The pore-forming subunit MCU of the mitochondrial Ca2+ uniporter is required for normal glucose-stimulated insulin secretion in vitro and in vivo in mice.

AIMS/HYPOTHESIS: Mitochondrial oxidative metabolism is central to glucose-stimulated insulin secretion (GSIS). Whether Ca2+ uptake into pancreatic beta cell mitochondria potentiates or antagonises this process is still a matter of debate. Although the mitochondrial Ca2+ importer (MCU) complex is thought to represent the main route for Ca2+ transport across the inner mitochondrial membrane, its role in beta cells has not previously been examined in vivo. METHODS: Here, we inactivated the pore-forming subunit of the MCU, encoded by Mcu, selectively in mouse beta cells using Ins1Cre-mediated recombination. Whole or dissociated pancreatic islets were isolated and used for live beta cell fluorescence imaging of cytosolic or mitochondrial Ca2+ concentration and ATP production in response to increasing glucose concentrations. Electrophysiological recordings were also performed on whole islets. Serum and blood samples were collected to examine oral and i.p. glucose tolerance. RESULTS: Glucose-stimulated mitochondrial Ca2+ accumulation (p< 0.05), ATP production (p< 0.05) and insulin secretion (p< 0.01) were strongly inhibited in beta cell-specific Mcu-null (ßMcu-KO) animals, in vitro, as compared with wild-type (WT) mice. Interestingly, cytosolic Ca2+ concentrations increased (p< 0.001), whereas mitochondrial membrane depolarisation improved in ßMcu-KO animals. ßMcu-KO mice displayed impaired in vivo insulin secretion at 5 min (p< 0.001) but not 15 min post-i.p. injection of glucose, whilst the opposite phenomenon was observed following an oral gavage at 5 min. Unexpectedly, glucose tolerance was improved (p< 0.05) in young ßMcu-KO (<12 weeks), but not in older animals vs WT mice. CONCLUSIONS/INTERPRETATION: MCU is crucial for mitochondrial Ca2+ uptake in pancreatic beta cells and is required for normal GSIS. The apparent compensatory mechanisms that maintain glucose tolerance in ßMcu-KO mice remain to be established.

Decreased STARD10 Expression Is Associated with Defective Insulin Secretion in Humans and Mice.

Genetic variants near ARAP1 (CENTD2) and STARD10 influence type 2 diabetes (T2D) risk. The risk alleles impair glucose-induced insulin secretion and, paradoxically but characteristically, are associated with decreased proinsulin:insulin ratios, indicating improved proinsulin conversion. Neither the identity of the causal variants nor the gene(s) through which risk is conferred have been firmly established. Whereas ARAP1 encodes a GTPase activating protein, STARD10 is a member of the steroidogenic acute regulatory protein (StAR)-related lipid transfer protein family. By integrating genetic fine-mapping and epigenomic annotation data and performing promoter-reporter and chromatin conformational capture (3C) studies in ß cell lines, we localize the causal variant(s) at this locus to a 5 kb region that overlaps a stretch-enhancer active in islets. This region contains several highly correlated T2D-risk variants, including the rs140130268 indel. Expression QTL analysis of islet transcriptomes from three independent subject groups demonstrated that T2D-risk allele carriers displayed reduced levels of STARD10 mRNA, with no concomitant change in ARAP1 mRNA levels. Correspondingly, ß-cell-selective deletion of StarD10 in mice led to impaired glucose-stimulated Ca2+ dynamics and insulin secretion and recapitulated the pattern of improved proinsulin processing observed at the human GWAS signal. Conversely, overexpression of StarD10 in the adult ß cell improved glucose tolerance in high fat-fed animals. In contrast, manipulation of Arap1 in ß cells had no impact on insulin secretion or proinsulin conversion in mice. This convergence of human and murine data provides compelling evidence that the T2D risk associated with variation at this locus is mediated through reduction in STARD10 expression in the ß cell.

Caffeine protects against experimental acute pancreatitis by inhibition of inositol 1,4,5-trisphosphate receptor-mediated Ca2+ release.

OBJECTIVE: Caffeine reduces toxic Ca2+ signals in pancreatic acinar cells via inhibition of inositol 1,4,5-trisphosphate receptor (IP3R)-mediated signalling, but effects of other xanthines have not been evaluated, nor effects of xanthines on experimental acute pancreatitis (AP). We have determined effects of caffeine and its xanthine metabolites on pancreatic acinar IP3R-mediated Ca2+ signalling and experimental AP. DESIGN: Isolated pancreatic acinar cells were exposed to secretagogues, uncaged IP3 or toxins that induce AP and effects of xanthines, non-xanthine phosphodiesterase (PDE) inhibitors and cyclic adenosine monophosphate and cyclic guanosine monophosphate (cAMP/cGMP) determined. The intracellular cytosolic calcium concentration ([Ca2+]C), mitochondrial depolarisation and necrosis were assessed by confocal microscopy. Effects of xanthines were evaluated in caerulein-induced AP (CER-AP), taurolithocholic acid 3-sulfate-induced AP (TLCS-AP) or palmitoleic acid plus ethanol-induced AP (fatty acid ethyl ester AP (FAEE-AP)). Serum xanthines were measured by liquid chromatography-mass spectrometry. RESULTS: Caffeine, dimethylxanthines and non-xanthine PDE inhibitors blocked IP3-mediated Ca2+ oscillations, while monomethylxanthines had little effect. Caffeine and dimethylxanthines inhibited uncaged IP3-induced Ca2+ rises, toxin-induced Ca2+ release, mitochondrial depolarisation and necrotic cell death pathway activation; cAMP/cGMP did not inhibit toxin-induced Ca2+ rises. Caffeine significantly ameliorated CER-AP with most effect at 25 mg/kg (seven injections hourly); paraxanthine or theophylline did not. Caffeine at 25 mg/kg significantly ameliorated TLCS-AP and FAEE-AP. Mean total serum levels of dimethylxanthines and trimethylxanthines peaked at >2 mM with 25 mg/kg caffeine but at <100 µM with 25 mg/kg paraxanthine or theophylline. CONCLUSIONS: Caffeine and its dimethylxanthine metabolites reduced pathological IP3R-mediated pancreatic acinar Ca2+ signals but only caffeine ameliorated experimental AP. Caffeine is a suitable starting point for medicinal chemistry.

Changes in the expression of the type 2 diabetes-associated gene VPS13C in the ß-cell are associated with glucose intolerance in humans and mice.

Single 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 ß-cells and other cell lines, the risk variant at rs7163757 lowered enhancer activity. Mice deleted for Vps13c selectively in the ß-cell were generated by crossing animals bearing a floxed allele at exon 1 to mice expressing Cre recombinase under Ins1 promoter control (Ins1Cre). Whereas Vps13c(fl/fl):Ins1Cre (ßVps13cKO) 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 ß-cell mass in KO mice vs. controls, and glucose-stimulated insulin secretion from isolated islets was not altered in vitro between control and ßVps13cKO mice. However, a tendency was observed in female null mice for lower insulin levels and ß-cell function (HOMA-B) in vivo. Furthermore, glucose-stimulated increases in intracellular free Ca(2+) were significantly increased in islets from female KO mice, suggesting impaired Ca(2+) 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.

Disallowance of Acot7 in ß-Cells Is Required for Normal Glucose Tolerance and Insulin Secretion.

Encoding acyl-CoA thioesterase-7 (Acot7) is one of ∼60 genes expressed ubiquitously across tissues but relatively silenced, or disallowed, in pancreatic ß-cells. The capacity of ACOT7 to hydrolyze long-chain acyl-CoA esters suggests potential roles in ß-oxidation, lipid biosynthesis, signal transduction, or insulin exocytosis. We explored the physiological relevance of ß-cell-specific Acot7 silencing by re-expressing ACOT7 in these cells. ACOT7 overexpression in clonal MIN6 and INS1(832/13) ß-cells impaired insulin secretion in response to glucose plus fatty acids. Furthermore, in a panel of transgenic mouse lines, we demonstrate that overexpression of mitochondrial ACOT7 selectively in the adult ß-cell reduces glucose tolerance dose dependently and impairs glucose-stimulated insulin secretion. By contrast, depolarization-induced secretion was unaffected, arguing against a direct action on the exocytotic machinery. Acyl-CoA levels, ATP/ADP increases, membrane depolarization, and Ca(2+) fluxes were all markedly reduced in transgenic mouse islets, whereas glucose-induced oxygen consumption was unchanged. Although glucose-induced increases in ATP/ADP ratio were similarly lowered after ACOT7 overexpression in INS1(832/13) cells, changes in mitochondrial membrane potential were unaffected, consistent with an action of Acot7 to increase cellular ATP consumption. Because Acot7 mRNA levels are increased in human islets in type 2 diabetes, inhibition of the enzyme might provide a novel therapeutic strategy.

The two pore channel TPC2 is dispensable in pancreatic ß-cells for normal Ca²âº dynamics and insulin secretion.

Ca(2+) signals are central to the stimulation of insulin secretion from pancreatic ß-cells by glucose and other agents, including glucagon-like peptide-1 (GLP-1). Whilst Ca(2+) influx through voltage-gated Ca(2+) channels on the plasma membrane is a key trigger for glucose-stimulated secretion, mobilisation of Ca(2+) from acidic stores has been implicated in the control of more localised Ca(2+) changes and membrane potential. Nicotinic acid adenine dinucleotide phosphate (NAADP), generated in ß-cells in response to high glucose, is a potent mobiliser of these stores, and has been proposed to act through two pore channels (TPC1 and TPC2, murine gene names Tpcn1 and Tpcn2). Whilst the role of TPC1 in the control of Ca(2+) mobilisation and insulin secretion was recently confirmed, conflicting data exist for TPC2. Here, we used the selective and efficient deleter strain, Ins1Cre to achieve ß-cell selective deletion of the Tpcn2 gene in mice. ßTpcn2 KO mice displayed normal intraperitoneal and oral glucose tolerance, and glucose-stimulated Ca(2+) dynamics and insulin secretion from islets were similarly normal. GLP-1-induced Ca(2+) increases involved an increase in oscillation frequency from 4.35 to 4.84 per minute (p=0.04) at 8mM glucose, and this increase was unaffected by the absence of Tpcn2. The current data thus indicate that TPC2 is not absolutely required for normal glucose- or incretin-stimulated insulin secretion from the ß-cell. Our findings suggest that TPC1, whose expression tended to increase in Tpcn2 null islets, might be sufficient to support normal Ca(2+) dynamics in response to stimulation by nutrients or incretins.

Molecular Genetic Regulation of Slc30a8/ZnT8 Reveals a Positive Association With Glucose Tolerance.

Zinc transporter 8 (ZnT8), encoded by SLC30A8, is chiefly expressed within pancreatic islet cells, where it mediates zinc (Zn(2+)) uptake into secretory granules. Although a common nonsynonymous polymorphism (R325W), which lowers activity, is associated with increased type 2 diabetes (T2D) risk, rare inactivating mutations in SLC30A8 have been reported to protect against T2D. Here, we generate and characterize new mouse models to explore the impact on glucose homeostasis of graded changes in ZnT8 activity in the ß-cell. Firstly, Slc30a8 was deleted highly selectively in these cells using the novel deleter strain, Ins1Cre. The resultant Ins1CreZnT8KO mice displayed significant (P < .05) impairments in glucose tolerance at 10 weeks of age vs littermate controls, and glucose-induced increases in circulating insulin were inhibited in vivo. Although insulin release from Ins1CreZnT8KO islets was normal, Zn(2+) release was severely impaired. Conversely, transgenic ZnT8Tg mice, overexpressing the transporter inducibly in the adult ß-cell using an insulin promoter-dependent Tet-On system, showed significant (P < .01) improvements in glucose tolerance compared with control animals. Glucose-induced insulin secretion from ZnT8Tg islets was severely impaired, whereas Zn(2+) release was significantly enhanced. Our findings demonstrate that glucose homeostasis in the mouse improves as ß-cell ZnT8 activity increases, and remarkably, these changes track Zn(2+) rather than insulin release in vitro. Activation of ZnT8 in ß-cells might therefore provide the basis of a novel approach to treating T2D.

Mechanism of mitochondrial permeability transition pore induction and damage in the pancreas: inhibition prevents acute pancreatitis by protecting production of ATP.

OBJECTIVE: Acute pancreatitis is caused by toxins that induce acinar cell calcium overload, zymogen activation, cytokine release and cell death, yet is without specific drug therapy. Mitochondrial dysfunction has been implicated but the mechanism not established. DESIGN: We investigated the mechanism of induction and consequences of the mitochondrial permeability transition pore (MPTP) in the pancreas using cell biological methods including confocal microscopy, patch clamp technology and multiple clinically representative disease models. Effects of genetic and pharmacological inhibition of the MPTP were examined in isolated murine and human pancreatic acinar cells, and in hyperstimulation, bile acid, alcoholic and choline-deficient, ethionine-supplemented acute pancreatitis. RESULTS: MPTP opening was mediated by toxin-induced inositol trisphosphate and ryanodine receptor calcium channel release, and resulted in diminished ATP production, leading to impaired calcium clearance, defective autophagy, zymogen activation, cytokine production, phosphoglycerate mutase 5 activation and necrosis, which was prevented by intracellular ATP supplementation. When MPTP opening was inhibited genetically or pharmacologically, all biochemical, immunological and histopathological responses of acute pancreatitis in all four models were reduced or abolished. CONCLUSIONS: This work demonstrates the mechanism and consequences of MPTP opening to be fundamental to multiple forms of acute pancreatitis and validates the MPTP as a drug target for this disease.

Incretin-modulated beta cell energetics in intact islets of Langerhans.

Incretins such as glucagon-like peptide 1 (GLP-1) are released from the gut and potentiate insulin release in a glucose-dependent manner. Although this action is generally believed to hinge on cAMP and protein kinase A signaling, up-regulated beta cell intermediary metabolism may also play a role in incretin-stimulated insulin secretion. By employing recombinant probes to image ATP dynamically in situ within intact mouse and human islets, we sought to clarify the role of GLP-1-modulated energetics in beta cell function. Using these techniques, we show that GLP-1 engages a metabolically coupled subnetwork of beta cells to increase cytosolic ATP levels, an action independent of prevailing energy status. We further demonstrate that the effects of GLP-1 are accompanied by alterations in the mitochondrial inner membrane potential and, at elevated glucose concentration, depend upon GLP-1 receptor-directed calcium influx through voltage-dependent calcium channels. Lastly, and highlighting critical species differences, beta cells within mouse but not human islets respond coordinately to incretin stimulation. Together, these findings suggest that GLP-1 alters beta cell intermediary metabolism to influence ATP dynamics in a species-specific manner, and this may contribute to divergent regulation of the incretin-axis in rodents and man.

Fatty acid ethyl ester synthase inhibition ameliorates ethanol-induced Ca2+-dependent mitochondrial dysfunction and acute pancreatitis.

OBJECTIVE: Non-oxidative metabolism of ethanol (NOME) produces fatty acid ethyl esters (FAEEs) via carboxylester lipase (CEL) and other enzyme action implicated in mitochondrial injury and acute pancreatitis (AP). This study investigated the relative importance of oxidative and non-oxidative pathways in mitochondrial dysfunction, pancreatic damage and development of alcoholic AP, and whether deleterious effects of NOME are preventable. DESIGN: Intracellular calcium ([Ca(2+)](C)), NAD(P)H, mitochondrial membrane potential and activation of apoptotic and necrotic cell death pathways were examined in isolated pancreatic acinar cells in response to ethanol and/or palmitoleic acid (POA) in the presence or absence of 4-methylpyrazole (4-MP) to inhibit oxidative metabolism. A novel in vivo model of alcoholic AP induced by intraperitoneal administration of ethanol and POA was developed to assess the effects of manipulating alcohol metabolism. RESULTS: Inhibition of OME with 4-MP converted predominantly transient [Ca(2+)](C) rises induced by low ethanol/POA combination to sustained elevations, with concurrent mitochondrial depolarisation, fall of NAD(P)H and cellular necrosis in vitro. All effects were prevented by 3-benzyl-6-chloro-2-pyrone (3-BCP), a CEL inhibitor. 3-BCP also significantly inhibited rises of pancreatic FAEE in vivo and ameliorated acute pancreatic damage and inflammation induced by administration of ethanol and POA to mice. CONCLUSIONS: A combination of low ethanol and fatty acid that did not exert deleterious effects per se became toxic when oxidative metabolism was inhibited. The in vitro and in vivo damage was markedly inhibited by blockade of CEL, indicating the potential for development of specific therapy for treatment of alcoholic AP via inhibition of FAEE generation.

Cryo-electron microscopy of coagulation Factor VIII bound to lipid nanotubes.

Factor VIII (FVIII) is a key protein in blood coagulation, deficiency or malfunction of which causes Haemophilia A. The sole cure for this condition is intravenous administration of FVIII, whose membrane-bound structure we have studied by Cryo-electron microscopy and image analysis. Self-assembled lipid nanotubes were optimised to bind FVIII at close to native conditions. The tubes diameter was constant at 30 nm and the lipid bilayer resolved. The FVIII molecules were well defined, forming an 8.5 nm thick outer layer, and appeared to reach the hydrophobic core of the bilayer. The two known FVIII atomic models were superimposed with the averaged 2D protein densities. The insertion of the FVIII within the membrane was evaluated, reaffirming that the membrane-binding C2 or C1-C2 domain(s) fully penetrate the outer leaflet of the lipid layer. The presented results lay the basis for new models of the FVIII overall orientation and membrane-binding mechanism.