1 Human Muscle Metabolism Research Group, Loughborough University, Loughborough LE11 3TU; 2 School of Biomedical Sciences, Queens Medical Centre, Nottingham NG7 2UH; 3 Sunderland Royal Hospital, Sunderland SR4 7TP; and 4 Sport and Exercise Research Centre, South Bank University, London SE1 0AA, United Kingdom
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ABSTRACT |
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The aims of the present
study were twofold: first to investigate whether TCA cycle intermediate
(TCAI) pool expansion at the onset of moderate-intensity exercise in
human skeletal muscle could be enhanced independently of pyruvate
availability by ingestion of glutamine or ornithine -ketoglutarate,
and second, if it was, whether this modification of TCAI pool expansion
had any effect on oxidative energy status during subsequent exercise.
Seven males cycled for 10 min at ~70% maximal O2 uptake
1 h after consuming either an artificially sweetened placebo (5 ml/kg body wt solution, CON), 0.125 g/kg body wt
L-(+)-ornithine
-ketoglutarate dissolved in 5 ml/kg body
wt solution (OKG), or 0.125 g/kg body wt L-glutamine dissolved in 5 ml/kg body wt solution (GLN). Vastus lateralis muscle
was biopsied 1 h postsupplement and after 10 min of exercise. The
sum of four measured TCAI (
TCAI; citrate, malate, fumarate, and
succinate, ~85% of total TCAI pool) was not different between conditions 1 h postsupplement. However, after 10 min of exercise,
TCAI (mmol/kg dry muscle) was greater in the GLN condition
(4.90 ± 0.61) than in the CON condition (3.74 ± 0.38, P < 0.05) and the OKG condition (3.85 ± 0.28).
After 10 min of exercise, muscle phosphocreatine (PCr) content was
significantly reduced (P < 0.05) in all conditions,
but there was no significant difference between conditions. We conclude
that the ingestion of glutamine increased TCAI pool size after 10 min
of exercise most probably because of the entry of glutamine carbon at
the level of
-ketoglutarate. However, this increased expansion in
the TCAI pool did not appear to increase oxidative energy production,
because there was no sparing of PCr during exercise.
tricarboxylic acid cycle intermediates; exercise; glutamate; phosphocreatine
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INTRODUCTION |
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THE TOTAL CONTENT of the tricarboxylic acid (TCA) cycle intermediates (TCAI) has been shown to expand during the initial few minutes of muscular contraction (15, 18, 32, 33). However, it is still not clear whether this expansion is of any functional significance to oxidative energy production. For example, recent studies have suggested that the expansion of TCAI may merely reflect the increase in pyruvate availability that results from the mismatch between the rate of pyruvate formation, via glycolysis, and the rate of oxidation of acetyl units in the TCA cycle (7, 16). Alternatively, the increase in TCAI pool size has been reported to be necessary for increased TCA cycle flux and, hence, oxidative energy production during intense muscular contraction (27, 33).
The expansion of the TCAI pool at the onset of skeletal muscle
contraction is due primarily to an increase in the rate of anaplerosis
(replenishment of TCAI) at the onset of exercise (15, 33,
36). Indeed, it has previously been suggested that TCAI pool
size is determined by the balance between the flux of carbon into and
out of the TCA cycle (27, 33, 36). During the initial 15 min of moderate-intensity exercise, when TCAI pool size expands by
~300%, muscle glutamate content decreases by ~60% and muscle alanine content increases by ~50% (4, 14). This
suggests that the alanine aminotransferase reaction (AAT:
glutamate + pyruvate
-ketoglutarate + alanine) is of
prime importance for providing anaplerotic carbon at the onset of
exercise (15, 33, 36), with carbon entering the TCA cycle
at the level of
-ketoglutarate.
More recently, the importance of acetyl units to the TCA cycle at the
onset of exercise has been demonstrated (7, 39, 40). TCAI
pool size and the supply of acetyl units to the TCA cycle have
previously been manipulated pharmacologically by infusion of
dichloroacetate (DCA), which causes activation of the pyruvate dehydrogenase complex (PDC). The delivery of acetyl units to the TCA
cycle was increased both at rest and at the onset of exercise after
activation of PDC by DCA (23). After activation of PDC by
DCA infusion in resting human skeletal muscle, TCAI pool size was
decreased in conjunction with the accumulation of acetyl-CoA and
acetylcarnitine (7, 17). However, after DCA infusion, TCAI
pool size after 1 min of exercise was not different from control values
(17). This suggests that once contraction was initiated,
glycolysis was accelerated to an extent that pyruvate availability was
not limiting anaplerosis via the AAT reaction. This observation also
raises the question as to where the limitation to anaplerosis resides
at the onset of exercise (14). One aim of the present
study, therefore, was to determine whether nutritional intervention
could be used to modify TCAI pool size. Specifically, was the extent of
TCAI pool expansion at the start of exercise limited by
-ketoglutarate availability and, hence, altered by the ingestion of
glutamine or ornithine
-ketoglutarate before exercise.
The nonessential amino acid glutamine is readily taken up into skeletal
muscle via the high-capacity sodium-dependent system Nm
(1), resulting in an increased intramuscular glutamine
content (42). The enzymes required to catalyze the
conversion of glutamine to -ketoglutarate, glutaminase (EC 3.5.1.2)
(38) and glutamate dehydrogenase (EC 1.4.1.2)
(37) or alanine aminotransferase (EC 2.6.1.2)
(28) or glutamine transaminase (EC 2.6.1.15) (38,
45) and
-amidase (EC 3.5.1.3) (38, 45), should exist in human skeletal muscle. It is feasible, therefore, that carbon
derived from glutamine could enter the TCA cycle at the level of
-ketoglutarate. Indeed, when glutamine was infused after exhaustive exercise, muscle glycogen storage was increased. There appeared to be an increase in the availability of carbon units for
incorporation into glycogen, presumably due to
-ketoglutarate feeding into the TCA cycle (42). This result was recently
replicated when glutamine was ingested after exhaustive exercise
(5).
A more direct approach to increasing TCAI pool size would be to provide
the substrate -ketoglutarate itself. Ornithine
-ketoglutarate (OKG), a salt formed of two molecules of ornithine and one molecule of
-ketoglutarate, is a precursor of glutamine, arginine, proline, and
polyamines (for review see Ref. 25). Supplementation with OKG has been shown to improve the nutritional status of hypercatabolic patients (8), improve nitrogen balance (12),
and restore the muscle glutamine pool (44). However, to
our knowledge, the effect of OKG ingestion on TCAI pool size in human
skeletal muscle has never been assessed.
It has recently been suggested that changes in TCAI are not causally linked to TCA cycle flux and oxidative energy production (7, 16); however, the precise functional significance of TCAI pool expansion is still under question. Therefore, if we were successful in augmenting TCAI pool expansion through nutritional intervention, a second aim was to determine the effect on energy metabolism. During the transition from rest to exercise, the increase in oxidative energy delivery is not sufficiently rapid to meet the energy demands of the exercising muscle. The magnitude of the decline in phosphocreatine (PCr) stores is indicative of the mismatch between oxidative metabolism and energy demand (24). It was hypothesized that if oxidative energy delivery were increased at the onset of exercise as a result of augmenting TCAI pool expansion, a concomitant reduction in muscle PCr degradation would be expected to occur.
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METHODS |
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Subjects.
Seven healthy male well-trained cyclists participated in three trials
separated by 2 wk. Their mean age, height, body mass, and maximal
O2 uptake (
O2 max) were
24 ± 1 yr, 180 ± 2 cm, 80 ± 2 kg, and 5.0 ± 0.2 l/min (62.5 ± 1.8 ml · min
1 · kg
1),
respectively. The experimental procedures and potential risks were
fully explained to the subjects before their participation in this
study, and all gave voluntary written consent. The experimental protocol was approved by the Ethics Committee of Loughborough University.
Preliminary tests.
Subjects reported to the laboratory ~1 wk before the experiment and
performed a submaximal test, to determine the relationship between
O2 uptake and work rate, and an incremental cycling test to
determine their O2 max on a
friction-braked cycle ergometer (Monark 824E, Varberg, Sweden). A
workload corresponding to 70%
O2 max
was then calculated for each subject.
Preexperimental procedures. Subjects were instructed to consume their habitual diet and refrain from exercise or strenuous physical activity for 48 h before each experiment. On the afternoon before the experiment, subjects performed a bout of glycogen-depleting exercise that had been used previously (5) and had been designed to deplete both type I and type II muscle fibers of glycogen as validated by Vøllestad et al. (43).
All food consumed after the glycogen-depleting exercise bout was prescribed for each subject and was identical before all trials for a given subject, providing 35 ± 2% carbohydrate, 56 ± 2% fat, and 11 ± 2% protein (~1,400 kcal; Compeat 5.0 Diet Analysis Software, Carlston Bengston Consultants). This ensured that only limited muscle glycogen resynthesis occurred before the 2nd day of the experiment and that the magnitude of resynthesis was the same across groups. This design was employed to ensure that muscle and hepatic glycogen stores, and hence pyruvate availability, were similar in all three conditions.Experimental protocol.
On arrival at the laboratory on the morning of the experiment, the
overnight-fasted subject rested in a supine position. A cannula was
inserted into an anticubital vein, and a resting blood sample was
obtained. Subjects then immediately consumed, in a double-blind
fashion, one of three solutions: 5 ml/kg body wt of an artificially
sweetened placebo (CON), 0.125 g/kg body wt L-(+)-ornithine
-ketoglutarate (Laboratoires Jacques Logeais, Paris, France)
dissolved in 5 ml/kg body wt of the artificially sweetened
placebo (OKG), or 0.125 g/kg body wt L-glutamine
(Sigma-Aldrich Chemicals, Dorset, UK) dissolved in 5 ml/kg body
wt of the artificially sweetened placebo (GLN). The solutions were
allocated by systematic rotation. After consumption of the assigned
solution, subjects rested for 60 min and then immediately cycled at
70%
O2 max for 10 min. This time
course was chosen because it has previously been shown that maximal
increase in TCAI pool size is achieved after ~10 min of submaximal
exercise in humans (18).
Blood analyses.
Venous blood samples were drawn and aliquoted for subsequent blood
analysis. An aliquot was used to immediately determine whole blood
lactate and glucose concentration by use of a YSI 2300 STATPLUS
analyzer (Yellow Springs Instruments, Yellow Springs, OH), because
there was a risk that subjects in the OKG condition might have
developed hypoglycemia (11). The remainder of the venous
blood sample was centrifuged at 4°C, and the supernatant was
collected and stored at 20°C and later analyzed for free amino
acids by HPLC (30).
Muscle analyses.
After removal from the leg, the muscle biopsy sample was immediately
frozen by plunging the needle into liquid nitrogen. The samples were
removed from the needle while still frozen and were subsequently
freeze-dried, dissected free from visible connective tissue and blood,
powdered, and stored at 80°C. Aliquots of the powdered muscle were
extracted with 0.5 M perchloric acid (containing 1 mM EDTA), and after
centrifugation, the supernatant was neutralized with 2.2 M
KHCO3. Extracts were assayed enzymatically for lactate, pyruvate, glutamate, glutamine, citrate, malate, fumarate, and succinate content (2) with a fluorometer (Hitachi F2000
fluorescence spectrophotometer, Hitachi Instruments). Intramuscular
metabolite contents were normalized for total creatine content (except lactate).
Statistics. The data were analyzed by two-way analysis of variance (ANOVA) for repeated measures (time × treatment). When the ANOVA resulted in a significant F ratio (P < 0.05), Fisher's post hoc test was used to locate differences between means. Values are presented as means ± SE.
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RESULTS |
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Cardiorespiratory, blood glucose, and blood lactate data. No differences were observed between conditions in any of the measured physiological variables during the glycogen-depleting exercise on the afternoon of the 1st day. During the main experimental trials, blood glucose, blood lactate, heart rate, respiratory exchange ratio, and expired ventilation showed main effects for time (P < 0.05). However, there were no significant differences between conditions (data not shown). The power output in W (CON: 241 ± 11, OKG: 243 ± 9, and GLN: 242 ± 10) and pulmonary O2 uptake in l/min (CON: 3.73 ± 0.16, OKG: 3.73 ± 0.15, and GLN: 3.72 ± 0.14) were not different between conditions.
Plasma and muscle amino acids.
The plasma concentration of both glutamine and glutamate was increased
1 h after GLN consumption (P < 0.05) compared
with both the CON and OKG conditions. Furthermore, the plasma
concentration of glutamine remained elevated at 10 min of exercise
(P < 0.05) compared with both the CON and OKG
conditions (Table 1). Plasma ornithine
concentration was higher 1 h after OKG consumption and remained
elevated (P < 0.05) throughout exercise compared with both the CON and OKG conditions. Plasma alanine concentration significantly increased (P < 0.05) in all conditions
during the 10 min of exercise relative to the basal value (Table 1). In addition, plasma alanine concentration was significantly higher in the
GLN and OKG conditions compared with the CON condition 1 h after
consumption of the supplements and at 10 min of exercise. Plasma
aspartate concentration was significantly higher in the GLN condition
1 h after ingestion of the supplement in the CON and OKG
conditions (P < 0.05) and at 10 min of exercise was
significantly higher in the GLN than in the OKG condition (P
< 0.05).
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TCAI.
The total content of the four measured TCAI (TCAI; citrate,
succinate, malate, and fumarate) was not different between conditions at rest (Fig. 1). The
TCAI at 10 min
of exercise was greater than at rest in all conditions
(P < 0.05) and was higher in the GLN condition
(4.90 ± 0.61 mmol/kg dry muscle) compared with the control
condition (3.74 ± 0.38 mmol/kg dry muscle, P < 0.05) and the OKG condition (3.85 ± 0.28 mmol/kg dry muscle) at
10 min of exercise (Fig. 1). However, there were no differences between conditions in the content of any of the individual TCAI (Table 2).
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Intramuscular metabolites. PCr content declined to a similar extent during the 10 min of exercise in all conditions (CON: 26 ± 6%, OKG 19 ± 7%, and GLN: 21 ± 6%; Table 2). Intramuscular lactate content was not different between conditions at rest. During exercise, the increase in intramuscular lactate content was similar in all conditions (Table 2). Intramuscular pyruvate content increased in all conditions during exercise (CON: 70 ± 39%, OKG: 134 ± 96%, and GLN: 171 ± 99%; Table 2). No significant differences in PCr content were observed between conditions at any time point.
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DISCUSSION |
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A main finding from the present investigation was that the
consumption of glutamine 1 h before exercise augmented the
increase in the TCAI in human skeletal muscle after 10 min of
moderate-intensity cycling exercise (399%) compared with control
(260%). This suggests that the availability of the breakdown products
of glutamine, namely glutamate and ultimately
-ketoglutarate, may
limit anaplerosis at the onset of exercise. Indeed, the increase in
TCAI pool size was concomitant with a ~60% decrease in intramuscular
glutamate content in all conditions. It is conceivable that this
decrease in intramuscular glutamate content increased flux through the glutaminase reaction, thus increasing the breakdown of glutamine to
form glutamate. Certainly, intramuscular glutamine content and plasma
glutamine concentration decreased in the GLN condition during the 10 min of exercise, presumably contributing to the larger expansion in
TCAI pool size.
It has been reported that ~50% of an enterally delivered dose of
glutamine is sequestered on first pass through the splanchnic bed
(22, 29). However, in the present study, the oral
provision of glutamine was able to increase plasma glutamine at the
measured peak concentration by 51%, demonstrating that a substantial
portion of the oral load escaped utilization by the gut mucosal cells and uptake by the kidneys and liver. The plasma glutamine will be
rapidly taken up into skeletal muscle through system Nm
(1), as demonstrated by the elevated muscle glutamine
content in the GLN condition relative to the CON and OKG conditions
1 h after the supplements were consumed. The glutamine can then be
deaminated to form glutamate, and then -ketoglutarate, through the
action of the enzymes glutaminase (EC 3.5.1.2) (38) and glutamate dehydrogenase (EC 1.4.1.2) (37) or alanine
aminotransferase (EC 2.6.1.2) (28); or glutamine
transaminase (EC 2.6.1.15) (38, 45) and
-amidase (EC
3.5.1.3) (38, 45), all of which exist in skeletal muscle.
It is therefore entirely feasible for
-ketoglutarate derived from
glutamine to enter the TCA cycle and increase the total content of TCAI
as observed in the present study.
In addition to the elevated intramuscular glutamine content after GLN
supplementation, plasma glutamate concentration was also significantly
higher in the GLN condition 1 h after consumption of the
supplement. It is likely that glutamine-glutamate cycling in the liver
or kidneys resulted in the higher plasma glutamate level. Plasma
glutamate concentration was not different between conditions at 10 min
of exercise. Presumably, the excess glutamate was taken up into
skeletal muscle (15, 33, 41) and converted to
-ketoglutarate either via the AAT reaction or the glutamate dehydrogenase reaction (glutamate + NAD+
-ketoglutarate + NH3 + NADH), thus
contributing to the greater increase in TCAI during the GLN condition.
In fact, the decline in intramuscular glutamine (~7 mmol/kg dry wt)
and glutamate (~11 mmol/kg dry wt) content during the first 10 min of
exercise was more than fourfold greater than the increase in the
measured TCAI (~4 mmol/kg dry wt) over the same time period in the
GLN trial. Similarly during the OKG and CON conditions, the decline in
muscle glutamate content was about threefold greater than the increase
in TCAI. There are two means by which this discrepancy might be
explained: first, drainage of the TCAI to take part in the many other
reactions in which they are involved, e.g., amino acid synthesis, and
second, glutamate utilization in reactions by which there is no net
production of TCAI. The aspartate aminotransferase (EC 2.6.1.1)
(34) is one such reaction in which glutamate donates its
amino group to oxaloacetate, resulting in the production of aspartate
and
-ketoglutarate (glutamate + oxaloacetate + NAD+
-ketoglutarate + aspartate + NADH).
The removal of oxaloacetate is balanced by the production of
-ketoglutarate; thus there is no net expansion of the TCAI pool.
Indeed, plasma aspartate concentration was elevated during the GLN
condition and may therefore account for a proportion of the
"missing" glutamate. Similarly, Graham et al. (20)
also found that ingestion of monosodium glutamate increased plasma
aspartate concentration, presumably via the aspartate aminotransferase reaction.
Gibala et al. (18) demonstrated that the sum of citrate,
succinate, malate, and fumarate accounts for 85% of the total TCAI
both at rest and during exercise in humans; thus we are confident that
our data represent an accurate quantitative and qualitative index of
the total TCAI pool.
-Ketoglutarate content was not measured in the
present study; however, previous studies have not observed an increase
in this TCAI during the initial minutes of moderate exercise (15,
18, 19). This phenomenon has been linked to the equilibrium
between
-ketoglutarate and glutamate via the glutamate dehydrogenase
reaction; thus
-ketoglutarate content may be influenced by the
decrease in glutamate content during the initial minutes of exercise
(15, 16, 33, 41). A disproportionate increase in the
contents of succinate, malate, and fumarate has been observed during
exercise in the present and previous studies (7, 16). An
increase in
-ketoglutarate content causes a rapid activation of the
-ketoglutarate dehydrogenase complex (6); therefore,
-ketoglutarate derived from exogenous glutamine is likely to be
rapidly converted to intermediates situated in the "second span" of
the TCA cycle.
Ingestion of OKG did not enhance TCAI expansion at the onset of
exercise, either directly via entry of -ketoglutarate into the TCA
cycle or indirectly via the elevation of the muscle glutamate or
glutamine pool. One possible explanation is that the exogenous
-ketoglutarate was not taken up into the skeletal muscle. However,
-ketoglutarate transport into neurons (35) and the
kidney (13) is via a Na+-dependent
high-affinity process, and
-ketoglutarate infusion has been shown to
increase the intramuscular
-ketoglutarate content in anesthetized
dogs (31). It is unlikely, therefore, that skeletal muscle
-ketoglutarate transport was the limiting factor. The more feasible
explanation is that, despite successfully increasing plasma ornithine
concentration (345% 1 h after ingestion of OKG), only a small
increase in plasma
-ketoglutarate occurred, as observed previously
(10, 11), and this increase was not sufficient to elevate
intramuscular
-ketoglutarate content. Certainly, a large proportion
of the OKG dose is likely to have been sequestered by the splanchnic
bed, as indicated by the observed increase in OKG metabolites (proline,
arginine, glutamate) in the splanchnic areas (46). In the
present study, the dose was limited to ~10 g, the dosage used in
clinical practice (26), because of reported gastric
problems associated with larger doses (21).
The -ketoglutarate and ornithine moieties of OKG interact to give a
different metabolic pattern from that observed when they are provided
individually as ornithine (as hydrochloride) or
-ketoglutarate (as a
calcium salt) (10). Ornithine and
-ketoglutarate share a common metabolic pathway, resulting in the diversion of ornithine and
-ketoglutarate metabolism to other pathways (i.e., glutamine synthesis) when the common pathway is saturated. Indeed, it has been
demonstrated that the administration of OKG to humans postsurgery effectively restores the muscle glutamine pool (44),
presumably due to OKG acting as a precursor to glutamine
(9). However, the majority of studies that have
demonstrated an anabolic effect of OKG has utilized hypercatabolic
states (burn injury, surgery, trauma), in which the intramuscular
glutamine pool is reduced. No increase was observed in muscle glutamine
content at rest in the present study after OKG supplementation compared
with the CON condition. This suggests that OKG may be effective only in the hypercatabolic state.
Previously, it has been suggested that anaplerosis is dependent on pyruvate availability (7). In the present study, therefore, subjects completed a glycogen-depletion exercise protocol on the day preceding each main trial and consumed a low-carbohydrate diet during the intervening 18-h period. This design was employed to ensure that muscle and hepatic glycogen stores, and hence pyruvate availability, in exercising skeletal muscle were similar for all three conditions. Certainly, the metabolic response during the glycogen-depleting exercise bout was identical between trials, confirming that the physiological status of each subject was similar before each trial. Although muscle glycogen content was not measured in the present study, the bout of glycogen-depleting exercise employed has previously been shown by our group to reduce muscle glycogen content to 13 mmol glycosyl U/kg wet wt (~55 mmol glycosyl U/kg dry weight) immediately after exercise (5). During the 18-h period between glycogen depletion and the main trial, subjects consumed on average ~123 g of carbohydrate (~1.5 g carbohydrate/kg body wt). It is likely, therefore, that both muscle and liver glycogen stores remained reduced at the start of the main trial. It is therefore interesting that the magnitude of TCAI pool expansion was similar to that observed in previous studies in which skeletal muscle glycogen content was not manipulated (18). Gibala et al. (14) introduced the concept of a critical minimum concentration of glycogen necessary to provide an adequate pyruvate flux to drive anaplerotic reactions. In the present study, the intramuscular pyruvate content was similar to that observed in normal glycogen studies, suggesting that the extent of glycogen depletion was not sufficient to reduce glycogenolysis at the start of exercise.
A second main finding was that, despite the exaggerated expansion of the TCAI pool after glutamine ingestion, the decline in PCr content and accumulation of muscle lactate were similar in the three experimental conditions. These results suggest that ATP production via oxidative phosphorylation was not enhanced by a further increase in TCAI pool size, because substrate-level phosphorylation was not different between conditions. It appears, therefore, that TCAI pool size does not limit oxidative energy production at the onset of exercise. Indeed, it has been demonstrated that a lag in acetyl group delivery to the TCA cycle is likely to limit oxidative energy delivery at the onset of exercise (39, 40).
Previous studies using DCA to increase the active fraction of PDC have been able to enhance oxidative energy delivery at the onset of exercise without a further expansion of the TCAI pool (17, 39, 40). This apparent dissociation between TCAI pool size and oxidative energy delivery suggests that TCA cycle flux is not limited by TCAI pool size. The activation of PDC at rest increased the supply of pyruvate-derived acetyl units, resulting in the stockpiling of acetylcarnitine and a decrease in the TCAI pool (7, 17, 39, 40). At the onset of exercise, a normal expansion of the TCAI pool occurred, but acetyl unit availability was enhanced. It was suggested that the decreased utilization of PCr and reduced muscle lactate accumulation that occurred during this period were facilitated by the increased availability of acetyl units. In the present study, also, a dissociation between TCAI pool expansion and oxidative energy delivery has been demonstrated.
In conclusion, we have demonstrated that the provision of glutamine 1 h before exercise is able to further increase TCAI pool expansion after 10 min of moderate-intensity exercise. However, despite this further increase in TCAI pool expansion, no reduction in PCr utilization and muscle lactate accumulation was observed during this initial period of exercise. This suggests that, at the onset of exercise, energy production is not limited by TCAI pool size but by some other factor, possibly muscle oxygen availability or delivery of acetyl groups to the TCA cycle.
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ACKNOWLEDGEMENTS |
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We thank the subjects for their time and effort.
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FOOTNOTES |
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This work was supported by The Nuffield Foundation, University of Nottingham, Loughborough University, and Laboratoires Jacques Logeais, Paris.
Address for reprint requests and other correspondence: J. L. Bowtell, Sport & Exercise Research Centre, School of Applied Science, South Bank Univ., London SE1 0AA, United Kingdom (E-mail:bowteljl{at}sbu.ac.uk).
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 11 July 2000; accepted in final form 4 December 2000.
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