Biology subjects

Withers, D. J.

Publications and source records attributed to Withers, D. J..

7 recordsLinked to original sources

bioRxiv2024

In vivo functional profiling and structural characterisation of the human Glp1r A316T variant

Glucagon-like peptide-1 receptor agonists (GLP-1RAs) are effective therapies for type 2 diabetes (T2D) and obesity, yet patient responses are variable. Variation in the human Glp1r gene might be directly linked to therapeutic responses. A naturally occurring missense variant, A316T, protects against T2D and cardiovascular disease. Here, we have generated and characterised a human Glp1r A316T mouse model. Human Glp1rA316T/A316T mice displayed lower fasting blood glucose versus wildtype littermates, even under metabolic stress, and exhibited alterations in islet cytoarchitecture and /{beta}-cell identity under a high-fat, high-sucrose diet. This was however associated with blunted responses to GLP-1RAs in vivo. Further investigations in rodent and human {beta}-cell models demonstrated that human Glp1r A316T exhibits characteristics of constitutive activation but dampened GLP-1RA responses. Results are further supported by cryo-EM analyses and molecular dynamics simulations of GLP-1R A316T structure, collectively demonstrating that the A316T variant governs basal GLP-1R activity and pharmacological responses to GLP-1R-targeting therapies. TeaserThe Glp1r A316T missense variant displays improved glucose tolerance but impaired pharmacological incretin responses in vivo.

cell biology↗

bioRxiv2024

An inter-organelle contact between endosomal GLP-1R, ER VAP-B, and the mitochondrial AKAP SPHKAP triggers PKA-dependent MIC19 phosphorylation and β-cell mitochondrial remodelling

Glucagon-like peptide-1 receptor (GLP-1R) agonists (GLP-1RAs) ameliorate mitochondrial health by increasing mitochondrial turnover in metabolically relevant tissues. Mitochondrial adaptation to metabolic stress is crucial to maintain pancreatic {beta}-cell function and prevent type 2 diabetes (T2D) progression. While the GLP-1R is well-known to stimulate cAMP production leading to Protein Kinase A (PKA) and Exchange Protein Activated by cyclic AMP 2 (Epac2) activation, there is a lack of understanding of the molecular mechanisms linking GLP-1R signalling with mitochondrial and {beta}-cell functional adaptation. Here, we present a comprehensive study in {beta}-cell lines and primary islets that demonstrates that, following GLP-1RA stimulation, GLP-1R-positive endosomes associate with the endoplasmic reticulum (ER) membrane contact site (MCS) tether VAPB at ER-mitochondria MCSs (ERMCSs), where active GLP-1R engages with SPHKAP, an A-kinase anchoring protein (AKAP) previously linked to T2D and adiposity risk in genome-wide association studies (GWAS). The inter-organelle complex formed by endosomal GLP-1R, ER VAPB and SPHKAP triggers a pool of ERMCS-localised cAMP/PKA signalling via the formation of a PKA-RI biomolecular condensate which leads to changes in mitochondrial contact site and cristae organising system (MICOS) complex phosphorylation, mitochondrial remodelling, and {beta}-cell functional adaptation, with important consequences for the regulation of {beta}-cell insulin secretion and survival to stress.

cell biology↗

bioRxiv2023

Ribosomal S6 kinase 1 regulates inflammaging via the senescence secretome

Inhibition of the nutrient-responsive mTOR (mammalian target of rapamycin) signalling pathway including the key downstream effector S6 kinase 1 (S6K1) extends lifespan and improves healthspan in mice. However, the underlying mechanisms contributing to the broad range of age-related benefits observed with loss of S6K1 signalling are unclear. Cellular senescence is a stable growth arrest accompanied by an inflammatory phenotype (termed the senescence-associated secretory phenotype, or SASP). While both cellular senescence and SASP-mediated chronic inflammation contribute to age-related pathology, the specific role of S6K1 signalling in these processes has not been determined. Here, focussing on mouse liver, a key target tissue for the beneficial metabolic effects of loss of S6K1 signalling, we show that S6K1 deletion does not reduce senescence but ameliorates inflammation and immune cell infiltration in aged livers. Using human and mouse models of senescence, we demonstrated that reduced inflammation is a liver-intrinsic effect associated with S6K deletion. Furthermore, gene expression analysis suggested that downregulated cGAS/STING and IRF3 activation might mediate the impaired SASP observed upon S6K deletion. Using a hepatic oncogene induced senescence model, we showed in vivo that S6K1 deletion results in reduced IRF3 activation, impaired production of cytokines such as IL1y and reduced immune infiltration. Overall, deletion of S6K reduces inflammation in the liver suggesting that suppression of the inflammatory SASP by loss of S6K could contribute to explain the beneficial effects of inhibiting this pathway on healthspan and lifespan.

physiology↗

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↗

bioRxiv2023

Establishment of beta cell heterogeneity via differential CpG methylation at Nnat

Aims/hypothesisBeta cells within the pancreatic islet represent a heterogenous population wherein individual sub-groups of cells make distinct contributions to the overall control of insulin secretion. These include a subpopulation of highly-connected hub cells, important for the propagation of intercellular Ca2+ waves. Functional subpopulations have also been demonstrated in human beta cells, with an altered subtype distribution apparent in type 2 diabetes. At present, the molecular mechanisms through which beta cell hierarchy is established are poorly understood. Changes at the level of the epigenome provide one such possibility which we explore here by focussing on the imprinted gene neuronatin (Nnat), which is required for normal insulin synthesis and secretion. MethodsSingle cell RNA-seq datasets were examined using Seurat 4.0 and ClusterProfiler running under R. Transgenic mice expressing eGFP under the control of the Nnat enhancer/promoter regions were generated for fluorescence-activated cell (FAC) sorting of beta cells and downstream analysis of CpG methylation by bisulphite and RNA sequencing, respectively. Animals deleted for the de novo methyltransferase, DNMT3A from the pancreatic progenitor stage were used to explore control of promoter methylation. Proteomics was performed using affinity purification mass spectrometry and Ca2+ dynamics explored by rapid confocal imaging of Cal-520 and Cal-590. Insulin secretion was measured using Homogeneous Time Resolved Fluorescence Imaging. ResultsNnat mRNA was differentially expressed in a discrete beta cell population in a developmental stage- and DNA methylation (DNMT3A)-dependent manner. Thus, pseudo-time analysis of embryonic data sets demonstrated the early establishment of Nnat-positive and negative subpopulations during embryogenesis. NNAT expression is also restricted to a subset of beta cells across the human islet that is maintained throughout adult life. NNAT+ beta cells also displayed a discrete transcriptome at adult stages, representing a sub-population specialised for insulin production, reminiscent of recently-described "{beta}HI" cells and were diminished in db/db mice. Hub cells were less abundant in the NNAT+ population, consistent with epigenetic control of this functional specialization. Conclusions/interpretationThese findings demonstrate that differential DNA methylation at Nnat represents a novel means through which beta cell heterogeneity is established during development. We therefore hypothesise that changes in methylation at this locus may thus contribute to a loss of beta cell hierarchy and connectivity, potentially contributing to defective insulin secretion in some forms of diabetes. Research in contextWhat is already known about this subject? - Neuronatin (Nnat/NNAT) is an imprinted gene in humans and mice and is required for glucose-stimulated insulin secretion in vivo - Pancreatic beta cells are functionally heterogeneous with specific highly-connected subpopulations known to coordinate islet wide Ca2+ dynamics - Functional subpopulations have been described in human beta cells and their distribution is altered in type 2 diabetes What is the key question? - Does NNAT mark a discrete subpopulation of functional beta cells and which epigenetic pathways coordinate its formation and maintenance? What are the new findings? - A subpopulation of NNAT+ beta cells is established prior to the first week of postnatal life in mice via de novo DNA methylation at the Nnat promoter - NNAT+ beta cells are transcriptionally highly differentiated and appear to be functionally specialised for insulin production, possibly corresponding to recently-described "{beta}HI" and "CD63hi" beta cells. NNAT is expressed in a subset of beta cells across the human islet, and its deficiency in human beta cells diminishes glucose-stimulated insulin secretion - NNAT+ cells are likelier to belong to the population of follower, rather than hub cells, consistent with a role in insulin production rather than glucose detection How might this impact on clinical practice in the foreseeable future? - Epigenome-modifying compounds may provide a way of enhancing beta cell function and the ensemble behaviour of the islet to stimulate insulin secretion

cell biology↗

bioRxiv2022

Reduced insulin signalling in neurons induces sex-specific health benefits

Reduced activity of the insulin/IGF signalling (IIS) network extends healthspan and lifespan in mammals and possibly humans. Loss of the Irs1 gene increases survival in mice and causes tissue-specific changes in gene expression. However, the tissues underlying IIS mediated longevity are currently unknown. Here we measured survival and healthspan in male and female animals lacking Irs1 activity specifically in the liver, muscle, fat and brain. Tissue-specific loss of IRS1 did not increase survival, suggesting that lack of Irs1 in more than one tissue is required for lifespan extension. Furthermore, loss of Irs1 in liver, muscle and fat did not improve health at old age. In contrast, loss of neuronal Irs1 increased energy expenditure, locomotion and insulin sensitivity, specifically in old males. Neuronal loss of IRS1 also caused male-specific mitochondrial dysfunction, activation of Atf4 and metabolic adaptations consistent with an activated integrated stress response at old age. Thus, we identified a male-specific brain signature of ageing in response to reduced IIS associated with improved health outcomes at old age.

neuroscience↗

bioRxiv2019

Mesocorticolimbic circuit mechanisms underlying the effects of ketamine on dopamine: a translational imaging study

Patients with schizophrenia show increased striatal dopamine synthesis capacity in imaging studies. However, the mechanism underlying this is unclear but may be due to N-methyl-D-aspartate receptor (NMDAR) hypofunction and parvalbumin (PV) neuronal dysfunction leading to disinhibition of mesostriatal dopamine neurons. Here, we test this in a translational mouse imaging study using a ketamine model. Mice were treated with sub-chronic ketamine (30mg/kg) or saline followed by in-vivo positron emission tomography of striatal dopamine synthesis capacity, analogous to measures used in patients. Locomotor activity was measured using the open field test. In-vivo cell-type-specific chemogenetic approaches and pharmacological interventions were used to manipulate neuronal excitability. Immunohistochemistry and RNA sequencing were used to investigate molecular mechanisms. Sub-chronic ketamine increased striatal dopamine synthesis capacity (Cohens d=2.5, P<0.001) and locomotor activity. These effects were countered by inhibition of midbrain dopamine neurons, and by activation of cortical and ventral subiculum PV interneurons. Sub-chronic ketamine reduced PV expression in these neurons. Pharmacological intervention with SEP-363856, a novel psychotropic agent with agonism at trace amine receptor 1 (TAAR1), significantly reduced the ketamine-induced increase in dopamine synthesis capacity. These results show that sub-chronic ketamine treatment in mice mimics the dopaminergic alterations in patients with psychosis, and suggest an underlying neurocircuit involving PV interneuron hypofunction in frontal cortex and hippocampus as well as activation of midbrain dopamine neurons. A novel TAAR1 agonist reversed the dopaminergic alterations suggesting a therapeutic mechanism for targeting presynaptic dopamine dysfunction in patients.

neuroscience↗