Mitochondrial NADPH Research Articles

A Role for Cytosolic Isocitrate Dehydrogenase as a Negative Regulator of Glucose Signaling for Insulin Secretion in Pancreatic ß-Cells

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/α–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.

Metabolic Reprogramming for Producing Energy and Reducing Power in Fumarate Hydratase Null Cells from Hereditary Leiomyomatosis Renal Cell Carcinoma

Fumarate hydratase (FH)-deficient kidney cancer undergoes metabolic remodeling, with changes in mitochondrial respiration, glucose, and glutamine metabolism. These changes represent multiple biochemical adaptations in glucose and fatty acid metabolism that supports malignant proliferation. However, the metabolic linkages between altered mitochondrial function, nucleotide biosynthesis and NADPH production required for proliferation and survival have not been elucidated. To characterize the alterations in glycolysis, the Krebs cycle and the pentose phosphate pathways (PPP) that either generate NADPH (oxidative) or do not (non-oxidative), we utilized [U-13C]-glucose, [U-13C,15N]-glutamine, and [1,2- 13C2]-glucose tracers with mass spectrometry and NMR detection to track these pathways, and measured the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of growing cell lines. This metabolic reprogramming in the FH null cells was compared to cells in which FH has been restored. The FH null cells showed a substantial metabolic reorganization of their intracellular metabolic fluxes to fulfill their high ATP demand, as observed by a high rate of glucose uptake, increased glucose turnover via glycolysis, high production of glucose-derived lactate, and low entry of glucose carbon into the Krebs cycle. Despite the truncation of the Krebs cycle associated with inactivation of fumarate hydratase, there was a small but persistent level of mitochondrial respiration, which was coupled to ATP production from oxidation of glutamine-derived α–ketoglutarate through to fumarate. [1,2- 13C2]-glucose tracer experiments demonstrated that the oxidative branch of PPP initiated by glucose-6-phosphate dehydrogenase activity is preferentially utilized for ribose production (56-66%) that produces increased amounts of ribose necessary for growth and NADPH. Increased NADPH is required to drive reductive carboxylation of α-ketoglutarate and fatty acid synthesis for rapid proliferation and is essential for defense against increased oxidative stress. This increased NADPH producing PPP activity was shown to be a strong consistent feature in both fumarate hydratase deficient tumors and cell line models.

A spontaneous mutation in the nicotinamide nucleotide transhydrogenase gene of C57BL/6J mice results in mitochondrial redox abnormalities

NADPH is the reducing agent for mitochondrial H2O2 detoxification systems. Nicotinamide nucleotide transhydrogenase (NNT), an integral protein located in the inner mitochondrial membrane, contributes to an elevated mitochondrial NADPH/NADP+ ratio. This enzyme catalyzes the reduction of NADP+ at the expense of NADH oxidation and H+ reentry to the mitochondrial matrix. A spontaneous Nnt mutation in C57BL/6J (B6J-NntMUT) mice arose nearly 3 decades ago but was only discovered in 2005. Here, we characterize the consequences of the Nnt mutation on the mitochondrial redox functions of B6J-NntMUT mice. Liver mitochondria were isolated both from an Nnt wild-type C57BL/6 substrain (B6JUnib-NntW) and from B6J-NntMUT mice. The functional evaluation of respiring mitochondria revealed major redox alterations in B6J-NntMUT mice, including an absence of transhydrogenation between NAD and NADP, higher rates of H2O2 release, the spontaneous oxidation of NADPH, the poor ability to metabolize organic peroxide, and a higher susceptibility to undergo Ca2+-induced mitochondrial permeability transition. In addition, the mitochondria of B6J-NntMUT mice exhibited increased oxidized/reduced glutathione ratios as compared to B6JUnib-NntW mice. Nonetheless, the maximal activity of NADP-dependent isocitrate dehydrogenase, which is a coexisting source of mitochondrial NADPH, was similar between both groups. Altogether, our data suggest that NNT functions as a high-capacity source of mitochondrial NADPH and that its functional loss due to the Nnt mutation results in mitochondrial redox abnormalities, most notably a poor ability to sustain NADP and glutathione in their reduced states. In light of these alterations, the potential drawbacks of using B6J-NntMUT mice in biomedical research should not be overlooked.

Expanding the Phenotypic Spectrum of Nicotinamide Nucleotide Transhydrogenase (NNT) Mutations and using Whole Exome Sequencing to Discover Potential Disease Modifiers

Mutations in the NNT gene (nicotinamide nucleotide transhydrogenase), which is involved in NADPH generation in mitochondria, have recently been described in familial glucocorticoid deficiency. We report two patients, one with isolated glucocorticoid deficiency and the other with a combined glucocorticoid and mineralocorticoid deficiency. Through whole exome sequencing, both cases were found to carry two different NNT mutations, confirming previous results for these patients. Each patient also carries multiple heterozygous protein-altering mutations in other genes involved in steroid hormone biogenesis and regulation. The patient with a combined glucocorticoid and mineralocorticoid deficiency is a compound heterozygote for common missense variants in the ME3 gene (mitochondrial malic enzyme 3), the product of which also generates NADPH in mitochondria. Mutations in NNT are the likely proximal cause of the glucocorticoid deficiency in both patients, but genetic background effects may be important in modulating the specific phenotype of adrenal insufficiency.

Acetylcholine Attenuates Hypoxia/ Reoxygenation-Induced Mitochondrial and Cytosolic ROS Formation in H9c2 Cells via M2 Acetylcholine Receptor

Background: The anti-infammatory and cardioprotective effect of acetylcholine (ACh) has been reported; nevertheless, whether and how ACh exhibits an antioxidant property against ischemia/reperfusion (I/R)-induced oxidative stress remains obscure. Methods: In the present study, H9c2 rat cardiomyocytes were exposed to hypoxia/reoxygenation (H/R) to mimic I/R injury. We estimated intracellular different sources of reactive oxygen species (ROS) by measuring mitochondrial ROS (mtROS), mitochondrial DNA (mtDNA) copy number, xanthine oxidase (XO) and NADPH oxidase (NOX) activity and expression of rac 1. Cell injury was determined by lactate dehydrogenase (LDH) release and cleaved caspase-3 expression. The siRNA transfection was performed to knockdown of M2 acetylcholine receptor (M2 AChR) expression. Results: 12-h hypoxia followed by 2-h reoxygenation resulted in an abrupt burst of ROS in H9c2 cells. Administration of ACh reduced the levels of ROS in a concentration-dependent manner. Compared to the H/R group, ACh decreased mtROS, recovered mtDNA copy number, diminished XO and NOX activity, rac 1 expression as well as cell injury. Co- treatment with atropine rather than hexamethonium abolished the antioxidant and cardioprotective effect of ACh. Moreover, knockdown of M2 AChR by siRNA showed the similar trends as atropine co-treatment group. Conclusions: ACh inhibits mitochondria-, XO- and NOX-derived ROS production thus protecting H9c2 cells against H/R-induced oxidative stress, and these benefcial effects are mainly mediated by M2 AChR. Our findings suggested that increasing ACh release could be a potential therapeutic strategy for treatment and prevention of I/R injury.

The Cellular Effects of Carbon Monoxide in the Airway

The notion of inhaled carbon monoxide (CO) being a toxic chemical under all circumstances is currently being challenged, as recent research has suggested that low concentrations of CO may have therapeutic value, especially in the airway. This review evaluates CO's effects on cellular functions that may result in beneficial outcomes in the settings of airway disease, inflammation, and injury. CO can modulate the stress response system of the cell by decreasing levels of reactive intermediates over time, produced by mitochondrial iNOS and NADPH oxidase. Intracellular stress-induced response factors (e.g., HIF-1 and HSP- 70) are induced in response to CO, possibly facilitating more rapid and effective defenses, in response to subsequent stressors. CO also can trigger changes in cellular functions downstream, protecting the cells from stress-associated events promoted in the airway, as a result of disease or injury, including reducing rates of apoptosis, proliferation, and inflammatory cellular infiltration, as well as preventing an imbalance in the extracellular matrix composition. CO has also been associated with maintaining homeostasis of ions essential for normal cellular functions (e.g., Na+, Fe2+,3+). CO also targets cell-specific functions of the airway, such as reduction of contractility of airway smooth muscle cells, and preservation of the innate defense mechanism of airway epithelial cells. Further understanding of CO's effects on fundamental cellular functions in the airway will likely hold significant value in future considerations of CO's role in airway therapy and health.

Reverse-Mode of the Mitochondrial Transhydrogenase Consumes NADPH and Provokes Oxidative Stress in Response to Elevated Cardiac Workload

Mitochondrial production of reactive oxygen species (ROS) contributes to the progression of heart failure, but the mechanisms of ROS generation are incompletely resolved. Superoxide (.O2−) is generated at the electron transport chain, dismutated to H2O2 and eliminated by enzymes that require NADPH. The nicotinamide nucleotide transhydrogenase (NNT) is highly expressed in the heart and catalyzes the reaction NADH+NADP+ to NADPH+NAD+. Since this reaction is coupled to the proton-motive force, it is perceived that the NNT prevents ROS production by regenerating mitochondrial NADPH. The exact role of the NNT in cardiomyocyte biology, however, has never been assessed. We took advantage of a loss-of-function mutation in the Nnt gene in C57BL/6J, but not C57BL/6N mice. In isolated cardiac myocytes exposed to a physiological increase in workload, β-adrenergic stimulation led to mitochondrial Ca2+-induced Krebs cycle activation with NADH regeneration, providing a substrate for the forward mode NNT reaction to regenerate NADPH. In contrast, under Ca2+-free conditions in isolated mitochondria, acceleration of NADH-coupled respiration by ADP favoured the reverse-mode NNT reaction, regenerating NADH by oxidizing NADPH. Accordingly, in response to an elevated workload in isolated hearts, the NADPH-coupled antioxidants glutathione and peroxiredoxin were oxidized through reverse-mode NNT reaction. This resulted in elevated mitochondrial formation of H2O2 in vivo after thoraco-aortic constriction (TAC) for 6 weeks. In NNT-deficient C57BL/6J mice, TAC-induced oxidative stress in vivo, cardiac fibrosis, left ventricular dysfunction and early mortality were ameliorated. Furthermore, scavenging mitochondrial ROS with the peptide SS-31 in vivo reduced TAC-induced mortality in C57BL/6N mice to levels observed in C57BL/6J mice. In conclusion, we believe that we discovered the mechanism how an inadequate increase in cardiac workload produces mitochondrial oxidative stress that leads to maladaptive cardiac remodelling and cardiac decompensation.

Redox Signaling in Adult Stem Cell Biology: A New Target Controlling Pluripotency and Differentiation. What about Iron Chelators?.

AbstractAbstract 2299▪▪This icon denotes a clinically relevant abstract IntroductionHematopoietic stem cells (HSCs) constitute a reservoir of undifferentiated cells that can be committed, upon appropriate stimuli, in the haematic lineages. Although residing in a bone-marrow hypoxic microenvironment (niche) and mainly relying on anaerobic glycolysis, HSCs are endowed with mitochondria. Recently, specific interest has been focused on HSCs mitochondria and on their role as reactive oxygen species (ROS) generators during the early phases of commitment. Indeed, consolidated evidences highlight the importance of redox signalling in the homeostasis of fundamental processes in cell adaptive biology and particularly in controlling the balance between self-renewal and differentiation of stem cells. HSCs constitutively generate low levels of ROS produced by both mitochondrial respiratory chain and NADPH oxidases (NOXs). ROS would act as secondary messengers, modulating the expression of master transcription factors leading or (pre)conditioning stem cells towards differentiation. Myelodisplastic syndrome (MDS) is characterized by disturbance of the HSC differentiation and most MDS patients are treated with iron chelators to compensate for the iron overload consequent to the blood cell transfusion-based standard therapy. Intriguingly, a robust percentage of patients, treated with the iron chelator deferasirox (DFX), recover correct HSCs differentiation whereas other chelators, like deferoxamine (DFO) did not. In the presented study we investigated the effect of DFX and DFO on the redox homeostasis of hematopoietic stem/progenitor cells (HSPCs) in order to get insights on the differential effect of iron chelators in rescuing altered hematopoiesis. Materials and MethodsHuman HSPCs were isolated from peripheral blood (PB) or bone marrow (BM) of G-CSF-treated or untreated healthy donors, respectively, by immuno-selection against the specific markers CD133 and CD34 and resulted >99% immunophenotypically homogeneous. Mitochondrial respiratory activity was measured in intact HSPCs by high resolution oxymetry. Morpho-functional features of HSPC-mitochondria, expression of the HSPC-surface commitment markers and ROS level were analyzed by laser scanning confocal microscopy (LSCM) and flow cytometry using specific probes or antibodies. HSPCs were treated with 100 mM DFX or DFO for 24 hrs. ResultsMeasurement of oxygen consumption rate as well as molecular analyses in PB-HSPCs confirmed a poor mitochondrial oxidative phosphorylation phenotype. LSCM imaging of mitochondria in either PB- and BM-HSCs displayed a punctuate rather than interconnected network. However, co-staining of mitochondria and CD34/CD133 stemness-markers revealed a striking inverse correlation. Finally HSPCs produced DCF-detectable and DPI-inhibitable ROS attributable to constitutive NOX activity and related to stabilization of the hypoxia-inducibile factor (HIF1a) under normoxic condition. DFX treatment of HSPCs resulted in a significant up-regulation of ROS level whereas no significant change was observed following DFO treatment. Importantly, the DFX-mediated ROS production was insensitive to treatment with low NOX-inhibiting concentration of DPI but was abrogated by high concentration of DPI thus pointing to mitochondria as ROS source. ConclusionsOur results show that HSCs in the early phase of commitment undergo a progressive increase of mitochondrial mass indicating the need of a bioenergetic up-regulation to cope with the oncoming energy-demanding proliferative/differentiative phenotype. Redox signaling, mediated by ROS production and likely triggered by changes in the environmental oxygen tension, appears to be essential in regulating HSC self-renewal and preservation of pluripotency. DFX treatment, by modulating ROS production, might lead to activation of redox-sensitive key factors able to restore the hematopoietic function in MDS patients. This effect seemingly is independent on the iron-chelating property of DFX but pertains to additional pharmacological properties that warren further investigation. Disclosures:No relevant conflicts of interest to declare.

Macrophage migration inhibitory factor antagonizes pressure overload-induced cardiac hypertrophy

Macrophage migration inhibitory factor (MIF) functions as a proinflammatory cytokine when secreted from the cell, but it also exhibits antioxidant properties by virtue of its intrinsic oxidoreductase activity. Since increased production of ROS is implicated in the development of left ventricular hypertrophy, we hypothesized that the redox activity of MIF protects the myocardium when exposed to hemodynamic stress. In a mouse model of myocardial hypertrophy induced by transverse aortic coarctation (TAC) for 10 days, we showed that growth of the MIF-deficient heart was significantly greater by 32% compared with wild-type (WT) TAC hearts and that fibrosis was increased by fourfold (2.62 ± 0.2% vs. 0.6 ± 0.1%). Circulating MIF was increased in TAC animals, and expression of MIF receptor, CD74, was increased in the hypertrophic myocardium. Gene expression analysis showed a 10-fold increase (P < 0.01) in ROS-generating mitochondrial NADPH oxidase and 2- to 3-fold reductions (P < 0.01) in mitochondrial SOD2 and mitochondrial aconitase activities, indicating enhanced oxidative injury in the hypertrophied MIF-deficient ventricle. Hypertrophic signaling pathways showed that phosphorylation of cytosolic glycogen synthase kinase-3α was greater (P < 0.05) at baseline in MIF-deficient hearts than in WT hearts and remained elevated after 10-day TAC. In the hemodynamically stressed MIF-deficient heart, nuclear p21(CIP1) increased sevenfold (P < 0.01), and the cytosolic increase of phospho-p21(CIP1) was significantly greater than in WT TAC hearts. We conclude that MIF antagonizes myocardial hypertrophy and fibrosis in response to hemodynamic stress by maintaining a redox homeostatic phenotype and attenuating stress-induced activation of hypertrophic signaling pathways.

Involvement of Nicotinamide Nucleotide Transhydrogenase in Cell Growth Regulation

Oxidative stress has been implicated in aging and pathogenesis of cancer and degenerative diseases. Cells are equipped with various antioxidant systems, some of which depend on NADPH for ultimate reducing equivalents. Nicotinamide nucleotide transhydrogenase represents an important source of NADPH in mitochondria. The physiological role of transhydrogenase in cell remains elusive. In the present study, we generated stable SK-Hep1 clones which express shRNA against NNT. The growth rate and colony forming ability of NNT knockdown cells were lower than those expressing scrambled shRNA controls. The migration and invasion abilities of the knockdown cells were lower than the control. When transplanted subcutaneously in immunodeficient mice, the NNT knockdown cells did not form tumors readily as the control cells did. The growth slowdown was accompanied by an increase in reactive oxygen species (ROS) regeneration as well as oxygen consumption and energy charge. But there are no differences of mitochondrial mass and membrane potential. In microarray experiment, we found that NNT deficiency decrease many tumorgenetic gene expression, such as DMBT1, MACC1, CABL2 and AHRR. These findings suggest that deficiency in NNT may cause growth cessation as well as increased ROS generation. Abstract

Redox Control of Leukemia: From Molecular Mechanisms to Therapeutic Opportunities

Reactive oxygen species (ROS) play both positive and negative roles in the proliferation and survival of a cell. This dual nature has been exploited by leukemia cells to promote growth, survival, and genomic instability-some of the hallmarks of the cancer phenotype. In addition to altered ROS levels, many antioxidants are dysregulated in leukemia cells. Together, the production of ROS and the expression and activity of antioxidant enzymes make up the primary redox control of leukemia cells. By manipulating this system, leukemia cells gain proliferative and survival advantages, even in the face of therapeutic insults. Standard treatment options have improved leukemia patient survival rates in recent years, although relapse and the development of resistance are persistent challenges. Therapies targeting the redox environment show promise for these cases. This review highlights the molecular mechanisms that control the redox milieu of leukemia cells. In particular, ROS production by the mitochondrial electron transport chain, NADPH oxidase, xanthine oxidoreductase, and cytochrome P450 will be addressed. Expression and activation of antioxidant enzymes such as superoxide dismutase, catalase, heme oxygenase, glutathione, thioredoxin, and peroxiredoxin are perturbed in leukemia cells, and the functional consequences of these molecular alterations will be described. Lastly, we delve into how these pathways can be potentially exploited therapeutically to improve treatment regimens and promote better outcomes for leukemia patients.

Effects of a Mitochondrial Mutator Mutation in Yeast POS5 NADH Kinase on Mitochondrial Nucleotides

Saccharomyces cerevisiae contains three NADH/NAD(+) kinases, one of which is localized in mitochondria and phosphorylates NADH in preference to NAD(+). Strand et al. reported that a yeast mutation in POS5, which encodes the mitochondrial NADH kinase, is a mutator, specific for mitochondrial genes (Strand, M. K., Stuart, G. R., Longley, M. J., Graziewicz, M. A., Dominick, O. C., and Copeland, W. C. (2003) Eukaryot. Cell 2, 809-820). Because of the involvement of NADPH in deoxyribonucleotide biosynthesis, we asked whether mitochondria in a pos5 deletion mutant contain abnormal deoxyribonucleoside triphosphate (dNTP) pools. We found the pools of the four dNTPs to be more than doubled in mutant mitochondrial extracts relative to wild-type mitochondrial extracts. This might partly explain the mitochondrial mutator phenotype. However, the loss of antioxidant protection is also likely to be significant. To this end, we measured pyridine nucleotide pools in mutant and wild-type mitochondrial extracts and found NADPH levels to be diminished by ∼4-fold in Δpos5 mitochondrial extracts, with NADP(+) diminished to a lesser degree. Our data suggest that both dNTP abnormalities and lack of antioxidant protection contribute to elevated mitochondrial gene mutagenesis in cells lacking the mitochondrial NADH kinase. The data also confirm previous reports of the specific function of Pos5p in mitochondrial NADP(+) and NADPH biosynthesis.

Regulation by mitochondrial superoxide and nadph oxidase of cellular formation of nitrated cyclic GMP: Potential implications for NO and ROS signaling

8-Nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP) is a nitrated derivative of cGMP, which can function as a unique electrophilic second messenger involved in regulation of oxidative stress response in cells. In the present study, we investigated chemical and biochemical regulatory mechanisms involved in 8-nitro-cGMP formation, with particular focus on the roles of reactive oxygen species (ROS). Chemical analyses demonstrated that peroxynitrite-dependent oxidation and myeloperoxidase-dependent oxidation of nitrite in the presence of hydrogen peroxide were two major pathways for guanine nucleotide nitration. Among the guanine nucleotides examined, GTP was the most sensitive to peroxynitrite-mediated nitration. Immunocytochemical and tandem mass spectrometric analyses revealed that formation of 8-nitro-cGMP in rat C6 glioma cells stimulated with lipopolysaccharide plus pro-inflammatory cytokines depended on production of both superoxide and hydrogen peroxide. Using the mitochondria-targeted chemical probe MitoSOX Red, we found that mitochondria-derived superoxide can act as a direct determinant of 8-nitro-cGMP formation. Furthermore, we demonstrated that NADPH oxidase 2 (Nox2)-generated hydrogen peroxide regulated mitochondria-derived superoxide production, which suggests the importance of cross-talk between Nox2-dependent hydrogen peroxide production and mitochondrial superoxide production. The results of the present study suggest that 8-nitro-cGMP can serve as a unique second messenger that may be implicated in regulating ROS signaling in the presence of NO.

Advanced glycation end-products induce injury to pancreatic beta cells through oxidative stress

This study evaluated the direct effects of advanced glycation end-products (AGEs) on pancreatic β cells, including cellular viability, generation of reactive oxygen species (ROS) and insulin secretion, and also looked for the main source of ROS in INS-1 cells and the possible molecular mechanism(s) of cell injury by AGEs. INS-1 cells were cultured with 100, 200 and 500 mg/L of AGEs for specific periods of time. Cell apoptosis was determined by ELISA and real-time PCR assays. ROS were detected by DCFH-DA and MitoSOX Red probes with a flow cytometer, NADPH oxidase activity was measured by lucigenin chemiluminescence and MAPK phosphorylation was measured by Western blot tests. Both cell apoptosis and ROS generation increased in AGE-treated cells in a dose-dependent way, and both the mitochondrial electron transport chain and NADPH oxidase pathway participated in ROS generation, although the role of the mitochondrial pathway was earlier and more important. AGEs exerted a toxic effect on insulin secretion that could be largely reversed by inhibiting ROS. AGEs injured INS-1 cells by oxidative stress mainly through the mitochondrial pathway, although the JNK and p38 MAPK signaling pathways were also key modulators in ROS-mediated β-cell death.

NADPH Oxidase-derived Reactive Oxygen Species Increases Expression of Monocyte Chemotactic Factor Genes in Cultured Adipocytes

Excess glucose and free fatty acids delivered to adipose tissue causes local inflammation, which contributes to insulin resistance. Glucose and palmitate generate reactive oxygen species (ROS) in adipocytes, leading to monocyte chemotactic factor gene expression. Docosahexaenoate (DHA) has the opposite effect. In this study, we evaluated the potential sources of ROS in the presence of excess nutrients. Differentiated 3T3-L1 adipocytes were exposed to palmitate and DHA (250 μM) in either 5 or 25 mM glucose to evaluate the relative roles of mitochondrial electron transport and NADPH oxidases (NOX) as sources of ROS. Excess glucose and palmitate did not increase mitochondrial oxidative phosphorylation. However, glucose exposure increased glycolysis. Of the NOX family members, only NOX4 was expressed in adipocytes. Moreover, its activity was increased by excess glucose and palmitate and decreased by DHA. Silencing NOX4 inhibited palmitate- and glucose-stimulated ROS generation and monocyte chemotactic factor gene expression. NADPH, a substrate for NOX, and pentose phosphate pathway activity increased with glucose but not palmitate and decreased with DHA exposure. Inhibition of the pentose phosphate pathway by glucose-6-phosphate dehydrogenase inhibitors and siRNA suppressed ROS generation and monocyte chemotactic factor gene expression induced by both glucose and palmitate. Finally, both high glucose and palmitate induced NOX4 translocation into lipid rafts, effects that were blocked by DHA. Excess glucose and palmitate generate ROS via NOX4 rather than by mitochondrial oxidation in cultured adipocytes. NOX4 is regulated by both NADPH generated in the PPP and translocation of NOX4 into lipid rafts, leading to expression of monocyte chemotactic factors.

Regulation by mitochondrial superoxide and NADPH oxidase of cellular formation of nitrated cyclic GMP: potential implications for ROS signalling

8-Nitro-cGMP (8-nitroguanosine 3',5'-cyclic monophosphate) is a nitrated derivative of cGMP, which can function as a unique electrophilic second messenger involved in regulation of an antioxidant adaptive response in cells. In the present study, we investigated chemical and biochemical regulatory mechanisms involved in 8-nitro-cGMP formation, with particular focus on the roles of ROS (reactive oxygen species). Chemical analyses demonstrated that peroxynitrite-dependent oxidation and myeloperoxidase-dependent oxidation of nitrite in the presence of H2O2 were two major pathways for guanine nucleotide nitration. Among the guanine nucleotides examined, GTP was the most sensitive to peroxynitrite-mediated nitration. Immunocytochemical and tandem mass spectrometric analyses revealed that formation of 8-nitro-cGMP in rat C6 glioma cells stimulated with lipopolysaccharide plus pro-inflammatory cytokines depended on production of both superoxide and H2O2. Using the mitochondria-targeted chemical probe MitoSOX Red, we found that mitochondria-derived superoxide can act as a direct determinant of 8-nitro-cGMP formation. Furthermore, we demonstrated that Nox2 (NADPH oxidase 2)-generated H2O2 regulated mitochondria-derived superoxide production, which suggests the importance of cross-talk between Nox2-dependent H2O2 production and mitochondrial superoxide production. The results of the present study suggest that 8-nitro-cGMP can serve as a unique second messenger that may be implicated in regulating ROS signalling in the presence of NO.

Silencing of nicotinamide nucleotide transhydrogenase impairs cellular redox homeostasis and energy metabolism in PC12 cells

Mitochondrial NADPH generation is largely dependent on the inner-membrane nicotinamide nucleotide transhydrogenase (NNT), which catalyzes the reduction of NADP+ to NADPH utilizing the proton gradient as the driving force and NADH as the electron donor. Small interfering RNA (siRNA) silencing of NNT in PC12 cells results in decreased cellular NADPH levels, altered redox status of the cell in terms of decreased GSH/GSSG ratios and increased H2O2 levels, thus leading to an increased redox potential (a more oxidized redox state). NNT knockdown results in a decrease of oxidative phosphorylation while anaerobic glycolysis levels remain unchanged. Decreased oxidative phosphorylation was associated with a) inhibition of mitochondrial pyruvate dehydrogenase (PDH) and succinyl-CoA:3-oxoacid CoA transferase (SCOT) activity; b) reduction of NADH availability, c) decline of mitochondrial membrane potential, and d) decrease of ATP levels. Moreover, the alteration of redox status actually precedes the impairment of mitochondrial bioenergetics. A possible mechanism could be that the activation of the redox-sensitive c-Jun N-terminal kinase (JNK) and its translocation to the mitochondrion leads to the inhibition of PDH (upon phosphorylation) and induction of intrinsic apoptosis, resulting in decreased cell viability. This study supports the notion that oxidized cellular redox state and decline in cellular bioenergetics – as a consequence of NNT knockdown – cannot be viewed as independent events, but rather as an interdependent relationship coordinated by the mitochondrial energy-redox axis. Disruption of electron flux from fuel substrates to redox components due to NNT suppression induces not only mitochondrial dysfunction but also cellular disorders through redox-sensitive signaling.

Crosstalk Between Mitochondrial ROS and NADPH Oxidases in Cardiovascular and Degenerative Diseases: Application of Mitochondria-Targeted Antioxidants

Crosstalk Between Mitochondrial ROS and NADPH Oxidases in Cardiovascular and Degenerative Diseases: Application of Mitochondria-Targeted Antioxidants

Neutral sphingomyelinase, NADPH oxidase and reactive oxygen species. Role in acute hypoxic pulmonary vasoconstriction

The molecular mechanisms underlying hypoxic pulmonary vasoconstriction (HPV) are not yet properly understood. Mitochondrial electron transport chain (ETC) and NADPH oxidase have been proposed as possible oxygen sensors, with derived reactive oxygen species (ROS) playing key roles in coupling the sensor(s) to the contractile machinery. We have recently reported that activation of neutral sphingomyelinase (nSMase) and protein kinase C ζ (PKCζ) participate in the signalling cascade of HPV. Herein, we studied the significance of nSMase in controlling ROS production rate in rat pulmonary artery (PA) smooth muscle cells and thereby HPV in rat PA. ROS production (analyzed by dichlorofluorescein and dihydroethidium fluorescence) was increased by hypoxia in endothelium-denuded PA segments and their inhibition prevented hypoxia-induced voltage-gated potassium channel (K(V) ) inhibition and pulmonary vasoconstriction. Consistently, H(2) O(2) , or its analogue t-BHP, decreased K(V) currents and induced a contractile response, mimicking the effects of hypoxia. Inhibitors of mitochondrial ETC (rotenone) and NADPH oxidase (apocynin) prevented hypoxia-induced ROS production, K(V) channel inhibition and vasoconstriction. Hypoxia induced p47(phox) phosphorylation and its interaction with caveolin-1. Inhibition of nSMase (GW4869) or PKCζ prevented p47(phox) phosphorylation and ROS production. The increase in ceramide induced by hypoxia (analyzed by immunocytochemistry) was inhibited by rotenone. Exogenous ceramide increased ROS production in a PKCζ sensitive manner. We propose an integrated signalling pathway for HPV which includes nSMase-PKCζ-NADPH oxidase as a necessary step required for ROS production and vasoconstriction.

Monoamine oxidase activity promotes oxidative stress and cell death in cardiomyocytes exposed to high glucose

Mitochondrial respiratory chain and NADPH oxidase are described as major sources of reactive oxygen species (ROS) in diabetic cardiomyopathy. Oddly, the role of monoamine oxidase (MAO) has been overlooked so far. These mitochondrial flavoenzymes are responsible for oxidative stress and cell death in failing and ischemic/reperfused hearts. Here we assessed whether MAO contribute to high glucose (HG) induced cell damage. Neonatal rat cardiomyocytes were cultured in high glucose (HG, 30 mM) for 24 or 48 hours in the absence or presence of MAO inhibitor pargyline and compared to those cultured in normal (NG, 5 mM) or high mannitol (HM, 30mM) containing media. Mitochondrial ROS production, detected by Mitotracker Red, was significantly increased in HG myocytes (+40%, p&lt;0.001) as compared to other cells. Pargyline completely prevented this increase. Apoptosis and necrosis, assessed by bisbenzimide and propidium iodide staining respectively, were 1.8 and 2‐fold higher in HG (p&lt;0.05). MAO inhibition significantly reduced cell death both after 24 and 48 hours of culture in HG. In conclusion, here we demonstrate that MAO play a major role in the oxidative stress and cell death in cardiomyocytes exposed to HG. MAO may amplify the effects of existing ROS triggers or HG may directly enhance MAO activity. Both phenomena are likely to be relevant for the onset and progression of diabetic cardiomyopathy.