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1 From the Departments of Surgery and Molecular & Cellular Biochemistry, The Ohio State University Medical Center, Columbus, and the Department of Physiology, University of Kuopio, Kuopio, Finland.
2 Presented at the workshop Role of Dietary Supplements for Physically Active People, held in Bethesda, MD, June 3–4, 1996.
3 Supported by the Finnish Ministry of Education, Juho Vainio Foundation, Helsinki, and the National Institutes of Health (DK 50430).
4 Address reprint requests to CK Sen, 512 Heart & Lung Institute, The Ohio State University Medical Center, 437 West 12th Avenue, Columbus, OH 43210-1252. E-mail: sen-1{at}medctr.osu.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONCENTRAL ROLE OF THIOLS...ENDOGENOUS BIOTHIOLSEXERCISE-INDUCED OXIDATIVE...SKELETAL MUSCLE GLUTATHIONETRAINING AND GLUTATHIONE...EXERCISE-INDUCED CHANGES IN...EXERCISE-INDUCED CHANGES IN...MANIPULATION OF TISSUE...THIOL MANIPULATION AND EXERCISEHUMAN DISEASESIGNAL TRANSDUCTIONCONCLUSIONSDIRECTIONS FOR FUTURE RESEARCHREFERENCES |
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-lipoic acid hold the most promise. These agents may have antioxidant effects at the biochemical level but are also known to influence redox-sensitive cell signaling.
Key Words: Antioxidant • N-acetyl-L-cysteine • glutathione • fatigue • lipoic acid • muscle • nutrition • oxidative stress • performance • redox • training
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONCENTRAL ROLE OF THIOLS...ENDOGENOUS BIOTHIOLSEXERCISE-INDUCED OXIDATIVE...SKELETAL MUSCLE GLUTATHIONETRAINING AND GLUTATHIONE...EXERCISE-INDUCED CHANGES IN...EXERCISE-INDUCED CHANGES IN...MANIPULATION OF TISSUE...THIOL MANIPULATION AND EXERCISEHUMAN DISEASESIGNAL TRANSDUCTIONCONCLUSIONSDIRECTIONS FOR FUTURE RESEARCHREFERENCES |
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Thiols are ubiquitously distributed in aerobic life forms and have multifaceted functions, including a pivotal role in antioxidant defense (Figure 1
). Added protection against oxygen toxicity is provided by exogenous antioxidants obtained primarily as nutrients or nutritional supplements (9). The present paper introduces thiols, then focuses on their significance relative to exercise-induced oxidative stress and nutritional supplements.
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Another characteristic feature of most thiols is their ability to act as reducing agents. Reactive oxygen species have a strong tendency to transfer electrons to other species (ie, to oxidize). Reducing agents such as thiols have negative standard reduction potentials and thus act as prompt electron acceptors (Table 1). Therefore, in the case of an oxidant-thiol interaction, the oxidant is neutralized to a relatively less toxic byproduct at the expense of the reducing power of thiol, which itself gets oxidized to a disulfide (C-S-S-C). A thiyl radical (C-S•) is produced when a thiol (C-SH) loses the H atom from the -SH group or loses an electron from the sulfur, followed by a proton. Under conditions of physiologic pH, thiyl radicals are unstable and may recombine to form the corresponding disulfide (12). In biological systems, there are specific reductases that recycle disulfides to thiols using cellular-reducing equivalents such as NADH or the corresponding phosphorylated form (NADPH) (Figure 1
). In this way the power of cellular metabolism is coupled to maintain a favorable oxidoreductive (or redox) state of thiols.
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CENTRAL ROLE OF THIOLS IN THE ANTIOXIDANT NETWORK |
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TOPABSTRACTINTRODUCTIONCENTRAL ROLE OF THIOLS...ENDOGENOUS BIOTHIOLSEXERCISE-INDUCED OXIDATIVE...SKELETAL MUSCLE GLUTATHIONETRAINING AND GLUTATHIONE...EXERCISE-INDUCED CHANGES IN...EXERCISE-INDUCED CHANGES IN...MANIPULATION OF TISSUE...THIOL MANIPULATION AND EXERCISEHUMAN DISEASESIGNAL TRANSDUCTIONCONCLUSIONSDIRECTIONS FOR FUTURE RESEARCHREFERENCES |
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). For example, vitamin E is a major lipid-phase antioxidant that protects against oxidative lipid damage. Although vitamin E can effectively terminate lipid peroxidation chain reactions, during the course of such reactions vitamin E can itself be oxidized to a tocopheroxyl radical. Inadequate recycling of this oxidized form of vitamin E to the potent reduced form may lead to accumulation of oxidized vitamin E, which may even lead to the initiation of pathologic processes (2, 13). In biological membranes, vitamin E is present in a low molar ratio in contrast with phospholipids, which are abundant and highly susceptible to oxidative damage. For every 1000–2000 molecules of phospholipid, it is estimated that only 1 molecule of vitamin E is present for antioxidant defense. In addition, lipid peroxyl radicals, reactive oxygen species that can trigger lipid peroxidation reactions, are continuously produced in biological membranes (14). The ability of vitamin E to protect membranes under such remarkably adverse conditions can be explained by the continuous recycling of this vitamin as it acts as an antioxidant (2–8), which permits it to regain antioxidant potency. Thiols such as reduced glutathione (GSH) and dihydrolipoate support the recycling of vitamins C and E (Figure 1). In addition to their central role in antioxidant biochemistry, thiols have such functions as protein synthesis and structure, redox-sensitive signal transduction, cell growth and proliferation, regulation of programmed cell death, xenobiotic metabolism, and immune regulation (Figure 2).
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ENDOGENOUS BIOTHIOLS |
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TOPABSTRACTINTRODUCTIONCENTRAL ROLE OF THIOLS...ENDOGENOUS BIOTHIOLSEXERCISE-INDUCED OXIDATIVE...SKELETAL MUSCLE GLUTATHIONETRAINING AND GLUTATHIONE...EXERCISE-INDUCED CHANGES IN...EXERCISE-INDUCED CHANGES IN...MANIPULATION OF TISSUE...THIOL MANIPULATION AND EXERCISEHUMAN DISEASESIGNAL TRANSDUCTIONCONCLUSIONSDIRECTIONS FOR FUTURE RESEARCHREFERENCES |
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).
The GSH-dependent detoxification of reactive oxygen species is accomplished through 2 general mechanisms: 1) direct or spontaneous reaction with reactive oxygen species and 2) glutathione peroxidase catalyzed decomposition of reactive oxygen species both of which produce glutathione disulfide (GSSG, also known as oxidized glutathione) (Figure 1). Intracellular GSSG, thus formed, may be reduced back to GSH by glutathione disulfide reductase (GSSG reductase) or released to the extracellular compartment. Another family of GSH-dependent enzymes, glutathione transferases, catalyzes the detoxification of electrophilic xenobiotics, rendering them more water soluble for excretion (25). Thioredoxin and glutaredoxin are other endogenous biothiols known to participate in important redox processes; both are small proteins with active center dithiols [-(SH)2] in their reduced forms (26). This center, by way of rapid thiol-disulfide interchange, participates in redox reactions.
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EXERCISE-INDUCED OXIDATIVE STRESS |
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TOPABSTRACTINTRODUCTIONCENTRAL ROLE OF THIOLS...ENDOGENOUS BIOTHIOLSEXERCISE-INDUCED OXIDATIVE...SKELETAL MUSCLE GLUTATHIONETRAINING AND GLUTATHIONE...EXERCISE-INDUCED CHANGES IN...EXERCISE-INDUCED CHANGES IN...MANIPULATION OF TISSUE...THIOL MANIPULATION AND EXERCISEHUMAN DISEASESIGNAL TRANSDUCTIONCONCLUSIONSDIRECTIONS FOR FUTURE RESEARCHREFERENCES |
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Does the contraction of skeletal muscle with the associated enhanced consumption of oxygen increase the generation of reactive oxygen species? Numerous experimental studies have addressed this question with use of the contracting muscle model. For example, Diaz et al (29) observed that hydroxyl radical, a highly reactive oxidant, is generated in fatiguing contractions. In addition, Reid et al (30, 31) observed that superoxides are produced intracellularly in muscle cells and then released to the extracellular medium. Hydrogen peroxide, produced in the contracting muscle cell, readily crosses the cell membrane to reach the extracellular medium, where it contributes to the formation of hydroxyl radicals. In this way, relatively less damaging forms of reactive oxygen such as superoxides and hydrogen peroxide can be released from the cells to produce more damaging hydroxyl radicals. Barclay and Hansel (32) showed that superoxide radicals attenuate the function and enhance the rate of fatigue of contracting muscles. Other studies indicate that strenuous exercise augments oxidative stress and that exercise-induced oxidative stress may damage biological components, eg, lipids and proteins, as well as the genetic material (8, 33, 34). Various mechanisms that may contribute to exercise-induced oxidative stress were reviewed recently (35).
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SKELETAL MUSCLE GLUTATHIONE |
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TOPABSTRACTINTRODUCTIONCENTRAL ROLE OF THIOLS...ENDOGENOUS BIOTHIOLSEXERCISE-INDUCED OXIDATIVE...SKELETAL MUSCLE GLUTATHIONETRAINING AND GLUTATHIONE...EXERCISE-INDUCED CHANGES IN...EXERCISE-INDUCED CHANGES IN...MANIPULATION OF TISSUE...THIOL MANIPULATION AND EXERCISEHUMAN DISEASESIGNAL TRANSDUCTIONCONCLUSIONSDIRECTIONS FOR FUTURE RESEARCHREFERENCES |
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TRAINING AND GLUTATHIONE-DEPENDENT TISSUE ANTIOXIDANT DEFENSES |
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TOPABSTRACTINTRODUCTIONCENTRAL ROLE OF THIOLS...ENDOGENOUS BIOTHIOLSEXERCISE-INDUCED OXIDATIVE...SKELETAL MUSCLE GLUTATHIONETRAINING AND GLUTATHIONE...EXERCISE-INDUCED CHANGES IN...EXERCISE-INDUCED CHANGES IN...MANIPULATION OF TISSUE...THIOL MANIPULATION AND EXERCISEHUMAN DISEASESIGNAL TRANSDUCTIONCONCLUSIONSDIRECTIONS FOR FUTURE RESEARCHREFERENCES |
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Lew and Quintanilha (43) observed that for the same amount of submaximal exercise, endurance-trained rats improved their ability to maintain tissue glutathione redox status (as reflected by the ratio of GSSG to total glutathione) in comparison with their untrained counterparts. The endurance training program significantly increased the activities of glutathione peroxidase, GSSG reductase, and glucose-6-phosphate 1-dehydrogenase in the skeletal muscle and heart tissues. A favorable change of the glutathione redox cycle in response to exercise training has also been observed in the brain, particularly in the cortex and brain stem (44).
The effects of aging and exercise training on antioxidant enzyme activities in rat skeletal muscle were tested by Ji et al (45). Superficial glycolytic and deep oxidative vastus lateralis muscles were collected from rats ranging in age from 2.5 mo (young) to 27.5 mo (senescent). Old rats had significantly lower glutathione peroxidase activity in the deep vastus lateralis muscle. After progressive treadmill training, the activity of the hydroperoxide-metabolizing enzyme in deep vastus lateralis muscle increased significantly to a point where it was greater than that observed in sedentary young rats. The authors concluded that although aging can adversely influence antioxidant enzyme capacity in skeletal muscle, regular exercise can preserve such protective function. Similarly, in an earlier study by Ji et al (46), an increase was seen in rat skeletal muscle glutathione peroxidase activity from endurance treadmill training. In a different model, Kanter et al (47) found consistently that swim training enhanced the activity of glutathione peroxidase in the blood and liver. In contrast, Tiidus and Houston (48) observed that in female rats, 6 wk of treadmill training did not significantly influence glutathione peroxidase activity in skeletal muscle, heart, and liver. In a human study, however, endurance training increased erythrocyte glutathione peroxidase activity (49).
Much of the research in our laboratory has been directed toward the study of tissue GSH metabolism in response to exercise and training. In one study (50), treadmill training of rats increased skeletal muscle citrate synthase activity, indicating enhanced oxidative capacity. In addition, total glutathione content of the liver was elevated in the trained rats. However, such an effect was not observed in any of the skeletal muscles studied, eg, red gastrocnemius, mixed vastus lateralis, and longissimus dorsi. Glutathione peroxidase activity in leg muscle was higher in trained rats than in untrained rats, however. Treadmill training decreased GSSG reductase activity in red gastrocnemius muscle, a finding that may be related to the high intensity (2.1 km/h, 2 h/d, 5 d/wk, 8 wk) of training, which may have increased flavoprotein turnover and breakdown in the muscle. Endurance training also increased the activity of 
-glutamyltransferase in both leg muscles, the effect being more pronounced in red gastrocnemius. In the trained leg muscles, activated -glutamyltransferase may facilitate the import of substrates required for GSH generation (Figure 3). Thus, in the presence of extracellular cysteine, availability of this amino acid in the cell is increased.
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In the treadmill study, decreased -glutamyltransferase activity was observed in the control leg muscles after exercise (50), but this effect was not observed in the trained leg muscles, indicating that during exercise trained muscles have a more active substrate import system for GSH generation than untrained muscles. -Glutamyltransferase activity of the trained liver decreased (
50%) after the exercise bout; this response might ensure that fewer -glutamyl compounds are re-trapped in the liver when the active peripheral tissues have acute needs. The contention that exercise training strengthens GSH-dependent antioxidant defenses is supported by a more recent study in which swim training of rats was associated with a marked increase in the activities of glutathione peroxidase and GSSG reductase in the skeletal muscle, heart, and liver (53).
GSH-dependent antioxidant protection in the skeletal muscle is influenced by the state of physical activity: endurance training enhances and restriction of chronic activity diminishes such protection (50). Beagles, commonly used as a laboratory animal, possess a well-developed musculoskeletal system apparently suited for running, and thus we studied the influence of treadmill training in these dogs. Training on the treadmill (5.5–6.8 km/h, 40 km/d, 5 d/wk, 15% uphill grade, for 40 wk) increased the oxidative capacity of red gastrocnemius, extensor carpi radialis, and triceps muscles of the leg. Training-induced changes in the components of GSH metabolism were most pronounced in the red gastrocnemius muscle, which is predominantly oxidative by composition. Total glutathione in the liver and red gastrocnemius was elevated in response to training. In all 3 leg muscles noted above, training increased glutathione peroxidase activity, and again this effect was most pronounced in the red gastrocnemius muscle. GSSG reductase activities in extensor carpi radialis and triceps muscles were higher in the trained dogs. Trained animals with higher total glutathione reserves in the liver also had higher glutathione transferase activity, indicating that the liver of the trained animals had a higher detoxicant status. Training effects were not observed in the splenius muscle of the neck and trunk region.
In a separate experiment (50), the effect of chronically restricting activity on the red gastrocnemius muscle of beagles was studied by immobilizing the knee and ankle joint of the right pelvic limb of each dog for 11 wk in a light fiberglass cast. The left leg was used as the paired control. Chronic physical inactivity did not influence the activity of GSH-dependent enzymes, but the total amount of glutathione in the red gastrocnemius muscle was remarkably decreased in the immobilized leg. In another study, decreased total glutathione and increased GSSG were associated with atrophy of skeletal muscle (54).
In a 55-wk endurance training study with beagles, it was observed that physical training may enhance hepatic glutathione transferase activity (50). Glutathione transferases are a family of GSH-dependent enzymes that play a central role in drug detoxification and xenobiotic metabolism. In addition, glutathione transferases may contribute to hydroperoxide metabolism because they have glutathione peroxidase activity that does not depend on selenium. More recently, Veera Reddy et al (25) confirmed in a rat model that swim training results in higher hepatic glutathione transferase activity than in untrained controls. Electrophoretic and western blot analyses revealed that a Ya-sized subunit of the transferase is specifically induced by exercise training. Analyses of affinity-purified glutathione transferases further revealed that a Ya1 subunit of Ya was most sensitive to exercise training. Untrained control rats had Ya subunits predominantly made up of Ya2, whereas the trained animals had a 4.3-fold increase in Ya1. Glutathione transferases of exercise-trained animals had increased peroxidase activity, an effect that was consistent with the changes in subunit composition. Studies on the regulation of Ya gene expression have revealed that the gene contains a regulatory sequence known as the antioxidant response element, or ARE, in the 5'-flanking region. Transcription of Ya is activated by oxidants such as hydrogen peroxide by a mechanism acting through the ARE (55). Ya1 is induced in hydroperoxide overload situations, such as selenium deficiency (56). Thus, exercise-induced regulation of Ya1 may be expected to be oxidant mediated.
Powers et al (57) investigated the effect of intensity and duration of exercise on training-induced antioxidant enzyme responses; rats were exercised at low, moderate, or high intensity for 1 of 3 time periods (30, 60, or 90 min/d). The costal and crural diaphragm, plantaris muscle, and parasternal intercostal muscles were studied. Training effects were highly tissue specific. All training programs markedly increased glutathione peroxidase activity in the costal diaphragm but not in the crural diaphragm. The intensity or duration of exercise did not have a major influence on training-induced elevation of glutathione peroxidase activity in the costal diaphragm. In the crural diaphragm, however, moderate- and high-intensity exercise training decreased tissue glutathione peroxidase activity when daily exercise lasted as long as 90 min. None of the training programs influenced glutathione peroxidase activity of the parasternal intercostal muscle, but in the plantaris muscle, longer duration of daily exercise triggered a more marked response.
Results of a similar study support the idea that training effects are indeed highly tissue specific (58). In this study, although exercise training increased glutathione peroxidase activity in the red gastrocnemius muscle of rats, such effects were not seen consistently in the soleus or even white gastrocnemius muscles. As with the previous results for the plantaris muscle, the duration of daily exercise markedly affected the response in glutathione peroxidase activity. In another study, Criswell et al (59) found that high-intensity training was superior to moderate-intensity training in elevating glutathione peroxidase activity in the soleus muscle of rats.
Studies investigating the influence of physical training on tissue antioxidant status generally tested endurance training, which enhances tissue oxidative capacity. Comparable information on the effect of sprint training, which relies primarily on nonoxidative metabolism, is scanty. Atalay et al (60) examined the effect of a sprint training regimen on the rat skeletal muscle and heart GSH system. Soleus muscle, made up predominantly of slow-oxidative fibers, was studied as representative of slow-twitch muscle; plantaris and extensor digitorum longus muscles, consisting mainly of glycolytic fibers, and the superficial white portion of the quadriceps femoris muscle, consisting mainly of fast-oxidative glycolytic fibers, were studied as representative of fast-twitch muscle. Mixed gastrocnemius muscle was examined as an antagonist of extensor digitorum longus muscle. Lactate dehydrogenase and citrate synthase enzyme activities were measured in muscle to test the effects of training on glycolytic and oxidative metabolism, respectively. The efficacy and specificity of the 6-wk sprint training protocol was attested by markedly increased anaerobic but not aerobic metabolic capacity in primarily mixed and fast-twitch fiber muscles. Endurance training consistently upregulated GSH-dependent defenses and other antioxidant enzymes, with effects most marked in highly oxidative muscle (38, 50, 59, 61–63). In contrast, sprint training enhanced antioxidant defenses primarily in fast glycolytic muscle. Compared with that in the control group, glutathione peroxidase activities in gastrocnemius, extensor digitorum longus muscles, and the heart increased after sprint training. The training program also increased GSSG reductase activity in the extensor digitorum longus muscle and heart. Sprint training did not influence the amount of glutathione or GSH-related enzymes in the oxidative soleus muscle.
The effect of intermittent sprint cycle training on the degree of muscle antioxidant enzyme protection was also investigated in humans (64). Resting muscle biopsies obtained before and after 6 wk of training and 3 h, 24 h, and 72 h after the final session of an additional 1 wk of more frequent training were analyzed for activities of the antioxidant enzymes glutathione peroxidase, GSSG reductase, and superoxide dismutase. Intermittent sprint cycle training, which enhances the capacity to generate anaerobic energy, also improved the amount of antioxidant protection in the muscle. Thus, depending on the type of work program, GSH metabolism of specific tissues may be expected to respond to physical training.
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EXERCISE-INDUCED CHANGES IN TISSUE GLUTATHIONE |
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TOPABSTRACTINTRODUCTIONCENTRAL ROLE OF THIOLS...ENDOGENOUS BIOTHIOLSEXERCISE-INDUCED OXIDATIVE...SKELETAL MUSCLE GLUTATHIONETRAINING AND GLUTATHIONE...EXERCISE-INDUCED CHANGES IN...EXERCISE-INDUCED CHANGES IN...MANIPULATION OF TISSUE...THIOL MANIPULATION AND EXERCISEHUMAN DISEASESIGNAL TRANSDUCTIONCONCLUSIONSDIRECTIONS FOR FUTURE RESEARCHREFERENCES |
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, where a trend is seen for an age-dependent increase in the ratio of blood GSSG to total glutathione. In the same study, plasma was shown to contain only 0.4% of total blood GSH. Compared with adults, children (median age: 13.3 y) had lower total glutathione, but not GSH, concentrations in blood. Individuals who practiced habitual physical activity had higher blood concentrations of both GSH and total glutathione. Smoking and alcohol consumption were associated with elevated blood GSH, perhaps as a defense against chronic oxidative stress exposure. The use of oral contraceptives tended to lower blood glutathione concentrations, but the relation was not statistically significant. Interestingly, in the population as a whole, total blood glutathione concentration was positively correlated with cholesterol and calcium concentrations (66).
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). However, Ji et al (68), who exercised 8 healthy male cyclists at 70% of maximal oxygen uptake, found that a bout of exercise lasting >2 h did not elevate blood GSSG. Studies involving human blood GSH oxidation during exercise are limited, but previous studies showed that exhaustive exercising of rats remarkably increases GSSG concentrations in plasma (69).
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14 min) and, 1 wk apart, 2 bouts of 30 min of exercise at their aerobic and anaerobic thresholds (70). Blood samples were drawn before, immediately after, and 24 h after tests. In line with the observation of Gohil et al (67), all 4 test exercise bouts notably increased the concentration of blood GSSG. Exercise-induced perturbations in blood glutathione redox status and plasma lipid peroxide concentration were no longer observed in the 24-h postexercise recovery samples. In another study, Viguie et al (71) found no evidence of persistent or cumulative effects of repeated leg cycling exercise (at 65% of peak oxygen uptake, for 90 min, for 3 consecutive days) on blood glutathione redox status. In moderately trained men, a 50% decline in the blood GSH concentration was observed during the first 15 min of exercise, which was accompanied by an increased blood concentration of GSSG. Total glutathione concentration in the blood did not change significantly during the exercise, and the blood GSH concentration returned to baseline after 15 min of recovery.
Although running at high speed (intervals of 20 s) to exhaustion did not influence blood GSH oxidation, Sastre et al (72) observed that in trained men, blood GSSG concentrations were 72% higher immediately after exercise than at rest. Accurately determining the ratio of GSSG to GSH is challenging primarily because GSH is unstable during sample processing. This issue was addressed by Vina et al (73), who used a modified analytic approach in showing that, compared with preexercise values, exhaustive exercise may increase blood GSSG concentrations in humans and rats by 1-fold and 3.5-fold, respectively. Human blood GSH oxidation has proven to be a consistent response to exercise. In young men, intermittent exercise bouts to exhaustion increased blood GSSG by 35% (49). More recently, Laaksonen et al (74) observed exercise-induced blood GSH oxidation in young men with type 1 diabetes and in corresponding healthy control subjects. Thus, from results obtained so far we may conclude that physical exercise may enhance the use of blood GSH, resulting in a decreased ratio of GSH to GSSG.
Studies in other tissues
Information on the possible effect of physical exercise on the GSH status of human tissues such as the skeletal muscles, heart, or liver is currently not available, but several studies have investigated this issue in experimental animals. Physical exercise clearly influences GSH metabolism in the skeletal muscles and liver of rats (39, 75). Lew et al (69) reported that exhaustive exercise consistently decreases both liver and muscle GSH. In investigating the influence of an exhaustive treadmill run on the tissue GSH status of rats (50), we found that the exercise decreased the total glutathione reserves of the liver and the active skeletal muscles red gastrocnemius and mixed vastus lateralis, but we did not observe this effect in the less active longissimus dorsi muscle. Exercise-induced decreases in the total glutathione pool in the liver, red gastrocnemius muscle, mixed vastus lateralis muscle, and the heart of rats were also seen in another independent study we carried out, but this effect was not seen in the lung (76). Duarte et al (77) confirmed that a single bout of exercise results in GSH loss from skeletal muscle; in their study, exercising resulted in a 50% decrease in the total glutathione content of the left soleus muscle, an effect that was interpreted as an index of oxidative stress. Recovery of the muscle GSH concentration was slow in the postexercise recovery period. This recovery was remarkably faster in mice that were supplemented with allopurinol, an inhibitor of the superoxide-producing enzyme xanthine oxidase. Hellsten (78) suggested that exercise-induced increases in superoxides generated by xanthine oxidase causes oxidative stress to muscle tissues located nearby and that this stress is manifested as a loss of tissue GSH.
Exhaustive treadmill exercise (24.1 m/min, 15% uphill grade) induces GSH oxidation in the plasma, skeletal muscle, and liver of rats (69). This effect was confirmed in our rat studies, in which exhaustive treadmill exercise markedly increased the amounts of GSSG in the liver, red gastrocnemius muscle, mixed vastus lateralis muscle, blood, and plasma (76). After oxidant challenge, GSH is transformed within the cell to GSSG. When the rate of oxidation is low, much of the GSSG thus produced may be enzymatically reduced by GSSG reductase activity to GSH. However, with a more severe oxidative stress, the rate of GSSG reduction cannot match the rate of its formation, which may result in the accumulation of intracellular GSSG. High concentrations of intracellular GSSG may be cytotoxic. In erythrocytes, cardiac muscle cells, and skeletal muscle cells, research has shown that excess intracellular GSSG is pumped out of the cell by an energy-dependent mechanism (79–81); such efflux of GSSG from oxidatively stressed tissues may account for the exercise-induced decrease in the total glutathione pools of these tissues, as discussed above.
In rats, the abilities of exhaustive physical exercise and intraperitoneal hydroperoxide injection to cause oxidative stress have been compared (82); an exhaustive treadmill run increased amounts of GSH in deep vastus lateralis muscle, but hydroperoxide injection had no effect. In that study, hepatic GSH amounts were not influenced by exercise. The exercise bout increased the amount of skeletal muscle GSSG, but no such effect was observed in the liver. Based on the muscle GSH oxidation results, it was concluded that the bout of exercise induced more oxidative stress than did the bolus of oxidant.
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EXERCISE-INDUCED CHANGES IN PROTEIN SULFHYDRYLS |
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TOPABSTRACTINTRODUCTIONCENTRAL ROLE OF THIOLS...ENDOGENOUS BIOTHIOLSEXERCISE-INDUCED OXIDATIVE...SKELETAL MUSCLE GLUTATHIONETRAINING AND GLUTATHIONE...EXERCISE-INDUCED CHANGES IN...EXERCISE-INDUCED CHANGES IN...MANIPULATION OF TISSUE...THIOL MANIPULATION AND EXERCISEHUMAN DISEASESIGNAL TRANSDUCTIONCONCLUSIONSDIRECTIONS FOR FUTURE RESEARCHREFERENCES |
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Protein sulfhydryls are highly unstable and, if not adequately processed, are likely to be oxidized during sample processing or assays. Thus, it is essential that membrane-permeable thiol probes be used to process protein sulfhydryls in the intact tissue. Sulfhydryl status and oxidative damage of microsomes derived from skeletal muscle in swim-exercised rats were studied, but this work (85) suffers from the fact that microsomal sulfhydryls were reacted with a thiol-detecting agent, dithionitrobenzoic acid, only after tissue homogenization, differential centrifugation, and denaturation with detergent. It is unlikely that the redox state of microsomal sulfhydryls would resist all such tissue-processing procedures. Swim exercise induced oxidative lipid damage, but this was accompanied by an increase in tissue sulfhydryls (85). Seward et al (86) observed in rats that although exhaustive exercise is followed by a remarkable decrease in heart nonprotein GSH content, there are no changes in tissue total soluble thiol status. More recently, plasma protein-bound thiols were studied in healthy young men who participated in a full marathon race (42.195 km) after 6 mo of training (87). Plasma protein thiol concentrations were markedly decreased immediately after the race, suggesting that protein sulfhydryls were oxidized during the competition. In contrast with the oxidation of blood GSH during exercise, which is known to recover rapidly during the postexercise period, plasma protein thiol concentrations remained low even after 24 and 48 h of postmarathon recovery.
Acute exercise has been shown to markedly decrease phosphofructokinase activity, the rate-limiting enzyme of glycolysis, in white (rich in type IIb fiber ) and red (rich in type IIa fiber) gastrocnemius muscles. In both old and young age groups, such an effect appears to depend on the muscle fiber type [IIb > IIa > I (soleus)]. The degree of phosphofructokinase down-regulation was inversely related to the activities of superoxide dismutase and glutathione peroxidase in the tissue, indicating that the down-regulation process may have been driven by secondary changes triggered as a response to oxidative stress. Mammalian phosphofructokinase is rich in exposed sulfhydryl groups that make the enzyme more prone to oxidant attack (88). It has also been observed that in cells derived from skeletal muscle, certain membrane K+ transport proteins are highly sensitive to oxidant exposure (89).
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MANIPULATION OF TISSUE GLUTATHIONE |
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TOPABSTRACTINTRODUCTIONCENTRAL ROLE OF THIOLS...ENDOGENOUS BIOTHIOLSEXERCISE-INDUCED OXIDATIVE...SKELETAL MUSCLE GLUTATHIONETRAINING AND GLUTATHIONE...EXERCISE-INDUCED CHANGES IN...EXERCISE-INDUCED CHANGES IN...MANIPULATION OF TISSUE...THIOL MANIPULATION AND EXERCISEHUMAN DISEASESIGNAL TRANSDUCTIONCONCLUSIONSDIRECTIONS FOR FUTURE RESEARCHREFERENCES |
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Selenium
GSH-dependent enzymatic metabolism of hydroperoxides involves glutathione peroxidase activity. Selenium acts as a cofactor of glutathione peroxidase and thus selenium is necessary to maintain full-strength GSH-dependent antioxidant defense (90). Dietary deficiency of selenium remarkably lowers tissue glutathione peroxidase activity and thus makes the tissue more susceptible to oxidative damage. In one study, chronic dietary deficiency of selenium did not affect the body weight or endurance capacity of rats even though the activity of selenium-dependent glutathione peroxidase in the liver and skeletal muscles decreased by >80% (91). The authors explained that weakening of the glutathione peroxidase–dependent antioxidant protection may have activated alternative antioxidant pathways as a compensatory response. However, selenium-independent glutathione peroxidase activity was not increased in selenium-deficient rats. The liver vitamin E content of selenium-deficient rats was significantly decreased, indicating enhanced consumption of this lipophilic antioxidant. Selenium supplements appeared to have a sparing effect on the dietary requirement for vitamin E.
In another study (61), dietary selenium deficiency remarkably decreased selenium-dependent glutathione peroxidase activity in the liver and skeletal muscle. Selenium-independent glutathione peroxidase activity was barely affected by the dietary restriction. Thus, dietary selenium insufficiency may limit GSH-dependent tissue antioxidant defense. The effects of dietary selenium supplementation (0.5 ppm) and of selenium deprivation on the liver, muscle, and blood of rats in a swimming protocol have also been tested (92). Tissue glutathione peroxidase activity sensitively responded to dietary selenium status, and selenium deficiency was associated with increased oxidative lipid damage in tissues. In a double-blind human study, 180 µg selenomethionine supplement every day for 10 wk increased plasma glutathione peroxidase activity but had no effect on physical performance (49).
Riboflavin
Regeneration of GSH from GSSG inside the cell depends on GSSG reductase activity in the presence of NADPH, and activity of this enzyme requires the tissue to have adequate riboflavin status. Thus, nutritional vitamin B supply may influence tissue GSH redox cycle activity. Because GSSG reductase activity is sensitive to the riboflavin status of the tissue, tissue GSSG reductase activity is often used as a marker of riboflavin availability in tissues (93). Regular physical exercise decreases riboflavin excretion and enhances its retention in tissues (93, 94); in this way more riboflavin retained in tissues can favorably influence GSSG reductase activity.
Strategies to increase cellular glutathione
Enhancing tissue GSH reserve in mammals is a challenging task, particularly because GSH per se is not available to most tissues when administered orally or injected intraperitoneally. The net amount of tissue GSH depends primarily on 2 factors: GSH neosynthesis and GSH regeneration from GSSG (95). Synthesis of GSH in the cell is rate-limited by the availability of the amino acid cysteine, which, in its reduced form, is highly unstable. Thus, >90% of cysteine in the human circulation is present in the oxidized cystine form (96). In cell culture media, this amino acid is present only in the cystine form. Thus, one strategy to enhance intracellular GSH is to improve cysteine availability within the cell (Figure 3). For example, addition of a reducing agent such as ß-mercaptoethanol to cell culture media will enhance cell GSH content (95, 97). Other ways to boost GSH synthesis in the cell include enhancing the activity of GSH-synthesizing enzymes by gene transfer. In Escherichia coli these specific genes have been isolated and used to transform the wild strain to overexpress the synthetase enzymes (98). Similar work with mammalian cell systems has been performed (99, 100), but at present this approach is far from being clinically relevant.
Glutathione esters
The cell GSH reserve may be directly elevated by administering GSH esters. Both GSH mono(glycyl)esters and GSH diethyl esters have been used for this purpose (101). Unlike GSH itself, which is a lipophobic molecule that cannot penetrate the cell membrane, esterified GSH is lipophilic and thus membrane permeable. However, using such esters to boost the tissue GSH pool is not without limitations. For example, metal ion contamination of GSH monoesters remarkably decreases the capacity of the compound to serve as a GSH-delivering agent. In addition, certain forms of esterified GSH, such as the GSH dimethyl ester, appear to be toxic to mice (102). Furthermore, some esters are deesterified by esterase activity in the plasma and lose their ability to permeate the cell membrane. Levy et al (102) reported that GSH diethyl ester may be the GSH delivery agent of choice, especially for those species (eg, humans, not rats or mice) that lack GSH diester -esterase in the plasma (102). GSH diester has been found to be nontoxic to mice and hamsters, but has yet to be tested in vivo in humans. Therefore, although the use of such forms of esterified GSH is appealing, at present it may be premature to run clinical trials to test the efficiency of such esters in managing exercise-induced oxidative stress.
N-acetyl-L-cysteine
Improving cysteine availability for the biosynthesis of GSH is the most extensively studied approach for enhancing the cell GSH pool. Among the agents tested are N-acetyl-L-cysteine (NAC), lipoic acid, cysteamine, and 2-oxothiazolidine 4-carboxylate. Other reducing agents, such as dithiocarbamate, ß-mercaptoethanol, and dithiothreitol, may also improve GSH synthesis through the extracellular reduction of cystine to cysteine. NAC and -lipoic acid have generated the most interest because of their proven clinical safety features and efficacy in vivo (16, 95, 96, 103–110). Oral NAC (2-mercapto-propionyl glycine) was found to elevate GSH in the plasma and bronchoalveolar lavage fluid (107). NAC effectively controls perturbations in the thiol redox status after acetaminophen toxicity and has been successfully used for clinical purposes (105).
-Lipoic acid
-Lipoic acid is also known as thioctic acid, 1,2-dithiolane-3-pentanoic acid, 1,2-dithiolane-3-valeric acid, and 6,8-thioctic acid. At physiologic pH, lipoic acid is anionic and in this form it is commonly called lipoate. As early as the 1950s, -lipoate was seen as an essential cofactor in oxidative metabolism (16, 83, 95, 109–111). Biologically, lipoate exists as lipoamide in 
5 proteins, where it is covalently linked to a lysyl residue. Four of these proteins are found in -ketoacid dehydrogenase complexes: the pyruvate dehydrogenase complex, the branched-chain keto-acid dehydrogenase complex, and the -ketoglutarate dehydrogenase complex. Three of the lipoamide-containing proteins are present in the E2 enzyme dihydrolipoamide S-acetyltransferase, which is different in each of the complexes and is specific for the substrate of the complex. One lipoyl residue is found in protein X, which is the same in each complex. The fifth lipoamide residue is present in the glycine cleavage system (111).
Lipoic acid is detected in the form of lipoyllysine in various natural sources (112). When expressed as weight per dry weight of lyophilized vegetables, the relative abundance of naturally existing lipoate was found to be spinach > > broccoli floral buds > tomato fruit > garden peas and Brussels sprouts > rice bran. Lipoyllysine concentrations were not detectable in acetone powders of banana, orange peel, soybean, and horseradish. In animal tissues, the abundance of lipoyllysine in bovine acetone powders was determined to be kidney > heart > liver > spleen > brain > pancreas > lung. Concentrations of lipoyllysine in spinach and bovine kidney were 3.15 ± 1.11 and 2.64 ± 1.23 µg/g dry wt, respectively (112).
Lipoate has generated considerable clinical interest as a thiol-replenishing and redox-modulating agent (16, 17, 83, 95). Recently, it was also observed that lipoate treatment selectively facilitates the death of cancer cells by potentiating the inducible activity of caspase 3, also known as death protease (113). A unique property of lipoate is that it is a "metabolic antioxidant"; enzymes in human cells accept it as a substrate for reduction. Accordingly, supplemented lipoate is promptly taken up by cells and reduced to dihydrolipoate at the expense of cellular-reducing equivalents such as NADH and NADPH (114, 115). As more of these reducing equivalents are used, the rate of cellular metabolism is expedited to cater to the enhanced demand (115). Dihydrolipoate is a powerful reducing agent (Table 1) with many antioxidant properties that mediates the pro-GSH effects of lipoate (Figure 3). Thus, a unique property of supplemental lipoate is harnessing the power of the cell's own metabolic processes for its recycling and potency. In addition to its remarkable effect in strengthening antioxidant defenses, lipoate is known to promote the efficiency of glucose uptake by cultured skeletal muscle cells at a magnitude comparable to that of insulin (116, 117). Interestingly, lipoate was shown to retain its ability to stimulate glucose uptake even where L6 myotubes were insulin resistant (117). Whether such an insulin-mimetic property of lipoate influences muscle bioenergetics during exercise is an open question.
After being enzymatically generated inside the cell, dihydrolipoate rapidly escapes to the extracellular culture medium (114). To improve retention in cells, we recently modified the -lipoic acid molecule to confer a positive charge at physiologic pH (118); the protonated form of the new molecule is called LA-Plus. We found the uptake of LA-Plus by human T cells to be higher than the uptake of lipoate. In addition, amounts of DHLA-Plus, the corresponding reduced form of LA-Plus, were several times higher in cells treated with LA-Plus than were amounts of dihydrolipoate in cells treated with lipoate. Furthermore, on a concentration basis, LA-Plus was more biologically potent than lipoate (118, 119). These promising results set the stage for further testing of the possible beneficial role of LA-Plus as an antioxidant supplement.
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THIOL MANIPULATION AND EXERCISE |
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TOPABSTRACTINTRODUCTIONCENTRAL ROLE OF THIOLS...ENDOGENOUS BIOTHIOLSEXERCISE-INDUCED OXIDATIVE...SKELETAL MUSCLE GLUTATHIONETRAINING AND GLUTATHIONE...EXERCISE-INDUCED CHANGES IN...EXERCISE-INDUCED CHANGES IN...MANIPULATION OF TISSUE...THIOL MANIPULATION AND EXERCISEHUMAN DISEASESIGNAL TRANSDUCTIONCONCLUSIONSDIRECTIONS FOR FUTURE RESEARCHREFERENCES |
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5 min) to induce oxidative stress. More complete studies are necessary to explain how exogenous GSH may enhance exercise performance. To test the time-dependent distribution of intraperitoneally administered GSH, we administered the thiol (1 g/kg body wt) to male Wistar rats (76). Injection of GSH solution resulted in a rapid appearance (>102 times 0.5 h after administration) of glutathione in the plasma. After such a response, a rapid clearance of plasma total glutathione was observed. Twenty-four hours after the injection, plasma total glutathione was restored to the preinjection control concentration. Excess postinjection plasma GSH was rapidly oxidized, as detected by the presence of GSSG. Supplemented GSH was not available to tissues such as the liver, skeletal muscles, lung, kidney, or heart. However, after repeated injection of GSH for 3 consecutive days, total amounts of glutathione in blood and kidney increased. No such effect was observed in the liver, red gastrocnemius muscle, mixed vastus lateralis muscle, heart, or lung (76).
Rats treated with a single injection of GSH or NAC (1 g/kg body wt) were subjected to exhaustive treadmill exercise 0.5 h after the intraperitoneal injection (76). The exercise induced GSH oxidation in several tissues, including the skeletal muscle, lung, blood, and plasma. GSH supplementation did not protect against exercise-associated changes in GSH status and lipid peroxidation in the liver, skeletal muscle, heart, and lung of rats. Exercise caused blood GSH oxidation and NAC supplementation protected against such oxidation. NAC also appeared to protect against exercise-induced perturbation of GSH redox status in the lung. Neither GSH nor NAC supplementation had any influence on endurance during an exhaustive long distance treadmill run (76). In a separate study in which GSH supplementation was studied in endurance-trained rats, such supplementation appeared effective in decreasing exercise-induced leakage of mitochondrial superoxide dismutase protein from tissues to the plasma (122).
The influence of supplementation with GSH (1 g/kg body wt), NAC (1 g/kg), or vitamin C (0.5 g/kg) for 1 wk on exercise-induced blood GSH oxidation was also investigated in rats. GSH supplementation had no significant effect, but both NAC and vitamin C supplementations partially decreased blood GSH oxidation after treadmill exercise (72).
Although it has been argued that lipoate supplementation may improve mitochondrial function by facilitating the activity of lipoyllysine-containing enzymes (123), there is no evidence to support this contention. The first study testing the possible effects of oral -lipoic acid supplementation as well as the effect of a single bout of strenuous exercise and endurance exercise training on the lipoyllysine content of skeletal muscle and liver tissues in rats was reported recently (124). Incorporation of the lipoyl moiety to tissue protein was not increased by dietary lipoate. Interestingly, endurance exercise training markedly increased lipoyllysine content in the liver at rest, and a bout of exhaustive exercise also increased hepatic lipoyllysine content. A significant interaction between exhaustive exercise and training in increasing tissue lipoyllysine content was evident. In vastus lateralis skeletal muscle, training did not influence tissue lipoyllysine content. A single bout of exhaustive exercise, however, clearly increased the amount of lipoyllysine in the muscle. Comparison of tissue lipoyllysine data with results for free or loosely bound lipoate showed a clear lack of association between the 2 apparently related parameters. Thus, the tightly protein-bound lipoyllysine pool in tissues is independent of the loosely bound or free lipoate status in the tissue (124).
In the
) and women (
) of different age groups. GSSG data were calculated [2 GSSG = total glutathione - GSH] from results published by Michelet et al (66). A trend was seen that with increasing age, more of total glutathione in the human blood is present in the oxidized form. No sex-specific differences in blood glutathione redox state were observed.
O2peak, peak oxygen uptake. *Significantly different from resting values, P < 0.05. Reproduced with permission from Gohil et al (67).