GRK Inhibition Potentiates Glucagon-Like Peptide-1 Action

Abstract

The glucagon-like peptide-1 receptor (GLP-1R) is a G-protein-coupled receptor (GPCR) whose activation results in suppression of food intake and improvement of glucose metabolism. Several receptor interacting proteins regulate the signaling of GLP-1R such as G protein-coupled receptor kinases (GRK) and β-arrestins. Here we evaluated the physiological and pharmacological impact of GRK inhibition on GLP-1R activity leveraging small molecule inhibitors of GRK2 and GRK3. We demonstrated that inhibition of GRK: i) inhibited GLP-1-mediated β-arrestin recruitment, ii) enhanced GLP-1-induced insulin secretion in isolated islets and iii) has additive effect with dipeptidyl peptidase 4 in mediating suppression of glucose excursion in mice. These findings highlight the importance of GRK to modulate GLP-1R function in vitro and in vivo. GRK inhibition is a potential therapeutic approach to enhance endogenous and pharmacologically stimulated GLP-1R signaling.

Keywords: CKD - chronic kidney disease; GLP-1; GRK2 = G protein–coupled receptor kinase 2; NASH; diabetes; insulin secretion; obesity.

Figure 1

GLP-1-stimulated recruitment of β-arrestin in PathHunter^® eXpress GLP1R CHO-K1 β-arrestin cells. (A) Inhibition of GLP-1-stimulated recruitment of β-arrestin by Cpd A; (B) Inhibition of GLP-1-stimulated recruitment of β-arrestin by Cpd B; (C) IC₅₀ determination of Cpd A and Cpd B Values represent the mean of three data points ± SEM. For (A, B) Statistical comparison are assessed by one-way ANOVA with Dunnett’s multiple comparisons test. ****P < 0.0001; ns, not significant.

Figure 2

GLP-1 mediated insulin secretion in INS-1 832/13 cells. (A) GLP-1 mediated insulin secretion; (B) Effect of Cpd A on 10 nM GLP-1 mediated insulin secretion at 5 mM glucose; (C) Effect of Cpd B on 10 nM GLP-1 mediated insulin secretion at 5 mM glucose. Values represent the mean of five replicates ± SEM. Statistical comparison for more than 2 datasets are assessed by one-way ANOVA with Dunnett’s multiple comparisons test. *P = 0.01-0.1, **P = 0.001-0.01, ****P < 0.0001; ns, not significant. Unpaired t-test was performed for 2 datasets.

Figure 3

Real-time PCR assay of mRNA abundance of GRK1-6 in mouse pancreatic islets. Cyclophilin A (PPIA) was used as a housekeeping gene, and quantifications were conducted using the 2ΔΔCt method. Samples were run in duplicates.

Figure 4

GLP-1 mediated insulin secretion in mouse pancreatic islets. (A) Effect of Cpd A on 20 nM GLP-1 mediated insulin secretion at 2 mM glucose or 10 mM glucose; (B) Effect of Cpd B on 20 nM GLP-1 mediated insulin secretion at 2 mM glucose or 10 mM glucose. Values represent the mean of eight replicates ± SEM. Statistical comparison for more than 2 datasets are assessed by one-way ANOVA with Dunnett’s multiple comparisons test. **P = 0.001–0.01, ***P < 0.001; ns, not significant. An unpaired t-test is performed for 2 datasets.

Figure 5

OGTT in C57BL/6J mice. (A) Effect of Cpd A on DPP4i-mediated glucose excursion in C57BL/6J mice; (B) Effect of Cpd B on DPP4i-mediated glucose excursion in C57BL/6J mice; (C) Results of A expressed as Area under the curve (AUC); (D) Results of A expressed as AUC. Data are expressed as mean values ± SD of 6 animals in each group. For Figures (A, B) differences between group means are assessed by Ordinary 2-way ANOVA followed by Tukey’s multiple comparison test. The analysis result was shown under each figure. For Figures (C, D), differences between group means are assessed by Ordinary one-way ANOVA followed by Tukey’s multiple comparison test. **P = 0.001-0.01, ***P = 0.0001-0.001, ****P < 0.0001; ns, not significant.