcription. Exendin-(9-39)-amide reduced these effects. In conclusion, exendin-4 is an agonist and exendin-(9-39)-amide is a specific GLP-1 receptor antagonist.

Topics: Animals; Binding, Competitive; Cell Line; Cell Membrane; Cyclic AMP; Exenatide; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Insulin; Insulin Secretion; Insulinoma; Kinetics; Lizards; Lung; Pancreatic Neoplasms; Peptide Fragments; Peptides; Rats; Receptors, Cell Surface; Receptors, Glucagon; Tumor Cells, Cultured; Venoms

The effect of dexamethasone on binding of glucagon-like peptide-1(7-36)amide (GLP-1(7-36)amide) to rat insulinoma-derived cells (RINm5F) was investigated. Preincubation of RINm5F cells with dexamethasone (100 nmol/l) for 24 h resulted in a decrease of GLP-1(7-36)amide binding to 55.0 +/- 8.16% (mean +/- S.E.M.), incubation for 48 h to 39.1 +/- 1.76%, and for 72 h to 15.5 +/- 4.35% of maximal binding. The GLP-1(7-36)amide-induced stimulation of cyclic AMP (cAMP) production was significantly decreased to 61.03 +/- 7.4% of maximum production in cells pretreated with dexamethasone (100 nmol/l) for 48 h. The decreased binding was due to a reduction of the receptor number while the receptor affinity remained unchanged. These inhibitory effects on binding and cAMP formation induced by dexamethasone were completely abolished when the antiglucocorticoid RU 38486 (100 nmol/l) was added during preincubation with dexamethasone. RU 38486 alone had no effects. Our data suggest that the biological action of GLP-1(7-36)amide at the B-cell may be modified by glucocorticoids.

Topics: Animals; Cyclic AMP; Dexamethasone; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Glucocorticoids; Insulinoma; Mifepristone; Pancreatic Neoplasms; Peptide Fragments; Peptides; Protein Binding; Rats; Time Factors; Tumor Cells, Cultured

Glucagon-like peptide-1(7-36)amide [GLP-1(7-36)amide], probably representing an important incretin, binds to receptors on RINm5F cells resulting in an adenosine 3',5'-cyclic monophosphate increase. Guanine nucleotides (GTP, GTP-gamma-S, GDP-beta-S) decreased the binding of GLP-1(7-36)amide to receptors on RINm5F cell membranes. Further analysis revealed that GTP (10(-4) M) decreased the receptor affinity with an increase of the Kd from 2.5 +/- 0.99 x 10(-10) M to 9.43 +/- 2.16 x 10(-10) M. In cross-linking experiments the amount of labeled peptide linked to receptors was reduced in the presence of GTP (10(-4) M). Further studies investigated the involvement of membrane depolarization or changes in the cytosolic free calcium level in the intracellular signaling of GLP-1(7-36)amide-induced insulin secretion. In contrast to fuel and nonfuel secretagogues, GLP-1(7-36)amide did not cause a depolarization of the membrane potential. This was unaffected by various glucose concentrations (0-20 mM) or by previous cell depolarization by D-glyceraldehyde. Similarly, the cytosolic calcium concentration remained unchanged after addition of GLP-1(7-36)amide (10(-12)-10(-8) M). The effect of guanine nucleotides on binding of GLP-1(7-36)amide indicates that the action of the peptide is mediated by the adenylate cyclase system. GLP-1(7-36)amide binding neither changed the membrane potential nor altered the intracellular calcium concentration, making an involvement of the inositol 1,4,5-trisphosphate pathway or an activation of protein kinase C in the postreceptor signaling after GLP-1(7-36)amide binding unlikely.

Topics: Adenoma, Islet Cell; Animals; Calcium; Cell Line; Cell Membrane; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Insulinoma; Membrane Potentials; Peptide Fragments; Peptides; Rats; Signal Transduction; Tumor Cells, Cultured

Glucagon-like peptide-1(7-36)amide [GLP-1(7-36)amide], a new important incretin candidate, binds to specific high-affinity receptors on rat insulinoma-derived beta-cells (RINm5F). In the present study, the effect of somatostatin-14 on the GLP-1(7-36)amide-induced insulin release and cAMP generation in this cell line was investigated. Somatostatin did not decrease basal insulin release of RINm5F cells. The GLP-1(7-36)amide-induced insulin release was decreased concentration dependently by somatostatin. Somatostatin, 1 microM reduced the maximally GLP-1(7-36)amide-stimulated (0.1 microM) insulin release to basal insulin levels. The GLP-1(7-36)amide-induced cAMP production was significantly decreased by somatostatin in a concentration-dependent manner. The GLP-1(7-36)amide concentration causing half-maximal cAMP production was 2.98 +/- 1.56 nM. Somatostatin left the EC50 unaltered but decreased the maximal GLP-1(7-36)amide effect for 32% in the presence of 1 nM somatostatin and for 50% at 1 microM. In additional experiments, the interaction of both hormones was evaluated in the perfused pancreas as a nontumor model. Somatostatin (1 nM, 1 microM) inhibited the glucose-induced (6.7 mM) and GLP-1(7-36)amide-potentiated (0.05, 0.5, and 5 nM) insulin release dose dependently. The biphasic pattern of insulin release remained preserved. The GLP-1(7-36)amide-induced insulin release is potently inhibited by somatostatin-14. This effect was demonstrated in different model systems for beta-cell function studies. The present data allow the conclusion that the somatostatin action upon GLP-1(7-36)amide effects is at least partly related to regulation of intracellular cyclic nucleotides.

Topics: Adenoma, Islet Cell; Animals; Cell Line; Cyclic AMP; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Insulin; Insulinoma; Male; Pancreas; Peptide Fragments; Peptides; Rats; Somatostatin; Tumor Cells, Cultured

Specific binding of 125I-labelled glucagon-like peptide-1(7-36)amide (GLP-1(7-36)amide) to rat insulinoma-derived RINm5F cells was dependent upon time and temperature and was proportional to cell concentration. Binding of radioactivity was inhibited in a concentration-dependent manner by GLP-1(7-36) amide consistent with the presence of a single class of binding site with a dissociation constant (Kd) of 204 +/- 8 pmol/l (mean +/- S.E.M.). Binding of the peptide resulted in a dose-dependent increase in cyclic AMP concentrations (half maximal response at 250 +/- 20 pmol/l). GLP-1(1-36)amide was approximately 200 times less potent than GLP-1(7-36)amide in inhibiting the binding of 125I-labelled GLP-1(7-36)amide to the cells (Kd of 45 +/- 6 nmol/l). Binding sites for GLP-1 (7-36)amide were not present on dispersed enterocytes from porcine small intestine.

Topics: Adenoma, Islet Cell; Animals; Binding, Competitive; Cell Line; Cyclic AMP; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Insulinoma; Intestinal Mucosa; Jejunum; Pancreatic Neoplasms; Peptide Fragments; Peptides; Rats; Receptors, Cell Surface; Receptors, Glucagon