Enteroendocrine K-cells exert complementary effects to control bone quality and mass in mice

International audienceThe involvement of a gut-bone axis in controlling bone physiology has been long suspected, although the exact mechanisms are unclear. We explored whether glucose-dependent insulinotropic polypeptide (GIP)-producing enteroendocrine K-cells were involved in this process. The bone phenotype of transgenic mouse models lacking GIP secretion (GIP-GFP-KI) or enteroendocrine K-cells (GIP-DT) was investigated. Mice deficient in GIP secretion exhibited lower bone strength, trabecular bone mass, trabecula number and cortical thickness, notably due to higher bone resorption. Alterations of microstructure, modifications of bone compositional parameters, represented by lower collagen cross-linking were also apparent. None of these alterations were observed in GIP-DT mice lacking enteroendocrine K-cells, suggesting that other K-cell secretory product acts to counteract GIP action. To assess this, stable analogues of the known K-cell peptide hormones, xenin and GIP, were administered to mature NIH Swiss male mice. Both were capable of modulating bone strength mostly by altering bone microstructure, bone gene expression and bone compositional parameters. However, the two molecules exhibited opposite actions on bone physiology, with evidence that xenin effects are mediated indirectly, possibly via neural networks. Our data highlight a previously unknown interaction between GIP and xenin, which both moderate gut-bone connectivity

Varicella zoster virus glycoprotein C increases chemokine-mediated leukocyte migration

Varicella zoster virus (VZV) is a highly prevalent human pathogen that establishes latency in neurons of the peripheral nervous system. Primary infection causes varicella whereas reactivation results in zoster, which is often followed by chronic pain in adults. Following infection of epithelial cells in the respiratory tract, VZV spreads within the host by hijacking leukocytes, including T cells, in the tonsils and other regional lymph nodes, and modifying their activity. In spite of its importance in pathogenesis, the mechanism of dissemination remains poorly understood. Here we addressed the influence of VZV on leukocyte migration and found that the purified recombinant soluble ectodomain of VZV glycoprotein C (rSgC) binds chemokines with high affinity. Functional experiments show that VZV rSgC potentiates chemokine activity, enhancing the migration of monocyte and T cell lines and, most importantly, human tonsillar leukocytes at low chemokine concentrations. Binding and potentiation of chemokine activity occurs through the C-terminal part of gC ectodomain, containing predicted immunoglobulin-like domains. The mechanism of action of VZV rSgC requires interaction with the chemokine and signalling through the chemokine receptor. Finally, we show that VZV viral particles enhance chemokine-dependent T cell migration and that gC is partially required for this activity. We propose that VZV gC activity facilitates the recruitment and subsequent infection of leukocytes and thereby enhances VZ

Perspective: implications of ligand-receptor binding kinetics for therapeutic targeting of G protein-coupled receptors

The concept of ligand-receptor binding kinetics has been broadly applied in drug development pipelines focusing on G protein-coupled receptors (GPCRs). The ligand residence time (RT) for a receptor describes how long a ligand-receptor complex exists, and is defined as the reciprocal of the dissociation rate constant (k off). RT has turned out to be a valuable parameter for GPCR researchers focusing on drug development as a good predictor of in vivo efficacy. The positive correlation between RT and in vivo efficacy has been established for several drugs targeting class A GPCRs (e.g., the neurokinin-1 receptor (NK1R), the β2 adrenergic receptor (β2AR), and the muscarinic 3 receptor (M3R)) and for drugs targeting class B1 (e.g., the glucagon-like peptide 1 receptor (GLP-1R)). Recently, the association rate constant (k on) has gained similar attention as another parameter affecting in vivo efficacy. In the current perspective, we address the importance of studying ligand-receptor binding kinetics for therapeutic targeting of GPCRs, with an emphasis on how binding kinetics can be altered by subtle molecular changes in the ligands and/or the receptors and how such changes affect treatment outcome. Moreover, we speculate on the impact of binding kinetic parameters for functional selectivity and sustained receptor signaling from endosomal compartments; phenomena that have gained increasing interest in attempts to improve therapeutic targeting of GPCRs.Toxicolog

Molecular and in vivo phenotyping of missense variants of the human glucagon receptor

Naturally occurring missense variants of G protein–coupled receptors with loss of function have been linked to metabolic disease in case studies and in animal experiments. The glucagon receptor, one such G protein–coupled receptor, is involved in maintaining blood glucose and amino acid homeostasis; however, loss-of-function mutations of this receptor have not been systematically characterized. Here, we observed fewer glucagon receptor missense variants than expected, as well as lower allele diversity and fewer variants with trait associations as compared with other class B1 receptors. We performed molecular pharmacological phenotyping of 38 missense variants located in the receptor extracellular domain, at the glucagon interface, or with previously suggested clinical implications. These variants were characterized in terms of cAMP accumulation to assess glucagon-induced Gα(s) coupling, and of recruitment of β-arrestin-1/2. Fifteen variants were impaired in at least one of these downstream functions, with six variants affected in both cAMP accumulation and β-arrestin-1/2 recruitment. For the eight variants with decreased Gα(s) signaling (D63(ECD)N, P86(ECD)S, V96(ECD)E, G125(ECD)C, R225(3.30)H, R308(5.40)W, V368(6.59)M, and R378(7.35)C) binding experiments revealed preserved glucagon affinity, although with significantly reduced binding capacity. Finally, using the UK Biobank, we found that variants with wildtype-like Gα(s) signaling did not associate with metabolic phenotypes, whereas carriers of cAMP accumulation-impairing variants displayed a tendency toward increased risk of obesity and increased body mass and blood pressure. These observations are in line with the essential role of the glucagon system in metabolism and support that Gα(s) is the main signaling pathway effecting the physiological roles of the glucagon receptor

The European research network on signal transduction (ERNEST): toward a multidimensional holistic understanding of G protein-coupled receptor signaling

G protein-coupled receptors (GPCRs) are intensively studied due to their therapeutic potential as drug targets. Members of this large family of transmembrane receptor proteins mediate signal transduction in diverse cell types and play key roles in human physiology and health. In 2013 the research consortium GLISTEN (COST Action CM1207) was founded with the goal of harnessing the substantial growth in knowledge of GPCR structure and dynamics to push forward the development of molecular modulators of GPCR function. The success of GLISTEN, coupled with new findings and paradigm shifts in the field, led in 2019 to the creation of a related consortium called ERNEST (COST Action CA18133). ERNEST broadens focus to entire signaling cascades, based on emerging ideas of how complexity and specificity in signal transduction are not determined by receptor-ligand interactions alone. A holistic approach that unites the diverse data and perspectives of the research community into a single multidimensional map holds great promise for improved drug design and therapeutic targeting.The authors are grateful for the continued support of the European Cooperation in Science and Technology (COST) through Actions CM1207 GLISTEN and CA18133 ERNEST. On behalf of ERNEST, M.E.S. thanks the Max Delbrück Center for Molecular Medicine Berlin for support in managing the Action. M.E.S. is supported by the Deutsche Forschungsgemeinschaft (DFG) (SO1037/1-3) and the Berlin Institute of Health (Delbrück Fellowship BIH_PRO_314). J.S. acknowledges support from the Instituto de Salud Carlos III FEDER (PI18/00094) and the ERA-NET NEURON & Ministry of Economy, Industry and Competitiveness (AC18/00030). J.C. receives funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (Grant Agreement No. 715052). D.E.G. is supported by the Lundbeck Foundation (R313- 2019-526) and Novo Nordisk Foundation (NNF17OC0031226). G.M.K. is funded by the National Brain Research Program (2017-1.2.1-NKP-2017-00002). M.K. acknowledges support from the Israel Science Foundation (Grants 1454/13 and 3512/19) and the DS Research Center at the University of Haifa. S.M. is supported by the Alfred Benzon Foundation (ABF-0-0-312) and Polish National Science Center (HARMONIA 2015/18/M/NZ2/00423). M.M.R. acknowledges support from the European Research Council: VIREX Grant agreement 682549, Call ERC-2105- CoG, the Independent Research Fund Denmark, the NovoNordisk Foundation (NNF17OC0029222:) and the Lundbeck Foundation (R268-2017-409). E.S. thanks the Xunta de Galicia (Centro singular de Investigacion de Galicia ́ acreditacion 2019-2022, ED431G 2019/03 and GI-1597 2017- ́ 2019 ED431B2017/70) and the European Union (European Regional Development Fund - ERDF) for financial support. J.K.S.T. acknowledges support from the DFG (HI1502/1-2) and the Novo Nordisk Foundation (Challenge Grant PRISM). N.V. is funded by grants from the Slovenian Research Agency (P3-310, J3-7605, BI-DE/18-19-015). P.K. is supported by the DFG (KO4095/4-1 and Heisenberg professorship KO4095/5- 1). All coauthors thank the stellar organizers of the eight GLISTEN meetings for their vital contributions and their associated institutes and companies for support, including the University of Warsaw (Poland), Pompeu Fabra University and Autonomous University of Barcelona (Spain), Research Centre for Natural Sciences of the Hungarian Academy of Sciences (Budapest, Hungary), Actelion Pharmaceuticals (Allschwil, Switerland), Vrije Universiteit (Amsterdam, The Netherlands), Friedrich Alexander University Erlangen and Philipps-University Marburg (Germany), University of Chemistry and Technology Prague (Czech Republic), the University of Porto (Portugal), and Sosei Heptares (Cambridge, UK). Parts of this paper are derived from the Memorandum of Understanding for the implementation of the COST Action “European Research Network on Signal Transduction” (ERNEST) CA18133

Neprilysin Inhibition Increases Glucagon Levels in Humans and Mice With Potential Effects on Amino Acid Metabolism

peer reviewedCONTEXT: Inhibitors of the protease neprilysin (NEP) are used for treating heart failure, but are also linked to improvements in metabolism. NEP may cleave proglucagon-derived peptides, including the glucose and amino acid (AA)-regulating hormone glucagon. Studies investigating NEP inhibition on glucagon metabolism are warranted. OBJECTIVE: This work aims to investigate whether NEP inhibition increases glucagon levels. METHODS: Plasma concentrations of glucagon and AAs were measured in eight healthy men during a mixed meal with and without a single dose of the NEP inhibitor/angiotensin II type 1 receptor antagonist, sacubitril/valsartan (194 mg/206 mg). Long-term effects of sacubitril/valsartan (8 weeks) were investigated in individuals with obesity (n = 7). Mass spectrometry was used to investigate NEP-induced glucagon degradation, and the derived glucagon fragments were tested pharmacologically in cells transfected with the glucagon receptor (GCGR). Genetic deletion or pharmacological inhibition of NEP with or without concomitant GCGR antagonism was tested in mice to evaluate effects on AA metabolism. RESULTS: In healthy men, a single dose of sacubitril/valsartan significantly increased postprandial concentrations of glucagon by 228%, concomitantly lowering concentrations of AAs including glucagonotropic AAs. Eight-week sacubitril/valsartan treatment increased fasting glucagon concentrations in individuals with obesity. NEP cleaved glucagon into 5 inactive fragments (in vitro). Pharmacological NEP inhibition protected both exogenous and endogenous glucagon in mice after an AA challenge, while NEP-deficient mice showed elevated fasting and AA-stimulated plasma concentrations of glucagon and urea compared to controls. CONCLUSION: NEP cleaves glucagon, and inhibitors of NEP result in hyperglucagonemia and may increase postprandial AA catabolism without affecting glycemia