1 Department of Medicine, McMaster University, Hamilton, Ontario L8N 3Z5; 2 Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada N1G 2W1; and 3 Department of Clinical Chemistry, Huddinge University Hospital, Karolinska Institute, S-141 Stockholm 86, Sweden
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ABSTRACT |
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During the onset of exercise in hypoxia, the increased lactate accumulation is associated with a delayed activation of pyruvate dehydrogenase (PDH; Parolin ML, Spreit LL, Hultman E, Hollidge-Horvat MG, Jones NL, and Heigenhauser GJF. Am J Physiol Endocrinol Metab 278: E522-E534, 2000). The present study investigated whether activation of PDH with dichloroacetate (DCA) before exercise would reduce lactate accumulation during exercise in acute hypoxia by increasing oxidative phosphorylation. Six subjects cycled on two occasions for 15 min at 55% of their normoxic maximal oxygen uptake after a saline (control) or DCA infusion while breathing 11% O2. Muscle biopsies of the vastus lateralis were taken at rest and after 1 and 15 min of exercise. DCA increased PDH activity at rest and at 1 min of exercise, resulting in increased acetyl-CoA concentration and acetylcarnitine concentration at rest and at 1 min. In the first minute of exercise, there was a trend toward a lower phosphocreatine (PCr) breakdown with DCA compared with control. Glycogenolysis was lower with DCA, resulting in reduced lactate concentration ([lactate]), despite similar phosphorylase a mole fractions and posttransformational regulators. During the subsequent 14 min of exercise, PDH activity was similar, whereas PCr breakdown and muscle [lactate] were reduced with DCA. Glycogenolysis was lower with DCA, despite similar mole fractions of phosphorylase a, and was due to reduced posttransformational regulators. The results from the present study support the hypothesis that lactate production is due in part to metabolic inertia and cannot solely be explained by an oxygen limitation, even under conditions of acute hypoxia.
pyruvate dehydrogenase; glycogen phosphorylase; lactate metabolism; glycogenolysis; glycolysis; oxidative phosphorylation; phosphocreatine
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INTRODUCTION |
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EXERCISE DURING ACUTE HYPOXIA is
associated with increased lactate accumulation in the blood and
muscle compared with exercise at the same power output during normoxia
(4, 12, 19, 23). Two conflicting theories have been
proposed to explain this enhanced lactate response to hypoxia. The
first and perhaps most obvious explanation is that an O2
limitation may exist at the mitochondria (see Ref. 20 for review). The
second explanation implies that there is no limitation of
O2 supply and that lactate production is the consequence of
metabolic inertia at the onset of exercise related to the delayed
activation of pyruvate dehydrogenase (PDH), enzymes of the
tricarboxylic acid (TCA) cycle and -oxidation, which provide
substrate for aerobic ATP production (15, 16, 31-33).
A third possibility is that a combination of both an O2 limitation and metabolic inertia is responsible for the enhanced lactate accumulation observed during hypoxic exercise
(34). In a recent study in our laboratories, we examined
glycogen phosphorylase and PDH, which are the rate-limiting enzymes
involved in glycogen metabolism, and compared the regulation of these
enzymes during exercise in normoxia and acute hypoxia
(23). In the transition from rest to the first minute of
exercise, the increased lactate accumulation observed in hypoxia was
associated with both an increased glycogenolytic flux and a delayed
activation of PDH. This delay in PDH activation may have reduced the
availability of oxidative substrate at the onset of exercise and thus
reduced the contribution of oxidative phosphorylation to ATP
resynthesis. Consequently, in hypoxia, a greater reliance on substrate
level phosphorylation from glycolysis ("anaerobic glycolysis")
resulted in significant lactate accumulation compared with little or no
lactate accumulation in normoxia.
Recent studies have shown that activation of PDH by dichloroacetate (DCA) infusion before exercise subsequently reduced the accumulation of lactate during exercise (15, 31-33). This effect has been demonstrated during exercise at moderate power outputs both in normoxia (15) and under conditions of O2 limitation such as partial ischemia (31-33). Increasing the activity of PDH and the subsequent availability of oxidative substrate for the TCA cycle before exercise reduced the reliance on substrate level phosphorylation from phosphocreatine (PCr) breakdown and glycolysis and consequently reduced both glycogen utilization and lactate accumulation.
As an extension of these previous studies, the present study was conducted to test the hypothesis that activation of PDH with DCA before exercise in hypoxia would reduce the reliance on substrate level phosphorylation at the onset of exercise and consequently reduce lactate accumulation. If this hypothesis is verified, then PCr breakdown and glycogen utilization will be reduced at the onset of exercise with a concomitant reduction in lactate accumulation. These results would support the theory that the increased lactate production associated with exercise in hypoxia is in part the consequence of increased glycogenolysis as a result of reduced substrate delivery to the TCA cycle due to a delayed activation of PDH in hypoxia and is not solely due to a reduced O2 supply.
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METHODS |
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Subjects
Six healthy nonsmoking male subjects volunteered to participate in the present study. Their mean ± SE age, height, weight, and maximal O2 uptake (DCA
Monosodium DCA was obtained from TCI America (Portland, OR). The DCA salt was made in a 100 mg/ml solution (pH 7.0), filtered, and assayed to ensure sterility and lack of pyrogens by the pharmacy at the McMaster University Medical Center; the DCA salt was checked for purity by HPLC in an independent laboratory. The prepared solution was infused intravenously at a dose of 100 mg/kg body wt in ~500 ml of normal saline solution over the course of 1 h immediately before exercise.Preexperimental Protocol
Experimental Protocol
The experimental protocol was conducted on two separate occasions 1-2 wk apart, where subjects received either DCA or saline (control) infusion (~500 ml) in a randomized order. Both trials were performed under hypoxic conditions. The subjects consumed a high-carbohydrate diet (80% carbohydrate, 9% fat, and 11% protein) during the 48 h preceding the first trial and were asked to replicate this diet before the second trial. Subjects were also instructed to abstain from the consumption of caffeine and alcohol and to refrain from strenuous exercise for 24 h before both trials, which were conducted at the same time of day for each subject.The experimental protocol is summarized in Fig.
1. Before beginning the protocol, a
catheter was inserted in the antecubital vein of the forearm and was
maintained patent with saline. A preinfusion blood sample was taken
before the DCA or control infusion, which was started 60 min before the
beginning of exercise while the subject rested quietly on the bed.
After 20 min, one thigh was prepared for needle biopsies of the vastus
lateralis. Three incisions were made through the skin to the deep
fascia under local anesthesia (2% lidocaine without epinephrine) as
described by Bergström (3). A second blood sample
was taken after 40 min, before subjects began breathing a hypoxic gas
mixture during an equilibration period in the final 20 min of the
infusion period (Fig. 1). During both trials, subjects breathed from a
mouthpiece attached to a 120-liter Tissot spirometer containing a
hypoxic gas mixture [fraction of inspired O2
(FIO2) = 10.9%, balance
N2], with a nose clip in place. At the end of the infusion
period, a resting venous blood sample and muscle biopsy were taken
while the subject rested on the bed before being moved to the cycle
ergometer. While continuing to breathe the hypoxic gas mixture,
the subject cycled for 15 min at ~55% of the
O2 max determined during normoxia (138 ± 11 W). Muscle biopsies were taken after 1 and 15 min of exercise while the subject remained on the cycle ergometer (Fig. 1).
Venous blood was sampled after 5, 10, and 14 min of exercise. Throughout the protocol, expired gases were continuously sampled. O2 uptake, CO2 output, respiratory exchange
ratio (RER), ventilation, tidal volume (VT), fraction of
expired O2 (FEO2),
fraction of expired CO2
(FECO2), and end-tidal
PCO2 (PETCO2) were
measured from expired gases using a Quinton metabolic cart. Data were
averaged over 15-s intervals. FIO2 was
also monitored to ensure that the subjects were effectively breathing
the hypoxic gas mixture from the Tissot spirometer. Throughout the
protocol, blood pressure, heart rate, and fingertip arterial oxygen
saturation (INVIVO 4500 Plus 1 Pulse Oximeter; INVIVO Research, Broken
Arrow, OK) were monitored.
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Blood Handling and Analysis
Venous blood samples were drawn into heparinized plastic syringes and placed on ice. A 400-µl aliquot of blood was deproteinized in 800 µl of 0.5 M HClO4, vortexed, and centrifuged at 10,000 rpm for 1 min. The supernatant was stored atMuscle Handling and Analysis
Muscle biopsies were immediately frozen in liquid N2, removed from the needle while frozen, and stored in liquid N2 until analyzed. A 5- to 10-mg piece of muscle was chipped from each biopsy under liquid N2 and was dissected free of blood and connective tissue for the determination of the active form of PDH (PDHa), as described by Putman et al. (26). The remaining muscle was freeze-dried, dissected free of blood, connective tissue, and fat, and stored dry atOne aliquot of powdered muscle was used for the determination of
glycogen phosphorylase activity as described by Young et al.
(36). Briefly, 3-4 mg of powdered muscle were
homogenized at 25°C in 200 µl of buffer containing 100 mM Tris,
60% glycerol, 50 mM KF, and 10 mM EDTA (pH 7.5). Homogenates were then
diluted with 800 µl of the above buffer without glycerol and
homogenized further at 0°C. Total (a + b)
phosphorylase activity (in the presence of 3 mM AMP) and phosphorylase
a activity (in the absence of AMP) were measured by
following the production of glucose 1-phosphate (G-1-P)
spectrophotometrically at 30°C. Maximal velocity and the mole
fraction of phosphorylase a and a + b were calculated from the measured activities as described
by Chasiotis et al. (7). Resting measurements of
phosphorylase a were not obtained to minimize the total
number of biopsies in the study. An accurate estimate of the active
form of phosphorylase at rest requires that biopsies be frozen in
liquid N2 after a 30-s delay at room temperature (27) and would have necessitated two additional biopsies.
Previous measurements have given values of ~10% for the mole
fraction of phosphorylase a at rest (8, 22).
A second aliquot of powdered muscle was assayed enzymatically for glycogen as described by Harris et al. (13). The remaining dry powdered muscle was extracted in a solution of 0.5 M HClO4 and 1 mM EDTA and neutralized with 2.2 M KHCO3. These extracts were assayed for ATP, PCr, creatine, pyruvate, lactate, glucose, G-1-P, glucose 6-phosphate (G-6-P), fructose 6-phosphate (F-6-P), and glycerol 3-phosphate (Gly-3-P) as described by Bergmeyer (2) and acetyl-CoA and acetylcarnitine as described by Cederblad et al. (5). Muscle metabolites and enzyme activities were corrected to the highest total creatine content for each subject.
Calculations
Arterial PCO2 was estimated from PETCO2 and VT according to Jones et al. (18). Intracellular H+ concentration ([H+]) was calculated from muscle lactate concentration ([lactate]) and pyruvate concentration ([pyruvate]) according to Sahlin et al. (30). The concentrations of free ADP and free AMP were calculated from the near-equilibrium reactions of creatine kinase and adenylate kinase, respectively (9). The concentration of free Pi was calculated as the difference between resting and exercise PCr concentration ([PCr]), less the accumulation of G-6-P, F-6-P, and Gly-3-P, plus the assumed resting concentration of 10.8 mmol/kg dry wt (9).Total PDH flux during the control trial was estimated from PDHa, since previous studies have shown that flux through PDH is equivalent to its level of activity in whole body exercise in humans (11, 16, 25). The portion of total PDH flux in the control trial directed to oxidative phosphorylation was calculated as the difference between total PDH flux and PDH flux directed to acetylcarnitine formation. During the DCA trial, total PDH flux was estimated from the sum of the "baseline" (estimated from the control trial) PDH flux to oxidative phosphorylation, plus the calculated flux to acetylcarnitine, plus the "extra" PDH flux to oxidative phosphorylation. The extra PDH flux to oxidative phosphorylation was calculated based on the assumptions that the ATP turnover was equal in both trials and that the reduced ATP provision from substrate level phosphorylation (PCr breakdown and glycolysis) in the DCA trial was met by increased or extra oxidative phosphorylation. This was calculated from the difference in ATP provision from substrate phosphorylation from muscle and blood lactate accumulation and PCr breakdown between the two conditions. This difference was converted to pyruvate flux through PDH, assuming an ATP yield of 15 mmol ATP/mmol of pyruvate oxidized.
The rate of pyruvate flux through PDH was equivalent to the total flux through PDH as estimated above and was converted to units of millimoles per kilogram dry weight per minute, assuming a wet-to-dry muscle ratio of 4:1 at rest and 4.5:1 during exercise (24). The rate of pyruvate production was calculated from the accumulation of muscle lactate and pyruvate and blood lactate plus the flux of pyruvate through PDH. The rate of lactate production was calculated from the accumulation of muscle and blood lactate. The distribution volume of blood lactate was assumed to be 0.64 × body weight (1).
Estimates of the glycogenolytic rate at 1 min of exercise were derived from the accumulation of muscle G-6-P, F-6-P, Gly-3-P, pyruvate, lactate, and blood lactate plus the flux of pyruvate through PDH from rest to 1 min of exercise. The glycogenolytic rate during the subsequent 14 min of exercise was calculated from total glycogen utilization during 15 min of exercise, minus the estimated glycogen utilization in the first minute, and divided by time.
The rate of ATP turnover from PCr was calculated from the breakdown of PCr, whereas the rate of ATP turnover from glycolysis was calculated from the accumulation of muscle and blood lactate and the flux of pyruvate through PDH. The rate of ATP turnover from oxidative phosphorylation originating from carbohydrate sources was calculated from total acetyl-CoA production as the area under the PDH curves directed to oxidative phosphorylation; 1 mmol of acetyl-CoA was equal to 15 mmol ATP.
Statistical Analysis
All data are presented as means ± SE. Data were analyzed by a two-way ANOVA with repeated measures over time. When a significant F ratio was found, the Newman-Keuls post hoc test was used to compare the means. Cardiorespiratory parameters and net changes in glycogen concentration ([glycogen]) and [PCr] during control and DCA were compared using a two-tailed paired dependent-samples t-test. Results were considered significant at P < 0.05. ![]() |
RESULTS |
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Cardiorespiratory Parameters
The cardiorespiratory parameters were similar during the control and DCA conditions at rest and after 15 min of hypoxic exercise, with the exception of a higher heart rate in DCA at rest and a lower VT in DCA after 15 min of exercise compared with control (Table 1). All variables changed significantly from rest during exercise under both conditions with the exception of RER, FEO2, and FECO2, which did not change under either condition, and PETCO2, which did not change during control conditions.
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Glycogen Phosphorylase and PDHa
The mole fraction of phosphorylase a did not change from 1 to 15 min of exercise and was similar between the two conditions at both time points (Fig. 2).
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DCA infusion resulted in a significant increase in PDHa at
rest compared with control, which remained constant throughout exercise
(Fig. 3). In the control condition,
PDHa increased significantly from rest after 1 min of
exercise but was lower compared with DCA and increased further after 15 min of exercise to a level not different from DCA.
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Muscle Metabolites
Glycogen. [Glycogen] was not significantly different between conditions at rest (479 ± 22 vs. 562 ± 44, DCA vs. control) or after 15 min of exercise (409 ± 27 vs. 444 ± 43 mmol/kg dry wt), but total glycogen utilization was significantly lower in DCA compared with control (70 ± 12 vs. 118 ± 5 mmol/kg dry wt).
Glycolytic intermediates and H+.
DCA infusion had no effect on resting concentrations of the glycolytic
intermediates or H+. Muscle glucose concentration
([glucose]) did not change with exercise and was similar between
conditions (Table 2). G-1-P concentration ([G-1-P]) was unaltered by exercise in DCA,
unlike the control condition where [G-1-P] was similar to
DCA at 1 min but increased significantly after 15 min of exercise and
was greater compared with DCA. G-6-P concentration
([G-6-P]) did not change at 1 min but increased
significantly after 15 min of exercise with DCA. In the control
condition, [G-6-P] increased significantly after 1 min of
exercise in hypoxia and was similar to DCA but increased further after
15 min of exercise to a greater extent than DCA. F-6-P
concentration did not change at 1 min but increased significantly after
15 min of exercise in both conditions and was greater in control
compared with DCA. Gly-3-P concentration increased
significantly after 1 min and further after 15 min of exercise in both
conditions and was similar between conditions at both time
points. [Pyruvate] did not change at 1 min but increased significantly after 15 min of exercise with DCA. In the control condition, [pyruvate] increased significantly from rest after 1 min,
did not change after 15 min of exercise, and was similar between
conditions at all time points (Table 2). Muscle [lactate] did not
change after 1 min but increased significantly after 15 min of exercise
with DCA. In the control condition, muscle [lactate] increased
significantly after 1 min and further after 15 min of exercise and was
significantly greater compared with DCA at both time points (Fig.
4). [H+] did not change
after 1 min but increased significantly after 15 min of exercise in
both conditions and was significantly higher in control compared with
DCA (Table 2).
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High-energy phosphates.
ATP concentration ([ATP]) was unaltered by exercise and was similar
between conditions (Table 3). [PCr] was
similar at rest and decreased significantly to similar levels at 1 min.
[PCr] decreased further after 15 min of exercise in both conditions but was significantly lower in control compared with DCA (Fig. 5). Free ADP concentration ([ADP]) and
free AMP concentration ([AMP]) were similar at 1 min and increased
significantly after 15 min of exercise under both conditions but were
higher in control compared with DCA (Table 3). The accumulation of free
Pi was reciprocal to PCr breakdown and was similar at 1 min
in both conditions. Free Pi concentration
([Pi]) increased after 15 min of exercise during both
conditions but was higher in control compared with DCA (Table 3).
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Acetyl group accumulation.
Acetyl-CoA concentration ([acetyl-CoA]) increased significantly at
rest as a result of the DCA infusion and remained elevated throughout
exercise. In the control condition, [acetyl-CoA] increased significantly from rest after 1 min but was lower compared with DCA and
increased further after 15 min of exercise to a level not different
from DCA (Fig. 6A).
Acetylcarnitine concentration ([acetylcarnitine]) was significantly
elevated at rest as a result of the DCA infusion, remained constant at
1 min, and increased significantly above the resting level after 15 min
of exercise. In the control condition, [acetylcarnitine] increased
significantly from rest after 1 min and increased further after 15 min
of exercise but was lower in control compared with DCA at both time
points (Fig. 6B).
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Blood Metabolites
Resting blood [lactate] was similar under both conditions before infusion but decreased significantly after 40 min of DCA infusion and remained depressed during the equilibration period. In the control condition, blood [lactate] did not change throughout the infusion or the equilibration periods and was significantly greater compared with DCA after 40 and 60 min of infusion. Blood [lactate] increased progressively with exercise under both conditions and was significantly lower with DCA compared with control after 10 and 14 min of exercise (Table 4). Blood [glucose] was similar at all time points and between conditions (Table 4).
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Glycogenolysis
The rate of muscle glycogenolysis decreased significantly from the first minute during the subsequent 14 min of exercise with DCA (8.4 ± 1.8 vs. 4.4 ± 0.8 mmol glucosyl units · kg dry wtATP Turnover
During the first minute of exercise in control, substrate phosphorylation accounted for 48% of the total ATP turnover, with 17 and 31% from PCr breakdown and glycolysis, respectively, whereas in DCA, substrate phosphorylation accounted for only 33% of the total ATP turnover during the first minute of exercise, with 11 and 21% from PCr breakdown and glycolysis, respectively (Fig. 7). In the control condition, oxidative phosphorylation contributed the remaining 52% of the total ATP turnover compared with 67% in DCA (Fig. 7).
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PDH Flux
Total PDH flux was similar in both conditions during the first minute of exercise (Table 5). In the DCA condition, 83 ± 5% of this total flux was directed to oxidative phosphorylation compared with only 56 ± 12% in the control condition (Table 5). In the subsequent 14 min of exercise, total PDH flux was also similar between conditions, but with nearly all of this flux directed to oxidative phosphorylation under both conditions (Table 5).
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DISCUSSION |
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The present study examined the effects of a DCA infusion given before exercise on the regulation of skeletal muscle metabolism at rest and during 15 min of submaximal exercise under conditions of acute hypoxia. At rest, after the DCA infusion, PDHa, [acetyl-CoA], and [acetylcarnitine] increased to levels approaching those observed with maximal exercise (22, 25), whereas [PCr] and [lactate] were unchanged. In the initial minutes of exercise, PDHa remained high in the DCA condition; although PDHa increased significantly in the control condition, it remained significantly lower than after DCA. PCr breakdown was similar in the first minute, whereas glycogenolysis and [lactate] were reduced with DCA compared with control, despite similar mole fractions of phosphorylase a and similar concentrations of the posttransformational regulators of phosphorylase, free Pi (substrate), and free AMP (positive allosteric modulator). During the subsequent 14 min, PDHa increased significantly in the control condition to become similar to the DCA condition, but there were significant reductions in PCr breakdown, glycogenolysis, and lactate accumulation with DCA compared with control. Despite similar mole fractions of phosphorylase a, the rate of glycogenolysis was significantly depressed with DCA compared with control and was associated with reductions in concentrations of posttransformational regulators, free Pi, and AMP.
During the transition from rest to exercise, there is a transient
mismatch between ATP utilization and ATP production from oxidative
phosphorylation as the rate of oxidative phosphorylation adapts to a
higher energy demand. This transient mismatch in ATP provision from
oxidative sources is bridged by PCr breakdown and glycolysis. At the
onset of exercise, the acceleration of oxidative phosphorylation could
in theory be limited by the following two factors: first, an
O2 limitation at the mitochondria, and second, a metabolic
inertia related to the delayed activation of PDH, enzymes of the TCA
cycle, and enzymes of -oxidation. A combination of these factors may
be responsible (see Ref. 34 for review).
During exercise under hypoxic conditions, it is generally thought that there is an O2 limitation at the onset of exercise as demonstrated by slowed O2 on-kinetics (17, 21) and increased lactate production (20). However, inertia in aerobic metabolism is expected to have the same effect. In a recent study from our laboratory, we observed a delayed activation of PDH at the onset of exercise in hypoxia, which strongly suggested that the provision of substrate may also be limiting oxidative phosphorylation under hypoxic conditions (23). Thus the present study was designed to determine whether activating PDH with DCA before exercise would reduce the effects of metabolic inertia and accelerate the onset of oxidative phosphorylation at the initiation of exercise in hypoxia.
In the present study, DCA was successful in increasing PDH relative to control at rest and during the first minute of exercise. The total calculated fluxes through PDH were similar with and without DCA (Table 5), resulting in a greater proportion of the total PDH flux being directed toward oxidative phosphorylation in the DCA trial, as was found previously in normoxia (15). When the rate of acetyl-CoA production from PDH exceeds its rate of utilization in the TCA cycle, the acetyl groups that are formed may be buffered as acetylcarnitine, thereby freeing the acetyl-CoA reductase stores. The acetyl groups derived from acetylcarnitine may then enter the TCA cycle, or acetyl-CoA may enter the TCA cycle directly from PDH. In the present study, during the initial minutes of exercise in the control condition, 44 ± 12% of the total flux was diverted to acetylcarnitine formation compared with only 17 ± 5% in the DCA condition. Because the acetylcarnitine stores were already large at the onset of exercise in the DCA condition, a greater proportion of the total flux (83 ± 5%) was directed to oxidative phosphorylation compared with only 56 ± 12% in the control condition.
In both canine (32) and human models (31), Timmons et al. showed that DCA reduced PCr breakdown and lactate accumulation at the onset of ischemic exercise. They postulated that the decreased reliance on substrate level phosphorylation was due to the increased availability of oxidative substrate, acetyl-CoA, and acetylcarnitine before exercise. Similar observations were made by Howlett et al. (15) under normoxic conditions in human subjects exercising at a relative workload that was comparable to that in the present study. The reduced reliance on substrate level phosphorylation was equivalent to 29.4 mmol ATP/kg dry wt during the initial 30 s of exercise (15). In the present study, during the initial minutes of exercise, activation of PDH with DCA also reduced the reliance on substrate level phosphorylation by 15.5 mmol ATP/kg dry wt compared with control. It therefore appears that DCA was more effective in reducing reliance on substrate phosphorylation during the onset of exercise under conditions of normoxia than in hypoxia.
The rate of oxidative phosphorylation is regulated by the ratios
NAD+ concentration/NADH concentration ([NADH]) and
[ATP]/[ADP][Pi], as well as the availability of
O2, according to the following reaction
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Richardson et al. (29) demonstrated that the PO2 inside the cell was lower during hypoxia compared with normoxia at rest and remained lower during exercise in hypoxia compared with normoxia. Yet this lower PO2 remained above the critical PO2 for optimal in vitro mitochondrial function (6). However, other studies have shown that mitochondria are sensitive to PO2 well above the critical PO2 (35). Wilson (35) has shown that a decrease in PO2 is countered by increases in [NADH], [ADP], and [Pi] to provide the same driving force for oxidative phosphorylation. Furthermore, as [ADP] and [Pi] increase, the Michaelis constant for PO2 in the mitochondria is reduced (35). Hence, the effect of PO2 is sensitive to the phosphorylation potential and can vary within a range of PO2 without affecting oxidative phosphorylation.
The paradigm of oxidative phosphorylation described by Wilson (35) appeared to apply in our previous study (23). Exercise during hypoxia was associated with a tendency toward increased PCr breakdown compared with normoxia, which resulted in a greater free [Pi], leading to increased glycogenolysis and pyruvate production (23). The increased lactate accumulation observed with hypoxia may thus be attributed to a mass action effect as a result of increased glycolytic pyruvate production and reduced pyruvate oxidation, which resulted from a delayed activation of PDH at the onset of exercise in hypoxia.
In our previous study, during the initial minutes of exercise in hypoxia, the increased reliance on substrate level phosphorylation from PCr breakdown and glycolysis was equivalent to 32 mmol ATP/kg dry wt, which corresponded to a calculated O2 deficit of 268 ml greater than the O2 deficit in normoxia (23). In the present study, the additional ATP that was produced from oxidative phosphorylation in the DCA condition reduced the calculated O2 deficit compared with control by 128 ml O2, suggesting that there was sufficient O2 to support a greater rate of oxidative phosphorylation. Hence, although PO2 is low in hypoxia, increasing oxidative substrate delivery to the TCA cycle can accelerate the rate of oxidative phosphorylation and reduce the O2 deficit by adjustments in the redox and phosphorylation potentials. The same was shown to be true at the onset of exercise under normoxic conditions (15). However, increased flux through PDH with DCA was not sufficient to decrease PCr breakdown and lactate production completely in the present and previous studies (15, 31, 32). Thus O2 may still be limiting at the onset of exercise because of a delay in diffusive or convective O2 delivery (34), or yet another component of metabolic inertia could be responsible for the remaining substrate phosphorylation not reduced by DCA.
After the initial few minutes of exercise, previous studies have shown, the effect of DCA is reduced, the [PCr] is normalized, and there are no differences in lactate production (15, 31). During normoxia and under conditions of partial ischemia, it appears that DCA plays a role only at the onset of exercise (15, 31). However, in the present study, the effects of DCA were pronounced during the subsequent 14 min of exercise; PCr and glycogen were significantly spared, and lactate accumulation was significantly reduced compared with control, because a greater proportion of total flux was still being maintained in the direction of oxidative phosphorylation with DCA compared with control (98 ± 1 vs. 95 ± 1%). Increasing oxidative phosphorylation by a small amount can significantly reduce substrate phosphorylation from PCr breakdown and glycolysis due to a 12-fold greater ATP yield from the complete oxidation of pyruvate. During exercise in hypoxia alone, Katz and Sahlin (19) demonstrated an increase in [NADH] compared with normoxia, and, as shown in our previous study, to drive oxidative phosphorylation during hypoxia, an increase in free Pi and ADP is also required (23). Thus, in the present study, increased delivery of oxidative substrate due to DCA would have increased the provision of reducing equivalents compared with control and allowed a lower free Pi and ADP to drive oxidative phosphorylation at the same ATP turnover rate as the control condition. As a result, the posttransformational regulators of glycogen phosphorylase, free [Pi] and [AMP], were significantly reduced during the subsequent 14 min of exercise with DCA. This resulted in the reduced stimulation of glycogenolysis compared with control under hypoxic conditions despite similar mole fractions of phosphorylase a, thereby reducing the accumulation of lactate. However, DCA was not able to fully recover PCr and [lactate] to those levels found in normoxia (23). The studies of Howlett et al. (15) and Timmons et al. (31) took place under conditions of normoxia and partial ischemia produced by vascular occlusion. Under conditions of acute respiratory hypoxia, alkalosis induced by hyperventilation may also be a significant contributor to lactate production (10). In a recent study from our laboratory (14), induced metabolic alkalosis was associated with increased PCr breakdown, glycogenolysis, and lactate accumulation. Therefore, PCr may not have been spared by DCA in the present study to the same extent as normoxia due to the effects of the respiratory alkalosis.
From the previous study by Howlett et al. (15), it appears that, once oxygen delivery and/or metabolic inertia has been normalized within the initial few minutes of exercise, DCA has little effect on the source of ATP production. However, in the present study, the persistently lower PO2 may have allowed DCA to have a continued positive effect on oxidative phosphorylation throughout a longer period of exercise.
The mechanism of lactate production during acute hypoxia may be summarized as follows. At the onset of exercise in hypoxia, there is a transient mismatch between ATP demand at the contractile apparatus and oxidative ATP provision. PCr is primarily used as the immediate energy source, since oxidative substrate seems to be limited by a slowed activation of PDH or reduced PO2. As a result of the enhanced PCr breakdown, free Pi, ADP, and AMP accumulate and drive oxidative phosphorylation. The increases in free Pi and AMP also serve as stimuli for increased flux through glycogen phosphorylase to first increase ATP regeneration from substrate level phosphorylation from glycolysis and second to increase pyruvate provision for PDH, leading to increased provision of oxidative substrate to the TCA cycle.
In the present study, DCA reduced a portion of the need for substrate phosphorylation but was not able to completely alleviate the reliance on substrate level phosphorylation to the degree that was observed in normoxia (23). At the onset of exercise in hypoxia, activation of PDH by DCA increased oxidative phosphorylation and reduced the calculated O2 deficit compared with control by approximately one-half. This suggests that O2 supply limitation cannot account in full for limiting the onset of oxidative phosphorylation in hypoxia. The fact that DCA was not as effective in reducing PCr breakdown at the onset of exercise as later in exercise, unlike previous studies, suggests that either PO2 or metabolic inertia might still be limiting at the onset of exercise under hypoxic conditions.
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ACKNOWLEDGEMENTS |
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We thank Tina Bragg for excellent technical assistance. The help of Dr. Gary Lopaschuk of the University of Alberta is also gratefully acknowledged for HPLC analysis of the DCA solutions.
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FOOTNOTES |
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This study was supported by operating grants from the Medical Research and Natural Sciences and Engineering Research Councils of Canada. M. L. Parolin was supported by a Natural Sciences and Engineering Research Council scholarship and by the Ontario Thoracic Society. M. G. Hollidge-Horvat was supported by a Medical 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: G. J. F. Heigenhauser, Dept. of Medicine, McMaster Univ. Medical Centre, 1200 Main St. W., Hamilton, Ontario, Canada L8N 3Z5 (E-mail: heigeng{at}fhs.csu.mcmaster.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 17 November 1999; accepted in final form 16 May 2000.
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