Enzymes Involved in Ketone Utilization in Different Types of Muscle: Adaptation to Exercise

Transcription

1 Eur. J. Biochem. 47, (1974) Enzymes Involved in Ketone Utilization in Different Types of Muscle: Adaptation to Exercise William W. WINDER, Kenneth M. BALDWIN, and John 0. HOLLOSZY Department of Preventive Medicine, Washington University School of Medicine, St. Louis, Missouri (Received March 30/June 15, 1974) Activity levels of the enzymes involved in ketone utilization were compared in heart, fast-twitch red, slow-twitch red, and fast-twitch white types of muscles in rats. 3-Hydroxybutyrate dehydrogenase, 3-ketoacid CoA-transferase, and acetoacetyl-coa thiolase activities were lowest in white muscle, higher in slow red than in fast red muscle, and highest in the heart. The large differences between the four muscle types in the levels of these enzymes reflects differences in both mitochondrial content and composition. Differences in composition were evidenced by large dissimilarities between the different muscle types in the ratios of the activities of the enzymes of ketone oxidation to each other and to citrate synthase and cytochrome c. Of particular interest is the finding that, despite the fact that in the rat slow red muscle has a lower content of mitochondria than fast red muscle, 3-hydroxybutyrate dehydrogenase activity is 140% higher and 3-ketoacid CoA-transferase is 70% higher in slow red than in fast red muscle. A 14-week-long program of treadmill running induced increases in the levels of ketone utilization enzymes in all three types of skeletal muscle but not in heart; 3-hydroxybutyrate dehydrogenase became measurable, though at very low levels, in white muscle; increased 2.6-fold in slow red muscle, and 6-fold in fast red muscle. 3-Ketoacid CoA-transferase increased 2-fold in both fast red and white types of muscle, but only 26 % in slow red muscle. Acetoacetyl-CoA thiolase activity increased % in all three types of skeletal muscle. In contrast, citrate synthase and cytochrome c increased approximately 2-fold in all three types of skeletal muscle. These adaptive changes tend to make skeletal muscles more like heart muscle in their enzyme patterns and may help to explain why physically trained, as compared to untrained, individuals do not develop post-exercise ketosis. Skeletal muscle adapts to a chronic increase in contractile activity with increases in the capacities to oxidize pyruvate and fatty acids [l- 31. Underlying this response are increases in the levels of activity of the mitochondrial enzymes involved in the activation, transport and oxidation of fatty acids [2], the enzymes of the citrate cycle [4] and the enzymes of the mitochondrial respiratory chain [l]. In addition, there is a change in mitochondrial composition [5,6]. Recently it was found that homogenates of gastrocnemius muscles from rats that had adapted to a program of running oxidized 3-hydroxybutyrate 2 to 3 times as rapidly as those from sedentary animals [7]. This observation, together with the finding of Pette Enzymes. Citrate synthase (EC ); acetyl-coa acetyltransferase or acetoacetyl-coa thiolase (EC ) ; D-3-hydroxybutyrate dehydrogenase (EC ); 3-ketoacid CoA-transferase (EC ). and coworkers 181 that 3-ketoacid CoA-transferase activity increased in rabbit extensor digitorum longus muscles in response to 28 days of electrical stimulation, provides evidence that the capacity to utilize ketones also increases in response to a chronic increase in contractile activity. Mammalian skeletal muscles are mixtures of three fiber types which differ in morphology and biochemical properties [3, In rodents there are the fasttwitch white muscle fibers which have a low respiratory capacity, a high glycogenolytic capacity, and high myosin ATPase activity; the fast-twitch red fibers which have a high respiratory capacity, a high glycolytic capacity, and high myosin ATPase activity; and the slow-twitch red fibers which have a moderately high respiratory capacity, a low glycogenolytic capacity, and low myosin ATPase activity [3, The muscle fiber types also differ in their

2 462 Enzymes Involved in Ketone Utilization in Muscle capacities to metabolize ketones. Histochemical studies have shown that the staining intensity of 3-hydroxybutyrate dehydrogenase is low in white relative to slow-twitch red muscle [14]. Subsequently, Kark et al. [15] reported that slow-twitch red muscle slices oxidized 3-hydroxybutyrate nineteen times more rapidly than slices of white muscle. No information was obtained on fast-twitch red muscle. In this context, we have compared the levels of the enzymes involved in ketone utilization in the different types of muscle in sedentary and chronically exercised rats. Our results show that there are major differences in mitochondrial composition, with respect to these enzymes, in the different types of muscle. Exercise resulted in increases in the levels of the enzymes of ketone oxidation in skeletal muscle ; the proportions of these enzymes to each other and to the respiratory chain and citrate cycle enzymes were altered in such a way as to diminish the differences in mitochondrial composition between the various types of muscle. MATERIALS AND METHODS Treatment of Animals Male, specific pathogen- free, Wistar rats weighing 100 g were obtained from Carworth farms and divided into three groups. An exercising group was trained 5 days per week by means of a 12-week-long program of treadmill running described previously [l], at the end of which the rats were running continuously for 120 rnin daily at 31 m per min up an 8" incline, with 12 intervals of running at 42 m per min, each lasting 60 s, spaced 10 min apart through the exercise session. The animals were maintained at this work level until they were killed. The exercising animals were provided with food ad libitum. A paired weight sedentary group had their food intake restricted so as to maintain their body weights the same as those of the runners. A freely eating sedentary group was fed ad libitum. Unless otherwise stated, animals were maintained on a diet of Purina chow and water. Homogenate Preparation and Assay Methods Rats were killed by decapitation. Animals were not exercised for 48 h prior to the time they were killed. For studies of the fast-red and the white types of muscle, the quadriceps muscles were dissected out, freed of fat and connective tissue, and separated into a superficial white portion, which consists entirely of white fibers [3], and a deep red portion which consists predominantly of fast-red fibers [3]. These fast-red and white muscle samples each weighed roughly 500 mg. The middle, mixed portion of the quadriceps weighing approximately 1500 mg was discarded. The soleus muscle, which consists predominantly of slowtwitch red fibers [3] was used for studies on slow-red muscle. Tissues were minced and then homogenized in a glass Potter-Elvehjem homogenizer immersed in ice water. Homogenates to be used for measurement of 3-hydroxybutyrate dehydrogenase activity were prepared in 10 mm succinate containing 2 mm NADH, 2 mm ATP, and 1 mm EDTA, ph 7.4. Homogenates for assays of the other enzymes were prepared in 175 mm KCl containing 10 mm glutathione and 2 mm EDTA, ph 7.4. All the homogenates were frozen and thawed three times to disrupt the mitochondria prior to the enzyme assays. Spectrophotometric assays were performed in a Gilford spectrophotometer model 240 in 1 -ml cuvettes of 1-cm light path at 30 "C. Initial reaction rates were determined from a segment of the linear portion of the change in absorbance and corrected for the rates of any nonspecific activity. Enzymatic activities are reported as pmol substrate utilized per min at 30 "C. 3-Ketoacid CoA-transferase activity was measured by following the succinate-dependent decrease in acetoacetyl-coa concentration at 323 nm as described by Benson and Boyer [16], with the modification that initial acetoacetyl-coa concentration was 0.1 mm. Assays were run on whole homogenates which were diluted in 50 mm Tris-HC1, ph 8.5, immediately prior to the assay. Reaction rates were corrected for acetoacetyl-coa disappearance rates in the absence of succinate. Acetoacetyl-CoA thiolase activity was determined by following the CoA-dependent loss of acetoacetyl CoA at 313 nm [17,18]. The reaction mixture contained 50 mm Tris-HC1, ph 8.1; 5 mm MgCl,; mm acetoacetyl-coa ; and 0.2 mm CoA. Acetoacetyl-CoA disappearance was also measured in the absence of CoA (spontaneous hydrolysis plus "acetoacetyl-coa deacylase" activity). Assays were run on whole homogenates which were diluted in 50 mm Tris-HC1, ph 8.1, immediately prior to the assay. Acetoacetyl-CoA concentrations were calculated using amolar absorptioncoefficient at 313 nmof 11.9 mm-' x cm-' [18]. 3-Hydroxybutyrate dehydrogenase activity was determined on tissue homogenates by measuring the rate of 3-hydroxybutyrate-dependent acetoacetate accumulation (cf. [17]). The reaction mixture contained in a final volume of 4 ml, 40 mm Tris-HC1, ph 8.5; 2.4 mm NAD; 2mM succinate; 0.2 mm EDTA; 0.4 mm ATP; 20 mm ~,~-3-hydroxybutyrate; and tissue homogenate. Aliquots of the reaction mixture were removed after 30 and 60 min and deproteinized

3 W. W. Winder, K. M. Baldwin, and J. 0. Holloszy 463 with 6% HC104. After removal of protein by centrifugation, the supernatant was neutralized and analyzed for acetoacetate by the method of Walker [19]. Citrate synthase activity was measured by the method of Srere [20] using 5,5-dithiobis-(2-nitrobenzoate). Protein was measured with the biuret method [21]. Homogenates were prepared for protein assay as described by Cleland and Slater [22]. RESULTS Group Comparisons No significant differences were found between the paired weight and the freely eating sedentary animals in the levels of activity of any of the enzymes measured. The results obtained on these two groups have, therefore, been combined under the heading sedentary controls in the following sections. The concentrations of protein in the different types of muscle were not significantly different, averaging approximately 200 mg/g fresh muscle; total muscle protein concentration was not significantly changed by the exercise. The water content of muscle (78%) is also the same in the exercised and sedentary animals [l]. Thus, the relationships between the different types of muscles, and between the exercised and sedentary animals, with respect to the levels of enzyme activity, are the same whether the results are expressed per mg muscle protein, per mg dry weight or per g fresh weight. In the following sections we have expressed enzymatic activity per g fresh tissue, as this seems the most meaningful reference when relating enzyme content to functional capacity. Ketone Oxidative Enzymes in the DiLferent Types of Muscle The levels of activity of 3-hydroxybutyrate dehydrogenase differ greatly in the four types of muscles, ranging from not detectable by our assay method in white skeletal muscle of sedentary animals to approximately 4 U per g in heart muscle (Table 1, column 2). Slow-twitch red skeletal muscle has approximately 2.5 times as much 3-hydroxybutyrate dehydrogenase activity as fast-twitch red muscle in sedentary animals. 3-Ketoacid CoA-transferase and acetoacetyl-coa thiolase activities are also lowest in white muscle, higher in slow-red than in fast-red muscle, and highest in the heart (Table 1, columns 4 and 6). However, the differences between the four types of muscle are somewhat less extreme for 3-ketoacid CoA-transferase and much less extreme for acetoacetyl-coa thiolase than for 3-hydroxybutyrate dehydrogenase. Differences in the Composition of Mitochondria in the Various Tjps of Muscle The large differences in the levels of activity of 3-hydroxybutyrate dehydrogenase and 3-ketoacid CoA-transferase in the four types of muscle reflect not only differences in mitochondrial content but also major differences in mitochondrial composition. For example, 3-ketoacid CoA-transferase activity was 40 times higher in heart than in white muscle in the sedentary animals (Table 1, column 4), while 3-hydroxybutyrate dehydrogenase activity was at least 140 times higher in heart than in white muscle (Table 1, columns 1 and 2). In contrast, the level of citrate synthase, which was used as a marker for the citrate cycle, was 20-fold higher, and the level of cytochrome c, which was used as a marker for the respiratory chain, was only 16 times higher in heart than in white muscle in the sedentary rats (Table 1, columns 8 and 10). A similar disproportion is seen when the levels of the enzymes involved in ketone oxidation are compared to mitochondrial protein concentration. The protein content of the mitochondrial pellet obtained by differential centrifugation of muscle homogenates [6], prepared in 300 mm sucrose containing 2 mm EDTA, averaged mg per g white muscle, 3.07 f 0.06 mg per g slow red muscle, mg per g fast red muscle, and 16.9 k 1.4 mg per g heart muscle in 7 animals. Based on comparisons of levels of enzyme activity on whole homogenates and on the mitochondrial pellet, these protein values appear to represent a yield of between 15% and 30% of the total mitochondrial content of the muscles; the apparent yield of mitochondria varies considerably with the mitochondrial marker measured. The differences in composition between the mitochondria in the various muscle types with respect to the enzymes involved in ketone oxidation are clearly brought out using the approach of Pette and coworkers [ This involves relating the levels of these enzymes to other mitochondrial enzymes in terms of an enzyme activity ratio, as illustrated for citrate synthase in Table 2. Clearly, the ratio of citrate synthase to 3-hydroxybutyrate dehydrogenase is, using the terminology of Pette and coworkers [25-271, a variable or discriminative ratio which demonstrates large differences in the composition of the mitochondria of the four muscle types. The differences with respect to the ratio of citrate synthase to 3-ketoacid CoA-transferase, while much less marked, do clearly discriminate between the slow-twitch red and

4 Enzymes Involved in Ketone Utilization in Muscle -. M v) W 0 1 the fast-twitch (both red and white) types of muscle (Table 2, column 4), but not between the fast-red and fast white types. Citrate synthase and acetoacetyl- CoA thiolase activities roughly parallel each other except in slow-red muscle which shows a lower ratio of citrate synthase to acetoacetyl-coa thiolase than do the other muscle types (Table 2, column 6). Similar patterns are evident in the ratios of the ketoneutilizing enzymes to cytochrome c or to mitochondria1 protein concentration. Adaptive Responses of the Different Types of Muscle to Exercise I v1 2 d E!m Ih * 2 8 a 8-4 I W x z M X 4 I z C X - 0 % 1 m - r - +I +I +I +I 3-Hydroxybutyrate dehydrogenase activity became measurable, though at a very low level, in white muscle, and increased 2.6-fold in slow red muscle in response to the exercise. An approximately 6-fold increase in 3-hydroxybutyrate dehydrogenase activity occurred in fast red muscle, which attained levels comparable to those in slow red muscle (Table 1, column 2). Because 3-hydroxybutyrate dehydrogenase increased to a much greater extent than did the enzymes of the citrate cycle and respiratory chain in fast red muscle, the relative proportions of these enzymes became more like those found in slow red and heart muscles. This is demonstrated by the decrease in the activity ratio of citrate synthase to 3-hydroxybutyrate dehydrogenase in fast-red muscle (Table 2, column 3). 3-Ketoacid CoA-transferase activity increased 2-fold in both the white and fast red types of muscle, but only 26% in slow red muscle (Table 1, column 5). As a result of this relatively small increase in 3-ketoacid CoA-transferase in slow red muscle, an increase occurred in the activity ratio of citrate synthase to 3-ketoacid CoA-transferase to make it comparable to that found in heart muscle (Table 2, column 5). Acetoacetyl-CoA thiolase activity increased slightly, and to approximately the same extent (40 % to 45 %) in all three types of skeletal muscle (Table 1, column 7). No significant changes in the levels of any of the enzymes measured occurred in heart muscle. Brain, Kidney and Liver It has been reported that the ketosis associated with a high fat diet induces increases in the levels of activity of acetoacetyl-coa thiolase in the liver [18] and kidney [17,18], and of 3-ketoacid CoA-transferase in the kidney [18]. Since exercise has also been shown to produce ketosis [28-301, it seemed of interest to examine the effects of the running program on the levels of the enzymes involved in ketone metabolism in these organs. As shown in Table 3, no significant adaptive changes in the levels of these enzymes occurred in liver, kidney or brain. Eur. J. Biochern. 47 (1974)

5 W. W. Winder, K. M. Baldwin, and J. 0. Holloszy 465 Table 2. Relation of the levels of citrate synthase activity to the activities of the ketone oxidative enzymes in the different types of muscle Muscle type Ratio of activity of citrate synthase to ~- ~~ 3-hydroxybutyrate 3-ketoacid acetoacetyl-coa thiolase dehydrogenase CoA-transferase sedentary runners sedentary runners sedentary runners White Fast-red Slow-red Heart Table 3. Levels of activity of 3-hydroxybutyrate dehydrogenase, 3-ketoacid CoA-trunsferase and acetoucetyl-coa thiolase in bruin, kidney und liver of sedentary and exercised ruts Values are expressed per g fresh tissue and are means f S.E. The number of animals per group is given in parentheses Tissue 3-Hydroxybut yrate CoA-transferase Thiolase dehydrogenase sedentary runners sedentary runners sedentary runners pmol x min-' x g tissue-' Kidney f & & & 2.5 (8) (8) (7) (8) (6) (5) Liver 17 *I 1 4 k & f f f 2.4 (9) (9) (4) (4) (4) (4) Brain 0.47 j~ f & & (7) (7) (4) (4) DISCUSSION The influence of exercise-training on skeletal muscle mitochondria has been shown to include increases in the enzymes of the mitochondrial respiratory chain [I], citrate cycle [4], and fatty acid oxidation pathway [2]. The present results show that the enzymes of the ketone oxidation pathway also participate in this response to exercise. Of particular interest is the finding that the levels of these enzymes do not increase in parallel, and that the levels of activity of the enzymes of ketone oxidation can be regulated independently of each other and of the enzymes of the citrate cycle and respiratory chain. The changes in the levels of the enzymes involved in ketone oxidation, together with the other previously reported adaptive changes in mitochondrial composition induced in skeletal muscle by exercise [l -61, tend to make skeletal muscle mitochondria more like cardiac mitochondria in their enzyme pattern. Heart muscle contracts continually and has the highest capacity for aerobic metabolism of any mammalian muscle; it seems reasonable that the enzyme pattern found in heart mitochondria is the optimal one for the oxidation of all the commonly available substrates. Since the level of contractile activity strongly influences a muscle's content of mitochondria [l - 8, , it seems possible that the differences in mitochondrial enzyme levels between fast-twitch red and fast-twitch white types of muscle could be due largely to differences in the amount of contractile activity. Red muscle fibers, which have small motor neurons that require less excitatory input for discharge than the larger motor neurons innervating white fibers, are preferentially recruited during work of light and moderate intensity [ As a result, white fibers appear to contract infrequently during exercise that can be maintained for a long time, such as the running program used in these studies [34]. This may explain the very small absolute increases in the levels of the mitochondrial enzymes measured in this and previous studies in white as compared to red muscles.

6 466 Enzymes Involved in Ketone Utilization in Muscle On the other hand, the differences in mitochondria1 content and composition between fast-twich red and slow-twitch red fibers cannot be explained by differences in the amount of contractile activity alone. Despite the fact that, in rodents, fast-twitch red muscle has a higher content of mitochondria, and higher levels of respiratory chain, citrate cycle, and fattyacid oxidative enzymes than slow-twitch red muscle [3,11], as shown in the present study, slow red muscle has much higher levels of 3-hydroxybutyrate dehydrogenase and 3-ketoacid-CoA transferase than fast red muscle. Clearly, if the greater capacity of fasttwitch red muscle to oxidize pyruvate and fatty acids is due to more frequent contractile activity, then the greater capacity of slow twitch muscle to oxidize ketones must be mediated by some other means (or vice versa). The other factor responsible for this difference could relate to the differences in the type of motor neuron innervating fast-twitch and slow-twitch red muscle fibers, with differences in firing pattern, imposed contractile response, specific mechanical stress and resulting metabolic response [35,37]. In physically untrained individuals blood ketone levels increase moderately during prolonged exercise and then rise sharply after exercise when blood freefatty-acid levels increase [ In contrast, little or no elevation in blood ketones occurs in athletes during or after exercise [ Tolerance to an administered dose of ketones is also greater in athletes than in sedentary individuals during and after exercise [30]. Our finding that the levels of activity of the enzymes involved in ketone oxidation undergo an adaptive increase in muscle in response to exercise training may help to explain why physically trained individuals do not develop post-exercise ketosis. The present results show that the level of activity of 3-hydroxybutyrate dehydrogenase in skeletal muscle is very low compared to 3-ketoacid CoA-transferase and acetoacetyl-coa thiolase activities, particularly in fibers of the fast-twitch red and white types. This is true even when one takes into account that 3-ketoacid CoA-transferase was measured in the direction of acetoacetate formation which gives values about 5 times higher than measurement in the physiological direction [17]. The possibility must, therefore, be considered that 3-hydroxybutyrate dehydrogenase may be rate-limiting for 3-hydroxybutyrate oxidation in skeletal muscle. Supporting this view is the observation by Ruderman and coworkers [38,39] that the rate of 3-hydroxybutyrate oxidation at a given concentration was only one-fifth that for acetoacetate in the isolated perfused rat hindquarters. They further found that the ratio of 3-hydroxybutyrate to acetoacetate was higher in muscle than in blood. Additional evidence that 3-hydroxybutyrate dehydrogenase may be rate-limiting is the finding that during starvation 3-hydroxybutyrate concentration increases approximately 4 times as much as acetoacetate concentration in blood 401. In this context, it seems reasonable that the large exercise-induced increases in 3-hydroxybutyrate dehydrogenase activity in skeletal muscles, particularly the approximately 6-fold increase in the fast-twitch red type, may have resulted in an increase in the capacity to oxidize 3-hydroxybutyrate in our exercise-trained rats. We wish to thank Mrs May Chen for technical assistance and Miss Sara Watson for assistance in the preparation of this manuscript. This investigation was supported by United States Public Health Service Research Grant HD01613 and Training Grant AM05341, and by a Grant from the Missouri Heart Association. W. W. Winder was supported by U.S. Public Health Service Postdoctoral Research Fellowship AM K. M. Baldwin was a Postdoctoral Trainee in Nutrition supported by Training Grant AM J. 0. Holloszy was the recipient of U.S. Public Health Service Research Career Development Award K4-HD REFERENCES 1. Holloszy, J. 0. (1967) J. Biol. Chem. 242, Molt, P. A., Oscai, L. B. & Holloszy, J. 0. (1971) J. Clin. Invest. 50, Baldwin, K. M., Klinkerfuss, G. H., Terjung, R. L. Mole, P. A. & Holloszy, J. 0. (1972) Am. J. Physiol. 222, Holloszy, J. O., Oscai, L. B., Don, I. J. & Mole, P. A. (1970) Biochem. Biophys. Res. Commun. 40, Holloszy, J. 0. & Oscai, L. B. (1969) Arch. Biochem. Biophys. 130, Oscai, L. B. & Holloszy, J. 0. (1971) J. Biol. Chem. 246, Winder, W. W., Baldwin, K. M. & Holloszy, J. 0. (1973) Proc. SOC. Exp. Biol. Med. 143, Pette, D., Smith, M. E., Staudte, H. W. & Vrbova, G. (1973) Pfliigers Arch. 338, Gauthier, G. F. (1970) in The Physiology and Biochemistry of Muscle as a Food (Briskey, E. J., Cassens, R. G. & Marsh, B. B., eds) 2nd edn, pp , University of Wisconsin Press, Madison. 10. 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