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Am J Physiol Regul Integr Comp Physiol 293: R1335-R1341, 2007. First published June 20, 2007; doi:10.1152/ajpregu.00115.2007
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ENVIRONMENTAL, EXERCISE AND RESPIRATORY PHYSIOLOGY

Exercise training increases branched-chain oxoacid dehydrogenase kinase content in human skeletal muscle

Krista R. Howarth, Kirsten A. Burgomaster, Stuart M. Phillips, and Martin J. Gibala

Exercise Metabolism Research Group, Department of Kinesiology, McMaster University, Hamilton, Ontario, Canada

Submitted 14 February 2007 ; accepted in final form 15 June 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The branched-chain oxoacid dehydrogenase complex (BCOAD) is rate determining for the oxidation of branched-chain amino acids (BCAAs) in skeletal muscle. Exercise training blunts the acute exercise-induced activation of BCOAD (BCOADa) in human skeletal muscle (McKenzie S, Phillips SM, Carter SL, Lowther S, Gibala MJ, Tarnopolsky MA. Am J Physiol Endocrinol Metab 278: E580–E587, 2000); however, the mechanism is unknown. We hypothesized that training would increase the muscle protein content of BCOAD kinase, the enzyme responsible for inactivation of BCOAD by phosphorylation. Twenty subjects [23 ± 1 yr; peak oxygen uptake (VO2peak) = 41 ± 2 ml·kg–1·min–1] performed 6 wk of either high-intensity interval or continuous moderate-intensity training on a cycle ergometer (n = 10/group). Before and after training, subjects performed 60 min of cycling at 65% of pretraining VO2peak, and needle biopsy samples (vastus lateralis) were obtained before and immediately after exercise. The effect of training was demonstrated by an increased VO2peak, increased citrate synthase maximal activity, and reduced muscle glycogenolysis during exercise, with no difference between groups (main effects, P < 0.05). BCOADa was lower after training (main effect, P < 0.05), and this was associated with a ~30% increase in BCOAD kinase protein content (main effect, P < 0.05). We conclude that the increased protein content of BCOAD kinase may be involved in the mechanism for reduced BCOADa after exercise training in human skeletal muscle. These data also highlight differences in models used to study the regulation of skeletal muscle BCAA metabolism, since exercise training was previously reported to increase BCOADa during exercise and decrease BCOAD kinase content in rats (Fujii H, Shimomura Y, Murakami T, Nakai N, Sato T, Suzuki M, Harris RA. Biochem Mol Biol Int 44: 1211–1216, 1998).

protein metabolism; branched-chain amino acids; enzyme regulation

THE BRANCHED-CHAIN 2-oxoacid dehydrogenase complex (BCOAD) is rate determining for the oxidation of branched-chain amino acids (BCAAs; leucine, isoleucine, valine) in skeletal muscle (12, 13, 19, 25, 28, 29). Following a reversible transamination reaction in which the amino group from a BCAA is transferred onto 2-oxoglutarate to form glutamate, BCOAD commits the resulting branched-chain oxoacid (BCOA) to oxidation. BCOAD activity is controlled by covalent modification, with phosphorylation of the E1{alpha}-subunit by a specific kinase causing inactivation and dephosphorylation by a specific phosphatase causing activation (12, 28, 29). Little is known about the phosphatase; however, a large body of evidence suggests that the kinase plays an important role in the regulation of BCAA metabolism, with an inverse relation suggested between BCOAD complex activity and BCOAD kinase activity (29).

Acute exercise increases the active form of BCOAD in both rat (9, 16, 17, 36) and human skeletal muscle (15, 20, 26, 32–34); however, the specific mechanism involved is unclear. Several animal studies have implicated an important role for BCOAD kinase in regulating conversion of the BCOAD complex from the less active to more active form during exercise (27, 36). Shimomura et al. (27) showed, using an electrically stimulated muscle contraction model, that BCOAD activation was associated with an increased concentration of {alpha}-ketoisocaproic acid (KIC), which is a potent inhibitor of the kinase (12, 28, 29). More recently, Xu et al. (36) reported that BCOAD activation during exercise was associated with a decrease in the amount of kinase bound to the enzyme complex, possibly due to an increased concentration of KIC, which promotes dissociation of the kinase from the BCOAD complex in vitro (21). Other putative modulators of BCOAD complex activity during exercise include a change in cellular energy state (ATP/ADP ratio) and the concentrations of various metabolites including glycogen, pyruvate, and acetyl-CoA (15, 17, 20, 22, 32, 33).

Chronic exercise training alters the acute exercise-induced activation of BCOAD in skeletal muscle, but there are limited and equivocal data regarding the nature of this adaptive response (8, 20). Fujii et al. (8) showed increased skeletal muscle activation of BCOAD (BCOADa) during wheel running in endurance-trained compared with sedentary rats and proposed that training increased the utilization of BCAA for fuel. These authors also reported that training decreased BCOAD kinase content in skeletal muscle and suggested this might be involved in the mechanism for greater activation of BCOADa during exercise (8). In contrast to data obtained from rodents (8), McKenzie et al. (20) showed that 38 days of combined continuous and interval training attenuated BCOADa in human skeletal muscle during matched-work exercise, and this was associated with a reduced rate of whole body leucine oxidation. These authors (20) speculated that the training-induced decrease in BCOADa was related to an increased muscle oxidative capacity and reduced cellular energy disturbance. However, given the inverse relation suggested between BCOAD complex activity and BCOAD kinase activity (28, 29), the training-induced decrease in BCOADa in human skeletal muscle (20) could also be related to a change in the muscle content of BCOAD kinase.

The main purpose of the present study was to determine whether exercise training altered the protein content of BCOAD kinase in human skeletal muscle. In addition, given that McKenzie et al. (20) previously used a combined training approach, we sought to separately examine the effect of high-intensity interval and continuous moderate-intensity training on BCOADa during matched-work exercise. Both training strategies have been reported to induce rapid metabolic adaptations in human skeletal muscle, despite large differences in total exercise volume and time commitment (10). We hypothesized that 1) BCOADa would be reduced after both interval and continuous training and 2) the reduced BCOADa would be associated with a training-induced increase in BCOAD kinase protein content.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Subjects

Twenty young healthy men and women volunteered for the study (Table 1). A preliminary screening process was employed to establish that subjects were 1) free of risk factors associated with cardiovascular, pulmonary, or metabolic disease; 2) deemed safe to begin a physical activity program; and 3) aside from activities of daily living, not engaged in a regular exercise training program (i.e., ≤2 sessions/wk and ≤30 min/session, for at least 1 yr before the study). The experimental procedures and potential risks were fully explained to the subjects before the study, and all subjects provided written, informed consent. The experimental protocol was approved by the McMaster University and Hamilton Health Sciences Research Ethics Board.


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  		Table 1. Subject characteristics and exercise trial data

 
Preexperimental Procedures

Subjects initially performed a progressive exercise test (increasing 1 W every 2 s) on an electronically braked cycle ergometer (Lode, Excalibur Sport V2.0) to determine their peak oxygen uptake (VO2peak) using an online gas collection system (Moxus Modular VO2 System; AEI Technologies, Pittsburgh, PA). The value used for VO2peak corresponded to the highest value achieved over a 30-s collection period. Subjects subsequently performed a familiarization ride to determine the workload that elicited ~65% of their VO2peak. All subjects also performed a 30-s all-out effort (Wingate test) on the same cycle ergometer against a resistance equivalent to 0.075 kg/kg body mass. After the familiarization procedures, subjects were assigned to either a sprint training group or an endurance training group in a matched fashion based on sex and VO2peak (Table 1).

Experimental Protocol

Each subject served as their own control and performed two experimental trials, before and after a 6-wk exercise training intervention (see below). On arrival at the laboratory, the lateral portion of one thigh was prepared for the extraction of needle biopsy samples from the vastus lateralis muscle (2). Two small incisions were made in the skin and overlying fascia after injection of a local anesthetic (2% lidocaine). A biopsy was obtained at rest, and then subjects commenced cycling for 60 min on an electronically braked cycle ergometer (Lode) at a workload designed to elicit ~65% of pretraining VO2peak. Expired gases were collected during the exercise for the determination of oxygen consumption (VO2), carbon dioxide production (VCO2), and respiratory exchange ratio using a metabolic cart (Moxus Modular VO2 System, AEI Technologies), and heart rate was determined using telemetry (Polar Electro, Woodbury, NY). A second muscle biopsy was obtained immediately following exercise. The second experimental trial was performed 96 h following the final exercise training session and was identical in all respects to the first experimental trial, including power output, which was set at the same absolute workload (i.e., 65% of pretraining VO2peak).

Training Protocol

The training protocols were initiated several days after the first experimental trial. Endurance training consisted of continuous cycling on an ergometer (Lode) 5 days/wk (Monday through Friday) for 6 wk, at a power output corresponding to ~65% VO2peak. Subjects performed 40 min of exercise per training session for the first 2 wk. Exercise time was increased to 50 min per session during weeks 3 and 4, and during the final 2 wk, subjects performed 60 min of exercise per session. VO2peak tests were conducted after 3 wk of training, and training loads were adjusted to maintain a training intensity equivalent to 65% VO2peak. Sprint training consisted of repeated 30-s "all-out" bouts of cycling (Wingate tests) on an ergometer (Lode) 3 days/wk (Monday, Wednesday, Friday) for 6 wk. The number of Wingate tests performed during each training session increased from four during wk 1–2 to five during wk 3–4 and, finally, to six during wk 5–6. For all training sessions, the recovery interval between Wingate tests was fixed at 4.5 min, during which time subjects cycled at a low cadence (<50 rpm) against a light resistance (30 W) to reduce venous pooling in the lower extremities and minimize feelings of light-headedness or nausea. The endurance training program was based on general guidelines recommended by leading public health agencies (1), whereas the sprint training program was modeled after recent studies conducted in our laboratory that have examined metabolic and performance adaptations to low-volume, high-intensity interval training (4, 5, 10). By design, the protocols differed substantially in terms of total exercise training volume and time commitment to evaluate adaptations in skeletal muscle protein metabolism to two diverse training impulses.

Physical Activity and Nutritional Controls

Subjects were instructed to continue their normal dietary and physical activity practices throughout the experiment but refrain from alcohol and exercise for 48 h before each trial. Exercise was performed 3 h following a standardized meal. Subjects recorded their dietary intake for 24 h before the pretrial so that their individual pattern of food intake could be replicated in the 24 h before the posttrial. Subsequent dietary analyses (Nutritionist Five; First Data Bank, San Bruno, CA) revealed no differences in total energy intake or macronutrient composition before the two trials and no differences between groups (Table 2).


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  		Table 2. Nutritional data

 
Muscle Analysis

On removal from the leg, a 15- to 40-mg piece of wet muscle was quickly sectioned from the biopsy sample, blotted with gauze to remove excess blood, weighed, and placed in a buffer for the determination of both actual and total BCOAD activity. The remainder of the biopsy sample was quickly frozen in liquid nitrogen and used for the subsequent determination of BCOAD kinase by Western blotting and glycogen content by fluorometry. In addition, a 10-mg piece of each resting muscle sample was used for the determination of citrate synthase maximal activity.

BCOAD activity. Fresh wet muscle was homogenized in ice-cold buffer (0.25 M sucrose, 10 mM Tris base, and 2 mM EDTA, pH 7.4) and used for the determination of BCOAD activity using the method described by Wagenmakers et al. (33, 34). This method allows for measurement of both the total activity and the active (i.e., dephosphorylated) form of the enzyme. Briefly, an aliquot of homogenate was preincubated for 5 min at 37°C with ADP (8.3 mM) and NaF (83.3 mM) for the measurement of the active fraction of BCOAD, and a second aliquot was preincubated with ADP only for total BCOAD activity. After preincubation, 100 µl of 0.5 mM 2-oxo-[1-14C]isocaproate with (total activity) or without (active fraction) NaF were added to the sealed reaction vials. The samples were then incubated for 10 min at 37°C, and the reaction was terminated by injection of 3 M perchloric acid. The 14CO2 produced during the reaction was collected with an ethanolamine-ethylene glycol (1:2, vol/vol) solution located in a separate compartment within the sealed reaction vials. The vials were then placed on ice for 90 min. The ethanolamine-ethylene glycol solution was then removed and added to 10 ml of scintillation cocktail. The specific activity of the 14CO2 ethanolamine-ethylene glycol was then determined in a liquid scintillation beta-counter, and the amount of CO2 produced during the 10-min incubation period was calculated. Activities were determined in duplicate or triplicate and were subsequently normalized to total protein within the homogenate, measured using a Bradford assay (3).

BCOAD kinase protein content. A 4- to 5-mg aliquot of freeze-dried muscle was added to 250 µl of homogenizing buffer (50 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 50 mM NaF, 5 mM Na pyrophosphatase, 1 mM DTT) with 2 µl of protease inhibitor cocktail (PIC) and homogenized on ice, using a Polytron PT 1200E homogenizer, until completely homogenized. The sample was centrifuged at 13,000 rpm for 5 min and the supernatant collected. The protein content of the supernatant was determined using a commercial assay (Pierce BCA Protein Assay), and all samples were subsequently diluted to 3 mg/ml protein using homogenizing buffer. A 200-µl aliquot of sample was mixed with 50 µl of loading buffer and heated at 100°C for 5 min. For each blot, a standard and a positive control (10 µg/µl protein from rat heart) was loaded along with 40 µl of each sample onto a 5% polyacrylamide stacking gel and separated using a 10% polyacrylamide separating gel of 1.5-mm thickness at 180 V with a running time of 45 min in Tris-glycine electrophoresis buffer. The gels were electroblotted onto nitrocellulose membranes in transfer buffer (37 mM Tris base, 140 mM glycine, 20% methanol) for 90 min at 90 V in a cold room. Membranes were incubated in Tris-buffered saline-Tween (TBST; 10 mM Tris base, 150 mM NaCl, 0.05% Tween 20) with 5% skim milk for 1 h and washed twice with TBST for 3 min each. Membranes were incubated overnight in a cold room with the mouse-derived antibody of BCOAD kinase (Kamiya Biomedical, Seattle, WA) at a 1:1,500 dilution in PBS with 1% BSA. Following incubation with the primary antibody, the membranes were rewashed four times with TBST for 5 min each and then incubated with horseradish peroxidase (HRP) anti-mouse antibody (Cell Signaling Technology, Danvers, MA) at a 1:10,000 dilution in PBS with 1% BSA for 45 min. Membranes were rewashed five times for 8 min each with TBST before being exposed to a chemiluminescent liquid (Immuno-Star HRP Substrate Kit, Bio-Rad) for 2 min. Membranes were exposed using a Bio-Rad Chemi-Doc System for 5 min, and the density of the bands was determined using the associated image analysis software.

Glycogen content. A ~10-mg piece of wet muscle was freeze-dried, powdered, and incubated in 2.0 N HCl and heated for 2 h at 100°C to hydrolyze the glycogen to glucosyl units. The solution was subsequently neutralized with an equal volume of 2.0 N NaOH and analyzed for glucose using an enzymatic assay adapted for fluorometry (23).

Citrate synthase maximal activity. A 10- to 15-mg piece of frozen wet muscle sample was homogenized (14), and the maximal activity of citrate synthase was determined on a spectrophotometer (Ultrospec 3000 pro UV/Vis) using a method described by Carter et al. (6).

Statistical Analyses

BCOAD activity was analyzed using a three-factor analysis of variance (ANOVA), with the factors "group" (sprint interval vs. endurance training), "training" (pre- vs. posttraining), and "time" (rest vs. 60 min). BCOAD kinase and citrate synthase maximal activity were analyzed using a two-factor ANOVA, with the factors group and training. Linear regression analysis was used to examine the relationship between BCOAD kinase content and BCOAD activity. The level of significance for all analyses was set at P < 0.05, and significant main effects and interactions were further analyzed using a Tukey's honestly significant difference post hoc test. All values are presented as means ± standard error (SE).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Exercise Trial Data and Evidence of a Training Effect

VO2peak (Table 1) and citrate synthase maximal activity (Fig. 1) increased after training, with no difference between groups. Absolute exercise workload was the same before and after training for each group and corresponded to 65% of pretraining VO2peak (127 ± 10 and 139 ± 13 W for sprint interval and endurance training, respectively). Oxygen uptake during exercise was not different after training in either group; however, exercise heart rate was reduced (Table 1). Muscle glycogen content was higher after training (main effect, P < 0.05; Fig. 2); however, net glycogenolysis during exercise was reduced (P < 0.05).


Figure 1
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  		Fig. 1. Maximal activity of citrate synthase measured in resting biopsy samples before (pre) and after (post) 6 wk of sprint interval (SIT) or endurance training (ET). Values are means ± SE; n = 10/group. ww, Wet weight. *Main effect for training (post > pre; P < 0.05).

 

Figure 2
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  		Fig. 2. Muscle glycogen concentration ([glycogen]) at rest and after 60 min of exercise, before (pre) and after (post) 6 wk of SIT or ET. Values are means ± SE; n = 9 for SIT, and n = 10 for ET. dw, Dry weight. *Main effect for training (post > pre; P < 0.05). {dagger}Main effect for time (60 min < rest; P < 0.05).

 
BCOAD Activity

BCOADa was higher during exercise compared with rest (main effect for time, P < 0.05); however, BCOADa was attenuated after training (main effect for training, P < 0.05), with no difference between groups (Fig. 3). Total BCOAD activity was not different at any time point (Table 3). Percent enzyme activation, or BCOADa expressed relative to total BCOAD, was higher during exercise compared with rest (main effect for time, P < 0.05) but attenuated after training (main effect for training, P < 0.05).


Figure 3
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  		Fig. 3. Active form of branched-chain oxoacid dehydrogenase (BCOADa) at rest and after 60 min of exercise, before (pre) and after (post) 6 wk of SIT or ET. Values are means ± SE; n = 9 for SIT, and n = 10 for ET. *Main effect for training (post < pre; P < 0.05). {dagger}Main effect for time (60 min > rest; P < 0.05).

 

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  		Table 3. Total BCOAD activity

 
BCOAD Kinase Protein Content

The muscle content of BCOAD kinase increased after training (main effect for training, P < 0.05), with no difference between groups (Fig. 4). BCOAD kinase content measured in each group before and after training was inversely correlated (P ≤ 0.05) with BCOAD activity, expressed as the average percent activation measured in the rest and exercise samples (Fig. 5).


Figure 4
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  		Fig. 4. Muscle content of BCOAD kinase measured in resting biopsy samples obtained before (pre) and after (post) 6 wk of either SIT or ET. Values are means ± SE; n = 10 for SIT, and n = 9 for ET. *Main effect for training (post > pre; P < 0.05).

 

Figure 5
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  		Fig. 5. Relationship between BCOAD kinase content and BCOAD activity, expressed as the average %activation measured in the rest and exercise samples in each group (sprint and endurance) before and after training.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major novel finding from the present study was that exercise training increased the content of BCOAD kinase protein in human skeletal muscle, and this was associated with a training-induced decrease in BCOAD activity. This is also the first study to show that brief, intense interval training attenuates BCOADa similar to continuous moderate-intensity training. Finally, given that exercise training was previously reported to increase BCOAD activity and decrease BCOAD kinase content in rodent skeletal muscle (8), the present data highlight differences between models used to study the regulation of skeletal muscle BCAA metabolism.

In contrast to carbohydrate and fat metabolism, relatively few data are available regarding the effect of exercise training on the regulation of amino acid metabolism in human skeletal muscle. In the only other study of its kind, McKenzie et al. (20) reported that 38 days of exercise training blunted the acute exercise-induced increase in BCOADa, and this was associated with a reduced rate of whole body leucine oxidation. The training protocol in that study (20) consisted of both continuous and interval training, with subjects cycling for 1 h/day at 60% of VO2peak, 5 days/wk, and also performing repeated 1-min efforts at 100% of VO2peak with 4 min of recovery, for a total of 30 min, 1 day/wk. In the present study, we separately examined the effect of these two training strategies, and subjects performed either continuous moderate-intensity training for 5 days/wk or high-intensity interval training for 3 days/wk. Despite large differences in total exercise and training time commitment between groups, BCOAD activation was reduced to a similar extent after training (Fig. 3). These data therefore extend the work of McKenzie et al. (20) and suggest that both traditional endurance training and brief, intense interval training induce metabolic adaptations in human skeletal muscle that attenuate the activation state of BCOAD. One discrepancy from the work of McKenzie et al. (20) is that, in the present study, training did not increase the total activity of BCOAD even though the maximal activity of citrate synthase was increased in both studies. The difference may be related to the overall volume and duration of exercise training performed, which were greater in the study by McKenzie et al. (20). Consistent with this interpretation, Fujii et al. (9) previously showed in rats that 5 wk of training increased citrate synthase maximal activity but not total BCOAD complex activity, whereas after 12 wk of training, significant increases in both enzymes were detected.

The precise factors responsible for the reduction in BCOAD complex activation following exercise training in human skeletal muscle remain to be firmly established. However, consistent with our primary hypothesis, exercise training increased the skeletal muscle protein content of BCOAD kinase (Fig. 4), and the activation of the BCOAD complex was inversely related with BCOAD kinase content (Fig. 5). We did not measure BCOAD kinase activity directly, as the assay is based on measurement of the BCOAD complex, and the latter activity is too low in skeletal muscle to measure the kinase activity (36). However, experimental interventions in other tissues such as rat liver have demonstrated that chronic changes in BCOAD kinase protein content parallel changes in BCOAD kinase activity (24), and the activity state of the kinase is inversely related to the BCOAD complex activity (36). BCOAD kinase has also been shown to exist in two forms, free and tightly bound to the BCOAD complex, with the latter being associated with inactivation of the enzyme complex (28, 29). Xu et al. (36) reported that the increase in BCOAD activity during acute exercise in rat skeletal muscle was associated with a reduced amount of kinase protein bound to the complex, possibly due to increased KIC, which promotes dissociation of the kinase from the BCOAD complex in vitro (21). Although speculative, a training-induced change in total BCOAD kinase protein content, as shown in the present study, could potentially alter BCOAD activation by changing the proportion of free and bound forms of the kinase during exercise. Nonetheless, because this is the first study to measure the content of BCOAD kinase in human skeletal muscle, further research is necessary to establish whether the training-induced increase in BCOAD kinase content is causally related to the decreased activation state of the BCOAD complex.

In addition to a change in BCOAD kinase protein content, several allosteric regulators have been proposed to influence the activation state of the BCOAD complex during exercise (15, 17, 20, 22, 32, 33). McKenzie et al. (20) suggested that the training-induced decrease in BCOADa was related to an increased muscle oxidative capacity and improved cellular energy charge (17). Consistent with the work of McKenzie et al. (20), both the interval and continuous training programs in the present study increased muscle oxidative capacity as reflected by the maximal activity of citrate synthase. However, the precise role of cellular energy charge in regulating BCOAD activity is equivocal (17, 30), due in part to the lack of information regarding changes in the mitochondrial concentrations of adenine nucleotides of the ATP/ADP ratio. Kasparek (17) reported a strong inverse correlation between whole muscle ATP content and BCOAD activation during exercise and recovery in rats, whereas Shimomura et al. (30) reported a large exercise-induced increase in BCOAD activity despite no change in the muscle concentrations of ATP or ADP. Consistent with the latter finding, human studies have reported no relationship between the extent of muscle BCOAD activation during exercise and whole muscle or estimated cytosolic free concentrations of adenine nucleotides (15, 26).

Other putative modulators of skeletal muscle BCOAD activity during exercise include the availability of glycogen, pyruvate, acetyl-CoA, and BCOAs (15, 17, 20, 22, 32, 33). However, there are limited and equivocal data regarding the importance of these various metabolites during acute exercise, and thus the significance of potential training-induced changes is very speculative. Several investigators have proposed that BCOAD activation during exercise is inversely related to muscle glycogen availability (32, 33), and in the present study, the reduced BCOAD activation after training was accompanied by an increased muscle glycogen content. However, in a human study that directly investigated this issue, Jackman et al. (15) manipulated BCOAD activity during exercise using a dietary intervention and showed no relation between the activity state of the enzyme and either initial glycogen content or the rate of glycogenolysis during exercise. In addition, Jackman et al. (15) showed that BCOAD activation during exercise was unrelated to changes in the muscle contents of pyruvate or acetyl-CoA. With respect to BCOAs, some investigators have reported that the contraction-induced increase in BCOADa is associated with an increased muscle content of KIC in rats (30). However, others have found no correlation between BCOAD activation and the muscle contents of BCOAs and have concluded that these metabolites are not important regulators during exercise (16). Fielding et al. (7) reported an increased content of KIC after intense exercise in human skeletal muscle; however, no study has examined the relationship between BCOAs and BCOAD activation during exercise in humans, and additional work on this topic is warranted.

Finally, the present data are in contrast to work by Fujii et al. (8), who reported that BCOAD activation by exercise was higher in trained vs. untrained rats, and this was associated with a decreased muscle content of BCOAD kinase. Whether the discrepancy between studies represents a true species difference remains to be determined. Variations in muscle fiber type and recruitment pattern may explain part of the difference, since Fujii et al. (8) studied the effect of run training on rat gastrocnemius muscle (red or white portion was not specified), whereas the present data are based on human vastus lateralis muscle after cycle exercise training. However, Kasparek and Snider (18) showed no difference between the gastrocnemius and quadriceps muscle groups in total BCOAD activity or the active fraction of BCOAD at rest or during exercise. These findings suggest that the divergent training response between the human and rat cannot readily be attributed to potential variations in the muscle fiber sample used for analyses. Alternatively, the discrepancy may reflect a basic species difference in the regulation of skeletal muscle amino acid metabolism (11). Graham and MacLean (11) reviewed various models used to study skeletal muscle amino acid metabolism and highlighted potential differences between rat and human muscle in the regulation of both ammonia and inosine monophosphate metabolism. It is also possible that species differences in the relative balance between BCAA transaminase (BCAAT) activity, which catalyzes the reversible transamination of BCAA, and BCOAD may be important. Suryawan et al. (31) reported that rat skeletal muscle BCAAT activity is 30-fold higher than that of BCOAD, and in the liver, BCOAD activity is 11-fold higher than that of BCAAT. Hence, skeletal muscle acts as a net exporter of BCOA to the liver. However, in human beings, BCAAT activity is higher than BCOAD in both liver and muscle, and BCOAD generally operates at or below its Km so that any increase in BCOA is likely to increase BCAA oxidation. Thus it seems that BCOAD activity would be of greater importance in regulating BCAA oxidation in human than in rat skeletal muscle.

In conclusion, we have shown for the first time that exercise training increased the content of BCOAD kinase in human skeletal muscle, and this was associated with reduced BCOADa during matched-work exercise. The present data also demonstrate that brief bouts of high-intensity interval training reduce BCOADa, similar to the effect seen after traditional high-volume endurance training. Finally, given that exercise training has been reported to increase skeletal muscle BCOAD activity and decrease BCOAD kinase content in rodents (8), the present data highlight differences in models used to study the regulation of skeletal muscle BCAA metabolism. Additional studies are warranted to determine whether the increased kinase content is causally related to the reduced BCOAD activity, as well as reasons for the apparent species difference in the response of BCOAD to exercise training.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This project was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC). K. R. Howarth was supported by an Ontario Graduate Scholarship, and K. A. Burgomaster held an NSERC Canada Graduate Scholarship. S. M. Phillips is a Canadian Institutes of Health Research New Investigator.


   ACKNOWLEDGMENTS
 
We thank Mark Rakobowchuk and Dr. Maureen MacDonald for assistance with study design and implementation. Alicia Jury, Natalie Moreau, Todd Prior, Sophie Tanguay, Terrence Ho, Lindsay Gurr, and Amy Solheim assisted with experimental trials and data analyses. We are especially indebted to Drs. Sean McGee and Mark Hargreaves from the University of Melbourne for expert guidance regarding the Western blotting analyses. Finally, we thank our subjects for their time, effort, and outstanding commitment throughout the study.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. J. Gibala, Exercise Metabolism Research Group, Dept. of Kinesiology, IWC Rm. 219, McMaster Univ., Hamilton, ON, Canada L8S 4K1 (e-mail: gibalam{at}mcmaster.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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