Role of mitochondria in toxic oxidative stress

Oxidative stress and mitochondrial oxidative damage have been implicated in the etiology of numerous common diseases. The critical mitochondrial events responsible for oxidative stress-mediated cell death (toxic oxidative stress), however, have yet to be defined. Several oxidative events implicated in toxic oxidative stress include alterations in mitochondrial lipids (e.g., cardiolipin), mitochondrial DNA, and mitochondrial proteins (eg. aconitase and uncoupling protein 2). Furthermore, recent findings indicate the enrichment of mitochondrial membranes with vitamin E protects cells against the toxic effects of oxidative stress. This review briefly summarizes the role of these mitochondrial events in toxic oxidative stress, including: 1) the protective role of mitochondrial vitamin E in toxic oxidative stress, 2) the role of mitochondrial DNA in toxic oxidative stress, 3) the interaction between cardiolipin and cytochrome c in mitochondrial regulation of apoptosis, 4) the role of mitochondrial aconitase in oxidative neurodegeneration, and 5) the role of mitochondrial uncoupling protein 2 in the pathogenesis of type 2 diabetes.     

Inhibition of ROS production through mitochondria-targeted antioxidant and mitochondrial uncoupling increases post-thaw sperm viability in yellow catfish

Reactive oxygen species (ROS) are one of the main causes for decreased viability in cryopreserved sperm. Many studies have reported the beneficial effect of antioxidant supplements in freezing media for post-thaw sperm quality. In the present study, we explored two new approaches of ROS inhibition in sperm cryopreservation of yellow catfish, namely mitochondrial-targeted antioxidant and metabolic modulator targeting mitochondrial uncoupling pathways. Our study revealed that addition of MitoQ, a compound designed to deliver ubiquinone into mitochondria, significantly decreased ROS production, as well as lipid peroxidation, and increased post-thaw viability. Similarly, sperm incubated with 2,4-dinitrophenol (DNP), a chemical protonophore that induces mitochondrial uncoupling, also had reduced ROS production, as well as lipid peroxidation, and increased post-thaw sperm viability. Conversely, activation of uncoupling protein (UCP2) by 4-hydroxynonenal (HNE) neither reduced ROS production nor increased post-thaw sperm viability. Our findings indicate that ROS inhibition through mitochondrial-targeted antioxidant or mild mitochondrial uncoupling is beneficial for sperm cryopreservation in yellow catfish. Our study provides novel methods to mitigate oxidative stress induced damage in cryopreserved sperm for future applications.     

Mitochondrial superoxide: production, biological effects, and activation of uncoupling proteins

Mitochondria are potent producers of cellular superoxide, from complexes I and III of the electron transport chain, and mitochondrial superoxide production is a major cause of the cellular oxidative damage that may underlie degradative diseases and aging. This superoxide production is very sensitive to the proton motive force, so it can be strongly decreased by mild uncoupling. Superoxide and the lipid peroxidation products it engenders, including hydroxyalkenals such as hydroxynonenal, are potent activators of proton conductance by mitochondrial uncoupling proteins such as UCP2 and UCP3, although the mechanism of activation has yet to be established. These observations suggest a hypothesis for the main, ancestral function of uncoupling proteins: to cause mild uncoupling and so diminish mitochondrial superoxide production, hence protecting against disease and oxidative damage at the expense of a small loss of energy. We review the growing evidence for this hypothesis, in mitochondria, in cells, and in vivo. More recently evolved roles of uncoupling proteins are in adaptive thermogenesis (UCP1) and perhaps as part of a signaling pathway to regulate insulin secretion in pancreatic beta cells (UCP2).     

Induction of mitochondrial uncoupling enhances VEGF₁₂₀ but reduces MCP-1 release in mature 3T3-L1 adipocytes: possible regulatory mechanism through endogenous ER stress and AMPK-related pathways

Although white adipocytes contain a larger number of mitochondria per cytoplasmic volume, adipocyte mitochondrial uncoupling to reduce the efficiency of ATP production on cellular function including secretory regulation of bioactive molecules such as VEGF and MCP-1 remains to be elucidated. Here we induce mitochondrial uncoupling under hypoxia-independent conditions in mature 3T3-L1 adipocytes using a metabolic uncoupler, dinitrophenol (DNP). MCP-1 release was significantly decreased by 26% (p<0.01) in 24h DNP (30 μmol/L)-treated adipocytes compared to control cells. In contrast, secreted VEGF(120) lacking a heparin-binding domain was markedly increased 2.0-fold (p<0.01). CHOP content in these cells also were augmented (p<0.01), but no significant increase of endogenous oxidative stress was observed. Treatment with thapsigargin, which can induce exogenous endoplasmic reticulum (ER) stress, clearly attenuated MCP-1 release (p<0.01), but exhibited no effects on VEGF(120) secretion. On the other hand, exogenous H(2)O(2) amplified both MCP-1 and VEGF(120) secretion (p<0.05). In addition, under chronic activation of AMPK by AICAR, MCP-1 release was significantly diminished (p<0.05) but VEGF(120) secretion was increased (p<0.01). JNK phosphorylation in mature adipocytes was decreased by treatment with either DNP or AICAR (p<0.01). Enhanced VEGF(120) secretion with either DNP or AICAR was markedly suppressed by PI3K inhibitor LY294002 (p<0.01). Thus, induced mitochondrial uncoupling in adipocytes can reduce MCP-1 release through induction of endogenous ER stress and by reduced JNK activities via chronic activation of AMPK. Under this condition, VEGF(120) secretion was increased through PI3K-dependent pathways, which were chronically activated by AMPK, and not through ER stress. Because the decrease of MCP-1 secretion under mitochondrial uncoupling might attenuate chronic low-grade inflammation by suppressing macrophages recruitment to adipose tissue, clarification of the mechanism might reveal novel therapeutic targets for ameliorating obesity-associated insulin resistance in metabolic syndrome and type 2 diabetes.     

Dinitrophenol-induced mitochondrial uncoupling in vivo triggers respiratory adaptation in HepG2 cells

Here, we show that 3 days of mitochondrial uncoupling, induced by low concentrations of dinitrophenol (10 and 50 microM) in cultured human HepG2 cells, triggers cellular metabolic adaptation towards oxidative metabolism. Chronic respiratory uncoupling of HepG2 cells induced an increase in cellular oxygen consumption, oxidative capacity and cytochrome c oxidase activity. This was associated with an upregulation of COXIV and ANT3 gene expression, two nuclear genes that encode mitochondrial proteins involved in oxidative phosphorylation. Glucose consumption, lactate and pyruvate production and growth rate were unaffected, indicating that metabolic adaptation of HepG2 cells undergoing chronic respiratory uncoupling allows continuous and efficient mitochondrial ATP production without the need to increase glycolytic activity. In contrast, 3 days of dinitrophenol treatment did not change the oxidative capacity of human 143B.TK(-) cells, but it increased glucose consumption, lactate and pyruvate production. Despite a large increase in glycolytic metabolism, the growth rate of 143B.TK(-) cells was significantly reduced by dinitrophenol-induced mitochondrial uncoupling. We propose that chronic respiratory uncoupling may constitute an internal bioenergetic signal, which would initiate a coordinated increase in nuclear respiratory gene expression, which ultimately drives mitochondrial metabolic adaptation within cells.     

Effects of Purified Helminthosporium maydis Race T Toxin on the Structure and Function of Corn Mitochondria and Protoplasts

A toxin preparation from Helminthosporium maydis Race T containing several closely related molecules with apparently identical biological activities was highly active against mitochondria and protoplasts from Texas male-sterile (T) cytoplasm corn (T mitochondria and T protoplasts, respectively) but had no effect on their male-fertile (N) cytoplasm counterparts. The toxin preparation caused multiple changes in isolated T mitochondria, including uncoupling of oxidative phosphorylation, stimulation of succinate and NADH respiration, inhibition of malate respiration, increased swelling, loss of matrix density, and unfolding of the inner membrane. Only 6 to 7 nanograms toxin per milligram mitochondrial protein (1.8 nanogram per milliliter) were required to fully uncouple oxidative phosphorylation and to completely inhibit malate respiration in isolated T mitochondria. Similar low concentrations of toxin caused collapse of T protoplasts after several days of culture. Severe ultrastructural damage to mitochondria in T protoplasts was observed within 20 minutes; no changes in other cellular components were observed at this time. These observations on the cytoplasmic specificity, multiple effects, and high activity of the toxin at the mitochondrial and cellular levels highlight its biological significance and potential usefulness in determining the molecular basis of southern corn leaf blight disease.     

Metabolism of round spermatids: gossypol induces uncoupling of respiratory chain and oxidative phosphorylation

The effect of gossypol on energy metabolism of round spermatids of rats was examined. When spermatids were treated with various concentrations of gossypol for 30 min at 32 degrees C, a biphasic response (stimulation at low concentrations and inhibition at high concentrations) was seen in pyruvate and CO2 production from lactate. At the early period of incubation, gossypol at even high concentrations stimulated CO2 production to an extent similar to that stimulated by 2,4-dinitrophenol (DNP). At longer periods of incubation, however, the rates of CO2 production from lactate dropped to those seen in the rotenone-treated cells. The rates of oxygen consumption were not increased further by DNP when cells were pretreated with gossypol. The adenosine triphosphate (ATP) content in spermatids was reduced markedly, although lactate oxidation to CO2 and mitochondrial respiration were stimulated by gossypol. These results suggest that gossypol probably exerts its effect on spermatids by uncoupling respiratory chain and oxidative phosphorylation.     

Hydroxynonenal and uncoupling proteins: a model for protection against oxidative damage

In this mini review we summarize recent studies from our laboratory that show the involvement of superoxide and the lipid peroxidation product 4-hydroxynonenal in the regulation of mitochondrial uncoupling. Superoxide produced during mitochondrial respiration is a major cause of the cellular oxidative damage that may underlie degenerative diseases and ageing. Superoxide production is very sensitive to the magnitude of the mitochondrial protonmotive force, so can be strongly decreased by mild uncoupling. Superoxide is able to give rise to other reactive oxygen species, which elicit deleterious effects primarily by oxidizing intracellular components, including lipids, DNA and proteins. Superoxide-induced lipid peroxidation leads to the production of reactive aldehydes, including 4-hydroxynonenal. These aldehydic lipid peroxidation products are in turn able to modify proteins such as mitochondrial uncoupling proteins and the adenine nucleotide translocase, converting them into active proton transporters. This activation induces mild uncoupling and so diminishes mitochondrial superoxide production, hence protecting against disease and oxidative damage at the expense of energy production.     

Mitochondrial oxidative stress and inflammation: an slalom to obesity and insulin resistance

Mitochondria, in addition to energy transformation, play a role in important metabolic tasks such as apoptosis, cellular proliferation, heme/steroid synthesis as well as in the cellular redox state regulation. The mitochondrial phosphorylation process is very efficient, but a small percentage of electrons may prematurely reduce oxygen forming toxic free radicals potentially impairing the mitochondria function. Furthermore, under certain conditions, protons can reenter the mitochondrial matrix through different uncoupling proteins (UCPs), affecting the control of free radicals production by mitochondria. Disorders of the mitochondrial electron transport chain, overgeneration of reactive oxygen species (ROS) and lipoperoxides or impairments in antioxidant defenses have been reported in situations of obesity and type-2 diabetes. On the other hand, obesity has been associated to a low degree pro-inflammatory state, in which impairments in the oxidative stress and antioxidant mechanism could be involved. Indeed, reactive oxygen species have been attributed a causal role in multiple forms of insulin resistance. The scientific evidence highlights the importance of investigating the relationships between oxidative stress and inflammation with obesity/diabetes onset and underlines the need to study in mitochondria from different tissues, the interactions of such factors either as a cause or consequence of obesity and insulin resistance.     

Reexamination of respiration-impairing effect of bikaverin on isolated mitochondria

Summary  Kovac et al (1) have shown that bikaverin uncouples oxidative phosphorylation of mitochondria at 20μg/ml (52 nmol/ml) in the reaction system without magnesium (Mg²⁺). In the present study, the effects of bikaverin on mitochondrial reactions were reexamined in detail at various concentrations both in the reaction systems with and without magnesium, using isolated rat liver mitochondria and submitochondrial particles (SMP) to characterize its mode of actions to mitochondrial respiration at low concentrations (<30nmol/mg mitochondrial protein). It was concluded that bikaverin showed no uncoupling effect (no decrease in P/O ratio) at low concentrations and did it at high concentrations in consequence of disturbing the ion permeability in the mitochondrial inner membranes. At low concentrations, bikaverin repressed both NAD- and succinate-linked respirations, but did not interfere with electron transport and energy transfer systems of mitochondria.     

Analysis of the effect of α-dinitrophenol on cleavage and development of the sea urchin embryo

Summary The influence of DNP on the developing sea urchin embryo was studied by means of time-lapse cinematography and staining methods. In the concentrations of DNP which affect oxidative phosphorylation cleavage is rapidly brought to a standstill. DNP inflicts morphological changes of the mitochondria, which also show a lowered affinity for Janus green B. Some new aspects on the effect of DNP on morphogenesis are discussed.     

Reversible mitochondrial uncoupling in the cold phase during liver preservation/reperfusion reduces oxidative injury in the rat model

Reversible uncoupling of the mitochondrial electron-transport chain may be one strategy to prevent intracellular oxidative stress during liver cold preservation/warm reperfusion (CP/WR) injury. 2,4-Dinitrophenol (DNP) is a potent water-soluble uncoupling agent for supplementation of the hepatic CP solution. The aim of this work was to investigate the possible influence of DNP in the CP solution on the isolated rat liver state during CP/WR. Livers were subjected to CP at 4 °C in sucrose–phosphate based solution (SPS) for 18 h, followed by WR for 60 min in vitro. The final concentration of DNP was 100 μM. DNP presence during the CP stage led to partial ATP level decrease accompanied by a significant diminution in liver TBARS and a prevention of antioxidant enzyme activity decrease (catalase, GSH-PO, GSH-Red) when compared with livers stored without DNP. After DNP wash-out during WR, an improvement in the mitochondrial functional state (higher respiratory control indices and ATP levels, and a decrease in V₄ respiration rates) were observed. This was concurrent with lower TBARS levels, higher antioxidant enzyme activities and significant increase of bile production. The results suggest that reversible uncoupling may be one way to influence oxidative stress associated with hepatic cold preservation.     

Chronic mitochondrial uncoupling treatment prevents acute cold-induced oxidative stress in birds

Endotherms have evolved two major types of thermogenesis that allow them to actively produce heat in response to cold exposure, either through muscular activity (i.e. shivering thermogenesis) or through futile electro-chemical cycles (i.e. non-shivering thermogenesis). Amongst the latter, mitochondrial uncoupling is of key importance because it is suggested to drive heat production at a low cost in terms of oxidative stress. While this has been experimentally shown in mammals, the oxidative stress consequences of cold exposure and mitochondrial uncoupling are clearly less understood in the other class of endotherms, the birds. We compared metabolic and oxidative stress responses of zebra finches chronically treated with or without a chemical mitochondrial uncoupler (2,4-dinitrophenol: DNP), undergoing an acute (24 h) and a chronic (4 weeks) cold exposure (12 °C). We predicted that control birds should present at least a transient elevation of oxidative stress levels in response to cold exposure. This oxidative stress cost should be more pronounced in control birds than in DNP-treated birds, due to their lower basal uncoupling state. Despite similar increase in metabolism, control birds presented elevated levels of DNA oxidative damage in response to acute (but not chronic) cold exposure, while DNP-treated birds did not. Plasma antioxidant capacity decreased overall in response to chronic cold exposure. These results show that acute cold exposure increases oxidative stress in birds. However, uncoupling mitochondrial functioning appears as a putative compensatory mechanism preventing cold-induced oxidative stress. This result confirms previous observations in mice and underlines non-shivering thermogenesis as a putative key mechanism for endotherms in mounting a response to cold at a low oxidative cost.     

A Metabolomics-driven Elucidation of the Anti-obesity Mechanisms of Xanthohumol

Mild, mitochondrial uncoupling increases energy expenditure and can reduce the generation of reactive oxygen species (ROS). Activation of cellular, adaptive stress response pathways can result in an enhanced capacity to reduce oxidative damage. Together, these strategies target energy imbalance and oxidative stress, both underlying factors of obesity and related conditions such as type 2 diabetes. Here we describe a metabolomics-driven effort to uncover the anti-obesity mechanism(s) of xanthohumol (XN), a prenylated flavonoid from hops. Metabolomics analysis of fasting plasma from obese, Zucker rats treated with XN revealed decreases in products of dysfunctional fatty acid oxidation and ROS, prompting us to explore the effects of XN on muscle cell bioenergetics. At low micromolar concentrations, XN acutely increased uncoupled respiration in several different cell types, including myocytes. Tetrahydroxanthohumol also increased respiration, suggesting electrophilicity did not play a role. At higher concentrations, XN inhibited respiration in a ROS-dependent manner. In myocytes, time course metabolomics revealed acute activation of glutathione recycling and long term induction of glutathione synthesis as well as several other changes indicative of short term elevated cellular stress and a concerted adaptive response. Based on these findings, we hypothesize that XN may ameliorate metabolic syndrome, at least in part, through mitochondrial uncoupling and stress response induction. In addition, time course metabolomics appears to be an effective strategy for uncovering metabolic events that occur during a stress response.     

A signalling role for 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling

Oxidative stress and mitochondrial dysfunction are associated with disease and aging. Oxidative stress results from overproduction of reactive oxygen species (ROS), often leading to peroxidation of membrane phospholipids and production of reactive aldehydes, particularly 4-hydroxy-2-nonenal. Mild uncoupling of oxidative phosphorylation protects by decreasing mitochondrial ROS production. We find that hydroxynonenal and structurally related compounds (such as trans-retinoic acid, trans-retinal and other 2-alkenals) specifically induce uncoupling of mitochondria through the uncoupling proteins UCP1, UCP2 and UCP3 and the adenine nucleotide translocase (ANT). Hydroxynonenal-induced uncoupling was inhibited by potent inhibitors of ANT (carboxyatractylate and bongkrekate) and UCP (GDP). The GDP-sensitive proton conductance induced by hydroxynonenal correlated with tissue expression of UCPs, appeared in yeast mitochondria expressing UCP1 and was absent in skeletal muscle mitochondria from UCP3 knockout mice. The carboxyatractylate-sensitive hydroxynonenal stimulation correlated with ANT content in mitochondria from Drosophila melanogaster expressing different amounts of ANT. Our findings indicate that hydroxynonenal is not merely toxic, but may be a biological signal to induce uncoupling through UCPs and ANT and thus decrease mitochondrial ROS production.     

Specific induction of metallothionein synthesis by mitochondrial oxidative stress.

Metallothionein (MT), a sulfhydryl-rich protein, may be increased by administration of a variety of agents, including metals, cytokines and oxidative stress agents. Mitochondria are a major source of reactive oxygen species, but antioxidant systems against mitochondrial free radicals are not fully understood. In this study, we examined the induction of MT synthesis by administration of mitochondrial-specific reactive oxygen generators such as antimycin A (AA), an electron transfer inhibitor, and 2,4-dinitrophenol (DNP), an uncoupling agent. Subcutaneous administration of AA to mice significantly increased the hepatic MT concentration in a dose- and time-dependent manner. AA slightly elevated glutathione peroxidase (GSHPx) activity, but the rate of increase in GSHPx (1.3-fold) was smaller than that in MT (11.8-fold). Other antioxidants such as catalase, manganese-superoxide dismutase (Mn-SOD), copper/zinc-superoxide dismutase (Cu/Zn-SOD) and GSHPx were not activated by AA treatment. Moreover, administration of DNP induced the synthesis of MT in the liver. Although DNP slightly elevated Mn-SOD activity, the rate of increase in Mn-SOD (1.3-fold) was smaller than that in MT (3.7-fold). Other antioxidants such as catalase, Cu/Zn-SOD and GSHPx were not activated by DNP treatment. These data suggest that MT plays a major role in protection against oxidative stress induced in mitochondria.     

Dinitrophenol-induced mitochondrial uncoupling in vivo triggers respiratory adaptation in HepG2 cells

Here, we show that 3 days of mitochondrial uncoupling, induced by low concentrations of dinitrophenol (10 and 50 μM) in cultured human HepG2 cells, triggers cellular metabolic adaptation towards oxidative metabolism. Chronic respiratory uncoupling of HepG2 cells induced an increase in cellular oxygen consumption, oxidative capacity and cytochrome c oxidase activity. This was associated with an upregulation of COXIV and ANT3 gene expression, two nuclear genes that encode mitochondrial proteins involved in oxidative phosphorylation. Glucose consumption, lactate and pyruvate production and growth rate were unaffected, indicating that metabolic adaptation of HepG2 cells undergoing chronic respiratory uncoupling allows continuous and efficient mitochondrial ATP production without the need to increase glycolytic activity. In contrast, 3 days of dinitrophenol treatment did not change the oxidative capacity of human 143B.TK⁻ cells, but it increased glucose consumption, lactate and pyruvate production. Despite a large increase in glycolytic metabolism, the growth rate of 143B.TK⁻ cells was significantly reduced by dinitrophenol-induced mitochondrial uncoupling. We propose that chronic respiratory uncoupling may constitute an internal bioenergetic signal, which would initiate a coordinated increase in nuclear respiratory gene expression, which ultimately drives mitochondrial metabolic adaptation within cells.     

Overexpression of wheat mitochondrial uncoupling protein in rice plants confers tolerances to oxidative stresses promoted by exogenous hydrogen peroxide and low temperature

To obtain a better understanding of the function of mitochondrial uncoupling protein (UCP) in higher plants, the wheat gene for mitochondrial uncoupling protein (WhUCP) in rice was overexpressed by Agrobacterium-mediated transformation with a construct containing the WhUCP ORF under control of the 35S promoter. The transgenic rice plants showed a significant increase in tolerance against oxidative stress promoted by exogenous hydrogen peroxide at the seedling stage. The transgenic rice plants overexpressing WhUCP also exhibited greater tolerance against cold stress than did the wild-type plants. These results demonstrated that the mitochondrial UCP in higher plants is positively involved in the pathway for abiotic stress tolerance, probably through a decrease in cellular oxidative damage, and that controlled uncoupling by UCP could be used for improvement of stress tolerance in higher plants.Kenjirou Ozawa and Seiji Murayama are contributed equally to the work     

Mitochondrial uncoupling proteins: new insights from functional and proteomic studies

Mitochondria are the major sites of ATP synthesis through oxidative phosphorylation, a process that is weakened by proton leak. Uncoupling proteins are mitochondrial membrane proteins specialized in inducible proton conductance. They dissipate the proton electrochemical gradient established by the respiratory chain at the expense of reducing substrates. Several physiological roles have been suggested for uncoupling proteins, including roles in the control of the cellular energy balance and in preventive action against oxidative stress. This review focuses on new leads emerging from comparative proteomics about the involvement of uncoupling protein in the mitochondrial physiology. A brief overview on uncoupling proteins and on proteomics applied to mitochondria is also presented herein.