INTRODUCTION

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ONE OF THE HALLMARK ADAPTATIONS to endurance training is a decrease in carbohydrate utilization and an increase in fatty acid oxidation during exercise of the same absolute intensity after training (9, 11). The glucose-fatty acid cycle hypothesis [i.e., increased fatty acid oxidation suppressing carbohydrate utilization by inhibition of key enzymes of the glycolytic pathway, resulting in increased glucose 6-phosphate (G-6-P) concentration (18)] has been proposed to explain the adaptations in substrate utilization with training (8). However, Jansson and Kaijser (11) and, more recently, Coggan et al. (2) found that muscle G-6-P concentration was lower during exercise in the trained state, indicating that the shift in substrate utilization after training is unlikely to be explained by the glucose-fatty acid cycle concept as proposed by Randle et al. (18).

Alternatively, the increase in fatty acid oxidation with endurance training may be mediated by the decrease in carbohydrate utilization. From work in vitro it has been suggested that increased pyruvate availability increases malonyl-CoA formation, which in turn inhibits carnitine palmitoyltransferase (CPT) I, thereby decreasing fatty acid oxidation (16). Consistent with this hypothesis, evidence has recently been provided that increased carbohydrate flux decreases fat oxidation by inhibition of fatty acid entry into the mitochondria both at rest (24) and during exercise (23). Because training reduces the rate of carbohydrate flux during exercise, it has been hypothesized (3) that the difference in fatty acid oxidation during exercise of the same absolute intensity in the trained vs. the untrained state may be due to accelerated long-chain fatty acid entry into the mitochondria.

We have investigated the regulation of fatty acid oxidation during exercise on a cycle ergometer at an oxygen consumption (O₂) of ~2 l/min in five sedentary men and five well-trained cyclists. [1-¹³C]oleate and [1-¹⁴C]octanoate were infused to trace the metabolism of the long- (LCFA) and medium-chain fatty acids (MCFA), respectively. Octanoate, unlike oleate, freely enters the cell and crosses the inner mitochondrial membrane; hence, if fatty acid oxidation is higher in the trained state due to increased mitochondrial LCFA uptake, then oleate oxidation and the percentage of [1-¹³C]oleate taken up by tissues that is oxidized should be higher in the trained group. Octanoate oxidation and the percentage of [1-¹⁴C]octanoate uptake that is oxidized, however, would not be expected to differ between the two groups.

	METHODS

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Volunteers. Five sedentary male volunteers and five endurance-trained cyclists participated in this study. Volunteer characteristics are presented in Table 1. All volunteers were healthy, as indicated by comprehensive history, physical examination, and standard blood and urine tests, and they consented to participate in this study, which was approved by the Institutional Review Board and the General Clinical Research Center of the University of Texas Medical Branch at Galveston.

Experimental design. All experiments were performed in the morning after the volunteers had fasted overnight (i.e., 12 h). Trained volunteers were instructed to limit the duration (to <45 min) as well as the intensity of their final training bout, which was performed 24 h before the study. Teflon catheters were placed percutaneously into an antecubital vein for isotope infusion and into a contralateral dorsal hand vein, which was heated, for sampling of arterialized venous blood. After catheter placement, each volunteer sat quietly on a cycle ergometer for 15-30 min. Blood and breath samples were then obtained for the determination of background enrichments, after which the sedentary volunteer exercised for 30 min and the trained volunteer for 60 min at a O₂ of ~2.0 l/min. This represented ~80 and 40% of peak O₂ (O_{2 peak}) for the sedentary and trained volunteers, respectively. At the beginning of exercise, infusions of [1-¹⁴C]octanoate (prime = 16.0 nCi/kg, constant infusion = 1.0 nCi · kg¹ · min¹) and [1-¹³C]oleate (no prime, constant infusion = 0.15 µmol · kg¹ · min¹) were started and continued for the duration of the exercise by use of syringe pumps (Harvard Apparatus, Natick, MA). The bicarbonate pools were primed via bolus infusion of NaH¹⁴CO₃ (40 nCi/kg) and NaH¹³CO₃ (7.0 µmol/kg) at the beginning of the study. In the sedentary group only, a lipid emulsion (0.5 ml · kg¹ · h¹) was infused together with heparin (bolus of 7.0 U/kg; continuous infusion of 7.0 U · kg¹ · h¹) to prevent the expected decline in plasma free fatty acid (FFA) concentration observed during strenuous exercise (20). It was not necessary to infuse MCFA during exercise in either group, because there is virtually no octanoate in plasma, and thus the specific activity (SA) of plasma octanoate should be the same as that of the octanoate infusate in both groups.

Materials. [1-¹³C]oleate, 99% enriched, was obtained from MSD Isotopes (Montreal, Canada), [1-¹⁴C]octanoate from Du Pont (Boston, MA), human albumin (5%) from Baxter Healthcare (Glendale, CA), the lipid emulsion [Intralipid, 20%, containing linoleic (50%), oleic (26%), palmitic (10%), linolenic (9%), and stearic (3.5%) acids] from Kabi (Clayton, NC), and heparin from Elkins Sinn, Cherry Hill, NJ.

Assays. Expired air was collected in 3-liter anesthesia bags, and ¹⁴CO₂ carbon SA was determined using a liquid scintillation counter, as previously described (21). For determination of breath CO₂ carbon enrichment, 10 ml of expired air were injected into evacuated tubes, and the ¹³CO₂-to-¹²CO₂ ratio (tracer-to-tracee ratio; enrichment) was determined using isotope ratio mass spectrometry (SIRA VG Isotech, Cheshire, UK) as previously described (21).

Calculations. Plasma oleate and breath CO₂ carbon enrichment (¹³C-to-¹²C ratio) and SA (¹⁴C-to-¹²C ratio) reached plateau over at least the last 15 min of exercise (Figs. 1 and 2, respectively). The average enrichment values during the last 10 min of exercise were used for the calculation of substrate kinetics and oxidation. Substrate oxidation calculated at 20-30 min was not different from that calculated at 50-60 min of exercise at ~40% O_{2 peak} in the trained group. Therefore, we compared substrate oxidation values calculated for the last 10 min of exercise in the sedentary and trained groups.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Plasma oleate carbon enrichment (13C-to-12C ratio) in sedentary (A) and endurance-trained volunteers (B) during the last 25 min of cycle ergometer exercise at an oxygen consumption (O2) of ~2.0 l/min. Values (means ± SE for 5 volunteers) of the last 10 min were used for calculation of oleate kinetics.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Breath CO2 carbon specific activity (14C-to-12C ratio from [1-14C]octanoate infusion; A) and enrichment (13C-to-12C ratio from [1-13C]oleate infusion; B) in sedentary and endurance-trained volunteers during the last 25 min of cycle ergometer exercise at a O2 of ~2.0 l/min. Values (means ± SE for 5 volunteers) of the last 10 min were used for calculation of substrate oxidation and percent uptake oxidized.

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Statistical analysis. Differences between the two groups were identified using an unpaired t-test. Statistical significance was set at P < 0.05.

	RESULTS

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Plasma substrate concentrations are presented in Table 2 as the average of the last 10 min of exercise. By infusing lipids and heparin in the sedentary volunteers, we were able to maintain similar plasma oleate and total FFA concentrations in the two groups. Plasma glucose concentration was also similar in the two groups; however, plasma lactate concentration was significantly higher in the sedentary volunteers (P < 0.05). Similarly, carbohydrate oxidation was significantly higher in the sedentary group (196 ± 9 vs. 102 ± 17 µmol · kg¹ · min¹, P < 0.05).

The R_a of oleate in plasma was similar in the two groups, as was the R_a of total FFA (Table 3). Oleate oxidation (Fig. 3) and plasma fatty acid oxidation (tracer methodology; Table 3) were significantly higher in the trained group, whereas octanoate oxidation rate was not different in the two groups (1.2e⁰⁵ ± 9.3e⁰⁷ vs. 8.7e⁰⁶ ± 2.8e⁰⁷ µmol · kg¹ · min¹ in the sedentary and trained volunteers, respectively). Total fatty acid oxidation, calculated using indirect calorimetry and representing the sum of plasma derived and nonplasma fatty acids (intramuscular, fatty acids that are oxidized without first entering the plasma), was 16.7 ± 4.4 µmol · kg¹ · min¹ in the trained and 0 µmol · kg¹ · min¹ in the untrained. Given that there was significant plasma fatty acid oxidation even in the untrained volunteers, as measured using the labeled oleate (Fig. 3), indirect calorimetry significantly underestimated fat oxidation in this group of untrained volunteers.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Plasma oleate oxidation during cycle ergometer exercise at a O2 of ~2.0 l/min in sedentary and endurance-trained volunteers. Lipids and heparin were infused in the sedentary group to maintain plasma free fatty acid concentration constant in the 2 groups. Values are means ± SE for 5 volunteers. * P < 0.05 vs. sedentary.

The percent [1-¹³C]oleate uptake that was oxidized was higher in the trained (P < 0.05; Fig. 4) group, whereas the percent [1-¹⁴C]octanoate uptake that was oxidized was similar in the two groups (Fig. 4). The ratio of percent [1-¹³C]oleate to percent [1-¹⁴C]octanoate oxidized was also higher in the trained group (Fig. 5), suggesting that fatty acid oxidation is higher in the trained partly because of accelerated entry of fatty acids into the mitochondria. Interestingly, unlike the sedentary group, in the trained group the ratio of percent [1-¹³C]oleate to percent [1-¹⁴C]octanoate oxidized was not significantly different from one, suggesting that mitochondrial fatty acid uptake is not the rate-limiting step in endurance-trained cyclists exercising at a O₂ of ~2 l/min.

View larger version (40K): [in this window] [in a new window]   Fig. 4.   Percentage of oleate and octanoate tracer taken up by tissues and oxidized during cycle ergometer exercise at a O2 of ~2.0 l/min in sedentary and endurance-trained volunteers. Values are means ± SE for 5 volunteers. * P < 0.05 vs. sedentary.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Ratio of percent oleate uptake oxidized to percent octanoate taken up and oxidized during cycle ergometer exercise at a O2 of ~2.0 l/min in sedentary and endurance-trained volunteers. A ratio significantly different from unity suggests that mitochondrial fatty acid uptake is rate limiting for fatty acid oxidation. Values are means ± SE for 5 volunteers. * P < 0.05 vs. unity.

	DISCUSSION

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Using isotopically labeled LCFA and MCFA, we have provided evidence that mitochondrial fatty acid uptake is rate limiting for fatty acid oxidation during high-intensity exercise in sedentary individuals. In contrast, during exercise of the same absolute intensity in trained subjects, fatty acid entry into the mitochondria was enhanced and was apparently no longer limiting for fatty acid oxidation. These findings suggest that, during exercise of the same intensity, fat oxidation is higher in endurance-trained vs. sedentary individuals in part because of accelerated fatty acid entry into the mitochondria.

The comparison of oleate to octanoate oxidation requires the assumption that the main difference between the fate of the two tracers is the need for oleate to utilize CPT for transport across the mitochondrial membrane. McGarry and Foster (15) tested this assumption by comparing relative rates of oxidation of octanoate, oleate, and ()-octanoylcarnitine in perfused rat livers. ()-Octanoylcarnitine, in contrast to octanoate, is transported across the mitochondrial membrane utilizing CPT but enters the same -oxidation pathway as octanoate once inside the mitochondria. It was found that the relative rate of ()-octanoylcarnitine oxidation followed that observed for oleate rather than octanoate in livers from fed and fasted rats, suggesting that the main difference in the oxidation of oleate and octanoate is the need for oleate to utilize CPT I for transport across the mitochondrial membrane. However, there is still the possibility that LCFA and MCFA may also differ in their mode of transport across the sarcolemmal membrane. Unlike MCFA, which are thought to be able to diffuse across the membrane, LCFA may require a transport system to gain access into cells (1, 10). Therefore, the increase in the capacity of the trained volunteers to oxidize LCFA could also be interpreted as reflecting a training-induced increase of the capacity of muscle for uptake of fatty acids. Whereas this is certainly a possibility that cannot be excluded from our data, it could not explain the increased oxidation of fatty acids derived from triacylglycerols stored inside the cell (intramuscular triacylglycerols) that was observed in the trained volunteers (Table 3).

The relative concentration of various fatty acids in plasma may affect their kinetics and oxidation. To prevent the effect of different concentrations of oleate on oleate uptake and oxidation, we infused lipids and heparin during exercise in the sedentary group to increase oleate availability to the same level as in the trained group. This was necessary because peripheral lipolytic rate decreases significantly during high-intensity exercise (20). By matching oleate availability in the sedentary and trained groups, we ensured a valid comparison for oleate oxidation between them. On the other hand, oleate and octanoate concentrations were significantly different, which raises the possibility that the observed differences in their kinetics and oxidation may have been due to differences in their relative concentrations. We have previously tested this possibility (23). We increased the R_a of octanoate in plasma during exercise at 40 and 80% O_{2 peak} by exogenous infusion of unlabeled octanoate to match the R_a of oleate. In these studies we did not observe any significant changes in the percent octanoate tracer oxidized between the "low" and "high" octanoate availability experiments, suggesting that the observed differences between octanoate and oleate tracer oxidation are not due to differences in their relative concentrations (23).

The fact that the sedentary volunteers oxidized a greater percentage of octanoate than oleate during high-intensity exercise supports the concept that flux through CPT I may be rate limiting for fat oxidation during exercise when the rate of carbohydrate utilization is very high (4, 23). However, this did not appear to be true in trained volunteers exercising at the same absolute intensity. In these subjects, the percentage of oleate uptake that was oxidized was similar to that of octanoate, such that the ratio of their relative rates of oxidation did not differ significantly from unity. Thus some factor other than mitochondrial fatty acid uptake was apparently rate limiting for fat oxidation under these conditions in the trained volunteers. Possibly, during the early stages of low-intensity exercise, fat oxidation is limited by the rate of lipolysis and thus fatty acid availability.

The exact mechanism explaining the higher fatty acid oxidation in the trained volunteers cannot be determined from the present study. However, several possibilities can be suggested. It is well established that activated LCFA must bind to carnitine, a reaction catalyzed by the enzyme CPT I, to gain access into the mitochondrial matrix (6). The product of this reaction, fatty acyl-carnitine, is transported across the inner mitochondrial membrane via the carnitine-acylcarnitine translocase mechanism (17). Studies in vitro (16) and in vivo (23, 24) have suggested that accelerated carbohydrate flux inhibits CPT I, possibly via malonyl-CoA (16), and, as a consequence, fatty acid uptake into the mitochondria and fatty acid oxidation decrease. Our data suggest that the increase in fat oxidation after training is at least in part due to accelerated entry of fatty acids into the mitochondria. This is perhaps mediated via increased CPT I activity as a consequence of decreased carbohydrate flux. Alternatively, it is well established that endurance training increases the number of mitochondria in trained muscles (see Ref. 8). Thus training may enhance fatty acid entry into the mitochondria by increasing the number of mitochondria without any change in CPT I activity per mitochondrion. Furthermore, chronic increased fatty acid availability to the mitochondria, as is probably the case in endurance training, may also increase CPT I [rat hepatocytes (14)] and -hydroxyl-CoA-dehydrogenase [human vastus lateralis; (7)] activities, explaining the ability of the trained volunteers to oxidize more fatty acids.

In the present study we found that 58 ± 3% and 76 ± 7% (P < 0.05) of oleate tracer were oxidized in the sedentary and trained volunteers, respectively. Martin et al. (13) did not see any difference in the percentage of LCFA tracer oxidized before and after 12 wk of endurance training. Similarly, Kanaley et al. (12) did not observe any difference between marathon runners and moderately trained runners in the percentage of fatty acid uptake oxidized. In the above mentioned studies (12, 13), the differences in oxidative capacity between the two groups [i.e., before and after training (13) and marathoners vs. moderately trained runners (12)] may not have been as large as in the present study. It is possible that a difference in the percentage fatty acid uptake oxidized between trained and untrained persons may only become obvious, or measurable, when the difference in oxidative capacity is very large, as was probably the case in our two groups of volunteers.

There was a clear discrepancy between fatty acid oxidation calculated using indirect calorimetry and tracer methodology during exercise in the untrained group (Table 3). Indirect calorimetry suggested that there was virtually no fatty acid oxidation during exercise, whereas tracer estimates indicated that fatty acid oxidation contributed significantly to energy production. High-intensity exercise is often associated with a progressive increase in plasma lactate concentration, progressive hyperventilation, and a decrease in the bicarbonate pool as reflected in plasma bicarbonate concentrations (25). Under such conditions indirect calorimetry may not be valid in estimating substrate oxidation, because changes in the bicarbonate pool preclude reliable measurements of tissue CO₂ by breath CO₂ analyses. On the other hand, if lactate concentration increases in the initial phase of the exercise but remains stable subsequently, as is the case with highly trained cyclists exercising at ~80% maximal aerobic capacity (19), indirect calorimetry may still be valid in estimating whole body fatty acid oxidation (19).

Because muscle glycogen concentrations were not measured in this study, it is possible that the two groups differ in their preexercise glycogen stores (lower in trained), which could potentially affect the interpretation of the findings of the present study. However, it seems unlikely that potential differences in preexercise glycogen stores have significantly influenced the pattern of glycogen use in these two groups, because on the basis of the literature, the trained volunteers exercising at 40% O_{2 peak} used significantly less muscle glycogen than the sedentary volunteers exercising at 80% O_{2 peak}. However, it is still possible that the observed differences in fatty acid oxidation between the two groups reflect genetic variation in physiological characteristics of the subjects, such as fiber type, percent body fat, or hormonal responses. As a cross-sectional design, the present study shares all the limitations inherent in this type of research design.

In summary, the results of the present study suggest that endurance training may increase fat oxidation during exercise in part by enhancing fatty acid entry into the mitochondria. This may be due to decreased carbohydrate flux and thus increased CPT I activity per mitochondrion, or simply to increased mitochondrial number and thus increased total CPT I. Furthermore, our data suggest that in untrained human volunteers exercising at a O₂ of 2 l/min, mitochondrial fatty acid uptake is one factor limiting the rate of fat oxidation. However, when trained volunteers exercise at the same absolute intensity, fatty acid uptake by the mitochondria does not appear to be limiting fat oxidation.