RAPID COMMUNICATION
Muscle acetyl group availability is a major determinant of oxygen deficit in humans during submaximal exercise

James A. Timmons1, Thomas Gustafsson2, Carl Johan Sundberg2, Eva Jansson3, and Paul L. Greenhaff1

1 Department of Physiology and Pharmacology, University Medical School, Queen's Medical Centre, Nottingham N97 2UH, United Kingdom; 2 Section of Environmental Physiology, Department of Physiology and Pharmacology, Karolinska Institute, S-171 77 Stockholm; and 3 Department of Clinical Physiology, Huddinge University Hospital, Karolinska Institute, S-141 86 Huddinge, Sweden

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

The delay in skeletal muscle mitochondrial ATP production at the onset of exercise is thought to be a function of a limited oxygen supply. The delay, termed the oxygen deficit, can be quantified by assessing the above baseline oxygen consumption during the first few minutes of recovery from exercise. During submaximal exercise, the oxygen deficit is reflected by the extent of muscle phosphocreatine (PCr) breakdown. In the present study, nine male subjects performed 8 min of submaximal, single leg knee extension exercise after saline (Control) and dichloroacetate (DCA) infusion on two separate occasions. Administration of DCA increased resting skeletal muscle pyruvate dehydrogenase complex activation status threefold (Control = 0.4 ± 0.1 vs. DCA = 1.3 ± 0.1 mmol acetyl-CoA · min-1 · kg wet muscle-1 at 37°C, P < 0.01) and elevated acetylcarnitine concentration fivefold (Control = 2.2 ± 0.5 vs. DCA = 10.9 ± 1.2 mmol/kg dry mass, P < 0.01). During exercise, PCr degradation was reduced by ~50% after DCA (Control = 33.2 ± 7.1 vs. DCA = 18.4 ± 7.1 mmol/kg dry mass, P < 0.05). It would appear, therefore, that in humans acetyl group availability is a major determinant of the rate of increase in mitochondrial respiration at the onset of exercise and hence the oxygen deficit.

acetylcarnitine; pyruvate dehydrogenase complex; phosphocreatine

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

A MAJOR UNANSWERED QUESTION that has been asked by physiologists since the start of this century is, What determines the rate of increase in oxidative energy provision (mitochondrial ATP production) in the face of an increased external workload (8, 10, 12, 14)? ATP resynthesis by substrate level phosphorylation at the onset of exercise has been termed the oxygen deficit and is thought to be a function of a limited oxygen supply (14, 19). Classically, the oxygen deficit has been measured by assessing the amount of oxygen consumed above basal in the first few minutes postexercise (12). The magnitude of the oxygen deficit is a function of exercise intensity and, during intense exercise, can be directly quantified by assessing the extent of skeletal muscle phosphocreatine (PCr) degradation and muscle lactate production (11, 19). During submaximal exercise, the oxygen deficit is primarily reflected by the extent of muscle PCr breakdown (11). Originally, the development of an oxygen deficit was attributed to a lag in skeletal muscle blood flow and hence oxygen delivery at the onset of exercise. However, it has been concluded more recently that this may not be the case (8, 18), such that the physiological determinants of the oxygen deficit remain to be identified.

In resting skeletal muscle, the pyruvate dehydrogenase complex (PDC) is thought to be involved in glucose homeostasis, with its inactivation preventing unnecessary glucose oxidation (6, 16). During skeletal muscle contraction, PDC is activated (5, 13), and the rate of this activation process (dephosphorylation) appears to be an important determinant of the oxygen deficit and hence canine skeletal muscle contractile function during ischemia (21). Under ischemic conditions, after PDC activation before contraction, there was a reduction in muscle PCr degradation and lactate accumulation despite a greater contractile function being maintained (21, 22). In the present study, we set out to establish whether the activation status of PDC could influence PCr kinetics during submaximal voluntary exercise in humans. In doing so we have demonstrated that the availability of acetyl groups to the mitochondria is an important determinant of the oxygen deficit in exercising humans.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Nine healthy male volunteers participated in the present study, which was approved by the Ethical Committee of the Karolinska Institute. The average (range) age, height, body mass, and physical activity levels of the subjects were 22 (21-25) yr, 182 (176-186) cm, 77 (70-89) kg, and 4.5 (2-8) h/wk of moderate exercise, respectively. Before the study, the experimental protocol was explained to all subjects and their written consent was obtained. Dichloroacetate (DCA) was purchased from Sigma Chemical (Sweden) and prepared for sterile infusion by Apoteksbolaget Umeå, Sweden. The aqueous solution was sterilized by filtration and had a pH of 7.0 and serum osmolality of 550 mosmol/kg.

Exercise model. At least 1 wk before the first experiment, each subject was thoroughly familiarized with the experimental procedures and the exercise model, which consisted of 8 min of single leg knee extension using a modified electrically braked cycle ergometer at an individually determined submaximal workload. During familiarization, maximal one-leg performance capacity was determined. Familiarization commenced with 4 min at 5 W, and the workload was increased by 5 W at 1-min intervals to the point of volitional fatigue. During the experiment, the fixed workload was 20 ± 2 W (60 revolutions/min), which equaled ~45% of the final workload achieved during the incremental exercise protocol. Each voluntary contraction extended the leg from 70 to 150° (vertical to horizontal at the knee joint). Flexion was performed passively using the ergometer flywheel momentum to reposition the leg for the next extension.

Study protocol. Each subject rested in a supine position, and a 21-gauge cannula was placed in an antecubital vein of each arm for infusion or blood collection. They received, in a randomized, double-blind, crossover fashion, 50 mg/kg body mass DCA (50 mg/ml, sodium salt, pH 7.0) or an equivalent volume of saline intravenously over a 30-min period, commencing 90 min before the start of exercise. Five minutes before the onset of exercise, a resting muscle biopsy from vastus lateralis was taken using a Bergström needle (11). Blood pressure and heart rate were determined manually every 15 min throughout the infusion period while the subject was resting supine. During exercise, heart rate was derived continuously from an electrocardiogram monitor and blood pressure was obtained every 2 min using automated sphygmomanometry (Criticon Exercise Monitor 1165, Sweden). The subject exercised for 8 min, after which a second muscle biopsy was obtained. Seven to 10 days later, the same protocol was repeated using the same leg, but on this occasion the alternative infusate was administered.

Blood and muscle analysis. Venous blood was obtained at predetermined intervals throughout the experiment for the assessment of blood glucose and lactate concentrations as described previously (23). Muscle biopsy samples were freeze-dried, dissected free from visible blood and connective tissue, and powdered, and ~10 mg were extracted in 0.5 M perchloric acid containing 1 mM EDTA. After centrifugation, the supernatant was neutralized with 2.2 M KHCO3 and used for spectrophotometric determination of ATP, PCr, creatine (Cr), and lactate (9). The supernatant was also used for the radioisotopic determination of acetylcarnitine (2). Briefly, for the determination of acetylcarnitine, the acetyl group was transferred to CoA-SH in a reaction catalyzed by carnitine acetyltransferase to form acetyl-CoA. Acetyl-CoA so formed was condensed with [14C]oxaloacetate to form [14C]citrate, which was then measured. Freeze-dried muscle powder was also used for the determination of muscle glycogen (9). Intramuscular metabolites were corrected for total muscle creatine concentration. In the resting muscle biopsy procedure, sufficient muscle was obtained to assess PDC activation status (PDCa) and the samples were stored wet in liquid nitrogen until analysis was performed. Briefly, activity was measured by the addition of NaF and DCA to the extraction buffer, and then the rate of production of acetyl-CoA, when incubated with pyruvate, was measured as previously described (5).

Statistics. All data are reported as means ± SE. Comparisons between treatments (saline-control and DCA infusion) were made using analysis of variance on the changes in muscle metabolite concentration from rest to exercise. When a significant F ratio was found, a Student's paired t-test was used to locate the difference in treatment effect between DCA and Control. Significance was accepted at the 5% level. Linear relationships were examined using the Pearson product moment correlation.

    RESULTS
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Methods
Results
Discussion
References

DCA infusion did not alter blood glucose concentration at any time during the experiment but did reduce resting and exercise blood lactate concentration when compared with the control visit (Table 1). DCA infusion did not alter heart rate or mean arterial pressure.

                              
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Table 1.   Resting venous blood glucose and lactate concentrations and cardiovascular parameters pre- and postinfusion and at end of exercise period

Resting muscle metabolites and PDCa. There were no differences in muscle ATP, PCr, and lactate concentrations between control and DCA-treated muscles at rest (Table 2). DCA did, however, result in a marked increase in acetylcarnitine concentration (P < 0.01). In addition, a threefold increase in the activation status of resting skeletal muscle PDC was achieved after DCA (Control = 0.4 ± 0.1 vs. DCA = 1.3 ± 0.1 mmol acetyl-CoA · min-1 · kg wet muscle-1 at 37°C, P < 0.01).

                              
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Table 2.   Skeletal muscle metabolites in saline and dichloroacetate pretreated subjects at rest and after submaximal knee extension exercise

Muscle metabolites during exercise. DCA reduced the extent of PCr degradation by ~50% when compared with the control visit (Fig. 1, P < 0.05). There were no significant differences in muscle ATP and lactate concentrations when the control visit was compared with the DCA visit, with both groups demonstrating only modest changes compared with the respective resting values (Table 2). Acetylcarnitine accumulation during exercise was 10-fold lower after DCA treatment. However, the absolute concentration still remained higher than that measured during the control visit (Table 2). There was a significant inverse linear relationship between the extent of skeletal muscle PCr degradation and the extent of acetylcarnitine accumulation after 8 min of contraction (Pearson correlation coefficient: r = -0.61, P < 0.05).


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Fig. 1.   Skeletal muscle phosphocreatine (PCr) degradation was measured after 8 min of submaximal exercise after pretreatment with saline (Control) or dichloroacetate. * P < 0.01 indicates that dichloroacetate significantly reduced PCr degradation compared with control visit (values are mmol/kg dry mass, means ± SE).

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Mitochondrial ATP regeneration is the principal mechanism in mammalian skeletal muscle for meeting the energy requirements for sustained force production and calcium homeostasis (see Ref. 15). One of the remarkable features of skeletal muscle is the large range over which it can vary its demands for ATP regeneration. Since the start of this century, indirect examination of the utilization of molecular oxygen by skeletal muscle as the final substrate for ATP regeneration led to the belief that when external work is performed a limit exists in the provision of oxygen to the mitochondria during the first minutes of exercise (10, 12, 14). This proposed limitation resulted in the introduction of the concept termed the oxygen deficit (see Ref. 19), which has been established as being of finite capacity and being related to the exercise intensity, the breakdown of PCr (11), and hence the development of muscle fatigue (19). However, overall regulation of skeletal muscle cellular energetics is extremely complex and has yet to be fully elucidated. On the basis of in vitro analysis, it appears that the processes of ATP utilization and regeneration both independently regulate the energetics of ATP homeostasis, and hence it has not been possible to clearly establish which factors dominate in vivo (3, 15).

In the present experiment, conducted under double-blind crossover conditions, we activated in vivo the inner mitochondrial membrane-bound enzyme complex, PDC, in resting skeletal muscle by pharmacologically dephosphorylating pyruvate dehydrogenase (20), resulting in a threefold increase in PDC activation status. This eliminated any potential lag in substrate flux through the reaction attributable to the activation status of the enzyme complex per se (6, 21). Activation of PDC in resting skeletal muscle also resulted in a fivefold accumulation of acetylcarnitine formed via the flux of pyruvate through the PDC reaction, which is then transferred intramitochondrially to carnitine via the carnitine acetyltransferase reaction (1). The acetylcarnitine thus acts as a substrate store for the mitochondria at the onset of exercise (22). In addition, acetylation of intramuscular carnitine should also reduce the proposed competition for acetyl-CoA between the carnitine acetyltransferase reaction and the tricarboxylic acid cycle at the initiation of contraction (4, 21). It is also important to note that DCA has no vascular effects in skeletal muscle tissue at rest or during contraction (21, 22).

In the present study there was an inverse linear relationship between the change in muscle PCr concentration and acetylcarnitine accumulation during exercise, i.e., the greater the decline in PCr, and hence the greater the metabolic disturbance, the greater the accumulation of acetylcarnitine. We have also demonstrated this inverse linear relationship between the extent of muscle PCr degradation and the accumulation of acetylcarnitine during contraction in a previous study (23). Hence, once steady state is achieved, the concentration of muscle carnitine, which facilitates the PDC reaction (1, 4), will have been reduced in proportion to the rate of pyruvate production in excess of tricarboxylic acid flux. Classically, this would have been interpreted to mean that the availability of acetyl groups exceeds the demands of the mitochondria during exercise. However, it has also been noted that after DCA pretreatment, acetylcarnitine concentration falls (21, 22) such that we have proposed that initially, at the onset of exercise under control conditions, there is a lag in acetyl group availability that partly determines the extent of PCr degradation (21). Overall, this would mean that the pattern of change in acetylcarnitine concentration, from rest to steady-state exercise, follows a biphasic response whereby a period of insufficient acetyl group production is followed by a period of excess production until steady-state metabolic control is restored.

The exercise protocol in the present study led to a small decline in ATP concentration and a slight increase in muscle lactate concentration and hence little change in muscle pH. This is particularly important if we consider that the creatine kinase isoenzymes, which catalyze the reversible exchange of "high-energy" phosphates between PCr and ATP via the reaction: PCr + MgADP + H+ left-right-arrow  MgATP + Cr, are thought to be a near-equilibrium reaction under almost all but the most extreme metabolic conditions (17). Under the present submaximal exercise conditions, the creatine kinase reaction should have remained as a near-equilibrium reaction at all times such that the decline in skeletal muscle PCr concentration after 8 min of exercise would directly reflect the rate of onset of mitochondrial respiration at the start of exercise (3, 15, 21). We also used a nonfatiguing protocol to ensure that the decline in PCr concentration would constitute the most significant component of the oxygen deficit and that no differences in contractile efficiency would exist between the conditions (7, 11). The present data clearly demonstrate that the extent of the decline in skeletal muscle PCr stores was reduced when the supply of oxidative substrate to the tricarboxylic acid cycle was increased before the initiation of contraction. Overall, this indicates that, during the transition from rest to work, a faster rate of onset of mitochondrial respiration occurred, resulting in closer coupling between ATP consumption and oxidative regeneration mechanisms.

In summary, the present observations highlight a physiological compromise between PDC's role at rest with that required at the onset of exercise. PDC is an important determinant of carbohydrate homeostasis at rest, since its high degree of inactivation limits carbohydrate oxidation. At the onset of skeletal muscle contraction, however, PDC plays a critical role as a rapid provider of oxidative substrate. It seems, therefore, that the oxygen deficit characterized by Hill et al. (10) and Krogh and Lindhard (12) earlier this century is partly determined by a lag in the onset of oxidative substrate delivery to the tricarboxylic acid cycle and that this can be substantially reduced by the prior activation of PDC.

    ACKNOWLEDGEMENTS

J. A. Timmons was supported by the Defence Evaluation Research Agency, Centre for Human Sciences during this study.

    FOOTNOTES

This study was supported by grants from the Swedish Medical Research Council (MFR 4494) Karolinska Institute research funds and the Swedish National Centre for Research in Sports.

Address for reprint requests: J. A. Timmons, Discovery Biology III, Central Research, Pfizer Limited, Sandwich, Kent CT13 9NJ, UK.

Received 2 September 1997; accepted in final form 31 October 1997.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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