Skeletal muscle malonyl-CoA content at the onset of exercise at varying power outputs in humans

L. Maureen Odland, Richard A. Howlett, George J. F. Heigenhauser, Eric Hultman, and Lawrence L. Spriet

Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario N1G 2W1; Department of Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5; and Department of Clinical Chemistry, Huddinge University Hospital, S 141 86 Huddinge, Sweden

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

To investigate the regulation of intramuscular fuel selection, we measured the malonyl-CoA (M-CoA) content in human skeletal muscle at three exercise power outputs [35, 65, and 90% maximal rate of O2 consumption (VO2 max)]. Four males and four females cycled for 10 min at one power output on three separate occasions with muscle biopsies sampled at rest and at 1 and 10 min. The respiratory exchange ratio was 0.84 ± 0.03, 0.92 ± 0.02, and >1.0 at 35, 65 and 90% VO2 max, respectively. Muscle lactate content increased and phosphocreatine content decreased as a function of power output. Pyruvate dehydrogenase a activity increased from 0.40-0.64 mmol · kg wet muscle-1 · min-1 at rest to 1.57 ± 0.28, 2.80 ± 0.41, and 3.28 ± 0.27 mmol · kg wet muscle-1 · min-1 after 1 min of cycling at the three power outputs, respectively. Mean resting M-CoA contents were similar at all power outputs (1.85-1.98 µmol/kg dry muscle). During exercise at 35% VO2 max, M-CoA decreased from rest at 1 min (1.85 ± 0.29 to 1.20 ± 0.12 µmol/kg dry muscle) but returned to rest level by 10 min (1.86 ± 0.25 µmol/kg dry muscle). M-CoA content did not decrease during cycling at 65% VO2 max. At 90% VO2 max, M-CoA did not increase despite significant acetyl-CoA accumulation (the substrate for M-CoA synthesis). The data suggest that a decrease in M-CoA content is not required for the increase in free fatty acid uptake and oxidation that occurs during exercise at 35 and 65% VO2 max. Furthermore, M-CoA content does not increase during exercise at 90% VO2 max and does not contribute to the lower rate of fat oxidation at this power output.

fatty acid oxidation; acetyl-coenzyme A; acetyl-coenzyme A carboxylase; carnitine palmitoyltransferase I; high-performance liquid chromatography; malonyl-coenzyme A

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

MALONYL-COA (M-CoA) has recently been the subject of much interest due to its potential role in intramuscular fuel selection during exercise. Carnitine palmitoyltransferase (CPT) I, the key enzyme responsible for the transport of fatty acids into mitochondria for oxidation, is potently inhibited by M-CoA in vitro (3, 21, 27). It is currently believed that resting levels of M-CoA in rodent muscle are sufficiently high to inhibit excessive entry of free fatty acids (FFA) into the mitochondria at rest. During exercise, when the demand for energy from fat oxidation increases, rat skeletal muscle M-CoA content decreases during treadmill running and in response to electrical stimulation (9, 28, 35). This contraction-induced decrease in M-CoA has been postulated to relieve CPT-I inhibition and allow increased transport of FFA into the mitochondria for oxidation (35). To date, however, no direct measurements of fatty acid oxidation or any aspect of fat metabolism have been correlated with M-CoA contents in rodent muscle during exercise. Furthermore, research investigating M-CoA content in humans is extremely limited.

A recent investigation from our laboratory examining both rodent and human skeletal muscle (22) reported a 52% reduction in rodent red gastrocnemius M-CoA content in response to electrical stimulation at 0.7 Hz, which agrees with previous reports in rodent skeletal muscle (9). However, the M-CoA content in human vastus lateralis muscle remained constant during cycle exercise for 10 min at 40% maximal rate of O2 consumption (VO2 max) and for 60 min at 65% VO2 max. This occurred despite significant increases in fat utilization from rest to exercise and over time during exercise at 65% VO2 max (22). On the basis of these results, we concluded that measured levels of M-CoA in exercising muscle did not predict fatty acid oxidation rates in human muscle during low- and moderate-intensity exercise. However, we suggested that a more detailed time course of the human skeletal muscle M-CoA response to exercise was required.

Saddik et al. (26) reported that heart muscle M-CoA content increased concomitantly with the carbohydrate (CHO)-derived acetyl-CoA level. It is well known that acetyl-CoA accumulates rapidly in human skeletal muscle at the onset of high-intensity exercise (6, 10). It is possible that the increase in acetyl-CoA stimulates the activity of acetyl-CoA carboxylase (ACC), the enzyme responsible for M-CoA production, leading to an increase in M-CoA content (rather than a decrease) at high power outputs. Previous studies in rodent skeletal muscle, however, have reported reductions in M-CoA with contraction, even when CHO availability was enhanced (11), but M-CoA measurements at different power outputs have not been conducted.

Due to the importance of exercise intensity in the determination of intramuscular fuel selection (19, 30), the purpose of this study was to examine the time course of M-CoA kinetics early in exercise in human skeletal muscle at varying power outputs. It was hypothesized that M-CoA would be unaffected at 1 and 10 min of exercise at 35 and 65% VO2 max and may increase as a function of increasing muscle acetyl-CoA levels at 90% VO2 max.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Subjects

Four males and four females volunteered to participate in the study. The subjects were not well trained, but all were healthy and physically active (age, 22.1 ± 0.3 yr; weight, 77.7 ± 3.6 kg, VO2 max, 3.5 ± 0.3 l/min). The experimental procedures, including possible risks and benefits, were explained to each subject before written consent was obtained. The study received approval from the Human Ethics Committees of the University of Guelph and McMaster University.

Experimental Protocol

Before the experiment, all participants underwent a continuous incremental VO2 max test on a cycle ergometer. Each subject also participated in two practice trials on separate days to confirm the power outputs required to elicit 35, 65, and 90% VO2 max and to ensure that all subjects could complete 10 min of cycling at the high power output.

The experimental trials were performed on 2 days, separated by 2-3 wk. On each test day, subjects reported to the laboratory at the same time of day having eaten a meal high in CHO 2-4 h before the experiment. Daily food records were kept for 48 h preceding the initial trial, and subjects were instructed to replicate their consumption before the second trial. CHO intake represented 60.0 ± 4.3% of the total caloric intake of the pretrial diet, whereas fat and protein comprised 25.0 ± 2.4 and 15.0 ± 2.0%, respectively.

On one of the test days, exercise consisted of 10 min of cycling on an electronically braked ergometer (Excalibur; Quinton Instrument, Seattle, WA) at 35% VO2 max, followed by at least 1 h of rest and then 10 min of cycling at 65% VO2 max. On the other day, subjects cycled for 10 min at 90% VO2 max. Test days were randomly assigned. Before each exercise bout, one leg was prepared for percutaneous needle biopsy of the vastus lateralis with three incisions of the skin through to the deep fascia, under local anesthesia (2% lidocaine without epinephrine) as described by Bergstrom (2). Biopsy samples were obtained at rest after at least 30 min of complete rest on a bed and were immediately frozen in liquid N2. Exercise biopsy samples were obtained at 1 and 10 min at each power output. Rate of O2 consumption (VO2), CO2 output (VCO2), and the respiratory exchange ratio (RER) were determined using a metabolic cart (Quinton Q-plex 1; Quinton Instrument). At 35 and 65% VO2 max, expired gases were collected between 4 and 8 min of exercise, whereas at 90% VO2 max, gas was collected between 2-4 and 7-9 min. The rate of fat oxidation (g/min) was calculated from the VCO2 and VO2 using the RER (12).

Muscle Sampling and Analyses

A piece of frozen wet muscle (20-30 mg) was removed under liquid N2 for the determination of the activity of pyruvate dehydrogenase (PDH) in its active form (PDHa), as described previously (7, 24).

The remainder of the muscle samples were freeze-dried, dissected free of blood and connective tissue, and powdered. A portion of dry muscle (~5 mg) was extracted in 0.5 M perchloric acid and 1 mM EDTA, neutralized to pH 7.0 with 2.2 M KHCO3, and analyzed for acetyl-CoA and acetylcarnitine (5) as well as phosphocreatine (PCr), creatine, ATP, glucose, and lactate (1, 16).

M-CoA was extracted from aliquots of dry muscle (~8-14 mg) with ice-cold 0.5 M PCA in the ratio of 10 µl/mg dry muscle as previously described (22). M-CoA esters were separated and quantified as described in previous reports (18, 26).

All muscle metabolites were normalized to the highest total creatine content for a given individual's biopsies to correct for nonmuscle contamination. This correction averaged 9.2 ± 1.2%.

Statistical Analysis

Data are expressed as means ± SE. A paired t-test was used to compare RER data from eight subjects at 35 and 65% VO2 max. Due to the large amount of tissue required to measure M-CoA, missing data points precluded the use of a two-way (time vs. power output) repeated-measures analysis of variance (ANOVA). Significant differences between means over time at each power output were thus determined using a one-way repeated-measures ANOVA for all metabolites. At 35, 65, and 90% VO2 max, complete data sets were obtained for seven, six, and eight subjects, respectively. Tukey post hoc analysis was used to determine the location of significant differences. Results were considered significant at P < 0.05.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Respiratory Gas Exchange

During exercise at 35 and 65% VO2 max, VO2 was 1.32 ± 0.12 and 2.18 ± 0.19 l/min, respectively. At 90% VO2 max, VO2 drifted from 2.75 ± 0.26 (2-4 min) to 3.29 ± 0.31 l/min (7-9 min). RER was significantly lower at 35 compared with 65% VO2 max (0.84 ± 0.3 vs. 0.92 ± 0.02, P < 0.001). RER was consistently above 1.0 during exercise at 90% VO2 max and therefore did not provide an accurate representation of substrate use but indicated a large reliance on CHO.

Muscle Metabolites

PCr, ATP, glucose, and lactate. Muscle ATP remained unchanged from rest during exercise at 35, 65, and at 1 min at 90% VO2 max but decreased at 10 min at 90% VO2 max (Table 1, P < 0.05). PCr was significantly reduced in a stepwise manner after 1 min of exercise at all power outputs (35%, P < 0.006; 65 and 90%, P < 0.001), and lactate was increased above rest at 1 and 10 min at 65% (P < 0.005) and 90% VO2 max (P < 0.001).

                              
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Table 1.   Muscle metabolites during cycle exercise at varying power outputs

M-CoA. At rest, muscle M-CoA data were similar between power outputs (35%, 1.85 ± 0.29; 65%, 1.85 ± 0.46; 90% VO2 max, 1.98 ± 0.26 µmol/kg dry muscle; Fig. 1). M-CoA was significantly reduced from rest at 1 min during exercise at 35% VO2 max but returned to rest level by 10 min (Fig. 1, P < 0.02). No differences in M-CoA content occurred during exercise at 65 or 90% VO2 max.


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Fig. 1.   Muscle malonyl-CoA content in human skeletal muscle at rest and during exercise at varying power outputs. Values are means ± SE. * Significantly different from rest and 10 min at same power output. dm, Dry muscle.

Acetyl group accumulation. Muscle acetyl-CoA remained constant during exercise at 35% VO2 max and was elevated above rest at 10 min at 65% VO2 max (P < 0.06) and at 1 and 10 min at 90% VO2 max (Fig. 2A, P < 0.001). Acetylcarnitine increased at 10 min during exercise at all three power outputs (Fig. 2B; 35%, P < 0.002; 65 and 90%, P < 0.001).


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Fig. 2.   Muscle acetyl-CoA (A) and acetylcarnitine (acetylcarn; B) content at rest and during exercise at varying power outputs. Values are means ± SE. * Significantly different from rest at same power output; # significantly different from 1 min.

PDH activity. Resting PDHa activity was similar before all exercise trials and increased above rest after 1 min of cycling as a function of power output (Fig. 3, P < 0.001). PDHa activity did not change from 1 to 10 min at the lower two power outputs but increased at 90% VO2 max.


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Fig. 3.   Activity of muscle puruvate dehydrogenase in its active form (PDHa) at rest and during exercise at varying power outputs. Values are means ± SE. * Significantly different from rest at same power output; # significantly different from 1 min.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

The primary purpose of this investigation was to measure M-CoA in human skeletal muscle biopsy samples at rest and during the onset of exercise at varying power outputs. At rest, individual M-CoA contents ranged from 0.78 to 3.41 µmol/kg dry muscle, but mean values were similar across power outputs (Fig. 1). Generally, M-CoA did not change in response to exercise at the lower two power outputs, although M-CoA fell significantly at 1 min at 35% VO2 max and then returned to rest level by 10 min. M-CoA did not increase at the high power output despite significant activation of PDH and accumulations of acetyl-CoA and acetylcarnitine.

M-CoA and Intramuscular Fuel Selection

Observed decreases in M-CoA content during exercise have led to the suggestion that it is a regulator of fat-CHO interaction in rodent skeletal muscle at rest and during exercise (20, 28, 35). It has also been postulated to play the same role in contracting human skeletal muscle, although M-CoA was not measured in these investigations (17, 32). Two of the main regulatory enzymes responsible for substrate choice for oxidation are PDH and CPT-I, which control the entry of CHO and FFA into the mitochondria, respectively. M-CoA is a potent inhibitor of CPT-I in vitro (3, 27). Because the M-CoA content may be influenced by acetyl-CoA, which is an end product of the PDH reaction and a substrate for M-CoA formation, alterations in M-CoA content could provide a mechanism to control the balance between CHO and fat oxidation.

It has long been established that exercise intensity is an important factor responsible for determining the amount of fat or CHO utilization during exercise (19, 30). A recent study (25) with well-trained, fasted cyclists demonstrated that at low power outputs (25% VO2 max), 90% of the energy expenditure was derived from fat. At 65% VO2 max, fat oxidation fell to 50% of total energy expenditure, but absolute fat oxidation rates were greater than at 25% VO2 max, since energy demand also increased. Finally, at high power outputs (>85% VO2 max), both the absolute and relative contribution of fat decreased (25). Less information is available regarding intramuscular fuel selection in untrained, fed subjects, although a greater reliance on CHO would be expected at exercise intensities above 50% VO2 max (30).

During low to moderate submaximal exercise, when fat provides a significant proportion of the fuel required for contraction, a reduction in M-CoA may relieve inhibition at CPT-I and allow more FFA transport into the mitochondria for oxidation. During high-intensity exercise (i.e., 85% VO2 max or greater), when the contribution from fat to total energy supply is reduced, high PDHa activity leads to CHO-derived accumulations of acetyl-CoA and acetylcarnitine. If a portion of this acetyl-CoA accumulation occurs in the cytoplasm and stimulates ACC to produce M-CoA, then M-CoA-induced inhibition of CPT-I may contribute to the reduced FFA oxidation during high-intensity exercise.

In the present study, the RER data suggested that exercise at 35 and 65% VO2 max was associated with a large energy contribution from fat oxidation. On the basis of previous measurements with rodent skeletal muscle, it was expected that M-CoA content would decrease at these workloads. However, our previous work with M-CoA after 10 min of exercise suggested that a decrease in M-CoA was not involved in the increase in FFA transport and oxidation. In the present study, M-CoA fell significantly only at 1 min at 35% VO2 max and then returned to preexercise level by 10 min. At 65% VO2 max, M-CoA remained unchanged throughout exercise. The calculated fat oxidation rates were 0.24 ± 0.04 g/min at 35% and 0.18 ± 0.03 g/min at 65% VO2 max despite similar M-CoA contents. At 90% VO2 max, a level at which fat contributed less energy, M-CoA also remained constant.

The early decline in M-CoA at 35% VO2 max may be explained by fiber-type recruitment. In rodent skeletal muscle, M-CoA content was reported to vary among different fiber types and to correlate with mitochondrial content of muscle fibers (36). In resting rats, M-CoA concentration in the white region of the quadriceps (fast glycolytic) was only 28% of the deep red quadriceps content (slow oxidative). Furthermore, during treadmill running at 21 m/min up a 15% grade, the most rapid decrease in rodent muscle M-CoA occurred in the red quadriceps, whereas the slowest decrease occurred in white quadriceps (36). Although regions of rat skeletal muscle are homogeneous with respect to fiber type, human skeletal muscle is heterogeneous. Human vastus lateralis muscle is composed of ~50% type I, 40% type IIa, and 10% type IIb fibers (31). The M-CoA content within specific human fiber types is not known, but the contents of many metabolites in human type I and II fibers are similar (8, 13, 33). However, if there were specific recruitment of mainly type I fibers during exercise at 35% VO2 max, a decrease in the M-CoA content of ~67% in these fibers would be required to account for the early ~33% decrease in total muscle M-CoA at this power output.

Fiber-type recruitment cannot, however, explain the return of M-CoA to resting levels by 10 min of exercise at 35% VO2 max. It is possible that the early decline at 35% "primes" the fatty acid oxidation machinery at the onset of exercise and that the mechanism responsible for the early decline at 35% VO2 max is overridden by other regulators over time and at higher power outputs. In addition, there is the possibility that the M-CoA-induced inhibition of CPT-I is maximal at rest, such that further increases are not necessary during high-intensity exercise.

M-CoA Interaction With CPT-I

M-CoA content did not correlate with fat utilization during exercise at varying power outputs, but the possibility of M-CoA-induced inhibition of CPT-I and subsequent regulation of fatty acid oxidation cannot be dismissed. M-CoA-induced inhibition of CPT-I may be reduced independent of a change in M-CoA, as other factors could account for the reduction in inhibition. Certainly, pH is known to affect the sensitivity of CPT-I to inhibition by M-CoA. At pH 6.8, rodent muscle CPT-I has been shown to bind M-CoA more efficiently and to have a lower IC50 for M-CoA (concentration of M-CoA required for 50% inhibition of CPT-I activity), a higher Michaelis constant (Km) for carnitine, and a lower maximal velocity than at higher pH (21). If human muscle CPT-I is also sensitive to pH, this may help explain why M-CoA did not change at the higher power outputs, at a time when fatty acid utilization was reduced. Certainly the M-CoA-CPT-I interaction in skeletal muscle is complex, as in vitro experiments predict that CPT-I activity should be fully inhibited at resting and exercise M-CoA concentrations (3, 27), and this is not the case in vivo.

At present, very little is known regarding the regulatory properties of the muscle isoform of CPT-I, particularly in humans. Both acetyl-CoA and free CoA were reported to antagonize the inhibitory effect of M-CoA on total CPT activity in human skeletal muscle homogenates by increasing the inhibitory constant (Ki) for M-CoA (39). It should be noted, however, that, despite increasing the Ki for M-CoA, addition of both acetyl-CoA and free CoA acted in a synergistic manner with M-CoA to increase (not decrease) inhibition of total CPT activity. Results from this study are difficult to interpret, however, since total CPT activity (i.e., CPT-I and CPT-II) was measured rather than CPT-I activity alone (39). Further in vitro experimentation is required to determine the human CPT-I response to physiological concentrations of various CoA esters. Specifically, inhibitor studies of human muscle CPT-I are needed to determine whether M-CoA binds directly at the catalytic site or at a separate regulatory site.

Regulation of Muscle ACC Activity

Tissue levels of M-CoA will be dependent upon the activity of ACC and the rate of M-CoA degradation. Tissue-specific isoforms of ACC have been identified, and human and rat skeletal muscle appear to contain a unique 272- to 275-kDa isoform (ACC-beta ; see Refs. 34 and 38). Unlike the liver isoform (ACC-alpha ), which is well characterized and undergoes complex allosteric regulation and hormone-induced enzyme phosphorylation (15), little is known concerning the regulatory properties of ACC-beta . It was recently hypothesized that ACC-beta may be anchored to the outer mitochondrial membrane (14). This binding of ACC-beta was suggested to control CPT-I activity by generating M-CoA at or near the M-CoA-binding site of CPT-I, potentially increasing the local concentration as much as 1,000-fold (14). At present, no direct evidence is available to determine the existence or the extent of tissue-specific differences in CPT-I.

In vitro investigations have reported ACC-beta to have a higher Km for acetyl-CoA than ACC-alpha (4), and research in perfused working heart muscle has suggested that muscle ACC-beta may be substrate regulated (26). In the present study, M-CoA did not increase above resting level in any subject, at any time point or power output, despite concomitant increases in acetyl-CoA and acetylcarnitine at 65 and 90% VO2 max. This result suggests that M-CoA production in human skeletal muscle during moderate- to high-intensity exercise is not regulated by acetyl-CoA content.

Unfortunately, measurements of acetyl-CoA and acetylcarnitine in biopsy samples are made on total tissue content, making it difficult to determine what proportion of the measured acetyl groups are mitochondrial or cytosolic. Clearly, in order to affect ACC, some acetyl-CoA must be in the cytoplasm. It is likely that cytoplasmic acetyl-CoA content increases to some extent during exercise, as the large increases in acetylcarnitine are believed to be primarily cytosolic.

Recent studies of rodent soleus muscle incubated at rest with glucose and insulin reported increased M-CoA formation modulated by changes in the cytosolic concentration of citrate rather than by stable increases in ACC-beta activity (29). In contrast, it was suggested that ACC-beta activity was reduced after 5 and 30 min of treadmill exercise, despite a significant increase in muscle citrate (37). In the present study, citrate was not determined, but previous studies with similar protocols have established that significant increases in citrate occur during exercise in humans (10, 23). It is possible that 1) significant increases in muscle citrate (and acetyl-CoA) offset a potential contraction-induced decrease in ACC-beta activity, and 2) ACC-beta may be regulated by fuel supply at rest, but this control mechanism is overridden during exercise. Saha et al. (28) suggested that fuel supply and contraction-induced alterations in M-CoA were regulated by different systems in isolated rodent muscle.

In summary, human skeletal muscle M-CoA content remained remarkably constant during exercise at varying power outputs. Regardless of the mechanism(s) regulating M-CoA formation and/or degradation, M-CoA content was not correlated with fatty acid oxidation rate. Therefore, if M-CoA is involved in CPT-I regulation during exercise at 35 and 65% VO2 max, it is not primarily due to decreases in its concentration. Conversly, M-CoA content did not increase during exercise at 90% VO2 max and therefore did not contribute to the lower rate of fat oxidation at this power output. In conclusion, other factors may be present during exercise that interact with M-CoA and CPT-I and override its inhibitory effects on CPT-I activity.

    ACKNOWLEDGEMENTS

We thank Drs. David Dyck and Jack Rosenfeld for invaluable technical assistance.

    FOOTNOTES

This work was supported by operating grants from the Natural Sciences and Engineering Research and Medical Research Councils of Canada and by a student grant from the Gatorade Sports Science Institute, Barrington, IL. G. J. F. Heigenhauser is a Career Investigator of the Heart and Stroke Foundation of Ontario.

Address for reprint requests: L. L. Spriet, Dept. of Human Biology and Nutritional Sciences, Univ. of Guelph, Guelph, Ontario, Canada N1G 2W1.

Received 29 September 1997; accepted in final form 18 February 1998.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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