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Exercise-Induced Increase in the Capacity of Rat Skeletal Muscle to Oxidize Ketones1 W. W. WINDER,^ K. M. BALD WIN,^'^ A N D J . 0. HOLLOSZY~ Department ofPre~lentiveMedicine, Washington University School of Medicine, S t . Louis, Missouri63110 Received August 20,1974
WINDER, W. W., BALDWIN, K. M., and H ~ L L O S ZJ.Y0. , 1975. Exercise-induced increase in the capacity of rat skeletal muscle to oxidize ketones. Can. J. Physiol. Pharmacol. 53,86-91. During and after strenuous prolonged exercise, sedentary individuals develop high blood levels of acetoacetate and P-hydroxybutyrate whereas exercise-trained animals and human subjects do not. We have investigated the possibility that exercise training can increase the capacity of skeletal muscle to oxidize ketones. In this study we measured rates of ~-/3-[3-'~C]hydroxybutyrate and [3-14C]acetoacetate oxidation, and the levels of activity of the enzymes involved in the oxidation of ketones in homogenates of gastrocnemius muscles of exercisetrained and of untrained male rats. The trained animals had markedly lower blood ketone levels immediately and 60 min after a 90 min long bout of exercise than did the sedentary animals. The and [3-14C]acetoacetate oxidation were twice as high in rates of ~-/3-[3-~~C]hydroxybutyrate homogenates of muscles from the trained as compared to the sedentary rats. The increases in levels of activity in gastrocnemius muscle in response to the exercise program were: P-hydroxybutyrate dehydrogenase threefold; 3-ketoacid CoA-transferase twofold; and acetoacetyl-CoA thiolase 55%. This exercise-induced increase in the capacity of skeletal muscle to oxidize ketones could play a role in preventing development of ketosis in the physically trained animal during and following prolonged strenuous exercise.
K. M. et HOLLOS~Y, J. 0. 1975. Exercise-induced increase in the WINDER, W. W., BALDWIN, capacity of rat skeletal muscle to oxidize ketones. Can. J . Physiol. Pharmacol. 53,86-91. Chez les individus sedentaires, l'exercice intense et prolonge provoque une augmentation importante des taux sanguins d'adtoacetate et de 0-hydroxybutyrate, alors que, chez les animaux et les hommes entraines a l'effort, il n'y a pas de semblable augmentation. Nous explorons ici la possibilite que I'entrainement augmente la capacite des muscles squelettiques d'oxyder les corps cetoniques. Dans cette etude, nous mesurons l'oxydation du D-/3[3-'4C]hydroxybutyrate et de le 13-I4C]acetoacetate, ainsi que l'activite des enzymes impliquts dans I'oxydation des cetones par les homogenats de muscles jumeaux preleves chez des rats entraines a l'exercice et chez des rats sedentaires. Immediatement apres un exercice de 90 min, et 6Q min apres, les animaux entraines ont des taux citoniques beaucoup moins eleves que les animaux sedentaires. L'oxydation du phydroxybutyrate 3-14C et de le 13-14C]acetoacetateest deux fois plus elevee dans les homogenats preleves chez les rats entraines qu'elle ne I'est chez les sedentaires. L'activite de la 0-hydroxybutyrate deshydrogtnase est augmentee trois fois, celle de la 3 cetoacyl CoA-transferase deux fois, et celle de l'ac~toacetyl-CoAthiolase de 55% dans les muscles jumeaux, en reponse au programme d'exercice. Cette augmentation de l'oxydation des cetones par le muscle squelettique provoguee par I'exercice joue peut-2tre un r6le dans la prevention de la cktose, pendant et apres l'exercice intense et prolonge, chez les animaux physiquement entrainks. [Traduit par le journal]
Inhodncltiorn A marked difference has been observed betwecn endurance athletes, such as distance runners, and sedentary subjects with respect to the metabolism sf ketones (Johnson and 'This research was supported by Research Grant HHb 01 413 and Training Grant AM 05348 from the National Institutes of Health, and by a grant from the Missouri Heart Association. W. W. Winder was a Postdoctoral Research Fellow s~~pportedl by Research Fellowship AM 51241 from the National Institutes of FIealth.
Walton 1974, 197%; Johnson et wl. 1969a, 8 969 b ) . In play sically untrained individuals, blood ketones increase moderately during grolonged cxercisc and then increase sharply after cessation of cxercise (Johnson and Walton 19'7%; Johnson et QI. 1 9 6 9 ~1969b). ~ In individuals who have adaptcd to endurance "K. M. Baldwin was a Postdoctoral Research Trainee supported by Training Grant AM 05341. 'Present address, Department of Physiology, University sf California at Hrvirae. Irvine, California 92664. 3. 0. Hslloszy was the recipient of Research Career ilevelopmene Award K4-HD 19573.
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WINDER ET AL.: INCREASED KETONE OXIDATION IN MUSCLE
exercise, blood ketones are maintained at or near resting levels during and after a prolonged strenuous exercise bout (Johnson and Walton 1972; Johnson et 01. 1969a, 1969b). This difference may be due to differences in rates of hepatic ketogcnesis and/or in the rates of ketone oxidation. In a preliminary study it was found that homogenates of gastrocnemius muscles from chronically exercised rats oxidized D-P-hydroxybutyrate at a significantly increased rate (Winder et a/. 1973). This observation was confirmed in the present detailed study in which it was further found that the levels of activity of the enzymes involved in ketone oxidation were significantly increased in leg muscles of rats subjected to a program of running. The rate of acetoacetate oxidation by gastrocnemius nluscle from the trained animals was also significantly increased.
Methods Trc)atment of Animals Male Wistar rats (specific pathogen free CFN rats from Carworth Farms) weighing approximately 100 g were divided into three groups. A running group was trained by means of a 12-week program of treadmill running described previously (Pattengale and Holloszy 3967) at the end of which, rats were running continuously for 2 h daily at 1.2 miles/h (m.p.h.) 41 n3.p.h. 0.45 m/s) up an 8 deg. (1 deg. 0.017 rad) incline with 12, 30-s periods of running at 1.6 m.p.h. spaced 10 nlin apart throughout the exercise sessions. The rats were maintained at this level of training 5 days per week until the time of sacrifice; this period varied from 2 t o 8 weeks. The rtlnners were provided with food ad libitum. A freely eating sedentary group was also provided with food UCIlibitum. A paired weight sedentary group had their food intake restricted so as to maintain body weights the same as those of the runners. All animals were maintained on a diet of P~arinachow and water.
-
Post-Ext,rci.sc K'c2r~sis The sedentary rats that were used in the studies on the development of post-exercise ketosis were taught to run on the treadmill for 10-15 min daily at 0.6 m.p.k. for 2 weeks prior to the test. All rats were given 20 g Purina chow the night prior to the test. The next morning trained and untrained rats were exercised on the treadmill for 90 rnin at 0.6 1n.p.h. tip an 8 deg. incline. Immediately following the exercise, rats were anesthetized lightly with ether and a 0.8 rnl blood sarrmple was obtained by cardiac puncture and processed for plasma free fatty acid and blood ketone determinations. Rats were then placed in cages without access to food for 1 h, at which time another blood sample was obtained and processed. Blood was also collected from rats, not
87
involved in the exercise test that day, for the purpose of determining resting blood ketones and plasma free fatty acids. In addition, blood ketones and plasma free fatty acids were determined on trained animals subjected to a 90 min bout of exercise at 1.2 mp.h. up an 8 deg. incline. Plasma free fatty acids were determined by the method of Novak (1965). Blood ketones were determined by the method of Williamson et al. (1962). Homogcnatc Preparaticrn and Assay hlethods Rats were killed by decapitation 24-72 h following the runners' last training session. The gastrocnenlius muscles were removed and placed in small beakers on ice until the time of homogenization. Muscles were cleaned of connective tissue and minced thoroughly with scissors prior to homogenization. Homogenates were prepared using a Potter-Elvehjem homogenizer immersed in ice water. Homogenates for all assays with the exception of p-hydroxybutyrate dehydrogenase were prepared in 175 m M KC1 containing 10 mM GSH and 2 m M EDTA, pH 7.4. The rate of oxidation of ~-p-[3-~'C]hydroxybutyrate by fresh whole homogenates of gastrocnemius muscle was assessed as described previously (Winder et al. 1973 ) . The rate of oxidation of [3-14Clacetoacetate by whole homogenates of gastrocnemius muscle was measured in the same system used to measure D-phydroxybutyrate oxidation. This system contained 0.1 mil4 or 0.5 mM acetoacetate in place of DL-phydroxybutyrate, and 0.05 [3-14C]acetoacetate (Mallinckrodt, St. 1-ouis) in place of labeled @-hydroxybutyrate. T o correct for chemical decomposition of acetoacetate, radioactivity trapped in hyamine in the absence of homogenate was subtracted from radioactivity trapped in the presence of homogenate. Homogenates to be used for enzyme assays were frozen and thawed three times prior to the assays, for the purpose of disrupting the mitochondria. 3-Ketoacid CoA-tsansferase (EC 2.8.3.5) activity was assessed spectrophotometrically by measuring the late of succinate-dependent loss of acetoacetyl-CoA (Sigma, St. 1-ouis) at 313 nm as described by Benson and Boyer (1969) with the modification that the initial acetoacetyl-CoA concentration was 0.1 maw. Acetoacetyl-CoA thiolase (EC 2.3,1.9) activity was determined spectrophotometrically on diluted hornogenates by measuring CoA-dependent loss of acetoaeetyl-CoA at 3 13 nm (Dierks-Ventling and Cone 1971; Williamson et al. 1971 ). A millimoIar extinction coefficient at 313 wm of 11.9 for acetoacetyl-Cora was used for calculation of enzyme activity (Dierks-Ventling and Cone 1971) . Citrate syrnthase was assayed by the method of Srere ( 1969 ) with the use of 5,5'dithiobis (2-nitrokenzoic acid 1. Homogenates for p-hydroxybutrate dehydrogenase OX I .I .1.30) determinations were prepared in 10 m M ~uccinlatecontaining 2 m M DPN, 2 mika ATP, and 1 mM EDTA, pH 7.4. p-Hydroxybutyrate dehydrogenase activity of homogenates was determined by measuring the rate of 8-hydroxybutyrate-dependent acetoacetate accumuHadion (WiHliamson a t a / . 1971 ). AH1 spectrophotonaetric assays were rum on a
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CAN. J. PWYSIOL. PHARMACOL. VOL. 53, 1975
Gilford spectrophotometer, model 240, with a thermostated cell compartment, at 30 "C. Enzyme activities are reported as micromoles of substrate utilized per minute at 30 " C .
10
2:
35 (ELL
Results Cornyarisolz of Sedentary Groups No diflerences were noted between the freely eating sedentary control animals and the paired weight sedentary animals, with respect to enzyme activities and ketone oxidation rates in the tissues studied. Therefore, the results obtained on these two groups have been cornbined. Post-exercise Ketosis Exercise-trained and untrained rats were compared as to their capacity to maintain low blood ketones during and following exercise. When both groups were subjected to a sirlgle 90-min long bout of mild exercise (0.6 m.p.h. up an 8 deg. incline), blood ketone concentration (acetoacetate and p-hydroxybutyrate) was thrce times as high in untrained animals as in the trained exercising aninaals 60 inin following the exercise (Fig. 1 ) - p-Hydroxybutyrate was also significantly higher in untrained than in trained animals immediately following the exercise bout (0.99 -C 0.12 m M VS. 0.6% 9 0.06 m M ; P < 0.05), but acetoacetate was not significantly different in runners compared to untrained controls at this time. Post-exercise blood ketone levels were much lower in the trained than in the untrained animals, even when thc exercise-trained rats ran twice as fast as their untrained controls (Fig. 1 .). Plasma free fatty acid levels were also considerably lower, immediately and 60 min post-exercise, in the trained than in thc untrained animals (Fig. 1) . D-@-Hydroxybutyrateand Acetoacetate Oxidation by Skeletal Muscle The rate of oxidation of [3-1TC]acetoacetate by gastrocnemius muscle homogenates was approximately twofold greater in the trained than in the untrained animals at two physiological concentrations of substrate (Table 1). We found in a preliminary study (Winder et al. 1973) that exercise training increases the capacity sf skeletal muscle to oxidize p-hydroxybutyrate. In the present study we again measured p-hydroxybutj~ateoxidation in order to compare directly the rates of p-hydroxybuty-
Rats not adapted to endurance runnlng w 4
8 u& I
Rats adapted to endurance rwnnlng
(0.6rn.p.h.) (1.2 m.p.h.1
Rots adapted to endurance running (0.6rn.ph.1
at
20
46
60
80
100
120
140
TIME FOLLOWING BEGlNldlNG OF EXERCISE (min$
FIG. 1 . Effect s f 90 min of treadmill running on plasma FFA and blood kctones of rats previously trained by means of a program of prolonged treadmill running, compared t o rats not adapted to endurance running. Endurance-trained rats ran at 0.6 6 ) and unm.p.h. ( n = 7) or at 1.2 m.p.h. ( n trained rats ran at 0.6 n1.p.h. ( n - 7 ) during the 90 minm exercise period (1 m.p.h. = 0.45 m i s ) . S.E.M. is indicated by the vertical bars.
-
rate and acetoacetate oxidation in the same muscle homogenates. As shown in Table 1, the rate of oxidation of D-p-hydroxybutyrate was again two- to threefold greater in gastrocnemius homogenates of the traincd animals than in sedentary controls. At identical substrate concentrations, the rate of acetoacetate oxidation was greater than the rate of 11-p-hydroxybutyrate oxidation in n~usclchomogenates of both sedentary and exercise-trained rats (Table 1 ) . Effects of the Runnirzg Prc?grant on Ketone Oxidation Enzymes of Skeletal Muscle Levels of activity of the three enzymes specifically involvcd in oxidation of @-hydroxyksutyrate and acetoacetate increased in gastrocnemius muscle in response to the exercise program. p-Hydroxybutyrate dchydrogenase actitity increased threefold in gastrocnemius muscle, 3-ketoacid CoA-transferase activity increased twofold, while acetoacetyl-CsA
WlNDER ET AE.: INCREASED KETONE OXlDATlON 1N MUSCLE
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TABLE1. Effects of therunning program on rates* of oxidation of D-@-[~-'~C]hydroxybutyrnte and of [3-14C]ncetoacetate by homogenates of rat gastrocnemnus muscle Oxidation rate ( m e 1 min-I (g muscle)Substrate DL-/3-Hydrsxybutyrate Acetoacetate
Concentration 0.21I .O$ 0.1 0.5
l)
Exercising
Sedentary
18+ 1 (12)s 43 k 3 (12)§ 38_+2(11)5 5 9 + 2 (11)s
7 + 1 (14) 1 9 k 2 (13) 20+1(13) 32&2 (13)
*Expressed as nanomoles of I>-j3-hydroxybutyrate or acetoacetate ox~dizedto C O , per rninute per graIa1 of gastrocnemius muscle (wet wcight); values are means tThe number of animals in each group IS given in parentheses. +Equivalent to 0.1 mM D-fi-hydroxybutyrate, the iitili7abIe natural irorner. $Equivalent to 0.5 m M D-0-hydroxyk?utysate. §P < 0.001, exercising ts. sedentary.
S.E.W.
TABLE 2. Effects of the running program on ketone oxidation enzymes* of gastrocnemius rnuscle of the rat
Enzyme /I-Hydrsxybutyrate dehydrogenase 3-Ketoacid COB-transfcrase Acetoacetyl-CsA thiolase Citrate synthase
Exercising 0.27+0.02 25.9 k1.1 5.7 f 0 . 5 37 k3.0
(12)t (14)t (13)t (9)T
Sedentary O.OS_+O.Ol(13) 13.1 k 8 . 5 (15) 3.7 k 0 . 3 (14) 20 f l . O (10)
*Activities of e ~ ~ ~ y expressed mes in terms of micromoles per gram wet weight of muscle per minute; mean S.E.M.; numbers of observations are indicated in parentheses. +r< 0.001.
thiolase activity increased approximately 55 % (Table 2 ) . Transferase activities reported here are considerably higher than those reported by previous investigators (Williamson et al. 1971 ) . This difference is likely due to the fact that Williamson et ad. ( 1971) used a highspeed supernatant sf a sonicated muscle homogenate which in our hands yields consislerably lower activity than does the frozenthawed whole hcsmogenate. Efiect of the Running Program opt Cilmfe Synthase Activity A well docaimented adaptation of muscle to endurance training is an increase in the mitochondsial enzymes of the citric acid cycle, electron transport chain, and fatty acid oxidation (Dohm et ul. 1973; Holloszy et a!. 1970; Mo16 et ~ 1 . I991 ). Citrate synthase (EC 4.1.3.7) was measured for the purpose of providing an index of the level of training of these animals. and with the intent of comparing the response of ketone oxidation enzymes to the exercise program, with the response sf citrate synthase. As in previous studies (Holloszy ed ul. 1970), citrate synthase activity increased
+
approximately twofold (Table 2). The magnitude sf the increase in citrate synthase activity was similar to that seen for 3-ketoacid CoAtransferase.
Discussion The observation that physically untrained individuals develop post-exercise ketosis was first reported by Courtice and Douglas ( 1936). This phenomenon has been studied extensively by Johnson and co-workers (1971, 11972, 1969a, 1969b) who found that trained athletes, in contrast to untrained individuals, maintain relatively low blood ketone levels during and after exercise. Jt was also Eolrnd that tolerance to ingested acetoacetate is lower in untrained than in trained men following a bout of exercise (Johnson and Walton 1972). Since, in their studies, the blood concentratiorls of free fatty acids (FFA) and ketones tended to parallel each other, thcsc investigators suggested that the availability of FFA may be an important factor in the development of posta=xercisa:ketosis (Johnson and Walton 1972; Johnson et ak. 1 9 6 9 ~ ) .
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CAN. J. PHYSIOL. PHARMACOL.
Elevation of blood FFA levels is generally associated with increased hepatic ketogenesis (Fritz and Lee 1972; Williamson and Hems 1970). In the present study on rats, as in previous studies on men (Johnson and Walton 1972; Johnson et al. 1969a, 1969b), postexercise plasma FFA levels were higher in the untrained than in the trained state. This relationship favors the possibility that a greater rate of hepatic kctogenesis may play a role in accounting for the higher post-exercise blood ketone levels in the untrained state. The results of the present study show that exercise-training can induce significant increases in the levels OF the enzymes involved in the oxidation of p-hydroxybutyrate and acetoacetate in skeletal muscle. Associated with this increase in enzyme levels is an increase in the ability of skeletal muscle homogenates to oxidize ketones. Thus, it seems clear that the poteiltial of skeletal muscle for ketone oxidation is enhanced by exercise training. However, the extent to which this increase in the capacity to oxidize ketones is responsible for protecting trained individuals against post-exercise ketosis is not clear from available information. It will require p-hydroxybutyrate and acetoacctate turnover studies to determine the relative contributions of decreased synthesis and of increased oxidation of ketones to the protective effect of exercise training against post-exercise ketosis. However, since post-exercise blood FFA levels are lower, and the capacity of skeletal muscle to utilize ketones is greater, in the trained than in the untrained state, we think it is likely that both decreased synthesis and increased oxidation play important roles. It does not seem likely that the increase in the capacity of skeletal muscle to oxidize ketones that is induccd by exercise training is of much importance relative to the substrate mixture oxidized by muscle during exercise. While it seems well documented that ketones can serve as major substrates in resting inuscle (Ruderman and Goodman 1973; Ruderman et al. 197 1 ; Beatty et crl. 1963), available evidence indicates that ketones play a very minor ].ole as a fuel for respiration during exercise (Hagenfeldt and Wahren 1968; Paul 1975; Rowel1 197%).It appears that ketone oxidatioil can account for less than 2% of the total substrate oxidized during exercise (Paul 1975; Rowel1 1971) .
VOL.
53, 1975
Elevated blood ketone levels have a hypoglycemic action (Balasse and Ooms 1968; Balasse et al. 1967; Felts et al. 1964), which appears to be mediated via stimulation of insulin secretion (Balasse et al. 1967; Felts et al. 1964). It seems possible that the maintenance of low post-exercise blood ketone levels in physically trained individuals could have the physiologically beneficial effect of protecting against the development of hypoglycemia during prolonged, intermittent exercise. In summary, we have demonstrated that untrained rats have higher blood ketone levels than do exercise-trained animals following prolonged exercise. Exercise training induced significant increases in the levels s f activity of the three enzymes specifically involved in ketone oxidation in gastrocnemius muscle. The rates of oxidation of p-hydroxybutyrate and acetoacetate were significantly higher in homogenates of gastrocnemius muscles from the exereisetrained as compared to the control animals. This adaptation may help to explain why exercise-trained individuals have lower blood kctones during and following exercise. We wish to thank Mrs. May Chen for skillful fechilical assistance and Ms. Sandra Zigler for secretarial assistance.
E., COUTLJRPER, E., and FRANCKSON, J . R. M. BALASSE, 1967. Influence of sodium @-hydroxybutyrate on glucose and free fatty acid metabolism in normal dogs. Diabetologia, 3,488-493. BALASSE, E . , and OOMS,H. A. 1968. Changes in the concentrations of glucose, free fatty acids, insulin, and ketone bodies in the blood during sodium P-hydroxybutyrate infusions in man. Diabetologia, 4, 133-135. R. D., and BOCEK,R.M. 1963. BEATTY.C. H., PETERSON, Metabolism of red and white muscle fiber groups. Am. J. Physiol. 204,939-942. BENSON,R. W., and BOYER,P. D. 1969. The participation of an enzyme hound oxygen group in a coenzyme A transferase reaction. J. Biol. Chem. 246,2366-2371. COURTICE, F. C., and DOUGLAS,C. G. 1936. Effects of prolonged muscular exercise on metaholism. R o c . R. Soc. Lond. Ser. B, 119.381439. DIERKS-VENTLING, C.. and CONE,A. L. 1971. Ketone body enzymes in mammalian tissues. Effect of high fat diet. J. Biol. Chem. 246,5533-5534. DBHM,6. L., HUSTON,R. L., ASKEW, E. W., and FLESHQOD, H. L. 1973. Effects of exercise, training and diet on muscle citric acid cycle enzyme activity. Can. J. Biochem. 51,849-854. FELTS,P. W., CROFFORD,0. B., and PARK,C. R. 1964. Effect of infused ketone bodies on glucose utilization in the dog. J. Clin. Invest. 43,638446. FRITZ,I. B., and LEE, L. P. K. 1972. Fat mobilization and
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WINDER E T AL.: INCREASED KETONE OXIDATION IN MUSCLE
ketogenesis. I n Handbook s f physiology. Sect, 7. Vol. I. Edited by D. F. Steiner and N. Freinkel. American Physiological Society, Washington. D.C. pp. 549-596. MAGENFELDT, L., and WAHREN, J. 1968. Human forearm muscle metabolism. Scand. J. Clin. Lab. Invest. 21, 3 14-320. HOLLOSZY, J. O., OSCAI,L. B., DON,I. J., and M C P LP. ~, A. 1970. Mitochondria1 citric acid cycle and related enzymes: ad-aptive response t o exercise. Biochem. Biophys. Res. Commun. 40. 1368-1343. JOHNSON.R. H., and WALTON.J. L. 1971. Fitness, fatness, and post-exercise ketosis. Lancet. 1,566-568. 1972. The effect of exercise upon acetoacetate metabolism in athletes and non-athletes. Q, J. Exp. Physiol. 57,73-79. JOHNSON,R. H.. WALTON,J. L., KREBS,H. A., and WILLIAMSON. D. H. 1 9 6 9 ~Metabolic . fuels duiing and after severe exercise in athletes and non-athletes. Lancet, 2,452455. 1969b. Post-exercise ketosis. Lancet, 2,1383-1385. J. , 0. 1971. MOLE. P. A., OSCAI,L. B., and H ~ L L O S Z Y Adaptation of muscle to exercise. Increase in levels of palmityl CoA synthetase, camitine palmityltransferase and palmityl CoA dehydrogenase, and in the capacity to oxidize fatty acids. J. Clin. Invest. 50. 2323-2330. NOVAK, M. 1965. Colorimetric ultramicro method for the determination of free fatty acids. J. Lipid Res. 6 , 43 1 4 3 3 . PATTENGALE, P. K., and HOLLOSZY, J. 0. 1%7. Augmentation of skeletal muscle myoglobin by a program of treadmill running. Am. J. Physiol. 213,783-785. PAUL,P. 1975. Effects of long lasting physical exercise and
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training on lipid metabolism. In Metabolic adaptation to prolonged exercise. Second International Symposium on Biochemistry of Exercise. In press. ROWELL,L. B. f 971 The liver as an energy source in man during exercise. In Muscle metabolism during exercise. Edited by B . Pernow and B. Saltin. Plenum Publishing Csrp., New York, N.Y. pp. 127-141. RUDERMAN, N. B.. and GOODMAN,M. N. 1973. Regulation of ketone body metabolism in skeletal muscle. Am. J . Physiol. 224, 1391-1397. RUDERMAN, N . B., HOUCHTON,C. R. S., and HEMS,R. 1971. Evaluation of the isolated perfused rat hindquarter for the study of muscle metabolism. Biochem. J. 124,639-561. SRERE,P. A. 1969. Citrate synthase. Methods Enzymol. P3,3-5. WILLIAMSON, D. H . , BATES, M. W., PAGE,M. A . , and KREBS,H. A. 1971. Activities of enzymes involved in acetoacetate utilization in adult mammalian tissues. Biochem. J. 1 2 1 , 4 1 4 7 . WILLIAMSON, D. PI., and HEMS,R. 1970. Metabolism and function of ketone bodies. I n Essays in cell metabolism. Edited by W. Bartley, H. L. Kornberg, and J. R . Quayle. Interscience, London. pp. 257-28 1. D. H., MELLANBY, J.. and KREBS,H. A. WILLIAMSON, 1962. Enzymic determination of u(-)-p-hydroxybutyric acid and acetoacetic acid in blood. Biochem. J. 82,W-96. WINDER,W. W., BALDWIN, IC. M., and H o r , ~ o s z J. ~ ,0. 1973. Exercise-induced adaptive increase in rate of oxidation of 0-hydroxybutyrate by skeletal muscle. R o c . Soc. Exp. Biol. Med. 143,753-755.
Exercise-Induced Increase in the Capacity of Rat Skeletal Muscle to Oxidize Ketones1 W. W. WINDER,^ K. M. BALD WIN,^'^ A N D J . 0. HOLLOSZY~ Department ofPre~lentiveMedicine, Washington University School of Medicine, S t . Louis, Missouri63110 Received August 20,1974
WINDER, W. W., BALDWIN, K. M., and H ~ L L O S ZJ.Y0. , 1975. Exercise-induced increase in the capacity of rat skeletal muscle to oxidize ketones. Can. J. Physiol. Pharmacol. 53,86-91. During and after strenuous prolonged exercise, sedentary individuals develop high blood levels of acetoacetate and P-hydroxybutyrate whereas exercise-trained animals and human subjects do not. We have investigated the possibility that exercise training can increase the capacity of skeletal muscle to oxidize ketones. In this study we measured rates of ~-/3-[3-'~C]hydroxybutyrate and [3-14C]acetoacetate oxidation, and the levels of activity of the enzymes involved in the oxidation of ketones in homogenates of gastrocnemius muscles of exercisetrained and of untrained male rats. The trained animals had markedly lower blood ketone levels immediately and 60 min after a 90 min long bout of exercise than did the sedentary animals. The and [3-14C]acetoacetate oxidation were twice as high in rates of ~-/3-[3-~~C]hydroxybutyrate homogenates of muscles from the trained as compared to the sedentary rats. The increases in levels of activity in gastrocnemius muscle in response to the exercise program were: P-hydroxybutyrate dehydrogenase threefold; 3-ketoacid CoA-transferase twofold; and acetoacetyl-CoA thiolase 55%. This exercise-induced increase in the capacity of skeletal muscle to oxidize ketones could play a role in preventing development of ketosis in the physically trained animal during and following prolonged strenuous exercise.
K. M. et HOLLOS~Y, J. 0. 1975. Exercise-induced increase in the WINDER, W. W., BALDWIN, capacity of rat skeletal muscle to oxidize ketones. Can. J . Physiol. Pharmacol. 53,86-91. Chez les individus sedentaires, l'exercice intense et prolonge provoque une augmentation importante des taux sanguins d'adtoacetate et de 0-hydroxybutyrate, alors que, chez les animaux et les hommes entraines a l'effort, il n'y a pas de semblable augmentation. Nous explorons ici la possibilite que I'entrainement augmente la capacite des muscles squelettiques d'oxyder les corps cetoniques. Dans cette etude, nous mesurons l'oxydation du D-/3[3-'4C]hydroxybutyrate et de le 13-I4C]acetoacetate, ainsi que l'activite des enzymes impliquts dans I'oxydation des cetones par les homogenats de muscles jumeaux preleves chez des rats entraines a l'exercice et chez des rats sedentaires. Immediatement apres un exercice de 90 min, et 6Q min apres, les animaux entraines ont des taux citoniques beaucoup moins eleves que les animaux sedentaires. L'oxydation du phydroxybutyrate 3-14C et de le 13-14C]acetoacetateest deux fois plus elevee dans les homogenats preleves chez les rats entraines qu'elle ne I'est chez les sedentaires. L'activite de la 0-hydroxybutyrate deshydrogtnase est augmentee trois fois, celle de la 3 cetoacyl CoA-transferase deux fois, et celle de l'ac~toacetyl-CoAthiolase de 55% dans les muscles jumeaux, en reponse au programme d'exercice. Cette augmentation de l'oxydation des cetones par le muscle squelettique provoguee par I'exercice joue peut-2tre un r6le dans la prevention de la cktose, pendant et apres l'exercice intense et prolonge, chez les animaux physiquement entrainks. [Traduit par le journal]
Inhodncltiorn A marked difference has been observed betwecn endurance athletes, such as distance runners, and sedentary subjects with respect to the metabolism sf ketones (Johnson and 'This research was supported by Research Grant HHb 01 413 and Training Grant AM 05348 from the National Institutes of Health, and by a grant from the Missouri Heart Association. W. W. Winder was a Postdoctoral Research Fellow s~~pportedl by Research Fellowship AM 51241 from the National Institutes of FIealth.
Walton 1974, 197%; Johnson et wl. 1969a, 8 969 b ) . In play sically untrained individuals, blood ketones increase moderately during grolonged cxercisc and then increase sharply after cessation of cxercise (Johnson and Walton 19'7%; Johnson et QI. 1 9 6 9 ~1969b). ~ In individuals who have adaptcd to endurance "K. M. Baldwin was a Postdoctoral Research Trainee supported by Training Grant AM 05341. 'Present address, Department of Physiology, University sf California at Hrvirae. Irvine, California 92664. 3. 0. Hslloszy was the recipient of Research Career ilevelopmene Award K4-HD 19573.
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WINDER ET AL.: INCREASED KETONE OXIDATION IN MUSCLE
exercise, blood ketones are maintained at or near resting levels during and after a prolonged strenuous exercise bout (Johnson and Walton 1972; Johnson et 01. 1969a, 1969b). This difference may be due to differences in rates of hepatic ketogcnesis and/or in the rates of ketone oxidation. In a preliminary study it was found that homogenates of gastrocnemius muscles from chronically exercised rats oxidized D-P-hydroxybutyrate at a significantly increased rate (Winder et a/. 1973). This observation was confirmed in the present detailed study in which it was further found that the levels of activity of the enzymes involved in ketone oxidation were significantly increased in leg muscles of rats subjected to a program of running. The rate of acetoacetate oxidation by gastrocnemius nluscle from the trained animals was also significantly increased.
Methods Trc)atment of Animals Male Wistar rats (specific pathogen free CFN rats from Carworth Farms) weighing approximately 100 g were divided into three groups. A running group was trained by means of a 12-week program of treadmill running described previously (Pattengale and Holloszy 3967) at the end of which, rats were running continuously for 2 h daily at 1.2 miles/h (m.p.h.) 41 n3.p.h. 0.45 m/s) up an 8 deg. (1 deg. 0.017 rad) incline with 12, 30-s periods of running at 1.6 m.p.h. spaced 10 nlin apart throughout the exercise sessions. The rats were maintained at this level of training 5 days per week until the time of sacrifice; this period varied from 2 t o 8 weeks. The rtlnners were provided with food ad libitum. A freely eating sedentary group was also provided with food UCIlibitum. A paired weight sedentary group had their food intake restricted so as to maintain body weights the same as those of the runners. All animals were maintained on a diet of P~arinachow and water.
-
Post-Ext,rci.sc K'c2r~sis The sedentary rats that were used in the studies on the development of post-exercise ketosis were taught to run on the treadmill for 10-15 min daily at 0.6 m.p.k. for 2 weeks prior to the test. All rats were given 20 g Purina chow the night prior to the test. The next morning trained and untrained rats were exercised on the treadmill for 90 rnin at 0.6 1n.p.h. tip an 8 deg. incline. Immediately following the exercise, rats were anesthetized lightly with ether and a 0.8 rnl blood sarrmple was obtained by cardiac puncture and processed for plasma free fatty acid and blood ketone determinations. Rats were then placed in cages without access to food for 1 h, at which time another blood sample was obtained and processed. Blood was also collected from rats, not
87
involved in the exercise test that day, for the purpose of determining resting blood ketones and plasma free fatty acids. In addition, blood ketones and plasma free fatty acids were determined on trained animals subjected to a 90 min bout of exercise at 1.2 mp.h. up an 8 deg. incline. Plasma free fatty acids were determined by the method of Novak (1965). Blood ketones were determined by the method of Williamson et al. (1962). Homogcnatc Preparaticrn and Assay hlethods Rats were killed by decapitation 24-72 h following the runners' last training session. The gastrocnenlius muscles were removed and placed in small beakers on ice until the time of homogenization. Muscles were cleaned of connective tissue and minced thoroughly with scissors prior to homogenization. Homogenates were prepared using a Potter-Elvehjem homogenizer immersed in ice water. Homogenates for all assays with the exception of p-hydroxybutyrate dehydrogenase were prepared in 175 m M KC1 containing 10 mM GSH and 2 m M EDTA, pH 7.4. The rate of oxidation of ~-p-[3-~'C]hydroxybutyrate by fresh whole homogenates of gastrocnemius muscle was assessed as described previously (Winder et al. 1973 ) . The rate of oxidation of [3-14Clacetoacetate by whole homogenates of gastrocnemius muscle was measured in the same system used to measure D-phydroxybutyrate oxidation. This system contained 0.1 mil4 or 0.5 mM acetoacetate in place of DL-phydroxybutyrate, and 0.05 [3-14C]acetoacetate (Mallinckrodt, St. 1-ouis) in place of labeled @-hydroxybutyrate. T o correct for chemical decomposition of acetoacetate, radioactivity trapped in hyamine in the absence of homogenate was subtracted from radioactivity trapped in the presence of homogenate. Homogenates to be used for enzyme assays were frozen and thawed three times prior to the assays, for the purpose of disrupting the mitochondria. 3-Ketoacid CoA-tsansferase (EC 2.8.3.5) activity was assessed spectrophotometrically by measuring the late of succinate-dependent loss of acetoacetyl-CoA (Sigma, St. 1-ouis) at 313 nm as described by Benson and Boyer (1969) with the modification that the initial acetoacetyl-CoA concentration was 0.1 maw. Acetoacetyl-CoA thiolase (EC 2.3,1.9) activity was determined spectrophotometrically on diluted hornogenates by measuring CoA-dependent loss of acetoaeetyl-CoA at 3 13 nm (Dierks-Ventling and Cone 1971; Williamson et al. 1971 ). A millimoIar extinction coefficient at 313 wm of 11.9 for acetoacetyl-Cora was used for calculation of enzyme activity (Dierks-Ventling and Cone 1971) . Citrate syrnthase was assayed by the method of Srere ( 1969 ) with the use of 5,5'dithiobis (2-nitrokenzoic acid 1. Homogenates for p-hydroxybutrate dehydrogenase OX I .I .1.30) determinations were prepared in 10 m M ~uccinlatecontaining 2 m M DPN, 2 mika ATP, and 1 mM EDTA, pH 7.4. p-Hydroxybutyrate dehydrogenase activity of homogenates was determined by measuring the rate of 8-hydroxybutyrate-dependent acetoacetate accumuHadion (WiHliamson a t a / . 1971 ). AH1 spectrophotonaetric assays were rum on a
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CAN. J. PWYSIOL. PHARMACOL. VOL. 53, 1975
Gilford spectrophotometer, model 240, with a thermostated cell compartment, at 30 "C. Enzyme activities are reported as micromoles of substrate utilized per minute at 30 " C .
10
2:
35 (ELL
Results Cornyarisolz of Sedentary Groups No diflerences were noted between the freely eating sedentary control animals and the paired weight sedentary animals, with respect to enzyme activities and ketone oxidation rates in the tissues studied. Therefore, the results obtained on these two groups have been cornbined. Post-exercise Ketosis Exercise-trained and untrained rats were compared as to their capacity to maintain low blood ketones during and following exercise. When both groups were subjected to a sirlgle 90-min long bout of mild exercise (0.6 m.p.h. up an 8 deg. incline), blood ketone concentration (acetoacetate and p-hydroxybutyrate) was thrce times as high in untrained animals as in the trained exercising aninaals 60 inin following the exercise (Fig. 1 ) - p-Hydroxybutyrate was also significantly higher in untrained than in trained animals immediately following the exercise bout (0.99 -C 0.12 m M VS. 0.6% 9 0.06 m M ; P < 0.05), but acetoacetate was not significantly different in runners compared to untrained controls at this time. Post-exercise blood ketone levels were much lower in the trained than in the untrained animals, even when thc exercise-trained rats ran twice as fast as their untrained controls (Fig. 1 .). Plasma free fatty acid levels were also considerably lower, immediately and 60 min post-exercise, in the trained than in thc untrained animals (Fig. 1) . D-@-Hydroxybutyrateand Acetoacetate Oxidation by Skeletal Muscle The rate of oxidation of [3-1TC]acetoacetate by gastrocnemius muscle homogenates was approximately twofold greater in the trained than in the untrained animals at two physiological concentrations of substrate (Table 1). We found in a preliminary study (Winder et al. 1973) that exercise training increases the capacity sf skeletal muscle to oxidize p-hydroxybutyrate. In the present study we again measured p-hydroxybutj~ateoxidation in order to compare directly the rates of p-hydroxybuty-
Rats not adapted to endurance runnlng w 4
8 u& I
Rats adapted to endurance rwnnlng
(0.6rn.p.h.) (1.2 m.p.h.1
Rots adapted to endurance running (0.6rn.ph.1
at
20
46
60
80
100
120
140
TIME FOLLOWING BEGlNldlNG OF EXERCISE (min$
FIG. 1 . Effect s f 90 min of treadmill running on plasma FFA and blood kctones of rats previously trained by means of a program of prolonged treadmill running, compared t o rats not adapted to endurance running. Endurance-trained rats ran at 0.6 6 ) and unm.p.h. ( n = 7) or at 1.2 m.p.h. ( n trained rats ran at 0.6 n1.p.h. ( n - 7 ) during the 90 minm exercise period (1 m.p.h. = 0.45 m i s ) . S.E.M. is indicated by the vertical bars.
-
rate and acetoacetate oxidation in the same muscle homogenates. As shown in Table 1, the rate of oxidation of D-p-hydroxybutyrate was again two- to threefold greater in gastrocnemius homogenates of the traincd animals than in sedentary controls. At identical substrate concentrations, the rate of acetoacetate oxidation was greater than the rate of 11-p-hydroxybutyrate oxidation in n~usclchomogenates of both sedentary and exercise-trained rats (Table 1 ) . Effects of the Runnirzg Prc?grant on Ketone Oxidation Enzymes of Skeletal Muscle Levels of activity of the three enzymes specifically involvcd in oxidation of @-hydroxyksutyrate and acetoacetate increased in gastrocnemius muscle in response to the exercise program. p-Hydroxybutyrate dchydrogenase actitity increased threefold in gastrocnemius muscle, 3-ketoacid CoA-transferase activity increased twofold, while acetoacetyl-CsA
WlNDER ET AE.: INCREASED KETONE OXlDATlON 1N MUSCLE
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TABLE1. Effects of therunning program on rates* of oxidation of D-@-[~-'~C]hydroxybutyrnte and of [3-14C]ncetoacetate by homogenates of rat gastrocnemnus muscle Oxidation rate ( m e 1 min-I (g muscle)Substrate DL-/3-Hydrsxybutyrate Acetoacetate
Concentration 0.21I .O$ 0.1 0.5
l)
Exercising
Sedentary
18+ 1 (12)s 43 k 3 (12)§ 38_+2(11)5 5 9 + 2 (11)s
7 + 1 (14) 1 9 k 2 (13) 20+1(13) 32&2 (13)
*Expressed as nanomoles of I>-j3-hydroxybutyrate or acetoacetate ox~dizedto C O , per rninute per graIa1 of gastrocnemius muscle (wet wcight); values are means tThe number of animals in each group IS given in parentheses. +Equivalent to 0.1 mM D-fi-hydroxybutyrate, the iitili7abIe natural irorner. $Equivalent to 0.5 m M D-0-hydroxyk?utysate. §P < 0.001, exercising ts. sedentary.
S.E.W.
TABLE 2. Effects of the running program on ketone oxidation enzymes* of gastrocnemius rnuscle of the rat
Enzyme /I-Hydrsxybutyrate dehydrogenase 3-Ketoacid COB-transfcrase Acetoacetyl-CsA thiolase Citrate synthase
Exercising 0.27+0.02 25.9 k1.1 5.7 f 0 . 5 37 k3.0
(12)t (14)t (13)t (9)T
Sedentary O.OS_+O.Ol(13) 13.1 k 8 . 5 (15) 3.7 k 0 . 3 (14) 20 f l . O (10)
*Activities of e ~ ~ ~ y expressed mes in terms of micromoles per gram wet weight of muscle per minute; mean S.E.M.; numbers of observations are indicated in parentheses. +r< 0.001.
thiolase activity increased approximately 55 % (Table 2 ) . Transferase activities reported here are considerably higher than those reported by previous investigators (Williamson et al. 1971 ) . This difference is likely due to the fact that Williamson et ad. ( 1971) used a highspeed supernatant sf a sonicated muscle homogenate which in our hands yields consislerably lower activity than does the frozenthawed whole hcsmogenate. Efiect of the Running Program opt Cilmfe Synthase Activity A well docaimented adaptation of muscle to endurance training is an increase in the mitochondsial enzymes of the citric acid cycle, electron transport chain, and fatty acid oxidation (Dohm et ul. 1973; Holloszy et a!. 1970; Mo16 et ~ 1 . I991 ). Citrate synthase (EC 4.1.3.7) was measured for the purpose of providing an index of the level of training of these animals. and with the intent of comparing the response of ketone oxidation enzymes to the exercise program, with the response sf citrate synthase. As in previous studies (Holloszy ed ul. 1970), citrate synthase activity increased
+
approximately twofold (Table 2). The magnitude sf the increase in citrate synthase activity was similar to that seen for 3-ketoacid CoAtransferase.
Discussion The observation that physically untrained individuals develop post-exercise ketosis was first reported by Courtice and Douglas ( 1936). This phenomenon has been studied extensively by Johnson and co-workers (1971, 11972, 1969a, 1969b) who found that trained athletes, in contrast to untrained individuals, maintain relatively low blood ketone levels during and after exercise. Jt was also Eolrnd that tolerance to ingested acetoacetate is lower in untrained than in trained men following a bout of exercise (Johnson and Walton 1972). Since, in their studies, the blood concentratiorls of free fatty acids (FFA) and ketones tended to parallel each other, thcsc investigators suggested that the availability of FFA may be an important factor in the development of posta=xercisa:ketosis (Johnson and Walton 1972; Johnson et ak. 1 9 6 9 ~ ) .
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CAN. J. PHYSIOL. PHARMACOL.
Elevation of blood FFA levels is generally associated with increased hepatic ketogenesis (Fritz and Lee 1972; Williamson and Hems 1970). In the present study on rats, as in previous studies on men (Johnson and Walton 1972; Johnson et al. 1969a, 1969b), postexercise plasma FFA levels were higher in the untrained than in the trained state. This relationship favors the possibility that a greater rate of hepatic kctogenesis may play a role in accounting for the higher post-exercise blood ketone levels in the untrained state. The results of the present study show that exercise-training can induce significant increases in the levels OF the enzymes involved in the oxidation of p-hydroxybutyrate and acetoacetate in skeletal muscle. Associated with this increase in enzyme levels is an increase in the ability of skeletal muscle homogenates to oxidize ketones. Thus, it seems clear that the poteiltial of skeletal muscle for ketone oxidation is enhanced by exercise training. However, the extent to which this increase in the capacity to oxidize ketones is responsible for protecting trained individuals against post-exercise ketosis is not clear from available information. It will require p-hydroxybutyrate and acetoacctate turnover studies to determine the relative contributions of decreased synthesis and of increased oxidation of ketones to the protective effect of exercise training against post-exercise ketosis. However, since post-exercise blood FFA levels are lower, and the capacity of skeletal muscle to utilize ketones is greater, in the trained than in the untrained state, we think it is likely that both decreased synthesis and increased oxidation play important roles. It does not seem likely that the increase in the capacity of skeletal muscle to oxidize ketones that is induccd by exercise training is of much importance relative to the substrate mixture oxidized by muscle during exercise. While it seems well documented that ketones can serve as major substrates in resting inuscle (Ruderman and Goodman 1973; Ruderman et al. 197 1 ; Beatty et crl. 1963), available evidence indicates that ketones play a very minor ].ole as a fuel for respiration during exercise (Hagenfeldt and Wahren 1968; Paul 1975; Rowel1 197%).It appears that ketone oxidatioil can account for less than 2% of the total substrate oxidized during exercise (Paul 1975; Rowel1 1971) .
VOL.
53, 1975
Elevated blood ketone levels have a hypoglycemic action (Balasse and Ooms 1968; Balasse et al. 1967; Felts et al. 1964), which appears to be mediated via stimulation of insulin secretion (Balasse et al. 1967; Felts et al. 1964). It seems possible that the maintenance of low post-exercise blood ketone levels in physically trained individuals could have the physiologically beneficial effect of protecting against the development of hypoglycemia during prolonged, intermittent exercise. In summary, we have demonstrated that untrained rats have higher blood ketone levels than do exercise-trained animals following prolonged exercise. Exercise training induced significant increases in the levels s f activity of the three enzymes specifically involved in ketone oxidation in gastrocnemius muscle. The rates of oxidation of p-hydroxybutyrate and acetoacetate were significantly higher in homogenates of gastrocnemius muscles from the exereisetrained as compared to the control animals. This adaptation may help to explain why exercise-trained individuals have lower blood kctones during and following exercise. We wish to thank Mrs. May Chen for skillful fechilical assistance and Ms. Sandra Zigler for secretarial assistance.
E., COUTLJRPER, E., and FRANCKSON, J . R. M. BALASSE, 1967. Influence of sodium @-hydroxybutyrate on glucose and free fatty acid metabolism in normal dogs. Diabetologia, 3,488-493. BALASSE, E . , and OOMS,H. A. 1968. Changes in the concentrations of glucose, free fatty acids, insulin, and ketone bodies in the blood during sodium P-hydroxybutyrate infusions in man. Diabetologia, 4, 133-135. R. D., and BOCEK,R.M. 1963. BEATTY.C. H., PETERSON, Metabolism of red and white muscle fiber groups. Am. J. Physiol. 204,939-942. BENSON,R. W., and BOYER,P. D. 1969. The participation of an enzyme hound oxygen group in a coenzyme A transferase reaction. J. Biol. Chem. 246,2366-2371. COURTICE, F. C., and DOUGLAS,C. G. 1936. Effects of prolonged muscular exercise on metaholism. R o c . R. Soc. Lond. Ser. B, 119.381439. DIERKS-VENTLING, C.. and CONE,A. L. 1971. Ketone body enzymes in mammalian tissues. Effect of high fat diet. J. Biol. Chem. 246,5533-5534. DBHM,6. L., HUSTON,R. L., ASKEW, E. W., and FLESHQOD, H. L. 1973. Effects of exercise, training and diet on muscle citric acid cycle enzyme activity. Can. J. Biochem. 51,849-854. FELTS,P. W., CROFFORD,0. B., and PARK,C. R. 1964. Effect of infused ketone bodies on glucose utilization in the dog. J. Clin. Invest. 43,638446. FRITZ,I. B., and LEE, L. P. K. 1972. Fat mobilization and
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WINDER E T AL.: INCREASED KETONE OXIDATION IN MUSCLE
ketogenesis. I n Handbook s f physiology. Sect, 7. Vol. I. Edited by D. F. Steiner and N. Freinkel. American Physiological Society, Washington. D.C. pp. 549-596. MAGENFELDT, L., and WAHREN, J. 1968. Human forearm muscle metabolism. Scand. J. Clin. Lab. Invest. 21, 3 14-320. HOLLOSZY, J. O., OSCAI,L. B., DON,I. J., and M C P LP. ~, A. 1970. Mitochondria1 citric acid cycle and related enzymes: ad-aptive response t o exercise. Biochem. Biophys. Res. Commun. 40. 1368-1343. JOHNSON.R. H., and WALTON.J. L. 1971. Fitness, fatness, and post-exercise ketosis. Lancet. 1,566-568. 1972. The effect of exercise upon acetoacetate metabolism in athletes and non-athletes. Q, J. Exp. Physiol. 57,73-79. JOHNSON,R. H.. WALTON,J. L., KREBS,H. A., and WILLIAMSON. D. H. 1 9 6 9 ~Metabolic . fuels duiing and after severe exercise in athletes and non-athletes. Lancet, 2,452455. 1969b. Post-exercise ketosis. Lancet, 2,1383-1385. J. , 0. 1971. MOLE. P. A., OSCAI,L. B., and H ~ L L O S Z Y Adaptation of muscle to exercise. Increase in levels of palmityl CoA synthetase, camitine palmityltransferase and palmityl CoA dehydrogenase, and in the capacity to oxidize fatty acids. J. Clin. Invest. 50. 2323-2330. NOVAK, M. 1965. Colorimetric ultramicro method for the determination of free fatty acids. J. Lipid Res. 6 , 43 1 4 3 3 . PATTENGALE, P. K., and HOLLOSZY, J. 0. 1%7. Augmentation of skeletal muscle myoglobin by a program of treadmill running. Am. J. Physiol. 213,783-785. PAUL,P. 1975. Effects of long lasting physical exercise and
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training on lipid metabolism. In Metabolic adaptation to prolonged exercise. Second International Symposium on Biochemistry of Exercise. In press. ROWELL,L. B. f 971 The liver as an energy source in man during exercise. In Muscle metabolism during exercise. Edited by B . Pernow and B. Saltin. Plenum Publishing Csrp., New York, N.Y. pp. 127-141. RUDERMAN, N. B.. and GOODMAN,M. N. 1973. Regulation of ketone body metabolism in skeletal muscle. Am. J . Physiol. 224, 1391-1397. RUDERMAN, N . B., HOUCHTON,C. R. S., and HEMS,R. 1971. Evaluation of the isolated perfused rat hindquarter for the study of muscle metabolism. Biochem. J. 124,639-561. SRERE,P. A. 1969. Citrate synthase. Methods Enzymol. P3,3-5. WILLIAMSON, D. H . , BATES, M. W., PAGE,M. A . , and KREBS,H. A. 1971. Activities of enzymes involved in acetoacetate utilization in adult mammalian tissues. Biochem. J. 1 2 1 , 4 1 4 7 . WILLIAMSON, D. PI., and HEMS,R. 1970. Metabolism and function of ketone bodies. I n Essays in cell metabolism. Edited by W. Bartley, H. L. Kornberg, and J. R . Quayle. Interscience, London. pp. 257-28 1. D. H., MELLANBY, J.. and KREBS,H. A. WILLIAMSON, 1962. Enzymic determination of u(-)-p-hydroxybutyric acid and acetoacetic acid in blood. Biochem. J. 82,W-96. WINDER,W. W., BALDWIN, IC. M., and H o r , ~ o s z J. ~ ,0. 1973. Exercise-induced adaptive increase in rate of oxidation of 0-hydroxybutyrate by skeletal muscle. R o c . Soc. Exp. Biol. Med. 143,753-755.
