sites of superoxide and hydrogen peroxide production by muscle

Sites of Superoxide and Hydrogen Peroxide Production byMuscle Mitochondria Assessed ex Vivo under ConditionsMimicking Rest and Exercise*

Received for publication, October 14, 2014, and in revised form, November 6, 2014 Published, JBC Papers in Press, November 11, 2014, DOI 10.1074/jbc.M114.619072

Renata L. S. Goncalves1, Casey L. Quinlan2, Irina V. Perevoshchikova1, Martin Hey-Mogensen3,and Martin D. Brand1

From the Buck Institute for Research on Aging, Novato, California 94945

Background: Ten mitochondrial sites of superoxide/H2O2 generation are known, but their contributions in vivo areundefined.Results: We assessed their rates ex vivo in conditions mimicking rest and exercise.Conclusion: Sites IQ and IIF generated half the signal at rest. During exercise, rates were lower, and site IF dominated.Significance: Contributing sites ex vivo probably reflect those in vivo.

The sites and rates of mitochondrial production of superoxideand H2O2 in vivo are not yet defined. At least 10 different mito-chondrial sites can generate these species. Each site has a differ-ent maximum capacity (e.g. the outer quinol site in complex III(site IIIQo) has a very high capacity in rat skeletal muscle mito-chondria, whereas the flavin site in complex I (site IF) has a verylow capacity). The maximum capacities can greatly exceed theactual rates observed in the absence of electron transport chaininhibitors, so maximum capacities are a poor guide to actualrates. Here, we use new approaches to measure the rates atwhich different mitochondrial sites produce superoxide/H2O2

using isolated muscle mitochondria incubated in media mim-icking the cytoplasmic substrate and effector mix of skeletalmuscle during rest and exercise. We find that four or five sitesdominate during rest in this ex vivo system. Remarkably, thequinol site in complex I (site IQ) and the flavin site in complex II(site IIF) each account for about a quarter of the total measuredrate of H2O2 production. Site IF, site IIIQo, and perhaps site EF inthe �-oxidation pathway account for most of the remainder.Under conditions mimicking mild and intense aerobic exercise,total production is much less, and the low capacity site IF dom-inates. These results give novel insights into which mitochon-drial sites may produce superoxide/H2O2 in vivo.

Mitochondrial generation of superoxide and hydrogen per-oxide was discovered in the 1960s and 1970s (1, 2) and has beenwell studied (3–15). It is not a single process; the signal obtained

is the sum of rates from different sites that prematurely reduceoxygen to superoxide or H2O2. Each site has its own uniqueproperties. The sites can be distinguished and studied in situ byproviding electrons from appropriate substrates and using spe-cific electron transport inhibitors to isolate them pharmacolog-ically. In this way, at least 10 distinct sites have been character-ized in rat skeletal muscle mitochondria (16). These sites arerepresented as red circles in Fig. 1. In order of their maximumcapacities, they are as follows: IIIQo

4 in complex III (17); IQ (18,19) and IIF (20) in complexes I and II; OF, PF, and BF in the2-oxoglutarate, pyruvate, and branched-chain 2-oxoaciddehydrogenase complexes (16); GQ in mitochondrial glyc-erol phosphate dehydrogenase (21); IF in complex I (16); EFin ETF/ETF:Q oxidoreductase (22); and DQ in dihydrooro-tate dehydrogenase (23).

The rates of superoxide/H2O2 production under native con-ditions (i.e. in the absence of added inhibitors) are much lessthan these maximum capacities (24). Therefore, the maximumcapacities of the sites are not necessarily related to the actualengagement and rate of each site under native conditions. Toestablish the native rate from each site, we devised novel meth-ods based on measurements of the redox states of endogenousreporters in isolated mitochondria oxidizing conventional sub-strates (24, 25). These studies led to two striking conclusions.First, the overall rates of H2O2 production differed 5-foldbetween different conventional substrates; they were muchhigher with succinate than with glutamate plus malate as sub-

* This work was supported, in whole or in part, by National Institutes of HealthGrant TL1 AG032116 (to C. L. Q.). This work was also supported by theBrazilian Government through the Coordenacao de Aperfeicoamento dePessoal de Nível Superior (CAPES) e ao Conselho de Nacional de Desenvol-vimento Científico e Tecnologico programa Ciencias Sem Fronteiras(CNPq-CSF) (to R. L. S. G.), the Glenn Foundation (to R. L. S. G. and I. V. P.),and the Carlsberg Foundation (to M. H.-M.).

1 To whom correspondence should be addressed: Buck Institute for Researchon Aging, 8001 Redwood Blvd., Novato, CA 94945. Tel.: 415-209-2000; Fax:415-209-2235; E-mail: [email protected].

2 Present address: Oncology Research Unit, Pfizer Inc., La Jolla, CA 92121.3 Present address: Obesity Biology, Novo Nordisk A/S, Novo Nordisk Park,

2760 Måløv, Denmark.

4 The abbreviations used are: site IIIQo, outer quinol-oxidizing site of respira-tory complex III; site IF, flavin in the NADH-oxidizing site of respiratorycomplex I; site IQ, ubiquinone-reducing site of respiratory complex I; site IIF,flavin site of respiratory complex II; site OF, flavin in the 2-oxoglutaratedehydrogenase complex; site PF, flavin in the pyruvate dehydrogenasecomplex; site BF, flavin in the branched-chain 2-oxoacid (or �-ketoacid)dehydrogenase complex; site GQ, quinone reducing site in mitochondrialglycerol 3-phosphate dehydrogenase; ETF, electron-transferring flavopro-tein; ETF:QOR, ETF:ubiquinone oxidoreductase; site EF, site in ETF:QOR,probably the flavin of ETF; site DQ, quinone reducing site in dihydroorotatedehydrogenase; CDNB, 1-chloro-2,4-dinitrobenzene; VO2max, whole bodymaximal O2 consumption rate; CN-POBS, N-cyclohexyl-4-(4-nitrophenoxy)benzenesulfonamide; Q, ubiquinone; QH2, ubiquinol; Eh, operating redoxpotential; ANOVA, analysis of variance.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 1, pp. 209 –227, January 2, 2015© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

JANUARY 2, 2015 • VOLUME 290 • NUMBER 1 JOURNAL OF BIOLOGICAL CHEMISTRY 209

by guest on January 28, 2018http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

strate. Second, the relative contribution of each site was com-pletely dependent on the substrate being oxidized. With succi-nate as substrate, site IQ was dominant; with glutamate plusmalate, sites IF, IIIQo, and OF all contributed; with palmitoyl-carnitine, site IIF was also important; and with glycerol 3-phos-phate, five sites contributed significantly, including GQ (25).Thus, the relative and absolute contribution of each specific siteto the production of superoxide/H2O2 in isolated mitochondriadepends very strongly on which conventional substrate is beingoxidized.

In complex systems, such as intact cells, different substratesare metabolized simultaneously. The main groups of substratesoxidized by mitochondria are represented in Fig. 1 and Table 1by colored boxes. In skeletal muscle, oxidation of ketone bodies,amino acids, tricarboxylic acid cycle intermediates, glycerol3-phosphate, and fatty acids feeds electrons into multiple sitesin the matrix dehydrogenases and electron transport chain.Therefore, in vivo, it is likely that several sites produce super-oxide/H2O2 simultaneously at different rates.

The conditions experienced by muscle mitochondria withincells differ substantially between rest and exercise. In particular,ADP supply, free Ca2�, cytosolic pH, and the availability of differ-ent substrates are very different, and this will have profound effectson the mitochondrial production of superoxide and H2O2. Duringexercise, the levels of both reactive oxygen species and reactivenitrogen species are increased (26). The production of such speciesis crucial for the beneficial effects of exercise (27–29) and for forcedevelopment (30). However, excess production is detrimental forskeletal muscle performance (30, 31). Mitochondria are leadingcandidates for the increased production of these reactive speciesduring exercise (30, 32), although others argue for non-mitochon-drial sources (26, 32).

Several different probes can be used in intact cells to reportchanges in reactive oxygen species (33, 34), but they cannot reli-ably distinguish the mitochondrial sites active under particularphysiological or pathological conditions. This is because pharma-cological or genetic manipulation of particular mitochondrial sitesinvariably alters the redox states of other sites, changing their con-tributions to overall production of superoxide/H2O2 and makinginterpretation unreliable. Also, these probes invariably competewith the endogenous antioxidant defenses and measure steadystate levels rather than rates of radical production. On the otherhand, the rate from each site can now be quantified using isolatedmitochondria (24, 25). However, given the strong substratedependence of their superoxide/H2O2 production, isolatedmitochondria oxidizing conventional substrates are not suf-ficiently physiologically relevant.

To improve the physiological relevance of the more amena-ble mitochondrial system, in the present work, we develop an exvivo system in which isolated muscle mitochondria are incu-bated acutely in novel complex media. These media contain themeasured physiological concentrations of all metabolites andeffectors assumed to be relevant in skeletal muscle cytosol atrest and during mild and intense aerobic exercise. Using thissystem, we quantify the contribution of each mitochondrial siteto total H2O2 production to gain novel insights into the ratesand sites of mitochondrial superoxide/H2O2 production inskeletal muscle during rest and exercise.

EXPERIMENTAL PROCEDURES

Animals, Mitochondria, and Reagents—Female Wistar ratswere from Charles River Laboratories, age 5–10 weeks, and fedchow ad libitum with free access to water. Mitochondria wereisolated from hind limb skeletal muscle at 4 °C in Chappell-Perry buffer (CP1; 100 mM KCl, 50 mM Tris, 2 mM EGTA, pH7.4, at 4 °C) by standard procedures (35) and kept on ice duringthe assays (�5 h). Protein was measured by the biuret method.The animal protocol was approved by the Buck Institute Ani-mal Care and Use Committee in accordance with IACUCstandards. Reagents were from Sigma except for AmplexUltraRed, from Invitrogen.

Oxygen Consumption and Superoxide/H2O2 Production—Skeletal muscle mitochondria (0.3 mg of protein�ml�1) wereincubated at 37 °C for 4 –5 min in the appropriate “basicmedium” (Table 1) mimicking the cytosol of skeletal muscleduring rest (basic “rest” medium plus oligomycin), mild aerobicexercise (basic “mild aerobic exercise” medium with no furtheradditions), or intense aerobic exercise (basic “intense aerobicexercise” medium plus glucose and sufficient hexokinase aftertitration (about 0.08 units�ml�1) to give 90% of the maximumstate 3 oxygen consumption rate). ATP (6 mM) was injected intothe chamber, and then after 1 min, the appropriate “complexsubstrate mix” was added (Table 1). Oxygen consumption rateswere measured using a Clark-type electrode fitted in a water-jacketed chamber (Rank Brothers, Bottisham, UK). Rates ofsuperoxide/H2O2 production were measured collectively asrates of H2O2 production as two superoxide molecules are dis-mutated by endogenous or exogenous superoxide dismutase toyield one H2O2. H2O2 was detected using 5 units�ml�1 horse-radish peroxidase and 50 �M Amplex UltraRed in the presenceof 25 units�ml�1 superoxide dismutase (36) in a Varian CaryEclipse spectrofluorometer (�excitation � 560 nm, �emission �590 nm) with constant stirring and calibrated with knownamounts of H2O2 in the presence of all relevant additionsbecause some of them quenched fluorescence (36).

NAD(P)H and Cytochrome b566 Redox State—The reductionstate of endogenous NAD(P)H was determined by autofluores-cence in mitochondria incubated as described for H2O2 pro-duction (most of the signal is from bound NADH in the matrix,and NADPH hardly changes in the present experiments, but fortransparency we call it “NAD(P)H”) using a ShimadzuRF5301-PC dual wavelength spectrophotometer at �excitation �365 nm, �emission � 450 nm (18, 24). NAD(P)H was assumed tobe 0% reduced after 5 min without added substrate. 100%reduction was established internally by adding saturating con-ventional substrate (e.g. 5 mM malate plus 5 mM glutamate) and4 �M rotenone at the end of each run. Intermediate values ofNAD(P)H reduction were measured at 3– 4 min after the addi-tion of the appropriate mix of substrates and were determinedas percentage of reduced NAD(P)H relative to the 0 and 100%values. The reduction state of endogenous cytochrome b566 wasmeasured using 1.5 mg of mitochondrial protein�ml�1 withconstant stirring at 37 °C in an Olis DW 2 dual wavelengthspectrophotometer at A566 nm–A575 nm (17). This signal reports�75% cytochrome b566 and �25% cytochrome b562 (17, 37).Cytochrome b566 was assumed to be 0% reduced after 5 min

Mitochondrial Sites of Superoxide/H2O2 Production ex Vivo

210 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 1 • JANUARY 2, 2015


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

without added substrate. 100% reduction was established inseparate cuvettes with 5 mM succinate and 2 �M antimycin A.Intermediate values of cytochrome b566 reduction were mea-sured over 45 s, �20 s after the addition of the appropriatecomplex substrate mix, and were determined as percentage ofreduced cytochrome b566 relative to the 0 and 100% values.

Correction for Matrix Peroxidase Activity—H2O2 productionrates in Figs. 3 and 12 (but not in other figures) were correctedfor losses of H2O2 caused by peroxidase activity in the matrix togive a better estimate of actual superoxide/H2O2 productionrates (38). Rates were mathematically corrected to those thatwould have been observed after pretreatment with 1-chloro-2,4-dinitrobenzene (CDNB) to deplete glutathione and decreaseglutathione peroxidase and peroxiredoxin activity, using anempirical equation,

�CDNB � �control � �100 � �control�/�72.6 � �control� (Eq. 1)

where � is the rate of H2O2 production in pmol ofH2O2�min�1�mg protein�1.

Due to the non-linearity of the curve, the correction is differ-ent at different overall rates. Therefore, the total H2O2 produc-tion rates were first corrected using Equation 1. The correctedrate from each site (and its S.E.) was then back-calculated basedon its relative contribution in a given condition (Table 3). Allsites produce superoxide/H2O2 exclusively into the mitochon-drial matrix, except for IIIQo and GQ, which generate �50% ofthe superoxide/H2O2 to the cytosol (24). For these sites, only50% of the rate was corrected.

Curve Fitting—Data were fit by exponential functions in Figs.5C, 6C, and 10 (C and F) to yield the parameter values in Equa-tions 2–5, respectively,

�H2O2 (%NAD(P)H) � 21.22 � e0.038 %NAD�P�H (Eq. 2)

�H2O2 �%b566� � �40.64 � 40.64 e0.028 � %b566 (Eq. 3)

�H2O2 (%NAD(P)H) � 15.78 � 0.017 e0.104 NAD�P�H (Eq. 4)

�H2O2 �%b566� � 4.09 e0.054 %b566 4.09 (Eq. 5)

where �H2O2 is the rate of H2O2 production.Statistics—The calibration curves in Figs. 5C, 6C, and 10 (C

and F) were used to calculate the rates of superoxide/H2O2production at sites IF and IIIQo, both directly when assessing therates from these sites and indirectly when correcting for theeffects on these sites of inhibition at other sites (Table 3).The errors in the calibration curves were taken into accountwhen calculating the S.E. values of the assessed rates. This errorwas calculated by error propagation as described previously(24).

Because error propagation was used to include the uncer-tainty from the calibration curve in the assessed rates, themean S.E. values plotted do not represent individual values.Therefore, the statistics were calculated using the averaged val-ues S.E. and the number of observations. The significances ofdifferences between the total measured rates and the assessedrates were calculated using Welch’s t test. For the regular mul-tiple comparison tests in Figs. 2 and 4, a one-way ANOVA wasused, followed by Tukey’s post hoc test. When comparing the

values with omission of single substrates with the total values,Student’s t test was used. p � 0.05 was considered significant.

RESULTS

Media Mimicking Skeletal Muscle Cytosol during Rest, MildAerobic Exercise, and Intense Aerobic Exercise ex Vivo—In vivo,the substrates for mitochondrial metabolism come primarilyfrom the catabolism of sugars, proteins, and fats. In the cytosol,they are available to the mitochondria as metabolites that canbe categorized into five groups according to their origins:ketone bodies, amino acids, tricarboxylic acid cycle intermedi-ates, glycerol 3-phosphate, and acylcarnitines (Table 1). Themajor metabolic fates of these substrates are indicated by thecolored boxes leading to the mitochondrial dehydrogenases inFig. 1. These metabolites connect to the 10 sites in skeletalmuscle mitochondria known to have significant capacity toproduce superoxide or H2O2. In Fig. 1, these sites are groupedin planes reflecting their operating redox potentials. The topplane represents the NADH/NAD� isopotential group, con-taining the dehydrogenases that reduce NAD� and the sites incomplex I that oxidize NADH. From complex I, the electronsdrop down in energy to a more positive redox potential in theubiquinone pool. The bottom plane represents the QH2/Q iso-potential group, containing the ubiquinone oxidoreductasesthat reduce ubiquinone and the sites in complex III that oxidizeubiquinol. The electrons are then transferred to cytochrome cand on to the final acceptor, O2, to generate H2O (not shown).

Table 1 lists the consensus concentrations in rat skeletalmuscle cytosol of all metabolites and effectors thought to bepotentially relevant to mitochondrial electron transport andproduction of superoxide/H2O2. The values were taken fromthe extensive literature on rat skeletal muscle obtained mostlyby freeze-clamp followed by enzymatic or chromatographicquantitation, both in vivo and in isolated muscle preparations.Where appropriate, they were corrected using consensus val-ues for extracellular contamination and for the estimated com-partmentation between the mitochondrial matrix and the cyto-sol. Values are listed for resting muscle and for two exerciseconditions: submaximal stimulation, representing mild aerobicexercise, and extensive stimulation, representing intense aero-bic exercise. We assumed that the exact K� and Cl� concen-trations were unimportant and therefore used KCl to bring theosmolarity to the physiological value of 290 mosM.

These literature values enabled us to prepare three differentmedia for mitochondrial incubations ex vivo, containing therelevant metabolites and effectors at the concentrations thatwould be encountered by mitochondria in vivo during rest andmild and intense aerobic exercise. ATP turnover and hencesteady-state ADP concentrations were set based on respirationrates as described below. These media are the first that we knowof to be carefully designed to mimic the substrate and effectormix in the cytosol of skeletal muscle cells during “rest,” “mildaerobic exercise,” and “intense aerobic exercise” (Table 1). Theyenabled us to assess the production of H2O2 from isolated mito-chondria in an ex vivo system that closely mimicked the rele-vant aspects of the cytosol of rat muscle cells in vivo.




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

TABLE 1Concentrations of substrates and effectors during rest, mild aerobic exercise, and intense aerobic exercise in rat skeletal muscle cytosol andcompositions of the three media mimicking these conditions ex vivoData are for all metabolites assumed to be relevant, from the literature for rat skeletal muscle as indicated (values for carnitine and acetylcarnitine include some human data;values for free Ca2� include some mouse data). Where appropriate, calculations used Equation 3 of Ref. 60 and assumed that muscle wet weight is 77% water (60 – 64) and81% intracellular (60 – 62, 64 – 66) or that 18% of muscle wet weight is protein (66, 67). Published values were corrected for plasma concentrations where appropriate andfor the distribution of intracellular metabolites between cytosol and mitochondria, using the following matrix/cytosol ratios from heart and liver (or the assumed values formetabolites in parentheses): glycerol 3-phosphate, glutamine (dihydroxyacetone phosphate, taurine): 0 (68, 69); citrate, isocitrate: 10 (69 –71); malate (succinate): 4 (69, 70,72, 73); 2-oxoglutarate: 7 (69 –73); pyruvate (acetoacetate, 3-hydroxybutyrate, arginine, lysine): 2 (70, 73); aspartate (neutral amino acids, acylcarnitines): 1 (69, 70, 72, 74);glutamate: 3.5 (69, 70, 72, 74); carnitine: 0.74 (75); ATP: 0.25 (69); phosphate: 5 (76). Mitochondrial volume was assumed to be 10% of intracellular (77– 82). Values wererounded to convenient whole numbers. Total Mg2� and Ca2� concentrations to give the targeted free values were calculated using the software MaxChelator. Targeted freeCa2� concentrations were 0.05 �M (rest), 1 �M (mild aerobic exercise), and 1 �M (intense aerobic exercise) (83– 86). Targeted free Mg2� concentrations were 600 �M (rest),600 �M (mild aerobic exercise), and 1000 �M (intense aerobic exercise) (40, 87– 89). Targeted Na� concentrations were 16 mM for all media (65, 89). Media had K� and Cl�adjusted to give an osmolarity of 290 mosM (90 –92). Total K� concentrations were 80 mM (rest), 77 mM (mild aerobic exercise), and 74 mM (intense aerobic exercise). The“basic media” had the compositions shown; the “complex substrate mixes” contained all of the substrates in the colored boxes. *, no data found, so values are assumed; **,maximum value to avoid mitochondrial uncoupling in vitro.




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Setting ATP Demand during “Rest”, “Mild Aerobic Exercise,”and “Intense Aerobic Exercise”—To deplete endogenous sub-strates, rat skeletal muscle mitochondria were incubated for4 –5 min in the appropriate basic medium lacking all respira-tory substrates (Table 1). ATP was added 1 min before respira-tion was initiated, to minimize the time available for ATPhydrolysis. To initiate respiration, the appropriate complex mixof substrates mimicking rest or mild or intense aerobic exercise(Table 1) was then added (Fig. 2A).

“Rest”—In resting muscle in vivo, the rates of respiration andATP synthesis are low (39). To mimic rest ex vivo, a low rate ofATP synthesis by mitochondria incubated in the “rest” medium(Table 1) was achieved by including oligomycin in the mediumto fully inhibit the mitochondrial ATP synthase. This was not ideal,because it implies no ATP turnover at rest, but it was necessarybecause the relatively high contaminating ATPase activity presentin the mitochondrial preparation caused an intermediate respira-tion rate. The rate of oxygen consumption at “rest” was less than10% of the maximum rate (Fig. 2, A and B, red).

“Mild Aerobic Exercise”—During exercise in vivo, respiratoryrates increase due to increased ATP demand and altered sub-strate supply and concentrations of effectors (pH, Ca2�). Mildexercise induces 45% of whole body maximal O2 consump-tion rate, VO2max (39). To mimic mild aerobic exercise ex vivo,

mitochondria were incubated in the basic “mild aerobic exer-cise” medium in the absence of oligomycin, followed by ATPand then the appropriate complex mix of relevant substrates(Table 1). The initial fast rate of respiration required to rephos-phorylate ADP formed from the added ATP (Fig. 2A) wasignored. After this fast phase was complete and ATP/ADP settledto a steady state value, ATP demand and respiration rate ran at anintermediate rate limited by the supply of ADP from contaminat-ing extramitochondrial ATPases. In this phase, the respiratory ratewas 22% of the maximum rate (Fig. 2, A and B, green).

“Intense Aerobic Exercise”—Metabolic demand increaseswith exercise intensity. During intense exercise, respiration is�65% of VO2max (39). ADP level increases significantly but isstill an order of magnitude lower than the ATP level (40). Cyto-solic pH drops to 6.7 due to increased CO2 and lactate produc-tion. To mimic intense aerobic exercise ex vivo, mitochondriawere incubated in the basic “intense aerobic exercise” medium,followed by ATP and then the appropriate complex mix of rel-evant substrates. To achieve high respiratory rates but keepADP levels relatively low, in separate experiments, the O2 con-sumption rate was titrated to 90% of the maximum rate usinghexokinase in the presence of glucose to set the rate of extram-itochondrial ATP hydrolysis high but still submaximal in eachmitochondrial preparation, and this amount of hexokinase

FIGURE 1. Sites of superoxide and H2O2 production during electron flow from different blocks of metabolites through the mitochondrial electrontransport chain. Metabolic substrates are grouped into five blocks colored to match Table 1: ketone bodies, amino acids, tricarboxylic acid (TCA) cycle, glycerol3-phosphate (GP) shuttle, and �-oxidation. Unfilled boxes represent intermediate metabolites not added in the media. Electrons from the oxidation of thesereduced substrates enter the mitochondrial electron transport chain through different isopotential groups of redox centers, denoted by the two planes, eachoperating at about the same redox potential (Eh): NADH/NAD� at Eh ��280 mV and QH2/Q at Eh ��20 mV (59). The flow of electrons through NADH to theQ pool at complex I and from the Q pool to cytochrome c (cyt c) at complex III is indicated by the large green arrows dropping down through the isopotentialplanes. Enzymes that feed electrons into each isopotential group are represented as ovals, electron transport inhibitors are drawn with red blunted arrows, andCN-POBS, a suppressor of electron leak at site IQ that does not inhibit electron transport, is drawn with a green blunted arrow. Electrons from NAD-linkedsubstrates enter the NADH/NAD� pool through appropriate NAD-linked dehydrogenases (DH), including those for branched-chain 2-oxoacids (BCOADH),pyruvate (PDH), 2-oxoglutarate (OGDH), and others that are grouped together because there is no evidence that they produce superoxide/H2O2. Electrons fromNADH flow into complex I (site IF) and then drop down via site IQ to QH2/Q in the next isopotential pool, providing the energy to generate protonmotive force(pmf). Q oxidoreductases, including complex II, mitochondrial glycerol 3-phosphate dehydrogenase (mGPDH), ETF:QOR, and dihydroorotate dehydrogenase(DHODH), can also pass electrons into the Q pool. Electrons flow from QH2 through complex III to cytochrome c and finally to oxygen (not shown), againpumping protons and generating pmf. The redox state of NADH (outlined in blue) reports the redox state of the first isopotential group. The redox state ofcytochrome b566 (outlined in blue) reports the redox state of the second isopotential group (24). Red circles indicate sites of superoxide/H2O2 production: theflavin/lipoate of the dehydrogenases for branched-chain 2-oxoacids (BF), pyruvate (PF), and 2-oxoglutarate (OF), the complex I flavin (IF) and Q-binding site (IQ),the flavin site of complex II (IIF), the quinone site of mitochondrial glycerol 3-phosphate dehydrogenase (GQ), the flavin site of ETF:QOR (EF), the quinone site ofdihydroorotate dehydrogenase (DQ), and the outer quinol-binding site of complex III (IIIQo).




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

(about 0.08 units�ml�1) was included in the “intense aerobicexercise” medium (Fig. 2, A and B, blue). Maximum phosphor-ylating respiration was set by adding excess hexokinase (Fig. 2B,white).

Mitochondrial H2O2 Production during “Rest,” “Mild AerobicExercise,” and “Intense Aerobic Exercise”—The rate of genera-tion of superoxide and H2O2 was measured under the threeconditions described above as the rate of extramitochondrialH2O2 production in the presence of exogenous superoxide dis-mutase to convert any superoxide to H2O2 for assay. Fig. 2Cshows that rates were linear for 2–3 min after the addition ofthe complex substrate mixes and then decreased. Some metab-olites were present at very low concentrations (Table 1) andwere likely to be consumed quickly, so ideally we would mea-sure initial rates, but because the system was not at steady-stateover the first minute in the “exercise” medium (Fig. 2A), wecalculated the rates as the pseudolinear rate between 1 and 3min (dotted boxes in Fig. 2, A and C). Fig. 2D shows that thehighest rate of H2O2 production was observed when mitochon-dria were incubated in the medium mimicking the cytosol ofskeletal muscle at rest (336 pmol of H2O2�min�1�mg of pro-tein�1). In the media mimicking mild aerobic exercise andintense aerobic exercise, the rates were significantly lower.

Unphysiological concentrations of conventional substratesand the presence of appropriate electron transport inhibitorsfavor high rates of mitochondrial superoxide/H2O2 produc-

tion. These rates can be up to 2% of the total respiration rate ofuninhibited resting mitochondria (2, 41). Under more realisticnon-inhibited (native) conditions with conventional substrates,the percentage of electron leak to H2O2 is only �0.15% (42, 43).Table 2 shows the % electron leak when mitochondria were incu-bated under conditions mimicking rest, mild aerobic exercise, andintense aerobic exercise. At “rest,” the electron leak was 0.35%.During “exercise,” it was 10–35-fold lower: only 0.03% for “mildaerobic exercise” and 0.01% for “intense aerobic exercise.”

The ex vivo data in Fig. 2D and Table 2 suggest that skeletalmuscle mitochondria in vivo are unlikely to contribute to theoverall increase in reactive oxygen species production observedduring exercise, supporting the conclusions of others (26, 32).Instead, the mitochondrial production rate may decrease dur-ing exercise, because the redox centers that donate electrons toO2 become more oxidized during exercise (see below).

Sites of Superoxide/H2O2 Production—Ten mitochondrialsites associated with the tricarboxylic acid cycle and electrontransport chain are known to produce superoxide or H2O2 (Fig.1). Their maximum capacities are shown in Fig. 3, on the samescale as the ex vivo rates from Fig. 2D after all of the values havebeen corrected for consumption of intramitochondrial H2O2by matrix peroxidases. Clearly, the maximum capacities of sev-eral sites are much higher than the ex vivo rates, making itimpossible to predict a priori which sites contribute most to thetotal signal ex vivo or in vivo. The rates of H2O2 production

FIGURE 2. Oxygen consumption and H2O2 generation by isolated skeletal muscle mitochondria incubated in media mimicking the cytosol of skeletalmuscle during rest, mild aerobic exercise, or intense aerobic exercise. A, representative traces of oxygen consumption. The dashed box indicates theinterval over which the rates were analyzed. Numbers by the traces indicate mean rates in nmol of O�min�1�mg of protein�1. B, rates of oxygen consumption.The maximum (state 3) rate “Max” was measured in the presence of 20 mM glucose and excess (2.5 units�ml�1) hexokinase. C, representative traces of H2O2generation. The dashed box indicates the interval over which the rates were analyzed. Numbers by the traces indicate mean rates in pmol of H2O2�min�1�mg ofprotein�1. D, rates of H2O2 generation. In each panel, mitochondria were incubated in media mimicking rest, mild aerobic exercise, or intense aerobic exercise (Table1), as indicated. Values are means S.E. (error bars) (n � 3–20 biological replicates). *, p � 0.05; ***, p � 0.0001, one-way ANOVA with Tukey’s post hoc test.




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

measured under the different conditions in Fig. 2D are the sumsof the rates from different sites. Our goal here was to determinethe contribution of each site during “rest,” “mild aerobic exer-cise,” and “intense aerobic exercise” in the tractable ex vivo sys-tem because no adequate methods exist to address this questionin vivo or in intact cells. We assume that the sites contributingex vivo in media containing the physiological cytosolic concen-trations of all substrates and effectors thought to be relevantwill approximate the sites contributing in vivo.

Conventionally, complexes I and III are thought to be thedominant sources of mitochondrial superoxide/H2O2 in iso-lated mitochondria and cells (10, 13). Complex I producessuperoxide at two distinct sites: site IQ, a high capacity site,most obviously active during reverse transfer of electrons fromubiquinol to NADH (18, 19, 44, 45); and site IF (18), now knownto have much lower capacity (16). Complex III can producesuperoxide at high rates from site IIIQo (2, 17). The rate ofsuperoxide production from site IF depends on the redox stateof the complex I flavin, which in turn has a unique relationshipto the redox state of mitochondrial NAD(P)H. Similarly, therate of superoxide production from site IIIQo is related to theredox state of cytochrome b566 (17). The redox states ofNAD(P)H and cytochrome b566 were therefore used as endog-enous reporters of the rates of superoxide production at sites IFand IIIQo, respectively (24, 25).

Fig. 4 and Table 3 show the reduction levels of NAD(P)H andcytochrome b566 when mitochondria were incubated in the threemedia. During “rest,” NAD(P)H was nearly 100% reduced, and

cytochrome b566 was 30% reduced. During “exercise,” both weresignificantly more oxidized. To assess the rate of superoxide pro-duction by sites IF and IIIQo, calibration curves were built to estab-lish the relationships between superoxide production from thesesites and the reduction levels of the endogenous reporters.

Flavin of Complex I, Site IF, during “Rest”—NAD(P)H redoxstate was calibrated as a reporter of the rate of superoxide pro-duction at site IF, as described previously (24). Fig. 5A shows theobserved rate of H2O2 production from site IF as a function ofthe concentration of malate in the presence of rotenone (with

FIGURE 3. Maximum capacities for superoxide/H2O2 production of the 10 characterized sites in the mitochondrial electron transport chain and matrixcompared with the native ex vivo rates using isolated mitochondria incubated in the absence of inhibitors in media mimicking the cytosol of skeletalmuscle during rest, mild aerobic exercise, or intense aerobic exercise. The sites in the NADH/NAD� isopotential group (Fig. 1) are OF (flavin site of the2-oxoglutarate dehydrogenase complex), PF (flavin site of the pyruvate dehydrogenase complex); BF (flavin site of the branched-chain 2-oxoacid dehydro-genase complex); and IF (flavin site of complex I). Site IQ of complex I is between the two isopotential groups. The sites in the QH2/Q isopotential groups are IIIQo(outer ubiquinone binding site of complex III); IIF (flavin site of complex II); GQ (quinone site of site mitochondrial glycerol 3-phosphate dehydrogenase); EF(flavin site of the electron-transferring flavoprotein/ETF:ubiquinone oxidoreductase system), and DQ (quinone site of dihydroorotate dehydrogenase). The firstnine bars are replotted from Ref. 16, and the DQ bar is replotted from Ref. 23. Ex vivo native rates are from Fig. 2D. All rates were corrected for matrix peroxidasesusing Equation 1. Values are means S.E. (error bars) (n � 3–20).

FIGURE 4. Reduction state of NAD(P)H and cytochrome b566. Mitochondriawere incubated in media mimicking rest, mild aerobic exercise, or intenseaerobic exercise (Table 1), as indicated. Values are means S.E. (error bars)(n � 3–20). *, p � 0.05; **, p � 0.01; ***, p � 0.0001, one-way ANOVA withTukey’s post hoc test.

TABLE 2Electron leak during “rest,” “mild aerobic exercise,” and “intense aerobic exercise”Rates were calculated per nmol e� by multiplying the rates of H2O2 production in Fig. 2D in pmol of H2O2�min�1�mg of protein�1 by 0.002 and rates of oxygen consumptionin Fig. 2B in nmol of O�min�1�mg of protein�1 by 2. Values are means S.E. (n � 3 for oxygen consumption rates; n � 3–20 for H2O2 production rates).

“Rest” “Mild aerobic exercise” “Intense aerobic exercise”

Rate of H2O2 production (nmol e��min�1�mg of protein�1) 0.68 0.04 0.16 0.01 0.10 0.01Rate of oxygen consumption (nmol e��min�1�mg of protein�1) 196 40 492 88 2032 73Percentage electron leak (100�H2O2 production rate/respiration rate) 0.35% 0.03% 0.01%




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

added ATP and aspartate to minimize the contribution of siteOF (16)). Fig. 5B shows the NAD(P)H reduction state under thesame conditions. Fig. 5C replots the data from Fig. 5, A and B, toshow the dependence of the measured rate of H2O2 productionarising from superoxide production at site IF on the NAD(P)Hreduction state. As shown in Fig. 4, NAD(P)H during “rest” wasalmost 100% reduced. From Fig. 5C, the contribution of site IFduring “rest” was assessed to be 65 12 pmol ofH2O2�min�1�mg of protein�1 (mean S.E.; n � 8). The contri-

butions of site IF and other relevant values developed below aresummarized in Table 3.

Site IF accounted for 20 3% of the total rate of H2O2 pro-duction measured during “rest” (Fig. 5D). Therefore, other sitesalso produced superoxide/H2O2 at rest.

Outer Ubiquinone Binding Site of Complex III, Site IIIQo, dur-ing “Rest”—Cytochrome b566 redox state was calibrated as areporter of the rate of superoxide production at site IIIQo asdescribed previously (24). Fig. 6A shows the observed myx-

FIGURE 5. Contribution of site IF at “rest.” Mitochondria were incubated in medium mimicking rest (Table 1). A, rate of superoxide production from site IF,defined by the presence of 4 �M rotenone, 1.5 mM aspartate, and 2.5 mM ATP, at different malate concentrations. B, dependence of NAD(P)H reduction level onmalate concentration under the same conditions (100% reduction was established by adding 5 mM malate plus 5 mM glutamate). C, calibration curve obtainedby combining the y axes from A and B, showing the dependence of the rate of superoxide production from site IF on the NAD(P)H reduction level. D, total rateof H2O2 production at “rest” (from Fig. 2D) and the contribution of site IF assessed from the reduction state of NAD(P)H in Fig. 4 and the calibration curve in C(green; the inverted error bar indicates the propagated error for site IF). Values are means S.E. (error bars) (n � 3–20). p � 0.0001 by Welch’s t test.

TABLE 3Corrections applied for changes in redox state of NAD(P)H and cytochrome b566, and rate of superoxide/H2O2 production from each site during“rest,” “mild aerobic exercise,” and “intense aerobic exercise”� control values in columns B and C were used to correct the observed changes in H2O2 production rate in column A for changes in the redox states of NAD(P)H andcytochrome b566, as shown in the last column.

a 2.5 �M CN-POBS decreased IQ superoxide production by only 65%; therefore, values were scaled to account for CN-POBS potency.b 10 �M CN-POBS reduced IQ superoxide production by only 75%; therefore, values were corrected for CN-POBS potency.c The relative contribution was calculated based on the CDNB corrected rates for each site. The S.E. was scaled according to the internal error for each site. DHAP, dihy-

droxyacetone phosphate; nd, not determined.




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

othiazol-sensitive rate of H2O2 production from site IIIQo as afunction of the ratio of added succinate and malonate in thepresence of rotenone. At each point, the rate of H2O2 produc-tion from site IF was calculated from the observed NAD(P)Hreduction level using Fig. 5C and subtracted from the totalobserved rate to give the rate specifically from site IIIQo (Table3). Fig. 6B shows the cytochrome b566 redox state under thesame conditions. Fig. 6C replots the data from Fig. 6, A and B, toshow the dependence of the rate of H2O2 production arisingfrom superoxide production at site IIIQo on the cytochromeb566 reduction state. Fig. 4 and Table 3 show that cytochromeb566 was 30% reduced during “rest.” Using the calibration curvein Fig. 6C, the contribution of site IIIQo was 55 7 pmol ofH2O2�min�1�mg of protein�1 (mean S.E.; n � 12) (Fig. 6D).

Site IIIQo accounted for 15 2% of the total rate of H2O2production measured during “rest” (Fig. 6D and Table 3).After accounting for the contributions of sites IF and IIIQo tothe total rate of H2O2 production, there was still a significantdifference between the total rates measured and assessed

(Fig. 6D). Therefore, other sites also produced superoxide/H2O2 at rest.

Ubiquinone Binding Site of Complex I, Site IQ, during “Rest”—Site IQ has a high capacity for superoxide production duringreverse electron transport when succinate is oxidized (19, 43)(Fig. 3) and during forward electron flow from NAD-linkedsubstrates under particular conditions (44). However, theimportance of this site in vivo and under semiphysiological con-ditions (25, 46, 47) is unknown. The contribution of site IQduring “rest” ex vivo was assessed by adding rotenone to inhibitelectron transport at this site, followed by correction for conse-quent changes in the rate of superoxide production from sites IFand IIIQo. Rotenone was added 1 min prior to the substrate mixand after correction significantly decreased the rate of superox-ide/H2O2 production at “rest” (Fig. 7A). The rate attributed tosite IQ was 79 23 pmol of H2O2�min�1�mg of protein�1

(mean S.E.; n � 6).CN-POBS at 2.5 �M selectively blocks 65% of superoxide

production from site IQ with no effects on oxidative phosphor-

FIGURE 6. Contribution of site IIIQo at “rest.” Mitochondria were incubated in medium mimicking rest (Table 1). A, rate of superoxide production from siteIIIQo, defined as the rate in the presence of 4 �M rotenone sensitive to 4 �M myxothiazol, at different ratios of succinate/malonate (total concentration 5 mM).Data were corrected for changes in the contribution of site IF using changes in the NAD(P)H redox state and the calibration curve in Fig. 5C (Table 3) (24). B,dependence of cytochrome b566 reduction state on the succinate/malonate ratio under the same conditions (100% reduction was established by adding 5 mM

succinate plus 2 �M antimycin A). C, calibration curve obtained by combining the y axes from A and B showing the dependence of the rate of superoxideproduced from site IIIQo on the cytochrome b566 reduction level. D, total rate of H2O2 production at “rest” (from Fig. 2D) and the contributions of sites IF (fromFig. 5D) and IIIQo (assessed from the reduction state of cytochrome b566 in Fig. 4 and the calibration curve in C). Inverted error bars indicate the propagated errorsfor each site, and the conventional error bar indicates the propagated sum of these errors. Values are means S.E. (error bars) (n � 3–20). p � 0.0001 by Welch’st test.

FIGURE 7. Contributions of sites IQ and IIF at “rest.” Mitochondria were incubated in medium mimicking rest (Table 1). A, total rate of H2O2 production andthe rates in the presence of a IQ electron transport inhibitor (4 �M rotenone) or a suppressor of IQ superoxide production (2.5 �M CN-POBS) after correction forchanges in the rates of sites IF and IIIQo assessed by changes in NAD(P)H and cytochrome b566 redox state (Table 3). The yellow hatched area represents thecontribution of site IQ. B, total rate of H2O2 production at “rest” (from Fig. 2D) and the contributions of sites IF (from Fig. 5D), IIIQo (from Fig. 6D), and IQ (from A).Inverted error bars indicate the propagated errors for each site, and the conventional error bar indicates the propagated sum of these errors. C, total rate of H2O2production and the rate in the presence of 4 �M malonate to inhibit the flavin site of complex II, after correction for changes in the rates of sites IF, IQ, and IIIQo(Table 3). The red hatched area represents the contribution of site IIF. D, total rate of H2O2 production at “rest” (from Fig. 2D) and the contributions of sites IF (fromFig. 5D), IIIQo (from Fig. 6D), IQ (from A), and IIF (from C). Inverted error bars indicate the propagated errors for each site, and the conventional error bar indicatesthe propagated sum of these errors. Values are means S.E. (error bars) (n � 3–20). **, p � 0.005; ***, p � 0.0001 by one-way ANOVA followed by Tukey’s posthoc test. *, p � 0.05 by Student’s t test. Welch’s t test was used to determine p � 0.003 and p � 0.39.




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

ylation, although at higher concentrations, it is less selective(48). CN-POBS was added 1 min prior to the substrate mix andsignificantly decreased the rate of superoxide/H2O2 productionat “rest.” The rate attributed to site IQ after correction for the65% effect of CN-POBS was 66 29 pmol H2O2�min�1�mg ofprotein�1 (mean S.E.; n � 6) (Fig. 7A), indistinguishable fromthe estimate made using rotenone.

From the data obtained with CN-POBS and corroboratedusing rotenone, site IQ accounted for 23 11% of the total rateof H2O2 production measured during “rest” (Fig. 7B and Table3). This is the first evidence in any system that site IQ may be animportant contributor to mitochondrial superoxide/H2O2 pro-duction ex vivo and in vivo. The sum of the rates of superoxide/H2O2 production from sites IF, IIIQo, and IQ was significantlydifferent from the total rate of H2O2 production measured dur-ing “rest” (Fig. 7B), suggesting that yet other sites also producedsuperoxide/H2O2 at rest.

Flavin of Complex II, Site IIF, during “Rest”—The flavin site ofcomplex II, site IIF, has a high capacity for superoxide/H2O2production (20) (Fig. 3). When downstream electron transportis prevented, there is a peak of superoxide/H2O2 productionfrom this site at succinate concentrations close to the physio-logical range (20), indicating that site IIF might also contributeto H2O2 generation at rest. The contribution of site IIF during“rest” ex vivo was assessed by adding malonate to inhibit elec-tron transport at this site, followed by correction for conse-quent changes in the rate of superoxide production from sitesIF, IIIQo, and IQ. The addition of malonate significantlydecreased the rates of H2O2 production at “rest” (Table 3). Thechange in rate attributed to site IIF after correction was 80 35pmol H2O2�min�1�mg of protein�1 (mean S.E.; n � 7) (Fig.7C and Table 3). Site IIF accounted for 24 10% of the total rateof H2O2 production measured during “rest” (Fig. 7D and Table3). This is the first evidence in any system that site IIF may be animportant contributor to mitochondrial superoxide/H2O2 pro-duction ex vivo and in vivo.

The sum of the rates of superoxide/H2O2 production fromsites IF, IIIQo, IQ, and IIF was not significantly different from thetotal rate of H2O2 production measured during “rest” (Fig. 7D).However, we still assessed the potential contributions of theremaining sites.

Electron-transferring Flavoprotein (ETF) and ETF:Q Oxi-doreductase (ETF:QOR), Site EF, during “Rest”—�-Oxidation offatty acids, primarily palmitoylcarnitine, is an important sourceof ATP for resting skeletal muscle. During oxidation of palmi-toylcarnitine, electrons are transferred partly to NAD� andpartly to ETF and then through ETF:QOR to the Q-pool.Superoxide/H2O2 production in the ETF/ETF:QOR system(site EF) probably occurs at the flavin site of ETF (22). Site EFproduces superoxide/H2O2 at a maximum rate of 210 pmol ofH2O2�min�1�mg of protein�1 (22). However, during oxidationof palmitoylcarnitine plus carnitine as the sole added substrateunder native conditions without inhibitors, the observed H2O2production was mainly from sites IF, IIIQo, and IIF, leaving asmall (but not statistically significant) shortfall that might havebeen from other sites, such as ETF (22). The contribution of siteEF during “rest” ex vivo was assessed by omitting palmitoylcar-nitine from the substrate mix, followed by correction for

consequent changes in the rate of superoxide production fromsites IF and IIIQo. Palmitoylcarnitine omission significantlydecreased the rate of H2O2 production (Fig. 8A). There was noconsequent change in the rates from sites IF and IIIQo (Table 3).The possibility that palmitoylcarnitine withdrawal could alterthe rate of superoxide/H2O2 production from site IIF was testedby adding malonate in a mix without palmitoylcarnitine, whichdecreased H2O2 production to the same extent as in the presenceof palmitoylcarnitine, showing that palmitoylcarnitine omissiondid not affect the rate from site IIF (data not shown). The rateattributed to site EF was 46 21 pmol of H2O2�min�1�mg ofprotein�1 (mean S.E.; n � 8) (Fig. 8A and Table 3). Site EFaccounted for 13 6% of the total rate of H2O2 productionmeasured during “rest” (Fig. 8B and Table 3).

Other Sites, PF, OF, GQ, BF, and DQ, during “Rest”—The pyru-vate dehydrogenase complex, site PF, and the 2-oxoglutaratedehydrogenase complex, site OF, have the greatest capacitiesfor superoxide/H2O2 production in the NADH/NAD� isopo-tential group (16) (Figs. 1 and 3). Omission of pyruvate from thesubstrate mixture slightly slowed the rate of H2O2 productionduring “rest,” although this effect was not statistically signifi-cant (p � 0.5). Fig. 8C shows that after correcting for conse-quent changes at sites IF, IIIQo, and IQ (Table 3), site PF was notsignificantly active at rest. Arithmetically, omission of pyruvategave small negative rates for site PF after correction (Fig. 8D andTable 3), presumably reflecting noise in the assays and smallerrors in the assumptions.

Similarly, omission of 2-oxoglutarate or the glycerol 3-phos-phate/dihydroxyacetone phosphate couple from the substratemixture resulted in non-significant small increases in the totalrates of H2O2 production after correction for consequentchanges in other sites (Fig. 8C and Table 3). Therefore, thesesites were also unlikely to contribute to the measured rates ofH2O2 production. Again, these assessments gave small negativerates for sites OF and GQ after correction(Fig. 8D and Table 3).

Site DQ in dihydroorotate dehydrogenase (23) was consid-ered to make zero contribution to total H2O2 production in ouranalysis because this site has low maximum capacity, theenzyme has low activity in skeletal muscle (49), and its sub-strate, dihydroorotate, is present at very low concentrations inrat skeletal muscle and was therefore not included in the sub-strate mix (Table 1). Similarly, site BF of the branched-chain2-oxoacid dehydrogenase (16) was considered to make zerocontribution because (i) branched-chain 2-oxoacids are pres-ent at very low concentrations in rat skeletal muscle mitochon-dria at rest and were therefore not included in the substrate mix(Table 1) and (ii) the addition of the transaminase inhibitoraminooxyacetate at 1 mM did not alter the measured rate ofH2O2 production (data not shown), indicating that transami-nation of the added branched-chain amino acids to generatebranched-chain 2-oxoacids and produce superoxide/H2O2 atsite BF was not a major pathway.

Fig. 8D shows the final sum of the assessed rates of superox-ide/H2O2 production from all of the sites, plotted both as astack of positive and notionally negative contributions and asthe sum of these values. This final value was indistinguishablefrom the total measured rate of H2O2 production (Fig. 8D andTable 3), so the observed rate of H2O2 production was entirely




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

accounted for, within experimental error, by the sum of theassessed rates from each site.

In summary, when rat skeletal muscle mitochondria wereincubated in a complex medium mimicking the cytosol of ratskeletal muscle at rest, containing physiological concentrationsof all substrates and effectors thought to be relevant, the totalrate of H2O2 production was 337 21 pmol of H2O2�min�1�mgof protein�1 (mean S.E.; n � 11). Ranked by magnitude, thesites that contributed to this H2O2 production were sites IIF(24 10%), IQ (23 11%), IF (20 3%), IIIQo (15 2%), and EF(13 6%).

“Mild Aerobic Exercise”—During mild aerobic exercise, sub-strate concentrations are different, and the free Ca2� concen-tration is 20-fold higher compared with rest (Table 1). Whenmitochondria were incubated in the medium mimicking mildaerobic exercise, ATP turnover was driven by extramitochon-drial ATPases, respiration ran at 22% of the maximum rate (Fig.2, A and B), and NAD(P)H and cytochrome b566 reduction lev-

els were significantly lower than at “rest” (Fig. 4 and Table 3).Fig. 2, C and D, shows that the measured rate of H2O2 produc-tion during “mild aerobic exercise” was only a quarter of therate at “rest.”

The contribution of each site to superoxide/H2O2 produc-tion during “mild aerobic exercise” was assessed exactly asdescribed above for “rest” (Table 3). The contributions of sitesIF and IIIQo were assessed using the calibration curves in Figs.5C and 6C. Fig. 9 shows the final sum of the assessed rates ofsuperoxide/H2O2 production from all sites, plotted both as astack of positive and notionally negative contributions and asthe sum of these values. This final value was indistinguishablefrom the total measured rate of H2O2 production (Fig. 9 andTable 3), so the observed rate of H2O2 production was entirelyaccounted for, within experimental error, by the sum of theassessed rates from each site. Compared with “rest,” the abso-lute contribution of each site decreased. The decrease was mostmarked for the sites in or connected to the QH2/Q isopotential

FIGURE 8. Contributions of sites EF, PF, OF, and GQ at “rest.” Mitochondria were incubated in medium mimicking rest (Table 1). A, total rate of H2O2production and the rate in the absence of palmitoylcarnitine after correction for changes in the rates of sites IF and IIIQo (Table 3). The olive hatched arearepresents the contribution of site EF. B, total rate of H2O2 production at “rest” (from Fig. 2D) and the contributions of sites IF (from Fig. 5D), IIIQo (from Fig. 6D),IQ (from Fig. 7A), IIF (from Fig. 7C), and EF (from A). Inverted error bars indicate the propagated errors for each site, and the conventional error bar indicates thepropagated sum of these errors. C, total rate of H2O2 production and the rates in the absence of pyruvate, 2-oxoglutarate, or glycerol 3-phosphate plusdihydroxyacetone phosphate (DHAP) to assess the contributions of sites PF, OF, and GQ. D, total rate of H2O2 production at “rest” (from Fig. 2D) and thecontributions of sites IF (from Fig. 5D); IIIQo (from Fig. 6D); IQ (from Fig. 7A); IIF (from Fig. 7C); EF (from A); and GQ, PF, and OF (from C). Inverted error bars indicatethe propagated errors for each site. Because some of the assessed contributions were negative, the sum of the contributions by each site was also plotted (errorbar indicates the propagated sum of the errors for each assessed site) and used for the statistical test. Values are means S.E. (error bars) (n � 3–20). *, p � 0.05by Student’s t test. NS, not significantly different using one-way ANOVA followed by Tukey’s post hoc test. Welch’s t test was used to determine p � 0.42 andp � 0.15.




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

group (Fig. 1), reflecting their sensitivity to the reduction levelof the Q pool, which dropped by 50% (Fig. 4). Site IQ is also verysensitive to the protonmotive force (19), which will havedecreased when ATP turnover increased. The absolute contri-bution of the only site in the NADH/NAD� isopotential groupthat made a large contribution to H2O2 production at rest, siteIF, decreased much less, because the reduction level ofNAD(P)H dropped by only 20% (Fig. 4). As a result, the relativecontribution of site IF to the rate of superoxide/H2O2 produc-tion increased significantly from 20% during “rest” to 44% dur-ing “mild aerobic exercise” (Table 3).

In summary, when rat skeletal muscle mitochondria wereincubated in a complex medium mimicking the cytosol of ratskeletal muscle during mild aerobic exercise, containing phys-iological concentrations of all substrates and effectors thoughtto be relevant, the total rate of H2O2 production decreased toabout 25% of the rate in the medium mimicking rest. The sitesthat contributed to this H2O2 production were, ranked by mag-nitude, sites IF (44 4%), IQ (18 15%), IIIQo (15 4%), andmaybe EF (12 15%) and GQ (5 7%).

“Intense Aerobic Exercise”—During intense aerobic exercise,free Ca2� concentration is high; ATP concentration is main-tained almost constant (�6 mM); ADP concentration, which ismore than 1 order of magnitude lower than ATP concentration,can increase 5-fold; and creatine phosphate hydrolysis gener-ates an increased concentration of Pi (40). CO2 and lactate pro-duction increase, lowering intracellular pH (40, 50, 51), andsubstrate and effector concentrations also change (Table 1).When mitochondria were incubated in the medium mimickingintense aerobic exercise, ATP turnover was driven by sufficientextramitochondrial hexokinase to cause respiration to run at90% of the maximum rate (Fig. 2, A and B), and NAD(P)H andcytochrome b566 reduction levels were significantly lower thanat “rest” (Fig. 4 and Table 3). NAD(P)H reduction level was alsosignificantly lower than during “mild aerobic exercise” (Fig. 4and Table 3). Fig. 2, C and D, shows that the measured rate of

H2O2 production during “intense aerobic exercise” was onlyone-sixth of the rate at “rest,” but there was no significant dif-ference in rates between “mild aerobic exercise” and “intenseaerobic exercise.”

The contribution of each site to superoxide/H2O2 produc-tion during “intense aerobic exercise” was assessed as describedabove for “rest” and “mild aerobic exercise” (Table 3). The con-tributions of sites IF and IIIQo were assessed using the new cal-ibration curves in Fig. 10, C and F, because we found that thecalibrations were different in this medium compared withthe other two media (Figs. 5C and 6C), probably because of thelower pH.

Fig. 10, A and B, show the dependences of the observed rateof H2O2 production from site IF and the NAD(P)H redox stateon the concentration of malate in the medium mimickingintense aerobic exercise. Fig. 10C replots the data from Fig. 10,A and B, to show the dependence of the measured rate of H2O2production arising from superoxide production at site IF onNAD(P)H reduction state in this medium. Similarly, Fig. 10Dshows the observed rate of H2O2 production from site IIIQo as afunction of the ratio of added succinate and malonate in thepresence of rotenone and myxothiazol. At each point, the rateof H2O2 production from site IF was calculated from theobserved NAD(P)H reduction level using Fig. 10C and sub-tracted from the total observed rate to give the rate specificallyfrom site IIIQo. Fig. 10E shows the cytochrome b566 redox stateunder the same conditions. Fig. 10F replots the data from Fig.10, D and E, to show the dependence of the rate of H2O2 pro-duction arising from superoxide production at site IIIQo on thecytochrome b566 reduction state in this medium.

Fig. 11 shows the final sum of the assessed rates of superox-ide/H2O2 production from all sites, plotted both as a stack ofpositive and notionally negative contributions and as the sum ofthese values. This final value was indistinguishable from thetotal measured rate of H2O2 production (p � 0.46) (Fig. 11 andTable 3), so the observed rate of H2O2 production was entirelyaccounted for, within experimental error, by the sum of theassessed rates from each site. However, because of the lowrate of H2O2 production during “intense aerobic exercise,” theexperimental errors were relatively high, particularly for thesites making smaller contributions (Table 3). Under this condi-tion, site IF was the major contributor (99 26%) and possiblythe only one (Fig. 11). However, we could not discard the pos-sibility that other sites were also active. Note that the totalmeasured rates of H2O2 production were indistinguishablebetween “mild aerobic exercise” and “intense aerobic exercise”(Fig. 2D), but the sites contributing under each conditionappeared to differ (Table 3).

In summary, when rat skeletal muscle mitochondria wereincubated in a complex medium mimicking the cytosol of ratskeletal muscle during intense aerobic exercise, containingphysiological concentrations of all substrates and effectorsthought to be relevant, the total rate of H2O2 productiondecreased to about 15% of the rate in the medium mimickingrest. The main site that contributed to this H2O2 productionwas site IF (99 26% of the total measured rate and 42 10% ofthe sum of assessed rates).

FIGURE 9. Contributions of different sites during “mild aerobic exercise.”Mitochondria were incubated in medium mimicking mild aerobic exercise(Table 1). The total rate of H2O2 production (from Fig. 2D) and the contribu-tions of sites IF, IIIQo, IQ, EF, GQ, PF, IIF, and OF were assessed in the same way asfor “rest” in Fig. 8 using the calibration curves in Figs. 5C and 6C. Inverted errorbars indicate the propagated errors for each site. Values are means S.E.(error bars) (n � 3–9). Because some of the assessed contributions were neg-ative, the sum of the contributions by each site was also plotted (error barindicates the propagated sum of the errors for each assessed site) and usedfor the statistical test. Welch’s t test was used to determine p � 0.95.




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Rates of H2O2 Production after Correction for Losses of H2O2

in the Mitochondrial Matrix—Peroxidases in the mitochon-drial matrix degrade some H2O2 before it can escape and beregistered by the extramitochondrial horseradish peroxidase/Amplex UltraRed assay. The losses can be greatly decreased bydepleting mitochondrial glutathione. Previous work has estab-lished the relationship between observed rates of H2O2 produc-tion and total rates after correcting for H2O2 losses (24, 38), sowe corrected the data presented above using Equation 1 to givea more realistic estimate of the true total rates and the ratesfrom each site. Most of the sites produce superoxide/H2O2

exclusively to the matrix, so their relative contributions will notchange substantially after correction. Importantly, however,�50% of the superoxide/H2O2 from sites IIIQo and GQ is pro-duced toward the cytosol and avoids matrix peroxidase scav-enging (17, 21). Therefore, the contribution of these sitesbecomes relatively smaller after this correction. Fig. 12 showsthe corrected data and represents our best estimate of the totalrate of mitochondrial superoxide/H2O2 production ex vivo andthe contribution of each site during rest and mild and intenseaerobic exercise. At “rest,” more than 50% of the total rate of

FIGURE 10. Calibration of the reporters of sites IF and IIIQo during “intense aerobic exercise.” Mitochondria were incubated in the basic medium mimick-ing intense aerobic exercise (Table 1). A–C, construction of the calibration curve showing the dependence of the rate of superoxide production from site IF onNAD(P)H reduction level exactly as in Fig. 5, A–C, but using the basic medium mimicking intense aerobic exercise. D–F, construction of the calibration curveshowing the dependence of the rate of superoxide production from site IIIQo on the cytochrome b566 reduction level exactly as in Fig. 6, A–C, but using the basicmedium mimicking intense aerobic exercise. Values are means S.E. (error bars) (n � 3).

FIGURE 11. Contributions of different sites during “intense aerobic exer-cise.” Mitochondria were incubated in medium mimicking intense aerobicexercise (Table 1). The total rate of H2O2 production (from Fig. 2D) and thecontributions of sites IF, IIIQo, IIF, EF, OF, GQ, IQ, and PF were assessed in the sameway as for “rest” in Fig. 8 using the calibration curves in Fig. 10, C and F.Inverted error bars indicate the propagated errors for each site. Values aremeans S.E. (error bars) (n � 3). Because some of the assessed contribu-tions were negative, the sum of the contributions by each site was alsoplotted (error bar indicates the propagated sum of the errors for eachassessed site) and used for the statistical test. Welch’s t test was used todetermine p � 0.46.




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

superoxide/H2O2 production was shared between site IQ andsite IIF, previously not considered to be physiologically relevant.During “exercise,” superoxide/H2O2 production decreasedsubstantially, and the low capacity site IF accounted for half ormore of the superoxide produced.

DISCUSSION

Mitochondria can produce superoxide or H2O2 from at least10 different sites (16, 23). Which sites are the major producersin vivo is unknown. Currently, there are no methods availablethat can be used in intact cells or tissues or in vivo to identifyunambiguously which sites are active or to measure the rates ofproduction from each site. However, we recently developedmethods to identify and quantify the rates from these sites inisolated mitochondria (24, 25). From these studies, we con-cluded that the overall rates and relative contributions of eachsite are strongly dependent on the substrate being oxidized. Asa consequence, it is unreasonable to extrapolate from isolatedmitochondria to tissues in vivo when the results in vitro areobtained using single conventional substrates at unphysiologi-cal concentrations.

The work described here represents a first step toward char-acterizing the mitochondrial sites of superoxide and H2O2 pro-duction in skeletal muscle in vivo. We analyzed the sites ex vivoin media designed to mimic the in vivo concentrations of allsubstrates and effectors we thought likely to be important indetermining mitochondrial superoxide and H2O2 productionin muscle at rest and during exercise. With this approach,extrapolations from ex vivo to in vivo are much more reasona-ble. Of course, the approach can still be criticized for assumingthat other effects, such as fragmentation of mitochondria dur-ing isolation, do not substantially affect mitochondrial function

and that other effectors that are not included in the media, suchas components of unknown signaling pathways from the cyto-sol that do not work by altering the concentrations of theexplicit metabolites, can be ignored. We used the approachspecifically for rat skeletal muscle mitochondria, but it couldbe used with mitochondria from any cell type or any tissuefrom control or disease models as long as all metabolites andeffectors are present at their physiological or pathologicalconcentrations.

In the current paper, we identified and quantified for the firsttime all mitochondrial sites that are likely to contribute to mito-chondrial superoxide and H2O2 production in skeletal muscleat rest and under mild aerobic and intense aerobic exercise. Wefound that the maximal capacities of the sites did not correlatewith their native rates of superoxide and H2O2 production exvivo. At “rest,” half of the total rate of H2O2 production wasfrom sites IQ and IIF, two sites whose relevance in vivo was notpreviously appreciated. Also, the low capacity site IF producedH2O2 as fast as the highest capacity site, IIIQo.

In isolated mitochondria, site IQ can produce superoxide/H2O2 at high rates during both the forward and reverse reac-tions (43, 44). The contribution of site IQ in cells and in vivo isunknown. In some cells, the addition of rotenone decreasescellular production of reactive oxygen species (29, 52–54),which is consistent with a substantial contribution of site IQ.However, if the cells were oxidizing predominantly NAD-linked substrates, the addition of rotenone would block reduc-tion of ubiquinone and decrease superoxide/H2O2 productionfrom sites in the QH2/Q isopotential group, particularly sitesIIIQo and IIF. If the cells were running reverse electron transport(29), the addition of rotenone would block reduction of NAD�

and decrease superoxide/H2O2 production from sites in theNADH/NAD� isopotential group, particularly sites IF, PF, andOF. These alternative explanations greatly weaken any conclu-sion from rotenone inhibition experiments that site IQ is activein cells. Conversely, in many other cells, rotenone increasescellular production of reactive oxygen species (55–57), whichappears to be inconsistent with a substantial role for site IQ.However, if the cells were oxidizing primarily NAD-linked sub-strates, the addition of rotenone would cause reduction of theNADH/NAD� isopotential group and increase superoxide/H2O2 production from sites IF, PF, OF, etc., masking anydecrease in the rate from site IQ. In general, whether rotenoneaddition causes an increase or decrease in observed productionof reactive oxygen species may depend upon the balance ofrates and capacities of sites in the NADH/NAD� and QH2/Qisopotential groups and not only on the activity of site IQ itself.These considerations highlight the problems of valid interpre-tation when using electron transport chain inhibitors or geneticmanipulations to define mitochondrial sites of superoxide/H2O2 production in cells if changes in the redox states of allother relevant sites are not properly accounted for as in Table 3.

Here, we used two different approaches to assess the contri-bution of site IQ. First, we used rotenone to inhibit the Q-site ofcomplex I and corrected the observed overall changes in H2O2production for the changes caused by alterations in the redoxstates of the NADH/NAD� isopotential pool (site IF) andQH2/Q isopotential pool (site IIIQo), exposing the contribution

FIGURE 12. Contributions of different sites to superoxide and H2O2 pro-duction by isolated skeletal muscle mitochondria ex vivo in media mim-icking rest, mild aerobic exercise, and intense aerobic exercise. Positivevalues taken from Figs. 8D, 9, and 11 were corrected for the losses of H2O2caused by the activity of mitochondrial matrix peroxidases using Equation 1.Because of their topology, only 50% of the signal assessed for sites IIIQo andGQ was corrected, slightly diminishing their contributions relative to othersites. Inverted error bars indicate the propagated errors for each site, and con-ventional error bars indicate the propagated sum of these errors. Values aremeans S.E. (error bars) (n � 3–20).




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

of site IQ. Second, we used CN-POBS, which specifically sup-presses superoxide production from site IQ without inhibitingelectron transport (48) or altering the redox state of the NADH/NAD� and QH2/Q isopotential pools (Table 3). Both approachesgave the same result (Fig. 7A). This is the first evidence that site IQis active under a semiphysiological condition and therefore mayalso be significantly active in vivo.

The physiological contribution of superoxide/H2O2 produc-tion from site IIF has also been unappreciated, mostly becausethis site was not recognized as an important potential sourceuntil recently. However, the capacity of site IIF to producesuperoxide/H2O2 in muscle mitochondria is great and is sec-ond only to site IIIQo (Fig. 3) (20). When succinate is used as asingle substrate by isolated skeletal muscle mitochondria, therate of production of superoxide/H2O2 by site IIF is highlydependent on the concentration of succinate, rising to a peak atabout 400 �M (20), a concentration that approximates the phys-iological cytosolic level in skeletal muscle (Table 1), and thenfalling away at higher concentrations as succinate becomesinhibitory for this reaction. Our data indicate that at “rest,”�25% of the total H2O2 produced by muscle mitochondriaoriginates from site IIF, indicating that this site is an importantsource of superoxide/H2O2 in vivo. Although the raw data aresparse, the concentration of succinate in skeletal muscle mayrise from about 200 �M at rest to about 300 �M during exercise(Table 1). However, this increase ex vivo was not associatedwith a corresponding increase in the contribution of site IIF in“aerobic exercise” (Fig. 12), presumably because the activatoryeffect of higher succinate concentration was more than com-pensated for by the inhibitory effect of oxidation of the QH2/Qpool during “exercise” (Fig. 4). Nonetheless, under conditionsin which succinate concentration rises, such as hypoxia-reper-fusion (58), but the QH2/Q pool may not become oxidized, thecontribution of site IIF in cardiac muscle or brain may be evengreater.

The mechanisms underlying the decrease in total mitochon-drial superoxide/H2O2 production during “aerobic exercise”are clear from our results. Despite increases in the concentra-tions of several substrates and effectors in the media mimickingexercise, including citrate, malate, pyruvate, succinate, glycerol3-phosphate, acetylcarnitine, and free Ca2� (Table 1), the redoxcenters in both the NADH/NAD� and QH2/Q isopotentialgroups became more oxidized (Fig. 4 and Table 3). This showsthat the dominant effect of the media mimicking exercise wasincreased supply of ADP caused by turnover of ATP, leading toan increased respiration rate and a more oxidized electrontransport chain. Because of the steep dependence of superox-ide/H2O2 production on the redox state of electron transportchain components (Figs. 5, 6, and 10), this led to the steepdecline in total mitochondrial superoxide/H2O2 productionseen in “exercise” in Fig. 2.

Similarly, the mechanisms underlying the decreased relativecontributions of some sites are also clear. Because the oxidationof the NADH/NAD� pool was less severe than the oxidation ofthe QH2/Q pool (Fig. 4 and Table 3), the decrease in the rate ofsuperoxide production by site IF was less than the decrease atthe other major sites, which are all linked to the QH2/Q pool,and the relative contribution of site IF increased. Although

complex I has a high capacity for superoxide production at siteIQ, the flavin site (site IF) has one of the lowest capacities inskeletal muscle mitochondria (Fig. 3). During “aerobic exercise”when the protonmotive force decreases and the NADH andQH2 pools become more oxidized, other sites contribute muchless to overall H2O2 production, allowing the low capacity siteIF to dominate (Fig. 12).

CONCLUSIONS

There is a lack of information about the specific mitochon-drial sites of superoxide and hydrogen peroxide productionthat are physiologically or pathologically active in vivo or inintact cells. Our data provide the first realistic estimate of thesites active in skeletal muscle in vivo under three physiologicalconditions, rest, mild aerobic exercise, and intense aerobicexercise, and provide the first evidence that in addition to thesites currently recognized (sites IIIQo and IF), sites IQ and IIFmay also be very important. These results illuminate the spe-cific sites that may need to be normalized to prevent excessivemitochondrial superoxide and H2O2 production both physio-logically and in many disease states.

REFERENCES1. Loschen, G., Flohe, L., and Chance, B. (1971) Respiratory chain linked

H2O2 production in pigeon heart mitochondria. FEBS Lett. 18, 261–2642. Boveris, A., and Chance, B. (1973) The mitochondrial generation of hy-

drogen peroxide. General properties and effect of hyperbaric oxygen.Biochem. J. 134, 707–716

3. Cadenas, E., and Davies, K. J. (2000) Mitochondrial free radical genera-tion, oxidative stress, and aging. Free Radic. Biol. Med. 29, 222–230

4. Turrens, J. F. (2003) Mitochondrial formation of reactive oxygen species.J. Physiol. 552, 335–344

5. Brand, M. D., Buckingham, J. A., Esteves, T. C., Green, K., Lambert, A. J.,Miwa, S., Murphy, M. P., Pakay, J. L., Talbot, D. A., and Echtay, K. S.(2004) Mitochondrial superoxide and aging: uncoupling-protein activityand superoxide production. Biochem. Soc. Symp. 71, 203–213

6. Balaban, R. S., Nemoto, S., and Finkel, T. (2005) Mitochondria, oxidants,and aging. Cell 120, 483– 495

7. Andreyev, A. Y., Kushnareva, Y. E., and Starkov, A. A. (2005) Mitochon-drial metabolism of reactive oxygen species. Biochemistry 70, 200 –214

8. Brookes, P. S. (2005) Mitochondrial H� leak and ROS generation: an oddcouple. Free Radic. Biol. Med. 38, 12–23

9. Starkov, A. A. (2008) The role of mitochondria in reactive oxygen speciesmetabolism and signaling. Ann. N.Y. Acad. Sci. 1147, 37–52

10. Kowaltowski, A. J., de Souza-Pinto, N. C., Castilho, R. F., and Vercesi,A. E. (2009) Mitochondria and reactive oxygen species. Free Radic. Biol.Med. 47, 333–343

11. Lambert, A. J., and Brand, M. D. (2009) Reactive oxygen species produc-tion by mitochondria. Methods Mol. Biol. 554, 165–181

12. Murphy, M. P. (2009) How mitochondria produce reactive oxygen spe-cies. Biochem. J. 417, 1–13

13. Brand, M. D. (2010) The sites and topology of mitochondrial superoxideproduction. Exp. Gerontol. 45, 466 – 472

14. Drose, S., and Brandt, U. (2012) Molecular mechanisms of superoxideproduction by the mitochondrial respiratory chain. Adv. Exp. Med. Biol.748, 145–169

15. Sies, H. (2014) Role of metabolic H2O2 generation: redox signaling andoxidative stress. J. Biol. Chem. 289, 8735– 8741

16. Quinlan, C. L., Goncalves, R. L., Hey-Mogensen, M., Yadava, N., Bunik,V. I., and Brand, M. D. (2014) The 2-oxoacid dehydrogenase complexesin mitochondria can produce superoxide/hydrogen peroxide at muchhigher rates than complex I. J. Biol. Chem. 289, 8312– 8325

17. Quinlan, C. L., Gerencser, A. A., Treberg, J. R., and Brand, M. D. (2011)The mechanism of superoxide production by the antimycin-inhibited




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

mitochondrial Q-cycle. J. Biol. Chem. 286, 31361–3137218. Treberg, J. R., Quinlan, C. L., and Brand, M. D. (2011) Evidence for two

sites of superoxide production by mitochondrial NADH-ubiquinone ox-idoreductase (complex I). J. Biol. Chem. 286, 27103–27110

19. Lambert, A. J., and Brand, M. D. (2004) Superoxide production by NADH:ubiquinone oxidoreductase (complex I) depends on the pH gradient acrossthe mitochondrial inner membrane. Biochem. J. 382, 511–517

20. Quinlan, C. L., Orr, A. L., Perevoshchikova, I. V., Treberg, J. R., Ackrell,B. A., and Brand, M. D. (2012) Mitochondrial complex II can generatereactive oxygen species at high rates in both the forward and reversereactions. J. Biol. Chem. 287, 27255–27264

21. Orr, A. L., Quinlan, C. L., Perevoshchikova, I. V., and Brand, M. D. (2012)A refined analysis of superoxide production by mitochondrial sn-glycerol3-phosphate dehydrogenase. J. Biol. Chem. 287, 42921– 42935

22. Perevoshchikova, I. V., Quinlan, C. L., Orr, A. L., Gerencser, A. A., andBrand, M. D. (2013) Sites of superoxide and hydrogen peroxide produc-tion during fatty acid oxidation in rat skeletal muscle mitochondria. FreeRadic. Biol. Med. 61, 298 –309

23. Hey-Mogensen, M., Goncalves, R. L., Orr, A. L., and Brand, M. D. (2014)Production of superoxide/H2O2 by dihydroorotate dehydrogenase in ratskeletal muscle mitochondria. Free Radic. Biol. Med. 72, 149 –155

24. Quinlan, C. L., Treberg, J. R., Perevoshchikova, I. V., Orr, A. L., andBrand, M. D. (2012) Native rates of superoxide production from multiplesites in isolated mitochondria measured using endogenous reporters.Free Radic. Biol. Med. 53, 1807–1817

25. Quinlan, C. L., Perevoshchikova, I. V., Hey-Mogensen, M., Orr, A. L., andBrand, M. D. (2013) Sites of reactive oxygen species generation by mito-chondria oxidizing different substrates. Redox Biol. 1, 304 –312

26. Sakellariou, G. K., Vasilaki, A., Palomero, J., Kayani, A., Zibrik, L.,McArdle, A., and Jackson, M. J. (2013) Studies of mitochondrial andnonmitochondrial sources implicate nicotinamide adenine dinucleotidephosphate oxidase(s) in the increased skeletal muscle superoxide gener-ation that occurs during contractile activity. Antioxid. Redox Signal. 18,603– 621

27. Vanden Hoek, T. L., Becker, L. B., Shao, Z., Li, C., and Schumacker, P. T.(1998) Reactive oxygen species released from mitochondria during briefhypoxia induce preconditioning in cardiomyocytes. J. Biol. Chem. 273,18092–18098

28. Powers, S. K., Duarte, J., Kavazis, A. N., and Talbert, E. E. (2010) Reactiveoxygen species are signalling molecules for skeletal muscle adaptation.Exp. Physiol. 95, 1–9

29. Lee, S., Tak, E., Lee, J., Rashid, M. A., Murphy, M. P., Ha, J., and Kim, S. S.(2011) Mitochondrial H2O2 generated from electron transport chaincomplex I stimulates muscle differentiation. Cell Res. 21, 817– 834

30. Powers, S. K., and Jackson, M. J. (2008) Exercise-induced oxidative stress:cellular mechanisms and impact on muscle force production. Physiol.Rev. 88, 1243–1276

31. Callahan, L. A., She, Z. W., and Nosek, T. M. (2001) Superoxide, hydroxylradical, and hydrogen peroxide effects on single-diaphragm fiber con-tractile apparatus. J. Appl. Physiol. 90, 45–54

32. Sakellariou, G. K., Jackson, M. J., and Vasilaki, A. (2014) Redefining themajor contributors to superoxide production in contracting skeletalmuscle: the role of NAD(P)H oxidases. Free Radic. Res. 48, 12–29

33. Wardman, P. (2007) Fluorescent and luminescent probes for measure-ment of oxidative and nitrosative species in cells and tissues: progress,pitfalls, and prospects. Free Radic. Biol. Med. 43, 995–1022

34. Halliwell, B., and Whiteman, M. (2004) Measuring reactive species andoxidative damage in vivo and in cell culture: how should you do it andwhat do the results mean? Br. J. Pharmacol. 142, 231–255

35. Affourtit, C., Quinlan, C. L., and Brand, M. D. (2012) Measurement ofproton leak and electron leak in isolated mitochondria. Methods Mol.Biol. 810, 165–182

36. Quinlan, C. L., Perevoschikova, I. V., Goncalves, R. L., Hey-Mogensen,M., and Brand, M. D. (2013) The determination and analysis of site-specific rates of mitochondrial reactive oxygen species production.Methods Enzymol. 526, 189 –217

37. Crofts, A. R., Meinhardt, S. W., Jones, K. R., and Snozzi, M. (1983) Therole of the quinone pool in the cyclic electron-transfer chain of Rhodop-

seudomonas sphaeroides: a modified Q-cycle mechanism. Biochim. Bio-phys. Acta 723, 202–218

38. Treberg, J. R., Quinlan, C. L., and Brand, M. D. (2010) Hydrogen peroxideefflux from muscle mitochondria underestimates matrix superoxideproduction: a correction using glutathione depletion. FEBS J. 277,2766 –2778

39. Brooks, G. A., and Mercier, J. (1994) Balance of carbohydrate and lipidutilization during exercise: the “crossover” concept. J. Appl. Physiol. 76,2253–2261

40. Cieslar, J. H., and Dobson, G. P. (2000) Free [ADP] and aerobic musclework follow at least second order kinetics in rat gastrocnemius in vivo.J. Biol. Chem. 275, 6129 – 6134

41. Chance, B., Sies, H., and Boveris, A. (1979) Hydroperoxide metabolism inmammalian organs. Physiol. Rev. 59, 527– 605

42. St-Pierre, J., Buckingham, J. A., Roebuck, S. J., and Brand, M. D. (2002)Topology of superoxide production from different sites in the mitochon-drial electron transport chain. J. Biol. Chem. 277, 44784 – 44790

43. Hansford, R. G., Hogue, B. A., and Mildaziene, V. (1997) Dependence ofH2O2 formation by rat heart mitochondria on substrate availability anddonor age. J. Bioenerg. Biomembr. 29, 89 –95

44. Lambert, A. J., and Brand, M. D. (2004) Inhibitors of the quinone-bindingsite allow rapid superoxide production from mitochondrial NADH:ubiquinone oxidoreductase (complex I). J. Biol. Chem. 279, 39414–39420

45. Hinkle, P. C., Butow, R. A., Racker, E., and Chance, B. (1967) Partialresolution of the enzymes catalyzing oxidative phosphorylation. XV. Re-verse electron transfer in the flavin-cytochrome beta region of the respi-ratory chain of beef heart submitochondrial particles. J. Biol. Chem. 242,5169 –5173

46. Starkov, A. A., and Fiskum, G. (2003) Regulation of brain mitochondrialH2O2 production by membrane potential and NAD(P)H redox state.J. Neurochem. 86, 1101–1107

47. Muller, F. L., Liu, Y., Abdul-Ghani, M. A., Lustgarten, M. S., Bhattacha-rya, A., Jang, Y. C., and Van Remmen, H. (2008) High rates of superoxideproduction in skeletal-muscle mitochondria respiring on both complexI- and complex II-linked substrates. Biochem. J. 409, 491– 499

48. Orr, A. L., Ashok, D., Sarantos, M. R., Shi, T., Hughes, R. E., and Brand,M. D. (2013) Inhibitors of ROS production by the ubiquinone-bindingsite of mitochondrial complex I identified by chemical screening. FreeRadic. Biol. Med. 65, 1047–1059

49. Loffler, M., Becker, C., Wegerle, E., and Schuster, G. (1996) Catalyticenzyme histochemistry and biochemical analysis of dihydroorotate de-hydrogenase/oxidase and succinate dehydrogenase in mammalian tis-sues, cells and mitochondria. Histochem. Cell Biol. 105, 119 –128

50. Hultman, E., and Spriet, L. L. (1986) Skeletal muscle metabolism, con-traction force and glycogen utilization during prolonged electrical stim-ulation in humans. J. Physiol. 374, 493–501

51. Horska, A., Brant, L. J., Ingram, D. K., Hansford, R. G., Roth, G. S., andSpencer, R. G. (1999) Effect of long-term caloric restriction and exerciseon muscle bioenergetics and force development in rats. Am. J. Physiol.276, E766 –E773

52. Zamzami, N., Marchetti, P., Castedo, M., Decaudin, D., Macho, A.,Hirsch, T., Susin, S. A., Petit, P. X., Mignotte, B., and Kroemer, G. (1995)Sequential reduction of mitochondrial transmembrane potential andgeneration of reactive oxygen species in early programmed cell death. J.Exp. Med. 182, 367–377

53. Chandel, N. S., Maltepe, E., Goldwasser, E., Mathieu, C. E., Simon, M. C.,and Schumacker, P. T. (1998) Mitochondrial reactive oxygen speciestrigger hypoxia-induced transcription. Proc. Natl. Acad. Sci. U.S.A. 95,11715–11720

54. Aon, M. A., Cortassa, S., Marban, E., and O’Rourke, B. (2003) Synchro-nized whole cell oscillations in mitochondrial metabolism triggered by alocal release of reactive oxygen species in cardiac myocytes. J. Biol. Chem.278, 44735– 44744

55. Barrientos, A., and Moraes, C. T. (1999) Titrating the effects of mito-chondrial complex I impairment in the cell physiology. J. Biol. Chem.274, 16188 –16197

56. Nakamura, K., Bindokas, V. P., Kowlessur, D., Elas, M., Milstien, S.,Marks, J. D., Halpern, H. J., and Kang, U. J. (2001) Tetrahydrobiopterin




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

scavenges superoxide in dopaminergic neurons. J. Biol. Chem. 276,34402–34407

57. Li, N., Ragheb, K., Lawler, G., Sturgis, J., Rajwa, B., Melendez, J. A., andRobinson, J. P. (2003) Mitochondrial complex I inhibitor rotenone in-duces apoptosis through enhancing mitochondrial reactive oxygen spe-cies production. J. Biol. Chem. 278, 8516 – 8525

58. Ariza, A. C., Deen, P. M., and Robben, J. H. (2012) The succinate receptoras a novel therapeutic target for oxidative and metabolic stress-relatedconditions. Front. Endocrinol. (Lausanne) 3, 22

59. Nicholls, D. G., Ferguson, S. J. (2002) Bioenergetics 3, pp. 105–106, Aca-demic Press, London

60. Owen, O. E., Markus, H., Sarshik, S., and Mozzoli, M. (1973) Relationshipbetween plasma and muscle concentrations of ketone bodies and freefatty acids in fed, starved and alloxan-diabetic states. Biochem. J. 134,499 –506

61. Cieslar, J., Huang, M. T., and Dobson, G. P. (1998) Tissue spaces in ratheart, liver, and skeletal muscle in vivo. Am. J. Physiol. 275,R1530 –R1536

62. Ardawi, M. S., and Jamal, Y. S. (1990) Glutamine metabolism in skeletalmuscle of glucocorticoid-treated rats. Clin. Sci. 79, 139 –147

63. Adibi, S. A. (1971) Interrelationships between level of amino acids inplasma and tissues during starvation. Am. J. Physiol. 221, 829 – 838

64. Børsheim, E., Kobayashi, H., Traber, D. L., and Wolfe, R. R. (2006) Com-partmental distribution of amino acids during hemodialysis-induced hy-poaminoacidemia. Am. J. Physiol. Endocrinol. Metab. 290, E643–E652

65. Williams, J. A., Withrow, C. D., and Woodbury, D. M. (1971) Effects ofouabain and diphenylhydantoin on transmembrane potentials, intracel-lular electrolytes, and cell pH of rat muscle and liver in vivo. J. Physiol.212, 101–115

66. Heymsfield, S. B., Stevens, V., Noel, R., McManus, C., Smith, J., andNixon, D. (1982) Biochemical composition of muscle in normal andsemistarved human subjects: relevance to anthropometric measure-ments. Am. J. Clin. Nutr. 36, 131–142

67. Vinnakota, K. C., and Bassingthwaighte, J. B. (2004) Myocardial densityand composition: a basis for calculating intracellular metabolite concen-trations. Am. J. Physiol. Heart. Circ. Physiol. 286, H1742–H1749

68. Brocks, D. G., Siess, E. A., and Wieland, O. H. (1980) Validity of thedigitonin method for metabolite compartmentation in isolated hepato-cytes. Biochem. J. 188, 207–212

69. Wiesner, R. J., Kreutzer, U., Rosen, P., and Grieshaber, M. K. (1988)Subcellular distribution of malate-aspartate cycle intermediates duringnormoxia and anoxia in the heart. Biochim. Biophys. Acta 936, 114 –123

70. Siess, E. A., Brocks, D. G., Lattke, H. K., and Wieland, O. H. (1977) Effectof glucagon on metabolite compartmentation in isolated rat liver cellsduring gluconeogenesis from lactate. Biochem. J. 166, 225–235

71. Kauppinen, R. A., Hiltunen, J. K., and Hassinen, I. E. (1982) Compart-mentation of citrate in relation to the regulation of glycolysis and themitochondrial transmembrane proton electrochemical potential gradi-ent in isolated perfused rat heart. Biochim. Biophys. Acta 681, 286 –291

72. Soboll, S., Horst, C., Hummerich, H., Schumacher, J. P., and Seitz, H. J.(1992) Mitochondrial metabolism in different thyroid states. Biochem. J.281, 171–173

73. Sundqvist, K. E., Heikkila, J., Hassinen, I. E., and Hiltunen, J. K. (1987)Role of NADP� (corrected)-linked malic enzymes as regulators of thepool size of tricarboxylic acid-cycle intermediates in the perfused ratheart. Biochem. J. 243, 853– 857

74. Hensgens, H. E., Meijer, A. J., Williamson, J. R., Gimpel, J. A., and Tager,J. M. (1978) Proline metabolism in isolated rat liver cells. Biochem. J. 170,699 –707

75. Idell-Wenger, J. A., Grotyohann, L. W., and Neely, J. R. (1978) CoenzymeA and carnitine distribution in normal and ischemic hearts. J. Biol. Chem.253, 4310 – 4318

76. Akerboom, T. P., Bookelman, H., Zuurendonk, P. F., van der Meer, R.,and Tager, J. M. (1978) Intramitochondrial and extramitochondrial con-centrations of adenine nucleotides and inorganic phosphate in isolatedhepatocytes from fasted rats. Eur. J. Biochem. 84, 413– 420

77. Schiaffino, S., Hanzlıkova, V., and Pierobon, S. (1970) Relations betweenstructure and function in rat skeletal muscle fibers. J. Cell Biol. 47,

107–11978. Mathieu-Costello, O., Ju, Y., Trejo-Morales, M., and Cui, L. (2005)

Greater capillary-fiber interface per fiber mitochondrial volume in skel-etal muscles of old rats. J. Appl. Physiol. 99, 281–289

79. Desplanches, D., Kayar, S. R., Sempore, B., Flandrois, R., and Hoppeler, H.(1990) Rat soleus muscle ultrastructure after hindlimb suspension.J. Appl. Physiol. 69, 504 –508

80. Kirkwood, S. P., Munn, E. A., and Brooks, G. A. (1986) Mitochondrialreticulum in limb skeletal muscle. Am. J. Physiol. 251, C395–C402

81. van Ekeren, G. J., Sengers, R. C., and Stadhouders, A. M. (1992) Changesin volume densities and distribution of mitochondria in rat skeletal mus-cle after chronic hypoxia. Int. J. Exp. Pathol. 73, 51– 60

82. Else, P. L., and Hulbert, A. J. (1985) Mammals: an allometric study ofmetabolism at tissue and mitochondrial level. Am. J. Physiol. 248,R415–R421

83. Westerblad, H., and Allen, D. G. (1993) The contribution of [Ca2�]i tothe slowing of relaxation in fatigued single fibres from mouse skeletalmuscle. J. Physiol. 468, 729 –740

84. Ingalls, C. P., Warren, G. L., and Armstrong, R. B. (1999) IntracellularCa2� transients in mouse soleus muscle after hindlimb unloading andreloading. J. Appl. Physiol. 87, 386 –390

85. Bruton, J., Tavi, P., Aydin, J., Westerblad, H., and Lannergren, J. (2003)Mitochondrial and myoplasmic [Ca2�] in single fibres from mouse limbmuscles during repeated tetanic contractions. J. Physiol. 551, 179 –190

86. Berchtold, M. W., Brinkmeier, H., and Muntener, M. (2000) Calcium ionin skeletal muscle: its crucial role for muscle function, plasticity, anddisease. Physiol. Rev. 80, 1215–1265

87. Aragon, J. J., and Lowenstein, J. M. (1980) The purine-nucleotide cycle.Comparison of the levels of citric acid cycle intermediates with the op-eration of the purine nucleotide cycle in rat skeletal muscle during exer-cise and recovery from exercise. Eur. J. Biochem. 110, 371–377

88. Pichard, C., Vaughan, C., Struk, R., Armstrong, R. L., and Jeejeebhoy,K. N. (1988) Effect of dietary manipulations (fasting, hypocaloric feeding,and subsequent refeeding) on rat muscle energetics as assessed by nu-clear magnetic resonance spectroscopy. J. Clin. Invest. 82, 895–901

89. MacDermott, M. (1990) The intracellular concentration of free magne-sium in extensor digitorum longus muscles of the rat. Exp. Physiol. 75,763–769

90. Baraban, S. C., Bellingham, M. C., Berger, A. J., and Schwartzkroin, P. A.(1997) Osmolarity modulates K� channel function on rat hippocampalinterneurons but not CA1 pyramidal neurons. J. Physiol. 498, 679 – 689

91. Gillin, A. G., and Sands, J. M. (1992) Characteristics of osmolarity-stim-ulated urea transport in rat IMCD. Am. J. Physiol. 262, F1061–F1067

92. Makara, J. K., Petheo, G. L., Toth, A., and Spat, A. (2000) Effect of osmo-larity on aldosterone production by rat adrenal glomerulosa cells. Endo-crinology 141, 1705–1710

93. Parrilla, R. (1978) The effect of starvation in the rat on metabolite con-centrations in blood, liver and skeletal muscle. Pflugers Arch. 374, 9 –14

94. Chen, V., Ianuzzo, C. D., Fong, B. C., and Spitzer, J. J. (1984) The effectsof acute and chronic diabetes on myocardial metabolism in rats. Diabetes33, 1078 –1084

95. Fellenius, E., Bjorkroth, U., and Kiessling, K. H. (1973) Metabolic changesinduced by ethanol in muscle and liver tissue of the rat in vivo. ActaChem. Scand. 27, 2361–2366

96. Paul, H. S., and Adibi, S. A. (1978) Leucine oxidation in diabetes andstarvation: effects of ketone bodies on branched-chain amino acid oxi-dation in vitro. Metabolism 27, 185–200

97. Konijn, A. M., Carmel, N., and Kaufmann, N. A. (1976) The redox stateand the concentration of ketone bodies in tissues of rats fed carbohydratefree diets. J. Nutr. 106, 1507–1514

98. Zorzano, A., Balon, T. W., Brady, L. J., Rivera, P., Garetto, L. P., Young,J. C., Goodman, M. N., and Ruderman, N. B. (1985) Effects of starvationand exercise on concentrations of citrate, hexose phosphates and glyco-gen in skeletal muscle and heart: evidence for selective operation of theglucose-fatty acid cycle. Biochem. J. 232, 585–591

99. Brass, E. P., and Hoppel, C. L. (1978) Carnitine metabolism in the fastingrat. J. Biol. Chem. 253, 2688 –2693

100. Scharff, R., and Wool, I. G. (1966) Effect of diabetes on the concentration




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

of amino acids in plasma and heart muscle of rats. Biochem. J. 99,173–178

101. Goodman, M. N., Ruderman, N. B., and Aoki, T. T. (1978) Glucose andamino acid metabolism in perfused skeletal muscle: effect of dichloroac-etate. Diabetes 27, 1065–1074

102. Dohm, G. L., Beecher, G. R., Warren, R. Q., and Williams, R. T. (1981)Influence of exercise on free amino acid concentrations in rat tissues.J. Appl. Physiol. 50, 41– 44

103. Olde Damink, S. W., de Blaauw, I., Deutz, N. E., and Soeters, P. B. (1999)Effects in vivo of decreased plasma and intracellular muscle glutamineconcentration on whole-body and hindquarter protein kinetics in rats.Clin. Sci. 96, 639 – 646

104. Ruderman, N. B., Schmahl, F. W., and Goodman, M. N. (1977) Regula-tion of alanine formation and release in rat muscle in vivo: effect ofstarvation and diabetes. Am. J. Physiol. 233, E109 –E114

105. Manchester, K. L., and Wool, I. G. (1963) Insulin and incorporation ofamino acids into protein of muscle. 1. Accumulation and incorporationstudies with the perfused rat heart. Biochem. J. 89, 202–209

106. Goldstein, L., Perlman, D. F., McLaughlin, P. M., King, P. A., and Cha,C. J. (1983) Muscle glutamine production in diabetic ketoacidotic rats.Biochem. J. 214, 757–767

107. Hutson, S. M., Zapalowski, C., Cree, T. C., and Harper, A. E. (1980)Regulation of leucine and �-ketoisocaproic acid metabolism in skeletalmuscle. Effects of starvation and insulin. J. Biol. Chem. 255, 2418 –2426

108. Garber, A. J., Karl, I. E., and Kipnis, D. M. (1976) Alanine and glutaminesynthesis and release from skeletal muscle. I. Glycolysis and amino acidrelease. J. Biol. Chem. 251, 826 – 835

109. Karl, I. E., Garber, A. J., and Kipnis, D. M. (1976) Alanine and glutaminesynthesis and release from skeletal muscle. III. Dietary and hormonalregulation. J. Biol. Chem. 251, 844 – 850

110. Millward, D. J., Nnanyelugo, D. O., James, W. P., and Garlick, P. J. (1974)Protein metabolism in skeletal muscle: the effect of feeding and fasting onmuscle RNA, free amino acids and plasma insulin concentrations. Br. J.Nutr. 32, 127–142

111. Turinsky, J., and Long, C. L. (1990) Free amino acids in muscle: effect ofmuscle fiber population and denervation. Am. J. Physiol. 258, E485–E491

112. Pastoris, O., Dossena, M., Foppa, P., Arnaboldi, R., Gorini, A., Villa, R. F.,and Benzi, G. (1995) Modifications by chronic intermittent hypoxia anddrug treatment on skeletal muscle metabolism. Neurochem. Res. 20,143–150

113. Wijekoon, E. P., Skinner, C., Brosnan, M. E., and Brosnan, J. T. (2004)Amino acid metabolism in the Zucker diabetic fatty rat: effects of insulinresistance and of type 2 diabetes. Can. J. Physiol. Pharmacol. 82, 506 –514

114. Bertocci, L. A., and Lujan, B. F. (1999) Incorporation and utilization of[3-13C]lactate and [1,2-13C]acetate by rat skeletal muscle. J. Appl. Physiol.86, 2077–2089

115. Spydevold, S., Davis, E. J., and Bremer, J. (1976) Replenishment and de-pletion of citric acid cycle intermediates in skeletal muscle. Indication ofpyruvate carboxylation. Eur. J. Biochem. 71, 155–165

116. Lee, S. H., and Davis, E. J. (1979) Carboxylation and decarboxylationreactions. Anaplerotic flux and removal of citrate cycle intermediates inskeletal muscle. J. Biol. Chem. 254, 420 – 430

117. Garber, A. J. (1978) Skeletal muscle protein and amino acid metabolismin experimental chronic uremia in the rat: accelerated alanine and gluta-mine formation and release. J. Clin. Invest. 62, 623– 632

118. Garber, A. J. (1978) The regulation of skeletal muscle alanine and gluta-mine formation and release in experimental chronic uremia in the rat:subsensitivity of adenylate cyclase and amino acid release to epinephrineand serotonin. J. Clin. Invest. 62, 633– 641

119. Goodman, M. N., and Lowenstein, J. M. (1977) The purine nucleotidecycle: studies of ammonia production by skeletal muscle in situ and inperfused preparations. J. Biol. Chem. 252, 5054 –5060

120. Walser, M., Lund, P., Ruderman, N. B., and Coulter, A. W. (1973) Syn-thesis of essential amino acids from their �-[keto] analogues by perfusedrat liver and muscle. J. Clin. Invest. 52, 2865–2877

121. Albe, K. R., Butler, M. H., and Wright, B. E. (1990) Cellular concentra-tions of enzymes and their substrates. J. Theor. Biol. 143, 163–195

122. Tischler, M. E., Henriksen, E. J., and Cook, P. H. (1988) Role of glucocor-

ticoids in increased muscle glutamine production in starvation. MuscleNerve 11, 752–756

123. Kasperek, G. J. (1989) Regulation of branched-chain 2-oxo acid dehydro-genase activity during exercise. Am. J. Physiol. 256, E186 –E190

124. Hutson, S. M., and Harper, A. E. (1981) Blood and tissue branched-chainamino and �-[keto] acid concentrations: effect of diet, starvation, anddisease. Am. J. Clin. Nutr. 34, 173–183

125. May, R. C., Hara, Y., Kelly, R. A., Block, K. P., Buse, M. G., and Mitch,W. E. (1987) Branched-chain amino acid metabolism in rat muscle: ab-normal regulation in acidosis. Am. J. Physiol. 252, E712–E718

126. Berger, M., Hagg, S. A., Goodman, M. N., and Ruderman, N. B. (1976)Glucose metabolism in perfused skeletal muscle: effects of starvation,diabetes, fatty acids, acetoacetate, insulin and exercise on glucose uptakeand disposition. Biochem. J. 158, 191–202

127. Maizels, E. Z., Ruderman, N. B., Goodman, M. N., and Lau, D. (1977)Effect of acetoacetate on glucose metabolism in the soleus and extensordigitorum longus muscles of the rat. Biochem. J. 162, 557–568

128. Dawson, K. D., Baker, D. J., Greenhaff, P. L., and Gibala, M. J. (2005) Anacute decrease in TCA cycle intermediates does not affect aerobic energydelivery in contracting rat skeletal muscle. J. Physiol. 565, 637– 643

129. Goodman, M. N., Berger, M., and Ruderman, N. B. (1974) Glucose me-tabolism in rat skeletal muscle at rest. Effect of starvation, diabetes, ke-tone bodies and free fatty acids. Diabetes 23, 881– 888

130. Saha, A. K., Laybutt, D. R., Dean, D., Vavvas, D., Sebokova, E., Ellis, B.,Klimes, I., Kraegen, E. W., Shafrir, E., and Ruderman, N. B. (1999) Cyto-solic citrate and malonyl-CoA regulation in rat muscle in vivo. Am. J.Physiol. 276, E1030 –E1037

131. Hintz, C. S., Chi, M. M., Fell, R. D., Ivy, J. L., Kaiser, K. K., Lowry, C. V.,and Lowry, O. H. (1982) Metabolite changes in individual rat musclefibers during stimulation. Am. J. Physiol. 242, C218 –C228

132. Rennie, M. J., Winder, W. W., and Holloszy, J. O. (1976) A sparing effectof increased plasma fatty acids on muscle and liver glycogen content inthe exercising rat. Biochem. J. 156, 647– 655

133. Hagg, S. A., Taylor, S. I., and Ruberman, N. B. (1976) Glucose metabolismin perfused skeletal muscle: pyruvate dehydrogenase activity in starva-tion, diabetes and exercise. Biochem. J. 158, 203–210

134. Sacktor, B., Wormser-Shavit, E., and White, J. I. (1965) Diphosphopyri-dine nucleotide-linked cytoplasmic metabolites in rat leg muscle in situduring contraction and recovery. J. Biol. Chem. 240, 2678 –2681

135. Howarth, R. E., and Baldwin, R. L. (1971) Concentrations of selectedenzymes and metabolites in rat skeletal muscle: effects of food restric-tion. J. Nutr. 101, 485– 494

136. Dale, R. A. (1965) Effects of sampling procedures on the contents of someintermediate metabolities of glycolysis in rat tissues. J. Physiol. 181,701–711

137. Kondoh, Y., Kawase, M., Kawakami, Y., and Ohmori, S. (1992) Concen-trations of D-lactate and its related metabolic intermediates in liver,blood, and muscle of diabetic and starved rats. Res. Exp. Med. (Berl.) 192,407– 414

138. Koves, T. R., Ussher, J. R., Noland, R. C., Slentz, D., Mosedale, M., Il-kayeva, O., Bain, J., Stevens, R., Dyck, J. R., Newgard, C. B., Lopaschuk,G. D., and Muoio, D. M. (2008) Mitochondrial overload and incompletefatty acid oxidation contribute to skeletal muscle insulin resistance. CellMetab. 7, 45–56

139. Minatogawa, Y., and Hue, L. (1984) Fructose 2,6-bisphosphate in ratskeletal muscle during contraction. Biochem. J. 223, 73–79

140. Peterson, R. D., Gaudin, D., Bocek, R. M., and Beatty, C. H. (1964) �-glyc-erophosphate metabolism in muscle under aerobic and hypoxic condi-tions. Am. J. Physiol. 206, 599 – 602

141. Klingenberg, M., and Buecher, T. (1960) Biological oxidations. Annu.Rev. Biochem. 29, 669 –708

142. Veech, R. L., Raijman, L., Dalziel, K., and Krebs, H. A. (1969) Disequilib-rium in the triose phosphate isomerase system in rat liver. Biochem. J.115, 837– 842

143. Veech, R. L., Lawson, J. W., Cornell, N. W., and Krebs, H. A. (1979)Cytosolic phosphorylation potential. J. Biol. Chem. 254, 6538 – 6547

144. Arnold, H., and Pette, D. (1970) Binding of aldolase and triosephosphatedehydrogenase to F-actin and modification of catalytic properties of al-




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

dolase. Eur. J. Biochem. 15, 360 –366145. Bhuiyan, A. K., Bartlett, K., Sherratt, H. S., and Agius, L. (1988) Effects of

ciprofibrate and 2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate(POCA) on the distribution of carnitine and CoA and their acyl-estersand on enzyme activities in rats: relation between hepatic carnitine con-centration and carnitine acetyltransferase activity. Biochem. J. 253,337–343

146. Noland, R. C., Koves, T. R., Seiler, S. E., Lum, H., Lust, R. M., Ilkayeva, O.,Stevens, R. D., Hegardt, F. G., and Muoio, D. M. (2009) Carnitine insuf-ficiency caused by aging and overnutrition compromises mitochondrialperformance and metabolic control. J. Biol. Chem. 284, 22840 –22852

147. Marquis, N. R., and Fritz, I. B. (1964) Enzymological determination offree carnitine concentrations in rat tissues. J. Lipid Res. 5, 184 –187

148. Pearson, D. J., and Tubbs, P. K. (1967) Carnitine and derivatives in rattissues. Biochem. J. 105, 953–963

149. Howarth, K. R., LeBlanc, P. J., Heigenhauser, G. J., and Gibala, M. J. (2004)Effect of endurance training on muscle TCA cycle metabolism duringexercise in humans. J. Appl. Physiol. 97, 579 –584

150. Thyfault, J. P., Cree, M. G., Tapscott, E. B., Bell, J. A., Koves, T. R., Il-kayeva, O., Wolfe, R. R., Dohm, G. L., and Muoio, D. M. (2010) Metabolicprofiling of muscle contraction in lean compared with obese rodents.Am. J. Physiol. 299, R926 –R934

151. Challiss, R. A., Vranic, M., and Radda, G. K. (1989) Bioenergetic changesduring contraction and recovery in diabetic rat skeletal muscle. Am. J.Physiol. 256, E129 –E137

152. Essen-Gustavsson, B., and Blomstrand, E. (2002) Effect of exercise onconcentrations of free amino acids in pools of type I and type II fibres inhuman muscle with reduced glycogen stores. Acta Physiol. Scand. 174,275–281

153. Awapara, J. (1956) The taurine concentration of organs from fed andfasted rats. J. Biol. Chem. 218, 571–576

154. Hitchins, S., Cieslar, J. M., and Dobson, G. P. (2001) 31P NMR quantita-tion of phosphorus metabolites in rat heart and skeletal muscle in vivo.Am. J. Physiol. Heart Circ. Physiol. 281, H882–H887

155. Kushmerick, M. J., and Meyer, R. A. (1985) Chemical changes in rat legmuscle by phosphorus nuclear magnetic resonance. Am. J. Physiol. 248,C542–C549

156. Dumas, J. F., Bielicki, G., Renou, J. P., Roussel, D., Ducluzeau, P. H.,Malthiery, Y., Simard, G., and Ritz, P. (2005) Dexamethasone impairsmuscle energetics, studied by 31P NMR, in rats. Diabetologia 48,328 –335

157. Geers, C., and Gros, G. (1990) Effects of carbonic anhydrase inhibitors oncontraction, intracellular pH and energy-rich phosphates of rat skeletalmuscle. J. Physiol. 423, 279 –297

158. Munkvik, M., Lunde, P. K., and Sejersted, O. M. (2009) Causes of fatiguein slow-twitch rat skeletal muscle during dynamic activity. Am. J. Physiol.Regul. Integr. Comp. Physiol. 297, R900 –R910

159. Chung, Y., Sharman, R., Carlsen, R., Unger, S. W., Larson, D., and Jue, T.(1998) Metabolic fluctuation during a muscle contraction cycle. Am. J.Physiol. 274, C846 –C852




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Hey-Mogensen and Martin D. BrandRenata L. S. Goncalves, Casey L. Quinlan, Irina V. Perevoshchikova, Martin

under Conditions Mimicking Rest and Exerciseex VivoAssessed Sites of Superoxide and Hydrogen Peroxide Production by Muscle Mitochondria

doi: 10.1074/jbc.M114.619072 originally published online November 11, 20142015, 290:209-227.J. Biol. Chem.

10.1074/jbc.M114.619072Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

http://www.jbc.org/content/290/1/209.full.html#ref-list-1

This article cites 158 references, 60 of which can be accessed free at


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M114.619072

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;290/1/209&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/290/1/209

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=290/1/209&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/290/1/209

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/290/1/209.full.html#ref-list-1

http://www.jbc.org/

sites of superoxide and hydrogen peroxide production by muscle

Documents