GLP-1 action in L6 myotubes is via a receptor different from the pancreatic GLP-1 receptor

The incretin hormone glucagon-like peptide-1 (GLP-1)-(736)amide is best known for its antidiabetogenic actions mediated via aGLP-1 receptor present on pancreatic endocrine cells. To investigatethe molecular mechanisms of GLP-1 action in muscle, we used cultured L6myotubes. In L6 myotubes, GLP-1 enhanced insulin-stimulated glycogensynthesis by 140% while stimulatingCO₂ production and lactateformation by 150%. In the presence of IBMX, GLP-1 diminished cAMPlevels to 83% of IBMX alone. In L6 myotubes transfected with pancreatic GLP-1 receptor, GLP-1 increased cAMP levels and inhibited glycogen synthesis by 60%. An antagonist of pancreatic GLP-1 receptor, exendin-4-(939), inhibited GLP-1-mediated glycogen synthesis in GLP-1receptor-transfected L6 myotubes. However, in parental L6 myotubes,exendin-4-(939) and GLP-1-(136) amide, an inactive peptide onpancreatic GLP-1 receptor, displaced¹²⁵I-labeled GLP-1binding and stimulated glycogen synthesis by 186 and 130%,respectively. These results suggest that the insulinomimetic effects ofGLP-1 in L6 cells are likely to be mediated by a receptor that isdifferent from the GLP-1 receptor found in the pancreas.

     

PACAP contributes to insulin secretion after gastric glucose gavage in mice

Pituitary adenylate cyclase-activating polypeptide (PACAP) is localized to pancreatic ganglia governing the parasympathetic nerves, which contribute to prandial insulin secretion. We hypothesized that this contribution involves PACAP and show here that the PACAP receptor antagonist PACAP-(6---27) (1.5 nmol/kg iv) reduces the 15-min insulin response to gastric glucose (150 mg/mouse) by 18% in anesthetized mice (P = 0.041). The reduced insulinemia was not due to inhibited release of the incretin factor glucagon-like peptide 1 (GLP-1) because PACAP-(6---27) enhanced the GLP-1 response to gastric glucose. Furthermore, the GLP-1 antagonist exendin-3-(9---39) (30 nmol/kg) exerted additive inhibitory effect on the insulin response when combined with PACAP-(6---27). The PACAP antagonism was specific because intravenous PACAP-(6---27) inhibited the insulin response to intravenous PACAP-27 plus glucose without affecting the insulin response to intravenous glucose alone (1 g/kg) or glucose together with other insulin secretagogues of potential incretin relevance of intestinal (GLP-1, gastric inhibitory polypeptide, cholecystokinin) and neural (vasoactive intestinal peptide, gastrin-releasing peptide, cholinergic agonism) origin. We conclude that PACAP contributes to the insulin response to gastric glucose in mice and suggest that PACAP is involved in the regulation of prandial insulin secretion.     

GLP-1-analogues resistant to degradation by dipeptidyl-peptidase IV in vitro

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.     

Glucagon-Like Peptide-1 Receptor Ligand Interactions: Structural Cross Talk between Ligands and the Extracellular Domain

Activation of the glucagon-like peptide-1 receptor (GLP-1R) in pancreatic β-cells potentiates insulin production and is a current therapeutic target for the treatment of type 2 diabetes mellitus (T2DM). Like other class B G protein-coupled receptors (GPCRs), the GLP-1R contains an N-terminal extracellular ligand binding domain. N-terminal truncations on the peptide agonist generate antagonists capable of binding to the extracellular domain, but not capable of activating full length receptor. The main objective of this study was to use Hydrogen/deuterium exchange (HDX) to identify how the amide hydrogen bonding network of peptide ligands and the extracellular domain of GLP-1R (nGLP-1R) were altered by binding interactions and to then use this platform to validate direct binding events for putative GLP-1R small molecule ligands. The HDX studies presented here for two glucagon-like peptide-1 receptor (GLP-1R) peptide ligands indicates that the antagonist exendin-4_[9-39] is significantly destabilized in the presence of nonionic detergents as compared to the agonist exendin-4. Furthermore, HDX can detect stabilization of exendin-4 and exendin-4_[9-39] hydrogen bonding networks at the N-terminal helix [Val19 to Lys27] upon binding to the N-terminal extracellular domain of GLP-1R (nGLP-1R). In addition we show hydrogen bonding network stabilization on nGLP-1R in response to ligand binding, and validate direct binding events with the extracellular domain of the receptor for putative GLP-1R small molecule ligands.     

Biological activity of GLP-1-analogues with N-terminal modifications

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. Therefore, various GLP-1 analogues with alterations in cleavage positions were synthesized. GLP-1-receptor binding was investigated in RINm5F cells. Biological activity of the GLP-1 analogues was investigated in vitro by measuring cAMP production in RINm5F cells. GLP-1 analogues with modifications in position 2 were not cleaved by DPP IV and showed receptor affinity and in vitro biological activity comparable to native GLP-1. Analogues with alterations in positions 2 and 8, 2 and 9 or 8 and 9 showed a significant decrease in receptor affinity and biological activity. In vivo biological activity was tested in pigs. GLP-1 analogues were administered subcutaneously followed by an intravenous bolus injection of glucose. Plasma glucose and insulin were monitored over 4 h. Compared to native GLP-1, analogues with an altered position 2 showed similar or increased potency and biological half-time. Other GLP-1 analogues were less active. Despite the lack of degradation of these GLP-1 analogues by DPP IV in vitro, their biological action is as short as that of GLP-1, except for desamino-GLP-1, indicating that other degradation enzymes are important in vivo. Alterations of GLP-1 in positions 8 or 9 result in a loss of biological activity without extending biological half-time.     

Human Y-79 Retinoblastoma Cells Exhibit Specific Insulin Receptors

The presence of insulin receptors was investigated in human Y-79 retinoblastoma cells grown in suspension culture. The binding of [125I] insulin to these cells was time, temperature, and pH dependent, was competed for by insulin and proinsulin but not other peptides, and was inhibited by antibodies against the insulin receptor. The Scatchard plot of insulin competition data was curvilinear and was resolved into a high-affinity (KD approximately 0.5 X 10(-9) M)/low-capacity (approximately 3,000 sites/cell) and a low-affinity (KD approximately 1 X 10(-7) M)/high-capacity (approximately 155,000 sites/cell) component. Negative cooperativity was not found, in agreement with other studies in rodent neural cells. However, in contrast to studies with rodent cells, insulin specifically down-regulated its receptor on human Y-79 cells after prolonged exposure. In conclusion, these data show for the first time the presence of specific insulin receptors in human Y-79 retinoblastoma cells. Because these cells were previously shown to have several characteristics typical of neural cells, we propose their use as a model to study the effects of insulin on neural and retinal tissues of human origin.     

Solubilization of active GLP-1 (7–36)amide receptors from RINm5F plasma membranes

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-β-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 conentration-dependently by addition of unlabeled peptide. Scatchard analysis of binding data revealed a single class of binding sites with K_a= 0.26 ± 0.03 nM and B_max= 65.4 ± 21.24 fmol/mg of protein for the membrane-bound receptor and K_a= 22.54 ± 4.42 μM and B_max= 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-γ-S nor GDP-β-S affected binding or 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.     

Enhanced protection against cytokine- and fatty acid-induced apoptosis in pancreatic beta cells by combined treatment with glucagon-like peptide-1 receptor agonists and insulin analogues.

We recently showed that insulin analogues exhibit a beta-cell protective function. The aim of this study was to test if the anti-apoptotic activity of GLP-1 agonists and insulin analogues is mediated by different pathways and if combined treatment may provide augmented protection against beta-cell death. Incubation of INS-1 cells with cytokines or fatty acids increased the number of apoptotic cells and caspase 3 activity, which was reduced by pretreatment with GLP-1 and its receptor agonists exendin-4 and AVE0010 by 50-60%. Similar effects (about 40% reduction) were observed after pretreatment with several insulin analogues. Combined treatment revealed additive activity and resulted in prevention of both cytokine- and fatty acid-induced apoptosis by up to 80%. No acute Akt-phosphorylation in response to GLP-1 receptor agonists could be observed, however, it became detectable after 24-hour stimulation. Gene silencing of Akt2 increased cytokine-induced apoptosis 2-fold. Under these conditions the beta-cell protective activity of AVE0010 remained completely unaltered. We show here that the anti-apoptotic activity of GLP-1 and its receptor agonists AVE0010 and exendin-4 is enhanced by addition of insulin analogues and that the anti-apoptotic action of GLP-1 mimetics is mostly unrelated to Akt2 signaling. It is suggested that combination of GLP-1 receptor agonists and insulin analogues, specifically insulin glargine, may represent a new therapeutic option for preservation of beta-cell mass in type 2 diabetic patients.     

Galanin: structural requirements for binding and signal transduction in RINm5F insulinoma cells.

Receptors for galanin, a neuropeptide inhibiting insulin release, have been described on RINm5F insulinoma cells. To characterize structural requirements for binding and biological activity of galanin, we studied binding and inhibition of hormone stimulated intracellular cAMP-production of N-terminal galanin fragments and -analogues in RINm5F cells. Half-maximal binding and potency were the same for all peptides used. Active peptides had the following rank of potency: galanin = galanin(1-22(23)Cys) greater than galanin(1-29(4)NLe) greater than galanin(1-18) greater than galanin(1-29(7)DAla) greater than galanin(1-29(2)DTrp4NLe7DAla) greater than galanin(1-29(2)DTrp). Galanin(3-29) was inactive. Therefore the first two amino acids of the galanin molecule with the indole side chain of the tryptophane residue in the right steric position are crucial for receptor binding.     

The isolated N-terminal domain of the glucagon-like peptide-1 (GLP-1) receptor binds exendin peptides with much higher affinity than GLP-1

Two fragments of the receptor for glucagon-like peptide-1 (GLP-1), each containing the N-terminal domain, were expressed and characterized in either bacterial or mammalian cells. The first fragment, rNT-TM1, included the N-terminal domain and first transmembrane helix and was stably expressed in the membrane of human embryonic kidney 293 cells. The second, 6H-rNT, consisted of only the N-terminal domain of the receptor fused with a polyhistidine tag at its N terminus. The latter fragment was expressed in Escherichia coli in the form of inclusion bodies from which the protein was subsequently purified and refolded in vitro. Although both receptor fragments displayed negligible (125)I-labeled GLP-1(7-36)amide-specific binding, they both displayed high affinity for the radiolabeled peptide antagonist (125)I-exendin-4(9-39). Competition binding studies demonstrated that the N-terminal domain of the GLP-1 receptor maintains high affinity for the agonist exendin-4 as well as the antagonists exendin-4(3-39) and exendin-4(9-39) whereas, in contrast, GLP-1 affinity was greatly reduced. This study shows that although the exendin antagonists are not dependent upon the extracellular loops and transmembrane helices for maintaining their normal high affinity binding, the endogenous agonist GLP-1 requires regions outside of the N-terminal domain. Hence, distinct structural features in exendin-4, between residues 9 and 39, provide additional affinity for the N-terminal domain of the receptor. These data are consistent with a model for the binding of peptide ligands to the GLP-1 receptor in which the central and C-terminal regions of the peptides bind to the N terminus of the receptor, whereas the N-terminal residues of peptide agonists interact with the extracellular loops and transmembrane helices.     

Crystal Structure of Glucagon-like Peptide-1 in Complex with the Extracellular Domain of the Glucagon-like Peptide-1 Receptor

GLP-1 (glucagon-like peptide-1) is an incretin released from intestinal L-cells in response to food intake. Activation of the GLP-1 receptor potentiates the synthesis and release of insulin from pancreatic β-cells in a glucose-dependent manner. The GLP-1 receptor belongs to class B of the G-protein-coupled receptors, a subfamily characterized by a large N-terminal extracellular ligand binding domain. Exendin-4 and GLP-1 are 50% identical, and exendin-4 is a full agonist with similar affinity and potency for the GLP-1 receptor. We recently solved the crystal structure of the GLP-1 receptor extracellular domain in complex with the competitive antagonist exendin-4(9–39). Interestingly, the isolated extracellular domain binds exendin-4 with much higher affinity than the endogenous agonist GLP-1. Here, we have solved the crystal structure of the extracellular domain in complex with GLP-1 to 2.1 Åresolution. The structure shows that important hydrophobic ligand-receptor interactions are conserved in agonist- and antagonist-bound forms of the extracellular domain, but certain residues in the ligand-binding site adopt a GLP-1-specific conformation. GLP-1 is a kinked but continuous α-helix from Thr¹³ to Val³³ when bound to the extracellular domain. We supplemented the crystal structure with site-directed mutagenesis to link the structural information of the isolated extracellular domain with the binding properties of the full-length receptor. The data support the existence of differences in the binding modes of GLP-1 and exendin-4 on the full-length GLP-1 receptor.     

Role of glucagon-like peptide-1 and its agonists on early prevention of cardiac remodeling in type 1 diabetic rat hearts

Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted from intestinal L cells upon nutrients ingestion, and is currently used for treating diabetes mellitus. It plays an important role in receptor modulation and cross talk with insulin at the coronary endothelium (CE) and cardiomyocytes (CM) in diabetic type 1 rat heart model. We studied the effects of insulin, GLP-1 analogues (exendin-4), and dipeptidyl peptidase-IV (DPP-IV) inhibitor on GLP-1 cardiac receptor modulation. The binding affinity of GLP-1 to its receptor on CE and CM was calculated using a rat heart perfusion model with [(125)I]-GLP-1(7-36). Tissue samples from the heart were used for immunostaining and Western blot analyses. GLP-1 systemic blood levels were measured using ELISA. GLP-1 binding affinity (τ) increased on the CE (0.33 ± 0.01 vs. 0.25 ± 0.01 min; p < 0.001) and decreased on the CM (0.29 ± 0.02 vs. 0.43 ± 0.02 min; p < 0.001) in the diabetic non-treated rats when compared to normal. There was normalization of τ back to baseline on the CE and CM levels with insulin and DPP-IV inhibitor treatment, respectively. Histological sections and immunofluorescence showed receptor up-regulation in diabetic rats with significant decrease and even normalization with the different treatment strategies. Systemic GLP-1 levels increased after 14 days of diabetes induction (10 ± 3.7 vs. 103 ± 58 pM; p = 0.0005). In conclusion, there is a significant GLP-1 receptor affinity modulation on the CE and CM levels in rats with diabetes type 1, and a cross talk with GLP-1 analogues in early prevention of cardiac remodeling.     

Effects of Selenium and Exendin-4 on Glucagon-Like Peptide-1 Receptor, IRS-1, and Raf-1 in the Liver of Diabetic Rats

Selenium and exendin-4 exert antidiabetic effects by unknown mechanisms. Herein, we investigated their effects on the expression of glucagon-like peptide-1 receptor (GLP-1R), insulin receptor substrate-1 (IRS-1), and Raf-1 in the livers of rats with streptozotocin-induced diabetes. Diabetic rats were injected intraperitoneally with exendin-4 (0.03?μg/kg body weight) twice daily or treated with 5?ppm selenium as sodium selenite in drinking water for 4?weeks. Both selenium and exendin-4 reduced the hyperglycemia in diabetic rats. Induction of diabetes mellitus resulted in decreased level of GLP-1R and increased levels of IRS-1 and Raf-1 in the liver. Treatment of diabetic rats with selenium or exendin-4 resulted in increased level of GLP-1R and decreased levels of IRS-1 and Raf-1 in the liver, compared with the levels in diabetic rats. Therefore, the antidiabetic actions of selenium and exendin-4 involve their effects on GLP-1R, IRS-1, and Raf-1 levels in the liver.     

Glucagon-like peptide-1 receptor signaling modulates beta cell apoptosis

Glucagon-like peptide-1 (GLP-1) stimulates insulin secretion and augments beta cell mass via activation of beta cell proliferation and islet neogenesis. We examined whether GLP-1 receptor signaling modifies the cellular susceptibility to apoptosis. Mice administered streptozotocin (STZ), an agent known to induce beta cell apoptosis, exhibit sustained improvement in glycemic control and increased levels of plasma insulin with concomitant administration of the GLP-1 agonist exendin-4 (Ex-4). Blood glucose remained significantly lower for weeks after cessation of exendin-4. STZ induced beta cell apoptosis, which was significantly reduced by co-administration of Ex-4. Conversely, mice with a targeted disruption of the GLP-1 receptor gene exhibited increased beta cell apoptosis after STZ administration. Exendin-4 directly reduced cytokine-induced apoptosis in purified rat beta cells exposed to interleukin 1beta, tumor necrosis fator alpha, and interferon gamma in vitro. Furthermore, Ex-4-treated BHK-GLP-1R cells exhibited significantly increased cell viability, reduced caspase activity, and decreased cleavage of beta-catenin after treatment with cycloheximide in vitro. These findings demonstrate that GLP-1 receptor signaling directly modifies the susceptibility to apoptotic injury, and provides a new potential mechanism linking GLP-1 receptor activation to preservation or enhancement of beta cell mass in vivo.     

Inhibition of insulin secretion by KN-62, a specific inhibitor of the multifunctional Ca2+/calmodulin-dependent protein kinase II.

The effects of KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (CamPKII), on insulin secretion and protein phosphorylation were studied in rat pancreatic islets and RINm5F cells. KN-62 was found to dose-dependently inhibit autophosphorylation of CamPKII in subcellular preparations of RINm5F cells (K0.5 = 3.1 +/- 0.3 microM), but had no effect on protein kinase C or myosin light chain kinase activity. KN-62, but not the inactive analogue KN-04, dose-dependently inhibited glucose-induced insulin release (K0.5 = 1.5 +/- 0.5 microM) in a manner similar to the inhibition of CamPKII autophosphorylation. KN-62 (10 microM) inhibited carbachol (in the presence of 8 mM glucose) and potassium-stimulated insulin secretion from islets by 53% and 59%, respectively. These results support a role of CamPKII in glucose-sensitive insulin secretion.     

Exendin-4, a glucagon-like peptide-1 receptor agonist, inhibits cell apoptosis induced by lipotoxicity in pancreatic β-cell line

Lipotoxicity plays an important role in the underlying mechanism of type 2 diabetes mellitus. Prolonged exposure of pancreatic β-cells to elevated concentrations of fatty acid is associated with β-cell apoptosis. Recently, glucagon-like peptide-1 (GLP-1) receptor agonists have been reported to have direct beneficial effects on β-cells, such as anti-apoptotic effects, increased β-cell mass, and improvement of β-cell function. The mechanism of GLP-1 receptor agonists' protection of pancreatic β-cells against lipotoxicity is not completely understood. We investigated whether the GLP-1 receptor agonist exendin-4 promoted cell survival and attenuated palmitate-induced apoptosis in murine pancreatic β-cells (MIN6). Exposure of MIN6 cells to palmitate (0.4mM) for 24h caused a significant increase in cell apoptosis, which was inhibited by exendin-4. Exposure of MIN6 cells to exendin-4 caused rapid activation of protein kinase B (PKB) under lipotoxic conditions. Furthermore, LY294002, a PI3K inhibitor, abolished the anti-lipotoxic effect of exendin-4 on MIN6 cells. Exendin-4 also inhibited the mitochondrial pathway of apoptosis and down-regulated Bax in MIN6 cells. Exendin-4 enhanced glucose-stimulated insulin secretion in the presence of palmitate. Our findings suggest that exendin-4 may prevent lipotoxicity-induced apoptosis in MIN6 cells through activation of PKB and inhibition of the mitochondrial pathway.     

Exendin-4 inhibits structural remodeling and improves Ca2+ homeostasis in rats with heart failure via the GLP-1 receptor through the eNOS/cGMP/PKG pathway

The glucagon-like peptide-1 receptor (GLP-1R) agonist exendin-4 is a long-acting analog of GLP-1, which stimulates insulin secretion and is clinically used in the treatment of type 2 diabetes. Previous studies have demonstrated that GLP-1 agonists and analogs serve as cardioprotective factors in various conditions. Disturbances in calcium cycling are characteristic of heart failure (HF); therefore, the aim of this study was to investigate the effect of exendin-4 (a GLP-1 mimetic) on the regulation of calcium handling and to identify the underlying mechanisms in an HF rat model after myocardial infarction (MI). Rats underwent surgical ligation of the left anterior descending coronary artery or sham surgery prior to infusion with vehicle, exendin-4, or exendin-4 and exendin9-39 for 4 weeks. Exendin-4 treatment decreased MI size, suppressed chamber dilation, myocyte hypertrophy, and fibrosis and improved in vivo heart function in the rats subjected to MI. Exendin-4 resulted in an increase in circulating GLP-1 and GLP-1R in ventricular tissues. Additionally, exendin-4 activated the eNOS/cGMP/PKG signaling pathway and inhibited the Ca²⁺/calmodulin-dependent kinase II (CaMKII) pathways. Myocytes isolated from exendin-4-treated hearts displayed higher Ca²⁺ transients, higher sarcoplasmic reticulum Ca²⁺ content, and higher l-type Ca²⁺ current densities than MI hearts. Exendin-4 treatment restored the protein expression of sarcoplasmic reticulum Ca²⁺ uptake ATPase (SERCA2a), phosphorylated phospholamban (PLB) and Cav1.2 and decreased the levels of phosphorylated ryanodine receptor (RyR). Moreover, the favorable effects of exendin-4 were significantly inhibited by exendin9-39 (a GLP-1 receptor antagonist). Exendin-4 treatment of an HF rat model after MI inhibited cardiac and cardiomyocytes progressive remodeling. In addition, Ca²⁺ handling and its molecular modulation were also improved by exendin-4 treatment. The beneficial effects of exendin-4 on cardiac remodeling may be mediated through activation of the eNOS/cGMP/PKG pathway.