Biology subjects

Bloom, S. R.

Publications and source records attributed to Bloom, S. R..

13 recordsLinked to original sources

bioRxiv2024

The physiological impact of an N-terminal Halo-tag on GIPR function in mice

AimsG protein-coupled receptors (GPCRs) modified with self-labelling enzymatic tags (e.g. Halo, SNAP, CLIP) have enabled the in vitro study of receptor expression and trafficking. We developed a GiprHalo/Halo mouse model to gain insights into endogenous glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) signalling, an important modulator of glucose and appetite homeostasis. MethodsGiprHalo/Halo mice were generated through ES cell-based gene targeting and physiologically characterised via body weight, oral glucose tolerance test and intraperitoneal glucose tolerance test analysis. Live cell imaging was used to measure cAMP responses in dispersed pancreatic islets from GiprWt/Wt and GiprHalo/Halo littermates in response to GIP. cAMP accumulation and surface receptor expression were assessed in AD293 cells transiently transfected with mouse and human untagged GIPR and Halo-GIPR. Endogenous pancreatic islet expression of Halo-GIPR was determined by immunohistochemistry. ResultsGiprHalo/Halo mice displayed comparable body weights and responses to oral glucose administration as GiprHalo/Wt and GiprWt/Wt littermates. However, GiprHalo/Halo mice only responded to high dose human GIP during intraperitoneal glucose tolerance testing, and cAMP responses to GIP were impaired in GiprHalo/Halo islets. Despite unimpaired surface expression, in vitro cAMP responses were lessened in AD293 cells expressing Halo-GIPR compared to untagged GIPR. Anti-Halo staining in mouse GiprHalo/Wt pancreatic islets displayed detectable signal. ConclusionsWhile the GiprHalo/Halo mouse model could be used to study endogenous GIPR expression, due to reduced responses we advise against its use for endogenous GIPR trafficking and signalling. Our study highlights the importance of carefully phenotyping mouse models in which modifications to endogenous proteins have been introduced.

cell biology↗

bioRxiv2022

Intra-islet glucagon signalling regulates pulsatile insulin secretion and glucose homeostasis

BackgroundType 2 diabetes (T2D) is characterised by the loss of pulsatile insulin secretion. We studied mice with {beta}-cell specific loss of the glucagon receptor (Gcgr fl/fl X Ins-1Cre), to investigate the role of intra-islet glucagon receptor signalling on pan-islet calcium oscillations and insulin pulsatility. MethodsFrequently sampled intravenous glucose tolerance tests were conducted on Gcgr {beta}-cell-/- and littermate controls. Crossing with GCaMP6f (STOP flox) animals further allowed for {beta}-cell specific expression of a fluorescent calcium indicator. These islets were functionally imaged in vitro and in vivo. Wild-type mice were transplanted with islets expressing GCaMP6f in {beta}-cells into the anterior eye chamber and placed on a high fat diet. Part of the cohort received a glucagon analogue (GCG-analogue) for 40 days and the control group were fed to achieve weight matching. Calcium imaging was performed regularly during the development of hyperglycaemia and in response to GCG-analogue treatment. ResultsGcgr {beta}-cell-/- mice exhibited impaired glucose tolerance following intraperitoneal glucose challenge (control 12.7mmol/L {+/-}0.6 vs. Gcgr {beta}-cell-/- 15.4mmol/L {+/-}0.0 at 15 min, p=0.002); fasting glycaemia was not different to controls. In vitro, Gcgr {beta}-cell-/- islets showed profound loss of synchronised calcium waves in response to glucose which was only partially rescued in vivo. First-phase insulin pulsatility on peripheral blood sampling (n=5) was significantly disordered in Gcgr {beta}-cell-/- mice (burst mass Gcgr {beta}-cell-/- 0.30 {+/-}0.03 versus 0.84 {+/-}0.23 for controls p=0.04). Diet induced obesity and hyperglycaemia resulted in a loss of co-ordinated [Ca2+]I waves in transplanted islets. This was reversed with GCG-analogue treatment, independently of weight-loss (n=8). ConclusionThese data provide novel evidence for the role of intra-islet GCGR signalling in sustaining synchronised calcium oscillations and support a possible therapeutic role for glucagonergic agents to restore the insulin pulsatility lost in T2D.

physiology↗

bioRxiv2021

Hepatocyte cholesterol content modulates glucagon receptor signalling

Glucagon decreases liver fat, and non-alcoholic fatty liver disease (NAFLD) is associated with hepatic glucagon resistance. Increasingly it is recognised that the function of G protein-coupled receptors can be regulated by their local plasma membrane lipid environment. The aim of this study was to evaluate the effects of experimentally modulating hepatocyte cholesterol content on the function of the glucagon receptor (GCGR). We found that glucagon-mediated cAMP production is inversely proportional to cholesterol content of human hepatoma and primary mouse hepatocytes after treatment with cholesterol-depleting and loading agents, with ligand internalisation showing the opposite trend. Mice fed a high cholesterol diet had increased hepatic cholesterol and a blunted hyperglycaemic response to glucagon, both of which were partially reversed by simvastatin. Molecular dynamics simulations identified potential membrane-exposed cholesterol binding sites on the GCGR. Overall, our data suggest that increased hepatocyte membrane cholesterol could directly contribute to glucagon resistance in NAFLD.

pharmacology and toxicology↗

bioRxiv2021

Receptor Activity-Modifying Protein 2 (RAMP2) alters glucagon receptor trafficking in hepatocytes with functional effects on receptor signalling

ObjectivesReceptor Activity-Modifying Protein 2 (RAMP2) is a chaperone protein which allosterically binds to and interacts with the glucagon receptor (GCGR). The aims of this study were to investigate the effects of RAMP2 on GCGR trafficking and signalling in the liver, where glucagon is important for carbohydrate and lipid metabolism. MethodsSubcellular localisation of GCGR in the presence and absence of RAMP2 was investigated using confocal microscopy, trafficking assays and radioligand binding assays in human embryonic kidney (HEK293T) and human hepatoma (Huh7) cells. Mouse embryonic fibroblasts (MEFs) lacking Wiskott Aldrich Syndrome protein and scar homologue (WASH) complex were used to investigate the effect of a halt in recycling of internalised proteins on GCGR signalling in the absence of RAMP2. NanoBiT complementation and cyclic AMP assays were used to study the functional effect of RAMP2 on recruitment and activation of GCGR signalling mediators. Response to hepatic RAMP2 up-regulation in lean and obese adult mice using a bespoke adeno-associated viral vector was also studied. ResultsGCGR is predominantly localised at the plasma membrane in the absence of RAMP2 and exhibits remarkably slow internalisation in response to agonist stimulation. Rapid intracellular retention of glucagon-stimulated GCGR in cells lacking WASH complex indicates that activated GCGRs undergo continuous cycles of internalisation and recycling despite apparent GCGR plasma membrane localisation up to 40 minutes post-stimulation. Co-expression of RAMP2 induces GCGR internalisation both basally and in response to agonist-stimulation. The intracellular retention of GCGR in the presence of RAMP2 confers a bias away from {beta}-arrestin-2 recruitment coupled to increased activation of Gs proteins at endosomes. This is associated with increased short-term efficacy for glucagon-stimulated cAMP production, although long-term signalling is dampened by increased receptor lysosomal targeting for degradation. Despite these signalling effects, only minor disturbance of carbohydrate metabolism was observed in mice with up-regulated hepatic RAMP2. ConclusionsBy retaining GCGR intracellularly, RAMP2 alters the spatiotemporal pattern of GCGR signalling. Further exploration of the effects of RAMP2 on GCGR in vivo is warranted. Graphical abstractIcons sourced from [1] O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

cell biology↗

bioRxiv2021

Evaluation of efficacy- versus affinity-driven agonism with biased GLP-1R ligands P5 and exendin-F1

The glucagon-like peptide-1 receptor (GLP-1R) is an important regulator of glucose homeostasis and has been successfully targeted for the treatment of type 2 diabetes. Recently described biased GLP-1R agonists with selective reductions in {beta}-arrestin versus G protein coupling show improved metabolic actions in vivo. However, two prototypical G protein-favouring GLP-1R agonists, P5 and exendin-F1, are reported to show divergent effects on insulin secretion. In this study we aimed to resolve this discrepancy by performing a side-by-side characterisation of these two ligands across a variety of in vitro and in vivo assays. Exendin-F1 showed reduced acute efficacy versus P5 for several readouts, including recruitment of mini-G proteins, G protein-coupled receptor kinases (GRKs) and {beta}-arrestin-2. Maximal responses were also lower for both GLP-1R internalisation and the presence of active GLP-1R-mini-Gs complexes in early endosomes with exendin-F1 treatment. In contrast, prolonged insulin secretion in vitro and sustained anti-hyperglycaemic efficacy in mice were both greater with exendin-F1 than with P5. We conclude that the particularly low acute efficacy of exendin-F1 and associated reductions in GLP-1R downregulation appear to be more important than preservation of endosomal signalling to allow sustained insulin secretion responses. This has implications for the ongoing development of affinity- versus efficacy-driven biased GLP-1R agonists as treatments for metabolic disease.

pharmacology and toxicology↗

bioRxiv2021

Acylation of the incretin peptide exendin-4 directly impacts GLP-1 receptor signalling and trafficking

The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor and mainstay therapeutic target for the treatment of type 2 diabetes and obesity. Recent reports have highlighted how biased agonism at the GLP-1R affects sustained glucose-stimulated insulin secretion through avoidance of desensitisation and downregulation. A number of GLP-1R agonists (GLP-1RAs) feature a fatty acid moiety to promote albumin binding in order to prolong their pharmacokinetics, but the potential for these ligand changes to influence GLP-1R signalling has rarely been investigated beyond potency assessments for cyclic adenosine monophosphate (cAMP). In this work we directly compare the prototypical GLP-1RA exendin-4 with its C-terminally acylated analogue, exendin-4-C16, for their relative propensities to recruit and activate G proteins and {beta}-arrestins, endocytic and post-endocytic trafficking profiles, and interactions with model and cellular membranes. Both ligands had similar cAMP potency but the exendin-4-C16 showed [~]2.5-fold bias towards G protein recruitment and a [~]60% reduction in {beta}-arrestin-2 recruitment efficacy compared to exendin-4, as well as reduced GLP-1R endocytosis and preferential targeting towards recycling pathways. These effects were associated with a reduced ability to promote the movement of the GLP-1R extracellular domain, as determined using a conformational biosensor approach, and a [~]70% increase in insulin secretion. Interactions with plasma membrane lipids were enhanced by the acyl chain. Exendin-4-C16 showed extensive albumin binding and was highly effective for lowering of blood glucose in mice over at least 72 hours. Overall, our study highlights the importance of a broad approach to the evaluation of GLP-1RA pharmacology. Significance statementAcylation is a common strategy to enhance the pharmacokinetics of peptide-based drugs. Our work shows how acylation can also affect various other pharmacological parameters, including biased agonism, receptor trafficking and interactions with the plasma membrane, which may be therapeutically important.

pharmacology and toxicology↗

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

The Vagus Nerve Mediates the Physiological but not Pharmacological Effects of PYY3-36 on Food Intake

Peptide YY (PYY3-36) is a post-prandially released gut hormone with potent appetite-reducing activity mediated by the neuropeptide Y (NPY) Y2 receptor (Y2R). However, the neuronal pathways by which PYY3-36 acts to supress appetite are unclear. Determining how the PYY3-36 system physiologically regulates food intake may help exploit its therapeutic potential. Here we demonstrate that germline and post-natal targeted knockdown of the Y2R in the afferent vagus nerve inhibits the anorectic effects of physiologically-released PYY3-36, but not peripherally-administered higher doses. Post-natal knockdown of the Y2R results in a transient body weight phenotype that is compensated for in the germline model. Loss of vagal Y2R signalling also alters meal patterning and accelerates gastric emptying. These results may facilitate the design of PYY-based anti-obesity agents. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/241851v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@bef424org.highwire.dtl.DTLVardef@8e7d68org.highwire.dtl.DTLVardef@cb6757org.highwire.dtl.DTLVardef@1edb059_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2020

Impact of N-terminally substituted glucagon family receptor agonists on signal bias, trafficking and downstream responses

Receptors for the peptide hormones glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and glucagon (GCG) are important regulators of insulin secretion and energy metabolism. Recently described GLP-1 receptor agonists showing signal bias in favour of cyclic AMP over {beta}-arrestin-2 recruitment have delivered promising results in preclinical studies. Here we first sought to establish the role of {beta}-arrestins in the control of intracellular signalling and trafficking responses at the closely related GLP-1, GIP and GCG receptors, through studies performed in cells depleted of both {beta}-arrestin isoforms. We also generated analogues of GLP-1, GCG and GIP which in some cases showed selective reduction in {beta}-arrestin-2 recruitment versus cAMP signalling compared to the parent peptide. Despite reduced acute signalling potency and/or efficacy, some biased GLP-1 and GIP analogues increased maximal sustained insulin secretion from INS-1 832/3 clonal beta cells, although only at high agonist concentrations. Biased GCG analogues did not affect maximal insulin release, or glucose output in hepatocytes.

pharmacology and toxicology↗

bioRxiv2020

The influence of peptide context on signalling and trafficking of glucagon-like peptide-1 receptor biased agonists

Signal bias and membrane trafficking have recently emerged as important considerations in the therapeutic targeting of the glucagon-like peptide-1 receptor (GLP-1R) in type 2 diabetes and obesity. In the present study, we have evaluated a peptide series with varying sequence homology between native GLP-1 and exendin-4, the archetypal ligands on which approved GLP-1R agonists are based. We find notable differences in agonist-mediated signalling, endocytosis and recycling, dependent both on the introduction of a His [->] Phe switch at position 1 and the specific mid-peptide helical regions and C-termini of the two agonists. These observations were linked to insulin secretion in a beta cell model and provide insights into how ligand factors influence GLP-1R function at the cellular level. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/961524v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@cb425borg.highwire.dtl.DTLVardef@719e37org.highwire.dtl.DTLVardef@14a45b9org.highwire.dtl.DTLVardef@127afd1_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2019

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

ObjectiveTo determine how pharmacokinetically advantageous acylation impacts on glucagon-like peptide-1 receptor (GLP-1R) signal bias, trafficking, anti-hyperglycaemic efficacy and appetite suppression. MethodsIn vitro signalling responses were measured using biochemical and biosensor assays. GLP-1 receptor 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. ResultsA C-terminally acylated ligand, exendin-phe1-C16, was identified with undetectable {beta}-arrestin recruitment and GLP-1R internalisation. Depending on the cellular system used, this molecule was up to 1000-fold less potent than the comparator exendin-asp-3-C16 for cyclic AMP signalling, yet was considerably more effective in vivo, particularly for glucose regulation. ConclusionsC-terminal acylation of biased GLP-1R agonists increases their degree of signal bias in favour of cAMP production and improves their therapeutic potential.

pharmacology and toxicology↗

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↗