Cytosolic Reactive Oxygen Species Production Research Articles

Cytosolic ROS production by NADPH oxidase 2 regulates muscle glucose uptake during exercise

Reactive oxygen species (ROS) act as intracellular compartmentalized second messengers, mediating metabolic stress-adaptation. In skeletal muscle fibers, ROS have been suggested to stimulate glucose transporter 4 (GLUT4)-dependent glucose transport during artificially evoked contraction ex vivo, but whether myocellular ROS production is stimulated by in vivo exercise to control metabolism is unclear. Here, we combined exercise in humans and mice with fluorescent dyes, genetically-encoded biosensors, and NADPH oxidase 2 (NOX2) loss-of-function models to demonstrate that NOX2 is the main source of cytosolic ROS during moderate-intensity exercise in skeletal muscle. Furthermore, two NOX2 loss-of-function mouse models lacking either p47phox or Rac1 presented striking phenotypic similarities, including greatly reduced exercise-stimulated glucose uptake and GLUT4 translocation. These findings indicate that NOX2 is a major myocellular ROS source, regulating glucose transport capacity during moderate-intensity exercise.

N-Acetyl cysteine ameliorates hyperglycemia-induced cardiomyocyte toxicity by improving mitochondrial energetics and enhancing endogenous Coenzyme Q9/10 levels.

The diabetic heart has been linked with reduced endogenous levels of coenzyme Q9/10 (CoQ), an important antioxidant and component of the electron transport chain. Although CoQ has displayed cardioprotective potential in experimental models of diabetes, the impact of N-acetyl cysteine (NAC) on mitochondrial energetics and endogenous levels of CoQ remains to be clarified. To explore these effects, high glucose-exposed H9c2 cardiomyocytes were used as an experimental model of hyperglycemia-induced cardiac injury. The results showed that high glucose exposure caused an increased production of reactive oxygen species (ROS), which was associated with impaired mitochondrial energetics as confirmed by a reduction of maximal respiration rate and depleted ATP levels. These detrimental effects were consistent with significantly reduced endogenous CoQ levels and accelerated cell toxicity. Although metformin demonstrated similar effects on mitochondrial energetics and cell viability, NAC demonstrated a more pronounced effect in ameliorating cytosolic and mitochondrial ROS production. Interestingly, the ameliorative effects of NAC against hyperglycemia-induced injury were linked with its capability to enhance endogenous CoQ levels. Although such data are to be confirmed in other models, especially in vivo studies, the overall findings provide additional evidence on the therapeutic mechanisms by which NAC protects against diabetes-induced cardiac injury.

The lipid peroxidation product 4-hydroxynonenal contributes to oxidative stress-mediated deterioration of the ageing oocyte

An increase in intraovarian reactive oxygen species (ROS) has long been implicated in the decline in oocyte quality associated with maternal ageing. Oxidative stress (OS)-induced lipid peroxidation and the consequent generation of highly electrophilic aldehydes, such as 4-hydroxynonenal (4-HNE), represents a potential mechanism by which ROS can inflict damage in the ageing oocyte. In this study, we have established that aged oocytes are vulnerable to damage by 4-HNE resulting from increased cytosolic ROS production within the oocyte itself. Further, we demonstrated that the age-related induction of OS can be recapitulated by exposure of germinal vesicle (GV) oocytes to exogenous H2O2. Such treatments stimulated an increase in 4-HNE generation, which remained elevated during in vitro oocyte maturation to metaphase II. Additionally, exposure of GV oocytes to either H2O2 or 4-HNE resulted in decreased meiotic completion, increased spindle abnormalities, chromosome misalignments and aneuploidy. In seeking to account for these data, we revealed that proteins essential for oocyte health and meiotic development, namely α-, β-, and γ-tubulin are vulnerable to adduction via 4-HNE. Importantly, 4-HNE-tubulin adduction, as well as increased aneuploidy rates, were resolved by co-treatment with the antioxidant penicillamine, demonstrating a possible therapeutic mechanism to improve oocyte quality in older females.

Measurement of Mitochondrial Mass by Flow Cytometry during Oxidative Stress.

Properly assessing mitochondrial health is crucial to understand their role in disease. MitoTracker green (MTG) and nonylacridine orange (NAO) are fluorescent probes which have been commonly used to assess mitochondrial mass. This is based on the assumption that both MTG and NAO accumulate in mitochondria regardless of the mitochondrial transmembrane potential (ΔΨm). Here, we utilized flow cytometry to evaluate the performance of these probes for assessment of mitochondrial mass relative to forward (FSC) and side scatter (SSC) in human peripheral blood lymphocytes (PBL). In isolated mitochondria, two subpopulations were identified by FSC and SSC measurements which were matched to subpopulations stained by MTG and NAO. The performance of these dyes was examined under oxidative and nitrosative stress induced by rotenone and NOC-18 while N-acetylcysteine (NAC) was employed as an antioxidant. Production of reactive oxygen species (ROS) and ΔΨm were monitored in parallel. With respect to representation of mitochondrial mass, neither MTG nor NAO was affected by ΔΨm. However, MTG showed significant correlation with cytosolic and mitochondrial ROS production and nitrosative stress. Our data suggest that NAO may be more suitable than MTG for assessment of mitochondrial mass by flow cytometry during oxidative stress.

Mechanisms of modulation of brain microvascular endothelial cells function by thrombin

Brain microvascular endothelial cells are a critical component of the blood-brain barrier. They form a tight monolayer which is essential for maintaining the brain homeostasis. Blood-derived proteases such as thrombin may enter the brain during pathological conditions like trauma, stroke, and inflammation and further disrupts the permeability of the blood-brain barrier, via incompletely characterized mechanisms. We examined the underlying mechanisms evoked by thrombin in rat brain microvascular endothelial cells (RBMVEC). Our results indicate that thrombin, acting on protease-activated receptor 1 (PAR1) increases cytosolic Ca2+ concentration in RBMVEC via Ca2+ release from endoplasmic reticulum through inositol 1,4,5-trisphosphate receptors and Ca2+ influx from extracellular space. Thrombin increases nitric oxide production; the effect is abolished by inhibition of the nitric oxide synthase or by antagonism of PAR1 receptors. In addition, thrombin increases mitochondrial and cytosolic reactive oxygen species production via PAR1-dependent mechanisms. Immunocytochemistry studies indicate that thrombin increases F-actin stress fibers, and disrupts the tight junctions. Thrombin increased the RBMVEC permeability assessed by a fluorescent flux assay. Taken together, our results indicate multiple mechanisms by which thrombin modulates the function of RBMVEC and may contribute to the blood-brain barrier dysfunction.

Reactive oxygen species contribute toward Smac mimetic/temozolomide-induced cell death in glioblastoma cells.

Small-molecule inhibitors of Inhibitor of Apoptosis proteins such as Smac mimetics have been reported to provide a promising tool to sensitize glioblastoma (GBM) cells to cytotoxic therapies including chemotherapeutic drugs. However, the underlying molecular mechanisms of action have not yet been fully unraveled. In the present study, we therefore investigated the role of reactive oxygen species (ROS) in the regulation of Smac mimetic/temozolomide (TMZ)-induced cell death in GBM cells. Here, we show that the Smac mimetic BV6 and TMZ act in concert to stimulate the production of both cytosolic and mitochondrial ROS. This accumulation of ROS contributes toward the activation of the proapoptotic factor BAX upon BV6/TMZ cotreatment as several ROS scavengers (i.e. N-acetyl-L-cysteine, MnTBAP, or α-tocopherol) protect GBM cells against BV6/TMZ-mediated BAX activation. In addition, ROS scavengers significantly rescue GBM cells from BV6/TMZ-triggered cell death, indicating that ROS generation is required for the induction of cell death. By showing that ROS play an important role in the regulation of Smac mimetic/TMZ-induced cell death, our work sheds light on the crucial role of the oxidative system in the cooperative antitumor activity of Smac mimetic/TMZ combination therapy against GBM cells.

Abstract 369: Protein Kinase C Epsilon-dependent Nox Activation Mediates Resistin-induced Cardiovascular Disease

Background: Adipokine resistin induces vascular smooth muscle cell (VSMC) dysfunction, implying a vital role in cardiovascular disease. In this study, we examined the in vivo and in vitro effects of resisin and investigated the underlying mechanisms involving protein kinase C epsilon (PKCε) and reactive oxygen species (ROS). Methods: After guide-wire injury of distal left common carotid artery, ApoE-/- mice fed with a western diet were treated with or without subcutaneous infusion of resistin in the presence or absence of PKCε inhibitor for 4 weeks. The progress of intimal hyperplasia was monitored using an ultrasound micro-image system. NADPH oxidase (Nox) activity in the mouse coronary arteries was measured using a chemiluminescence assay. Additionally, we treated human coronary artery smooth muscle with resistin in the presence or absence of PKCε and Nox-specific inhibitors to determine mechanistic and functional effects of resistin in VSMCs functions. Reactive oxygen species (ROS) production was analyzed using confocal microscopy. Results: Resistin significantly enhanced injury-induced intimal hyperplasia in our murine model, which was mitigated by the PKCε inhibitor. Nox activity was also substantially augmented in the injured artery in a resistin-PKCε dependent manner. On the cellular level, resistin-induced ROS production was time-dependent and mitochondrial ROS seemed to be the trigger for cytosolic Nox-induced ROS. Inhibition of Nox completely abolished resistin-exaggerated VSMC proliferation, migration and dedifferentiation. Consistently, inhibition of PKCε significantly reduced resistin-induced cytosolic ROS production, Nox activity, and VSMC dysfunction. Conclusion: This is the first study showing that resistin-induced VSMC dysfunction and intimal hyperplasia is mediated through PKCε-dependent Nox activation pathway. These findings suggest potential molecular targets for cardiovascular disease.

N-acetyl cysteine reduces oxidative toxicity, apoptosis, and calcium entry through TRPV1 channels in the neutrophils of patients with polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is a common inflammatory and oxidant disease with an uncertain pathogenesis. N-acetyl cysteine (NAC) decreases oxidative stress, intracellular free calcium ion [Ca2+]i, and apoptosis levels in human neutrophil. We aimed to investigate the effects of NAC on apoptosis, oxidative stress, and Ca2+ entry through transient receptor potential vanilloid 1 (TRPV1) and TRP melastatin 2 (TRPM2) channels in neutrophils from patients with PCOS. Neutrophils isolated from PCOS group were investigated in three settings: (1) after incubation with TRPV1 channel blocker capsazepine or TRPM2 channel blocker 2-aminoethyl diphenylborinate (2-APB), (2) after supplementation with NAC (for 6 weeks), and (3) with combination (capsazepine + 2-APB + NAC) exposure. The neutrophils in TRPM2 and TRPV1 experiments were stimulated by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP; 1 μM) and capsaicin (10 μM) as concentration agonists, respectively. Neutrophil lipid peroxidation and capsaicin-induced increase in [Ca2+]i concentrations were reduced by capsazepine and NAC treatments. However, the [Ca2+]i concentration did not change by fMLP stimulation. Neutrophil lipid peroxidation, apoptosis, caspase-3, caspase-9, cytosolic reactive oxygen species production, and mitochondrial membrane depolarization values were decreased by NAC treatment although neutrophil glutathione peroxidase and reduced glutathione levels were increased by the NAC treatment. Serum lipid peroxidation, luteinizing hormone, testosterone, insulin, interleukin-1 beta, and homocysteine levels were decreased by NAC treatment although serum vitamin A, beta-carotene, vitamin E, and total antioxidant status were increased by the NAC treatment. In conclusion, NAC reduced oxidative stress, apoptosis, cytokine levels, and Ca2+ entry through TRPV1 channel, which provide supportive evidence that oxidative stress and TRPV1 channel plays a key role in etiology of PCOS.

C-Peptide Activates AMPKα and Prevents ROS-Mediated Mitochondrial Fission and Endothelial Apoptosis in Diabetes

Vasculopathy is a major complication of diabetes; however, molecular mechanisms mediating the development of vasculopathy and potential strategies for prevention have not been identified. We have previously reported that C-peptide prevents diabetic vasculopathy by inhibiting reactive oxygen species (ROS)-mediated endothelial apoptosis. To gain further insight into ROS-dependent mechanism of diabetic vasculopathy and its prevention, we studied high glucose–induced cytosolic and mitochondrial ROS production and its effect on altered mitochondrial dynamics and apoptosis. For the therapeutic strategy, we investigated the vasoprotective mechanism of C-peptide against hyperglycemia-induced endothelial damage through the AMP-activated protein kinase α (AMPKα) pathway using human umbilical vein endothelial cells and aorta of diabetic mice. High glucose (33 mmol/L) increased intracellular ROS through a mechanism involving interregulation between cytosolic and mitochondrial ROS generation. C-peptide (1 nmol/L) activation of AMPKα inhibited high glucose–induced ROS generation, mitochondrial fission, mitochondrial membrane potential collapse, and endothelial cell apoptosis. Additionally, the AMPK activator 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside and the antihyperglycemic drug metformin mimicked protective effects of C-peptide. C-peptide replacement therapy normalized hyperglycemia-induced AMPKα dephosphorylation, ROS generation, and mitochondrial disorganization in aorta of diabetic mice. These findings highlight a novel mechanism by which C-peptide activates AMPKα and protects against hyperglycemia-induced vasculopathy.

Ketone Supplementation Reduces Superoxide Production in Cultured Primary Human Dermal Fibroblasts: Comparison of Young vs Aged Cells

In the United States, it is estimated that there are 1.8 million new cases of chronic wounds annually, with the majority occurring in the elderly. Reactive oxygen species (ROS) damage is augmented in aged patients leading to redox imbalance and is a key characteristic in impaired wound healing. Ketones are an energy substrate that reduce ROS production in the brain and heart, and thus may augment wound healing. We hypothesized that Beta‐Hydroxybutyrate (BHB; 5mM) would reduce cytosolic and mitochondrial ROS production in both young and aged primary human dermal fibroblasts. Primary human dermal fibroblasts (HDFs, passaged 2–8) from young and aged donors (24 to 88 years) were incubated in the absence or presence of BHB for 72 hours prior to stress with 100uM tert‐buytl‐hydrogen peroxide for 3 hours. ROS production was measured fluorescently with dihydroethidium (DHE, cytosolic) and Mitosox Red (mitochondrial) in triplicate, N=6. In the cytosol, aged HDFs produced 28% (p=<0.001)) more ROS basally compared to their young counterparts. When incubated in BHB for 72 hours, ROS production in the young HDFs decreased by 40% (p=<0.0001) and by 41% (p=<0.0001) in the aged. In the mitochondria, aged HDFs produced 27% (p=<0.0001) more ROS at baseline than the young HDFs, consistent with observations in cytosol. When incubated in BHB, ROS production was reduced in the young by 29% (p<0.0001) and in the aged by 51% (p<0.0001); the aged ROS production was no longer significantly different from the young. Ketone supplementation did not significantly change proliferation in the HDFs, but did increase migration in both young and aged HDFs. We conclude that ketone supplementation may protect against redox stress and augment wound healing, shifting redox homeostasis in HDFs, especially in the elderly. Funding: ONR, VA Merit Review.

Studies on the relationship between pulsed UV light irradiation and the simultaneous occurrence of molecular and cellular damage in clinically-relevant Candida albicans

This constitutes the first study to report on the relationship between pulsed UV light (PL) irradiation and the simultaneous occurrence of molecular and cellular damage in clinical strains of Candida albicans. Microbial protein leakage and propidium iodide (PI) uptake assays demonstrated significant increases in cell membrane permeability in PL-treated yeast that depended on the amount of UV pulses applied. This finding correlated well with the measurement of increased levels of lipid hydroperoxidation in the cell membrane of PL-treated yeast. PL-treated yeast cells also displayed a specific pattern of intracellular reactive oxygen species (ROS) generation, where ROS were initially localised in the mitochondria after low levels of pulsing (UV dose 0.82 μJ/cm 2) before more wide-spread cytosolic ROS production occurred with enhanced pulsing. Intracellular ROS levels were measured using the specific mitochondrial peroxide stain dihydrorhodamine 123 and the cytosolic oxidation stain dichloroflurescin diacetate. Use of the dihydroethidium stain also revealed increased levels of intracellular superoxide as a consequence of augmented pulsing. The ROS bursts observed during the initial phases of PL treatment was consistent with the occurrence of apoptotic cells as confirmed by detection of specific apoptotic markers, abnormal chromatin condensation and externalisation of cell membrane lipid phosphatidylserine. Increased amount of PL-irradiation (ca. UV does 1.24–1.65 μJ/cm 2) also resulted in the occurrence of late apoptotic and necrotic yeast phenotypes, which coincided with the transition from mitochondrial to cytosolic localisation of ROS and with irreversible cell membrane leakage. Use of the comet assay also revealed significant nuclear damage in similarly treated PL samples. Although some level of cellular repair was observed in all test strains during sub-lethal exposure to PL-treatments (≤ 20 pulses or UV dose 0.55 μJ/cm 2), this was absent in similar samples exposed to increased amounts of pulsing. This study showed that PL-irradiation inactivates C. albicans test strains through a multi-targeted process with no evidence of microbial ability to support cell growth after ≤ 20 pulses. Implications of our findings in terms of application of PL for contact-surface disinfection are discussed.

Mitochondrial redox potential during contraction in single intact muscle fibers

Although the production of reactive oxygen species (ROS) during muscle contractile activity has been linked to both positive and negative adaptive responses, the sites for ROS generation within working muscle are not clearly defined. We assessed cytosolic ROS production and mitochondrial redox potential with a targeted redox-sensitive green fluorescent protein during repetitive field stimulation of single mature myofibers. Cytosolic ROS production increased by 94%, an effect that was abolished by pretreatment with the reducing agent dithiothreitol. Mitochondrial redox potential was not altered during muscle contraction. In contrast, activity-dependent ROS production was ablated by an inhibitor of NADPH oxidase. We provide the first report on dynamic ROS production from mitochondria in single living myofibers and suggest that the mitochondria are not the major source of ROS during skeletal muscle contraction. Alternatively, our data support a role for NADPH oxidase-derived ROS during contractile activity.

Mechanisms Underlying Fructose-Induced Oxidative Stress in the Cytosol and Mitochondria

High levels of dietary fructose are increasingly recognized as an important nutritional factor in the development of nonalcoholic fatty liver disease and intestinal inflammation in both humans and animal models. The signals linking excessive fructose intake on one hand and the onset of these pathologies are not known. We have employed genetically encoded biosensors sensitive to changes in superoxide anion (O2- ) or hydrogen peroxide (H2O2) concentrations to test the hypothesis that fructose metabolism increases the production of reactive oxygen species (ROS) in hepatocytes or intestinal cells. The data indicate that acute treatment with physiological concentrations of fructose (1 - 10 mM) significantly increased the formation of mitochondrial O2- as well as mitochondrial and cytosolic H2O2. The xanthine oxidase inhibitor, allopurinol, inhibited fructose-induced increases in cytosolic H2O2, but was unaffected by apocynin, a NADPH oxidase inhibitor. These data are consistent with the well known effects of acute fructose treatment to reduce ATP levels and stimulate the breakdown of purines. Fructose-induced ROS production in the mitochondria was not altered by allopurinol treatment, whereas apocynin strongly suppressed mitochondrial derived ROS. Fructose administration transiently increased ROS formation in all mitochondria regardless of the subcellular localization and this was paralleled by a sustained rise in mitochondrial membrane potential and an increase in pyridine nucleotide fluorescence. The activation of mitochondrial metabolism was followed by large amplitude O2- bursts in a subset of mitochondria. The addition of mitochondrial respiration inhibitors blocked the effects of fructose on mitochondrial, but not cytosolic ROS production. Taken together, these data indicate that fructose treatment stimulates mitochondrial metabolism leading to an increase in ROS production through an apocynin sensitive pathway. Finally, we show that chronic consumption of fructose results in markedly higher baseline levels of mitochondrial ROS in hepatocytes.

RAGE-Induced Cytosolic ROS Promote Mitochondrial Superoxide Generation in Diabetes

Damaged mitochondria generate an excess of superoxide, which may mediate tissue injury in diabetes. We hypothesized that in diabetic nephropathy, advanced glycation end-products (AGEs) lead to increases in cytosolic reactive oxygen species (ROS), which facilitate the production of mitochondrial superoxide. In normoglycemic conditions, exposure of primary renal cells to AGEs, transient overexpression of the receptor for AGEs (RAGE) with an adenoviral vector, and infusion of AGEs to healthy rodents each induced renal cytosolic oxidative stress, which led to mitochondrial permeability transition and deficiency of mitochondrial complex I. Because of a lack of glucose-derived NADH, which is the substrate for complex I, these changes did not lead to excess production of mitochondrial superoxide; however, when we performed these experiments in hyperglycemic conditions in vitro or in diabetic rats, we observed significant generation of mitochondrial superoxide at the level of complex I, fueled by a sustained supply of NADH. Pharmacologic inhibition of AGE-RAGE-induced mitochondrial permeability transition in vitro abrogated production of mitochondrial superoxide; we observed a similar effect in vivo after inhibiting cytosolic ROS production with apocynin or lowering AGEs with alagebrium. Furthermore, RAGE deficiency prevented diabetes-induced increases in renal mitochondrial superoxide and renal cortical apoptosis in mice. Taken together, these studies suggest that AGE-RAGE-induced cytosolic ROS production facilitates mitochondrial superoxide production in hyperglycemic environments, providing further evidence of a role for the advanced glycation pathway in the development and progression of diabetic nephropathy.

TNF-α/cycloheximide-induced apoptosis in intestinal epithelial cells requires Rac1-regulated reactive oxygen species

Previously we have shown that both Rac1 and c-Jun NH(2)-terminal kinase (JNK1/2) are key proapoptotic molecules in tumor necrosis factor (TNF)-alpha/cycloheximide (CHX)-induced apoptosis in intestinal epithelial cells, whereas the role of reactive oxygen species (ROS) in apoptosis is unclear. The present studies tested the hypothesis that Rac1-mediated ROS production is involved in TNF-alpha-induced apoptosis. In this study, we showed that TNF-alpha/CHX-induced ROS production and hydrogen peroxide (H(2)O(2))-induced oxidative stress increased apoptosis. Inhibition of Rac1 by a specific inhibitor NSC23766 prevented TNF-alpha-induced ROS production. The antioxidant, N-acetylcysteine (NAC), or rotenone (Rot), the mitochondrial electron transport chain inhibitor, attenuated mitochondrial ROS production and apoptosis. Rot also prevented JNK1/2 activation during apoptosis. Inhibition of Rac1 by expression of dominant negative Rac1 decreased TNF-alpha-induced mitochondrial ROS production. Moreover, TNF-alpha-induced cytosolic ROS production was inhibited by Rac1 inhibition, diphenyleneiodonium (DPI, an inhibitor of NADPH oxidase), and NAC. In addition, DPI inhibited TNF-alpha-induced apoptosis as judged by morphological changes, DNA fragmentation, and JNK1/2 activation. Mitochondrial membrane potential change is Rac1 or cytosolic ROS dependent. Lastly, all ROS inhibitors inhibited caspase-3 activity. Thus these results indicate that TNF-alpha-induced apoptosis requires Rac1-dependent ROS production in intestinal epithelial cells.

Verapamil-induced changes in central conduction in patients with multiple sclerosis.

The electrophysiological characteristics of demyelinated axons are sensitive to changes in plasma calcium concentration. This study investigated the effect of verapamil, a calcium antagonist drug, on brainstem auditory, visual, and somatosensory evoked potentials in multiple sclerosis patients. Eight clinically stable patients with abnormal visual and/or brainstem auditory evoked potentials and four normal volunteers were studied. During intravenous infusions of verapamil (mean plasma concentration = 130.0 +/- 56.4 ng/ml), the latencies of peaks III and V were shortened (p less than 0.05) in multiple sclerosis patients with abnormally prolonged BAEPs. The I-III (delta = 0.08 ms), III-V (delta = 0.46 ms), and I-V (delta = 0.53 ms) interpeak intervals, and the P100 latency (delta = 10.15 ms) of the visual evoked potential were similarly affected in these patients. In contrast, normal evoked potentials of both multiple sclerosis patients and control subjects were not altered compared to baseline recordings obtained 24 hours earlier. Intravenous verapamil, therefore, alters the BAEPs and VEPs of some multiple sclerosis patients with demyelinated auditory and visual pathways by shortening pathologically prolonged latencies toward normal. The present study suggests pharmacological manipulation of calcium-dependent processes, possibly at the level of the demyelinated axon, can acutely facilitate central conduction of electrical impulses in some patients with clinically stable multiple sclerosis.