Acta Phystol Scand 1991, 143, 367-372
Acetyl group accumulation and pyruvate dehydrogenase activity in human muscle during incremental exercise D. C O N S T A N T I N - T E O D O S I U , J. I. C A R L I N , G. C E D E R B L A D " , R. C. H A R R I S t and E. H U L T M A N Departments of Clinical Chemistry I" and 11, Huddinge University Hospital, Karolinska Institutet, S-141 86 Huddinge, Sweden CONSTANTIN-TEODOSIU, D., CARLIN, J. I., CEDERBLAD, G., HARRIS, R. C. & HULTMAN, E. 1991. Acetyl group accumulation and pyruvate dehydrogenase activity in human muscle during incremental exercise. Acta P h y d &and 143, 367-372. Received 10 April 1991, accepted 31 July 1991. ISBN 0001-6772. Departments of Clinical Chemistry I and 11, Huddinge University Hospital, Karolinska Institutet, Sweden The changes in the muscle contents of CoASH and carnitine and their acetylated forms, lactate and the active form of pyruvate dehydrogenase complex were studied during incremental dynamic exercise. Eight subjects exercised for 3 4 minutes on a bicycle ergometer at work loads corresponding to 30, 60 and 90% of their ~o,,,,. Muscle samples were obtained by percutaneous needle biopsy technique at rest, at the end of each work period and after 10 minutes of recovery. During the incremental exercise test there was a continuous increase in muscle lactate, from a basal value of 4.5 mmol kg-' dry weight to 83 mmol kg-' at the end of the final period. The active form of pyruvate dehydrogenase complex increased from 0.37 mmol acetyl-CoA formed per minute per kilogram wet weight at rest to 0.80 at 3004 l&max, 1.28 and 1.25 at 60 and 9004 respectively. Both acetyl-CoA and acetylcarnitine increased at the two highest work loads. The increase of acetyl-CoA was from 12.5 pmol k g ' dry weight at rest to 27.3 after the highest work load and for acetylcarnitine from 6.0 mmol kg-' dry weight to 15.2. The CoASH and free carnitine contents fell correspondingly. There was a close relationship between acetyl-CoA and acetylcarnitine accumulation in muscle during exercise, with a binding of 500 mol acetyl groups to carnitine for each mole of acetylCoA accumulated. The results imply that the carnitine store in muscle functions as a buffer for excess formation of acetyl groups from pyruvate catalyzed by the pyruvate dehydrogenase complex.
vo,,,,,
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Key Words: Acetylcarnitine. acetyl-CoA, carnitine, coenzyme A, dynamic exercise, pyruvate dehydrogenase
The three enzymes which together constitute the pyruvate dehydrogenase complex (PDC) (pyruvate dehydrogenase El-EC 1 .2.4.1, dihydrolipoyl transacetylase E,-EC 2 . 3 . 1 .12, dihydrolipoyl dehydrogenase E,-EC 1 .8.1.4) catalyse Correspondence : Prof. Eric Hultman, Dept. of Clinical Chemistry 11, Huddinge University Hospital, S-141 86 Huddinge, Sweden. t Present address: (R. C. Harris) Department of Comparative Physiology, The Animal Health Trust, Newmarket, Suffolk, England.
the oxidation of pyruvate to acetyl-CoA. The acetyl-CoA formed condenses with oxaloacetate to give citric acid which then undergoes further degradation in the citric acid cycle. Activation of the pyruvate dehydrogenase component of the complex (PDH, EC 1.2.4.1) is brought about through dephosphorylation mediated by a protein phosphatase, while the enzyme is deactivated by phosphorylation by protein kinase. This activation of the P D H component transforms PDC from the inactive to the active form, i.e. PDCa. Effectors of the two interconverting
367
368
D.Constantin- Teodosiu et al.
enz! nits include pyruvate and ADP which inhibit the kinase and Ca'* and Mg'+ which both actiwte the phosphatasc and inhibit the kinase (Denton e/ rrl. 19i.5). I t has also been s h o w that the P I X transformation to active form in kidney and heart preparations is inhibited bl- increasing ratios of YL4D€1/X.4D and acetyl-Co.4/Co.4 (Cooper et a / . 197.i, Pettit et a / . 197.5), and that the catalytic acti\-ity of the PDCa enzyme is regulated by end-product inhibition b!- acetylCo.4 and N-lDH (for references see Wieland 1983). Recent studies in humans and equines h a w demonstratcd a marked increase in the acetl-Icarnitine content of skeletal muscle during high intensity exercise of short duration (Lennon et ill. 198.3, Carlin el u / . 1986, Foster & Harris 1987, tiarris et a(. 1987, Harris & Foster 1990). Studies of acetylcarnitine formation during human exercise with increasing work intensity h a w also been presented showing large increases in acetylcarnitine at 7.j0, and 100°, C;z, (Sahlin 1990). These findings are consistent with a role for carnitine in the regulation of the mitochondria1 acetyl-CoL4/CoASH ratio by buffering excess acctl-l-CoA production from pyruvate (-4lkonyi et a / . 1975, Childress et a / . 1966). By acting as an acceptor for acetyl groups from acetyl-CoA, carnitine will help in maintaining the catalytic activitl- of PDCa under conditions where the rate of acetyl-CoA condensation with oxaloacetatc is less than its rate of formation from pyruvate. In a recent stud!-, formation of both acetyl-CoA and acetylcarnitine was investigated during treadmill exercise of increasing intensit?. in thoroughbred horses (Carlin et a / . 1990). At high work intensities a plateau of both compounds was reached. There was a significant relationship between the contents of the two compounds in contracting muscle. T h e aim of the present study was to investigate PDC transformation during incremental work in human subjects, and to relate this to the accumulation of acetyl groups and the buffering function of carnitine.
M A T E R I A L S A N D METHODS Eight healthy men participated in this study. Their mean (range) age, height, weight and &',,,,, were 27 (22-32) years, 180 (175-188) cm, 73 (67-82) kg and 3.9 ( 3 . j 4..5)I min-', respectivell-. The subjects were physicall! active and pcrformed exercise on a regular
basis though none of them was a competitive athlete. .\I1 subjects were familiarized with the testing procedures before the start of the study including an initial bicycle exercise test. The subjects were informed about the possible risks of the study hefore giving their voluntary consent. The experiincntal protocol was approved by the Ethics Committee of the Karolinska Institute, Stockholm, Sweden. Experimental procedure. Maximal oxygen uptake capacity (I;',,,,,,) was determined during progressive workloads on a bicycle ergometer. Oxygen consumption (Q, carbon dioxide production (&,,) and the respiratory exchange rate (REK) were monitored e\-ery 30 s using an oxygen analyser (Oxycon-4, llijnhardt, Runnik, Netherlands) calibrated with known gases (0, and CO,). For the eight subjects mean work ,loads ( fSD) corresponding to 30, 60 and 909, of V,,,,,x were 8 9 f l l W, 189+12 W and 267 34 W, icspectively. \Lluscle biopsy samples were obtained from thc vastus lateralis by a percutaneous needle biopsy technique according to Bergstrom (1962) at rest, after 4 min rxercise at work loads corresponding to 30 and 60°, F,2n,c,x, and after 3 rnin at 90"1~, V",,,,,,, and after 10 min rest. Each tissue sample waB immediately frozen b>- insertion of the needle in liquid nitrogen. The delay between freezing of the 'exercise '-samples and the end of the exercise was 5-10 s. Aiia/ytrcal methods. Muscle samples were divided under liquid N,into two parts. One part of the sample was freeze-dried, dissected free from visible connective tissue and blood, and powdered. Seven to 12 mg of the muscle power was analysed for carnitine, acetylcarnitine, CoASH and acetyl-CoA on the same extract by the radioisotopic methods described by Cederblad et a/. (1982, 1990). The other part (20-30 mg) of the muscle sample was used for determination of pyruvate dehydrogenase complex in principle according to the method described by Dohm et al. (1986). Instead of measurement of "CO, formation in this method the production of acetyl-CoA from added pyruvate was analysed by the method described by Cederblad et 61. (1990). Lactate was determined in the same extract by the method described by 1Iarris et al. (1974).
RESULTS T h e contents of lactate, acetyl-CoA, CoASH, acctj-lcarnitine and carnitine as well as PDCa activity are given in Table 1. There was no significant accumulation of lactate or acetyl groups in the muscle tissue after exercise at a work load corresponding to V, maI (Fig. 1). At the higher work intensities of 60bo and 900j0 ~ o j o ? n , 6muscle x, lactatc increased to 29.8 and 82.7 mmol kg-' dry wt, respectively, and both acetyl-CoA and acetylcarnitine contents
Acetyl group accumulation during exercise
369
Table 1. Muscle concentrations of CoASH, carnitine, their acetylated forms and lactate and the measured activity of the active form of pyruvate dehydrogenase complex at rest and after exercise with different intensities ~~~
Work load, yo of
~o',,,,,
30
60
90
10 min post
~-
Variable ~~~~
At rest ~
Lactate mmol kg-' dry wt PDCa' CoASH pmol kg-l dry wt Acetyl-CoA pmol kg-' dry wt Free carnitine mmol kg-' dry wt Acetylcarnitine mmol kg-' dry wt
4.5f1.1
9.3+4.5*
29.8+ 17.2"'
82.7i30.3""
25.3k 16.4"*
0.3750.22 36.7k8.6
0.80+0.26*" 33.5k8.6
1.28+0.39*" 29.0 & 7.1
1.25f0.53*" 26.6 _+ 7.3"
0.60+0.28 37.nk 9.3
12.5f4.3
12.5k4.1
18.7k4.7**
27.3+7.5*"
13.6k5.0
15.2f2.2
12.9f 3.8
10.7 k 2.7""
6.5k 1.5'"
12.1 1.6*
+
6.2 2.3
15.2*2.8**
10.7k2.5"
6.0 2.7
+
10.1 f2.4'
mmol acetyl-CoA formed min-' and kg wet wt at 307 "C, n = 6. Comparison (Student's paired t-test; n = 8) between values at rest and after the three periods of exercise and after 10 min rest; * .2P < 0.05, ** .2P < 0.01. Values are given as mean+SD. +
were increased significantly at the end of the work periods reaching after 90% ~o',,,,, 27.3 pmol kg-' dry wt and 15.2 mmol kg-' dry wt, respectively. The concentrations of CoASH and free carnitine decreased correspondingly. These changes were reversed during a I0-min recovery period at the end of which metabolite contents were approaching those found prior to the start of exercise. There was a significant correlation between contents of acetyl-CoA and acetylcarnitine in muscle tissue at rest and during exercise (Fig. 2). Measured PDCa activity increased from 0.37 mmol acetyl-CoA min-' kg-' wet wt to 0.80 mmol at the end of the 4 min work at 30% Yo,,,,. The activity increased further to 1.28 pmol acetyl-CoA min-' kg-' wet wt at 60% Vo2max and showed the same value at the highest work load. Ten min after exercise the PDCa activity had decreased to 0.60 mmol min-l kg-I wet wt. DISCUSSION The absence of significant lactate accumulation at 30% ~o,,a, indicates that at this low work load pyruvate formation from glucolysis was equally matched by the rate of decarboxylation by PDC, and in addition minimum disturbance in the cytoplasmic redox potential had occurred.
The absence of acetyl-CoA, and particularly acetylcarnitine accumulation signify that the turnover rate of the citric acid cycle and the rate of NADH oxidation by the electron transport chain must have been sufficient in matching the rate of formation of acetyl-CoA from pyruvate. Previous studies of Sahlin et al. (1987) have shown that the concentration of NADH in muscle may actually decrease during exercise at low work intensities approaching 30 yo VoZmax. The rate of formation of acetyl-CoA calculated from the amount of PDC in the activated form (PDCa), and assuming full catalytic activity was 0.8 mmol acetyl-CoA min-' kg-' wet wt, consistent with a glucolytic flux rate of 0.4 mmol glucosyl units min-' kg-'. This is of the same order of magnitude as previously observed at 30% ~ o , m a x ; i.e. 0.3 mmol glucosyl units min-l kg-I (Saltin & Karlsson, 1971). At the higher work loads, 60% and 90% of Vo20pmax, the rate of pyruvate formation from glucolysis was higher than the catalytic rate of PDC as shown by accumulation of lactate. The accumulation of lactate also indicates an increase in the redox potential within the cytoplasmic compartment and is consistent with increases in the muscle NADH concentration shown by Sahlin et al. (1987) to occur during exercise of similar intensities. Accumulation of acetyl-CoA and acetylcarnitine during exercise at work loads
-
D.Constantin-Teodosiu et al.
370
100,
80 6 0 40
20 0
04 0
20 Acetyl-CoA
10
ipmOl
kg
1
30
3
d.wf.1
Fig. 2. The relationship between the contents of wet! lcarnitine and acet) I-CoA in the vastus lateralis and during exercise (m). The muscle at rest (0) regression line is: JJ = 0.49x+0.89; Y = 0.899.
, 1
1
I
1
90%
E
VO, m a x
0
J 0
0
4
8
11
21 mln
2
Y
Fig. 1. Thc concentrations of lactate, CoASH, carnitine and their acetylated forms (mean SEXI) and the activity of PDCa in vastus lateralis muscle at rest, at the end of the three periods of exercise and after a 10 rnin rest period. m'ork loads (OOC;)l,,,Rs) are indicated.
corresponding to 60 and 9001~V,,~,n,,, indicates that the rate of condensation of acetyl groups with oxaloacetate is less than its rate of formation. The limitation here could be enzyme activities of the citric acid cycle or availability of substrates or of cofactors of the cycle. Thus a lack of NAD+ due to insufficient electron transport activity or even the lack of CoASH itself acting at the level of oxoglutarate dehydrogenase could limit the catalytic activity of the cycle. Transformation of PDC to the active form (PDCa) was related to the intensity of the exercise, increasing from 0.4 pmol acetyl-CoA min-' kg-' wet wt at rest to 0.8 at 30y/0 I',,,,,, and to 1.3 at the two highest work loads. ?'he activation of PDC by exercise has been shown earlier (Ward et ul. 1982, Dohm e t ul. 1986, Brozinick et ul. 1988) but the mechanism for the activation is not known. Hennig et ul. (1975) showed a relationship in time between PCr degradation and PDC transformation but there is no obvious casual relationship between PCr decrease and the activities of the PDC transforming kinase and phosphatase enzymes. However, it is known that transformation of the cnzl-me to the active form can be induced Zn vitro by increase in Ca2+and Mg". T h e La2+release into the sarcoplasm which determines exercise intensity could serve as a physiological regulator of PDC transformation during exercise. However, this infers that a relation exists betwecn sarcoplasmic and intramitochondrial Ca2+ concentrations. Anothcr possible mechanism is
Acetyl group accumulation during exercise regulation by the A D P concentration in the mitochondria. A D P is known to inhibit the kinase which inactivates PDC. If P D C phosphatase is active an inhibition of the kinase by ADP would be sufficient to regulate PDCa formation in relation to the work load. While work load determines the A T P turnover rate this in turn will regulate the A D P concentration in the sarcoplasm and in the mitochondria. Also increased concentration of pyruvate inhibits the PDC kinase and stimulates the transformation of PDC to active form. I n earlier studies of PDC activity of isolated pyruvate dehydrogenase preparations from bovine kidney and heart (Pettit et a/. 1975) and pig heart (Cooper et al. 1975), it was shown that the transformation of PDC is also dependent on the N A D H / N A D + and acetyl-CoA/CoASH ratios, with increasing ratios inhibiting the transformation to active form. I n the present study on human muscle tissue during contraction, this inhibition of the transformation was not observed. Apparently the contraction induced stimulation of the PDC transformation via increased concentrations of Ca2+, pyruvate and ADP overcomes the inhibitory effects of acetylCoA accumulation and results in simultaneous increases of the PDCa form and accumulation of acetyl-CoA (Fig. I). I t is also known that the catalytic activity of the transformed PDC complex can be inhibited by acetyl-CoA and N A D H accumulation (for references see Wieland 1983). This mechanism regulates the PDC activity in concert with the turnover rate of the citric acid cycle and of the electron transport chain. I f the PDCa during exercise (see Table 1) is fully active the maximum catalytic rate would correspond to a pyruvatc degradation of rate 20-25 pmol s-l kg-' wet wt. This rate is supported by a CoASH concentration in the muscle at rest of 9 p m o l kg-' wet wt (dry wt concentration divided by 4.2; see Table 1) with the result that the entire store of CoASH in the muscle could theoretically be acetylated within less than one second. If this occurred the result would be the immediate inhibition of PDC as well as of the citric acid cycle at the level of oxoglutarate dehydrogenase. T h e concentration of carnitine in wet muscle tissue at rest was 3600 pmol kg-' thus 400 times higher. T h e same relation was observed between the acetylated products of CoA and carnitine both at rest and during exercise (Fig. 2), indicating a rapid
37 1
transfer of intramitochondrially formed acetyl groups primarily bound to CoA and thereafter distributed to the carnitine store in the whole cell. T h i s rapid transfer is mediated via carnitineacetyl transferase (CAT) and carnitineacetylcarnitine translocase. These enzymes have been shown to have high activities in skeletal muscle (Pande & Parvin 1976, Bieber et a/. 1982) and enables carnitine to act as a buffer for excess acetyl-CoA production (Lysiak et a!. 1988, Uziel et a/. 1988) opposing the depletion of CoASH. At the same time the buffering capacity of carnitine protects against an excessive acetylCoA accumulation which otherwise may inhibit the catalytic activity of PDC. T h e P D C / C A T system is likely to be of greatest importance during activity necessitating frequent bursts of near maximal effort, when it will serve to 'smooth out' extremes in the acetyl-CoA accumulation. Acetyl units stored as acetylcarnitine during periods of heavy exercise will be available for utilization during rest periods or if exercise intensity is decreased. In this way the P D C / C A T system is potentially an important element in the overall metabolic architecture of the mitochondrion. The authors gratefully acknowledge support for this study by way of grants from the Swedish Research Council (02647 and 07136), the Karolinska Institutet, the Swedish Sports Research Council, and the Swedish Work Environment Fund (81/0173). Roger Harris gratefully acknowledges financial support from Sigma Tau s.p.a., Rome, and the Horse-race Betting Levy Board, London. REFERENCES ALKONYI, I., KERNER, J. & SANDOR,A. 1975. The possible role of carnitine and carnitine acetyltransferase in the contracting frog skeletal muscle. F E B S Lett 52, 265-268. BERGSTROM, J. 1962. Muscle electrolytes in man. Determined by neutron activation analysis on needle biopsy specimens. A study on normal subjects, kidney patients, and patients with chronic diarrhoea. Scand 3 Cldn Lab Invest 14, Suppl 68, 1-110. BIEBER, L.L., EMAUS,R., VALKNER, K. & FARREL, S. 1982. Possible functions of short-chain and medium-chain carnitine acyltransferases. Fed Proc 41, 2858-2861. BROZINICK JR, J.T., PATEL, V.K. & DOHM, G.L. 1988. Effects of fasting and training on pyruvate dehydrogenase activation during exercise. Znt 3 Biochem 20, 297-301.
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J.I., R E D I ) . ~W.G., ~, SANJAK,M.& 1 iOD.4CH, HARRIS, R.C. & FOSTER, C.V.L. 1990. Changes in muscle free carnitine and acetylcarnitine with R . 1986. Carnitine metabolism during prolonged increasing work intensity in the thoroughbred horsc. exercise and recovery in humans.3 ,4ppl Ph,ysd 61, Eur 3 Appl Ph,blsiol 60, 81-85. 1275-1 278. I G , G., LOFFLER, G. & WIELAND, O.H. 1975. (;ARI.IN, J.I., HARRIS,R.C., CtDERBL.4D. G., ice and inactive forms of pyruvatedehydroCONST.ANTIN-TEODOSIU, D., SNOW, D.H. & genasc in skeletal muscle as related to the metabolic HULTVAX,E. 1990. Associated between muscle and functional stare of the muscle cell. F E B S Lett aceryl-Co.4 and acetylcarnitine levels in the exer59, 142-14.5. cising horse. 3 .4ppI Physiol 69, 4 2 4 5 . ON, D.I..F., STRATMAN, F.W., SHRAGO, E., 'TROM, 0. & .\I.~RTENSSO\, J. N'AGL~, F.J., ~ I A D D E NM., , HANSON, P. & CARTER, 1982. Urinar! excretion of carnitine and its A.1,. 1983. Effects of' acute moderate-intensity deri\ati\es in newborns. Biochem .+fed 27, 26C265. exercise on carnitine metabolism in men and C:MXRHL.AD. G . , CIRLIS,J.I., COXSTAYTIN-TEODOSIU, women. J Appl Ph~ysiul55, 489495. D., I h R P t x , P. & WLLT%~.AN, E. 1990. Radioisotopic LYSIAK, W., LILLY,K., TOTH, Y.P. & BIEBER, L.L. assa! s of Co..\SH and carnitine and their acetylated 1988. Effect of the concentration of carnitine on forms in human skeletal muscle. .4na/ Biorhem 185, acetylcarnitine production by rat heart mitochon?/4--2i8. dria oxidizing pyruvate. Nutrition 4, 215-219. C:HILDRtS3, (;. c., SACKTOR, B. & ~ R . ~ Y W R D.R. , PANDL, S.V. & PARVIN, R. 1976. Characterization of 1966. Function of carnitine in thc fatty acid oridasecarnitinc acetylcarnitine translocase system of heart mitochondria. 3 Biol Chem 251, 6683-6601. deficient insect flight muscle. 3 Biol Chem 242, PETTIT, F.H., PELLEJ,J.W. & REED, I-. 1975. 7.54-7hO. Regulation of pyruvate kinase and phosphatase by COOPER, R.H., RANDLE,P. J. & DENTOS,K..\1. 197.5. acetyl-CoA/CoA and NADH/NAD ratios. Stimulation of phosphorylation and inactivation of Biorhrm Bioph,ys Res Comm 65, 575-582. pyruvate dehydrogenase by Physiological inhibitors SAHLIN, K., KATZ,'4. & HENRIKSSON, J. 1987. Redox of the pyuvate dehydrogenase reaction. Strrurr state and lactate accumulation in human skeletal 257, 808-809. I h v r o \ ; , R . M . RAKDL.E, P.J., RRIGDES,€3. J., COOPLR, muscle during dynamic exercise. Biochem 3 245,
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K . H . , KERREY,AL., P.ASK, H.T., S ~ V ~ R SD.L., ON, STANSBIE, D. & WHITEHOCSE, S. 197.5. Regulation of mammalian pyruvate dehl-drogenasc. .21ol Cell Bioi.hem 9. 27-53. DOHM, G.I.., P.ATEL,\.-.K. & KASPARF.K, G.J. 1986. Regulation of muscle p! ruvate metabolism during exercise. Riochem Med .Ilrt Biol 35, 26C266. FOSTER, C.\.-.L.& H.4RRIS, R.C. 1987. Formation of acetylcarnitine in muscle of horse during high intensity exercise. Eur 3 .-lppl Ph,ysiol 56, 639-642. I $ 4 R R I S , R.C., HULTMAS, E. & XORDESJO,L.-O. l9i4. Glycogen, glycolytic intermediates and high-energy phosphates determined in biopsy samples of musculus quadriceps femoris of man at rest. Methods and variance of values. Srond 3 Clin Lob In7'est 33, 109-120. HARRIS, R.C., FOSTER, C.V.1,. & HVI,TMAS, E. 1987. 4cetylcarnitine formation during intense muscular contraction in humans. j"4ppf Ph.ysinf 63, 440442.
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S.mLis, K. 1990. Muscle carnitine metabolism during incremental dynamic exercise in humans. Acta Physiol Srand 138, 259-262. S 4 L T I S , B. & KARI.SSON, J. 1971. Muscle glycogen utilization during work of different intensities. In : B. Pernow & B. Saltin (eds) Muscle Metabolism During Exercise, Vol. 11, pp. 289-300, Plenum Press, New York. LZIEL, G., GARAVAGLIA, B. & DI DONATO, S. 1988. Carnitine stimulation of pyruvate dehydrogenase complex (PDHC) in isolated human skeletal muscle mitochondria. Muscle & Nerce 11, 72&724. WARD,G. R., SUTTON, J.R., JONES,N.L. & TOEWS, C.J. 1982. Activation by exercise of human skeletal muscle pyruvate dehydrogenase in aiuo. Clin Sci 63, 87-92. WIELANU,O.H. 1983. T h e mammalian pyruvatc dehydrogenase complex :structure and regulation. Rer Phyriol Rinchm Pharmacol 96, 123-170.
Acetyl group accumulation and pyruvate dehydrogenase activity in human muscle during incremental exercise D. C O N S T A N T I N - T E O D O S I U , J. I. C A R L I N , G. C E D E R B L A D " , R. C. H A R R I S t and E. H U L T M A N Departments of Clinical Chemistry I" and 11, Huddinge University Hospital, Karolinska Institutet, S-141 86 Huddinge, Sweden CONSTANTIN-TEODOSIU, D., CARLIN, J. I., CEDERBLAD, G., HARRIS, R. C. & HULTMAN, E. 1991. Acetyl group accumulation and pyruvate dehydrogenase activity in human muscle during incremental exercise. Acta P h y d &and 143, 367-372. Received 10 April 1991, accepted 31 July 1991. ISBN 0001-6772. Departments of Clinical Chemistry I and 11, Huddinge University Hospital, Karolinska Institutet, Sweden The changes in the muscle contents of CoASH and carnitine and their acetylated forms, lactate and the active form of pyruvate dehydrogenase complex were studied during incremental dynamic exercise. Eight subjects exercised for 3 4 minutes on a bicycle ergometer at work loads corresponding to 30, 60 and 90% of their ~o,,,,. Muscle samples were obtained by percutaneous needle biopsy technique at rest, at the end of each work period and after 10 minutes of recovery. During the incremental exercise test there was a continuous increase in muscle lactate, from a basal value of 4.5 mmol kg-' dry weight to 83 mmol kg-' at the end of the final period. The active form of pyruvate dehydrogenase complex increased from 0.37 mmol acetyl-CoA formed per minute per kilogram wet weight at rest to 0.80 at 3004 l&max, 1.28 and 1.25 at 60 and 9004 respectively. Both acetyl-CoA and acetylcarnitine increased at the two highest work loads. The increase of acetyl-CoA was from 12.5 pmol k g ' dry weight at rest to 27.3 after the highest work load and for acetylcarnitine from 6.0 mmol kg-' dry weight to 15.2. The CoASH and free carnitine contents fell correspondingly. There was a close relationship between acetyl-CoA and acetylcarnitine accumulation in muscle during exercise, with a binding of 500 mol acetyl groups to carnitine for each mole of acetylCoA accumulated. The results imply that the carnitine store in muscle functions as a buffer for excess formation of acetyl groups from pyruvate catalyzed by the pyruvate dehydrogenase complex.
vo,,,,,
-
Key Words: Acetylcarnitine. acetyl-CoA, carnitine, coenzyme A, dynamic exercise, pyruvate dehydrogenase
The three enzymes which together constitute the pyruvate dehydrogenase complex (PDC) (pyruvate dehydrogenase El-EC 1 .2.4.1, dihydrolipoyl transacetylase E,-EC 2 . 3 . 1 .12, dihydrolipoyl dehydrogenase E,-EC 1 .8.1.4) catalyse Correspondence : Prof. Eric Hultman, Dept. of Clinical Chemistry 11, Huddinge University Hospital, S-141 86 Huddinge, Sweden. t Present address: (R. C. Harris) Department of Comparative Physiology, The Animal Health Trust, Newmarket, Suffolk, England.
the oxidation of pyruvate to acetyl-CoA. The acetyl-CoA formed condenses with oxaloacetate to give citric acid which then undergoes further degradation in the citric acid cycle. Activation of the pyruvate dehydrogenase component of the complex (PDH, EC 1.2.4.1) is brought about through dephosphorylation mediated by a protein phosphatase, while the enzyme is deactivated by phosphorylation by protein kinase. This activation of the P D H component transforms PDC from the inactive to the active form, i.e. PDCa. Effectors of the two interconverting
367
368
D.Constantin- Teodosiu et al.
enz! nits include pyruvate and ADP which inhibit the kinase and Ca'* and Mg'+ which both actiwte the phosphatasc and inhibit the kinase (Denton e/ rrl. 19i.5). I t has also been s h o w that the P I X transformation to active form in kidney and heart preparations is inhibited bl- increasing ratios of YL4D€1/X.4D and acetyl-Co.4/Co.4 (Cooper et a / . 197.i, Pettit et a / . 197.5), and that the catalytic acti\-ity of the PDCa enzyme is regulated by end-product inhibition b!- acetylCo.4 and N-lDH (for references see Wieland 1983). Recent studies in humans and equines h a w demonstratcd a marked increase in the acetl-Icarnitine content of skeletal muscle during high intensity exercise of short duration (Lennon et ill. 198.3, Carlin el u / . 1986, Foster & Harris 1987, tiarris et a(. 1987, Harris & Foster 1990). Studies of acetylcarnitine formation during human exercise with increasing work intensity h a w also been presented showing large increases in acetylcarnitine at 7.j0, and 100°, C;z, (Sahlin 1990). These findings are consistent with a role for carnitine in the regulation of the mitochondria1 acetyl-CoL4/CoASH ratio by buffering excess acctl-l-CoA production from pyruvate (-4lkonyi et a / . 1975, Childress et a / . 1966). By acting as an acceptor for acetyl groups from acetyl-CoA, carnitine will help in maintaining the catalytic activitl- of PDCa under conditions where the rate of acetyl-CoA condensation with oxaloacetatc is less than its rate of formation from pyruvate. In a recent stud!-, formation of both acetyl-CoA and acetylcarnitine was investigated during treadmill exercise of increasing intensit?. in thoroughbred horses (Carlin et a / . 1990). At high work intensities a plateau of both compounds was reached. There was a significant relationship between the contents of the two compounds in contracting muscle. T h e aim of the present study was to investigate PDC transformation during incremental work in human subjects, and to relate this to the accumulation of acetyl groups and the buffering function of carnitine.
M A T E R I A L S A N D METHODS Eight healthy men participated in this study. Their mean (range) age, height, weight and &',,,,, were 27 (22-32) years, 180 (175-188) cm, 73 (67-82) kg and 3.9 ( 3 . j 4..5)I min-', respectivell-. The subjects were physicall! active and pcrformed exercise on a regular
basis though none of them was a competitive athlete. .\I1 subjects were familiarized with the testing procedures before the start of the study including an initial bicycle exercise test. The subjects were informed about the possible risks of the study hefore giving their voluntary consent. The experiincntal protocol was approved by the Ethics Committee of the Karolinska Institute, Stockholm, Sweden. Experimental procedure. Maximal oxygen uptake capacity (I;',,,,,,) was determined during progressive workloads on a bicycle ergometer. Oxygen consumption (Q, carbon dioxide production (&,,) and the respiratory exchange rate (REK) were monitored e\-ery 30 s using an oxygen analyser (Oxycon-4, llijnhardt, Runnik, Netherlands) calibrated with known gases (0, and CO,). For the eight subjects mean work ,loads ( fSD) corresponding to 30, 60 and 909, of V,,,,,x were 8 9 f l l W, 189+12 W and 267 34 W, icspectively. \Lluscle biopsy samples were obtained from thc vastus lateralis by a percutaneous needle biopsy technique according to Bergstrom (1962) at rest, after 4 min rxercise at work loads corresponding to 30 and 60°, F,2n,c,x, and after 3 rnin at 90"1~, V",,,,,,, and after 10 min rest. Each tissue sample waB immediately frozen b>- insertion of the needle in liquid nitrogen. The delay between freezing of the 'exercise '-samples and the end of the exercise was 5-10 s. Aiia/ytrcal methods. Muscle samples were divided under liquid N,into two parts. One part of the sample was freeze-dried, dissected free from visible connective tissue and blood, and powdered. Seven to 12 mg of the muscle power was analysed for carnitine, acetylcarnitine, CoASH and acetyl-CoA on the same extract by the radioisotopic methods described by Cederblad et a/. (1982, 1990). The other part (20-30 mg) of the muscle sample was used for determination of pyruvate dehydrogenase complex in principle according to the method described by Dohm et al. (1986). Instead of measurement of "CO, formation in this method the production of acetyl-CoA from added pyruvate was analysed by the method described by Cederblad et 61. (1990). Lactate was determined in the same extract by the method described by 1Iarris et al. (1974).
RESULTS T h e contents of lactate, acetyl-CoA, CoASH, acctj-lcarnitine and carnitine as well as PDCa activity are given in Table 1. There was no significant accumulation of lactate or acetyl groups in the muscle tissue after exercise at a work load corresponding to V, maI (Fig. 1). At the higher work intensities of 60bo and 900j0 ~ o j o ? n , 6muscle x, lactatc increased to 29.8 and 82.7 mmol kg-' dry wt, respectively, and both acetyl-CoA and acetylcarnitine contents
Acetyl group accumulation during exercise
369
Table 1. Muscle concentrations of CoASH, carnitine, their acetylated forms and lactate and the measured activity of the active form of pyruvate dehydrogenase complex at rest and after exercise with different intensities ~~~
Work load, yo of
~o',,,,,
30
60
90
10 min post
~-
Variable ~~~~
At rest ~
Lactate mmol kg-' dry wt PDCa' CoASH pmol kg-l dry wt Acetyl-CoA pmol kg-' dry wt Free carnitine mmol kg-' dry wt Acetylcarnitine mmol kg-' dry wt
4.5f1.1
9.3+4.5*
29.8+ 17.2"'
82.7i30.3""
25.3k 16.4"*
0.3750.22 36.7k8.6
0.80+0.26*" 33.5k8.6
1.28+0.39*" 29.0 & 7.1
1.25f0.53*" 26.6 _+ 7.3"
0.60+0.28 37.nk 9.3
12.5f4.3
12.5k4.1
18.7k4.7**
27.3+7.5*"
13.6k5.0
15.2f2.2
12.9f 3.8
10.7 k 2.7""
6.5k 1.5'"
12.1 1.6*
+
6.2 2.3
15.2*2.8**
10.7k2.5"
6.0 2.7
+
10.1 f2.4'
mmol acetyl-CoA formed min-' and kg wet wt at 307 "C, n = 6. Comparison (Student's paired t-test; n = 8) between values at rest and after the three periods of exercise and after 10 min rest; * .2P < 0.05, ** .2P < 0.01. Values are given as mean+SD. +
were increased significantly at the end of the work periods reaching after 90% ~o',,,,, 27.3 pmol kg-' dry wt and 15.2 mmol kg-' dry wt, respectively. The concentrations of CoASH and free carnitine decreased correspondingly. These changes were reversed during a I0-min recovery period at the end of which metabolite contents were approaching those found prior to the start of exercise. There was a significant correlation between contents of acetyl-CoA and acetylcarnitine in muscle tissue at rest and during exercise (Fig. 2). Measured PDCa activity increased from 0.37 mmol acetyl-CoA min-' kg-' wet wt to 0.80 mmol at the end of the 4 min work at 30% Yo,,,,. The activity increased further to 1.28 pmol acetyl-CoA min-' kg-' wet wt at 60% Vo2max and showed the same value at the highest work load. Ten min after exercise the PDCa activity had decreased to 0.60 mmol min-l kg-I wet wt. DISCUSSION The absence of significant lactate accumulation at 30% ~o,,a, indicates that at this low work load pyruvate formation from glucolysis was equally matched by the rate of decarboxylation by PDC, and in addition minimum disturbance in the cytoplasmic redox potential had occurred.
The absence of acetyl-CoA, and particularly acetylcarnitine accumulation signify that the turnover rate of the citric acid cycle and the rate of NADH oxidation by the electron transport chain must have been sufficient in matching the rate of formation of acetyl-CoA from pyruvate. Previous studies of Sahlin et al. (1987) have shown that the concentration of NADH in muscle may actually decrease during exercise at low work intensities approaching 30 yo VoZmax. The rate of formation of acetyl-CoA calculated from the amount of PDC in the activated form (PDCa), and assuming full catalytic activity was 0.8 mmol acetyl-CoA min-' kg-' wet wt, consistent with a glucolytic flux rate of 0.4 mmol glucosyl units min-' kg-'. This is of the same order of magnitude as previously observed at 30% ~ o , m a x ; i.e. 0.3 mmol glucosyl units min-l kg-I (Saltin & Karlsson, 1971). At the higher work loads, 60% and 90% of Vo20pmax, the rate of pyruvate formation from glucolysis was higher than the catalytic rate of PDC as shown by accumulation of lactate. The accumulation of lactate also indicates an increase in the redox potential within the cytoplasmic compartment and is consistent with increases in the muscle NADH concentration shown by Sahlin et al. (1987) to occur during exercise of similar intensities. Accumulation of acetyl-CoA and acetylcarnitine during exercise at work loads
-
D.Constantin-Teodosiu et al.
370
100,
80 6 0 40
20 0
04 0
20 Acetyl-CoA
10
ipmOl
kg
1
30
3
d.wf.1
Fig. 2. The relationship between the contents of wet! lcarnitine and acet) I-CoA in the vastus lateralis and during exercise (m). The muscle at rest (0) regression line is: JJ = 0.49x+0.89; Y = 0.899.
, 1
1
I
1
90%
E
VO, m a x
0
J 0
0
4
8
11
21 mln
2
Y
Fig. 1. Thc concentrations of lactate, CoASH, carnitine and their acetylated forms (mean SEXI) and the activity of PDCa in vastus lateralis muscle at rest, at the end of the three periods of exercise and after a 10 rnin rest period. m'ork loads (OOC;)l,,,Rs) are indicated.
corresponding to 60 and 9001~V,,~,n,,, indicates that the rate of condensation of acetyl groups with oxaloacetate is less than its rate of formation. The limitation here could be enzyme activities of the citric acid cycle or availability of substrates or of cofactors of the cycle. Thus a lack of NAD+ due to insufficient electron transport activity or even the lack of CoASH itself acting at the level of oxoglutarate dehydrogenase could limit the catalytic activity of the cycle. Transformation of PDC to the active form (PDCa) was related to the intensity of the exercise, increasing from 0.4 pmol acetyl-CoA min-' kg-' wet wt at rest to 0.8 at 30y/0 I',,,,,, and to 1.3 at the two highest work loads. ?'he activation of PDC by exercise has been shown earlier (Ward et ul. 1982, Dohm e t ul. 1986, Brozinick et ul. 1988) but the mechanism for the activation is not known. Hennig et ul. (1975) showed a relationship in time between PCr degradation and PDC transformation but there is no obvious casual relationship between PCr decrease and the activities of the PDC transforming kinase and phosphatase enzymes. However, it is known that transformation of the cnzl-me to the active form can be induced Zn vitro by increase in Ca2+and Mg". T h e La2+release into the sarcoplasm which determines exercise intensity could serve as a physiological regulator of PDC transformation during exercise. However, this infers that a relation exists betwecn sarcoplasmic and intramitochondrial Ca2+ concentrations. Anothcr possible mechanism is
Acetyl group accumulation during exercise regulation by the A D P concentration in the mitochondria. A D P is known to inhibit the kinase which inactivates PDC. If P D C phosphatase is active an inhibition of the kinase by ADP would be sufficient to regulate PDCa formation in relation to the work load. While work load determines the A T P turnover rate this in turn will regulate the A D P concentration in the sarcoplasm and in the mitochondria. Also increased concentration of pyruvate inhibits the PDC kinase and stimulates the transformation of PDC to active form. I n earlier studies of PDC activity of isolated pyruvate dehydrogenase preparations from bovine kidney and heart (Pettit et a/. 1975) and pig heart (Cooper et al. 1975), it was shown that the transformation of PDC is also dependent on the N A D H / N A D + and acetyl-CoA/CoASH ratios, with increasing ratios inhibiting the transformation to active form. I n the present study on human muscle tissue during contraction, this inhibition of the transformation was not observed. Apparently the contraction induced stimulation of the PDC transformation via increased concentrations of Ca2+, pyruvate and ADP overcomes the inhibitory effects of acetylCoA accumulation and results in simultaneous increases of the PDCa form and accumulation of acetyl-CoA (Fig. I). I t is also known that the catalytic activity of the transformed PDC complex can be inhibited by acetyl-CoA and N A D H accumulation (for references see Wieland 1983). This mechanism regulates the PDC activity in concert with the turnover rate of the citric acid cycle and of the electron transport chain. I f the PDCa during exercise (see Table 1) is fully active the maximum catalytic rate would correspond to a pyruvatc degradation of rate 20-25 pmol s-l kg-' wet wt. This rate is supported by a CoASH concentration in the muscle at rest of 9 p m o l kg-' wet wt (dry wt concentration divided by 4.2; see Table 1) with the result that the entire store of CoASH in the muscle could theoretically be acetylated within less than one second. If this occurred the result would be the immediate inhibition of PDC as well as of the citric acid cycle at the level of oxoglutarate dehydrogenase. T h e concentration of carnitine in wet muscle tissue at rest was 3600 pmol kg-' thus 400 times higher. T h e same relation was observed between the acetylated products of CoA and carnitine both at rest and during exercise (Fig. 2), indicating a rapid
37 1
transfer of intramitochondrially formed acetyl groups primarily bound to CoA and thereafter distributed to the carnitine store in the whole cell. T h i s rapid transfer is mediated via carnitineacetyl transferase (CAT) and carnitineacetylcarnitine translocase. These enzymes have been shown to have high activities in skeletal muscle (Pande & Parvin 1976, Bieber et a/. 1982) and enables carnitine to act as a buffer for excess acetyl-CoA production (Lysiak et a!. 1988, Uziel et a/. 1988) opposing the depletion of CoASH. At the same time the buffering capacity of carnitine protects against an excessive acetylCoA accumulation which otherwise may inhibit the catalytic activity of PDC. T h e P D C / C A T system is likely to be of greatest importance during activity necessitating frequent bursts of near maximal effort, when it will serve to 'smooth out' extremes in the acetyl-CoA accumulation. Acetyl units stored as acetylcarnitine during periods of heavy exercise will be available for utilization during rest periods or if exercise intensity is decreased. In this way the P D C / C A T system is potentially an important element in the overall metabolic architecture of the mitochondrion. The authors gratefully acknowledge support for this study by way of grants from the Swedish Research Council (02647 and 07136), the Karolinska Institutet, the Swedish Sports Research Council, and the Swedish Work Environment Fund (81/0173). Roger Harris gratefully acknowledges financial support from Sigma Tau s.p.a., Rome, and the Horse-race Betting Levy Board, London. REFERENCES ALKONYI, I., KERNER, J. & SANDOR,A. 1975. The possible role of carnitine and carnitine acetyltransferase in the contracting frog skeletal muscle. F E B S Lett 52, 265-268. BERGSTROM, J. 1962. Muscle electrolytes in man. Determined by neutron activation analysis on needle biopsy specimens. A study on normal subjects, kidney patients, and patients with chronic diarrhoea. Scand 3 Cldn Lab Invest 14, Suppl 68, 1-110. BIEBER, L.L., EMAUS,R., VALKNER, K. & FARREL, S. 1982. Possible functions of short-chain and medium-chain carnitine acyltransferases. Fed Proc 41, 2858-2861. BROZINICK JR, J.T., PATEL, V.K. & DOHM, G.L. 1988. Effects of fasting and training on pyruvate dehydrogenase activation during exercise. Znt 3 Biochem 20, 297-301.
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