Divergent acute versus prolonged pharmacological GLP-1R responses in adult beta cell-selective β-arrestin 2 knockout mice

Abstract

The 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 effects of GLP-1RAs. 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 molecular consequences 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-specific β-arrestin 2 KO mouse model. KO mice displayed worse exendin-4 responses acutely that then improved after glucose rechallenge 6 hours post-agonist injection, an effect mirrored by differences in plasma insulin levels and in ex vivo islet 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 the 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 preserved in beta cell β-arrestin 2 KO but lost in control islets imaged in vivo following implantation in mouse eyes. While islet GLP-1R internalisation was not affected by β-arrestin 2 KO, both recycling and lysosomal targeting were significantly impaired, with the receptor instead redirected to the TGN. Trafficking results were replicated in INS-1 832/3 β-arrestin 2 knock-down cells, where we also detected 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.

Abolishing β‐arrestin recruitment is necessary for the full metabolic benefits of G protein‐biased glucagon‐like peptide‐1 receptor agonists

“…However, β-cellspecific conditional knockout studies of β-arrestin-1 and -2 in mice showed no acute insulin secretory deficit in response to GLP-1R agonists, 41,42 although we recently found that loss of β-arrestin-2 may lead to reduced GLP-1R agonist-induced insulin secretion in female mice and DIO male mice, omitted from earlier studies. 43 With this in mind, we aimed to test whether partial or complete loss of GLP-1R agonist-induced β-arrestin recruitment is optimal for enhancing insulin secretion using SRB106 and SRB107 The rapid expansion of next-generation sequencing technology has highlighted extensive genomic coding variation in drug targets. 17 Current GLP-1R agonists are effective for many patients, but a significant minority fail to achieve treatment targets.…”

An examination of the divergent spatiotemporal signaling of GLP-1R versus GIPR in pancreatic beta cells

“…Here, we again observe reduced propensity for b-arrestin 2 recruitment by the GIPR, this time in a beta cell context. The role of b-arrestins on incretin receptor trafficking and signaling has previously been investigated by us from several angles, for example by the use of biased agonists with different capabilities for b-arrestin recruitment [8,26], using in vivo conditional b-arrestin 2 knockout mouse models [21], or with in vitro cell systems with deleted b-arrestin 1/2 expression [13,26]. In all these instances, b-arrestin recruitment closely correlated with the degree of incretin receptor internalization, but alterations in b-arrestin expression levels or complete b-arrestin deletion did not lead to .…”

“…Here, we again observe reduced propensity for β-arrestin 2 recruitment by the GIPR, this time in a beta cell context. The role of β-arrestins on incretin receptor trafficking and signaling has previously been investigated by us from several angles, for example by the use of biased agonists with different capabilities for β-arrestin recruitment [8, 26], using in vivo conditional β-arrestin 2 knockout mouse models [21], or with in vitro cell systems with deleted β-arrestin 1/2 expression [13, 26]. In all these instances, β-arrestin recruitment closely correlated with the degree of incretin receptor internalization, but alterations in β-arrestin expression levels or complete β-arrestin deletion did not lead to significant effects in receptor endocytosis, but rather resulted in the prolongation of cAMP/PKA signaling duration, suggesting that the main effect of this important signaling mediator lies in the steric hindrance caused by its binding to the receptor, leading to reduced access of Gα S to its binding pocket and promoting homologous receptor desensitization [30].…”

“…Of note, similar reduced GIPR versus GLP-1R β-arrestin recruitment and internalization into a Rab5-positive endosomal compartment were also apparent in a separate study [31], although in this instance the authors focused on the comparison between responses from single and dual agonists such as tirzepatide or MAR709 for each receptor rather than performing a direct comparison of both receptor responses. Also interestingly, we previously observed that in vivo GIPR responses were less affected by β-arrestin 2 deletion specifically from pancreatic beta cells [21], suggesting a reduced reliance on β-arrestin 2 to regulate GIPR signaling effects in the pancreas.…”

“…Imaging of INS-1 832/3 cells or whole-islet Ca 2+ dynamics was performed as previously described [21]. Cells or Matrigel-encased islets from individual animals were loaded with the Ca 2+ responsive dye Cal-520 AM (AAT Bioquest), pre-incubated for 1 hr in KRBH G6, and imaged in MatTek glass bottom dishes every 6 sec at 488 nm using a Nikon Eclipse Ti microscope with an ORCA-Flash 4.0 camera (Hamamatsu) and Metamorph software (Molecular Devices) while maintained at 37°C on a heated stage.…”