Imperial College London

DrBenJones

Faculty of MedicineDepartment of Metabolism, Digestion and Reproduction

Senior Clinical Research Fellow
 
 
 
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ben.jones

 
 
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Hammersmith HospitalHammersmith Campus

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Summary

 

Publications

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56 results found

McGlone ER, Manchanda Y, Jones B, Pickford P, Inoue A, Carling D, Bloom S, Tan T, Tomas Aet al., 2021, Receptor Activity-Modifying Protein 2 (RAMP2) alters glucagon receptor trafficking in hepatocytes with functional effects on receptor signalling, Molecular Metabolism, Vol: 53, Pages: 1-11, ISSN: 2212-8778

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 (GCG) is important for carbohydrate and lipid metabolism.MethodsSubcellular localisation of GCGR in the presence and absence of RAMP2 was investigated using confocal microscopy, trafficking 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 and the trafficking inhibitor monensin were used to investigate the effect of a halt in recycling of internalised proteins on GCGR subcellular localisation and signalling in the absence of RAMP2. NanoBiT complementation and cyclic AMP assays were used to study the functional effect of RAMP2 on the 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 accumulation of GCG-stimulated GCGR in cells lacking WASH complex or in the presence of monensin 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 β-arrestin-2 recruitment coupled to increased activation of Gαs proteins at endosomes. This is associated with increased short-term efficacy for glucagon-stimulated

Journal article

Pickford P, Lucey M, Rujan R-M, McGlone ER, Bitsi S, Ashford FB, Corrêa IR, Hodson DJ, Tomas A, Deganutti G, Reynolds CA, Owen BM, Tan TM, Minnion J, Jones B, Bloom SRet al., 2021, Partial agonism improves the anti-hyperglycaemic efficacy of an oxyntomodulin-derived GLP-1R/GCGR co-agonist, Molecular Metabolism, Vol: 51, ISSN: 2212-8778

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 β-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 effects on glucose homeostasis and weight loss. RESULTS: Ligand-specific reductions in β-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 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 β-arrestin-2 recruitment at both receptors and were best classified as partial agonists for the two pathways measured. CONCLUSIONS: Diminishing β-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.

Journal article

Marzook A, Chen S, Pickford P, Lucey M, Wang Y, Corrêa Jr IR, Broichhagen J, Hodson DJ, Salem V, Rutter GA, Tan TM, Bloom SR, Tomas A, Jones Bet al., 2021, Evaluation of efficacy- versus affinity-driven agonism with biased GLP-1R ligands P5 and exendin-F1, Biochemical Pharmacology, Vol: 190, Pages: 1-12, ISSN: 0006-2952

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 β-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 β-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.

Journal article

Lucey M, Ashik T, Marzook A, Wang Y, Goulding J, Oishi A, Broichhagen J, Hodson D, Minnion J, Elani Y, Jockers R, Briddon S, Bloom S, Tomas A, Jones Bet al., 2021, Acylation of the incretin peptide exendin-4 directly impacts GLP-1 receptor signalling and trafficking, Molecular Pharmacology, ISSN: 0026-895X

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 prolong their pharmacokinetics via increased albumin binding, but the potential for these chemical changes to influence GLP-1R function has rarely been investigated beyond potency assessments for cyclic adenosine monophosphate (cAMP). Here we directly compare the prototypical GLP-1RA exendin-4 with its C-terminally acylated analogue, exendin-4-C16. We examine relative propensities of each ligand to recruit and activate G proteins and β-arrestins, endocytic and post-endocytic trafficking profiles, and interactions with model and cellular membranes in HEK293 and HEK293T cells. Both ligands had similar cAMP potency but exendin-4-C16 showed ~2.5-fold bias towards G protein recruitment and a ~60% reduction in β-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 reduced movement of the GLP-1R extracellular domain measured using a conformational biosensor approach, and a ~70% increase in insulin secretion in INS-1 832/3 cells. 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. Our study highlights the importance of a broad approach to the evaluation of GLP-1RA pharmacology.

Journal article

Marzook A, Tomas A, Jones B, 2021, The interplay of glucagon-like peptide-1 receptor trafficking and signalling in pancreatic beta cells, Frontiers in Endocrinology, Vol: 12, Pages: 1-12, ISSN: 1664-2392

The glucagon-like peptide 1 receptor (GLP-1R) is a class B G protein-coupled receptor (GPCR) which mediates the effects of GLP-1, an incretin hormone secreted primarily from L-cells in the intestine and within the central nervous system. The GLP-1R, upon activation, exerts several metabolic effects including the release of insulin and suppression of appetite, and has, accordingly, become an important target for the treatment for type 2 diabetes (T2D). Recently, there has been heightened interest in how the activated GLP-1R is trafficked between different endomembrane compartments, controlling the spatial origin and duration of intracellular signals. The discovery of “biased” GLP-1R agonists that show altered trafficking profiles and selective engagement with different intracellular effectors has added to the tools available to study the mechanisms and physiological importance of these processes. In this review we survey early and recent work that has shed light on the interplay between GLP-1R signalling and trafficking, and how it might be therapeutically tractable for T2D and related diseases.

Journal article

Jones B, 2021, The therapeutic potential of GLP-1 receptor biased agonism, British Journal of Pharmacology, ISSN: 0007-1188

Glucagon-like peptide-1 (GLP-1) receptor agonists are effective treatments for type 2 diabetes as they stimulate insulin release and promote weight loss through appetite suppression. Their main side effect is nausea. All approved GLP-1 agonists are full agonists across multiple signalling pathways. However, selective engagement with specific intracellular effectors, or biased agonism, has been touted as a means to improve GLP-1 agonists therapeutic efficacy. In this review, I critically examine how GLP-1 receptor-mediated intracellular signalling is linked to physiological responses and discuss the implications of recent studies investigating the metabolic effects of biased GLP-1 agonists. Overall, there is little conclusive evidence that beneficial and adverse effects of GLP-1 agonists are attributable to distinct, nonoverlapping signalling pathways. Instead, G protein-biased GLP-1 agonists appear to achieve enhanced anti-hyperglycaemic efficacy by avoiding GLP-1 receptor desensitisation and downregulation, partly via reduced β-arrestin recruitment. This effect seemingly applies more to insulin release than to appetite regulation and nausea, possible reasons for which are discussed. At present, most evidence derives from cellular and animal studies, and more human data are required to determine whether this approach represents a genuine therapeutic advance.

Journal article

Arcones AC, Vila-Bedmar R, Mirasierra M, Cruces-Sande M, Vallejo M, Jones B, Tomas A, Mayor F, Murga Cet al., 2021, GRK2 regulates GLP-1R-mediated early phase insulin secretion in vivo, BMC Biology, Vol: 19, ISSN: 1741-7007

BACKGROUND: Insulin secretion from the pancreatic β-cell is finely modulated by different signals to allow an adequate control of glucose homeostasis. Incretin hormones such as glucagon-like peptide-1 (GLP-1) act as key physiological potentiators of insulin release through binding to the G protein-coupled receptor GLP-1R. Another key regulator of insulin signaling is the Ser/Thr kinase G protein-coupled receptor kinase 2 (GRK2). However, whether GRK2 affects insulin secretion or if GRK2 can control incretin actions in vivo remains to be analyzed. RESULTS: Using GRK2 hemizygous mice, isolated pancreatic islets, and model β-cell lines, we have uncovered a relevant physiological role for GRK2 as a regulator of incretin-mediated insulin secretion in vivo. Feeding, oral glucose gavage, or administration of GLP-1R agonists in animals with reduced GRK2 levels (GRK2+/- mice) resulted in enhanced early phase insulin release without affecting late phase secretion. In contrast, intraperitoneal glucose-induced insulin release was not affected. This effect was recapitulated in isolated islets and correlated with the increased size or priming efficacy of the readily releasable pool (RRP) of insulin granules that was observed in GRK2+/- mice. Using nanoBRET in β-cell lines, we found that stimulation of GLP-1R promoted GRK2 association to this receptor and that GRK2 protein and kinase activity were required for subsequent β-arrestin recruitment. CONCLUSIONS: Overall, our data suggest that GRK2 is an important negative modulator of GLP-1R-mediated insulin secretion and that GRK2-interfering strategies may favor β-cell insulin secretion specifically during the early phase, an effect that may carry interesting therapeutic applications.

Journal article

Manchanda Y, Bitsi S, Kang Y, Jones B, Tomas Aet al., 2021, Spatiotemporal control of GLP-1 receptor activity, Current Opinion in Endocrine and Metabolic Research, Vol: 16, Pages: 19-27, ISSN: 2451-9650

Pleiotropic signalling by G protein–coupled receptors is subject to spatiotemporal regulation, which allows precise control over a diverse range of cellular outputs. The glucagon-like peptide-1 receptor (GLP-1R), a class B G protein–coupled receptor important in the control of blood glucose and energy homeostasis, is subject to redistribution within nanoregions of the plasma membrane and throughout the endocytic network, enabling complex patterns of signalling at different locations. Nanodomain segregation of GLP-1Rs promotes the formation of functionally important homo-oligomers and hetero-oligomers and increases proximity to cytosolic effectors. Persistent signalling from GLP-1Rs within the endosomal compartment has also been described. These processes can be dramatically altered by biased and/or modified orthosteric GLP-1R agonists and allosteric modulators. GLP-1R signalling is not a monotonic process and fine-tuning of cellular responses in space and time may provide a means to improve the therapeutic efficacy of GLP-1R agonists.

Journal article

Jones B, McGlone ER, Fang Z, Pickford P, Corrêa IR, Oishi A, Jockers R, Inoue A, Kumar S, Görlitz F, Dunsby C, French PMW, Rutter GA, Tan TM, Tomas A, Bloom SRet al., 2021, Genetic and biased agonist-mediated reductions in β-arrestin recruitment prolong cAMP signalling at glucagon family receptors, Journal of Biological Chemistry, Vol: 296, Pages: 1-15, ISSN: 0021-9258

Receptors for the peptide hormones glucagon-like peptide-1 (GLP-1R), glucose-dependent insulinotropic polypeptide (GIPR) and glucagon (GCGR) are important regulators of insulin secretion and energy metabolism. GLP-1R agonists have been successfully deployed for the treatment of type 2 diabetes, but it has been suggested that their efficacy is limited by target receptor desensitisation and downregulation due to recruitment of β-arrestins. Indeed, recently described GLP-1R agonists with reduced β-arrestin-2 recruitment have delivered promising results in preclinical and clinical studies. We therefore aimed to determine if the same phenomenon could apply to the closely related GIPR and GCGR. In HEK293 cells depleted of both β-arrestin isoforms the duration of G protein-dependent cAMP/PKA signalling was increased in response to the endogenous ligand for each receptor. Moreover, in wild-type cells, “biased” GLP-1, GCG and GIP analogues with selective reductions in β-arrestin-2 recruitment led to reduced receptor endocytosis and increased insulin secretion over a prolonged stimulation period, although the latter effect was only seen at high agonist concentrations. Biased GCG analogues increased the duration of cAMP signalling, but this did not lead to increased glucose output from hepatocytes. Our study provides a rationale for development of GLP-1R, GIPR and GCGR agonists with reduced β-arrestin recruitment, but further work is needed to maximally exploit this strategy for therapeutic purposes.

Journal article

Jones B, Fang Z, Chen S, Manchanda Y, Bitsi S, Pickford P, David A, Shchepinova MM, Corrêa Jr IR, Hodson DJ, Broichhagen J, Tate EW, Reimann F, Salem V, Rutter GA, Tan T, Bloom SR, Tomas Aet al., 2020, Ligand-specific factors influencing GLP-1 receptor post-endocytic trafficking and degradation in pancreatic beta cells, International Journal of Molecular Sciences, Vol: 212, Pages: 1-24, ISSN: 1422-0067

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.

Journal article

Ast J, Arvaniti A, Fine NHF, Nasteska D, Ashford FB, Stamataki Z, Koszegi Z, Bacon A, Jones BJ, Lucey MA, Sasaki S, Brierley DI, Hastoy B, Tomas A, D'Agostino G, Reimann F, Lynn FC, Reissaus CA, Linnemann AK, D'Este E, Calebiro D, Trapp S, Johnsson K, Podewin T, Broichhagen J, Hodson DJet al., 2020, Author Correction: Super-resolution microscopy compatible fluorescent probes reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics., Nature Communications, Vol: 11, Pages: 1-1, ISSN: 2041-1723

Correction to: Nature Communications https://doi.org/10.1038/s41467-020-14309-w, published online 24 January 2020.

Journal article

Anand U, Jones B, Korchev Y, Bloom S, Pacchetti B, Anand P, Sodergren Met al., 2020, CBD effects on TRPV1 signaling pathways in cultured DRG neurons, Journal of Pain Research, Vol: 2020, Pages: 2269-2278, ISSN: 1178-7090

Introduction: Cannabidiol (CBD) is reported to produce pain relief, but the clinically relevant cellular and molecular mechanisms remain uncertain. The TRPV1 receptor integrates noxious stimuli and plays a key role in pain signaling. Hence, we conducted in vitro studies, to elucidate the efficacy and mechanisms of CBD for inhibiting neuronal hypersensitivity in cultured rat sensory neurons, following activation of TRPV1. Methods: Adult rat dorsal root ganglion (DRG) neurons were cultured, and supplemented with the neurotrophic factors NGF and GDNF, in an established model of neuronal hypersensitivity. 48 h after plating, neurons were stimulated with CBD (Adven 150, EMMAC Life Sciences) at 1, 10, 100 nMol/L and 1, 10 and 50 µMol/L. In separate experiments, DRG neurons were also stimulated with capsaicin with or without CBD (1 nMol/L to10 µMol/L), in a functional calcium imaging assay. The effects of the adenylyl cyclase activator forskolin and the calcineurin inhibitor cyclosporin were determined. We also measured forskolin-stimulated cAMP levels, without and after treatment with CBD, using a homogenous time resolved fluorescence (HTRF) assay. The results were analysed using Student’s t-test. Results: DRG neurons treated with 10 and 50 µMol/L CBD showed calcium influx, but not at lower doses. Neurons treated with capsaicin demonstrated robust calcium influx, which was dose-dependently reduced in the presence of low dose CBD (IC50 = 100 nMol/L). The inhibition or desensitization by CBD was reversed in the presence of forskolin and cyclosporin. Forskolin stimulated cAMP levels were significantly reduced in CBD treated neurons.Conclusions: CBD at low doses corresponding to plasma concentrations observed physiologically, inhibits or desensitizes neuronal TRPV1 signalling by inhibiting the adenylyl cyclase – cAMP pathway, which is essential for maintaining TRPV1 phosphorylation and sensitization. CBD also facilitated calcineurin-med

Journal article

Pickford P, Lucey M, Fang Z, Bitsi S, Bernardino de la Serna J, Broichhagen J, Hodson DJ, Minnion J, Rutter GA, Bloom SR, Tomas A, Jones Bet al., 2020, Signalling, trafficking and glucoregulatory properties of glucagon-like peptide-1 receptor agonists exendin-4 and lixisenatide., British Journal of Pharmacology, Vol: 177, Pages: 3905-3923, ISSN: 0007-1188

BACKGROUND AND PURPOSE: Amino 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. 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 5-fold less potent for cAMP signalling. Both peptides induced extensive GLP-1R clustering in the plasma membrane and were rapidly endocytosed, but the GLP-1R 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-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.

Journal article

Poc P, Gutzeit VA, Ast J, Lee J, Jones BJ, D'Este E, Mathes B, Lehmann M, Hodson DJ, Levitz J, Broichhagen Jet al., 2020, Interrogating surfaceversusintracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates, Chemical Science, Vol: 11, Pages: 7871-7883, ISSN: 2041-6520

Employing self-labelling protein tags for the attachment of fluorescent dyes has become a routine and powerful technique in optical microscopy to visualize and track fused proteins. However, membrane permeability of the dyes and the associated background signals can interfere with the analysis of extracellular labelling sites. Here we describe a novel approach to improve extracellular labelling by functionalizing the SNAP-tag substrate benzyl guanine (“BG”) with a charged sulfonate (“SBG”). This chemical manipulation can be applied to any SNAP-tag substrate, improves solubility, reduces non-specific staining and renders the bioconjugation handle impermeable while leaving its cargo untouched. We report SBG-conjugated fluorophores across the visible spectrum, which cleanly label SNAP-fused proteins in the plasma membrane of living cells. We demonstrate the utility of SBG-conjugated fluorophores to interrogate class A, B and C G protein-coupled receptors (GPCRs) using a range of imaging approaches including nanoscopic superresolution imaging, analysis of GPCR trafficking from intra- and extracellular pools, in vivo labelling in mouse brain and analysis of receptor stoichiometry using single molecule pull down.

Journal article

Jones B, Pickford P, Lucey M, Tomas-Catala A, Minnion J, Bitsi S, Ungewiss J, Schoeneberg K, Rutter G, Bloom Set al., 2020, Disconnect between signalling potency and in vivo efficacy of pharmacokinetically optimised biased glucagon-like peptide-1 receptor agonists, Molecular Metabolism, Vol: 37, ISSN: 2212-8778

ObjectiveThe 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.MethodsIn 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.ResultsA C-terminally acylated ligand, [F1,K⁴⁰.C16 diacid]exendin-4, was identified that showed undetectable β-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,K⁴⁰.C16 diacid]exendin-4 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.

Journal article

Jones B, McGlone ER, Fang Z, Pickford P, Corrêa IR, Oishi A, Jockers R, Inoue A, Kumar S, Görlitz F, Dunsby C, French PMW, Rutter GA, Tan T, Tomas A, Bloom SRet al., 2020, Signal bias at glucagon family receptors: rationale and downstream impacts

<jats:title>Abstract</jats:title><jats:p>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 β-arrestin-2 recruitment have delivered promising results in preclinical studies. Here we first sought to establish the role of β-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 β-arrestin isoforms. We also generated analogues of GLP-1, GCG and GIP which in some cases showed selective reduction in β-arrestin-2 recruitment <jats:italic>versus</jats:italic> 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.</jats:p>

Journal article

Ma Y, Ratnasabapathy R, De Backer I, Izzi-Engbeaya C, Nguyen-Tu M-S, Cuenco J, Jones B, John C, Lam B, Rutter G, Yeo G, Dhillo W, Gardiner Jet al., 2020, Glucose in the hypothalamic paraventricular nucleus regulates GLP-1 release, JCI insight, Vol: 5, ISSN: 2379-3708

Glucokinase (GK) is highly expressed in the hypothalamic paraventricular nucleus (PVN); however, its role is currently unknown. We found that GK in the PVN acts as part of a glucose-sensing mechanism within the PVN that regulates glucose homeostasis by controlling glucagon-like peptide 1 (GLP-1) release. GLP-1 is released from enteroendocrine L cells in response to oral glucose. Here we identify a brain mechanism critical to the release of GLP-1 in response to oral glucose. We show that increasing expression of GK or injection of glucose into the PVN increases GLP-1 release in response to oral glucose. On the contrary, decreasing expression of GK or injection of nonmetabolizable glucose into the PVN prevents GLP-1 release. Our results demonstrate that gluco-sensitive GK neurons in the PVN are critical to the response to oral glucose and subsequent release of GLP-1.

Journal article

Fang Z, Chen S, Pickford P, Broichhagen J, Hodson DJ, Corrêa IR, Kumar S, Görlitz F, Dunsby C, French PMW, Rutter GA, Tan T, Bloom SR, Tomas A, Jones Bet al., 2020, The influence of peptide context on signaling and trafficking of glucagon-like peptide-1 receptor biased agonists, ACS Pharmacology & Translational Science, Vol: 3, Pages: 345-360, ISSN: 2575-9108

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 cyclic AMP signaling, recruitment of β-arrestins, endocytosis, and recycling, dependent both on the introduction of a His → Phe switch at position 1 and the specific midpeptide 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.

Journal article

Tomas A, Jones B, Leech C, 2020, New insights into beta cell GLP-1 receptor and cAMP signaling, Journal of Molecular Biology, Vol: 432, Pages: 1347-1366, ISSN: 0022-2836

Harnessing the translational potential of the GLP-1/GLP-1R system in pancreatic beta cells has led to the development of established GLP-1R-based therapies for the long-term preservation of beta cell function. In this review, we discuss recent advances in the current research on the GLP-1/GLP-1R system in beta cells, including the regulation of signaling by endocytic trafficking as well as the application of concepts such as signal bias, allosteric modulation, dual agonism, polymorphic receptor variants, spatial compartmentalization of cAMP signaling and new downstream signaling targets involved in the control of beta cell function.

Journal article

Poc P, Gutzeit VA, Ast J, Lee J, Jones BJ, DEste E, Mathes B, Hodson DJ, Levitz J, Broichhagen Jet al., 2020, Interrogating surface versus intracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates

<jats:title>Abstract</jats:title><jats:p>Employing self-labelling protein tags for the attachment of fluorescent dyes has become a routine and powerful technique in optical microscopy to visualize and track fused proteins. However, membrane permeability of the dyes and the associated background signals can interfere with the analysis of extracellular labeling sites. Here we describe a novel approach to improve extracellular labeling by functionalizing the SNAP-tag substrate benzyl guanine (“BG”) with a charged sulfonate (“SBG”). This chemical manipulation improves solubility, reduces non-specific staining and renders the bioconjugation handle impermeable while leaving its cargo untouched. We report SBG-conjugated fluorophores across the visible spectrum, which cleanly label SNAP-fused proteins in the plasma membrane of living cells. We demonstrate the utility of SBG-conjugated fluorophores to interrogate class A, B and C G protein-coupled receptors (GPCRs) using a range of imaging approaches including nanoscopic super-resolution imaging, analysis of GPCR trafficking from intra- and extracellular pools, <jats:italic>in vivo</jats:italic> labelling in mouse brain and analysis of receptor stoichiometry using single molecule pull down.</jats:p>

Journal article

Ast J, Arvaniti A, Fine NHF, Nasteska D, Ashford FB, Stamataki Z, Koszegi Z, Bacon A, Jones BJ, Lucey MA, Sasaki S, Brierley DI, Hastoy B, Tomas A, D'Agostino G, Reimann F, Lynn FC, Reissaus CA, Linnemann AK, D'Este E, Calebiro D, Trapp S, Johnsson K, Podewin T, Broichhagen J, Hodson DJet al., 2020, Super-resolution microscopy compatible fluorescent probes reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics, Nature Communications, Vol: 11, ISSN: 2041-1723

The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in metabolism. Presently, its visualization is limited to genetic manipulation, antibody detection or the use of probes that stimulate receptor activation. Herein, we present LUXendin645, a far-red fluorescent GLP1R antagonistic peptide label. LUXendin645 produces intense and specific membrane labeling throughout live and fixed tissue. GLP1R signaling can additionally be evoked when the receptor is allosterically modulated in the presence of LUXendin645. Using LUXendin645 and LUXendin651, we describe islet, brain and hESC-derived β-like cell GLP1R expression patterns, reveal higher-order GLP1R organization including membrane nanodomains, and track single receptor subpopulations. We furthermore show that the LUXendin backbone can be optimized for intravital two-photon imaging by installing a red fluorophore. Thus, our super-resolution compatible labeling probes allow visualization of endogenous GLP1R, and provide insight into class B GPCR distribution and dynamics both in vitro and in vivo.

Journal article

Koch M, Cegla J, Jones B, Lu Y, Mallar Z, Blagborough A, Angrisano F, Baum Jet al., 2019, The effects of dyslipidaemia and cholesterol modulation on erythrocyte susceptibility to malaria parasite infection, Malaria Journal, Vol: 18, ISSN: 1475-2875

BackgroundMalaria disease commences when blood-stage parasites, called merozoites, invade human erythrocytes. Whilst the process of invasion is traditionally seen as being entirely merozoite-driven, emerging data suggests erythrocyte biophysical properties markedly influence invasion. Cholesterol is a major determinant of cell membrane biophysical properties demanding its interrogation as a potential mediator of resistance to merozoite invasion of the erythrocyte. MethodsBiophysical measurements of erythrocyte deformability by flicker spectroscopy were used to assess changes in erythrocyte bending modulus on forced integration of cholesterol and how these artificial changes affect invasion by human Plasmodium falciparum merozoites. To validate these observations in a natural context, either murine Plasmodium berghei or human Plasmodium falciparum merozoites were tested for their ability to invade erythrocytes from a hypercholesterolaemic mouse model or human clinical erythrocyte samples deriving from patients with a range of serum cholesterol concentrations, respectively. ResultsErythrocyte bending modulus (a measure of deformability) was shown to be markedly affected by artificial modulation of cholesterol content and negatively correlated with merozoite invasion efficiency. In an in vitro infection context, however, erythrocytes taken from hypercholesterolaemic mice or from human clinical samples with varying serum cholesterol levels showed little difference in their susceptibility to merozoite invasion. Explaining this, membrane cholesterol levels in both mouse and human hypercholesterolaemia erythrocytes were subsequently found to be no different from matched normal serum controls.ConclusionsBased on these observations, serum cholesterol does not appear to impact on erythrocyte susceptibility to merozoite entry. Indeed, no relationship between serum cholesterol and cholesterol content of the erythrocyte is apparent. This work, nonetheless, suggests that native p

Journal article

Lucey M, Pickford P, Minnion J, Ungewiss J, Schoeneberg K, Rutter GA, Bloom SR, Tomas A, Jones Bet al., 2019, Disconnect between signalling potency and in vivo efficacy of pharmacokinetically optimised biased glucagon-like peptide-1 receptor agonists

<jats:title>Abstract</jats:title><jats:sec><jats:title>Objective</jats:title><jats:p>To determine how pharmacokinetically advantageous acylation impacts on glucagon-like peptide-1 receptor (GLP-1R) signal bias, trafficking, anti-hyperglycaemic efficacy and appetite suppression.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p><jats:italic>In vitro</jats:italic> 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.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>A C-terminally acylated ligand, exendin-phe1-C16, was identified with undetectable β-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 <jats:italic>in vivo</jats:italic>, particularly for glucose regulation.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>C-terminal acylation of biased GLP-1R agonists increases their degree of signal bias in favour of cAMP production and improves their therapeutic potential.</jats:p></jats:sec>

Journal article

Fremaux J, Venin C, Mauran L, Zimmer R, Koensgen F, Rognan D, Bitsi S, Lucey MA, Jones B, Tomas A, Guichard G, Goudreau SRet al., 2019, Ureidopeptide GLP-1 analogues with prolonged activity in vivo via signal bias and altered receptor trafficking, Chemical Science, Vol: 42, Pages: 9872-9879, ISSN: 2041-6520

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.

Journal article

Pickford P, Lucey M, Fang Z, Bitsi S, Broichhagen J, Hodson DJ, Minnion J, Rutter GA, Bloom SR, Tomas A, Jones Bet al., 2019, Differences in signalling, trafficking and glucoregulatory properties of glucagon-like peptide-1 receptor agonists exendin-4 and lixisenatide

<jats:title>Abstract</jats:title><jats:sec><jats:title>Background and purpose</jats:title><jats:p>Amino 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<jats:italic>in vivo</jats:italic>. 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.</jats:p></jats:sec><jats:sec><jats:title>Experimental approach</jats:title><jats:p>Exendin-4, lixisenatide, and N-terminally substituted analogues with biased signalling characteristics were compared across a range of<jats:italic>in vitro</jats:italic>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.</jats:p></jats:sec><jats:sec><jats:title>Key results</jats:title><jats:p>Lixisenatide 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.</jats:p></jats:sec><jats:sec><

Journal article

Buenaventura T, Bitsi S, Laughlin WE, Burgoyne T, Lyu Z, Oqua AI, Norman H, McGlone ER, Klymchenko AS, Corrêa IR, Walker A, Inoue A, Hanyaloglu A, Grimes J, Koszegi Z, Calebiro D, Rutter GA, Bloom SR, Jones B, Tomas Aet al., 2019, Agonist-induced membrane nanodomain clustering drives GLP-1 receptor responses in pancreatic beta cells., PLoS Biology, Vol: 17, Pages: 1-40, ISSN: 1544-9173

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.

Journal article

Humphries SE, Cooper JA, Capps N, Durrington PN, Jones B, McDowell IFW, Soran H, Neil AHWet al., 2019, Coronary heart disease mortality in severe vs. non-severe familial hypercholesterolaemia in the Simon Broome Register, Atherosclerosis, Vol: 281, Pages: 207-212, ISSN: 0021-9150

Background and aimsThe International Atherosclerosis Society (IAS) has proposed that patients with “severe” FH (SFH) would warrant early and more aggressive cholesterol-lowering treatment such as with PCSK9 inhibitors. SFH is diagnosed if LDL-cholesterol (LDLC) > 10 mmol/L, or LDLC >8.0 mmol/L plus one high-risk feature, or LDLC >5 mmol/L plus two high-risk features. Here we compare CHD mortality in SFH and non-SFH (NSFH) patients in the UK prospective Simon Broome Register since 1991, when statin use became routine.Methods2929 definite or possible PFH patients (51% women) aged 20–79 years were recruited from 21 UK lipid clinics and followed prospectively between 1992 and 2016. The excess CHD standardised mortality ratio (SMR) compared to the England and Wales population was calculated (with 95% confidence intervals).Results1982 (67.7%) patients met the SFH definition. Compared to the non-SFH, significantly (p < 0.001) more SFH patients had diagnosed CHD at baseline (24.6% vs. 17.5%), were current smokers (21.9% vs 10.2%) and had a BMI > 30 kg/m2 (14.9% vs. 7.8%). The SMR for CHD mortality was significantly (p = 0.007) higher for SFH (220 (184–261) (34,134 person years, 129 deaths observed, vs. 59 expected) compared to NSFH of 144 (98–203) (15,432 person years, 32 observed vs. 22 expected). After adjustment for traditional risk factors, the Hazard Ratio for CHD mortality in SFH vs. NSFH was 1.22 (0.80–1.87) p = 0.36, indicating that the excess risk was largely accounted for by these factors.ConclusionsCHD mortality remains elevated in treated FH, especially for SFH, emphasising the importance of optimal lipid-lowering and management of other risk factors.

Journal article

Buenaventura T, Laughlin WE, Bitsi S, Burgoyne T, Lyu Z, Oqua AI, Norman H, McGlone ER, Klymchenko AS, Corrêa IR, Walker A, Inoue A, Hanyaloglou A, Rutter GA, Bloom SR, Jones B, Tomas Aet al., 2018, Agonist binding affinity determines palmitoylation of the glucagon-like peptide-1 receptor and its functional interaction with plasma membrane nanodomains in pancreatic beta cells, Publisher: Cold Spring Harbor Laboratory

<jats:p>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.</jats:p>

Working paper

Humphries SE, Cooper JA, Seed M, Capps N, Durrington PN, Jones B, McDowell IFW, Soran H, Neil HAWet al., 2018, Coronary heart disease mortality in treated familial hypercholesterolaemia: Update of the UK Simon Broome FH register, ATHEROSCLEROSIS, Vol: 274, Pages: 41-46, ISSN: 0021-9150

Journal article

Anand U, Yiangou Y, Akbar A, Quick T, MacQuillan A, Fox M, Sinisi M, Korchev YE, Jones B, Bloom SR, Anand Pet al., 2018, Glucagon-like peptide 1 receptor (GLP-1R) expression by nerve fibres in inflammatory bowel disease and functional effects in cultured neurons, PLoS ONE, Vol: 13, ISSN: 1932-6203

IntroductionGlucagon like-peptide 1 receptor (GLP-1R) agonists diminish appetite and may contribute to the weight loss in inflammatory bowel disease (IBD).ObjectivesThe aim of this study was to determine, for the first time, the expression of GLP-1R by colon nerve fibres in patients with IBD, and functional effects of its agonists in cultured rat and human sensory neurons.MethodsGLP-1R and other nerve markers were studied by immunohistochemistry in colon biopsies from patients with IBD (n = 16) and controls (n = 8), human dorsal root ganglia (DRG) tissue, and in GLP-1R transfected HEK293 cells. The morphological effects of incretin hormones oxyntomodulin, exendin-4 and glucagon were studied on neurite extension in cultured DRG neurons, and their functional effects on capsaicin and ATP signalling, using calcium imaging.ResultsSignificantly increased numbers of colonic mucosal nerve fibres were observed in IBD biopsies expressing GLP-1R (p = 0.0013), the pan-neuronal marker PGP9.5 (p = 0.0008), and sensory neuropeptide CGRP (p = 0.0014). An increase of GLP-1R positive nerve fibres in IBD colon was confirmed with a different antibody to GLP-1R (p = 0.016). GLP-1R immunostaining was intensely positive in small and medium-sized neurons in human DRG, and in human and rat DRG cultured neurons. Co-localization of GLP-1R expression with neuronal markers in colon and DRG confirmed the neural expression of GLP-1R, and antibody specificity was confirmed in HEK293 cells transfected with the GLP-1R. Treatment with oxyntomodulin, exendin-4 and GLP-1 increased neurite length in cultured neurons compared with controls, but did not stimulate calcium influx directly, or affect capsaicin responses. However, exendin-4 significantly enhanced ATP responses in human DRG neurons.ConclusionOur results show that increased GLP-1R innervation in IBD bowel could mediate enhanced visceral afferent signalling, and provide a peripheral target for therapeutic intervention. The differential effect of

Journal article

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