Download Tricarboxylic acid cycle intermediate pool size and estimated cycle

Tricarboxylic acid cycle intermediate pool size and
estimated cycle flux in human muscle during exercise
MARTIN J. GIBALA,1 DAVE A. MACLEAN,1 TERRY E. GRAHAM,2 AND BENGT SALTIN1
Muscle Research Centre, Rigshospitalet, DK-2200 Copenhagen N, Denmark; and
2Department of Human Biology and Nutritional Sciences, University of Guelph,
Guelph, Ontario, Canada N1G 2W1
1Copenhagen
pyruvate dehydrogenase complex; muscle oxygen uptake;
amino acids; metabolism
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).
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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-tointense 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
0193-1849/98 $5.00 Copyright r 1998 the American Physiological Society
E235
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Gibala, Martin J., Dave A. MacLean, Terry E. Graham, and Bengt Saltin. Tricarboxylic acid cycle intermediate pool size and estimated cycle flux in human muscle during
exercise. Am. J. Physiol. 275 (Endocrinol. Metab. 38): E235–
E242, 1998.—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 6 0.5 min). The sum of seven measured TCAI (STCAI)
increased (P # 0.05) from 1.39 6 0.11 at rest to 2.88 6 0.31
after 10 min and to 5.38 6 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 STCAI during
exercise may primarily reflect an imbalance between the rate of
pyruvate production and its rate of oxidation in the TCA cycle.
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ANAPLEROSIS AND TCA CYCLE FLUX IN HUMAN SKELETAL MUSCLE
PDH (PDHa ) in biopsy samples obtained from the same
muscle.
METHODS
RESULTS
Cardiorespiratory, leg blood flow, muscle O2 uptake,
and TCA cycle flux data. Changes in heart rate, pulmonary O2 uptake, and ventilation during exercise are
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Subjects. Six healthy males, with a mean age, height, and
mass of 23.3 6 1.1 yr, 181.2 6 3.1 cm, and 79.3 6 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 6 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 to $220 mmHg. Muscle biopsy samples
were obtained at rest, after 5 and 10 min of exercise, and at
exhaustion. Heart rate, pulmonary O2 uptake, and ventilation were measured at rest and continually during exercise
(MedGraphics CPX System, Klampenborg, Denmark).
Blood 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 280°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 280°C. A 15to 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.
The remaining portion of each biopsy sample was freeze
dried, powdered to dissect out nonmuscle elements, and
stored at 280°C. Aliquots of freeze-dried muscle were extracted with 0.5 M perchloric acid (containing 1 mM EDTA),
neutralized with 2.2 M KHCO3, and assayed for citrate,
isocitrate, 2-oxoglutarate, succinate, fumarate, malate, and
oxalacetate (4, 27, 36) by fluorometric procedures as previously described (13). Succinyl-CoA was not determined because of its extremely low concentration; however, it is likely
that the remaining seven intermediates comprise .99% of
total TCAI and thus provide a quantitative index of total pool
size. A portion of the muscle extract was also used for the
fluorometric determination of pyruvate (27).
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 6
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 3 4; time) repeatedmeasures ANOVA. PDHa data were analyzed using a onefactor (1 3 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 6 SE unless otherwise noted.
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ANAPLEROSIS AND TCA CYCLE FLUX IN HUMAN SKELETAL MUSCLE
Table 1. Cardiorespiratory, blood flow, muscle O2 uptake, and estimated TCA cycle flux data
at rest and during exercise
Heart rate, beats/min
Pulmonary V̇O2 , l/min
Ventilation, l/min
Muscle blood flow, l · min21 · kg21
Muscle V̇O2 , ml O2 · min21 · kg21
TCA cycle flux, mmol · min21 · kg21
Rest
5 min
10 min
Exh
64 6 4
0.30 6 0.04
12.1 6 2.3
0.06 6 0.01
361
0.04 6 0.01
101 6 6*
1.04 6 0.06*
36.5 6 4.1*
1.67 6 0.12*
205 6 15*
2.69 6 0.20*
102 6 5*
1.12 6 0.09*
39.9 6 6.3*
1.68 6 0.13*
215 6 17*
2.81 6 0.23*
137 6 4*†
2.01 6 0.25*†
86.1 6 15.3*†
2.20 6 0.22*†
306 6 33*†
4.02 6 0.43*†
Values are means 6 SE; n 5 6. Muscle blood flow, muscle V̇O2 , and tricarboxylic acid (TCA) cycle flux data are expressed per wet weight of
quadriceps muscle. V̇O2, rate of O2 consumption; Exh, exhaustion. * P # 0.05 vs. rest. † P # 0.05 vs. 10 min.
the uptake at Exh was also higher (P # 0.05) compared
with values at 5 and 10 min of exercise (Table 2).
Intramuscular TCA cycle intermediates. The total
intramuscular concentration of the seven measured
TCAI (STCAI) increased (P # 0.05) approximately
onefold above rest levels during the submaximal exercise period (Fig. 1). The STCAI 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 5
0.873x 1 1.024; r 2 5 0.75; P # 0.0001) or polynomial
( y 5 0.072x2 1 0.556x 1 1.199; r 2 5 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).
The changes in individual TCAI during exercise are
summarized in Table 3. Citrate, isocitrate, fumarate,
malate, and oxalacetate were higher (P # 0.05) at all
times during exercise compared with rest, whereas
succinate was only higher (P # 0.05) at Exh compared
with rest. The concentrations of malate, fumarate,
Table 2. Arterial concentrations and net
uptake/release of metabolites and some plasma
amino acids at rest and during exercise
Rest
5 min
10 min
Exh
Arterial concentration
Lactate, mM
Ammonia, µM
Alanine, µM
Glutamine, µM
Glutamate, µM
0.6460.10 1.7660.25* 1.6860.26*
22.962.9 24.762.5
25.562.5
316657
356639
362641
619652
623641
601635
6264
4564*
4163*
3.5360.35*†
45.065.4*†
421647*†
634638
4163*
Net uptake/release
Lactate
Ammonia
Alanine
Glutamine
Glutamate
0.060.0
0.460.3
2361
2361
361
21.360.3*
21964*
267620*
269616*
1563*
20.960.3*
21864*
283617*
278614*
1162*
23.560.3*†
2110614*†
2103613*
289611*
1864*†
Values are means 6 SE; n 5 6. Net uptake/release values are in
µmol·min21 ·kg wet wt21 except for lactate which is in mmol·min21 ·kg
wet wt21. A negative value indicates a release. * P # 0.05 vs. rest.
† P # 0.05 vs. 10 min.
Fig. 1. Total muscle concentration of 7 measured tricarboxylic acid
(TCA) cycle intermediates (TCAI; citrate, isocitrate, 2-oxoglutarate,
succinate, fumarate, malate, and oxalacetate) at rest, after 5 and 10
min of submaximal exercise (60% of maximum), and at exhaustion
(Exh) after maximal dynamic knee extensor exercise. Values are
means 6 SE; n 5 6. * P # 0.05 vs. rest. 1 P , 0.05 vs. 10 min.
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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).
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
E238
ANAPLEROSIS AND TCA CYCLE FLUX IN HUMAN SKELETAL MUSCLE
succinate, and isocitrate were also higher (P # 0.05) at
Exh compared with 5 and 10 min of exercise. 2-Oxoglutarate was the only TCAI that decreased during exercise and was lower (P # 0.05) at all times compared
with rest.
The overall changes in TCAI pool size during exercise
were primarily due to increases in the concentration of
malate. This intermediate accounted for over one-half of
the increase in pool size during each transition in
workload and, at Exh, was more than sixfold higher
than at rest (Table 3). Succinate and fumarate also
demonstrated relatively large concentration changes
during exercise, and together these three TCAI accounted for a progressively larger portion of the total
pool size at each work intensity; i.e., the total concentration of malate, fumarate, and succinate comprised 61 6
4% of total pool size at rest, but this proportion
increased to 67 6 3 and 68 6 2% after 5 and 10 min of
Fig. 3. Relationship between TCAI pool size and TCA cycle flux at
rest and during exercise. Values are means 6 SE; n 5 6.
DISCUSSION
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., DTCAI pool size/Dcycle 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
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Fig. 2. Relationship between TCAI pool size and calculated TCA
cycle flux. Individual data are plotted for each subject at rest and
during exercise. Values are means 6 SE; n 5 6. Explained variance
was not different when relationship was described based on a linear
(r 2 5 0.75) or polynomial (r 2 5 0.76) regression analysis. See RESULTS
for further explanation.
submaximal exercise, respectively, and was 81 6 2% at
Exh. Thus, in both an absolute and relative sense, most
of the anaplerotic carbon that entered TCA cycle was
directed toward increasing the TCAI in the second
‘‘span’’ of the cycle, i.e., the span from 2-oxoglutarate to
oxalacetate (Fig. 4).
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 6 0.09 mmol · kg
wet wt21 · min21 ) was severalfold higher than TCA cycle
flux; however, PDHa after 5 min of exercise (2.07 6
0.11) and at Exh (3.60 6 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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ANAPLEROSIS AND TCA CYCLE FLUX IN HUMAN SKELETAL MUSCLE
Table 3. Intramuscular concentrations of individual TCA cycle intermediates at rest and during exercise
Citrate
Isocitrate
2-Oxoglutarate
Succinate
Fumarate
Malate
Oxalacetate
Rest
5 min
10 min
Exh
0.362 6 0.047
0.085 6 0.013
0.050 6 0.004
0.368 6 0.076
0.087 6 0.006
0.365 6 0.037
0.012 6 0.003
0.658 6 0.077*
0.194 6 0.022*
0.036 6 0.005*
0.567 6 0.115
0.198 6 0.029*
1.163 6 0.203*
0.030 6 0.005*
0.631 6 0.052*
0.200 6 0.022*
0.038 6 0.005*
0.609 6 0.118
0.195 6 0.032*
1.182 6 0.160*
0.027 6 0.006*
0.676 6 0.079*
0.305 6 0.040*†
0.030 6 0.005*
1.257 6 0.188*†
0.361 6 0.046*†
2.723 6 0.155*†
0.027 6 0.004*
Values are means 6 SE in mmol/kg dry wt; n 5 6. * P # 0.05 vs. rest. † P # 0.05 vs. 10 min.
Fig. 4. Total concentration of TCAI in span I of TCA cycle [i.e., citrate
(Cit), isocitrate (Iso), and 2-oxoglutarate (2OG)] and span II of cycle
[i.e., succinate (Suc), fumarate (Fum), malate (Mal), and oxalacetate
(Oxa)] at rest, after 5 min of submaximal exercise (60% of maximum),
and at exhaustion after maximal dynamic knee extensor exercise.
Values are means 6 SE; n 5 6.
tion 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 1 glutamate = 2-oxoglutarate 1 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 coworkers (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 (V̇O2max)] that did not cause an increase in intramus-
Fig. 5. Measured active fraction of pyruvate dehydrogenase (PDHa )
and estimated flux through TCA cycle at rest, after 5 min of
submaximal exercise (60% of maximum), and at exhaustion after
maximal dynamic knee extensor exercise. Values are means 6 SE;
n 5 6. * P # 0.05 vs. rest. 1 P , 0.05 vs. 5 min.
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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 contrac-
E240
ANAPLEROSIS AND TCA CYCLE FLUX IN HUMAN SKELETAL MUSCLE
cular 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 V̇O2 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
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Fig. 6. Intramuscular pyruvate at rest and during exercise. Values
are means 6 SE; n 5 6. * P # 0.05 vs. rest. 1 P , 0.05 vs. 10 min.
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 welldocumented 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
ANAPLEROSIS AND TCA CYCLE FLUX IN HUMAN SKELETAL MUSCLE
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 1 ammonia 1 ATP = glutamine 1 ADP 1 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.
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.
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.
Address for reprint requests: M. J. Gibala, Copenhagen Muscle
Research Centre, Rigshospitalet, Section 7652, Tagensvej 20, DK2200 Copenhagen N, Denmark.
Received 10 February 1998; accepted in final form 21 April 1998.
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