Abstract
The glucagon-like peptide-1 receptor (GLP-1R) is a GPCR from the glucagon receptor family with important roles in the regulation of beta cell function and feeding behaviours. After ligand-stimulated G protein binding, active GLP-1Rs are rapidly desensitised by GRKs, followed by recruitment of β-arrestins, scaffolding proteins that terminate G protein interaction through steric hindrance but also act as independent signalling mediators. GLP-1R agonists (GLP-1RAs) are well-established therapeutics in type 2 diabetes and obesity that are nevertheless associated with dose-related gastrointestinal side effects affecting ∼50% of patients. Exploiting the power of ligand-directed signalling bias with modified β-arrestin engagement is a promising approach to favour therapeutically beneficial over harmful GLP-1RA effects. Although GLP-1R interacts with both β-arrestin isoforms 1 and 2 with similar affinities, expression of the latter is greatly enhanced in beta cells, making this the most functionally relevant isoform. To increase our understanding of the effect of β-arrestin 2 activity in beta cell GLP-1R function, we have assessed in vivo glycaemic responses to the pharmacological GLP-1RA exendin-4 in an adult beta cell-selective β-arrestin 2 KO mouse model. Lean female KO mice and mice of both sexes fed a high-fat, high-sucrose diet displayed worse acute exendin-4 responses versus control littermates, an effect that was reversed 6 hours post-agonist injection, mirrored by differences in plasma insulin levels and ex vivo calcium and insulin secretion responses. Similar effects were observed for semaglutide and tirzepatide, two clinically relevant GLP-1RAs, but not for the less β-arrestin 2-reliant biased agonist exendin-phe1. Acute exendin-4-induced cAMP was impaired, but cAMP responses to GLP-1 following overnight exendin-4 exposure tended to improve in KO versus control islets. Acute signalling defects were attributed to the concerted effect of phosphodiesterase PDE4 and β-arrestin 1, as beta cell β-arrestin 2 KO islets regained cAMP responsivity with either β-arrestin 1 knockdown or PDE4 inhibition. Cell-cell connectivity was prolonged in beta cell β-arrestin 2 KO compared with control islets imaged in vivo following implantation in mouse eyes. While islet GLP-1R internalisation was not affected by β-arrestin 2 deletion, both recycling and lysosomal targeting were significantly impaired, with active receptors redirected instead to the trans-Golgi network (TGN). Results were replicated in INS-1 832/3 β-arrestin 2 knockdown cells, where we also measured increased levels of exendin-4-induced TGN signalling as well as reduced GLP-1R ubiquitination and recruitment of the E3 ubiquitin ligase NEDD4, suggesting a role for this post-translational modification in β-arrestin 2-dependent GLP-1R trafficking. The present study represents the first in-depth in vivo and ex vivo analysis of the effects of beta cell β-arrestin 2 gene ablation on acute versus sustained pharmacological GLP-1R responses.
Competing Interest Statement
AT and BJ have received grant funding from Eli Lilly.
