Skeletal muscle oxidative capacity, antioxidant enzymes, and exercise training M. H. LAUGHLIN, T. SIMPSON, W. L. SEXTON, 0. R. BROWN, J. K. SMITH, AND R. J. KORTHUIS of Veterinary Biomedical Sciences and Medical Physiology and Dalton Departments University of Missouri, Columbia, Missouri 65211; and Department of Physiology, Louisiana State University Medical Center, Shreveport, Louisiana 71130
M. H., T. SIMPSON, W. L. SEXTON, 0. R. K. SMITH, AND R. J. KORTHUIS. Skeletal muscle oxidative capacity, antioxidant enzymes, and exercise training. J. Appl. Physiol. 68(6): 2337-2343,1990.-The purposes of this study were to determine whether exercise training induces increases in skeletal muscle antioxidant enzymes and to further characterize the relationship between oxidative capacity and antioxidant enzyme levels in skeletal muscle. Male SpragueDawley rats were exercise trained (ET) on a treadmill 2 h/day at 32 m/min (8% incline) 5 days/wk or were cage confined (sedentary control, S) for 12 wk. In both S and ET rats, catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX) activities were directly correlated with the percentages of oxidative fibers in the six skeletal muscle samples studied. Muscles of ET rats had increased oxidative capacity and increased GPX activity compared with the same muscles of S rats. However, SOD activities were not different between ET and S rats, but CAT activities were lower in skeletal muscles of ET rats than in S rats. Exposure to 60 min of ischemia and 60 min of reperfusion (I/R) resulted in decreased GPX and increased CAT activities but had little or no effect on SOD activities in muscles from both S and ET rats. The I/R-induced increase in CAT activity was greater in muscles of ET than in muscles of S rats. Xanthine oxidase (X0), xanthine dehydrogenase (XD), and X0 + XD activities after I/R were not related to muscle oxidative capacity and were similar in muscles of ET and S rats. It is concluded that although antioxidant enzyme activities are related to skeletal muscle oxidative capacity, the effects of exercise training on antioxidant enzymes in skeletal muscle cannot be predicted by measured changes in oxidative capacity. LAUGHLIN, BROWN, J.
oxygen radicals; xanthine oxidase; superoxide lase; glutathione peroxidase; citrate synthase
dismutase;
cata-
eficial effects of exercise on various disease. Skeletal muscle contains severa mechanisms of protection from inju oxygen metabolites. These protect clude the enzymes superoxide dismu (CAT), and glutathione peroxidase lyzes the dismutation of superoxi which CAT converts to water and O glutathione can reduce HzOp to for fide and water (15, 21). Free-radical-mediated events ar volved in ischemia-reperfusion injur heart, kidney, pancreas, small intes (21,22). Several investigators have idant enzymes are related to the skeletal muscle (15) and/or have p training increases the resistance of potentially toxic effects of oxygen r 24, 26). Therefore we proposed th induces adaptations in the content enzymes in association with increa pacity and that these changes may cle from damage by active oxygen s radicals. Results of experiments des exercise training increases the resist cle to ischemia-reperfusion injury companion paper (28). The experiments outlined herein the following hypotheses. 1) Antio skeletal muscle are related to ox Training will increase antioxidant muscles in proportion to the trainin in oxidative capacity. 3) SOD, CAT in skeletal muscle tissue will be dec of the muscle to ischemia-reperfus zymes may be inactivated by rea generated when blood flow is reins reperfusion will result in the con dehydrogenase (XD) to xanthine ox
NOTION that exercise training induces adaptive responses that provide protection from various forms of injury and disease is widely accepted in the field of exercise science. Although training-induced adaptations in the cardiorespiratory system are well known, the mechanisms responsible for improvements in health and Downloaded from www.physiology.org/journal/jappl ${individualUser.surname} resistance byto${individualUser.givenNames} disease associated with exercise training (163.015.154.053) on September 13, 2018. Copyright © 1990 American Physiological Society. All rights reserved. still are not established. There is a growing body of THE
2338
TRAINING-INDUCED
BIOCHEMICAL
rats and housed two per cage (8 x 7 X 17 in.) in a room with controlled temperature (22 t 2OC) and light (12-h light-dark cycle). Rats had free access to food and water. One week after arrival, the rats were introduced to treadmill exercise with lo-min exercise bouts, at 15-30 m/ min 0% incline, 5 days/wk, for 2 wk. The six rats most resistant to treadmill exercise were omitted from the study. The remaining 20 rats were randomly divided into sedentary control (S) and exercise-trained (ET) groups. The S rats were confined to their cages throughout the remainder of the study, whereas the ET rats were trained on a modified Stanhope (Stanhope Scientific, Davis, CA) rodent treadmill. Over the first 2-6 wk of training the rats were gradually conditioned to run 32 m/min up an 8% incline for 2 h/day. During the last 6 wk the rats ran 32 m/min, up an 8% incline for 2 h/day. Experimental design. This study is composed of three series of experiments that were conducted on three paired groups of 10 ET and 10 S rats (total of 6 groups, 3 ET and 3 S). Two of these series of experiments were conducted on rats also included in the ischemia-reperfusion experiments outlined in the companion paper (28). To determine whether the exercise-training program described above would have an effect on the levels of free radical scavenger enzymes in skeletal muscle, biochemical assays were performed on biopsies taken from brachial muscles. In the first series of experiments, biochemical assays were not conducted on the hindlimb muscles of ET and S because these muscles were exposed to ischemia and reperfusion (28). Since the first series of experiments indicated that training induced some changes in scavenger enzymes in the brachial muscles (Fig. 3), it was important to determine whether similar changes occurred in hindlimb muscles. Therefore, in a second series of experiments on ET and S rats, biochemical assays were performed on biopsies of hindlimb muscles following ischemia-reperfusion. Finally, in a third series of experiments, the effects of ischemia-reperfusion injury on scavenger enzymes in hindlimb skeletal muscles of ET and S rats were determined. Muscle sampling procedures. In all three series of experiments samples (complete cross sections) of medial head of triceps brachii and long head of triceps brachii (red and white portions) were removed from the forelimbs of ET and S rats. In addition, the hearts were isolated and the atria and great vessels were removed. In the second and third series of experiments, samples of soleus and red and white portions of gastrocnemius muscles were also removed from the hindlimbs. Finally, in the third series of experiments, samples of vastus intermedius and red, middle, and white portions of vastus lateralis muscles were removed from the hindlimbs. All muscle samples were weighed, frozen in liquid Nz, and stored at -70°C until assayed. Subsequently, muscle samples were thawed, homogenized at 4°C in 0.1 M potassium phosphate buffer (pH 7.0), centrifuged, and dialyzed before analysis for enzyme activities as described below. Protein was determined with the Bradford dye-binding procedure with bovine albumin as the standard (4). Oxidative enzymes. Succinate dehydrogenase (SDH; EC 1.3991) activity was determined by following cyto-
ADAPTATION
chrome c reduction at 22°C with the procedure of Cooperstein et al. (5). Citrate synthase (CS; EC 4.1.3.7) activity was measured with the method of Srere (27). One unit of SDH activity is equal to the amount of enzyme present that reduces 1 pmol of cytochrome co min-’ . g wet wt-l. Antioxidant enzymes. SOD (EC 1.15.1.1) activity was determined using the method of McCord and Fridovich (23) and Flohe (9). One unit of SOD activity is the amount of enzyme present that inhibits the rate of cytochrome c reduction by 50%. CAT (EC 1.11.1.6) activity was determined by monitoring the decrease in absorbance at 240 nm in the presence of 10 mM H202. One unit of CAT activity is the amount of enzyme present that decomposes 1 M H20Jmin at 25OC. GPX (EC 1.11.1.19) activity was determined as described by Flohe and Gunzler (10). One unit of GPX activity is the amount of enzyme that oxidizes 1 pmol of glutathione/min at 25”C, pH 7.0. Xanthine oxidase and xanthine dehydrogenase. X0 and XD activities were assayed using a modification of the method described by Roy and McCord (25, 31). These enzymes were assayed in the vastus intermedius and vastus lateralis (red, white, and middle portions) muscles of the hindlimbs of rats following ischemia-reperfusion (28). At the completion of the ischemia-reperfusion experiments, the muscles were removed and frozen as described above. Samples were homogenized in potassium phosphate buffer (pH 7.8) containing phenylmethylsulfonyl fluoride and dithioerythreitol. The homogenate was centrifuged, and the supernatant was applied to a Sephadex G-25 column to remove low-molecular-weight inhibitors. The enzyme was assayed with xanthine as the substrate in a phosphate buffer (pH 7.8) containing NAD+. The addition of NAD+ to the assay allows measurement of total xanthine oxidase activity, i.e., the sum of XD and X0 activities. By excluding NAD+ from the assay, true oxidase activity can be measured. XD activity can be estimated by subtracting true X0 activity from total xanthine oxidase (X0 + XD) activity. The rate of reaction was monitored at 295 nm. The rate of change in absorbance reflects the rate of formation of uric acid. Allopurinol (50 PM) was added to the solution after the initial rate was obtained to ensure that the velocity represents X0 activity. Specific activities are expressed as milliunits per gram wet weight. One unit of activity is equal to the number of micromoles of urate formed per minute at 25°C. Data analysis. Data were analyzed using a two-way analysis of variance and Duncan’s new multiple range test (32). The specific activities of antioxidant enzymes were regressed on the SDH activities of the muscles to test for a relationship between oxidative capacity and antioxidant enzyme activities (32). Paired analyses were used where appropriate (32). Significance was defined as P < 0.05. RESULTS
The exercise-training program used in duced increases in the oxidative capacity muscles of the ET rats (Fig. 1). The CS red portion of the long head of the triceps
this study proof the skeletal activity of the brachii muscle
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TRAINING-INDUCED
BIOCHEMICAL
of ET rats was nearly twice that of the S rats, and in the white portion of the long head of triceps brachii of ET rats it was 20% greater than in S rats. Similarly, the SDH activities of these muscles from ET rats were greater than sedentary control in the red (150%) and white (20%) portions of the long head of triceps brachii. Effects of training on antioxidant enzymes. As shown in Fig. 2, there. were no differences between control and trained SOD activities in any sampled extensor muscle loogo-
I
Sedentary
8070605040302010
-
0L Red
White Long
Medial
Head
Head
FIG. 1. Effects of exercise training on citrate synthase activities of triceps brachii muscles of rats. Means k SE are presented; n = 18 in each group. * Trained value significantly greater than control, P 5 0.05.
300
Triceps
Brachii
of the forelimb or leg. Sedentary control and ET rats also had similar (P < 0.05) levels of myocardial SOD activity (S = 124 t 4 and ET = 126 t 4 U/mg protein). In contrast, four of six muscles from the ET rats had lower CAT activities than S rats (Fig. 3). Three of the muscles in which training induced a decrease in CAT activity were leg extensors (Fig. 3). The myocardial levels of CAT activity were slightly greater (P < 0.05) in ET rats (22.9 t 0.3 U/mg protein) compared with S rats (19.7 t 0.3). Exercise training produced significant increases in GPX activities in soleus and in red and white portions of the gastrocnemius muscle (Fig. 4) and the heart (S = 0.24 t 0.002 and ET = 0.32 t 0.005 U/mg protein). Effects of ischemia-reperfusion on antioxidant enzymes. Sixty minutes of ischemia followed by 60 min of reperfusion (ischemia-reperfusion) had no effect on SOD activities in any of the ET muscles sampled (Fig. 5). Only the red gastrocnemius sample of the S rats had greater SOD after ischemia-reperfusion. CAT activities were greater in soleus and both the red and white portions of the gastrocnemius muscle of both ET and S rats after ischemia-reperfusion (Fig. 5). The relative ischemia-reperfusion-related increases in CAT activities in the muscles of ET rats were greater than in the muscles of S rats (soleus muscle: ET 62% increase, S 6% increase; gastrocnemius muscle: red ET 75% increase, S 40% increase, and white ET 189% increase, S 60% increase). Since both the red and white portions of the gastrocnemius Brachii
Muscles
*al
% 61
J
.
Triceps
c
Muscles
1
250-
2339
ADAPTATION
I
Sedentary
EI
Trained
200 -
Red
White Long
Medial
Head
Head
Calf Muscles
241 White
Red Long
.-
Medial
Head
I
Sedentary
El
Trained
Head
Calf Muscles
C
; 100 k F! 50 L z.L -.-ul 0 c 3
b a -c 3
a-
White
Red
Soleus
Gastrocnemius 2. Superoxide dismutase activities in extensor muscles of forelimb (n = 18 in each group; triceps brachii muscles) and leg (n = 9 for trained, n = 10 for sedentary; calf muscles) of rats. Means k SE are presented. There were no statistically significant differences between sedentarv control and trained values in anv muscle sample. FIG.
i
m
6 * 03 White
Red
Soleus
Gastrocnemius FIG. 3. Catalase activities in extensor muscles of forelimb and leg of rats. Means t SE are presented; n, same as in Fig. 2. * Trained value is different from sedentary control, P 5 0.05.
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TRAINING-INDUCED
2340 Triceps
Brachii
BIOCHEMICAL
Superoxide
Muscles
0.5
.g ?I ‘L
100
0.4
D
Sedentary
ezzl
Trained
.E 2 (! a
0.3
F 2 * .e
0.2
=
0.1
ADAPTATION
IT $ a m Y =
ON ezd I/R
80
60
4o 20
0
0.0 White
Red Long
Medial
Head
Dismutase
SED
SED
ET
White
Head
ET
Red
SED
E-T
Soleus
Gastrocnemius 0.5
.G 3 !! 0.
0.4
Calf Muscles
1
Catalase 25
*
20 .i
0.3 1
% FL
F h v) Y
0.2
F
=
0.1
ki CL m Y 5
1
15
10
5
0.0 / White Gastrocnemius
Red
0
Soleus
FIG. 4. Glutathione peroxidase activities in extensor muscles of forelimb and leg of rats. Means + SE are presented; n, same as in Fig. 2. * Trained value is different from sedentary control, P 5 0.05.
muscle are primarily composed of fast-twitch muscle fibers (3) and the soleus is primarily slow twitch (3), these data suggest that the effects of ischemia-reperfusion on CAT activity is different in fast- and slow-twitch skeletal muscle. CAT activity appeared to be increased more in fast-twitch muscle after ischemia-reperfusion than in slow skeletal muscle (soleus). Ischemia-reperfusion had no effect on GPX activities of white gastrocnemius muscle (Fig. 6). GPX activities of the red gastrocnemius and soleus muscles from S rats were lower after ischemia-reperfusion (Fig. 6). Thus ischemia-reperfusion appears to produce greater decreases in GPX activities in high oxidative muscles of both sedentary and ET rats than in the less oxidative white gastrocnemius muscle (3). Xanthine oxidase and xanthine dehydrogenase activities. After exposure to ischemia-reperfusion there were
no differences among X0 activities in the four samples of thigh muscles in either group of rats (Table 1). However, the XD and X0 + XD activities were greater in the vastus intermedius muscles of sedentary rats than in the vastus lateralis muscle tissue. X0 + XD activities of vastus intermedius muscle were also greater in the ET rats. Although X0 + XD activities tended to increase with increasing oxidative capacity of the muscles (i.e.,
SED
ET
SED
White
ET
Red
:D
ET
Soleus
Gastrocnemius FIG. 5. Superoxide dismutase and catalase activities of leg muscles of exercise-trained (ET; n = 7) and sedentary control (SED; n = 8) rats. N, nonischemic; I/R, after ischemia-reperfusion. Means + SE are presented. * I/R values are different from N, P 5 0.05.
0.40 0.35 5
0.30
g
0.25
F ii a
0.20
$
0.10
UN CIia
I/R
0.15
0.05 0.00
SED
ET
SED
ET
Red
White
SE0
Soleus
Gastrocnemius FIG. 6. Glutathione peroxidase activities of leg muscles of exercisetrained (ET; n = 7) and sedentary control (SED; n = 8) rats. N, nonischemic; I/R, after ischemia-reperfusion. Means If: SE are presented. * I/R values are different from N, P 5 0.05.
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TRAINING-INDUCED
BIOCHEMICAL
Long
1. Specific activities of X0, XD, and X0 + XD in rat skeletal muscles after ischemia-reperfusion TABLE
Muscle Sample
Vastus lateralis White Middle Red Vastus intermedius
x0
T S T
XD
XO+XD
% Total as X0
T S T
7.6t0.9 7.7kO.9 7.8t1.5 7.3kl.l 7.8t0.4 8.1Gl.2 10.9t2.3
7.3t1.4 6.3kl.0 8.0t1.5 7.6rt1.5 9.1t0.7 7.6k0.9 11.6t1.5
14.9k1.6 14.0t1.0 15.8t2.5 14.9k1.9 16.9kO.9 15.7t1.2 22.5&2.2*
51k7 46k2 51k6 47k6
S
7.9kO.9
12.4k1.2"
20.4tl.O*
39t4
S
5226 56&6
49&5
Values are mea ns t SE expressed in m U/g wet w t for muscles from 7 trained (T) and 7 sedentary control (S) rats. X0, xanthine oxi .dase; XD, xanthine dehydrogenase. The muscles were isolated following exposure to 60 min of ischemia and 60 min of reperfusion (28). * Vastus intermedius enzyme activity is greater than enzyme activities of other muscles, P 5 0.05. ‘There were no differences between trained and sedentary control data.
white vastus activity < middle vastus c red vastus c vastus intermedius), none of the values was significantly different among the vastus lateralis samples.
2341
ADAPTATION Head
of Triceps
Brachii
Muscle
200
iii
150
0 -w
is bv1 \ E Ql fJ 100
0
8
lz ‘$
c" it 5
50
0
0.30 ii g
0.25
T t
1
DISCUSSION
This study evolved from our interest in the effects of ischemia-reperfusion-induced injury to the microvasculature of skeletal muscle (7, 21, 22, 28-31). Reports of exercise training-induced increases in the specific activities of SOD, CAT, and GPX in skeletal muscle and the fact that SOD and CAT provide protection from microvascular damage induced by ischemia-reperfusion (22) indicate that exercise training could induce adaptations within the muscle resulting in increased resistance to the damaging effects of ischemia-reperfusion. The results of this study indicate that antioxidant enzyme activities vary within and among extensor muscles of rats. The specific activities of SOD, CAT, and GPX are greater in skeletal muscles composed primarily of oxidative fiber types of both sedentary and exercise-trained rats. Both slow-twitch oxidative (i.e., soleus) (3) and fast-twitch oxidative (i.e., red gastrocnemius) (3) fiber types had elevated antioxidant enzyme levels compared with fasttwitch glycolytic fiber types (i.e., white gastrocnemius) (3). Since the generation of ATP via oxidative metabolism is one normal source of reactive oxygen metabolites, one would expect that antioxidant enzyme levels and the levels of enzymes involved in oxidative metabolism would be related as proposed by Jenkins and Tengi (16) and Higuchi et al. (12). The analysis of our data among muscle samples is consistent with this hypothesis. However, when the relationship between oxidative capacity and antioxidant enzymes was explored within muscles (in contrast to among muscles) with linear regression analysis, no relationship was found between oxidative capacity and SOD (e.g., Fig. 7, top) or CAT activities. In contrast, GPX activity seems to be related to SDH activity both within and among skeletal muscles (Figs. 4 and 7, bottom). Thus, although SOD and CAT activities are related to oxidative capacity of skeletal muscle when
0
1 Succinate
2 Dehydrogenase
3
4
(units/mg) FIG. 7. Superoxide dismutase (top) and glutathione peroxidase (bottom) activities as functions of succinate dehydrogenase (SDH) activity of red and white portions of long head of triceps brachii muscle. Superoxide dismutase and catalase (not shown) activities were not related to SDH activity. However, glutathione peroxidase activity (bottom) is related to SDH activity (glutathione peroxidase activity = 0.02 SDH + 0.04, r = 0.56, P 5 0.001, n = 32).
examined among muscles of various fiber type composition, the coupling of SOD and CAT activities to oxidative capacity of muscle is less apparent when examined within muscles. GPX activity appears to be more closely linked to oxidative capacity both among muscles of various fiber type composition and within muscles. The tight coupling between oxidative capacity and GPX activity within and among muscles may be related to the fact that GPX was the only antioxidant enzyme that was upregulated in the muscle of the exercise-trained rats. Effects of exercise training. As expected, exercise training produced increases in oxidative capacity in all muscle samples investigated. However, exercise training had no effect on SOD levels in any of the six extensor muscle samples (Fig. 2). Our results are in agreement with those of Ji et al. (17,18) and Alessio and Goldfarb (2). However, Higuchi et al. (12) reported 20-30s greater SOD activities in the soleus and red vastus lateralis muscles of trained Wistar rats. Jenkins (15) has also measured higher SOD levels in the skeletal muscles of trained human subjects and in the soleus muscle of trained rats. It might be suggested that these disparate results are related to differences in training intensity, duration, or
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2342
TRAINING-INDUCED
BIOCHEMICAL
mode (treadmill running vs. swimming). However, the results obtained by Higuchi et al. (12) disagree with the results obtained in this study (Fig. 2) and those of Alessio and Goldfarb (2); similar training programs were used in all three studies. Thus the reason for the disparate results among these studies remains unclear. Jenkins (15) indicated, in a recent review of free radical biochemistry and exercise, that animal studies investigating the effects of training on CAT activity in skeletal muscle have also produced conflicting results. CAT activities in four of the muscle samples of the exercisetrained rats were lower than control (Fig. 2). These results are in agreement with those of Alessio and Goldfarb (Z), who recently reported that CAT activity was 30-40s lower in the red and white portions of vastus lateralis muscle of trained rats. Higuchi et al. (12) reported that training did not produce any change in soleus CAT activity. Thus available data indicate that exercise training either does not change CAT activity in skeletal muscle or produces decreasesin CAT activities. Our data indicate that some of the controversy concerning the effects of training on CAT activity in skeletal muscle may be attributable to the fact that the effects of training on CAT activities are not uniform among muscle tissue of differing fiber type composition. Our data indicate that exercise training has no effect on myocardial SOD activity, a result which is consistent with the results of Kanter et al. (19) and Higuchi et al. (12). Kihlstrom et al. (20) recently reported that swim training produced decreases in myocardial SOD activity. Thus training does not produce increases in SOD activity in cardiac muscle. Similarly, Higuchi et al. (12) and Kanter et al. (19) found that myocardial CAT activity was not altered by exercise training of rats and mice, respectively. These results are consistent with ours in that we measured a small increase in CAT activity in hearts of trained rats. In contrast, Kihlstrom et al. (20) found rats trained with swimming had lower cardiac CAT activities than control rats. Treadmill training may affect the expression of antioxidant enzymes in cardiac tissue (12, 19) in a manner different from the effects of swim training (20). The GPX activities in skeletal muscles (Fig. 4) and hearts of trained rats tended to be greater than the GPX activities of these muscles from sedentary rats. These data are consistent with the reports of others investigating the effects of exercise training on GPX activity (2, 16, 17, 19, 24). Our hypothesis was that exercise training would induce increases in skeletal muscle antioxidant enzymes in proportion to the training-induced increase in oxidative capacity. This hypothesis was based on a rationale similar to that presented above in relation to the apparent coupling of the enzymes of oxidative metabolism and antioxidant enzymes. The results indicate that the effects of training on antioxidant enzymes varied both among the three enzymes investigated as well as within and among the muscles sampled. Therefore the data do not support our hypothesis. Effects of ischemia and reperfusion. Sixty minutes of ischemia followed by 60 min of reperfusion had no effect on SOD activities in skeletal muscles from trained or
ADAPTATION
sedentary rats. Similarly, Smith et al. (30) reported that 4 h of ischemia followed by 1 h of reperfusion had no effect on SOD or CAT activities of dog gracilis muscle. In the present study, ischemia-reperfusion produced increases in CAT activity in soleus muscle and the red and white portions of the gastrocnemius muscle in both trained and sedentary rats. The increase in CAT activity induced by ischemia-reperfusion was consistently greater in muscles isolated from the trained rats compared with sedentary controls (Fig. 5). The effects of ischemiareperfusion on the activities of SOD and CAT in skeletal muscle appear similar to the acute effects of sustained exercise on these enzymes. For example, Alessio and Goldfarb (2) reported that an acute bout of exercise (20 min) produced increases in the CAT activity of skeletal muscle but had no effect on SOD activity. Also, Alessio and Goldfarb (2) observed that the increase in CAT activity produced by acute exercise was greater in exercise-trained rats. We do not know of an established mechanism for an ischemia-reperfusion-induced increase in CAT activity in skeletal muscle or the fact that exercise training exacerbates this effect of ischemiareperfusion. GPX activities appeared to be decreased after ischemia-reperfusion in muscles of both sedentary and exercise-trained rats. This is similar to the effects of ischemia-reperfusion on GPX activity of dog gracilis muscle as reported by Smith et al. (30). The relative decrease in GPX activity produced by ischemia-reperfusion was similar in trained and sedentary rats despite the fact that the trained rats generally had higher GPX levels in nonischemic muscle (Fig. 6). X0-derived oxidants are believed to have a role in the genesis of ischemia-reperfusion injury in skeletal muscle (21, 30, 31). This notion is based in part on the observation that XD is converted to X0 in skeletal muscle subjected to ischemia-reperfusion. In addition, ischemiareperfusion injury in skeletal muscle is attenuated by either X0 inhibition (using oxypurinol) or depletion (using tungsten-supplemented diets) (31). The results presented in Table 1 indicate that these enzymes do not vary with muscle fiber composition in the vastus lateralis muscle although the vastus intermedius muscle had greater XD (in sedentary only) and X0 + XD activities than the vastus lateralis muscle (Table 1). We previously reported XD + X0 values of 10.9 t 0.5 with 30% of total activity in the X0 form in normal (nonischemic) vastus lateralis muscle (31). In this previous study, X0 activity represented 59% of total (X0 + XD) activity after 2 h of ischemia and 30 min of reperfusion, suggesting that conversion of XD to X0 had occurred (31). Our data (Table 1) indicate that a similar amount of XD-to-X0 conversion occurred during 1 h of ischemia followed by 1 h of reperfusion in that the vastus lateralis had ~50% of total activity in the X0 form. The data also suggest that XD-to-X0 conversion is greater after ischemiareperfusion in fast-twitch glycolytic (FG) muscle, since the white vastus muscle (97% FG fibers) (3) tended to have the largest percentage of total activity in the X0 form. Exercise training did not appear to influence these enzymes.
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TRAINING-INDUCED
BIOCHEMICAL
ou r results support several conclusions 1) Antioxidant enzymes appear to be rel .ated to the 0 ixidative capacity of muscle tissue when compared among skeletal muscles of various fiber type composition. However, the relationship between antioxidant enzymes and oxidative enzymes is not as obvious when examined within muscles. 2) The effects of exercise training on antioxidant enzyme levels cannot be predicted solely from knowledge of the training-induced increases in oxidative capacity of the muscle. For example, exercise training did not produce increases in SOD or CAT activities in skeletal muscl .e with training induced increases in oxidative capacity. In fact, CAT activities were decreased in the muscles of trained rats. Training induced increases in GPX activities in both skeletal muscle and heart. 3) Exposure of skeletal muscle to 60 min of ischemia followed by 60 min of reperfusion resulted in increased CAT activities, decreased GPX activities, and had no effect on SOD activities. The effects of ischemia-reperfusion on CAT and GPX activities in skeletal muscle were greater in the trained rats than in control. Finally, 4) exercise training did not alter the activities of X0, XD, and X0 + XD or the apparent amount of conversion of XD to X0 in the skeletal muscle tissue during ischemia-reperfusion. The authors thank Donna Baumgartner and M. E. Schaefer for exercise training the rats and Greg Kelly for work in data analysis and creating the illustrations. This study was supported by National Heart, Lung, and Blood Institute Grants HL-36088 and HL-36069. M. H. Laughlin is the recipient of National Heart, Lung, and Blood Institute Research Career Development Award HL-01774, and R. J. Korthuis is an Established Investigator of the American Heart Association (880197). Address for reprint requests: M. H. Laughlin, University of Missouri, W116 Vet. Med. Bldg., Columbia, MO 65211. Received
4 October
1989; accepted
in final
form
29 January
1990.
REFERENCES 1. AEBI, H. Catalase in vitro. Methods Enzymol. 105: 121-126, 1984. 2. ALESSIO, H. M., AND A. H. GOLDFARB. Lipid peroxidation and scavenger enzymes during exercise: adaptive response to training. J. Appl. Physiol. 64: 1333-1336, 1988. 3. ARMSTRONG, R. B., AND R. 0. PHELPS. Muscle fiber type composition of the rat hindlimb. Am. J. Anat. 171: 259-272, 1984. 4. BRADFORD, M. A rapid and sensitive method for the quantitation [sic] of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254, 1976. 5. COOPERSTEIN, S. J., A. LAZAROW, AND N. J. KURFESS. A microspectrophotometric method for the determination of succinic dehydrogenase. J. Biol. Chem. 186: 129-139, 1950. 6. DAVIES, K. J. A., A. T. QUINTANILHA, G. A. BROOKS, AND L. PACKER. Free radicals and tissue damage produced by exercise. Biochem. Biophys. Rex Commun. 107: 1198-1205, 1982. 7. DIANA, J. N., AND M. H. LAUGHLIN. Effect of ischemia on capillary pressure and equivalent pore radius in capillaries of the isolated dog hindlimb. Circ. Res. 35: 77-101, 1974. 8. DUDLEY, G. A., W. M. ABRAHAM, AND R. J. TERJUNG. Influence of exercise intensity and duration on biochemical adaptations in skeletal muscle. J. Appl. Physiol. 53: 844-850, 1982. 9. FLOHJ?, L. Superoxide dismutase assay: cytochrome c reduction. Methods Enzymol. 105: 101-104, 1984.
ADAPTATION
2343
10. FLOH& L., AND W. A. GUNZLER. Assay of glutathione peroxidase assay. Methods Enzymol. 105: 114-121, 1984. 11. GEE, D. L., AND A. I. TAPPEL. The effect of exhaustive exercise on expired pentane as a measure of in vivo lipid peroxidation in the rat. Life Sci. 28: 2425-2429, 1981. 12. HIGUCHI, M., L. CARTIER, M. CHEN, AND J. 0. HOLLOSZY. Superoxide dismutase and catalase in skeletal muscle: adaptive response to exercise. J. Gerontol. 40: 281-286, 1985. 13. HOLLOSZY, J. 0. Adaptations of muscular tissue to training. Prog. Cardiovasc. Dis. 18: 445-458, 1976. E. K., AND I. FRIDOVICH. The interaction of bovine 14. HODGSON, erythrocyte superoxide dismutase with hydrogen peroxide: inactivation of the enzyme. Biochemistry 14: 5295-5298,1975. 15. JENKINS, R. R. Free radical chemistry: relationship to exercise. Sports Med. 5: 156-170,1988. 16. JENKINS, R. R., AND J. TENGI. Catalase activity in skeletal muscle of varying fiber types (Abstract). Experientia 37: 67, 1981. 17. JI, L. L., F. W. STRATMAN, AND H. A. LARDY. Enzymatic down regulation with exercise in rat skeletal muscle. Arch. Biochem. Biophys. 263: 137-149, 1988. 18. JI, L. L., F. W. STRATMAN, AND H. A. LARDY. Antioxidant enzyme systems in rat liver and skeletal muscle. Arch. Biochem. Biophys. 263: 150-160,1988. 19. KANTER, M. M., R. L. HAMLIN, D. V. UNVERFERTH, H. W. DAVIS, AND A. J. MEROLA. Effect of exercise training on antioxidant enzymes and cardiotoxicity of doxorubicin. J. Appl. Physiol. 59: 1298-1303, 1985. 20. KIHLSTROM, M., J. OJALA, AND A. SALMINEN. Decreased level of cardiac antioxidants in endurance-trained rats. Acta Physiol. Stand. 135: 549-554, 1989. 21. KORTHUIS, R. J., AND D. N. GRANGER. Ischemia-reperfusion injury: role of oxygen-derived free radicals. In: Physiology of Oxygen Radicals, edited by A. E. Taylor, S. Matalon, and P. A. Ward. Bethesda, MD: Am. Physiol. Sot., 1986, p. 217-249. (Clin. Physiol. Ser.) 22. KORTHUIS, R. J., D. N. GRANGER, M. I. TOWNSLEY, AND A. E. TAYLOR. The role of oxygen-derived free radicals in ischemiainduced increases in canine skeletal muscle vascular permeability. Circ. Res. 57: 599-609, 1985. 23. MCCORD, J. M., AND I. FRIDOVICH. Superoxide dismutase. An enzymatic function for erythrocuprien (hemeocuprien). J. Biol. Chem. 244: 6049-6055,1969. 24. QUINTANILHA, A. T. Effects of physical exercise and/or vitamin E on tissue oxidative metabolism. Biochem. Sot. Trans. 12: 403-404, 1984. 25. ROY, R. S., AND J. M. MCCORD. Superoxide and ischemia: conversion of xanthine dehydrogenase to xanthine oxidase. In: Oxy Radicals and Their Scavenger Systems. Cellular and Medical Aspects, edited by R. A. Greenwald and G. Cohen. New York: Elsevier/ North-Holland, 1983, vol. II, p. 143-153. 26. SALMINEN, A., AND V. VIHKO. Endurance training reduces the susceptibility of mouse skeletal muscle to lipid peroxidation in vitro. Acta Physiol. Stand. 177: 105-110, 1983. 27. SRERE, P. A. Citrate synthase. Methods Enzymol. 13: 3-5, 1969. 28. SEXTON, W. L., R. J. KORTHUIS, AND M. H. LAUGHLIN. Microvascular injury after ischemia and reperfusion in skeletal muscle of exercise-trained rats. J. Appl. Physiol. 68: 2329-2336, 1990. 29. SMITH, J. K., D. L. CARDEN, M. B. GRISHAM, D. N. GRANGER, AND R. J. KORTHUIS. Role of iron in portischemic microvascular injury. Am. J. Physiol. 256 (Heart Circ. Physiol. 25): H1472-H1477, 1989. 30. SMITH, J. K., M. B. GRISHAM, D. N. GRANGER, AND R. J. KORTHUIS. Free radical defense mechanisms and neutrophil infiltration in postischemic skeletal muscle. Am. J. Physiol. 256 (Heart Circ. Physiol. 25): H789-H793, 1989. 31. SMITH, J. K., D. L. CARDEN, AND R. J. KORTHUIS. Role of xanthine oxidase in postischemic microvascular injury in skeletal muscle. Am. J. Physiol. 257 (Heart Circ. Physiol. 26): H1782-H1789, 1989. 32. STEEL, R. G. D., AND J. H. TORRIE. Principles and Procedures of Statistics. New York: McGraw-Hill, 1960, p. 161-182.
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M. H., T. SIMPSON, W. L. SEXTON, 0. R. K. SMITH, AND R. J. KORTHUIS. Skeletal muscle oxidative capacity, antioxidant enzymes, and exercise training. J. Appl. Physiol. 68(6): 2337-2343,1990.-The purposes of this study were to determine whether exercise training induces increases in skeletal muscle antioxidant enzymes and to further characterize the relationship between oxidative capacity and antioxidant enzyme levels in skeletal muscle. Male SpragueDawley rats were exercise trained (ET) on a treadmill 2 h/day at 32 m/min (8% incline) 5 days/wk or were cage confined (sedentary control, S) for 12 wk. In both S and ET rats, catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX) activities were directly correlated with the percentages of oxidative fibers in the six skeletal muscle samples studied. Muscles of ET rats had increased oxidative capacity and increased GPX activity compared with the same muscles of S rats. However, SOD activities were not different between ET and S rats, but CAT activities were lower in skeletal muscles of ET rats than in S rats. Exposure to 60 min of ischemia and 60 min of reperfusion (I/R) resulted in decreased GPX and increased CAT activities but had little or no effect on SOD activities in muscles from both S and ET rats. The I/R-induced increase in CAT activity was greater in muscles of ET than in muscles of S rats. Xanthine oxidase (X0), xanthine dehydrogenase (XD), and X0 + XD activities after I/R were not related to muscle oxidative capacity and were similar in muscles of ET and S rats. It is concluded that although antioxidant enzyme activities are related to skeletal muscle oxidative capacity, the effects of exercise training on antioxidant enzymes in skeletal muscle cannot be predicted by measured changes in oxidative capacity. LAUGHLIN, BROWN, J.
oxygen radicals; xanthine oxidase; superoxide lase; glutathione peroxidase; citrate synthase
dismutase;
cata-
eficial effects of exercise on various disease. Skeletal muscle contains severa mechanisms of protection from inju oxygen metabolites. These protect clude the enzymes superoxide dismu (CAT), and glutathione peroxidase lyzes the dismutation of superoxi which CAT converts to water and O glutathione can reduce HzOp to for fide and water (15, 21). Free-radical-mediated events ar volved in ischemia-reperfusion injur heart, kidney, pancreas, small intes (21,22). Several investigators have idant enzymes are related to the skeletal muscle (15) and/or have p training increases the resistance of potentially toxic effects of oxygen r 24, 26). Therefore we proposed th induces adaptations in the content enzymes in association with increa pacity and that these changes may cle from damage by active oxygen s radicals. Results of experiments des exercise training increases the resist cle to ischemia-reperfusion injury companion paper (28). The experiments outlined herein the following hypotheses. 1) Antio skeletal muscle are related to ox Training will increase antioxidant muscles in proportion to the trainin in oxidative capacity. 3) SOD, CAT in skeletal muscle tissue will be dec of the muscle to ischemia-reperfus zymes may be inactivated by rea generated when blood flow is reins reperfusion will result in the con dehydrogenase (XD) to xanthine ox
NOTION that exercise training induces adaptive responses that provide protection from various forms of injury and disease is widely accepted in the field of exercise science. Although training-induced adaptations in the cardiorespiratory system are well known, the mechanisms responsible for improvements in health and Downloaded from www.physiology.org/journal/jappl ${individualUser.surname} resistance byto${individualUser.givenNames} disease associated with exercise training (163.015.154.053) on September 13, 2018. Copyright © 1990 American Physiological Society. All rights reserved. still are not established. There is a growing body of THE
2338
TRAINING-INDUCED
BIOCHEMICAL
rats and housed two per cage (8 x 7 X 17 in.) in a room with controlled temperature (22 t 2OC) and light (12-h light-dark cycle). Rats had free access to food and water. One week after arrival, the rats were introduced to treadmill exercise with lo-min exercise bouts, at 15-30 m/ min 0% incline, 5 days/wk, for 2 wk. The six rats most resistant to treadmill exercise were omitted from the study. The remaining 20 rats were randomly divided into sedentary control (S) and exercise-trained (ET) groups. The S rats were confined to their cages throughout the remainder of the study, whereas the ET rats were trained on a modified Stanhope (Stanhope Scientific, Davis, CA) rodent treadmill. Over the first 2-6 wk of training the rats were gradually conditioned to run 32 m/min up an 8% incline for 2 h/day. During the last 6 wk the rats ran 32 m/min, up an 8% incline for 2 h/day. Experimental design. This study is composed of three series of experiments that were conducted on three paired groups of 10 ET and 10 S rats (total of 6 groups, 3 ET and 3 S). Two of these series of experiments were conducted on rats also included in the ischemia-reperfusion experiments outlined in the companion paper (28). To determine whether the exercise-training program described above would have an effect on the levels of free radical scavenger enzymes in skeletal muscle, biochemical assays were performed on biopsies taken from brachial muscles. In the first series of experiments, biochemical assays were not conducted on the hindlimb muscles of ET and S because these muscles were exposed to ischemia and reperfusion (28). Since the first series of experiments indicated that training induced some changes in scavenger enzymes in the brachial muscles (Fig. 3), it was important to determine whether similar changes occurred in hindlimb muscles. Therefore, in a second series of experiments on ET and S rats, biochemical assays were performed on biopsies of hindlimb muscles following ischemia-reperfusion. Finally, in a third series of experiments, the effects of ischemia-reperfusion injury on scavenger enzymes in hindlimb skeletal muscles of ET and S rats were determined. Muscle sampling procedures. In all three series of experiments samples (complete cross sections) of medial head of triceps brachii and long head of triceps brachii (red and white portions) were removed from the forelimbs of ET and S rats. In addition, the hearts were isolated and the atria and great vessels were removed. In the second and third series of experiments, samples of soleus and red and white portions of gastrocnemius muscles were also removed from the hindlimbs. Finally, in the third series of experiments, samples of vastus intermedius and red, middle, and white portions of vastus lateralis muscles were removed from the hindlimbs. All muscle samples were weighed, frozen in liquid Nz, and stored at -70°C until assayed. Subsequently, muscle samples were thawed, homogenized at 4°C in 0.1 M potassium phosphate buffer (pH 7.0), centrifuged, and dialyzed before analysis for enzyme activities as described below. Protein was determined with the Bradford dye-binding procedure with bovine albumin as the standard (4). Oxidative enzymes. Succinate dehydrogenase (SDH; EC 1.3991) activity was determined by following cyto-
ADAPTATION
chrome c reduction at 22°C with the procedure of Cooperstein et al. (5). Citrate synthase (CS; EC 4.1.3.7) activity was measured with the method of Srere (27). One unit of SDH activity is equal to the amount of enzyme present that reduces 1 pmol of cytochrome co min-’ . g wet wt-l. Antioxidant enzymes. SOD (EC 1.15.1.1) activity was determined using the method of McCord and Fridovich (23) and Flohe (9). One unit of SOD activity is the amount of enzyme present that inhibits the rate of cytochrome c reduction by 50%. CAT (EC 1.11.1.6) activity was determined by monitoring the decrease in absorbance at 240 nm in the presence of 10 mM H202. One unit of CAT activity is the amount of enzyme present that decomposes 1 M H20Jmin at 25OC. GPX (EC 1.11.1.19) activity was determined as described by Flohe and Gunzler (10). One unit of GPX activity is the amount of enzyme that oxidizes 1 pmol of glutathione/min at 25”C, pH 7.0. Xanthine oxidase and xanthine dehydrogenase. X0 and XD activities were assayed using a modification of the method described by Roy and McCord (25, 31). These enzymes were assayed in the vastus intermedius and vastus lateralis (red, white, and middle portions) muscles of the hindlimbs of rats following ischemia-reperfusion (28). At the completion of the ischemia-reperfusion experiments, the muscles were removed and frozen as described above. Samples were homogenized in potassium phosphate buffer (pH 7.8) containing phenylmethylsulfonyl fluoride and dithioerythreitol. The homogenate was centrifuged, and the supernatant was applied to a Sephadex G-25 column to remove low-molecular-weight inhibitors. The enzyme was assayed with xanthine as the substrate in a phosphate buffer (pH 7.8) containing NAD+. The addition of NAD+ to the assay allows measurement of total xanthine oxidase activity, i.e., the sum of XD and X0 activities. By excluding NAD+ from the assay, true oxidase activity can be measured. XD activity can be estimated by subtracting true X0 activity from total xanthine oxidase (X0 + XD) activity. The rate of reaction was monitored at 295 nm. The rate of change in absorbance reflects the rate of formation of uric acid. Allopurinol (50 PM) was added to the solution after the initial rate was obtained to ensure that the velocity represents X0 activity. Specific activities are expressed as milliunits per gram wet weight. One unit of activity is equal to the number of micromoles of urate formed per minute at 25°C. Data analysis. Data were analyzed using a two-way analysis of variance and Duncan’s new multiple range test (32). The specific activities of antioxidant enzymes were regressed on the SDH activities of the muscles to test for a relationship between oxidative capacity and antioxidant enzyme activities (32). Paired analyses were used where appropriate (32). Significance was defined as P < 0.05. RESULTS
The exercise-training program used in duced increases in the oxidative capacity muscles of the ET rats (Fig. 1). The CS red portion of the long head of the triceps
this study proof the skeletal activity of the brachii muscle
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TRAINING-INDUCED
BIOCHEMICAL
of ET rats was nearly twice that of the S rats, and in the white portion of the long head of triceps brachii of ET rats it was 20% greater than in S rats. Similarly, the SDH activities of these muscles from ET rats were greater than sedentary control in the red (150%) and white (20%) portions of the long head of triceps brachii. Effects of training on antioxidant enzymes. As shown in Fig. 2, there. were no differences between control and trained SOD activities in any sampled extensor muscle loogo-
I
Sedentary
8070605040302010
-
0L Red
White Long
Medial
Head
Head
FIG. 1. Effects of exercise training on citrate synthase activities of triceps brachii muscles of rats. Means k SE are presented; n = 18 in each group. * Trained value significantly greater than control, P 5 0.05.
300
Triceps
Brachii
of the forelimb or leg. Sedentary control and ET rats also had similar (P < 0.05) levels of myocardial SOD activity (S = 124 t 4 and ET = 126 t 4 U/mg protein). In contrast, four of six muscles from the ET rats had lower CAT activities than S rats (Fig. 3). Three of the muscles in which training induced a decrease in CAT activity were leg extensors (Fig. 3). The myocardial levels of CAT activity were slightly greater (P < 0.05) in ET rats (22.9 t 0.3 U/mg protein) compared with S rats (19.7 t 0.3). Exercise training produced significant increases in GPX activities in soleus and in red and white portions of the gastrocnemius muscle (Fig. 4) and the heart (S = 0.24 t 0.002 and ET = 0.32 t 0.005 U/mg protein). Effects of ischemia-reperfusion on antioxidant enzymes. Sixty minutes of ischemia followed by 60 min of reperfusion (ischemia-reperfusion) had no effect on SOD activities in any of the ET muscles sampled (Fig. 5). Only the red gastrocnemius sample of the S rats had greater SOD after ischemia-reperfusion. CAT activities were greater in soleus and both the red and white portions of the gastrocnemius muscle of both ET and S rats after ischemia-reperfusion (Fig. 5). The relative ischemia-reperfusion-related increases in CAT activities in the muscles of ET rats were greater than in the muscles of S rats (soleus muscle: ET 62% increase, S 6% increase; gastrocnemius muscle: red ET 75% increase, S 40% increase, and white ET 189% increase, S 60% increase). Since both the red and white portions of the gastrocnemius Brachii
Muscles
*al
% 61
J
.
Triceps
c
Muscles
1
250-
2339
ADAPTATION
I
Sedentary
EI
Trained
200 -
Red
White Long
Medial
Head
Head
Calf Muscles
241 White
Red Long
.-
Medial
Head
I
Sedentary
El
Trained
Head
Calf Muscles
C
; 100 k F! 50 L z.L -.-ul 0 c 3
b a -c 3
a-
White
Red
Soleus
Gastrocnemius 2. Superoxide dismutase activities in extensor muscles of forelimb (n = 18 in each group; triceps brachii muscles) and leg (n = 9 for trained, n = 10 for sedentary; calf muscles) of rats. Means k SE are presented. There were no statistically significant differences between sedentarv control and trained values in anv muscle sample. FIG.
i
m
6 * 03 White
Red
Soleus
Gastrocnemius FIG. 3. Catalase activities in extensor muscles of forelimb and leg of rats. Means t SE are presented; n, same as in Fig. 2. * Trained value is different from sedentary control, P 5 0.05.
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TRAINING-INDUCED
2340 Triceps
Brachii
BIOCHEMICAL
Superoxide
Muscles
0.5
.g ?I ‘L
100
0.4
D
Sedentary
ezzl
Trained
.E 2 (! a
0.3
F 2 * .e
0.2
=
0.1
ADAPTATION
IT $ a m Y =
ON ezd I/R
80
60
4o 20
0
0.0 White
Red Long
Medial
Head
Dismutase
SED
SED
ET
White
Head
ET
Red
SED
E-T
Soleus
Gastrocnemius 0.5
.G 3 !! 0.
0.4
Calf Muscles
1
Catalase 25
*
20 .i
0.3 1
% FL
F h v) Y
0.2
F
=
0.1
ki CL m Y 5
1
15
10
5
0.0 / White Gastrocnemius
Red
0
Soleus
FIG. 4. Glutathione peroxidase activities in extensor muscles of forelimb and leg of rats. Means + SE are presented; n, same as in Fig. 2. * Trained value is different from sedentary control, P 5 0.05.
muscle are primarily composed of fast-twitch muscle fibers (3) and the soleus is primarily slow twitch (3), these data suggest that the effects of ischemia-reperfusion on CAT activity is different in fast- and slow-twitch skeletal muscle. CAT activity appeared to be increased more in fast-twitch muscle after ischemia-reperfusion than in slow skeletal muscle (soleus). Ischemia-reperfusion had no effect on GPX activities of white gastrocnemius muscle (Fig. 6). GPX activities of the red gastrocnemius and soleus muscles from S rats were lower after ischemia-reperfusion (Fig. 6). Thus ischemia-reperfusion appears to produce greater decreases in GPX activities in high oxidative muscles of both sedentary and ET rats than in the less oxidative white gastrocnemius muscle (3). Xanthine oxidase and xanthine dehydrogenase activities. After exposure to ischemia-reperfusion there were
no differences among X0 activities in the four samples of thigh muscles in either group of rats (Table 1). However, the XD and X0 + XD activities were greater in the vastus intermedius muscles of sedentary rats than in the vastus lateralis muscle tissue. X0 + XD activities of vastus intermedius muscle were also greater in the ET rats. Although X0 + XD activities tended to increase with increasing oxidative capacity of the muscles (i.e.,
SED
ET
SED
White
ET
Red
:D
ET
Soleus
Gastrocnemius FIG. 5. Superoxide dismutase and catalase activities of leg muscles of exercise-trained (ET; n = 7) and sedentary control (SED; n = 8) rats. N, nonischemic; I/R, after ischemia-reperfusion. Means + SE are presented. * I/R values are different from N, P 5 0.05.
0.40 0.35 5
0.30
g
0.25
F ii a
0.20
$
0.10
UN CIia
I/R
0.15
0.05 0.00
SED
ET
SED
ET
Red
White
SE0
Soleus
Gastrocnemius FIG. 6. Glutathione peroxidase activities of leg muscles of exercisetrained (ET; n = 7) and sedentary control (SED; n = 8) rats. N, nonischemic; I/R, after ischemia-reperfusion. Means If: SE are presented. * I/R values are different from N, P 5 0.05.
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TRAINING-INDUCED
BIOCHEMICAL
Long
1. Specific activities of X0, XD, and X0 + XD in rat skeletal muscles after ischemia-reperfusion TABLE
Muscle Sample
Vastus lateralis White Middle Red Vastus intermedius
x0
T S T
XD
XO+XD
% Total as X0
T S T
7.6t0.9 7.7kO.9 7.8t1.5 7.3kl.l 7.8t0.4 8.1Gl.2 10.9t2.3
7.3t1.4 6.3kl.0 8.0t1.5 7.6rt1.5 9.1t0.7 7.6k0.9 11.6t1.5
14.9k1.6 14.0t1.0 15.8t2.5 14.9k1.9 16.9kO.9 15.7t1.2 22.5&2.2*
51k7 46k2 51k6 47k6
S
7.9kO.9
12.4k1.2"
20.4tl.O*
39t4
S
5226 56&6
49&5
Values are mea ns t SE expressed in m U/g wet w t for muscles from 7 trained (T) and 7 sedentary control (S) rats. X0, xanthine oxi .dase; XD, xanthine dehydrogenase. The muscles were isolated following exposure to 60 min of ischemia and 60 min of reperfusion (28). * Vastus intermedius enzyme activity is greater than enzyme activities of other muscles, P 5 0.05. ‘There were no differences between trained and sedentary control data.
white vastus activity < middle vastus c red vastus c vastus intermedius), none of the values was significantly different among the vastus lateralis samples.
2341
ADAPTATION Head
of Triceps
Brachii
Muscle
200
iii
150
0 -w
is bv1 \ E Ql fJ 100
0
8
lz ‘$
c" it 5
50
0
0.30 ii g
0.25
T t
1
DISCUSSION
This study evolved from our interest in the effects of ischemia-reperfusion-induced injury to the microvasculature of skeletal muscle (7, 21, 22, 28-31). Reports of exercise training-induced increases in the specific activities of SOD, CAT, and GPX in skeletal muscle and the fact that SOD and CAT provide protection from microvascular damage induced by ischemia-reperfusion (22) indicate that exercise training could induce adaptations within the muscle resulting in increased resistance to the damaging effects of ischemia-reperfusion. The results of this study indicate that antioxidant enzyme activities vary within and among extensor muscles of rats. The specific activities of SOD, CAT, and GPX are greater in skeletal muscles composed primarily of oxidative fiber types of both sedentary and exercise-trained rats. Both slow-twitch oxidative (i.e., soleus) (3) and fast-twitch oxidative (i.e., red gastrocnemius) (3) fiber types had elevated antioxidant enzyme levels compared with fasttwitch glycolytic fiber types (i.e., white gastrocnemius) (3). Since the generation of ATP via oxidative metabolism is one normal source of reactive oxygen metabolites, one would expect that antioxidant enzyme levels and the levels of enzymes involved in oxidative metabolism would be related as proposed by Jenkins and Tengi (16) and Higuchi et al. (12). The analysis of our data among muscle samples is consistent with this hypothesis. However, when the relationship between oxidative capacity and antioxidant enzymes was explored within muscles (in contrast to among muscles) with linear regression analysis, no relationship was found between oxidative capacity and SOD (e.g., Fig. 7, top) or CAT activities. In contrast, GPX activity seems to be related to SDH activity both within and among skeletal muscles (Figs. 4 and 7, bottom). Thus, although SOD and CAT activities are related to oxidative capacity of skeletal muscle when
0
1 Succinate
2 Dehydrogenase
3
4
(units/mg) FIG. 7. Superoxide dismutase (top) and glutathione peroxidase (bottom) activities as functions of succinate dehydrogenase (SDH) activity of red and white portions of long head of triceps brachii muscle. Superoxide dismutase and catalase (not shown) activities were not related to SDH activity. However, glutathione peroxidase activity (bottom) is related to SDH activity (glutathione peroxidase activity = 0.02 SDH + 0.04, r = 0.56, P 5 0.001, n = 32).
examined among muscles of various fiber type composition, the coupling of SOD and CAT activities to oxidative capacity of muscle is less apparent when examined within muscles. GPX activity appears to be more closely linked to oxidative capacity both among muscles of various fiber type composition and within muscles. The tight coupling between oxidative capacity and GPX activity within and among muscles may be related to the fact that GPX was the only antioxidant enzyme that was upregulated in the muscle of the exercise-trained rats. Effects of exercise training. As expected, exercise training produced increases in oxidative capacity in all muscle samples investigated. However, exercise training had no effect on SOD levels in any of the six extensor muscle samples (Fig. 2). Our results are in agreement with those of Ji et al. (17,18) and Alessio and Goldfarb (2). However, Higuchi et al. (12) reported 20-30s greater SOD activities in the soleus and red vastus lateralis muscles of trained Wistar rats. Jenkins (15) has also measured higher SOD levels in the skeletal muscles of trained human subjects and in the soleus muscle of trained rats. It might be suggested that these disparate results are related to differences in training intensity, duration, or
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2342
TRAINING-INDUCED
BIOCHEMICAL
mode (treadmill running vs. swimming). However, the results obtained by Higuchi et al. (12) disagree with the results obtained in this study (Fig. 2) and those of Alessio and Goldfarb (2); similar training programs were used in all three studies. Thus the reason for the disparate results among these studies remains unclear. Jenkins (15) indicated, in a recent review of free radical biochemistry and exercise, that animal studies investigating the effects of training on CAT activity in skeletal muscle have also produced conflicting results. CAT activities in four of the muscle samples of the exercisetrained rats were lower than control (Fig. 2). These results are in agreement with those of Alessio and Goldfarb (Z), who recently reported that CAT activity was 30-40s lower in the red and white portions of vastus lateralis muscle of trained rats. Higuchi et al. (12) reported that training did not produce any change in soleus CAT activity. Thus available data indicate that exercise training either does not change CAT activity in skeletal muscle or produces decreasesin CAT activities. Our data indicate that some of the controversy concerning the effects of training on CAT activity in skeletal muscle may be attributable to the fact that the effects of training on CAT activities are not uniform among muscle tissue of differing fiber type composition. Our data indicate that exercise training has no effect on myocardial SOD activity, a result which is consistent with the results of Kanter et al. (19) and Higuchi et al. (12). Kihlstrom et al. (20) recently reported that swim training produced decreases in myocardial SOD activity. Thus training does not produce increases in SOD activity in cardiac muscle. Similarly, Higuchi et al. (12) and Kanter et al. (19) found that myocardial CAT activity was not altered by exercise training of rats and mice, respectively. These results are consistent with ours in that we measured a small increase in CAT activity in hearts of trained rats. In contrast, Kihlstrom et al. (20) found rats trained with swimming had lower cardiac CAT activities than control rats. Treadmill training may affect the expression of antioxidant enzymes in cardiac tissue (12, 19) in a manner different from the effects of swim training (20). The GPX activities in skeletal muscles (Fig. 4) and hearts of trained rats tended to be greater than the GPX activities of these muscles from sedentary rats. These data are consistent with the reports of others investigating the effects of exercise training on GPX activity (2, 16, 17, 19, 24). Our hypothesis was that exercise training would induce increases in skeletal muscle antioxidant enzymes in proportion to the training-induced increase in oxidative capacity. This hypothesis was based on a rationale similar to that presented above in relation to the apparent coupling of the enzymes of oxidative metabolism and antioxidant enzymes. The results indicate that the effects of training on antioxidant enzymes varied both among the three enzymes investigated as well as within and among the muscles sampled. Therefore the data do not support our hypothesis. Effects of ischemia and reperfusion. Sixty minutes of ischemia followed by 60 min of reperfusion had no effect on SOD activities in skeletal muscles from trained or
ADAPTATION
sedentary rats. Similarly, Smith et al. (30) reported that 4 h of ischemia followed by 1 h of reperfusion had no effect on SOD or CAT activities of dog gracilis muscle. In the present study, ischemia-reperfusion produced increases in CAT activity in soleus muscle and the red and white portions of the gastrocnemius muscle in both trained and sedentary rats. The increase in CAT activity induced by ischemia-reperfusion was consistently greater in muscles isolated from the trained rats compared with sedentary controls (Fig. 5). The effects of ischemiareperfusion on the activities of SOD and CAT in skeletal muscle appear similar to the acute effects of sustained exercise on these enzymes. For example, Alessio and Goldfarb (2) reported that an acute bout of exercise (20 min) produced increases in the CAT activity of skeletal muscle but had no effect on SOD activity. Also, Alessio and Goldfarb (2) observed that the increase in CAT activity produced by acute exercise was greater in exercise-trained rats. We do not know of an established mechanism for an ischemia-reperfusion-induced increase in CAT activity in skeletal muscle or the fact that exercise training exacerbates this effect of ischemiareperfusion. GPX activities appeared to be decreased after ischemia-reperfusion in muscles of both sedentary and exercise-trained rats. This is similar to the effects of ischemia-reperfusion on GPX activity of dog gracilis muscle as reported by Smith et al. (30). The relative decrease in GPX activity produced by ischemia-reperfusion was similar in trained and sedentary rats despite the fact that the trained rats generally had higher GPX levels in nonischemic muscle (Fig. 6). X0-derived oxidants are believed to have a role in the genesis of ischemia-reperfusion injury in skeletal muscle (21, 30, 31). This notion is based in part on the observation that XD is converted to X0 in skeletal muscle subjected to ischemia-reperfusion. In addition, ischemiareperfusion injury in skeletal muscle is attenuated by either X0 inhibition (using oxypurinol) or depletion (using tungsten-supplemented diets) (31). The results presented in Table 1 indicate that these enzymes do not vary with muscle fiber composition in the vastus lateralis muscle although the vastus intermedius muscle had greater XD (in sedentary only) and X0 + XD activities than the vastus lateralis muscle (Table 1). We previously reported XD + X0 values of 10.9 t 0.5 with 30% of total activity in the X0 form in normal (nonischemic) vastus lateralis muscle (31). In this previous study, X0 activity represented 59% of total (X0 + XD) activity after 2 h of ischemia and 30 min of reperfusion, suggesting that conversion of XD to X0 had occurred (31). Our data (Table 1) indicate that a similar amount of XD-to-X0 conversion occurred during 1 h of ischemia followed by 1 h of reperfusion in that the vastus lateralis had ~50% of total activity in the X0 form. The data also suggest that XD-to-X0 conversion is greater after ischemiareperfusion in fast-twitch glycolytic (FG) muscle, since the white vastus muscle (97% FG fibers) (3) tended to have the largest percentage of total activity in the X0 form. Exercise training did not appear to influence these enzymes.
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TRAINING-INDUCED
BIOCHEMICAL
ou r results support several conclusions 1) Antioxidant enzymes appear to be rel .ated to the 0 ixidative capacity of muscle tissue when compared among skeletal muscles of various fiber type composition. However, the relationship between antioxidant enzymes and oxidative enzymes is not as obvious when examined within muscles. 2) The effects of exercise training on antioxidant enzyme levels cannot be predicted solely from knowledge of the training-induced increases in oxidative capacity of the muscle. For example, exercise training did not produce increases in SOD or CAT activities in skeletal muscl .e with training induced increases in oxidative capacity. In fact, CAT activities were decreased in the muscles of trained rats. Training induced increases in GPX activities in both skeletal muscle and heart. 3) Exposure of skeletal muscle to 60 min of ischemia followed by 60 min of reperfusion resulted in increased CAT activities, decreased GPX activities, and had no effect on SOD activities. The effects of ischemia-reperfusion on CAT and GPX activities in skeletal muscle were greater in the trained rats than in control. Finally, 4) exercise training did not alter the activities of X0, XD, and X0 + XD or the apparent amount of conversion of XD to X0 in the skeletal muscle tissue during ischemia-reperfusion. The authors thank Donna Baumgartner and M. E. Schaefer for exercise training the rats and Greg Kelly for work in data analysis and creating the illustrations. This study was supported by National Heart, Lung, and Blood Institute Grants HL-36088 and HL-36069. M. H. Laughlin is the recipient of National Heart, Lung, and Blood Institute Research Career Development Award HL-01774, and R. J. Korthuis is an Established Investigator of the American Heart Association (880197). Address for reprint requests: M. H. Laughlin, University of Missouri, W116 Vet. Med. Bldg., Columbia, MO 65211. Received
4 October
1989; accepted
in final
form
29 January
1990.
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