title: Over-expression of the glucagon-like peptide-1 receptor on INS-1 cells confers autocrine stimulation of insulin gene promoter activity: a strategy for production of pancreatic beta-cell lines for use in transplantation.
abstract: 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.
authors.affiliation: Department of Physiology and Neuroscience, New York University School of Medicine, New York, NY 10016, USA.
authors.forename: Oleg G
authors.initials: OG
authors.lastname: Chepurny
authors.forename: George G
authors.initials: GG
authors.lastname: Holz
mesh_terms: D000818:Animals; D019898:Autocrine Communication; D002460:Cell Line; D003920:Diabetes Mellitus / Q000628:therapy; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000077270:Exenatide; D005786:Gene Expression Regulation; D005818:Genetic Engineering; D005934:Glucagon / Q000378:metabolism*; D052216:Glucagon-Like Peptide 1; D007211:Indoles / Q000494:pharmacology; D007328:Insulin / Q000235:genetics*; D007340:Insulinoma / Q000473:pathology; D007515:Islets of Langerhans / Q000473:pathology; D016381:Islets of Langerhans Transplantation; D010446:Peptide Fragments / Q000378:metabolism*; D010455:Peptides / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011498:Protein Precursors / Q000378:metabolism*; D051381:Rats; D018000:Receptors, Peptide / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012107:Research Design; D015290:Second Messenger Systems; D014407:Tumor Cells, Cultured; D014688:Venoms / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D007211:Indoles; D007328:Insulin; D010446:Peptide Fragments; D010455:Peptides; D011498:Protein Precursors; D018000:Receptors, Peptide; D011994:Recombinant Proteins; D014688:Venoms; D052216:Glucagon-Like Peptide 1; D005934:Glucagon; D000077270:Exenatide; D011493:Protein Kinase C; C064758:Ro 31-8220