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1.  Mitochondrial Reactive Oxygen Species Production in Excitable Cells: Modulators of Mitochondrial and Cell Function 
Antioxidants & Redox Signaling  2009;11(6):1373-1414.
The mitochondrion is a major source of reactive oxygen species (ROS). Superoxide (O2•−) is generated under specific bioenergetic conditions at several sites within the electron-transport system; most is converted to H2O2 inside and outside the mitochondrial matrix by superoxide dismutases. H2O2 is a major chemical messenger that, in low amounts and with its products, physiologically modulates cell function. The redox state and ROS scavengers largely control the emission (generation scavenging) of O2•−. Cell ischemia, hypoxia, or toxins can result in excess O2•− production when the redox state is altered and the ROS scavenger systems are overwhelmed. Too much H2O2 can combine with Fe2+ complexes to form reactive ferryl species (e.g., Fe(IV) = O•). In the presence of nitric oxide (NO•), O2•− forms the reactant peroxynitrite (ONOO−), and ONOOH-induced nitrosylation of proteins, DNA, and lipids can modify their structure and function. An initial increase in ROS can cause an even greater increase in ROS and allow excess mitochondrial Ca2+ entry, both of which are factors that induce cell apoptosis and necrosis. Approaches to reduce excess O2•− emission include selectively boosting the antioxidant capacity, uncoupling of oxidative phosphorylation to reduce generation of O2•− by inducing proton leak, and reversibly inhibiting electron transport. Mitochondrial cation channels and exchangers function to maintain matrix homeostasis and likely play a role in modulating mitochondrial function, in part by regulating O2•− generation. Cell-signaling pathways induced physiologically by ROS include effects on thiol groups and disulfide linkages to modify posttranslationally protein structure to activate/inactivate specific kinase/phosphatase pathways. Hypoxia-inducible factors that stimulate a cascade of gene transcription may be mediated physiologically by ROS. Our knowledge of the role played by ROS and their scavenging systems in modulation of cell function and cell death has grown exponentially over the past few years, but we are still limited in how to apply this knowledge to develop its full therapeutic potential. Antioxid. Redox Signal. 11, 1373–1414.
Focus of review
Ambiguities about ROS
Overview of Mitochondrial Structure and Function
Mitochondrial Sources and Mechanism of ROS Generation
Requirement for charged membrane, electron flux, and O2
Reactive and nonreactive O2 species and reactants
Assessing ROS generation
Sites and conditions for mitochondrial ROS generation
Complex I (NADH ubiquinone oxidoreductase)
Complex III (co-enzyme Q, bc1 complex, ubiquinone/cytochrome c reductase)
Pathologic Induction of Mitochondrial ROS Release
ROS-induced ROS release
ROS-induced Ca2+ loading
ROS generation during tissue ischemia and hypoxia
Very low po2 and lack of mitochondrial ROS generation
Antioxidant Defenses Against Pathologic ROS Formation
SODs, catalase, cytochrome c, GSH, and TRXSH2, and other linked redox couples
Regulation of genes encoding mitochondrial antioxidant systems
ROS generation versus ROS scavenging
MPT pore opening and cytochrome c
Targets of Excess ROS Emission
DNA, proteins, and phospholipids
Role of cardiolipin
Approaches to Reduce Excess ROS
Capacity of mitochondrial and cell reductants
Exogenous SODs and catalase
Proton leak to modulate superoxide generation
Uncoupling proteins
HNE-induced proton leak
ROS-induced proton leak
Physiologic Modulation of Mitochondrial ROS Emission
H2O2 and ONOO− as chemical effectors
ROS modulation by cations
K+: A modulator of ROS generation?
Biphasic effect of KCa channels on ROS generation
KATP channel opening and ROS
Direct Ca2+-induced ROS unlikely
Rate of oxidative phosphorylation and ROS generation
Role of ROS in Triggering or Effecting Cardioprotection
Pathways and mechanisms
Inhibiting complex I and cardioprotection
Regulation of Cellular Processes by Mitochondria-Derived ROS
Cell signaling by oxidative modifications and redox systems
Examples of signaling by ROS
Importance of cysteine thiols in ROS-induced signaling
ROS oxidation reactions
O2 sensors
Hypoxia-inducible factors
ROS as O2 sensors
Peroxide-induced TCA shunts
Difficulties in understanding the role of ROS
Future directions
PMCID: PMC2842133  PMID: 19187004
2.  Mitochondrial Dysfunction in Diabetes: From Molecular Mechanisms to Functional Significance and Therapeutic Opportunities 
Antioxidants & Redox Signaling  2010;12(4):537-577.
Given their essential function in aerobic metabolism, mitochondria are intuitively of interest in regard to the pathophysiology of diabetes. Qualitative, quantitative, and functional perturbations in mitochondria have been identified and affect the cause and complications of diabetes. Moreover, as a consequence of fuel oxidation, mitochondria generate considerable reactive oxygen species (ROS). Evidence is accumulating that these radicals per se are important in the pathophysiology of diabetes and its complications. In this review, we first present basic concepts underlying mitochondrial physiology. We then address mitochondrial function and ROS as related to diabetes. We consider different forms of diabetes and address both insulin secretion and insulin sensitivity. We also address the role of mitochondrial uncoupling and coenzyme Q. Finally, we address the potential for targeting mitochondria in the therapy of diabetes. Antioxid. Redox Signal. 12, 537–577.
Basic Physiology
Electron transport
Reactive oxygen species and mitochondria
Mitochondrial nitric oxide
Role of calcium and the mitochondrial permeability transition pore
Assessing Mitochondrial Function
Respiration and potential
ATP production and the proton leak
ROS production by isolated mitochondria
Site specificity of mitochondrial superoxide production
Mitochondrial ROS production in intact cells
Oxidative damage to mitochondria in intact cells
Mitochondrial Metabolism and Diabetes
General considerations
Mitochondrial diabetes
Type 1 and type 2 diabetes
Mitochondrial number and morphology
Mitochondrial biogenesis
Mitochondrial function in type 2 diabetes and insulin-resistant states
Is mitochondrial impairment a cause of insulin resistance?
Mitochondrial respiratory coupling and insulin release
Mitochondrial function in insulin-deficient diabetes
Diabetes and mitochondrial function in non–insulin-sensitive tissues
Mitochondria and cell-fuel selectivity
Diabetic cardiomyopathy and mitochondrial function
Mitochondrial ROS and Diabetes
ROS production and the cause of diabetes
Oxidative damage and pancreatic islet β cells
ROS and oxidative damage in insulin-sensitive target tissues
ROS and the complications of diabetes
Non–insulin-sensitive tissues (retina, renal, neural cells)
ROS and vascular cells
Mitochondrial Membrane Potential and Diabetes
Role of uncoupling proteins
Does membrane potential actually protect against superoxide production?
Coenzyme Q and Diabetes
Therapeutic Implications
Improving mitochondrial metabolism
Lifestyle modification
Pharmacologic intervention
Controlling ROS production and oxidative damage
Mitochondria-targeted antioxidants
Metabolic effects of mitochondria-targeted antioxidants
Mitochondria-targeted antioxidant peptides
Targeting superoxide
PMCID: PMC2824521  PMID: 19650713
3.  Control mechanisms in mitochondrial oxidative phosphorylation☆ 
Neural Regeneration Research  2013;8(4):363-375.
Distribution and activity of mitochondria are key factors in neuronal development, synaptic plasticity and axogenesis. The majority of energy sources, necessary for cellular functions, originate from oxidative phosphorylation located in the inner mitochondrial membrane. The adenosine-5’- triphosphate production is regulated by many control mechanism–firstly by oxygen, substrate level, adenosine-5’-diphosphate level, mitochondrial membrane potential, and rate of coupling and proton leak. Recently, these mechanisms have been implemented by “second control mechanisms,” such as reversible phosphorylation of the tricarboxylic acid cycle enzymes and electron transport chain complexes, allosteric inhibition of cytochrome c oxidase, thyroid hormones, effects of fatty acids and uncoupling proteins. Impaired function of mitochondria is implicated in many diseases ranging from mitochondrial myopathies to bipolar disorder and schizophrenia. Mitochondrial dysfunctions are usually related to the ability of mitochondria to generate adenosine-5’-triphosphate in response to energy demands. Large amounts of reactive oxygen species are released by defective mitochondria, similarly, decline of antioxidative enzyme activities (e.g. in the elderly) enhances reactive oxygen species production. We reviewed data concerning neuroplasticity, physiology, and control of mitochondrial oxidative phosphorylation and reactive oxygen species production.
PMCID: PMC4107533  PMID: 25206677
neural regeneration; reviews; mitochondria; metabolic pathway; membrane potential; oxidative phosphorylation; electron transport chain complex; reactive oxygen species; respiratory state; calcium; uncoupling protein; fatty acid; neuroregeneration
4.  Reactive Oxygen Species Production by Forward and Reverse Electron Fluxes in the Mitochondrial Respiratory Chain 
PLoS Computational Biology  2011;7(3):e1001115.
Reactive oxygen species (ROS) produced in the mitochondrial respiratory chain (RC) are primary signals that modulate cellular adaptation to environment, and are also destructive factors that damage cells under the conditions of hypoxia/reoxygenation relevant for various systemic diseases or transplantation. The important role of ROS in cell survival requires detailed investigation of mechanism and determinants of ROS production. To perform such an investigation we extended our rule-based model of complex III in order to account for electron transport in the whole RC coupled to proton translocation, transmembrane electrochemical potential generation, TCA cycle reactions, and substrate transport to mitochondria. It fits respiratory electron fluxes measured in rat brain mitochondria fueled by succinate or pyruvate and malate, and the dynamics of NAD+ reduction by reverse electron transport from succinate through complex I. The fitting of measured characteristics gave an insight into the mechanism of underlying processes governing the formation of free radicals that can transfer an unpaired electron to oxygen-producing superoxide and thus can initiate the generation of ROS. Our analysis revealed an association of ROS production with levels of specific radicals of individual electron transporters and their combinations in species of complexes I and III. It was found that the phenomenon of bistability, revealed previously as a property of complex III, remains valid for the whole RC. The conditions for switching to a state with a high content of free radicals in complex III were predicted based on theoretical analysis and were confirmed experimentally. These findings provide a new insight into the mechanisms of ROS production in RC.
Author Summary
Respiration at the level of mitochondria is considered as delivery of electrons and protons from NADH or succinate to oxygen through a set of transporters constituting the respiratory chain (RC). Mitochondrial respiration, dealing with transfer of unpaired electrons, may produce reactive oxygen species (ROS) such as O2− and subsequently H2O2 as side products. ROS are chemically very active and can cause oxidative damage to cellular components. The production of ROS, normally low, can increase under stress to the levels incompatible with cell survival; thus, understanding the ways of ROS production in the RC represents a vital task in research. We used mathematical modeling to analyze experiments with isolated brain mitochondria aimed to study relations between electron transport and ROS production. Elsewhere we reported that mitochondrial complex III can operate in two distinct steady states at the same microenvironmental conditions, producing either low or high levels of ROS. Here, this property of bistability was confirmed for the whole RC. The associations between measured ROS production and computed individual free radical levels in complexes I and III were established. The discovered phenomenon of bistability is important as a basis for new strategies in organ transplantation and therapy.
PMCID: PMC3068929  PMID: 21483483
5.  Respiratory uncoupling by increased H+ or K+ flux is beneficial for heart mitochondrial turnover of reactive oxygen species but not for permeability transition 
BMC Cell Biology  2013;14:40.
Ischemic preconditioning has been proposed to involve changes in mitochondrial H+ and K+ fluxes, in particular through activation of uncoupling proteins and ATP-sensitive K+ channels (MitoKATP). The objectives of the present study were to explore how increased H+ and K+ fluxes influence heart mitochondrial physiology with regard to production and scavenging of reactive oxygen species (ROS), volume changes and resistance to calcium-induced mitochondrial permeability transition (mPT).
Isolated rat heart mitochondria were exposed to a wide concentration range of the protonophore CCCP or the potassium ionophore valinomycin to induce increased H+ and K+ conductance, respectively. Simultaneous monitoring of mitochondrial respiration and calcium retention capacity (CRC) demonstrated that the relative increase in respiration caused by valinomycin or CCCP correlated with a decrease in CRC, and that no level of respiratory uncoupling was associated with enhanced resistance to mPT. Mitochondria suspended in hyperosmolar buffer demonstrated a dose-dependent reduction in CRC with increasing osmolarity. However, mitochondria in hypoosmolar buffer to increase matrix volume did not display increased CRC. ROS generation was reduced by both K+- and H+-mediated respiratory uncoupling. The ability of heart mitochondria to detoxify H2O2 was substantially greater than the production rate. The H2O2 detoxification was dependent on respiratory substrates and was dramatically decreased following calcium-induced mPT, but was unaffected by uncoupling via increased K+ and H+ conductance.
It is concluded that respiratory uncoupling is not directly beneficial to rat heart mitochondrial resistance to calcium overload irrespective of whether H+ or K+ conductance is increased. The negative effects of respiratory uncoupling thus probably outweigh the reduction in ROS generation and a potential positive effect by increased matrix volume, resulting in a net sensitization of heart mitochondria to mPT activation.
PMCID: PMC3849260  PMID: 24053891
Ischemic preconditioning; Mitochondrial permeability transition; Potassium channels; Respiratory uncoupling; Reactive oxygen species
6.  Stimulation of mitochondrial proton conductance by hydroxynonenal requires a high membrane potential 
Bioscience Reports  2008;28(Pt 2):83-88.
Mild uncoupling of oxidative phosphorylation, caused by a leak of protons back into the matrix, limits mitochondrial production of ROS (reactive oxygen species). This proton leak can be induced by the lipid peroxidation products of ROS, such as HNE (4-hydroxynonenal). HNE activates uncoupling proteins (UCP1, UCP2 and UCP3) and ANT (adenine nucleotide translocase), thereby providing a negative feedback loop. The mechanism of activation and the conditions necessary to induce uncoupling by HNE are unclear. We have found that activation of proton leak by HNE in rat and mouse skeletal muscle mitochondria is dependent on incubation with respiratory substrate. In the presence of HNE, mitochondria energized with succinate became progressively more leaky to protons over time compared with mitochondria in the absence of either HNE or succinate. Energized mitochondria must attain a high membrane potential to allow HNE to activate uncoupling: a drop of 10–20 mV from the resting value is sufficient to blunt induction of proton leak by HNE. Uncoupling occurs through UCP3 (11%), ANT (64%) and other pathways (25%). Our findings have shown that exogenous HNE only activates uncoupling at high membrane potential. These results suggest that both endogenous HNE production and high membrane potential are required before mild uncoupling will be triggered to attenuate mitochondrial ROS production.
PMCID: PMC2518262  PMID: 18384278
adenine nucleotide translocase (ANT); 4-hydroxynonenal (HNE); mitochondria; proton leak; uncoupling protein 3 (UCP3); ANT, adenine nucleotide translocase; HNE, 4-hydroxynonenal; KO, knockout; ROS, reactive oxygen species; TPMP, triphenylmethylphosphonium; UCP, uncoupling protein; WT, wild-type.
7.  Mitochondrial handling of excess Ca2+ is substrate-dependent with implications for reactive oxygen species generation 
The mitochondrial electron transport chain is the major source of reactive oxygen species (ROS) during cardiac ischemia. Several mechanisms modulate ROS production; one is mitochondrial Ca2+ uptake. Here we sought to elucidate the effects of extra-mitochondrial Ca2+ (e[Ca2+]) on ROS production (measured as H2O2 release) from complexes I and III.
Mitochondria, isolated from guinea pig hearts, were pre-incubated with increasing concentrations of CaCl2 and then energized with the complex I substrate Na+-pyruvate or the complex II substrate Na+-succinate. Mitochondrial H2O2 release rates were assessed after giving either rotenone or antimycin A to inhibit complex I or III, respectively. After pyruvate, mitochondria maintained a fully polarized membrane potential (Δψ, assessed using rhodamine 123) and were able to generate NADH (assessed using autofluorescence) even with excess e[Ca2+] (assessed using CaGreen-5N), whereas they remained partially depolarized and did not generate NADH after succinate. This partial Δψ depolarization with succinate was accompanied by a large release of H2O2 (assessed using amplex red/horseradish peroxidase) with later addition of antimycin A. In the presence of excess e[Ca2+], adding cyclosporine A to inhibit mitochondrial permeability transition pore (mPTP) opening restored Δψ and significantly decreased antimycin A-induced H2O2 release.
Succinate accumulates during ischemia to become the major substrate utilized by cardiac mitochondria. The inability of mitochondria to maintain a fully polarized Δψ under excess e[Ca2+] when succinate, but not pyruvate, is the substrate may indicate a permeabilization of the mitochondrial membrane which enhances H2O2 emission from complex III during ischemia.
PMCID: PMC3542420  PMID: 23010495
complex III; mitochondrial permeability transition pore; succinate; Ca2+
8.  Ca2+‐induced High Amplitude Swelling and Cytochrome c Release From Wheat (Triticum aestivum L.) Mitochondria Under Anoxic Stress 
Annals of Botany  2002;90(4):509-516.
Under stress conditions, mitochondria sense metabolic changes, e.g. in pH, cytoplasmic Ca2+, energy status, and reactive oxygen species (ROS), and respond by induction of the permeability transition pore (PTP) and by releasing cytochrome c, thus initiating the programmed cell death (PCD) cascade in animal cells. In plant cells, the presence of all the components of the cascade has not yet been shown. In wheat (Triticum aestivum L.) root mitochondria, the onset of anoxia caused rapid dissipation of the inner membrane potential, initial shrinkage of the mitochondrial matrix and the release of previously accumulated Ca2+. Ca2+ uptake by mitochondria was dependent on the presence of inorganic phosphate. Treatment of mitochondria with high micromolar and millimolar Ca2+ (but not Mg2+) concentrations induced high amplitude swelling, indicative of PTP opening. Alterations in mitochondrial volume were confirmed by transmission electron microscopy. Mitochondrial swelling was not sensitive to cyclosporin A (CsA)—an inhibitor of mammalian PTP. The release of cytochrome c was monitored under lack of oxygen. Anoxia alone failed to induce cytochrome c release from mitochondria. Oxygen deprivation and Ca2+ ions together caused cytochrome c release in a CsA‐insensitive manner. This process correlated positively with Ca2+ concentration and required Ca2+ localization in the mitochondrial matrix. Functional characteristics of wheat root mitochondria, such as membrane potential, Ca2+ transport, swelling, and cytochrome c release under lack of oxygen are discussed in relation to PCD.
PMCID: PMC4240387  PMID: 12324275
Anoxia; apoptosis; Ca2+; calcium; oxygen deprivation; permeability transition; plant mitochondria; programmed cell death (PCD); reactive oxygen species (ROS); Triticum aestivum; wheat
9.  The sites and topology of mitochondrial superoxide production 
Experimental gerontology  2010;45(7-8):466-472.
Mitochondrial superoxide production is an important source of reactive oxygen species in cells, and may cause or contribute to ageing and the diseases of ageing. Seven major sites of superoxide production in mammalian mitochondria are known and widely accepted. In descending order of maximum capacity they are the ubiquinone binding sites in complex I (site IQ) and complex III (site IIIQo), glycerol 3-phosphate dehydrogenase, the flavin in complex I (site IF), the electron transferring flavoprotein:Q oxidoreductase (ETFQOR) of fatty acid beta oxidation, and pyruvate and 2-oxoglutarate dehydrogenases. None of these sites is fully characterized and for some we only have sketchy information. The topology of the sites is important because it determines whether or not a site will produce superoxide in the mitochondrial matrix and be able to damage mitochondrial DNA. All sites produce superoxide in the matrix; site IIIQo and glycerol 3-phosphate dehydrogenase also produce superoxide to the intermembrane space. The relative contribution of each site to mitochondrial reactive oxygen species generation in the absence of electron transport inhibitors is unknown in isolated mitochondria, in cells or in vivo, and may vary considerably with species, tissue, substrate, energy demand and oxygen tension.
PMCID: PMC2879443  PMID: 20064600
Reactive oxygen species; ROS; electron transport; semiquinone; complex I; complex III; glycerol phosphate dehydrogenase; ETFQOR
10.  Human neuronal uncoupling proteins 4 and 5 (UCP4 and UCP5): structural properties, regulation, and physiological role in protection against oxidative stress and mitochondrial dysfunction 
Brain and Behavior  2012;2(4):468-478.
Uncoupling proteins (UCPs) belong to a large family of mitochondrial solute carriers 25 (SLC25s) localized at the inner mitochondrial membrane. UCPs transport protons directly from the intermembrane space to the matrix. Of five structural homologues (UCP1 to 5), UCP4 and 5 are principally expressed in the central nervous system (CNS). Neurons derived their energy in the form of ATP that is generated through oxidative phosphorylation carried out by five multiprotein complexes (Complexes I–V) embedded in the inner mitochondrial membrane. In oxidative phosphorylation, the flow of electrons generated by the oxidation of substrates through the electron transport chain to molecular oxygen at Complex IV leads to the transport of protons from the matrix to the intermembrane space by Complex I, III, and IV. This movement of protons to the intermembrane space generates a proton gradient (mitochondrial membrane potential; MMP) across the inner membrane. Complex V (ATP synthase) uses this MMP to drive the conversion of ADP to ATP. Some electrons escape to oxygen-forming harmful reactive oxygen species (ROS). Proton leakage back to the matrix which bypasses Complex V resulting in a major reduction in ROS formation while having a minimal effect on MMP and hence, ATP synthesis; a process termed “mild uncoupling.” UCPs act to promote this proton leakage as means to prevent excessive build up of MMP and ROS formation. In this review, we discuss the structure and function of mitochondrial UCPs 4 and 5 and factors influencing their expression. Hypotheses concerning the evolution of the two proteins are examined. The protective mechanisms of the two proteins against neurotoxins and their possible role in regulating intracellular calcium movement, particularly with regard to the pathogenesis of Parkinson's disease are discussed.
PMCID: PMC3432969  PMID: 22950050
Energy homeostasis; mitochondrial dysfunction; neurodegeneration; neuroprotection; oxidative stress; uncoupling proteins
11.  Isoflurane differentially modulates mitochondrial reactive oxygen species production via forward versus reverse electron transport flow: Implications for preconditioning 
Anesthesiology  2011;115(3):531-540.
Reactive oxygen species (ROS) mediate the effects of anesthetic precondition to protect against ischemia and reperfusion injury, but the mechanisms of ROS generation remain unclear. In this study, we investigated if mitochondria-targeted antioxidant (mitotempol) abolishes the cardioprotective effects of anesthetic preconditioning. Further, we investigated the mechanism by which isoflurane alters ROS generation in isolated mitochondria and submitochondrial particles.
Rats were pretreated with 0.9% saline, 3.0 mg/kg mitotempol in the absence or presence of 30 min exposure to isoflurane. Myocardial infarction was induced by left anterior descending artery occlusion for 30 min followed by reperfusion for 2h and infarct size measurements. Mitochondrial ROS production was determined spectrofluorometrically. The effect of isoflurane on enzymatic activity of mitochondrial respiratory complexes was also determined.
Isoflurane reduced myocardial infarct size (40±9 % = mean±SD) compared to control experiments (60±4 %). Mitotempol abolished the cardioprotective effects of anesthetic preconditioning (60±9%). Isoflurane enhanced ROS generation in submitochondrial particles with NADH, but not with succinate, as substrate. In intact mitochondria, isoflurane enhanced ROS production in the presence of rotenone, antimycin A, or ubiquinone when pyruvate and malate were substrates, but isoflurane attenuated ROS production when succinate was substrate. Mitochondrial respiratory experiments and electron transport chain complex assays revealed that isoflurane inhibited only complex I activity.
The results demonstrated that isoflurane produces ROS at complex I and III of the respiratory chain via the attenuation of complex I activity. The action on complex I decreases unfavorable reverse electron flow and ROS release in myocardium during reperfusion.
PMCID: PMC3337729  PMID: 21862887
12.  Endothelial Cell and Platelet Bioenergetics: Effect of Glucose and Nutrient Composition 
PLoS ONE  2012;7(6):e39430.
It has been suggested that cells that are independent of insulin for glucose uptake, when exposed to high glucose or other nutrient concentrations, manifest enhanced mitochondrial substrate oxidation with consequent enhanced potential and generation of reactive oxygen species (ROS); a paradigm that could predispose to vascular complications of diabetes. Here we exposed bovine aortic endothelial (BAE) cells and human platelets to variable glucose and fatty acid concentrations. We then examined oxygen consumption and acidification rates using recently available technology in the form of an extracellular oxygen and proton flux analyzer. Acute or overnight exposure of confluent BAE cells to glucose concentrations from 5.5 to 25 mM did not enhance or change the rate of oxygen consumption (OCR) under basal conditions, during ATP synthesis, or under uncoupled conditions. Glucose also did not alter OCR in sub-confluent cells, in cells exposed to low serum, or in cells treated with added pyruvate. Likewise, overnight exposure to fatty acids of varying saturation had no such effects. Overnight exposure of BAE cells to low glucose concentration decreased maximal uncoupled respiration, but not basal or ATP related oxygen consumption. Labeled glucose oxidation to CO2 increased, but only marginally after high glucose exposure while oleate oxidation to CO2 decreased. Overnight exposure to linolenic acid, but not oleic or linoleic acid increased extracellular acidification consistent with enhanced glycolytic metabolism. We were unable to detect an increase in production of reactive oxygen species (ROS) from BAE cells exposed to high medium glucose. Like BAE cells, exposure of human platelets to glucose did not increase oxygen consumption. As opposed to BAE cells, platelet mitochondria demonstrate less respiratory reserve capacity (beyond that needed for basal metabolism). Our data do not support the concept that exposure to high glucose or fatty acids accelerates mitochondrial oxidative metabolism in endothelial cells or platelets.
PMCID: PMC3382132  PMID: 22745753
13.  Mitochondrial function and redox control in the aging eye: Role of MsrA and other repair systems in cataract and macular degenerations 
Experimental eye research  2008;88(2):195-203.
Oxidative stress occurs when the level of prooxidants exceeds the level of antioxidants in cells resulting in oxidation of cellular components and consequent loss of cellular function. Oxidative stress is implicated in wide range of age related disorders including Alzheimer's disease, Parkinson's disease amyotrophic lateral sclerosis (ALS), Huntington's disease and the aging process itself (Lin and Beal, 2006). In the anterior segment of the eye, oxidative stress has been linked to lens cataract (Truscott, 2005) and glaucoma (Tezel, 2006) while in the posterior segment of the eye oxidative stress has been associated with macular degeneration (Hollyfield et al., 2008). Key to many oxidative stress conditions are alterations in the efficiency of mitochondrial respiration resulting in superoxide (O2-) production. Superoxide production precedes subsequent reactions that form potentially more dangerous reactive oxygen species (ROS) species such as the hydroxyl radical (˙OH), hydrogen peroxide (H2O2) and peroxynitrite (OONO-). The major source of ROS in the mitochondria, and in the cell overall, is leakage of electrons from complexes I and III of the electron transport chain. It is estimated that 0.2-2% of oxygen taken up by cells is converted to ROS, through mitochondrial superoxide generation, by the mitochondria (Hansford et al., 1997). Generation of superoxide at complex I and III has been shown to occur at both the matrix side of the inner mitochondrial membrane and the cytosolic side of the membrane (Kakkar and Singh 2007). While exogenous sources of ROS such as UV light, visible light, ionizing radiation, chemotherapeutics, and environmental toxins may contribute to the oxidative milieu, mitochondria are perhaps the most significant contribution to ROS production affecting the aging process. In addition to producing ROS, mitochondria are also a target for ROS which in turn reduces mitochondrial efficiency and leads to the generation of more ROS in a vicious self-destructive cycle. Consequently, the mitochondria have evolved a number of antioxidant and key repair systems to limit the damaging potential of free oxygen radicals and to repair damaged proteins (Figure 1.0). The aging eye appears to be at considerable risk from oxidative stress. This review will outline the potential role of mitochondrial function and redox balance in age-related eye diseases, and detail how the methionine sulfoxide reductase (Msr) protein repair system and other redox systems play key roles in the function and maintenance of the aging eye.
PMCID: PMC2683477  PMID: 18588875
Mitochondria; Cataract; Macular Degeneration; Oxidative Stress; Reactive Oxygen Species; Aging; Methionine Sulfoxide Reductase
14.  Localization of superoxide anion production to mitochondrial electron transport chain in 3-NPA-treated cells 
Mitochondrion  2006;6(5):235-244.
3-Nitropropionic acid (3-NPA), an inhibitor of succinate dehydrogenase (SDH) at complex II of the mitochondrial electron transport chain induces cellular energy deficit and oxidative stress-related neurotoxicity. In the present study, we identified the site of reactive oxygen species production in mitochondria. 3-NPA increased O2•− generation in mitochondria respiring on the complex I substrates pyruvate + malate, an effect fully inhibited by rotenone. Antimycin A increased O2•− production in the presence of complex I and/or II substrates. Addition of 3-NPA markedly increased antimycin A-induced O2•− production by mitochondria incubated with complex I substrates, but 3-NPA inhibited O2•− formation driven with the complex II substrate succinate. At 0.6 μM, myxothiazol inhibits complex III, but only partially decreases complex I activity, and allowed 3-NPA-induced O2•− formation; however, at 40 μM myxothiazol (which completely inhibits both complexes I and III) eliminated O2•− production from mitochondria respiring via complex I substrates. These results indicate that in the presence of 3-NPA, mitochondria generate O2•− from a site between the ubiquinol pool and the 3-NPA block in the respiratory complex II.
PMCID: PMC3031911  PMID: 17011837
3-NPA; Superoxide anion; Mitochondrial respiratory complexes
15.  Type 1 Diabetic Akita Mouse Hearts Are Insulin Sensitive but Manifest Structurally Abnormal Mitochondria That Remain Coupled Despite Increased Uncoupling Protein 3 
Diabetes  2008;57(11):2924-2932.
OBJECTIVE— Fatty acid–induced mitochondrial uncoupling and oxidative stress have been proposed to reduce cardiac efficiency and contribute to cardiac dysfunction in type 2 diabetes. We hypothesized that mitochondrial uncoupling may also contribute to reduced cardiac efficiency and contractile dysfunction in the type 1 diabetic Akita mouse model (Akita).
RESEARCH DESIGN AND METHODS— Cardiac function and substrate utilization were determined in isolated working hearts and in vivo function by echocardiography. Mitochondrial function and coupling were determined in saponin-permeabilized fibers, and proton leak kinetics was determined in isolated mitochondria. Hydrogen peroxide production and aconitase activity were measured in isolated mitochondria, and total reactive oxygen species (ROS) were measured in heart homogenates.
RESULTS— Resting cardiac function was normal in Akita mice, and myocardial insulin sensitivity was preserved. Although Akita hearts oxidized more fatty acids, myocardial O2 consumption was not increased, and cardiac efficiency was not reduced. ADP-stimulated mitochondrial oxygen consumption and ATP synthesis were decreased, and mitochondria showed grossly abnormal morphology in Akita. There was no evidence of oxidative stress, and despite a twofold increase in uncoupling protein 3 (UCP3) content, ATP-to-O ratios and proton leak kinetics were unchanged, even after perfusion of Akita hearts with 1 mmol/l palmitate.
CONCLUSIONS— Insulin-deficient Akita hearts do not exhibit fatty acid–induced mitochondrial uncoupling, indicating important differences in the basis for mitochondrial dysfunction between insulin-responsive type 1 versus insulin-resistant type 2 diabetic hearts. Increased UCP3 levels do not automatically increase mitochondrial uncoupling in the heart, which supports the hypothesis that fatty acid–induced mitochondrial uncoupling as exists in type 2 diabetic hearts requires a concomitant increase in ROS generation.
PMCID: PMC2570388  PMID: 18678617
16.  Glucose metabolism determines resistance of cancer cells to bioenergetic crisis after cytochrome-c release 
How can cancer cells survive the consequences of cyt-c release? Huber et al provide a quantitative analysis of the protective role of enhanced glucose utilization in cancer cells and investigate the impact of cell-to-cell heterogeneity in mitochondrial bioenergetics.
How can cancer cells survive the consequences of cyt-c release? Huber et al provide a quantitative analysis of the protective role of enhanced glucose utilization in cancer cells and investigate the impact of cell-to-cell heterogeneity in mitochondrial bioenergetics.
We combine ordinary differential equations based computational modelling, single-cell microscopy and in biochemistry assays to provide the first integrated system study to portray the bioenergetic crisis in cell populations subsequent to cytochrome-c (cyt-c) release; a hallmark during chemotherapeutically induced cell death.We experimentally identified a cell-to-cell heterogeneity in the dynamics of the ATP synthase subsequent to cyt-c release, which the model explained by variations in (i) accessible cytochrome-c after release and (ii) the cell's glycolytic capacity.Our model predicted, and single-cell imaging confirmed, that high (increasing) glucose in media was able to sustain (repolarise) ΔΨm in HeLa cervical cancer and MCF-7 breast cancer cells, suggesting that they might recover from bioenergetic crisis upon elevation of glucose. However, no significant repolarisation was found in non-transformed human epithelial CRL-1807 cells. Therefore, this mechanism may provide cancer cells with a competitive advantage to evade cell death induced by anticancer drugs or other stress conditions when compared with non-transformed cells.
How can cells cope with a bioenergetic crisis? In particular, how can cancer cells survive the bioenergetic consequences of cyt-c release that are often induced by chemotherapeutic agents, and that lead to depolarisation of the mitochondrial membrane potential ΔΨm, result in loss of ionic homeostasis and induce cell death? Is there an inherent population heterogeneity that can lead to a non-synchronous response to above cell death stimuli, thereby aggravating treatment and contributing to clinical relapse? Do cancer cells have a competitive advantage to non-transformed cells in averting such a bioenergetic crisis after cyt-c release. We have investigated these questions in our study, which we regard as the first rigorous system study of single-cell bioenergetics subsequent to cyt-c release and one that bridges single-cell microscopy, in vitro analysis with ordinary differential equations (ODE) based modelling of bioenergetics pathways in the mitochondria and the cytosol.
Several laboratories have so far investigated cyt-c release experimentally (Slee et al, 1999; Atlante et al, 2000; Goldstein et al, 2000; Luetjens et al, 2001; Plas et al, 2001; Waterhouse et al, 2001; Ricci et al, 2003; Colell et al, 2007; Dussmann et al, 2003a, 2003b) and isolated mitochondria (Chinopoulos and Adam-Vizi, 2009; Kushnareva et al, 2002; Kushnareva et al, 2001). However, the cause and mechanistic of several key findings remain elusive and need a system level understanding of post-cyt-c release bioenergetic and its potential cross-talk to apoptosis signalling.
Ricci et al (2003) have identified that the cell death-inducing protease caspase-3, which get activated upon cyt-c release, can further impair mitochondrial function by cleaving and deactivating respiratory complexes I and II. We addressed the question of how such a mechanism could potentiate a bioenergetic crisis. To do so, we first assembled our ODE-based model by integrating approaches from metabolic modelling (Beard, 2005; Beard and Qian, 2007; Dash and Beard, 2008) and calibrated the model to literature data that describe bioenergetic state variables (mitochondrial membrane potential ΔΨm, mitochondrial transmembrane membrane ΔpH, respiration ratio between respiring and resting state mitochondria). By remodelling cyt-c release as observed experimentally and integrating it into our model as input, the single-cell model was able to correctly describe the kinetics of ΔΨm depolarisation and allowed its quantification. Moreover, it suggested that an additional complex I/II cleavage may further impair respiration and depolarise ΔΨm by approximately further 10%.
It was further reported that ATP synthase reversal, a change of direction in the ATP-producing enzyme that leads to pumping of protons from the mitochondrial matrix into the intramembrane space, can stabilise ΔΨm. By a remnant ΔΨm polarisation, cycling of Na+, Ca2+, K+, Cl− ions and protons across the mitochondrial and the plasma membranes is preserved, and ionic homeostasis can be maintained (Nicholls and Budd, 2000; Dussmann et al, 2003a; Chinopoulos and Adam-Vizi, 2009; Garedew et al, 2010). Our model confirmed that ATP synthase activity was reversed 10 min after onset of cyt-c release, predicted that ATP synthase reversal consumed ATP and that glycolysis was required and sufficient to provide the necessary ATP to sustain this reversal. Reverting back to our single-cell HeLa system, we confirmed the presence of ATP synthase reversal. However, reversal was only present in 20% of cells, 65% of cells showed no detectable reaction and even 15% maintained ATP synthase in forward direction.
To explain this cell-to-cell heterogeneity, we modelled that a cyt-c fraction remains accessible by respiratory complexes and for respiration subsequent to release, which we denoted as ‘respiration-accessible cyt-c'. Our model suggested that small variations in such levels can sufficiently explain the experimentally detected population heterogeneity in the direction and amount of ATP synthase proton flux (Figure 6AB). Variations in respiration-accessible cyt-c may arise from incomplete mitochondrial release. Such incomplete release has been associated with failure of cristae remodelling in the absence of the BH3-only family member BID or the intramitochondrial protein OPA1 (Frezza et al, 2006; Scorrano et al, 2002).
As the model identified glycolysis as necessary for sustaining ATP synthase reversal, we next investigated cells cultured in a medium that contained Na-pyruvate instead of glucose and which consequently were not able to perform glycolysis. We found that such cell populations had a significantly higher fraction of cells that maintained ATP synthase in forward mode consistent with our model predictions. The common influence of respiration-accessible cyt-c and the cell's ability to perform glycolysis is summarised in Figure 7A.
Because glycolysis was able to influence ATP synthase proton pumping, which can affect ΔΨm levels, we investigating the effect of higher glucose in single cells. Our model predicted that an increase in glucose utilisation that generates higher cytosolic ATP levels is able to stabilise and repolarise ΔΨm and after release. This mechanism is independent from ATP synthase direction. For cells that have ATP synthase in reverse mode, elevated ATP leads to increased proton efflux from the matrix, cell populations that maintain ATP synthase in forward mode achieve a similar result through a reduction of proton influx at increased ATP. In both cases, the proton gradient along the inner membrane, and therefore ΔΨm, is increased as a consequence of ATP elevation. The mechanism is depicted in Figure 7B.
We confirmed our model predictions that high glucose was able to stabilise (cells maintained in high-glucose media) and/or to repolarise (cells where glucose was added subsequent to release) ΔΨm (Figure 6). While a similar recovery was also present in MCF7 breast cancer cell lines, no significant effect of elevated glucose was found in non-transformed CRL-1807 cells. In conjunction with an impairment of caspase-dependent cell death observed in many human cancers, cancer cells may use this mechanism, and this mechanism may provide cancer cells with a competitive advantage to evade cell death induced by anticancer drugs or other stress conditions when compared with non-transformed cells.
Many anticancer drugs activate caspases via the mitochondrial apoptosis pathway. Activation of this pathway triggers a concomitant bioenergetic crisis caused by the release of cytochrome-c (cyt-c). Cancer cells are able to evade these processes by altering metabolic and caspase activation pathways. In this study, we provide the first integrated system study of mitochondrial bioenergetics and apoptosis signalling and examine the role of mitochondrial cyt-c release in these events. In accordance with single-cell experiments, our model showed that loss of cyt-c decreased mitochondrial respiration by 95% and depolarised mitochondrial membrane potential ΔΨm from −142 to −88 mV, with active caspase-3 potentiating this decrease. ATP synthase was reversed under such conditions, consuming ATP and stabilising ΔΨm. However, the direction and level of ATP synthase activity showed significant heterogeneity in individual cancer cells, which the model explained by variations in (i) accessible cyt-c after release and (ii) the cell's glycolytic capacity. Our results provide a quantitative and mechanistic explanation for the protective role of enhanced glucose utilisation for cancer cells to avert the otherwise lethal bioenergetic crisis associated with apoptosis initiation.
PMCID: PMC3094064  PMID: 21364572
apoptosis; bioenergetics; cancer; ODE; single-cell imaging
17.  Loss of UCP2 Attenuates Mitochondrial Dysfunction without Altering ROS Production and Uncoupling Activity 
PLoS Genetics  2014;10(6):e1004385.
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.
Author Summary
Mitochondria produce the majority of the energy needed for numerous cell functions through oxidative phosphorylation. However, this comes with the cost in the form of potentially harmful reactive oxygen species (ROS) that could damage all kinds of biological macromolecules. Changes in mitochondrial membrane potential through mild uncoupling could alter ROS production in the cell (“uncoupling to survive”). Mitochondrial uncoupling proteins (UCPs) are believed to play a central role in this process. We detected increased amounts of UCP2 in mtDNA mutator mice, a model for premature aging. Depletion of UCP2 in mtDNA mutator mice led to further shortening of the lifespan with earlier signs of mitochondrial cardiomyopathy accompanied with high systemic lactic acidosis, often used as a marker of mitochondrial diseases. Remarkably, our results demonstrate that the presence of UCP2 wields beneficial effect on respiratory deficient mitochondria without affecting ROS production or uncoupling. Instead, UCP2 protein seems to mediate a valuable upregulation of fatty acid metabolism detected in mtDNA mutator hearts. Our results provide a novel mechanism of adaptation of mitochondria to respiratory deficiency mediated by UCP2 that clearly argues against the “uncoupling to survive” theory.
PMCID: PMC4063685  PMID: 24945157
18.  Prohibitin reduces mitochondrial free radical production and protects brain cells from different injury modalities 
The Journal of Neuroscience  2012;32(2):583-592.
Prohibitin is an essential mitochondrial protein that has been implicated in a wide variety of functions in many cell types, but its role in neurons remains unclear. In a proteomic screen of rat brains in which ischemic tolerance was induced by electrical stimulation of the cerebellar fastigial nucleus, we found that prohibitin is upregulated in mitochondria. This observation prompted us to investigate the role of prohibitin in neuronal death and survival. We found that prohibitin is upregulated also in the ischemic tolerance induced by transient ischemia in vivo, or oxygen-glucose deprivation in neuronal cultures. Cell fractionation and electron microscopic immunolabeling studies demonstrated that prohibitin is localized to neuronal mitochondria. Upregulation of prohibitin in neuronal cultures or hippocampal slices was markedly neuroprotective, whereas prohibitin gene-silencing increased neuronal vulnerability, an effect associated with loss of mitochondrial membrane potential and increased mitochondrial production of reactive oxygen species. Prohibitin upregulation was associated with reduced production of reactive oxygen species in mitochondria exposed to the complex I inhibitor rotenone. In addition, prohibitin protected complex I activity from the inhibitory effects of rotenone. These observations, collectively, establish prohibitin as an endogenous neuroprotective protein involved in ischemic tolerance. Prohibitin exerts beneficial effects on neurons by reducing mitochondrial free radical production. The data with complex I activity suggest that prohibitin may stabilize the function of complex I. The protective effect of prohibitin has potential translational relevance in diseases of the nervous system associated with mitochondrial dysfunction and oxidative stress.
PMCID: PMC3287080  PMID: 22238093
19.  Melatonin combats molecular terrorism at the mitochondrial level 
Interdisciplinary Toxicology  2010;1(2):137-149.
The intracellular environmental is a hostile one. Free radicals and related oxygen and nitrogen-based oxidizing agents persistently pulverize and damage molecules in the vicinity of where they are formed. The mitochondria especially are subjected to frequent and abundant oxidative abuse. The carnage that is left in the wake of these oxygen and nitrogen-related reactants is referred to as oxidative damage or oxidative stress. When mitochondrial electron transport complex inhibitors are used, e.g., rotenone, 1-methyl-1-phenyl-1,2,3,6-tetrahydropyridine, 3-nitropropionic acid or cyanide, pandemonium breaks loose within mitochondria as electron leakage leads to the generation of massive amounts of free radicals and related toxicants. The resulting oxidative stress initiates a series of events that leads to cellular apoptosis. To alleviate mitochondrial destruction and the associated cellular implosion, the cell has at its disposal a variety of free radical scavengers and antioxidants. Among these are melatonin and its metabolites. While melatonin stimulates several antioxidative enzymes it, as well as its metabolites (cyclic 3-hydroxymelatonin, N1-acetyl-N2-formyl-5-methoxykynuramine and N1-acetyl-5-methoxykynuramine), likewise effectively neutralize free radicals. The resulting cascade of reactions greatly magnifies melatonin's efficacy in reducing oxidative stress and apoptosis even in the presence of mitochondrial electron transport inhibitors. The actions of melatonin at the mitochondrial level are a consequence of melatonin and/or any of its metabolites. Thus, the molecular terrorism meted out by reactive oxygen and nitrogen species is held in check by melatonin and its derivatives.
PMCID: PMC2993480  PMID: 21218104
melatonin; mitochondria; free radicals; oxidative stress; mitochondrial complex inhibitors
20.  Reactive oxygen species regulation by AIF- and complex I-depleted brain mitochondria 
Free radical biology & medicine  2009;46(7):939-947.
Apoptosis-inducing factor (AIF)-deficient harlequin (Hq) mice undergo neurodegeneration associated with a 40–50% reduction in complex I level and activity. We tested the hypothesis that AIF and complex I regulate reactive oxygen species (ROS) production by brain mitochondria. Isolated Hq brain mitochondria oxidizing complex I substrates displayed no difference compared to wild type (WT) in basal ROS production, H2O2 removal, or ROS production stimulated by complex I inhibitors rotenone or 1-methyl-4-phenylpyridinium. In contrast, ROS production caused by reverse electron transfer to complex I was attenuated by ~50% in Hq mitochondria oxidizing the complex II substrate succinate. Basal and rotenone-stimulated rates of H2O2 release from in situ mitochondria did not differ between Hq and WT synaptosomes metabolizing glucose, nor did the level of in vivo oxidative protein carbonyl modifications detected in synaptosomes, brain mitochondria, or homogenates. Our results suggest that AIF does not directly modulate ROS release from brain mitochondria. In addition, they demonstrate that in contrast to ROS produced by mitochondria oxidizing succinate, ROS release from in situ synaptosomal mitochondria or from isolated brain mitochondria oxidizing complex I substrates is not proportional to the amount of complex I. These findings raise the important possibility that complex I contributes less to physiological ROS production by brain mitochondria than previously suggested.
PMCID: PMC2775507  PMID: 19280713
apoptosis-inducing factor; electron transport chain; neurodegeneration; oxidative stress; protein carbonyl; synaptosome
21.  The mitochondrial uncoupling proteins 
Genome Biology  2002;3(12):reviews3015.1-reviews3015.9.
The uncoupling proteins are transporters, present in the mitochondrial inner membrane, that mediate a regulated discharge of the proton gradient that is generated by the respiratory chain, to serve functions such as thermogenesis, maintenance of the redox balance, or reduction in the production of reactive oxygen species.
The uncoupling proteins (UCPs) are transporters, present in the mitochondrial inner membrane, that mediate a regulated discharge of the proton gradient that is generated by the respiratory chain. This energy-dissipatory mechanism can serve functions such as thermogenesis, maintenance of the redox balance, or reduction in the production of reactive oxygen species. Some UCP homologs may not act as true uncouplers, however, and their activity has yet to be defined. The UCPs are integral membrane proteins, each with a molecular mass of 31-34 kDa and a tripartite structure in which a region of around 100 residues is repeated three times; each repeat codes for two transmembrane segments and a long hydrophilic loop. The functional carrier unit is a homodimer. So far, 45 genes encoding members of the UCP family have been described, and they can be grouped into six families. Most of the described genes are from mammals, but UCP genes have also been found in fish, birds and plants, and there is also functional evidence to suggest their presence in fungi and protozoa. UCPs are encoded in their mature form by nuclear genes and, unlike many nuclear-encoded mitochondrial proteins, they lack a cleavable mitochondrial import signal. The information for mitochondrial targeting resides in the first loop that protrudes into the mitochondrial matrix; the second matrix loop is essential for insertion of the protein into the inner mitochondrial membrane. UCPs are regulated at both the transcriptional level and by activation and inhibition in the mitochondrion.
PMCID: PMC151194  PMID: 12537581
22.  Bistability of Mitochondrial Respiration Underlies Paradoxical Reactive Oxygen Species Generation Induced by Anoxia 
PLoS Computational Biology  2009;5(12):e1000619.
Increased production of reactive oxygen species (ROS) in mitochondria underlies major systemic diseases, and this clinical problem stimulates a great scientific interest in the mechanism of ROS generation. However, the mechanism of hypoxia-induced change in ROS production is not fully understood. To mathematically analyze this mechanism in details, taking into consideration all the possible redox states formed in the process of electron transport, even for respiratory complex III, a system of hundreds of differential equations must be constructed. Aimed to facilitate such tasks, we developed a new methodology of modeling, which resides in the automated construction of large sets of differential equations. The detailed modeling of electron transport in mitochondria allowed for the identification of two steady state modes of operation (bistability) of respiratory complex III at the same microenvironmental conditions. Various perturbations could induce the transition of respiratory chain from one steady state to another. While normally complex III is in a low ROS producing mode, temporal anoxia could switch it to a high ROS producing state, which persists after the return to normal oxygen supply. This prediction, which we qualitatively validated experimentally, explains the mechanism of anoxia-induced cell damage. Recognition of bistability of complex III operation may enable novel therapeutic strategies for oxidative stress and our method of modeling could be widely used in systems biology studies.
Author Summary
The levels of reactive oxygen species (ROS) that are generated as a side product of mitochondrial respiratory electron transport largely define the extent of oxidative stress in living cells. Free radicals formed in electron transport, such as ubisemiquinone, could pass their non-paired electron directly to oxygen, thus producing superoxide radical that gives rise to a variety of ROS. It is well known in clinical practice that upon recommencing oxygen supply after anoxia a tissue produces much more ROS than before the anoxia, and the state of high ROS production is stable. The mechanism of switching from low to high ROS production by temporal anoxia was unknown, in part because of the lack of detailed mathematical description of hundreds of redox states of respiratory complexes, which are formed in the process of electron transport. A new methodology of automated construction of large systems of differential equations allowed us to describe the system in detail and predicts that the mechanism of paradoxical effect of anoxia-reoxygenation could be defined by the properties of complex III of mitochondrial respiratory chain. Our experiments confirmed that the effect of hypoxia-reoxygenation is confined by intramitochondrial processes since it is observed in isolated mitochondria.
PMCID: PMC2789320  PMID: 20041200
23.  UCP2 overexpression worsens mitochondrial dysfunction and accelerates disease progression in a mouse model of amyotrophic lateral sclerosis 
Mitochondrial dysfunction leading to deficits in energy production, Ca2+ uptake capacity, and free radical generation has been implicated in the pathogenesis of familial amyotrophic lateral sclerosis (ALS) caused by mutations in Cu, Zn superoxide dismutase (SOD1). Numerous studies link UCP2, a member of the uncoupling protein family, to protection of neurons from mitochondrial dysfunction and oxidative damage in various mouse models of acute stress and neurodegeneration, including Parkinson’s disease. Here, we tested the potential neuroprotective effects of UCP2 and its ability to modulate mitochondrial function, in the G93A mutant SOD1 mouse model of familial ALS. Disease phenotype, mitochondrial bioenergetics, and Ca2+ uptake capacity were investigated in the central nervous system of double transgenic mice, expressing both human mutant G93A SOD1 and human UCP2 (hUCP2). Unexpectedly, hUCP2 expression accelerated the disease course of SOD1 mutant mice. In addition, we did not observe a classical uncoupling effect of hUCP2 in G93A brain mitochondria, although we did detect a decrease in reactive oxygen species (ROS) production from mitochondria challenged with the respiratory chain inhibitors rotenone and antimycin A. We also found that mitochondrial Ca2+ uptake capacity was decreased in the double transgenic mice, as compared to G93A mice. Taken together our results indicate that the neuroprotective role of UCP2 in neurodegeneration is disease-specific and that, while a mild uncoupling by UCP2 in brain mitochondria may protect against neurodegeneration in some injury paradigms, the mitochondrial damage and the disease caused by mutant SOD1 cannot be ameliorated by UCP2 overexpression.
PMCID: PMC3891658  PMID: 24141050
ALS; mitochondria; UCP2; SOD1
24.  Brown adipose tissue mitochondria: modulation by GDP and fatty acids depends on the respiratory substrates 
Bioscience Reports  2011;32(Pt 1):53-59.
The UCP1 [first UCP (uncoupling protein)] that is found in the mitochondria of brown adipocytes [BAT (brown adipose tissue)] regulates the heat production, a process linked to non-shivering thermogenesis. The activity of UCP1 is modulated by GDP and fatty acids. In this report, we demonstrate that respiration and heat released by BAT mitochondria vary depending on the respiratory substrate utilized and the coupling state of the mitochondria. It has already been established that, in the presence of pyruvate/malate, BAT mitochondria are coupled by faf-BSA (fatty-acid-free BSA) and GDP, leading to an increase in ATP synthesis and mitochondrial membrane potential along with simultaneous decreases in both the rates of respiration and heat production. Oleate restores the uncoupled state, inhibiting ATP synthesis and increasing the rates of both respiration and heat production. We now show that in the presence of succinate: (i) the rates of uncoupled mitochondria respiration and heat production are five times slower than in the presence of pyruvate/malate; (ii) faf-BSA and GDP accelerate heat and respiration as a result and, in coupled mitochondria, these two rates are accelerated compared with pyruvate/malate; (iii) in spite of the differences in respiration and heat production noted with the two substrates, the membrane potential and the ATP synthesized were the same; and (iv) oleate promoted a decrease in heat production and respiration in coupled mitochondria, an effect different from that observed using pyruvate/malate. These effects are not related to the production of ROS (reactive oxygen species). We suggest that succinate could stimulate a new route to heat production in BAT mitochondria.
PMCID: PMC3198502  PMID: 21561434
brown adipose tissue (BAT); heat release; oxygen consumption; mitochondria; thermogenesis; BAT, brown adipose tissue; faf-BSA, fatty-acid-free BSA; HRP, horseradish peroxidase; NEFA, non-esterified fatty acid; ROS, reactive oxygen species; UCP, uncoupling protein; UCP1, first UCP
25.  Reversible inactivation of dihydrolipoamide dehydrogenase by mitochondrial hydrogen peroxide 
Free radical research  2012;47(2):123-133.
Under oxidative stress conditions, mitochondria are the major site for cellular production of reactive oxygen species (ROS) such as superoxide anion and H2O2 that can attack numerous mitochondrial proteins including dihydrolipoamide dehydrogenase (DLDH). While DLDH is known to be vulnerable to oxidative inactivation, the mechanisms have not been clearly elucidated. The present study was therefore designed to investigate the mechanisms of DLDH oxidative inactivation by mitochondrial reactive oxygen species (ROS). Mitochondria, isolated from rat brain, were incubated with mitochondrial respiratory substrates such as pyruvate/malate or succinate in the presence of electron transport chain inhibitors such as rotenone or antimycin A. This is followed by enzyme activity assay and gel-based proteomic analysis. The present study also examined whether ROS-induced DLDH oxidative inactivation could be reversed by reducing reagents such as DTT, cysteine, and glutathione. Results show that DLDH could only be inactivated by complex III- but not complex I-derived ROS; and the accompanying loss of activity due to the inactivation could be restored by cysteine and glutathione, indicating that DLDH oxidative inactivation by complex III-derived ROS was a reversible process. Further studies using catalase indicate that it was H2O2 instead of superoxide anion that was responsible for DLDH inactivation. Moreover, using sulfenic acid-specific labeling techniques in conjunction with two-dimensional Western blot analysis, we show that protein sulfenic acid formation (also known as sulfenation) was associated with the loss of DLDH enzymatic activity observed under our experimental conditions. Additionally, such oxidative modification was shown to be associated with preventing DLDH from further inactivation by the thiol-reactive reagent N-ethylmaleimide. Taken together, the present study provides insights into the mechanisms of DLDH oxidative inactivation by mitochondrial H2O2.
PMCID: PMC3690130  PMID: 23205777
brain; dihydrolipoamide dehydrogenase; H2O2; mitochondria; reactive oxygen species; reversible inactivation; sulfenic acid; sulfenation

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