Free radicals are highly reactive atoms or molecules that have an unpaired electron in their outer orbital (9,11). Recent evidence indicates that muscular exercise results in the production of radicals and other reactive oxygen species within skeletal muscle (3,8,13,27,42,57,72,87,88). Specifically, muscular contraction has been shown to generate several reactive radicals, such as superoxide (O•−2), hydrogen peroxide (H2O2), nitric oxide (NO.), and hydroxyl radicals (HO.) (8,13,27,42,57,72,87,88). It seems likely that the majority of the oxidants produced within the contracting myocyte are due to an elevated rate of mitochondrial respiration (reviewed in 15,28). However, other potential sources of oxidants during exercise include the xanthine oxidase pathway, prostanoid metabolism, and calcium-mediated radical production (reviewed in 28,81). Further, NO. is also produced by the mitochondria (18), and several oxidants as well as chlorinating species are produced by phagocytes (24), which may have implications in the etiology of exercise-induced oxidative damage.
Unscavenged oxidants can modify macromolecules in the cell including nucleic acids, proteins, and lipids (21,101). This oxidation of cellular components (oxidative stress) can occur when an imbalance exists between oxidants and antioxidants. Oxidative stress occurs under conditions when local antioxidant defenses are depleted because of oxidants or when the rate constants of the radical reactions are greater than the rate constants of the antioxidant defense mechanisms (10). This could occur in skeletal muscle during acute exercise under conditions when oxidant/antioxidant balance shifts toward the pro-oxidant state.
Indeed, intense or prolonged muscular exercise can result in oxidative injury to lipids, proteins, and DNA within skeletal muscle myocytes (2,23,47,49,90,97,98). Moreover, evidence exists implicating oxidants as a contributor to both exercise-induced bioenergetic enzyme down-regulation and muscle fatigue (4,7,15,29,47,48,50,68,69,71,87-89,93). Given the potential role oxidants play in cellular damage, it is not surprising that myocytes and other cells contain several naturally occurring antioxidant defense mechanisms to prevent oxidative injury. Two major classes of endogenous protective mechanisms work together to reduce the harmful effects of oxidants in the cell: 1) enzymatic defenses (e.g., superoxide dismutase, glutathione peroxidase, and catalase); and 2) nonenzymatic antioxidants (e.g., glutathione). The purpose of this review is to discuss the impact of endurance exercise training on the antioxidant capacity of skeletal muscle. Specifically, we will focus on exercise training-induced changes in the activities of primary antioxidant enzymes and the glutathione antioxidant defense system.
OVERVIEW OF ANTIOXIDANT ENZYMES
There are three primary antioxidant enzymes in cells: 1) superoxide dismutase (SOD); 2) glutathione peroxidase (GPX); and 3) catalase (CAT). Each of these enzymes is capable of producing other less reactive species or neutralizing reactive oxygen metabolites. SOD promotes the dismutation of superoxide radical (O•−2) and forms hydrogen peroxide (H2O2) and oxygen. GPX utilizes reduced glutathione (GSH) as a reducing equivalent to reduce H2O2 to form oxidized glutathione and water. CAT converts H2O2 to water and oxygen. A brief overview of the structure, cellular location, and catalytic function of each of these enzymes follows.
Superoxide dismutase. The first defense against superoxide radicals is the enzyme superoxide dismutase (reaction 1). SOD dismutates superoxide radicals to form hydrogen peroxide and O2(21): Equation [1]
In mammals, two isozymes of SOD exist in skeletal muscle; they vary in both cellular locations as well as the metal ion bound to its active site. The Cu-Zn SOD is primarily located in the cytosol whereas the Mn-SOD is principally found in the mitochondrial matrix (21). Both enzymes catalyze the dismutation of superoxide anions with similar efficiency (76). The Cu-Zn SOD is a dimer with a molecular weight of ∼32,000 kDa whereas the Mn SOD is a tetramer (MW = ∼88,000 kDa) (76).
The distribution of the SOD isoforms varies from tissue to tissue. In skeletal muscle, 15-35% of the total SOD activity is in the mitochondria with the remaining 65-85% being in the cytosol (53). The literature suggests that SOD activity is greatest in highly oxidative muscles (i.e., a high percentage of Type I and IIa fibers) compared with muscles with low oxidative capacity (i.e., a high percentage of Type IIb fibers) (12,83,84).
Numerous techniques to determine SOD activity in tissues have been developed. However, the direct measurement of SOD activity in tissues is difficult because both the substrate and product of this reaction are unstable. Therefore, a common approach to assay SOD activity is to employ a system for the production of superoxide, e.g., xanthine/xanthine oxidase reaction, in the presence of SOD. The approach is to then measure the reduction of an optically sensitive compound such as cytochrome c or nitroblue tetrazolium. Other approaches have employed autoxidation techniques using epinephrine or sulfite. Using these assay techniques, the two isoforms of SOD can be distinguished via inhibition of Cu-Zn SOD by cyanide.
Glutathione peroxidase. GPX catalyzes the reduction of H2O2 or organic hydroperoxide to H2O (reaction 2) and alcohol (reaction 3), respectively, using reduced glutathione (GSH) as the electron donor (21). In these reactions GSH is oxidized to form glutathione disulfide (GSSG): Equations [2] and [3]
GPX is a selenium dependent enzyme that exists in one isoform (33,37). Although GPX is highly specific for its electron donor (GSH), this enzyme has a low specificity for hydroperoxides. In this regard, GPX will reduce a wide variety of hydroperoxides ranging from H2O2 to numerous complex organic hydroperoxides (10,101). This favorable characteristic makes GPX an important cellular protectant against reactive oxygen species-mediated damage to membrane lipids, proteins, and nucleic acids.
GPX activity varies between muscle fiber types with Type I fibers containing the highest activity and Type IIb fibers possessing the lowest activity (31,84). Similar to SOD, GPX is located in both the cytosol and the mitochondria. In skeletal muscle, approximately 45% of the GPX activity is found in the cytosol; the remaining 55% of GPX activity is found in the mitochondria (31). The location of GPX in both the mitochondria and cytosol allows it to reach a number of sources of hydroperoxide generation (37).
To function, GPX requires GSH as the electron donor. Because GSH is oxidized by GPX to form GSSH, cells must possess a pathway capable of regenerating GSH. This is accomplished by the enzyme glutathione reductase (GR), which uses NADPH as the reducing power of the reaction (reaction 4): Equation [4]
In many tissues, NADPH is largely produced by glucose-6-phosphate dehydrogenase via the pentose pathway (64). However, in skeletal muscle, NADPH is primarily produced by isocitrate dehydrogenase (48,80).
GR has a cellular distribution similar to GPX, and its activity is also greater in highly oxidative muscles. Although GR is not considered to be a primary antioxidant enzyme, it is essential for the normal antioxidant function of GPX. The interaction of GPX, GR, and GSH will be discussed in more detail in a later section.
GPX is commonly assayed by using GSH and H2O2 as substrates, with a coupled reaction involving both GPX and GR (see reactions 2 and 4). GR uses NADPH to regenerate GSH from GSSG. The rate of change in NADPH oxidation is monitored spectrophotometrically at a wavelength of 340 nm.
Catalase. The primary function of CAT is to catalyze the decomposition of H2O2(reaction 5): Equation [5]
CAT is a tetramer with a molecular weight of ∼240,000 kDa. Iron (Fe3+) is a required cofactor that must be bound to the enzyme's active site (21). Although there is some overlap between the function of CAT and GPX, the two enzymes differ in their affinity for H2O2 as a substrate. Mammalian GPX has a much greater affinity for H2O2 at low concentrations compared with CAT (e.g., GPX Km = 1μM vs CAT Km = 1mM). This means that at low H2O2 concentrations GPX plays a more active role in removing H2O2 from the muscle cell.
CAT is widely distributed in the cell with high concentrations found in the peroxisomes and smaller concentrations in the mitochondria (21). Similar to SOD and GPX, CAT activity is highest in highly oxidative muscles and lowest in muscle with a large percentage of fast (Type II) fibers (84). CAT activity is commonly assayed via spectrophotometric techniques using H2O2 as a substrate (37). A review of the literature reveals that a large range of CAT activities have been reported in mammalian skeletal muscle. This wide variance in CAT activity probably results from the fact that the turnover rate varies with both the amount of active CAT protein present and also with the concentration of H2O2 in the reaction medium. Therefore, CAT activity in tissues can only be compared between studies in which the conditions of the assay are carefully defined (37).
Glutathione. GSH is a thiol-containing tripeptide (L-γ-glutamyl-L-cysteinyl-glycine) found in virtually all animal and plant cells as well as in some bacteria (65-67). GSH serves multiple roles in cellular antioxidant defenses (14,37,38,45,63,65,67). The most important antioxidant function of GSH is to remove hydrogen peroxide and organic peroxides (e.g., lipid peroxide) catalyzed by the selenium-dependent enzyme GPX, forming water or alcohol, respectively. By donating a pair of hydrogen ions, GSH is oxidized to GSSG. As mentioned earlier, reduction of GSSG is catalyzed by GR, a flavin-containing enzyme in which NADPH is used as a reducing substrate. This reaction takes place in conjunction with GPX, thus providing a redox cycle for the regeneration of GSH (17,67). NADPH is supplied by the hexose monophosphate pathway or by reactions catalyzed by isocitrate dehydrogenase and malic enzyme, depending on the tissue (Fig. 1)(37,38,67).
;)
Figure 1: Illustration of the glutathione redox cycle. The reduction of glutathione disulfide (GSSG) is catalyzed by the enzyme glutathione reductase (GR), and nicotinamide-adenine dinucleotide phosphate (NADPH) is used as a reducing substrate. This reaction takes place in conjunction with GPX, thus providing a redox cycle for the regeneration of GSH. Cellular NADPH can be produced by both glucose-6-phosphate dehydrogenase (G6PDH) via the pentose pathway or by isocitrate dehydrogenase (ICDH).
GSSG levels in most tissues are kept very low, and the tissue-specific intracellular ratio of GSH:GSSG may be much higher than reported in the literature (6,67,99). Cells are capable of exporting GSSG to maintain the GSH:GSSG ratio and to alleviate the potential toxic effect of GSSG. This has been demonstrated in skeletal muscle, liver, heart, and erythrocytes (1,40,41,44,65).
Within the cell, GSH is most likely involved in reducing a variety of antioxidants to their native structure. For example, GSH has been postulated to reduce vitamin E radicals (α-tocopheroxyl) that are formed in the chain-breaking reactions with alkoxyl or lipid peroxyl radicals (21,70,101). GSH may also reduce semi-dehydroascorbate radical (the vitamin C radical) derived in the recycling of vitamin E and can reduce α-lipoic acid to dihydrolipoate. The latter may play an important role in the recycling of ascorbic acid (21,70,101). Together, these reactions efficiently regenerate vitamin E and C at the expense of GSH.
GSH is the most abundant short-chain peptide and non-protein thiol source in the cell (65-67). GSH concentration in the cell is in the millimolar range with variable amounts of GSH in different organs due to their function and oxidative capacity. The liver has one of the highest concentrations of GSH (5-7 mM) in the body as only the lens of the eye has a greater content (21). Other important organs such as the lung, kidney, and heart contain 2-3 mM of GSH. It is noteworthy that the red blood cell contains a high level of GSH (∼2 mM) compared with blood plasma (<0.05 mM) to protect against oxidative stress (34,43,58,61).
Skeletal muscle GSH concentration varies depending on muscle fiber type and species (35-37). Type I muscle (slow-twitch oxidative), such as the soleus, contains six-fold higher GSH content (∼3 mM) than the Type IIb muscle, white vastus lateralis (fast-twitch glycolytic) in rats. However, the GSH:GSSG ratio appears remarkably consistent across various fiber types (37).
Intracellular GSH levels depend on GSH uptake from the blood, intracellular GSH synthesis, and GSH utilization and regeneration by GPX and GR (reviewed in 37 and 67). Although much of the de novo synthesis of GSH occurs in the liver, many other cell types can also synthesize GSH de novo. The de novo synthesis of GSH occurs via several enzymes known as the γ-glutamyl cycle. γ-Glutamyl-transpeptidase (GGT) is bound to the plasma membrane and controls the cleavage of GSH and the subsequent translocation of amino acids across the cell membrane (67). Glutamylcysteine synthetase (GCS) catalyzes the formation of an initial peptide bond between cysteine and glutamate. This rate-limiting step of GSH synthesis is inhibited by negative feedback of GSH on this enzyme. The final step of GSH synthesis is catalyzed by GSH synthetase (GS) (Fig. 2).
Figure 2: Schematic of the de novo synthesis of glutathione (GSH) and transport of GSH out of the cell. Synthesis of GSH occurs via several enzyme systems known as the γ-glutamyl cycle. γ-Glutamyltranspeptidase (GGT) is bound to the plasma membrane and regulates the breakdown of GSH and the movement of amino acids across the cell membrane. γ-Glutamylcysteine synthetase (GCS), the rate-limiting enzyme, catalyzes the formation of the peptide bond between cysteine and glutamate. The final step of GSH synthesis is catalyzed by glutathione synthetase (GS). GSH can be transported out of the cell via a GSH transporter (GSHT). Other abbreviations include: AA, amino acids; 5-OP, oxoprolinase; Cys2, cystine; cysH, cysteine; and Gly, glycine.
Tissues synthesize GSH at different rates depending on the presence of synthesizing enzymes. For instance, skeletal muscle (62), intestinal mucosa (94), macrophages (19), and the liver (53,54,56) all contain relatively high γ-glutamyl-cysteine synthetase (GCS) activities; this is significant because GCS is the rate limiting enzyme in the γ-glutamyl cycle. Note that the rate of GSH synthesis can be altered under certain physiological conditions. For example, exercise training in dogs significantly increased GCS by 1.5- to 2-fold and glutathione synthetase 3-fold (62). Also, macrophages increase the level of intracellular glutathione by de novo synthesis upon stimulation by oxidized low-density lipoprotein (LDL) (19).
The liver contains a major reserve of GSH and supplies a large amount of the circulating GSH (reviewed in 67). Hepatic GSH synthesis is controlled by amino acid availability and hormonal regulation (14,53,56). Fasting decreases liver GSH content and refeeding quickly restores GSH levels to control levels (53,56). Insulin and glucocorticoids stimulate hepatic GSH synthesis by an induction of GCS (59,60). In contrast, glucagon and several other cAMP-stimulating agents down-regulate hepatic GSH synthesis by phosphorylating and inhibiting GCS (60). Liver efflux of GSH is enhanced by an increase in adrenaline and glucagon levels (59,60,86,95).
GSH turns over at a significant rate in virtually all mammalian tissues. It is estimated that the turnover rate amounts to 4.5, 2.7, and 1.6 μmol·h−1 for the liver, kidney, and skeletal muscle, respectively (20). GSH turnover rate is low in the skeletal muscle, partly due to a low GGT activity (51,91). However, the skeletal muscle as a whole is an important GSH pool because of the large muscle mass (∼40% of the body weight), which may exert an important influence on plasma GSH levels and GSH turnover under certain physiological conditions (44).
Intracellular GSH distribution is especially important for some organelles such as the mitochondria in which the membrane is an important source of free radical generation. Because mitochondria lack the enzymes of the γ-glutamyl cycle, mitochondrial GSH is transported from the cytosol by specific carriers (16,63). In this regard, Fernandez et al. (16) identified the mitochondrial GSH carrier, which displays functional characteristics distinct from other plasma membrane GSH carriers, such as its ATP dependency, inhibitor specificity, and the size class of mRNA that encode the carrier.
The physiological role of GSH is best illustrated when tissue GSH homeostasis is perturbed by physiological, nutritional, and pharmacological interventions and then subjected to an oxidative challenge. For example, a procedure to deplete tissue GSH is through the administration of L-buthionine SR-sulfoximine (BSO), an irreversible inhibitor of GCS (66). Rats receiving BSO by i.p. injections experienced a 50% decrease in running endurance time, which suggests that GSH plays an essential role in maintaining exercise performance during high-intensity treadmill running (92). Moreover, GSH supplementation has shown to be effective in increasing exercise performance in mice (56,71).
EXERCISE TRAINING AND SKELETAL MUSCLE ANTIOXIDANT ENZYME ACTIVITY
The antioxidant capacity of mammalian organ systems is well matched to the rates of oxygen consumption and radical production. Body tissues with the highest oxygen consumption, e.g., liver, brain, and kidney, have the greatest antioxidant enzyme activity. Further, skeletal muscles with high oxidative capacities possess higher antioxidant capacities compared with those muscles with lower oxidative potential.
It is well established that the antioxidant defense systems of many mammalian tissues are capable of adaptation in response to chronic exposure to oxidants. For example, it has been shown that irradiation of the mouse heart results in an increased expression of Mn-SOD (73). Because prolonged exercise results in an increased production of oxidants in skeletal muscle, regular exercise training should up-regulate muscle antioxidant enzyme activities. Collectively, there is strong evidence that endurance exercise training results in an increase in skeletal muscle antioxidant enzyme activity. Nonetheless, a few studies have failed to find augmented muscle antioxidant activity after exercise training. In this section, we will summarize the major findings regarding the effects of endurance exercise training on the activities of primary antioxidant enzymes in skeletal muscle.
Superoxide dismutase.Table 1 contains a collection of representative reports regarding the effects of endurance exercise on skeletal muscle total SOD activity. Although some investigators have reported that endurance training does not promote an increase in SOD activity in skeletal muscles (2,31,46), many studies have reported a training-induced increase in total SOD activity (26,30,51-55,74,75,82-84).
TABLE 1: Effects of endurance training on total skeletal muscle superoxide dismutase (SOD) activity; these studies are a representative sample of published reports regarding the effects of endurance exercise on SOD activity in both human and rat skeletal muscle.
A definitive explanation for the lack of agreement among studies is not available. Nonetheless, differences in the techniques used to assay SOD activity, variances in the type of exercise training paradigm, and fiber composition of the muscles investigated could be key factors. For example, the relative sensitivity to detect SOD activity differs widely between assays. Indeed, when comparing seven common assays to estimate SOD activity, Oyanagui (77) reported that a 10-fold difference in relative sensitivity exists between methods. Therefore, investigations using SOD procedures with low sensitivity could fail to observe small-to-moderate training-induced changes in tissue SOD activity due to the lack of assay sensitivity.
Another potential explanation for the variable SOD findings is the large differences in the type of exercise training protocols used by investigators. Studies that have reported training-induced increases in muscle antioxidant enzymes have generally employed rigorous exercise training programs (i.e., high intensity and long duration of exercise). This observation suggests that rigorous exercise training programs may be required to promote antioxidant enzyme activity in skeletal muscle. To test the hypothesis that high-intensity and long duration-exercise training is required to increase SOD activity in skeletal muscle, Powers et al. (84) experimentally analyzed the relationship between the magnitude of the training stimulus (i.e., exercise intensity and daily duration) and the activity of SOD in locomotor skeletal muscles. Nine groups of rats ran at three different daily durations (i.e., 30, 60, 90 min·d−1) and three different exercise intensities (i.e., 55, 65, and 75% of V̇O2max). The results for the soleus muscle are illustrated in Figure 3. These data clearly show that high intensity exercise training is superior to low-intensity exercise in the up-regulation of skeletal muscle SOD activity.
Figure 3: The effects of training intensity and duration on soleus superoxide dismutase activity (SOD) of exercise trained rats. Animals were trained for 10 wk on a treadmill at varying duration and intensities (i.e., 30, 60, 90 min·d
−1 and 55 (low), 65 (med), and 75% (high) of V̇O
2max). Values are means. Data are from Powers et al. (
84: Powers, S., D. Criswell, J. Lawler, et al. Influence of exercise and fiber type on antioxidant enzyme activity in rat skeletal muscle.
Am. J. Physiol. 266:R375-R380, 1994). * Different (
P < 0.05) from medium- and high-intensity exercise training group.
Further, a critical analysis of training studies reveals that exercise induction of SOD may be fiber type specific with highly oxidative muscles being most responsive (12,31,46,84). A study that illustrates this point was performed by Powers et al. (84), and the results are summarized in Figure 4. In these experiments, adult rats were exercise trained for 60 min·d−1 (∼75% V̇O2max) for 10 wk. Upon completion of the training program, the soleus, red gastrocnemius, and white gastrocnemius were removed, homogenized, and assays performed to determine SOD activity. The results revealed that training promoted an increase in SOD activity in both the soleus and red gastrocnemius. In contrast, SOD activity in the white gastrocnemius was not altered by training. This is significant because the soleus (primarily Type I fibers) and red gastrocnemius (primarily Type IIa fibers) are composed of highly oxidative fibers, but the white gastrocnemius (primarily Type IIb fibers) is composed of highly glycolytic fibers (5).
Figure 4: The effects of endurance exercise training on the up-regulation of superoxide dismutase activity (SOD) in muscles differing in fiber composition. Values are means. Data are from Powers et al. (
84: Powers, S., D. Criswell, J. Lawler, et al. Influence of exercise and fiber type on antioxidant enzyme activity in rat skeletal muscle.
Am. J. Physiol. 266:R375-R380, 1994). * Indicates different (
P < 0.05) from untrained.
It seems possible that differences in fiber recruitment patterns (i.e., size principle) may account for these differences. However, the direct influence of exercise intensity and fiber recruitment on the up-regulation of SOD activity in individual muscle fibers is difficult to assess. The impact of exercise on a muscle fiber is determined by a variety of complex factors including the force and frequency of contraction as well as trophic hormonal influences (79,96). Hence, at present, it is unknown as to whether the selective up-regulation of antioxidant enzyme activity in highly oxidative muscles is due to ordered fiber type recruitment alone or due to fiber type regulated differences in antioxidant enzyme activities.
At present, only a few studies have examined which SOD isoforms in skeletal muscle are up-regulated after endurance exercise training, and the findings are inconsistent. For example, work by Higuchi et al. (26), Pereira et al. (78), and Vincent et al. (100) indicates that exercise training results in an increase in the activity of MnSOD only. In contrast, Leeuwenburgh et al. (55) reported that endurance training promotes an increase in the Cu-Zn isoform of SOD in locomotor muscle. Two recent studies have provided new evidence that intense exercise training can result in an up-regulation of both MnSOD and Cu-ZnSOD in rat skeletal muscle. First, studies at the University of Florida (85) recently examined the effects of intense endurance training on the activity of SOD isoforms in skeletal muscles of rats. Similar to the findings of Oh-Ishi et al. (75), these investigators reported that regular endurance training increases the activity of both MnSOD and Cu-ZnSOD in highly oxidative rat locomotor muscles (i.e., red gastrocnemius). In summary, although the literature lacks consistency, there is mounting evidence that endurance exercise training can result in increased activities of both MnSOD and Cu-ZnSOD in rodent skeletal muscle.
Endurance training promotes an increase in the activity of both oxidative enzymes and SOD; however, is the increase in oxidative capacity matched by a proportional increase in SOD activity? The answer to this question is no. Several studies have shown that endurance training does not result in parallel increases in both oxidative and antioxidant enzymes; indeed, the training induced increase in antioxidant enzyme activity is generally lower than the increase in oxidative enzyme activity (12,22,83,84). For example, the correlation between oxidative enzyme activity (e.g., Krebs cycle enzyme) and SOD activity is relatively low (r = 0.17) when compared across several rodent hindlimb muscles after exercise training (22). The physiological significance of this oxidative vs antioxidant mismatch is unclear and is worthy of further investigation.
Glutathione peroxidase.Table 2 contains numerous representative studies that have investigated the effects of endurance exercise on GPX activity in skeletal muscle. Note that the literature concerning the effects of endurance training on skeletal muscle GPX activity is generally consistent and that most studies indicate that regular endurance exercise training results in increased GPX activity in active skeletal muscles (12,25,31,51,55,83,84,92,97,98). Importantly, endurance training promotes an increase in both cytosolic and mitochondrial GPX activity with the greater increase occurring in the mitochondrial fraction (31). The obvious benefit of this adaptation is that GPX would be available to remove hydroperoxides from both the mitochondria and cytosol.
TABLE 2: Endurance training-induced changes in glutathione peroxidase (GPX) activity in skeletal muscle; these studies are a representative sample of published reports regarding the effects of endurance exercise on GPX activity in skeletal muscles of human and other animals.
Similar to SOD, the magnitude of the training-induced increase in muscle GPX activity is impacted by both the intensity and (daily) duration of exercise. Indeed, compared with low-to-moderate intensity exercise, high-intensity exercise promotes a greater increase in muscle GPX activity (12,83,84). Further, long-duration exercise training is superior to short-duration exercise training in the up-regulation of muscle GPX activity (83,84)(Fig. 5).
Figure 5: The effects of daily training duration on glutathione peroxidase (GPX) activity in the red gastrocnemius muscle of exercise trained rats. Animals were trained for 10 wk on a treadmill at varying daily durations at ∼75% of V̇O
2max. Values are means. Data are from Powers et al. (
84: Powers, S., D. Criswell, J. Lawler, et al. Influence of exercise and fiber type on antioxidant enzyme activity in rat skeletal muscle.
Am. J. Physiol. 266:R375-R380, 1994). *
P < 0.05, sedentary vs animals.
Finally, the endurance training-induced up-regulation of GPX activity is generally limited to oxidative skeletal muscles (i.e., Type I and IIa fibers) (12,84) and training does not result in parallel and predictable increases in oxidative enzyme activity and GPX activity (22). In general, training results in greater percent increases in muscle oxidative enzyme activity compared to GPX activity (12,22,83,84). As mentioned previously, the physiological significance of this mismatch between oxidative versus antioxidant capacity is worthy of study.
Catalase.Table 3 contains several representative studies investigating the effects of endurance exercise on skeletal muscle CAT activity. As illustrated in Table 3, there is little evidence to suggest that exercise training promotes an increase in catalase activity in skeletal muscle (26,51,83,84). In fact, several studies have shown that exercise training results in reduced catalase activity in some locomotor muscles (2,46,55). The explanation for the training-induced reduction in catalase activity in selected muscles remains unclear.
TABLE 3: Training-induced changes in the total catalase activity in skeletal muscle; these studies are a representative sample of published reports regarding the effects of endurance exercise on catalase activity in rat skeletal muscle.
EXERCISE TRAINING ELEVATES SKELETAL MUSCLE GLUTATHIONE LEVELS
The effect of training on GSH content appears to vary between animal species and tissues (37,38,91). In regard to locomotor skeletal muscle, there is growing evidence that regular endurance exercise training promotes an increase in GSH content. Specifically, high-intensity and long-duration endurance training has been shown to increase GSH content in the hindlimb muscles of both rats and dogs (51,55,62,91). What is the mechanism to explain this training-induced elevation in muscle GSH? A definitive answer to this question is not available. The increase in GSH content in trained muscles may be explained by an enhanced synthesis and/or the ability to take up greater amounts of GSH from the blood. The enhanced uptake may be due, at least in part, to increased activities of gamma-glutamyltranspeptidase, whereas the increase in GSH synthesis may be due to increases in gamma-glutamylcysteine synthase, and GSH synthase (62,91).
Careful examination of the literature reveals that training adaptation of GSH in muscle is fiber type specific. The elucidation for this muscle fiber type variation in adaptation may be related to the rate of GSH utilization versus the capacity of GSH uptake within each fiber type. In this regard, activities of the gamma-glutamyl cycle enzymes may play an important role. For instance, endurance training results in significant increases in GSH within the deep vastus laterialis muscle of rats (51,55). Conversely, the GSH level in the soleus muscle was not elevated in response to training (51,55). A potential explanation for these findings is that, compared to the soleus muscle, the deep vastus laterialis muscle has a higher gamma-glutamyltranspeptidase activity (55). Interestingly, no significant differences existed in gamma-glutamylcysteine synthase activity between these skeletal muscles; this suggests that the translocation of amino acids across the muscle sarcolemma may be the limiting factor in the intracellular assembly of GSH (37).
Although it appears clear that endurance training promotes an increase in skeletal muscle GSH content, results on the effects of training on GR (required to recycle GSSG back to GSH) are less clear. Although some investigators report that endurance training results in a decrease or no change in GR activity in locomotor muscles (51,55,91), others report that training promotes in increases in GR activity in skeletal muscle of trained rats (98). The explanations for these divergent findings are unclear, but determination of the enzyme's Km and measurements of mRNA may be more informative.
TRAINING-INDUCED IMPROVEMENTS IN SKELETAL MUSCLE ANTIOXIDANT CAPACITY: PHYSIOLOGICAL SIGNIFICANCE
Most investigations concerning the effects of endurance training on skeletal muscle antioxidant capacity have measured low molecular weight nonenzymatic antioxidants (e.g., GSH) and/or activities of primary antioxidant enzymes. Clearly, measurement of selected antioxidants in tissue provides limited information regarding the total antioxidant status of the tissue. Indeed, this approach does not permit a complete appraisal of the synergistic cooperation of the various antioxidant systems. Therefore, an obvious pragmatic question is: "Does the training-induced increase in muscle antioxidant capacity provide increased protection against exercise-induced oxidative stress?" The general approach to resolve this question has been to challenge tissues of control and trained animals with specific free radicals in vitro to determine overall antioxidant defense capacity. Three recent studies have performed such experiments. Venditti and Di Meo (97,98) have reported that 10 wk of swimming training results in a significant improvement in the in vitro antioxidant capacity of skeletal muscles. Further, these investigators reported that skeletal muscles of trained rats experienced less lipid peroxidation after a bout of exhaustive exercise (97,98). Similarly, recent work by Vincent et al. (100) also indicates that regular endurance training increases the antioxidant capacity of the diaphragm and protects the muscle against contractile-induced oxidative stress. Collectively, these observations suggest that endurance exercise training results in increased protection against in vitro oxidative stress.
Several investigators have proposed a link between reactive oxidants and muscle fatigue (reviewed in 15 and 50). If oxidants are involved in muscle fatigue, training-induced improvements in skeletal muscle antioxidant capacity could result in improved muscular endurance. Although it is well established that endurance training results in improved muscle endurance, the available literature does not demonstrate that training-induced improvements in muscle endurance are due solely to improvements in muscle antioxidant capacity. Designing experiments to address this issue will be complex because regular endurance exercise results in numerous changes in skeletal muscle that could influence muscular performance.
SUMMARY
Growing evidence indicates that endurance exercise training promotes an increase in both total SOD and GPX activity in skeletal muscles. In this regard, it appears that high-intensity exercise training is generally superior to low-intensity exercise in the up-regulation of muscle SOD and GPX activities. Also, training-induced up-regulation of antioxidant enzymes is limited to highly oxidative skeletal muscles. In contrast to the observation that endurance exercise increases SOD and GPX activities, training does not result in an increase in muscle catalase activity. GSH plays a pivotal role in the maintenance of the intracellular redox status and antioxidant enzyme function during acute and chronic exercise. Regular endurance training improves the GSH antioxidant reserve in active skeletal muscles by increasing GSH levels. The mechanism responsible for this increase is unknown and remains an active area of research. Finally, recent evidence suggests that the exercise training-induced increase in the antioxidant capacity of rat skeletal muscles is physiologically significant as trained animals experience less oxidative damage in skeletal muscles after an in vitro oxidative challenge or a bout of exhaustive exercise.
RECOMMENDATIONS FOR FURTHER STUDY
Numerous key questions concerning exercise and skeletal muscle antioxidant capacity remain unanswered. First, what is the explanation for the observation that exercise training up-regulates muscle SOD and GPX activities but does not up-regulate muscle CAT activity? An answer to this question would improve our basic knowledge concerning the interactive roles of these three primary antioxidant enzymes in removing exercise-produced oxidants.
An important mechanistic question is which exercise-induced intracellular signals are responsible for the increased gene expression of SOD and GPX? Although it is clear that many genes are redox sensitive, the exact factors that regulate SOD and GPX gene expression in skeletal muscle during exercise remain largely unknown. Improving our understanding of the exercise-induced up-regulation of antioxidant enzyme gene expression is a key area for future research.
At present, there is a paucity of data regarding the effects of exercise on skeletal muscle levels of nonenzymatic antioxidants such as beta carotene, vitamin C, etc. Additional experimental attention in this area is warranted to determine if regular endurance exercise alters muscle levels of these important antioxidants; this information is essential to determine if regular exercise training alters the need for dietary antioxidants.
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