Michail Tonkonogi

The role of phosphorylcreatine and creatine in the regulation of mitochondrial respiration in human skeletal muscle.

1 The role of phosphorylcreatine (PCr) and creatine (Cr) in the regulation of mitochondrial respiration was investigated in permeabilised fibre bundles prepared from human vastus lateralis muscle. 2 Fibre respiration was measured in the absence of ADP (V̇0) and after sequential additions of submaximal ADP (0.1 mm ADP, V̇submax), PCr (or Cr) and saturating [ADP] (V̇max). 3 V̇ submax increased by 55% after addition of saturating creatine (P < 0.01; n= 8) and half the maximal effect was obtained at 5 mm[Cr]. In contrast, V̇submax decreased by 54% after addition of saturating phosphorylcreatine (P < 0.01; n= 8) and half the maximal effect was obtained at 1 mm[PCr]. V̇max was not affected by Cr or PCr. 4 V̇ submax was similar when PCr and Cr were added simultaneously at concentrations similar to those in muscle at rest (PCr/Cr = 2) and at low‐intensity exercise (PCr/Cr = 0.5). At conditions mimicking high‐intensity exercise (PCr/Cr = 0.1), V̇submax increased to 60% of V̇max (P < 0.01) vs. rest and low‐intensity exercise). 5 Eight of the subjects participated in a 16 day Cr supplementation programme. Following Cr supplementation, V̇0 decreased by 17% (P < 0.01) vs. prior to Cr supplementation), whereas ADP‐stimulated respiration (with and without Cr or PCr) was unchanged. 6 For the first time evidence is given that PCr is an important regulator of mitochondrial ADP‐stimulated respiration. Phosphorylcreatine decreases the sensitivity of mitochondrial respiration to ADP whereas Cr has the opposite effect. During transition from rest to high‐intensity exercise, decreases in the PCr/Cr ratio will effectively increase the sensitivity of mitochondrial respiration to ADP. The decrease in V̇0 after Cr supplementation indicates that intrinsic changes in membrane proton conductance occur.

Mitochondrial function and antioxidative defence in human muscle: effects of endurance training and oxidative stress

1 The influence of endurance training on oxidative phosphorylation and the susceptibility of mitochondrial oxidative function to reactive oxygen species (ROS) was investigated in skeletal muscle of four men and four women. Mitochondria were isolated from muscle biopsies taken before and after 6 weeks of endurance training. Mitochondrial respiration was measured before and after exposure of mitochondria to exogenous ROS (H2O2+ FeCl2). 2 Endurance training increased peak pulmonary O2 uptake (V̇O2,peak) by 24 % and maximal ADP‐stimulated mitochondrial oxygen consumption (state 3) by 40 % (P < 0.05). Respiration in the absence of ADP (state 4), the respiratory control ratio (RCR = state 3/state 4) and the ratio between added ADP and consumed oxygen (P/O) remained unchanged by the training programme. 3 Exposure to ROS reduced state 3 respiration but the effect was not significantly different between pre‐ and post‐training samples. State 4 oxygen consumption increased after exposure to ROS both before (+189 %, P < 0.05) and after training (+243 %, P < 0.05) and the effect was significantly higher after training (P < 0.05, pre‐ vs. post‐training). The augmented state 4 respiration could in part be attenuated by atractyloside, which indicates that ADP/ATP translocase was affected by ROS. The P/O ratio in ROS‐treated mitochondria was significantly lower (P < 0.05) compared to control conditions, both before (−18.6 ± 2.2 %) and after training (−18.5 ± 1.1 %). 4 Muscle activities of superoxide dismutase (mitochondrial and cytosolic), glutathione peroxidase and muscle glutathione status were unaffected by training. There was a positive correlation between muscle superoxide dismutase activity and age (r= 0.75; P < 0.05; range of age 20–37 years), which may reflect an adaptation to increased generation of ROS in senescent muscle. The muscle glutathione pool was more reduced in subjects with high activity of glutathione peroxidase (r= 0.81; P < 0.05). 5 The influence of short‐term training on mitochondrial oxygen consumption has for the first time been investigated in human skeletal muscle. The results showed that maximal mitochondrial oxidative power is increased after endurance training but that the efficiency of energy transfer (P/O ratio) remained unchanged. Antioxidative defence was unchanged after training when expressed relative to muscle weight. Although this corresponds to a reduced antioxidant protection per individual mitochondrion, the sensitivity of aerobic energy transfer to ROS was unchanged. However, the augmented ROS‐induced non‐coupled respiration after training indicates an increased susceptibility of mitochondrial membrane proton conductance to oxidative stress.

Effects of acute and chronic endurance exercise on mitochondrial uncoupling in human skeletal muscle

Mitochondrial proteins such as uncoupling protein 3 (UCP3) and adenine nucleotide translocase (ANT) may mediate back‐leakage of protons and serve as uncouplers of oxidative phosphorylation. We hypothesized that UCP3 and ANT increase after prolonged exercise and/or endurance training, resulting in increased uncoupled respiration (UCR). Subjects were investigated with muscle biopsies before and after acute exercise (75 min of cycling at 70% of ) or 6 weeks endurance training. Mitochondria were isolated and respiration measured in the absence (UCR or state 4) and presence of ADP (coupled respiration or state 3). Protein expression of UCP3 and ANT was measured with Western blotting. After endurance training , citrate synthase activity (CS), state 3 respiration and ANT increased by 24, 47, 40 and 95%, respectively (all P < 0.05), whereas UCP3 remained unchanged. When expressed per unit of CS (a marker of mitochondrial volume) UCP3 and UCR decreased by 54% and 18%(P < 0.05). CS increased by 43% after acute exercise and remained elevated after 3 h of recovery (P < 0.05), whereas the other muscle parameters remained unchanged. An intriguing finding was that acute exercise reversibly enhanced the capacity of mitochondria to accumulate Ca2+(P < 0.05) before opening of permeability transition pores. In conclusion, UCP3 protein and UCR decrease after endurance training when related to mitochondrial volume. These changes may prevent excessive basal thermogenesis. Acute exercise enhances mitochondrial resistance to Ca2+ overload but does not influence UCR or protein expression of UCP3 and ANT. The increased Ca2+ resistance may prevent mitochondrial degradation and the mechanism needs to be further explored.

Mitochondrial oxidative function in human saponin-skinned muscle fibres: effects of prolonged exercise.

1 The influence of prolonged exhaustive exercise on mitochondrial oxidative function was investigated in ten men. 2 Muscle biopsies were taken before and after exercise and mitochondrial respiration investigated in fibre bundles made permeable by pretreatment with saponin. 3 After exercise, respiration in the absence of ADP increased by 18 % (P < 0.01), but respiration at suboptimal ADP concentration (0.1 mM) and maximal ADP‐stimulated respiration (1 mM ADP) remained unchanged. 4 In the presence of creatine (20 mM), mitochondrial affinity for ADP increased markedly and respiration at suboptimal ADP concentration (0.1 mM) was similar (pre‐exercise) or higher (post‐exercise; P < 0.05) than with 1 mM ADP alone. The increase in respiratory rate with creatine was correlated to the relative type I fibre area (r= 0.84). Creatine‐stimulated respiration increased after prolonged exercise (P < 0.01). 5 The respiratory control index (6.8 ± 0.4, mean ± s.e.m.) and the ratio between respiration at 0.1 and 1 mM ADP (ADP sensitivity index, 0.63 ± 0.03) were not changed after exercise. The sensitivity index was negatively correlated to the relative type I fibre area (r=−0.86). 6 The influence of exercise on muscle oxidative function has for the first time been investigated with the skinned‐fibre technique. It is concluded that maximal mitochondrial oxidative power is intact or improved after prolonged exercise, while uncoupled respiration is increased. The latter finding may contribute to the elevated post‐exercise oxygen consumption. The finding that the sensitivity of mitochondrial respiration for ADP and creatine are related to fibre‐type composition indicates intrinsic differences in the control of mitochondrial respiration between fibres.

Physical exercise and mitochondrial function in human skeletal muscle.

TONKONOGI, M., and K. SAHLIN. Physical exercise and mitochondrial function in human skeletal muscle. Exerc. Sport Sci. Rev., Vol. 30, No. 3, pp. 129–137, 2002. Muscle adaptation to endurance training involves qualitative changes in intrinsic properties of mitochondria. After training, the ADP sensitivity of mitochondrion is decreased whereas the effect of creatine on respiration is increased. This results in an improved control of aerobic energy production. Acute exercise does not adversely affect mitochondrial function.

Effect of eccentric exercise on muscle oxidative metabolism in humans.

PURPOSE The purpose of this study was to evaluate the effects of eccentric exercise on muscle oxidative function. METHODS Thirteen subjects performed high-intensity eccentric cycling for 30 min. Muscle oxidative function in vastus lateralis was evaluated by measurements of respiration in permeabilized muscle fibers (skinned fibers) and from the kinetics of oxyhemoglobin (oxyHb) saturation measured with near infrared spectroscopy (NIRS). RESULTS After eccentric cycling, all subjects reported extensive delayed onset muscle soreness (DOMS), but plasma markers of muscle damage (creatine kinase and beta-glucuronidase activity) were not significantly altered. The half time of oxyHb desaturation after circulatory occlusion (128 +/- 11 s, mean +/- SE) and oxyHb resaturation after restoration of blood flow (13.8 +/- 0.7 s) were not significantly changed after eccentric cycling (N = 7). Respiration in skinned muscle fibers measured in the absence of ADP and in the presence of a submaximal (0.1 mM) or maximal ADP concentration (1 mM) was not significantly changed after eccentric cycling (N = 6). The sensitivity of respiration to ADP was not significantly changed after eccentric cycling. CONCLUSIONS Muscle oxidative function (maximal respiration and respiratory control by ADP) was not compromised after high-intensity eccentric cycle exercise. Furthermore, NIRS indicates that after eccentric cycling muscle oxygen utilization and local oxygen transport at rest are unchanged. It is concluded that eccentric cycling, although causing DOMS, does not negatively affect skeletal muscle oxidative function.

Ultraendurance exercise increases the production of reactive oxygen species in isolated mitochondria from human skeletal muscle

Exercise-induced oxidative stress is important for the muscular adaptation to training but may also cause muscle damage. We hypothesized that prolonged exercise would increase mitochondrial production of reactive oxygen species (ROS) measured in vitro and that this correlates with oxidative damage. Eight male athletes (24-32 yr) performed ultraendurance exercise (kayaking/running/cycling) with an average work intensity of 55% V(O(2peak)) for 24 h. Muscle biopsies were taken from vastus lateralis before exercise, immediately after exercise, and after 28 h of recovery. The production of H(2)O(2) was measured fluorometrically in isolated mitochondria with the Amplex red and peroxidase system. Succinate-supported mitochondrial H(2)O(2) production was significantly increased after exercise (73% higher, P = 0.025) but restored to the initial level at recovery. Plasma level of free fatty acids (FFA) increased fourfold and exceeded 1.2 mmol/l during the last 6 h of exercise. Plasma FFA at the end of exercise was significantly correlated to mitochondrial ROS production (r = 0.74, P < 0.05). Mitochondrial content of 4-hydroxy-nonenal-adducts (a marker of oxidative damage) was increased only after recovery and was not correlated with mitochondrial ROS production. Total thiol group level and glutathione peroxidase activity were elevated after recovery. In conclusion, ultraendurance exercise increases ROS production in isolated mitochondria, but this is reversed after 28 h recovery. Mitochondrial ROS production was not correlated with oxidative damage of mitochondrial proteins, which was increased at recovery but not immediately after exercise.

No evidence of an intracellular lactate shuttle in rat skeletal muscle

The concerted view is that cytosolic pyruvate is transferred into mitochondria and after oxidative decarboxylation further metabolized in the tricarboxylic acid cycle. Recently this view has been challenged. Based on experimental evidence from rat skeletal muscle it has been concluded that mitochondria predominantly oxidize lactate in vivo and that this constitutes part of an ‘intracellular lactate shuttle’. This view appears to be gaining acceptance in the scientific community and due to its conceptual importance, confirmation by independent experiments is required. We have repeated the experiments in mitochondria isolated from rat soleus muscle. Contrary to the previously published findings we cannot find any mitochondrial respiration with lactate. Analysis of lactate dehydrogenase (LDH) by spectrophotometry demonstrated that the activity in the mitochondrial fraction was only 0.7 % of total activity. However, even when external LDH was added to mitochondria, there were no signs of respiration with lactate. In the presence of conditions where lactate is converted to pyruvate (external additions of both LDH and NAD+), mitochondrial oxygen consumption increased. Furthermore, we provide theoretical evidence that direct mitochondrial lactate oxidation is energetically unlikely. Based on the present data we conclude that direct mitochondrial lactate oxidation does not occur in skeletal muscle. The presence of an ‘intracellular lactate shuttle’ can therefore be questioned.

Mitochondrial function in human skeletal muscle is not impaired by high intensity exercise.

Abstract The hypothesis that high-intensity (HI) intermittent exercise impairs mitochondrial function was investigated with different microtechniques in human muscle samples. Ten male students performed three bouts of cycling at 130% of peak O2 consumption (V·O2,peak). Muscle biopsies were taken from the vastus lateralis muscle at rest, at fatigue and after 110 min recovery. Mitochondrial function was measured both in isolated mitochondria and in muscle fibre bundles made permeable with saponin (skinned fibres). In isolated mitochondria there was no change in maximal respiration, rate of adenosine 5’-triphosphate (ATP) production (measured with bioluminescence) and respiratory control index after exercise or after recovery. The ATP production per consumed oxygen (P/O ratio) also remained unchanged at fatigue but decreased by 4% (P<0.05) after recovery. In skinned fibres, maximal adenosine 5’-diphosphate (ADP)-stimulated respiration increased by 23% from rest to exhaustion (P<0.05) and remained elevated after recovery, whereas the respiratory rates in the absence of ADP and at 0.1 mM ADP (submaximal respiration) were unchanged. The ratio between respiration at 0.1 and 1 mM ADP (ADP sensitivity index) decreased at fatigue (P<0.05) but after the recovery period was not significantly different from that at rest. It is concluded that mitochondrial oxidative potential is maintained or improved during exhaustive HI exercise. The finding that the sensitivity of mitochondrial respiration to ADP is reversibly decreased after strenuous exercise may indicate that the control of mitochondrial respiration is altered.

Endurance training increases stimulation of uncoupling of skeletal muscle mitochondria in humans by non-esterified fatty acids: an uncoupling-protein-mediated effect?

Uncoupled respiration (UCR) is an essential property of muscle mitochondria and has several functions in the cell. We hypothesized that endurance training may alter the magnitude and properties of UCR in human muscle. Isolated mitochondria from muscle biopsies taken before and after 6 weeks of endurance exercise training (n=8) were analysed for UCR. To investigate the role of uncoupling protein 2 (UCP2) and UCP3 in UCR, the sensitivity of UCR to UCP-regulating ligands (non-esterified fatty acids and purine nucleotides) and UCP2 and UCP3 mRNA expression in muscle were examined. Oleate increased the mitochondrial oxygen consumption rate, an effect that was not attenuated by GDP and/or cyclosporin A. The effect of oleate was significantly greater after compared with before training. Training had no effect on UCP2 or UCP3 mRNA levels, but after training the relative increase in respiration rate induced by oleate was positively correlated with the UCP2 mRNA level. In conclusion, we show that the sensitivity of UCR to non-esterified fatty acids is up-regulated by endurance training. This suggests that endurance training causes intrinsic changes in mitochondrial function, which may enhance the potential for regulation of aerobic energy production, prevent excess free radical generation and contribute to a higher basal metabolic rate.