glucagon-like-peptide-1 and Insulinoma

Tumor-induced hypoglycemia (TIH) is a rare clinical entity that may occur in patients with diverse kinds of tumor lineages and that may be caused by different mechanisms. These pathogenic mechanisms include the eutopic insulin secretion by a pancreatic islet β-cell tumor, and also the ectopic tumor insulin secretion by non-islet-cell tumor, such as bronchial carcinoids and gastrointestinal stromal tumors. Insulinoma is, by far, the most common tumor associated with clinical and biochemical hypoglycemia. Insulinomas are usually single, small, sporadic, and intrapancreatic benign tumors. Only 5-10% of insulinomas are malignant. Insulinoma may be associated with the multiple endocrine neoplasia type 1 in 4-6% of patients. Medical therapy with diazoxide or somatostatin analogs has been used to control hypoglycemic symptoms in patients with insulinoma, but only surgical excision by enucleation or partial pancreatectomy is curative. Other mechanisms that may, more uncommonly, account for tumor-associated hypoglycemia without excess insulin secretion are the tumor secretion of peptides capable of causing glucose consumption by different mechanisms. These are the cases of tumors producing IGF2 precursors, IGF1, somatostatin, and glucagon-like peptide 1. Tumor autoimmune hypoglycemia occurs due to the production of insulin by tumor cells or insulin receptor autoantibodies. Lastly, massive tumor burden with glucose consumption, massive tumor liver infiltration, and pituitary or adrenal glands destruction by tumor are other mechanisms for TIH in cases of large and aggressive neoplasias.

Topics: Autoantibodies; Bronchial Neoplasms; Carcinoid Tumor; Gastrointestinal Neoplasms; Gastrointestinal Stromal Tumors; Glucagon-Like Peptide 1; Humans; Hypoglycemia; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Insulinoma; Neoplasms; Pancreatic Neoplasms; Paraneoplastic Syndromes; Receptor, Insulin; Somatostatin

Glucagon-like peptide-1 (GLP-1) is a potent antihyperglycemic hormone. It can induce glucose-dependent insulin secretion. GLP-1 has a short half-life of less than 2 min in vivo due to degradation by the dipeptidyl peptidase-IV. GLP-l analogues, such as exendin-4 and exendin-3, have similar biological activity but a longer in-vivo half-life. Pancreatic β-cells and pancreatic islet cell tumors highly express GLP-1 receptors. Hence, radiolabeled GLP-1 analogues play a potential role in imaging and radiation therapy of pancreatic islet cell tumors as well as in the monitoring of pancreatic β-cell transplantation.

Topics: Animals; Female; Glucagon-Like Peptide 1; Humans; Insulin-Secreting Cells; Insulinoma; Male; Mice; Pancreatic Neoplasms; Positron-Emission Tomography; Radioisotopes; Radiopharmaceuticals; Tomography, Emission-Computed, Single-Photon

Diabetes has become the third largest cause of death in humans worldwide. Therefore, effective treatment for this disease remains a critical issue. Glucagon-like peptide-1 (GLP-1) plays an important role in glucose homeostasis, and therefore represents a promising candidate to use for the treatment of diabetes. Native GLP-1, however, is quickly degraded in in the circulatory system; which limits its clinical application. In the present study, a chemically-synthesized, modified analogue of human GLP-1 (mGLP-1) was designed. Our analyses indicated that, relative to native GLP-1, mGLP-1 is more resistant to trypsin and pancreatin degradation. mGLP-1 promotes mouse pancreatic β-cell proliferation by up-regulating the expression level of cyclin E, CDK2, Bcl-2 and down-regulating Bax, p21, and stimulates insulin secretion. An oral glucose tolerance test indicated that mGLP-1 significantly improved glucose tolerance in mice. Intraperitoneal injections of mGLP-1 into streptozotocin (STZ)-induced type 2 diabetic mice significantly reduced blood sugar levels and stimulated insulin secretion. Oral gavages of mGLP-1 in diabetic mice did not result in significant hypoglycemic activity.

Topics: Animals; Blood Glucose; Cell Line, Tumor; Diabetes Mellitus, Experimental; Gene Expression Regulation, Neoplastic; Glucagon-Like Peptide 1; Humans; Hypoglycemic Agents; Injections, Intraperitoneal; Insulin; Insulin Secretion; Insulinoma; Male; Mice; Pancreatic Neoplasms

Radiotracer diagnosis of insulinoma, can be done using somatostatin or glucagon-like peptide 1 (GLP-1). Performance of GLP-1 antagonists tends to be better than of agonists.. We investigated the uptake of the antagonist exendin (9-39), radiolabeled with technetium- 99m. Two different sites of the biomolecule were selected for chelator attachment.. HYNIC-βAla chelator attached to serine (C- terminus) of exendin, was associated with higher tumor uptake than to aspartate (N- terminus).. The chelator position in the biomolecule influenced receptor uptake.

Topics: Animals; Cell Line, Tumor; Chelating Agents; Chromatography, High Pressure Liquid; Glucagon-Like Peptide 1; Insulinoma; Male; Mice; Mice, SCID; Pancreatic Neoplasms; Peptide Fragments; Radionuclide Imaging; Radiopharmaceuticals; Sensitivity and Specificity; Technetium; Tissue Distribution

The aim of this study was to assess the utility of [Lys40(Ahx-HYNIC-99mTc/EDDA)NH2]-exendin-4 scintigraphy in the management of patients with hypoglycemia, particularly in the detection of occult insulinoma.. Forty patients with hypoglycemia and increased/confusing results of serum insulin and C-peptide concentration and negative/inconclusive results of other imaging examinations were enrolled in the study. In all patients GLP-1 receptor imaging was performed to localise potential pancreatic lesions.. Positive results of GLP-1 scintigraphy were observed in 28 patients. In 18 patients postsurgical histopathological examination confirmed diagnosis of insulinoma. Two patients had contraindications to the surgery, one patient did not want to be operated. One patient, who presented with postprandial hypoglycemia, with positive result of GLP-1 imaging was not qualified for surgery and is in the observational group. Eight patients were lost for follow up, among them 6 patients with positive GLP-1 scintigraphy result. One patient with negative scintigraphy was diagnosed with malignant insulinoma. In two patients with negative scintigraphy Munchausen syndrome was diagnosed (patients were taking insulin). Other seven patients with negative results of 99mTcGLP-1 scintigraphy and postprandial hypoglycemia with C-peptide and insulin levels within the limits of normal ranges are in the observational group. We would like to mention that 99mTc-GLP1-SPECT/CT was also performed in 3 pts with nesidioblastosis (revealing diffuse tracer uptake in two and a focal lesion in one case) and in two patients with malignant insulinoma (with the a focal uptake in the localization of a removed pancreatic headin one case and negative GLP-1 1 scintigraphy in the other patient).. 99mTc-GLP1-SPECT/CT could be helpful examination in the management of patients with hypoglycemia enabling proper localization of the pancreatic lesion and effective surgical treatment. This imaging technique may eliminate the need to perform invasive procedures in case of occult insulinoma.

Topics: Adolescent; Adult; Aged; Blood Glucose; Exenatide; Female; Glucagon-Like Peptide 1; Humans; Hypoglycemia; Insulinoma; Isotope Labeling; Male; Middle Aged; Organotechnetium Compounds; Peptides; Radionuclide Imaging; Venoms; Young Adult

Glucagon-like peptide-1 (GLP-1) has been shown to protect pancreatic β-cells against glucolipotoxicity via activation of the Akt pathway. The present study investigated the protective effects of the GLP-1 analog liraglutide against cholesterol-induced lipotoxicity and the mechanisms involved.. The mouse βTC-6 pancreatic β-cell line was preincubated for 30 min with 10 nmol/L liraglutide alone or in combination with the mammalian target of rapamycin (mTOR) inhibitor rapamycin (1 μmol/L) before being exposed to 5 mmol/L cholesterol for 6 h. 4',6'-Diamidino-2-phenylindole (DAPI) staining and Western blot analyses were used to assess the effects of liraglutide on cholesterol-induced apoptosis and the phosphorylation of Akt and mTOR.. Cholesterol significantly promoted cell apoptosis and attenuated the phosphorylation of Akt and mTOR, effects that were significantly reversed by liraglutide. Furthermore, rapamycin pretreatment alone significantly increased cholesterol-induced apoptosis compared with cholesterol-treated cells without any other pretreatment.. The data indicate that mTOR signaling is an essential mediator in the protection of pancreatic β-cells against cholesterol-induced apoptosis by a GLP-1 analog.

Topics: Animals; Apoptosis; Blotting, Western; Cholesterol; Electrophoresis, Polyacrylamide Gel; Glucagon-Like Peptide 1; Hypoglycemic Agents; Immunosuppressive Agents; Insulinoma; Liraglutide; Mice; Pancreatic Neoplasms; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

Insulin-like peptide 5 (INSL5), a member of the insulin/relaxin superfamily, can activate the G-protein-coupled receptor relaxin/insulin-like family peptide receptor 4 (RXFP4), but its precise biological functions are largely unknown. Recent studies suggest that INSL5/RXFP4 is involved in the control of food intake and glucose homoeostasis. We report in the present study that RXFP4 is present in the mouse insulinoma cell line MIN6 and INSL5 augments glucose-stimulated insulin secretion (GSIS) both in vitro and in vivo. RXFP4 is also expressed in the mouse intestinal L-cell line GLUTag and INSL5 is capable of potentiating glucose-dependent glucagon-like peptide-1 (GLP-1) secretion in GLUTag cells. We propose that the insulinotrophic effect of INSL5 is probably mediated through stimulation of insulin/GLP-1 secretion and the INSL5/RXFP4 system may be a potential therapeutic target for Type 2 diabetes.

Topics: Animals; Blood Glucose; Cell Line, Tumor; Dose-Response Relationship, Drug; Glucagon-Like Peptide 1; HEK293 Cells; Humans; Insulin; Insulinoma; Male; Mice; Mice, Inbred C57BL; Pancreatic Neoplasms; Peptide Hormones

A fluorescein-GLP-1 (7-37) analog was generated to determine GLP-1R distribution in various cell types of the pancreas in both strains of mice and receptor-specific uptake was confirmed by blocking with exendin-4. Biodistribution studies were carried out using 68Ga-labeled GLP-1(7-37) peptides in CD1 and C57BL/6 mice. In addition, immunocompromised mice bearing GLP-1R-expressing insulinomas were evaluated by positron emission tomography (PET) imaging and ex vivo biodistribution studies. The optical GLP-1 probe strongly colocalized with immunofluorescence for insulin and glucagon, and more weakly with amylase (exocrine pancreas) and cytokeratin 19 (ductal cells), confirming its application for in situ GLP-1R imaging in various pancreatic cell types. Insulinomas were clearly visualized by in vivo PET. Reducing the peptide positive charge decreased renal retention as well as tumor uptake. Results demonstrate the application of the developed GLP-1 peptide analogues for in situ (optical) and in vivo (PET) imaging of GLP-1R expression.

Topics: Animals; CHO Cells; Cricetinae; Cricetulus; Cyclic AMP; Gallium; Gallium Radioisotopes; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucose; Insulin; Insulinoma; Male; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Molecular Imaging; Neoplasm Transplantation; Peptides; Positron-Emission Tomography; Radioimmunoassay

Exendin-4 (Ex-4) is an anti-diabetic drug that is a potent agonist of the glucagon-like peptide-1 (GLP-1) receptor. It has already been approved for the treatment of type 2 diabetes mellitus, but its underlying mechanisms of action are not fully understood. Calcium/calmodulin-dependent serine protein kinase (CASK), which plays a vital role in the transport and release of neurotransmitters in neurons, is expressed in pancreatic islet cells and β-cells. This study aimed to investigate whether CASK is involved in the insulin secretagogue action induced by Ex-4 in INS-1 cells.. A glucose-stimulated insulin secretion (GSIS) assay was performed with or without siRNA treatment against CASK. The expression level and location of CASK were evaluated by real-time PCR, western blotting and immunofluorescence. With the use of a protein kinase A (PKA) inhibitor or an exchange protein directly activated by cAMP-2 (Epac2) agonist, immunoblotting was performed to establish the signaling pathway through which Ex-4 alters CASK expression.. Knock-down of CASK significantly attenuated the Ex-4-enhanced insulin release, and we showed that Ex-4 could increase transcription of CASK mRNA and expression of CASK protein but did not change the cellular location of CASK. A PKA inhibitor reduced the ability of Ex-4 to stimulate CASK expression, but an Epac2 agonist had no effect suggesting that regulation was mediated by the cAMP/PKA pathway.. Our study suggests that the stimulation of β-cell insulin secretion by Ex-4 is mediated, at least in part, by CASK via a novel signaling mechanism.

Topics: Animals; Blotting, Western; Cell Line, Tumor; Cyclic AMP-Dependent Protein Kinases; Diabetes Mellitus, Type 2; Exenatide; Gene Knockdown Techniques; Glucagon-Like Peptide 1; Guanylate Kinases; Hypoglycemic Agents; Insulin; Insulin Secretion; Insulinoma; Microscopy, Fluorescence; Pancreatic Neoplasms; Peptides; Rats; Real-Time Polymerase Chain Reaction; RNA, Small Interfering; Signal Transduction; Venoms

This study explores a new, non-invasive imaging method for the specific diagnosis of insulinoma by providing an initial investigation of the use of 125I-labelled molecules of the glucagon-like peptide-1 (GLP-1) analogue liraglutide for in vivo and in vitro small-animal SPECT/CT (single-photon emission computed tomography/computed tomography) imaging of insulinomas.. Liraglutide was labelled with 125I by the Iodogen method. The labelled 125I-liraglutide compound and insulinoma cells from the INS-1 cell line were then used for in vitro saturation and competitive binding experiments. In addition, in a nude mouse model, the use of 125I-liraglutide for the in vivo small-animal SPECT/CT imaging of insulinomas and the resulting distribution of radioactivity across various organs were examined.. The labelling of liraglutide with 125I was successful, yielding a labelling rate of approximately 95% and a radiochemical purity of greater than 95%. For the binding between 125I-liraglutide and the GLP-1 receptor on the surface of INS-1 cells, the equilibrium dissociation constant (Kd) was 128.8 ± 30.4 nmol/L(N = 3), and the half-inhibition concentration (IC50) was 542.4 ± 187.5 nmol/L(N = 3). Small-animal SPECT/CT imaging with 125I-liraglutide indicated that the tumour imaging was clearest at 90 min after the 125I-liraglutide treatment. An examination of the in vivo distribution of radioactivity revealed that at 90 min after the 125I-liraglutide treatment, the target/non-target (T/NT) ratio for tumour and muscle tissue was 4.83 ± 1.30(N = 3). Our study suggested that 125I-liraglutide was predominantly metabolised and cleared by the liver and kidneys.. The radionuclide 125I-liraglutide can be utilised for the specific imaging of insulinomas, representing a new non-invasive approach for the in vivo diagnosis of insulinomas.

Topics: Animals; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Humans; Insulinoma; Iodine Radioisotopes; Mice; Peptides; Radiography; Radioisotopes; Receptors, Glucagon; Tomography, Emission-Computed, Single-Photon

Topics: Diabetes Mellitus, Type 2; Female; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Humans; Hypoglycemia; Insulin; Insulinoma; Liraglutide; Middle Aged; Pancreatic Neoplasms; Receptors, Glucagon; Severity of Illness Index

The pathophysiological roles of thyroid hormones in glucose metabolism remain uncertain. Type 3 iodothyronine deiodinase (D3) converts thyroxine (T4) and 3,5,3'-triiodothyronine (T3) to 3,3',5'-triiodothyronine (rT3) and 3,3'-diiodothyronine (T2), respectively, inactivating thyroid hormones in a cell-specific fashion. In the present study, we identified D3 expression in MIN6 cells derived from a mouse insulinoma cell line and examined the mechanisms regulating D3 expression in these cells.. We characterized D3 activity using HPLC analysis, and examined the effect of GLP-1 or exendin-4 on D3 expression and cAMP accumulation in MIN6 cells. We also measured insulin secretion from MIN6 cells exposed to GLP-1 and T3.. We identified enzyme activity that catalyzes the conversion of T3 to T2 in MIN6 cells, which showed characteristics compatible with those for D3. D3 mRNA was identified in these cells using RT-PCR analysis. Forskolin rapidly stimulated D3 mRNA and D3 activity. Glucagon-like peptide-1 (GLP-1) increased D3 expression in a dose-dependent manner, and this effect was inhibited by the protein kinase A (PKA) inhibitor H-89. Exendin-4, a GLP-1 receptor agonist, also stimulated D3 expression in MIN6 cells. These results suggest that a cAMP-PKA-mediated pathway participates in GLP-1-stimulated D3 expression in MIN6 cells. Furthermore, GLP-1 stimulated insulin secretion was suppressed by the addition of T3 in MIN6 cells.. Our findings indicate that GLP-1 regulates intracellular T3 concentration in pancreatic β cells via a cAMP-PKA-D3-mediated pathway that may also regulate β-cell function.

Topics: Animals; Cell Line, Tumor; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Exenatide; Gene Expression Regulation, Neoplastic; Glucagon-Like Peptide 1; Insulin-Secreting Cells; Insulinoma; Iodide Peroxidase; Mice; Pancreatic Neoplasms; Peptides; RNA, Messenger; Signal Transduction; Triiodothyronine; Triiodothyronine, Reverse; Venoms

The pathophysiology of type 2 diabetes is progressive pancreatic beta cell failure with consequential reduced insulin secretion. Glucotoxicity results in the reduction of beta cell mass in type 2 diabetes by inducing apoptosis. Autophagy is essential for the maintenance of normal islet architecture and plays a crucial role in maintaining the intracellular insulin content by accelerating the insulin degradation rate in beta cells. Recently more attention has been paid to the effect of autophagy in type 2 diabetes. The regulatory pathway of autophagy in controlling pancreatic beta cells is still not clear. The aim of our study was to evaluate whether liraglutide can inhibit apoptosis and modulate autophagy in vitro in insulinoma cells (INS-1 cells).. INS-1 cells were incubated for 24 hours in the presence or absence of high levels of glucose, liraglutide (a long-acting human glucagon-like peptide-1 analogue), or 3-methyadenine (3-MA). Cell viability was measured using the Cell Counting Kit-8 (CCK8) viability assay. Autophagy of INS-1 cells was tested by monodansylcadaverine (MDC) staining, an autophagy fluorescent compound used for the labeling of autophagic vacuoles, and by Western blotting of microtubule-associated protein I light chain 3 (LC3), a biochemical markers of autophagic initiation.. The viability of INS-1 cells was reduced after treatment with high levels of glucose. The viability of INS-1 cells was reduced and apoptosis was increased when autophagy was inhibited. The viability of INS-1 cells was significantly increased by adding liraglutide to supplement high glucose level medium compared with the cells treated with high glucose levels alone.. Apoptosis and autophagy were increased in rat INS-1 cells when treated with high level of glucose, and the viability of INS-1 cells was significantly reduced by inhibiting autophagy. Liraglutide protected INS-1 cells from high glucose level-induced apoptosis that is accompanied by a significant increase of autophagy, suggesting that liraglutide plays a role in beta cell apoptosis by targeting autophagy. Thus, autophagy may be a new target for the prevention or treatment of diabetes.

Topics: Animals; Apoptosis; Autophagy; Cell Line, Tumor; Cell Survival; Glucagon-Like Peptide 1; Glucose; Insulinoma; Liraglutide; Rats

Insulinoma is a neuroendocrine tumor derived from the β cells of pancreatic islets. They are usually relatively inaccessible for surgical intervention. High expression levels of glucagon-like peptide-1 (GLP-1) receptor have been detected in insulinoma.. The aim of the study was to evaluate the potential of F-radiolabeled GLP-1 analog exendin-4 for the diagnosis of insulinoma using PET/computed tomography imaging.. The GLP-1 receptor-specific molecular probe [F]FB-exendin-4 was prepared by the conjugation of exendin-4 and N-succinimidyl-4-[F] fluorobenzoate ([F]SFB). High expression of GLP-1 by the RIN-m5f insulinoma line and GLP-1 receptor specificity were evaluated by determining the saturation curve for in-vitro binding of I-radiolabeled exendin-4 and by investigation of the competitive binding between I-radiolabeled and unlabeled exendin-4. Further, the in-vivo biodistribution and micro-PET/computed tomography images of insulinoma-bearing mice were studied.. An overall radiochemical yield of 35.6±2.3% (decay corrected, n=5) and specific radioactivity of around 30 GBq/µmol were achieved for [F]FB-exendin-4, and the radiochemical purity was over 98%. Both in-vitro and in-vivo studies confirmed the specificity of [F]FB-exendin-4 to insulinoma cells.. [F]FB-exendin-4 has been found to be an effective molecular imaging probe for detecting insulinomas.

Topics: Animals; Benzoates; Binding, Competitive; Exenatide; Female; Fluorine Radioisotopes; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Insulinoma; Isotope Labeling; Mice; Multimodal Imaging; Pancreatic Neoplasms; Peptides; Positron-Emission Tomography; Radiochemistry; Receptors, Glucagon; Succinimides; Tomography, X-Ray Computed; Venoms

(68)Gallium-PET ((68)Ga-PET) agents have significant clinical promise. The radionuclide can be produced from a (68)Ge/(68)Ga generator on site and is a convenient alternative to cyclotron-based PET isotopes. The short half-life of (68)Ga permits imaging applications with sufficient radioactivity while maintaining patient dose to an acceptable level. Furthermore, due to superior resolution, (68)Ga-PET agents have the ability to replace current SPECT agents in many applications. This article outlines the upcoming agents and challenges faced during the translational development of (68)Ga agents.

Topics: Amine Oxidase (Copper-Containing); Cell Adhesion Molecules; Gallium Radioisotopes; Glucagon-Like Peptide 1; Half-Life; Insulinoma; Neuroendocrine Tumors; Octreotide; Oligopeptides; Organometallic Compounds; Positron-Emission Tomography; Radionuclide Generators; Radiopharmaceuticals; Receptors, Bombesin; Receptors, Somatostatin; Single-Domain Antibodies; Somatostatin; Vascular Endothelial Growth Factor A

Glucagon-like peptide type 1 (GLP-1) is an incretin peptide that augments glucose-stimulated insulin release following oral consumption of nutrients. Its message is transmitted via a G protein-coupled receptor called GLP-1R, which is colocalized with pancreatic β-cells. The GLP-1 system is responsible for enhancing insulin release, inhibiting glucagon production, inhibiting hepatic gluconeogenesis, inhibiting gastric mobility, and suppression of appetite. The abundance of GLP-1R in pancreatic β-cells in insulinoma, a cancer of the pancreas, and the activity of GLP-1 in the cardiovascular system have made GLP-1R a target for molecular imaging.. We prepared (18)F radioligands for GLP-1R by the reaction of [(18)F]FBEM, a maleimide prosthetic group, with [Cys(0)] and [Cys(40)] analogs of exendin-4. The binding affinity, cellular uptake and internalization, in vitro stability, and uptake and specificity of uptake of the resulting compounds were determined in an INS-1 xenograft model in nude mice.. The [(18)F]FBEM-[Cys(x)]-exendin-4 analogs were obtained in good yield (34.3 ± 3.4%, n = 11), based on the starting compound [(18)F]FBEM), and had a specific activity of 45.51 ± 16.28 GBq/μmol (1.23 ± 0.44 Ci/μmol, n = 7) at the end of synthesis. The C-terminal isomer, [(18)F]FBEM-[Cys(40)]-exendin-4, had higher affinity for INS-1 tumor cells (IC(50) 1.11 ± 0.057 nM) and higher tumor uptake (25.25 ± 3.39 %ID/g at 1 h) than the N-terminal isomer, [(18)F]FBEM-[Cys(0)]-exendin-4 (IC(50) 2.99 ± 0.06 nM, uptake 7.20 ± 1.26 %ID/g at 1 h). Uptake of both isomers into INS-1 tumor, pancreas, stomach, and lung could be blocked by preinjection of nonradiolabeled [Cys(x)]-exendin-4 (p < 0.05).. [(18)F]FBEM-[Cys(40)]-exendin-4 and [(18)F]FBEM-[Cys(0)]-exendin-4 have high affinity for GLP-1R and display similar in vitro cell internalization. The higher uptake into INS-1 xenograft tumors exhibited by [(18)F]FBEM-[Cys(40)]-exendin-4 suggests that this compound would be the better tracer for imaging GLP-1R.

Topics: Amino Acid Sequence; Animals; Cell Line, Tumor; Cell Transformation, Neoplastic; Drug Stability; Exenatide; Female; Fluorine Radioisotopes; Glucagon-Like Peptide 1; Humans; Insulinoma; Mice; Molecular Sequence Data; Peptides; Positron-Emission Tomography; Rats; Venoms

Topics: Animals; Exenatide; Female; Fluorine Radioisotopes; Glucagon-Like Peptide 1; Humans; Insulinoma; Peptides; Positron-Emission Tomography; Venoms

Somatostatin receptor subtype 5 (SSTR5) mediates the inhibitory effect of somatostatin and its analogs on insulin expression/secretion and islet cell proliferation. We provide biochemical and genetic evidence that SSTR5 exerted its physiological actions via down-regulating pancreatic and duodenal homeobox-1 (PDX-1), a β-cell-specific homeodomain-containing transcription factor. Cotransfection of SSTR5 with PDX-1 resulted in dose-dependent inhibition of PDX-1 expression in human embryonic kidney 293 cells. SSTR5 agonist RPL-1980 inhibited PDX-1 expression and abolished glucagon-like peptide 1-stimulated PDX-1 expression in mouse insulinoma β-TC-6 cells. SSTR5 knockdown by short hairpin RNA led to increased PDX-1 expression that was accompanied by enhanced insulin secretion stimulated by high glucose in β-TC6 cells and alternated expressions of cell cycle proteins that favor cell proliferation in mouse insulinoma MIN6 cells. Quantitative RT-PCR analysis showed that cotransfected SSTR5 inhibited PDX-1 mRNA expression, whereas knockdown of SSTR5 increased PDX-1 mRNA expression. In addition, we found that cotransfected wild-type SSTR5 increased PDX-1 ubiquitination in human embryonic kidney 293 cells, whereas SSTR5 P335L, a hypofunctional single nucleotide polymorphism of SSTR5, inhibited PDX-1 ubiquitination. SSTR5 knockout resulted in increased expression of PDX-1, insulin, and proliferating cell nuclear antigen in the islets of sstr(-/-) mice. Immunohistochemistry analysis showed that SSTR5 P335L was associated with elevated expression of PDX-1 in human pancreatic neuroendocrine tumor. Taken together, our studies demonstrated that SSTR5 is a negative regulator for PDX-1 expression and that SSTR5 may mediate the inhibitory effects of somatostatin and its analogs on insulin expression/secretion and cell proliferation via down-regulating PDX-1 at both transcriptional and posttranslational levels.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Glucagon-Like Peptide 1; Glucose; HEK293 Cells; Homeodomain Proteins; Humans; Insulin; Insulin Secretion; Insulinoma; Mice; Mice, Knockout; Polymorphism, Single Nucleotide; Proliferating Cell Nuclear Antigen; Receptors, Somatostatin; RNA Interference; RNA, Messenger; RNA, Small Interfering; Somatostatin; Trans-Activators; Ubiquitination

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) potentiates glucose-stimulated insulin secretion from pancreatic β cells, yet does not directly stimulate secretion. The mechanisms underlying this phenomenon are incompletely understood. Here, we report that GLP-1 augments glucose-dependent rises in NAD(P)H autofluorescence in both βTC3 insulinoma cells and islets in a manner consistent with post-translational activation of glucokinase (GCK). GLP-1 treatment increased GCK activity and enhanced GCK S-nitrosylation in βTC3 cells. A 2-fold increase in S-nitrosylated GCK was also observed in mouse islets. Furthermore, GLP-1 activated a FRET-based GCK reporter in living cells. Activation of this reporter was sensitive to inhibition of nitric-oxide synthase (NOS), and incorporating the S-nitrosylation-blocking V367M mutation into this sensor prevented activation by GLP-1. GLP-1 potentiation of the glucose-dependent increase in islet NAD(P)H autofluorescence was also sensitive to a NOS inhibitor, whereas NOS inhibition did not affect the response to glucose alone. Expression of the GCK(V367M) mutant also blocked GLP-1 potentiation of the NAD(P)H response to glucose in βTC3 cells, but did not significantly affect metabolism of glucose in the absence of GLP-1. Co-expression of WT or mutant GCK proteins with a sensor for insulin secretory granule fusion also revealed that blockade of post-translational GCK S-nitrosylation diminished the effects of GLP-1 on granule exocytosis by ∼40% in βTC3 cells. These results suggest that post-translational activation of GCK is an important mechanism for mediating the insulinotropic effects of GLP-1.

Topics: Animals; Apoptosis; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Glucagon-Like Peptide 1; Glucokinase; Humans; Incretins; Insulin; Insulin-Secreting Cells; Insulinoma; Male; Mice; Mice, Inbred C57BL; Mutation; Nitric Oxide Synthase; Protein Processing, Post-Translational; Signal Transduction; Swine

Derived from endocrine pancreatic beta cells, insulinomas express glucagon-like peptide-1 (GLP-1) receptor with high density and incidence. In this study, we labeled a novel GLP-1 analogue, EM3106B, with (18)F and performed PET imaging to visualize insulinoma tumors in an animal model. A GLP-1 analogue that contains multiple lactam bridges, EM3106B, was labeled with (18)F through a maleimide-based prosthetic group, N-2-(4-(18)F-fluorobenzamido)ethylmaleimide ((18)F-FBEM). The newly developed radiotracer was characterized by cell based receptor-binding assay, cell uptake and efflux assay. The stability in serum was evaluated by radio-HPLC analysis. In vivo PET imaging was performed in nude mice bearing subcutaneous INS-1 insulinoma tumors and MDA-MB-435 tumors of melanoma origin. Ex vivo biodistribution study was performed to confirm the PET imaging data. EM3106B showed high binding affinity (IC(50) = 1.38 nM) and high cell uptake (5.25 ± 0.61% after 120 min incubation). (18)F-FBEM conjugation of EM3106B resulted in high labeling yield (24.9 ± 2.4%) and high specific activity (>75 GBq/μmol at the end of bombardment). EM3106B specifically bound and was internalized by GLP-1R positive INS-1 cells. After intravenous injection of 3.7 MBq (100 μCi) of (18)F-FBEM-EM3106B, the INS-1 tumors were clearly visible with high contrast in relation to the contralateral background on PET images, and tumor uptake of (18)F-FBEM-EM3106B was determined to be 28.5 ± 4.7 and 25.4 ± 4.1% ID/g at 60 and 120 min, respectively. (18)F-FBEM-EM3106B showed low uptake in MB-MDA-435 tumors with low level of GLP-1R expression. Direct tissue sampling biodistribution experiment confirmed high tracer uptake in INS-1 tumors and receptor specificity in both INS-1 tumor and pancreas. In conclusion, (18)F-FBEM-EM3106B exhibited GLP-1R-receptor-specific targeting properties in insulinomas. The favorable characteristics of (18)F-FBEM-EM3106B, such as high specific activity and high tumor uptake, and high tumor to nontarget uptake, demonstrate that it is a promising tracer for clinical insulinoma imaging.

Topics: Animals; Biological Transport; Cell Line, Tumor; Contrast Media; Drug Stability; Female; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Humans; Insulinoma; Islets of Langerhans; Lactams; Maleimides; Mice; Mice, Nude; Molecular Imaging; Neoplasm Proteins; Peptide Fragments; Peptides; Positron-Emission Tomography; Receptors, Glucagon; Tissue Distribution; Whole Body Imaging

A 61-year-old woman with fasting hypoglycemia following total gastrectomy was diagnosed as insulinoma. GIP and GLP-1 levels after a mixed meal were extremely increased. Administration of miglitol, an alpha-glucosidase inhibitor, suppressed the GIP and GLP-1 elevations.

Topics: 1-Deoxynojirimycin; Female; Gastrectomy; Gastric Inhibitory Polypeptide; Glucagon-Like Peptide 1; Glycoside Hydrolase Inhibitors; Humans; Hypoglycemia; Insulinoma; Middle Aged; Pancreatic Neoplasms; Postprandial Period

Topics: Fasting; Glucagon-Like Peptide 1; Humans; Insulinoma

In beta cells, both glucose and hormones, such as GLP-1, stimulate production of the second messenger cAMP, but glucose and GLP-1 elicit distinct cellular responses. We now show in INS-1E insulinoma cells that glucose and GLP-1 produce cAMP with distinct kinetics via different adenylyl cyclases. GLP-1 induces a rapid cAMP signal mediated by G protein-responsive transmembrane adenylyl cyclases (tmAC). In contrast, glucose elicits a delayed cAMP rise mediated by bicarbonate, calcium, and ATP-sensitive soluble adenylyl cyclase (sAC). This glucose-induced, sAC-dependent cAMP rise is dependent upon calcium influx and is responsible for the glucose-induced activation of the mitogen-activated protein kinase (ERK1/2) pathway. These results demonstrate that sAC-generated and tmAC-generated cAMP define distinct signaling cascades.

Topics: Adenylyl Cyclases; Cell Line; Cyclic AMP; Dose-Response Relationship, Drug; Glucagon-Like Peptide 1; Glucose; Humans; Insulinoma; Signal Transduction

cAMP activates multiple signal pathways, crucial for the pancreatic beta-cells function and survival and is a major potentiator of insulin release. A family of phosphodiesterases (PDEs) terminate the cAMP signals. We examined the expression of PDEs in rat beta-cells and their role in the regulation of insulin response. Using RT-PCR and Western blot analyses, we identified PDE3A, PDE3B, PDE4B, PDE4D, and PDE8B in rat islets and in INS-1E cells and several possible splice variants of these PDEs. Specific depletion of PDE3A with small interfering (si) RNA (siPDE3A) led to a small (67%) increase in the insulin response to glucose in INS-1E cells but not rat islets. siPDE3A had no effect on the glucagon-like peptide-1 (10 nmol/liter) potentiated insulin response in rat islets. Depletion in PDE8B levels in rat islets using similar technology (siPDE8B) increased insulin response to glucose by 70%, the potentiation being of similar magnitude during the first and second phase insulin release. The siPDE8B-potentiated insulin response was further increased by 23% when glucagon-like peptide-1 was included during the glucose stimulus. In conclusion, PDE8B is expressed in a small number of tissues unrelated to glucose or fat metabolism. We propose that PDE8B, an 3-isobutyl-1-methylxanthine-insensitive cAMP-specific phosphodiesterase, could prove a novel target for enhanced insulin response, affecting a specific pool of cAMP involved in the control of insulin granule trafficking and exocytosis. Finally, we discuss evidence for functional compartmentation of cAMP in pancreatic beta-cells.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Animals; Cell Line, Tumor; Cyclic Nucleotide Phosphodiesterases, Type 3; Diabetes Mellitus, Type 2; Gene Expression Regulation, Enzymologic; Glucagon-Like Peptide 1; Glucose; Insulin; Insulin Secretion; Insulin-Secreting Cells; Insulinoma; Male; Pancreatic Neoplasms; Rats; Rats, Wistar

Since its discovery glucagon-like peptide-1 (GLP-1) is investigated as a treatment for type II diabetes based on its major function as insulin secretagogue. A therapeutic use is, however, limited by its short biological half-life in the range of minutes, predominantly caused via degradation catalyzed by dipeptidyl peptidase IV (DPP-IV). Therefore, we aimed to design a GLP-1 analogue exhibiting resistance against DPP-IV-catalyzed inactivation while retaining its biological activity. By means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) we have studied the stability of the N-terminally blocked new analogue Ac-GLP-1-(7-34)-amide against DPP-IV and compared it with both unblocked GLP-1-(7-34)-amide and the major naturally occurring form GLP-1-(7-36)-amide. GLP-1-(7-36)-amide and the C-terminally two amino acid residues shorter GLP-1-(7-34)-amide rapidly generated peptide fragments truncated by the N-terminal dipeptide. In contrast, the N-terminal blocked Ac-GLP-1-(7-34)-amide was not degraded in the presence of DPP-IV over a period of at least two hours. Ac-GLP-1-(7-34)-amide induced a concentration-dependent increase of intracellular cAMP production and insulin release from rat insulinoma RIN-m5F cells to an extent comparable to that found for the N-terminally unblocked peptides GLP-1-(7-34)-amide and GLP-1-(7-36)-amide. Ac-GLP-1-(7-34)-amide may thus have the potential to act as a new long-acting GLP-1 analogue with significant resistance against DPP-IV and retained biological activity in vitro. Further research is required to investigate whether Ac-GLP-1-(7-34)-amide also exhibits its characteristics in animal models and humans.

Topics: Acetylation; Amides; Animals; Cells, Cultured; Cyclic AMP; Dipeptidyl Peptidase 4; Glucagon-Like Peptide 1; Insulin; Insulin Secretion; Insulinoma; Peptide Fragments; Rats; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Type 2 diabetes is a polygenic disorder characterized by increased insulin resistance, and impaired insulin secretion leading to abnormalities of glucose and lipid metabolism. Reduced responsiveness of the beta-cells to glucose is a critical feature of this syndrome. Glucagon-like peptide 1, a product of the pro-glucagon gene makes beta-cells competent and has many other anti-diabetic properties. We speculated whether GLP-1-based gene therapy could be an approach for treatment of type 2 diabetes. We started with a clone of rat insulinoma cells (S4 cells), which showed reduced responsiveness to glucose in terms of insulin secretion. We transfected these cells with a plasmid encoding a mutated form of GLP-1 (GLP-1-Gly8), which is resistant to the degrading enzyme dipeptidyl-peptidase IV. Activity of secreted GLP-1-Gly8 was assayed using Chinese hamster lung fibroblasts (CHL) cells that expressed cloned GLP-1 receptor and that were transfected with CRE-Luc. Stable cell lines (Glipsulin cells) obtained by this means produced and stored immunoreactive GLP-1-Gly8. In addition to insulin, the Glipsulin cells secreted the GLP-1-Gly8. The secreted GLP-1-Gly8 was active as evidenced by the ability of the conditioned media to elevate cAMP levels in CHL cells expressing GLP-1 receptors. Glipsulin cells responded to glucose with a 6.8 fold increase in insulin secretion compared to a 2.2 fold increase in the control cells. Our results demonstrate that prolonged exposure to GLP-1-Gly8 secreted by increases glucose-responsiveness of these cells. We speculate that engineering GLP-1-Gly8 secretion by beta-cells is a potential gene therapeutic strategy to treat diabetes.

Topics: Animals; Cell Line, Tumor; Clone Cells; Cricetinae; Cricetulus; Cyclic AMP; Dipeptidyl Peptidase 4; Genetic Engineering; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucose; Humans; Insulin; Insulin Secretion; Insulinoma; Islets of Langerhans; Peptide Fragments; Protein Precursors; Rabbits; Rats; Receptors, Glucagon; Transfection

Glucagon-like peptide-1 (GLP-1) is a peptide hormone secreted from the enteroendocrine L-cells of the gut and which acts primarily to potentiate the effects of glucose on insulin secretion from pancreatic beta-cells. It also stimulates insulin gene expression, proinsulin biosynthesis and affects the growth and differentiation of the islets of Langerhans. Previous studies on the mechanisms whereby GLP-1 regulates insulin gene transcription have focused on the rat insulin promoter. The aim of this study was to determine whether the human insulin promoter was also responsive to GLP-1, and if so to investigate the possible role of cAMP-responsive elements (CREs) that lie upstream (CRE1 and CRE2) and downstream (CRE3 and CRE4) of the transcription start site. INS-1 pancreatic beta-cells were transfected with promoter constructs containing fragments of the insulin gene promoter placed upstream of the firefly luciferase reporter gene. GLP-1 was found to stimulate the human insulin promoter, albeit to a lesser degree than the rat insulin promoter. Mutagenesis of CRE2, CRE3 and CRE4 blocked the stimulatory effect of GLP-1 while mutagenesis of CRE1 had no effect. Analysis of nuclear protein binding to the four CREs showed that, while they share some proteins, each CRE site is unique. Stimulation of transcription by GLP-1 through CRE2, CRE3 and CRE4 resulted in altered protein binding that was different for each of the CRE sites involved. Collectively, these data show that the four human CREs are not simply multiple copies of the rat CRE site and further emphasise that the human insulin promoter is distinct from the rodent promoter.

Topics: Animals; Cell Line, Tumor; Cyclic AMP; Electrophoretic Mobility Shift Assay; Gene Expression; Gene Expression Regulation; Glucagon; Glucagon-Like Peptide 1; Humans; Insulin; Insulinoma; Peptide Fragments; Promoter Regions, Genetic; Protein Precursors; Rats; Response Elements; Transcription Initiation Site; Transfection

The activation of the glucagon-like peptide-1 (GLP-1) receptor has been shown to have an important role in the functional activity of islet beta-cells and in the expansion of the islet cell mass. Constant remodeling of islet cell mass is mediated in vivo by proliferative and apoptotic stimuli to ensure a dynamic response to a changing demand for insulin. The present study was undertaken to investigate the biological activity of GLP-1 when cells were challenged by a proapoptotic stimulus. We have shown that activation of the GLP-1 receptor inhibits H(2)O(2)-induced apoptosis in a cultured mouse insulinoma cell line, termed MIN6. GLP-1 reduced DNA fragmentation and improved cell survival. This was mediated by an increased expression of the antiapoptotic proteins Bcl-2 and Bcl-xL. GLP-1 also prevented the H(2)O(2)-dependent cleavage of poly-(ADP-ribose)-polymerase. Inhibition of the GLP-1-dependent increase of cAMP by Rp-cAMP blocked the antiapoptotic action of GLP-1, as determined by DNA fragmentation and poly-(ADP-ribose)-polymerase assays and by detection of Bcl-2 and Bcl-xL protein levels. Investigation of the role of the protein kinases, PI-3 kinase (PI3K) and MAPK, by use of the inhibitors PD098059 and LY294002 demonstrated that the activation of PI3K, but not MAPK, was required to prevent proapoptotic events in cells exposed to H(2)O(2). The present study provides evidence that GLP-1 has an antiapoptotic action mediated by a cAMP- and PI3K-dependent signaling pathway.

Topics: Animals; Apoptosis; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; DNA; Glucagon; Glucagon-Like Peptide 1; Hydrogen Peroxide; Insulinoma; Islets of Langerhans; Mice; Oxidants; Peptide Fragments; Phosphatidylinositol 3-Kinases; Poly(ADP-ribose) Polymerases; Protein Precursors; Signal Transduction; Tumor Cells, Cultured

The cAMP-elevating pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates insulin release in pancreatic B-cells. Here, we have investigated its potentiating action in rat insulinoma INS-1 cells. In intact cells, PACAP-27 (100 nM) stimulated glucose-induced insulin secretion by >60%. Using the patch-clamp technique with single-cell exocytosis monitored as increases in cell capacitance, we observed that at 10 mM and 20 mM extracellular glucose, PACAP-27 acted mainly by a >50% enhancement of depolarization-elicited Ca(2+) entry, whereas at low (3 mM) glucose, the predominant effect of the peptide was a twofold increase in Ca(2+) sensitivity of insulin exocytosis. The latter effect was mimicked by glucose itself in a dose-dependent fashion. PACAP-27 exerts a prolonged effect on insulin secretion that is dissociated from changes of cytoplasmic cAMP. Whereas an elevation of cellular cAMP content (135%) could be observed 2 min after addition of PACAP-27, after 30 min preincubation with the peptide, cAMP concentrations were not different from basal. Yet, such pretreatment with PACAP-27 stimulated subsequent insulin release by congruent with60%. This sustained action is likely to reflect an increased degree of protein-kinase-A-dependent phosphorylation, and inhibitors of the kinase largely prevented the PACAP-mediated effects.

Topics: Action Potentials; Animals; Calcium; Calcium Channels; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Electrophysiology; Exocytosis; Glucagon; Glucagon-Like Peptide 1; Glucose; Insulin; Insulin Secretion; Insulinoma; Islets of Langerhans; Neuropeptides; Neurotransmitter Agents; Pancreatic Neoplasms; Patch-Clamp Techniques; Peptide Fragments; Pituitary Adenylate Cyclase-Activating Polypeptide; Protein Precursors; Rats; Tumor Cells, Cultured

Glucagon-like peptide-1 (GLP-1) is an incretin hormone derived from the proglucagon gene, capable of regulating the transcription of the three major genes that determine the pancreatic beta-cell-specific phenotype: insulin, GLUT-2, and glucokinase. The aim of this study was to investigate the potential role of GLP-1 for the gene therapy of glucose-insensitive pancreatic beta-cells. We transfected mouse insulinoma cells with a DNA fragment of the human proglucagon gene containing the nucleotide sequence encoding for human GLP-1 but lacking the coding region for glucagon. Two constructs were generated: In one, the expression of GLP-1 was under the control of the cytomegalovirus (CMV) promoter (CMV/GLP-1), and the second was regulated by the rat insulin II promoter (RIP)/GLP-1). Northern blot, HPLC, and RIA analyses confirmed that the minigene was transcribed and the protein appropriately translated, processed, and secreted in the extracellular environment. Gene expression studies revealed that although CMV/GLP-1 cells did not gain a greater glucose sensitivity as a result of the transfection with GLP-1, compared with cells transfected with the plasmid alone, RIP/GLP-1 was capable of regulating the gene expression of insulin and GLP-1 based on the concentration of glucose in the culture medium. Detection of the counterpart proteins (insulin and GLP-1) in the culture medium paralleled the observation derived from the Northern blot analysis. GLP-1 action was mediated by an IDX-1 (islet/duodenum homeobox-1) dependent transactivation of the endogenous insulin promoter, as demonstrated by gel shift analysis. This was further suggested by a significant increase of the glucose-dependent binding of IDX-1 to the insulin promoter in RIP/GLP-1 cells but not in CMV/GLP-1 cells or control cells. Finally, we observed that although the GLP-1-dependent secretion of insulin was mediated by an increase in cAMP levels, the transcription of the insulin gene, in response to GLP-1, was in large part cAMP independent. The present study lays the research foundation to investigate the potential use of GLP-1 for the gene or cell therapy of diabetes.

Topics: Animals; Blotting, Northern; Blotting, Western; Cyclic AMP; Cytomegalovirus; Fluorescent Antibody Technique; Glucagon; Glucagon-Like Peptide 1; Glucose; Humans; Insulin; Insulin Secretion; Insulinoma; Islets of Langerhans; Mice; Microscopy, Fluorescence; Pancreatic Neoplasms; Peptide Fragments; Proglucagon; Promoter Regions, Genetic; Protein Precursors; RNA, Messenger; Signal Transduction; Transfection; Tumor Cells, Cultured

Glucagon-like peptide-1 [GLP-1; formerly GLP-1(7-36)amide] and somatostatin (SS) are two postprandially or paracrine released peptide hormones that regulate insulin secretion from pancreatic islets. Using the rat insulinoma cell line RINm5F as a model, we investigated the effects of both peptides alone and in combination on insulin release, proliferation, and intracellular signal transduction. In addition, we determined the SS receptor subtypes expressed and involved by reverse transcription-polymerase chain reaction and use of selective SS agonists. GLP-1 stimulated insulin release, cell proliferation, intracellular cAMP accumulation and activation of the transcription factor cAMP-response element binding protein (CREB) which all could be reduced to basal values by co-incubation with SS. Incubation with SS alone did not affect basal levels. RINm5F cells express the somatostatin receptor (sst) subtypes sst1 and sst2 as well as traces of sst3. In accordance, the sst1- or sst2-selective non-peptide agonists L-797591 or L-054522 and peptide agonist octreotide (SMS 201995; sst2, sst3, and sst5 selective) potently inhibited GLP-1-induced insulin secretion whereas the sst3-selective agonist L-796778 showed little effect. Moreover, the sst1- and sst2-selective agonists slightly reduced also basal insulin release. The experiments show that GLP-1 and SS are perfect opponents for regulating pancreatic beta-cell insulin secretion.

Topics: Animals; Cell Division; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Disease Models, Animal; Drug Interactions; Glucagon; Glucagon-Like Peptide 1; Insulin; Insulin Secretion; Insulinoma; Pancreatic Neoplasms; Peptide Fragments; Protein Precursors; Rats; Somatostatin; Transcription Factors; Tumor Cells, Cultured

To develop transplantable beta-cell lines for the treatment of diabetes mellitus, we have taken advantage of the property of INS-1 cells to synthesize and secrete not only insulin, but also small quantities of the insulinotropic hormone glucagon-like peptide-1 (GLP-1). In INS-1 cells over-expressing the beta-cell GLP-1 receptor (GLP-1-R), we have shown, by radioimmune assay and bioassay of conditioned medium, that an autocrine signaling mechanism of hormone action exists whereby self-secreted GLP-1 acts as a competence factor in support of insulin gene transcription. INS-1 cells also exhibit insulin gene promoter activity, as assayed in cells transfected with a rat insulin gene I promoter-luciferase construct (RIP1-Luc). The GLP-1-R agonist exendin-4 stimulates RIP1-Luc activity in a glucose-dependent manner, an effect mediated by endogenous GLP-1-Rs, and is blocked by the serine/threonine protein kinase inhibitor Ro 31-8220. Over-expression of GLP-1-R in transfected INS-1 cells reduces the threshold for exendin-4 agonist action, whereas basal RIP1-Luc activity increases 2.5-fold in the absence of added agonist. The increase of basal RIP1-Luc activity is a consequence of autocrine stimulation by self-secreted GLP-1 and is blocked by introduction of (1) an inactivating W39A mutation in the N-terminus ligand-binding domain of GLP-1-R or (2) mutations in the third cytoplasmic loop that prevent G protein coupling. No evidence for constitutive ligand-independent signaling properties of the GLP-1-R has been obtained. Over-expression of GLP-1-R increases the potency and efficacy of D-glucose as a stimulator of RIP1-Luc. Thus, INS-1 cells over-expressing the GLP-1-R recapitulate the incretin hormone effect of circulating GLP-1, thereby providing a possible strategy by which beta-cell lines may be engineered for efficient glucose-dependent insulin biosynthesis and secretion.

Topics: Animals; Autocrine Communication; Cell Line; Diabetes Mellitus; Enzyme Inhibitors; Exenatide; Gene Expression Regulation; Genetic Engineering; Glucagon; Glucagon-Like Peptide 1; Indoles; Insulin; Insulinoma; Islets of Langerhans; Islets of Langerhans Transplantation; Peptide Fragments; Peptides; Promoter Regions, Genetic; Protein Kinase C; Protein Precursors; Rats; Receptors, Peptide; Recombinant Proteins; Research Design; Second Messenger Systems; Tumor Cells, Cultured; Venoms

IA-1, an insulinoma-associated cDNA-1, encodes a zinc-finger DNA-binding protein originally isolated from a human insulinoma subtraction library.. To demonstrate the restriction of IA-1 gene expression in human fetal pancreata of different gestational stages and to determine whether the expression of IA-1 gene is associated with rat AR42J cell differentiation into insulin-positive cells.. To examine whether the IA-1 gene is associated with pancreatic endocrine cell differentiation, we used a rat pancreatic amphicrine cell line, AR42J, to investigate whether the expression of the IA-1 gene coincides with AR42J cells converting into either endocrine or exocrine lineage. We also examined a set of islet transcription factors that regulate key differentiation steps involved in activating the genes that confer the specialized functions of terminally differentiated pancreatic islet cells.. When the AR42J cells were converted into insulin-positive cells induced by GLP-1, insulinoma conditioned-medium, or both, we observed a significant elevated expression of mRNA for IA-1 and islet-specific transcription factors such as Pdx-1, NeuroD/beta2, and Nkx6.1. In contrast, dramatically decreased expression of mRNA for IA-1 and islet-specific transcription factors was displayed when AR42J cells were converted into the acinar-like phenotype by dexamethasone.. IA-1 gene was shown to be developmentally regulated in fetal pancreatic cells, and its expression pattern is consistent with parallel changes in islet-specific transcription factors during the endocrine differentiation of AR42J cells.

Topics: Adult; Animals; Basic Helix-Loop-Helix Transcription Factors; Cell Differentiation; Culture Media, Conditioned; DNA-Binding Proteins; DNA, Complementary; Fetus; Gene Expression Regulation, Developmental; Glucagon; Glucagon-Like Peptide 1; Homeodomain Proteins; Humans; Insulin; Insulinoma; Islets of Langerhans; Mice; Peptide Fragments; Protein Precursors; Rats; Repressor Proteins; RNA, Messenger; Trans-Activators; Tumor Cells, Cultured; Zinc Fingers

The non-invasive detection of insulinomas remains a diagnostic problem that is not solved by means of somatostatin receptor scintigraphy. We investigated the biokinetics and specificity of uptake and degradation of the incretin hormone glucagon-like peptide-1 (GLP-1) in a rat insulinoma cell line (RINm5F) in order to ascertain whether radiolabelled GLP-1 may be suitable for specific visualisation of insulinomas in vivo. GLP-1 (7-36)amide was radioiodinated according to the iodogen method. The specificity of the uptake of [(125)I]GLP-1(7-36)amide by RINm5F cells was investigated. Degradation products of GLP-1 (7-36)amide in the cell medium were purified by HPLC. Their masses and amino acid sequences were determined by (252)Cf-plasma desorption mass spectrometry. Lysosomal degradation was inhibited and after differential centrifugation the amount of radiotracer incorporated into lysosomes was determined. Biodistribution studies were performed in a rat insulinoma model (NEDH rats and RINm5F cells) with [(123)I]GLP-1(7-36)amide and its more stable agonist [(123)I]exendin 3. The uptake of radiotracer into insulinoma cells reached a maximum within 5 min. It was inhibited by an excess of unlabelled peptide. [(125)I]GLP-1(7-36)amide accumulated in the cells if lysosomal degradation was inhibited. Degradation products of the peptide were found in the cell medium. We determined their mass and derived their amino acid sequence. Radiolabelling of exendin 3 was more difficult than that of GLP-1 because of the lack of tyrosine in its primary structure. Biodistribution studies showed rapid blood clearance and uptake of the radiotracer into the tumour and the pancreas. It was also possible to detect insulinomas in an animal model by external scintigraphy using radioiodinated GLP-1 (7-36)amide and exendin 3. GLP-1 (7-36)amide is specifically internalised into insulinoma cells by a receptor-mediated mechanism. Our results demonstrate that GLP-1 receptor-directed scintigraphy may be a new method for the detection of insulinomas in vivo. Due to the short half-life of GLP-1, its more stable analogue exendin 3 may better suit this purpose in vivo.

Topics: Animals; Chromatography, High Pressure Liquid; Feasibility Studies; Glucagon; Glucagon-Like Peptide 1; Insulinoma; Iodine Radioisotopes; Neoplasm Transplantation; Pancreatic Neoplasms; Peptide Fragments; Peptides; Protein Precursors; Radionuclide Imaging; Rats; Reproducibility of Results; Sensitivity and Specificity; Tissue Distribution; Tumor Cells, Cultured

Glucagon-like peptide-1 (GLP-1) stimulates insulin secretion and improves glycemic control in type 2 diabetes. In serum the peptide is degraded by dipeptidyl peptidase IV (DPP IV). The resulting short biological half-time limits the therapeutic use of GLP-1. DPP IV requires an intact alpha-amino-group of the N-terminal histidine of GLP-1 in order to perform its enzymatic activity. Therefore, the following GLP- analogues with alterations in the N-terminal position 1 were synthesized: N-methylated- (N-me-GLP-1), alpha-methylated (alpha-me-GLP-1), desamidated- (desamino-GLP-1) and imidazole-lactic-acid substituted GLP-1 (imi-GLP-1). All GLP-1 analogues except alpha-me-GLP-1 were hardly degraded by DPP IV in vitro. The GLP-1 analogues showed receptor affinity and in vitro biological activity comparable to native GLP-1 in RINm5F cells. GLP-1 receptor affinity was highest for imi-GLP-1, followed by alpha-me-GLP-1 and N-me-GLP-1. Only desamino-GLP-1 showed a 15-fold loss of receptor affinity compared to native GLP-1. All analogues stimulated intracellular cAMP production in RINm5F cells in concentrations comparable to GLP-1. N-terminal modifications might therefore be useful in the development of long-acting GLP-1 analogues for type 2 diabetes therapy.

Topics: Animals; Binding, Competitive; Chromatography, High Pressure Liquid; Cyclic AMP; Dipeptidyl Peptidase 4; Glucagon; Glucagon-Like Peptide 1; Insulinoma; Molecular Structure; Peptide Fragments; Protein Precursors; Rats; Receptors, G-Protein-Coupled; Receptors, Gastrointestinal Hormone; Tumor Cells, Cultured

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 1 (GLP-1), a hormonal activator of adenyl cyclase, stimulates insulin gene transcription, an effect mediated by the cAMP response element (CRE) of the rat insulin I gene promoter (RIP1). Here we demonstrate that the signaling mechanism underlying stimulatory effects of GLP-1 on insulin gene transcription results from protein kinase A (PKA)-independent activation of the RIP1 CRE. Although GLP-1 stimulates cAMP production in rat INS-1 insulinoma cells, we find accompanying activation of a -410-bp RIP1 luciferase construct (-410RIP1-LUC) to exist independently of this second messenger. GLP-1 produced a dose-dependent stimulation of -410RIP1-LUC (EC50 0.43 nmol/l), an effect reproduced by the GLP-1 receptor agonist exendin-4 and abolished by the antagonist exendin(9-39). Activation of RIP1 by GLP-1 was not affected by cotransfection with dominant-negative Gs alpha, was not blocked by cAMP antagonist Rp-cAMPS, and was insensitive to PKA antagonist H-89. Truncation of -410RIP1-LUC to generate -307-, -206-, and -166-bp constructs revealed 2 segments of RIP1 targeted by GLP-1. The first segment, not regulated by forskolin, was located between -410 and -307 bp of the promoter. The second segment, regulated by both GLP-1 and forskolin, included the CRE and was located between -206 and -166 bp. Consistent with these observations, stimulatory effects of GLP-1 at RIP1 were reduced after introduction of delta-182 and delta-183/180 inactivating deletions at the CRE. The action of GLP-1 at -410RIP1-LUC was also reduced by cotransfection with A-CREB, a genetically engineered isoform of the CRE binding protein CREB, which dimerizes with and prevents binding of basic-region-leucine-zipper (bZIP) transcription factors to the CRE. In contrast, the action of GLP-1 at the CRE was not blocked by cotransfection with M1-CREB, an isoform that lacks a consensus serine residue serving as substrate for PKA-mediated phosphorylation. On the basis of these studies, it is proposed that PKA-independent stimulatory actions of GLP-1 at RIP1 are mediated by bZIP transcription factors related in structure but not identical to CREB.

Topics: Animals; Base Sequence; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Gene Expression Regulation; Glucagon; Glucagon-Like Peptide 1; Insulin; Insulinoma; Luciferases; Pancreatic Neoplasms; Peptide Fragments; Promoter Regions, Genetic; Protein Precursors; Rats; Recombinant Fusion Proteins; Second Messenger Systems; Sequence Deletion; Transcription, Genetic; Tumor Cells, Cultured; Venoms

We have investigated intracellular Ca2+ mobilization in oscillations of cytoplasmic Ca2+ in response to glucagon-like peptide 1 (GLP-1) and glucose in clonal HIT insulinoma cells with a confocal laser-scanning microscope (CLSM). We also used electron probe X-ray microanalysis to determine the GLP-1- and glucose-induced changes in electrolyte levels in the cytoplasm and insulin granules of the cells. GLP-1 produced 10- to 35-s duration oscillations in cytoplasmic Ca2+ concentration ([Ca2+]i), both with and without Ca2+ in the extracellular solution, suggesting that Ca2+ is mobilized from intracellular Ca2+ stores, namely secretory granules. Glucose caused 1- to 3-min duration oscillatory increases in [Ca2+]i when the extracellular solution contained Ca2+. When the cells were cultured without Ca2+ (no Ca2+ added, 1 mM EGTA), an oscillatory [Ca2+]i increase of amplitude and short duration (12-35 s) was produced by 11 mM glucose, and the oscillation was inhibited by ruthenium red. X-ray microanalysis showed that stimulation with glucose increased the total Ca concentration in the cytoplasm and decreased it in the insulin granules with and without Ca2+ in the extracellular solution. The application of glucose significantly decreased K, and increased Na and C1 in the cytoplasm when the extracellular solution contained Ca2+. Our result also suggests that the [Ca2+]i oscillation induced by glucose is involved in the release of Ca2+ from intracellular Ca2+ stores through the ryanodine receptor, which is blocked by ruthenium red, and/or through the inositol trisphosphate receptor that may be present in the membrane of insulin granules.

Topics: Animals; Calcium; Cytoplasm; Cytoplasmic Granules; Electrolytes; Electron Probe Microanalysis; Extracellular Space; Glucagon; Glucagon-Like Peptide 1; Insulinoma; Islets of Langerhans; Microscopy, Confocal; Microscopy, Electron; Pancreatic Neoplasms; Peptide Fragments; Protein Precursors; Tumor Cells, Cultured

Glucagon-like peptide-1 (GLP-1) enhances insulin biosynthesis and secretion as well as transcription of the insulin, GLUT2 and glucokinase genes. The latter are also regulated by the PDX-1 homeoprotein. We investigated the possibility that GLP-1 may be having its long-term pleiotropic effects through a hitherto unknown regulation of PDX-1. We found that PDX-1 mRNA level was significantly increased (p<0.01) after 2 hours and insulin mRNA level was subsequently increased (p<0.01) after 3 hours of treatment with GLP-1 (10 nM) in RIN 1046-38 insulinoma cells. Under these experimental conditions, there was also a 1.6-fold increase in the expression of PDX-1 protein in whole cell and nuclear extracts. Overexpression of PDX-1 in these cells confirmed the finding of the wild type cells such that GLP-1 induced a 2-fold increase in whole cell extracts and a 3-fold increase in nuclear extracts of PDX-1 protein levels. The results of electrophoretic mobility shift experiments showed that PDX-1 protein binding to the Al element of the rat insulin II promoter was also increased 2 h post treatment with GLP-1. In summary, we have uncovered a previously unknown aspect to the regulation of PDX-1 in beta cells. This has important implications in the physiology of adult beta cells and the treatment of type 2 diabetes mellitus with GLP-1 or its analogs.

Topics: Animals; Glucagon; Glucagon-Like Peptide 1; Homeodomain Proteins; Insulinoma; Pancreatic Neoplasms; Peptide Fragments; Protein Precursors; Rats; RNA, Messenger; Trans-Activators; Tumor Cells, Cultured

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

The GLP-1 receptor (GLP-1R) mediates the insulinotropic effects of the incretion hormone glucagon-like peptide 1 (7-36) amide (GLP-1). Recently, we cloned the 5'-flanking region of the human GLP-1R gene. To characterize tissue- and cell-specific cis-regulatory elements, we constructed a series of 5'-deletions of the promoter. The activity of these constructs was tested in different cell lines. An element with high homology to PS1 was found to repress GLP-1R promoter activity in fibroblasts and pancreatic D-cells, but was not active in pancreatic A- and B-cells. PS1 was described to inhibit activation of a D-cell-specific enhancer. Cloning the PS1-like element upstream a heterologous promoter (SV40) revealed that it is functionally active independently from this enhancer. Our data suggest that basal activity of the GLP-1R promoter is silenced in a tissue- and cell-specific manner by negatively acting cis-regulatory elements, including a PS1-like element.

Topics: Animals; Base Sequence; Cell Line; Cell Nucleus; Cloning, Molecular; Cricetinae; Enhancer Elements, Genetic; Gene Expression Regulation; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagonoma; Humans; Insulinoma; Islets of Langerhans; Molecular Sequence Data; Mutagenesis, Site-Directed; Organ Specificity; Pancreatic Neoplasms; Peptide Fragments; Promoter Regions, Genetic; Protein Precursors; Receptors, Glucagon; Recombinant Proteins; Sequence Deletion; Sequence Homology, Nucleic Acid; Simian virus 40; Transfection; Tumor Cells, Cultured

Neuropeptide Y (NPY) has been shown to inhibit insulin secretion from the islets of Langerhans. We show that insulin secretion in the insulinoma cell line RIN 5AH is inhibited by NPY. 125I-Peptide YY (PYY) saturation and competition-binding studies using NPY fragments and analogues on membranes prepared from this cell line show the presence of a single class of NPY receptor with a Y1 receptor subtype-like profile. Inhibition of insulin secretion in this cell line by NPY fragments and analogues also shows a Y1 receptor-like profile. Both receptor binding and inhibition of insulin secretion showed the same orders of potency with NPY > [Pro34]-NPY > NPY 3-36 >> NPY 13-36. The Y1 receptor antagonist, BIBP 3226, blocks NPY inhibition of insulin secretion from, and inhibits 125I-PYY binding to, RIN 5AH cells. Northern blot analysis using a Y1-receptor specific probe shows that NPY Y1 receptors are expressed by RIN 5AH cells. Y5 receptors are not expressed in this cell line. Neuropeptide Y inhibition of insulin secretion is blocked by incubation with pertussis toxin, implying that the effect is via a G-protein (Gi or Go) coupled receptor. Neuropeptide Y inhibits the activation of adenylyl cyclase by isoprenaline in RIN 5AH cell lysates, and the stimulation of cAMP by glucagon-like peptide-1 (7-36) amide (GLP-1). It also blocks insulin secretion stimulated by GLP-1, but not by dibutyryl cyclic AMP. Hence, we suggest that NPY inhibits insulin secretion from RIN 5AH cells via a Y1 receptor linked through Gi to the inhibition of adenylyl cyclase.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Blotting, Northern; Cyclic AMP; Glucagon; Glucagon-Like Peptide 1; Glucose; Insulin; Insulin Secretion; Insulinoma; Iodine Radioisotopes; Isoproterenol; Neuropeptide Y; Pancreatic Neoplasms; Peptide Fragments; Peptide YY; Protein Precursors; Rats; Receptors, Neuropeptide Y; Swine; 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

Both glucagon and glucagon-like peptide-1 (GLP-1) play an important role in the regulation of nutrient homeostasis. In this study, the tissue distributions of the expression of receptor genes for glucagon and GLP-1 were examined. Expression of glucagon receptor gene was detected in liver, kidney, ileum and pancreatic islets but not in brain. In contrast, expression of GLP-1 receptor gene was detected in brain, pancreas and pancreatic islets but not in liver, kidney, or ileum. To investigate the existence and characteristics of glucagon and GLP-1 receptors on pancreatic beta cells, expression of the receptor genes and translational regulation of the expression of the receptor genes by glucose were analyzed in a mouse pancreatic beta cell line, MIN6 cells. In the cDNA pool of MIN6 cells, both glucagon and GLP-1 receptor genes were identified and showed higher expression level in MIN6 cells cultured under high glucose condition than in those cultured under low glucose condition. These results suggest that glucagon and GLP-1 receptor genes are expressed in pancreatic beta cells and their expression is upregulated by glucose.

Topics: Animals; DNA, Complementary; Gene Expression Regulation; Glucagon; Glucagon-Like Peptide 1; Glucose; Insulinoma; Islets of Langerhans; Mice; Mice, Inbred C57BL; Peptides; Protein Biosynthesis; Protein Precursors; RNA, Messenger; Tissue Distribution; 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

Maitotoxin (MTX) activates a Ca2+-dependent non-selective cation current (ICa-NS) in insulinoma cells whose time course is identical to non-selective cation currents activated by incretin hormones such as glucagon-like peptide-1 (GLP-1), which stimulate glucose-dependent insulin secretion by activating cAMP signaling pathways. We investigated the mechanism of activation of ICa-NS in insulinoma cells using specific pharmacological reagents, and these studies further support an identity between MTX- and GLP-1-activated currents. ICa-NS is inhibited by extracellular application of genistein, econazole, and SKF 96365. This inhibition by genistein suggests that tyrosine phophorylation may play a role in the activation of ICa-NS. ICa-NS is not inhibited by incubation of cells in glucose-free solution, by extracellular tetrodotoxin, nimodipine, or tetraethylammonium, or by intracellular dialysis with 4-aminopyridine, ATP, ryanodine, or heparin. ICa-NS is also not significantly inhibited by staurosporine, which does, however, partially inhibit the MTX-induced rise of intracellular Ca2+ concentration. These effects of staurosporine suggest that protein kinase C may not be involved in the activation of ICa-NS but that it may regulate intracellular Ca2+ release. Alternatively, ICa-NS may have a small component that is carried through separate divalent cation-selective channels that are inhibited by staurosporine. ICa-NS is neither activated nor inhibited by dialysis with KF, KF + AlF3 or GTPgammaS (guanosine 5'-O-(3-thiotriphosphate)), suggesting that GTP-binding proteins do not play a major role in the activation of this current.

Topics: Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Cricetinae; Egtazic Acid; Glucagon-Like Peptide 1; Imidazoles; Insulinoma; Islets of Langerhans; Marine Toxins; Membrane Potentials; Oxocins; Pancreatic Neoplasms; Peptides; Staurosporine; Tumor Cells, Cultured

Glucagon-like peptide-1 (GLP-1), secreted from intestine in response to food intake, enhances insulin secretion from pancreatic beta-cells. In this study, we evaluated the effects of stably transfecting the GLP-1 receptor into an insulinoma cell line, RIN 1046-38, on basal and glucose-mediated insulin secretion and on second messenger pathways involved in insulin secretion. The GLP-1 receptor transfected cells had similar insulin mRNA levels but higher insulin content compared with parental cells. In GLP-1 receptor transfected cells, glucose (0.5 mM)-mediated insulin release was increased compared with parental cells (4.52 +/- 0.79 pmol insulin/l per mg protein x h vs. 2.21 +/- 0.36 pmol insulin/l per mg protein x h; mean +/- S.E., n = 6, P = 0.015, in transfected vs. parental cells, respectively). By hemolytic plaque assay measuring single cell insulin secretion, we observed that in the GLP-1 receptor transfected cells versus parental cells the increased insulin secretion was due to the presence of more glucose-responsive cells as well as more insulin released in response to glucose per cell. Resting intracellular cAMP was higher in the GLP-1 transfected cells (35.96 +/- 3.88 vs. 18.6 +/- 2.01 nmol/l per mg protein x h; mean +/- S.E., n = 4, P = 0.039, in transfected vs. parental cells, respectively). In response to GLP-1, both GLP-1 receptor transfected cells and parental cells showed increased cAMP levels independent of glucose. Resting intracellular calcium was the same in both parental and GLP-1 receptor transfected cells. However, more cells were responsive to glucose in the GLP-1 receptor transfected cells and the calcium transients attained in the presence of glucose developed at a faster rate and reached a higher amplitude than in parental cells. We conclude that having an excess of GLP-1 receptors renders beta-cells more sensitive to glucose.

Topics: Animals; Calcium; Cyclic AMP; Gene Expression; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucose; Insulin; Insulin Secretion; Insulinoma; Ion Transport; Pancreatic Neoplasms; Peptides; Rats; Receptors, Glucagon; Transfection; Tumor Cells, Cultured

We investigated the possibility that tyrosine phosphorylation might play a role in insulin secretion in the insulinoma cell line, RIN 1046-38 cells. At least 4 proteins of 18, 25, 35, and 46 kDa size were found to be tyrosine phosphorylated in the presence of glucose and an insulin secretagogue, glucagon-like peptide-1 (GLP-1). The addition of glucose and GLP-1 to cells that were exposed to the tyrosine kinase inhibitor genistein resulted in a decrease in the extent of phosphorylation of the 18, 25, and 35 kDa proteins and a concomitant reduction in insulin secretion, whereas treatment with vanadate, a tyrosine phosphatase inhibitor, led to enhanced responses. Immunoprecipitation of cellular proteins with an anti-phosphotyrosine antibody followed by immunoblotting with a specific monoclonal antibody to SNAP-25 (synaptosome-associated protein of 25 kDa) revealed that the 25 kDa protein is SNAP-25. These results suggest that tyrosine phosphorylation of SNAP-25 may be involved in the regulation of insulin secretion in RIN 1046-38 cells.

Topics: Animals; Glucagon-Like Peptide 1; Glucose; Insulinoma; Membrane Proteins; Nerve Tissue Proteins; Pancreatic Neoplasms; Peptides; Phosphorylation; Rats; Synaptosomal-Associated Protein 25; Tumor Cells, Cultured

To evaluate the growth and insulin secretion from microencapsulated beta TC6-F7 cells in vitro and to assess the in vivo function of microencapsulated cells transplanted in rats with steptozotocin (STZ)-induced diabetes.. Alginate-poly-L-lysine encapsulated beta TC6-F7 cells were exposed to glucose, isobutylmethylxanthine (IBMX) and glucagon-like peptide I (7-36 amide) in a static in vitro challenge. In vivo, 2.5-3.5 x 10(7) encapsulated cells were implanted into diabetic rats. Graft function was evaluated by monitoring blood glucose concentrations and by an intraperitoneal glucose tolerance test.. The cell density (number of cells per capsule) of cultured microencapsulated beta TC6-F7 cells increased almost 35-fold over a 55 day observation period to reach a plateau of approximately 3500 cells/capsule. While insulin secretion per capsule remained unchanged over the first 21 days of culture, a 7-fold increase was observed during the last 14 days of the 55 day observation period. Intraperitoneal transplantation of 3.5 x 10(7) encapsulated cells into diabetic rats resulted, within 24 hours, in reversal of hyperglycemia for up to 60 days. Post-transplantation blood glucose concentrations varied between 2 and 4 mM. Glucose clearance rates evaluated by an intraperitoneal glucose tolerance test at 30 days post-transplantation resulted in a markedly flat glucose clearance curve with blood glucose never rising above 4 mM. The glucose challenge of microencapsulated cells recovered 30 days post-transplantation resulted in a 2-fold increase in insulin response at glucose concentrations greater than 5.5 mM as compared to glucose-free media. In addition, immunostaining of recovered grafted tissue for insulin, reveals a strong presence of the peptide within the cell population.. These data demonstrate the potential use of an immunoisolated beta-cell line for the treatment of diabetes.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Blood Glucose; Capsules; Cell Division; Cell Separation; Diabetes Mellitus, Experimental; Glucagon; Glucagon-Like Peptide 1; Glucose Tolerance Test; Insulin; Insulin Secretion; Insulinoma; Pancreas, Artificial; Pancreatic Neoplasms; Peptide Fragments; Protein Precursors; Rats; Rats, Wistar; Time Factors; Tumor Cells, Cultured

The gastrointestinal hormones glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP) strongly stimulate insulin release. Despite their high N-terminal sequence similarity, GLP-1 does not bind to the GIP receptor and vice versa. To characterize the domains required for interaction of the peptide ligands with their specific receptors, we performed displacement studies with various synthetic GLP-1/GIP hybrid peptides on RINm5F insulinoma cells. Displacement of 125I-GIP and 125I-GLP-1 was measured using GLP-1/GIP chimeras which comprised GIP and GLP-1 sequences at different positions. The binding affinity to the GLP-1 receptor was found to be sensitive to GIP-like exchanges in the N-terminal 22 amino acids as well as in positions 13 and 15 (loss of affinity 280-fold to more than 1000-fold). C-terminal substitution of the GLP-1 sequence by GIP diminished the affinity towards the GLP-1 receptor only 20-fold. All hybrid peptides investigated showed minimal binding affinity for the GIP receptor, indicating that the entire GIP-sequence (1-31) is important for receptor recognition. These findings provide insight into the structural requirements for the specific interaction of two important insulinotropic peptides with their specific receptors.

Topics: Amino Acid Sequence; Animals; Chloramines; Cyclic AMP; Dose-Response Relationship, Drug; Gastric Inhibitory Polypeptide; Gastrointestinal Agents; Glucagon; Glucagon-Like Peptide 1; Indicators and Reagents; Insulinoma; Iodine Radioisotopes; Molecular Sequence Data; Peptide Fragments; Protein Precursors; Radioligand Assay; Rats; Recombinant Fusion Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Structure-Activity Relationship; Tosyl Compounds; 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: Animals; Cyclic AMP; Gene Deletion; Glucagon; Glucagon-Like Peptide 1; Humans; Insulinoma; Mutation; Peptide Fragments; Phosphorylation; Protein Precursors; Receptors, Glucagon; Sensitivity and Specificity; Signal Transduction; Tumor Cells, Cultured

Topics: Colforsin; Electrophoresis, Gel, Two-Dimensional; Glucagon; Glucagon-Like Peptide 1; Insulinoma; Isoelectric Focusing; Molecular Weight; Peptide Fragments; Phosphoproteins; Phosphorylation; Protein Kinases; Protein Precursors; Receptors, Glucagon; Signal Transduction; 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

Glucagon-like peptide-1 (GLP-1) stimulates glucose-induced insulin secretion by binding to a specific G protein-coupled receptor linked to activation of the adenylyl cyclase pathway. Here, using insulinoma cell lines, we studied homologous and heterologous desensitization of GLP-1-induced cAMP production. Preexposure of the cells to GLP-1 induced a decrease in GLP-1-mediated cAMP production, as assessed by a 3- to 5-fold rightward shift of the dose-response curve and an approximately 20 percent decrease in the maximal production of cAMP. Activation of protein kinase C by the phorbol ester phorbol 12-myristate 13-acetate (PMA) also induced desensitization of the GLP-1-mediated response, leading to a 6- to 9-fold shift in the EC50 and a 30% decrease in the maximal production of cAMP. Both forms of desensitization were additive, and the protein kinase C inhibitor RO-318220 inhibited PMA-induced desensitization, but not agonist-induced desensitization. GLP-1- and PMA-dependent desensitization correlated with receptor phosphorylation, and the levels of phosphorylation induced by the two agents were additive. Furthermore, PMA-induced, but not GLP-1-induced, phosphorylation was totally inhibited by RO-318220. Internalization of the GLP-1 receptor did not participate in the desensitization induced by PMA, as a mutant GLP-1 receptor lacking the last 20 amino acids of the cytoplasmic tail was found to be totally resistant to the internalization process, but was still desensitized after PMA preexposure. PMA and GLP-1 were not able to induce the phosphorylation of a receptor deletion mutant lacking the last 33 amino acids of the cytoplasmic tail, indicating that the phosphorylation sites were located within the deleted region. The cAMP production mediated by this deletion mutant was not desensitized by PMA and was only poorly desensitized by GLP-1. Together, our results indicate that the production of cAMP and, hence, the stimulation of insulin secretion induced by GLP-1 can be negatively modulated by homologous and heterologous desensitization, mechanisms that involve receptor phosphorylation.

Topics: Amino Acid Sequence; Animals; Blotting, Western; Cyclic AMP; Enzyme Activation; Enzyme Inhibitors; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Indoles; Insulinoma; Molecular Sequence Data; Pancreatic Neoplasms; Peptide Fragments; Phosphorylation; Protein Kinase C; Protein Precursors; Rats; Receptors, Glucagon; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Glucagon-like peptide-I (GLP-I) is a potent incretin hormone and mediates its actions via the cyclic AMP (cAMP) pathway. The GLP-I receptor belongs to the family of seven-transmembrane domain receptors coupled to G proteins. We have analyzed the regulation of GLP-I receptor function and expression by its own ligand and the cAMP-dependent pathway in rat insulinoma-derived beta cells (RINm5F). The GLP-I receptor underwent rapid homologous desensitization, which occurred at the receptor level. This was characterized by a reduced binding capacity not mediated by protein kinase A (PKA). GLP-I receptor mRNA levels were down-regulated during incubation of cells by agents increasing cAMP levels including GLP-I itself. This effect was dependent upon time and concentration. Forskolin, the PKA activator 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole-3, 5-monophosphorothiotate, and GLP-I stabilized the GLP-I receptor mRNA. All induced down-regulation of the GLP-I receptor number within 3 h, a time point at which GLP-I receptor mRNA levels were not decreased. This effect was not influenced by cycloheximide. Therefore, in addition to transcriptional effects, posttranslational mechanisms exist to regulate GLP-I receptor numbers in insulin-secreting cells.

Topics: Animals; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cycloheximide; Enzyme Activation; Gene Expression; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Guanosine 5'-O-(3-Thiotriphosphate); Insulin; Insulin Secretion; Insulinoma; Islets of Langerhans; Pancreatic Neoplasms; Peptide Fragments; Protein Precursors; Protein Synthesis Inhibitors; Rats; Receptors, Glucagon; RNA, Messenger; Tumor Cells, Cultured

Glucagon-like peptide-1 (GLP-1) is an intestinally derived insulinotropic hormone that is currently under investigation for use in the treatment of diabetes mellitus. To investigate the Ca2+ signaling pathways by which GLP-1 may stimulate the secretion of insulin from pancreatic beta-cells, we examined its effects on the concentration of free intracellular Ca2+ ([Ca2+]i) while simultaneously determining what action it exerts on ion channel function. Measurements of [Ca2+]i were obtained from single rat beta-cells and from beta TC6 and HIT-T15 insulinoma cells loaded with the Ca2+ indicator fura-2, and changes in membrane potential and current were monitored using the perforated patch clamp technique. We report a previously undocumented action of GLP-1 and analogs of cAMP (8-bromo-cAMP, Sp- or Rp-adenosine 3',5'-cyclic monophosphothionate triethylamine) to raise [Ca2+]i that is attributable to the activation of a prolonged inward current designated here as IcAMP. Activation of IcAMP is associated with an increased membrane conductance, membrane depolarization, and triggers large increases of [Ca2+]i. IcAMP is primarily a Na+ current that is blocked by extracellularly applied La3+ or by intracellular administration of Ca2+ chelators (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl, EGTA) and which exhibits a reversal potential of about -26 mV. We propose that IcAMP results from the opening of nonselective cation channels that are activated by intracellular Ca2+ and cAMP and which might play an important role in the regulation of insulin secretion from pancreatic beta-cells.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Calcium; Cell Line; Cyclic AMP; Glucagon; Glucagon-Like Peptide 1; Insulinoma; Ion Channels; Islets of Langerhans; Membrane Potentials; Peptide Fragments; Protein Precursors; Rats; Signal Transduction; Tumor Cells, Cultured

It has been previously demonstrated that the enteric hormone glucagon-like peptide-1 (7-36 amide) (GLP-1) has acute effects on glucose-induced insulin secretion by RIN 1046-38 cells. In this study, we investigated the effects of extended exposure of RIN 1046-38 cells to GLP-1 and examine the mechanism by which GLP-1 synergizes with glucose in stimulating insulin secretion. Compared with cells cultured with glucose alone, incubation of cells with glucose plus 1 or 10 nM GLP-1 for 12 or 24 h significantly increased insulin release by about 3-fold, intracellular insulin content by 1.5-fold, and insulin messenger RNA (mRNA) by almost 2.5-fold. The insulinotropic effects of GLP-1 on RIN 1046-38 cells were accompanied by an up-regulation of both glucose transporter-1 (GLUT-1) and hexokinase I mRNA by about 2-fold. mRNA levels of GLUT-2 and glucokinase, which were low in controls, were unchanged by GLP-1 treatment. Treatment of cells with a transcription inhibitor, actinomycin D, demonstrated that elevated insulin mRNA levels after a GLP-1 exposure are mainly due to stabilization of the mRNA. In contrast, the elevated mRNA levels of GLUT-1 and hexokinase I are the result of increased transcription stimulated by GLP-1 exposure. Actinomycin D blunted the GLP-1 effect on insulin release but did not affect GLP-1 mediated elevation of insulin mRNA. This suggests that actinomycin D inhibits the transcription of the proteins necessary for insulin biosynthesis and insulin release, such as GLUT-1 and hexokinase I. Our study suggests that the mechanisms by which extended exposure of RIN 1046-38 cells to GLP-1 increases glucose-stimulated insulin secretion include significant up-regulation of glucose-sensing elements.

Topics: Animals; Culture Media, Conditioned; Cycloheximide; Dactinomycin; Drug Stability; Drug Synergism; Gene Expression Regulation; Glucagon; Glucagon-Like Peptide 1; Glucose; Glucose Transporter Type 1; Hexokinase; Insulin; Insulin Secretion; Insulinoma; Monosaccharide Transport Proteins; Pancreatic Neoplasms; Peptide Fragments; Protein Precursors; Rats; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured

Glucose-dependent insulinotropic polypeptide (GIP) plays an important role in the regulation of postprandial insulin secretion and proinsulin gene expression of pancreatic beta-cells. This study demonstrates the molecular cloning of a cDNA for the GIP-receptor from a human insulinoma lambda gt11 cDNA library. The cloned cDNA encoded a seven transmembrane domain protein of 466 amino acids which showed high homology (41%) to the human glucagon-like peptide 1 (GLP-1) receptor. Homology to the GIP receptor from rat or hamster was 79% and 81%, respectively. When transfected 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 stimulation with human GIP-1-42 (EC50 = 1.29 x 10(-13) M). The receptor accepted only human GIP 1-42 (Kd = 1.93 +/- 0.2 x 10(-8) M) and porcine truncated GIP 1-30 (Kd = 1.13 +/- 0.1 x 10(-8) M) as high affinity ligands. At 1 microM, exendin-4 and (9-39)amide weakly reduced GIP-binding (25%) whereas secretin, glucagon, glucagon-like peptide-1, vasoactive intestinal polypeptide, peptide histidine-isoleucine, and pituitary adenylyl cyclase activating peptide were without effect. In transfected CHL cells, GIP-1-42 did not increase intracellular calcium. Northern analysis revealed one transcript of human GIP receptor mRNA with an apparent size of 5.5 kb. The exact understanding of GIP receptor regulation and signal transduction will aid in the understanding of the incretin hormone's failure to exert its biological action at the pancreatic B-cell in type II diabetes mellitus.

Topics: Amino Acid Sequence; Animals; Base Sequence; Binding, Competitive; Clone Cells; Cricetinae; Gastric Inhibitory Polypeptide; Gene Library; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Humans; Insulinoma; Kinetics; Molecular Sequence Data; Pancreatic Neoplasms; Peptide Fragments; Protein Precursors; Rats; Receptors, Gastrointestinal Hormone; Receptors, Glucagon; Recombinant Proteins; Sequence Homology, Amino Acid; Signal Transduction; Substrate Specificity; Swine; Transfection; 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

The arylpiperazine L-686,398 was described as an oral hypoglycemic agent and is shown to be an insulin secretagogue in vitro. The characteristics of its activity were similar to those of the incretin glucagon-like peptide I (GLP-I). We demonstrate that both the peptide and L-686,398 increase the accumulation of cAMP in isolated ob/ob mouse pancreatic islet cells, but by different mechanisms. Although GLP-I activates adenylate cyclase, the arylpiperazine has no effect on this enzyme or on the binding of 125I-labeled GLP-I to its receptor on RINm5F rat insulinoma cell membranes. However, L-686,398 inhibits the total cAMP phosphodiesterase (PDE) activity in homogenates of ob/ob mouse pancreatic islets with an EC50 of approximately 50 mumol/l. To determine the mechanism of PDE inhibition by the arylpiperazine and to examine its specificity, we studied the kinetics of arylpiperazine inhibition of two recombinant PDEs. The arylpiperazine is a competitive inhibitor of both a human heart type III PDE and a rat type IV-D PDE. Inhibition of the type III and IV isozymes are characterized by Ki values of 27 and 5 mumol/l, respectively. Although not extremely potent, the arylpiperazine does exhibit modest selectivity between these PDEs. The observation that L-686,398 acts as a PDE inhibitor suggests that exploration for beta-cell-specific PDE isoforms may reveal novel PDEs as targets for the development of therapeutically useful glucose-dependent insulin secretagogues.

Topics: Animals; Calcium; Cells, Cultured; Cyclic AMP; Dose-Response Relationship, Drug; Glucagon; Glucagon-Like Peptide 1; Glucose; Insulin; Insulin Secretion; Insulinoma; Islets of Langerhans; Mice; Mice, Inbred C57BL; Mice, Obese; Pancreatic Neoplasms; Peptide Fragments; Phosphodiesterase Inhibitors; Piperazines; Protein Precursors; Tumor Cells, Cultured

Glucagon-like peptide-1 (GLP-1) is the major incretin hormone from the distal small intestine which stimulates basal and glucose-induced insulin secretion. Using the rat insulinoma cell line RINm5F (Gazdar et al. 1980) we investigated the effects of GLP-1 on insulin secretion, insulin content, and insulin receptor binding. During a 1 hour incubation, GLP-1 [1 nM] stimulated insulin secretion 2-fold (p < 0.01 vs controls). Incubating RINm5F for 24 h with GLP-1 [1 nM], a 1.6-fold higher cellular insulin content was observed (p < 0.01 vs controls). Moreover, GLP-1 induced a 2-fold higher capacity and a 15-fold higher affinity of 125I-insulin binding on the cell surface (p < 0.01 vs controls). Glucagon, known as a potent stimulator of insulin secretion, yielded a similar effect only in 1,000-fold higher concentrations, whereas the intracellular insulin content as well as insulin receptor binding was not increased. Taken together, in RINm5F insulinoma cells GLP-1 potently stimulates insulin secretion and insulin content, and improves insulin receptor binding.

Topics: Animals; Glucagon; Glucagon-Like Peptide 1; Insulin; Insulinoma; Iodine Radioisotopes; Microscopy, Electron; Pancreatic Neoplasms; Peptide Fragments; Protein Precursors; Rats; Receptor, Insulin; 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

Different glucagon-like peptide-1 (GLP-1) molecular forms are produced in the pancreas and the small intestine by differential processing of proglucagon. In this report, molecular forms of GLP-1 in two human pancreatic endocrine tumors were studied and compared with those in the pancreas and small intestine. A predominant GLP-1 immunoreactive form in the pancreas was eluted at the position of GLP-1(1-36) amide, whereas a predominant immunoreactive form in the ileal mucosa was eluted at the position of GLP-1(7-36) amide. In a glucagonoma, GLP-1 immunoreactive forms corresponding to GLP-1(7-36) amide and GLP-1(7-37) were predominant and immunoreactive forms at the position of GLP-1(1-36) amide and GLP-1(1-37) were minor. In another tumor, an insulinoma, immunoreactive forms were detected at the positions of GLP-1(7-36) amide, GLP-1(7-37), GLP-1(1-36) amide and GLP-1(1-37). Thus, the pattern of GLP-1 molecules in pancreatic tumors was not a pancreatic pattern and mimicked that found in the small intestine or consisted of both the patterns found in the small intestine and the pancreas. These data suggest that neoplastic transformation of the islet cells is associated with a switching in processing phenotype from islet (A) cells to intestinal (L) cells.

Topics: Chromatography, High Pressure Liquid; Glucagon; Glucagon-Like Peptide 1; Glucagonoma; Humans; Insulinoma; Pancreatic Neoplasms; Peptide Fragments; Protein Precursors; Radioimmunoassay; Reference Values

Glucagon-like peptide-1 is a gastrointestinal hormone that strongly stimulates insulin release via specific receptors on the pancreatic beta-cell. To characterize the side-chain groups required for interaction of glucagon-like peptide-1 with its receptor, we performed binding studies with alanine-substituted glucagon-like peptide-1 analogues on RINm5F insulinoma cells. The binding affinity and biological activity of glucagon-like peptide-1 have been found to be sensitive to alanine exchanges in the N-terminal positions 1, 4, 6 and the C-terminal positions 22 and 23. Alanine substitutions at positions 5, 8, 10-12, 14, 16-21 and 25-30 do not change receptor affinity. These findings could be exploited to design glucagon-like peptide-1 agonists and probably antagonists for further physiological studies.

Topics: Amino Acid Sequence; Animals; Binding Sites; Binding, Competitive; Cyclic AMP; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Insulinoma; Kinetics; Molecular Sequence Data; Pancreatic Neoplasms; Peptide Fragments; Protein Precursors; Rats; Receptors, Cell Surface; Receptors, Glucagon; Structure-Activity Relationship; Tumor Cells, Cultured

Glucagon-like peptide-1-(7-36) amide (GLP-1) and glucose-dependent insulinotropic peptide (GIP) are known incretin hormones, released from enteroendocrine cells in response to food, that enhance insulin secretion, but only in the presence of elevated blood glucose. We used a rat insulinoma cell line, RIN 1046-38, to study the mechanisms underlying the interaction of incretins and glucose. We measured insulin secretion using RIA and the reverse hemolytic plaque assay. GLP-1 stimulates insulin secretion, with a half-maximal concentration of 34 pM. GLP-1 is approximately 2 orders of magnitude more potent than GIP. GLP-1 and GIP have additive effects at submaximal concentrations, but probably not at maximal concentrations, suggesting a common signal transduction pathway. The glucose requirement for GLP-1 action can be replaced by cell membrane depolarization (20 mM KCl in the extracellular medium), suggesting that a rise of intracellular Ca2+ may be an early step required for GLP-1 action. GLP-1 stimulates insulin secretion by significantly increasing the maximum rate of insulin secretion from 10.3 +/- 2.25 to 25.2 +/- 2.94 ng insulin/mg protein.h. GLP-1 acts by recruiting 1.5-fold more cells to secrete insulin as well as enhancing insulin secretion by individual cells. Combinations of stimuli, such as glucose, cell membrane depolarization, and GLP-1, can recruit 90% of RIN 1046-38 cells to secrete insulin.

Topics: Animals; Calcium; Dose-Response Relationship, Drug; Gastric Inhibitory Polypeptide; Glucagon; Glucagon-Like Peptide 1; Glucose; Insulin; Insulin Secretion; Insulinoma; Kinetics; Membrane Potentials; Pancreatic Neoplasms; Peptide Fragments; Potassium Chloride; Protein Precursors; Rats; Signal Transduction; 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

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

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

We studied binding of 125I-labelled truncated-glucagon-like peptide-1 (proglucagon 78-107 amide) to a cloned rat insulin-producing cell line, RIN 5AH, in monolayer culture. Interaction of the peptide with pancreatic insulinoma cells was saturable and time dependent. Half-maximal binding was obtained when the cells were incubated in the presence of 3.3 x 10(-9) mol/l unlabelled truncated-glucagon-like peptide-1 (proglucagon 78-107 amide). Neither glucagon, full-length glucagon-like peptide-1 (proglucagon 72-107 amide) nor gastric inhibitory peptide competed for binding in concentrations up to 10(-6) mol/l.

Topics: Adenoma, Islet Cell; Animals; Binding, Competitive; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Insulin; Insulinoma; Intestinal Mucosa; Intestines; Kinetics; Pancreatic Neoplasms; Peptides; Rats; Receptors, Cell Surface; Receptors, Glucagon

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