Divergent acute versus prolonged pharmacological GLP-1R responses in adult ß cell-specific ß-arrestin 2 knockout mice.

The glucagon-like peptide-1 receptor (GLP-1R) is a major type 2 diabetes therapeutic target. Stimulated GLP-1Rs are rapidly desensitized by ß-arrestins, scaffolding proteins that not only terminate G protein interactions but also act as independent signaling mediators. Here, we have assessed in vivo glycemic responses to the pharmacological GLP-1R agonist exendin-4 in adult ß cell-specific ß-arrestin 2 knockout (KO) mice. KOs displayed a sex-dimorphic phenotype consisting of weaker acute responses that improved 6 hours after agonist injection. Similar effects were observed for semaglutide and tirzepatide but not with biased agonist exendin-phe1. Acute cyclic adenosine 5'-monophosphate increases were impaired, but desensitization reduced in KO islets. The former defect was attributed to enhanced ß-arrestin 1 and phosphodiesterase 4 activities, while reduced desensitization co-occurred with impaired GLP-1R recycling and lysosomal targeting, increased trans-Golgi network signaling, and reduced GLP-1R ubiquitination. This study has unveiled fundamental aspects of GLP-1R response regulation with direct application to the rational design of GLP-1R-targeting therapeutics.

Architecture of androgen receptor pathways amplifying glucagon-like peptide-1 insulinotropic action in male pancreatic ß cells.

Male mice lacking the androgen receptor (AR) in pancreatic ß cells exhibit blunted glucose-stimulated insulin secretion (GSIS), leading to hyperglycemia. Testosterone activates an extranuclear AR in ß cells to amplify glucagon-like peptide-1 (GLP-1) insulinotropic action. Here, we examined the architecture of AR targets that regulate GLP-1 insulinotropic action in male ß cells. Testosterone cooperates with GLP-1 to enhance cAMP production at the plasma membrane and endosomes via: (1) increased mitochondrial production of CO2, activating the HCO3--sensitive soluble adenylate cyclase; and (2) increased Gαs recruitment to GLP-1 receptor and AR complexes, activating transmembrane adenylate cyclase. Additionally, testosterone enhances GSIS in human islets via a focal adhesion kinase/SRC/phosphatidylinositol 3-kinase/mammalian target of rapamycin complex 2 actin remodeling cascade. We describe the testosterone-stimulated AR interactome, transcriptome, proteome, and metabolome that contribute to these effects. This study identifies AR genomic and non-genomic actions that enhance GLP-1-stimulated insulin exocytosis in male ß cells.

Enhanced Endosomal Signaling and Desensitization of GLP-1R vs GIPR in Pancreatic Beta Cells.

The incretin receptors, glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR), are prime therapeutic targets for the treatment of type 2 diabetes (T2D) and obesity. They are expressed in pancreatic beta cells where they potentiate insulin release in response to food intake. Despite GIP being the main incretin in healthy individuals, GLP-1R has been favored as a therapeutic target due to blunted GIPR responses in T2D patients and conflicting effects of GIPR agonists and antagonists in improving glucose tolerance and preventing weight gain. There is, however, a recently renewed interest in GIPR biology, following the realization that GIPR responses can be restored after an initial period of blood glucose normalization and the recent development of dual GLP-1R/GIPR agonists with superior capacity for controlling blood glucose levels and weight. The importance of GLP-1R trafficking and subcellular signaling in the control of receptor outputs is well established, but little is known about the pattern of spatiotemporal signaling from the GIPR in beta cells. Here, we have directly compared surface expression, trafficking, and signaling characteristics of both incretin receptors in pancreatic beta cells to identify potential differences that might underlie distinct pharmacological responses associated with each receptor. Our results indicate increased cell surface levels, internalization, degradation, and endosomal vs plasma membrane activity for the GLP-1R, while the GIPR is instead associated with increased plasma membrane recycling, reduced desensitization, and enhanced downstream signal amplification. These differences might have potential implications for the capacity of each incretin receptor to control beta cell function.

Partial agonism improves the anti-hyperglycaemic efficacy of an oxyntomodulin-derived GLP-1R/GCGR co-agonist.

OBJECTIVE: Glucagon-like peptide-1 and glucagon receptor (GLP-1R/GCGR) co-agonism can maximise weight loss and improve glycaemic control in type 2 diabetes and obesity. In this study, we investigated the cellular and metabolic effects of modulating the balance between G protein and ß-arrestin-2 recruitment at GLP-1R and GCGR using oxyntomodulin (OXM)-derived co-agonists. This strategy has been previously shown to improve the duration of action of GLP-1R mono-agonists by reducing target desensitisation and downregulation. METHODS: Dipeptidyl dipeptidase-4 (DPP-4)-resistant OXM analogues were generated and assessed for a variety of cellular readouts. Molecular dynamic simulations were used to gain insights into the molecular interactions involved. In vivo studies were performed in mice to identify the effects on glucose homeostasis and weight loss. RESULTS: Ligand-specific reductions in ß-arrestin-2 recruitment were associated with slower GLP-1R internalisation and prolonged glucose-lowering action in vivo. The putative benefits of GCGR agonism were retained, with equivalent weight loss compared to the GLP-1R mono-agonist liraglutide despite a lesser degree of food intake suppression. The compounds tested showed only a minor degree of biased agonism between G protein and ß-arrestin-2 recruitment at both receptors and were best classified as partial agonists for the two pathways measured. CONCLUSIONS: Diminishing ß-arrestin-2 recruitment may be an effective way to increase the therapeutic efficacy of GLP-1R/GCGR co-agonists. These benefits can be achieved by partial rather than biased agonism.

Ligand-Specific Factors Influencing GLP-1 Receptor Post-Endocytic Trafficking and Degradation in Pancreatic Beta Cells.

The glucagon-like peptide-1 receptor (GLP-1R) is an important regulator of blood glucose homeostasis. Ligand-specific differences in membrane trafficking of the GLP-1R influence its signalling properties and therapeutic potential in type 2 diabetes. Here, we have evaluated how different factors combine to control the post-endocytic trafficking of GLP-1R to recycling versus degradative pathways. Experiments were performed in primary islet cells, INS-1 832/3 clonal beta cells and HEK293 cells, using biorthogonal labelling of GLP-1R to determine its localisation and degradation after treatment with GLP-1, exendin-4 and several further GLP-1R agonist peptides. We also characterised the effect of a rare GLP1R coding variant, T149M, and the role of endosomal peptidase endothelin-converting enzyme-1 (ECE-1), in GLP1R trafficking. Our data reveal how treatment with GLP-1 versus exendin-4 is associated with preferential GLP-1R targeting towards a recycling pathway. GLP-1, but not exendin-4, is a substrate for ECE-1, and the resultant propensity to intra-endosomal degradation, in conjunction with differences in binding affinity, contributes to alterations in GLP-1R trafficking behaviours and degradation. The T149M GLP-1R variant shows reduced signalling and internalisation responses, which is likely to be due to disruption of the cytoplasmic region that couples to intracellular effectors. These observations provide insights into how ligand- and genotype-specific factors can influence GLP-1R trafficking.

The chemokine CXCL16 can rescue the defects in insulin signaling and sensitivity caused by palmitate in C2C12 myotubes.

In obesity, macrophages infiltrate peripheral tissues and secrete pro-inflammatory cytokines that impact local insulin sensitivity. Lipopolysaccharide (LPS) and the saturated fatty acid (FA) palmitate polarise macrophages towards a pro-inflammatory phenotype in vitro and indirectly cause insulin resistance (IR) in myotubes. In contrast, unsaturated FAs confer an anti-inflammatory phenotype and counteract the actions of palmitate. To explore paracrine mechanisms of interest, J774 macrophages were exposed to palmitate ± palmitoleate or control medium and the conditioned media generated were screened using a cytokine array. Of the 62 cytokines examined, 8 were significantly differentially expressed following FA treatments. Notably, CXCL16 secretion was downregulated by palmitate. In follow-up experiments using ELISAs, this downregulation was confirmed and reversed by simultaneous addition of palmitoleate or oleate, while LPS also diminished CXCL16 secretion. To dissect potential effects of CXCL16, C2C12 myotubes were treated with palmitate to induce IR, recombinant soluble CXCL16 (sCXCL16), combined treatment, or control medium. Palmitate caused the expected reduction of insulin-stimulated Akt activation and glycogen synthesis, whereas simultaneous treatment with sCXCL16 attenuated these effects. These data indicate a putative role for CXCL16 in preservation of Akt activation and insulin signaling in the context of chronic low-grade inflammation in skeletal muscle.

Signalling, trafficking and glucoregulatory properties of glucagon-like peptide-1 receptor agonists exendin-4 and lixisenatide.

BACKGROUND AND PURPOSE: Amino acid substitutions at the N-termini of glucagon-like peptide-1 (GLP-1) receptor agonist peptides result in distinct patterns of intracellular signalling, sub-cellular trafficking and efficacy in vivo. Here, we to determine whether sequence differences at the ligand C-termini of clinically approved GLP-1 receptor agonists exendin-4 and lixisenatide lead to similar phenomena. EXPERIMENTAL APPROACH: Exendin-4, lixisenatide and N-terminally substituted analogues with biased signalling characteristics were compared across a range of in vitro trafficking and signalling assays in different cell types. Fluorescent ligands and new time-resolved FRET approaches were developed to study agonist behaviours at the cellular and sub-cellular level. Anti-hyperglycaemic and anorectic effects of each parent ligand and their biased derivatives were assessed in mice. KEY RESULTS: Lixisenatide and exendin-4 showed equal binding affinity, but lixisenatide was fivefold less potent for cAMP signalling. Both peptides induced extensive GLP-1 receptor clustering in the plasma membrane and were rapidly endocytosed, but the GLP-1 receptor recycled more slowly to the cell surface after lixisenatide treatment. These combined deficits resulted in reduced maximal sustained insulin secretion and reduced anti-hyperglycaemic and anorectic effects in mice with lixisenatide. N-terminal substitution of His1 by Phe1 to both ligands had favourable effects on their pharmacology, resulting in improved insulin release and lowering of blood glucose. CONCLUSION AND IMPLICATIONS: Changes to the C-terminus of exendin-4 affect signalling potency and GLP-1 receptor trafficking via mechanisms unrelated to GLP-1 receptor occupancy. These differences were associated with changes in their ability to control blood glucose and therefore may be therapeutically relevant.

Disconnect between signalling potency and in vivo efficacy of pharmacokinetically optimised biased glucagon-like peptide-1 receptor agonists.

OBJECTIVE: The objective of this study was to determine how pharmacokinetically advantageous acylation impacts on glucagon-like peptide-1 receptor (GLP-1R) signal bias, trafficking, anti-hyperglycaemic efficacy, and appetite suppression. METHODS: In vitro signalling responses were measured using biochemical and biosensor assays. GLP-1R trafficking was determined by confocal microscopy and diffusion-enhanced resonance energy transfer. Pharmacokinetics, glucoregulatory effects, and appetite suppression were measured in acute, sub-chronic, and chronic settings in mice. RESULTS: A C-terminally acylated ligand, [F1,G40,K41.C16 diacid]exendin-4, was identified that showed undetectable ß-arrestin recruitment and GLP-1R internalisation. Depending on the cellular system used, this molecule was up to 1000-fold less potent than the comparator [D3,G40,K41.C16 diacid]exendin-4 for cyclic AMP signalling, yet was considerably more effective in vivo, particularly for glucose regulation. CONCLUSIONS: C-terminal acylation of biased GLP-1R agonists increases their degree of signal bias in favour of cAMP production and improves their therapeutic potential.

Agonist-induced membrane nanodomain clustering drives GLP-1 receptor responses in pancreatic beta cells.

The glucagon-like peptide-1 receptor (GLP-1R), a key pharmacological target in type 2 diabetes (T2D) and obesity, undergoes rapid endocytosis after stimulation by endogenous and therapeutic agonists. We have previously highlighted the relevance of this process in fine-tuning GLP-1R responses in pancreatic beta cells to control insulin secretion. In the present study, we demonstrate an important role for the translocation of active GLP-1Rs into liquid-ordered plasma membrane nanodomains, which act as hotspots for optimal coordination of intracellular signaling and clathrin-mediated endocytosis. This process is dynamically regulated by agonist binding through palmitoylation of the GLP-1R at its carboxyl-terminal tail. Biased GLP-1R agonists and small molecule allosteric modulation both influence GLP-1R palmitoylation, clustering, nanodomain signaling, and internalization. Downstream effects on insulin secretion from pancreatic beta cells indicate that these processes are relevant to GLP-1R physiological actions and might be therapeutically targetable.

Ureidopeptide GLP-1 analogues with prolonged activity in vivo via signal bias and altered receptor trafficking.

The high demand of the pharmaceutical industry for new modalities to address the diversification of biological targets with large surfaces of interaction led us to investigate the replacement of α-amino acid residues with ureido units at selected positions in peptides to improve potency and generate effective incretin mimics. Based on molecular dynamics simulations, N-terminally modified GLP-1 analogues with a ureido residue replacement at position 2 were synthesized and showed preservation of agonist activity while exhibiting a substantial increase in stability. This enabling platform was applied to exenatide and lixisenatide analogues to generate two new ureidopeptides with antidiabetic properties and longer duration of action. Further analyses demonstrated that the improvement was due mainly to differences in signal bias and trafficking of the GLP-1 receptor. This study demonstrates the efficacy of single α-amino acid substitution with ureido residues to design long lasting peptides.

Profound hyperglycemia in knockout mutant mice identifies novel function for POU4F2/Brn-3b in regulating metabolic processes.

The POU4F2/Brn-3b transcription factor has been identified as a potentially novel regulator of key metabolic processes. Loss of this protein in Brn-3b knockout (KO) mice causes profound hyperglycemia and insulin resistance (IR), normally associated with type 2 diabetes (T2D), whereas Brn-3b is reduced in tissues taken from obese mice fed on high-fat diets (HFD), which also develop hyperglycemia and IR. Furthermore, studies in C2C12 myocytes show that Brn-3b mRNA and proteins are induced by glucose but inhibited by insulin, suggesting that this protein is itself highly regulated in responsive cells. Analysis of differential gene expression in skeletal muscle from Brn-3b KO mice showed changes in genes that are implicated in T2D such as increased glycogen synthase kinase-3ß and reduced GLUT4 glucose transporter. The GLUT4 gene promoter contains multiple Brn-3b binding sites and is directly transactivated by this transcription factor in cotransfection assays, whereas chromatin immunoprecipitation assays confirm that Brn-3b binds to this promoter in vivo. In addition, correlation between GLUT4 and Brn-3b in KO tissues or in C2C12 cells strongly supports a close association between Brn-3b levels and GLUT4 expression. Since Brn-3b is regulated by metabolites and insulin, this may provide a mechanism for controlling key genes that are required for normal metabolic processes in insulin-responsive tissues and its loss may contribute to abnormal glucose uptake.