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Journal articleJohnston NR, Mitchell RK, Haythorne E, et al., 2016,
Beta cell hubs dictate pancreatic islet responses to glucose
, Cell Metabolism, Vol: 24, Pages: 389-401, ISSN: 1932-7420The arrangement of beta cells within islets of Langerhans is critical for insulin release through thegeneration of rhythmic activity. A privileged role for individual beta cells in orchestrating theseresponses has long-been suspected, but not directly demonstrated. We show here that the beta cellpopulation in situ is operationally heterogeneous. Mapping of islet functional architecturerevealed the presence of hub cells with pacemaker properties, which remain stable over recordingperiods of 2-3 hours. Using a dual optogenetic/photopharmacological strategy, silencing of hubsabolished coordinated islet responses to glucose, whereas specific stimulation restoredcommunication patterns. Hubs were metabolically-adapted and targeted by both proinflammatoryand glucolipotoxic insults to induce widespread beta cell dysfunction. Thus, theislet is wired by hubs, whose failure may contribute to type 2 diabetes mellitus.
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Journal articleMehta ZB, FIne N, Pullen TJ, et al., 2016,
Changes in the expression of the type 2 diabetes-associated gene VPS13C in the β cell are associated with glucose intolerance in humans and mice
, American Journal of Physiology-Endocrinology and Metabolism, Vol: 311, Pages: E488-E507, ISSN: 1522-1555Single nucleotide polymorphisms (SNPs) close to the VPS13C, C2CD4A and C2CD4B genes on chromosome 15q are associated with impaired fasting glucose and increased risk of type 2 diabetes. eQTL analysis revealed an association between possession of risk (C) alleles at a previously implicated causal SNP, rs7163757, and lowered VPS13C and C2CD4A levels in islets from female (n = 40, P < 0.041) but not from male subjects. Explored using promoter-reporter assays in β-cells and other cell lines, the risk variant at rs7163757 lowered enhancer activity. Mice deleted for Vps13c selectively in the β-cell were generated by crossing animals bearing a floxed allele at exon 1 to mice expressing Cre recombinase under Ins1 promoter control (Ins1Cre). Whereas Vps13cfl/fl:Ins1Cre (βVps13cKO) mice displayed normal weight gain compared with control littermates, deletion of Vps13c had little effect on glucose tolerance. Pancreatic histology revealed no significant change in β-cell mass in KO mice vs. controls, and glucose-stimulated insulin secretion from isolated islets was not altered in vitro between control and βVps13cKO mice. However, a tendency was observed in female null mice for lower insulin levels and β-cell function (HOMA-B) in vivo. Furthermore, glucose-stimulated increases in intracellular free Ca2+ were significantly increased in islets from female KO mice, suggesting impaired Ca2+ sensitivity of the secretory machinery. The present data thus provide evidence for a limited role for changes in VPS13C expression in conferring altered disease risk at this locus, particularly in females, and suggest that C2CD4A may also be involved.
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Journal articleFrank JA, Yushchenko DA, Hodson DJ, et al., 2016,
Photoswitchable diacylglycerols enable optical control of protein kinase C.
, Nature Chemical Biology, Vol: 12, Pages: 755-762, ISSN: 1552-4469Increased levels of the second messenger lipid diacylglycerol (DAG) induce downstream signaling events including the translocation of C1-domain-containing proteins toward the plasma membrane. Here, we introduce three light-sensitive DAGs, termed PhoDAGs, which feature a photoswitchable acyl chain. The PhoDAGs are inactive in the dark and promote the translocation of proteins that feature C1 domains toward the plasma membrane upon a flash of UV-A light. This effect is quickly reversed after the termination of photostimulation or by irradiation with blue light, permitting the generation of oscillation patterns. Both protein kinase C and Munc13 can thus be put under optical control. PhoDAGs control vesicle release in excitable cells, such as mouse pancreatic islets and hippocampal neurons, and modulate synaptic transmission in Caenorhabditis elegans. As such, the PhoDAGs afford an unprecedented degree of spatiotemporal control and are broadly applicable tools to study DAG signaling.
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Journal articleSolomou A, Philippe E, Chabosseau P, et al., 2016,
Over-expression of Slc30a8/ZnT8 selectively in the mouse α cell impairs glucagon release and responses to hypoglycemia
, Nutrition and Metabolism, Vol: 13, ISSN: 0029-6678BackgroundThe human SLC30A8 gene encodes the secretory granule-localised zinc transporter ZnT8 whose expression is chiefly restricted to the endocrine pancreas. Single nucleotide polymorphisms (SNPs) in the human SLC30A8 gene have been associated, through genome-wide studies, with altered type 2 diabetes risk. In addition to a role in the control of insulin release, recent studies involving targeted gene ablation from the pancreatic α cell (Solomou et al., J Biol Chem 290(35):21432-42) have also implicated ZnT8 in the control of glucagon release. Up to now, however, the possibility that increased levels of the transporter in these cells may impact glucagon secretion has not been explored.MethodsHere, we use a recently-developed reverse tetracyline transactivator promoter-regulated ZnT8 transgene to drive the over-expression of human ZnT8 selectively in the α cell in adult mice. Glucose homeostasis and glucagon secretion were subsequently assessed both in vivo during hypoglycemic clamps and from isolated islets in vitro.ResultsDoxyclin-dependent human ZnT8 mRNA expression was apparent in both isolated islets and in fluorescence-activated cell sorting- (FACS) purified α cells. Examined at 12 weeks of age, intraperitoneal glucose (1 g/kg) tolerance was unchanged in transgenic mice versus wild-type littermates (n = 8-10 mice/genotype, p > 0.05) and sensitivity to intraperitoneal insulin (0.75U/kg) was similarly unaltered in transgenic animals. In contrast, under hyperinsulinemic-hypoglycemic clamp, a ~45 % (p < 0.001) reduction in glucose infusion rate was apparent, and glucagon release was significantly (~40 %, p < 0.01) impaired, in transgenic mice. Correspondingly, examined in vitro, glucagon secretion was significantly reduced (~30 %, p < 0.05) from transgenic versus control islets at low, stimulatory glucose concentrations (1 mM, p < 0.05) but not at
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Journal articleGerber PA, Rutter GA, 2016,
The role of oxidative stress and hypoxia in pancreatic beta-cell dysfunction in diabetes mellitus.
, Antioxidants & Redox Signaling, Vol: 26, Pages: 501-518, ISSN: 1557-7716SIGNIFICANCE: Metabolic syndrome is a frequent precursor of type 2 diabetes mellitus (T2D), a disease that currently affects ∼8% of the adult population worldwide. Pancreatic beta-cell dysfunction and loss are central to the disease process, although understanding of the underlying molecular mechanisms is still fragmentary. RECENT ADVANCES: Oversupply of nutrients, including glucose and fatty acids, and the subsequent overstimulation of beta cells, are believed to be an important contributor to insulin secretory failure in T2D. Hypoxia has also recently been implicated in beta-cell damage. Accumulating evidence points to a role for oxidative stress in both processes. Although the production of reactive oxygen species (ROS) results from enhanced mitochondrial respiration during stimulation with glucose and other fuels, the expression of antioxidant defense genes is unusually low (or disallowed) in beta cells. CRITICAL ISSUES: Not all subjects with metabolic syndrome and hyperglycemia go on to develop full-blown diabetes, implying an important role in disease risk for gene-environment interactions. Possession of common risk alleles at the SLC30A8 locus, encoding the beta-cell granule zinc transporter ZnT8, may affect cytosolic Zn(2+) concentrations and thus susceptibility to hypoxia and oxidative stress. FUTURE DIRECTIONS: Loss of normal beta-cell function, rather than total mass, is increasingly considered to be the major driver for impaired insulin secretion in diabetes. Better understanding of the role of oxidative changes, its modulation by genes involved in disease risk, and effects on beta-cell identity may facilitate the development of new therapeutic strategies to this disease. Antioxid. Redox Signal. 00, 000-000.
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Journal articleRutter GA, Semplici F, Mondragon A, et al., 2016,
Cell type-specific deletion in mice reveals roles for PAS kinase in insulin and glucagon production
, Diabetologia, Vol: 59, Pages: 1938-1947, ISSN: 1432-0428Background and Aims. Per-Arnt-Sim domain containing kinase (PASK) is a nutrient regulated protein kinase previously implicated in the control of insulin gene expression and glucagon secretion. Here, we explore the roles of the kinase in the control of islet hormone release by generating mice deleted selectively for the Pask gene in pancreatic beta or alpha cells. Methods. Floxed alleles of Pask were produced by homologous recombination and animals bred with mice bearing beta (Ins1Cre, PaskBKO), or alpha (PPG-Cre; PaskAKO) cell selective Cre recombinase alleles. Glucose homeostasis and hormone secretion in vivo and in vitro, gene expression, and islet cell mass, were measured using standard techniques.Results. Ins1Cre-based recombination led to efficient beta cell targeted deletion of Pask. Beta cell mass was reduced by 36.5% (p<0.05) compared to controls in PaskBKO mice, as well as in global null Pask mice (38%, p<0.05). PaskBKO mice displayed normal body weight and fasting glycemia, but slightly impaired glucose tolerance, and beta cell proliferation, after maintenance on a high fat diet. Whilst glucose tolerance was unaffected in PaskAKO mice, glucose infusion rates were increased, and glucagon secretion tended to be lower, during hypoglycemic clamps. Though alpha cell mass was increased (21.9%, p<0.05), glucagon release at low glucose was impaired (p<0.05) in PaskAKO islets. Conclusions. The present findings demonstrate cell autonomous roles for PASK in the control of pancreatic endocrine hormone secretion. Differencesbetween the glycemic phenotype of global versus cell type specific null mice suggest important roles for tissue interactions in the control of glycemia by the kinase.
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Journal articleChabosseau P, Rutter GA, 2016,
Zinc and diabetes
, Archives of Biochemistry and Biophysics, Vol: 611, Pages: 79-85, ISSN: 1096-0384Zn(2+) ions are essential for the normal processing and storage of insulin and altered pancreatic insulin content is associated with all forms of diabetes mellitus. Work of the past decade has identified variants in the human SLC30A8 gene, encoding the zinc transporter ZnT8 which is expressed highly selectively on the secretory granule of pancreatic islet β and α cells, as affecting the risk of Type 2 Diabetes. Here, we review the regulation and roles of Zn(2+) ions in islet cells, the mechanisms through which SLC30A8 variants might affect glucose homeostasis and diabetes risk, and the novel technologies including recombinant targeted zinc probes and knockout mice which have been developed to explore these questions.
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Conference paperMondragon A, Hodson D, Semplici F, et al., 2016,
1964-P: PAS-domain containing protein kinase (PASK) is required for the regulation of food intake and glucose homeostasis
, Annual Professional Conference of the American Diabetes Association, Publisher: American Diabetes Association, Pages: A504-A504, ISSN: 0012-1797 -
Journal articleDenton RM, Pullen TJ, Armstrong CT, et al., 2016,
Calcium-insensitive splice variants of mammalian E1 subunit of 2-oxoglutarate dehydrogenase complex with tissue-specific patterns of expression
, Biochemical Journal, Vol: 473, Pages: 1165-1178, ISSN: 1470-8728The 2-oxoglutarate dehydrogenase (OGDH) complex is an important control point in vertebrate mitochondrial oxidative metabolism, including in the citrate cycle and catabolism of alternative fuels including glutamine. It is subject to allosteric regulation by NADH and the ATP/ADP ratio, and by Ca2+ through binding to the E1 subunit. The latter involves a unique Ca2+-binding site which includes D114ADLD (site 1). Here, we describe three splice variants of E1 in which either the exon expressing this site is replaced with another exon (loss of site 1, LS1) or an additional exon is expressed leading to the insertion of 15 amino acids just downstream of site 1 (Insert), or both changes occur together (LS1/Insert). We show that all three variants are essentially Ca2+-insensitive. Comparison of massive parallel sequence (RNA-Seq) databases demonstrates predominant expression of the Ca2+-sensitive archetype form in heart and skeletal muscle, but substantial expression of the Ca2+-insensitive variants in brain, pancreatic islets and other tissues. Detailed proteomic and activity studies comparing OGDH complexes from rat heart and brain confirmed the substantial difference in expression between these tissues. The evolution of OGDH variants was explored using bioinformatics, and this indicated that Ca2+-sensitivity arose with the emergence of chordates. In all species examined, this was associated with the co-emergence of Ca2+-insensitive variants suggesting a retained requirement for the latter in some settings. Tissue-specific expression of OGDH splice variants may thus provide a mechanism that tunes the control of the enzyme to the specialized metabolic and signalling needs of individual cell types.
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Journal articleYavari A, Stocker CJ, Ghaffari S, et al., 2016,
Chronic activation of γ2 AMPK induces obesity and reduces β cell function.
, Cell Metabolism, Vol: 23, Pages: 821-836, ISSN: 1932-7420Despite significant advances in our understanding of the biology determining systemic energy homeostasis, the treatment of obesity remains a medical challenge. Activation of AMP-activated protein kinase (AMPK) has been proposed as an attractive strategy for the treatment of obesity and its complications. AMPK is a conserved, ubiquitously expressed, heterotrimeric serine/threonine kinase whose short-term activation has multiple beneficial metabolic effects. Whether these translate into long-term benefits for obesity and its complications is unknown. Here, we observe that mice with chronic AMPK activation, resulting from mutation of the AMPK γ2 subunit, exhibit ghrelin signaling-dependent hyperphagia, obesity, and impaired pancreatic islet insulin secretion. Humans bearing the homologous mutation manifest a congruent phenotype. Our studies highlight that long-term AMPK activation throughout all tissues can have adverse metabolic consequences, with implications for pharmacological strategies seeking to chronically activate AMPK systemically to treat metabolic disease.
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