PDC activity and acetyl group accumulation in skeletal muscle during prolonged exercise D. CONSTANTIN-TEODOSIU, Department

of Clinical

S-141 86 Huddinge,

Chemistry, Sweden

G. CEDERBLAD, Huddinge

University

AND

E. HULTMAN

Hospital,

Karolinska

Institute,

CONSTANTIN-TEODOSIU,D., G.CEDERBLAD,AND E. HULTMAN. FDC activity and acetyl group accumulation in skeletal muscleduring prolongedexercise.J. Appl. Physiol. 73(6): 24032407, 1992.--Seven subjectscycled to exhaustion 158t 7 (SE) min] at -75% of their maximal oxygen uptake (vo2 maX). Needle biopsy sampleswere taken from the quadriceps femoris muscleat rest, after 3,10, and 40 min of exercise,at exhaustion, and after 10 min of recovery. After 3 min of exercise, a nearly complete transformation of the pyruvate dehydrogenasecomplex (PDC) into active form had occurred and wasmaintained throughout the exercise period. The total in vitro activated PDC was unchanged during exercise. The muscleconcentration of acetyl-CoA increasedfrom a resting value of 8.4 t 1.0to 31.6.zt3.3pmollkgdrywt at exhaust ion and that of acetylcarnitine from 2.9 -t 0.7 to 15.6 t 1.6 mmol/kg dry wt. This was accompanied by corresponding decreasesin reduced CoA (CoASH) from 45.3 t 3.1 to 25.9 k 3.1 pmol/kg dry wt and in free carnitine from 18.8 t 0.7 to 5.7 t 0.5 mmol/kg dry wt. Acetyl group accumulation, in the form of acetyl-CoA and acetylcarnitine, was maintained throughout exerciseto exhaustion while the glycogen content decreasedby 90%. This suggests that availability of acetyl groups was not limiting to exercise performance despite the nearly total depletion of the glycogen store. The increasedacetyl-CoA-to-CoASH ratio during exercise causedinhibition of neither the PDC transformation nor the calculated catalytic activity of active PDC.

sults in inactivation of the PDC. The total activity of PDC (PDCt) is assayed after complete in vitro transformation of the complex into PDCa either by activation of the endogenous phosphatase or by addition of exogenous phosphatase. The other regulatory mechanism is endproduct inhibition of the catalytic activity of PDCa by acetyl-CoA and NADH. In vitro studies of PDC have shown that both the transformation to the active form and the catalytic activity of PDCa can be inhibited by high ratios of acetyl-CoA to reduced CoA (CoASH) and NADH to NAD+ (12,25,29). The mechanism regulating the activity of PDC during exercise is still largely unknown. We have previously found a gradual increase in PDCa in human skeletal muscle during short-term exercise with increasing work loads (10). The purpose of the present study was to determine the transformation state of PDC and, if possible, its catalytic activity and the acetyl during aP rolonged submaximal ex!TsuP accumulation ercise continued to exhausti on. Another aim of the study was to analyze the acetyl group availability in muscle at the end of exercise when the glycogen store is near depletion.

carnitine metabolism;acetylcarnitine; acetyl-coenzyme A; pyruvate metabolism;glycogen metabolism

MATERIAL AND METHODS

THE ENERGY REQUIRED fur the resynthesis of ATP dur-

ing prqlonged exercise at 70-80% of maximal oxygen uptake tvo$j max) is to a large extent derived from muscle glycogen utilization. It is generally accepted that the availability of muscle glycogen limits the exercise capacity at this work intensity (3). The main pathways for the pyruvate produced from glycogen are reduction to lactate in the cytosol and oxidation to acetyl-CoA in the mitochondria. The latter reaction is catalyzed by pyruvate dehydrogenase complex (PDC). Exercise increases pyruvate utilization by enhancing glycogenolysis and pyruvate oxidation to acetyl-CoA by increasing the activity of PDC (5, 19, 30). The catalytic activity of PDC is controlled by two regulatory mechanisms: covalent -transfermation (23) and end-product inhibition (14,29). The first mechanism involves dephosphorylation of the complex by a specific phosphatase (EC 3.1.3.43), resulting in formation of the active form (PDCa). Conversely, phosphorylation of the complex by a kinase (EC 2.7.1.99) re-

Seven healthy male subjects participated in the present . study. Their mean (range) age, weight, height, and vo 2 maxwere 28 (23-35) yr, 79 (69-105) kg, 182 (172-193) cm, and 52 (42-58) ml 0,. mine1 . kg body wt-I, respectively. All subjects were informed of the purpose and nature of the experiment before their voluntary consent was obtained. The study is part of a project approved by the Ethics Committee of Karolinska Institute, Stockholm, Sweden. Experimental protocol. Vozrnax was determined in the weeks preceding the experiment by means of an electrically braked cycle ergometer (Siemens-Elema, Stockholm, Sweden) and a gradually increasing exercise protocol. Ventilation parameters, oxygen consumption (VO&, CO, production, and respiratory exchange ratio (RER) were measured during exercise using an on-line gas analysis system (Oxycon-4, Mijnhardt, The Netherlands) that was calibrated using certified 0, and CO, mixtures (AGA, Sweden). The results were used to calculate a work load that corresponded to 75% of each sub* ject’s VO, m8X. Subjects were requested not to perform any strenuous

0161-7567/92 $2.00 Copyright 0 1992 the AmericanPhysiological Society

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2404

PDC ACTIVITY

AND ACETYL

measured after incubation with Ca’+, Mg2+, DCA, glucose, and hexokinase to achieve total transformation of the enzyme complex. The acetyl-CoA formed in the PDC reaction was determined as described above. Statistid analysis. The data were analyzed by oneway analysis of variance (ANOVA) for repeated measurements. When the ANOVA resulted in a significant F ratio (P < 0.05), the location of significance was determined using Scheffh’s test. Values are means t SE unless otherwise indicated.

r z s F 21 F ‘t.I E z E Eo 0 P P 0

f

I

I

I

10

20

I

I

0

30

40

50

N-

exercise

at 75%

VO, max,

58*7

min

GROUP ACCUMULATION

I

1

60 -

70 min rest ) 10 min

RESULTS

1. Muscle activities of pyruvate dehydrogenase complex (PDC) at rest, during 58 k 7 min of exercise at 75% of maximal O2 consumption (VO 2 -), and after 10 min of recovery. PDCa and PDCt, active form and total PDC, respectively. Values are means k SE of 7 subjs.

The first 3 min of exercise resulted in a nearly complete transformation of PDC to PDCa (Fig. 1). This represented a four- to fivefold increase in PDCa compared with the preexercise level. After the initial 3 min of exercise, PDCa was maintained constant to the end of exercise (P > 0.05) and returned thereafter to the preexercise exercise on the day before and the day of the experiment. level within a IO-min recovery period. PDCt was not inOn the day of the study, all subjects reported to the laboratory after an overnight fast and were requested to cycle fluenced by the exercise (P > 0.05). The muscle glycogen content decreased continuously at a work load corresponding to ~75% of their VO, max during the exercise from a mean of 432 t 33 mmol glucountil exhaustion (the point at which a pedal frequency of syl units/kg dry wt at rest to 53 t 16 mmol glucosyl 50 rpm could not be maintained). The mean exercise units/kg dry wt at exhaustion (Fig. 2). The muscle lactate work load was 213 t 8 W. Exercise time to exhaustion concentration increased during the first 3 min of exercise averaged 58 -t 7 min (range 50-90 min). and decreased successively thereafter (Fig. 2). Muscle biopsy samples (2) were taken from the vastus The muscle acetyl-CoA and acetylcarnitine concentralateralis muscle at rest, after 3,10, and 40 min of exercise, tions increased rapidly at the onset of exercise; those of at exhaustion, and after 10 min of recovery. The muscle biopsy samples were immediately frozen by plunging the CoASH and free carnitine decreased correspondingly needle into liquid nitrogen. The time delay between the (Fig. 3). However, the initial mean increase in acetylinsertion of the needle into the muscle and freezing was CoA, -24 pmollkg dry wt, was twice the corresponding -5 s. Samples were stored in liquid nitrogen until the mean decrease in CoASH, i.e., -10 pmol. The acetylCoA-to-CoASH ratio increased almost five times, from analyses were undertaken. Analytic methods, Each frozen muscle sample was divided into two parts under liquid nitrogen. One part was freeze-dried, dissected free from visible connective tissue and blood, and powdered. Seven to ten milligrams of muscle powder were then extracted with 0.5 M perchloric acid (PCA) containing 1 mM EDTA; after centrifugation the supernatant was neutralized with 2.2 M KHCO, (17). Free carnitine, acetylcarnitine, CoASH, and acetyl-CoA were measured in the neutralized extract by enzymatic assays using radioisotopic substrates, as previously described (8, 9). Briefly, for the determination of CoASH, CoASH reacted with acetylphosphate in a reaction catalyzed by phosphotransacetylase to form acetyl-CoA. In 60 1 the assay for acetylcarnitine, acetylcarnitine reacted 50 -I T with CoASH in a reaction catalyzed by carnitine acetyltransferase (CAT) to form acetyl-CoA. The acetyl-CoA was determined as [~*C]citrate after condensation with [‘“C] oxaloacetate by citrate synthase. Lactate was determined as described earlier (17). For the determination of muscle glycogen content, 1.0-2.5 mg of muscle powder I t I were digested in 0.5 M NaOH at 80’C for 10 min and 1 o\ neutralized with HCl-citrate buffer (pH 4.9). After cenb trifugation, the glycogen present in the supernatant was 0 , 0 10 20 30 40 50 60 70 min hydrolyzed with a-amyloglucosidase and analyzed for exet’cise at 754b V 0, maX, 58+7 mln ------+d glucosyl units by an enzymatic method (17). The other part of the frozen muscle was used to deterFIG. 2. Muscle contents of glycogen and lactate at rest, during 58 + mine PDC activity (11). PDCa was measured after addi7 min of exercise at 75% b02max, and after 10 min of recovery. Values tion of NaF and dichloroacetate (DCA), and PDCt was are means t SE of 6 subjs for glycogen and 7 subjs for lactate. FIG.

I

1

I

J

I

f

I

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2405

PIE ACTIVITY AND ACETYL GROUP ACCUMULATION

ti I2

0 0 -

10I

I 20 exercise

I

30

I

I

40

at 75% V 0, m8x, 58*7

I

50 min

I

70 min

60 -

rest 10 min*

3. Muscle contents of reduced CoA (CoASH), carnitine, and their acetylated forms at rest, during 58 & 7 min of exercise at 75% v02 max9 and after 10 min of recovery. Values are means t SE of 7 subjs. FIG.

0.19 at rest to 0.92, and the acetylcarnitine-to-free carnitine ratio increased from 0.16 to 1.10 during the first, 3 min of exercise (Table 1). A further increase in these ratios to 1.29 and 2.19, respectively, was observed at 10 min of exercise. The ratios thereafter remained the same until exhaustion. During recovery, the muscle concentrations of acetyl-CoA and acetylcarnitine fell with corresponding increases in CoASH and free carnitine (Fig. 3), The sum of the muscle-free carnitine and acetylcarnitine concentrations was unchanged during exercise (Table 1). This was also true of the sum of the muscle CoASH and acetyl-CoA concentrations, except at 3 min of exercise, when it was slightly higher than at rest.

The present data demonstrate that there is a nearly complete transformation of PDC during prolonged submaximal exercise. This degree of transformation corresponds to a four- to fivefold increase in PDCa from the resting level. Transformation occurs during the very first minutes of exercise and is maintained until exhaustion. The increase in PDCa in response to exercise is in agree-

ment with previously reported results in rat skeletal muscle (5, 19) and human skeletal muscle (30). In a previous study we demonstrated that there was a gradual increase in PDCa in human skeletal muscle during shortterm exercise with increasing work loads (10). The content of PDCa is dependent on the relation between the activities of phosphatase and kinase. Calcium (13), pyruvate, ADP (21, 23), NAD+, and CoASH (25) have been shown to stimulate and NADH, acetyl-CoA (25), and ATP (21, 23) to inhibit the transformation of PDC in vitro, respectively. Calcium ions activate the phosphatase both by lowering the apparent Michaelis constant of the phosphatase for phosphorylated PDC (13,27) and by facilitating the binding of the phosphatase to the PDC (26). Experiments using isolated heart mitochondria (15) or fat cell preparations (24) have shown that an increase in the extramitochondrial free Ca2+ concentration to the range of 10B7 to lOa M results in an increase in PDCa. This Ca2+ concentration is of the same order of magnitude as the concentration necessary to induce muscle contraction. Pyruvate, at a concentration of 0.5 mM, has been shown to inhibit the kinase from cardiac muscle, kidney, and liver tissues by -50% (21,23). This would favor the transformation of PDC to PDCa. However, the pyruvate concentration in human skeletal muscle during moderate dynamic exercise is in the range of 0.1-0.2 mM (28, 30). A previous study by Linn et al. (23) showed that ADP inhibited the kinases from kidney, liver, and heart tissues, which would favor the transformation of PDC into the active form. The ADP inhibition on the kinases was competitive with respect to ATP. Small decreases in ATP-to-ADP quotients are known to occur during exercise (18), but the intramitochondrial concentrations during exercise are not known. It is therefore difficult to evaluate the effect of ATP-ADP changes on PDC transformation. An increase in the acetyl-CoA-to-CoASH ratio from 0 to 0.5 (12) and from 0.1 to 10 (25) has been shown to stimulate the kinase reaction and thus to induce a decrease in PDCa. In the present study with exercising muscle, the acetyl-CoA-to-CoASH ratio increased from -Ot2 at rest to - 1.0 after 3 min of exercise. A further increase in this ratio to 1.4 was observed at 40 min of exercise. Nevertheless, the exercise-induced PDC transformation remained constant, close to a maximum level during the entire exercise period. This has some bearing on the study by Ashour and Hansford (1) in rat skeletal muscle mitochondria, which showed that after the phosphatase activity was enhanced by Ca2+, the PDC trans-

1. Sums of muscle aeetyl-CoA and &ASH concenf;rutions, muscle acetyleurnitine and curnitine concentrations, and their ratios during exercise at 75% of VO, max: TABLE

Acetyl-CoA/CoASH Acetyl-CoA + CoASH, pmolfkg dry wt Acetylcarnitine/carnitine Acetylcarnitine + carnitine, mmol/kg dry wt

Rest

3 min

10 min

40 min

Exhaustion

Recovery

0.19-+u.o3 53.7*3*1 0.16tO.04

0.92tO.O9* 67.2*3.7* 1.10-t-o.i3* 22.0t1.3

1.29&U. 10* 63. W3.8 2.19-t0.29*t

1.43+0.10*~ 64.1t4.8 2.42-+0.35*-t

1.33*0.19* 57.5t3.9 2.95+0.46”?

0.69+0.12* 56.7k4.0 1.44t0.32*

19.7t1.2

22.0t1.2

21.3k1.4

2l.Ot1.5

21.6t0.8

Values are means + SE of 7 subjs. CoASH, reduced CoA; %B~ -,, maximal oxygen consumption. Values at exhaustion correspond to 58*7 min of exercise and those at recovery to IO min postexercise. * Significantly (P < 0.05) different from value at rest; t significantly (P < 0.05) different from value at 3 min of exercise. Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (192.236.036.029) on December 25, 2018.

2406

PDC ACTIVITY

AND ACETYL

formation remained elevated, even when the mitochondrial acetyl-CoA-to=CoASH ratio was high. Studies on purified preparations of PDC established that enzyme activity is inhibited by acetyl=CoA and NADH, the products of pyruvate oxidation (14,29). The data showed that acetyl=CoA inhibition was competitive with respect to CoASH (29). These data suggested that the acetyl-CoA-to-CoASH ratio could control the flux through PDC in vivo (31). The flux through PDC was calculated from the data obtained during the exercise period of 10 min to exhaustion when the acetyl group concentration (sum of acetyl=CoA and acetylcarnitine) was constant. Pyruvate formation from glycogen degradation was 2.46 mmol pyruvateemin-l. kg wet wt-l [calculated from the glycogenolytic rate of 5.30 mmol min-’ kg dry muscle-l (Fig. 2) and division of this number by 4.3 to express it per kilogram wet weight, i.e., 1.23 mol min-’ kg wet tissue-l]. Part of this pyruvate is transformed to lactate or alanine or is used by anaplerotic reactions. We did not measure lactate release during exercise but used the results of a similar study by Sahlin et al. (28). They found a lactate release from muscle corresponding to -0.4 mmol min-l kg? Over the same period of time the lactate content in muscle tissue fell by -0.1 mmol min-’ kg wet wt-I, which is similar to the value observed in our study (Fig. 2). The lactate formation rate thus corresponds to 0.4 - 0.1 = 0.3 mmol min-l . kg wet wt? In the same study by Sahlin et al. (as), alanine release and the anaplerotic reaction rate were calculated to account for -0.1 mmol pyruvate min-l kg wet wt? Thus of the total pyruvate formed from glycogenolysis, -0.4 mmol can be accounted for by reactions other than the PDC pathway, and the remaining 2.1 mm01 min-l skg wet wt-’ would correspond to the pyruvate flux through PDC. This is about the same as the in vitro measured PDCa activity, thus showing full catalytic activity of PDCa in muscle despite increased acetyl-CoAKoASH. During prolonged submaximal exercise some blood-borne glucose will also be taken up by the muscle and utilized. This contribution is not taken into account in the calculation. The amount of glucose taken up by leg muscle during moderate dynamic exercise is -0.1 mm01 . min-l kg wet wt-l after 5 min of exercise and increases during exercise up to -0.3 mmol +min-’ kg-l in the latter part of exercise (22). If the uptake is taken into account, the calculated flux through PDC will be slightly higher than the PDCa measured in the muscle, possibly because of some loss of PDC activity during muscle extraction. The sum of muscle acetyl-CoA and CoASH concentrations increased by 25% during the first minutes of exercise. This may indicate that CoA esters other than acetyl=CoA were metabolized and contributed to the CoASH pool. Muscle short-chain acyl-CoA is reported to be twice the sum of acetyl=CoA and CoASH in human muscle (7). Carnitine acetyltransferase catalyzes the reversible transfer of acetyl groups between CoA and carnitine. Acetylcarnitine can then be transported out of the mitochondria by a translocase (4). In this experiment, 360 pmol of acetylcarnitine were formed for each micromole of acetyl=CoA accumulated during the first 3 min of exercise. The relationship between acetyl groups bound to l

l

l

l

l

l

l

l

l

l

l

l

l

l

GROUP ACCUMULATION

CoA and to carnitine was almost constant throughout the exercise. An increase in the acetylcarnitine-to-free carnitine ratio in muscle during exercise has been observed in animals (6) and humans (10,16,20). The acetyl group buffering by carnitine is necessary for preservation of the mitochondrial CoASH and thus for continued pyruvate oxidation via PDC. During the first 3 min of exercise, - 11,000 pmol of acetyl groups have been transferred from the small pool of CoASH (-45 pmol) to carnitine. This demonstrates the important role of carnitine as a buffer for the PDC pathway, especially at the onset of exercise. The constancy of acetyl=CoA and acetylcarnitine concentrations between 10 min and exhaustion implies that the rate of acetyl=CoA formation was equal to the rate of acetyl=CoA oxidation by the tricarboxylic acid cycle. At exhaustion, when the glycogen store had decreased to -10% of the initial value, the acetyl group content in the cell was still of the same order of magnitude as that during the first one-half of the exercise period. It is generally accepted that depletion of the glycogen store limits the exercise capacity at this work intensity (3). The presence of a high concentration of acetyl=CoA at exhaustion indicates that the limitation observed in exercise performance depends on substrates or factors other than acetyl=CoA availability in the muscle. It has been suggested that carbohydrate depletion may impair aerobic energy production by reducing the level of tricarboxylic cycle intermediates (28), which could result in a reduced capacity for acetyl group oxidation. In conclusion, the present study showed that PDC transformation is nearly complete even after 3 min of exercise and is maintained throughout exercise until exhaustion despite acetyl group accumulation. The increase in acetyl-CoAKoASH during exercise did not seem to inhibit the catalytic activity of the enzyme. The high content of acetyl groups in muscle tissue at exhaustion with low availability of glycogen indicates that the presence of a high concentration of acetyl=CoA is not sufficient for continuation of the exercise. We thank Dr. Paul Greenhaff for advice concerning the vogrnax determinations and Agneta Laveskog for excellent technical assistance. This study was supported by grants from the Karolinska Institute, the Swedish Sports Research Council (75/91), and the Swedish Medical Research Council (2647 and 7136). Address for reprint requests: G. Cederblad, Dept. of Clinical Chemistry, Huddinge University Hospital, S-141 86 Huddinge, Sweden. Received 14 April 1992; accepted in final form 10 July 1992. REFERENCES ASHOUR, B., AND G. HANSFORD. Effect of fatty acids and ketones on the activity of pyruvate dehydrogenase in skeletal-muscle mitochondria. BE’ochem. J. 214: 725-736, 1983. 2. BERGSTROM, J. 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. Stand. J. Clin. Lab. Invest. 14, Suppl. 68: l-110, 1962. 3. BERGSTROM, J., AND E. HULTMAN. A study of the glycogen metabolism during exercise in man. Stand. J. Clin. Lab. b-west. 19: 21% 1.

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BIEBER, L. L.,R. EMANS, K. VALKNER,AND S.FARRELL. Possible functions of short-chain and medium-chain carnitine acyltransferases. Federation hoc. 41: 2858-2862, 1982. 5. BROZINICK, J. T., JR., V. K. PATEL, AND G. L. DOHM. Effects of 4.

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PDC ACTIVITY

AND ACETYL

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