Am J Physiol Endocrinol Metab
281: E1144–E1150, 2001.
Effects of acetate infusion and hyperoxia on muscle
substrate phosphorylation after onset of moderate exercise
MELISSA K. EVANS,1 INGRID SAVASI,1 GEORGE J. F. HEIGENHAUSER,2
AND LAWRENCE L. SPRIET1
1
Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario N1G 2W1;
and 2Department of Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5
Received 2 January 2001; accepted in final form 17 July 2001
acetylcarnitine; acetyl-coenzyme A; oxidative phosphorylation; pyruvate dehydrogenase activity; lactate; phosphocreatine; oxygen
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 tranTHE ACTIVATION OF MITOCHONDRIAL
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: [email protected]).
E1144
sient 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% V̇O2 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% V̇O2 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
(V̇O2) kinetics measured at the mouth, an indirect esThe 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
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0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society
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Evans, Melissa K., Ingrid Savasi, George J. F. Heigenhauser, and Lawrence L. Spriet. Effects of acetate infusion and hyperoxia on muscle substrate phosphorylation after onset of moderate exercise. Am J Physiol Endocrinol
Metab 281: E1144–E1150, 2001.—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 (V̇O2 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 ␮mol/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.
ACETATE INFUSION AND HYPEROXIA DURING ONSET OF EXERCISE
METHODS
Subjects. Eight healthy, moderately active male subjects
volunteered to participate in this study. Their mean (⫾ SE)
age, height, weight, and V̇O2 max were 22.9 ⫾ 1.0 yr, 180.0 ⫾
2.7 cm, 77.0 ⫾ 2.8 kg, and 54.7 ⫾ 3.0 ml 䡠 kg⫺1 䡠 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 V̇O2 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% V̇O2 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
V̇O2 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 carbohydraterich meal 2–4 h before the trial. Subjects were asked to
AJP-Endocrinol Metab • VOL
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-6P), 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
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timation of the O2 deficit. Hyperoxic conditions enhanced V̇O2 on-kinetics during intense (78–82%
V̇O2 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% V̇O2 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.
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E1146
ACETATE INFUSION AND HYPEROXIA DURING ONSET OF EXERCISE
10.8 mmol/kg dry weight (7) to the difference in PCr content
(⌬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.
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
RESULTS
Metabolite
Time, s
CON
ACE
ATP
0
30
120
23.2 ⫾ 1.0
24.4 ⫾ 1.1
23.0 ⫾ 1.4
Free ADP
0
30
120
77.9 ⫾ 5.0
148.4 ⫾ 13.2
234.6 ⫾ 60.6*
72.2 ⫾ 5.3
150.2 ⫾ 18.0
259.3 ⫾ 42.2*†
84.4 ⫾ 6.2
182.5 ⫾ 14.5*
240.4 ⫾ 44.4*
Free AMP
0
30
120
0.25 ⫾ 0.02
0.89 ⫾ 0.15
3.10 ⫾ 1.71*
0.21 ⫾ 0.03
1.01 ⫾ 0.26
3.35 ⫾ 1.12*
0.28 ⫾ 0.04
1.30 ⫾ 0.20
2.41 ⫾ 0.88
Free Pi
30
120
28.1 ⫾ 2.5
42.1 ⫾ 6.3†
34.0 ⫾ 4.5
51.4 ⫾ 6.1†
34.5 ⫾ 3.8
41.8 ⫾ 5.1
Glucose
0
30
120
2.21 ⫾ 0.33
4.90 ⫾ 1.05
5.20 ⫾ 0.86
3.40 ⫾ 1.05
5.71 ⫾ 1.60
6.00 ⫾ 1.23
2.67 ⫾ 0.73
4.94 ⫾ 1.53
5.19 ⫾ 0.53
G-6-P
0
30
120
0.19 ⫾ 0.04
1.39 ⫾ 0.47*
1.73 ⫾ 0.44*
0.58 ⫾ 0.10
1.60 ⫾ 0.66*
2.04 ⫾ 0.43*
0.35 ⫾ 0.17
1.27 ⫾ 0.18
1.49 ⫾ 0.27*
F-6-P
0
30
120
0.03 ⫾ 0.02
0.19 ⫾ 0.08
0.27 ⫾ 0.07*
0.20 ⫾ 0.10
0.32 ⫾ 0.09
0.27 ⫾ 0.07
0.10 ⫾ 0.04
0.22 ⫾ 0.10
0.24 ⫾ 0.04
23.6 ⫾ 0.7
23.4 ⫾ 0.9
23.6 ⫾ 0.8
HYP
25.0 ⫾ 0.7
25.7 ⫾ 1.1
26.5 ⫾ 0.9
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
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.
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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Values are mean ⫾ SE. CON, ACE, and HYP, control, acetate
infusion, and hyperoxic conditions, respectively. G-6-P and F-6-P,
glucose and fructose 6-phosphate, respectively. Units are mmol/kg
dry muscle except for free ADP and AMP (␮mol/kg dry muscle).
* Significantly different from rest for the same trial; † significantly
different from 30 s for the same trial.
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Power output and V̇O2. During each trial, subjects
cycled at a power output ranging from 155 to 220 W,
with a mean of 186 ⫾ 9 W. The average V̇O2 was 36.5 ⫾
1.6 ml 䡠 kg⫺1 䡠 min⫺1 or 64.8 ⫾ 0.9% V̇O2 max. V̇O2 measurements during the practice ride indicated that all
subjects reached a steady-state V̇O2 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.
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 ␮mol/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).
Muscle metabolites during exercise. There was no
significant difference in ATP content among trials or
ACETATE INFUSION AND HYPEROXIA DURING ONSET OF EXERCISE
E1147
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.
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.
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.
Fig. 6. Calculated substrate phosphorylation (from PCr degradation
and lactate accumulation) during 30 and 120 s of exercise in CON,
ACE, and HYP trials.
AJP-Endocrinol Metab • VOL
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).
DISCUSSION
This study determined that increasing the availability of acetylcarnitine (acetate infusion) and O2 (hyper-
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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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ACETATE INFUSION AND HYPEROXIA DURING ONSET OF EXERCISE
AJP-Endocrinol Metab • VOL
gen phosphorylase and phosphofructokinase. Consequently, PCr degradation and lactate accumulation are
reduced.
In contrast to the DCA studies, when extra acetylCoA 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% V̇O2 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% V̇O2 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 V̇O2 onkinetics (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
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oxia) 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% V̇O2 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 (acetylCoA) 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 acetylCoA 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 glyco-
ACETATE INFUSION AND HYPEROXIA DURING ONSET OF EXERCISE
AJP-Endocrinol Metab • VOL
fect 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%
V̇O2 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%
V̇O2 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% V̇O2 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.
We thank Dr. Eric Hultman for critical reading of the manuscript.
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).
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14.
ACETATE INFUSION AND HYPEROXIA DURING ONSET OF EXERCISE