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1Exercise Metabolism Research Group, Department of Kinesiology, and 2Departments of Neurology and Rehabilitation, Department of Medicine, McMaster University, Hamilton, Ontario, Canada L8S 4K1
Submitted 4 December 2002 ; accepted in final form 7 May 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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2-oxoglutarate + alanine). Eight women [22
± 1 yr, peak oxygen uptake
(
O2 peak) = 40.3 ±
4.6 ml · kg-1 ·
min-1] performed seven 45-min bouts of cycle exercise at
70% O2 peak over 9 days
(1 bout/day; rest only on days 2 and 8). During the first
and last bouts, biopsies (vastus lateralis) were obtained at rest and after 5
and 45 min of exercise. Muscle glycogen concentration was 
50% higher at
rest after STT (493 ± 38 vs. 330 ± 20 mmol/kg dry wt; P
0.05), and net glycogenolysis and lactate accumulation were reduced after
5 min of exercise by 59 and 49%, respectively (P 0.05). The net
increase in four measured TCAI was 40% lower (P 0.05)
during exercise after training (1.68 ± 0.60 vs. 2.71 ± 0.44
mmol/kg dry wt), and the net decrease in glutamate concentration was
attenuated (P 0.05). We conclude that 1) the
contraction-induced increase in flux through AAT is reduced after 5 days of
aerobic training and 2) the muscle glycogenolytic response during
exercise after STT in women is similar to that in men. metabolic regulation; glycogen; lactate; tricarboxylic acid cycle
2-oxoglutarate + alanine) appears quantitatively
most important for the rapid increase in TCAI at the start of exercise
(9,
27). The increase in AAT flux
appears to be driven by an increase in pyruvate availability, which occurs
when the rate of glycolytic flux transiently exceeds flux through the pyruvate
dehydrogenase (PDH) reaction. This leads to an increased production of
2-oxoglutarate, which expands the TCAI pool, as the amino group from glutamate
is transferred onto pyruvate, forming alanine
(2,
9). The mechanistic explanation for the net increase in muscle TCAI is generally accepted; however, the physiological significance of this phenomenon remains controversial. In 1990, two groups of investigators independently proposed that an increase in TCAI concentration is necessary to optimize aerobic energy provision during exercise (27, 31). This theory remains prevalent in the literature (30), although we are aware of no direct evidence to support it. Indeed, recent studies by one of the present authors (11) and others (4) demonstrated that augmenting the rate of TCAI expansion during the initial phase of exercise did not affect aerobic energy provision. These findings are consistent with an alternative explanation for anaplerosis; namely, changes in TCAI concentration during exercise reflect alterations in carbon flux into and out of the TCA cycle but are not causally related to TCA cycle turnover (8, 10).
It could be argued that a more appropriate method to investigate the physiological significance of anaplerosis would be to attenuate the magnitude of TCAI expansion during exercise and examine the effect on oxidative energy metabolism. Although attempts have been made with pharmacological (12) and nutritional interventions (11), no study has successfully attenuated the expansion of the TCAI pool during exercise. One potential strategy to achieve this goal would be to reduce glycolytic flux during exercise and thus pyruvate availability for the AAT reaction. In this regard, several studies have shown that 3-7 days of aerobic exercise training dramatically reduced muscle glycogenolysis and lactate accumulation during submaximal exercise (5, 16). In the present investigation, we used "short-term" training as a model to investigate the physiological significance of reduced TCAI expansion in a group of young women. We hypothesized that 5 days of aerobic training would attenuate muscle TCAI expansion during exercise, because of a better matching between pyruvate production and oxidation; however, this would not compromise muscle oxidative metabolism. In addition, because all comparable short-term training investigations of similar duration examined muscle metabolic adaptations in men (5, 7, 13-16, 26), we sought to determine whether changes in muscle glycogen and lactate metabolism induced by 5 days of aerobic training were similar in women.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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Overview of experimental design. Subjects reported to the
laboratory at least 3 days before the experiment and performed an incremental
exercise test on a cycle ergometer (Excalibur Sport ver. 2.0, Lode, Groningen,
The Netherlands) to determine their peak oxygen uptake
(O2 peak) by using an
on-line gas collection system (Moxus modular oxygen uptake system, AEI
Technologies, Pittsburgh, PA). The first three stages of the
O2 peak test consisted of
2-min intervals at 50, 100, and 150 W, respectively, and thereafter workload
was increased by 25 W every minute until volitional exhaustion. The value used
for O2 peak corresponded
to the highest value achieved over a 30-s collection period. Subjects were
instructed to refrain from exercise or strenuous physical activity on the day
before and day of the experiment. They were also advised to consume their
habitual diet and to refrain from the consumption of alcohol throughout the
duration of the study. All experimental trials were conducted during the
morning, 2-3 h after the consumption of a light breakfast of the
subjects' choosing. Subjects were instructed to consume the same types and
quantities of food during the 24 h before each experimental trial, and all
subjects maintained pretrial food diaries. The diaries were subsequently
analyzed for nutritional content (Nutritionist 5, ver. 1.7, First Data-Bank,
San Bruno, CA) to confirm that total energy intake and proportion of energy
from carbohydrate, fat, and protein were similar before each experimental
trial. The women were tested during the early follicular phase of their
menstrual cycle, and six of the eight subjects were taking oral contraceptives
(OCs).
Experimental protocol. On arrival at the laboratory for the first
experimental trial, subjects rested in the supine position, and the area over
the lateral aspect of one thigh was prepared for the extraction of needle
biopsy samples from the vastus lateralis muscle. After injection of a local
anesthesic into the skin and underlying fascia (1% wt/vol lignocaine
hydrochloride with epinephrine; Antigen Pharmaceuticals, Roscrea, Ireland),
three small incisions were made and a resting biopsy sample was obtained. The
incision used for the first biopsy was closed with a suture, and the remaining
two incisions were covered with sterile gauze and surgical tape. Subjects then
mounted an ergometer and cycled at a power output corresponding to 70%
O2 peak (163 ± 24
W) for 45 min. Needle biopsy samples were obtained after 5 and 45 min of
exercise, and cardiorespiratory measurements were obtained during the 20- to
25-min period of each work bout. On completion of the exercise bout, the
remaining two incisions were closed with sutures, and the subjects were
instructed to refrain from physical activity for the rest of the experimental
day and the following day (day 2). They reported back to the
laboratory on the third day and performed the first of five consecutive
exercise bouts over 5 days, i.e., the short-term training regime. Each bout of
exercise on days 3-7 consisted of 45 min of cycling at 70%
O2 peak, and all training
sessions were supervised by one of the investigators. Subjects were instructed
to refrain from exercise on day 8, and then they reported back to the
laboratory during the morning of day 9 for the second experimental
exercise trial, which was performed at the same absolute work intensity as the
first trial. Biopsy samples and cardiorespiratory measurements were obtained
as described for the first trial, except that the opposite leg was used for
muscle sampling. Two days after the second experimental exercise trial, the
subjects reported back to the laboratory and performed a second
O2 peak test using
procedures identical to those employed during the pretraining
O2 peak test.
Muscle analyses. Each biopsy sample was immediately frozen by plunging the needle into liquid nitrogen, and the samples were removed from the needle while still frozen. All resting samples were subsequently divided into two pieces. One piece from each resting sample was homogenized and analyzed for the maximal activities of citrate synthase and AAT (19) with an Ultrospec 3000 pro UV/Vis spectrophotometer. The remainder of the resting samples, and all exercise samples, were subsequently freeze-dried, powdered to dissect out nonmuscle elements, and stored at -86°C. Aliquots of powdered muscle were extracted and assayed for glycogen, lactate, phosphocreatine, citrate, isocitrate, malate, fumarate, alanine, and glutamate with enzymatic methods (1, 18, 25) adapted for fluorometry (Hitachi model F-2500, Hitachi Instruments, Tokyo, Japan). To correct for differences in blood or connective tissue between samples, muscle metabolites were corrected to the highest total creatine value obtained in all biopsy samples from a given subject.
Statistical analyses. All muscle metabolite data were analyzed by
using a two-factor (time x condition) repeated-measures ANOVA. Mean
cardiorespiratory data and net changes in muscle metabolites from rest to 5
min of exercise, including the sum total of the four measured TCAI
(
TCAI), were analyzed by using paired t-tests. Statistical
significance for all analyses was accepted as P 0.05. Data are
expressed as means ± SE unless otherwise noted.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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O2 peak
determined after the short-term training protocol was not different compared
with pretraining (Table 1).
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Muscle glycogen, lactate, phosphocreatine, and citrate synthase.
Resting muscle glycogen concentration increased (P 0.05) by 50%
after training (Fig. 1) and was
higher at all times during exercise (main effect for condition, P
0.05). Net glycogenolysis during the first 5 min of exercise was 59%
lower (P 0.05) after training (29 ± 12 vs. 70 ± 16
mmol/kg dry wt), and total glycogen degradation over the 45-min exercise bout
was reduced by 50% (52 ± 23 vs. 105 ± 30 mmol/kg dry wt;
P 0.05). Muscle lactate accumulation during the first 5 min of
exercise was reduced by 49% (P 0.05) after training
(Fig. 2). Phosphocreatine
degradation during exercise tended to be lower after training; however, the
changes were not statistically significant
(Table 2). The maximal
activities of citrate synthase (posttraining: 22.6 ± 1.8 vs.
pretraining: 22.2 ± 2.0 mmol · min-1
· kg wet wt-1) and AAT (posttraining: 3.0
± 0.6 vs. pretraining: 3.1 ± 0.5 mmol ·
min-1 · kg wet wt-1) were
not different between trials.
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Muscle TCAI, glutamate, and alanine. The concentrations of
citrate, isocitrate, malate and fumarate increased during exercise compared
with rest in both trials (main effect for time, P 0.05;
Table 3). There was no
significant interaction between trials for any of the individual TCAI;
however, the net increase in TCAI during exercise was reduced by
40% (P 0.05) after training
(Fig. 3). The attenuation of
TCAI expansion was primarily attributable to changes in malate concentration,
which accounted for >70% of the changes in TCAI during exercise. Net
changes in muscle glutamate concentration
(Fig. 4) and alanine
concentration (Table 2) during
exercise were attenuated by 40 and 25% respectively, posttraining, but only
the changes in glutamate were significant (P 0.05).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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Effect of short-term training on muscle TCAI. We hypothesized that
the balance between pyruvate production and oxidation would be improved after
short-term training, and this would attenuate the exercise-induced expansion
of the muscle TCAI pool by decreasing pyruvate flux through the AAT reaction.
There was no significant difference between trials in the individual
concentrations of TCAI; however, the net increase in the sum total of
the TCAI was indeed lower after training
(Fig. 3). Consistent with our
hypothesis and as discussed further below, muscle glycogenolysis and lactate
accumulation were reduced by 50% during exercise, which suggests there
was a reduced rate of pyruvate formation, and the net increase in muscle
glutamate was attenuated by 40%, which is indicative of a reduced flux
through AAT. Although the maximal activity of AAT was unchanged after
training, the analytic method employed does not permit the determination of
"active" enzyme activity at the time of tissue harvesting. We also
cannot discount the possibility that an increased rate of pyruvate oxidation
through the PDH reaction may have contributed to the reduction in TCAI
expansion. A previous study, however, showed that the active fraction of PDH
was not different during exercise after short-term training
(26).
Two previous studies showed that administration of the pharmacological agent dichloroacetate reduced the concentrations of TCAI in resting human muscle (8, 12), presumably by increasing PDH activity and the oxidative disposal of pyruvate, which diverted substrate away from anaplerotic pathways. However, this is the first study that has successfully attenuated the expansion of the muscle TCAI pool during exercise. Despite the blunted increase in TCAI concentration, aerobic energy provision was not compromised, as evidenced by the similar rate of phosphocreatine degradation during exercise after training. This finding appears at odds with the hypothesis that links changes in TCAI during exercise with the capacity for oxidative metabolism (27, 31). On the basis of this theory, one would presume that a given concentration of TCAI is required to sustain a given rate of oxidative phosphorylation during exercise. Indeed, a major tenet of the theory is that a decrease in muscle TCAI concentration during prolonged exercise compromises aerobic energy provision and leads to fatigue. However, in the present study, there was a clear dissociation between these variables, because subjects maintained the same power output (and presumably rate of oxidative ATP provision) after training despite a 40% decrease in the TCAI pool. The present data, therefore, support the hypothesis that changes in muscle TCAI during exercise primarily reflect changes in substrate availability for the AAT reaction and are not directly related to TCA cycle turnover.
Our laboratory (10) and others (8) have previously observed that the TCA cycle appears to operate as two distinct "spans" during exercise, and, on contraction, intermediates in the "second" span of the cycle (particularly malate) increase disproportionately. Consistent with this phenomenon, the blunted TCAI concentration response during exercise in the present study was primarily due to an attenuated rise in malate concentration. A notable discrepancy in the present study was that the resting concentration of malate was approximately twofold higher than values our laboratory has previously reported in men (9-12). To our knowledge, no study has specifically investigated potential gender differences in muscle TCAI metabolism, and we suspect that the discrepancy might simply reflect individual variability. We did not perform muscle fiber-type analyses, but Hintz et al. (20) showed that the resting concentration of malate varied by sevenfold in various fiber groups from the rat hindlimb, with higher values measured in more oxidative fibers. Although speculative, is also possible that a heightened sense of anxiety or nervousness by our subjects before the biopsy procedures may have influenced the resting concentrations of TCAI. We did not measure blood catecholamine concentrations, but epinephrine infusion has previously been shown to increase the resting intramuscular concentration of malate in humans, presumably by stimulating glycogenolytic rate and flux through AAT (28). Irrespective of the absolute resting concentration of malate, which was similar between trials at rest, the net increase in TCAI concentration was attenuated posttraining, and this was largely attributable to a reduction in malate concentration. Moreover, a subsequent study from our laboratory, which has appeared in abstract form (22), confirmed that the exercise-induced expansion of the muscle TCAI pool was attenuated after a more extensive 7-wk aerobic training program, and this was primarily due to a blunted rise in malate concentration.
Muscle glycogen and lactate concentrations after short-term training in
women. Several investigations have examined the effect of 3-7 consecutive
days of aerobic training at an intensity equivalent to 60-70%
O2 peak for 2 h each day
on muscle metabolic adaptations in men
(5,
7,
13-16,
26). The present study
examined metabolic adaptations in women in response to five consecutive bouts
of training at a similar work intensity (70%
O2 peak) but shorter bout
duration (45 min/day). Consistent with all but one of the studies cited above
(16), we observed a
significant effect of training on muscle glycogen content, such that glycogen
concentration was significantly increased at rest and remained higher during
exercise compared with pretraining values. The overall magnitude of increase
in resting glycogen concentration in our study (50%) was higher than
previously reported for men (average increase: 20%; range: 13-35%)
(5,
7,
13-15,
26). Also consistent with
several previous studies that sampled muscle during the initial few minutes of
exercise after short-term training
(7,
13-16),
we observed a marked decrease in muscle glycogenolysis and lactate
accumulation after 5 min of exercise posttraining. The potential mechanisms
responsible for the blunted glycogenolytic and glycolytic responses after
short-term training have been previously discussed
(13-16)
and appear to involve reductions in the putative modulators of glycogen
phosphorylase and phosphofructokinase (i.e., free ADP, free AMP, and
Pi). The present study, therefore, confirms that healthy,
recreationally active young women respond in a manner similar to men with
respect to resting and exercise-induced changes in muscle glycogen and lactate
concentrations after several consecutive days of aerobic training.
It should be noted that our subjects commenced the 9-day experimental protocol during the early follicular phase of the menstrual cycle, and six of the eight women were taking OCs. However, data regarding the effect of both menstrual cycle phase (6, 21) and OC use (3, 29) on substrate metabolism during exercise are equivocal and inconsistent. For example, two recent, carefully controlled studies that employed stable-isotope tracer techniques to examine the effect of menstrual cycle phase on substrate utilization during moderate-intensity exercise produced conflicting results: Campbell et al. (6) reported slightly higher rates of carbohydrate oxidation during the follicular compared with the luteal phase, whereas Horton et al. (21) reported no effect of menstrual cycle phase on glucose kinetics or whole body fuel oxidation. Similarly, studies that have directly examined the effect of menstrual cycle phase on muscle glycogen utilization during exercise have yielded equivocal data (17, 24). Thus, although we cannot rule out the possibility that fluctuations in the sex steroid environment may have influenced substrate utilization in the present study, it seems unlikely that hormonal changes alone would have produced the dramatic reductions in muscle glycogenolysis and lactate accumulation that were observed posttraining. With respect to OC use, although there may have been subtle differences in the individual responses between the users and nonusers in the present study, each subject served as her own control. Thus our experimental design controlled for between-subject variability, and we nonetheless detected a significant treatment effect.
Conclusion. In summary, the present study showed that the contraction-induced increase in muscle TCAI concentration was lower after 5 days of aerobic training, and this was likely due in part to a lower net flux through AAT during the rest-work transition. However, the attenuated rate of TCAI expansion did not appear to compromise aerobic energy delivery, since there was no difference between trials in phosphocreatine degradation during exercise, and subjects were able to maintain the same power output posttraining despite a 40% decrease in the TCAI pool. Although this is the first study to successfully attenuate muscle TCAI expansion during exercise, our findings are consistent with several recent reports that showed a dissociation between TCAI pool size and indirect markers of mitochondrial respiration (4, 11). Collectively, these data argue against the hypothesis that links changes in TCAI during exercise with the capacity for oxidative metabolism (27, 31). Finally, in addition to changes in TCAI metabolism, we also observed that resting muscle glycogen concentration was increased after training and that net glycogenolysis and lactate accumulation during exercise were reduced. We conclude that young healthy women demonstrate rapid shifts in carbohydrate metabolism after short-term exercise training, similar to those previously reported for men (13-16).
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DISCLOSURES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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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.
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REFERENCES |
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) in the total concentration of 4 measured
tricarboxylic acid cycle intermediates (TCAI; citrate, isocitrate, fumarate,
and malate) from rest to 5 min of exercise before and after 5 days of aerobic
training. Values are means ± SE. *P 