Relationship between fatty acid delivery and fatty acid oxidation during strenuous exercise. J. Appl. Physiol. 79(6): 1939- 1945, 1995.-To evaluate the extent to.
Relationship between fatty acid delivery oxidation during strenuous exercise J. A. ROMIJN,
E. F. COYLE,
L. S. SIDOSSIS,
and fatty acid
X.-J. ZHANG,
AND
R. R. WOLFE
Metabolism Unit, Shriners Burns Institute, Galveston 77550; Human Performance Laboratory, Department of Kinesiology and Health Education, The University of Texas at Austin, Austin 78712; and Departments of Anesthesiology, Biochemistry, and Surgery, University of Texas Medical Branch, Galveston, Texas 77550 Romijn, J. A., E. F. Coyle, L. S. Sidossis, X.-J. Zhang, and R. R. Wolfe. Relationship between fatty acid delivery and fatty acid oxidation during strenuous exercise. J. Appl. Physiol. 79(6): 1939- 1945, 1995.-To evaluate the extent to which decreased plasma free fatty acid (FFA) concentration contributes to the relatively low rates of fat oxidation during high-intensity exercise, we studied FFA metabolism in six endurance-trained cyclists during 20-30 min of exercise. [85% of maximal O2 uptake (VOLT,)]. They were studied on two occasions: once during a control trial when plasma FFA concentration is normally low and again when plasma FFA concentration was maintained between 1 and 2 mM by intravenous infusion of lipid (Intralipid) and heparin. During the 2030 min of exercise, fat and carbohydrate oxidation were measured by indirect calorimetry, and the rates of appearance (Ra) of plasma FFA and glucose were determined by the constant infusion of [6,6-2H2]glucose and [2H2]palmitate. Lipid-heparin infusion did not influence the Ra or rate of disappearance of glucose. During exercise in the control trial, Ra FFA failed to increase above resting levels (11.0 5 1.2 and 12.4 2 1.7 pmol kg-‘* min-’ for rest and exercise, respectively) and plasma FFA concentration dropped from a resting value of 0.53 ir 0.08 to 0.29 t 0.02 mM. The restoration of plasma FFA concentration resulted in a 27% increase in total fat oxidation (26.7 ? 2.6 vs. 34.0 2 4.4 pmol . kg-’ min-‘, P < 0.05) with a concomitant reduction in carbohydrate oxidation, apparently due to a 15% (P < 0.05) reduction in muscle glycogen utilization. However, the elevation of plasma FFA concentration during exercise at 85% Tjosmax only partially restored fat oxidation compared with the levels observed during exercise at 65% Vo2 max.These findings indicate that fat oxidation is normally impaired during exercise at 85% V02 maxbecause of the failure of FFA mobilization to increase above resting levels, but this explains only part of the decline in fat oxidation when exercise intensity is increased from 65 to 85% vo2 max. l
l
stable isotopes; carbohydrate oxidation; blood glucose; indirect calorimetry
muscle
glycogen;
FREE FATTY ACIDS (FFA) supply most of the substrate oxidized by muscle during low-intensity exercise at 25% of maximal oxygen uptake (VO,,,) (19). In endurance-trained cyclists, however, the mobilization or rate of appearance (Ra) of plasma FFA actually declines progressively as the intensity of exercise is increased (11, 19). Despite this reduced availability of plasma FFA, the greatest absolute rates of total fat oxidation occur during moderate-intensity exercise (i.e., 50-75% VO 2 max), with plasma FFA and intramuscular triacylglycerides generally contributing equally to oxidation (19). When exercise at a moderate intensity is performed for prolonged periods (i.e., >30 min), plasma FFA concentration increases progressively to l-2 mM, yet total fat oxidation does not increase proPLASMA
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$3.00
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0 1995
portionally, suggesting that total fat oxidation is not limited by plasma FFA availability under these conditions (5, 19). Additionally, when carbohydrate oxidation declines after 2-3 h of moderate-intensity exercise because of depletion of muscle glycogen and hypoglycemia, fat oxidation is insufficient to meet the energy requirements, and fatigue results, despite high levels of plasma FFA (5). Therefore, during moderate-intensity exercise in fasted subjects, fat oxidation appears to be limited by muscle’s oxidative ability and/or FFA transport capacity. This notion may explain the reason why further elevation of plasma FFA during low- to moderate-intensity exercise has failed to increase total fat oxidation in some (10, 17) but not all studies (4). The situation regarding the relationship between fatty acid availability and oxidation during exercise at high intensity appears to be different from that during lower-intensity exercise. For example, during exercise at 85% VO 2lllax, Ra FFA does not increase above fasting basal levels (19). As a result of a minimal increase in Ra FFA and a high oxidative demand for FFA, plasma FFA concentration declines precipitously by >50% and remains low throughout intense exercise. The comparison of exercise at 65 and 85% vo2,, showed that between 20 and 30 min of exercise total fat oxidation was decreased at the higher intensity (43 vs. 30 pmol kg-’ min-l, P < 0.05) (19). The purpose of the present study was to determine the extent to which decreased plasma FFA mobilization and availability contribute to the limitation in fat oxidation during high-intensity exercise. We studied six endurance-trained cyclists during 30 min of high-intensity exercise (85% V02max ) on two occasions. A control trial with low plasma FFA concentration was compared with the experimental trial during which triacylglycerides (Intralipid) and heparin were infused intravenously to increase plasma FFA concentration to l-2 mM. Heparin releases lipoprotein lipase from the endothelial walls, resulting in hydrolysis of intravascular triacylglycerides, which thereby increases FFA availability during an infusion of lipid emulsion. The data obtained between 20 and 30 min of exercise were used for comparison between the two studies. Glucose and fatty acid kinetics were assessed by primed continuous infusions of the stable isotopes [6,6-2H2]glucose and [2H2]palmitate, while substrate oxidation rates were determined by indirect calorimetry. l
l
METHODS
Subjects Six endurance-trained cyclists (age 28 t 2 yr, weight 75.8 I~I 3.2 kg, height 1.81 & 0.02 m, vo2,, 63.5 t 3.2 ml= the American
Physiological
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1940
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kg-‘*min-‘) volunteered for the study. All subjects were healthy, as indicated by medical history and physical examination. They were consuming a weight-maintaining diet containing ~300 g of carbohydrates daily. vo2max was determined using an incremental cycling protocol lasting 7-10 min. The study was approved by the Institutional Review Boards of the University of Texas at Galveston and at Austin.
heparin trial, but not in the control study, the rate of [2H2]palmitate infusion was doubled 5 min before exercise to minimize changes in substrate isotopic enrichment (25). In both trials, the rate of infusion of [6,6-2H2]glucosewas doubled at the start of exercise and, after the first 10 min of exercise, the rate was tripled, compared with the infusion rate at rest. Blood
Exercise
Protocol
On 2 consecutive days, the subjects were studied at rest and during 30 min of exercise at 8085% VO, max.In both trials, the subjects were studied in the postabsorptive state, 5-6 h after the last meal, which contained 2 g of high-glycemic carbohydrate per kilogram of body weight. One experiment involved only the intravenous infusion of tracers, whereas on the other day, lipid-heparin was also infused. The order of the two trials was randomly assigned.On each occasion, stable isotopes ([6,6-2H2]glucoseand [2H2]palmitate) were infused for the determination of substrate kinetics. Substrate oxidation rates were assessedby indirect calorimetry. We previously showed that basal fat metabolism is not affected by exercise on the previous day in well-trained subjects (19). In the lipid-heparin trial, a lipid emulsion (Intralipid 20%, KABI, Clayton, NC; 0.9 ml. kg-’ h-l) was infused for 2 h at rest together with heparin (Elkins Sinn, Cherry Hill, NJ; bolus of 7.4 U/kg, continuous infusion of 7.4 U kg-’ h-l). Five minutes before the start of exercise, after blood sampleshad been taken for measurement of resting concentrations and substrate kinetics, a secondbolus of heparin (7.4 U/kg) was given and the infusion rates of lipid (1.9 ml kg-’ h-l) and heparin (15.4 U kg-’ h-l) were doubled. l
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Indirect
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Calorimetry
Indirect calorimetry was performed at rest for ~15 min and throughout the 30 min of exercise. The resting values were obtained after the subjects were supine for r2 h. The values obtained from 20 to 30 min of exercise were used to calculate substrate oxidation rates during exercise. Inspired volumes of air were measured with a dry gas meter (model CD-4, Parkinson-Cowan). Aliquots of expired gas were sampled from a mixing chamber for O2(model S3A, Applied Electrochemistry) and CO2 (model LB-2, Beckman). Analog outputs from the gas analyzers and gas meter were directed to a laboratory computer for calculation of O2uptake and CO2 output. Isotope Infusion
A Teflon catheter was placed percutaneously in an antecubital vein for infusion of isotopes and lipid-heparin. A sampling catheter was inserted in the dorsal hand vein of the contralateral side. The heated-hand technique was used to obtain arterialized blood samples(13). The subjects lay on a bed for 1 h after catheter placement. Then, after a blood sample was drawn to determine background enrichment, primed constant infusions of [6,6-2H2]glucose(0.22 pmol kg-’ min-‘, prime 17.6 pmol/kg, 99% enriched; Merck, Rahway, NJ) and, 50 min later, [2H2]palmitate (0.04 pmol kg-’ min+ , no prime) were started using calibrated syringe pumps (Harvard Apparatus, Natick, MA). The exact infusion rate in each trial was determined by measuring the concentrations in the infusates. The palmitate tracer (99% enriched) was purchased from Tracer Technologies (Newton, MA). Palmitate was bound to albumin (Cutter Biological, Berkeley, CA) by following previously described procedures (26). After 2 h of infusion during rest, exercise was started. In the lipidl
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EXERCISE
Sampling
The first blood sampleswere withdrawn before the isotope infusion was started to determine background enrichment. Blood was also taken 105, 110, 115, and 120 min after the start of infusion to measure resting kinetics and after 5, 10, 15,20,25, and 30 min of exercise. All sampleswere collected in prechilled tubes containing lithium heparin and placed on ice. Plasma was separated in a refrigerated centrifuge within 5 min after collection and stored at -7OOC. Sample
Analysis
Plasma glucoseconcentration was measured on a glucose analyzer (Beckman Instruments) by useof the glucoseoxidase method. Plasma lactate concentration was measured enzymatically on a Yellow Springs Instruments analyzer. The enrichment of glucose in plasma resulting from the [6,6-2H2]glucosetracer was determined as previously described(25). FFA were extracted from plasma immediately after the sample was thawed, isolated by thin-layer chromatography, and derivatized to their methyl esters. Palmitate and total FFA concentrations were determined by gas chromatography (model 5890, Hewlett-Packard) with use of heptadecanoic acid as an internal standard (26). Isotopic enrichment of palmitate was measured by gas chromatography/mass spectrometry by analysis of the methyl ester derivatives on a Hewlett-Packard instrument (model 5992) (25). Calculations Indirect calorimetry. Carbohydrate and fat oxidation rates were calculated using stoichiometric equations (8). In a previous study, we showedthat indirect calorimetry provides valid substrate oxidation measurementsduring high-intensity exercise (80-85% Vo2 max ) in trained subjects(18). This reflects the fact that trained cyclists are able to maintain a physiological steady state of gas exchange (including lactate concentration) at that intensity of exercise. Nitrogen excretion rate was assumedto be 135 pmol* kg-’ min? This value was taken from the measured average values determined in another study performed in our laboratory (2). A 30% error in this assumedvalue (which exceedsthe total range of values in the previous study) would have had no significant effect on the calculated values of fat and carbohydrate oxidation in exercise in the present study. Fatty acid oxidation was determined by converting the rate of triacylglyceride oxidation (gokg-’ min-‘) to its molar equivalent, with the assumptionthat the average molecular weight of triacylglyceride is 860 g/mol (8) and multiplying the molar rate of triacylglyceride oxidation by 3 becauseeach mole contains 3 mol of fatty acids. Ra and rate of disappearance (Rd; reflecting rate of tissue uptake) of glucoseand palmitate at rest were calculated using the equation of Steele (22), as modified for use of stable isotopes (19). During exercise the non-steady-state approximation of Steele was used in a spline-fitting program (23). The effective volume of distribution was assumedto be 100 ml/ kg for glucose and 40 ml/kg for palmitate. The value for palmitate was chosenbecause acute changes in palmitate concentration are essentially restricted to plasma (being bound to albumin). The Ra FFA was calculated by dividing the Ra l
l
FAT
OXIDATION
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EXERCISE
1. Substrate metabolism at rest and during exercise in a control study and with Intravenous lipid-heparin &fusion TABLE
LipidHeparin Infusion
Control
Rest
OS-
0
a
LipWHeparin Control
Plasma glucose concn, mM Plasma FFA concn, mM Blood lactate concn, mM Ra glucose, pmol kg-’ min-’ Ra FFA, pmol kg-’ min-’ Total fat oxidation, pmol kg-’ Total carbohydrate oxidation, pmol kg-’ min-’
Infusion
l
l
0.0 -20
1 -10
I 0
I 10
Time
I
1
20
l
l
l
30
(min)
l
l
min-’
5350.3 0.5350.08 1.350.1 13.450.7 11.02 1.2 5.6t0.7
4.950.2 0.89+0.05* 1.01tO.l 14.25 1.1 19.22 1.0* 4.320.4
9.95 1.2
l
8.55 1.4 Exercise
Plasma glucose concn, mM Plasma FFA concn, mM Blood lactate concn, mM Rd glucose, pmol kg-’ min-’ Ra FFA, pmol kg-’ min-’ Total fat oxidation, pmol kg-’ Total carbohydrate oxidation, pmol kg-’ min-’ Muscle glycogen oxidation, pmol kg-’ min-’ l
l
l
l
l
I
0
Time
I
1
1
10
20
30
(min)
FIG. 1. Tracer-to-tracee ratios (means t SE) of glucose (A) and palmitate (B) during infusion of [6,6-2H2]glucose and [2H2]palmitate, respectively, measured at rest and during 30 min of exercise (85% of maximal O2 uptake). Analytic variation of enrichment measurements was -2%.
of palmitate by the fractional contribution of palmitate to the total FFA concentration, as determined by gas chromatography. The rate of glycogen oxidation was calculated by subtracting the rate of plasma glucose uptake from the total rate of carbohydrate oxidation. This is a minimal value of glycogen oxidation, inasmuch as the assumption is made that all plasma glucose is oxidized.
l
l
l
l
l
min-’
5.920.1 0.2920.02 7.221.1 40.754.6 12.45 1.7 26.7k2.6
5.7kO.l 2.13+0.23* 6.85 1.0 45.323.9 61.05 10.6* 34.0+4.4*
25958
230+9* -
21926
186?11*
Values are means t SE averaged during 20- to 30-min exercise period for 6 subjects. Subjects exercised for 30 min at 85% of maximal O2 uptake. Ra, rate of appearance; Rd, rate of disappearance; FFA, free fatty acid. *P < 0.05 vs. control.
The basal values are given in Table 1. At rest the plasma concentrations of glucose and lactate and Ra glucose (Fig. 2) were not different between the control and the lipid-heparin trials. In the lipid-heparin trial, plasma FFA concentrations (Fig. 3) and Ra FFA (Fig. 3) were -70% higher than in the control trial. Nonetheless, resting carbohydrate and fat oxidation rates were not different between the two trials. Ra FFA exceeded fat oxidation rates by twofold or more in both trials (Fig. 3). Exercise
Statistical
Analysis
Substrate kinetics and oxidation were calculated using the 20- to 30-min values. The results are presented as means t SE. The effect of lipid-heparin infusion on measured parameters was evaluated by the Wilcoxon test for matched pairs. The effect of time on the response during exercise was analyzed by two-way analysis of variance for a randomized block with the subjects as blocks and time as treatment. If necessary, the times were compared by Fisher’s least significant difference test (NCSS, Gainesville, UT). Statistical significance was set at P < 0.05. RESULTS
Resting State
The isotope enrichments for palmitate and glucose are shown in Fig. 1. A good isotopic plateau existed in the basal state, and the changes in infusion rate successfully minimized changes in enrichment at the start of exercise.
Constant values of O2 uptake and COZ output were obtained during the last 20 min of each trial, and the for each trial. Lactate intensity was 84 t 0.5% vo2,, concentrations increased initially during exercise in both trials but remained stable over the last 15 min of exercise and were not significantly affected by lipidheparin infusion (Table 1). In the control trial, plasma FFA concentrations decreased 45% during exercise compared with resting values, and remained at 0.2-0.3 mM throughout exercise (P < 0.05). This was explained by increased FFA clearance, because there was no significant change from the resting value of Ra FFA during exercise (Fig. 3, Table 1). In contrast, plasma FFA concentrations increased from 1 to 2 mM in the lipid-heparin trial and were sevenfold higher after 30 min of exercise than in the control trial. There were no significant differences between trials in plasma glucose concentrations or Ra glucose (Table
1942
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LipWHeparin Control
io
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A
Infusion T
T 0
aam
0A
0 1
0 1
i5
ii
is i0 Time (min)
EXERCISE
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1*
Lipid/Heparin Infusion -a- Control
T0
T b
0
3
OXIDATION
oo!.
0
1
10
1
20
1
30
Time (min) B
5
10
15
Time
T 0
20.
25
0
‘;
0
T
T 0
30
(min)
FIG. 2. Rates of appearance (Ra) of glucose (A) and palmitate (B) at rest (t = 0) and during last 15 min of exercise (85% of maximal O2 uptake) in 6 subjects (means ? SE) during a control trial and during lipid infusion.
1). During exercise, plasma glucose concentrations increased by lo- 15%, and Rd glucose increased threefold above resting values in both trials. During lipid-heparin infusion, fat oxidation rates increased by -27% and, conversely, carbohydrate oxidation rates decreased 11% (Table 1) compared with exercise during control. The contribution of fat oxidation to energy expenditure increased significantly from N 27% during control to N 35% during lipid-heparin infusion. The minimum amount of muscle glycogen oxidation decreased significantly from 219 t 6 to 186 t 11 pmol glycosyl units kg-’ min-’ (15%). During exercise in the control trial, Ra FFA was significantly lower than whole body fat oxidation (P < 0.05; Fig. 4B), the difference indicating the minimum contribution of intramuscular triacylglycerides to energy expenditure. In contrast, during the lipid-heparin trial, Ra FFA exceeded whole body FFA oxidation rates by almost twofold (P < 0.05), indicating that plasma FFA availability was not a factor limiting fat oxidation in that circumstance. l
l
DISCUSSION
Plasma FFA are an important substrate for oxidation by muscle during exercise. We recently showed, how-
Of 0
1
10
I 20
1 30
Time (min) FIG. 3. Plasma free fatty acid (FFA) concentrations (A) and Ra FFA (B) at rest (t = 0) and during 30 min of exercise (85% of maximal O2 uptake) in 6 subjects (means + SE) during a control trial and during lipid infusion. All values during lipid-heparin infusion were significantly higher than control (P < 0.05).
ever, that their mobilization decreases markedly as exercise intensity is increased (19). This was confirmed in the present study by our observation that the Ra of FFA did not increase above resting levels when these endurance-trained cyclists exercised at 85% VO, max. Thus a 15- to 20-fold increase in oxidative metabolism from rest to exercise failed to elicit increased plasma FFA mobilization (Ra FFA; Table 1). As a result, plasma FFA concentration decreased from 0.6 mM at rest to 0.2-0.3 mM throughout the 30-min control bout, a concentration that is severalfold lower than typically observed during exercise at 25-65% vo2,, (19). The major findings of the present study were that the increase in plasma FFA concentration to l-2 mM during infusion, reexercise at 85% V02 max, via lipid-heparin sulted in a 27% increase (P < 0.05) in total fat oxidation with a concomitant reduction in carbohydrate oxidation due solely to a reduction of muscle glycogen utilization (i.e., 15%; P < 0.05) with no apparent reduction in blood glucose disappearance. Recently, Zambon et al. (28) documented the effect of in vitro lipolysis in post-heparin samples. Although we did not add a specific inhibitor to prevent in vitro lipolysis, it is unlikely that the magnitude of any in vitro
FAT OXIDATION
A 20
Total Fat Oxidation
E 0111
E a# 10
P
DURING
t T
0
E s 1
Control
Lipid Infusion
B 80 E 60 E aA 40 a
l LI
0
I
t
Control
Lipid Infusion
FIG. 4. Rates of disappearance of plasma FFA and total fat oxidation in 6 subjects (means of: SE) during a control trial and during lipid-heparin infusion. At rest (A), Ra FFA exceeded fat oxidation rates in both trials and Ra FFA during lipid-heparin infusion was significantly higher than control (T P < 0.05). During exercise at 85% of maximal O2 uptake (B), fat oxidation rates were higher than Ra FFA in control study, whereas they were only -50% of Ra FFA during lipid-heparin infusion. During lipid-heparin infusion, Ra FFA (“fP < 0.05) and fat oxidation (* P < 0.05) were significantly higher than control.
lipolysis that might have occurred would be sufficient to affect our conclusion that limitation in plasma FFA availability is only in part responsible for the relatively low FFA oxidation rates during high-intensity exercise. Even if we overestimated Ra FFA, the true value of plasma FFA availability would still be well in excess of FFA oxidation. For instance, if we overestimated Ra FFA during lipid infusion by as much as 25%, Ra FFA was still elevated by 400% over the value without Intralipid, yet fat oxidation increased by only 26%. It is well known that the absolute rate of fat oxidation is highest during moderate-intensity exercise and that it progressively declines as intensity is increased (19). For example, we recently observed that well-trained cyclists, possessing very similar characteristics to the men in the present study, oxidized fat at 42.8 pmol kg-’ min-’ after 30 min at 65% vo2max compared with only 29.6 pmol kg-’ min-’ at 85% vo2,, (19). The latter value is similar to the control conditions of the present study (i.e., 26.7 t 2.6; Table 1). Because the increase in plasma FFA concentration to l-2 mM increased fat oxidation to 34 pm01 . kg -l*min-l during exercise at one-half the 85% vos,,,, it seems that approximately normal decline in fat oxidation when intensity is inl
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EXERCISE
creased from 65 to 85% vo2,, is due to limited availability of plasma FFA for oxidation by muscle. However, the failure of fat oxidation to increase to the higher levels capable by muscle (i.e., 43 pmol kg-’ min-’ at 65% vo 2max) indicates that fat oxidation at 85% vo2,, is not merely limited by plasma FFA supply. Plasma FFA supply most of the energy for exercise at 25% v02max and about one-half of the fat for oxidation at 65-85% VO 2 max(19). However, whether endogenous supply of FFA to muscle is always adequate and therefore whether exogenous supplementation, as in the present study, is ever of benefit in humans, as reflected by a reduction of muscle glycogen use, have been unclear. Clearly, when plasma FFA concentration and mobilization are impaired by nicotinic acid, fat oxidation is reduced and muscle glycogen use is increased (1, 9, 20). These observations indicate that there are critical levels of plasma FFA concentration below which total fat oxidation is impaired. Because plasma FFA appearance is lowest during high-intensity exercise and plasma FFA concentration drops to 0.2-0.5 mM (19), we thought it logical that FFA supplementation would be most beneficial under these conditions of impaired endogenous FFA mobilization. We interpret the literature reporting mixed results regarding the benefits of FFA supplementation to be a matter of whether plasma FFA concentration and availability are low enough to limit fat oxidation during the control trial (4, 10, 17). Ravussin et al. (17) measured respiratory exchange ratio (RER) during 2.5 h of exercise at 42-47% vo2,, during control conditions (i.e., saline infusion) and also when plasma FFA concentration was raised via intravenous lipid-heparin infusion. Taken in total, their data indicated that increasing plasma FFA concentration did not increase the rate of fat oxidation. However, close inspection of their data reveals that RER values were significantly higher in the lipid infusion group at some of the time points when plasma FFA concentrations were lowered by the ingestion of exogenous glucose. On the other hand, when plasma FFA concentration was >0.7 mM during control, RER was not significantly different from that with lipid-heparin infusion. Consistent with this finding, Hargreaves et al. (10) raised plasma FFA concentration from fasting control levels of 0.6 mM to >l mM, via lipid-heparin infusion, while measuring metabolism of the knee extensor muscles and arteriovenous differences. Lipid-heparin infusion had no significant effect on muscle glycogen use or respiratory quotient across the exercising leg, although Hargreaves et al. reported a lower rate of blood glucose uptake and a lower whole body RER during lipid-heparin infusion. Thus, whereas there might be an effect of changing FFA concentrations on glucose oxidation when FFA concentration is 0.6 mM, Costill et al. (4) found a significant effect of additional lipid when the plasma FFA concentration was low (i.e., 0.2 mM). This observation has been more recently made by Dyck l
Ra FFA
0m
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l
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OXIDATION
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et al. (6). The low FFA values in the study of Costill et al. were probably due to the fact that the subjects ate carbohydrate 5-6 h before exercise. We recently provided further support for the notion that low FFA concentration limits fat oxidation, inasmuch as we found that a reduction in fat oxidation occurred during exercise at 70% VO 2 IIlax after a preexercise meal when FFA concentration fell below 0.3-0.5 mM (14). However, this notion lacks direct support from studies in which plasma FFA concentration was manipulated in the range of 0.2-0.6 mM, while the availability of other substrates was kept constant. Thus, to keep the FFA concentration low enough in our study to maximize the chance of observing an effect of increasing FFA concentration on substrate oxidation, we followed the protocol of Costill et al. by giving the subjects a high-carbohydrate meal 6 h before the start of the experiment. The metabolic conditions in the study of Costill et al. (4) were similar to those in our present study, in that plasma FFA concentration was -0.2 mM during the control trial and high with lipid supplementation, and in each study the subjects were exercising at an intensity at which muscle relies heavily on glycogen for oxidation in the absence of lipid supplementation. Plasma triacylglycerides were elevated in the study of Costill et al. by ingestion of heavy cream, and subsequently plasma FFA concentration was increased by heparin injection, with FFA concentrations of 0.6-1.0 mM during exercise. Costill et al. found that lipid-heparin supplementation raised fat oxidation 32% and reduced muscle glycogen use 40%, which is generally similar to the effects we presently observed using stable isotope techniques. We interpret these observations to indicate that fat oxidation is somewhat impaired during moderate- to high-intensity exercise when plasma FFA concentration is only 0.2-0.3 mM and that total fat oxidation can be increased by exogenous supplements that raise plasma FFA concentration (to 0.6. 1 mM) and thus reduce muscle glycogen use. Although the mechanism by which the increases in plasma FFA availability and fat oxidation in the present study caused reductions in carbohydrate oxidation is not clear, it does not seem to be due to the classic glucose-fatty acid cycle, as proposed by Randle and coworkers (15). When studying heart muscle, Randle et al. (16) proposed that increased fat oxidation raises muscle citrate and acetyl-CoA levels, which then reduce activity of the enzymes phosphofructokinase and pyruvate dehydrogenase, causing reduced carbohydrate oxidation as a result of reduced blood glucose uptake by these tissues. However, in the present study, the reduction of carbohydrate oxidation when plasma FFA was elevated was not associated with a decrease in the isotopically measured rate of blood glucose disappearance (Rd glucose). Because 290% of muscle glucose uptake and Rd glucose is oxidized during exercise (3), it is reasonable to assume that in our study blood glucose uptake and oxidation were not decreased below control levels by lipid-heparin infusion. Alternatively, from our data, we cannot rule out the possibility that impairment of glycolysis by FFA during exercise directly limits the rate of glycogen breakdown. However,
STRENUOUS
EXERCISE
the recent study of Dyck et al. (6) makes this possibility unlikely, because they found that the glycogen-sparing effect of Intralipid infusion was not related to a change in muscle citrate or acetyl-CoA concentration. Another important observation in the present study was that the very high rates of Ra FFA and restoration of plasma FFA to l-2 mM with lipid-heparin infusion increased fat oxidation during 85% Vo2,, only from 26.7 to 34.0 pmol kg-’ min. This is about one-half of the difference between the control levels at 85% 00, max and the rate of fat oxidation during exercise at 65% i702max (i.e., 42 pmol kg-’ rnin after 30 min) (19). Because the muscle is capable of higher rates of fat oxidation than observed during 85% V02max when plasma FFA availability is very high, inadequate FFA supply is not the only factor responsible for the lessthan-maximal rates of fat oxidation during high-intensity exercise. One possibility is limited FFA transport into the mitochondria as a consequence of malonyl-CoA inhibition of carnitine acetyl transferase (12). Elayan and Winder (7) showed that glucose infusion in exercising rats attenuates the normal decline in malonyl-CoA in muscle and thus could reduce the reliance on fat for energy (24). It is therefore possible that fat oxidation declines as exercise intensity is increased as a result of accelerated glycogenolysis and pyruvate, acetyl-CoA, and malonyl-CoA production, which subsequently reduces fatty acid transport into the mitochondria, despite ample FFA availability. Therefore an alternative to the glucose-fatty acid hypothesis is that pyruvate availability is the factor that determines the balance between carbohydrate and fat oxidation during exercise of various intensities. This notion is supported by the results of a variety of studies we have performed in dogs, in which different combinations of glucose and lipid infusion were tested (21, 27). In all cases, the rate of carbohydrate oxidation was determined by the availability of glucose regardless of the plasma FFA level. This suggests that carbohydrate availability regulates fat oxidation, even when FFA is readily *available, as we observed during exercise at 85% V02max with lipid and heparin infusion. In summary, the results of the present study suggest that multiple factors regulate fat oxidation during high-intensity exercise. Plasma FFA mobilization (Ra FFA) fails to increase above resting basal levels during high-intensity exercise, which results in a dramatic lowering of plasma FFA concentration to levels (i.e., 0.2-0.3 mM) that impair fat oxidation and result in increased muscle glycogen use compared with that when plasma FFA is maintained above 1 mM via intravenous lipid-heparin infusion. However, the elevation of plasma FFA during exercise at 85% To2 maxonly partially restores fat oxidation compared with the levels observed during exercise at 65% 00, max, indicating that factors other than FFA availability also regulate fat oxidation during high-intensity exercise. l
l
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The technical help of Susan George, Jeff Horowitz, Jose Gonzalez Alonso, and Ricardo Mora is greatly appreciated. This study was supported by Grants DK-34817 and DK-46017 and General Clinical Research Center Grant 00073 from the National Institutes of Health and Shriners Hospital Grant 15849. J. A. Romijn
FAT
OXIDATION
DURING
was supported by a grant from the Netherlands Organization for Scientific Research (NWO). L. S. Sidossis was an “Alexander S. Onassis” Foundation Scholar. Present address of J. A. Romijn: Dept. of Intensive Care, Academisch Medisch Centrum, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Address for reprint requests: R. R. Wolfe, Shriners Burns Institute, 815 Market St., Galveston, TX 77550.
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14. 15.
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