Nonconventional glucagon and GLP-1 receptor agonist and antagonist interplay at the GLP-1 receptor revealed in high-throughput FRET assays for cAMP

Abstract

Edited by Jeffrey E. Pessin G protein-coupled receptors (GPCRs) for glucagon (GluR) and glucagon-like peptide-1 (GLP-1R) are normally considered to be highly selective for glucagon and GLP-1, respectively. However, glucagon secreted from pancreatic ␣-cells may accumulate at high concentrations to exert promiscuous effects at the ␤-cell GLP-1R, as may occur in the volume-restricted microenvironment of the islets of Langerhans. Furthermore, systemic administration of GluR or GLP-1R agonists and antagonists at high doses may lead to off-target effects at other receptors. Here, we used molecular modeling to evaluate data derived from FRET assays that detect cAMP as a read-out for GluR and GLP-1R activation. This analysis established that glucagon is a nonconventional GLP-1R agonist, an effect inhibited by the GLP-1R orthosteric antagonist exendin(9 -39) (Ex(9 -39)). The GluR allosteric inhibitors LY2409021 and MK 0893 antagonized glucagon and GLP-1 action at the GLP-1R, whereas des-His 1 -[Glu 9 ]glucagon antagonized glucagon action at the GluR, while having minimal inhibitory action versus glucagon or GLP-1 at the GLP-1R. When testing Ex(9 -39) in combination with des-His 1 -[Glu 9 ]glucagon in INS-1 832/13 cells, we validated a dual agonist action of glucagon at the GluR and GLP-1R. Hybrid peptide GGP817containing glucagon fused to a fragment of peptide YY (PYY) acted as a triagonist at the GluR, GLP-1R, and neuropeptide Y2 receptor (NPY2R). Collectively, these findings provide a new triagonist strategy with which to target the GluR, GLP-1R, and NPY2R. They also provide an impetus to reevaluate prior studies in which GluR and GLP-1R agonists and antagonists were assumed not to exert promiscuous actions at other GPCRs.

Glucagon modulates proliferation and differentiation of human adipose precursors

“…Similarly, the inhibitory effect GLP1 induced on ASC proliferation was reversible in the presence of GI. Since each antagonist has been demonstrated to be selective and specific for its respective receptor (Chepurny et al 2019), with no cross-activity, these findings suggest that both receptors are necessary for mediating both glucagon and GLP-1 in ASCs, independently of the ligand. This conclusion is further supported by the absence of any synergistic effects in the block of proliferation obtained when glucagon and native GLP-1 were simultaneously administered to ASCs.…”

“…An inhibitory effect similar to that observed with liraglutide had been observed with the cleaved GLP-1 form, GLP-1(9-36), which displays a reduced binding ability to the classical receptor. In order to better clarify the role of GLP-1R and GCGR in mediating the similar effects observed with GLP-1RAs and glucagon in ASCs, we used the specific glucagon receptor antagonist, des-His1-[Glu9]-glucagon(1-29) (GI) (Unson et al 1987, Post et al 1993, Chepurny et al 2019, along with the antagonist of GLP-1R exendin 9-39 (EX). When cell count experiments were repeated in the presence of glucagon alone or in combination with GI (10 nM) or EX (10 nM), the inhibitory effect exerted by glucagon on ASC proliferation was significantly reverted not only by GI (86, 103, 97% for 24, 48, 72-h treatment, respectively), but also by EX (88, 96, 91% for 24, 48, 72-h treatment, respectively), Fig.…”

“…Since we demonstrated the coexistence of both GCGR and GLP-1R in ASCs, we were interested in clarifying which receptor mediated the glucagon effects observed in the present study. To this purpose, we used the best available orthosteric antagonists for GCGR, des-His1-[Glu9]-glucagon (Unson et al 1987) and exendin 9-39 for GLP-1R (Thorensen et al 1993), whose selectivity and specificity have been demonstrated in other systems (Unson et al 1991, Post et al 1993, Chepurny et al 2019. The inhibitory effect exerted by glucagon on ASC proliferation was completely reverted by des-His1-[Glu9]glucagon (GI) and, to a lesser extent, by exendin 9-39.…”

New Insights into Beta-Cell GLP-1 Receptor and cAMP Signaling

“…Whilst a significant component of their overall metabolic effect depends on GCGR-mediated increases in energy expenditure to increase weight loss [85,86], recent studies highlight an important role for intra-islet GCG signaling in supporting insulin release [46]. This carries a caveat that the glucagon contribution to insulin secretion probably arises jointly from action at GCGR and GLP-1R, as glucagon is in fact a low affinity agonist at both human [87,88] and mouse GLP-1Rs [46]. Indeed, the cAMP-stimulating and insulinotropic effects of glucagon can be partially blocked by the GLP-1R antagonist exendin(9-39) in mice and in INS-1 832/3 rat beta cells [46,88].…”

“…This carries a caveat that the glucagon contribution to insulin secretion probably arises jointly from action at GCGR and GLP-1R, as glucagon is in fact a low affinity agonist at both human [87,88] and mouse GLP-1Rs [46]. Indeed, the cAMP-stimulating and insulinotropic effects of glucagon can be partially blocked by the GLP-1R antagonist exendin(9-39) in mice and in INS-1 832/3 rat beta cells [46,88]. However, data presented in the same studies using gcg -/islets and the orthosteric GCGR antagonist des-his 1 glu 9 -glucagon demonstrated that intact GCGR signaling is required for full glucagon action on beta cells.…”