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Structural insight into antibody-mediated antagonism of the Glucagon-like peptide-1 Receptor.

ABSTRACT: The Glucagon-like peptide-1 receptor (GLP-1R) is a member of the class B G protein-coupled receptor (GPCR) family and a well-established target for the treatment of type 2 diabetes. The N-terminal extracellular domain (ECD) of GLP-1R is important for GLP-1 binding and the crystal structure of the GLP-1/ECD complex was reported previously. The first structure of a class B GPCR transmembrane (TM) domain was solved recently, but the full length receptor structure is still not well understood. Here we describe the molecular details of antibody-mediated antagonism of the GLP-1R using both in vitro pharmacology and x-ray crystallography. We showed that the antibody Fab fragment (Fab 3F52) blocked the GLP-1 binding site of the ECD directly and thereby acts as a competitive antagonist of native GLP-1. Interestingly, Fab 3F52 also blocked a short peptide agonist believed to engage primarily the transmembrane and extracellular loop region of GLP-1R, whereas functionality of an allosteric small-molecule agonist was not inhibited. This study has implications for the structural understanding of the GLP-1R and related class B GPCRs, which is important for the development of new and improved therapeutics targeting these receptors.

SUBMITTER: Hennen S

PROVIDER: S-EPMC4872540 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Structural insight into antibody-mediated antagonism of the Glucagon-like peptide-1 Receptor.

Hennen Stephanie S Kodra János T JT Soroka Vladyslav V Krogh Berit O BO Wu Xiaoai X Kaastrup Peter P Ørskov Cathrine C Rønn Sif G SG Schluckebier Gerd G Barbateskovic Silvia S Gandhi Prafull S PS Reedtz-Runge Steffen S

Scientific reports 20160519

The Glucagon-like peptide-1 receptor (GLP-1R) is a member of the class B G protein-coupled receptor (GPCR) family and a well-established target for the treatment of type 2 diabetes. The N-terminal extracellular domain (ECD) of GLP-1R is important for GLP-1 binding and the crystal structure of the GLP-1/ECD complex was reported previously. The first structure of a class B GPCR transmembrane (TM) domain was solved recently, but the full length receptor structure is still not well understood. Here ...[more]

PMID: 27196125

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Project description:ObjectiveTreatment with glucagon receptor antagonists (GRAs) reduces blood glucose but causes dyslipidemia and accumulation of fat in the liver. We investigated the acute and chronic effects of glucagon on lipid metabolism in mice.MethodsChronic effects of glucagon receptor signaling on lipid metabolism were studied using oral lipid tolerance tests (OLTTs) in overnight fasted glucagon receptor knockout (Gcgr-/-) mice, and in C57Bl/6JRj mice treated with a glucagon receptor antibody (GCGR Ab) or a long-acting glucagon analogue (GCGA) for eight weeks. Following treatment, liver tissue was harvested for RNA-sequencing and triglyceride measurements. Acute effects were studied in C57Bl/6JRj mice treated with a GRA or GCGA 1 h or immediately before OLTTs, respectively. Direct effects of glucagon on hepatic lipolysis were studied using isolated perfused mouse liver preparations. To investigate potential effects of GCGA and GRA on gastric emptying, paracetamol was, in separate experiments, administered immediately before OLTTs.ResultsPlasma triglyceride concentrations increased 2-fold in Gcgr-/- mice compared to their wild-type littermates during the OLTT (P = 0.001). Chronic treatment with GCGR Ab increased, whereas GCGA treatment decreased, plasma triglyceride concentrations during OLTTs (P < 0.05). Genes involved in lipid metabolism were upregulated upon GCGR Ab treatment while GCGA treatment had opposite effects. Acute GRA and GCGA treatment, respectively, increased (P = 0.02) and decreased (P = 0.003) plasma triglyceride concentrations during OLTTs. Glucagon stimulated hepatic lipolysis, evident by an increase in free fatty acid concentrations in the effluent from perfused mouse livers. In line with this, GCGR Ab treatment increased, while GCGA treatment decreased, liver triglyceride concentrations. The effects of glucagon appeared independent of changes in gastric emptying of paracetamol.ConclusionsGlucagon receptor signaling regulates triglyceride metabolism, both chronically and acutely, in mice. These data expand glucagon´s biological role and implicate that intact glucagon signaling is important for lipid metabolism. Glucagon agonism may have beneficial effects on hepatic and peripheral triglyceride metabolism.

Project description:Aims/hypothesisGlucagon like peptide-1 (GLP-1) enhances glucose-dependent insulin secretion by binding to GLP-1 receptors (GLP1Rs) on pancreatic beta cells. GLP-1 mimetics are used in the clinic for the treatment of type 2 diabetes, but despite their therapeutic success, several clinical effects of GLP-1 remain unexplained at a mechanistic level, particularly in extrapancreatic tissues. The aim of this study was to generate and characterise a monoclonal antagonistic antibody for the GLP1R for use in vivo.MethodsA naive phage display selection strategy was used to isolate single-chain variable fragments (ScFvs) that bound to GLP1R. The ScFv with the highest affinity, Glp1R0017, was converted into a human IgG1 and characterised further. In vitro antagonistic activity was assessed in a number of assays: a cAMP-based homogenous time-resolved fluorescence assay in GLP1R-overexpressing cell lines, a live cell cAMP imaging assay and an insulin secretion assay in INS-1 832/3 cells. Glp1R0017 was further tested in immunostaining of mouse pancreas, and the ability of Glp1R0017 to block GLP1R in vivo was assessed by both IPGTT and OGTT in C57/Bl6 mice.ResultsAntibodies to GLP1R were selected from naive antibody phage display libraries. The monoclonal antibody Glp1R0017 antagonised mouse, human, rat, cynomolgus monkey and dog GLP1R. This antagonistic activity was specific to GLP1R; no antagonistic activity was found in cells overexpressing the glucose-dependent insulinotropic peptide receptor (GIPR), glucagon like peptide-2 receptor or glucagon receptor. GLP-1-stimulated cAMP and insulin secretion was attenuated in INS-1 832/3 cells by Glp1R0017 incubation. Immunostaining of mouse pancreas tissue with Glp1R0017 showed specific staining in the islets of Langerhans, which was absent in Glp1r knockout tissue. In vivo, Glp1R0017 reversed the glucose-lowering effect of liraglutide during IPGTTs, and reduced glucose tolerance by blocking endogenous GLP-1 action in OGTTs.Conclusions/interpretationGlp1R0017 is a monoclonal antagonistic antibody to the GLP1R that binds to GLP1R on pancreatic beta cells and blocks the actions of GLP-1 in vivo. This antibody holds the potential to be used in investigating the physiological importance of GLP1R signalling in extrapancreatic tissues where cellular targets and signalling pathways activated by GLP-1 are poorly understood.

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Structural insight into antibody-mediated antagonism of the Glucagon-like peptide-1 Receptor.

Publications

Structural insight into antibody-mediated antagonism of the Glucagon-like peptide-1 Receptor.

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