GLP-1 Receptor Expression Research Articles

Suppression of Pdx-1 perturbs proinsulin processing, insulin secretion and GLP-1 signalling in INS-1 cells

Mutations in genes encoding HNF-4alpha, HNF-1alpha and IPF-1/Pdx-1 are associated with, respectively, MODY subtypes-1, -3 and -4. Impaired glucose-stimulated insulin secretion is the common primary defect of these monogenic forms of diabetes. A regulatory circuit between these three transcription factors has also been suggested. We aimed to explore how Pdx-1 regulates beta cell function and gene expression patterns. We studied two previously established INS-1 stable cell lines permitting inducible expression of, respectively, Pdx-1 and its dominant-negative mutant. We used HPLC for insulin processing, adenovirally encoded aequorin for cytosolic [Ca2+], and transient transfection of human growth hormone or patch-clamp capacitance recordings to monitor exocytosis. Induction of DN-Pdx-1 resulted in defective glucose-stimulated and K+-depolarisation-induced insulin secretion in INS-1 cells, while overexpression of Pdx-1 had no effect. We found that DN-Pdx-1 caused down-regulation of fibroblast growth factor receptor 1 (FGFR1), and consequently prohormone convertases (PC-1/3 and -2). As a result, DN-Pdx-1 severely impaired proinsulin processing. In addition, induction of Pdx-1 suppressed the expression of glucagon-like peptide 1 receptor (GLP-1R), which resulted in marked reduction of both basal and GLP-1 agonist exendin-4-stimulated cellular cAMP levels. Induction of DN-Pdx-1 did not affect glucokinase activity, glycolysis, mitochondrial metabolism or ATP generation. The K+-induced cytosolic [Ca2+] rise and Ca2+-evoked exocytosis (membrane capacitance) were not abrogated. The severely impaired proinsulin processing combined with decreased GLP-1R expression and cellular cAMP content, rather than metabolic defects or altered exocytosis, may contribute to the beta cell dysfunction induced by Pdx-1 deficiency.

Distribution of glucokinase, glucose transporter GLUT2, sulfonylurea receptor-1, glucagon-like peptide-1 receptor and neuropeptide Y messenger RNAs in rat brain by quantitative real time RT-PCR

Glucokinase (GK), glucose transporter GLUT2, sulfonylurea receptor-1 (SUR1), glucagon-like peptide-1 receptor (GLP-1R) and neuropeptide Y (NPY) have been proposed to be involved in central glucose sensing or regulation of food intake. In this study, we combined tissue micropunch and real time reverse transcription polymerase chain reaction (RT-PCR), and measured GK, GLUT2, SUR1, GLP-1R and NPY mRNA expression in discrete areas in the hypothalamus and the hindbrain.

Insulinotropic hormone glucagon-like peptide-1 differentiation of human pancreatic islet-derived progenitor cells into insulin-producing cells.

Glucagon-like peptide-1 (GLP-1) is an intestinal incretin hormone, derived from the processing of proglucagon, that exerts insulinotropic actions on insulin-producing pancreatic islet beta-cells. Recently GLP-1 was shown to stimulate the growth and differentiation (neogenesis) of beta-cells and appears to do so by inducing the expression of the homeodomain protein IDX-1 (islet duodenum homeobox-1; also known as PDX-1, pancreatic and duodenal homeobox gene; and as IPF-1, insulin promoter factor), which is required for pancreas development and the expression of beta-cell-specific genes. Earlier we identified multipotential progenitor cells in the islet and ducts of the pancreas, termed nestin-positive islet-derived progenitor cells (NIPs). Here we report the expression of functional GLP-1 receptors on NIPs and that GLP-1 stimulates the differentiation of NIPs into insulin-producing cells. Furthermore, confluent NIP cultures express the proglucagon gene and secrete GLP-1. These findings suggest a model of islet development in which pancreatic progenitor cells express both GLP-1 receptors and proglucagon with the formation of GLP-1. Locally produced GLP-1 may act as an autocrine/paracrine developmental morphogen on receptors on NIPs, resulting in the activation of IDX-1 and the expression of the proinsulin gene conferring a beta-cell phenotype. GLP-1 may be an important morphogen both for the embryonic development of the pancreas and for the neogenesis of beta-cells in the islets of the adult pancreas.

Insulinotropic Hormone Glucagon-Like Peptide-1 Differentiation of Human Pancreatic Islet-Derived Progenitor Cells into Insulin-Producing Cells

Glucagon-like peptide-1 (GLP-1) is an intestinal incretin hormone, derived from the processing of proglucagon, that exerts insulinotropic actions on insulin-producing pancreatic islet β-cells. Recently GLP-1 was shown to stimulate the growth and differentiation (neogenesis) of β-cells and appears to do so by inducing the expression of the homeodomain protein IDX-1 (islet duodenum homeobox-1; also known as PDX-1, pancreatic and duodenal homeobox gene; and as IPF-1, insulin promoter factor), which is required for pancreas development and the expression of β-cell-specific genes. Earlier we identified multipotential progenitor cells in the islet and ducts of the pancreas, termed nestin-positive islet-derived progenitor cells (NIPs). Here we report the expression of functional GLP-1 receptors on NIPs and that GLP-1 stimulates the differentiation of NIPs into insulin-producing cells. Furthermore, confluent NIP cultures express the proglucagon gene and secrete GLP-1. These findings suggest a model of islet development in which pancreatic progenitor cells express both GLP-1 receptors and proglucagon with the formation of GLP-1. Locally produced GLP-1 may act as an autocrine/paracrine developmental morphogen on receptors on NIPs, resulting in the activation of IDX-1 and the expression of the proinsulin gene conferring a β-cell phenotype. GLP-1 may be an important morphogen both for the embryonic development of the pancreas and for the neogenesis of β-cells in the islets of the adult pancreas.

Glucagon-like Peptide (GLP)-2 Action in the Murine Central Nervous System Is Enhanced by Elimination of GLP-1 Receptor Signaling

Glucagon-like peptide-2 (GLP-2) regulates energy homeostasis via effects on nutrient absorption and maintenance of gut mucosal epithelial integrity. The biological actions of GLP-2 in the central nervous system (CNS) remain poorly understood. We studied the sites of endogenous GLP-2 receptor (GLP-2R) expression, the localization of transgenic LacZ expression under the control of the mouse GLP-2R promoter, and the actions of GLP-2 in the murine CNS. GLP-2R expression was detected in multiple extrahypothalamic regions of the mouse and rat CNS, including cell groups in the cerebellum, medulla, amygdala, hippocampus, dentate gyrus, pons, cerebral cortex, and pituitary. A 1.5-kilobase fragment of the mouse GLP-2R promoter directed LacZ expression to the gastrointestinal tract and CNS regions in the mouse that exhibited endogenous GLP-2R expression, including the cerebellum, amygdala, hippocampus, and dentate gyrus. Intracerebroventricular injection of GLP-2 significantly inhibited food intake during dark-phase feeding in wild-type mice. Disruption of glucagon-like peptide-1 receptor (GLP-1R) signaling with the antagonist exendin-(9-39) in wild-type mice or genetically in GLP-1R(-)/- mice significantly potentiated the anorectic actions of GLP-2. These findings illustrate that CNS GLP-2R expression is not restricted to hypothalamic nuclei and demonstrate that the anorectic effects of GLP-2 are transient and modulated by the presence or absence of GLP-1R signaling in vivo.

Expression of glucagon-like peptide-1 (GLP-1) receptor and the effect of GLP-1-(7-36) amide on insulin release by pancreatic islets during rat ontogenic development.

The expression of glucagon-like peptide-1 (GLP-1) receptor and the effects of GLP-1-(7-36) amide (t-GLP-1) on glucose metabolism and insulin release by pancreatic islets during rat development were studied. GLP-1 receptor mRNA was found in significant amounts in pancreatic islets from all age groups studied, GLP-1 receptor expression being maximal when pancreatic islets were incubated at physiological glucose concentration (5.5 mM), but decreasing significantly when incubated with either 1.67 or 16.7 mM glucose. Glucose utilization and oxidation by pancreatic islets from fetal and adult rats rose as a function of glucose concentration, always being higher in fetal than in adult islets. The addition of t-GLP-1 to the incubation medium did not modify glucose metabolism but gastric inhibitory polypeptide and glucagon significantly increased glucose utilization by fetal and adult pancreatic islets at 16.7 mM glucose. At this concentration, glucose produced a significant increase in insulin release by the pancreatic islets from 10-day-old and 20-day-old suckling rats and adult rats, whereas those from fetuses showed only a significant increase when glucose was raised from 1.67 to 5.5 mM. t-GLP-1 elicited an increase in insulin release by pancreatic islets from all the experimental groups when the higher glucose concentrations were used. Our findings indicate that GLP-1 receptors and the effect of t-GLP-1 on insulin release are already present in the fetus, and they therefore exclude the possibility that alterations in the action of t-GLP-1 are responsible for the unresponsiveness of pancreatic beta cells to glucose in the fetus, but stimulation of t-GLP-1 release by food ingestion in newborns may partially confer glucose competence on beta cells.

Ontogeny of the glucagon-like peptide-2 receptor axis in the developing rat intestine.

Glucagon-like peptide-2 (GLP-2) is secreted by enteroendocrine cells in the small and large intestines and exerts intestinotropic effects in the gastrointestinal mucosal epithelium of the adult rodent. The actions of GLP-2 are mediated by the GLP-2 receptor, a new member of the G protein-coupled receptor superfamily. To ascertain whether the GLP-2/GLP-2 receptor axis is expressed and functional in the developing intestine, we have studied the synthesis of GLP-2 and the expression of the GLP-2 receptor (GLP-2R) in the fetal and neonatal rat gut. GLP-2 immunoreactivity (GLP-2-IR) was detected in the fetal rat intestine, and fetal rat intestinal cell cultures secreted correctly processed GLP-2(1-33) into the medium. High levels of GLP-2(1-33) were also detected in the circulation of 13-day-old neonatal rats (P < 0.001 vs. adult). Analysis of GLP-2 receptor expression by RT-PCR demonstrated GLP-2R messenger RNA transcripts in fetal intestine and in neonatal stomach, jejunum, ileum, and colon. The levels of GLP-2R messenger RNA transcripts were comparatively higher in the fetal and neonatal intestine (P < 0.05-001 vs. adult) and declined to adult levels by postnatal day 21. Subcutaneous administration of a degradation-resistant GLP-2 analog, h[Gly2]-GLP-2 once daily for 10 days increased stomach (0.009 +/- 0.0003 vs. 0.007 +/- 0.002 g/g body mass, h[Gly2]-GLP-2-treated vs. controls; P < 0.05) and small bowel weight (0.043 +/- 0.0037 vs. 0.031 +/- 0.0030 g/g body mass; P < 0.05). h[Gly2]-GLP-2 also increased both small (2.4 +/- 0.05 vs. 1.8 +/- 0.17 cm/g body mass; P < 0.05) and large bowel length (0.32 +/- 0.01 vs. 0.25 +/- 0.02 cm/g body mass, h[Gly2]-GLP-2-treated vs. saline-treated controls, respectively; P < 0.05) in neonatal rats. These findings demonstrate that both components of the GLP-2/GLP-2 receptor axis are expressed in the fetal and neonatal intestine. The ontogenic regulation and functional integrity of this axis raises the possibility that GLP-2 may play a role in the development and/or maturation of the developing rat intestine.

Tissue-specific control of glucagon-like peptide-2 receptor expression in mice expressing a GLP-2R-Lacz transgene

Tissue-specific control of glucagon-like peptide-2 receptor expression in mice expressing a GLP-2R-Lacz transgene

Increased glucagon-like peptide-1 receptor expression in glia after mechanical lesion of the rat brain

Glucagon-like peptide-1 (GLP-1)(7–36) amide, a member of the glucagon and related peptides family, and its receptor have an anatomically specific expression in the brain. Furthermore, the GLP-1 receptor is expressed in both neurons and glia. Because after a penetrating injury a large population of astrocytes become activated and augment their expression of numerous substances, we have used in situ hybridization to determine whether the expression of the GLP-1 receptor increases in response to a penetrating injury. We have found that GLP-1 receptor expression increases dramatically along the border of the injury. Furthermore, this expression can be colocalized to glial fibrillary acidic protein (GFAP) and non-GFAP mRNA containing cells, suggesting that at least part of this increase is due to an increase in GLP-1 receptor expression in glial cells.

Tissue distribution of rat glucagon receptor and GLP-1 receptor gene expression

The regulation of glucose metabolism by glucagon and GLP-1 is well established, but novel functions for these and other proglucagon-derived peptides are less well defined. This paper highlights the diversity of both GLP-1 and glucagon activity by studying the tissue distribution of glucagon and GLP-1 receptor gene expression by both Southern blot analysis of RT-PCR products and nuclease protection assays. By Southern blot analysis of RT-PCR products, GLP-1 receptor mRNA was detected in lung, hypothalamus, hippocampus, cerebral cortex, kidney, pancreas, and throughout the gastrointestinal tract. Glucagon receptor expression was detected in liver, kidney, spleen, thymus, adrenal glands, pancreas, cerebral cortex, lung, and throughout the gastrointestinal tract. Nuclease protection assay revealed glucagon receptor expression to be highest in liver and kidney, whereas GLP-1 receptor expression was only detected by protection assay in lung, stomach, and large bowel. Despite previous evidence that other receptors for proglucagon-derived peptides may exist, no evidence of novel receptors or multiple isoforms of the glucagon and GLP-1 receptors was found, indicating that the two cloned receptors may mediate all the effects of proglucagon-derived peptides, or that novel receptors may share less homology with the glucagon and GLP-1 receptors than previously anticipated.

Glucagon-like peptide-1 receptor expression in Xenopus oocytes stimulates inositol trisphosphate-dependent intracellular Ca 2+ mobilization

The signal transduction pathway of the cloned human glucagon-like peptide-1 (GLP-1) receptor was studied in voltage-clamped Xenopus oocytes. Binding of GLP-1(7–36)amide was associated with cAMP production, increased [Ca 2+] i and activation of Ca 2+-dependent Cl − current. The effect of GLP-1(7–36)amide reflects intracellular Ca 2+ mobilization and was suppressed by injection of the Ca 2+ chelator BAPTA and the inositol trisphosphate receptor antagonist heparin. The responses were not mimicked by the adenylate cyclase activator forskolin and unaffected by the protein kinase A (PKA) inhibitor Rp-cAMPS. We conclude that GLP-1 receptor expression in Xenopus oocytes evokes inositol trisphosphate-dependent intracellular Ca 2+ mobilization independent of the cAMP/PKA signaling pathway.

Reciprocal cellular distribution of glucagon-like peptide-1 (GLP-1) immunoreactivity and GLP-1 receptor mRNA in pancreatic islets of rat.

The respective cellular distribution of glucagon-like peptide-1 (GLP-1) immunoreactivity and mRNA expression of the GLP-1 receptor was compared in rat pancreas by means of immunohistochemistry and in situ hybridization. GLP-1 immunoreactivity was present in the marginal zone of rat pancreatic islets. In contrast, GLP-1 receptor mRNA signals were confined to the central part of pancreatic islets. Neither GLP-1 immunoreactivity nor GLP-1 receptor mRNA signals were detected in the exocrine pancreatic acinar cells or duct cells. The differential distribution of GLP-1 immunoreactivity and GLP-1 receptor mRNA signals indicates that the GLP-1 amino acid sequence is present in the alpha-cell zone of pancreatic islets, whereas the GLP-1 receptor is expressed mainly by beta cells. Thus, our data by in situ hybridization demonstrates the significant expression of GLP-1 receptors on beta cells but makes a significant expression on alpha cells rather unlikely.

Cloning and functional expression of the human islet GLP-1 receptor. Demonstration that exendin-4 is an agonist and exendin-(9-39) an antagonist of the receptor

Cloning and functional expression of the human islet GLP-1 receptor. Demonstration that exendin-4 is an agonist and exendin-(9-39) an antagonist of the receptor

Cloning and Functional Expression of the Human Islet GLP-1 Receptor: Demonstration That Exendin-4 Is an Agonist and Exendin-(9–39) an Antagonist of the Receptor

A complementary DNA for a glucagon-like peptide-1 receptor was isolated from a human pancreatic islet cDNA library. The isolated clone encoded a protein with 90% identity to the rat receptor. In stably transfected fibroblasts, the receptor bound [125I]GLP-1 with high affinity (Kd = 0.5 nM) and was coupled to adenylate cyclase as detected by a GLP-1-dependent increase in cAMP production (EC50 = 93 pM). Two peptides from the venom of the lizard Heloderma suspectum, exendin-4 and exendin-(9-39), displayed similar ligand binding affinities to the human GLP-1 receptor. Whereas exendin-4 acted as an agonist of the receptor, inducing cAMP formation, exendin-(9-39) was an antagonist of the receptor, inhibiting GLP-1-induced cAMP production. Because GLP-1 has been proposed as a potential agent for treatment of NIDDM, our present data will contribute to the characterization of the receptor binding site and the development of new agonists of this receptor.