International Union of Pharmacology. XXXV. The glucagon receptor family.

Peptide hormones within the secretin-glucagon family are expressed in endocrine cells of the pancreas and gastrointestinal epithelium and in specialized neurons in the brain, and subserve multiple biological functions, including regulation of growth, nutrient intake, and transit within the gut, and digestion, energy absorption, and energy assimilation. Glucagon, glucagon-like peptide-1, glucagon-like peptide-2, glucose-dependent insulinotropic peptide, growth hormone-releasing hormone and secretin are structurally related peptides that exert their actions through unique members of a structurally related G protein-coupled receptor class 2 family. This review discusses advances in our understanding of how these peptides exert their biological activities, with a focus on the biological actions and structural features of the cognate receptors. The receptors have been named after their parent and only physiologically relevant ligand, in line with the recommendations of the International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR).

Heterologous desensitization of the glucagon-like peptide-1 receptor by phorbol esters requires phosphorylation of the cytoplasmic tail at four different sites.

Glucagon-like peptide-1 stimulates glucose-induced insulin secretion by binding to a specific G protein-coupled receptor that activates the adenylyl cyclase pathway. We previously demonstrated that heterologous desensitization of the receptor by protein kinase C correlated with phosphorylation in a 33-amino acid-long segment of the receptor carboxyl-terminal cytoplasmic tail. Here, we determined that the in vivo sites of phosphorylation are four serine doublets present at positions 431/432, 441/442, 444/445, and 451/452. In vitro phosphorylation of fusion proteins containing mutant receptor C-tails, however, indicated that whereas serines at position 431/432 were good substrates for protein kinase C (PKC), serines 444/445 and 451/452 were poor substrates, and serines 441/442 were not substrates. In addition, serine 416 was phosphorylated on fusion protein but not in intact cells. This indicated that in vivo a different PKC isoform or a PKC-activated kinase may phosphorylate the receptor. The role of phosphorylation on receptor desensitization was assessed using receptor mutants expressed in COS cells or Chinese hamster lung fibroblasts. Mutation of any single serine doublet to alanines reduced the extent of phorbol 12-myristate 13-acetate-induced desensitization, whereas substitution of any combination of two serine doublets suppressed it. Our data thus show that the glucagon-like peptide-1 receptor can be phosphorylated in response to phorbol 12-myristate 13-acetate on four different sites within the cytoplasmic tail. Furthermore, phosphorylation of at least three sites was required for desensitization, although maximal desensitization was only achieved when all four sites were phosphorylated.

Expression cloning of the pancreatic beta cell receptor for the gluco-incretin hormone glucagon-like peptide 1.

Glucagon-like peptide 1 (GLP-1) is a hormone derived from the preproglucagon molecule and is secreted by intestinal L cells. It is the most potent stimulator of glucose-induced insulin secretion and also suppresses in vivo acid secretion by gastric glands. A cDNA for the GLP-1 receptor was isolated by transient expression of a rat pancreatic islet cDNA library into COS cells; this was followed by binding of radiolabeled GLP-1 and screening by photographic emulsion autoradiography. The receptor transfected into COS cells binds GLP-1 with high affinity and is coupled to activation of adenylate cyclase. The receptor binds specifically GLP-1 and does not bind peptides of related structure and similar function, such as glucagon, gastric inhibitory peptide, vasoactive intestinal peptide, or secretin. The receptor is 463 amino acids long and contains seven transmembrane domains. Sequence homology is found only with the receptors for secretin, calcitonin, and parathyroid hormone, which form a newly characterized family of G-coupled receptors.

Glucose competence of the hepatoportal vein sensor requires the presence of an activated glucagon-like peptide-1 receptor.

Activation of the hepatoportal glucose sensors by portal glucose infusion leads to increased glucose clearance and induction of hypoglycemia. Here, we investigated whether glucagon-like peptide-1 (GLP-1) could modulate the activity of these sensors. Mice were therefore infused with saline (S-mice) or glucose (P-mice) through the portal vein at a rate of 25 mg/kg. min. In P-mice, glucose clearance increased to 67.5 +/- 3.7 mg/kg. min as compared with 24.1 +/- 1.5 mg/kg. min in S-mice, and glycemia decreased from 5.0 +/- 0.1 to 3.3 +/- 0.1 mmol/l at the end of the 3-h infusion period. Coinfusion of GLP-1 with glucose into the portal vein at a rate of 5 pmol/kg. min (P-GLP-1 mice) did not increase the glucose clearance rate (57.4 +/- 5.0 ml/kg. min) and hypoglycemia (3.8 +/- 0.1 mmol/l) observed in P-mice. In contrast, coinfusion of glucose and the GLP-1 receptor antagonist exendin-(9-39) into the portal vein at a rate of 0.5 pmol/kg. min (P-Ex mice) reduced glucose clearance to 36.1 +/- 2.6 ml/kg. min and transiently increased glycemia to 9.2 +/- 0.3 mmol/l at 60 min of infusion before it returned to the fasting level (5.6 +/- 0.3 mmol/l) at 3 h. When glucose and exendin-(9-39) were infused through the portal and femoral veins, respectively, glucose clearance increased to 70.0 +/- 4.6 ml/kg. min and glycemia decreased to 3.1 +/- 0.1 mmol/l, indicating that exendin-(9-39) has an effect only when infused into the portal vein. Finally, portal vein infusion of glucose in GLP-1 receptor(-/-) mice failed to increase the glucose clearance rate (26.7 +/- 2.9 ml/kg. min). Glycemia increased to 8.5 +/- 0.5 mmol/l at 60 min and remained elevated until the end of the glucose infusion (8.2 +/- 0.4 mmol/l). Together, our data show that the GLP-1 receptor is part of the hepatoportal glucose sensor and that basal fasting levels of GLP-1 sufficiently activate the receptor to confer maximum glucose competence to the sensor. These data demonstrate an important extrapancreatic effect of GLP-1 in the control of glucose homeostasis.

Exendin-(9-39) is an inverse agonist of the murine glucagon-like peptide-1 receptor: implications for basal intracellular cyclic adenosine 3',5'-monophosphate levels and beta-cell glucose competence.

The effect of exendin-(9-39), a described antagonist of the glucagon-like peptide-1 (GLP-1) receptor, was evaluated on the formation of cAMP- and glucose-stimulated insulin secretion (GSIS) by the conditionally immortalized murine betaTC-Tet cells. These cells have a basal intracellular cAMP level that can be increased by GLP-1 with an EC50 of approximately 1 nM and can be decreased dose dependently by exendin-(9-39). This latter effect was receptor dependent, as a beta-cell line not expressing the GLP-1 receptor was not affected by exendin-(9-39). It was also not due to the endogenous production of GLP-1, because this effect was observed in the absence of detectable preproglucagon messenger RNA levels and radioimmunoassayable GLP-1. Importantly, GSIS was shown to be sensitive to this basal level of cAMP, as perifusion of betaTC-Tet cells in the presence of exendin-(9-39) strongly reduced insulin secretion. This reduction of GSIS, however, was observed only with growth-arrested, not proliferating, betaTC-Tet cells; it was also seen with nontransformed mouse beta-cells perifused in similar conditions. These data therefore demonstrated that 1) exendin-(9-39) is an inverse agonist of the murine GLP-1 receptor; 2) the decreased basal cAMP levels induced by this peptide inhibit the secretory response of betaTC-Tet cells and mouse pancreatic islets to glucose; 3) as this effect was observed only with growth-arrested cells, this indicates that the mechanism by which cAMP leads to potentiation of insulin secretion is different in proliferating and growth-arrested cells; and 4) the presence of the GLP-1 receptor, even in the absence of bound peptide, is important for maintaining elevated intracellular cAMP levels and, therefore, the glucose competence of the beta-cells.

Glucagon-like peptide-1 protects beta-cells against apoptosis by increasing the activity of an IGF-2/IGF-1 receptor autocrine loop.

OBJECTIVE: The gluco-incretin hormones glucagon-like peptide (GLP)-1 and gastric inhibitory peptide (GIP) protect beta-cells against cytokine-induced apoptosis. Their action is initiated by binding to specific receptors that activate the cAMP signaling pathway, but the downstream events are not fully elucidated. Here we searched for mechanisms that may underlie this protective effect. RESEARCH DESIGN AND METHODS: We performed comparative transcriptomic analysis of islets from control and GipR(-/-);Glp-1-R(-/-) mice, which have increased sensitivity to cytokine-induced apoptosis. We found that IGF-1 receptor expression was markedly reduced in the mutant islets. Because the IGF-1 receptor signaling pathway is known for its antiapoptotic effect, we explored the relationship between gluco-incretin action, IGF-1 receptor expression and signaling, and apoptosis. RESULTS: We found that GLP-1 robustly stimulated IGF-1 receptor expression and Akt phosphorylation and that increased Akt phosphorylation was dependent on IGF-1 but not insulin receptor expression. We demonstrated that GLP-1-induced Akt phosphorylation required active secretion, indicating the presence of an autocrine activation mechanism; we showed that activation of IGF-1 receptor signaling was dependent on the secretion of IGF-2. We demonstrated, both in MIN6 cell line and primary beta-cells, that reducing IGF-1 receptor or IGF-2 expression or neutralizing secreted IGF-2 suppressed GLP-1-induced protection against apoptosis. CONCLUSIONS: An IGF-2/IGF-1 receptor autocrine loop operates in beta-cells. GLP-1 increases its activity by augmenting IGF-1 receptor expression and by stimulating secretion; this mechanism is required for GLP-1-induced protection against apoptosis. These findings may lead to novel ways of preventing beta-cell loss in the pathogenesis of diabetes.

Desensitization and phosphorylation of the glucagon-like peptide-1 (GLP-1) receptor by GLP-1 and 4-phorbol 12-myristate 13-acetate.

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.

Agonist-induced internalization and recycling of the glucagon-like peptide-1 receptor in transfected fibroblasts and in insulinomas.

Glucagon-like peptide-1 (GLP-1) is the most potent stimulator of glucose-induced insulin secretion and its pancreatic beta-cell receptor is a member of a new subfamily of G-protein-coupled receptors which includes the receptors for vasoactive intestinal polypeptide, secretin and glucagon. Here we studied agonist-induced GLP-1 receptor internalization in receptor-transfected Chinese hamster lung fibroblasts using three different approaches. First, iodinated GLP-1 bound at 4 degrees C to transfected cells was internalized with a t 1/2 of 2-3 min following warming up of the cells to 37 degrees C. Secondly, exposure to GLP-1 induced a shift in the distribution of the receptors from plasma membrane-enriched to endosomes-enriched membrane fractions, as assessed by Western blot detection of the receptors using specific antibodies. Thirdly, continuous exposure of GLP-1 receptor-expressing cells to iodinated GLP-1 led to a linear accumulation of peptide degradation products in the medium following a lag time of 20-30 min, indicating a continuous cycling of the receptor between the plasma membrane and endosomal compartments. Potassium depletion and hypertonicity inhibited transferrin endocytosis, a process known to occur via coated pit formation, as well as GLP-1 receptor endocytosis. In contrast to GLP-1, the antagonist exendin-(9-39) did not lead to receptor endocytosis. Surface re-expression following one round of GLP-1 receptor endocytosis occurred with a half-time of about 15 min. The difference in internalization and surface re-expression rates led to a progressive redistribution of the receptor in intracellular compartments upon continuous exposure to GLP-1. Finally, endogenous GLP-1 receptors expressed by insulinoma cells were also found to be internalized upon agonist binding. Together our data demonstrate that the GLP-1 receptor is internalized upon agonist binding by a route similar to that taken by single transmembrane segment receptors. The characterization of the pathway and kinetics of GLP-1-induced receptor endocytosis will be helpful towards understanding the role of internalization and recycling in the control of signal transduction by this receptor.

Glucagon-like peptide-1-(7-36) amide, oxyntomodulin, and glucagon interact with a common receptor in a somatostatin-secreting cell line.

Glucagon-like peptide-1(7-36)amide (tGLP-1), oxyntomodulin (OXM), and glucagon are posttranslational end products of the glucagon gene expressed in intestinal L-cells. In vivo, these peptides are potent inhibitors of gastric acid secretion via several pathways, including stimulation of somatostatin release. We have examined the receptors through which these peptides stimulate somatostatin secretion using the somatostatin-secreting cell line RIN T3. tGLP-1, OXM, and glucagon stimulated somatostatin release and cAMP accumulation in RIN T3 cells to similar maximum levels, with ED50 values close to 0.2, 2, and 50 nM and 0.02, 0.3, and 8 nM, respectively. Binding of [125I]tGLP-1, [125I]OXM, and [125I]glucagon to RIN T3 plasma membranes was inhibited by the three peptides, with relative potencies as follows: tGLP-1 > OXM > glucagon. Whatever the tracer used, the IC50 for tGLP-1 was close to 0.15 nM and was shifted rightward for OXM and glucagon by about 1 and 2-3 orders of magnitude, respectively. Scatchard analyses for the three peptides were compatible with a single class of receptor sites displaying a similar maximal binding close to 2 pmol/mg protein. In the hamster lung fibroblast cell line CCL39 transfected with the receptor for tGLP-1, binding of [125I]tGLP-1 was inhibited by tGLP-1, OXM, and glucagon, with relative potencies close to those obtained with RIN T3 membranes. Chemical cross-linking of [125I]tGLP-1, [125I]OXM, and [125I]glucagon revealed a single band at 63,000 mol wt, the intensity of which was dose-dependently reduced by all three peptides. These data suggest that in the somatostatin-secreting cell line RIN T3, OXM and glucagon stimulate somatostatin release through a tGLP-1-preferring receptor. This suggests that some biological effects, previously described for these peptides, might be due to their interaction with this receptor.

Signal transduction by the cloned glucagon-like peptide-1 receptor: comparison with signaling by the endogenous receptors of beta cell lines.

Glucagon-like peptide-1 (GLP-1) is a gastrointestinal hormone that potentiates glucose-induced insulin secretion by pancreatic beta cells. The mechanisms of interaction between GLP-1 and glucose signaling pathways are not well understood. Here we studied the coupling of the cloned GLP-1 receptor, expressed in fibroblasts or in COS cells, to intracellular second messengers and compared this signaling with that of the endogenous receptor expressed in insulinoma cell lines. Binding of GLP-1 to the cloned receptor stimulated formation of cAMP with the same dose dependence and similar kinetics, compared with the endogenous receptor of insulinoma cells. Compared with forskolin-induced cAMP accumulation, that induced by GLP-1 proceeded with the same initial kinetics but rapidly reached a plateau, suggesting fast desensitization of the receptor. Coupling to the phospholipase C pathway was assessed by measuring inositol phosphate production and variations in the intracellular calcium concentration. No GLP-1-induced production of inositol phosphates could be measured in the different cell types studied. A rise in the intracellular calcium concentration was nevertheless observed in transfected COS cells but was much smaller than that observed in response to norepinephrine in cells also expressing the alpha 1B-adrenergic receptor. Importantly, no such increase in the intracellular calcium concentration could be observed in transfected fibroblasts or insulinoma cells, which, however, responded well to thrombin or carbachol, respectively. Together, our data show that interaction between GLP-1 and glucose signaling pathways in beta cells may be mediated uniquely by an increase in the intracellular cAMP concentration, with the consequent activation of protein kinase A and phosphorylation of elements of the glucose-sensing apparatus or of the insulin granule exocytic machinery.

Expression of the glucagon-like peptide-1 receptor gene in rat brain.

Evidence that glucagon-like peptide-1 (GLP-1) (7-36) amide functions as a novel neuropeptide prompted us to study the gene expression of its receptor in rat brain. Northern blot analysis showed transcripts of similar size in RINm5F cells, hypothalamus, and brain-stem. First-strand cDNA was prepared by using RNA from hypothalamus, brainstem, and R1Nm5F cells and subsequently amplified by PCR. Southern blot analysis of the PCR products showed a major 1.4-kb band in all these preparations. PCR products amplified from hypothalamus were cloned, and the nucleotide sequence of one strand was identical to that described in rat pancreatic islets. In situ hybridization studies showed specific labeling in both neurons and glia of the thalamus, hypothalamus, hippocampus, primary olfactory cortex, choroid plexus, and pituitary gland. In the hypothalamus, ventromedial nuclei cells were highly labeled. These findings indicate that GLP-1 receptors are actually synthesized in rat brain. In addition, the colocalization of GLP-1 receptors, glucokinase, and GLUT-2 in the same areas supports the idea that these cells play an important role in glucose sensing in the brain.

Glucagon-like peptide-1 increases beta-cell glucose competence and proliferation by translational induction of insulin-like growth factor-1 receptor expression.

Glucagon-like peptide-1 (GLP-1) protects beta-cells against apoptosis, increases their glucose competence, and induces their proliferation. We previously demonstrated that the anti-apoptotic effect was mediated by an increase in insulin-like growth factor-1 receptor (IGF-1R) expression and signaling, which was dependent on autocrine secretion of insulin-like growth factor 2 (IGF-2). Here, we further investigated how GLP-1 induces IGF-1R expression and whether the IGF-2/IGF-1R autocrine loop is also involved in mediating GLP-1-increase in glucose competence and proliferation. We show that GLP-1 up-regulated IGF-1R expression by a protein kinase A-dependent translational control mechanism, whereas isobutylmethylxanthine, which led to higher intracellular accumulation of cAMP than GLP-1, increased both IGF-1R transcription and translation. We then demonstrated, using MIN6 cells and primary islets, that the glucose competence of these cells was dependent on the level of IGF-1R expression and on IGF-2 secretion. We showed that GLP-1-induced primary beta-cell proliferation was suppressed by Igf-1r gene inactivation and by IGF-2 immunoneutralization or knockdown. Together our data show that regulation of beta-cell number and function by GLP-1 depends on the cAMP/protein kinase A mediated-induction of IGF-1R expression and the increased activity of an IGF-2/IGF-1R autocrine loop.

Impaired glucose homeostasis in mice lacking the alpha1b-adrenergic receptor subtype.

To assess the role of the alpha1b-adrenergic receptor (AR) in glucose homeostasis, we investigated glucose metabolism in knockout mice deficient of this receptor subtype (alpha1b-AR-/-). Mutant mice had normal blood glucose and insulin levels, but elevated leptin concentrations in the fed state. During the transition to fasting, glucose and insulin blood concentrations remained markedly elevated for at least 6 h and returned to control levels after 24 h whereas leptin levels remained high at all times. Hyperinsulinemia in the post-absorptive phase was normalized by atropine or methylatropine indicating an elevated parasympathetic activity on the pancreatic beta cells, which was associated with increased levels of hypothalamic NPY mRNA. Euglycemic clamps at both low and high insulin infusion rates revealed whole body insulin resistance with reduced muscle glycogen synthesis and impaired suppression of endogenous glucose production at the low insulin infusion rate. The liver glycogen stores were 2-fold higher in the fed state in the alpha1b-AR-/- compared with control mice, but were mobilized at the same rate during the fed to fast transition or following glucagon injections. Finally, high fat feeding for one month increased glucose intolerance and body weight in the alpha1b-AR-/-, but not in control mice. Altogether, our results indicate that in the absence of the alpha1b-AR the expression of hypotalamic NPY and the parasympathetic nervous activity are both increased resulting in hyperinsulinemia and insulin resistance as well as favoring obesity and glucose intolerance development during high fat feeding.

Normal glucagon signaling and β-cell function after near-total α-cell ablation in adult mice.

OBJECTIVEEvaluate whether healthy or diabetic adult mice can tolerate an extreme loss of pancreatic α-cells and how this sudden massive depletion affects β-cell function and blood glucose homeostasis.RESEARCH DESIGN AND METHODSWe generated a new transgenic model allowing near-total α-cell removal specifically in adult mice. Massive α-cell ablation was triggered in normally grown and healthy adult animals upon diphtheria toxin (DT) administration. The metabolic status of these mice was assessed in 1) physiologic conditions, 2) a situation requiring glucagon action, and 3) after β-cell loss.RESULTSAdult transgenic mice enduring extreme (98%) α-cell removal remained healthy and did not display major defects in insulin counter-regulatory response. We observed that 2% of the normal α-cell mass produced enough glucagon to ensure near-normal glucagonemia. β-Cell function and blood glucose homeostasis remained unaltered after α-cell loss, indicating that direct local intraislet signaling between α- and β-cells is dispensable. Escaping α-cells increased their glucagon content during subsequent months, but there was no significant α-cell regeneration. Near-total α-cell ablation did not prevent hyperglycemia in mice having also undergone massive β-cell loss, indicating that a minimal amount of α-cells can still guarantee normal glucagon signaling in diabetic conditions.CONCLUSIONSAn extremely low amount of α-cells is sufficient to prevent a major counter-regulatory deregulation, both under physiologic and diabetic conditions. We previously reported that α-cells reprogram to insulin production after extreme β-cell loss and now conjecture that the low α-cell requirement could be exploited in future diabetic therapies aimed at regenerating β-cells by reprogramming adult α-cells.