Effects of acetate infusion and hyperoxia on muscle substrate phosphorylation after onset of moderate exercise

Melissa K. Evans1, Ingrid Savasi1, George J. F. Heigenhauser2, and Lawrence L. Spriet1

1 Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario N1G 2W1; and 2 Department of Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study investigated whether increased muscle acetylcarnitine provision (acetate infusion) or hyperoxia (100% O2) would increase the rate of oxidative phosphorylation and reduce the reliance on muscle substrate phosphorylation after the onset of moderate exercise. Eight subjects completed three randomized trials, each separated by 1 wk: 1) saline infusion for 1 h before exercise, while breathing room air for 20 min before exercise and during 120 s of cycling at 65% maximal exercise (VO2 max) (CON), 2) saline infusion with 4 mmol/kg body wt sodium acetate, while breathing room air before and during exercise (ACE), and 3) saline infusion and breathing 100% O2 before and during exercise (HYP). Muscle biopsies were sampled at rest and after 30 and 120 s of exercise. ACE increased muscle acetyl-CoA and acetylcarnitine contents at rest vs. CON and HYP [22.9 ± 2.8 vs. 8.9 ± 2.4 and 10.5 ± 1.8 µmol/kg dry muscle (dm); 11.0 ± 1.2 vs. 3.5 ± 1.3 and 4.0 ± 1.2 mmol/kg dm]. Acetate had no effect on resting pyruvate dehydrogenase activity in the active form (PDHa) among CON, ACE, and HYP. During exercise, acetyl-CoA and acetylcarnitine were unchanged in ACE but increased over time in the CON and HYP trials, and PDHa increased similarly in all trials. Muscle phosphocreatine use, lactate accumulation, and substrate phosphorylation energy provision after 30 or 120 s of exercise were similar in all trials. In summary, increased acetylcarnitine availability did not accelerate the rate of oxidative phosphorylation at the onset of exercise, suggesting that this is not a site of extra substrate. Hyperoxia had no effect on substrate phosphorylation, suggesting that O2 availability does not limit oxidative phsophorylation at the onset of moderate exercise.

acetylcarnitine; acetyl-coenzyme A; oxidative phosphorylation; pyruvate dehydrogenase activity; lactate; phosphocreatine; oxygen


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE ACTIVATION OF MITOCHONDRIAL oxidative phosphorylation at the onset of exercise is believed to follow an exponential time course, such that the rate of ATP hydrolysis initially exceeds the rate of ATP production from oxidative phosphorylation (1, 29, 38). This transient shortfall in oxidative ATP supply, termed the O2 deficit, is supplemented by substrate level phosphorylation, including phosphocreatine (PCr) utilization and ATP production in the glycolytic pathway with lactate formation. The delayed activation of oxidative phosphorylation at the onset of exercise is thought to be a function of metabolic inertia, including lags in enzyme activation or substrate availability and/or a limited oxygen supply at the mitochondria in some muscle fibers (2, 8, 9, 15, 34, 37).

Previous work supported the existence of metabolic inertia during the onset of submaximal exercise (50-65% VO2 max) in conditions of normoxia (12, 30) and partial ischemia (31-33). In these studies, the administration of dichloroacetate (DCA) increased the provision of acetyl-CoA for the tricarboxylic acid (TCA) cycle, resulting in less PCr degradation and lactate accumulation after the start of exercise. However, because DCA increases both pyruvate dehydrogenase (PDH) activity and the content of acetylated compounds, it has been debated whether the source of extra substrate was from increased flux through PDH (12) or from the elevated acetylcarnitine store (31-33). Infusion of sodium acetate has been used previously to increase resting acetylcarnitine and acetyl-CoA contents in human skeletal muscle without affecting PDH activity (13, 25). In this manner, it was possible to directly test whether elevated resting acetylcarnitne alone can supplement the provision of acetyl-CoA, increase oxidative phosphorylation, and decrease substrate phosphorylation at the onset of exercise.

We previously measured a 35% reduction in substrate phosphorylation with DCA infusion during the onset of exercise at 65% VO2 max in human skeletal muscle (12), suggesting a large metabolic inertia component. However, the greater substrate provision did not completely eliminate substrate phosphorylation, suggesting that O2 availability may have been suboptimal in some fibers. Investigations into the existence of an O2 limitation have been based mainly on estimations of energy demand and whole body O2 uptake (VO2) kinetics measured at the mouth, an indirect estimation of the O2 deficit. Hyperoxic conditions enhanced VO2 on-kinetics during intense (78-82% VO2 peak) submaximal exercise (20) but had no effect at moderate (below ventilatory threshold) power outputs (14, 21). One study made direct muscle measurements and reported less PCr degradation and lactate accumulation during cycling at 55% VO2 max with hyperoxia (18), but the measurements were made too late to assess the rest-to-exercise transition (4 min).

The first purpose of this study was to determine whether an elevated acetylcarnitine store would increase acetyl-CoA provision at the onset of moderate exercise, resulting in enhanced activation of oxidative phosphorylation and decreased reliance on substrate phosphorylation. The second purpose was to determine whether an O2 limitation also contributes to the delay in oxidative phosphorylation at the onset of moderate exercise, by having subjects breathe 100% O2 during exercise. We did not estimate the O2 deficit with power output and respiratory measures but directly measured substrate phosphorylation from PCr degradation and glycolytic ATP production. We assumed that any increase in oxidative phosphorylation after the onset of exercise would result in decreased substrate phosphorylation. We hypothesized that both acetate infusion and hyperoxia would decrease substrate phosphorylation at the onset of exercise.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects. Eight healthy, moderately active male subjects volunteered to participate in this study. Their mean (± SE) age, height, weight, and VO2 max were 22.9 ± 1.0 yr, 180.0 ± 2.7 cm, 77.0 ± 2.8 kg, and 54.7 ± 3.0 ml · kg-1 · min-1, respectively. Written informed consent was obtained from each subject after a thorough explanation of the study protocol and the associated risks. The study was approved by the ethics committees of both universities.

Experimental infusions. Sodium acetate (4 M) was obtained from the McMaster University Medical Center and administered to the subjects intravenously in a 500-ml saline solution at a dose of 4 mmol/kg body weight. The acetate solution was infused over 1 h immediately before exercise. For the control and hyperoxic trials, 500 ml of saline were infused over 1 h immediately before exercise. The acetate dose was smaller and was infused over a longer time period than previously used (25) to minimize any changes in plasma pH.

Preexperimental protocol. Subjects underwent a continuous incremental exercise test on a bicycle ergometer to determine VO2 max by analysis of expired breath for gas concentrations (O2 and CO2) and volume with a metabolic cart (Quinton Q-Plex 1, Quinton Instruments, Seattle, WA). From this test, power outputs eliciting 65% VO2 max were estimated, and the subjects returned on a separate day for a 5-min practice ride to confirm the power output. Subjects breathed room air through a mouthpiece for 20 min immediately before and during exercise to simulate testing conditions. Subjects pedaled between 90 and 100 rpm during the VO2 max test and practice ride and maintained this cadence during all three experimental trials.

Experimental protocol. On three separate experimental days (each separated by 1 wk), subjects arrived at the laboratory at the same time of day, having eaten a carbohydrate-rich meal 2-4 h before the trial. Subjects were asked to replicate their diet each week for the 24-h period before testing and to refrain from prolonged or intense physical activity for the 24 h before each trial. Caffeine consumption was maintained at each subject's normal daily intake.

The three experimental conditions were control (breathing room air and saline infusion), acetate (breathing room air and acetate infusion), and hyperoxia (breathing 100% O2 and saline infusion). The order of the trials was randomly assigned, and the subjects were blind to the treatment they received. On each test day, a catheter was inserted into a forearm vein, and 500 ml of either an acetate/saline or saline solution were infused over 1 h before exercise. During this hour, subjects rested quietly on a bed. At 30 min into the infusion, one leg was prepared for needle biopsy (4), with three incisions made through the skin superficial to the vastus lateralis muscle under local anesthesia (2% lidocaine without epinephrine). The contralateral leg was prepared for biopsies in trial 2, and the initial biopsied leg was repeated in trial 3. During the final 20 min of this hour, the subjects breathed either room air (21% O2) or hyperoxic air (100% O2). After 1 h, the catheter was removed, and a resting biopsy was taken. Subjects then moved to an electrically braked cycle ergometer (Excalibur, manufactored by Lode and distributed by Quinton Instruments, Seattle, WA) while continuing to breathe the specified gas mixture, and they began pedaling at the prescribed power output for 120 s. Exercise biopsies were taken at 30 and 120 s while the subject remained on the cycle ergometer. The stop time to allow sampling of the 30-s biopsy (stop to restart) was fixed at 30 s in all trials. Muscle biopsies were immediately frozen in liquid N2, removed from the needle while frozen, and stored in liquid N2 until analysis.

Analysis. A small piece of frozen wet muscle (8-15 mg) was removed for the determination of PDH activity in its active form (PDHa), as described by Constantin-Teodosiu et al. (6) and modified by Putman et al. (26). Total creatine concentrations were measured for each muscle homogenate, and PDHa values were corrected to the highest total creatine value in all biopsies from the same subject. The remainder of the biopsy sample was freeze-dried; dissected free of all visible blood, connective tissue, and fat; and powdered for subsequent analysis.

One aliquot of freeze-dried muscle (8-10 mg) was extracted with 0.5 M perchloric acid (PCA) (containing 1 mM EDTA) and neutralized with 2.2 M KHCO3. This extract was used for the determination of ATP, PCr, creatine (Cr), lactate, glucose 6-phosphate (G-6-P), fructose 6-phosphate (F-6-P), and free glucose by enzymatic spectrophotometric assays (3, 10). Pyruvate was analyzed on this extract by use of a fluorometric assay (24). Acetyl-CoA and acetylcarnitine contents were determined by radiometric measures (5). Muscle glycogen content was measured in duplicate on an additional aliquot of freeze-dried muscle (2-3 mg) from resting samples (10). All muscle measurements were corrected for the highest total creatine measured in the nine biopsies from each subject.

Calculations. Free ADP and AMP concentrations were calculated with the assumption of equilibrium of the creatine kinase and adenylate kinase reactions, as previously described (7). Free ADP was calculated using the measured ATP, Cr, and PCr values, an estimated H+ concentration, and the creatine kinase equilibrium constant of 1.66 × 109. The H+ concentration was estimated from the measured lactate and pyruvate content with the regression equation described by Sahlin et al. (28). Free AMP was calculated from the estimated free ADP and measured ATP content with the adenylate kinase equilibrium constant of 1.05. Free Pi was calculated by adding the estimated resting free phosphate of 10.8 mmol/kg dry weight (7) to the difference in PCr content (Delta PCr) minus the accumulation of the glycolytic intermediates G-6-P and F-6-P between rest and each exercise time point.

Substrate phosphorylation (anaerobic energy yield) in millimoles ATP per kilogram dry muscle was determined for each treatment at 0-30 and 30-120 s by adding the PCr utilization plus 1.5 times the lactate accumulation.

Statistics. All data are presented as means ± SE. For all metabolite contents except glycogen, a 2-way ANOVA (time × trial) with repeated measures was used to test for significance. Glycogen and anaerobic energy yield were analyzed using a 1-way ANOVA with repeated measures. Results were considered significant at P < 0.05, and a Tukey post hoc test was used to determine where the significant differences occurred.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Power output and VO2. During each trial, subjects cycled at a power output ranging from 155 to 220 W, with a mean of 186 ± 9 W. The average VO2 was 36.5 ± 1.6 ml · kg-1 · min-1 or 64.8 ± 0.9% VO2 max. VO2 measurements during the practice ride indicated that all subjects reached a steady-state VO2 between 100 and 120 s of exercise.

PDHa. There was no significant difference in PDHa among CON, ACE, and HYP at any time point (Fig. 1). PDHa activity increased significantly during exercise in all trials.


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Fig. 1.   Pyruvate dehydrogenase activation (PDHa) during control (CON), acetate infusion (ACE), and hyperoxia (HYP) trials. *Significantly different from rest for all trials; H, significantly different from 30 s for all trials.

Resting muscle metabolites. There were no significant differences between trials in all resting muscle metabolites except acetyl-CoA and acetylcarnitine (Table 1, Figs. 2-5). ACE infusion increased resting acetyl-CoA by two- to threefold (22.9 ± 2.8 vs. 8.9 ± 2.4 and 10.5 ± 1.8 µmol/kg dm) and acetylcarnitine by threefold (11.0 ± 1.2 vs. 3.5 ± 1.3 and 4.0 ± 1.2 mmol/kg dry matter) compared with the CON and HYP trials. Resting glycogen contents were also not different among trials (CON, 405 ± 22; ACE, 466 ± 55; HYP, 404 ± 16 mmol/kg dry matter).

                              
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Table 1.   Muscle metabolite concentrations at rest and after 30 and 120 s of cycling in control (CON), acetate infusion (ACE), and hyperoxia (HYP) conditions



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Fig. 2.   Phosphocreatine degradation during CON, ACE infusion, and HYP trials. *Significantly different from rest for all trials; H, significantly different from 30 s for all trials.



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Fig. 3.   Lactate accumulation during CON, ACE infusion, and HYP trials. *Significantly different from rest for all trials; H, significantly different from 30 s for CON and ACE.



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Fig. 4.   Pyruvate accumulation during CON, ACE infusion, and HYP trials. *Significantly different from rest for all trials. H, significantly different from 30 s for CON and ACE.



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Fig. 5.   Acetyl-CoA accumulation (A) and acetylcarnitine accumulation (B) during CON, ACE, and HYP trials. +Significantly different from rest for CON and HYP; H, significantly different from 30 s for CON and HYP.

Muscle metabolites during exercise. There was no significant difference in ATP content among trials or during exercise (Table 1). Levels of free ADP, free AMP, and free Pi were not significantly different among trials but increased during exercise in all trials (Table 1). PCr content decreased significantly at 30 s of exercise, with no difference among trials (Fig. 2). PCr decreased further at 120 s in the CON and ACE trials but not in HYP, such that PCr degradation in HYP was significantly less than in ACE. There was a significant increase in lactate accumulation by 30 s, with no difference among trials (Fig. 3). A further increase in lactate occurred at 120 s in both CON and ACE but not in HYP. However, the difference among trials at 120 s was not significant (P > 0.07).

Pyruvate content significantly increased during exercise in all trials (Fig. 4). By 120 s, pyruvate was significantly greater in ACE than in HYP. G-6-P and F-6-P showed no changes with exercise or among trials, and G-6-P increased over time in all trials (Table 1).

Acetyl-CoA content remained significantly elevated in ACE at 30 s compared with CON and HYP, and ACE showed no change in acetyl-CoA content over time (Fig. 5A). However, both CON and HYP showed a significant increase in acetyl-CoA from 30 to 120 s, such that there was no difference between trials at 120 s. Acetylcarnitine content was significantly elevated in ACE at all time points compared with CON and HYP and did not change over time (Fig. 5B). Acetylcarnitine content increased in CON and HYP over time but remained significantly less than ACE.

The lack of significant differences in PCr and lactate between trials resulted in no difference in anaerobic energy yield (substrate phosphorylation) among trials during 30 s of exercise or from 30 to 120 s of exercise (Fig. 6).


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Fig. 6.   Calculated substrate phosphorylation (from PCr degradation and lactate accumulation) during 30 and 120 s of exercise in CON, ACE, and HYP trials.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study determined that increasing the availability of acetylcarnitine (acetate infusion) and O2 (hyperoxia) at the onset of moderate exercise did not decrease substrate phosphorylation, thereby implying that the rate of oxidative phosphorylation was not increased. The findings imply that acetylcarnitine is not a viable source of extra acetyl-CoA (and NADH) for oxidative phosphorylation, and that O2 availability does not limit oxidative phosphorylation during the intial 2 min of cycling at 65% VO2 max.

Increased muscle acetylcarnitine store. The rate of skeletal muscle oxidative phosphorylation is controlled by the availability of its substrates and products; the NAD+/NADH concentration ratio, the ATP/ADP × Pi concentration ratio, and the availability of O2 (38). The rate of oxidative phosphorylation at any point in time is regulated by the relative changes in the redox potential, phosphorylation state (energy charge), and mitochondrial PO2. Acetate infusion significantly elevated muscle acetyl-CoA and acetylcarnitine levels at rest compared with control without affecting PDHa, as previously observed (13, 25). This achieved the aim of providing the potential for enhanced substrate (acetyl-CoA) provision at exercise onset. If the provision of acetyl-CoA, and ultimately NADH, is a site of metabolic inertia at the onset of exercise, increasing its availability may increase the rate at which oxidative phosphorylation turns on and may decrease the need for substrate phosphorylation. This has been demonstrated to occur with DCA administration, which both increases the muscle acetylcarnitine store and activates PDHa at rest (12, 30). However, despite the potential for enhanced provision of acetyl-CoA for the TCA cycle, at exercise onset in ACE there was no significant difference in substrate phosphorylation, calculated from PCr degradation and lactate accumulation. The results suggest that the acetylcarnitine store does not provide a significant source of acetyl-CoA during the onset of submaximal exercise for enhanced activation of oxidative phosphorylation. They also imply that the activation of PDH is critical for increasing the acetyl-CoA supply, increasing the rate of oxidative phosphorylation, and decreasing substrate phosphorylation at the onset of exercise after DCA administration (12, 30).

Previous studies from our laboratories have demonstrated that it is the provision of extra NADH after DCA infusion, both before exercise and at the onset of exercise, that reduces the metabolic inertia, increases oxidative phosphorylation, and reduces the need for substrate phosphorylation (12, 23). When DCA activates PDH at rest, NADH is produced directly in the PDH reaction, and the acetyl-CoA is converted to acetylcarnitine to increase this store. The enzyme is also already fully activated as the exercise begins and is able to produce acetyl-CoA more rapidly, which can be metabolized in the TCA cycle to provide additional NADH. These two factors appear to augment the rate of oxidative phosphorylation and reduce the need for substrate phosphorylation. Lower levels of free ADP, AMP, and Pi also signal the need for less substrate phosphorylation to the creatine phosphokinase reaction and the glycolytic pathway at the levels of glycogen phosphorylase and phosphofructokinase. Consequently, PCr degradation and lactate accumulation are reduced.

In contrast to the DCA studies, when extra acetyl-CoA is generated directly from acetate, no extra NADH is provided at rest or during exercise. In addition, 2 moles of ATP are required for every mole of acetyl-CoA produced, as the acetylcarnitine store is increased at rest during the acetate infusion. The potential for the acetylcarnitine store to provide extra acetyl-CoA, and ultimately NADH, did not occur during exercise, as judged from the PCr use, the accumulation of lactate, and the signals that regulate these processes.

The present results are in contrast to work by Timmons and coworkers (30-33), who argued that the muscle acetylcarnitine store is important for increased oxidative substrate availability at exercise onset. Much of their support arises from work with ischemic contracting muscle. The acetylcarnitine store decreased from DCA-elevated levels by 2-5 mmol/kg dry matter in contracting ischemic dog muscle (32, 33) and by 1 mmol/kg dry matter during submaximal ischemic human knee extensor exercise (31). The decreases in acetylcarnitine content were accompanied by significantly less PCr degradation and lactate accumulation compared with CON (31-33). The same argument was made during the initial 8 min of submaximal knee extension exercise with normal blood flow, but muscle measurements made after 8 min of exercise do not reflect the transition from rest to exercise (30). It is not presently clear why elevated acetylcarnitine spared substrate phosphorylation in the ischemia studies and not during the initial 2 min of submaximal exercise (65% VO2 max) with normal blood flow (12, and the present study).

Recent work reported no change in acetylcarnitine content with DCA during the initial minute of whole body cycling exercise (55% VO2 max) under hypoxic conditions, despite a significant decrease in the reliance on substrate level phosphorylation (23). It has been previously shown that hypoxic conditions are associated with decreased PDH activation (22), slowed VO2 on-kinetics (14, 21), and increased lactate production (16). When DCA is administered during hypoxia, increased PDH activation contributes to the reduced reliance on substrate level phosphorylation at the onset of exercise (23). However, it is possible that extra acetyl-CoA from acetylcarnitine also contributed to the increased oxidative phosphorylation in the face of an O2 limitation, because the effect of increasing PDHa or acetylcarnitine alone during hypoxia has not been tested.

It is believed that the main function of the carnitine acetyltransferase reaction and the production of acetylcarnitine is to buffer increases in mitochondrial acetyl-CoA. The near-equilibrium reaction forms acetylcarnitine and free CoA when acetyl-CoA production is higher than its use in the TCA cycle. The acetylcarnitine is believed to move into the cytoplasm to keep the reaction moving in the direction of acetylcarnitine formation. This ensures that the mitochondrial free CoA store will not be sequestered and will remain high enough to take part in the numerous mitochondrial reactions associated with energy production. The present results, and an earlier study in which carbohydrate availability was reduced during exercise, suggest that the main function of acetylcarnitine is to buffer increases in acetyl-CoA, as it cannot provide acetyl-CoA (reverse reaction) quickly during exercise, even when the demand for acetyl-CoA is great (26).

Hyperoxia. The DCA studies and the present study demonstrate that flux through PDH is a site of metabolic inertia at the onset of submaximal exercise (12, 23, 30). DCA administration decreased the reliance on substrate level phosphorylation by only ~35%, suggesting that PDH activation and substrate provision may not be the sole factor limiting the activation of oxidative phosphorylation. Both the lack of a significant decrease in elevated acetyl-CoA levels in ACE and the accumulation of acetyl-CoA in CON suggest that there is an inability to use the available store of acetyl-CoA at exercise onset. This could be due to metabolic inertia farther downstream than PDH (e.g., TCA cycle) or a mitochondrial O2 limitation. However, the present results demonstrate that breathing 100% O2 before and during 120 s of exercise at 65% VO2 max did not affect substrate phosphorylation, implying that oxidative phosphorylation was not enhanced. This was supported by an inability to better utilize the available acetylated compounds, with similar accumulations of acetyl-CoA and acetylcarnitine in HYP and CON trials.

The amount of O2 delivered to the mitochondria is determined by convective O2 delivery, including arterial oxygen content (CaO2) and blood flow, and diffusive O2 delivery, influenced by the PO2 gradient from the red blood cell to the mitochondria (35). Breathing 100% O2 increases CaO2 by ~8-10% and increases the PO2 of arterial blood by about sixfold (35). Whether hyperoxic conditions actually increase the convective delivery of O2 to the muscle is unknown. It has been reported that decreases in blood flow during hyperoxic breathing offset the 8-10% increase in CaO2, such that convective O2 delivery in HYP was not different from normoxic conditions (21% O2) (21, 36). However, other studies reported that hyperoxic conditions increased the arterial PO2 to ~500-600 mmHg, did not affect blood flow, and increased CaO2 during exercise (17, 27). This suggests that both the diffusive and convective components of O2 delivery are increased in some studies. Although the diffusive component was increased at the onset of exercise in the present study, it is not known whether convective O2 delivery was increased, because arterial O2 content and leg blood flow were not measured.

The lack of sparing of substrate level phosphorylation during submaximal (65% VO2 max) exercise with HYP in this study concurs with previous indirect measurements of O2 deficit (VO2 on-kinetics) during moderate submaximal exercise (14, 21). However, Linnarsson et al. (18) reported decreased PCr use and lactate accumulation after 4 min of exercise in hyperoxia at 55% VO2 max. The present results demonstrated no effect of hyperoxia during moderate exercise over a much shorter time course (initial 30 s). However, there was a trend for less PCr degradation and lactate and pyruvate accumulation after 120 s of cycling in HYP. This may suggest an increase in the rate of oxidative phosphorylation by 120 s of exercise compared with control as a result of enhanced O2 availability. In support of this, Hogan et al. (11) reported less PCr degradation during steady-state exercise in hyperoxia vs. normoxia. Measurements taken beyond 120 s of exercise at 65% VO2 max may demonstrate an improved phosphorylation or energetic state with hyperoxia. Thus, it is possible that a metabolic inertia initially limits the activation of oxidative phosphorylation at exercise onset such that enhanced O2 availability is only beneficial once the metabolic inertia has been overcome.

In summary, elevated levels of acetate-induced muscle acetylcarnitine at the onset of exercise at 65% VO2 max did not enhance the provision of acetyl-CoA for the TCA cycle or NADH for faster activation of oxidative phosphorylation, as implied by the lack of effect on directly measured muscle substrate phosphorylation. This confirms previous results that substrate provision from flux through PDH, and not acetylcarnitine, is a site of metabolic inertia at the onset of exercise, partially accounting for the lag in oxidative phosphorylation (12). Hyperoxic conditions (breathing 100% O2) had no effect on muscle substrate phosphorylation at the onset of exercise at 65% VO2 max, impying that the activation of oxidative phosphorylation was also unaffected. This finding argues that O2 availability is not limiting at the onset of submaximal exercise.


    ACKNOWLEDGEMENTS

We thank Dr. Eric Hultman for critical reading of the manuscript.


    FOOTNOTES

This study was supported by the Natural Sciences and Engineering and the Medical Research Councils of Canada. M. K. Evans was the recipient of a Gatorade Sport Sciences student award, and I. Savasi was supported by a Natural Sciences and Engineering Research Council scholarship. G. J. F. Heigenhauser is a career investigator of the Heart and Stroke Foundation of Ontario (no. I-2576).

Address for reprint requests and other correspondence: L. L. Spriet, Dept. of Human Biology and Nutritional Sciences, Univ. of Guelph, Guelph, ON, Canada N1G 2W1 (E-mail: lspriet{at}uoguelph.ca).

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. Section 1734 solely to indicate this fact.

Received 2 January 2001; accepted in final form 17 July 2001.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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