Biology subjects

Carling, D.

Publications and source records attributed to Carling, D..

11 recordsLinked to original sources

bioRxiv2026

Identification and functional assessment of GPCRs across human adipogenesis

Clinical obesity, defined as the presence of excess adiposity in conjunction with the presence of at least one clinical presentation of disease, remains a significant social and economic burden. Pharmacological weight loss agents based on glucagon-like peptide-1 receptor (GLP-1R) targeting are effective but exhibit significant side-effects leading to cessation of treatment, weight regain and, importantly, re-development of co-morbidities. Adipose tissue, now established as a central mediator of energy balance, endocrine signalling and inflammation, plays a significant role in the protection against metabolic disease onset. Loss of adipose tissue expandability, insulin sensitivity and function is thought to be a pivotal event in the transition to clinical obesity. Targeting adipose tissue dysfunction prior to or following development of metabolic disease remains a key therapeutic strategy either in conjunction with incretin-based weight loss therapy, or as a stand-alone therapy. The recent confirmation of functional glucose-dependent insulinotropic polypeptide receptor (GIPR) in mature adipocytes has led to a significant shift in our mechanistic understanding of dual GLP-1R/GIPR agonists such as tirzepatide, with the addition of adipocyte-specific targeting thought to underpin its clinical superiority to GLP-1R agonism alone. However, the expression, regulation and mechanistic targets of GIPR, and indeed many G protein-coupled receptors (GPCRs), in human adipocytes remains unclear, with adipocyte development being particularly under-studied in this regard. Here we use unbiased transcriptomic analyses of human adipocyte development with high temporal resolution to identify the onset of human GPCR expression, uncovering a previously undocumented surge in expression following adipogenic induction and elegant gene waves throughout adipocyte development that may provide attractive pharmacological targets for adipose tissue dysfunction with or without weight loss. We functionally characterise GIPR, GLP-1 and CALCR/RAMP (Amylin) receptor activation at key differentiation timepoints, and identify a novel amylin response in early adipogenesis, presenting committed adipogenic precursors as a primary target of amylin signalling. HighlightsO_LIComprehensive transcriptomic analysis of human adipogenesis with improved temporal resolution and depth C_LIO_LIIdentification and classification of >150 GPCRs differentially regulated across adipocyte differentiation C_LIO_LIFunctional validation highlighting targeting of adipose stem cells and adipocytes using clinically approved receptor agonists C_LIO_LIGeneration of novel human adipocyte stem cell lines from healthy and obese individuals to drive early target validation and exploration of adipocyte biology with increased pre-clinical power. C_LI SummaryComprehensive temporal RNA sequencing across human adipocyte development with a focus on G-protein coupled receptor expression and activity.

cell biology↗

bioRxiv2025

High-throughput behavioural phenotyping of 25 C. elegans disease models including patient-specific mutations

Genetic diagnosis is fast and cheap, challenging our capacity to evaluate the functional impact of novel disease-causing variants or identify potential therapeutics. Model organisms including C. elegans present the possibility of systematically modelling genetic diseases, yet robust, high-throughput methods have been lacking. Here we show that automated multi-dimensional behaviour tracking can detect phenotypes in 25 new C. elegans disease models spanning homozygous loss-of-function alleles and patient-specific single-amino-acid substitutions. We find that homozygous loss-of-function (LoF) mutants across diverse genetic pathways (including BORC, FLCN, and FNIP-2) exhibit strong, readily detectable abnormalities in posture, locomotion, and stimulus responses compared to wild-type animals. An smc-3 mutant strain--modelled by introducing a patient-identified missense change--causes developmental anomalies and distinct behavioural profiles even though complete loss of SMC-3 is lethal. In contrast, patient-derived missense mutations in another essential gene, tnpo-2, did not show a strong phenotype initially but it could be "sensitized" chemically (e.g., with aldicarb), potentially facilitating future drug screens. Our findings show that scalable behavioural phenotyping can capture a wide range of mutant effects--from strong to subtle--in patient-avatar worm lines. We anticipate that this standardized approach will enable systematic drug repurposing for rare genetic disorders as new disease variants are discovered.

animal behavior and cognition↗

bioRxiv2023

Pharmacological AMP-activated protein kinase activation suppresses low glucose-dependent macrophage migration inhibitory factor release from macrophages

Aims/hypothesisAcute hypoglycemia promotes pro-inflammatory cytokine production, increasing risk for cardiovascular events in diabetes. AMP-activated protein kinase (AMPK) is regulated by and influences production of pro-inflammatory cytokines. We tested the mechanistic role of AMPK in low glucose induced changes in the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF), which is elevated in patients with diabetes. MethodsMacrophage cell line Raw264.7 cells, primary macrophage bone marrow derived macrophages obtained from wild type mice or AMPK {gamma}1 gain-of-function mice were utilized, as were AMPK1/2 knockout mouse embryonic fibroblasts (MEF). Allosteric AMPK activators PF-06409577 and BI-9774 were used, in conjunction with inhibitor SBI-0206965 were also used. We examined changes in protein phosphorylation/expression using western blotting, and protein localisation using immunofluorescence. Metabolic function was assessed using extracellular flux analyses and luciferase-based ATP assay. Cytokine release was quantified by ELISA. Oxidative stress was detected using a fluorescence-based ROS assay, and cell viability was examined using flow cytometry. ResultsMacrophages exposed to low glucose showed a transient and modest activation of AMPK and a metabolic shift towards increased oxidative phosphorylation. Low glucose induced oxidative stress and increased release of macrophage migration inhibitory factor (MIF). Pharmacological activation of AMPK by PF-06409577 and BI-9774 attenuated low glucose-induced MIF release, with a similar trend noted with genetic activation using AMPK{gamma}1 gain-of-function (D316A) mice, which produced a mild effect on low glucose-induced MIF release. Inhibition of NFB signalling diminished MIF release and AMPK activation modestly but significantly reduced low glucose-induced nuclear translocation of NFB. AMPK activation did not alter low glucose-induced oxidative stress in macrophages but application of AMPK inhibitor SBI-0206965 enhanced oxidative stress in macrophages and in AMPK knockout MEFs, suggesting an AMPK-independent mechanism Conclusions/interpretationTaken together, these data indicate that pharmacological AMPK activation suppresses release of MIF from macrophages. This is mediated by reduced activation of NFB signalling in response to low glucose-induced oxidative stress and suggests that pharmacological AMPK activation could be a useful strategy for mitigating hypoglycemia-induced inflammation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/562445v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1d3364borg.highwire.dtl.DTLVardef@1919395org.highwire.dtl.DTLVardef@704923org.highwire.dtl.DTLVardef@1d1cc59_HPS_FORMAT_FIGEXP M_FIG Tweet Low glucose induces pro-inflammatory MIF release from macrophages, and pharmacological AMPK activation suppresses the release of MIF. AMPK/ NFB signalling pathway is involved, which may be a new strategy to attenuate the pro-inflammatory response in hypoglycemia. C_FIG

pharmacology and toxicology↗

bioRxiv2023

Inhibition of an immunometabolic axis of mTORC1 activation extends mammalian healthspan

Human ageing is associated with metabolic dysfunction, sarcopenia and frailty that taken together reduce healthspan. For age-associated diseases and lifespan, ERK, AMPK and mTORC1 represent critical pathways, across species1-7. Here we examined the hypothesis that IL11, recently shown to regulate ERK/mTORC1, is an inflammaging factor important for healthspan. As mice age, IL11 is progressively upregulated in liver, skeletal muscle, and fat to stimulate an ERK/AMPK/mTORC1 axis of cellular, tissue- and organismal-level ageing pathologies. In old mice, deletion of Il11 or Il11ra1 protects against metabolic multi-morbidity, sarcopenia, and frailty. Administration of anti-IL11 therapy to elderly mice for six months reactivates an age-repressed program of white fat beiging, reverses metabolic dysfunction, restores muscle function, and reduces frailty. Across studies, inhibition of IL11 lowers epigenetic age, reduces telomere attrition, and preserves mitochondrial function. Towards clinical translation, we generated, humanised, and engineered a neutralising, high-affinity IL11 antibody. These studies identify IL11 as a key inflammaging factor and therapeutic target for mammalian healthspan.

molecular biology↗

bioRxiv2022

The CDK7 inhibitor CT7001 (Samuraciclib) targets proliferation pathways to inhibit advanced prostate cancer

BackgroundCurrent strategies to inhibit the androgen receptor (AR) are circumvented in castration-resistant prostate cancer (CRPC). Cyclin-dependent kinase 7 (CDK7) promotes AR signalling, in addition to established roles in cell cycle and global transcription regulation, together, providing a rationale for its therapeutic targeting in CRPC. MethodsThe antitumour activity of CT7001, an orally bioavailable CDK7 inhibitor, was investigated across CRPC models in vitro and in xenograft models in vivo. Cell-based assays and transcriptomic analyses of treated xenografts were employed to investigate the mechanism driving activity of CT7001, alone and in combination with the antiandrogen enzalutamide. ResultsCT7001 selectively engages with CDK7 in prostate cancer cells, causing inhibition of proliferation and cell cycle arrest. Activation of p53, induction of apoptosis, and suppression of transcription mediated by full-length and constitutively active AR splice variants contribute to antitumour efficacy in vitro. Oral administration of CT7001 represses growth of CRPC xenografts and significantly augments growth inhibition achieved by enzalutamide. Transcriptome analyses of treated xenografts indicate cell cycle and AR inhibition as the mode of action of CT7001 in vivo. ConclusionsThis study supports CDK7 inhibition as a strategy to target deregulated cell proliferation and demonstrates CT7001 is a promising CRPC therapeutic, alone or in combination with AR-targeting compounds.

molecular biology↗

bioRxiv2022

IL11 stimulates ERK/P90RSK to inhibit LKB1/AMPK and activate mTOR in hepatocytes, the stroma and cancer cells

Interleukin 11 (IL11) stimulates stromal cell activation but also causes hepatocyte metabolic dysfunction. The mechanisms underlying these seemingly unrelated processes are not known. Here we report that IL11-stimulated ERK/P90RSK activity causes the sequential phosphorylation of LKB1 (STK11) at S325 and S428, leading to its inactivation. This leads to a reduction in AMPK activity whilst concomitantly activating mTOR in human fibroblasts, hepatic stellate cells, hepatocytes and cancer cells. In fibroblasts and hepatic stellate cells, IL11-mediated LKB1/AMPK inhibition causes myofibroblast transformation whereas in hepatocytes it inhibits autophagy and fatty acid oxidation and is toxic. Across cell types, the self-amplifying loop of autocrine IL11 activity was inhibited by AMPK activation with metformin, AICAR or 991. In mice on a western diet with fructose, anti-IL11 therapy or hepatocyte-specific deletion of Il11ra1 rescues LKB1/AMPK activity and reduces NASH. In contrast, restoration of IL11 signalling in hepatocytes of mice with global Il11ra1 deletion inactivates LKB1/AMPK and exacerbates NASH. These data show that LKB1, an important tumour suppressor and master kinase, is not constitutively active and identify the IL11/LKB1/AMPK/mTOR axis as a point of signalling convergence for epithelial homeostasis, fibrogenesis, immunometabolism and cancer.

molecular biology↗

bioRxiv2021

Hepatocyte cholesterol content modulates glucagon receptor signalling

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

pharmacology and toxicology↗

bioRxiv2021

Molecular dissection of pro-fibrotic signaling identifies the mechanism underlying IL11-driven fibrosis gene translation, reveals non-specific effects of STAT3 and suggests a new mechanism of action for nintedanib

In fibroblasts, TGF{beta}1 stimulates IL11 upregulation that leads to an autocrine loop of IL11-dependent pro-fibrotic protein translation. The signalling pathways downstream of IL11 are contentious and both STAT3 and ERK have been implicated. Here we show that TGF{beta}1- or IL11-induced ERK activation is consistently associated with fibrogenesis whereas STAT3 phosphorylation (pSTAT3) is unrelated to fibroblast activation. Surprisingly, recombinant human IL11, which has been used extensively in mouse experiments to infer STAT3 activity downstream of IL11, non-specifically increases pSTAT3 in Il11ra1 null mouse fibroblasts. Pharmacologic inhibition of STAT3 prevents TGF{beta}1-induced fibrogenesis but this effect was found to reflect fibroblast dysfunction due to severe proteotoxic ER stress. In contrast, inhibition of MEK/ERK prevented fibrosis in the absence of ER stress. TGF{beta}1-stimulated ERK/mTOR/P70RSK-driven protein translation was IL11-dependent and selectivity for pro-fibrotic protein synthesis was ascribed to an EPRS-related mechanism. In TGF{beta}1-stimulated fibroblasts, the anti-fibrotic drug nintedanib caused dose-dependent ER stress, reduced pSTAT/pERK and inhibited pro-fibrotic protein translation, similarly to generic STAT3 inhibitors or ER stressors. Pirfenidone, while anti-fibrotic, had no effect on ER stress whereas anti-IL11 inhibited the ERK/mTOR axis while reducing ER stress. These studies discount a specific role for STAT3 in pro-fibrotic signaling, suggest a novel mechanism of action for nintedanib and prioritise further the IL11 pathway as a therapeutic target for fibrosis.

molecular biology↗

bioRxiv2021

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

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

cell biology↗

bioRxiv2020

Indisulam targets RNA splicing and metabolism to serve as a novel therapeutic strategy for high-risk neuroblastoma

Neuroblastoma is the most common solid tumour in childhood and prognosis remains poor for high-risk cases despite the use of multimodal treatment. Analysis of public drug sensitivity data showed neuroblastoma lines to be particularly sensitive to indisulam, a molecular glue that selectively targets RNA splicing factor RBM39 for proteosomal degradation via DCAF15-E3-ubiquitin ligase. In neuroblastoma models, indisulam induced rapid loss of RBM39, accumulation of splicing errors and growth inhibition in a DCAF15- dependent manner. Integrative analysis of RNAseq and proteomics data highlighted a distinct disruption to cell cycle and metabolism. Metabolic profiling demonstrated metabolome perturbations and mitochondrial dysfunction resulting from indisulam. Complete tumour without relapse was observed in both xenografts and the Th-MYCN transgenic model of neuroblastoma after indisulam treatment, with RBM39 loss confirmed in vivo. Our data imply that dual targeting of metabolism and RNA splicing with anti-cancer sulphonamides such as indisulam is a promising therapeutic approach for high-risk neuroblastoma.

cancer biology↗

bioRxiv2020

Differences in mitochondrial activity trigger cell competition during early mouse development.

Cell competition is emerging as a quality control mechanism that eliminates unfit cells in a wide range of settings from development to the adult. However, the nature of the cells normally eliminated by cell competition and what triggers their elimination remains poorly understood. In mouse, prior to gastrulation 35% of epiblast cells are eliminated. Here we have performed single cell transcriptional profiling of these cells and find that they show the hallmarks of cell competition and have mitochondrial defects. We demonstrate that mitochondrial defects are common to a range of different loser cell types and that manipulating mitochondrial function is sufficient to trigger competition. Importantly, we show that in the embryo cell competition eliminates cells with mitochondrial DNA mutations and that even non-pathological changes in mitochondrial DNA sequence can induce cell competition. Our results therefore suggest that cell competition is a purifying selection that optimises mitochondrial performance prior to gastrulation.

developmental biology↗