No 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.

Intestinal fibrosis and stricture formation is an aggressive complication of Crohns disease (CD), linked to increased morbidity and costs. This study investigates the contribution of Wnt signalling to intestinal fibrogenesis, considers potential crosstalk between Wnt and TGFβ signalling pathways and assesses the therapeutic potential of small-molecule Wnt inhibitors.β-catenin expression was explored by immunohistochemistry in FFPE tissue from patient-matched non-strictured (NSCD) and strictured (SCD) intestine (n=6 pairs). Functional interactions between Wnt activation, TGFβ signalling and Collagen-I expression were explored in CCD-18Co cells and primary CD myofibroblast cultures established from surgical resection specimens (n=16) using small molecule Wnt inhibitors and molecular techniques, including siRNA-mediated gene knockdown, immunofluorescence, Wnt gene expression arrays and western blotting. Fibrotic SCD tissue was marked by an increase in β-catenin positive cells. In vitro, activation of Wnt-β-catenin signalling increased Collagen-I expression in CCD-18Co cells. Conversely, ICG-001, an inhibitor of β-catenin signalling, reduced Collagen-I expression in cell lines and primary CD myofibroblasts. TGFβ increased β-catenin protein levels but did not activate canonical Wnt signalling. Rather, TGFβ up-regulated WNT5B, a non-canonical Wnt ligand, and the Wnt receptor FZD8, which contributed directly to the up-regulation of Collagen-I through a β-catenin-independent mechanism. Treatment of CCD-18Co fibroblasts and patient-derived myofibroblasts with the FZD8 inhibitor 3235-0367 reduced extracellular matrix expression. Our data highlight small-molecule Wnt inhibitors of both canonical and non-canonical Wnt signalling, as potential anti-fibrotic drugs to treat SCD intestinal fibrosis. They also highlight the importance of the crosstalk between Wnt and TGFβ signalling pathways in CD intestinal fibrosis.

Aim: To determine the kinase activity profiles of human pancreatic beta cells downstream of GLP-1R balanced versus biased agonist stimulations. Materials and methods: This study analysed the kinomic profiles of human EndoC-βh1 cells following vehicle and glucagon-like peptide-1 receptor (GLP-1R) stimulation with the pharmacological agonist exendin-4, as well as exendin-4-based biased derivatives exendin-phe1 and exendin-asp3 for acute (10-minute) versus sustained (120-minute) responses, using PamChip protein tyrosine kinase (PTK) and serine/threonine kinase (STK) assays. The raw data were filtered and normalised using BioNavigator. The kinase analyses were conducted with R, mainly including kinase-substrate mapping and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Results: The present analysis reveals that kinomic responses are distinct for acute versus sustained GLP-1R agonist (GLP-1RA) exposure, with individual responses associated with agonists presenting specific bias profiles. According to pathway analysis, several kinases, including JNKs, PKCs, INSR and LKB1, are important GLP-1R signalling mediators, constituting potential targets for further research on biased GLP-1R downstream signalling. Conclusion: Results from this study suggest that differentially biased exendin-phe1 and exendin-asp3 can modulate distinct kinase interaction networks. Further understanding of these mechanisms will have important implications for the selection of appropriate anti-T2D therapies with optimised downstream kinomic profiles.