Evaluation of commercially available glucagon receptor antibodies and glucagon receptor expression
Anna BilleschouChristian D. JohansenJens Bager ChristensenSasha A. S. KjeldsenKatrine D. GalsgaardMarie Winther‐SørensenReza SerizawaMads HornumEsteban PorriniJens PedersenCathrine ØrskovLise Lotte GluudCharlotte Mehlin SørensenJens J. HolstReidar AlbrechtsenNicolai J. Wewer Albrechtsen
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Abstract Glucagon is a major regulator of metabolism and drugs targeting the glucagon receptor (GCGR) are being developed. Insight into tissue and cell-specific expression of the GCGR is important to understand the biology of glucagon and to differentiate between direct and indirect actions of glucagon. However, it has been challenging to localize the GCGR in tissue due to low expression levels and lack of specific methods. Immunohistochemistry has frequently been used for GCGR localization, but antibodies targeting G-protein-coupled-receptors may be inaccurate. We evaluated all currently commercially available GCGR antibodies. The antibody, ab75240 (Antibody no. 11) was found to perform best among the twelve antibodies tested and using this antibody we found expression of the GCGR in the kidney, liver, preadipocytes, pancreas, and heart. Three antibody-independent approaches all confirmed the presence of the GCGR within the pancreas, liver and the kidneys. GCGR expression should be evaluated by both antibody and antibody-independent approaches.Keywords:
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Glucagon exerts effects on the mammalian heart. These effects include alterations in the force of contraction, beating rate, and changes in the cardiac conduction system axis. The cardiac effects of glucagon vary according to species, region, age, and concomitant disease. Depending on the species and region studied, the contractile effects of glucagon can be robust, modest, or even absent. Glucagon is detected in the mammalian heart and might act with an autocrine or paracrine effect on the cardiac glucagon receptors. The glucagon levels in the blood and glucagon receptor levels in the heart can change with disease or simultaneous drug application. Glucagon might signal via the glucagon receptors but, albeit less potently, glucagon might also signal via glucagon-like-peptide-1-receptors (GLP1-receptors). Glucagon receptors signal in a species- and region-dependent fashion. Small molecules or antibodies act as antagonists to glucagon receptors, which may become an additional treatment option for diabetes mellitus. Hence, a novel review of the role of glucagon and the glucagon receptors in the mammalian heart, with an eye on the mouse and human heart, appears relevant. Mouse hearts are addressed here because they can be easily genetically modified to generate mice that may serve as models for better studying the human glucagon receptor.
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Peptidic glucagon antagonists have been shown to lower blood glucose levels in diabetic models (1-3), but attempts to identify small molecular weight glucagon receptor-binding antagonists have met with little success. Skyrin, a fungal bisanthroquinone, exhibits functional glucagon antagonism by uncoupling the glucagon receptor from adenylate cyclase activation in rat liver membranes (1). We have examined the effects of skyrin on cells transfected with the human glucagon receptor and on isolated rat and human hepatocytes. The skyrin used was isolated from Talaromyces wortmanni American Type Culture Collection 10517. In rat hepatocytes, skyrin (30 micromol/l) inhibited glucagon-stimulated cAMP production (53%) and glucose output (IC50 56 micromol/l). There was no detectable effect on epinephrine or glucagon-like peptide 1 (GLP-1) stimulation of these parameters, which demonstrates skyrin's selective activity. Skyrin was also evaluated in primary cultures of human hepatocytes. Unlike cell lines, which are largely unresponsive to glucagon, primary human hepatocytes exhibited glucagon-dependent cAMP production for 14 days in culture (EC50 10 nmol/l). Skyrin (10 micromol/l) markedly reduced glucagon-stimulated cAMP production (55%) and glycogenolysis (27%) in human hepatocytes. The inhibition of glucagon stimulation was a specific property displayed by skyrin and oxyskyrin but not shared by other bisanthroquinones. Skyrin is the first small molecular weight nonpeptidic agent demonstrated to interfere with the coupling of glucagon to adenylate cyclase independent of binding to the glucagon receptor. The data presented in this study indicate that functional uncoupling of the human glucagon receptor from cAMP production results in metabolic effects that could reduce hepatocyte glucose production and hence alleviate diabetic hyperglycemia.
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Hundred years after the discovery of glucagon, its biology remains enigmatic. Accurate measurement of glucagon has been essential for uncovering its pathological hypersecretion that underlies various metabolic diseases including not only diabetes and liver diseases but also cancers (glucagonomas). The suggested key role of glucagon in the development of diabetes has been termed the bihormonal hypothesis. However, studying tissue-specific knockout of the glucagon receptor has revealed that the physiological role of glucagon may extend beyond blood-glucose regulation. Decades ago, animal and human studies reported an important role of glucagon in amino acid metabolism through ureagenesis. Using modern technologies such as metabolomic profiling, knowledge about the effects of glucagon on amino acid metabolism has been expanded and the mechanisms involved further delineated. Glucagon receptor antagonists have indirectly put focus on glucagon’s potential role in lipid metabolism, as individuals treated with these antagonists showed dyslipidemia and increased hepatic fat. One emerging field in glucagon biology now seems to include the concept of hepatic glucagon resistance. Here, we discuss the roles of glucagon in glucose homeostasis, amino acid metabolism, and lipid metabolism and present speculations on the molecular pathways causing and associating with postulated hepatic glucagon resistance.
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Glucagon-secreting pancreatic α-cells can interact with β-cells. However, the long-term effect of glucagon on the function and phenotype of β-cells has remained elusive. Our new finding shows that long-term glucagon induces β-cell dedifferentiation in cultured β-cells. FoxO1 inhibitor mimicks whereas glucagon signaling blockage by GCGR mAb reverses the effect of glucagon. In type 2 diabetic mice, GCGR mAb increases β-cell area, improves β-cell function, and inhibits β-cell dedifferentiation, and the effect is partially mediated by FoxO1.
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Multiple bioactive peptides are produced from proglucagon encoded by glucagon gene ( Gcg ). Glucagon is produced in islet α ‐cells through processing by prohormone convertase 2 (Pcsk2) and exerts its action through the glucagon receptor (Gcgr). Although it is difficult to produce a genetic model that harbours isolated glucagon deficiency without affecting the production of other peptides derived from proglucagon, three different animal models that harbour deficiencies in glucagon signalling have been generated by gene targeting strategy. Although both Pcsk2 −/− and Gcgr −/− mice display lower blood glucose levels, homozygous glucagon‐GFP knock‐in mice ( Gcg gfp/gfp ) display normoglycaemia despite complete glucagon deficiency. In Gcg gfp/gfp mice, the metabolic impact of glucagon deficiency is probably ameliorated by lower plasma insulin levels and glucagon‐independent mechanisms that maintain gluconeogeneis. As both Pcsk2 −/− and Gcgr −/− mice exhibit increased production of glucagon‐like peptide‐1 (GLP‐1), which is absent in Gcg gfp/gfp , GLP‐1 is the likely cause of the difference in metabolic impact of glucagon deficiency in these animal models. Although all the three models display islet ‘ α ’‐cell hyperplasia, the mechanisms involved remain to be elucidated. Studies using Pcsk2 −/− , Gcgr −/− and Gcg gfp/gfp mice, especially in combination with α ‐cell ablation models such as pancreas‐specific aristaless‐related homeobox (ARX) knockout mice, should further clarify the physiological and pathological roles of glucagon in the regulation of metabolism and the control of islet cell differentiation and proliferation.
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Des-histidine-glucagon (DH-glucagon, glucagon(2-29)) does not activate the glucagon-sensitive adenylate cyclase system present in either liver plasma membranes or in fat-cell "ghosts", but inhibits the response of these systems to submaximal concentrations of glucagon. DH-glucagon also inhibits, competitively, the binding of [(125)I]glucagon to its receptor in liver plasma membranes. Amino-terminal fragments of glucagon (glucagon(1-21), glucagon(1-23)) and carboxy-terminal fragments (glucagon(20-29), glucagon(22-29)) failed to activate adenylate cyclase, to inhibit the response of the enzyme to glucagon, or to compete with labeled glucagon at its receptor. It is concluded that the amino-terminal histidine residue of glucagon is essential for biological activity and that a hydrophobic near-carboxy-terminal region (residues 22-27) is essential for binding of glucagon to its receptor. Amino-terminal histidine may also contribute to the binding of glucagon, since the apparent affinity of DH-glucagon for the receptor is only about one-sixth that of glucagon. Thus, essentially the entire molecule of glucagon must be considered to be the biologically active species.Because, as shown elsewhere, the binding of glucagon to its receptor shows characteristics of hydrophobic bonding, and because certain detergents induce conformational changes in the carboxy-terminal binding region of glucagon, the binding is probably of a lipophilic type.
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Hyperglucagonemia in type 2 diabetes (T2D) may result from impaired hepatic glucagon signaling and consequent reduced glucagon-induced amino acid (AA) turnover leading to higher AA concentrations and stimulation of glucagon secretion. In a double-blinded, cross-over study, blood was sampled from 10 overnight fasted patients with T2D (BMI [mean±SD]: 33.0±5.4 kg/m2; HbA1c: 46.2±6.1 mmol/mol, 6.4±0.6%) and 10 matched healthy controls (BMI: 31.7±4.2 kg/m2; HbA1c: 33.9±3.0 mmol/mol, 5.3±0.3%) after a single-dose of the glucagon receptor antagonist (GRA) LY2409021 or placebo. Total AA and glucagon concentrations are means of 3 samples (15 minutes apart) on 3 GRA and 3 placebo days. Fractioned AA were analyzed from one sample per day. Total AA concentrations were increased by GRA compared to placebo by 1.4 fold in T2D and 1.3 fold in controls (P≤0.001) with threonine, proline and the glucagonotropic AAs alanine and tyrosine exhibiting the greatest increases in T2D (1.6-2 fold) and controls (1.4-1.5 fold). GRA also increased plasma glucagon concentrations by more than 3-fold (P≤0.0001), and the glucagonotropic AAs alanine (R2 0.24, P=0.0012) and tyrosine (R2 0.30, P=0.0002) were correlated to glucagon. In conclusion, acute inhibition of glucagon receptor signaling by GRA causes hyperaminoacidemia linked to hyperglucagonemia, supporting the importance of the liver-alpha cell axis in regulating circulating glucagon and AA in humans. Disclosure S. Haedersdal: None. N.J. Wewer Albrechtsen: Research Support; Self; Mercodia, Novo Nordisk A/S. Speaker's Bureau; Self; Merck Sharp & Dohme Corp. A.B. Lund: None. K.D. Galsgaard: None. M. Winther-Soerensen: None. J.J. Holst: Advisory Panel; Self; Novo Nordisk A/S. F.K. Knop: Advisory Panel; Self; AstraZeneca, MedImmune, Merck Sharp & Dohme Corp., Mundipharma, Novo Nordisk A/S, Sanofi. Consultant; Self; Amgen Inc., Carmot Therapeutics, Novo Nordisk A/S. Research Support; Self; AstraZeneca, Novo Nordisk A/S. Speaker's Bureau; Self; AstraZeneca, MedImmune, Merck Sharp & Dohme Corp., Mundipharma, Norgine, Novo Nordisk A/S. T. Vilsbøll: None. Funding Novo Nordisk Foundation
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