Keywords

1 Generation of Reactive Oxygen Species in Exercise

Exercise leads to the production of reactive oxygen species (ROS), mainly via the mitochondrial electron transport chain, xanthine oxidase, and phagocytes (Fig. 15.1). A small percent of the oxygen utilized in the mitochondria is converted to superoxide during the electron transport chain reaction. It is known that oxygen consumption during aerobic exercise is elevated 10 to 20-fold in the whole body and over 100-fold in the skeletal muscle, which causes ROS generation that correlates with the duration and intensity of exercise (Blomstrand et al. 1997; Ferguson et al. 2001). In addition, xanthine oxidase is activated via the ischemia-reperfusion process during exercise, a process known as “blood flow redistribution,” resulting in the production of ROS by the capillary endothelium in contracting muscles (Duarte et al. 1993; Flamm et al. 1990). Oxidative stress generated during exercise also leads to further ROS production due to the invasion of phagocytes into the muscles after exercise via the redox-sensitive inflammatory cascade (Aoi et al. 2004).

Fig. 15.1
figure 1

Exercise leads to ROS production via mitochondrial electron transport chain, endothelium xanthine oxidase, and phagocytes in muscle tissues. Exercise elevates oxygen consumption over 100-fold in skeletal muscle, leading to production of reactive oxygen species (ROS) via mitochondrial electron transport chain. Xanthine oxidase is activated via the ischemia-reperfusion process during exercise, a process known as “blood flow redistribution,” resulting in the production of ROS by the capillary endothelium in contracting muscles. Following exercise, further ROS production is increased by an invasion of phagocytes into the muscles

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Previously, studies have shown elevated levels of ROS in response to exercise by using various methods. Electron-spin-resonance (ESR) studies have demonstrated that the ESR signal is markedly increased in muscle homogenates obtained immediately after exercise (Ashton et al. 1999; Davies et al. 1982). Other studies have shown a decrease in diet-derived antioxidants, and a reduced level of glutathione in tissues and blood (Aikawa et al. 1984; Liu et al. 2000; Rietjens et al. 2007), caused by its usage for ROS elimination. As in the above antioxidant capacity, ROS act as oxidative stressors and oxidize cell components such as proteins, lipids, and DNA in tissues such as skeletal muscle, blood, and internal organs, and oxidation results in various oxidative products. Specifically, peroxidation of polyunsaturated fatty acids such as arachidonic acid, which are a large constituent of the membranes around cells and their organelles, occurs via a radical chain reaction and has been used as a useful marker of exercise-induced oxidative stress. Numerous previous animal and human studies have shown that acute exercise such as running, cycling, and resistance exercise, markedly elevates thiobarbituric acid reactive substances (TBARS), one of the oldest and most frequently used methods for measuring the peroxidation of fatty acids in the skeletal muscle immediately after exercise and on the next day after the exercise (Aoi et al. 2004; Liao et al. 2010; Miyazaki et al. 2001). In addition, this elevation in TBARS is also shown in high-intensity exercise training (Chang et al. 2007; Couillard et al. 2003). On the other hand, dietary supplementation with antioxidants such as vitamin E, vitamin C, carotenoids, and polyphenols can decrease oxidative damage induced by acute and chronic exercises (Aoi et al. 2003, 2004; Bryer and Goldfarb 2006; Goldfarb et al. 2011). These observations clearly demonstrate that exercise induces oxidative stress and causes the oxidation of cellular components. Such oxidative stress is not always negative, and many evidences suggest that moderate oxidative stress is necessary for beneficial adaptations induced by dietary exercise, as mentioned below. The effect of oxidative stress on bodily functions in living body would be due to the extent of this stress, and whether it acts a damaging factor or a protective factor is dependent on the intensity, duration, frequency, and habits of exercise.

2 Involvement of Oxidative Stress on Fatigue and Damage in Muscle

The possibility that oxidative stress induced by exercise causes a health disorder is a debatable matter. However, it has been demonstrated that the excess ROS generated by exercise are involved in muscle fatigue and muscle damage. It is well established that ROS have important influences on force production in the skeletal muscle. A study using both spin traps and vitamin E in animals demonstrated that scavenging ROS in muscles during exercise delays the onset of muscular fatigue (Novelli et al. 1990). Moreover, many reports have shown that administration of the antioxidant N-acetylcysteine (NAC), which acts as a reduced thiol donor supporting glutathione re-synthesis, delays muscular fatigue during a variety of submaximal exercise tasks such as electrically stimulated fatigue of the muscle, cycling exercise, and repetitive handgrip exercise (Cobley et al. 2011; Matuszczak et al. 2005; McKenna et al. 2006; Medved et al. 2004; Reid et al. 1994). In animal studies, NAC administration has also been shown to delay fatigue in both in vitro and in situ muscle preparations (Kobzik et al. 1995; Perkins et al. 1997). Studies using excised muscle fiber bundles also revealed that force production during submaximal tetanic contractions is decreased by nitric oxide (NO) donors (Morrison et al. 1998; Richmonds and Kaminski 2001) and increased by nitric oxide synthase (NOS) inhibitors and NO scavengers (Joneschild et al. 1999; Kobzik et al. 1994).

Unaccustomed and strenuous exercise can cause muscle damage that presents clinically as muscular pain and involves protein degradation and ultrastructural changes. As such, muscle damage usually occurs sometime after exercise and not during or immediately after exercise; this is called as “delayed-onset muscle damage.” Previous studies have demonstrated that delayed-onset muscle damage is mainly induced by mechanical stress, especially eccentric muscle contraction (Komulainen et al. 1998; Proske and Morgan 2001), and disturbances in calcium homeostasis (Chen et al. 2007; Gissel and Clausen 2001). In addition, we demonstrated that delayed-onset muscle damage induced by prolonged exercise is partly related to inflammation via phagocyte infiltration caused by ROS generated during exercise (Aoi et al. 2004). In an in vitro study using myotube cells, addition of H2O2 induced the translocation of p65, a component of the redox-sensitive transcription nuclear factor-kappa B (NF-κB), into the nucleus and subsequently increased the expression of cytokine-induced neutrophil chemoattractant-1 (CINC-1) and monocyte chemoattractant protein-1 (MCP-1). Prolonged acute exercise caused an increase in the amount of nuclear p65, a constitutive protein of NF-κB, in rat gastrocnemius muscles at 1 h after exercise, which was similar to the in vitro results, and it caused muscle damage with neutrophil invasion on the next day. Therefore, delayed-onset muscle damage after prolonged exercise is related to inflammation secondary to phagocyte infiltration caused by ROS generated during exercise. In contrast, dietary antioxidants such as vitamin E and carotenoids can partly attenuate delayed-onset muscle damage along with inflammatory changes (Aoi et al. 2003, 2004; Rosa et al. 2009), though several studies reported that supplementation of dietary antioxidants does not have this preventative effect.

3 Oxidative Protein Modification in the Muscle Induced by Exercises

Numerous posttranslational modifications have been characterized as resulting either from direct modification of amino acid residues or through the formation of reactive intermediates by the oxidation of other cellular components (Naito and Yoshikawa 2009). The modification of 20 different of amino acids plays an important role in the manifestation of the function of many proteins. The modification can be subdivided into two general forms: reversible modification and irreversible modifications. Some of the lipid peroxidation products exhibit a facile reactivity with proteins, generating a variety of intra- and intermolecular covalent adducts. In addition, the oxidation of cysteine to sulfenic, sulfinic, and sulfonic acids has been shown to occur frequently, and these sulfenic and sulfinic acids can often be enzymatically reduced. Nitration by reactive nitrogen species, chlorination by hypochlorous acid, and bromination by hypobromous acid of the target protein are also the frequently detected modifications. Many evidences suggest that such protein modifications can be associated with the onset of various common diseases, including cancer, inflammation, and metabolic disorders (Bidasee et al. 2004; Hill and Bhatnagar 2012; Oya-Ito et al. 2011; Sultana and Butterfield 2009). Some muscular proteins are also targets modified by exercise-induced ROS or nitrogen oxide species (Aoi et al. 2003; Kato et al. 2000; Magherini et al. 2012; Veskoukis et al. 2008; Vassilakopoulos et al. 2003; Barreiro and Hussain 2010) (Table 15.1).

Table 15.1 Non-enzymatic oxidative protein modification induced by exercise/muscle contraction
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The influence of oxidative stress on the sarcoplasmic reticulum (SR), a subcellular organelle, which controls the contractile state of the muscle by regulating the calcium concentration in the cytosol, has been studied extensively in the skeletal muscle, and is associated with the oxidative modification of the membrane proteins (Anzueto et al. 1992; Salama et al. 1992; Xia et al. 2003). Muscle contraction is performed by increasing intracellular calcium concentrations, which are released from the SR via the ryanodine receptor (RyR) calcium-release channel following active potentials during the excitation-contraction coupling process. Afterwards, calcium is immediately taken into the SR via SR calcium-dependent ATPase (SERCA), which relaxes the muscle. It has been known that the responsive proteins in the SR are sensitive to redox modulation (Sun et al. 2001; Zhang et al. 1999). The RyR appears to be in close association with the NADP(H) oxidase(s) found in the SR, and locally generated superoxide appears to be the major ROS capable of influencing this channel (Xia et al. 2003). Each subunit of this large tetrameric protein contains a small number of regulatory cysteines. ROS and NO oxidize thiol residues on neighboring cysteines to form disulfide bonds, which induce channel opening. Disulfide formation is reversed by reducing agents, providing a mechanism for direct redox modulation of channel activity. The SERCA, another potential target, contains a small number of critical sulfhydryls near the SERCA active site, which have been shown to slow the reuptake of calcium into the SR (Daiho and Kanazawa 1994; Xu et al. 1997; Gutierrez-Martin et al. 2004). Exposure to elevated NO also inhibits SERCA activity via thiol oxidation and nitration of tyrosine residues (Viner et al. 1997, 2000). Consequently, oxidation of SR proteins tends to increase cytosolic calcium levels, which causes prevention of muscle relaxation. Therefore, the modification of calcium transport proteins in the SR can causes excess or chronic muscle contraction via increasing intracellular calcium levels, leading to muscle fatigue.

Muscle myofilaments are also sensitive to direct redox modification (Fedorova et al. 2009). Myosin heavy chains contain several sulfhydryl residues, which are useful sites for protein labeling; however, thiol modification generally does not dramatically alter myosin function (Crowder and Cooke 1984). On the other hand, Yamada et al. (2007) reported that a force reduction in the soleus muscle of hyperthyroid rat is associated with carbonylation of the myosin heavy chain. In addition, it has been suggested that the myosin heavy chain is easily glycosylated, which changes the structural and functional properties of the protein (Haus et al. 2007). However, the involvement of these modifications on exercise-induced fatigue is unclear. In contrast, myosin light chains, actin, and tropomyosin appear less sensitive to redox modulation (Liu et al. 1990; Williams and Swenson 1982).

In delayed-onset muscle damage, protein modification is also observed. We reported the elevation of 4-hydroxy-2-nonenal (4-HNE), a lipid peroxidation product that covalently modifies the proteins on cysteine, histidine, and lysine residues, in the damaged muscle obtained from mice on the next day after acute running (Aoi et al. 2003). Liu et al. (2005) reported a positive correlate between 4-HNE and creatine kinase activity in blood following strenuous exercise in humans. Recently, insulin receptor substrate-1 (IRS-1) was detected as a 4-HNE-targeted protein (Aoi et al. 2012). IRS-1 is upstream in the PI3K/Akt-dependent insulin-signaling pathway in muscle cells and regulates glucose uptake via glucose transporter 4. In the damaged muscle after strenuous exercise, insulin-stimulated glucose uptake is decreased along with a reduction of insulin signal transduction, which suggests that 4-HNE modification of IRS-1 is involved in the transient impairment in insulin sensitivity. Sahlin et al. (2010) showed a marked elevation of 4-HNE modification of mitochondrial protein after acute endurance exercise. In addition, Kato et al. (2000) have reported that Nε-(hexanoyl)lysine (HEL), which is generated from the reaction between the lysine moiety and 13-hydroperoxyoctadecadienoic acid (13-HPODE), is increased in the muscle obtained from rats, which have performed high-intensity exercise training for 3 weeks. In contrast, the administration of eriocitrin, a flavonoid contained in lemon and lime fruits, to exercise-trained rats suppressed the formation of HEL.

4 HEL-Modification of CPT-I and Lipid Metabolism in Exercise

A major source of ROS production during exercise is the mitochondrial electron transport. Thus, mitochondrial proteins could be a major target of posttranslational oxidative modification during exercise. The HEL moiety is a novel adduct formed by the reaction of linoleic acid hydroperoxide and lysine, and is a marker of lipid peroxidation-derived protein modification in the early stages after oxidative stress (Kato et al. 1999; Osawa and Kato 2005), and therefore may be useful in detecting oxidative modification of mitochondrial protein during acute exercise (Fig. 15.2). Thus, we hypothesized that proteins on the mitochondrial membrane are easily modified by lipid peroxide generated on the membrane. In an analysis of mouse gastrocnemius muscle obtained immediately after running, the modification of carnitine palmitoyltransferase I (CPT I) by HEL was detected (Aoi et al. 2008). CPT I is located on the mitochondrial membrane and is a rate-limiting step in fatty acyl-CoA entry into the mitochondria in the muscle (McGarry and Brown 1997). In contrast, astaxanthin, an antioxidant in the mitochondrial membrane, limits the modification of CPT I by HEL after exercise, suggesting that astaxanthin directly traps the oxygen radicals generated by exercise, and/or that astaxanthin promotes the activation of a defense mechanism such as the induction of antioxidative enzymes, and/or that astaxanthin furthers the excretion of HEL from the skeletal muscle by activation of the proteasome. We previously reported that orally administered astaxanthin is absorbed in the intestine and accumulates in the muscle tissues in mice (Aoi et al. 2008). In addition, Manabe et al. (2008) have demonstrated that astaxanthin accumulates in the mitochondrial fraction and inhibits the oxidative modification of mitochondrial proteins in mesangial cells. Thus, the mitochondria are considered to be a major target of astaxanthin.

Fig. 15.2
figure 2

Relationship among ROS, oxidative protein modification, and nutrient metabolism. ROS are generated via mitochondria and xanthine oxidase, and then oxidizes lipids during exercise, which results in elevation of Nε-(hexanoyl)lysine, an early stage lipid peroxidation marker. In recovery period following exercise, invasion of phagocytes into muscle tissues occurs, which progresses muscle damage via inflammation. In this process, lipid peroxide reaction is further progressed and aldehyde including 4-hydroxy-2-nonenal is produced. Such lipid peroxides modify particular metabolic protein posttranslationally, leading to modulation of nutrient metabolism

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Several studies (Campbell et al. 2004; Holloway et al. 2006) have shown that the fatty acid translocase/cluster of differentiation 36 (FAT/CD36) is associated with CPT I on the mitochondrial membrane and increases its function. We found that the interaction between CPT I and FAT/CD36 in the muscle during exercise was facilitated by astaxanthin (Aoi et al. 2008). Thus, modification of CPT I by HEL may alter the colocalization of CPT I with FAT/CD36 by changing the CPT I structure, which could lead to the regulation of lipid metabolism during exercise. Lipolysis in the body is important during exercise to facilitate lipid utilization in the muscle rather than release it from the adipose tissue. The utilization ratio of carbohydrates and lipids for energy generation is almost equal when exercise is of low to moderate intensity. A possible factor influencing the utilized ratio of these energy substrates is the colocalization of CPT I with FAT/CD36. Exercise-induced ROS may partly limit the utilization of fatty acids via diminishing the CPT I activity caused by HEL modification (Fig. 15.3). Indeed, we and another group found that inhibition of this modification by dietary astaxanthin increased fat utilization during exercise as compared with mice on a normal diet and prolonged the running time to exhaustion (Aoi et al. 2008; Ikeuchi et al. 2006). Therefore, HEL-modification of CPT I can partly suppress lipid metabolism during exercise, which would affect the endurance performance and efficiency of adipose tissue reduction with training.

Fig. 15.3
figure 3

HEL modification of CPT - I on mitochondrial membrane. Amount of FAT/CD36 that coimmunoprecipitated with CPT I (A) and HEL-modified CPT I (B) in skeletal muscle of ICR mice. A single bout of exercise was performed at 30 m/min for 30 min. Lysate protein from the muscle collected immediately after running was immunoprecipitated with CPT I antibody. Immunoprecipitates were separated by SDS-PAGE and membranes probed for FAT/CD36 (A) or HEL (B). Values are the mean ± SE. *, significant difference at the level of P < 0.05 (Data are from Aoi et al. 2008)

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5 Prospective

Many evidences have indicated that oxidative stress has both positive and negative effects. A moderate grade of oxidative stress enhances muscle force production, nutrient metabolism, and antioxidant enzymes (Ristow et al. 2009; Gomez-Cabrera et al. 2008; Powers and Jackson 2008), although excess ROS functions as a damaging factor in cellular components. In contrast, dietary vitamin C and E cancel many exercise-induced adaptive benefits, such as improvements in insulin sensitivity, blood pressure, and endurance capacity, which are caused by suppressing the expression of redox-sensitive proteins, including PPAR gamma coactivator-1 alpha, AMP activated kinase, and superoxide dismutase 2. Therefore, the dietary intake of antioxidants for exercise therapy for the treatment and prevention of diseases and for training to improve the athletic performance is a debatable matter. On the other hand, some antioxidants accelerate energy metabolism and insulin sensitivity induced by exercise via the elevation of key modulators (Aoi et al. 2008; Richards et al. 2010; Henriksen 2006; Dolinsky et al. 2012). Therefore, we cannot group all antioxidants together, and we should consider the respective properties of each antioxidant individually but not their absolute antioxidative capacity. This may be responsible for the beyond-antioxidant properties that each compound has specifically. Oxidative protein modification during early-stage oxidative damage, e.g. HEL, may be useful as a potential biomarker to examine the benefit of antioxidant compounds in the sporting scene as well as in regulating oxidative stress levels, although a study focusing on the relationship between protein modification and physiological function in exercise is underdevelopment. Further research is required regarding the functional analysis of modified proteins along with whether this can be a biomarker of athletic performance levels, fatigue, and health benefits obtained from dietary exercise.