Biology subjects

Bitsi, S.

Publications and source records attributed to Bitsi, S..

6 recordsLinked to original sources

bioRxiv2022

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

The incretin receptors, glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR), are class B GPCRs and 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 favoured versus GIPR as a therapeutic target due to GIPR responses being blunted in T2D patients and the 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 realisation 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 the control of 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 the main trafficking and signaling characteristics of both receptors in pancreatic beta cells, finding striking differences in their propensities for internalization, recycling, and degradation, as well as plasma membrane versus endosomal activity, with potential implications for receptor-specific control of beta cell function.

cell biology↗

bioRxiv2022

Divergent acute versus prolonged in vivo GLP-1R responses in β-arrestin 2-deleted primary beta cells

The glucagon-like peptide-1 receptor (GLP-1R) is a major therapeutic target in type 2 diabetes (T2D) and obesity. Following activation, GLP-1Rs are rapidly desensitised by {beta}-arrestins, scaffolding proteins that terminate G protein interactions but also act as independent signalling mediators. While GLP-1R interacts with {beta}-arrestins 1 and 2, expression of the latter is greatly enhanced in beta cells, making this the most relevant isoform. Here, we have assessed in vivo glycaemic responses to the pharmacological GLP-1R agonist exendin-4 in adult beta cell-selective {beta}-arrestin 2 knockout (KO) mice. Lean female and high-fat, high-sucrose-fed KO mice of both sexes displayed worse acute responses versus control littermates, an effect that was inverted 6 hours post-agonist injection, resulting in prolonged in vivo cell-cell connectivity in KO islets implanted in mouse eyes. Similar effects were observed for the clinically relevant semaglutide and tirzepatide but not with exendin-phe1, an agonist biased away from {beta}-arrestin recruitment. Ex vivo acute cAMP was impaired, but overnight desensitisation was reduced in KO islets. The acute signalling defect was attributed to enhanced {beta}-arrestin 1 and phosphodiesterase (PDE) 4 activity in the absence of {beta}-arrestin 2, while the reduced desensitisation correlated with altered GLP-1R trafficking, involving impaired recycling and lysosomal targeting and increased trans-Golgi network (TGN) localisation and signalling, as well as reduced GLP-1R ubiquitination by the E3 ubiquitin ligase NEDD4. This study has unveiled fundamental aspects of the role of {beta}-arrestin 2 in regulating pharmacological GLP-1R responses with direct application to the rational design of improved GLP-1R-targeting therapeutics.

cell biology↗

bioRxiv2021

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

ObjectiveGlucagon-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 activation and {beta}-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. MethodsDipeptidyl 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 effects on glucose homeostasis and weight loss. ResultsLigand-specific reductions in {beta}-arrestin-2 recruitment led to reduced 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 in spite of a lesser degree of food intake suppression. The compounds tested showed only a minor degree of biased agonism between G protein and {beta}-arrestin-2 recruitment at both receptors and were best classified as partial agonists for the two pathways measured. ConclusionsDiminishing {beta}-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.

pharmacology and toxicology↗

bioRxiv2020

Testosterone enhances GLP-1 efficacy at the plasma membrane and endosomes to augment insulin secretion in male pancreatic β cells

Male mice with elimination of the androgen receptor (AR) in islet {beta} cells ({beta}ARKO) exhibit blunted glucose-stimulated insulin secretion (GSIS), leading to hypoinsulinemia and hyperglycemia when challenged with a Western diet. Testosterone activation of an extranuclear AR in {beta} cells potentiates GSIS by amplifying the insulinotropic action of glucagon-like peptide-1 (GLP-1). Here, using a combination of {beta}ARKO and {beta} cell-selective GLP-1 receptor knockout mice and their islets, we show that AR activation in {beta} cells amplifies the insulinotropic effect of islet-derived GLP-1. In {beta} cell models expressing cAMP sensors, testosterone enhances the ability of GLP-1, but not that of glucose-dependent insulinotropic polypeptide or glucagon, to produce cAMP. Accordingly, testosterone selectively enhances the ability of GLP-1 to potentiate GSIS. Notably, testosterone enhances GLP-1 production of cAMP at the plasma membrane and endosomes. In male mouse and human islets, the insulinotropic effect of testosterone is abolished following inhibition of the membrane and endosomal cAMP-dependent protein kinase A and exchange protein activated by cAMP islet 2 pathways. Thus, membrane localization of AR enhances the ability of the GLP-1 receptor to produce cAMP, thus increasing glucose-stimulated insulin exocytosis. Significance StatementThis study reveals that testosterone, acting on the androgen receptor (AR) in insulin-producing {beta} cells amplifies the insulinotropic action of glucagon-like peptide-1 (GLP-1) by increasing GLP-1-mediated production of cAMP at the plasma membrane and endosomal compartments, to promote insulin vesicles exocytosis in human {beta} cells. This study establishes a novel biological paradigm in which membrane location of a steroid nuclear receptor enhances the ability of a G protein-coupled receptor to produce cAMP. It has exceptional clinical significance for targeted delivery of testosterone to {beta} cells in the large population of aging and androgen-deficient men who are at increased risk of diabetes.

physiology↗

bioRxiv2019

Differences in signalling, trafficking and glucoregulatory properties of glucagon-like peptide-1 receptor agonists exendin-4 and lixisenatide

Background and purposeAmino acid substitutions at the N-termini of glucagon-like peptide-1 receptor agonist (GLP-1RA) peptides result in distinct patterns of intracellular signalling, sub-cellular trafficking and efficacy in vivo. Here we aimed to determine whether sequence differences at the ligand C-termini of clinically approved GLP-1RAs exendin-4 and lixisenatide lead to similar phenomena. We also sought to establish the impact of the C-terminus on signal bias resulting from modifications elsewhere in the molecule. Experimental approachExendin-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 resultsLixisenatide and exendin-4 showed equal binding affinity, but lixisenatide was 5-fold less potent for cAMP signalling. Both peptides were rapidly endocytosed, but the GLP-1R recycled more slowly to the plasma membrane after lixisenatide treatment. These combined deficits resulted in reduced maximal sustained insulin secretion and reduced anti-hyperglycaemic and anorectic effects in mice. N-terminal substitutions to both ligands had favourable effects on their pharmacology, resulting in improved insulin release and lowering of blood glucose. Conclusion and implicationsChanges to the C-terminus of exendin-4 affect signalling potency and GLP-1R trafficking via mechanisms unrelated to GLP-1R occupancy. These differences were associated with changes in their ability to control blood glucose and therefore may be therapeutically relevant.

pharmacology and toxicology↗

bioRxiv2018

Agonist binding affinity determines palmitoylation of the glucagon-like peptide-1 receptor and its functional interaction with plasma membrane nanodomains in pancreatic beta cells

The glucagon-like peptide-1 receptor (GLP-1R), a key pharmacological target in type 2 diabetes and obesity, is known to undergo palmitoylation by covalent ligation of an acyl chain to cysteine 438 in its carboxyl-terminal tail. Work with other GPCRs indicates that palmitoylation can be dynamically regulated to allow receptors to partition into plasma membrane nanodomains that act as signaling hotspots. Here, we demonstrate that the palmitoylated state of the GLP-1R is increased by agonist binding, leading to its segregation and clustering into plasma membrane signaling nanodomains before undergoing internalization in a clathrin-dependent manner. Both GLP-1R signaling and trafficking are modulated by strategies targeting nanodomain segregation and cluster formation, including depletion of cholesterol or expression of a palmitoylation-defective GLP-1R mutant. Differences in receptor binding affinity exhibited by biased GLP-1R agonists, and modulation of binding kinetics with the positive allosteric modulator BETP, 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.

cell biology↗