A Dual GLP-1/GIP Receptor Agonist Does Not Antagonize Glucagon at Its Receptor but May Act as a Biased Agonist at the GLP-1 Receptor

Abstract

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are important regulators of metabolism, making their receptors (GLP-1R and GIPR) attractive targets in the treatment of type 2 diabetes mellitus (T2DM). GLP-1R agonists are used clinically to treat T2DM but the use of GIPR agonists remains controversial. Recent studies suggest that simultaneous activation of GLP-1R and GIPR with a single peptide provides superior glycemic control with fewer adverse effects than activation of GLP-1R alone. We investigated the signaling properties of a recently reported dual-incretin receptor agonist (P18). GLP-1R, GIPR, and the closely related glucagon receptor (GCGR) were expressed in HEK-293 cells. Activation of adenylate cyclase via Gα_s was monitored using a luciferase-linked reporter gene (CRE-Luc) assay. Arrestin recruitment was monitored using a bioluminescence resonance energy transfer (BRET) assay. GLP-1, GIP, and glucagon displayed exquisite selectivity for their receptors in the CRE-Luc assay. P18 activated GLP-1R with similar potency to GLP-1 and GIPR with higher potency than GIP. Interestingly, P18 was less effective than GLP-1 at recruiting arrestin to GLP-1R and was inactive at GCGR. These data suggest that P18 can act as both a dual-incretin receptor agonist, and as a G protein-biased agonist at GLP-1R.

Keywords: GIP; GLP-1; arrestin; glucagon; receptor.

Figure 1

Peptide ligands used in this study. Residues that are derived from those of glucagon are shown in black, from glucose-dependent insulinotropic polypeptide (GIP) in blue and from glucagon-like peptide-1 (GLP-1) and exendin-4 (Ex-4) in red. Residues shown in grey are shared by glucagon, GIP, GLP-1, and Ex-4. Residues shown in purple are unique to P18, X = aminoisobutyric acid. GLP-1 and Ex-4 are C-terminally amidated (Adapted from Finan et al., 2013 [8]).

Figure 2

Peptide ligand activity at the GLP-1 receptor expressed in HEK-293 cells. Data represent the mean ± S.E.M. from at least three independent experiments, each performed in triplicates. The counts were normalised to the maximum GLP-1 response.

Figure 3

Peptide ligand activity at the GIP receptor expressed in HEK-293 cells. Data represent the mean ± S.E.M. from at least three independent experiments, each performed in triplicates. The counts were normalised to the maximum GIP response.

Figure 4

Peptide ligand activity at the glucagon receptor expressed in HEK-293 cells. Data represent the mean ± S.E.M. from at least three independent experiments, each performed in triplicates. The counts were normalised to the maximum glucagon response.

Figure 5

Increasing concentrations of P18 did not inhibit the response to 1 nM glucagon at the glucagon receptor expressed in HEK-293 cells. P18 exerted no effect on stimulation with glucagon, except at concentrations of 1 µM, for which a small but significant (p < 0.05) increase in activity compared to 1 nM glucagon alone was observed. Data represent the mean ± S.E.M. from at least three independent experiments, each performed in triplicates. The counts were normalised to the maximum glucagon response.

Figure 6

Arrestin recruitment (represented by the BRET ratio) by GLP-1 or P18 to GLP-1R-SYFP2 expressed in Flip-In HEK-293 cells stably expressing Arr3-Nluc. Data represent the mean ± S.E.M. from at least three independent experiments, each performed in triplicates. The counts were normalised to the maximum GLP-1 response.

Figure 7

Gα_S recruitment (represented by the BRET ratio) by GLP-1 or P18 to GLP-1R-Nluc expressed in Flip-In HEK-293 cells. Data represent the mean ± S.E.M. from at least three independent experiments, each performed in triplicates. The counts were normalised to the maximum GLP-1 response.

Figure 8

Arrestin recruitment (represented by the BRET ratio) by glucagon or P18 to GCGR-SYFP2 expressed in Flip-In HEK-293 cells stably expressing Arr3-Nluc. Data represent the mean ± S.E.M. from at least three independent experiments, each performed in triplicates. The counts were normalised to the maximum glucagon response.