Am J Physiol Endocrinol Metab. 2005 Jan.
Malonyl-CoA and carnitine in regulation of fat oxidation in human skeletal muscle during exercise.
Roepstorff C, Halberg N, Hillig T, Saha AK, Ruderman NB, Wojtaszewski JF, Richter EA, Kiens B.
The Copenhagen Muscle Research Centre, Department of Human Physiology, Institute of Exercise and Sport Sciences, University of Copenhagen, Copenhagen, Denmark. [ PubMed ] [ Free full text ]

Abstract: Intracellular mechanisms regulating fat oxidation were investigated in human skeletal muscle during exercise. Eight young, healthy, moderately trained men performed bicycle exercise (60 min, 65% peak O2 consumption) on two occasions, where they ingested either 1) a high-carbohydrate diet (H-CHO) or 2) a low-carbohydrate diet (L-CHO) before exercise to alter muscle glycogen content as well as to induce, respectively, low and high rates of fat oxidation. Leg fat oxidation was 122% higher during exercise in L-CHO than in H-CHO (P < 0.001). In keeping with this, the activity of alpha2-AMP-activated protein kinase (alpha2-AMPK) was increased twice as much in L-CHO as in H-CHO (P < 0.01) at 60 min of exercise. However, acetyl-CoA carboxylase (ACC)beta Ser221 phosphorylation was increased to the same extent (6-fold) under the two conditions. The concentration of malonyl-CoA was reduced 13% by exercise in both conditions (P < 0.05). Pyruvate dehydrogenase activity was higher during exercise in H-CHO than in L-CHO (P < 0.01). In H-CHO only, the concentrations of acetyl-CoA and acetylcarnitine were increased (P < 0.001), and the concentration of free carnitine was decreased (P < 0.01), by exercise. The data suggest that a decrease in the concentration of malonyl-CoA, secondary to alpha2-AMPK activation and ACC inhibition (by phosphorylation), contributes to the increase in fat oxidation observed at the onset of exercise regardless of muscle glycogen levels. They also suggest that, with high muscle glycogen, the availability of free carnitine may limit fat oxidation during exercise, due to its increased use for acetylcarnitine formation.

Excerpts from the full text article:

Fat and carbohydrate are the main sources of fuel for ATP synthesis in human skeletal muscle. The ratio between fat and carbohydrate oxidation during exercise depends on preexercise substrate levels and exercise duration and intensity. However, the intracellular determinants of the use of these fuels are unclear.

It has been suggested that the fat oxidation rate in human skeletal muscle during exercise is regulated intracellularly at the entry of long-chain fatty acyl (LCFA)-CoA into the mitochondria. The transport of LCFA-CoA across the inner mitochondrial membrane is preceded by the transfer of the acyl moiety to carnitine, a process catalyzed by carnitine palmitoyltransferase 1 (CPT-1), an enzyme located in the outer mitochondrial membrane. Two mechanisms for the intracellular regulation of CPT-1 have been proposed; one involves malonyl-CoA, and the other involves the cytosolic concentration of carnitine.

Malonyl-CoA is an intermediate in the de novo synthesis of fatty acids (FA) and an allosteric inhibitor of CPT-1. Studies in rat muscle suggest that changes in the glucose supply and energy expenditure of the muscle cell regulate the concentration of malonyl-CoA, in keeping with its need to generate ATP from fat oxidation. Two types of regulation of malonyl-CoA have been described: one involving the cytosolic concentration of citrate and the other involving 5'-AMP-activated protein kinase (AMPK). Cytosolic citrate is both an allosteric activator of acetyl-CoA carboxylase (ACC), the key enzyme governing malonyl-CoA synthesis, and a substrate for the malonyl-CoA precursor, cytosolic acetyl-CoA. In rodents and humans, high glucose availability at rest has been shown to elevate the cytosolic citrate concentration and consequently the muscle malonyl-CoA concentration. This is likely the mechanism whereby fat oxidation is inhibited by high glucose availability in resting humans. AMPK in turn regulates the concentration of malonyl-CoA by phosphorylating and inhibiting ACC and possibly by activating malonyl-CoA decarboxylase (MCD), the major enzyme regulating malonyl-CoA turnover in muscle. During muscle contractions in rodents, AMPK is activated and ACC is phosphorylated and consequently inhibited, leading to a decrease in muscle malonyl-CoA concentration, which may induce the increase in fat oxidation at the onset of contraction. In contrast, several human studies have failed to show a reduction in the muscle malonyl-CoA concentration during moderate-intensity exercise with normal muscle glycogen stores when fat oxidation is increased from resting levels. Still, with low muscle glycogen content, there is a higher AMPK activation during exercise, which may decrease the malonyl-CoA level and thereby cause the higher fat oxidation seen during exercise with low vs. high muscle glycogen. So far, the muscle malonyl-CoA concentration has never been measured in humans during submaximal exercise with low muscle glycogen content.

Carnitine is a substrate for CPT-1. Consequently, the availability of cytosolic carnitine may limit fat oxidation. Another function of carnitine is to buffer accumulated acetyl-CoA in a reaction catalyzed by carnitine acetyltransferase (CAT). It has been proposed that, under exercise conditions with high glycolytic flux and therefore excess formation of acetyl-CoA compared with its utilization by the tricarboxylic acid cycle, carnitine serves to buffer the excess acetyl-CoA, which leaves less carnitine available to CPT-1. By this mechanism, carnitine may play a role in adjusting the rate of fat oxidation inversely to the rate of carbohydrate oxidation. Accordingly, in humans it was shown that fat oxidation and muscle carnitine concentration were tightly coupled during incremental exercise: with increasing exercise intensity, they both decreased, while, on the other hand, carbohydrate oxidation increased.

The present study investigated intracellular mechanisms to regulate fat oxidation in response to altered carbohydrate availability in human skeletal muscle during submaximal exercise. Healthy male subjects performed 60 min of submaximal bicycle exercise with either high (H-CHO) or low (L-CHO) preexercise muscle glycogen. During the two exercise protocols, with expected similar energy output but marked differences in the relative fat and carbohydrate oxidation, we measured in the same setup the malonyl-CoA, carnitine, acetylcarnitine, and acetyl-CoA concentrations, the pyruvate dehydrogenase (PDH) and AMPK activity, and the ACC Ser221 phosphorylation in skeletal muscle to examine the intramuscular mechanisms that regulate the ratio between fat and carbohydrate oxidation during exercise. To rule out marked differences between H-CHO and L-CHO conditions in blood-borne substrate and hormone levels and their possible confounding effects on the interpretations of the results, we allocated a light meal 4.5 h before exercise and infused glucose intravenously during exercise to keep the arterial blood glucose concentrations nearly constant and similar between the two conditions. By this approach, the levels of several other circulating metabolites and hormones also were kept nearly similar between H-CHO and L-CHO.

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Discussion

The intracellular mechanisms regulating the ratio between fat and carbohydrate oxidation in human skeletal muscle during exercise have not yet been fully clarified. A likely site of regulation of fat oxidation is CPT-1 at the entry of LCFA-CoA into mitochondria. The present study is the first to simultaneously measure several factors related to the possible role of malonyl-CoA and carnitine in regulation of fat oxidation at CPT-1 in humans during exercise. Exercise was performed under two conditions at comparable work loads but with marked differences in the relative fat and carbohydrate oxidation rates. The latter was accomplished by manipulating the muscle glycogen content to high (H-CHO) or low (L-CHO) levels before a 60-min bicycle exercise bout at 65% O2 peak.

It was previously suggested that low preexercise muscle glycogen content might be associated with high AMPK activity in rodents during muscle contraction in vitro and in humans during exercise. However, in the latter study, circulating epinephrine was also dependent on muscle glycogen content, wherefore the possible effect of muscle glycogen on 2-AMPK activity may have been confounded by adrenergic stimulation of 2-AMPK. Adrenergic stimulation of AMPK has been demonstrated in 3T3-L1 adipocytes (47) and has also been reported for rat skeletal muscle. In the present study, circulating epinephrine did not differ significantly between conditions, and, still, 2-AMPK activity during exercise was markedly higher in L-CHO than in H-CHO. This suggests that altered muscle glycogen alone can influence 2-AMPK activity in human skeletal muscle during exercise independently of adrenergic stimulation, in agreement with studies in rodent muscle. On this basis, AMPK could be a likely candidate mediating the effect of muscle glycogen on fat oxidation through regulation of ACC activity and malonyl-CoA formation, as suggested earlier. However, the similar ACC Ser221 phosphorylation and malonyl-CoA concentration between L-CHO and H-CHO in the present study suggest that the effect of muscle glycogen on fat oxidation occurs primarily by other mechanisms in human skeletal muscle during exercise.

AMPK Thr172 phosphorylation closely paralleled 2-AMPK activity in the present study, indicating that the markedly higher 2-AMPK activity during exercise in L-CHO than in H-CHO was due to a higher stimulatory effect on AMPK by an upstream AMPK kinase that phosphorylated 2-AMPK on Thr172. However, altered muscle glycogen did not influence the large increase in ACC Ser221 phosphorylation seen with exercise, suggesting that allosteric regulation of AMPK, which is not detected in the AMPK activity assay, may have overridden the covalent regulation of AMPK by an upstream AMPK kinase. This is in contrast to a previous study in which 2-AMPK activity and ACC Ser221 phosphorylation were closely associated during exercise with low vs. high muscle glycogen. The major difference between that study and the present study was that, in the present study, subjects ingested a preexercise meal and had glucose infused intravenously during exercise, which resulted in similar blood glucose and epinephrine levels between conditions. In the previous study, blood glucose and plasma epinephrine concentrations differed markedly between conditions. It follows that, in the present study, the different rates of intravenous glucose infusion in H-CHO and L-CHO leading to similar levels of circulating glucose and epinephrine between H-CHO and L-CHO may have contributed to the dissociation between measured 2-AMPK activity and ACC Ser221 phosphorylation. If so, the in vivo 2-AMPK activity in the present study may have been better reflected by the ACC Ser221 phosphorylation state than by the in vitro 2-AMPK activity. Alternatively, ACC Ser221 phosphorylation may have been affected by factors other than AMPK activity, such as spatial dissociation of AMPK and ACC or phosphatase activity acting on ACC Ser221. Those factors may in some cases render ACC Ser221 phosphorylation a not-so-optimal predictor of in vivo 2-AMPK activity. Altogether, the present study suggests that during exercise, covalent regulation of 2-AMPK on Thr172 is glycogen dependent, but that ACC Ser221 phosphorylation does not parallel 2-AMPK activity when circulating glucose and epinephrine levels are standardized. This is supported by findings in isolated contracting rodent skeletal muscle with high and low muscle glycogen levels, where differences in 2-AMPK activity did not translate into differences in ACC activity.

Despite markedly different 2-AMPK activity during exercise between H-CHO and L-CHO, ACC Ser221 phosphorylation and malonyl-CoA concentration did not differ significantly between L-CHO and H-CHO. Still, the fat oxidation rate was higher during exercise with low muscle glycogen even though 160% more glucose was infused intravenously under this condition. This suggests that the AMPK-ACC-malonyl-CoA pathway is not likely to mediate the regulation of fat oxidation by muscle glycogen availability in human skeletal muscle during prolonged exercise. This conclusion is also supported by other human exercise models where no association between malonyl-CoA content and fat oxidation rate was obtained in skeletal muscle during prolonged moderate-intensity exercise or during graded-intensity exercise. However, compartmentalization of malonyl-CoA may occur in skeletal muscle, i.e., concentrations of malonyl-CoA in the vicinity of CPT-1 may vary without any detectable changes in the measurement of total muscle malonyl-CoA concentration. In resting human skeletal muscle, changes in malonyl-CoA concentration have occurred with opposite changes in fat oxidation. Thus a significant negative correlation was obtained between muscle malonyl-CoA content and fat oxidation rate in healthy middle-aged men during a two-step euglycemic-hyperinsulinemic clamp. Furthermore, in healthy, young individuals, the inhibition of fat oxidation induced by hyperglycemia with hyperinsulinemia occurred together with a threefold increase in muscle malonyl-CoA concentration. These results suggest that, in resting human skeletal muscle, the downregulation of fat oxidation by insulin may be mediated by malonyl-CoA.

The absolute increase in fat oxidation rate that occurred from rest to exercise in both conditions in the present study was associated with increased ACC Ser221 phosphorylation and decreased malonyl-CoA concentration. This suggests that inactivation of ACC by AMPK and the consequent decrease in malonyl-CoA concentration from rest to exercise may have relieved malonyl-CoA inhibition of fat oxidation in both conditions. There was a tendency (P = 0.10) for malonyl-CoA to decrease more from rest to exercise in L-CHO than in H-CHO. This could have been due to lower availability of cytosolic acetyl-CoA, the precursor of malonyl-CoA, in L-CHO than in H-CHO. Alternatively, the activity of MCD, the enzyme catalyzing the turnover of malonyl-CoA, may have differed between the two conditions. It has been suggested that AMPK phosphorylates and activates MCD during muscle contraction. Consequently, the higher 2-AMPK activity in L-CHO than in H-CHO might have induced higher malonyl-CoA turnover in L-CHO via an effect on MCD. The tendency toward a more pronounced decrease in muscle malonyl-CoA by exercise in L-CHO than in H-CHO may have contributed to the difference between conditions in fat oxidation rate during exercise (Fig. 7). However, the increase in absolute fat oxidation rate by exercise was much more marked in L-CHO than in H-CHO. Therefore, factors other than malonyl-CoA seem to be more important in "fine tuning" the fat oxidation rate during prolonged exercise with different muscle glycogen stores. Availability of carnitine, a necessary substrate for CPT-1, could be one such factor. The importance of carnitine in fat oxidation is evidenced by the fact that, in the pathology of lipid storage myopathy, a marked reduction in skeletal muscle carnitine content by 85% was associated with a 75% reduction in palmitate and oleate oxidation in rectus femoris muscle homogenates. On the other hand, the usual fluctuations in free carnitine content in skeletal muscle of healthy humans between 1 and 4 mM are not expected to influence CPT-1 activity, since it has been reported that the Km of CPT-1 for carnitine is 0.5 mM at pH 7.4 in human skeletal muscle. Nevertheless, partitioning of free carnitine between the cytosol and the mitochondrial matrix makes it very difficult to estimate the absolute carnitine concentration in the vicinity of CPT-1. Furthermore, a drop in muscle pH during high-intensity exercise leads to an increase in the Km of CPT-1 for carnitine. Thus carnitine may still be a potent regulator of CPT-1 during exercise. In support, the skeletal muscle free carnitine concentration as well as the fat oxidation rate was lower during prolonged moderate-intensity exercise in H-CHO than in L-CHO in the present study. Also, in several previous human studies, similar alterations in muscle free carnitine content and fat oxidation rate occurred in parallel during exercise. Thus muscle carnitine content and fat oxidation rate increased to a similar extent during graded-intensity exercise. During bicycle exercise at 75% O2 peak to exhaustion with either high or low muscle glycogen, both muscle carnitine content and fat oxidation rate were markedly higher with low muscle glycogen. Taken together, the present and previous studies suggest a relation between muscle free carnitine availability and fat oxidation rate in human skeletal muscle during exercise.

Protein oxidation was not measured in the present study, and, consequently, absolute rates of fat and carbohydrate oxidation across the leg were calculated using the nonprotein respiratory quotient. Protein oxidation may have differed between L-CHO and H-CHO and, in that case, would have slightly confounded the fat and carbohydrate oxidation rates reported in Fig. 2. However, during moderate-intensity exercise, protein usually covers <5% of oxidative metabolism. Therefore, the fact that protein oxidation was ignored in the present study has probably not influenced the results and interpretations to any major extent.

Several potentially confounding blood metabolites and hormones were kept almost similar between L-CHO and H-CHO in the present study due to a light carbohydrate-rich preexercise meal and a variable intravenous glucose infusion during exercise. Thus the higher preexercise muscle glycogen content seems to have been the primary cause of the higher PDH activity seen in H-CHO vs. L-CHO. In accordance, it has previously been suggested that PDH activity is sensitive to the muscle glycogen level. PDH activity seems to be important in limiting glycolysis during exercise. In the present study, the high PDH activity during exercise in H-CHO may have, consequently, enhanced glycolytic flux and production of acetyl-CoA from carbohydrate compared with L-CHO (Fig. 7). The accumulation of acetyl-CoA in H-CHO compared with L-CHO most likely decreased the muscle free carnitine content via the carnitine acetyltransferase equilibrium. If muscle free carnitine is an important regulator of the fat oxidation rate during exercise, then PDH, acetyl-CoA, and acetylcarnitine could have linked the regulation of fat oxidation to carbohydrate availability and glycolytic flux (Fig. 7). The present findings therefore support the hypothesis that regulation of fat oxidation in response to carbohydrate availability in human skeletal muscle during exercise is at least partly mediated via the availability of free carnitine to CPT-1.

In conclusion, the present study is the first to show a decline in muscle malonyl-CoA concentrations from rest to moderate-intensity exercise in humans, which may contribute to the increase in absolute fat oxidation at the onset of exercise. However, malonyl-CoA does not seem to be a major factor in fine tuning fat oxidation in human skeletal muscle during prolonged exercise with different preexercise muscle glycogen content, since muscle malonyl-CoA concentrations did not depend on muscle glycogen levels despite marked differences in fat oxidation rates between low and high muscle glycogen conditions. On the other hand, the present findings support the assertion that the availability of free carnitine to CPT-1 may participate in regulation of fat oxidation in human skeletal muscle during prolonged moderate-intensity exercise, since muscle carnitine and fat oxidation rate were both lower during exercise with high compared with low glycogen.