The mechanisms regulating oxidative phosphorylation during exercise remain poorly defined; however, key mitochondrial proteins, including carnitine palmitoyltransferase-I (CPT-I) and adenine nucleotide translocase, have redox-sensitive sites. Interestingly, muscle contraction has recently been shown to increase mitochondrial membrane potential and reactive oxygen species (ROS) production; therefore, we aimed to determine if mitochondrial-derived ROS influences bioenergetic responses to exercise. Specifically, we examined the influence of acute exercise on mitochondrial bioenergetics in WT (wild type) and transgenic mice (MCAT, mitochondrial-targeted catalase transgenic) possessing attenuated mitochondrial ROS. We found that ablating mitochondrial ROS did not alter palmitoyl-CoA (P-CoA) respiratory kinetics or influence the exercise-mediated reductions in malonyl CoA sensitivity, suggesting that mitochondrial ROS does not regulate CPT-I. In contrast, while mitochondrial protein content, maximal coupled respiration, and ADP (adenosine diphosphate) sensitivity in resting muscle were unchanged in the absence of mitochondrial ROS, exercise increased the apparent ADP Km (decreased ADP sensitivity) ∼30% only in WT mice. Moreover, while the presence of P-CoA decreased ADP sensitivity, it did not influence the basic response to exercise, as the apparent ADP Km was increased only in the presence of mitochondrial ROS. This basic pattern was also mirrored in the ability of ADP to suppress mitochondrial H2O2 emission rates, as exercise decreased the suppression of H2O2 only in WT mice. Altogether, these data demonstrate that while exercise-induced mitochondrial-derived ROS does not influence CPT-I substrate sensitivity, it inhibits ADP sensitivity independent of P-CoA. These data implicate mitochondrial redox signaling as a regulator of oxidative phosphorylation.

Introduction

Classically, the capacity for oxidative phosphorylation has been considered paramount in the regulation of aerobic ATP (adenosine triphosphate) production [1]. While mitochondrial content is clearly important in the regulation of oxidative metabolism [1,2], external regulation of mitochondrial ADP transport likely exists, as acute exercise impairs mitochondrial ADP sensitivity in humans [3] and rodents [4,5]. In addition to the implications for aerobic respiration, the attenuation in mitochondrial ADP sensitivity appears intimately linked with exercise-mediated mitochondrial reactive oxygen species (ROS) production and the induction of mitochondrial biogenesis [5,6]. Therefore, the regulation of mitochondrial ADP sensitivity appears paramount in both acute and chronic responses to exercise.

Despite the fact that the transport of ADP into the mitochondria is a major regulatory control point of oxidative phosphorylation, very little is known about the mechanisms that influence this process. However, evidence has been generated to suggest that adenine nucleotide translocase (ANT), a protein required for ADP transport across the inner mitochondrial membrane [7], has several potential regulatory sites, including acetylation of lysine 23 [8], phosphorylation of tyrosine 194 [9], glutathionylation/carbonylation [10,11], and three matrix localized cysteine sites [12,13]. While the functional roles for these regulatory points remain unknown, indirect evidence suggests that ANT is inhibited through various redox modifications [10,12,13], raising the potential that exercise-induced impairments in mitochondrial ADP sensitivity result from oxidation of ANT. In addition, fatty acids, and in particular palmitoyl-CoA (P-CoA), have been shown to interact with ANT to reduce ADP sensitivity [14]. The sensitivity of ANT to P-CoA can be modified with chronic exercise training [15], and while intramuscular P-CoA content increases with acute exercise [16], it remains to be determined if potential alterations of ANT alter the inhibitory effect of P-CoA during exercise. While the processes that regulate fatty acid metabolism during exercise remain poorly defined, carnitine palmitoyltransferase-I (CPT-I) has an obligatory role in the movement of P-CoA into the mitochondria (as reviewed by ref. [17]), and acute exercise appears to promote CPT-I flux by attenuating malonyl CoA (M-CoA)-mediated inhibition [18]. The mechanism for this alteration remains unknown; however, similar to ANT, CPT-I is sensitive to post-translational redox modifications [19]. Altogether, the role of exercise-mediated mitochondrial ROS production in the regulation of CPT-I remains to be determined.

While classically mitochondria are not thought to be a major source of ROS during exercise, muscle contraction has recently been shown to increase mitochondrial membrane potential and ROS emission rates [5,6]. Therefore, the purpose of this study was to determine if mitochondrial ROS is a modulator of acute exercise-induced alterations in ADP and lipid supported mitochondrial respiration. To explore the relationship between ROS and oxidative phosphorylation, we utilized transgenic MCAT (mitochondrial-targeted catalase transgenic) mice, which overexpress human catalase ∼30-fold greater in the mitochondria of skeletal muscle compared with endogenous levels [20]. It was hypothesized that attenuating mitochondrial ROS would improve various aspects of mitochondrial respiratory control following acute exercise, implicating mitochondrial-derived ROS as a key determinant of energy homeostasis.

Methods

Mice

C57BL/6NJ (n = 16) and MCAT (n = 15) mice on the same C57BL/6NJ background were purchased from The Jackson Laboratory (Bar Harbor, U.S.A.) at 8 weeks of age. Animals were single housed on a 12 : 12-h light–dark cycle within a temperature-regulated environment with unrestricted access to a standard chow diet, and water available ad libitum, until they reached 15 weeks of age. All methodologies used during experimentation were approved by the Animal Care Committee at the University of Guelph, and were in compliance with the guide for the care and use of laboratory animals, as published by the National Institutes of Health.

Acute exercise protocol

Fifteen-week-old mice were familiarized to motorized rodent treadmills (Exer-3R treadmill, Columbus Instruments, Columbus, U.S.A.) for 10 min at 15 m/min and a grade of 5% over a 4-day period. Following familiarization, all mice underwent a run test to exhaustion, beginning at a moderate speed of 15 m/min (5% grade) for 90 min. Speed was then increased by 2 m/min every 5 min until volitional cessation. Mice were given 72 h to recover and then were randomly selected to remain sedentary (Sed) or to perform acute exercise (Ex). Exercising mice were run for a period of 90 min (15 m/min, and a grade of 5%). Mice were anesthetized with isoflurane and oxygen (2% : 98%), and the red gastrocnemius muscle was removed within 1 min of cessation of exercise and immediately placed in BIOPS preservation buffer for assessment of mitochondrial bioenergetics and mitochondrial H2O2 emission (details below).

Preparation of permeabilized muscle fibers

Saponin-permeabilized muscle fibers were generated as previously reported [35,15,18]. Briefly, muscles were trimmed of fat and connective tissue, and were separated in ice-cold BIOPS, containing CaK2-EGTA (2.77 mM), K2-EGTA (7.23 mM), Na2-ATP (5.77 mM), MgCl2·6H2O (6.56 mM), Na2-PCr (phosphocreatine) (15 mM), imidazole (20 mM), dithiothreitol (0.5 mM), taurine (20 mM), and MES (50 mM). Fiber bundles were treated with 40 µg/ml saponin and incubated on a rotor for 30 min at 4°C. Fiber bundles being used for analysis of mitochondrial respiration were washed in mitochondrial respiration buffer (MIR05), containing EGTA (0.5 mM), MgCl2·6H2O (3 mM), potassium lactobionate (60 mM), KH2PO4 (10 mM), HEPES (20 mM), sucrose (110 mM), taurine (20 mM), and fatty acid-free BSA (1 g/L), and fiber bundles being used for measurements of mitochondrial H2O2 emission were washed in buffer Z, containing K-MES (105 mM), KCl (30 mM), EGTA (1 mM), K2HPO4 (10 mM), MgCl2·6H2O (5 mM), glutamate (5 µM), malate (5 µM), and BSA (5 mg/ml).

High-resolution respirometry

Measurements of mitochondrial respiration using saponin-permeabilized muscle fibers were conducted in 2 ml of mitochondrial respiration buffer (MIR05) using high-resolution respirometry (Oroboros Oxygraph-2K: Oroboros Instruments, Innsbruck, Austria) at 37°C with constant stirring at 750 rpm. All experiments were conducted between 200 and 180 µM of oxygen, and the buffer was equilibrated to room air after the addition of each substrate. In addition, experiments were performed in the presence of 5 µM blebbistatin, a myosin II inhibitor, in order to better reflect an in vivo environment [21]. Cytochrome c (10 µM) was added as a measurement of mitochondrial membrane integrity, and all experiments demonstrated an increase in respiration of less than 10%. Respiratory control ratios, which are reflective of the coupling state of the mitochondria, were determined for all experiments, and fiber bundles were recovered, freeze-dried, and respiration data were normalized to fiber bundle weight.

P-CoA-stimulated respiration was determined in the presence of 1 mM malate and 5 mM ADP. Once stabilized, 2 mM l-carnitine was added, and P-CoA was then titrated at increasing concentrations (25, 50, 75, 100, 125, 150, 175, and 200 µM). To determine the inhibitory effect of M-CoA on P-CoA transport, a subset of experiments were completed in the presence of 7 µM M-CoA. In total, 10 mM glutamate and 10 mM succinate were added following P-CoA titrations to determine maximal respiration from complexes I and II. ADP-stimulated (25, 100, 175, 250, 500, 1000, 2000, 4000, 6000, 8000, and 10 000 µM) respiration was determined in the presence of 5 mM pyruvate and 1 mM malate. Both 10 mM glutamate and 10 mM succinate were added following ADP titrations to determine maximal mitochondrial respiration from complexes I and II. Separate experiments were completed in the presence of 20 µM and 60 µM P-CoA to determine the inhibitory effect on ADP sensitivity.

Mitochondrial H2O2 emission

To measure mitochondrial ROS production, we determined the rate of H2O2 release from permeabilized muscle fibers using Amplex Red fluorescence quantification (Invitrogen) at 37°C as previously reported [15]. Briefly, fiber bundles were added to a constantly stirring cuvette containing 10 µM Amplex Red reagent, 5 µM blebbistatin, 40 U/ml SOD, and 0.5 U/ml horseradish peroxidase. SOD was added to ensure the conversion of superoxide radicals to H2O2. In separate experiments, mitochondrial H2O2 emission was initiated by the addition of 20 mM succinate to a cuvette containing either 100 µM ADP, 60 µM P-CoA (palmitoyl-CoA), or 100 µM ADP and 60 µM P-CoA. Raw fluorescence was calibrated to a standard curve that was made for each experiment using known concentrations of H2O2, and the rate of mitochondrial H2O2 emission was calculated from the slope after subtracting background fluorescence. Fiber bundles were recovered, freeze-dried, and mitochondrial H2O2 emission data were then normalized to fiber bundle mass following each experiment.

Digestion of permeabilized muscle fibers for Western blotting

Freeze-dried permeabilized muscle fiber bundles were digested in a fiber lysis buffer, adapted from previously published methods [22,23]. Briefly, permeabilized muscle fibers were digested in a buffer solution containing 10% glycerol, 5% β-mercaptoethanol, 2.3% SDS in 62.5 mM Tris–HCl, and 0.01% bromophenol blue, for 1 h at 65°C with gentle shaking. To improve digestion, protein samples were vortexed for 3–5 s every 15 min. Approximately 5–10 µl of sample was then loaded for analysis by SDS–PAGE, as described below.

Western blotting

Digested fiber bundles were separated by electrophoresis at 150 V for 1 h on SDS–polyacrylamide gels, then transferred at 100 V for 1 h to polyvinylidene difluoride membranes. Commercially available antibodies were used to detect COXIV (5 µl protein; Invitrogen, Eugene, U.S.A.–A21347), OXPHOS (5 µl protein; Mitosciences, Eugene, U.S.A.—ab110413), PDH (5 µl protein; Invitrogen—459 400), ANT1 (10 µl protein; Abcam, Cambridge, U.S.A.—ab110322), ANT2 (10 µl protein; Abcam—ab118076), VDAC (5 µl protein; Abcam—ab14734), MiCK (5 µl protein; Abcam—ab131188), and α-tubulin (5 µl protein, Abcam—ab7291). All samples for each protein were loaded on the same membrane to limit variation. Western blots were quantified via chemiluminescence and the FluorChem HD imaging system (Alpha Innotech, Santa Clara, U.S.A.).

Statistics

Statistical analyses were completed using SigmaPlot software (SigmaPlot Software version 12.0, Systat Software, Inc.). Exercise tolerance, anthropometric measurements, and select mitochondrial comparisons where stated within results section were statistically analyzed using two-tailed independent Student's t-tests. All other parameters for sedentary and acute exercise states were analyzed using a two-way ANOVA in combination with a Newman–Keuls–Student (NK) post hoc analysis when appropriate. The apparent Michaelis–Menten constant (Km) for ADP was determined using GraphPad Prism software (GraphPad Software, Inc., LA Jolla, U.S.A.) as described previously [15]. Values for maximal respiration (Vmax) were represented by the highest respiratory value that was directly measured. Data were expressed as mean ± SEM (n = 7–8 per group). Values were statistically significant if P ≤ 0.05.

Results

Basic characterization of MCAT animals

MCAT animals had significantly lower maximal succinate-supported mitochondrial H2O2 emission, similar to data reported previously [24], validating our experimental model. MCAT mice did not show any significant differences in body weight, tibia length, or normalized heart weight (Table 1); however, MCAT mice displayed a ∼6% reduction in exercise tolerance (Table 1).

Table 1
WT and MCAT basic characterization
ParameterWTMCAT
Maximal mitochondrial H2O2 emission (pmol H2O2/min/mg dry weight) 200 ± 7 38 ± 4* 
Tibia length (cm) 1.8 ± 0.1 1.8 ± 0.1 
Body weight (g) 30 ± 1 32 ± 1 
Heart weight (mg) 122 ± 2.1 120 ± 2 
Body weight normalized to tibia length (g/cm) 17.1 ± 0.3 18.1 ± 0.3 
Heart weight normalized to tibia length (mg/cm) 68.8 ± 1.2 67.8 ± 1.2 
Time to exhaustion (min) 123 ± 2 116 ± 1* 
ParameterWTMCAT
Maximal mitochondrial H2O2 emission (pmol H2O2/min/mg dry weight) 200 ± 7 38 ± 4* 
Tibia length (cm) 1.8 ± 0.1 1.8 ± 0.1 
Body weight (g) 30 ± 1 32 ± 1 
Heart weight (mg) 122 ± 2.1 120 ± 2 
Body weight normalized to tibia length (g/cm) 17.1 ± 0.3 18.1 ± 0.3 
Heart weight normalized to tibia length (mg/cm) 68.8 ± 1.2 67.8 ± 1.2 
Time to exhaustion (min) 123 ± 2 116 ± 1* 
*

Significant difference from WT animals. N = 16 WT and 15 MCAT. Data are expressed as means ± SEM (P< 0.05).

Anthropometric data, maximal succinate-supported mitochondrial H2O2 emission and run to volitional exhaustion in WT and MCAT animals.

Markers of mitochondrial oxidative capacity

We used permeabilized muscle fibers and Western blotting to determine if MCAT animals had a reduced OXPHOS capacity. However, there were no significant changes in coupled respiration in the presence of various substrates (Figure 1A), or differences in the abundance of mitochondrial proteins (Figure 1B).

Markers of mitochondrial content.

Figure 1.
Markers of mitochondrial content.

Maximal non-ADP-stimulated respiration (PM: p = pyruvate, M = malate), ADP-stimulated respiration (PMD: D = ADP), maximal complex I stimulated respiration (PMDG: G = glutamate), and maximal complex I and II stimulated respiration (PMDGS: S = succinate) (A). Western blot analysis and representative images for OXPHOS proteins (CI-NDUFB8, CII-Core 2, CIII-30kDa, CIV-1, and CIV4) and PDH (B). α-Tubulin was used as a loading control. Data expressed as mean ± SEM (n = 7–8 per group).

Figure 1.
Markers of mitochondrial content.

Maximal non-ADP-stimulated respiration (PM: p = pyruvate, M = malate), ADP-stimulated respiration (PMD: D = ADP), maximal complex I stimulated respiration (PMDG: G = glutamate), and maximal complex I and II stimulated respiration (PMDGS: S = succinate) (A). Western blot analysis and representative images for OXPHOS proteins (CI-NDUFB8, CII-Core 2, CIII-30kDa, CIV-1, and CIV4) and PDH (B). α-Tubulin was used as a loading control. Data expressed as mean ± SEM (n = 7–8 per group).

CPT-I substrate sensitivity

We have previously shown reduced M-CoA sensitivity following acute exercise [18], and since it has recently been suggested that CPT-I can be inhibited in vitro by post-translational redox modification [19,25], we examined the influence of attenuating mitochondrial ROS on lipid-mediated adaptive responses to exercise. Specifically we examined various aspects of lipid-supported respiration, including determining CPT-I substrate sensitivity to P-CoA and M-CoA. Coupled respiration in the presence of P-CoA (Figure 2A) and P-CoA respiratory sensitivity (Figure 2B) were not influenced by exercise or genotype. In the presence of M-CoA, maximal respiration was similar between animals (Figure 2C); however, in contrast, P-CoA sensitivity was influenced by both genotype and exercise. Specifically, in the presence of M-CoA, MCAT mice displayed attenuated P-CoA sensitivity, while exercise improved P-CoA sensitivity regardless of genotype (main effect for apparent Km to be reduced: Figure 2D,E). The exercise-mediated attenuation in M-CoA sensitivity was not dependent on mitochondrial ROS, as exercise decreased the P-CoA apparent Km to the same magnitude (∼27 µM P-CoA) in both genotypes in the presence of M-CoA (Figure 2E). M-CoA appeared to have no effect after exercise, as while M-CoA increased the apparent P-CoA Km in resting muscle, after exercise, M-CoA did not alter the P-CoA respiratory sensitivity (Figure 2F). Altogether, these data suggest that mitochondrial ROS is not a primary regulator of CPT-I-dependent lipid transport during exercise.

The influence of exercise and attenuating mitochondrial ROS on CPT-I substrate sensitivity.

Figure 2.
The influence of exercise and attenuating mitochondrial ROS on CPT-I substrate sensitivity.

Maximal non-P-CoA-stimulated respiration (M: M = malate, D = ADP) and PCoA-stimulated respiration (MDLP: L = l-carnitine, P = PCoA). Thereafter, glutamate (MDLPG: G = glutamate) and succinate (MDLPGS: S = succinate) were sequentially added (A). P-CoA titrations displayed a Michaelis–Menten kinetic relationship (B), with unaltered maximal respiration and apparent P-CoA Km (inset in B). P-CoA sensitivity in the presence of M-CoA revealed unaltered maximal respiration (C), but dramatically different P-CoA sensitivity following exercise, independent of genotype (D). The x-axis from the Michaelis–Menten curve was rescaled to show the shifts in respiration of each group (D), and the calculated apparent P-CoA Km reported in (E). The ability of M-CoA to change the apparent P-CoA Km is reported in (F) and represents the difference between the Km reported in the absence of M-CoA in (B) and the presence of M-CoA in (E). Data expressed as mean ± SEM (n = 7–8 per group).

Figure 2.
The influence of exercise and attenuating mitochondrial ROS on CPT-I substrate sensitivity.

Maximal non-P-CoA-stimulated respiration (M: M = malate, D = ADP) and PCoA-stimulated respiration (MDLP: L = l-carnitine, P = PCoA). Thereafter, glutamate (MDLPG: G = glutamate) and succinate (MDLPGS: S = succinate) were sequentially added (A). P-CoA titrations displayed a Michaelis–Menten kinetic relationship (B), with unaltered maximal respiration and apparent P-CoA Km (inset in B). P-CoA sensitivity in the presence of M-CoA revealed unaltered maximal respiration (C), but dramatically different P-CoA sensitivity following exercise, independent of genotype (D). The x-axis from the Michaelis–Menten curve was rescaled to show the shifts in respiration of each group (D), and the calculated apparent P-CoA Km reported in (E). The ability of M-CoA to change the apparent P-CoA Km is reported in (F) and represents the difference between the Km reported in the absence of M-CoA in (B) and the presence of M-CoA in (E). Data expressed as mean ± SEM (n = 7–8 per group).

Mitochondrial ADP sensitivity

Since proteins involved in ADP transport, such as ANT1, are sensitive to post-translational redox modification [13], and exercise dramatically decreases ADP sensitivity, we examined the influence of local ROS production on ADP-stimulated respiration. Titrating ADP in the presence of pyruvate and malate generated the expected Michaelis–Menten kinetic curve (Figure 3A). While maximal respiration was not altered with exercise in either genotype (inset Figure 3A), the apparent Km for ADP was dramatically altered by exercise in a genotype-specific manner, as there was a significant interaction (P = 0.003). Specifically, while exercise impaired ADP sensitivity in WT (wild type) mice (apparent Km increased ∼33%; Figure 3B,C), exercise did not alter mitochondrial ADP sensitivity in MCAT mice (Figure 3B,C).

MCAT mice exhibit improved ADP sensitivity following acute exercise.

Figure 3.
MCAT mice exhibit improved ADP sensitivity following acute exercise.

ADP-stimulated respiration displayed a Michaelis–Menten kinetic relationship (A), with similar maximal respiration between groups (inset in A). The x-axis from the Michaelis–Menten curve was rescaled to show the shifts in respiration of each group (B), and the calculated apparent ADP Km (C). Asterisk denotes significant difference from sedentary control group of each genotype, and † denotes difference from WT animals within the same condition. Data expressed as mean ± SEM (n = 7–8 per group).

Figure 3.
MCAT mice exhibit improved ADP sensitivity following acute exercise.

ADP-stimulated respiration displayed a Michaelis–Menten kinetic relationship (A), with similar maximal respiration between groups (inset in A). The x-axis from the Michaelis–Menten curve was rescaled to show the shifts in respiration of each group (B), and the calculated apparent ADP Km (C). Asterisk denotes significant difference from sedentary control group of each genotype, and † denotes difference from WT animals within the same condition. Data expressed as mean ± SEM (n = 7–8 per group).

Since lipid availability increases during exercise, we also repeated these experiments in the presence of various concentrations of P-CoA, which is known to decrease ADP sensitivity through inhibition of ANT [4,14,15]. In the presence of 20 µM P-CoA, which is reflective of the concentration of lipid found in resting skeletal muscle [16], there remained an interaction for ADP sensitivity (P = 0.049), and basic t-test analysis revealed that 20 µM P-CoA did not affect the respiratory parameters that were measured (Figure 4 relative to Figure 3). In the presence of 60 µM P-CoA, a concentration that reflects exercised levels, ADP-supported respiration was not different between genotypes or exercise conditions (Figure 5A,B, inset). In addition, the presence of 60 µM P-CoA did not alter the response to exercise, as similar to other experiments there was an interaction for ADP sensitivity (P = 0.048) and only WT animals displayed an impairment in ADP sensitivity following exercise (Figure 5C,D). While 60 µM P-CoA did not affect the response to exercise, a basic t-test analysis revealed that this lipid concentration decreased maximal respiration ∼50% and increased the apparent ADP Km ∼50% (Figure 5E relative to Figure 3), regardless of genotype. Therefore, while a high-lipid environment attenuates ADP sensitivity, exercise and mitochondrial ROS do not influence this response.

The influence of 20 µM P-CoA on mitochondrial ADP respiratory sensitivity.

Figure 4.
The influence of 20 µM P-CoA on mitochondrial ADP respiratory sensitivity.

ADP-stimulated respiration in the presence of 20 µM P-CoA displayed a Michaelis–Menten kinetic relationship (A), with similar maximal respiration between groups (inset in A). The x-axis from the Michaelis–Menten curve was rescaled to show the shifts in ADP sensitivity of each group (B), and the calculated apparent ADP Km (C). Asterisk denotes significant difference from sedentary control group of each genotype, and † denotes difference from WT animals within the same condition. Data expressed as mean ± SEM (n = 7–8 per group).

Figure 4.
The influence of 20 µM P-CoA on mitochondrial ADP respiratory sensitivity.

ADP-stimulated respiration in the presence of 20 µM P-CoA displayed a Michaelis–Menten kinetic relationship (A), with similar maximal respiration between groups (inset in A). The x-axis from the Michaelis–Menten curve was rescaled to show the shifts in ADP sensitivity of each group (B), and the calculated apparent ADP Km (C). Asterisk denotes significant difference from sedentary control group of each genotype, and † denotes difference from WT animals within the same condition. Data expressed as mean ± SEM (n = 7–8 per group).

The influence of 60 µM P-CoA on mitochondrial ADP respiratory sensitivity.

Figure 5.
The influence of 60 µM P-CoA on mitochondrial ADP respiratory sensitivity.

Maximal non-ADP-stimulated respiration (PM: P = pyruvate, M = malate), ADP-stimulated respiration (PMD: D = ADP), maximal complex I stimulated respiration (PMDG: G = glutamate), and maximal complex I and II stimulated respiration (PMDGS: S = succinate) in the presence of 60 µM P-CoA (A). ADP-stimulated respiration in the presence of 60 µM P-CoA displayed a Michaelis–Menten kinetic relationship (B), with similar maximal respiration between groups (inset in A). The x-axis from the Michaelis–Menten curve was rescaled to show the shifts in ADP sensitivity of each group (C), and the calculated apparent ADP Km (D). The presence of 60 µM P-CoA attenuated maximal ADP-stimulated respiration and the P-CoA apparent Km similarly in all animals, regardless of exercise or genotype (E). Asterisk denotes significant difference from sedentary control group of each genotype, and † denotes difference from WT animals within the same condition. Data expressed as mean ± SEM (n = 7–8 per group).

Figure 5.
The influence of 60 µM P-CoA on mitochondrial ADP respiratory sensitivity.

Maximal non-ADP-stimulated respiration (PM: P = pyruvate, M = malate), ADP-stimulated respiration (PMD: D = ADP), maximal complex I stimulated respiration (PMDG: G = glutamate), and maximal complex I and II stimulated respiration (PMDGS: S = succinate) in the presence of 60 µM P-CoA (A). ADP-stimulated respiration in the presence of 60 µM P-CoA displayed a Michaelis–Menten kinetic relationship (B), with similar maximal respiration between groups (inset in A). The x-axis from the Michaelis–Menten curve was rescaled to show the shifts in ADP sensitivity of each group (C), and the calculated apparent ADP Km (D). The presence of 60 µM P-CoA attenuated maximal ADP-stimulated respiration and the P-CoA apparent Km similarly in all animals, regardless of exercise or genotype (E). Asterisk denotes significant difference from sedentary control group of each genotype, and † denotes difference from WT animals within the same condition. Data expressed as mean ± SEM (n = 7–8 per group).

Mitochondrial H2O2 emission and ADP transport proteins

To further investigate the interaction between mitochondrial ROS and ADP responsiveness, we examined the ability of ADP to attenuate mitochondrial ROS emission. Exercise did not alter maximal succinate-supported mitochondrial H2O2 emission (Figure 6A). In contrast, while the addition of 100 µM ADP significantly reduced the propensity for ROS generation in both WT and MCAT animals (Figure 6B), there was an interaction for this response (P = 0.03) such that exercise attenuated this response only in WT mice (i.e. H2O2 emission is higher after exercise), similar to the observed increase in the apparent ADP Km. To further establish that the alterations in ADP sensitivity occurred as a result of signaling events, we characterized key proteins involved in mitochondrial phosphate shuttling. Importantly, exercise and genotype did not influence the abundance of ANT1, ANT2, VDAC, or MiCK protein abundance (Figure 7A,B).

The influence of exercise on mitochondrial H2O2 emission in the presence and absence of ADP.

Figure 6.
The influence of exercise on mitochondrial H2O2 emission in the presence and absence of ADP.

Succinate-supported mitochondrial H2O2 emission in the absence (A) and presence of 100 µM ADP (B). There was a main effect of genotype on succinate-supported mitochondrial H2O2 emission. Asterisk denotes significant difference from sedentary control group of each genotype, and † denotes difference from WT animals within the same condition. Data expressed as mean ± SEM (n = 7–8 per group).

Figure 6.
The influence of exercise on mitochondrial H2O2 emission in the presence and absence of ADP.

Succinate-supported mitochondrial H2O2 emission in the absence (A) and presence of 100 µM ADP (B). There was a main effect of genotype on succinate-supported mitochondrial H2O2 emission. Asterisk denotes significant difference from sedentary control group of each genotype, and † denotes difference from WT animals within the same condition. Data expressed as mean ± SEM (n = 7–8 per group).

The absence of changes in mitochondrial proteins involved in ADP transport.

Figure 7.
The absence of changes in mitochondrial proteins involved in ADP transport.

Adenine nucleotide translocase 1 and 2 (ANT1 and ANT2), voltage-dependent anion channel (VDAC) and mitochondrial creatine kinase (MiCK) protein abundance (A) were not different between genotypes (WT and MCAT mice) or following acute exercise (Sed and Ex). Representative Western blots are shown in (B). Data expressed as mean ± SEM (n = 7–8 per group).

Figure 7.
The absence of changes in mitochondrial proteins involved in ADP transport.

Adenine nucleotide translocase 1 and 2 (ANT1 and ANT2), voltage-dependent anion channel (VDAC) and mitochondrial creatine kinase (MiCK) protein abundance (A) were not different between genotypes (WT and MCAT mice) or following acute exercise (Sed and Ex). Representative Western blots are shown in (B). Data expressed as mean ± SEM (n = 7–8 per group).

Discussion

The present data indicate that while mitochondrial ROS does not influence CPT-I substrate sensitivity, mitochondrial-derived ROS attenuates ADP sensitivity during exercise, as transgenic mice displaying lower mitochondrial ROS production (i.e. MCAT mice) retain ADP respiratory sensitivity, and the ability of ADP to suppress mitochondrial ROS emission, following a single bout of moderate-intensity exercise. In addition, the inhibitory effects of P-CoA on ADP transport were not altered with exercise or MCAT expression, suggesting that ROS does not influence the interaction between P-CoA and ANT. Altogether, the present data establish that exercise-mediated mitochondrial ROS production is a regulator of oxidative phosphorylation, and implicate mitochondrial ROS in the attenuation of ADP sensitivity that occurs with exercise.

Regulation of P-CoA sensitivity by ROS following acute exercise

Classically, the regulation of mitochondrial fatty acid transport has been attributed to a reduction in M-CoA content, which allows for an increase in flux through CPT-I [26]. However, previous work has emphasized that M-CoA content can remain unchanged following exercise [27,28], suggesting the existence of additional regulatory processes. In the present study, we report that M-CoA sensitivity is decreased after acute exercise, supporting previous work [18]. Specifically, we show that while M-CoA attenuated P-CoA sensitivity in sedentary animals, after exercise, M-CoA did not have an effect on P-CoA sensitivity. Consequently, the P-CoA sensitivity in the presence of M-CoA was improved with exercise, as the apparent P-CoA Km was reduced ∼40% in both genotypes. While it remains possible that the lack of difference between genotypes represents a type 2 error as a result of a low sample size, the reduction in the apparent P-CoA Km following exercise was almost identical between genotypes (∼28 µM P-CoA reduction: interaction P = 0.76), suggesting this is unlikely. Therefore, these data highlight the ability of CPT-I to modulate M-CoA sensitivity during exercise and suggest that mitochondrial-derived ROS does not influence CPT-I substrate sensitivity.

Regulation of ADP sensitivity by ROS during exercise

During exercise, cellular energy demand can be augmented over 100-fold, and ADP provision to the mitochondria must increase to sustain adequate ATP production via oxidative phosphorylation (reviewed in ref. [29]). In the present study, exercise consistently induced a decrease in mitochondrial ADP sensitivity in WT mice, as the apparent ADP Km increased in the presence and absence of P-CoA, and the ability of ADP to suppress mitochondrial H2O2 emission was attenuated, supporting previous research in rodents [4,5] and humans [3,30]. In contrast, exercise did not alter ADP sensitivity in MCAT mice in any of the four independent experiments conducted (three independent ADP titrations and ADP-mediated H2O2 suppression), suggesting exercise-induced mitochondrial ROS emission is a regulator of ADP sensitivity. While the exact mechanism through which ADP sensitivity was impaired during exercise is currently unknown, ANT contains redox-sensitive cysteine residues on its matrix-facing side, and the formation of a disulfide bridge between Cys 160 and 257 [10,12] causes a conformational change that decreases ADP-binding affinity [13]. Therefore, it is possible that mitochondrial ROS exerts post-translational regulatory control on ANT, explaining the current observations.

While the functional consequence to the impairment in mitochondrial ADP sensitivity that occurs during exercise remains debatable, in the current study exercise tolerance was marginally impaired in MCAT mice, which may suggest that the impairment in ADP sensitivity is required to optimize exercise performance. The reduction in mitochondrial ADP sensitivity that occurs during exercise does not likely compromise aerobic respiration, as whole body VO2 remains constant during prolonged exercise despite the reduction in ADP sensitivity [30]. Alternatively, the attenuation in ADP sensitivity may contribute to the well-characterized rise in cytosolic-free ADP that manifests during prolonged steady-state exercise [31]. While speculative, this increase in free ADP may be instrumental in optimizing substrate availability, as this response would be expected to activate the rate-limiting enzymes in glycogenolysis when glycogen granules become diminished (e.g. phosphorylase, phosphofructokinase, and pyruvate dehydrogenase) [32,33], while also modifying AMPK activity, a response associated with an increase in the capacity for plasma membrane substrate transport [34]. It should be noted that the impairment in exercise capacity in MCAT animals is modest; however, humans rely much more heavily on intramuscular glycogen during exercise [35,36] and therefore, fluctuations in mitochondrial ADP sensitivity may be important for optimizing exercise capacity. In addition, we have previously shown that attenuations in ADP sensitivity align with redox stress and the induction of mitochondrial biogenesis following acute and chronic exercise [5], also suggesting a key role for diminished ADP sensitivity in exercise-induced gene transcription. In support of this, in the present study, exercise-mediated impairments in ADP responsiveness aligned with increased succinate-supported ROS in WT mice following exercise. In this manner, an increase in exercise-induced mitochondrial-derived ROS may be required to attenuate mitochondrial ADP sensitivity, serving as a feedforward signal that exaggerates redox stress and induces mitochondrial biogenesis. To investigate the potential link between ROS and P-CoA inhibition of ANT, we also examined the impact of P-CoA on mitochondrial ADP sensitivity. Previously, in vitro work has established that P-CoA interacts with ANT via high-affinity binding on both its IMS and matrix-facing sides [37], and competitively inhibits ADP binding [14,38]. The current experiments were completed in the absence of l-carnitine, which prevented the facilitated transport of P-CoA across the inner mitochondrial membrane [15]. Therefore, the inhibitory effect of P-CoA was isolated to interactions with the intermembrane-facing side of ANT. In the present study, while P-CoA increased the apparent Km for ADP in all groups, the basic pattern in mitochondrial ADP sensitivity was similar in the presence and absence of P-CoA, suggesting that mitochondrial ROS and acute exercise do not modify the inhibitory effect of P-CoA.

Conclusions and perspectives

The present data indicate that mitochondrial-derived ROS contributes to the impairment in mitochondrial ADP sensitivity during exercise, as attenuating mitochondrial ROS through the overexpression of catalase within the matrix prevented exercise-mediated alterations in mitochondrial respiratory ADP sensitivity and mitochondrial H2O2 emission in the presence of ADP. Mechanistically, the location within the mitochondria where ROS is acting to promote these observations remains to be determined; however, ANT and ATP synthase are likely explanations. In the context of ATP synthase, redox-sensitive sites exist within the α-subunits and oxidation of these sites have been shown to reduce its activity [39]. However, oxidation of ATP synthase has been shown to affect maximal activity [39], while in the present study maximal respiration was not altered. It is therefore unlikely that oxidation of ATP synthase contributes to the impairment in ADP sensitivity observed during exercise. Alternatively, previous work has suggested that ROS can interact with redox-sensitive cysteine residues located on the matrix-facing side of ANT [13], which would decrease ADP-binding affinity in the intermembrane space. In addition, aging [11,40,41] and high fat diet [42] have both been associated with impaired mitochondrial ADP sensitivity in concert with attenuated carboxyatractyloside sensitivity and/or redox modifications of ANT. Carboxyatractyloside binds to the ADP-binding pocket on ANT [43], and a reduced sensitivity to this inhibitor suggests a structural change on ANT in diverse models linked to reduced ADP sensitivity. Nevertheless, a similar situation has not been examined following exercise, and therefore, it remains unknown if redox-mediated changes on ANT account for the attenuated ADP sensitivity observed in the present study. Altogether, while future work is required to elucidate the exact location of redox-mediated impairments in ADP sensitivity, the present study provides the necessary framework to further investigate redox signaling as a regulator of oxidative phosphorylation and cellular homeostasis during exercise.

Abbreviations

     
  • ADP

    adenosine diphosphate

  •  
  • ANT

    adenine nucleotide translocase

  •  
  • ATP

    adenosine triphosphate

  •  
  • CPT-I

    carnitine-palmitoyl transferase-I

  •  
  • M-CoA

    malonyl CoA

  •  
  • MCAT

    mitochondrial-targeted catalase transgenic mice

  •  
  • Mi-CK

    mitochondrial creatine kinase

  •  
  • P-CoA

    palmitoyl-CoA

  •  
  • ROS

    reactive oxygen species

  •  
  • WT

    wild type

Author Contribution

All authors analyzed data, interpreted results, and drafted the manuscript. P.-A.B. and G.P.H. designed the study, while P.-A.B. and P.M.M. conducted experiments. P.-A.B. wrote the first draft of the manuscript, but all authors edited and approved the final version for submission.

Funding

This work was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) (G.P.H.), and infrastructure was purchased with assistance from the Canadian Foundation for Innovation/Ontario Research Fund. P.M.M. is supported by an NSERC graduate scholarship.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

References

References
1
Dudley
,
G.A.
,
Tullson
,
P.C.
and
Terjung
,
R.L.
(
1987
)
Influence of mitochondrial content on the sensitivity of respiratory control
.
J. Biol. Chem.
262
,
9109
9114
PMID:
[PubMed]
2
Holloszy
,
J.O.
and
Coyle
,
E.F.
(
1984
)
Adaptations of skeletal muscle to endurance exercise and their metabolic consequences
.
J. Appl. Physiol.
56
,
831
838
3
Perry
,
C.G.
,
Kane
,
D.A.
,
Herbst
,
E.A.
,
Mukai
,
K.
,
Lark
,
D.S.
,
Wright
,
D.C.
et al
(
2012
)
Mitochondrial creatine kinase activity and phosphate shuttling are acutely regulated by exercise in human skeletal muscle
.
J. Physiol.
590
,
5475
5486
4
Miotto
,
P.M.
and
Holloway
,
G.P.
(
2016
)
In the absence of phosphate shuttling, exercise reveals the in vivo importance of creatine-independent mitochondrial ADP transport
.
Biochem. J.
473
,
2831
2843
5
Miotto
,
P.M.
and
Holloway
,
G.P.
(
2018
)
Exercise-induced reductions in mitochondrial ADP sensitivity contribute to the induction of gene expression and mitochondrial biogenesis through enhanced mitochondrial H2O2 emission
.
Mitochondrion
6
Place
,
N.
,
Ivarsson
,
N.
,
Venckunas
,
T.
,
Neyroud
,
D.
,
Brazaitis
,
M.
,
Cheng
,
A.J.
et al
 (
2015
)
Ryanodine receptor fragmentation and sarcoplasmic reticulum Ca2+ leak after one session of high-intensity interval exercise
.
Proc. Natl Acad. Sci. U.S.A.
112
,
15492
15497
7
Klingenberg
,
M.
(
2008
)
The ADP and ATP transport in mitochondria and its carrier
.
Biochim. Biophys. Acta, Biomembranes
1778
,
1978
2021
8
Mielke
,
C.
,
Lefort
,
N.
,
McLean
,
C.G.
,
Cordova
,
J.M.
,
Langlais
,
P.R.
,
Bordner
,
A.J.
et al
 (
2014
)
Adenine nucleotide translocase is acetylated in vivo in human muscle: Modeling predicts a decreased ADP affinity and altered control of oxidative phosphorylation
.
Biochemistry
53
,
3817
3829
9
Feng
,
J.
,
Zhu
,
M.
,
Schaub
,
M.C.
,
Gehrig
,
P.
,
Roschitzki
,
B.
,
Lucchinetti
,
E
et al et al (
2008
)
Phosphoproteome analysis of isoflurane-protected heart mitochondria: phosphorylation of adenine nucleotide translocator-1 on Tyr194 regulates mitochondrial function
.
Cardiovasc. Res.
80
,
20
29
10
Queiroga
,
C.S.F.
,
Almeida
,
A.S.
,
Martel
,
C.
,
Brenner
,
C.
,
Alves
,
P.M.
and
Vieira
,
H.L.
(
2010
)
Glutathionylation of adenine nucleotide translocase induced by carbon monoxide prevents mitochondrial membrane permeabilization and apoptosis
.
J. Biol. Chem.
285
,
17077
17088
11
Yan
,
L.-J.
and
Sohal
,
R.S.
(
1998
)
Mitochondrial adenine nucleotide translocase is modified oxidatively during aging
.
Proc. Natl Acad. Sci. U.S.A.
95
,
12896
12901
12
Costantini
,
P.
,
Belzacq
,
A.-S.
,
Vieira
,
H.L.
,
Larochette
,
N.
,
de Pablo
,
M.A.
,
Zamzami
,
N.
et al
 (
2000
)
Oxidation of a critical thiol residue of the adenine nucleotide translocator enforces Bcl-2-independent permeability transition pore opening and apoptosis
.
Oncogene
19
,
307
314
13
McStay
,
G.P.
,
Clarke
,
S.J.
and
Halestrap
,
A.P.
(
2002
)
Role of critical thiol groups on the matrix surface of the adenine nucleotide translocase in the mechanism of the mitochondrial permeability transition pore
.
Biochem. J.
367
,
541
548
14
Ho
,
C.H.
and
Pande
,
S.V.
(
1974
)
On the specificity of the inhibition of adenine nucleotide translocase by long chain acyl-coenzyme A esters
.
Biochim. Biophys. Acta, Lipids Lipid Metabol.
369
,
86
94
15
Ludzki
,
A.
,
Paglialunga
,
S.
,
Smith
,
B.K.
,
Herbst
,
E.A.F.
,
Allison
,
M.K.
,
Heigenhauser
,
G.J.
et al
 (
2015
)
Rapid repression of ADP transport by palmitoyl-CoA Is attenuated by exercise training in humans: A potential mechanism to decrease oxidative stress and improve skeletal muscle insulin signaling
.
Diabetes
64
,
2769
2779
16
Watt
,
M.J.
,
Heigenhauser
,
G.J.F.
,
O'Neill
,
M.
and
Spriet
,
L.L.
(
2003
)
Hormone-sensitive lipase activity and fatty acyl-CoA content in human skeletal muscle during prolonged exercise
.
J. Appl. Physiol.
95
,
314
321
17
McGarry
,
J.D.
,
Mills
,
S.E.
,
Long
,
C.S.
and
Foster
,
D.W.
(
1983
)
Observations on the affinity for carnitine, and malonyl-CoA sensitivity, of carnitine palmitoyltransferase I in animal and human tissues. Demonstration of the presence of malonyl-CoA in non-hepatic tissues of the rat
.
Biochem. J.
214
,
21
28
18
Miotto
,
P.M.
,
Steinberg
,
G.R.
and
Holloway
,
G.P.
(
2017
)
Controlling skeletal muscle CPT-I malonyl-CoA sensitivity: the importance of AMPK-independent regulation of intermediate filaments during exercise
.
Biochem. J.
474
,
557
569
19
Go
,
Y.-M.
,
Roede
,
J.R.
,
Orr
,
M.
,
Liang
,
Y.
and
Jones
,
D.P.
(
2014
)
Integrated redox proteomics and metabolomics of mitochondria to identify mechanisms of cd toxicity
.
Toxicol. Sci.
139
,
59
73
20
Schriner
,
S.E.
,
Linford
,
N.J.
,
Martin
,
G.M.
,
Treuting
,
P.
,
Ogburn
,
C.E.
,
Emond
,
M.
et al
 (
2005
)
Extension of murine life span by overexpression of catalase targeted to mitochondria
.
Science
308
,
1909
1911
21
Perry
,
C.G.R.
,
Kane
,
D.A.
,
Lin
,
C.-T.
,
Kozy
,
R.
,
Cathey
,
B.L.
,
Lark
,
D.S.
et al
 (
2011
)
Inhibiting myosin-ATPase reveals a dynamic range of mitochondrial respiratory control in skeletal muscle
.
Biochem. J.
437
,
215
222
22
Lally
,
J.S.V.
,
Herbst
,
E.A.F.
,
Matravadia
,
S.
,
Maher
,
A.C.
,
Perry
,
C.G.R.
,
Ventura-Clapier
,
R
et al et al (
2013
)
Over-expressing mitofusin-2 in healthy mature mammalian skeletal muscle does not alter mitochondrial bioenergetics
.
PLoS ONE
8
,
e55660
23
Herbst
,
E.A.F.
,
Paglialunga
,
S.
,
Gerling
,
C.
,
Whitfield
,
J.
,
Mukai
,
K.
,
Chabowski
,
A.
et al
(
2014
)
Omega-3 supplementation alters mitochondrial membrane composition and respiration kinetics in human skeletal muscle
.
J. Physiol.
592
,
1341
52
24
Anderson
,
E.J.
,
Lustig
,
M.E.
,
Boyle
,
K.E.
,
Woodlief
,
T.L.
,
Kane
,
D.A.
,
Lin
,
C.-T.
et al
(
2009
)
Mitochondrial H2O2 emission and cellular redox state link excess fat intake to insulin resistance in both rodents and humans
.
J. Clin. Invest.
119
,
573
581
25
Setoyama
,
D.
,
Fujimura
,
Y.
and
Miura
,
D.
(
2013
)
Metabolomics reveals that carnitine palmitoyltransferase-1 is a novel target for oxidative inactivation in human cells
.
Genes Cells
18
,
1107
1119
26
Winder
,
W.W.
(
1998
)
Intramuscular mechanisms regulating fatty acid oxidation during exercise
.
Adv. Exp. Med. Biol.
441
,
239
248
27
Odland
,
L.M.
,
Heigenhauser
,
G.J.
,
Lopaschuk
,
G.D.
and
Spriet
,
L.L.
(
1996
)
Human skeletal muscle malonyl-CoA at rest and during prolonged submaximal exercise
.
Am. J. Physiol.
270
,
E541
E544
28
Roepstorff
,
C.
,
Halberg
,
N.
,
Hillig
,
T.
,
Saha
,
A.K.
,
Ruderman
,
N.B.
,
Wojtaszewski
,
J.F.P.
et al
 (
2005
)
Malonyl-CoA and carnitine in regulation of fat oxidation in human skeletal muscle during exercise
.
Am. J. Physiol. Endocrinol. Metab.
288
,
E133
E142
29
Egan
,
B.
and
Zierath
,
J.R.
(
2013
)
Exercise metabolism and the molecular regulation of skeletal muscle adaptation
.
Cell Metab.
17
,
162
184
30
Tonkonogi
,
M.
,
Harris
,
B.
and
Sahlin
,
K.
(
1998
)
Mitochondrial oxidative function in human saponin-skinned muscle fibres: effects of prolonged exercise
.
J. Physiol.
510
,
279
286
31
Phillips
,
S.M.
,
Green
,
H.J.
,
Tarnopolsky
,
M.A.
,
Heigenhauser
,
G.J.
and
Grant
,
S.M.
(
1996
)
Progressive effect of endurance training on metabolic adaptations in working skeletal muscle
.
Am. J. Physiol.
270
,
E265
E272
32
Howlett
,
R.A.
,
Parolin
,
M.L.
,
Dyck
,
D.J.
,
Hultman
,
E.
,
Jones
,
N.L.
,
Heigenhauser
,
G.J
et al et al (
1998
)
Regulation of skeletal muscle glycogen phosphorylase and PDH at varying exercise power outputs
.
Am. J. Physiol.
275
,
R418
R425
PMID:
[PubMed]
33
Peters
,
S.J.
and
Spriet
,
L.L.
(
1995
)
Skeletal muscle phosphofructokinase activity examined under physiological conditions in vitro
.
J. Appl. Physiol.
78
,
1853
1858
34
Xiao
,
B.
,
Sanders
,
M.J.
,
Underwood
,
E.
,
Heath
,
R.
,
Mayer
,
F.V.
,
Carmena
,
D.
et al
 (
2011
)
Structure of mammalian AMPK and its regulation by ADP
.
Nature
472
,
230
233
35
Pederson
,
B.A.
,
Cope
,
C.R.
,
Schroeder
,
J.M.
,
Smith
,
M.W.
,
Irimia
,
J.M.
,
Thurberg
,
B.L.
et al
 (
2005
)
Exercise capacity of mice genetically lacking muscle glycogen synthase: in mice, muscle glycogen is not essential for exercise
.
J. Biol. Chem.
280
,
17260
17265
36
Bergström
,
J.
,
Hermansen
,
L.
,
Hultman
,
E.
and
Saltin
,
B.
(
1967
)
Diet, muscle glycogen and physical performance
.
Acta Physiol. Scand.
71
,
140
150
37
Woldegiorgis
,
G.
and
Shrago
,
E.
(
1979
)
The recognition of two specific binding sites of the adenine nucleotide translocase by palmitoyl CoA in bovine heart mitochondria and submitochondrial particles
.
Biochem. Biophys. Res. Commun.
89
,
837
844
38
Shug
,
A.L.
,
Shrago
,
E.
,
Bittar
,
N.
,
Folts
,
J.D.
and
Koke
,
J.R.
(
1975
)
Acyl-CoA inhibition of adenine nucleotide translocation in ischemic myocardium
.
Am. J. Physiol.
228
,
689
692
39
Bald
,
D.
,
Noji
,
H.
,
Yoshida
,
M.
,
Hirono-Hara
,
Y.
and
Hisabori
,
T.
(
2001
)
Redox regulation of the rotation of F1-ATP synthase
.
J. Biol. Chem.
276
,
39505
39507
40
Holloway
,
G.P.
,
Holwerda
,
A.M.
,
Miotto
,
P.M.
,
Dirks
,
M.L.
,
Verdijk
,
L.B.
and
van Loon
,
L.J.C.
(
2018
)
Age-associated impairments in mitochondrial ADP sensitivity contribute to redox stress in senescent human skeletal muscle
.
Cell Rep.
22
,
2837
2848
41
Gouspillou
,
G.
,
Bourdel-Marchasson
,
I.
,
Rouland
,
R.
,
Calmettes
,
G.
,
Franconi
,
J.-M.
,
Deschodt-Arsac
,
V
et al et al (
2010
)
Alteration of mitochondrial oxidative phosphorylation in aged skeletal muscle involves modification of adenine nucleotide translocator
.
Biochim. Biophys. Acta, Bioenerg.
1797
,
143
151
42
Miotto
,
P.M.
,
LeBlanc
,
P.J.
and
Holloway
,
G.P.
(
2018
)
High fat diet causes mitochondrial dysfunction as a result of impaired ADP sensitivity
.
Diabetes
.
In
press
43
Vignais
,
P.V.
,
Douce
,
R.
,
Lauquin
,
G.J.M.
and
Vignais
,
P.M.
(
1976
)
Binding of radioactively labeled carboxyatractyloside, atractyloside and bongkrekic acid to the ADP translocator of potato mitochondria
.
Biochim. Biophys. Acta, Bioenerg.
440
,
688
696