1 Copenhagen Muscle Research Centre, Rigshospitalet, DK-2200 Copenhagen N, Denmark; and 2 Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada N1G 2W1
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ABSTRACT |
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We examined the relationship between
tricarboxylic acid (TCA) cycle intermediate (TCAI) pool size, TCA cycle
flux (calculated from leg O2
uptake), and pyruvate dehydrogenase activity
(PDHa) in human skeletal muscle.
Six males performed moderate leg extensor exercise for 10 min, followed
immediately by intense exercise until exhaustion (3.8 ± 0.5 min).
The sum of seven measured TCAI (TCAI) increased
(P
0.05) from 1.39 ± 0.11 at
rest to 2.88 ± 0.31 after 10 min and to 5.38 ± 0.31 mmol/kg dry
wt at exhaustion. TCA cycle flux increased ~70-fold during submaximal
exercise and was ~100-fold higher than rest at exhaustion.
PDHa corresponded to 77 and 90%
of TCA cycle flux during submaximal and maximal exercise, respectively.
The present data demonstrate that a tremendous increase in TCA cycle
flux can occur in skeletal muscle despite a relatively small
change in TCAI pool size. It is suggested that the increase in
TCAI
during exercise may primarily reflect an imbalance between the
rate of pyruvate production and its rate of oxidation in the TCA cycle.
pyruvate dehydrogenase complex; muscle oxygen uptake; amino acids; metabolism
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INTRODUCTION |
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THE FACTORS that influence the combined pool size of tricarboxylic acid (TCA) cycle intermediates (TCAI) and the control of cycle activity have been characterized in the isolated, perfused rat heart (31). Because the total concentration of TCAI appears related to the energy state of the myocardium (22), it has been suggested that the size of the TCAI pool serves a regulatory role in cardiac energy metabolism (21). Considerably less information is available regarding the concentrations of TCAI and the factors that regulate TCAI pool size in mammalian skeletal muscle. Indeed, only one investigation has attempted to measure the pool of TCAI in rodent muscle (3), and although several studies have reported changes in specific intermediates in humans (e.g., Refs. 11, 17, 36), only recently has an effort been made to quantify the exercise-induced changes in total TCAI pool size (12, 13). Overall, these studies demonstrated that the total concentration of TCAI can increase severalfold during moderate-to-intense muscle contraction, and peak expansion of the TCAI pool occurs within the initial few minutes of exercise (12).
However, the precise physiological significance of changes in TCAI pool size during muscle contraction remains speculative. Flux through the TCA cycle is believed to be primarily regulated through allosteric control of the three nonequilibrium enzymes in the cycle, citrate synthase, isocitrate dehydrogenase, and 2-oxoglutarate dehydrogenase (18, 28, 41). In addition, it has been suggested that an increase in the total concentration of TCAI may be necessary to obtain optimal energy provision under conditions of increased energy demand (25, 36). Several investigators have implied that changes in the total concentration of TCAI are indicative of the capacity for TCA cycle flux in skeletal muscle (35, 36), and two theories that link a decrease in TCAI pool size with peripheral muscle fatigue during prolonged exercise in humans have been proposed (36, 40). Although these hypotheses are interesting, there is in fact little experimental evidence to support them. Indeed, the fundamental relationship between TCAI pool size and TCA cycle flux has not been clearly established for human skeletal muscle.
The primary purpose of the present investigation was therefore to examine the relationship between TCAI pool size and estimated TCA cycle flux in human skeletal muscle during exercise. It was hypothesized that a very large increase in TCA cycle flux can occur despite a relatively small increase in TCAI pool size. We utilized the one-legged knee extension exercise model, which essentially restricts work to the quadriceps femoris muscle (1) and permits TCA cycle turnover to be calculated from muscle O2 uptake based on the Fick principle (6, 9). These methods were recently employed by Blomstrand et al. (6) in a study that compared estimated TCA cycle flux with the maximal in vitro activities of three enzymes in the TCA cycle, citrate synthase, succinate dehydrogenase, and 2-oxoglutarate dehydrogenase. The results from that study were intriguing to us, since the calculated TCA cycle flux was considerably higher than the maximal in vitro activity previously reported for the pyruvate dehydrogenase enzyme complex (PDH) in human skeletal muscle (e.g., Refs. 7, 8, 10, 29, 30). PDH controls the flux of pyruvate-derived acetyl-CoA into the TCA cycle (40), and it is generally assumed that, during moderate-to-intense exercise when most of the energy is derived from carbohydrate sources, PDH activity is at all times equal to or greater than the rate of flux through the TCA cycle (23). Therefore, a secondary purpose of the present study was to compare the calculated rate of TCA cycle flux with the measured active fraction of PDH (PDHa) in biopsy samples obtained from the same muscle.
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METHODS |
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Subjects. Six healthy males, with a mean age, height, and mass of 23.3 ± 1.1 yr, 181.2 ± 3.1 cm, and 79.3 ± 4.7 kg, respectively, volunteered for the investigation. Four subjects were involved in recreational sport activities (e.g., soccer and bicycling), but none were engaged in any form of regular physical training. The subjects were advised of the purposes and associated risks of the study and gave written informed consent. The experimental protocol was approved by the Ethical Committee for Copenhagen and Frederiksberg communities.
Preexperimental procedures. The subjects were familiarized with the Krogh ergometer modified for one-legged knee extensor exercise as previously described (2). At least 3 days before the experiment, subjects performed an incremental exercise test with their dominant leg (kicking frequency 60/min) to determine the maximal exercise capacity of the knee extensors. This was defined as the highest workload that could be sustained while the desired kicking frequency was maintained. The mean peak workload for the group was 77 ± 2 W. Subjects were instructed to consume their habitual diet and refrain from exercise or strenuous physical activity for 48 h before the experiment.
Experimental protocol. Subjects arrived at the laboratory in the morning after an overnight fast. Teflon catheters were inserted into the femoral artery and vein of one leg, ~2 cm proximal and distal to the inguinal ligament, respectively. A thermistor for measurement of venous blood temperature was inserted through the venous catheter, and the tip was advanced ~8 cm proximal to the tip of the catheter. Subjects were moved to the exercise apparatus, where they rested supine for ~30 min before the initiation of the exercise test. During this time, the area over the vastus lateralis muscle of the leg to be exercised was prepared for the extraction of muscle biopsy samples (5).
The exercise protocol consisted of kicking at 60% of the predetermined one-legged maximal exercise capacity for 10 min, followed immediately by intense exercise at 100% of maximum until exhaustion (Exh), i.e., the point at which the subject could no longer maintain the desired kicking frequency of 60/min. The mean time to Exh, after the 10-min period of submaximal work, was 3.8 min (range 2.5-5.6 min). Arterial and venous blood samples were drawn simultaneously at rest, after 5 and 10 min of exercise, and immediately before exhaustion. Measurements of leg blood flow using the thermodilution technique (2) were made several times at rest and immediately before and after each blood sample during exercise. During blood sampling and blood flow measurements, an occlusion cuff positioned just below the knee was inflated toBlood analyses.
Blood samples were drawn with heparinized syringes.
O2 saturation,
O2 content, and hemoglobin were
measured using an OSM-2 hemoximeter (Radiometer, Copenhagen, Denmark).
Whole blood lactate concentrations were immediately determined with a
Yellow Springs lactate analyzer (Yellow Springs, OH). The remainder of
the arterial and venous blood samples was centrifuged, and the
supernatant was collected and stored at 80°C. Plasma samples
were subsequently analyzed for ammonia (4) with a fluorometer
(Perkin-Elmer model LS-50) and free amino acids by HPLC (20).
Muscle analyses.
Biopsy samples were immediately frozen (<5 s) by plunging the needle
into liquid nitrogen, removed from the needle while still frozen, and
stored at 80°C. A 15- to 30-mg piece of muscle was chipped
from each sample and used for the determination of the active fraction
of PDHa by using the method of
Constantin-Teodosiu et al. (8), as modified and described by Putman et
al. (30). Total creatine concentrations (TCr) were measured in
neutralized perchloric acid extracts of the
PDHa homogenates by using a
spectrophotometer (4). To correct for differences in blood or
connective tissue between samples,
PDHa values were adjusted to the
highest TCr value in all the biopsy samples obtained from each subject,
as described by Putman et al. (29). The mean TCr correction for all
PDHa determinations was 1.19. Due
to limited tissue in biopsy samples obtained from three subjects at the
10-min time point, complete PDHa
data for all six subjects were obtained only for the rest, 5 min, and
Exh time points.
Calculations. Thigh volume was estimated based on Simpson's rule, which includes thigh length, three circumferences of the thigh, and three skinfold measurements (24), and muscle mass was estimated from a regression equation (2). On the basis of these calculations, the estimated active mass of the quadriceps muscles used during exercise was 3.03 ± 0.21 kg. The uptake and/or release of O2, glucose, lactate, ammonia, and amino acids was calculated by multiplying the blood or plasma flow by the arteriovenous difference in concentration and expressed per kilogram wet weight of active muscle. Flux through the TCA cycle was calculated from the rate of muscle O2 uptake (6, 9), assuming that carbohydrate was the only substrate being oxidized. Briefly, on the basis of the stoichiometry of the pathway for glucose oxidation, the flux through the TCA cycle is equivalent to one-third of the O2 uptake. At a temperature of 37-38°C, 1 mol of O2 is equivalent to 25.4-25.5 liters according to the general law for gases.
Statistics.
Cardiorespiratory, blood, and muscle metabolite data were analyzed
using a one-factor (1 × 4; time) repeated-measures ANOVA. PDHa data were analyzed using a
one-factor (1 × 3; time) repeated-measures ANOVA. Linear and
polynomial regression analyses were used to examine the relationship
between TCAI pool size and estimated TCA cycle flux. Statistical
significance for all analyses was accepted as P 0.05, and significant main effects were further analyzed using a Tukey
honestly significant difference post hoc test. Data are expressed as
means ± SE unless otherwise noted.
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RESULTS |
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Cardiorespiratory, leg blood flow, muscle
O2 uptake, and TCA cycle flux data.
Changes in heart rate, pulmonary
O2 uptake, and ventilation during
exercise are summarized in Table 1. Muscle
blood flow was 27- and 35-fold higher
(P 0.05) than at rest during the submaximal exercise period and at Exh, respectively (Table 1). Muscle
O2 uptake and the estimated flux
through the TCA cycle increased (P
0.05) ~70-fold above rest during the submaximal exercise period and
reached values ~100-fold higher than rest at Exh (Table 1).
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Blood and plasma metabolites and flux data.
Changes in the arterial concentrations of lactate, ammonia, alanine,
glutamine, and glutamate are summarized in Table
2. The arterial lactate concentration and
lactate release were higher (P 0.05) at all times during exercise compared with rest, and the
respective values at Exh were also higher
(P
0.05) compared with values at 5 and 10 min of exercise (Table 2). Ammonia release was higher
(P
0.05) at all times during
exercise compared with rest; however, the efflux at Exh was
approximately fivefold higher (P
0.05) compared with values at 5 and 10 min of exercise (Table 2). The
release of alanine and glutamine was also higher (P
0.05)
during exercise compared with rest, but the respective values at Exh
were not significantly different compared with values at 5 and 10 min
of exercise (Table 2). Glutamate was the only amino acid that was taken
up in significant amounts during exercise, and the uptake at Exh was
also higher (P
0.05) compared with values at 5 and 10 min of exercise (Table 2).
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Intramuscular TCA cycle intermediates.
The total intramuscular concentration of the seven measured TCAI
(TCAI) increased (P
0.05)
approximately onefold above rest levels during the submaximal exercise
period (Fig. 1). The
TCAI showed a
further increase after the period of maximal work, and the value at Exh
was approximately threefold higher (P
0.05) than at rest (Fig. 1). There was a significant positive
correlation between total concentration of TCAI and the calculated flux
through the TCA cycle (Fig. 2). The
explained variance was not different when this relationship was
described based on a linear ( y = 0.873x + 1.024;
r2 = 0.75;
P
0.0001) or polynomial
( y = 0.072x2 + 0.556x + 1.199;
r2 = 0.76;
P
0.0001) regression analysis;
however, the pooled data suggest that a curvilinear relationship exists
between TCAI pool size and TCA cycle flux (Fig.
3).
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PDHa and intramuscular pyruvate.
PDHa increased progressively
during exercise and was 3.6- and 7.1-fold higher than at rest after 5 min of exercise and at Exh, respectively (Fig. 5).
PDHa at rest (0.45 ± 0.09 mmol · kg wet
wt1 · min
1)
was severalfold higher than TCA cycle flux; however,
PDHa after 5 min of exercise (2.07 ± 0.11) and at Exh (3.60 ± 0.21) corresponded to 77 and 90% of
the estimated TCA cycle flux, respectively. Intramuscular pyruvate was
higher (P
0.05) at all times during
exercise compared with rest, and the value at Exh was also higher
(P
0.05) compared with 5 and 10 min
of exercise (Fig.
6).
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DISCUSSION |
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The present results demonstrate that there is a significant, positive
correlation between TCAI pool size and estimated TCA cycle flux in
human skeletal muscle during dynamic leg extensor exercise. Although a
previous study (35) demonstrated that the total intramuscular
concentration of citrate, malate and fumarate increased progressively
during incremental cycle exercise, this is the first study to directly
compare changes in TCAI pool size with estimates of TCA cycle flux in
mammalian skeletal muscle. The relative changes in these variables were
very different, and the present data illustrate that a tremendous
increase in TCA cycle flux can occur despite only a modest elevation in
the total concentration of TCAI. For example, during the transition
from rest to submaximal exercise, the rate of TCA cycle flux increased by ~70-fold above rest, whereas the total concentration of TCAI only
doubled. In addition, these data suggest that the relationship between
TCAI pool size and TCA cycle flux is not linear (Fig. 3), since the
ratio of the change in TCAI pool size to change in cycle flux (i.e.,
TCAI pool size/
cycle flux) was larger during the second
transition in workload; i.e., when the exercise intensity was increased
from the submaximal to maximal workload, TCA cycle flux increased
another 30-fold above rest values (or one-half of initial relative
increase during first transition in workload), and yet the TCAI pool
size increased a further 2-fold.
It must be emphasized that a correlation between two variables does not imply cause and effect, and these data should not be interpreted to suggest that an increase in the total concentration of TCAI is necessary to increase cycle flux. Indeed, the mechanisms that control steady-state flux through the TCA cycle are extremely complex (18, 28, 41), and it remains speculative whether the size of the TCAI pool plays an important physiological role in this regard. Several investigators (35, 36, 39) have implied that changes in the total concentration of TCAI may be indicative of the capacity for TCA cycle flux in human skeletal muscle during exercise. However, although there is evidence to suggest that anaplerotic reactions may play a role in the maintenance of contractile function in isolated rat hearts oxidizing acetoacetate (32, 33), no such data have been presented for mammalian skeletal muscle. To properly investigate this question, it will be necessary to manipulate levels of TCAI and to determine what effect, if any, this has on TCA cycle flux, oxidative energy metabolism, and skeletal muscle function.
Nonetheless, the present findings demonstrate that there is a strong
correlation between changes in TCAI pool size and TCA cycle flux. There
appears to be a curvilinear relationship between these variables so
that, at high work intensities, there is a disproportionate increase in
TCAI pool size relative to the change in TCA cycle flux (Fig. 3). One
interpretation of these data is that, during the transition from rest
to moderate exercise, only a small increase in TCAI (relative to
resting concentrations) is necessary to accommodate a relatively large
change in TCA cycle flux. However, at more intense workloads, the TCAI
pool must expand to a larger extent to sustain smaller increments in
TCA cycle flux. Alternatively, it must also be considered that the
increase in TCAI may not represent an important regulatory signal but
may simply be a consequence of the huge increase in metabolic flux which occurs during exercise. Therefore, another possible explanation for anaplerosis is that the increase in TCAI during contraction primarily represents a sink for pyruvate when its rate of formation from glycolysis exceeds its rate of oxidation in the TCA cycle. This
interpretation is analogous to the "mass action" theory proposed to explain lactate accumulation in skeletal muscle during exercise (for
review, see Ref. 14). An increase in the concentration of pyruvate
appears necessary for anaplerosis, since many of the reactions that
could lead to a net influx of TCAI are directly or indirectly dependent
on the level of pyruvate, including those catalyzed by alanine
aminotransferase, phosphoenolpyruvate
carboxykinase, pyruvate carboxylase, and malic enzyme.
Although the precise mechanisms responsible for anaplerosis have not
been completely resolved, the alanine aminotransferase reaction
(pyruvate + glutamate 2-oxoglutarate + alanine) appears
quantitatively most important for the increase in TCAI at the onset of
exercise in humans (12, 36).
It therefore might be expected that an increase in TCAI should occur
only under conditions in which the rate of pyruvate production from
glycolysis exceeds its rate of oxidation in the TCA cycle. Sahlin and
co-workers (34, 35) have presented evidence, in separate studies,
suggesting that an increase in TCAI only takes place at exercise
intensities which result in an elevation of muscle pyruvate. When
healthy subjects cycled at a very low workload [~25% maximum
O2 consumption
(O2 max)] that did not
cause an increase in intramuscular pyruvate (34), there was no
significant increase in the intramuscular concentration of citrate,
malate, or fumarate (35). However, when subjects cycled at workloads corresponding to ~50 and ~80% of
O2 max, there were
significant elevations in both pyruvate (34) and the three measured
TCAI (35). These authors have also shown that patients with McArdle's disease, who lack the enzyme glycogen phosphorylase and cannot produce
pyruvate from glycogen, display markedly attenuated increases in these
TCAI during exercise (35). Finally, Spencer et al. (37) demonstrated
that epinephrine infusion caused a significant elevation in pyruvate in
resting human muscle, and this was associated with a doubling of
intramuscular citrate, malate, and fumarate.
Two spans of TCA cycle. The relative distribution of the individual TCAI during exercise also deserves comment. Malate consistently demonstrates the largest quantitative change of any measured TCAI during exercise (12, 13, 35, 36) and, in the present study, accounted for more than one-half of the net increase in TCAI pool size during each transition in workload. In addition to malate, fumarate and succinate also demonstrated relatively large concentration changes during exercise, and together these three TCAI accounted for a progressively larger portion of total pool size at each work intensity. Although the precise mechanisms responsible for the large increase in these three TCAI are not clear, the present data confirm the findings of our previous investigations (12, 13) and demonstrate that the vast majority of anaplerotic carbon which enters the TCA cycle during exercise is directed toward increasing the concentrations of the intermediates in the second span of the cycle (Fig. 4).
The disproportionate increase in the concentrations of malate, fumarate, and succinate can be reconciled if one considers the TCA cycle to consist of two physiological pathways: the span from acetyl-CoA to 2-oxoglutarate and the span from 2-oxoglutarate to oxalacetate (26, 31). As noted by Newsholme and Leech (26), in skeletal muscle during sustained exercise, this division of the cycle is only academic, since the flux through the two pathways must be identical and regulated in a concerted manner. Nonetheless, this division might explain how it is possible for carbon skeletons to feed into the cycle, e.g., at the level of 2-oxoglutarate through the alanine aminotransferase reaction (12), and accumulate at the level of malate, fumarate, and succinate. In principle, the carbon that enters could also be removed from the cycle at the level of malate via the reaction catalyzed by malic enzyme or at the level of oxalacetate via phosphoenolpyruvate carboxykinase (3, 38). However, the net accumulation of TCAI during exercise indicates that the removal of intermediates through these pathways was much slower than the rate of influx into the cycle. This could be due to the fact that the potential egress of TCAI through the reactions catalyzed by malic enzyme and phosphoenolpyruvate carboxykinase was prevented by the elevated intramuscular pyruvate concentration during exercise (Fig. 6).PDHa and TCA cycle flux. A second point that emerged from the present study was that PDHa at Exh after maximal leg extensor exercise, when the enzyme complex was likely fully transformed to its more active form (29), corresponded to only 90% of the calculated TCA cycle flux. This finding confirms the observation, made a priori based on data from separate studies, that the calculated maximal rate of flux through the TCA cycle in vivo (6) is higher than the maximal reported activity for PDH in vitro (e.g., Refs. 7, 8, 10, 29, 30). These observations are perplexing, given that, during moderate-to-intense exercise when most of the energy is derived from carbohydrate sources, it is generally assumed that PDHa is at all times equal to or greater than the rate of flux though the TCA cycle in skeletal muscle (23). Indeed, the explanation for the well-documented exercise-induced rise in acetylcarnitine concentration (7, 19, 30, 34) is that PDH flux exceeds TCA cycle flux, and carnitine functions to buffer the excess formation of acetyl groups generated from pyruvate through PDH. In doing so, carnitine serves to prevent the depletion of the mitochondrial CoASH pool, which would otherwise inhibit flux though the TCA cycle at the level of 2-oxoglutarate dehydrogenase (7, 19). Although this important role for carnitine is widely accepted, as noted earlier, this theory is not well supported by existing literature values for PDHa and TCA cycle flux in human muscle during maximal exercise.
The present study is the first attempt to directly compare estimates of TCA cycle flux with measurements of PDHa in biopsy samples obtained from the same muscle, and our data are consistent with existing literature values for these variables. The rate of TCA cycle flux that we calculated at Exh was ~10% lower than that reported by Blomstrand et al. (6) for maximal dynamic knee extensor exercise, mainly because our estimated quadriceps muscle mass (based on anthropometric measurements) was higher than that reported in their study (which utilized computer tomography). The values that we obtained for PDHa at Exh are similar to (10) or slightly higher than those previously reported for human skeletal muscle during intense dynamic exercise (29, 30), obtained with the same analytical procedures. The present data must therefore be considered valid and highlight the fact that although the reported changes in acetylcarnitine concentration (7, 19, 30, 34) suggest otherwise, in vitro measurements of PDHa in human muscle during maximal exercise are nonetheless lower than calculated rates of TCA cycle flux based on muscle O2 uptake. There are a number of explanations that could account for this discrepancy, including the accuracy of our analytical techniques. It is possible that the combined experimental variability associated with the measurement of both leg blood flow (which is used to derive muscle O2 uptake and TCA cycle flux) (2) and the determination of PDHa (8) is equal to or greater than the 10% difference observed between these variables in the present study. A second possibility is that measurements of PDHa are made under optimal in vitro conditions and in some situations may not represent the actual in vivo flux, since the latter is also determined in part by the availability of substrates and accumulation of products (30, 40). For example, when Putman et al. (30) attempted to calculate the in vivo rate of flux through PDHa during intense cycle exercise from measurements or estimates of glycogen degradation, glucose uptake, lactate production, leg blood flow, and active muscle mass, the value which they obtained was ~19% higher than their in vitro measurement of PDHa. It could also be argued that the use of leg O2 uptake may slightly overestimate the rate of TCA cycle flux in the quadriceps muscles, particularly during maximal work, since a substantial fraction of the limb blood flow is perfusing not only the knee extensors but also the hamstring and gluteal muscles (which may not be completely relaxed). Our subjects practiced the knee extensor exercise before the experiment to minimize this possibility, and it has previously been shown that familiarized subjects demonstrate little or no electromyographic activity in their noninvolved leg muscles (1). In addition, it has recently been demonstrated that derived measurements of muscle temperature reveal no elevation in the hamstrings, whereas the knee extensor muscles increase 1.0°C during this type of exercise (15). Finally, if we assume our measurements of PDHa and leg O2 uptake are precise indicators of in vivo fluxes, then it must be considered that, even during maximal leg extensor exercise, a small portion of the acetyl-CoA which entered the TCA cycle was derived from noncarbohydrate sources (e.g., intramuscular triglycerides or amino acid catabolism).Ammonia metabolism during exercise.
A final observation from the present study concerned the relative
changes in the flux of ammonia and the amino carriers, alanine and
glutamine. The efflux of all three compounds was higher after 5 and 10 min of submaximal exercise compared with rest; however, the arterial
concentrations did not change, indicating that their clearance (e.g.,
by liver) was well matched to muscle release. In addition, the increase
in ammonia efflux was much smaller than the relative changes in
glutamine and alanine release during the submaximal exercise period.
However, at exhaustion after the period of maximal work, the arterial
ammonia concentration doubled and ammonia release increased another
fourfold, whereas the arterial concentrations and efflux of alanine and
glutamine were unchanged compared with the respective values during
submaximal exercise. The relative changes in the efflux of glutamine
and ammonia at the two workloads may be due to the energetics of the
glutamine synthase reaction (glutamate + ammonia + ATP glutamine + ADP + Pi). Although
this reaction is quantitatively important for the clearance of muscle
ammonia during submaximal exercise (16), the data from the present
investigation suggest that this route of clearance was inhibited during
maximal exercise. This could be due to the fact that the glutamine
synthase reaction is energy requiring or possibly that there were other
demands on the intramuscular glutamate pool.
Conclusions. In summary, the results from the present investigation demonstrate there is a significant, positive correlation between the total concentration of TCAI and estimated TCA cycle flux in human skeletal muscle during exercise. The relative changes in TCAI pool size and cycle turnover rate were very different, however, and these data demonstrate that a tremendous increase in TCA cycle flux can occur despite a relatively small change in the total concentration of TCAI. The measured active fraction of PDH corresponded to 77 and 90% of the estimated flux through the TCA cycle during submaximal and maximal knee extensor exercise, respectively. Most of the anaplerotic carbon that entered the cycle during exercise was directed toward increasing the concentration of malate, and, overall, the TCAI in the second span of the TCA cycle (i.e., malate, fumarate, and succinate) accounted for a progressively larger portion of the TCAI pool at each work intensity. It remains speculative whether the increase in TCAI during exercise is important to augment cycle flux or is simply a consequence of the increase in pyruvate concentration that occurs when its rate of formation from glycolysis exceeds its rate of oxidation in the TCA cycle.
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ACKNOWLEDGEMENTS |
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The authors thank Karin Juel, Charlotte Mortensen, Carsten Nielsen, Maureen Odland, Lynda Powell, Premila Sathasivam, and Hana Villmusen for excellent technical assistance. We also thank our subjects for time and tremendous effort.
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FOOTNOTES |
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This project was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Danish National Research Foundation. M. J. Gibala was the recipient of an NSERC Postgraduate Scholarship, and D. A. MacLean was supported by a Medical Research Council of Canada Postdoctoral Fellowship.
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: M. J. Gibala, Copenhagen Muscle Research Centre, Rigshospitalet, Section 7652, Tagensvej 20, DK-2200 Copenhagen N, Denmark.
Received 10 February 1998; accepted in final form 21 April 1998.
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