Glutathionylation state of uncoupling protein-2 and the control of glucose-stimulated insulin secretion.
ABSTRACT: The role of reactive oxygen species (ROS) in glucose-stimulated insulin release remains controversial because ROS have been shown to both amplify and impede insulin release. In regard to preventing insulin release, ROS activates uncoupling protein-2 (UCP2), a mitochondrial inner membrane protein that negatively regulates glucose-stimulated insulin secretion (GSIS) by uncoupling oxidative phosphorylation. With our recent discovery that the UCP2-mediated proton leak is modulated by reversible glutathionylation, a process responsive to small changes in ROS levels, we resolved to determine whether glutathionylation is required for UCP2 regulation of GSIS. Using Min6 cells and pancreatic islets, we demonstrate that induction of glutathionylation not only deactivates UCP2-mediated proton leak but also enhances GSIS. Conversely, an increase in mitochondrial matrix ROS was found to deglutathionylate and activate UCP2 leak and impede GSIS. Glucose metabolism also decreased the total amount of cellular glutathionylated proteins and increased the cellular glutathione redox ratio (GSH/GSSG). Intriguingly, the provision of extracellular ROS (H(2)O(2), 10 ?M) amplified GSIS and also activated UCP2. Collectively, our findings indicate that the glutathionylation status of UCP2 contributes to the regulation of GSIS, and different cellular sites and inducers of ROS can have opposing effects on GSIS, perhaps explaining some of the controversy surrounding the role of ROS in GSIS.
Project description:We have recently shown that overnight exposure of INS-1E insulinoma cells to palmitate in the presence of high glucose causes defects in both mitochondrial energy metabolism and glucose-stimulated insulin secretion (GSIS). Here we report experiments designed to test the involvement of mitochondrial uncoupling protein-2 (UCP2) in these glucolipotoxic effects. Measuring real-time oxygen consumption in siRNA-transfected INS-1E cells, we show that deleterious effects of palmitate on the glucose sensitivity of mitochondrial respiration and on the coupling efficiency of oxidative phosphorylation are independent of UCP2. Consistently, palmitate impairs GSIS to the same extent in cells with and without UCP2. Furthermore, we knocked down UCP2 in spheroid INS-1E cell clusters (pseudoislets) to test whether or not UCP2 regulates insulin secretion during prolonged glucose exposure. We demonstrate that there are no differences in temporal GSIS kinetics between perifused pseudoislets with and without UCP2. We conclude that UCP2 is not involved in palmitate-induced impairment of GSIS in INS-1E insulinoma cells and is not needed for the amplification of insulin release. These conclusions inform ongoing debate on the disputed biochemical and physiological functions of the beta cell UCP2.
Project description:Glucose-stimulated insulin secretion (GSIS) from pancreatic ?-cells requires an increase in intracellular free Ca2+ concentration ([Ca2+]). Glucose uptake into ?-cells promotes Ca2+ influx and reactive oxygen species (ROS) generation. In other cell types, Ca2+ and ROS jointly induce Ca2+ release mediated by ryanodine receptor (RyR) channels. Therefore, we explored here if RyR-mediated Ca2+ release contributes to GSIS in ?-cell islets isolated from male rats. Stimulatory glucose increased islet insulin secretion, and promoted ROS generation in islets and dissociated ?-cells. Conventional PCR assays and immunostaining confirmed that ?-cells express RyR2, the cardiac RyR isoform. Extended incubation of ?-cell islets with inhibitory ryanodine suppressed GSIS; so did the antioxidant N-acetyl cysteine (NAC), which also decreased insulin secretion induced by glucose plus caffeine. Inhibitory ryanodine or NAC did not affect insulin secretion induced by glucose plus carbachol, which engages inositol 1,4,5-trisphosphate receptors. Incubation of islets with H2O2 in basal glucose increased insulin secretion 2-fold. Inhibitory ryanodine significantly decreased H2O2-stimulated insulin secretion and prevented the 4.5-fold increase of cytoplasmic [Ca2+] produced by incubation of dissociated ?-cells with H2O2. Addition of stimulatory glucose or H2O2 (in basal glucose) to ?-cells disaggregated from islets increased RyR2 S-glutathionylation to similar levels, measured by a proximity ligation assay; in contrast, NAC significantly reduced the RyR2 S-glutathionylation increase produced by stimulatory glucose. We propose that RyR2-mediated Ca2+ release, induced by the concomitant increases in [Ca2+] and ROS produced by stimulatory glucose, is an essential step in GSIS.
Project description:The circadian clock has been shown to regulate metabolic homeostasis. Mice with a deletion of Bmal1, a key component of the core molecular clock, develop hyperglycemia and hypoinsulinemia, suggesting ?-cell dysfunction. However, the underlying mechanisms are not fully known. In this study, we investigated the mechanisms underlying the regulation of ?-cell function by Bmal1. We studied ?-cell function in global Bmal1-/- mice, in vivo and in isolated islets ex vivo, as well as in rat insulinoma cell lines with shRNA-mediated Bmal1 knockdown. Global Bmal1-/- mice develop diabetes secondary to a significant impairment in glucose-stimulated insulin secretion (GSIS). There is a blunting of GSIS in both isolated Bmal1-/- islets and in Bmal1 knockdown cells, as compared to controls, suggesting that this is secondary to a loss of cell-autonomous effect of Bmal1. In contrast to previous studies, in these Bmal1-/- mice on a C57Bl/6 background, the loss of stimulated insulin secretion, interestingly, is with glucose but not to other depolarizing secretagogues, suggesting that events downstream of membrane depolarization are largely normal in Bmal1-/- islets. This defect in GSIS occurs as a result increased mitochondrial uncoupling with consequent impairment of glucose-induced mitochondrial potential generation and ATP synthesis, due to an upregulation of Ucp2. Inhibition of Ucp2, in isolated islets, leads to a rescue of the glucose-induced ATP production and insulin secretion in Bmal1-/- islets. Thus, Bmal1 regulates mitochondrial energy metabolism to maintain normal GSIS and its disruption leads to diabetes due to a loss of GSIS.
Project description:To investigate the anti-diabetic properties of chebulic acid (CA) associated with the prevention of methyl glyoxal (MG)-induced mitochondrial dysfunction in INS-1 pancreatic ?-cells, INS-1 cells were pre-treated with CA (0.5, 1.0, and 2.0 ?M) for 48 h and then treated with 2 mM MG for 8 h. The effects of CA and MG on INS-1 cells were evaluated using the following: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; glyoxalase 1 (Glo-1) expression via Western blot and enzyme activity assays; Nrf-2, nuclear factor erythroid 2-related factor 2 protein expression via Western blot assay; reactive oxygen species (ROS) production assay; mRNA expression of mitochondrial dysfunction related components (UCP2, uncoupling protein 2; VDAC1, voltage-dependent anion-selective channel-1; cyt c, cytochrome c via quantitative reverse transcriptase-PCR; mitochondrial membrane potential (MMP); adenosine triphosphate (ATP) synthesis; glucose-stimulated insulin secretion (GSIS) assay. The viability of INS-1 cells was maintained upon pre-treating with CA before exposure to MG. CA upregulated Glo-1 protein expression and enzyme activity in INS-1 cells and prevented MG-induced ROS production. Mitochondrial dysfunction was alleviated by CA pretreatment; this occurred via the downregulation of UCP2, VDAC1, and cyt c mRNA expression and the increase of MMP and ATP synthesis. Further, CA pre-treatment promoted the recovery from MG-induced decrease in GSIS. These results indicated that CA could be employed as a therapeutic agent in diabetes due to its ability to prevent MG-induced development of insulin sensitivity and oxidative stress-induced dysfunction of ?-cells.
Project description:The mitochondrial uncoupling proteins 2 and 3 (UCP2 and -3) are known to curtail oxidative stress and participate in a wide array of cellular functions, including insulin secretion and the regulation of satiety. However, the molecular control mechanism(s) governing these proteins remains elusive. Here we reveal that UCP2 and UCP3 contain reactive cysteine residues that can be conjugated to glutathione. We further demonstrate that this modification controls UCP2 and UCP3 function. Both reactive oxygen species and glutathionylation were found to activate and deactivate UCP3-dependent increases in non-phosphorylating respiration. We identified both Cys(25) and Cys(259) as the major glutathionylation sites on UCP3. Additional experiments in thymocytes from wild-type and UCP2 null mice demonstrated that glutathionylation similarly diminishes non-phosphorylating respiration. Our results illustrate that UCP2- and UCP3-mediated state 4 respiration is controlled by reversible glutathionylation. Altogether, these findings advance our understanding of the roles UCP2 and UCP3 play in modulating metabolic efficiency, cell signaling, and oxidative stress processes.
Project description:AMPK (AMP-activated protein kinase) signalling plays a key role in whole-body energy homoeostasis, although its precise role in pancreatic beta-cell function remains unclear. In the present study, we therefore investigated whether AMPK plays a critical function in beta-cell glucose sensing and is required for the maintenance of normal glucose homoeostasis. Mice lacking AMPK alpha2 in beta-cells and a population of hypothalamic neurons (RIPCre alpha2KO mice) and RIPCre alpha2KO mice lacking AMPK alpha1 (alpha1KORIPCre alpha2KO) globally were assessed for whole-body glucose homoeostasis and insulin secretion. Isolated pancreatic islets from these mice were assessed for glucose-stimulated insulin secretion and gene expression changes. Cultured beta-cells were examined electrophysiologically for their electrical responsiveness to hypoglycaemia. RIPCre alpha2KO mice exhibited glucose intolerance and impaired GSIS (glucose-stimulated insulin secretion) and this was exacerbated in alpha1KORIPCre alpha2KO mice. Reduced glucose concentrations failed to completely suppress insulin secretion in islets from RIPCre alpha2KO and alpha1KORIPCre alpha2KO mice, and conversely GSIS was impaired. Beta-cells lacking AMPK alpha2 or expressing a kinase-dead AMPK alpha2 failed to hyperpolarize in response to low glucose, although KATP (ATP-sensitive potassium) channel function was intact. We could detect no alteration of GLUT2 (glucose transporter 2), glucose uptake or glucokinase that could explain this glucose insensitivity. UCP2 (uncoupling protein 2) expression was reduced in RIPCre alpha2KO islets and the UCP2 inhibitor genipin suppressed low-glucose-mediated wild-type mouse beta-cell hyperpolarization, mimicking the effect of AMPK alpha2 loss. These results show that AMPK alpha2 activity is necessary to maintain normal pancreatic beta-cell glucose sensing, possibly by maintaining high beta-cell levels of UCP2.
Project description:<h4>Aims</h4>Pancreatic β-cell chronic lipotoxicity evolves from acute free fatty acid (FA)-mediated oxidative stress, unprotected by antioxidant mechanisms. Since mitochondrial uncoupling protein-2 (UCP2) plays antioxidant and insulin-regulating roles in pancreatic β-cells, we tested our hypothesis, that UCP2-mediated uncoupling attenuating mitochondrial superoxide production is initiated by FA release due to a direct H2O2-induced activation of mitochondrial phospholipase iPLA2γ.<h4>Results</h4>Pro-oxidant tert-butylhydroperoxide increased respiration, decreased membrane potential and mitochondrial matrix superoxide release rates of control but not UCP2- or iPLA2γ-silenced INS-1E cells. iPLA2γ/UCP2-mediated uncoupling was alternatively activated by an H2O2 burst, resulting from palmitic acid (PA) β-oxidation, and it was prevented by antioxidants or catalase overexpression. Exclusively, nascent FAs that cleaved off phospholipids by iPLA2γ were capable of activating UCP2, indicating that the previously reported direct redox UCP2 activation is actually indirect. Glucose-stimulated insulin release was not affected by UCP2 or iPLA2γ silencing, unless pro-oxidant activation had taken place. PA augmented insulin secretion via G-protein-coupled receptor 40 (GPR40), stimulated by iPLA2γ-cleaved FAs (absent after GPR40 silencing).<h4>Innovation and conclusion</h4>The iPLA2γ/UCP2 synergy provides a feedback antioxidant mechanism preventing oxidative stress by physiological FA intake in pancreatic β-cells, regulating glucose-, FA-, and redox-stimulated insulin secretion. iPLA2γ is regulated by exogenous FA via β-oxidation causing H2O2 signaling, while FAs are cleaved off phospholipids, subsequently acting as amplifying messengers for GPR40. Hence, iPLA2γ acts in eminent physiological redox signaling, the impairment of which results in the lack of antilipotoxic defense and contributes to chronic lipotoxicity.
Project description:<h4>Background and purpose</h4>Uncoupling protein-2 (UCP2) may regulate glucose-stimulated insulin secretion. The current study investigated the effects of berberine, an alkaloid found in many medicinal plants, on oxidative stress and insulin secretion through restoration of UCP2 expression in high glucose (HG)-treated INS-1E cells and rat islets or in db/db mouse islets.<h4>Experimental approach</h4>Mouse and rat pancreatic islets were isolated. Nitrotyrosine, superoxide dismutase (SOD)-1 and UCP2 expression and AMPK phosphorylation were examined by Western blotting. Insulin secretion was measured by ELISA. Mitochondrial reactive oxygen species (ROS) production was detected by confocal microscopy.<h4>Key results</h4>Incubation of INS-1E cells and rat islets with HG (30 mmol·L(-1); 8 h) elevated nitrotyrosine level, reduced SOD-1 and UCP2 expression and AMPK phosphorylation, and inhibited glucose-stimulated insulin secretion. HG also increased mitochondrial ROS in INS-1E cells. Co-treatment with berberine inhibited such effects. The AMPK inhibitor compound C, the UCP2 inhibitor genipin and adenovirus ucp2 shRNA inhibited these protective effects of berberine. Furthermore, compound C normalized berberine-stimulated UCP2 expression but genipin did not affect AMPK phosphorylation. Islets from db/db mice exhibited elevated nitrotyrosine levels, reduced expression of SOD-1 and UCP2 and AMPK phosphorylation, and decreased insulin secretion compared with those from db/m(+) mice. Berberine also improved these defects in diabetic islets and genipin blocked the effects of berberine.<h4>Conclusions and implications</h4>Berberine inhibited oxidative stress and restored insulin secretion in HG-treated INS-IE cells and diabetic mouse islets by activating AMPK and UCP2. UCP2 is an important signalling molecule in mediating anti-diabetic effects of berberine.
Project description:Although mitochondrial dysfunction is often accompanied by excessive reactive oxygen species (ROS) production, we previously showed that an increase in random somatic mtDNA mutations does not result in increased oxidative stress. Normal levels of ROS and oxidative stress could also be a result of an active compensatory mechanism such as a mild increase in proton leak. Uncoupling protein 2 (UCP2) was proposed to play such a role in many physiological situations. However, we show that upregulation of UCP2 in mtDNA mutator mice is not associated with altered proton leak kinetics or ROS production, challenging the current view on the role of UCP2 in energy metabolism. Instead, our results argue that high UCP2 levels allow better utilization of fatty acid oxidation resulting in a beneficial effect on mitochondrial function in heart, postponing systemic lactic acidosis and resulting in longer lifespan in these mice. This study proposes a novel mechanism for an adaptive response to mitochondrial cardiomyopathy that links changes in metabolism to amelioration of respiratory chain deficiency and longer lifespan.
Project description:Deterioration of pancreatic beta-cells plays a critical role in the development of type 2 diabetes. Among the various stressors contributing to these deleterious effects, glucotoxicity and superoxides have been proposed as major players. In this context, the mitochondrial uncoupling protein UCP2 is regularly associated with the stress response. In the present study, we tested the effects of UCP2 upregulation in mouse islets with beta-cell specific overexpression of UCP2 (RIP-UCP2). Islets were subjected to both chronic glucotoxicity (7 days at 30mM glucose) and acute oxidative stress (200µM H2O2 for 10min). Increased UCP2 expression did not alter mitochondrial potential and ATP generation but protected against glucotoxic effects. Glucose-stimulated insulin secretion was altered by both glucotoxicity and oxidative stress, in particular through higher basal insulin release at non-stimulatory glucose concentrations. The secretory response to glucose stimulation was partially preserved in beta-cells overexpressing UCP2. The higher rate of cell death induced by chronic high glucose exposure was lower in RIP-UCP2 islets. Finally, superoxide production was reduced by high glucose, both under acute and chronic conditions, and not modified by UCP2 overexpression. In conclusion, upregulation of UCP2 conferred protective effects to the stressed beta-cell through mechanisms not directly associated with superoxide production.