Department of Human Biology and Nutritional Sciences, University of Guelph, Ontario N1G 2W1; Department of Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5; and Department of Physiology, The University of Melbourne, Parkville, 3052, Australia
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ABSTRACT |
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The
present study examined the sensitivity of carnitine
palmitoyltransferase I (CPT I) activity to its inhibitor malonyl-CoA (M-CoA), and simulated metabolic conditions of rest and exercise, in
aerobically trained and untrained humans. Maximal CPT I activity was
measured in mitochondria isolated from resting human skeletal muscle.
Mean CPT I activity was 492.8 ± 72.8 and 260.8 ± 33.6 µmol · min1 · kg
wet muscle
1 in trained and untrained subjects,
respectively (pH 7.0, 37°C). The sensitivity to M-CoA was greater
in trained muscle; the IC50 for M-CoA was 0.17 ± 0.04 and
0.49 ± 0.17 µM in trained and untrained muscle, respectively. The
presence of acetyl-CoA, free coenzyme A (CoASH), and acetylcarnitine,
in concentrations simulating rest and exercise conditions did not
release the M-CoA-induced inhibition of CPT I activity. However, CPT I
activity was reduced at pH 6.8 vs. pH 7.0 in both trained and untrained
muscle in the presence of physiological concentrations of M-CoA. The
results of this study indicate that aerobic training is associated with
an increase in the sensitivity of CPT I to M-CoA. Accumulations of
acetyl-CoA, CoASH, and acetylcarnitine do not counteract the
M-CoA-induced inhibition of CPT I activity. However, small decreases in
pH produce large reductions in the activity of CPT I and may contribute
to the decrease in fat metabolism that occurs during moderate and intense aerobic exercise intensities.
long-chain fatty acids; fatty acid transport; mitochondria; aerobic
training; -oxidation; carnitine palmitoyltransferase I; malonyl-coenzyme A
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INTRODUCTION |
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THE CARNITINE PALMITOYLTRANSFERASE (CPT) complex,
consisting of CPT I, acylcarnitine translocase, and CPT II, has a
pivotal role in the transport of long-chain fatty acids in the
mitochondria for subsequent -oxidation in skeletal muscle, as well
as in other tissues (20). CPT I, located on the outer surface of the
outer mitochondrial membrane, catalyzes the transfer of a variety of long-chain fatty acyl groups from free coenzyme A (CoASH) to carnitine. The generated acylcarnitine can then permeate the inner membrane via
the acylcarnitine/carnitine translocase. The acyl-CoA moiety is then
reformed in the matrix of the mitochondria by the action of CPT II.
This enzyme is located on the inner mitochondrial membrane and
catalyses the transfer of the acyl group from carnitine to CoASH. The
reformed acyl-CoA then enters the
-oxidation pathway (20).
CPT I is considered the rate-limiting step in the oxidation of long-chain fatty acids and is reversibly inhibited by malonyl-CoA (M-CoA), the first committed intermediate in fatty acid synthesis (21, 31). Thus there has been speculation as to the potential role for M-CoA in fuel selection in skeletal muscle. Decreases in muscle M-CoA during exercise in rat skeletal muscle have led to the suggestion that M-CoA levels regulate the rate of fatty acid oxidation in muscle (39). However, existing in vitro work with rat skeletal muscle suggests that CPT I would be fully inhibited at the M-CoA concentrations that exist in the intact cell (P. M. Berthon, R. A. Howlett, G. J. F. Heigenhauser, and L. L. Spriet, unpublished observation and Refs. 12, 21, 22). In addition, it has been demonstrated that M-CoA levels did not change during exercise in human skeletal muscle despite large increases in fatty acid oxidation rates (25, 26). These uncertainties regarding the interaction between CPT I activity and M-CoA inhibition during exercise suggest that in vitro investigations of CPT I in human skeletal muscle are warranted.
A number of animal studies have attempted to define the sensitivity of CPT I activity to M-CoA (12, 21, 23). However, this has not been attempted in human skeletal muscle. Many previous studies of the entire CPT complex in human skeletal muscle have used muscle homogenates or freeze-thawed mitochondria (1, 8, 17, 33, 37, 40, 41). Because the mitochondria are not preserved intact, a clear measurement of CPT I activity alone is not possible. However, Berthon et al. (3) recently modified the existing radiometric technique to allow for CPT I measurements in intact mitochondria isolated from needle biopsy samples of human skeletal muscle.
In addition to the relationship between CPT I activity and M-CoA, other factors may be important in the regulation of CPT I and fuel selection at rest and during exercise. There is in vitro evidence in skeletal muscle that other metabolic intermediates, such as acetyl-CoA and CoASH, may affect the activity of CPT I (19, 22, 41). In addition, reductions in pH decreased the activity of CPT I in rat skeletal muscle (Berthon et al., unpublished observation and Refs. 12, 23). Again, these effects have not been investigated in human skeletal muscle.
The aims of the present study were 1) to characterize the sensitivity of skeletal muscle CPT I to M-CoA in resting biopsies from aerobically trained and untrained humans; 2) to determine the effect of the metabolites acetyl-CoA, CoASH, and acetylcarnitine, present in concentrations simulating rest and exercise conditions, on CPT I activity in the presence of physiological levels of M-CoA; and 3) to examine the effect of pH on CPT I activity in the presence of physiological levels of M-CoA.
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METHODS |
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Subjects.
Experiments were conducted on two subject groups. Six males and five
females were classified as aerobically trained, and six males and six
females were classified as untrained (Table
1). Trained subjects engaged in four or
more aerobic workouts per week, whereas untrained subjects engaged in
one workout per week or minimal activity. Subjects were fully informed
of the purpose of the experiments and of any possible risk before
giving written consent to participate. The study was approved by the
University of Guelph Ethics Committee.
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Experimental protocols.
Before the experiment, peak pulmonary O2 uptake
(O2 peak) was
measured with a metabolic cart (SensorMedics model 2900; Yorba Linda,
CA) during incremental exercise on a cycle ergometer (Excalibur, Quinton Instruments, Seattle, WA; Table 1). On the day of the experiment, a resting muscle sample was obtained from the vastus lateralis under local anesthesia (2% lidocaine without epinephrine) using the percutaneous needle biopsy technique described by
Bergström (2). Visible fat and connective tissue were dissected
free from the muscle, and it was blotted to remove excess blood. The muscle sample (~60-100 mg) was divided into two portions; the first (~50-90 mg) was used for the immediate isolation of
mitochondria for the determination of CPT I activity, and the second
(~10 mg) was frozen in liquid nitrogen for the later analysis of
citrate synthase (CS) and
-hydroxyacyl-CoA-dehydrogenase (
-HAD) activity.
Isolation of mitochondria. The procedure for the mitochondrial isolation has been previously described by Jackman and Willis (15). The entire procedure was performed at 0-4°C. The buffer solutions used contained the following: solution I: 100 mM KCl, 40 mM Tris · HCl, 10 mM Tris base, 5 mM MgCl2, 1 mM EDTA and 1 mM ATP, pH 7.4; solution II: 100 mM KCl, 40 mM Tris · HCl, 10 mM Tris base, 1 mM MgSO4, 0.1 mM EDTA, 0.2 mM ATP, and 1.5% fatty acid free BSA, pH 7.4; solution III: as per solution II without BSA.
Muscle samples were weighed, finely minced, and gently homogenized with a manual glass homogenizer in 20 vol/wt of solution I and then centrifuged at 700 g for 10 min. The supernatant was centifuged at 14,000 g for 10 min, and the pellet containing the mitochondria was then resuspended in 10 vol/wt of solution II and centrifuged at 7,000 g for 10 min. The pellet was resuspended in 10 vol/wt of solution III and centrifuged at 3,500 g for 10 min. This final pellet was resuspended in 1 vol/wt of a mannitol/sucrose buffer (220 mM sucrose, 70 mM mannitol, 10 mM Tris · HCl, and 1 mM EDTA, pH 7.4), gently rendered homogenous, and kept on ice for the subsequent determination of CPT I and intramitochondrial CS activities. This procedure extracts mainly subsarcolemmal mitochondria (as a protease was not used) that contain very low levels of contaminants, as previously described in heart (27) and skeletal muscle (4, 16).Determination of CPT I (EC 2.3.1.21) activity. The forward radioisotope assay for the determination of CPT I activity from needle biopsy samples has been previously described (3, 21). However, two significant changes were made to the method for the present study. The respective concentrations of the substrates palmitoyl-CoA and L-carnitine were increased to 300 µM and 5 mM, and the assay was performed at 37°C. Previous work with rat skeletal muscle suggested that these substrate concentrations were required for maximal CPT I activity and that 300 µM palmitoyl-CoA did not exert a detergent effect on the mitochondrial membranes (Berthon et al., unpublished observation and Ref. 24). Briefly, labeled palmitoyl-L-carnitine was measured after it was generated for 6 min after the addition of 10 µl of mitochondrial suspension (1:3 dilution) to 90 µl of the following standard reaction medium: 117 mM Tris · HCl (pH 7.0), 0.28 mM reduced glutathione, 4.4 mM ATP, 4.4 mM MgCl2, 16.7 mM KCl, 2.2 mM KCN, 40 mg/l rotenone, 0.5% BSA, 300 µM palmitoyl-CoA, and 5 mM L-carnitine with 1 µCi of L-[3H]carnitine. The reaction was stopped after 6 min with the addition of 60 µl of ice-cold 1 M HCl. Palmitoyl-[3H]carnitine formed during the reaction was extracted in 400 µl of water-saturated butanol in a process involving washes with distilled water and subsequent recentrifugation to separate the butanol phase. Finally, radioactivity was assayed in 100 µl of the butanol phase in 5 ml of scintillation cocktail. Assays were performed in duplicate, and blanks were subtracted.
To express CPT I activity in terms of the whole muscle (µmol · minDetermination of CS (EC 4.1.3.7) and -HAD (EC
1.1.1.35) activities.
CS activity was assayed spectrophotometrically at 25°C as
previously described (36). Total muscle CS activity was assayed in a
portion of the original muscle sample (~10 mg) and homogenized in 100 vol/wt of a 100 mM potassium phosphate buffer solution. CS activity of
intact mitochondria in the suspension was determined by first assaying
the extramitochondrial fraction in the suspension (1:20 dilution) and
then assaying the total CS activity of the suspension (1:20 dilution)
after lysing the mitochondria with 0.04% Triton X-100 and repeated
freeze thawing. The difference in these activities gives the
intramitochondrial fraction.
-HAD was assayed spectrophotometrically
at 25°C as previously described (18).
Data analysis.
All data are reported as means ± SE. The IC50, defined as
the M-CoA concentration causing 50% of the maximal inhibition of CPT I
activity, was calculated from plots of CPT I activity vs. log[M-CoA] for each subject (12). The effect of each
additional metabolite on CPT I activity was compared with the activity
of CPT I in the presence of 0.7 µm M-CoA with a paired
t-test. Comparisons between groups were made with unpaired
t-tests. Linear regression analysis was performed using the
method of least squares. The reliability of the assay was assessed with
the coefficient of variation (CV) of CPT I activities determined from
the same extracted mitochondria. The formula used to determine CV was
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RESULTS |
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O2 peak was
significantly greater in the trained group vs. the untrained group
(Table 1). The activities of CS (23.7 ± 3.5 vs. 16.1 ± 4.2 mmol/kg
wet muscle) and
HAD (3.98 ± 0.96 vs. 2.15 ± 0.92 mmol/kg wet
muscle) were also significantly higher in the trained vs. untrained
groups. The maximal CPT I activity measured in resting biopsies was
significantly higher in the trained group (n = 11) than in the
untrained group (n = 12; 492.8 ± 72.8 vs. 260.8 ± 33.6 µmol · min
1 · kg
wet muscle
1, respectively). There were significant
correlations between CPT I activity and relative
O2 peak (r =
0.46, P = 0.02), CS activity (r = 0.62, P = 0.003), and
-HAD activity (r = 0.75, P = 0.002)
M-CoA sensitivity.
The mean IC50 for M-CoA was significantly lower in the
trained than in the untrained group (0.17 ± 0.04 vs. 0.49 ± 0.17 µM, respectively; Fig. 1).
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Effects of additional metabolites on CPT I activity.
There was no effect of adding resting levels of acetyl-CoA (4 µM) and
CoASH (17 µM) and exercise levels (acetyl-CoA: 12 µM, CoASH: 9 µM) on the activity of CPT I in the presence of 0.7 µM M-CoA in
either trained or untrained groups (Figs. 2
and 3). Resting levels of
acetylcarnitine (2 mM) alone and resting levels of all three
metabolites together reduced the activity of CPT I below values
measured in the presence of 0.7 µM M-CoA in both trained and
untrained subjects (Fig. 2). However, exercise levels of
acetylcarnitine (6 mM) alone and exercise levels of all three
metabolites together had no effect (Fig. 3).
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Effect of pH on CPT I activity.
CPT I activity was markedly decreased when the pH of the reaction
medium was reduced from 7.0 to 6.8 in the presence of 0.7 µM M-CoA in
both trained (7.0: 406.5 ± 71.5 vs. 6.8: 251.0 ± 67.9) and
untrained muscle (7.0: 195.6 ± 30.2 vs. 6.8: 116.3 ± 20.7 µmol · min1 · kg
wet muscle
1; Fig.
4).
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DISCUSSION |
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The results of the present cross-sectional study indicate a clear relationship between the training status of the individual and the activity of CPT I measured in intact subsarcolemmal mitochondria from resting human skeletal muscle biopsy samples. This is in agreement with previous findings (3). The sensitivity of CPT I to M-CoA was higher in aerobically trained vs. untrained subjects. However, there was no clear influence of resting or exercise concentrations of acetyl-CoA, CoASH, or acetylcarnitine on the inhibition of physiological levels of M-CoA (0.7 µM) on CPT I activity. Only resting levels of acetylcarnitine (2 mM) caused a further reduction in CPT I activity in both trained and untrained individuals. Finally, the pH of the reaction medium had a marked affect on CPT I activity. A small reduction in pH from 7.0 to 6.8 reduced the activity of CPT I by ~40% in both trained and untrained individuals in the presence of physiological levels of M-CoA.
Measurement of in vitro CPT I activity.
The absolute values for maximal CPT I activity for both untrained and
trained groups were two- to threefold higher in the present study
compared with previously reported values from our laboratory (3). The
reason for the higher values in the present study relates to
alterations in the conditions used in the assay to measure CPT I
activity. The previous study employed substrate concentrations of 100 µM palmitoyl-CoA and 0.4 mM L-carnitine and a temperature
of 30°C (3). In the present study, the palmitoyl-CoA concentration
was increased to 300 µM, and the carnitine concentration was
increased to 5 mM, because previous experiments with rodent skeletal
muscle indicated that these concentrations were required to achieve
maximal CPT I activity (Berthon et al., unpublished observation). The
temperature employed in the assay was also increased from 30 to
37°C to simulate the in vivo condition. Although the altered assay
conditions resulted in higher CPT I activities, the linear relationship
between measures of the training status of the individual (-HAD, CS
activities) and maximal CPT I activity remained. The correlations
between CPT I activity and CS activity and also between CPT I and
relative O2 uptake were not as strong in the present study,
possibly due to a smaller range of O2 uptake values
(37-65 ml
O2 · min
1 · kg
body mass
1) in the present subjects. In the previous
study, we purposely included subjects at the extreme ends of the
O2 uptake spectrum (28-72 ml
O2 · min
1 · kg
body mass
1), and this appears to have strengthened
the above relationships.
Sensitivity of CPT I to M-CoA. The existing data on the relationship between CPT I activity and M-CoA concentration in skeletal muscle comes mainly from rodent tissue. IC50 values for M-CoA in rat skeletal muscle have been reported ranging from 0.015 to 0.22 µM (Berthon, unpublished observation and Refs. 12, 21, 23). This wide range is likely due to a combination of varying factors, such as the pH and temperature of the assay medium (12) and the different muscles (fiber types) examined. Berthon et al. (unpublished observation) reported an IC50 for M-CoA of 0.22 ± 0.04 µM in untrained rat soleus muscle at pH 7.0. It appears that CPT I from human skeletal muscle is less sensitive to M-CoA than rat skeletal muscle. The IC50 for M-CoA in untrained humans reported in the present study was 0.49 ± 0.17 µM, which is approximately twofold higher than reported for untrained rat skeletal muscle.
In the present study, the IC50 for M-CoA in aerobically trained human muscle is ~70% lower than that from untrained muscle. This finding suggests that aerobic training is associated with an increase in the sensitivity of skeletal muscle CPT I to M-CoA. In the presence of physiological levels of M-CoA (0.7 µM), the activity of CPT I was 284.2 ± 75.9 in the trained group and 196.0 ± 30.2 µmol · minEffect of acetyl-CoA, CoASH, and acetylcarnitine on CPT I activity. There is some evidence in the literature that compounds structurally related to M-CoA such as acetyl-CoA and CoASH can inhibit CPT activity in rat skeletal muscle (19, 22, 41). In addition to binding at their own sites with an inhibitory effect that is not as great as M-CoA, these compounds appear to also compete with M-CoA at its binding site on CPT I (22). It is possible that the presence of these compounds in the muscle cell might have a role in the regulation of CPT I activity by competing with M-CoA for the binding site on CPT I. In the presence of M-CoA, they may act as partial agonists, increasing CPT I activity since they are less potent inhibitors than M-CoA. The present study examined the effect of acetyl-CoA, CoASH, and acetylcarnitine on the activity of CPT I in the presence of physiological levels of M-CoA in concentrations simulating rest (4 µM, 17 µM, and 2 mM, respectively) and exercise conditions in the muscle cell (12 µM, 9 µM, and 6 mM, respectively). However, there was no effect of any of these compounds on CPT I activity, except for 2 mM acetylcarnitine. This further reduced the activity of CPT I below that of 0.7 µM M-CoA alone, in both trained and untrained muscle. However, when the concentration of acetylcarnitine was increased to 6 mM, the effect was not maintained in either group.
The physiological significance of this acetylcarnitine effect is unclear. The level of acetylcarnitine increases in muscle during exercise (10, 29). Perhaps the additional inhibition imposed on CPT I by lower levels of acetylcarnitine at rest is relieved during moderate exercise, and this contributes to the increased catabolism of fatty acids observed during exercise at this intensity (30). However, the increase in acetylcarnitine is less during submaximal exercise at the same absolute workload after training (28) and therefore would seem to be an unlikely contributor to the increased reliance on fatty acid oxidation in trained individuals during exercise. Another factor confounding the interpretation of the metabolite results is the uncertainty regarding the cytoplasmic and mitochondrial concentrations of acetylcarnitine, acetyl-CoA, CoASH, and M-CoA. We predict that most of the acetylcarnitine is outside the mitochondria, and most of the CoASH and acetyl-CoA are inside the mitochondria. We would also expect that most of the M-CoA is cytoplasmic, given that acetyl-CoA carboxylase, the enzyme that produces most of the cellular M-CoA, is cytoplasmic. However, to our knowledge, this information is not available in rodent or human skeletal muscle. Therefore, the concentrations used in our assays may be higher than the true cellular cytoplasmic concentrations. However, since no effects were found with these concentrations, it seems unlikely that there would be any effects at lower concentrations.Effect of pH on CPT I activity. A small decline in pH from 7.0 to 6.8 in the present study resulted in a significant decrease (~50%) in the activity of CPT I in both trained and untrained muscle in the presence of physiological levels of M-CoA. We were not able to measure whether M-CoA binding to CPT I (IC50) was decreased at pH 6.8 in the trained or untrained groups of this study, as the availability of muscle was limited. However, although a portion or all of the pH effect could be explained by decreased M-CoA binding to CPT I , we found no difference between IC50 values at pH 7 vs. 6.8 in rat skeletal muscle (Berthon, unpublished observation).
It seems likely that the small decline in pH that normally occurs during moderate and intense aerobic exercise would cause a significant reduction in the activity of CPT I in both trained and untrained individuals. Howlett et al. (13) reported muscle lactate levels of 38 and 108 mmol/kg dry muscle in untrained men after only 10 min of cycle exercise at 65 and 90% ![]() |
ACKNOWLEDGEMENTS |
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We acknowledge the assistance of Dr. Mark Hargreaves.
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FOOTNOTES |
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This study was supported by operating grants from the Natural Sciences and Engineering Research and Medical Research Councils of Canada. E. Starritt was supported by an Australian Postgraduate Award. G. J. F. Heigenhauser is a Career Investigator of the Heart and Stroke Foundation of Ontario (no. I-2576).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: Dr. Lawrence L. Spriet, Department of Human Biology and Nutrition Science, University of Guelph, Guelph, Ontario, N1G 2W1, Canada (E-mail: lspriet.ns{at}aps.uoguelph.ca).
Received 2 June 1999; accepted in final form 11 October 1999.
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