Glutathione and antioxidant enzymes in skeletal muscle: effects of fiber type and exercise intensity LI LI JI, RONGGEN
FU, AND
EDNA
W. MITCHELL
Department of Kinesiology and Division of Nutritional Urbana, Illinois 61801
Sciences, University of Illinois,
JI, LI LI, RONGGEN Fu, AND EDNA W. MITCHELL. Glututhiand a major source of nonprotein thiol in the body, and one and antioxidant enzymes in skeletal muscle: effects of fiber tissue GSH content seems to be related to its oxidative type and exercise intensity. J. Appl. Physiol. 73(5): 1854-1859, capacity (9). Thus, eye lens and the liver have the highest
1992.-Glutathione status and antioxidant enzymesin various types of rat skeletal musclewere studied after an acute bout of exercise (Ex) at different intensities. Glutathione (GSH) and glutathione disulfide (GSSG) concentrations were the highest in soleus(SO) muscle, followed by those in deep (DVL) and then superficial (SVL) portions of vastus lateralis. In DVL, but not in SO or SVL, muscleGSH increasedproportionally with Ex intensity and reached 1.8 t 0.08 pmollg wet wt compared with 1.5 t 0.03 (P < 0.05)in resting controls (R). GSSG in DVL was increasedfrom 0.10 & 0.01 pmol/g wet wt in R to 0.14 t 0.01 (P < 0.05) after Ex. Total glutathione (GSH + GSSG) contents in DVL were also significantly elevated with Ex, whereasGSH/GSSG ratio was unchanged. Activities of GSH peroxidase(GPX), GSSG reductase (GR), and catalase (CAT) were significantly higher in SO than in DVL and SVL, but there was no difference in superoxide dismutaseactivity between the three muscle types. Furthermore, Ex at moderate intensities elicited significant increasesin GPX, GR, and CAT activities in DVL muscle.None of the antioxidant enzymeswas affected by exercisein SO. It is concludedthat rat DVL muscle is particularly vulnerable to exercise-inducedfree radical damage and that a disturbance of muscleGSH status is indicative of an oxidative stress. glutathione disulfide; oxidative stress;vastus lateralis; soleus
THE
ROLE
OF
GLUTATHIONE
(y-glutamylcysteinylgly-
tine) in protecting biological tissues from exercise-induced oxidative damage has received increasing attention in recent years (8, 13, 17, 25). The concentrations and the ratio of reduced (GSH) vs. oxidized glutathione (GSSG) can undergo dynamic changes under various physiological and pathological conditions and are often documented as sensitive measures of tissue oxidative stress (1,6,9,27). A number of investigators have shown that plasma or blood GSH and GSSG levels can be disturbed by a single bout of exercise in rats (8, 17) and humans (25). Alterations of skeletal muscle and hepatic GSH systems after prolonged exercise have also been reported (13,17,21,23). Associated with the exercise-induced disturbance of tissue GSH status are the responses of cellular antioxidant enzyme systems (10-17). These findings are consistent with the concept that oxygen free radical generation may be enhanced in various body tissues during prolonged strenuous exercise (10, 22). GSH is one of the most abundant short-chain peptides 1854
GSH concentration (-10 mM) among all tissues, whereas skeletal muscle as a whole has one of the lowest GSH (- 1 mM). Furthermore, cellular GSH/GSSG ratio in most tissues is well maintained (9). However, skeletal muscles are highly heterogeneous in a variety of metabolic properties, such as oxidative potential, patterns of fuel utilization, and antioxidant enzyme activity (16,26). Therefore, the levels of muscle GSH and GSSG, as well as GSH/GSSG ratio, are expected to reflect these metabolic characteristics. To date only scarce information is available in the literature regarding GSH concentration and oxidoreductive (redox) status in different muscle types. It has been noticed that the rate of free radical generation in a biological tissue is closely related to its oxygen consumption, because under physiological conditions the majority of reactive oxygen species are produced in mitochondria, the cellular site of oxidative phosphorylation (5). It is well known that, in skeletal muscle, oxygen consumption is a direct function of work load. Thus, exercise intensity is expected to have a strong influence on the extent of oxidative stress in muscle and hence on the alteration of GSH status and antioxidant enzyme systems. Therefore, the primary purpose of the present study was to investigate whether GSH and GSSG concentrations and ratio varied within different types of rat skeletal muscle and how GSH status responded to exercise at different intensities. A secondary purpose was to investigate the major antioxidant enzymes in various types of muscle in response to the exercise stresses imposed. METHODS
AND
PROCEDURE
Animals and exercise protocol. Male Sprague-Dawley rats (age 10 wk, body wt 250 g) were purchased from Halen’s Sprague-Dawley (Indianapolis, IN). Rats were individually housed and maintained on a Purina Rat Chow diet ad libitum in a temperature-controlled (22°C) room at a 12:12 h dark-light cycle. All rats were subjected to a 2-wk acclimatization program as previously described (13). At the end of the 2-wk period, rats were randomly divided into six groups: five exercise (Ex) groups and a resting control group (R). Rats in each of the Ex groups were subjected to an acute
0161-7567/92 $2.00 Copyright 0 1992 the American Physiological Society
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GLUTATHIONE,
MUSCLE
FIBER
bout of treadmill run on a Quinton rodent treadmill immediately before being killed. The speeds and inclines for these exercise bouts were as follows: 15 or 20 m/min, 0% grade for 1 h (E, and EJ; 25 m/min, 0% grade until exhaustion (E3, endurance time 56.8 t 4.9 min); 25 m/min, 5 or 10% grade until exhaustion (E, and E,, endurance times 55.2 t 6.2 and 51.8 t 3.6 min, respectively). The relative work loads [percentage of maximal oxygen consumption (VO, m8x)] of these exercise protocols were not measured. However, they were intended to have the major energy source of exercise derived from aerobic metabolism, because free radical generation does not increase during maximal anaerobic exercise (8, 22). Thus the highest work intensity (E5) was chosen to correspond to ~80-85% vo 2maxin the adult male rats (3). We also intended to keep exercise time relatively constant (-60 min) while varying treadmill speed and grade so as to elicit various levels of metabolic stress to the animals (3). Although not all rats could complete the 60-min exercise sessions, the average running time was fairly similar among the various Ex groups. The R rats were killed in the resting state at the same time of day. Tissue preparation. Rats were killed by decapitation. After the rats were exsanguinated, superficial and deep portions of the vastus lateralis muscle (SVL and DVL) and soleus (SO) muscle of one hindlimb were quickly dissected and submerged in liquid nitrogen. The time between decapitation and freezing muscle samples was 4 min. The muscle tissues were kept in liquid nitrogen until various assays were performed. For GSH analysis, a portion of the frozen muscle tissues was directly transferred to a medium containing 7% perchloric acid and 2 mM phenanthroline (pH < 2.0) with a weight-to-volume ratio of 1:lO. For antioxidant enzyme assays, a portion of the various muscle samples was thawed in 0.1 M phosphate buffer (pH 7.4, wt/vol l:lO), minced, and homogenized with a polytron (model PT-10, Brinkman Instruments, Westbury, NY) at 0-4OC. The resulting muscle homogenates were stored at -8OOC. Biochemical analysis. GSH and GSSG concentrations in various muscle tissues were analyzed with a high-performance liquid chromatography method (24) with slight modifications (13). Activities of muscle antioxidant enzymes were determined as previously described (11): superoxide dismutase (SOD, EC 1.15.l.l), assayed according to Misra and Fridovich (20) at 30°C; catalase (CAT, EC 1.11.1.6), assayed at 20°C according to Aebi (2), with slight modification (12); glutathione peroxidase (GPX, EC 1.11.1.9), assayed at 37OC according to Flohe and Gunzler (7), with H,O, as substrate; and glutathione reductase (GR, EC 1.6.4.2), measured at 30°C according to Carlberg and Mannivik (4). Activities of citrate synthase (CS, EC 4.1.3.7) and phosphofructokinase (PFK, EC 2.7.1.11) were determined according to previously cited methods (12). Lipid peroxidation was determined by measuring malondialdehyde content in frozen muscle tissues according to Uchiyama and Mihara (28). Protein content was analyzed with the Bradford method. Statistics. Data were analyzed with a two -way analysis of variance method. When a significant F value was found in a specific set of data, Duncan’s multiple range
TYPE,
AND
EXERCISE
R
El
1855
4 F 3 z * c 2 0 E 51 0 0 E3
E2
E4
E5
FIG. 1. Glutathione (GSH) response to exercise at various intensities in superficial vastus lateralis (open bars), deep vastus lateralis (hatched bars), and soleus (solid bars). Error bars are SE. R, rested; El, 15 m/min, 0% grade; E,, 20 m/min, 0% grade; E,, 25 m/min, 0% grade; E4, 25 m/min, 5% grade; and E,, 25 m/min, 10% grade. GSH concentrations are significantly (P < 0.01) different between superficial vastus lateralis, deep vastus lateralis, and soleus. Exercise vs. R in deep vastus lateralis: * P < 0.05.
comparison was performed to test the si.gnificant levels of differences between mea ns. P < 0.05 was consi .dered statistically significant. RESULTS
To verify that the selected muscle tissues were truly characteristic of their phenotypes, activities of CS (indication of muscle oxidative potential) and PFK (indication of anaerobic potential) were measured in the various muscle samples. Resting CS activity was 8.5 t 0.4,25.8 + 1.6, and 28.0 t 0.8 pmol min-’ g wet wt-l in SVL, DVL, and SO muscles, respectively; whereas PFK activity was measured as 72 t 3, 46 t 4, and 18 t 2 pmol.min-‘0 g wet wt?. GSH concentration in SO muscle was significantly higher (P < 0.01) than those in SVL and DVL muscle (Fig. 1). Resting GSH content in SO was more than fivefold and twofold, respectively, of that in SVL and DVL. GSH in DVL was also significantly higher than that in SVL (P < 0.05). In DVL muscle, GSH content was significantly higher in E, and E, compared with that in R (P < 0.05). There was no significant change in GSH in SVL or SO muscle between treatment groups. SO muscle had significantly higher GSSG concentration than DVL and SVL muscle (Fig. 2). The differences of GSSG between DVL and SVL were also significant (P < 0.05). In DVL, the E, and E, groups had significantly higher GSSG than the R group (P < 0.05). No significant alteration in GSSG was found in SVL. In SO, however, GSSG was significantly increased in only E, group (P < 0.05). Muscle content of total glutathione (GSH + GSSG) was also significantly higher in SO than in DVL, which in turn was higher than in SVL (Fig. 3). As was GSH, GSH + GSSG in DVL was significantly increased in E, and E, rats, but no change in GSH + GSSG was observed in SO or SVL muscle. l
l
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1856
GLUTATHIONE,
MUSCLE
FIBER TYPE,
*
AND EXERCISE
30
T
R El E2 E3 E4 E5 2. Glutathione disulfide (GSSG) response to exercise at various intensities in superficial vastus lateralis (open bars), deep vastus lateralis (hatched bars), and soleus (solid bars). Error bars are SE. See Fig. 1 legend for definitions for R and E,-E,. GSSG concentrations are significantly (P < 0.01) different between superficial vastus lateralis, deep vastus lateralis, and soleus. Exercise vs. R in respective muscles: * P < 0.05. FIG.
R El E2 E3 E4 E5 -1. - glutathione (GSH GSSG) in 4. Ratio of reaucea vs. oxicuze( response to exercise at various intensities in superficial vastus lateralis (open bars), deep vastus lateralis (hatched bars), and soleus (solid bars). Error bars are SE. See Fig. 1 legend for definitions for R and El-ES. FIG.
1
1
(P < 0.05) higher GPX and GR activities than the R rats. E, rats also had significantly higher CAT activity. In SVL muscle, exercise had no effect on CAT or SOD, but GPX and GR activities were significantly elevated in E, and E, rats, respectively, vs. that in R. None of these antioxidant enzymes in SO muscle showed a significant response to various intensities of exercise. Lipid peroxidation showed no significant difference between DVL and SVL (Table 2). In both of the two measured muscle types, E, and E, rats had significantly higher rates of lipid peroxidation than their resting counterparts. Limited by the quantity of muscle samples, lipid peroxidation in SO was not determined. Protein content was not significantly different among the treatment groups in any muscle type. DISCUSSION
E3 E4 ES R El E2 3. Response of total glutathione (GSH + GSSG) to exercise at various intensities in superficial vastus lateralis (open bars), deep vastus lateralis (hatched bars), and soleus (solid bars). Error bars are SE. See Fig. 1 legend for definitions for R and El--E,. GSH + GSSG concentrations are significantly (P < 0.01) different between superficial vastus lateralis, deep vastus lateralis, and soleus. Exercise vs. R in deep vastus lateralis: * P < 0.05. FIG.
There were no significant differences in GSH/GSSG ratio among the three muscle types in the R rats (Fig. 4). In the various Ex groups, there tended to be increasing differences in GSH/GSSG between SVL vs. DVL or SO as exercise intensity increased, hut these were not significant. Activities of GPX, GR, and CAT were significantly higher (P < 0.01) in SO than in DVL and SVL (Table 1). Activities of these antioxidant enzymes were also significantly higher in DVL than in SVL (P < 0.05). There was no significant difference in SOD activity among the three muscle types. In DVL muscle, the E, and E, rats had significantly
Ghtathione status. Different types of skeletal muscles are known to possess distinctively different morphological, physiological, and biochemical properties. The three muscle types we chose to study in the present investigation, i.e., SO, DVL, and SVL of rat hindlimb, represent slow-twitch oxidative (type l), fast-twitch oxidative (type 2a), and fast-twitch glycolytic (type 2b) fibers, respectively (26). Furthermore, the analyses of the muscle mitochondrial oxidative capacity, glycolytic potential, and antioxidant capacity by measuring activities of citrate synthase, PFK, and various antioxidant enzymes, respectively, confirmed the biochemical properties of these selected muscle fiber types (11, 16, 26). Despite the well-characterized metabolic profiles, little attention has been given to the GSH status in various muscle types. Data in the present study demonstrate that muscle fibers with greater oxidative capacity (e.g., mitochondrial enzyme activity) also have much higher levels of GSH and total glutathione contents than those with lower oxidative potential. In fact, SO muscle was found to have a GSH concentration of -3 mM, which is
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GLUTATHIONE,
of antioxidant
TABLE 1. Activities
MUSCLE
FIBER TYPE, AND EXERCISE
enzymes in rut skeletal muscle Exercise
GPX* SVL DVL SO GR* SVL DVL so SOD SVL
DVL so CAT* SVL DVL SO
1857
Intensity
R
E*
0.82~0.07
0.88HUI3 2.71t0.57 13.o-Fo.59
0.95t0.11 2.75+0,43t 12.4kO.68
1.21*0.17~ 3.07?0.58-312.7kl.07
0.75t0.08 2.00t0.19 13.4t0.72
0.82t0.02
1.86t0.26 12.8t0.46 0.25kO.03 0.35-+0.04 0.76t0.06
0.26kO.04 0.43t0.07 0.80t0.05
0.24t0.03 0.44+0.05t 0.70t0.07
0.29t0.02 0.47+0.03p
0.26t0.03 0.4lt0*05 0.82kO.08
0.33t0.02”f 0.39kO.03 0.76kO.07
&
Es
0.80+0.03
1,398-t116 1,308+124 1,309&167
1,296*125 1,418+211 1,301&223
1,278+69 1,606zkl37 1,442&82
1,381+38 1,522+109 1,447&117
14.8kO.7 17.9tl.5 60.9t9.2
14.7kl.7 22.222.5 62.923.4
16.Ok2.9 21.5k2.5 62.3klO.6
13.9kl.5 26.8-tZ.3t 56.W4.0
E.4
1,376+90 1,386klll 1,499*108 15.5k5.7 21.7~13.7 62.8t8.9
Es
2.06t0.38 12.3t0.57
1,255+31 1,413&152 1,598&39 15.6kl.2 23.3rt6.5 63.2k6.9
Values are means t SE given in units/g wet muscle wt for 6 animals in each group. GPX, glutathione peroxidase; GR, glutathione reductase; SOD, superoxide dismutase; CAT, catalase. SVL, superficial vastus lateralis; DVL, deep vastus lateralis; SO, soleus; R, rested; E,, 15 m/min, 0% grade; E,, 20 m/min, 0% grade; E,, 25 m/min, 0% grade; E,, 25 m/min, 5% grade; E,, 25 m/min, 10% grade. * Significant differences between SVL, DVL, and SO: P < 0.01. “f Exercise vs. R in respective enzyme and muscle: P < 0.05.
greater than those in erythrocytes, lung, and brain (9). One of the primary functions of GSH in the body is to serve as a substrate for GPX, an important antioxidant enzyme in most tissues. As a major thiol source in the body, GSH is also a scavenger of singlet oxygen and hydroxyl radicals (9, 19). Thus SO muscle, which displayed the greatest GSH level and GPX activity, was most resistent to an exercise-induced oxidative stress, whereas the modest levels of GSH and GPX in DVL and SVL were associated with a greater vulnerability to lipid peroxidation under exercise stress. The present study confirmed our previous observations (13) that GSH levels were elevated during a prolonged exercise bout in the DVL muscle and that GSSG levels were also significantly increased. However, two additional findings were apparent in the current study. First, the effect of an acute exercise bout on muscle GSH status was directly related to the exercise intensity. Significant alterations of GSH and GSSG occurred only at work loads >25 m/min and 5% grade (Ed), corresponding to -75% ofV0 2maxin rats (3). At this work intensity, a 16% increase in GSH and a 42% increase in GSSG were observed in DVL muscle at exhaustion (endurance ~55 min). The magnitudes of these augmentations were simiTABLE 2. Lipid peroxidatiun in rat skeletal muscle SVL
R El E2 E3 Ed Es-
0.27t0.02 0.26t0.02 0.32-+0.03 0.34t0.01” 0.32t0.03 0.35&0.01*
DVL 0.2220.02 0.22t0.02 0.25t0.01 0.27t0.02* 0.26t0.03 0.27t0.01*
Values are means k SE given in nmol malonaldehyde/mg protein for 6 animals in each group. Measurements were not made in SO. See Table 1 footnote for definition of abbreviations. Exercise vs. R, * P < 0.05.
lar to those found in the aforementioned study, wherein rats ran at 20 m/min and 0% grade to exhaustion (endurante time 81 t 4 min). It is interesting to note that E, rats, which ran at the same treadmill speed and slope as our previous study (20 m/min and 0% grade) but for less time (60 vs. 81 min), showed no significant alteration in muscle GSH status. These data suggest that both work load and exercise duration may be important in eliciting a disturbance to muscle GSH status. Second, significant alteration in GSH status were found mainly in DVL muscle. These muscle fiber-specific changes may reflect the differences in recruitment patterns as well as metabolic and antioxidant capacities of the muscles during prolonged exercise. DVL in rat hindlimbs are typically involved in endurance exercise. A combination of a greater oxygen consumption, and hence free radical generation (5), with rather modest activities of antioxidant enzymes may render the DVL muscle more vulnerable to oxidative damage. In contrast, SVL is not actively recruited in endurance exe rcise until the very late stage. Generation of free radical species may be minimum despite a low antioxidant capacity. The- SO muscle, although actively contracting during endurance exercise, has a high GSH concentration and sufficient antioxidant enzyme activities and therefore may be better protected from free radical damage. . Although an increased GSSG during exercise is be11.eved to be elicited by an excessive production of reactive oxygen species (e.g., H,O,) or a decreased capacity of GR, or both, little is known about the mechanism(s) for the observed elevation of GSH in DVL muscle after exercise. We have hypothesized that the increased muscle GSH might be from blood circulation (13). Liver is known to export a large quantity of GSH to the plasma in respo nse to hormonal release, such as glucagon (18), which increases its plasma concentration during prolonged exercise. Circulating GSH can be utilized by various nonhepatic tissues (6,19). Whether or not exogenous GSH can
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1858
GLUTATHIONE,
MUSCLE
FIBER TYPE, AND EXERCISE
be taken up by muscle during exercise remains to be investigated. Antioxidant enzymes. An important finding of the present investigation was that the activities of GPX, GR, and CAT in skeletal muscle were related to fiber types, whereas the activity of SOD was not. It has been noted for some time that the maximal activities of several antioxidant enzymes, e.g., SOD and CAT, correlate with aerobic capacity (VO, max) among various tissues (10). In this regard, tissues with high oxygen consumption, e.g., liver, heart, and brain, have relatively high antioxidant enzyme levels, whereas skeletal muscle is among the lowest on the list. However, not much is known in this aspect between different muscle fibers. The current study demonstrates that there can be as much as a l&fold difference in GPX activity and a 3- to $-fold difference in GR and CAT activities between SO and SVL, whereas differences in these enzymes between DVL and SVL are relatively small. These findings seem to be consistent with the report by Laughlin et al. (16) on several antioxidant enzymes in rat gastrocnemius muscle. It is noteworthy that GPX activity in SO muscle approaches that in rat myocardium, one of the most aerobic tissues in the body (12). In contrast, SOD displayed little interfiber difference among SO, DVL, and SVL. In fact, resting SOD activities in all three types of muscles are similar to that found in rat heart (12). These data imply that skeletal muscles probably have sufficient reserve in SOD activity and that the elimination of superoxide radicals may not be a limiting factor in muscle’s defense against oxygen free radicals. An acute bout of exercise significantly increased activities of GPX, GR, and CAT in the DVL muscle, whereas these enzymes in SVL and SO muscles showed little alteration at any exercise intensity. These data were compatible with the patterns of GSH response in the current study. It is evident that DVL muscle is more susceptible to exercise-induced oxidative stress. However, in contrast to GSH and GSSG, increases of antioxidant enzyme activities in DVL muscle seem to be independent of exercise intensity. It is interesting to note that in our previous study (13), rats were run to exhaustion at an intensity identical to E,, and we found similar changes in activities of these enzymes in the DVL muscle. Results from both studies suggest that an acute bout of exercise at moderate intensity may be sufficient to trigger a disturbance of muscle antioxidant enzymes, the mechanism for which remains to be elucidated. Summary. We have investigated the GSH status and antioxidant enzyme systems in various types of rat skeletal muscle in response to an acute bout of exercise at different intensities. GSH concentration and activities of most antioxidant enzymes were the highest in SO muscle, followed by DVL and then SVL muscle. DVL muscle demonstrated an exercise intensity-dependent increase in GSH and GSSG, whereas little change was found in SO or SVL muscle. Activation of antioxidant enzymes was observed after exercise bouts at only modest intensities and only in DVL muscle. Thus DVL muscle seems to be more susceptible to exercise-induced oxidative stress.
The technical assistance of Raj Chandwaney and Margaret Griffiths is acknowledged. This research project was supported in part by a grant from the American Federation for Aging Research. Present address of R. G. Fu: Medical Center of Endocrinology, Metabolism, and Nutrition, Northwestern University School of Medicine, Chicago, IL 60611. Address for reprint requests: L. L. Ji, 125 Freer Hall, 906 South Goodwin Ave., Urbana, IL 61801. Received 26 February 1992; accepted in final form 8 June 1992. REFERENCES J. D., B. H. LAUTERBURG, AND J. R. MITCHELL. Plasma glutathione and gfutathione disulfide in the rat: regulation and response to oxidative stress. J. Pharmacol. Exp. Ther. 227: 749-754,
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FU, AND
EDNA
W. MITCHELL
Department of Kinesiology and Division of Nutritional Urbana, Illinois 61801
Sciences, University of Illinois,
JI, LI LI, RONGGEN Fu, AND EDNA W. MITCHELL. Glututhiand a major source of nonprotein thiol in the body, and one and antioxidant enzymes in skeletal muscle: effects of fiber tissue GSH content seems to be related to its oxidative type and exercise intensity. J. Appl. Physiol. 73(5): 1854-1859, capacity (9). Thus, eye lens and the liver have the highest
1992.-Glutathione status and antioxidant enzymesin various types of rat skeletal musclewere studied after an acute bout of exercise (Ex) at different intensities. Glutathione (GSH) and glutathione disulfide (GSSG) concentrations were the highest in soleus(SO) muscle, followed by those in deep (DVL) and then superficial (SVL) portions of vastus lateralis. In DVL, but not in SO or SVL, muscleGSH increasedproportionally with Ex intensity and reached 1.8 t 0.08 pmollg wet wt compared with 1.5 t 0.03 (P < 0.05)in resting controls (R). GSSG in DVL was increasedfrom 0.10 & 0.01 pmol/g wet wt in R to 0.14 t 0.01 (P < 0.05) after Ex. Total glutathione (GSH + GSSG) contents in DVL were also significantly elevated with Ex, whereasGSH/GSSG ratio was unchanged. Activities of GSH peroxidase(GPX), GSSG reductase (GR), and catalase (CAT) were significantly higher in SO than in DVL and SVL, but there was no difference in superoxide dismutaseactivity between the three muscle types. Furthermore, Ex at moderate intensities elicited significant increasesin GPX, GR, and CAT activities in DVL muscle.None of the antioxidant enzymeswas affected by exercisein SO. It is concludedthat rat DVL muscle is particularly vulnerable to exercise-inducedfree radical damage and that a disturbance of muscleGSH status is indicative of an oxidative stress. glutathione disulfide; oxidative stress;vastus lateralis; soleus
THE
ROLE
OF
GLUTATHIONE
(y-glutamylcysteinylgly-
tine) in protecting biological tissues from exercise-induced oxidative damage has received increasing attention in recent years (8, 13, 17, 25). The concentrations and the ratio of reduced (GSH) vs. oxidized glutathione (GSSG) can undergo dynamic changes under various physiological and pathological conditions and are often documented as sensitive measures of tissue oxidative stress (1,6,9,27). A number of investigators have shown that plasma or blood GSH and GSSG levels can be disturbed by a single bout of exercise in rats (8, 17) and humans (25). Alterations of skeletal muscle and hepatic GSH systems after prolonged exercise have also been reported (13,17,21,23). Associated with the exercise-induced disturbance of tissue GSH status are the responses of cellular antioxidant enzyme systems (10-17). These findings are consistent with the concept that oxygen free radical generation may be enhanced in various body tissues during prolonged strenuous exercise (10, 22). GSH is one of the most abundant short-chain peptides 1854
GSH concentration (-10 mM) among all tissues, whereas skeletal muscle as a whole has one of the lowest GSH (- 1 mM). Furthermore, cellular GSH/GSSG ratio in most tissues is well maintained (9). However, skeletal muscles are highly heterogeneous in a variety of metabolic properties, such as oxidative potential, patterns of fuel utilization, and antioxidant enzyme activity (16,26). Therefore, the levels of muscle GSH and GSSG, as well as GSH/GSSG ratio, are expected to reflect these metabolic characteristics. To date only scarce information is available in the literature regarding GSH concentration and oxidoreductive (redox) status in different muscle types. It has been noticed that the rate of free radical generation in a biological tissue is closely related to its oxygen consumption, because under physiological conditions the majority of reactive oxygen species are produced in mitochondria, the cellular site of oxidative phosphorylation (5). It is well known that, in skeletal muscle, oxygen consumption is a direct function of work load. Thus, exercise intensity is expected to have a strong influence on the extent of oxidative stress in muscle and hence on the alteration of GSH status and antioxidant enzyme systems. Therefore, the primary purpose of the present study was to investigate whether GSH and GSSG concentrations and ratio varied within different types of rat skeletal muscle and how GSH status responded to exercise at different intensities. A secondary purpose was to investigate the major antioxidant enzymes in various types of muscle in response to the exercise stresses imposed. METHODS
AND
PROCEDURE
Animals and exercise protocol. Male Sprague-Dawley rats (age 10 wk, body wt 250 g) were purchased from Halen’s Sprague-Dawley (Indianapolis, IN). Rats were individually housed and maintained on a Purina Rat Chow diet ad libitum in a temperature-controlled (22°C) room at a 12:12 h dark-light cycle. All rats were subjected to a 2-wk acclimatization program as previously described (13). At the end of the 2-wk period, rats were randomly divided into six groups: five exercise (Ex) groups and a resting control group (R). Rats in each of the Ex groups were subjected to an acute
0161-7567/92 $2.00 Copyright 0 1992 the American Physiological Society
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GLUTATHIONE,
MUSCLE
FIBER
bout of treadmill run on a Quinton rodent treadmill immediately before being killed. The speeds and inclines for these exercise bouts were as follows: 15 or 20 m/min, 0% grade for 1 h (E, and EJ; 25 m/min, 0% grade until exhaustion (E3, endurance time 56.8 t 4.9 min); 25 m/min, 5 or 10% grade until exhaustion (E, and E,, endurance times 55.2 t 6.2 and 51.8 t 3.6 min, respectively). The relative work loads [percentage of maximal oxygen consumption (VO, m8x)] of these exercise protocols were not measured. However, they were intended to have the major energy source of exercise derived from aerobic metabolism, because free radical generation does not increase during maximal anaerobic exercise (8, 22). Thus the highest work intensity (E5) was chosen to correspond to ~80-85% vo 2maxin the adult male rats (3). We also intended to keep exercise time relatively constant (-60 min) while varying treadmill speed and grade so as to elicit various levels of metabolic stress to the animals (3). Although not all rats could complete the 60-min exercise sessions, the average running time was fairly similar among the various Ex groups. The R rats were killed in the resting state at the same time of day. Tissue preparation. Rats were killed by decapitation. After the rats were exsanguinated, superficial and deep portions of the vastus lateralis muscle (SVL and DVL) and soleus (SO) muscle of one hindlimb were quickly dissected and submerged in liquid nitrogen. The time between decapitation and freezing muscle samples was 4 min. The muscle tissues were kept in liquid nitrogen until various assays were performed. For GSH analysis, a portion of the frozen muscle tissues was directly transferred to a medium containing 7% perchloric acid and 2 mM phenanthroline (pH < 2.0) with a weight-to-volume ratio of 1:lO. For antioxidant enzyme assays, a portion of the various muscle samples was thawed in 0.1 M phosphate buffer (pH 7.4, wt/vol l:lO), minced, and homogenized with a polytron (model PT-10, Brinkman Instruments, Westbury, NY) at 0-4OC. The resulting muscle homogenates were stored at -8OOC. Biochemical analysis. GSH and GSSG concentrations in various muscle tissues were analyzed with a high-performance liquid chromatography method (24) with slight modifications (13). Activities of muscle antioxidant enzymes were determined as previously described (11): superoxide dismutase (SOD, EC 1.15.l.l), assayed according to Misra and Fridovich (20) at 30°C; catalase (CAT, EC 1.11.1.6), assayed at 20°C according to Aebi (2), with slight modification (12); glutathione peroxidase (GPX, EC 1.11.1.9), assayed at 37OC according to Flohe and Gunzler (7), with H,O, as substrate; and glutathione reductase (GR, EC 1.6.4.2), measured at 30°C according to Carlberg and Mannivik (4). Activities of citrate synthase (CS, EC 4.1.3.7) and phosphofructokinase (PFK, EC 2.7.1.11) were determined according to previously cited methods (12). Lipid peroxidation was determined by measuring malondialdehyde content in frozen muscle tissues according to Uchiyama and Mihara (28). Protein content was analyzed with the Bradford method. Statistics. Data were analyzed with a two -way analysis of variance method. When a significant F value was found in a specific set of data, Duncan’s multiple range
TYPE,
AND
EXERCISE
R
El
1855
4 F 3 z * c 2 0 E 51 0 0 E3
E2
E4
E5
FIG. 1. Glutathione (GSH) response to exercise at various intensities in superficial vastus lateralis (open bars), deep vastus lateralis (hatched bars), and soleus (solid bars). Error bars are SE. R, rested; El, 15 m/min, 0% grade; E,, 20 m/min, 0% grade; E,, 25 m/min, 0% grade; E4, 25 m/min, 5% grade; and E,, 25 m/min, 10% grade. GSH concentrations are significantly (P < 0.01) different between superficial vastus lateralis, deep vastus lateralis, and soleus. Exercise vs. R in deep vastus lateralis: * P < 0.05.
comparison was performed to test the si.gnificant levels of differences between mea ns. P < 0.05 was consi .dered statistically significant. RESULTS
To verify that the selected muscle tissues were truly characteristic of their phenotypes, activities of CS (indication of muscle oxidative potential) and PFK (indication of anaerobic potential) were measured in the various muscle samples. Resting CS activity was 8.5 t 0.4,25.8 + 1.6, and 28.0 t 0.8 pmol min-’ g wet wt-l in SVL, DVL, and SO muscles, respectively; whereas PFK activity was measured as 72 t 3, 46 t 4, and 18 t 2 pmol.min-‘0 g wet wt?. GSH concentration in SO muscle was significantly higher (P < 0.01) than those in SVL and DVL muscle (Fig. 1). Resting GSH content in SO was more than fivefold and twofold, respectively, of that in SVL and DVL. GSH in DVL was also significantly higher than that in SVL (P < 0.05). In DVL muscle, GSH content was significantly higher in E, and E, compared with that in R (P < 0.05). There was no significant change in GSH in SVL or SO muscle between treatment groups. SO muscle had significantly higher GSSG concentration than DVL and SVL muscle (Fig. 2). The differences of GSSG between DVL and SVL were also significant (P < 0.05). In DVL, the E, and E, groups had significantly higher GSSG than the R group (P < 0.05). No significant alteration in GSSG was found in SVL. In SO, however, GSSG was significantly increased in only E, group (P < 0.05). Muscle content of total glutathione (GSH + GSSG) was also significantly higher in SO than in DVL, which in turn was higher than in SVL (Fig. 3). As was GSH, GSH + GSSG in DVL was significantly increased in E, and E, rats, but no change in GSH + GSSG was observed in SO or SVL muscle. l
l
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1856
GLUTATHIONE,
MUSCLE
FIBER TYPE,
*
AND EXERCISE
30
T
R El E2 E3 E4 E5 2. Glutathione disulfide (GSSG) response to exercise at various intensities in superficial vastus lateralis (open bars), deep vastus lateralis (hatched bars), and soleus (solid bars). Error bars are SE. See Fig. 1 legend for definitions for R and E,-E,. GSSG concentrations are significantly (P < 0.01) different between superficial vastus lateralis, deep vastus lateralis, and soleus. Exercise vs. R in respective muscles: * P < 0.05. FIG.
R El E2 E3 E4 E5 -1. - glutathione (GSH GSSG) in 4. Ratio of reaucea vs. oxicuze( response to exercise at various intensities in superficial vastus lateralis (open bars), deep vastus lateralis (hatched bars), and soleus (solid bars). Error bars are SE. See Fig. 1 legend for definitions for R and El-ES. FIG.
1
1
(P < 0.05) higher GPX and GR activities than the R rats. E, rats also had significantly higher CAT activity. In SVL muscle, exercise had no effect on CAT or SOD, but GPX and GR activities were significantly elevated in E, and E, rats, respectively, vs. that in R. None of these antioxidant enzymes in SO muscle showed a significant response to various intensities of exercise. Lipid peroxidation showed no significant difference between DVL and SVL (Table 2). In both of the two measured muscle types, E, and E, rats had significantly higher rates of lipid peroxidation than their resting counterparts. Limited by the quantity of muscle samples, lipid peroxidation in SO was not determined. Protein content was not significantly different among the treatment groups in any muscle type. DISCUSSION
E3 E4 ES R El E2 3. Response of total glutathione (GSH + GSSG) to exercise at various intensities in superficial vastus lateralis (open bars), deep vastus lateralis (hatched bars), and soleus (solid bars). Error bars are SE. See Fig. 1 legend for definitions for R and El--E,. GSH + GSSG concentrations are significantly (P < 0.01) different between superficial vastus lateralis, deep vastus lateralis, and soleus. Exercise vs. R in deep vastus lateralis: * P < 0.05. FIG.
There were no significant differences in GSH/GSSG ratio among the three muscle types in the R rats (Fig. 4). In the various Ex groups, there tended to be increasing differences in GSH/GSSG between SVL vs. DVL or SO as exercise intensity increased, hut these were not significant. Activities of GPX, GR, and CAT were significantly higher (P < 0.01) in SO than in DVL and SVL (Table 1). Activities of these antioxidant enzymes were also significantly higher in DVL than in SVL (P < 0.05). There was no significant difference in SOD activity among the three muscle types. In DVL muscle, the E, and E, rats had significantly
Ghtathione status. Different types of skeletal muscles are known to possess distinctively different morphological, physiological, and biochemical properties. The three muscle types we chose to study in the present investigation, i.e., SO, DVL, and SVL of rat hindlimb, represent slow-twitch oxidative (type l), fast-twitch oxidative (type 2a), and fast-twitch glycolytic (type 2b) fibers, respectively (26). Furthermore, the analyses of the muscle mitochondrial oxidative capacity, glycolytic potential, and antioxidant capacity by measuring activities of citrate synthase, PFK, and various antioxidant enzymes, respectively, confirmed the biochemical properties of these selected muscle fiber types (11, 16, 26). Despite the well-characterized metabolic profiles, little attention has been given to the GSH status in various muscle types. Data in the present study demonstrate that muscle fibers with greater oxidative capacity (e.g., mitochondrial enzyme activity) also have much higher levels of GSH and total glutathione contents than those with lower oxidative potential. In fact, SO muscle was found to have a GSH concentration of -3 mM, which is
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GLUTATHIONE,
of antioxidant
TABLE 1. Activities
MUSCLE
FIBER TYPE, AND EXERCISE
enzymes in rut skeletal muscle Exercise
GPX* SVL DVL SO GR* SVL DVL so SOD SVL
DVL so CAT* SVL DVL SO
1857
Intensity
R
E*
0.82~0.07
0.88HUI3 2.71t0.57 13.o-Fo.59
0.95t0.11 2.75+0,43t 12.4kO.68
1.21*0.17~ 3.07?0.58-312.7kl.07
0.75t0.08 2.00t0.19 13.4t0.72
0.82t0.02
1.86t0.26 12.8t0.46 0.25kO.03 0.35-+0.04 0.76t0.06
0.26kO.04 0.43t0.07 0.80t0.05
0.24t0.03 0.44+0.05t 0.70t0.07
0.29t0.02 0.47+0.03p
0.26t0.03 0.4lt0*05 0.82kO.08
0.33t0.02”f 0.39kO.03 0.76kO.07
&
Es
0.80+0.03
1,398-t116 1,308+124 1,309&167
1,296*125 1,418+211 1,301&223
1,278+69 1,606zkl37 1,442&82
1,381+38 1,522+109 1,447&117
14.8kO.7 17.9tl.5 60.9t9.2
14.7kl.7 22.222.5 62.923.4
16.Ok2.9 21.5k2.5 62.3klO.6
13.9kl.5 26.8-tZ.3t 56.W4.0
E.4
1,376+90 1,386klll 1,499*108 15.5k5.7 21.7~13.7 62.8t8.9
Es
2.06t0.38 12.3t0.57
1,255+31 1,413&152 1,598&39 15.6kl.2 23.3rt6.5 63.2k6.9
Values are means t SE given in units/g wet muscle wt for 6 animals in each group. GPX, glutathione peroxidase; GR, glutathione reductase; SOD, superoxide dismutase; CAT, catalase. SVL, superficial vastus lateralis; DVL, deep vastus lateralis; SO, soleus; R, rested; E,, 15 m/min, 0% grade; E,, 20 m/min, 0% grade; E,, 25 m/min, 0% grade; E,, 25 m/min, 5% grade; E,, 25 m/min, 10% grade. * Significant differences between SVL, DVL, and SO: P < 0.01. “f Exercise vs. R in respective enzyme and muscle: P < 0.05.
greater than those in erythrocytes, lung, and brain (9). One of the primary functions of GSH in the body is to serve as a substrate for GPX, an important antioxidant enzyme in most tissues. As a major thiol source in the body, GSH is also a scavenger of singlet oxygen and hydroxyl radicals (9, 19). Thus SO muscle, which displayed the greatest GSH level and GPX activity, was most resistent to an exercise-induced oxidative stress, whereas the modest levels of GSH and GPX in DVL and SVL were associated with a greater vulnerability to lipid peroxidation under exercise stress. The present study confirmed our previous observations (13) that GSH levels were elevated during a prolonged exercise bout in the DVL muscle and that GSSG levels were also significantly increased. However, two additional findings were apparent in the current study. First, the effect of an acute exercise bout on muscle GSH status was directly related to the exercise intensity. Significant alterations of GSH and GSSG occurred only at work loads >25 m/min and 5% grade (Ed), corresponding to -75% ofV0 2maxin rats (3). At this work intensity, a 16% increase in GSH and a 42% increase in GSSG were observed in DVL muscle at exhaustion (endurance ~55 min). The magnitudes of these augmentations were simiTABLE 2. Lipid peroxidatiun in rat skeletal muscle SVL
R El E2 E3 Ed Es-
0.27t0.02 0.26t0.02 0.32-+0.03 0.34t0.01” 0.32t0.03 0.35&0.01*
DVL 0.2220.02 0.22t0.02 0.25t0.01 0.27t0.02* 0.26t0.03 0.27t0.01*
Values are means k SE given in nmol malonaldehyde/mg protein for 6 animals in each group. Measurements were not made in SO. See Table 1 footnote for definition of abbreviations. Exercise vs. R, * P < 0.05.
lar to those found in the aforementioned study, wherein rats ran at 20 m/min and 0% grade to exhaustion (endurante time 81 t 4 min). It is interesting to note that E, rats, which ran at the same treadmill speed and slope as our previous study (20 m/min and 0% grade) but for less time (60 vs. 81 min), showed no significant alteration in muscle GSH status. These data suggest that both work load and exercise duration may be important in eliciting a disturbance to muscle GSH status. Second, significant alteration in GSH status were found mainly in DVL muscle. These muscle fiber-specific changes may reflect the differences in recruitment patterns as well as metabolic and antioxidant capacities of the muscles during prolonged exercise. DVL in rat hindlimbs are typically involved in endurance exercise. A combination of a greater oxygen consumption, and hence free radical generation (5), with rather modest activities of antioxidant enzymes may render the DVL muscle more vulnerable to oxidative damage. In contrast, SVL is not actively recruited in endurance exe rcise until the very late stage. Generation of free radical species may be minimum despite a low antioxidant capacity. The- SO muscle, although actively contracting during endurance exercise, has a high GSH concentration and sufficient antioxidant enzyme activities and therefore may be better protected from free radical damage. . Although an increased GSSG during exercise is be11.eved to be elicited by an excessive production of reactive oxygen species (e.g., H,O,) or a decreased capacity of GR, or both, little is known about the mechanism(s) for the observed elevation of GSH in DVL muscle after exercise. We have hypothesized that the increased muscle GSH might be from blood circulation (13). Liver is known to export a large quantity of GSH to the plasma in respo nse to hormonal release, such as glucagon (18), which increases its plasma concentration during prolonged exercise. Circulating GSH can be utilized by various nonhepatic tissues (6,19). Whether or not exogenous GSH can
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1858
GLUTATHIONE,
MUSCLE
FIBER TYPE, AND EXERCISE
be taken up by muscle during exercise remains to be investigated. Antioxidant enzymes. An important finding of the present investigation was that the activities of GPX, GR, and CAT in skeletal muscle were related to fiber types, whereas the activity of SOD was not. It has been noted for some time that the maximal activities of several antioxidant enzymes, e.g., SOD and CAT, correlate with aerobic capacity (VO, max) among various tissues (10). In this regard, tissues with high oxygen consumption, e.g., liver, heart, and brain, have relatively high antioxidant enzyme levels, whereas skeletal muscle is among the lowest on the list. However, not much is known in this aspect between different muscle fibers. The current study demonstrates that there can be as much as a l&fold difference in GPX activity and a 3- to $-fold difference in GR and CAT activities between SO and SVL, whereas differences in these enzymes between DVL and SVL are relatively small. These findings seem to be consistent with the report by Laughlin et al. (16) on several antioxidant enzymes in rat gastrocnemius muscle. It is noteworthy that GPX activity in SO muscle approaches that in rat myocardium, one of the most aerobic tissues in the body (12). In contrast, SOD displayed little interfiber difference among SO, DVL, and SVL. In fact, resting SOD activities in all three types of muscles are similar to that found in rat heart (12). These data imply that skeletal muscles probably have sufficient reserve in SOD activity and that the elimination of superoxide radicals may not be a limiting factor in muscle’s defense against oxygen free radicals. An acute bout of exercise significantly increased activities of GPX, GR, and CAT in the DVL muscle, whereas these enzymes in SVL and SO muscles showed little alteration at any exercise intensity. These data were compatible with the patterns of GSH response in the current study. It is evident that DVL muscle is more susceptible to exercise-induced oxidative stress. However, in contrast to GSH and GSSG, increases of antioxidant enzyme activities in DVL muscle seem to be independent of exercise intensity. It is interesting to note that in our previous study (13), rats were run to exhaustion at an intensity identical to E,, and we found similar changes in activities of these enzymes in the DVL muscle. Results from both studies suggest that an acute bout of exercise at moderate intensity may be sufficient to trigger a disturbance of muscle antioxidant enzymes, the mechanism for which remains to be elucidated. Summary. We have investigated the GSH status and antioxidant enzyme systems in various types of rat skeletal muscle in response to an acute bout of exercise at different intensities. GSH concentration and activities of most antioxidant enzymes were the highest in SO muscle, followed by DVL and then SVL muscle. DVL muscle demonstrated an exercise intensity-dependent increase in GSH and GSSG, whereas little change was found in SO or SVL muscle. Activation of antioxidant enzymes was observed after exercise bouts at only modest intensities and only in DVL muscle. Thus DVL muscle seems to be more susceptible to exercise-induced oxidative stress.
The technical assistance of Raj Chandwaney and Margaret Griffiths is acknowledged. This research project was supported in part by a grant from the American Federation for Aging Research. Present address of R. G. Fu: Medical Center of Endocrinology, Metabolism, and Nutrition, Northwestern University School of Medicine, Chicago, IL 60611. Address for reprint requests: L. L. Ji, 125 Freer Hall, 906 South Goodwin Ave., Urbana, IL 61801. Received 26 February 1992; accepted in final form 8 June 1992. REFERENCES J. D., B. H. LAUTERBURG, AND J. R. MITCHELL. Plasma glutathione and gfutathione disulfide in the rat: regulation and response to oxidative stress. J. Pharmacol. Exp. Ther. 227: 749-754,
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