oxyntomodulin and Insulinoma

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

The observations that glucagon binds to glucagon-like peptide-1 (tGLP-1) receptors have raised the question of whether glucagon receptors mediate the insulinotropic effect of glucagon. We have investigated the presence and selective activation of glucagon and tGLP-1 receptors on tumor-derived cells. Northern blot analysis detected either glucagon or tGLP-1 receptor messenger RNA in hamster (HIT) and mouse (beta TC3) beta-cell lines, respectively, whereas both receptor messenger RNA were revealed in Syrian hamster insulinoma. Their expression in insulinoma plasma membranes was confirmed by specific covalent labeling with either [125I]glucagon or [125I]tGLP-1. Both glucagon and tGLP-1 receptors showed a single class of high affinity binding sites with respective Kd values of 1.11 +/- 0.11 and 0.82 +/- 0.11 nM. [125I]tGLP binding was dose dependently inhibited with a hierarchy of exendin-4 > tGLP-1 > exendin-(9-39) > oxyntomodulin > glucagon. [125I]Glucagon binding was only inhibited by glucagon and oxyntomodulin. Both glucagon and tGLP-1 increased cAMP formation in insulinoma plasma membranes in a dose-dependent manner, with ED50 values of 170.0 +/- 25.0 and 3.1 +/- 0.4 pM, respectively. Exendin-(9-39), a tGLP-1 receptor antagonist, inhibited tGLP-1-induced, but not glucagon-induced, cAMP formation. Our data demonstrate on hamster insulinoma the presence of high affinity glucagon and tGLP-1 receptors selectively coupled to adenylyl cyclase. The observed low affinity of tGLP-1 receptors for glucagon sustains the idea that each hormone has a direct insulinotropic effect.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Cell Line; Cell Membrane; Cricetinae; Cross-Linking Reagents; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Insulinoma; Islets of Langerhans; Mice; Pancreatic Neoplasms; Peptide Fragments; Peptides; Receptors, Glucagon; RNA, Messenger

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

To elucidate the mechanism of the differential processing of proglucagon, we analyzed the processing products of proglucagon in three types of rodent endocrine cells and their relation to prohormone convertases PC1 (PC3) and PC2. Proglucagon gene was transfected into AtT-20 cells and GH3 cells, which are derived from pituitary tumors. InR1-G9 cells, which are insulinoma-derived cells, express an endogenous proglucagon gene. Oxyntomodulin was the predominant processing product in AtT-20 cells, which contained abundant PC1 mRNA. In contrast, glucagon was the major product in GH3 cells, which expressed PC2 mRNA. Oxyntomodulin and glucagon were produced in equal amounts in InR1-G9 cells, which expressed both PC1 and PC2 mRNAs. These findings suggest that PC1 and PC2 preferentially cleave proglucagon into oxyntomodulin and glucagon, respectively, thus contributing to the cell-specific processing of proglucagon.

Topics: Animals; Aspartic Acid Endopeptidases; Base Sequence; Blotting, Northern; Cell Line; Chromatography, High Pressure Liquid; DNA Primers; Glucagon; Glucagon-Like Peptides; Insulinoma; Molecular Sequence Data; Oxyntomodulin; Pancreatic Neoplasms; Peptide Fragments; Pituitary Neoplasms; Polymerase Chain Reaction; Proglucagon; Proprotein Convertase 2; Proprotein Convertases; Protein Precursors; Protein Processing, Post-Translational; Rats; RNA, Messenger; Subtilisins; Transfection

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

The interactions of glucagon-like peptide-I(7-37)/(7-36)amide (GLP-I) and somatostatin-14 were characterized on the cyclic adenosine monophosphate (cAMP)-dependent signal transduction pathway and on proinsulin gene expression using mouse insulinoma beta TC-1 cells. GLP-I stimulated the activity of adenylate cyclase maximally at 1 mumol/L (151%). This effect was inhibited by 1 mumol/L somatostatin (119%). Forskolin also stimulated adenylate cyclase activity (10 mumol/L forskolin, 265%), and this action was inhibited by somatostatin (220%). Somatostatin alone left the basal adenylate cyclase activity unaltered. Somatostatin reduced the GLP-I-stimulated increase of intracellular cAMP levels (100 nmol/L GLP-I, 141%; 100 nmol/L GLP-I + 1 mumol/L somatostatin, 110%). GLP-I stimulated concentration-dependently the activity of protein kinase A (PKA), with a maximum at 10 nmol/L (181%). This action was inhibited by 100 nmol/L somatostatin (118%), but somatostatin did not influence the basal PKA activity. Furthermore, somatostatin reduced the GLP-I-induced stimulation of proinsulin gene expression (10 nmol/L GLP-I, 176%; 10 nmol/L GLP-I + 1 mumol/L somatostatin, 77%). Somatostatin itself inhibited concentration-dependently proinsulin gene expression (1 mumol/L somatostatin, 53%). These data demonstrate that GLP-I increases the activities of both adenylate cyclase and cAMP-dependent PKA, whereas somatostatin counteracts the stimulatory effect of GLP-I on adenylate cyclase activity, cAMP generation, PKA activity, and proinsulin gene expression. The interaction of both hormones occurs at the level of adenylate cyclase. Therefore, the interaction of both peptide hormones regulates downstream events, including gene expression.

Topics: Adenylyl Cyclases; Animals; Colforsin; Cyclic AMP; Drug Interactions; Gene Expression; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Insulinoma; Mice; Pancreatic Neoplasms; Peptides; Proinsulin; Receptors, Cell Surface; Receptors, Glucagon; Signal Transduction; Somatostatin; Tumor Cells, Cultured

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

PTH-related protein (PTHrP), originally identified through its causative role in human humoral hypercalcemia of malignancy, is now known to be a normal gene product expressed in a wide variety of neuroendocrine, epithelial, and mesoderm-derived tissues. PTHrP gene expression has recently been demonstrated in fetal and adult, benign and malignant, as well as human and rodent pancreatic islets. As in other tissues, the role of PTHrP expression in the normal islet is only beginning to be explored. In the current report, PTHrP expression in the normal rat pancreatic islet was confirmed using an affinity-purified antiserum directed against the N-terminal, biologically active region of the molecule. The effects of PTHrP on the islet were then explored using rat insulinoma (RIN m5F) cells. Synthetic PTHrP-(1-36) bound specifically, but with low affinity (Kd, approximately 10(-7) M) to RIN cell membranes. PTHrP-(1-36) failed to stimulate cAMP production in RIN cells, although RIN cells displayed a normal adenylate cyclase response to glucagon-like peptide-1-(7-36). In contrast, PTHrP-(1-36) induced a rapid dose-dependent rise in intracellular calcium in RIN cells in doses as low as 10(-12)-10(-10) M. These findings 1) confirm that PTHrP is expressed by islet cells, 2) demonstrate that the effects of PTHrP on the pancreatic islet are mediated, as in keratinocytes and lymphocytes, by a receptor related to but distinct from the PTH receptor, and 3) suggest that PTHrP functions in the islet as an autocrine or paracrine factor. Further studies are required to determine the physiological consequences of PTHrP expression by the pancreatic islet.

Topics: Animals; Binding Sites; Calcium; Cell Membrane; Cyclic AMP; Cytosol; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Glyburide; Insulinoma; Kinetics; Pancreatic Neoplasms; Parathyroid Hormone; Parathyroid Hormone-Related Protein; Peptide Fragments; Peptides; Proteins; Rats; Rats, Inbred WF; Tumor Cells, Cultured

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

Glucagon-like peptide-I(7-37) [GLP-I(7-37)] is an intestinal peptide hormone that is released in response to oral nutrients and that potently augments glucose-mediated insulin secretion. GLP-I(7-37) has potent insulin-releasing activities in vivo in response to oral nutrients, in situ in the isolated perfused pancreas, and in vitro in cultured pancreatic B-cells. As such GLP-I(7-37) is a potent hormonal mediator in the enteroinsular axis involved in the regulation of glucose homeostasis. We now show that in addition to stimulating the release of insulin, GLP-I(7-37) stimulates proinsulin gene expression at the levels of gene transcription and cellular levels of proinsulin messenger RNA as well as the translational biosynthesis of proinsulin. These findings of the positive anabolic actions of GLP-I(7-37) on the synthesis of insulin in B-cells support the notion that GLP-I(7-37) may be of therapeutic use in stimulating the production of insulin in patients with noninsulin-dependent diabetes mellitus and that overproduction of insulin with subsequent hypoglycemia will not occur in response to the administration of GLP-I(7-37). Furthermore, these positive actions of GLP-I(7-37) on insulin production obviate the possibility of B-cell exhaustion in response to such a potent secretagogue.

Topics: Animals; Chloramphenicol O-Acetyltransferase; Cyclic AMP; Diabetes Mellitus, Type 2; Gene Expression; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Insulin; Insulin Secretion; Insulinoma; Mice; Pancreatic Neoplasms; Peptide Fragments; Peptides; Proinsulin; Promoter Regions, Genetic; RNA, Messenger; Tumor Cells, Cultured

Glucagon-like peptide-1 (7-36)amide (GLP-1 (7-36)amide) represents a physiologically important incretin in mammals including man. Receptors for GLP-1 (7-36)amide have been described in RINm5F cells. We have solubilized active GLP-1(7-36)amide receptors from RINm5F cell membranes utilizing the detergents octyl-beta-glucoside and CHAPS; Triton X-100 and Lubrol PX were ineffective. Binding of radiolabeled GLP-1(7-36)amide to the solubilized receptor was inhibited concentration-dependently by addition of unlabeled peptide. Scatchard analysis of binding data revealed a single class of binding sites with Kd = 0.26 +/- 0.03 nM and Bmax = 65.4 +/- 21.24 fmol/mg of protein for the membrane-bound receptor and Kd = 22.54 +/- 4.42 microM and Bmax = 3.9 +/- 0.79 pmol/mg of protein for the solubilized receptor. The binding of the radiolabel to the solubilized receptor was dependent both on the concentrations of mono- and divalent cations and the protein/detergent ratio in the incubation buffer. The membrane bound receptor is sensitive to guanine-nucleotides, however neither GTP-gamma-S nor GDP-beta-S affected binding of labeled peptide to solubilized receptor. These data indicate that the solubilized receptor may have lost association with its G-protein. In conclusion, the here presented protocol allows solubilization of the GLP-1(7-36)amide receptor in a functional state, and opens up the possibility for further molecular characterization of the receptor protein.

Topics: Animals; Cations; Detergents; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Insulinoma; Pancreatic Neoplasms; Peptide Fragments; Peptides; Protein Binding; Rats; Receptors, Cell Surface; Receptors, Glucagon; Solubility; Thionucleotides; Tumor Cells, Cultured

Oxyntomodulin (OXM), a glucagon-containing peptide extended at its C-terminal end by an octapeptide, is a potent inhibitor of gastric acid secretion in rat and man. OXM appears to act on gastric mucosa at least partially through a stimulation of gastric somatostatin release. We have investigated the effects of OXM on a somatostatin-secreting cell line (RIN T3) derived from a radiation-induced rat insulinoma and characterized specific binding sites for this peptide. OXM increased somatostatin release with an ED50 of 2.3 nM. OXM also stimulated the cAMP accumulation in intact RIN T3 cells and adenylate cyclase activity in RIN T3 cell membranes with ED50 values of 0.5 and 11 nM, respectively. On these parameters, glucagon was 10-30 times less potent than OXM. Forskolin, isobutylmethylxanthine, and 8-bromo-cAMP mimicked the effect of OXM on somatostatin release. Specific binding for mono-[125I]OXM was dependent upon time and membrane concentration. Binding of mono-[125I]OXM was inhibited by OXM and glucagon in a concentration-dependent manner, with dissociation constants (Kd) of 4.5 and 43 nM, respectively. The nonhydrolyzable analogs of GTP (guanosine 5',3-O-(thio)triphosphate and guanosine 5' (beta,gamma-imino)triphosphate decreased the binding of mono-[125I]OXM to its binding sites. Covalent cross-linking of mono-[125I]OXM or mono-[125I]glucagon to RIN T3 cell membranes followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated a single radiolabeled band at 63,000 mol wt, which differed from that observed after cross-linking with liver plasma membranes (55,000 mol wt). These results demonstrate the presence of specific high affinity binding sites for OXM in a somatostatin-secreting cell line (RIN T3) and their coupling to adenylate cyclase via guanine nucleotide-binding proteins.

Topics: 1-Methyl-3-isobutylxanthine; 8-Bromo Cyclic Adenosine Monophosphate; Adenylyl Cyclases; Animals; Binding Sites; Colforsin; Cyclic AMP; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Glucagon; Glucagon-Like Peptides; Insulinoma; Iodine Radioisotopes; Oxyntomodulin; Rats; Somatostatin; Tumor Cells, Cultured

The neuropeptide hormone galanin, released by sympathetic stimulation of nerve terminals in the endocrine pancreas, inhibits insulin secretion via a receptor-linked pertussis toxin-sensitive (Gi) transmembrane signaling pathway. Glucagon-like peptide-I(7-37) [GLP-I(7-37)] is an intestinal hormone shown to have potent insulin-releasing activities in pancreatic B-cells and is believed to serve a physiological role in the augmentation of nutrient-induced insulin release. GLP-I(7-37) binds to specific Gs- and adenylate cyclase-coupled receptors on pancreatic B-cells and directly stimulates proinsulin gene transcription, thereby increasing cellular levels of proinsulin messenger RNA (mRNA) and proinsulin biosynthesis. This study examines the effects of galanin on GLP-I(7-37)-stimulated proinsulin gene expression in mouse beta TC1 cells. The degree of proinsulin gene transcription was assessed by measuring the activity of chloramphenicol acetyl transferase (CAT) expressed from a CAT reporter plasmid linked to the rat insulin-1 gene promoter transferred to beta TC1 cells and by measuring proinsulin mRNA levels by Northern blot analysis. Galanin inhibited both CAT activity and the rise in proinsulin mRNA levels stimulated by either GLP-I(7-37) or forskolin (0.1 microM). Notably, galanin was without effect on CAT activity induced by the cAMP analog, 8-bromo-cAMP, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, or higher concentrations of forskolin. The inhibitory effects of galanin on GLP-I(7-37) and forskolin-induced CAT activity were reversed by the addition of pertussis toxin, a toxin that inactivates inhibitory G-proteins (Gi). We conclude that galanin inhibits GLP-I(7-37)-stimulated proinsulin gene expression by inhibiting the activation of adenylate cyclase by GLP-I(7-37) and subsequently the production of cAMP in B-cells. Further, our data suggest that these actions of galanin are mediated by a pertussis toxin sensitive pathway involving one or more Gis that inhibit adenylate cyclase. Thus, in addition to its well known inhibitory effects on insulin secretion galanin can inhibit proinsulin gene expression stimulated by GLP-I(7-37) activation of the cAMP signaling pathway. These findings may be a unique demonstration of the inhibition of proinsulin gene expression by a substance (galanin) released endogenously within the pancreas.

Topics: Adenylate Cyclase Toxin; Animals; Blotting, Northern; Cell Line; Chloramphenicol O-Acetyltransferase; Colforsin; Galanin; Gene Expression; Gene Expression Regulation, Neoplastic; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Insulinoma; Kinetics; Mice; Models, Biological; Pancreatic Neoplasms; Peptide Fragments; Peptides; Pertussis Toxin; Proinsulin; RNA, Neoplasm; Transfection; Virulence Factors, Bordetella

We previously reported that GLP-1(7-36)amide had glucagonostatic action as well as insulinotropic action in the perfused rat pancreas. In this study, we examined the effect of GLP-1(7-36)amide on glucagon secretion and cAMP concentration in glucagon-secreting cell line, In-R1-G9. GLP-1(7-36)amide (1nM) significantly suppressed glucagon secretion and decreased cAMP concentration in the cells. GLP-1(1-37) did not affect glucagon secretion. It is suggested that inhibitory effect of GLP-1(7-36)amide on glucagon secretion is at least partly mediated by adenylate cyclase system.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Clone Cells; Cricetinae; Cyclic AMP; Dose-Response Relationship, Drug; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Insulinoma; Kinetics; Pancreatic Neoplasms; Peptide Fragments; Peptides

We studied the effects of oxyntomodulin (OXM), of its C-terminal (19-37) fragment (OXM (19-37)) and of glucagon (GLU) on somatostatin release, cyclic AMP accumulation and inositol phosphate turnover in somatostatin-secreting RIN T3 cells in culture. Rapid changes in cellular free Ca2+ were also measured using fura-2. Carbachol was used as a control test agent for the parameters involving the inositol phosphate/Ca2+ cascade. OXM, GLU and OXM (19-37) were all able to stimulate somatostatin release with relative ED50 of approx. 1, 22 and 45, respectively. OXM and GLU stimulated cyclic AMP levels with relative ED50 of approx. 1 and 30, respectively, whereas OXM (19-37) was totally ineffective on this parameter. In contrast to carbachol, none of the peptides significantly modified the inositol phosphate turnover or induced rapid changes in cellular free Ca2+. We conclude that the RIN T3 cells contain a receptor-cyclic AMP system similar to that found in gastric mucosa and that this system is linked to somatostatin release. Another receptor-second messenger mechanism linked to somatostatin release is triggered by the (19-37) fragment. This mechanism is not the inositol phosphate/Ca2+ cascade triggered in the same cells by cholinergic agents.

Topics: Animals; Calcium; Carbachol; Cyclic AMP; Dose-Response Relationship, Drug; Glucagon; Glucagon-Like Peptides; In Vitro Techniques; Inositol Phosphates; Insulinoma; Oxyntomodulin; Peptide Fragments; Rats; Secretory Rate; Somatostatin; Time Factors; Tumor Cells, Cultured

Glucagon-like peptide 1-(7-37) [GLP-I-(7-37)] is a 31-amino acid hormone which may have an important role in the regulation of insulin secretion, It is processed from preproglucagon and found in the pancreas, brain, and, in highest quantity, intestine. In previous studies we found that GLP-I-(7-37) is a potent insulin secretagogue, and its effect was indistinguishable from that of GLP-I-(7-36) amide at concentrations of 10(-11) M. Herein we report insulinotropic effects of additional GLP-I analogs. GLP-I-(7-34) had no stimulatory effect on insulin release at 10(-10) M, but had a partial effect at 10(-9) M and was as active as GLP-I-(7-37) at 10(-8) M. GLP-I-(7-33) had no effect at any concentration tested. GLP-I-(8-37) caused no significant effect on insulin release at 10(-9) and 10(-8) M, but did have an effect at the high concentration of 10(-7) M. Similar results were found with cAMP formation in the beta TC1 line. In this system GLP-I-(7-34) was less potent than GLP-I-(7-37) at a concentration of 5 x 10(-9) M. GLP-I-(7-33) had only about 0.1% the potency of GLP-I-(7-37); thus, there is good agreement between cAMP formation in the beta-cell line and insulin secretion from the perfused pancreas experiments. We conclude that histidine in the 7 position in the N-terminus of GLP-I-(7-37) is crucial for cAMP formation and insulin secretion, and that removal of the last three C-terminus residues of GLP-I-(7-37) results in only partial loss of activity; the residue in the 34 position is, however, essential for the insulinotropic action.

Topics: Animals; Cell Line; Cyclic AMP; Dose-Response Relationship, Drug; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Insulin; Insulin Secretion; Insulinoma; Male; Pancreas; Peptide Fragments; Peptides; Rats; Rats, Inbred Strains

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

125I-Labelled glucagon-like peptide-1(7-36)amide was cross-linked to a specific binding protein in plasma membranes prepared from RINm5F rat insulinoma-derived cells using disuccinimidyl suberate. Consistent with the presence of a single class of binding site on the surface of intact cells, only a single radiolabelled band at Mr63,000 was identified by SDS-PAGE after solubilization of the ligand-binding protein complex. The band was not observed when 10nM glucagon-like peptide-1(7-36)amide was included in the binding assay, but 1 microM concentrations of glucagon-like peptide-1(1-36)amide, glucagon-like peptide-2 and glucagon did not decrease the intensity of labelling. No change in the mobility of the band was observed under reducing conditions, suggesting that the binding protein in the receptor is not attached to other subunits via disulphide bonds. In control incubations using plasma membranes from pig intestinal epithelial cells, which do not contain specific binding sites for glucagon-like peptide-1(7-36)amide, no cross-linked ligand-binding protein complex was observed.

Topics: Adenoma, Islet Cell; Animals; Cell Line; Cell Membrane; Cross-Linking Reagents; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Insulinoma; Peptides; Rats; Receptors, Cell Surface; Receptors, Glucagon

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] is supposed to be an important physiologic incretin. Recently, high affinity receptors for GLP-1(7-36)amide have been demonstrated on rat insulinoma-derived RINm5F cells. The present study examined the internalization and degradation of the GLP-1-receptor complex. Internalization of the peptide was time- and temperature-dependent. At 37 degrees C binding and internalization was rapid. At 60 min 35% of 125I-labeled GLP-1(7-36)amide was internalized. Incubation in the presence of increasing concentrations of non-labeled GLP-1(7-36)amide resulted in a decrease of internalization of 125I-labeled peptide indicating that this process is saturable. Incubation in the presence of 0.2 mM chloroquine, an inhibitor of intracellular hormone degradation, resulted in intracellular accumulation of 125I-GLP-1(7-36)amide. HPLC-supported analysis of cell content after internalization of 125I-GLP-1(7-36)amide during a 60-min incubation period at 37 degrees C revealed an elution profile showing two maxima of radioactivity: one represented intact labeled GLP-1(7-36)amide, the other an intracellular degradation product of the peptide. Chloroquine caused a 5-fold increase of the peak representing intact 125I-GLP-1(7-36)amide thus demonstrating inhibition of degradation of labelled peptide. Furthermore, a 4-fold increase of the other peak occurred possibly mirroring a delay of release of degradation products by chloroquine. It was excluded that chloroquine is able to interfere with GLP-1(7-36)amide-binding to its receptor.

Topics: Adenoma, Islet Cell; Animals; Chloroquine; Chromatography, High Pressure Liquid; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Insulinoma; Kinetics; Pancreatic Neoplasms; Peptides; Rats; Receptors, Cell Surface; Receptors, Glucagon; Temperature; 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

Topics: APUD Cells; Apudoma; Gastric Inhibitory Polypeptide; Gastrins; Glucagon; Glucagon-Like Peptides; Humans; Insulin; Insulinoma; Motilin; Pancreatic Neoplasms; Secretin