Ca2+, NAD(P)H and membrane potential changes in pancreatic beta-cells by methyl succinate: comparison with glucose.
ABSTRACT: The present study was undertaken to determine the main metabolic secretory signals generated by the mitochondrial substrate MeS (methyl succinate) compared with glucose in mouse and rat islets and to understand the differences. Glycolysis and mitochondrial metabolism both have key roles in the stimulation of insulin secretion by glucose. Both fuels elicited comparable oscillatory patterns of Ca2+ and changes in plasma and mitochondrial membrane potential in rat islet cells and clonal pancreatic beta-cells (INS-1). Saturation of the Ca2+ signal occurred between 5 and 6 mM MeS, while secretion reached its maximum at 15 mM, suggesting operation of a K(ATP)-channel-independent pathway. Additional responses to MeS and glucose included elevated NAD(P)H autofluorescence in INS-1 cells and islets and increases in assayed NADH and NADPH and the ATP/ADP ratio. Increased NADPH and ATP/ADP ratios occurred more rapidly with MeS, although similar levels were reached after 5 min of exposure to each fuel, whereas NADH increased more with MeS than with glucose. Reversal of MeS-induced cell depolarization by Methylene Blue completely inhibited MeS-stimulated secretion, whereas basal secretion and KCl-induced changes in these parameters were not affected. MeS had no effect on secretion or signals in the mouse islets, in contrast with glucose, possibly due to a lack of malic enzyme. The data are consistent with the common intermediates being pyruvate, cytosolic NADPH or both, and suggest that cytosolic NADPH production could account for the more rapid onset of MeS-induced secretion compared with glucose stimulation.
Project description:OBJECTIVE:The glucose stimulation of insulin secretion (GSIS) by pancreatic ?-cells critically depends on increased production of metabolic coupling factors, including NADPH. Nicotinamide nucleotide transhydrogenase (NNT) typically produces NADPH at the expense of NADH and ?pH in energized mitochondria. Its spontaneous inactivation in C57BL/6J mice was previously shown to alter ATP production, Ca2+ influx, and GSIS, thereby leading to glucose intolerance. Here, we tested the role of NNT in the glucose regulation of mitochondrial NADPH and glutathione redox state and reinvestigated its role in GSIS coupling events in mouse pancreatic islets. METHODS:Islets were isolated from female C57BL/6J mice (J-islets), which lack functional NNT, and genetically close C57BL/6N mice (N-islets). Wild-type mouse NNT was expressed in J-islets by adenoviral infection. Mitochondrial and cytosolic glutathione oxidation was measured with glutaredoxin 1-fused roGFP2 probes targeted or not to the mitochondrial matrix. NADPH and NADH redox state was measured biochemically. Insulin secretion and upstream coupling events were measured under dynamic or static conditions by standard procedures. RESULTS:NNT is largely responsible for the acute glucose-induced rise in islet NADPH/NADP+ ratio and decrease in mitochondrial glutathione oxidation, with a small impact on cytosolic glutathione. However, contrary to current views on NNT in ?-cells, these effects resulted from a glucose-dependent reduction in NADPH consumption by NNT reverse mode of operation, rather than from a stimulation of its forward mode of operation. Accordingly, the lack of NNT in J-islets decreased their sensitivity to exogenous H2O2 at non-stimulating glucose. Surprisingly, the lack of NNT did not alter the glucose-stimulation of Ca2+ influx and upstream mitochondrial events, but it markedly reduced both phases of GSIS by altering Ca2+-induced exocytosis and its metabolic amplification. CONCLUSION:These results drastically modify current views on NNT operation and mitochondrial function in pancreatic ?-cells.
Project description:Exposure to chemotherapeutic agents has been linked to an increased risk of type 2 diabetes (T2D), a disease characterized by both the peripheral insulin resistance and impaired glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells. Using the rat β-cell line INS-1 832/13 and isolated mouse pancreatic islets, we investigated the effect of the chemotherapeutic drug doxorubicin (Adriamycin) on pancreatic β-cell survival and function. Exposure of INS-1 832/13 cells to doxorubicin caused impairment of GSIS, cellular viability, an increase in cellular toxicity, as soon as 6 h post-exposure. Doxorubicin impaired plasma membrane electron transport (PMET), a pathway dependent on reduced equivalents NADH and NADPH, but failed to redox cycle in INS-1 832/13 cells and with their lysates. Although NADPH/NADP(+ )content was unaffected, NADH/NAD(+ )content decreased at 4 h post-exposure to doxorubicin, and was followed by a reduction in ATP content. Previous studies have demonstrated that doxorubicin functions as a topoisomerase II inhibitor via induction of DNA cross-linking, resulting in apoptosis. Doxorubicin induced the expression of mRNA for mdm2, cyclin G1, and fas whereas downregulating p53, and increased the melting temperature of genomic DNA, consistent with DNA damage and induction of apoptosis. Doxorubicin also induced caspase-3 and -7 activity in INS-1 832/13 cells and mouse islets; co-treatment with the pan-caspase inhibitor Z-VAD-FMK temporarily attenuated the doxorubicin-mediated loss of viability in INS-1 832/13 cells. Together, these data suggest that DNA damage, not H2O2 produced via redox cycling, is a major mechanism of doxorubicin toxicity in pancreatic β-cells.
Project description:Cytosolic NADPH may act as one of the signals that couple glucose metabolism to insulin secretion in the pancreatic ß-cell. NADPH levels in the cytoplasm are largely controlled by the cytosolic isoforms of malic enzyme and isocitrate dehydrogenase (IDHc). Some studies have provided evidence for a role of malic enzyme in glucose-induced insulin secretion (GIIS) via pyruvate cycling, but the role of IDHc in ß-cell signaling is unsettled. IDHc is an established component of the isocitrate/?-ketoglutarate shuttle that transfers reducing equivalents (NADPH) from the mitochondrion to the cytosol. This shuttle is energy consuming since it is coupled to nicotinamide nucleotide transhydrogenase that uses the mitochondrial proton gradient to produce mitochondrial NADPH and NAD(+) from NADP(+) and NADH. To determine whether flux through IDHc is positively or negatively linked to GIIS, we performed RNAi knockdown experiments in ß-cells. Reduced IDHc expression in INS 832/13 cells and isolated rat islet ß-cells resulted in enhanced GIIS. This effect was mediated at least in part via the KATP-independent amplification arm of GIIS. IDHc knockdown in INS 832/13 cells did not alter glucose oxidation but it reduced fatty acid oxidation and increased lipogenesis from glucose. Metabolome profiling in INS 832/13 cells showed that IDHc knockdown increased isocitrate and NADP(+) levels. It also increased the cellular contents of several metabolites linked to GIIS, in particular some Krebs cycle intermediates, acetyl-CoA, glutamate, cAMP and ATP. The results identify IDHc as a component of the emerging pathways that negatively regulate GIIS.
Project description:Hyperandrogenism is associated with hyperinsulinemia and insulin resistance in adult females. We tested whether androgens dysregulate pancreatic beta cell function to induce hyperinsulinemia through transcriptional regulation of insulin gene (Ins) in the islets. Adult female Wistar rats implanted with dihydrotestosterone (DHT; 7.5-mg, 90-d release) or placebo pellets were examined after 10 weeks. DHT exposure increased plasma DHT levels by 2-fold similar to that in polycystic ovary syndrome in women. DHT exposure induced hyperinsulinemia with increased HOMA-IR index in fasting state and glucose intolerance and exaggerated insulin responses following glucose tolerance test. DHT females had no change in islet number, size and beta cell proliferation/apoptosis but exhibited significant mitochondrial dysfunction (higher ADP/ATP ratio, decreased mtDNA copy number, increased reactive oxygen production and downregulation of mitochondrial biogenesis) and enhanced glucose-stimulated insulin secretion. Ins expression was increased in DHT islets. In vitro incubation of control islets with DHT dose dependently stimulated Ins transcription. Analysis of Ins1 gene revealed a putative androgen responsive element in the promoter. Chromatin-immunoprecipitation assays showed that androgen receptors bind to this element in response to DHT stimulation. Furthermore, reporter assays showed that the promoter element is highly responsive to androgens. Insulin-stimulated glucose uptake in skeletal muscle was decreased with associated decrease in IRβ expression in DHT females. Our studies identified a novel androgen-mediated mechanism for the control of Ins expression via transcriptional regulation providing a molecular mechanism linking elevated androgens and hyperinsulemia. Decreased IRβ expression in the skeletal muscles may contribute, in part, to glucose intolerance in this model.
Project description:AIM:We previously found that chronic tuberous sclerosis protein 2 (TSC2) deletion induces activation of mammalian target of rapamycin Complex 1 (mTORC1) and leads to hypertrophy of pancreatic beta cells from pancreatic beta cell-specific TSC2 knockout (?TSC2(-/-)) mice. The present study examines the effects of TSC2 ablation on insulin secretion from pancreatic beta cells. METHODS:Isolated islets from ?TSC2(-/-) mice and TSC2 knockdown insulin 1 (INS-1) insulinoma cells treated with small interfering ribonucleic acid were used to investigate insulin secretion, ATP content and the expression of mitochondrial genes. RESULTS:Activation of mTORC1 increased mitochondrial DNA expression, mitochondrial density and ATP production in pancreatic beta cells of ?TSC2(-/-) mice. In TSC2 knockdown INS-1 cells, mitochondrial DNA expression, mitochondrial density and ATP production were increased compared with those in control INS-1 cells, consistent with the phenotype of ?TSC2(-/-) mice. TSC2 knockdown INS-1 cells also exhibited augmented insulin secretory response to glucose. Rapamycin inhibited mitochondrial DNA expression and ATP production as well as insulin secretion in response to glucose. Thus, ?TSC2(-/-) mice exhibit hyperinsulinemia due to an increase in the number of mitochondria as well as enlargement of individual beta cells via activation of mTORC1. CONCLUSION:Activation of mTORC1 by TSC2 ablation increases mitochondrial biogenesis and enhances insulin secretion from pancreatic beta cells.
Project description:Pancreatic beta-cells couple the oxidation of glucose to the secretion of insulin. Apart from the canonical K(ATP)-dependent glucose-stimulated insulin secretion (GSIS), there are important K(ATP)-independent mechanisms involving both anaplerosis and mitochondrial GTP (mtGTP). How mtGTP that is trapped within the mitochondrial matrix regulates the cytosolic calcium increases that drive GSIS remains a mystery. Here we have investigated whether the mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK-M) is the GTPase linking hydrolysis of mtGTP made by succinyl-CoA synthetase (SCS-GTP) to an anaplerotic pathway producing phosphoenolpyruvate (PEP). Although cytosolic PEPCK (PEPCK-C) is absent, PEPCK-M message and protein were detected in INS-1 832/13 cells, rat islets, and mouse islets. PEPCK enzymatic activity is half that of primary hepatocytes and is localized exclusively to the mitochondria. Novel (13)C-labeling strategies in INS-1 832/13 cells and islets measured substantial contribution of PEPCK-M to the synthesis of PEP. As high as 30% of PEP in INS-1 832/13 cells and 41% of PEP in rat islets came from PEPCK-M. The contribution of PEPCK-M to overall PEP synthesis more than tripled with glucose stimulation. Silencing the PEPCK-M gene completely inhibited GSIS underscoring its central role in mitochondrial metabolism-mediated insulin secretion. Given that mtGTP synthesized by SCS-GTP is an indicator of TCA flux that is crucial for GSIS, PEPCK-M is a strong candidate to link mtGTP synthesis with insulin release through anaplerotic PEP cycling.
Project description:BACKGROUND AND PURPOSE:Quinic acid (QA) is an abundant natural compound from plant sources which may improve metabolic health. However, little attention has been paid to its effects on pancreatic beta-cell functions, which contribute to the control of metabolic health by lowering blood glucose. Strategies targeting beta-cell signal transduction are a new approach for diabetes treatment. This study investigated the efficacy of QA to stimulate beta-cell function by targeting the basic molecular machinery of metabolism-secretion coupling. EXPERIMENTAL APPROACH:We measured bioenergetic parameters and insulin exocytosis in a model of insulin-secreting beta-cells (INS-1E), together with Ca2+ homeostasis, using genetically encoded sensors, targeted to different subcellular compartments. Islets from mice chronically infused with QA were also assessed. KEY RESULTS:QA triggered transient cytosolic Ca2+ increases in insulin-secreting cells by mobilizing Ca2+ from intracellular stores, such as endoplasmic reticulum. Following glucose stimulation, QA increased glucose-induced mitochondrial Ca2+ transients. We also observed a QA-induced rise of the NAD(P)H/NAD(P)+ ratio, augmented ATP synthase-dependent respiration, and enhanced glucose-stimulated insulin secretion. QA promoted beta-cell function in vivo as islets from mice infused with QA displayed improved glucose-induced insulin secretion. A diet containing QA improved glucose tolerance in mice. CONCLUSIONS AND IMPLICATIONS:QA modulated intracellular Ca2+ homeostasis, enhancing glucose-stimulated insulin secretion in both INS-1E cells and mouse islets. By increasing mitochondrial Ca2+ , QA activated the coordinated stimulation of oxidative metabolism, mitochondrial ATP synthase-dependent respiration, and therefore insulin secretion. Bioactive agents raising mitochondrial Ca2+ in pancreatic beta-cells could be used to treat diabetes.
Project description:Fibroblast growth factor-21 (FGF21) has therapeutic potential for metabolic syndrome due to positive effects on fatty acid metabolism in liver and white adipose tissue. FGF21 also improves pancreatic islet survival in excess palmitate; however, much less is known about FGF21-induced metabolism in this tissue. We first confirmed FGF21-dependent activity in islets by identifying expression of the cognate coreceptor Klotho?, and by measuring a ligand-stimulated decrease in acetyl-CoA carboxylase expression. To further reveal the effect of FGF21 on metabolism, we employed a unique combination of two-photon and confocal autofluorescence imaging of the NAD(P)H and mitochondrial NADH responses while holding living islets stationary in a microfluidic device. These responses were further correlated to mitochondrial membrane potential and insulin secretion. Glucose-stimulated responses were relatively unchanged by FGF21. In contrast, responses to glucose in the presence of palmitate were significantly reduced compared to controls showing diminished NAD(P)H, mitochondrial NADH, mitochondrial membrane potential, and insulin secretion. Consistent with the glucose-stimulated responses being smaller due to continued fatty acid oxidation, mitochondrial membrane potential was increased in FGF21-treated islets by using the fatty acid transport inhibitor etomoxir. Citrate-stimulated NADPH responses were also significantly larger in FGF21-treated islets suggesting preference for citrate cycling rather than acetyl-CoA carboxylase-dependent fatty acid synthesis. Overall, these data show a reduction in palmitate-induced potentiation of glucose-stimulated metabolism and insulin secretion in FGF21-treated islets, and establish the use of autofluorescence imaging and microfluidic devices to investigate cell metabolism in a limited amount of living tissue.
Project description:Mitochondria and NADPH oxidase are important sources of reactive oxygen species in particular the superoxide radical (ROS) in pancreatic islets. These molecules derived from molecular oxygen are involved in pancreatic ?-cells signaling and control of insulin secretion. We examined the involvement of ROS produced through NADPH oxidase in the leucine- and/or glucose-induced insulin secretion by pancreatic islets from fed or 48-hour fasted rats. Glucose-stimulated insulin secretion (GSIS) in isolated islets was evaluated at low (2.8 mM) or high (16.7 mM) glucose concentrations in the presence or absence of leucine (20 mM) and/or NADPH oxidase inhibitors (VAS2870-20 ?M or diphenylene iodonium-DPI-5 ?M). ROS production was determined in islets treated with dihydroethidium (DHE) or MitoSOX Red reagent for 20 min and dispersed for fluorescence measurement by flow cytometry. NADPH content variation was examined in INS-1E cells (an insulin secreting cell line) after incubation in the presence of glucose (2.8 or 16.7 mM) and leucine (20 mM). At 2.8 mM glucose, VAS2870 and DPI reduced net ROS production (by 30%) and increased GSIS (by 70%) in a negative correlation manner (r = -0.93). At 16.7 mM glucose or 20 mM leucine, both NADPH oxidase inhibitors did not alter insulin secretion neither net ROS production. Pentose phosphate pathway inhibition by treatment with DHEA (75 ?M) at low glucose led to an increase in net ROS production in pancreatic islets from fed rats (by 40%) and induced a marked increase (by 144%) in islets from 48-hour fasted rats. The NADPH/NADP+ ratio was increased when INS-1E cells were exposed to high glucose (by 4.3-fold) or leucine (by 3-fold). In conclusion, increased ROS production through NADPH oxidase prevents the occurrence of hypoglycemia in fasting conditions, however, in the presence of high glucose or high leucine levels, the increased production of NADPH and the consequent enhancement of the activity of the antioxidant defenses mitigate the excess of ROS production and allow the secretory process of insulin to take place.
Project description:When the extracellular concentration of glucose was raised from 3 mM to 7 mM (the concentration interval in which beta-cell depolarization and the major decrease in K+ permeability occur), the cytosolic free [NADPH]/[NADP+] ratio in mouse pancreatic islets increased by 29.5%. When glucose was increased to 20 mM, a 117% increase was observed. Glucose had no effect on the cytosolic free [NADH]/[NAD+] ratio. Neither the cytosolic free [NADPH]/[NADP+] ratio nor the corresponding [NADH]/[NAD+] ratio was affected when the islets were incubated with 20 mM-fructose or with 3 mM-glucose + 20 mM-fructose, although the last-mentioned condition stimulated insulin release. The insulin secretagogue leucine (10 mM) stimulated insulin secretion, but lowered the cytosolic free [NADPH]/[NADP+] ratio; 10 mM-leucine + 10 mM-glutamine stimulated insulin release and significantly enhanced both the [NADPH]/[NADP+] ratio and the [NADH]/[NAD+] ratio. It is concluded that the cytosolic free [NADPH]/[NADP+] ratio may be involved in coupling beta-cell glucose metabolism to beta-cell depolarization and ensuing insulin secretion, but it may not be the sole or major coupling factor in nutrient-induced stimulation of insulin secretion.