Imperial College London

Dr Alejandra Tomas

Faculty of MedicineDepartment of Metabolism, Digestion and Reproduction

Senior Lecturer
 
 
 
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Contact

 

+44 (0)20 7594 3364a.tomas-catala Website CV

 
 
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Location

 

329ICTEM buildingHammersmith Campus

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Summary

 

Publications

Publication Type
Year
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108 results found

Hinds CE, Peace E, Chen S, Davies I, El Eid L, Tomas A, Tan T, Minnion J, Jones B, Bloom SRet al., 2024, Abolishing β-arrestin recruitment is necessary for the full metabolic benefits of G protein-biased glucagon-like peptide-1 receptor agonists, Diabetes, Obesity and Metabolism: a journal of pharmacology and therapeutics, Vol: 26, Pages: 65-77, ISSN: 1462-8902

AimEarlier studies have shown that peptide glucagon-like peptide-1 receptor (GLP-1R) agonists with reduced β-arrestin recruitment show enhanced anti-hyperglycaemic efficacy through avoidance of GLP-1R desensitization. However, the ligand modifications needed to decrease β-arrestin recruitment usually also reduces GLP-1R affinity, therefore higher doses are needed. Here we aimed to develop new, long-acting, G protein-biased GLP-1R agonists with acute signalling potency comparable with semaglutide, to provide insights into specific experimental and therapeutic scenarios.Materials and MethodsNew GLP-1R agonist peptides were assessed using a variety of in vitro and in vivo assays.ResultsFirst, we show that very substantial reductions in β-arrestin recruitment efficacy are required to realize fully the benefits of GLP-1R agonism on blood glucose lowering in mice, with more moderate reductions being less effective. Secondly, our lead compound (SRB107) performs substantially better than semaglutide for effects on blood glucose and weight loss, which may be jointly attributable to its biased agonist action and protracted pharmacokinetics. Thirdly, we show that biased agonist-specific GLP-1R internalization profiles occur at clinically relevant pharmacological concentrations. Finally, we show that SRB107 cAMP signalling is differentially modulated by single and double GLP1R coding variants seen in human populations, with implications for GLP-1R agonist pharmacogenomics.ConclusionsCompletely abolishing β-arrestin recruitment improves the anti-hyperglycaemic effects of GLP-1R agonists in mice.

Journal article

Lagou V, Jiang L, Ulrich A, Zudina L, González KSG, Balkhiyarova Z, Faggian A, Maina JG, Chen S, Todorov PV, Sharapov S, David A, Marullo L, Mägi R, Rujan R-M, Ahlqvist E, Thorleifsson G, Gao Η, Εvangelou Ε, Benyamin B, Scott RA, Isaacs A, Zhao JH, Willems SM, Johnson T, Gieger C, Grallert H, Meisinger C, Müller-Nurasyid M, Strawbridge RJ, Goel A, Rybin D, Albrecht E, Jackson AU, Stringham HM, Corrêa IR, Farber-Eger E, Steinthorsdottir V, Uitterlinden AG, Munroe PB, Brown MJ, Schmidberger J, Holmen O, Thorand B, Hveem K, Wilsgaard T, Mohlke KL, Wang Z, GWA-PA Consortium, Shmeliov A, den Hoed M, Loos RJF, Kratzer W, Haenle M, Koenig W, Boehm BO, Tan TM, Tomas A, Salem V, Barroso I, Tuomilehto J, Boehnke M, Florez JC, Hamsten A, Watkins H, Njølstad I, Wichmann H-E, Caulfield MJ, Khaw K-T, van Duijn CM, Hofman A, Wareham NJ, Langenberg C, Whitfield JB, Martin NG, Montgomery G, Scapoli C, Tzoulaki I, Elliott P, Thorsteinsdottir U, Stefansson K, Brittain EL, McCarthy MI, Froguel P, Sexton PM, Wootten D, Groop L, Dupuis J, Meigs JB, Deganutti G, Demirkan A, Pers TH, Reynolds CA, Aulchenko YS, Kaakinen MA, Jones B, Prokopenko I, Meta-Analysis of Glucose and Insulin-Related Traits Consortium MAGICet al., 2023, GWAS of random glucose in 476,326 individuals provide insights into diabetes pathophysiology, complications and treatment stratification, Nature Genetics, Vol: 55, Pages: 1448-1461, ISSN: 1061-4036

Conventional measurements of fasting and postprandial blood glucose levels investigated in genome-wide association studies (GWAS) cannot capture the effects of DNA variability on 'around the clock' glucoregulatory processes. Here we show that GWAS meta-analysis of glucose measurements under nonstandardized conditions (random glucose (RG)) in 476,326 individuals of diverse ancestries and without diabetes enables locus discovery and innovative pathophysiological observations. We discovered 120 RG loci represented by 150 distinct signals, including 13 with sex-dimorphic effects, two cross-ancestry and seven rare frequency signals. Of these, 44 loci are new for glycemic traits. Regulatory, glycosylation and metagenomic annotations highlight ileum and colon tissues, indicating an underappreciated role of the gastrointestinal tract in controlling blood glucose. Functional follow-up and molecular dynamics simulations of lower frequency coding variants in glucagon-like peptide-1 receptor (GLP1R), a type 2 diabetes treatment target, reveal that optimal selection of GLP-1R agonist therapy will benefit from tailored genetic stratification. We also provide evidence from Mendelian randomization that lung function is modulated by blood glucose and that pulmonary dysfunction is a diabetes complication. Our investigation yields new insights into the biology of glucose regulation, diabetes complications and pathways for treatment stratification.

Journal article

Xiao J, El Eid L, Buenaventura T, Boutry R, Bonnefond A, Jones B, Rutter GA, Froguel P, Tomas Aet al., 2023, Control of human pancreatic beta cell kinome by glucagon-like peptide-1 receptor biased agonism, Diabetes, Obesity and Metabolism, Vol: 25, Pages: 2105-2119, ISSN: 1462-8902

AimTo determine the kinase activity profiles of human pancreatic beta cells downstream of glucagon-like peptide-1 receptor (GLP-1R) balanced versus biased agonist stimulations.Materials and MethodsThis study analysed the kinomic profiles of human EndoC-βh1 cells following vehicle and GLP-1R stimulation with the pharmacological agonist exendin-4, as well as exendin-4–based biased derivatives exendin-phe1 and exendin-asp3 for acute (10-minute) versus sustained (120-minute) responses, using PamChip protein tyrosine kinase and serine/threonine kinase assays. The raw data were filtered and normalized using BioNavigator. The kinase analyses were conducted with R, mainly including kinase-substrate mapping and Kyoto Encyclopedia of Genes and Genomes pathway analysis.ResultsThe present analysis reveals that kinomic responses are distinct for acute versus sustained GLP-1R agonist exposure, with individual responses associated with agonists presenting specific bias profiles. According to pathway analysis, several kinases, including JNKs, PKCs, INSR and LKB1, are important GLP-1R signalling mediators, constituting potential targets for further research on biased GLP-1R downstream signalling.ConclusionThe results from this study suggest that differentially biased exendin-phe1 and exendin-asp3 can modulate distinct kinase interaction networks. Further understanding of these mechanisms will have important implications for the selection of appropriate anti-type 2 diabetes therapies with optimized downstream kinomic profiles.

Journal article

Xu W, Qadir MMF, Nasteska D, Mota de Sa P, Gorvin CM, Blandino-Rosano M, Evans CR, Ho T, Potapenko E, Veluthakal R, Ashford FB, Bitsi S, Fan J, Bhondeley M, Song K, Sure VN, Sakamuri SSVP, Schiffer L, Beatty W, Wyatt R, Frigo DE, Liu X, Katakam PV, Arlt W, Buck J, Levin LR, Hu T, Kolls J, Burant CF, Tomas A, Merrins MJ, Thurmond DC, Bernal-Mizrachi E, Hodson DJ, Mauvais-Jarvis Fet al., 2023, Architecture of androgen receptor pathways amplifying glucagon-like peptide-1 insulinotropic action in male pancreatic β cells, Cell Reports, Vol: 42, Pages: 1-29, ISSN: 2211-1247

Male mice lacking the androgen receptor (AR) in pancreatic β cells exhibit blunted glucose-stimulated insulin secretion (GSIS), leading to hyperglycemia. Testosterone activates an extranuclear AR in β 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 β 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 β cells.

Journal article

Adriaenssens A, Broichhagen J, de Bray A, Ast J, Hasib A, Jones B, Tomas A, Burgos NF, Woodward O, Lewis J, O'Flaherty E, Kimberley E, Cui C, Harada N, Inagaki N, Campbell J, Brierley D, Hodson DJ, Samms R, Gribble F, Reimann Fet al., 2023, Hypothalamic and brainstem glucose- dependent insulinotropic polypeptide receptor neurons employ distinct mechanisms to affect feeding, JCI INSIGHT, Vol: 8

Journal article

Manchanda Y, Bitsi S, Chen S, Broichhagen J, Bernardino de la Serna J, Jones B, Tomas Aet al., 2023, Enhanced endosomal signaling and desensitization of GLP-1R versus GIPR in pancreatic beta cells, Endocrinology, Vol: 164, Pages: 1-15, ISSN: 0013-7227

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 versus 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.

Journal article

Tomas Catala A, Bitsi S, Manchanda Y, El Eid L, Oqua A, Mohamed N, Hansen B, Suba K, Rutter G, Jones B, Salem Vet al., 2023, Divergent acute versus prolonged pharmacological GLP-1R responses in adult beta cell-specific β-arrestin 2 knockout mice, Science Advances, Vol: 9, Pages: 1-23, ISSN: 2375-2548

The glucagon-like peptide-1 receptor (GLP-1R) is a major type 2 diabetes therapeutic target. Stimulated GLP-1Rs are rapidly desensitized by β-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 β cell–specific β-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 β-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.

Journal article

Tomas Catala A, Austin G, 2023, Variation in responses to incretin therapy: modifiable and non-modifiable factors, Frontiers in Molecular Biosciences, Vol: 10, Pages: 1-10, ISSN: 2296-889X

Type 2 diabetes (T2D) and obesity have reached epidemic proportions. Incretin therapy is the second line of treatment for T2D, improving both blood glucose regulation and weight loss. Glucagon-like peptide-1 (GLP-1) and glucose-stimulated insulinotropic polypeptide (GIP) are the incretin hormones that provide the foundations for these drugs. While these therapies have been highly effective for some, the results are variable. Incretin therapies target the class B G protein-coupled receptors GLP-1R and GIPR, expressed mainly in the pancreas and the hypothalamus, while some therapeutical approaches include additional targeting of the related glucagon receptor (GCGR) in the liver. The proper functioning of these receptors is crucial for incretin therapy success and here we review several mechanisms at the cellular and molecular level that influence an individual’s response to incretin therapy.

Journal article

Davies A, Tomas A, 2023, Appreciating the potential for GPCR crosstalk with ion channels., Pages: 101-120

G protein-coupled receptors (GPCRs) are expressed by most tissues in the body and are exploited pharmacologically in a variety of pathological conditions including diabetes, cardiovascular disease, neurological diseases, and cancers. Numerous cell signaling pathways can be regulated by GPCR activation, depending on the specific GPCR, ligand and cell type. Ion channels are among the many effector proteins downstream of these signaling pathways. Saliently, ion channels are also recognized as druggable targets, and there is evidence that their activity may regulate GPCR function via membrane potential and cytoplasmic ion concentration. Overall, there appears to be a large potential for crosstalk between ion channels and GPCRs. This might have implications not only for targeting GPCRs for drug development, but also opens the possibility of co-targeting them with ion channels to achieve improved therapeutic outcomes. In this review, we highlight the large variety of possible GPCR-ion channel crosstalk modes.

Book chapter

El Eid L, Reynolds CA, Tomas A, Ben Joneset al., 2022, Biased agonism and polymorphic variation at the GLP-1 receptor: Implications for the development of personalised therapeutics., Pharmacol Res, Vol: 184

Glucagon-like peptide-1 receptor (GLP-1R) is a well-studied incretin hormone receptor and target of several therapeutic drugs for type 2 diabetes (T2D), obesity and, more recently, cardiovascular disease. Some signalling pathways downstream of GLP-1R may be responsible for drug adverse effects such as nausea, while others mediate therapeutic outcomes of incretin-based T2D therapeutics. Understanding the interplay between different factors that alter signalling, trafficking, and receptor activity, including biased agonism, single nucleotide polymorphisms and structural modifications is key to develop the next-generation of personalised GLP-1R agonists. However, these interactions remain poorly described, especially for novel therapeutics such as dual and tri-agonists that target more than one incretin receptor. Comparison of GLP-1R structures in complex with G proteins and different peptide and non-peptide agonists has revealed novel insights into important agonist-residue interactions and networks crucial for receptor activation, recruitment of G proteins and engagement of specific signalling pathways. Here, we review the latest knowledge on GLP-1R structure and activation, providing structural evidence for biased agonism and delineating important networks associated with this phenomenon. We survey current biased agonists and multi-agonists at different stages of development, highlighting possible challenges in their translational potential. Lastly, we discuss findings related to non-synonymous genomic variants of GLP1R and the functional importance of specific residues involved in GLP-1R function. We propose that studies of GLP-1R polymorphisms, and specifically their effect on receptor dynamics and pharmacology in response to biased agonists, could have a significant impact in delineating precision medicine approaches and development of novel therapeutics.

Journal article

McGlone ER, Dunsterville C, Carling D, tomas A, Bloom S, Tan T, Jones Bet al., 2022, Hepatocyte cholesterol content modulates glucagon receptor signalling, Molecular Metabolism, Vol: 63, ISSN: 2212-8778

ObjectiveTo determine whether glucagon receptor (GCGR) actions are modulated by cellular cholesterol levels.MethodsWe determined the effects of experimental cholesterol depletion and loading on glucagon-mediated cAMP production, ligand internalisation and glucose production in human hepatoma cells, mouse and human hepatocytes. GCGR interactions with lipid bilayers were explored using coarse-grained molecular dynamic simulations. Glucagon responsiveness was measured in mice fed a high cholesterol diet with or without simvastatin to modulate hepatocyte cholesterol content.ResultsGCGR cAMP signalling was reduced by higher cholesterol levels across different cellular models. Ex vivo glucagon-induced glucose output from mouse hepatocytes was enhanced by simvastatin treatment. 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. Simulations identified likely membrane-exposed cholesterol binding sites on the GCGR, including a site where cholesterol is a putative negative allosteric modulator.ConclusionsOur results indicate that cellular cholesterol content influences glucagon sensitivity and indicate a potential molecular basis for this phenomenon. This could be relevant to the pathogenesis of non-alcoholic fatty liver disease, which is associated with both hepatic cholesterol accumulation and glucagon resistance.

Journal article

Manchanda Y, Bitsi S, Chen S, Broichhagen J, de la Serna JB, Jones B, Tomas Aet al., 2022, An examination of the divergent spatiotemporal signaling of GLP-1R <i>versus</i> GIPR in pancreatic beta cells

<jats:title>Abstract</jats:title><jats:p>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 <jats:italic>versus</jats:italic> 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 <jats:italic>versus</jats:italic> endosomal activity, with potential implications for receptor-specific control of beta cell function.</jats:p>

Journal article

Jones B, Burade V, Akalestou E, Manchanda Y, Ramchunder Z, Carrat G, Nguyen-Tu M-S, Marchetti P, Piemonti L, Leclerc I, Thennati R, Vilsboll T, Thorens B, Tomas A, Rutter GAet al., 2022, In vivo and in vitro characterization of GL0034, a novel long-acting glucagon-like peptide-1 receptor agonist, Diabetes, Obesity and Metabolism: a journal of pharmacology and therapeutics, Vol: 24, Pages: 2090-2101, ISSN: 1462-8902

AimsTo describe the in vitro characteristics and antidiabetic in vivo efficacy of the novel glucagon-like peptide-1 receptor agonist (GLP-1RA) GL0034.Materials and MethodsGlucagon-like peptide-1 receptor (GLP-1R) kinetic binding parameters, cyclic adenosine monophosphate (cAMP) signalling, endocytosis and recycling were measured using HEK293 and INS-1832/3 cells expressing human GLP-1R. Insulin secretion was measured in vitro using INS-1832/3 cells, mouse islets and human islets. Chronic administration studies to evaluate weight loss and glycaemic effects were performed in db/db and diet-induced obese mice.ResultsCompared to the leading GLP-1RA semaglutide, GL0034 showed increased binding affinity and potency-driven bias in favour of cAMP over GLP-1R endocytosis and β-arrestin-2 recruitment. Insulin secretory responses were similar for both ligands. GL0034 (6 nmol/kg) led to at least as much weight loss and lowering of blood glucose as did semaglutide at a higher dose (14 nmol/kg).ConclusionsGL0034 is a G protein-biased agonist that shows powerful antidiabetic effects in mice, and may serve as a promising new GLP-1RA for obese patients with type 2 diabetes.

Journal article

Cheung R, Pizza G, Chabosseau P, Rolando D, Tomas A, Burgoyne T, Wu Z, Salowka A, Thapa A, Macklin A, Cao Y, Nguyen-Tu M-S, Dickerson MT, Jacobson DA, Marchetti P, Shapiro J, Piemonti L, de Koning E, Leclerc I, Bouzakri K, Sakamoto K, Smith DM, Rutter GA, Martinez-Sanchez Aet al., 2022, Glucose-Dependent miR-125b Is a Negative Regulator of β-Cell Function, DIABETES, Vol: 71, Pages: 1525-1545, ISSN: 0012-1797

Journal article

Rodriguez T, 2022, DRP1 levels determine the apoptotic threshold during embryonic differentiation through a mitophagy dependent mechanism, Developmental Cell, Vol: 57, Pages: 1316-1330.e7, ISSN: 1534-5807

The changes that drive differentiation facilitate the emergence of abnormal cells that need to be removed before they contribute to further development or the germline. Consequently, in mice in the lead-up to gastrulation, ∼35% of embryonic cells are eliminated. This elimination is caused by hypersensitivity to apoptosis, but how it is regulated is poorly understood. Here, we show that upon exit of naive pluripotency, mouse embryonic stem cells lower their mitochondrial apoptotic threshold, and this increases their sensitivity to cell death. We demonstrate that this enhanced apoptotic response is induced by a decrease in mitochondrial fission due to a reduction in the activity of dynamin-related protein 1 (DRP1). Furthermore, we show that in naive pluripotent cells, DRP1 prevents apoptosis by promoting mitophagy. In contrast, during differentiation, reduced mitophagy levels facilitate apoptosis. Together, these results indicate that during early mammalian development, DRP1 regulation of mitophagy determines the apoptotic response.

Journal article

Georgiadou E, Muralidharan C, Martinez M, Chabosseau P, Akalestou E, Tomas A, Yong Su Wern F, Stylianides T, Wretlind A, Legido-Quigley C, Jones B, Lopez Noriega L, Xu Y, Gu G, Alsabeeh N, Cruciani-Guglielmacci C, Magnan C, Ibberson M, Leclerc I, Ali Y, Soleimanpour SA, Linnemann AK, Rodriguez TA, Rutter GAet al., 2022, Mitofusins Mfn1 and Mfn2 are required to preserve glucose- but not incretin-stimulated beta cell connectivity and insulin secretion, Diabetes, Vol: 71, Pages: 1472-1489, ISSN: 0012-1797

Mitochondrial glucose metabolism is essential for stimulated insulin release from pancreatic beta cells. Whether mitofusin gene expression, and hence mitochondrial network integrity, is important for glucose or incretin signalling has not previously been explored. Here, we generated mice with beta cell-selective, adult-restricted deletion of the mitofusin genes Mfn1 and Mfn2 (βMfn1/2 dKO). βMfn1/2 dKO mice displayed elevated fed and fasted glycaemia and a >five-fold decrease in plasma insulin. Mitochondrial length, glucose-induced polarisation, ATP synthesis, cytosolic and mitochondrial Ca2+ increases were all reduced in dKO islets. In contrast, oral glucose tolerance was more modestly affected in βMfn1/2 dKO mice and GLP-1 or GIP receptor agonists largely corrected defective GSIS through enhanced EPAC-dependent signalling. Correspondingly, cAMP increases in the cytosol, as measured with an Epac-camps based sensor, were exaggerated in dKO mice. Mitochondrial fusion and fission cycles are thus essential in the beta cell to maintain normal glucose, but not incretin, sensing. These findings broaden our understanding of the roles of mitofusins in beta cells, the potential contributions of altered mitochondrial dynamics to diabetes development and the impact of incretins on this process.

Journal article

Bitsi S, Manchanda Y, ElEid L, Mohamed N, Hansen B, Suba K, Rutter GA, Salem V, Jones B, Tomas Aet al., 2022, Divergent acute <i>versus</i> prolonged pharmacological GLP-1R responses in adult beta cell-selective β-arrestin 2 knockout mice

<jats:title>Abstract</jats:title><jats:p>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 β-arrestins, scaffolding proteins that terminate G protein interactions but also act as independent signalling mediators. While GLP-1R interacts with β-arrestins 1 and 2, expression of the latter is greatly enhanced in beta cells, making this the most relevant isoform. Here, we have assessed <jats:italic>in vivo</jats:italic> glycaemic responses to the pharmacological GLP-1R agonist exendin-4 in adult beta cell-selective β-arrestin 2 knockout (KO) mice. Lean female and high-fat, high-sucrose-fed KO mice of both sexes displayed worse acute responses <jats:italic>versus</jats:italic> control littermates, an effect that was inverted 6 hours post-agonist injection, resulting in prolonged <jats:italic>in vivo</jats:italic> 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 β-arrestin recruitment. <jats:italic>Ex vivo</jats:italic> acute cAMP was impaired, but overnight desensitisation was reduced in KO islets. The acute signalling defect was attributed to enhanced β-arrestin 1 and phosphodiesterase (PDE) 4 activity in the absence of β-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 β-arrestin 2 in regulating pharmacological GLP-1R responses with direct application to the rational design of improved GLP-1R-targeting therape

Journal article

Manchanda Y, Ramchunder Z, Shchepinova MM, Rutter GA, Inoue A, Tate EW, Jones B, Tomas Aet al., 2021, Expression of mini-G proteins specifically halt cognate GPCR trafficking and intracellular signalling

<jats:title>Abstract</jats:title><jats:p>Mini-G proteins are engineered thermostable variants of Gα subunits designed to specifically stabilise G protein-coupled receptors (GPCRs) in their active conformation for structural analyses. Due to their smaller size and ease of use, they have become popular tools in recent years to assess specific GPCR behaviours in cells, both as reporters of receptor coupling to each G protein subtype and for in-cell assays designed to quantify compartmentalised receptor signalling from a range of subcellular locations. Here, we describe a previously unappreciated consequence of the co-expression of mini-G proteins with their cognate GPCRs, namely a profound disruption in GPCR trafficking and intracellular signalling caused by the co-expression of the specific mini-G subtype coupled to the affected receptor. We studied the Gαs-coupled pancreatic beta cell class B GPCR glucagon-like peptide-1 receptor (GLP-1R) as a model to describe in detail the molecular consequences derived from this effect, including a complete halt in β-arrestin-2 recruitment and receptor internalisation, despite near-normal levels of receptor GRK2 recruitment and lipid nanodomain segregation, as well as the disruption of endosomal GLP-1R signalling by mini-G<jats:sub>s</jats:sub> co-expression. We also extend our analysis to a range of other prototypical GPCRs covering the spectrum of Gα subtype coupling preferences, to unveil a widely conserved phenomenon of GPCR internalisation blockage by specific mini-G proteins coupled to a particular receptor. Our results have important implications for the design of methods to assess intracellular GPCR signalling. We also present an alternative adapted bystander intracellular signalling assay for the GLP-1R in which we substitute the mini-G<jats:sub>s</jats:sub> by a nanobody, Nb37, with specificity for active Gαs:GPCR complexes and no deleterious effect o

Journal article

McGlone ER, Ansell TB, Dunsterville C, Song W, Carling D, Tomas A, Bloom SR, Sansom MSP, Tan T, Jones Bet al., 2021, Hepatocyte cholesterol content modulates glucagon receptor signalling

<jats:title>Summary</jats:title><jats:p>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.</jats:p>

Working paper

de Jesus DS, Mak TCS, Wang Y-F, von Ohlen Y, Bai Y, Kane E, Chabosseau P, Chahrour CM, Distaso W, Salem V, Tomas A, Stoffel M, Rutter GA, Latreille Met al., 2021, Dysregulation of the Pdx1/Ovol2/Zeb2 axis in dedifferentiated β-cells triggers the induction of genes associated with epithelial-mesenchymal transition in diabetes, Molecular Metabolism, Vol: 53, ISSN: 2212-8778

OBJECTIVE: β-cell dedifferentiation has been revealed as a pathological mechanism underlying pancreatic dysfunction in diabetes. We previously showed that increased miR-7 levels trigger β-cell dedifferentiation and diabetes. We used β-cell-specific miR-7 overexpressing mice (Tg7) to test the hypothesis that loss of β-cell identity triggered by miR-7 overexpression alters islet gene expression and islet microenvironment in diabetes. METHODS: We performed bulk and single-cell RNA sequencing (RNA-seq) in islets obtained from β-cell-specific miR-7 overexpressing mice (Tg7). We carried out loss- and gain-of-function experiments in MIN6 and EndoC-bH1 cell lines. We analysed previously published mouse and human T2D data sets. RESULTS: Bulk RNA-seq revealed that β-cell dedifferentiation is associated with the induction of genes associated with epithelial-to-mesenchymal transition (EMT) in prediabetic (2-week-old) and diabetic (12-week-old) Tg7 mice. Single-cell RNA-seq (scRNA-seq) indicated that this EMT signature is enriched specifically in β-cells. These molecular changes are associated with a weakening of β-cell: β-cell contacts, increased extracellular matrix (ECM) deposition, and TGFβ-dependent islet fibrosis. We found that the mesenchymal reprogramming of β-cells is explained in part by the downregulation of Pdx1 and its inability to regulate a myriad of epithelial-specific genes expressed in β-cells. Notable among genes transactivated by Pdx1 is Ovol2, which encodes a transcriptional repressor of the EMT transcription factor Zeb2. Following compromised β-cell identity, the reduction in Pdx1 gene expression causes a decrease in Ovol2 protein, triggering mesenchymal reprogramming of β-cells through the induction of Zeb2. We provided evidence that EMT signalling associated with the upregulation of Zeb2 expression is a molecular feature of islets in T2D subjects. CONCLUSIONS: Our study indicates that m

Journal article

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

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, Vol: 100, Pages: 319-334, 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

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

Martinez-Sanchez A, Cheung R, Pizza G, Rolando D, Chabosseau P, Tomas A, Salowska A, Burgoyne T, Leclerc I, Rutter GAet al., 2021, Beta cell miR-125b controls glucose homeostasis by targeting lysosomal and mitochondrial genes, Publisher: SPRINGER, Pages: 158-158, ISSN: 0012-186X

Conference paper

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

Ast J, Novak AN, Podewin T, Fine NHF, Jones B, Tomas Catala A, Birke R, Roßmann K, Mathes B, Eichhorst J, Lehmann M, Linnemann AK, Hodson DJ, Broichhagen Jet al., 2021, An expanded LUXendin color palette for GLP1R detection and visualization in vitro and in vivo, Publisher: ChemRxiv

The glucagon-like peptide-1 receptor (GLP1R) is expressed in peripheral tissues and the brain, where it exerts pleiotropic actions on metabolic and inflammatory processes. Detection and visualization of GLP1R remains challenging, partly due to a lack of validated reagents. Previously, we generated LUXendins, antagonistic red and far-red fluorescent probes for specific labeling of GLP1R in live and fixed cells/tissue. We now extend this concept to the green and near-infrared color ranges by synthesizing and testing LUXendin492, LUXendin551, LUXendin615 and LUXendin762. All four probes brightly and specifically label GLP1R in cells and pancreatic islets. Further, LUXendin551 acts as chemical beta cell reporter in preclinical rodent models, while LUXendin762 allows non-invasive imaging, highlighting differentially-accessible GLP1R populations. We thus expand the color palette of LUXendins to seven different spectra, opening up a range of experiments using widefield microscopy available in most labs through super-resolution imaging and whole animal imaging. With this, we expect that LUXendins will continue to generate novel and specific insight into GLP1R biology.

Working paper

Cheung R, Pizza G, Chabosseau P, Rolando D, Tomas A, Burgoyne T, Salowka A, Macklin A, Cao Y, Nguyen-Tu M-S, Marchetti P, Shapiro J, Piemonti L, de Koning E, Leclerc I, Sakamoto K, Smith DM, Rutter GA, Martinez-Sanchez Aet al., 2021, Glucose-dependent miR-125b is a negative regulator of β-cell function, BioRxiv

<jats:title>SUMMARY</jats:title><jats:p>Impaired pancreatic β-cell function and insulin secretion are hallmarks of type 2 diabetes. MicroRNAs are short non-coding RNAs that silence gene expression, vital for the development and function of β-cells. MiR-125b-5p (miR-125b), a highly conserved miRNA, is abundant in β-cells, though its role in these cells is unclear. Here we show that miR-125b levels in human islets correlate with body mass index (BMI), and its expression is regulated by glucose in an AMP-activated protein kinase-dependent manner. An unbiased high-throughput screen identified multiple miR-125b targets, including the transporter of lysosomal hydrolases <jats:italic>M6pr</jats:italic> and the mitochondrial fission regulator <jats:italic>Mtfp1</jats:italic>. Inactivation of miR-125b in human β-cells shortened mitochondria and enhanced glucose-stimulated insulin secretion, whilst mice over-expressing miR-125b selectively in β-cells were glucose intolerant. β-cells from these animals contained enlarged lysosomal structures and showed reduced insulin content and secretion. Thus, we identify miR125b as a glucose-controlled regulator of organelle dynamics that modulates insulin secretion.</jats:p><jats:sec id="s1"><jats:title>Highlights</jats:title><jats:list list-type="bullet"><jats:list-item><jats:p>Islet miR-125b correlates with BMI and is regulated by glucose via AMP-activated protein kinase in β-cells</jats:p></jats:list-item><jats:list-item><jats:p>miR-125b targets dozens of genes including several involved in the regulation of mitochondrial (<jats:italic>Mtfp1</jats:italic>) and lysosomal (<jats:italic>M6pr</jats:italic>) morphology or function</jats:p></jats:list-item><jats:list-item><jats:p>Deletion of miR-125b results in shorter mitochondria an

Journal article

Imbernon M, Saponaro C, Helms HCC, Zubiaga L, Bitsi S, Duquenne M, Tomas A, Chen S, Salem V, Deligia E, Gmyr V, Denis R, Kerr-Conte J, Chao DHM, Beiroa D, Steals B, Pattou F, Pfrieger F, Brodin B, Jones B, Luquet S, Bonner C, Prevot Vet al., 2021, Tanycytes Control the Hypothalamic Uptake and Metabolic Actions of Liraglutide, Publisher: WILEY, Pages: E237-E237, ISSN: 0894-1491

Conference paper

Manchanda Y, Ben J, Carrat G, Ramchunder Z, Marchetti P, Leclerc I, Thennati R, Burade VS, Tomas A, Rutter GAet al., 2021, Binding Kinetics, Bias, Receptor Internalization, and Effects on Insulin Secretion for a Novel GLP1R-GIPR Dual Agonist, HISHS-2001, Publisher: AMER DIABETES ASSOC, ISSN: 0012-1797

Conference paper

Bitsi S, Suba K, Mohamed N, Leclerc I, Rutter GA, Salem V, Jones B, Tomas Aet al., 2021, β-arrestin-2 Deletion Influences GLP-1 Receptor Signaling in Pancreatic β Cells In Vivo, 81st Virtual Scientific Sessions of the American-Diabetes-Association (ADA), Publisher: AMER DIABETES ASSOC, ISSN: 0012-1797

Conference paper

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