Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario N1G 2W1; Department of Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5; and Department of Clinical Chemistry, Huddinge University Hospital, Karolinska Institute, S-141 86 Huddinge, Sweden
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ABSTRACT |
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This study
investigated whether dichloroacetate (DCA) decreases the reliance on
substrate level phosphorylation during the transition from rest to
moderate-intensity exercise in humans. Nine subjects cycled at ~65%
of maximal oxygen uptake
(O2 max) after a
saline or DCA (100 mg/kg body wt) infusion, with muscle biopsies taken
at rest and at 30 s and 2 and 10 min of exercise. DCA infusion
increased pyruvate dehydrogenase (PDH) activation at rest (4.0 ± 0.3 vs. 0.9 ± 0.1 mmol · kg wet
wt
1 · min
1)
and at 30 s (3.6 ± 0.2 vs. 2.5 ± 0.4 mmol · kg
1 · min
1)
of exercise. As a result, acetyl-CoA (45.9 ± 5.9 vs. 11.3 ± 1.5 µmol/kg dry wt) and acetylcarnitine (13.1 ± 1.0 vs. 1.6 ± 0.3 mmol/kg dry wt) were markedly increased by DCA infusion at rest. These
differences were maintained at 30 s and 2 min for both acetyl-CoA and
acetylcarnitine. Resting muscle lactate and phosphocreatine (PCr) were
not different between trials, but DCA infusion resulted in lower
lactate accumulation throughout exercise, especially at 2 min (21.6 ± 3.1 vs. 44.6 ± 8.0 mmol/kg dry wt). PCr utilization in the initial 30 s (16.9 ± 0.4 vs. 31.7 ± 2.6 mmol/kg dry wt) and 2 min (27.8 ± 4.7 vs. 45.1 ± 2.6 mmol/kg dry wt) of exercise was decreased with
DCA. This resulted in a lower accumulation of free inorganic phosphate
at 30 s (25.4 ± 2.0 vs. 36.4 ± 2.8 mmol/kg dry wt) and 2 min (34.6 ± 4.7 vs. 50.5 ± 2.2 mmol/kg dry wt) with DCA and
decreased glycogenolysis over 10 min. The data from this study support
the hypothesis that increased provision of substrate by DCA infusion
increases oxidative metabolism during the rest-to-work transition,
resulting in decreased PCr utilization and an improved cellular energy
state at the onset of exercise. The transitory improvement in energy
state decreased glycogenolysis and lactate accumulation during
moderate-intensity exercise.
glycogenolysis; lactate; phosphocreatine; inorganic phosphate; oxidative metabolism; pyruvate dehydrogenase activity; acetyl-coenzyme A; acetylcarnitine
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INTRODUCTION |
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THE CAUSES OF LACTATE PRODUCTION during steady-state,
moderate power output exercise are controversial (7), with two main mechanisms being proposed. Increases in lactate have been suggested to
be due to limitations in O2
delivery to the mitochondrial electron transport chain (19, 34); others
hypothesize that O2 is never limiting and that increased lactate content is simply a consequence of
the reduced energy state of the cell (6, 36). The latter hypothesis,
often called the "mass action effect," suggests that the rate of
lactate production is determined by the balance between pyruvate
production and oxidation. When pyruvate production exceeds the rate at
which it can be converted to acetyl-CoA via pyruvate dehydrogenase
(PDH), it begins to accumulate, and because lactate dehydrogenase is a
near-equilibrium enzyme, it is converted to lactate. In support of this
hypothesis, a recent study at 65% maximal oxygen uptake
(O2 max) showed that
significant lactate accumulation occurred in the working muscle, even
when PDH was not totally transformed to its active form. Therefore,
glycogenolytic flux was greater than PDH flux, even though PDH could
potentially have oxidized more pyruvate (17).
During the transition from rest to exercise, there is a transient mismatch between the rates of ATP degradation and aerobic synthesis. To maintain ATP levels, substrate level phosphorylation of ADP occurs from both glycogenolysis and phosphocreatine (PCr) breakdown, resulting in accumulation of free inorganic phosphate (Pi), ADP, AMP, and lactate. The magnitude of this mismatch determines how great the PCr utilization and free metabolite accumulations are, and these in turn determine the rate of glycogenolysis and lactate accumulation. The lag between the onset of ATP breakdown and the activation of oxidative ATP production will be one factor in determining the severity of this mismatch.
Dichloroacetic acid (DCA) increases the activation of PDH to its active "a" form (PDHa) by inhibiting PDH kinase (35). It has been used experimentally in animals (20) and clinically in humans for the treatment of lactic acidosis (29). Previous human studies utilizing DCA have shown reduced blood lactate accumulations in patients with lactic acidosis (29) and healthy volunteers during exercise (3), suggesting that enhanced PDH activation decreased lactate production and/or enhanced lactate clearance. Until recently, little or no work had been done with skeletal muscle to determine the biochemical mechanisms by which these changes occur. In a recent series of studies by Timmons et al. (30-33), DCA was used to test the hypothesis that substrate availability determines the rate at which aerobic ATP production begins, and that increased oxidative substrate at the start of exercise, specifically acetyl-CoA derived from acetylcarnitine, decreased the reliance on substrate level phosphorylation from PCr and glycogenolysis. Using contracting dog muscle (32, 33), contracting ischemic human muscle (31), and healthy human subjects performing leg-extensor exercise (30), these studies showed that DCA infusion resulted in increased resting PDH activation, less PCr utilization, and decreased glycogen breakdown. The decreased glycogenolysis decreased the amount of lactate accumulation, presumably as a better match between pyruvate production and oxidation occurred.
The present study was also designed to test the above hypothesis during the onset (first 2 min) of whole body, moderate-intensity aerobic exercise with normal blood flow in humans. Rapid biopsy sampling and a higher DCA dose (100 mg/kg) were selected to provide additional information on the time course (i.e., the rest-to-work transition) of the effects and to increase resting PDHa to maximal exercise levels.
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METHODS |
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Subjects.
Nine (2 female, 7 male) healthy subjects volunteered to participate in
this study. Their mean (±SE) age, height, weight, and O2 max were 21.8 ± 0.6 yr, 178.6 ± 2.3 cm, 72.6 ± 4.2 kg, and 3.7 ± 0.3 l/min (50.5 ± 2.4 ml · kg
1 · min
1),
respectively. None of the subjects were well trained, nor were they
regularly active (i.e., <2 aerobic workouts per week). Subjects were
informed of possible risks involved in the study, and informed consent
was received from all subjects. The study was approved by the ethics
committees of both universities.
DCA.
DCA (monosodium salt) was obtained from TCI America (Portland, OR)
under the supervision of Dr. N. L. Jones. It was prepared at a
concentration of 100 mg/ml (pH 7.0) by the pharmacy at McMaster University Medical Center, filtered, assayed to ensure sterility and
lack of pyrogens, and checked for purity by HPLC in an independent laboratory. It was delivered intravenously to subjects in the dose of
100 mg/kg by use of ~500 ml of normal saline solution over the course
of 1 h immediately before exercise. Although the infusion of DCA was
designed to fully activate PDH, it is possible that it exerted effects
on other enzymes. However, it is unlikely that the effect of DCA on
other enzymes would be important during 10 min of exercise at ~65%
O2 max in skeletal muscle.
Preexperimental protocol.
Subjects underwent a continuous incremental exercise test on a bicycle
ergometer (Excalibur, Quinton Instruments, Seattle, WA) to determine
O2 max with a
metabolic cart (model 2900, SensorMedics, Yorba Linda, CA). From this
test, the power output required to elicit ~65% of
O2 max was
calculated. On a separate day, subjects cycled for the required 10 min
to confirm that the correct percentage of
O2 max was reached.
Mean power output for the trials was 169 ± 14 W. It was assumed that
basal oxygen uptake (
O2)
and
O2 max were not
altered by DCA.
Experimental protocol.
On two separate experimental days (1-2 wk apart), subjects arrived
at the laboratory at the same time of day. Subjects ate a
high-carbohydrate meal 2 h previous to arriving at the laboratory, as
they would do in preparation for exercise. They were also asked to
consume their normal diet before the test days and to refrain from
strenuous exercise for 24 h before each trial. On test days, the
subjects received either DCA or saline infusion in a randomized order.
One hour before each exercise trial, a catheter was inserted into the
antecubital vein of each subject, and a preinfusion blood sample was
taken. The control or DCA infusion was then started, and the subject
rested quietly on a bed. A second blood sample was taken at 30 min of
infusion, at which time subjects had one leg prepared for needle
biopsies, with four incisions made through the skin superficial to the
vastus lateralis muscle under local anesthesia (2% lidocaine without
epinephrine) (2). A resting biopsy and blood sample were taken at 60 min, and then subjects moved to an electronically braked cycle
ergometer and began pedaling at the prescribed power output. Exercise
biopsies were taken at 30 s, 2 min, and 10 min while the subject
remained on the cycle ergometer. Samples were immediately frozen in
liquid N2 (3-5 s from the
insertion of the needle), removed from the needle, and stored in liquid
N2 until analysis. Exercise blood
samples were taken at 3 and 9 min. Expired gases were collected to
measure O2 and carbon
dioxide production between 4 and 6 min.
Analyses. A small piece of frozen wet muscle (20-30 mg) was removed under liquid N2 for the determination of PDH activation (PDHa), as described by Constantin-Teodosiu et al. (9) and modified by Putman et al. (25). The remainder of the biopsy sample was freeze-dried, dissected of all visible blood, connective tissue, and fat, and powdered for subsequent analysis.
One aliquot of freeze-dried muscle was extracted with 0.5 M HClO4 (containing 1 mM EDTA) and neutralized with 2.2 M KHCO3. This extract was used for determination of creatine, PCr, ATP, glucose 6-phosphate (G-6-P), lactate, glycerol 3-phosphate (G-3-P), and glucose by enzymatic spectrophotometric assays (1, 16). Pyruvate was determined on this extract fluorometrically (21). Acetyl-CoA and acetylcarnitine were determined by radiometric measures (4). Muscle glycogen content was determined on a second aliquot of freeze-dried muscle from resting and 10-min samples (16). Plasma FFA concentrations were determined using a WAKO nonesterified fatty acid (NEFA C) assay kit (WAKO Chemicals, Osaka, Japan). Whole blood glucose and lactate were determined on the PCA extract samples by the methods of Bergmeyer (1).Calculations. Free ADP and AMP concentrations were calculated by assuming equilibrium of the creatine kinase and adenylate kinase reactions as previously described (12). Free ADP was calculated using the measured ATP, PCr, and creatine content, and H+ concentration was estimated from the muscle lactate content according to the regression equation of Sahlin et al. (28). Free AMP was calculated from the estimated free ADP and ATP using the adenylate kinase reaction. Free Pi was calculated by adding the estimated resting free phosphate of 10.8 mmol/kg dry measure to the difference in PCr content minus the accumulation of glycolytic intermediates G-6-P and G-3-P between rest and each exercise time point. All metabolite contents and the activity of PDHa were normalized to the highest total creatine measurement in the eight biopsies from each subject.
PDH flux was calculated for the first 30 s and subsequent 90 s of exercise. It was assumed that 1) ATP turnover was equal in both trials as power output was the same and 2) any reduction in ATP from substrate level phosphorylation was due to oxidative ATP production. First, the "baseline" PDH flux to oxidative metabolism was estimated, with the assumption that it was equal to the average PDHa in the control (CON) trial, as previously shown by others (10, 17, 23, 25). Next, the amount of pyruvate directed to oxidative phosphorylation to account for the reduction in substrate-level phosphorylation during the DCA trial was calculated by adding the ATP equivalents of the decrease in PCr utilization and lactate accumulation, with the assumption of an ATP yield of 15 mmol of ATP per millimole of pyruvate. Finally, this "extra" PDH flux in the DCA trial was added to the flux that was directed to the increased accumulation of acetylcarnitine. The sum of these three fluxes is equal to the total PDH flux in the two trials.Statistics.
All data are presented as means ± SE. For net glycogen usage between
trials, a paired t-test was used to
determine significant difference. For all other dependent variables, a
two-way ANOVA (time × trial) with repeated measures was employed.
Significance was set at = 0.05, and when obtained, Tukey's post
hoc test was used to identify where significant differences occurred.
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RESULTS |
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O2 and respiratory
exchange ratio.
During the DCA trial,
O2 was
2.21 ± 0.16 l/min or 60.4 ± 1.1%
O2 max, whereas CON
O2 was 2.27 ± 0.15 l/min or
62.3 ± 1.3%
O2 max.
Respiratory exchange ratio values between 4 and 6 min in the DCA and
CON trials were 1.00 ± 0.01 vs. 0.97 ± 0.01, respectively.
PDHa.
DCA infusion at rest resulted in a very marked (4.04 ± 0.32 vs. 0.9 ± 0.11 mmol · kg wet
wt1 · min
1) and significant
increase in resting PDHa compared
with CON (Fig. 1). Resting
PDHa in the DCA trial was similar
to that reported for maximum total PDH activity during intense activity
(14, 17, 22, 23). During exercise, despite a small decrease in PDHa from rest in the DCA trial,
the significant difference between trials remained at 30 s. However,
there was no difference in PDHa at
subsequent time points, as PDHa in
the CON trial increased to DCA trial levels after 2 min of exercise.
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Muscle metabolites.
ATP levels were not significantly different at any time, regardless of
trial (Table 1). Resting PCr was similar
between trials but was significantly lower at both 30 s and 2 min
during exercise in CON (Fig. 2). Resting lactate levels
were similar between trials, but there was a significant main effect
for lower lactate accumulation during exercise over 10 min in the DCA
trial, with a significant interaction at 2 min (Fig. 3).
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Blood metabolites.
Resting blood lactate was higher in the CON trial than the DCA trial
after 30 and 60 min of infusion. During exercise, blood lactate was
significantly lower in the DCA than in the CON trial at 9 min. Blood
glucose and plasma FFA were not different between trials at any time
(Table 2).
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PDH flux.
Total PDH flux during the first 30 s of exercise was 46% higher in the
DCA trial compared with CON (Table 3). Of
this increase in total PDH flux, nearly all was directed toward
oxidative metabolism to reduce the dependence on substrate level
phosphorylation, as the contribution to acetylcarnitine accumulation
was virtually identical between trials (Tables 3 and 4).
However, during the subsequent 90 s of exercise, total PDH flux was
actually 16% higher in the CON trial (Table 3), as the flux directed
to "sparing" oxidative phosphorylation fell in the DCA trial, and
large amounts of flux were directed to increasing acetylcarnitine
accumulation in the CON trial (Tables 3 and 4).
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DISCUSSION |
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DCA infusion before moderate-intensity whole body exercise resulted in maximal activation of PDH to its active form (PDHa) and subsequent acetylation of the free CoA and carnitine pools to levels seen at maximal exercise (8, 23). During the initial 2 min of exercise, DCA infusion resulted in decreased PCr breakdown and Pi accumulation and decreased glycogenolysis and lactate accumulation. These results are consistent with previous results from studies utilizing different models (30-33), which demonstrated that increasing the amount of carbohydrate-derived substrate that can be oxidized during the transition from rest to exercise improves the energetic state of the working muscle. This improved energy state results in lower glycogenolysis and decreased lactate accumulation in the muscle. The greater substrate availability increased the amount of oxidative metabolism used in the work-to-rest transition, decreasing the reliance on substrate level phosphorylation and resulting in the above changes.
In the present study, the 100 mg/kg dose of DCA produced resting
PDHa activation values (4.04 ± 0.32 mmol · kg wet
wt1 · min
1)
that were higher than the significant increases previously reported in
humans (1.3 ± 0.1 to 2.2 ± 0.3 mmol · kg wet
wt
1 · min
1)
with a lower DCA dose (50 mg/kg) (11, 13, 30). The larger dose in this
study successfully increased resting
PDHa to levels seen during maximal
exercise (14, 17, 22, 23). The resting acetylcarnitine contents were
similar to previous DCA studies on humans (30, 31), and resting muscle
acetyl-CoA content was also similar to maximal exercise values (23).
During exercise, the decrease in PCr degradation in the present study was significantly lower with DCA, similar to previous studies on human volunteers (30). The accompanying decrease in lactate accumulation seen in the present study was not observed with DCA infusion at 8 min in humans undergoing voluntary exercise, although they were using lower-intensity leg extensor exercise (30). However, decreased muscle lactate has been observed previously with DCA infusion (31).
Increased muscle lactate content has often been assumed to be a result of tissue oxygen insufficiency (19, 34). However, another line of evidence suggests that lactate accumulation results from an imbalance between pyruvate oxidation and pyruvate production (6). Pyruvate oxidation is controlled by the entry of pyruvate into the mitochondria through PDH, whereas pyruvate production during exercise is a function of glycogenolysis, or flux through glycogen phosphorylase. Therefore, the regulation of these enzymes is crucial in the control of lactate production.
PDH exists in active and inactive forms, acutely regulated by an allosterically modulated phosphatase/kinase system in human skeletal muscle (18, 35). During exercise, PDH is transformed to its active "a" form at the start of exercise, primarily by increases in Ca2+ and pyruvate. The extent of PDH activation is highly dependent on power output; thus, during exercise, independently calculated PDH flux has been shown to correlate with the catalytic activity of PDHa (10, 17, 23, 25). Because skeletal muscle pyruvate (27) and Ca2+ contents increase with increased power output, they provide possible signals for increased PDH activation during exercise. In the present study, DCA infusion transformed PDH maximally at rest (22), resulting in complete acetylation of the carnitine and free CoA pools, and allowing maximal acetyl-CoA formation from pyruvate at the onset of exercise. When tricarboxylic acid (TCA) cycle flux during maximal exercise was compared with the catalytic activity of PDHa, it was very similar (14), suggesting that PDHa could be limiting for the entry of pyruvate-derived substrate into the TCA cycle (18).
Using the data from the present study and the correlation between PDH flux and PDHa measurements (10, 17, 23, 25), we estimated total PDH flux and the portion of this flux that went to various cellular fates for the initial 30 s and the subsequent 90 s of exercise (Table 3). The decreased reliance on substrate level phosphorylation, calculated from lactate accumulation and PCr utilization and expressed in ATP equivalents, resulted in a difference between DCA and CON trials of 29.4 mmol/kg dry wt at 30 s and 41.4 mmol/kg dry wt at 2 min.
During the first 30 s of exercise, PDH flux was 46% higher in the DCA trial (Table 3). This increase in PDH flux was directed to oxidative phosphorylation, and not acetylcarnitine production (Table 4), as the rate of acetylcarnitine accumulation was similar between trials despite the very different resting acetylcarnitine contents. These data suggest that the provision of substrate is limiting the increase in oxidative phosphorylation in the CON trial, as increased flux through PDH during the DCA trial allowed for greater oxidative phosphorylation, accounting for the decreased substrate level phosphorylation during the first 30 s of exercise.
Conversely, for the following 90 s, from 30 s to 2 min of exercise, the results are quite different. As most (71%) of the sparing of nonoxidative ATP production occurred during the first 30 s, the dependence on substrate level phosphorylation was much lower during the subsequent 90 s. Therefore, much of the increase in PDH flux in the CON trial was directed at increasing the acetylcarnitine pool. Because this pool was already very high in the DCA trial, and less extra flux to oxidative phosphorylation is required to account for the further attenuation of substrate level ATP production, the total PDH flux in this trial was lower than in CON. However, the percentage of this flux to oxidative phosphorylation was still higher in the DCA trial than in CON, as much of the PDH flux in the latter was used for acetylcarnitine production (Table 4). These data suggest that, during this 90-s period, there is no limitation in substrate utilization in the CON trial, but the use of PDH flux to increase acetylcarnitine limits the amount of PDH flux that can be used for oxidative phosphorylation. The lower amount of PDH flux committed to acetylcarnitine during DCA allowed for further decreases in substrate level phosphorylation during this time period, despite a lower total PDH flux.
Increased flux through PDH at the start of exercise may not be the only factor in the improved oxidative metabolism. The large resting acetyl-CoA and acetylcarnitine contents have been hypothesized to increase the substrate available for the TCA cycle and oxidative metabolism. DCA infusion has resulted in a decrease in the high acetyl-CoA levels early in exercise in some (31-33) but not all studies (30). In the present study, we observed no decrease in the acetylcarnitine content at any time point in either trial, but because very little acetylcarnitine utilization would be required for large changes in oxidative metabolism, it is possible that our measurements were not sensitive enough to detect the small changes over a shorter time course than previous studies.
Unfortunately, it is not possible to determine with the present results whether maximal PDH activation at the start of exercise causes the adjustments in cell metabolism via the greater pyruvate availability during the work-to-rest transition, or whether the increased oxidative substrate (acetyl-CoA and acetylcarnitine) produced at rest is responsible. A previous study involving acetate infusion caused large increases in acetyl-CoA and acetylcarnitine at rest without subsequent differences in the muscle energetic state during exercise (24). However, in that case, the increased acetyl-CoA also decreased PDHa at rest (but not during exercise), possibly counteracting the increases in resting substrate.
Flux through glycogen phosphorylase (glycogenolysis) is determined by two factors. The first is the extent of activation of phosphorylase to its more active "a" form by activation of phosphorylase kinase. The second level of regulation is via the concentrations of other modulators, especially free AMP and free Pi, a phosphorylase a allosteric modulator and a phosphorylase substrate, respectively (26). Because power outputs were the same between trials in the present study, DCA infusion would not affect the extent of phosphorylase activation, as the initial burst in activation is primarily due to Ca2+. Likewise, it has recently been shown that the activation state of phosphorylase is not affected by power output and is not a good indicator of phosphorylase flux (17). However, previous studies have shown a strong correlation between free Pi content and the glycogenolytic rate in contracting skeletal muscle, as Pi is a substrate for the glycogen phosphorylase reaction (17, 26). By decreasing the degradation of PCr, DCA infusion in the present study caused a decreased accumulation of free Pi and a subsequent decrease in the breakdown of glycogen compared with the CON trial. Likewise, free AMP is an allosteric activator of phosphorylase a, and there was a strong trend toward an attenuated rise (~50%) in free AMP during DCA.
Our data and those of others (30-33) suggest that the ability of metabolic processes to be activated limits O2 use in the transition from rest to steady-state aerobic exercise in the control state. DCA infusion improves the rate at which oxidative metabolism is activated. However, the degree to which DCA improves the cellular energy state was not as large as expected. Despite impressive resting changes in acetyl-CoA and acetylcarnitine, and total PDH activation, there was still significant PCr utilization and lactate production. There appears to be an inability to totally utilize the preexercise stores of acetylated compounds. It appears that this inability could be related to oxygen limitation, slower TCA cycle flux increases, and/or mass-action effects.
It is often assumed that O2 is not limiting at moderate power outputs (5), but during the rest-to-work transition, it is possible that there is a lag in O2 provision, or that mitochondria are sensitive to O2 pressure levels well above the level assumed to be limiting for cytochrome turnover (36). Any substrate level ATP production and the subsequent PCr breakdown and lactate formation would tend to blunt the effect of DCA. There is also the possibility of a lag in increasing TCA cycle flux, despite the high resting acetyl-CoA content. Although citrate synthase, the enzyme that utilizes acetyl-CoA, is not considered a near-equilibrium enzyme, its flux appears to be highly dependent on delivery of oxaloacetate, its other substrate (15). Resting oxaloacetate concentrations are extremely low and rise quickly with exercise via anaplerotic reactions (15), but the two carbon acetyl-CoA cannot increase the amount of TCA intermediates at rest. In fact, because pyruvate appears to be very important in increasing TCA intermediates, the increased oxidation of pyruvate with DCA could actually decrease anaplerosis, although this has presently been demonstrated only in resting skeletal muscle (11). Finally, it is possible that the lactate accumulation seen even with DCA administration at this power output, despite increased PDHa, is merely a consequence of mass action. Accumulations of free ADP, AMP, and Pi determine the rate of oxidative phosphorylation (36) but also regulate the glycogenolytic rate. As the rate of oxidative phosphorylation increases, there is a necessary increase in the delivery of pyruvate (30). Because of the near-equilibrium nature of lactate dehydrogenase as the concentration of pyruvate rises, there may be an obligatory increase in lactate content.
In summary, the present study demonstrated that DCA infusion increased
resting PDH activation and resting muscle contents of acetyl-CoA and
acetylcarnitine to values indicative of maximal exercise. These resting
changes, which increase the amount of substrate available for oxidative
metabolism, caused a blunting in PCr degradation and the accumulation
of free ADP, AMP, and Pi during
the initial 2 min of the transition to steady state during whole body
exercise at ~65%
O2 max. The improved
cellular energy state decreased glycogen breakdown and subsequent
lactate accumulation. These data concur with the findings of Timmons et al. (30-33), suggesting that the provision of oxidative substrate is one factor limiting oxidative metabolism early in exercise, and that
increasing the availability of substrate early in exercise allows for
increased oxidative metabolism and decreased reliance on substrate
level phosphorylation.
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ACKNOWLEDGEMENTS |
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We thank Tanya Pehleman, Andrew Patterson, and Alex Trochanowski for excellent technical assistance. We are also grateful to Dr. Gary Lopaschuk for help with this study.
![]() |
FOOTNOTES |
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This experiment was supported by operating grants from the Natural Sciences and Engineering Research and Medical Research Councils of Canada. R. A. Howlett was supported by a Gatorade Sports Science Institute student research award. G. J. F. Heigenhauser is a Career Investigator of the Heart and Stroke Foundation of Ontario (no. I-2576).
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. §1734 solely to indicate this fact.
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.NS{at}APS.uoGUELPH.CA).
Received 8 October 1998; accepted in final form 9 March 1999.
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