Glucagon Binding Research Articles

Antagonistic Effect and In Vitro Activity of Dauricine on Glucagon Receptor.

Abnormal increases in glucagon (GCG) are the primary cause of type II diabetes mellitus. When GCG interacts with a glucagon receptor (GCGR), GCG can increase the blood glucose level. In this paper, a compound that could interfere with the binding of GCG and GCGR to inhibit the increase of blood glucose was investigated. First, molecular docking was used to conduct preliminary screening of compounds whose active components could combine with GCGR by AutoDock Vina. The binding of the receptor-ligand complex was analyzed by PyMOL. Results showed that dauricine could tightly bind to the receptor pocket. Second, the plasmid pcDNA3.1(+)-GCGR containing the target gene was transfected into HEK293 cells for expression, which was the cell model established to screen GCGR antagonist. Dauricine, the lead compound of glucagon receptor antagonist (GRA), was screened using the GRA screening model in vitro. Finally, using [Des-His1, Glu9]-Glucagon amide as the positive control, flow cytometry was used to express the antagonistic effect of the compound. Consequently, dauricine can antagonize the GCGR.

Delineating the activation mechanism and conformational landscape of a class B G protein-coupled receptor glucagon receptor

Class B G protein-coupled receptors (GPCRs) are important targets in the treatment of metabolic syndrome and diabetes. Although multiple structures of class B GPCRs–G protein complexes have been elucidated, the detailed activation mechanism of the receptors remains unclear. Here, we combine Gaussian accelerated molecular dynamics simulations and Markov state models (MSM) to investigate the activation mechanism of a canonical class B GPCR, human glucagon receptor–GCGR, including the negative allosteric modulator-bound inactive state, the agonist glucagon-bound active state, and both glucagon- and Gs-bound fully active state. The free-energy landscapes of GCGR show the conformational ensemble consisting of three activation-associated states: inactive, active, and fully active. The structural analysis indicates the high dynamics of GCGR upon glucagon binding with both active and inactive conformations in the ensemble. Significantly, the H8 and TM6 exhibits distinct features from the inactive to the active states. The additional simulations demonstrate the role of H8 in the recruitment of Gs. Gs binding presents a crucial function of stabilizing the glucagon binding site and MSM highlights the absolute requirement of Gs to help the GCGR reach the fully active state. Together, our results reveal the detailed activation mechanism of GCGR from the view of conformational dynamics.

In silico evaluation of the downstream effect of mutated glucagon is consistent with higher blood glucose homeostasis in Galliformes and Strigiformes

In contrast to mammals, glucagon is reported as a much more potent blood glucose modulator in birds. Interestingly, we have found p.Thr16Ser mutation, a variation in the highly conserved glucagon hormone, in Galliformes as well as Strigiformes. To check the effect of this mutation on the receptor binding of glucagon, we predicted the ancestral glucagon receptor sequence of all available Galliformes and Strigiformes species. Subsequently, we analysed their binding to the mutated and wild type glucagon (ancestral) by molecular dynamics simulation. At first, we made a model of ancestral glucagon receptor and ancestral mutated, and wild type glucagon in the order Galliformes and Strigiformes. Then we performed molecular dynamics for each Galliformes and Strigiformes receptor as well as each glucagon peptide, respectively. The final structures were used for docking simulation of glucagon to their receptors. The results of the docking simulations showed a stronger binding affinity of mutated glucagon to glucagon receptors. Afterward, we obtained blood glucose concentrations of all available Galliformes members, as well as all available members of its only taxonomic neighbour (order Anseriformes) in superorder Galloanserae. Interestingly the p.Thr16Ser mutation could finely cluster these two orders into two groups: higher blood glucose concentration (order Galliformes, 17.64 ± 1.66 mMol/L) and lower blood glucose concentration (order Anseriformes, 11.34 ± 1.11 mMol/L). Strigiformes which carry the mutated glucagon peptide show also high blood glucose concentrations (17.40 ± 1.51 mMol/L). Therefore, the results suggest this mutation, which leads to stronger binding affinity of mutated glucagon to its receptor, may be a driving force for higher blood glucose homeostasis in the related birds.

682-P: Novel Dual Glucagon and Glucagon-Like Peptide-1 Receptor Agonist LY3305677 Improves Glucose Control, Reduces Body Weight, and Increases Energy Expenditure in Mice

LY3305677 (LY) is a synthetic peptide analog of mammalian oxyntomodulin with a fatty acyl side chain to extend time action. LY functions are mediated by binding and activation of both glucagon (GcgR) and glucagon-like peptide-1 (GLP-1R) receptors. LY is bound with potent binding affinity (Ki) to human and mouse GcgR (Ki: 17.7 nM and 15.9 nM, respectively) and GLP-1R (Ki: 28.6 nM and 25.1 nM, respectively). LY dose-dependently stimulated insulin secretion in mouse pancreatic islets (EC50: 5.2 nM) and in mice. In oral glucose tolerance tests of diet-induced obese (DIO) and streptozotocin-induced diabetic mice, a single subcutaneous (SC) dose of LY significantly reduced glucose area under the curve by up to 65%. SC injections of LY to DIO mice dose-dependently reduced serum triglycerides and PCSK9 (proprotein convertase subtilisin/kexin type 9) levels and dose-dependently increased FGF21 (fibroblast growth factor 21) levels. In DIO mice treated over 18 days, LY 30 nmol/kg reduced body weight to a greater extent (33%) than semaglutide 60 nmol/kg (12%), and decreased food intake by 50% compared to vehicle. At Day 18, LY 30 nmol/kg group lost 33% of initial bodyweight, which was primarily due to 70% reduced fat mass. LY reduced liver triglycerides by greater than 70% and increased serum ketones by more than 3-fold compared to vehicle, suggestive of increased fatty acid oxidation. LY decreased body weight in both GcgR knock-out (KO) and GLP-1R KO mice, indicating that activation of both receptors contributed to greater weight loss. To assess potential changes in energy expenditure, DIO mice were treated chronically with SC LY (15 nmol/kg), semaglutide (60 nmol/kg), or vehicle. LY, but not semaglutide, increased energy expenditure as compared with vehicle. These preclinical studies indicate that novel dual GcgR and GLP-1R agonist LY may have potential to enhance metabolic health by improving dyslipidemia, hyperglycemia, or obesity. Disclosure Y. Chen: None. H. Qu: Employee; Self; Eli Lilly and Company, Stock/Shareholder; Self; Eli Lilly and Company. A. Mezo: None. T. Coskun: Employee; Self; Eli Lilly and Company, Stock/Shareholder; Self; Eli Lilly and Company. M. Song: None. W. C. Roell: Employee; Self; Eli Lilly and Company, Stock/Shareholder; Self; Eli Lilly and Company. K. B. Bokvist: Employee; Self; Eli Lilly and Company, Sanofi-Aventis Deutschland GmbH, Stock/Shareholder; Self; Johnson & Johnson, Sanofi-Aventis Deutschland GmbH, Stock/Shareholder; Spouse/Partner; Novo Nordisk A/S. J. S. Moyers: Employee; Self; Eli Lilly and Company, Stock/Shareholder; Self; Eli Lilly and Company. M. K. Thomas: Employee; Self; Eli Lilly and Company, Stock/Shareholder; Self; Eli Lilly and Company. F. Valenzuela: None. Funding Eli Lilly and Company

Molecular cloning, sequence characteristics, and tissue expression analysis of glucagon receptor gene in Bama minipig

Recent studies have shown that the glucagon receptor (GCGR) plays an important role in the development of type 2 diabetes mellitus. Both pigs and humans exhibit significantly similar behaviors in their glucose and lipid metabolism. In this study, the obtained Bama minipig GCGR coding sequence was 1437 bp encoding 479 amino acids (AA), which demonstrated higher sequence homology with humans than other species. It showed the highest expression profile in the liver, followed by the lung and kidney. In addition, the three-dimensional structure analysis showed that the porcine GCGR protein also had a classic sevenfold transmembrane region and a stalk region at the N-terminus for ligand binding. The stalk region of GCGR possessed five AA variations. The ligand binding pocket of GCGR has one AA variation in the key region, none of which affected the glucagon binding verified by the crystal structure mutagenesis in humans. There was no variation found in the region of membrane anchoring, hydrophobic bond, salt bridge, and hydrogen bond. However, the Gly40Ser mutation in mice resulted in major diseases, meaning that pigs are more suitable for the evaluation of GCGR-related drugs than mice.

Mechanochemical Activation of Class-B G-Protein-Coupled Receptor upon Peptide-Ligand Binding.

Glucagon binding to the class-B G-protein-coupled glucagon receptor (GCGR) triggers the release of glucose from the liver during fasting. Recently, GCGR crystal structures have highlighted the conformation and molecular details of inactive and active receptor states. However, the dynamics of the conformational changes accompanying GCGR activation remains unclear. Here, we use multiplex force-distance curve-based atomic force microscopy (FD-based AFM) to probe in situ glucagon binding to individual GCGRs and monitor dynamically the transition to the active conformer. After a "dock" step, in which glucagon is partially bound to the GCGR extracellular domain, further interactions of the N-terminus with the transmembrane domain trigger an increase in the stiffness of the complex, adopting a highly stable and rigid "lock" conformer. This mechanotransduction is key for G-protein recruitment.

Identification of plasma binding proteins for glucose-dependent insulinotropic polypeptide.

Glucose-dependent insulinotropic polypeptide (GIP), secreted from enteroendocrine K cells, has potent insulin-releasing and extrapancreatic glucoregulatory activities. However, exogenous GIP has less potent biological effects compared with another incretin hormone, GLP-1, which limits its use for the treatment of type 2 diabetes. The fate and secretion of administered native GIP remain unclear. The aim of this study was to identify plasma binding proteins for human GIP. Fluorescent-labelled GIP was added to fresh human plasma and subjected to clear native polyacrylamide gel electrophoresis (CN-PAGE). Then fluorescent protein bands were in-gel trypsin-digested and subjected to liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis, revealing the presence of albumin, immunoglobulin G (IgG) and transferrin. In contrast to GIP, the binding of fluorescent GLP-1 and glucagon to plasma protein fractions were minimal. CN-PAGE analysis of synthetic GIP incubated with human serum albumin, purified IgG or transferrin, and subsequent western blot analysis revealed that GIP binds to each of these proteins. Taken together, these results indicate that GIP readily binds to albumin, IgG and transferrin, three plasma proteins highly abundant in the human peripheral circulation. Separation of protein complexes using CN-PAGE and the identification of in-gel digested proteins by LC-MS/MS analysis provide a promising strategy to identify plasma binding proteins for bioactive peptides.

A double negative: inhibition of hepatic Gi signaling improves glucose homeostasis.

Hepatic glucose production (HGP) is a key determinant of glucose homeostasis. Glucagon binding to its cognate seven-transmembrane Gs-coupled receptor in hepatocytes stimulates cAMP production, resulting in increased HGP. In this issue of the JCI, Rossi and colleagues tested the hypothesis that activation of hepatic Gi-coupled receptors, which should inhibit cAMP production, would oppose the cAMP-inducing action of glucagon and thereby decrease HGP. Surprisingly, however, the opposite occurred: activation of Gi signaling increased HGP via a novel mechanism, while inhibition of Gi signaling reduced HGP. These results define a new physiologic role for hepatic Gi signaling and identify a potential therapeutic target for HGP regulation.

Activation and conformational dynamics of a class B G-protein-coupled glucagon receptor.

The human glucagon receptor (GCGR) is a class B G-protein-coupled receptor (GPCR). The GCGR can be activated by glucagon and regulates the release of glucose. The GCGR has been proposed to be an important drug target for type 2 diabetes. Based on the structural model of a full-length glucagon-bound GCGR (glu-GCGR), we performed accelerated molecular dynamics (aMD) simulations, potential of mean force (PMF) calculations, cross-correlation analysis and community network analysis to study the activation mechanism and the conformational dynamics during the activation process. The PMF map depicts three different conformational states of the GCGR: the inactive, intermediate and active states. The activation of the GCGR is characterized by the outward movement of the intracellular side of helix VI. In the active state of the GCGR, the Arg173(2.46)-Ser350(6.41) and Glu245(3.50)-Thr351(6.42) hydrogen bonds break, and the χ1 rotamer of Phe322(5.54) changes from perpendicular to parallel to helix VI. The binding of the agonist glucagon decreases the correlated motions of the extracellular loops (ELCs) and the helices around the glucagon-binding site. During the activation of the GCGR, the connections between the intracellular sides of helices become weaker, and the connections between glucagon and ECLs and the extracellular sides of helices become stronger. These facilitate G-protein coupling on the intracellular side and glucagon binding on the extracellular side, and stabilize the GCGR in the active state. We expect that this study can provide useful information on the activation mechanism of the GCGR and facilitate the future design of GCGR inhibitors.

Computational identification of novel natural inhibitors of glucagon receptor for checking type II diabetes mellitus.

BackgroundInteraction of the small peptide hormone glucagon with glucagon receptor (GCGR) stimulates the release of glucose from the hepatic cells during fasting; hence GCGR performs a significant function in glucose homeostasis. Inhibiting the interaction between glucagon and its receptor has been reported to control hepatic glucose overproduction and thus GCGR has evolved as an attractive therapeutic target for the treatment of type II diabetes mellitus.ResultsIn the present study, a large library of natural compounds was screened against 7 transmembrane domain of GCGR to identify novel therapeutic molecules that can inhibit the binding of glucagon with GCGR. Molecular dynamics simulations were performed to study the dynamic behaviour of the docked complexes and the molecular interactions between the screened compounds and the ligand binding residues of GCGR were analysed in detail. The top scoring compounds were also compared with already documented GCGR inhibitors- MK-0893 and LY2409021 for their binding affinity and other ADME properties. Finally, we have reported two natural drug like compounds PIB and CAA which showed good binding affinity for GCGR and are potent inhibitor of its functional activity.ConclusionThis study contributes evidence for application of these compounds as prospective small ligand molecules against type II diabetes. Novel natural drug like inhibitors against the 7 transmembrane domain of GCGR have been identified which showed high binding affinity and potent inhibition of GCGR

Inhibitory Mechanism of an Allosteric Antibody Targeting the Glucagon Receptor

Elevated glucagon levels and increased hepatic glucagon receptor (GCGR) signaling contribute to hyperglycemia in type 2 diabetes. We have identified a monoclonal antibody that inhibits GCGR, a class B G-protein coupled receptor (GPCR), through a unique allosteric mechanism. Receptor inhibition is mediated by the binding of this antibody to two distinct sites that lie outside of the glucagon binding cleft. One site consists of a patch of residues that are surface-exposed on the face of the extracellular domain (ECD) opposite the ligand-binding cleft, whereas the second binding site consists of residues in the αA helix of the ECD. A docking model suggests that the antibody does not occlude the ligand-binding cleft. We solved the crystal structure of GCGR ECD containing a naturally occurring G40S mutation and found a shift in the register of the αA helix that prevents antibody binding. We also found that alterations in the αA helix impact the normal function of GCGR. We present a model for the allosteric inhibition of GCGR by a monoclonal antibody that may form the basis for the development of allosteric modulators for the treatment of diabetes and other class B GPCR-related diseases.

Same Hormone, Different Actions

Glucagon signaling in the hypothalamus limits hepatic glucose production, rather than stimulating glucose production as this hormone does in the liver.

Anti-Diabetic Efficacy and Impact on Amino Acid Metabolism of GRA1, a Novel Small-Molecule Glucagon Receptor Antagonist

Hyperglucagonemia is implicated in the pathophysiology of hyperglycemia. Antagonism of the glucagon receptor (GCGR) thus represents a potential approach to diabetes treatment. Herein we report the characterization of GRA1, a novel small-molecule GCGR antagonist that blocks glucagon binding to the human GCGR (hGCGR) and antagonizes glucagon-induced intracellular accumulation of cAMP with nanomolar potency. GRA1 inhibited glycogenolysis dose-dependently in primary human hepatocytes and in perfused liver from hGCGR mice, a transgenic line of mouse that expresses the hGCGR instead of the murine GCGR. When administered orally to hGCGR mice and rhesus monkeys, GRA1 blocked hyperglycemic responses to exogenous glucagon. In several murine models of diabetes, acute and chronic dosing with GRA1 significantly reduced blood glucose concentrations and moderately increased plasma glucagon and glucagon-like peptide-1. Combination of GRA1 with a dipeptidyl peptidase-4 inhibitor had an additive antihyperglycemic effect in diabetic mice. Hepatic gene-expression profiling in monkeys treated with GRA1 revealed down-regulation of numerous genes involved in amino acid catabolism, an effect that was paralleled by increased amino acid levels in the circulation. In summary, GRA1 is a potent glucagon receptor antagonist with strong antihyperglycemic efficacy in preclinical models and prominent effects on hepatic gene-expression related to amino acid metabolism.

Molecular basis for negative regulation of the glucagon receptor

Members of the class B family of G protein-coupled receptors (GPCRs) bind peptide hormones and have causal roles in many diseases, ranging from diabetes and osteoporosis to anxiety. Although peptide, small-molecule, and antibody inhibitors of these GPCRs have been identified, structure-based descriptions of receptor antagonism are scarce. Here we report the mechanisms of glucagon receptor inhibition by blocking antibodies targeting the receptor's extracellular domain (ECD). These studies uncovered a role for the ECD as an intrinsic negative regulator of receptor activity. The crystal structure of the ECD in complex with the Fab fragment of one antibody, mAb1, reveals that this antibody inhibits glucagon receptor by occluding a surface extending across the entire hormone-binding cleft. A second antibody, mAb23, blocks glucagon binding and inhibits basal receptor activity, indicating that it is an inverse agonist and that the ECD can negatively regulate receptor activity independent of ligand binding. Biochemical analyses of receptor mutants in the context of a high-resolution ECD structure show that this previously unrecognized inhibitory activity of the ECD involves an interaction with the third extracellular loop of the receptor and suggest that glucagon-mediated structural changes in the ECD accompany receptor activation. These studies have implications for the design of drugs to treat class B GPCR-related diseases, including the potential for developing novel allosteric regulators that target the ECDs of these receptors.

Identification of Key Residues for the Binding of Glucagon to the N-Terminal Domain of its Receptor: An Alanine Scan and Modeling Study

Glucagon plays an essential role in the glycemia maintenance during fasting, but also aggravates hyperglycemia in diabetic patients. A series of analogues of glucagon were synthesized replacing each amino acid of the C-terminal region (residues 15-29) with alanine. The residues affecting the binding to the glucagon receptor are found to be located on one face of the glucagon helix. Several 3-dimensional models of the N-terminal domain of the glucagon receptor in complex with its ligand peptide were built and used to analyze the peptide-receptor interface in terms of the nature of the peptide residues and the interactions they form with the receptor. The models suggest that glucagon keeps its native helical structure upon binding, and that a large part of the interface formed with the receptor is hydrophobic. We find that in the C-terminal region, F22, V23, M27, and D15 are the most important residues for peptide binding. They bury a large portion of their solvent accessible surface area and make numerous interactions with the receptor mainly of the hydrophobic type.

Analysis of the glucagon receptor first extracellular loop by the substituted cysteine accessibility method

Glucagon is an important hormone for the prevention of hypoglycemia, and contributes to the hyperglycemia observed in diabetic patients, yet very little is known about its receptor structure and the receptor–glucagon interaction. In related receptors, the first extracellular loop, ECL1, is highly variable in length and sequence, suggesting that it might participate in ligand recognition. We applied a variant of the SCAM (Substituted Cysteine Accessibility Method) to the glucagon receptor ECL1 and sequentially mutated positions 197 to 223 to cysteine. Most of the mutations (15/27) affected the glucagon potency, due either to a modification of the glucagon binding site, or to the destabilization of the active receptor conformation. We reasoned that side chains accessible to glucagon must also be accessible to large, hydrophilic cysteine reagents. We therefore evaluated the accessibility of the introduced cysteines to maleimide-PEO2-biotin ((+)-biotinyl-3-maleimido-propionamidyl-3,6-dioxa-octanediamine), and tested the effect of pretreatment of intact cells with a large cationic cysteine reagent, MTSET ([2-(trimethylammonium)ethyl]methanethiosulfonate bromide), on glucagon potency. Our results suggest that the second and third transmembrane helices (TM2 and TM3) are extended to position 202 and from position 215, respectively, and separated by a short β stretch (positions 203–209). Glucagon binding induced a conformational change close to TM2: L198C was accessible to the biotin reagent only in the presence of glucagon. Most other mutations affected the receptor activation rather than glucagon recognition, but S217 and D218 (at the top of TM3) were good candidates for glucagon recognition and V221 was very close to the binding site.

Mutational and Cysteine Scanning Analysis of the Glucagon Receptor N-terminal Domain

The glucagon receptor belongs to the B family of G-protein coupled receptors. Little structural information is available about this receptor and its association with glucagon. We used the substituted cysteine accessibility method and three-dimensional molecular modeling based on the gastrointestinal insulinotropic peptide and glucagon-like peptide 1 receptor structures to study the N-terminal domain of this receptor, a central element for ligand binding and specificity. Our results showed that Asp(63), Arg(116), and Lys(98) are essential for the receptor structure and/or ligand binding because mutations of these three residues completely disrupted or markedly impaired the receptor function. In agreement with these data, our models revealed that Asp(63) and Arg(116) form a salt bridge, whereas Lys(98) is engaged in cation-π interactions with the conserved tryptophans 68 and 106. The native receptor could not be labeled by hydrophilic cysteine biotinylation reagents, but treatment of intact cells with [2-(trimethylammonium)ethyl]methanethiosulfonate increased the glucagon binding site density. This result suggested that an unidentified protein with at least one free cysteine associated with the receptor prevented glucagon recognition and that [2-(trimethylammonium)ethyl]methanethiosulfonate treatment relieved this inhibition. The substituted cysteine accessibility method was also performed on 15 residues selected using the three-dimensional models. Several receptor mutants, despite a relatively high predicted cysteine accessibility, could not be labeled by specific reagents. The three-dimensional models show that these mutated residues are located on one face of the protein. This could be part of the interface between the receptor and the unidentified inhibitory protein, making these residues inaccessible to biotinylation compounds.

Discordance between glucokinase activity and insulin and glucagon receptor changes occurring during liver regeneration in the rat.

During regeneration of rat livers following 70% hepatectomy, insulin binding sites on hepatocyte plasma membranes are increased after 24-48 hours, glucagon binding sites are reduced on days 2-8, and the resultant insulin/glucagon binding ratio is markedly increased. An apparent paradox was the finding of a depression of the activity of an insulin associated enzyme, glucokinase, at a time when the number of insulin binding sites was increased.

Glucagon receptors.

Glucagon is a pancreatic peptide hormone that, as a counterregulatory hormone for insulin, stimulates glucose release by the liver and maintains glucose homeostasis. First described as a glucagon binding entity functionally linked to adenylyl cyclase, the glucagon receptor is a member of the family B receptors within the G protein coupled superfamily of seven transmembrane-spanning receptors. During the past decade, considerable progress has been made in the identification of the molecular determinants of the glucagon receptor that are important for ligand binding and signal transduction, in the development of glucagon analogs and of nonpeptide small molecules acting as receptor antagonists, and in the characterization of the mechanisms involved in the regulation of expression of the glucagon receptor gene. In the present review, the current knowledge of glucagon receptor structure, function and expression is described, with emphasis on the metabolic fate of glucagon and on the endocytosis and cell itinerary of both ligand and receptor.

Glucagon-stimulated but not Isoproterenol-stimulated Glucose Formation Inhibition by Interleukin-6 in Primary Cultured Rat Hepatocytes

During prolonged sepsis, impairment of glucose supply by the liver leads to hypoglycemia. Our aim was to investigate whether proinflammatory cytokine interleukin-6, a major mediator of the hepatic acute phase reaction, could contribute to this impairment by inhibiting hepatic glucose production stimulated by glucagon or isoproterenol in rat hepatocytes. Interleukin-6 inhibited the stimulation of glucose formation from glycogen by glucagon but not by isoproterenol in cultured rat hepatocytes. This was confirmed in the perfused rat liver. In cultured hepatocytes, the increase in cyclic adenosine-3',5'-monophosphate formation by glucagon was inhibited by interleukin-6, which was probably due to attenuation of glucagon binding to the glucagon receptor. The increase in cyclic adenosine-3',5'-monophosphate stimulated by isoproterenol was not affected by interleukin-6. However, the cytokine inhibited both expression of the key gluconeogenic control enzyme, phosphoenolpyruvate carboxykinase, stimulated by glucagon and isoproterenol. Thus, while increased glucose demand during the acute-phase reaction might initially be accomplished by catecholamine-mediated stimulation of glucose formation from glycogen, inhibition of gluconeogenesis by interleukin-6 may contribute to the impairment of glucose homeostasis during the prolonged acute phase reaction.