- Email: [email protected]
Exercise as an antioxidant: it up-regulates important enzymes for cell adaptations to exercise
Science & Sports 21 (2006) 85–89 http://france.elsevier.com/direct/SCISPO/
Article original
Exercise as an antioxidant: it up-regulates important enzymes for cell adaptations to exercise Effet anti-oxydant de l’exercice : up-régulation d’enzymes clés de l’adaptation cellulaire à l’exercice M.C. Gomez-Cabrera a, E. Domenech C, L.L. Ji b, J. Viña c,* a
Catholic University of Valencia, Valencia, Spain University of Wisconsin-Madison, Wisconsin, USA c Department of Physiology, Faculty of Medicine, University of Valencia, Avda. Blasco Ibanez 17, 46010 Valencia, Spain b
Accepted 27 June 2005 Available online 04 April 2006
Abstract Aims. – To assess the role of the reactive oxygen species (ROS) in cell signalling and in the regulation of gene expression. Methods. – Exercise causes oxidative stress only when exhaustive. Strenuous exercise causes oxidation of glutathione, release of cytosolic enzymes, and other signs of cell damage. We have tested this hypothesis by studying the effect of inhibition of ROS production by allopurinol (an inhibitor of xanthine oxidase, a free radical generating enzyme) on cell signalling pathways in marathon runners and in rats submitted to exhaustive exercise by running on a treadmill. Results. – Exercise caused an activation of NF-κB in lymphocytes from marathon runners which was completely prevented by allopurinol. In the rat model exercise caused an activation of MAP kinases in gastrocnemius muscle. This in turn activated the NF-κB pathway and consequently the expression of important enzymes associated with defence against ROS (superoxide dismutase) and adaptation to exercise (eNOS and iNOS). All these changes were abolished when ROS production was prevented by allopurinol. Conclusion. – Thus we report evidence that ROS act as signals in exercise because decreasing their formation prevents activation of important signalling pathways which cause useful adaptations in cells. Because these signals result in an up-regulation of powerful antioxidant enzymes, exercise itself can be considered as an antioxidant. © 2006 Elsevier SAS. All rights reserved. Résumé Objectifs. – Déterminer le rôle des espèces oxygénées réactives (ROS) dans la signalisation cellulaire et dans la régulation de l’expression des gènes. Matériel et méthodes. – L’exercice est à l’origine d’un stress oxydatif seulement lorsqu’il est maximal. Il provoque l’oxydation du glutathion, la libération d’enzymes cytosoliques, accompagnée par d’autres signes de dommages cellulaires. Nous avons testé cette hypothèse en étudiant l’effet de l’inhibition de la production de ROS par l’allopurinol (un inhibiteur de la xanthine oxydase, enzyme générateur de radicaux libres) sur les voies de la signalisation cellulaire chez des marathoniens et chez des rats soumis à un exercice épuisant sur tapis roulant. Résultats. – L’exercice provoque une activation de la voie NF-κB dans les lymphocytes des marathoniens, qui est totalement bloquée par l’allopurinol. Chez le Rat, l’exercice entraîne une activation des MAP-kinases du gastrocnémien, ce qui active la voie NF-κB et, par conséquent, l’expression d’enzymes clés de la défense contre les ROS (superoxyde dismutase) et de l’adaptation à l’exercice (eNOS et iNOS). Ces effets sont supprimés si la production de ROS est bloquée par l’allopurinol.
* Corresponding
author. Tel.: +34 96 386 4646; fax: +34 96 386 4642. E-mail address: [email protected] (J. Viña).
0765-1597/$ - see front matter © 2006 Elsevier SAS. All rights reserved. doi:10.1016/j.scispo.2005.06.012
86
M.C. Gomez-Cabrera et al. / Science & Sports 21 (2006) 85–89
Conclusion. – Ce travail met donc en évidence le rôle de signalisation des ROS, dans la mesure où l’arrêt de leur production empêche l’activation de voies de signalisation utiles à l’adaptation cellulaire. Le résultat final étant une « up-régulation » d’enzyme antioxydants, l’exercice lui-même peut être considéré comme un antioxydant. © 2006 Elsevier SAS. All rights reserved. Keywords: Oxidative stress; Adaptation; Cell signalling; Xanthine oxidase; Hormesis Mots clés: Stress oxidative; Adaptation; Signalisation cellulaire; Xanthine oxidase; Hormesis
1. Introduction Over the past two decades it was shown that unaccustomed exercise can increase the generation of reactive oxygen species (ROS) in biological tissues specially skeletal muscle and myocardium [1–4]. Work from our group revealed that exercise generates oxidative stress only when it is exhaustive [5]. Non-exhaustive exercise causes an increased production of ROS which can be counterbalanced by the concurrent increase in antioxidant defences [6]. Thus it is important to distinguish between non-exhaustive exercise, which is not accompanied by oxidative stress, and exercise to exhaustion, which causes oxidative stress. There are several sources of free radicals in exercise. We have focused in the role of xanthine oxidase (XO) an enzyme involved in the ischemia–repefusion syndrome, that is located in the vascular endothelial cells in skeletal muscle [7]. Administration of allopurinol, widely used inhibitor of the enzyme, to rats at a concentration of 32 mg/kg by intraperitoneal injection prevented the oxidation of glutathione, protein oxidation and lipid peroxidation associated with exhaustive exercise [6,8]. This indicated that XO might be a relevant source of ROS in exercise. 2. Methods and results To test this hypothesis in vivo, we performed experiments with the cyclists of the team US Postal during the Tour of France 2001 [9]. Oral administration of a dose of 300 mg of allopurinol during 13 days prevented the increase in the activities of creatine kinase and aspartate-aminotransferase in plasma (markers of muscle damage) only at the stage where participants performed at their peak level of exertion, the Team Time Trial stage. We also found evidence of an increase in plasma malondialdehyde levels in all participants at the end of the race, but the increase was significantly greater in placebo group than in the allopurinol group. These results suggested that XO is involved in the tissue damage associated to exhaustive physical exercise in vivo. We confirmed these data in a different study with marathon runners recruited from participants in the 23rd Marathon of Valencia. As shown in Fig. 1, marathon running induces a significant increase in plasma malondialdehyde levels. This is prevented by treatment with allopurinol. However free radicals not only cause damage but do have a role in cell signalling, receptor stimulation and enzymatic stimulation [10,11]. In Fig. 2 we report that ROS generated in exercise activate an important signalling pathway, the mitogen
activated protein kinases (p38 and ERK1/ERK2) that can activate transcription factors and regulate protein expression in skeletal muscle. Administration of allopurinol prevents this effect. This demonstrates the role of XO derived ROS in the activation of this signalling pathway. There is growing evidence that the continuous presence of small stimuli such as low concentrations of ROS is in fact able to induce the expression of antioxidant enzymes, DNA repair molecules and protein degrading enzymes, resulting in decreases in the incidence of oxidative stress-related diseases and retardation of the aging process [12]. The basis of this phenomenon may be encompassed by the concept of hormesis [13], which can be characterised as a particular dose–response relationship in which a low dose of a substance is stimulatory and a high dose is inhibitory. In this context, radicals may be considered to be beneficial since they act as signals to enhance defences rather than deleterious (as they are when cells are exposed to high levels of ROS). The concept of hormesis has been applied to the beneficial effects of moderate drinking of alcohol [14] and caloric restriction in experimental animals to prevent disease and promote longevity [15]. Recently the hormesis theory has been extended to the ROS generating effects of exercise [12,16,17]. In skeletal muscle hydrogen peroxide at a low concentration increases Ca2+ release from the sarcoplasmic reticulum and force production, whereas a massive increase in hydrogen peroxide concentration results in a sharp decrease in force output [18]. Animals frequently ex-
Fig. 1. NF-κB activation in lymphocytes and plasma MDA levels after marathon running are blocked by treatment with allopurinol. Values are mean ± S.D. Placebo group (n = 11). Allopurinol-treated group (n = 14). (**) indicates P < 0.01 vs. before the marathon. (*) indicates P < 0.05 vs. before the marathon.
M.C. Gomez-Cabrera et al. / Science & Sports 21 (2006) 85–89
87
Fig. 2. Exercise activates p38 and ERK1/ERK2 phosphorylations. Prevention by allopurinol administration. Exercise activates p38 and ERK1/ERK2 phosphorylation in the cytosolic fraction of rat gastrocnemius muscle. Prevention by allopurinol administration.
posed to exercise (chronic training) have shown less oxidative damage after exhaustive exercise than untrained ones. This is largely attributed to the up-regulation of endogenous antioxidant enzymes such as mitochondrial superoxide dismutase (MnSOD), glutathione peroxidase, and γ-glutamylcysteine synthetase (GCS) [19]. Since the adaptive response results from the cumulative effects of repeated exercise bouts, the initial signal for the stimulation leading to the long-term modula-
tion must occur after each individual exercise bout [20]. Several oxidative stress-sensitive signalling pathways are operational in mammalian systems and play an important role in maintaining cellular oxidant–antioxidant balance. One of the most important pathways that may be activated by ROS involves the transcription factor NF-κB [21]. Several antioxidant enzymes contain NF-κB binding sites in their gene promoter region, such as MnSOD, inducible nitric oxide synthetase
Fig. 3. Exercise activates NF-κB and induces up-regulation of Mn-SOD and NO synthases. Prevention by allopurinol administration. (a) EMSA analysis of NF-κB in the nuclear extracts of rat gastrocnemius (Ø competition assay). (b) Expression of Mn-SOD, iNOS and eNOS measured by real time RT-PCR from gastrocnemius muscle of rats at rest, after exercise and after exercise but pretreated with allopurinol (N = 9).
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M.C. Gomez-Cabrera et al. / Science & Sports 21 (2006) 85–89
Different research groups have shown that vitamin C supplementation during training reduces oxidative stress but at the same time attenuates adaptive responses to oxidants [26] and do not improve VO2max or maximal cardiac output [27]. The practical implication is that decreasing ROS levels with antioxidants may hinder beneficial cell adaptations during exercise. Therefore the common practice of taking antioxidant supplements during training should be seriously questioned. Références
Fig. 4. Proposed mechanism of the role of ROS in signalling of cell adaptations after exercise.
(iNOS) and GCS [22]. Therefore, they can be potential targets for exercise-activated up-regulation via NF-κB signalling pathway. Hollander et al. [23] first reported that an acute bout of treadmill running activated MnSOD gene expression in rat skeletal muscle, along with enhanced NF-κB binding in muscle nuclear extracts ~2 h after exercise. Vider et al. [24] showed that physical exercise (80% of maximal O2 consumption for 1 h) resulted in NF-κB activation in peripheral blood lymphocytes of physically fit young men. Plasma levels of TNF and IL-2 receptor in these subjects were also elevated. However our findings go further. Recently, we have investigated the effect of rigorous muscular contraction on NF-κB signalling pathway in two separate studies: in rat skeletal muscle [8,25] and in peripheral lymphocytes of marathon runners. As shown in Fig. 1, marathon running induces activation of the p50 subunit of the NF-κB complex in lymphocytes. This is prevented by treatment with allopurinol. In this study, we showed that XO derived ROS activates NF-κB signalling pathway in peripheral blood lymphocytes after running a marathon. For obvious ethical reasons, we could not test the explanation for this fact in humans and we had to turn to rats. In Fig. 2, we report that ROS generated in exercise activate MAPKs (p38 and ERK1/ERK2) which in turn activate NF-κB which results in an increased expression of important enzymes associated with cell defence (MnSOD) and adaptation to exercise (eNOS and iNOS). Prevention of ROS formation by inhibition of XO abolishes these effects (Fig. 3). Schematic representation highlighting the role of ROS in the regulation of cell function during exercise is shown in Fig. 4. We indicate that when oxidants are produced at moderate levels they act as signals to adapt cells to exercise. However, when they are overproduced they cause cellular damage.
[1] Davies KJ, Quintanilha AT, Brooks GA, Packer L. Free radicals and tissue damage produced by exercise. Biochem Biophys Res Commun 1982; 107:1198–205. [2] Jackson MJ, Edwards RH, Symons MC. Electron spin resonance studies of intact mammalian skeletal muscle. Biochim Biophys Acta 1985;847: 185–90. [3] Reid MB, et al. Reactive oxygen in skeletal muscle. I. Intracellular oxidant kinetics and fatigue in vitro. J Appl Physiol 1992;73:1797–804. [4] Bejma J, Ramires P, Ji LL. Free radical generation and oxidative stress with aging and exercise: differential effects in the myocardium and liver. Acta Physiol Scand 2000;169:343–51. [5] Sastre J, et al. Exhaustive physical exercise causes oxidation of glutathione status in blood: Prevention by antioxidant administration. Am J Physiol 1992;32:R992–R995. [6] Viña J, et al. Free radicals in exhaustive physical exercise: mechanism of production, and protection by antioxidants. IUBMB Life 2000;50:271–7. [7] Hellsten-Westing Y. Immunohistochemical localization of xanthine oxidase in human cardiac and skeletal muscle. Histochemistry 1993;100: 215–22. [8] Gomez-Cabrera MC, et al. Decreasing Xanthine Oxidase Mediated Oxidative Stress Prevents Useful Cellular Adaptations to Exercise in Rats. J Physiol 2005;567:113–20. [9] Gomez-Cabrera MC, Pallardo FV, Sastre J, Vina J, Garcia-del-Moral L. Allopurinol and markers of muscle damage among participants in the Tour de France. Jama 2003;289:2503–4. [10] Reid MB, Shoji T, Moody MR, Entman ML. Reactive oxygen in skeletal muscle. II. Extracellular release of free radicals. J Appl Physiol 1992;73: 1805–9. [11] Jackson MJ. Free radicals in skin and muscle: damaging agents or signals for adaptation? Proc Nutr Soc 1999;58:673–6. [12] Radak Z, Chung HY, Goto S. Exercise and hormesis: Oxidative stressrelated adaptation for successful aging. An Hypothesis. Biogerontology 2005;6(1):71–5. [13] Calabrese EJ, Baldwin LA. Hormesis: the dose-response revolution. Annu Rev Pharmacol Toxicol 2003;43:175–97. [14] Calabrese EJ, Baldwin LA. Ethanol and hormesis. Crit Rev Toxicol 2003;33:407–24. [15] Sohal RS, Weindruch R. Oxidative stress, caloric restriction, and aging. Science 1996;273:59–63. [16] Ji, L.L., Gómez-Cabrera, M.C., Vina, J., Exercise and Hormesis: Activation of Cellular Antioxidant Signaling Pathway. Ann. New. York. Acad. Sci. (IN PRESS). [17] Vina J, Borras C, Gomez-Cabrera MC, Orr WC. Importance of reactive oxygen species to modulate molecular and cellular response to stress. Free Rad Res 2006;40(2):111–9. [18] Andrade FH, Reid MB, Westerblad H. Contractile response of skeletal muscle to low peroxide concentrations: myofibrillar calcium sensitivity as a likely target for redox-modulation. FASEB J 2001;15:309–11. [19] Salminen A, Vihko V. Endurance training reduces the susceptibility of mouse skeletal muscle to lipid peroxidation in vitro. Acta Physiol Scand 1983;117:109–13.
M.C. Gomez-Cabrera et al. / Science & Sports 21 (2006) 85–89 [20] Hollander J, et al. Superoxide dismutase gene expression is activated by a single bout of exercise. Pflug Arch 2001;442:426–34 (Eur J Physiol). [21] Baeuerle PA, Baltimore D. Activation of DNA binding activity in an apparently cytoplasmic precursor of the NF-κB transcriptional factor. Cell 1998;53:211–7. [22] Laughlin MH, et al. Skeletal muscle oxidative capacity, antioxidant enzymes, and exercise training. JApplPhysiol 1990;68:2337–43. [23] Hollander J, et al. Superoxide dismutase gene expression in skeletal muscle: fiber-specific adaptation to endurance training. Am J Physiol 1999; 277:R856–R862.
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[24] Vider J, et al. Physical Exercise Induces Activation of NF-κB in Human Peripheral Blood Lymphocytes. Antiox Redox Signal 2001;3:1131–7. [25] Ji LL, Gomez-Cabrera MC, Steinhafel N, Vina J. Acute exercise activates nuclear factor (NF-κB) signalling pathway in rat skeletal muscle. FASEB J 2004;18:1499–506. [26] Khassaf M, et al. Effect of vitamin C supplements on antioxidant defence and stress proteins in human lymphocytes and skeletal muscle. J Physiol 2003;549:645–52. [27] Bell C, Carson JM, Motte NW, Seals DR. Ascorbic acid does not affect the age-associated reduction in maximal cardiac output and oxygen consumption in healthy adults. JApplPhysiol 2005;98:845–9.
Article original
Exercise as an antioxidant: it up-regulates important enzymes for cell adaptations to exercise Effet anti-oxydant de l’exercice : up-régulation d’enzymes clés de l’adaptation cellulaire à l’exercice M.C. Gomez-Cabrera a, E. Domenech C, L.L. Ji b, J. Viña c,* a
Catholic University of Valencia, Valencia, Spain University of Wisconsin-Madison, Wisconsin, USA c Department of Physiology, Faculty of Medicine, University of Valencia, Avda. Blasco Ibanez 17, 46010 Valencia, Spain b
Accepted 27 June 2005 Available online 04 April 2006
Abstract Aims. – To assess the role of the reactive oxygen species (ROS) in cell signalling and in the regulation of gene expression. Methods. – Exercise causes oxidative stress only when exhaustive. Strenuous exercise causes oxidation of glutathione, release of cytosolic enzymes, and other signs of cell damage. We have tested this hypothesis by studying the effect of inhibition of ROS production by allopurinol (an inhibitor of xanthine oxidase, a free radical generating enzyme) on cell signalling pathways in marathon runners and in rats submitted to exhaustive exercise by running on a treadmill. Results. – Exercise caused an activation of NF-κB in lymphocytes from marathon runners which was completely prevented by allopurinol. In the rat model exercise caused an activation of MAP kinases in gastrocnemius muscle. This in turn activated the NF-κB pathway and consequently the expression of important enzymes associated with defence against ROS (superoxide dismutase) and adaptation to exercise (eNOS and iNOS). All these changes were abolished when ROS production was prevented by allopurinol. Conclusion. – Thus we report evidence that ROS act as signals in exercise because decreasing their formation prevents activation of important signalling pathways which cause useful adaptations in cells. Because these signals result in an up-regulation of powerful antioxidant enzymes, exercise itself can be considered as an antioxidant. © 2006 Elsevier SAS. All rights reserved. Résumé Objectifs. – Déterminer le rôle des espèces oxygénées réactives (ROS) dans la signalisation cellulaire et dans la régulation de l’expression des gènes. Matériel et méthodes. – L’exercice est à l’origine d’un stress oxydatif seulement lorsqu’il est maximal. Il provoque l’oxydation du glutathion, la libération d’enzymes cytosoliques, accompagnée par d’autres signes de dommages cellulaires. Nous avons testé cette hypothèse en étudiant l’effet de l’inhibition de la production de ROS par l’allopurinol (un inhibiteur de la xanthine oxydase, enzyme générateur de radicaux libres) sur les voies de la signalisation cellulaire chez des marathoniens et chez des rats soumis à un exercice épuisant sur tapis roulant. Résultats. – L’exercice provoque une activation de la voie NF-κB dans les lymphocytes des marathoniens, qui est totalement bloquée par l’allopurinol. Chez le Rat, l’exercice entraîne une activation des MAP-kinases du gastrocnémien, ce qui active la voie NF-κB et, par conséquent, l’expression d’enzymes clés de la défense contre les ROS (superoxyde dismutase) et de l’adaptation à l’exercice (eNOS et iNOS). Ces effets sont supprimés si la production de ROS est bloquée par l’allopurinol.
* Corresponding
author. Tel.: +34 96 386 4646; fax: +34 96 386 4642. E-mail address: [email protected] (J. Viña).
0765-1597/$ - see front matter © 2006 Elsevier SAS. All rights reserved. doi:10.1016/j.scispo.2005.06.012
86
M.C. Gomez-Cabrera et al. / Science & Sports 21 (2006) 85–89
Conclusion. – Ce travail met donc en évidence le rôle de signalisation des ROS, dans la mesure où l’arrêt de leur production empêche l’activation de voies de signalisation utiles à l’adaptation cellulaire. Le résultat final étant une « up-régulation » d’enzyme antioxydants, l’exercice lui-même peut être considéré comme un antioxydant. © 2006 Elsevier SAS. All rights reserved. Keywords: Oxidative stress; Adaptation; Cell signalling; Xanthine oxidase; Hormesis Mots clés: Stress oxidative; Adaptation; Signalisation cellulaire; Xanthine oxidase; Hormesis
1. Introduction Over the past two decades it was shown that unaccustomed exercise can increase the generation of reactive oxygen species (ROS) in biological tissues specially skeletal muscle and myocardium [1–4]. Work from our group revealed that exercise generates oxidative stress only when it is exhaustive [5]. Non-exhaustive exercise causes an increased production of ROS which can be counterbalanced by the concurrent increase in antioxidant defences [6]. Thus it is important to distinguish between non-exhaustive exercise, which is not accompanied by oxidative stress, and exercise to exhaustion, which causes oxidative stress. There are several sources of free radicals in exercise. We have focused in the role of xanthine oxidase (XO) an enzyme involved in the ischemia–repefusion syndrome, that is located in the vascular endothelial cells in skeletal muscle [7]. Administration of allopurinol, widely used inhibitor of the enzyme, to rats at a concentration of 32 mg/kg by intraperitoneal injection prevented the oxidation of glutathione, protein oxidation and lipid peroxidation associated with exhaustive exercise [6,8]. This indicated that XO might be a relevant source of ROS in exercise. 2. Methods and results To test this hypothesis in vivo, we performed experiments with the cyclists of the team US Postal during the Tour of France 2001 [9]. Oral administration of a dose of 300 mg of allopurinol during 13 days prevented the increase in the activities of creatine kinase and aspartate-aminotransferase in plasma (markers of muscle damage) only at the stage where participants performed at their peak level of exertion, the Team Time Trial stage. We also found evidence of an increase in plasma malondialdehyde levels in all participants at the end of the race, but the increase was significantly greater in placebo group than in the allopurinol group. These results suggested that XO is involved in the tissue damage associated to exhaustive physical exercise in vivo. We confirmed these data in a different study with marathon runners recruited from participants in the 23rd Marathon of Valencia. As shown in Fig. 1, marathon running induces a significant increase in plasma malondialdehyde levels. This is prevented by treatment with allopurinol. However free radicals not only cause damage but do have a role in cell signalling, receptor stimulation and enzymatic stimulation [10,11]. In Fig. 2 we report that ROS generated in exercise activate an important signalling pathway, the mitogen
activated protein kinases (p38 and ERK1/ERK2) that can activate transcription factors and regulate protein expression in skeletal muscle. Administration of allopurinol prevents this effect. This demonstrates the role of XO derived ROS in the activation of this signalling pathway. There is growing evidence that the continuous presence of small stimuli such as low concentrations of ROS is in fact able to induce the expression of antioxidant enzymes, DNA repair molecules and protein degrading enzymes, resulting in decreases in the incidence of oxidative stress-related diseases and retardation of the aging process [12]. The basis of this phenomenon may be encompassed by the concept of hormesis [13], which can be characterised as a particular dose–response relationship in which a low dose of a substance is stimulatory and a high dose is inhibitory. In this context, radicals may be considered to be beneficial since they act as signals to enhance defences rather than deleterious (as they are when cells are exposed to high levels of ROS). The concept of hormesis has been applied to the beneficial effects of moderate drinking of alcohol [14] and caloric restriction in experimental animals to prevent disease and promote longevity [15]. Recently the hormesis theory has been extended to the ROS generating effects of exercise [12,16,17]. In skeletal muscle hydrogen peroxide at a low concentration increases Ca2+ release from the sarcoplasmic reticulum and force production, whereas a massive increase in hydrogen peroxide concentration results in a sharp decrease in force output [18]. Animals frequently ex-
Fig. 1. NF-κB activation in lymphocytes and plasma MDA levels after marathon running are blocked by treatment with allopurinol. Values are mean ± S.D. Placebo group (n = 11). Allopurinol-treated group (n = 14). (**) indicates P < 0.01 vs. before the marathon. (*) indicates P < 0.05 vs. before the marathon.
M.C. Gomez-Cabrera et al. / Science & Sports 21 (2006) 85–89
87
Fig. 2. Exercise activates p38 and ERK1/ERK2 phosphorylations. Prevention by allopurinol administration. Exercise activates p38 and ERK1/ERK2 phosphorylation in the cytosolic fraction of rat gastrocnemius muscle. Prevention by allopurinol administration.
posed to exercise (chronic training) have shown less oxidative damage after exhaustive exercise than untrained ones. This is largely attributed to the up-regulation of endogenous antioxidant enzymes such as mitochondrial superoxide dismutase (MnSOD), glutathione peroxidase, and γ-glutamylcysteine synthetase (GCS) [19]. Since the adaptive response results from the cumulative effects of repeated exercise bouts, the initial signal for the stimulation leading to the long-term modula-
tion must occur after each individual exercise bout [20]. Several oxidative stress-sensitive signalling pathways are operational in mammalian systems and play an important role in maintaining cellular oxidant–antioxidant balance. One of the most important pathways that may be activated by ROS involves the transcription factor NF-κB [21]. Several antioxidant enzymes contain NF-κB binding sites in their gene promoter region, such as MnSOD, inducible nitric oxide synthetase
Fig. 3. Exercise activates NF-κB and induces up-regulation of Mn-SOD and NO synthases. Prevention by allopurinol administration. (a) EMSA analysis of NF-κB in the nuclear extracts of rat gastrocnemius (Ø competition assay). (b) Expression of Mn-SOD, iNOS and eNOS measured by real time RT-PCR from gastrocnemius muscle of rats at rest, after exercise and after exercise but pretreated with allopurinol (N = 9).
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M.C. Gomez-Cabrera et al. / Science & Sports 21 (2006) 85–89
Different research groups have shown that vitamin C supplementation during training reduces oxidative stress but at the same time attenuates adaptive responses to oxidants [26] and do not improve VO2max or maximal cardiac output [27]. The practical implication is that decreasing ROS levels with antioxidants may hinder beneficial cell adaptations during exercise. Therefore the common practice of taking antioxidant supplements during training should be seriously questioned. Références
Fig. 4. Proposed mechanism of the role of ROS in signalling of cell adaptations after exercise.
(iNOS) and GCS [22]. Therefore, they can be potential targets for exercise-activated up-regulation via NF-κB signalling pathway. Hollander et al. [23] first reported that an acute bout of treadmill running activated MnSOD gene expression in rat skeletal muscle, along with enhanced NF-κB binding in muscle nuclear extracts ~2 h after exercise. Vider et al. [24] showed that physical exercise (80% of maximal O2 consumption for 1 h) resulted in NF-κB activation in peripheral blood lymphocytes of physically fit young men. Plasma levels of TNF and IL-2 receptor in these subjects were also elevated. However our findings go further. Recently, we have investigated the effect of rigorous muscular contraction on NF-κB signalling pathway in two separate studies: in rat skeletal muscle [8,25] and in peripheral lymphocytes of marathon runners. As shown in Fig. 1, marathon running induces activation of the p50 subunit of the NF-κB complex in lymphocytes. This is prevented by treatment with allopurinol. In this study, we showed that XO derived ROS activates NF-κB signalling pathway in peripheral blood lymphocytes after running a marathon. For obvious ethical reasons, we could not test the explanation for this fact in humans and we had to turn to rats. In Fig. 2, we report that ROS generated in exercise activate MAPKs (p38 and ERK1/ERK2) which in turn activate NF-κB which results in an increased expression of important enzymes associated with cell defence (MnSOD) and adaptation to exercise (eNOS and iNOS). Prevention of ROS formation by inhibition of XO abolishes these effects (Fig. 3). Schematic representation highlighting the role of ROS in the regulation of cell function during exercise is shown in Fig. 4. We indicate that when oxidants are produced at moderate levels they act as signals to adapt cells to exercise. However, when they are overproduced they cause cellular damage.
[1] Davies KJ, Quintanilha AT, Brooks GA, Packer L. Free radicals and tissue damage produced by exercise. Biochem Biophys Res Commun 1982; 107:1198–205. [2] Jackson MJ, Edwards RH, Symons MC. Electron spin resonance studies of intact mammalian skeletal muscle. Biochim Biophys Acta 1985;847: 185–90. [3] Reid MB, et al. Reactive oxygen in skeletal muscle. I. Intracellular oxidant kinetics and fatigue in vitro. J Appl Physiol 1992;73:1797–804. [4] Bejma J, Ramires P, Ji LL. Free radical generation and oxidative stress with aging and exercise: differential effects in the myocardium and liver. Acta Physiol Scand 2000;169:343–51. [5] Sastre J, et al. Exhaustive physical exercise causes oxidation of glutathione status in blood: Prevention by antioxidant administration. Am J Physiol 1992;32:R992–R995. [6] Viña J, et al. Free radicals in exhaustive physical exercise: mechanism of production, and protection by antioxidants. IUBMB Life 2000;50:271–7. [7] Hellsten-Westing Y. Immunohistochemical localization of xanthine oxidase in human cardiac and skeletal muscle. Histochemistry 1993;100: 215–22. [8] Gomez-Cabrera MC, et al. Decreasing Xanthine Oxidase Mediated Oxidative Stress Prevents Useful Cellular Adaptations to Exercise in Rats. J Physiol 2005;567:113–20. [9] Gomez-Cabrera MC, Pallardo FV, Sastre J, Vina J, Garcia-del-Moral L. Allopurinol and markers of muscle damage among participants in the Tour de France. Jama 2003;289:2503–4. [10] Reid MB, Shoji T, Moody MR, Entman ML. Reactive oxygen in skeletal muscle. II. Extracellular release of free radicals. J Appl Physiol 1992;73: 1805–9. [11] Jackson MJ. Free radicals in skin and muscle: damaging agents or signals for adaptation? Proc Nutr Soc 1999;58:673–6. [12] Radak Z, Chung HY, Goto S. Exercise and hormesis: Oxidative stressrelated adaptation for successful aging. An Hypothesis. Biogerontology 2005;6(1):71–5. [13] Calabrese EJ, Baldwin LA. Hormesis: the dose-response revolution. Annu Rev Pharmacol Toxicol 2003;43:175–97. [14] Calabrese EJ, Baldwin LA. Ethanol and hormesis. Crit Rev Toxicol 2003;33:407–24. [15] Sohal RS, Weindruch R. Oxidative stress, caloric restriction, and aging. Science 1996;273:59–63. [16] Ji, L.L., Gómez-Cabrera, M.C., Vina, J., Exercise and Hormesis: Activation of Cellular Antioxidant Signaling Pathway. Ann. New. York. Acad. Sci. (IN PRESS). [17] Vina J, Borras C, Gomez-Cabrera MC, Orr WC. Importance of reactive oxygen species to modulate molecular and cellular response to stress. Free Rad Res 2006;40(2):111–9. [18] Andrade FH, Reid MB, Westerblad H. Contractile response of skeletal muscle to low peroxide concentrations: myofibrillar calcium sensitivity as a likely target for redox-modulation. FASEB J 2001;15:309–11. [19] Salminen A, Vihko V. Endurance training reduces the susceptibility of mouse skeletal muscle to lipid peroxidation in vitro. Acta Physiol Scand 1983;117:109–13.
M.C. Gomez-Cabrera et al. / Science & Sports 21 (2006) 85–89 [20] Hollander J, et al. Superoxide dismutase gene expression is activated by a single bout of exercise. Pflug Arch 2001;442:426–34 (Eur J Physiol). [21] Baeuerle PA, Baltimore D. Activation of DNA binding activity in an apparently cytoplasmic precursor of the NF-κB transcriptional factor. Cell 1998;53:211–7. [22] Laughlin MH, et al. Skeletal muscle oxidative capacity, antioxidant enzymes, and exercise training. JApplPhysiol 1990;68:2337–43. [23] Hollander J, et al. Superoxide dismutase gene expression in skeletal muscle: fiber-specific adaptation to endurance training. Am J Physiol 1999; 277:R856–R862.
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