- Email: [email protected]
Progressive changes in fatty acid metabolism in rat liver and muscle during exercise
RIOCHEMI(‘A1.
MEDI(‘INE
29. ?#-3()h
( 19831
Progressive Changes in Fatty Acid Metabolism in Rat Liver and Muscle during Exercise HISHAM A. BARAKAT,’ GEORGEJ. KASPEREK,AND G. LYNIS DOHM Department of Biochemistry. School of Medicine. East Carolinu University. Greenville. North Carolina 27834 Received May 28, 1982
Although it is well established that fatty acids are prominent sources of energy during prolonged periods of exercise (I-6) fewer studies reporting the influence of an acute bout of exercise on synthesis of fatty acids by liver or oxidation of these compounds by liver and muscle have appeared. Askew et al. (7) reported that exercise of rats to exhaustion immediately prior to sacrifice significantly decreased lipogenesis from glucose and fatty acid synthetase activity in adipose tissue from trained but not untrained animals. Liver fatty acid synthetase was reported not to be influenced by exhaustive exercise (7). Tate et al. (8) recently reported that substrate oxidation by liver was enhanced after a single bout of exercise. Early in vitro studies showed that fatty acid oxidation by muscle preparations of rats subjected to a single exhaustive bout of exercise was lower than that of the unexercised controls (9). More recently, we reported that the activities of several enzymes involved in lipogenesis in liver were decreased with exhaustive exercise (10). Furthermore, fatty acid oxidation by liver preparations was increased, but oxidation was decreased in muscle homogenates of exhaustively exercised rats (10). These observations show that the influence of a single bout of exercise on fatty acid oxidation by muscle is different from that seen in training, and led to the hypothesis that the observed decrease may be a factor in exhaustion and fatigue. This study was undertaken to determine the influence of an exhaustive bout of exercise on fatty acid metabolism in liver and muscle preparations. To that end, the capacity of liver to synthesize fatty acids was assessed by measuring the activities of lipogenic enzymes. In addition, the capacity of liver and muscle to oxidize fatty acids was also determined. Through ’ To whom correspondence
should be addressed. 298
0006-2944/83 $3 .OO Copyright All rights
Q 1983 by Academic Press. Inc. of reproduction in any form reserved
FATTY
ACID
METABOLISM
DURING
EXCERCISE
299
these studies answers to two questions were sought. The first question is: Does the decrease in the capacity of muscle to oxidize fatty acids occur only at exhaustion, i.e., is this decrease a factor in fatigue? To answer this question, we determined the capacity of muscle to oxidize fatty acids after progressive increases in exercise time. The second question is: Are the effects of exhaustion permanent? If not, how long does it take the exhausted animals to recover from the effects of the exhaustive bout? To answer this question, we examined the capacity of liver and muscle to oxidize fatty acids during various periods of recovery. METHODS Male Holtzman rats (Holtzman Co., Madison, Wise.) weighing 250300 g were housed in individual cages in a room maintained at 20-23°C and kept on a 12 hr dark-light cycle (6 AM--~ PM light). The animals were given water and commercial lab chow (Wayne Blox, Allied Mills, Inc., Chicago, Ill.) ad libitum until the start of the experimental protocol. The results reported here are from two related experiments. In Experiment I, rats were divided into the following groups: (i) exercised for 1 hr; (ii) exercised for 2 hr; and (iii) exercised until exhaustion. There were 8 animals in each group along with a control group (10 animals). In Experiment II, rats were divided into the following groups: (i) exercised to exhaustion; (ii) exhausted-1Zhr rested; (iii) exhausted-24-hr rested; and (iv) exhausted-72-hr rested. Exercised animals were run on a rodent treadmill between 6 and ‘11 AM and were made to run at 28 m/min (0% grade) until they could no longer continue to run (220 & 19 min, x t SEM). Each of the exercised groups (with eight animals to a group) had an unexercised control group that was of comparable weight. In order to minimize the effects of variation in food consumption on fatty acid metabolism, the food intake of the controls was restricted to match that of the exercised group, i.e., pair fed. Food was removed from the cages of the control rats during the time that the exercised rats were on the treadmill. When the last rat was exhausted, the exercised group was given lab chow ad libitum, but the rats in the control group were given half of their daily allowance. The other half of their daily allowance was given between 11 and 12 PM so that the rats would not be totally without food for an extended period of time. Following the desired exercise period, both the experimental animals and their respective controls were killed by decapitation. The liver and muscle were then excised and placed in 0.15 M KC1 over ice. To assay for lipogenic enzymes, livers were homogenized in a glassTeflon Potter-Elvehjem homogenizer (1: 10 w/v) in 0.05 M potassium phosphate buffer, pH 7.4, containing 1 mM EDTA and 1 mM dithiothreitol. The particle-free supernatant fraction was prepared by centrifuging the
300
BARAKAT.KASPEREK.AND
DOHM
homogenates at 105,OOOg for 60 min followed by filtration through cheesecloth. Lipogenic enzymes in the particle-free supernatant solutions were assayed spectrophotometrically (I 1). Fatty acid synthetase was assayed according to the procedure of Arslanian and Wakil (12), ATP citrate lyase was assayed as described by Takeda rt ~1. (13). malic enzyme was assayed according to Hsu and Lardy (14), and glucose-6-phosphate dehydrogenase was assayed according to the method of Langdon (15). For the determination of the rate of fatty acid oxidation, liver was homogenized in 0.25 M sucrose (I : 10 w/v) in a glass-Teflon PotterElvehjem homogenizer then filtered through cheesecloth. Muscle was pressed through a tissue press, then homogenized in Chapell-Perry (16) buffer, pH 7.4, (1: 10 w/v) in a glass-glass homogenizer then filtered through cheesecloth. The capacity of liver and muscle homogenates to oxidize [I-‘4C]palmitate was determined in duplicate by collecting and counting 14C02 produced during the incubation period. A 2-ml incubation volume was used containing the following cofactors (pH 7.3): 2.0 mM ATP, 0.05 mM coenzyme A, 1.0 mM dithiothreitol, 0.1 mM malate, 1.0 mM MgC&. 0.072 mM bovine serum albumin (fatty acid free), 0.1 mM NAD, 100 mM sucrose, 100 mM K,HPO,, 80 mM KCI, 0.1 mM EDTA, 1.0 mM m-carnitine and 0.2 mM [l-‘4C]palmitate (I $Zi). The reaction was initiated by the addition of sample, and the contents were gassed with 95% 02, 5% CO? and stoppered with a rubber septum stopper containing a polypropylene center cup. The flasks were incubated at 37°C for 30 min with gentle shaking. Immediately prior to the termination of the incubation, 0.2 ml of ethanolamine:methyl Cellosolve (I :2) was injected into the hanging centerwell. The reaction was stopped by injecting 0.2 ml of 4.0 N H,SO, into the contents of the. flask. Flasks were shaken for 60 min to collect the evolved 14C0,. An aliquot of the ethanolamine:methyl Cellosolve was then taken and counted. All assays were performed under conditions of substrate saturation, with the amount of enzyme as the rate-limiting component of the reaction. Protein concentrations of all preparations were determined by the biuret method (17). The results of these experiments were analyzed by a one-way analysis of variance (18). Comparisons of means were tested for significant differences by a Newman-Keuls procedure (18). The level of statistical significance chosen for these experiments was P < 0.05. RESULTS AND DISCUSSION
The effect of progressively increasing exercise time on the activities of the lipogenic enzymes is shown in Fig. I. All four enzymes showed the same effect in that their activity decreased progressively. The lowest activity obtained was at the exhaustion point. when the animals could
FATTY ACID METABOLISM
DURING
301
EXCERCISE
A. MALIC
ENZYME
B. CITRATE-ATP
LYASE
C
GLUb-P-IIEHYGRGGENASE
0.
FATTY ACID
50 25 0
I
SYNTHETASE
I lOO75-I so--
I
l I I 1
2
EXH.
12
TIME
OIRS).
250
1 EXERCISE
PERIGD
I
,
I
1
I
24
36
48
80
72
RECOVERY
PERIOD
FIG. 1. Activities of hpogenic enzymes in liver of the experimental animals. The activity of each enzyme was measured in the high-speed supematant fraction of liver after 1 hr of exercise, 2 hr of exercise, or at exhaustion (Exercise Period) and at 12 hr of rest, 24 hr of rest, or 72 hr of rest after exhaustion (Recovery Period). Each point represents the average of eight observations expressed as percentage of control (tOO%). A shows the changes in the activity of mahc enzyme where the control value was 50.3 + 4.2 nmo~e/~n/~ protein; B, ATP citrate lyase where the control value was 24.6 t 1.3 nmole/min/mg protein; C, glucose-(i-phosphate dehydrogenase where control vaiue was 72.3 d 6.1 nmole/min/mg protein; and D, fatty acid synthetase where control value was 7.52 -c 0.43 nmole/min/mg protein. *Denotes statistically significant differences (P < .OS).
302
HAKAKAI’,
KASPEREK.
AND
DONM
not run any longer. Although no significant changes were seen upon exhaustion in the activity of fatty acid synthetase, glucose-&phosphate dehydrogenase or ATP citrate lyase, the activity of malic enzyme was significantly depressed. The lack of significant differences that was obtained in this experiment is different from what we reported earlier f 19) where the activities of all four enzymes were significantly depressed at exhaustion. Although the magnitude of depression in the activities of these enzymes is similar to that reported earlier, the results did not show significant differences here because of a wider variability than the previous experiment, No apparent cause of the variability is readily apparent at the present. However, it should be noted that the same trends were seen here as in th previous experiment. The progressive decrease in the activities of the enzymes of fatty acid synthesis may be related to the loss of hepatic proteins that we reported earlier (19). In that study, we reported that Iiver protein loss is increased with exhaustive exercise, including soluble proteins. The variation in the degree of loss of individual enzyme activity may be retated to turnover rates that are characteristic of each enzyme. This point is supported by examining the activities of the enzymes during the recovery periods. Whereas malic enzyme activity was depressed the most at exhaustion, it returned to normal levels by 12 hr after recovery. Fatty acid synthetase activity, which was depressed at a slower rate, took longer than 12 hr to get back to almost normal activity. Thus, it appears that exercise accelerates the rate of decay of the Iipogenic enzymes, and recovery from exercise depends on synthesis of new enzyme protein. This pattern of changes fits into “phase II” of the biphasic hormonal nature of stress that was recently described by Bessman and Renner (20). In this phase, the hormones that play a major role in metabolism are growth hormone, ACTH. and cortisol. In the early stages of this phase, the above hormones induce the synthesis of protease as well as the synthesis of the enzymes of gluconeogenesis. The results are increased degradation of proteins with the concomitant release of amino acids. Some of these amino acids are used as substrates for gluconeogenesis, which leads to the elevation in bl~-~ucose levels. The elevated bled-~ucose Ieve will subsequently cause the release of insulin which in turn will stimulate protein synthesis again. The phase II hormones take longer than “phase I” (the catecholamine phase) hormones to exert their effects on the organism. It thus seems likely that the changes in the activities of the enzymes that we observed may be under the influence of the phase II hormones. Fatty acid oxidation by liver preparations (Fig. 2) remains unchanged within the first hour of exercise, but rises steadily after that, until it reaches a maximum at exhaustion (approximately 180% of controls). After 12 hr of rest, however. fatty acid oxidation returns to normal levels
FATTY
ACID METABOLISM
DURING
303
EXCERCISE
I
I 1
2
12
24
36
46
60
12
EXH. EXERCISE
PERIOD
REcDvERY TIME
PERIOD
0iRS).
FIG. 2. [l-‘4C]Palmitate oxidation by liver homogenates of the experimental animals. Palmitate oxidation was measured in the same experimental animals described in Fig. 1. Each point represents the average of eight observations expressed as percentage of control value (100%) which was 0.084 k 0.011 nmole/min/mg protein. *Denotes statistically significant differences (P < .OS).
and remains close to normal during the subsequent periods of recovery. The rise in the oxidative capacity of liver with exercise may be related to changes in the levels of certain hormones, particularly insulin and glucagon. It has been reported that one of the controlling factors that regulates the process of fatty acid oxidation in liver is the insulin:glucagon ratio (21). Since variations in the levels of these hormones have been reported to occur with exercise (22), it is tempting to speculate that the elevation in the capacity of liver to oxidize fatty acids may be related to alterations in the ratio of these hormones. Upon reestablishment of normal levels of the hormones, such as during recovery from exercise, fatty acid oxidation returns to normal. Oxidation of fatty acids by muscle homogenates was depressed by approximately 80% at 1 hr of exercise, by 70% at 2 hr of exercise, and by 60% at exhaustion (Fig. 3). Similar decreases in fatty acid oxidation resulting from exhaustive exercise have been reported earlier (9), but the cause of such changes is not known. Since the mechanisms underlying the cause of such changes is not within the scope of this paper, no attempts are made to explain our observations. It is worthy to note, however, that such dramatic decreases in the capacity of muscle to oxidize fatty acids are observed within a short period of time (1 hr) and that an apparent recovery during prolonged periods of exercise occurs. Nonetheless, oxidation remains depressed at exhaustion. The recovery from exhaustion is fairly fast, so that within 12 hr, oxidation by the
304
BAKAKAT.
1 EXERCISE
2
KASPEKEK.
EXH.
12
PERIOD
AND DOHM
24
36
RECOVERY TIME
48
60
70
PERIOD
(HRS).
FIG. 3. [l-‘4C]Palmitate oxidation by muscle homogenates of the experimental animals. Palmitate oxidation was measured in the same experimental animals described in Fig. I. Each point represents the average of eight observations expressed as percentage of control value (100%) which was 0.096 * 0.014 nmole/min/mg protein. *Denotes statistically significant differences (P < .05).
exhausted-12-hr rested animals is approximately 80% that of the controls. It appears that by 72 hr recovery from exhaustive exercise, in relation to muscle’s capacity to oxidize fatty acids, is complete. The results of this study answer the major questions that were posed. It appears that exhaustive exercise influences both the synthesis and oxidation of fatty acids rather dramatically. The capacity of liver to synthesize fatty acids decreased with increasing the time of exercise. At the same time oxidation of fatty acids by liver was maximal at the time of exhaustion. In contrast, oxidation of fatty acids by muscle preparations was the lowest at 1 hr of exercise and started to recover after 2 hr and until the animals were exhausted. Thus, the decrease in the oxidation of fatty acids at exhaustion is not an abrupt process and cannot be a factor in fatigue. These studies also show that the effect of exhaustive exercise on the biochemical processes that were examined is not an irreversible phenomenon, as evidenced by the short period of recovery time which was required for these processes to return to control levels. SUMMARY
This study was undertaken to determine the effects of increasing exercise time until exhaustion on the capacity of liver to synthesize and oxidize fatty acids, as well as the capacity of muscle to oxidize fatty acids. Additionally, the same parameters were examined during various periods of recovery after exhaustion. These studies were prompted by the scarcity of information relating the effects of exercise on the metabolism of fatty acids in liver, and by our findings and those of others where exhaustive
FATTY
ACID METABOLISM
DURING
EXCERCISE
305
exercise was reported to cause a decrease in the capacity of muscle to oxidize fatty acids. The latter led to the hypothesis that the reported decrease in fatty acid oxidation may be a factor in fatigue. Thus the purpose of the present studies was threefold. The first was to determine changes in fatty acid metabolism in liver during exercise and recovery: the second was to test the above hypothesis by following time-course changes of fatty acid oxidation by muscle; and the third was to determine whether or not the effects of exhaustion are reversible. The results obtained showed that fatty acid synthesis in liver decreased with increasing time but returned to normal levels by approximately 12 hr of rest after exhaustion. Oxidation of fatty acids by liver remained unchanged during the first hour of exercise but steadily increased afterwards until it reached a maximum at exhaustion, Oxidation returned to control level after 12 hr of rest and remained so during the subsequent recovery period, Fatty acid oxidation by muscle was maximally depressed after 1 hr of exercise. yet the animals continued to run, thus ruling out the possibility that the decreased oxidation of fatty acids with exhaustion is a factor in fatigue, Finally, recovery studies showed that the biochemical processes that were examined returned to control levels within 12-24 hr after exercise demonstrating that these effects are not permanent. REFERENCES I. 2. 3. 4. 5. 6.
Havel, R. J., Naimark. A.. and Borchgrevink, C. F., J. C/in. Invest. 23, 1054 (1963). Carlson, L. A., Froberg, S., and Persson. S., Acta Physio/. Scund. 63, 434 (1965). Paul, P., and issekutz, B., J. Appl. Physiol. 22, 612 (11967). Essen, B., Ann. N.Y. Acad. Sri. 301, 30 (1977). Gollnick, P. I)., Ann. N. Y. Acad. Sri. 301, 64 f 1977). Costill, D. L., Fink, W. J., Getchell, L. H.. Ivy, J. L., and Witzman, F. A,, J. Appl. Physiol.
47, 787 (1979).
7. Askew, E. W.. Barakat. H. A., Kuhl. G. L., and Dohm, G. L., Lipids 10,491 (1975). 8. Tate, C. A., Scott, K. S., Wolkowicz, P. E.. and McMillin-Wood, J., J. Med. Sci. Sports Exer. 13, 118 (1981). 9. Dohm, G. L., Huston. R. L., Askew, E. W.. and Weiser, P. C.. Amer. J. Physial. 223, 783 (1972). IO. Barakat, H. A., Kasperek, G. J., Dohm, G. L., Tapscott, E. B., and Snider, R. D.. Fed. Pruc. 41, 1752 (1982). 1 I. Barakat. H. A., Brown, W. E., and Henry, S. D., Life Sci. 23, 1835 (1978). 12. Arslanian, M. J., and Wakil, S. J., ~ef~~d~ E~zy~o~. 35, 59 (19753. 13. Takeda, Y., Suzuki, F., and Inoue, H., Methods Enzymof. 13, 153 (19691. 14. Hsu, R. Y., and Lardy, H. A., Methods Enzymof. 13, 230 (1969). 15. Langdon, R. G., Methods Enzymol. 9, 126 (1966). 16. Chapell, J. B., and Perry, S. V., Nature (London) 173, 1094 (1954). 17. Gomall, A. G.. Bardawill, C.. and David, M. M., J. Biol. Chem. 177, 751 (1949). New 18. Weiner, B. J., “Statistical Principles in Experimental Design.” McGraw-Hill, York, 1971. 19. Kasperek. G. J., Dohm, G. L.. Barakat, H. A., Strausbauch, P. H. Barnes, D. W., and Snider, R. D., Biorhem. J. 202, 281 (1982).
306
BARAKAT.
KASPEREK.
AND DOHM
20. Bessman, S. P.. and Renner, V. J.. in “Pathology of Shock. Anoxia and Ischemia” (R. A. Cowley and B. F. Trump. Eds.). pp. 60-65. Williams & Wilkens. Baltimore.
1982. 21. McGarry, J. D., and Foster, D. W.. Ann. Rev. Biodwrn. 49. 395( 1980). 22. Fell. R. D., McLane, J. A., Winder, W. W.. and Holloszy. J. 0.. Ann. J. fl~~,~;,~/.
238.328(1980).
MEDI(‘INE
29. ?#-3()h
( 19831
Progressive Changes in Fatty Acid Metabolism in Rat Liver and Muscle during Exercise HISHAM A. BARAKAT,’ GEORGEJ. KASPEREK,AND G. LYNIS DOHM Department of Biochemistry. School of Medicine. East Carolinu University. Greenville. North Carolina 27834 Received May 28, 1982
Although it is well established that fatty acids are prominent sources of energy during prolonged periods of exercise (I-6) fewer studies reporting the influence of an acute bout of exercise on synthesis of fatty acids by liver or oxidation of these compounds by liver and muscle have appeared. Askew et al. (7) reported that exercise of rats to exhaustion immediately prior to sacrifice significantly decreased lipogenesis from glucose and fatty acid synthetase activity in adipose tissue from trained but not untrained animals. Liver fatty acid synthetase was reported not to be influenced by exhaustive exercise (7). Tate et al. (8) recently reported that substrate oxidation by liver was enhanced after a single bout of exercise. Early in vitro studies showed that fatty acid oxidation by muscle preparations of rats subjected to a single exhaustive bout of exercise was lower than that of the unexercised controls (9). More recently, we reported that the activities of several enzymes involved in lipogenesis in liver were decreased with exhaustive exercise (10). Furthermore, fatty acid oxidation by liver preparations was increased, but oxidation was decreased in muscle homogenates of exhaustively exercised rats (10). These observations show that the influence of a single bout of exercise on fatty acid oxidation by muscle is different from that seen in training, and led to the hypothesis that the observed decrease may be a factor in exhaustion and fatigue. This study was undertaken to determine the influence of an exhaustive bout of exercise on fatty acid metabolism in liver and muscle preparations. To that end, the capacity of liver to synthesize fatty acids was assessed by measuring the activities of lipogenic enzymes. In addition, the capacity of liver and muscle to oxidize fatty acids was also determined. Through ’ To whom correspondence
should be addressed. 298
0006-2944/83 $3 .OO Copyright All rights
Q 1983 by Academic Press. Inc. of reproduction in any form reserved
FATTY
ACID
METABOLISM
DURING
EXCERCISE
299
these studies answers to two questions were sought. The first question is: Does the decrease in the capacity of muscle to oxidize fatty acids occur only at exhaustion, i.e., is this decrease a factor in fatigue? To answer this question, we determined the capacity of muscle to oxidize fatty acids after progressive increases in exercise time. The second question is: Are the effects of exhaustion permanent? If not, how long does it take the exhausted animals to recover from the effects of the exhaustive bout? To answer this question, we examined the capacity of liver and muscle to oxidize fatty acids during various periods of recovery. METHODS Male Holtzman rats (Holtzman Co., Madison, Wise.) weighing 250300 g were housed in individual cages in a room maintained at 20-23°C and kept on a 12 hr dark-light cycle (6 AM--~ PM light). The animals were given water and commercial lab chow (Wayne Blox, Allied Mills, Inc., Chicago, Ill.) ad libitum until the start of the experimental protocol. The results reported here are from two related experiments. In Experiment I, rats were divided into the following groups: (i) exercised for 1 hr; (ii) exercised for 2 hr; and (iii) exercised until exhaustion. There were 8 animals in each group along with a control group (10 animals). In Experiment II, rats were divided into the following groups: (i) exercised to exhaustion; (ii) exhausted-1Zhr rested; (iii) exhausted-24-hr rested; and (iv) exhausted-72-hr rested. Exercised animals were run on a rodent treadmill between 6 and ‘11 AM and were made to run at 28 m/min (0% grade) until they could no longer continue to run (220 & 19 min, x t SEM). Each of the exercised groups (with eight animals to a group) had an unexercised control group that was of comparable weight. In order to minimize the effects of variation in food consumption on fatty acid metabolism, the food intake of the controls was restricted to match that of the exercised group, i.e., pair fed. Food was removed from the cages of the control rats during the time that the exercised rats were on the treadmill. When the last rat was exhausted, the exercised group was given lab chow ad libitum, but the rats in the control group were given half of their daily allowance. The other half of their daily allowance was given between 11 and 12 PM so that the rats would not be totally without food for an extended period of time. Following the desired exercise period, both the experimental animals and their respective controls were killed by decapitation. The liver and muscle were then excised and placed in 0.15 M KC1 over ice. To assay for lipogenic enzymes, livers were homogenized in a glassTeflon Potter-Elvehjem homogenizer (1: 10 w/v) in 0.05 M potassium phosphate buffer, pH 7.4, containing 1 mM EDTA and 1 mM dithiothreitol. The particle-free supernatant fraction was prepared by centrifuging the
300
BARAKAT.KASPEREK.AND
DOHM
homogenates at 105,OOOg for 60 min followed by filtration through cheesecloth. Lipogenic enzymes in the particle-free supernatant solutions were assayed spectrophotometrically (I 1). Fatty acid synthetase was assayed according to the procedure of Arslanian and Wakil (12), ATP citrate lyase was assayed as described by Takeda rt ~1. (13). malic enzyme was assayed according to Hsu and Lardy (14), and glucose-6-phosphate dehydrogenase was assayed according to the method of Langdon (15). For the determination of the rate of fatty acid oxidation, liver was homogenized in 0.25 M sucrose (I : 10 w/v) in a glass-Teflon PotterElvehjem homogenizer then filtered through cheesecloth. Muscle was pressed through a tissue press, then homogenized in Chapell-Perry (16) buffer, pH 7.4, (1: 10 w/v) in a glass-glass homogenizer then filtered through cheesecloth. The capacity of liver and muscle homogenates to oxidize [I-‘4C]palmitate was determined in duplicate by collecting and counting 14C02 produced during the incubation period. A 2-ml incubation volume was used containing the following cofactors (pH 7.3): 2.0 mM ATP, 0.05 mM coenzyme A, 1.0 mM dithiothreitol, 0.1 mM malate, 1.0 mM MgC&. 0.072 mM bovine serum albumin (fatty acid free), 0.1 mM NAD, 100 mM sucrose, 100 mM K,HPO,, 80 mM KCI, 0.1 mM EDTA, 1.0 mM m-carnitine and 0.2 mM [l-‘4C]palmitate (I $Zi). The reaction was initiated by the addition of sample, and the contents were gassed with 95% 02, 5% CO? and stoppered with a rubber septum stopper containing a polypropylene center cup. The flasks were incubated at 37°C for 30 min with gentle shaking. Immediately prior to the termination of the incubation, 0.2 ml of ethanolamine:methyl Cellosolve (I :2) was injected into the hanging centerwell. The reaction was stopped by injecting 0.2 ml of 4.0 N H,SO, into the contents of the. flask. Flasks were shaken for 60 min to collect the evolved 14C0,. An aliquot of the ethanolamine:methyl Cellosolve was then taken and counted. All assays were performed under conditions of substrate saturation, with the amount of enzyme as the rate-limiting component of the reaction. Protein concentrations of all preparations were determined by the biuret method (17). The results of these experiments were analyzed by a one-way analysis of variance (18). Comparisons of means were tested for significant differences by a Newman-Keuls procedure (18). The level of statistical significance chosen for these experiments was P < 0.05. RESULTS AND DISCUSSION
The effect of progressively increasing exercise time on the activities of the lipogenic enzymes is shown in Fig. I. All four enzymes showed the same effect in that their activity decreased progressively. The lowest activity obtained was at the exhaustion point. when the animals could
FATTY ACID METABOLISM
DURING
301
EXCERCISE
A. MALIC
ENZYME
B. CITRATE-ATP
LYASE
C
GLUb-P-IIEHYGRGGENASE
0.
FATTY ACID
50 25 0
I
SYNTHETASE
I lOO75-I so--
I
l I I 1
2
EXH.
12
TIME
OIRS).
250
1 EXERCISE
PERIGD
I
,
I
1
I
24
36
48
80
72
RECOVERY
PERIOD
FIG. 1. Activities of hpogenic enzymes in liver of the experimental animals. The activity of each enzyme was measured in the high-speed supematant fraction of liver after 1 hr of exercise, 2 hr of exercise, or at exhaustion (Exercise Period) and at 12 hr of rest, 24 hr of rest, or 72 hr of rest after exhaustion (Recovery Period). Each point represents the average of eight observations expressed as percentage of control (tOO%). A shows the changes in the activity of mahc enzyme where the control value was 50.3 + 4.2 nmo~e/~n/~ protein; B, ATP citrate lyase where the control value was 24.6 t 1.3 nmole/min/mg protein; C, glucose-(i-phosphate dehydrogenase where control vaiue was 72.3 d 6.1 nmole/min/mg protein; and D, fatty acid synthetase where control value was 7.52 -c 0.43 nmole/min/mg protein. *Denotes statistically significant differences (P < .OS).
302
HAKAKAI’,
KASPEREK.
AND
DONM
not run any longer. Although no significant changes were seen upon exhaustion in the activity of fatty acid synthetase, glucose-&phosphate dehydrogenase or ATP citrate lyase, the activity of malic enzyme was significantly depressed. The lack of significant differences that was obtained in this experiment is different from what we reported earlier f 19) where the activities of all four enzymes were significantly depressed at exhaustion. Although the magnitude of depression in the activities of these enzymes is similar to that reported earlier, the results did not show significant differences here because of a wider variability than the previous experiment, No apparent cause of the variability is readily apparent at the present. However, it should be noted that the same trends were seen here as in th previous experiment. The progressive decrease in the activities of the enzymes of fatty acid synthesis may be related to the loss of hepatic proteins that we reported earlier (19). In that study, we reported that Iiver protein loss is increased with exhaustive exercise, including soluble proteins. The variation in the degree of loss of individual enzyme activity may be retated to turnover rates that are characteristic of each enzyme. This point is supported by examining the activities of the enzymes during the recovery periods. Whereas malic enzyme activity was depressed the most at exhaustion, it returned to normal levels by 12 hr after recovery. Fatty acid synthetase activity, which was depressed at a slower rate, took longer than 12 hr to get back to almost normal activity. Thus, it appears that exercise accelerates the rate of decay of the Iipogenic enzymes, and recovery from exercise depends on synthesis of new enzyme protein. This pattern of changes fits into “phase II” of the biphasic hormonal nature of stress that was recently described by Bessman and Renner (20). In this phase, the hormones that play a major role in metabolism are growth hormone, ACTH. and cortisol. In the early stages of this phase, the above hormones induce the synthesis of protease as well as the synthesis of the enzymes of gluconeogenesis. The results are increased degradation of proteins with the concomitant release of amino acids. Some of these amino acids are used as substrates for gluconeogenesis, which leads to the elevation in bl~-~ucose levels. The elevated bled-~ucose Ieve will subsequently cause the release of insulin which in turn will stimulate protein synthesis again. The phase II hormones take longer than “phase I” (the catecholamine phase) hormones to exert their effects on the organism. It thus seems likely that the changes in the activities of the enzymes that we observed may be under the influence of the phase II hormones. Fatty acid oxidation by liver preparations (Fig. 2) remains unchanged within the first hour of exercise, but rises steadily after that, until it reaches a maximum at exhaustion (approximately 180% of controls). After 12 hr of rest, however. fatty acid oxidation returns to normal levels
FATTY
ACID METABOLISM
DURING
303
EXCERCISE
I
I 1
2
12
24
36
46
60
12
EXH. EXERCISE
PERIOD
REcDvERY TIME
PERIOD
0iRS).
FIG. 2. [l-‘4C]Palmitate oxidation by liver homogenates of the experimental animals. Palmitate oxidation was measured in the same experimental animals described in Fig. 1. Each point represents the average of eight observations expressed as percentage of control value (100%) which was 0.084 k 0.011 nmole/min/mg protein. *Denotes statistically significant differences (P < .OS).
and remains close to normal during the subsequent periods of recovery. The rise in the oxidative capacity of liver with exercise may be related to changes in the levels of certain hormones, particularly insulin and glucagon. It has been reported that one of the controlling factors that regulates the process of fatty acid oxidation in liver is the insulin:glucagon ratio (21). Since variations in the levels of these hormones have been reported to occur with exercise (22), it is tempting to speculate that the elevation in the capacity of liver to oxidize fatty acids may be related to alterations in the ratio of these hormones. Upon reestablishment of normal levels of the hormones, such as during recovery from exercise, fatty acid oxidation returns to normal. Oxidation of fatty acids by muscle homogenates was depressed by approximately 80% at 1 hr of exercise, by 70% at 2 hr of exercise, and by 60% at exhaustion (Fig. 3). Similar decreases in fatty acid oxidation resulting from exhaustive exercise have been reported earlier (9), but the cause of such changes is not known. Since the mechanisms underlying the cause of such changes is not within the scope of this paper, no attempts are made to explain our observations. It is worthy to note, however, that such dramatic decreases in the capacity of muscle to oxidize fatty acids are observed within a short period of time (1 hr) and that an apparent recovery during prolonged periods of exercise occurs. Nonetheless, oxidation remains depressed at exhaustion. The recovery from exhaustion is fairly fast, so that within 12 hr, oxidation by the
304
BAKAKAT.
1 EXERCISE
2
KASPEKEK.
EXH.
12
PERIOD
AND DOHM
24
36
RECOVERY TIME
48
60
70
PERIOD
(HRS).
FIG. 3. [l-‘4C]Palmitate oxidation by muscle homogenates of the experimental animals. Palmitate oxidation was measured in the same experimental animals described in Fig. I. Each point represents the average of eight observations expressed as percentage of control value (100%) which was 0.096 * 0.014 nmole/min/mg protein. *Denotes statistically significant differences (P < .05).
exhausted-12-hr rested animals is approximately 80% that of the controls. It appears that by 72 hr recovery from exhaustive exercise, in relation to muscle’s capacity to oxidize fatty acids, is complete. The results of this study answer the major questions that were posed. It appears that exhaustive exercise influences both the synthesis and oxidation of fatty acids rather dramatically. The capacity of liver to synthesize fatty acids decreased with increasing the time of exercise. At the same time oxidation of fatty acids by liver was maximal at the time of exhaustion. In contrast, oxidation of fatty acids by muscle preparations was the lowest at 1 hr of exercise and started to recover after 2 hr and until the animals were exhausted. Thus, the decrease in the oxidation of fatty acids at exhaustion is not an abrupt process and cannot be a factor in fatigue. These studies also show that the effect of exhaustive exercise on the biochemical processes that were examined is not an irreversible phenomenon, as evidenced by the short period of recovery time which was required for these processes to return to control levels. SUMMARY
This study was undertaken to determine the effects of increasing exercise time until exhaustion on the capacity of liver to synthesize and oxidize fatty acids, as well as the capacity of muscle to oxidize fatty acids. Additionally, the same parameters were examined during various periods of recovery after exhaustion. These studies were prompted by the scarcity of information relating the effects of exercise on the metabolism of fatty acids in liver, and by our findings and those of others where exhaustive
FATTY
ACID METABOLISM
DURING
EXCERCISE
305
exercise was reported to cause a decrease in the capacity of muscle to oxidize fatty acids. The latter led to the hypothesis that the reported decrease in fatty acid oxidation may be a factor in fatigue. Thus the purpose of the present studies was threefold. The first was to determine changes in fatty acid metabolism in liver during exercise and recovery: the second was to test the above hypothesis by following time-course changes of fatty acid oxidation by muscle; and the third was to determine whether or not the effects of exhaustion are reversible. The results obtained showed that fatty acid synthesis in liver decreased with increasing time but returned to normal levels by approximately 12 hr of rest after exhaustion. Oxidation of fatty acids by liver remained unchanged during the first hour of exercise but steadily increased afterwards until it reached a maximum at exhaustion, Oxidation returned to control level after 12 hr of rest and remained so during the subsequent recovery period, Fatty acid oxidation by muscle was maximally depressed after 1 hr of exercise. yet the animals continued to run, thus ruling out the possibility that the decreased oxidation of fatty acids with exhaustion is a factor in fatigue, Finally, recovery studies showed that the biochemical processes that were examined returned to control levels within 12-24 hr after exercise demonstrating that these effects are not permanent. REFERENCES I. 2. 3. 4. 5. 6.
Havel, R. J., Naimark. A.. and Borchgrevink, C. F., J. C/in. Invest. 23, 1054 (1963). Carlson, L. A., Froberg, S., and Persson. S., Acta Physio/. Scund. 63, 434 (1965). Paul, P., and issekutz, B., J. Appl. Physiol. 22, 612 (11967). Essen, B., Ann. N.Y. Acad. Sri. 301, 30 (1977). Gollnick, P. I)., Ann. N. Y. Acad. Sri. 301, 64 f 1977). Costill, D. L., Fink, W. J., Getchell, L. H.. Ivy, J. L., and Witzman, F. A,, J. Appl. Physiol.
47, 787 (1979).
7. Askew, E. W.. Barakat. H. A., Kuhl. G. L., and Dohm, G. L., Lipids 10,491 (1975). 8. Tate, C. A., Scott, K. S., Wolkowicz, P. E.. and McMillin-Wood, J., J. Med. Sci. Sports Exer. 13, 118 (1981). 9. Dohm, G. L., Huston. R. L., Askew, E. W.. and Weiser, P. C.. Amer. J. Physial. 223, 783 (1972). IO. Barakat, H. A., Kasperek, G. J., Dohm, G. L., Tapscott, E. B., and Snider, R. D.. Fed. Pruc. 41, 1752 (1982). 1 I. Barakat. H. A., Brown, W. E., and Henry, S. D., Life Sci. 23, 1835 (1978). 12. Arslanian, M. J., and Wakil, S. J., ~ef~~d~ E~zy~o~. 35, 59 (19753. 13. Takeda, Y., Suzuki, F., and Inoue, H., Methods Enzymof. 13, 153 (19691. 14. Hsu, R. Y., and Lardy, H. A., Methods Enzymof. 13, 230 (1969). 15. Langdon, R. G., Methods Enzymol. 9, 126 (1966). 16. Chapell, J. B., and Perry, S. V., Nature (London) 173, 1094 (1954). 17. Gomall, A. G.. Bardawill, C.. and David, M. M., J. Biol. Chem. 177, 751 (1949). New 18. Weiner, B. J., “Statistical Principles in Experimental Design.” McGraw-Hill, York, 1971. 19. Kasperek. G. J., Dohm, G. L.. Barakat, H. A., Strausbauch, P. H. Barnes, D. W., and Snider, R. D., Biorhem. J. 202, 281 (1982).
306
BARAKAT.
KASPEREK.
AND DOHM
20. Bessman, S. P.. and Renner, V. J.. in “Pathology of Shock. Anoxia and Ischemia” (R. A. Cowley and B. F. Trump. Eds.). pp. 60-65. Williams & Wilkens. Baltimore.
1982. 21. McGarry, J. D., and Foster, D. W.. Ann. Rev. Biodwrn. 49. 395( 1980). 22. Fell. R. D., McLane, J. A., Winder, W. W.. and Holloszy. J. 0.. Ann. J. fl~~,~;,~/.
238.328(1980).