• View in gallery

    Mean ± SE expression of MCT1 (A) and MCT4 (B) mRNA in biopsy specimens of a gluteus medius muscle immediately prior to (baseline) and at 0 (immediately after), 3, 6, and 24 hours after an IET for 12 Thoroughbreds (age, 3 to 4 years) before (untrained; white bars) and after (trained; black bars) an 18-week period of HIT. The IET was performed on a treadmill set at a 6% incline. For the IET, the treadmill was initially set at a speed of 1.8 m/s for 2 minutes (walking), 3.6 m/s for 5 minutes (trotting), 6 m/s for 1 minute, and 8 m/s for 1 minute. For the IET before HIT, the treadmill speed was then increased by 2 m/s every minute to 10 m/s, whereas for the IET after HIT, the treadmill speed was then increased by 2 m/s every minute to 12 m/s. For both IETs, the treadmill speed was subsequently increased by 1 m/s every minute until the horse could not maintain its position on the treadmill because of exhaustion. The HIT was also performed on a treadmill set at a 6% incline and consisted of a warmup period (1.7 m/s for 1 minute and 3.5 m/s for 3 minutes), a 3-minute period of running, and a cooldown period (1.7 m/s for 3 minutes). During the initial 10 weeks, the running speed for each horse was that calculated to correspond to 90% of its o2max, whereas during the subsequent 8 weeks, the running speed for each horse was that calculated to correspond to 110% of its o2max. The HIT was performed 5 d/wk. Expression of MCT mRNA was determined via a real-time reverse transcriptase PCR assay. *Within training status (untrained or trained), value differs significantly (P < 0.05) from that at baseline. AU = Arbitrary units. GAPDH = Glyceraldehyde-3-phosphate dehydrogenase.

  • View in gallery

    Mean ± SE MCT1 (A) and MCT4 (B) protein content in biopsy specimens of a gluteus medius muscle immediately prior to (baseline) and at 0 (immediately after), 3, 6, and 24 hours after an IET for 12 Thoroughbreds (age, 3 to 4 years) before (untrained; white bars) and after (trained; black bars) an 18-week period of HIT. The MCT protein content was determined via a western blotting technique. At each time, the value for untrained horses differs significantly (P < 0.05) from that for trained horses. *Within training status (untrained or trained), value differs significantly (P < 0.05) from that at baseline. See Figure 1 for remainder of key.

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Effect of acute exercise on monocarboxylate transporters 1 and 4 in untrained and trained Thoroughbreds

Yu Kitaoka PhD1, Yukari Endo MSc2, Kazutaka Mukai DVM, PhD3, Hiroko Aida DVM, PhD4, Atsushi Hiraga DVM, PhD5, Tohru Takemasa PhD6, and Hideo Hatta PhD7
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  • 1 Department of Sports Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan.
  • | 2 Department of Sports Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan.
  • | 3 Equine Research Institute, Japan Racing Association, 321-4 Tokami-cho, Utsunomiya, Tochigi 320-0856, Japan.
  • | 4 Equine Research Institute, Japan Racing Association, 321-4 Tokami-cho, Utsunomiya, Tochigi 320-0856, Japan.
  • | 5 Equine Research Institute, Japan Racing Association, 321-4 Tokami-cho, Utsunomiya, Tochigi 320-0856, Japan.
  • | 6 Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tennodai 1-1-1 Tsukuba, Ibaraki 305-8574, Japan.
  • | 7 Department of Sports Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan.

Abstract

Objective—To evaluate the effects of a single incremental exercise test (IET) on mRNA expression and protein content of monocarboxylate transporter (MCT) 1 and MCT4 in the gluteus medius muscle of Thoroughbreds.

Animals—12 Thoroughbreds (6 males and 6 females; age, 3 to 4 years).

Procedures—Horses underwent an IET before and after 18 weeks of high-intensity exercise training (HIT). Horses were exercised at 90% of maximal oxygen consumption for 3 minutes during the initial 10 weeks of HIT and 110% of maximal oxygen consumption for 3 minutes during the last 8 weeks of HIT. Gluteus medius muscle biopsy specimens were obtained from horses before (baseline), immediately after, and at 3, 6, and 24 hours after the IET.

Results—Expression of MCT1 and MCT4 mRNA was upregulated at 3 and 6 hours after the IET in muscle specimens obtained from horses prior to HIT (untrained horses) and at 6 hours after the IET in muscle specimens obtained from horses after HIT (trained horses). For both untrained and trained horses, MCT1 and MCT4 protein contents were increased at 6 hours after the IET and did not differ at 24 hours after the IET, compared with those at baseline.

Conclusions and Clinical Relevance—Results indicated that a single IET resulted in transient increases in MCT1 and MCT4 mRNA expression and protein content in untrained and trained horses. These results may be important for the elucidation of exercise-induced alterations in lactate metabolism.

Abstract

Objective—To evaluate the effects of a single incremental exercise test (IET) on mRNA expression and protein content of monocarboxylate transporter (MCT) 1 and MCT4 in the gluteus medius muscle of Thoroughbreds.

Animals—12 Thoroughbreds (6 males and 6 females; age, 3 to 4 years).

Procedures—Horses underwent an IET before and after 18 weeks of high-intensity exercise training (HIT). Horses were exercised at 90% of maximal oxygen consumption for 3 minutes during the initial 10 weeks of HIT and 110% of maximal oxygen consumption for 3 minutes during the last 8 weeks of HIT. Gluteus medius muscle biopsy specimens were obtained from horses before (baseline), immediately after, and at 3, 6, and 24 hours after the IET.

Results—Expression of MCT1 and MCT4 mRNA was upregulated at 3 and 6 hours after the IET in muscle specimens obtained from horses prior to HIT (untrained horses) and at 6 hours after the IET in muscle specimens obtained from horses after HIT (trained horses). For both untrained and trained horses, MCT1 and MCT4 protein contents were increased at 6 hours after the IET and did not differ at 24 hours after the IET, compared with those at baseline.

Conclusions and Clinical Relevance—Results indicated that a single IET resulted in transient increases in MCT1 and MCT4 mRNA expression and protein content in untrained and trained horses. These results may be important for the elucidation of exercise-induced alterations in lactate metabolism.

During exercise, lactate is produced primarily in glycolytic muscle fibers. It is subsequently oxidized in the heart and oxidative muscle fibers or absorbed by the liver for gluconeogenesis.1 During the postexercise recovery period, lactate is oxidized by oxidative as well as glycolytic muscle fibers.2 Currently, lactate production by muscle is considered to be essential for sustained, repeated, intense muscle contraction.3 The transmembrane movement of lactate is mediated by a family of proteins called MCTs, of which MCT1 and MCT4 are the 2 primary MCTs found in the skeletal muscle of mammals.4,5 On the basis of values for Michaelis constant, MCT1 facilitates the uptake of lactate into cells with low lactate concentration, whereas MCT4 facilitates the extrusion of lactate out of cells with high lactate concentration.6,7 Results of studies8–14 involving rodents and humans indicate that muscle activity strongly influences the expression of MCT. For example, exercise training results in increased protein contents of MCT1 and MCT4 in muscle,8,9 whereas denervation decreases those protein contents.10 Although many studies have investigated the effects of exercise training on MCT protein contents, only a few reports describe the changes in MCT protein contents after a single exercise session. In 1 study,11 MCT1 and MCT4 were upregulated in rodents immediately after 2 hours of running. Results of some studies12,13 that involved humans indicate that acute exercise results in increased protein contents of MCTs, whereas those of another study14 indicate that acute exercise results in decreased MCT1 and MCT4 protein contents. Those inconsistent results may be the result of limited sampling of muscle tissue and study subjects that were at various stages of training. Thus, the effect of a single exercise session on the changes in MCT protein contents in skeletal muscle remains unclear. Additionally, the extent of increased mRNA expression and protein contents of MCT after exercise in well-trained athletes is unknown.

Thoroughbred horses are recognized as elite athletes. Their high exercise capacity is achieved by an extreme capability for energy production via aerobic and anaerobic metabolism. Results of 1 study15 suggest that plasma lactate concentration in Thoroughbreds increases to > 30 mmol/L during exercise; thus, Thoroughbreds are suitable subjects for the study of lactate metabolism. Investigators of other studies16,17 have documented the presence of MCT1 and MCT4 in equine skeletal muscle, and work by our laboratory group indicates that HIT increases the protein contents of MCT1 and MCT4 in muscle specimens.18

The purpose of the study reported here was to evaluate changes in MCT expression following a single exercise session in Thoroughbreds and determine whether those changes were dependent on training status. We hypothesized that a single exercise session would result in the rapid upregulation of both MCT1 and MCT4 and that the response would be greater in untrained horses.

Materials and Methods

Animals—Twelve Thoroughbreds (6 males and 6 females; age, 3 to 4 years) were evaluated in the study. The pedigrees of all horses were known, and the horses were raised for research purposes in accordance with the standard procedures of the Japan Racing Association Equine Research Institute. The horses were acclimated to handling and riding in accordance with typical acclimation procedures in Japan.19 After acclimation, the horses did not undergo any training before the study except for voluntary exercise while they were housed in a 2-hectare pasture. Horses were fed concentrate (oats and a pelleted ration) and hay twice daily, and water was available ad libitum in accordance with National Research Council feeding standards for horses. All study protocols were reviewed and approved by the Animal Welfare and Ethics Committee of the Japan Racing Association Equine Research Institute.

IET—Following a carotid loop surgery and familiarization with the treadmill, each horse was subjected to an IET on a treadmilla that was set at a 6% incline before and after 18 weeks of HIT. For each horse, an 18-gauge, 6.4-cm polytetrafluoroethylene catheterb was placed in a carotid artery immediately prior to the horse being led onto the treadmill for each IET. For the IET, the treadmill was initially set at a speed of 1.8 m/s for 2 minutes (walking), 3.6 m/s for 5 minutes (trotting), 6 m/s for 1 minute, and 8 m/s for 1 minute. For the IET before HIT, the treadmill speed was then increased by 2 m/s every minute to 10 m/s, whereas for the IET after HIT, the treadmill speed was then increased by 2 m/s every minute to 12 m/s. For both IETs, the treadmill speed was subsequently increased by 1 m/s every minute until the horse could not maintain its position on the treadmill because of exhaustion.

Blood sample collection—For each horse, a blood sample (10 mL) was obtained from the catheter in a carotid artery via a syringe that had been rinsed with heparin immediately before (baseline), during (when treadmill speed was 6 m/s, 8 m/s, and 10 m/s), immediately after (exhaustion), and at 1, 3, 5, and 10 minutes after the IET. Blood samples were stored on ice until all samples had been collected for that particular horse and IET. The blood samples were centrifuged at 1,800 × g for 10 minutes at 4°C, and plasma lactate concentration was measured in each sample via an automated blood lactate analyzer.c The o2 max of each horse was measured via an open-flow system as described.18

HIT—Horses underwent 18 weeks of HIT. The training program consisted of a warmup period (1.7 m/s for 1 minute and 3.5 m/s for 3 minutes), a 3-minute period of running, and a cooldown period (1.7 m/s for 3 minutes). During the initial 10 weeks, the running speed for each horse was that calculated to correspond to 90% of its o2 max, whereas during the subsequent 8 weeks, the running speed for each horse was that calculated to correspond to 110% of its o2max. Training was performed 5 d/wk on a treadmill set at a 6% incline, and horses were housed in a 2-hectare pasture on the days that they did not train.

Muscle biopsy—For each biopsy performed, the area surrounding the proposed biopsy site was anesthetized via local infusion of a 2% lidocaine solution.d A 2-mm-diameter biopsy needlee was inserted into a gluteus medius muscle to a depth of 5 cm from the skin surface, and a muscle biopsy specimen was obtained with the horse at rest before and immediately after the IET and at 3, 6, and 24 hours after the IET. Biopsy specimens were frozen in melting isopentane cooled by liquid nitrogen and stored at −80°C until analysis.

Quantification of MCT mRNA—A commercial real-time PCR systemf,g was used as described20,21 to detect MCT mRNA in muscle biopsy specimens. Briefly, from each specimen, total RNA was isolatedh and complimentary DNA was synthesized via reverse transcriptasei by use of primersj (Appendix) in accordance with the manufacturer's instructions. The samples were denatured at 95°C for 15 seconds and then annealed and elongated at 60°C for 1 minute. A dissociation curve analysis was used to determine the relative amount of MCT mRNA in each sample via the standard curve method. Signals were normalized to glyceraldehyde-3-phosphate dehydrogenase. All specimens were assayed in duplicate for each gene (MCT1 and MCT4).

Determination of MCT protein contents—For each muscle biopsy specimen, proteins were separated by size via sodium dodecyl sulfate polyacrylamide gel electrophoresis and a western blotting technique was used to detect MCT1 and MCT4. For each batch analyzed by the western blotting technique, equal quantities of protein were loaded into each lane, and a common standard was included to normalize data among the various lanes (ie, samples) as described.18,22 Antibodies used in the western blot analysis were developed in rabbits and directed against the oligopeptide that corresponded to the C-terminus regions of human MCT1 and MCT4.j The specificity of these antibodies against MCT1 and MCT4 of horses was verified via a peptide-blocking method as described.18 The western blots were developed via enhanced chemiluminescence,k and the MCT protein contents were quantified with a chemiluminescent imaging system.l Densitometric analyses of the recorded images were performed with commercially available software.m

Statistical analysis—Results were expressed as mean ± SEM. Data were analyzed via a 2-way ANOVA for repeated measures and the Bonferroni post hoc test. For all analyses, values of P < 0.05 were considered significant.

Results

Animals—The mean body weight of the horses did not vary significantly during the study (Table 1). For the IET performed after 18 weeks of HIT (ie, for trained horses), the o2max and peak running speed were significantly increased, compared with those for the IET performed before HIT (ie, for untrained horses). The mean distance run by trained horses during the IET did not differ significantly, compared with the mean distance run by untrained horses during the IET.

Table 1—

Mean ± SE body weight, o2max, and peak running speed and running distance of 12 Thoroughbreds (age, 3 to 4 years) during an IET performed before (untrained) and after (trained) an 18-week period of HIT.

GroupBody weight (kg)o2 max (mL/kg/min)Peak speed (m/s)Distance (m)
Untrained496.5 ± 12.3142.9 ± 5.911.0 ± 0.44,374 ± 490
Trained503.5 ± 12.9164.2 ± 3.5*12.3 ± 0.2*4,901 ± 302

The IET was performed on a treadmill set at a 6% incline. For the IET, the treadmill was initially set at a speed of 1.8 m/s for 2 minutes (walking), 3.6 m/s for 5 minutes (trotting), 6 m/s for 1 minute, and 8 m/s for 1 minute. For the IET before HIT, the treadmill speed was then increased by 2 m/s every minute to 10 m/s, whereas for the IET after HIT, the treadmill speed was then increased by 2 m/s every minute to 12 m/s. For both IETs, the treadmill speed was subsequently increased by 1 m/s every minute until the horse could not maintain its position on the treadmill because of exhaustion. The HIT was also performed on a treadmill set at a 6% incline and consisted of a warmup period (1.7 m/s for 1 minute and 3.5 m/s for 3 minutes), a 3-minute period of running, and a cooldown period (1.7 m/s for 3 minutes). During the initial 10 weeks, the running speed for each horse was that calculated to correspond to 90% of its o2max, whereas during the subsequent 8 weeks, the running speed for each horse was that calculated to correspond to 110% of its o2max. The HIT was performed 5 d/wk.

Value differs significantly (P < 0.01) from that for untrained horses.

Plasma lactate concentration—The mean plasma lactate concentrations at running speeds of 8 and 10 m/s during the IET administered to trained horses were significantly decreased, compared with those at running speeds of 8 and 10 m/s during the IET administered to untrained horses (Table 2). Mean plasma lactate concentration at exhaustion and during the 10 minutes after the IET did not differ between trained and untrained horses. The decrease in the plasma lactate concentration between 1 minute (ie, peak plasma lactate concentration) and 10 minutes (6.2 ± 0.9 mmol/L) after the IET was significantly higher for trained horses, compared with that (4.1 ± 1.0 mmol/L) for untrained horses.

Table 2—

Mean ± SE plasma lactate concentration immediately before (baseline), during (when treadmill speed was 6 m/s, 8 m/s, and 10 m/s), immediately after (exhaustion), and at 1, 3, 5, and 10 minutes after an IET for 12 Thoroughbreds (age, 3 to 4 years) before (untrained) and after (trained) an 18-week period of HIT.

 Plasma lactate concentration (mmol/L)
Time relative to IETUntrained horsesTrained horses
Immediately before (baseline)0.7 ± 0.10.8 ± 0.1
During (treadmill speed, 6 m/s)2.3 ± 0.031.7 ± 0.2
During (treadmill speed, 8 m/s)6.0 ± 0.83.0 ± 0.3*
During (treadmill speed, 10 m/s)13.0 ± 1.27.7 ± 1.4*
Immediately after (exhaustion)18.1 ± 2.119.1 ± 2.6
1 min after18.9 ± 2.421.5 ± 2.9
3 min after18.3 ± 2.820.3 ± 3.1
5 min after17.8 ± 2.919.5 ± 3.4
10 min after14.9 ± 3.615.3 ± 3.8

See Table 1 for remainder of key.

MCT mRNA quantification—In untrained horses, the amount of MCT1 mRNA in muscle biopsy specimens was significantly greater at 3 (134%) and 6 (268%) hours after the IET, compared with that immediately prior to the IET (baseline; Figure 1). In trained horses, MCT1 mRNA was significantly upregulated at 6 hours after the IET (106%), compared with that at baseline. Similarly, compared with MCT4 mRNA expression at baseline, MCT4 mRNA was significantly upregulated at 3 (89%) and 6 (66%) hours after IET in untrained horses and at 6 (138%) hours after the IET in trained horses. The amount of MCT1 mRNA and MCT4 mRNA did not differ significantly between trained and untrained horses at any time during the observation period.

Figure 1—
Figure 1—

Mean ± SE expression of MCT1 (A) and MCT4 (B) mRNA in biopsy specimens of a gluteus medius muscle immediately prior to (baseline) and at 0 (immediately after), 3, 6, and 24 hours after an IET for 12 Thoroughbreds (age, 3 to 4 years) before (untrained; white bars) and after (trained; black bars) an 18-week period of HIT. The IET was performed on a treadmill set at a 6% incline. For the IET, the treadmill was initially set at a speed of 1.8 m/s for 2 minutes (walking), 3.6 m/s for 5 minutes (trotting), 6 m/s for 1 minute, and 8 m/s for 1 minute. For the IET before HIT, the treadmill speed was then increased by 2 m/s every minute to 10 m/s, whereas for the IET after HIT, the treadmill speed was then increased by 2 m/s every minute to 12 m/s. For both IETs, the treadmill speed was subsequently increased by 1 m/s every minute until the horse could not maintain its position on the treadmill because of exhaustion. The HIT was also performed on a treadmill set at a 6% incline and consisted of a warmup period (1.7 m/s for 1 minute and 3.5 m/s for 3 minutes), a 3-minute period of running, and a cooldown period (1.7 m/s for 3 minutes). During the initial 10 weeks, the running speed for each horse was that calculated to correspond to 90% of its o2max, whereas during the subsequent 8 weeks, the running speed for each horse was that calculated to correspond to 110% of its o2max. The HIT was performed 5 d/wk. Expression of MCT mRNA was determined via a real-time reverse transcriptase PCR assay. *Within training status (untrained or trained), value differs significantly (P < 0.05) from that at baseline. AU = Arbitrary units. GAPDH = Glyceraldehyde-3-phosphate dehydrogenase.

Citation: American Journal of Veterinary Research 74, 4; 10.2460/ajvr.74.4.642

MCT protein contents—At 6 hours after IET, MCT1 protein content was significantly greater, compared with that at baseline for both untrained (42%) and trained horses (42%; Figure 2). Similarly, MCT4 protein content was significantly greater at 6 hours after IET, compared with that at baseline for both untrained (35%) and trained horses (30%). At 24 hours after the IET, MCT1 and MCT4 protein contents did not differ significantly from those at baseline for trained or untrained horses. Protein contents of MCT1 and MCT4 were significantly greater for trained horses, compared with those for untrained horses at all points during the observation period.

Figure 2—
Figure 2—

Mean ± SE MCT1 (A) and MCT4 (B) protein content in biopsy specimens of a gluteus medius muscle immediately prior to (baseline) and at 0 (immediately after), 3, 6, and 24 hours after an IET for 12 Thoroughbreds (age, 3 to 4 years) before (untrained; white bars) and after (trained; black bars) an 18-week period of HIT. The MCT protein content was determined via a western blotting technique. At each time, the value for untrained horses differs significantly (P < 0.05) from that for trained horses. *Within training status (untrained or trained), value differs significantly (P < 0.05) from that at baseline. See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 74, 4; 10.2460/ajvr.74.4.642

Discussion

Results of the present study indicated that a single IET caused a transient increase in MCT1 and MCT4 protein contents in the gluteus medius muscle of Thoroughbreds. Increased MCT1 and MCT4 protein contents were detected at 6 hours after the IET; however, at 24 hours after the IET, MCT1 and MCT4 protein contents were similar to those immediately prior to the IET (baseline).

In studies23–25 involving rodents and humans, mRNA and protein contents of metabolic genes are increased immediately or within 6 hours after exercise. Results of a study26 involving Thoroughbreds indicate that a single exercise session increases mRNA expression of genes involved in mitochondrial biogenesis. For the horses of the present study, expression of lactate transporters (ie, MCT1 and MCT4) increased after a single exercise session. Similarly, a single exercise session resulted in increased expression of glucose transporters in rats27 and horses28 and fatty acid transporters in humans.29 Results regarding MCT protein contents in the present study were consistent with those of other studies.11,12 In 1 study11 that involved rats, MCT1 and MCT4 protein contents increased after 2 hours of exercise on a treadmill. In a study12 that involved humans, MCT4 protein content increased after 16 hours of intense, intermittent exercise in a standardized cycle test; MCT1 protein content also increased in that study, but the increase from the MCT1 protein content obtained prior to exercise did not quite reach significance (P = 0.10). Thus, it appears that MCT1 and MCT4 are rapidly upregulated by a single exercise session. Variations in the timing and magnitude of MCT1 and MCT4 upregulation among studies may be caused by variations in duration and intensity of exercise or species-specific effects.

In studies30,31 involving humans who underwent strength and exercise training, changes in MCT1 and MCT4 expression in skeletal muscle were dependent on exercise intensity. Therefore, the increase in MCT1 and MCT4 protein contents in the skeletal muscle of the horses of the present study during the IET may have been the result of the increased exercise intensity (ie, increased treadmill speed) as the IET progressed. A similar association between increased exercise intensity and upregulation of glucose transporter type 4 occurs in the skeletal muscle of humans.32 Further research is necessary to determine what type of exercise results in the upregulation of MCT1 and MCT4. Results of the present study indicated that horses had lower plasma lactate concentrations and higher protein contents of MCT1 and MCT4 in skeletal muscle after 18 weeks of HIT than they did before the HIT; therefore, future experiments should be designed carefully so as to ensure that the results reflect only the effects of the exercise session in question and not those of previous exercise training.

In the present study, acute exercise induced a transient increase in MCT1 and MCT4 protein contents; however, the adaptation (ie, training) to acute exercise had no effect on the magnitude of the increase in MCT1 and MCT4 protein contents. Results of another study33 indicate that exercise-induced metabolic signalling responses such as adenosine monophosphate–activated protein kinase were greater in untrained subjects than those in trained subjects immediately after exercise. Furthermore, transient increases in mRNA of metabolic genes (eg, pyruvate dehydrogenase kinase 4 and hexokinase 2) after exercise are reduced after a training period.34,35 Exercise-induced changes in peroxisome proliferator–activated receptor gamma coactivator 1α, a crucial regulator of mitochondrial biogenesis, are more dependent on relative rather than absolute exercise intensity.36 In humans, MCT1 protein content increased as a result of a supermaximal running test to exhaustion both before and after a 6-week period of sprint training, although MCT1 protein content was only determined at 2 hours after the running test.13 In the present study, changes in MCT1 and MCT4 protein contents during the 24-hour observation period after the IET were similar for trained and untrained horses. At 3 hours after the IET, expression of MCT1 and MCT4 mRNA was significantly increased from that at baseline only in untrained horses; however, at 6 hours after the IET, expression of MCT1 and MCT4 mRNA was significantly increased from that at baseline in both untrained and trained horses. These results suggested that MCT mRNA expression may decrease to some extent in response to training. Results of studies that involved elite skiers37 and well-trained horses18 indicate that moderate-intensity training after HIT results in decreased or no change in MCT protein content. Thus, exercise-induced changes in MCT expression may depend on the exercise training history and initial fitness of subjects. In the present study, expression of MCT1 and MCT4 mRNA at rest (ie, baseline) in horses after the 18-week period of HIT did not differ significantly, compared with that in the horses before the HIT, whereas MCT1 and MCT4 protein contents at baseline in horses after HIT were significantly higher, compared with those in horses before HIT. On the basis of these results, we speculated that the repeated transient increase in expression of MCT mRNA during each exercise session was necessary for the training-induced increase in MCT1 and MCT4 protein contents.

Results of other studies4,38 indicate that the rate of lactate flux into and out of skeletal muscle is correlated with the protein contents of MCT1 and MCT4 in muscle. In the present study, after horses underwent 18 weeks of HIT, baseline MCT1 and MCT4 protein contents in muscle were increased, compared with those in horses prior to HIT. Also, for horses after the HIT, plasma lactate concentration during the IET was decreased and the rate of plasma lactate clearance after the IET was increased, compared with those in the horses before HIT. Following an exercise training period, plasma lactate concentration immediately after exercise is reduced, compared with that prior to the exercise training period, and is an effect of increased lactate clearance, one of the first responses by the body to exercise training.39,40 In another study,41 expression of MCT1 was positively associated with blood lactate clearance after a single exercise session in humans. Results of yet another study12 indicated that a single exercise session in which the exercise intensity remained constant resulted in decreased blood lactate concentration for 2 days after the exercise session, compared with that prior to exercise, and was associated with a concurrent increase in MCT1 and MCT4 protein contents and no change in glycogen concentration or mitochondrial oxidative potential in skeletal muscle. Thus, we speculated that the rapid increase in MCT1 and MCT4 protein contents after acute exercise contributed to increased rates of lactate flux, as evidenced by an increase in baseline MCT1 and MCT4 protein contents in skeletal muscle in response to HIT.

Results of the present study indicated that expression of MCT mRNA and MCT protein contents in skeletal muscle of Thoroughbreds increased during the 6 hours immediately after a single IET. The magnitude of increase in MCT mRNA expression and protein contents was similar for both untrained and trained horses. Thus, MCT1 and MCT4 are genes that are rapidly upregulated in response to exercise. High-intensity exercise training caused an increase in baseline protein contents of MCT1 and MCT4 but had no effect on baseline expression of MCT1 and MCT4 mRNA. These results suggested that transient increases in MCT mRNA expression in response to repeated sessions of HIT may represent a mechanical adaptation to lactate transport and metabolism.

ABBREVIATIONS

HIT

High-intensity training

IET

Incremental exercise test

M

CT Monocarboxylate transporter

o2max

Maximal oxygen consumption

a.

Mustang, Kagra AG, Fahrwangen, Switzerland.

b.

Surflo, Telmo, Tokyo, Japan.

c.

YSI 2300 STAT Plus, Yellow Springs Instruments, Yellow Springs, Ohio.

d.

Lidocaine, Fujisawa Pharmaceutical Co, Osaka, Japan.

e.

Disposable Biopsy System, Sheen Man Co, Osaka, Japan.

f.

7500 Real-time PCR System, Applied Biosystems, Foster City, Calif.

g.

Power SYBR Green PCR Master Mix, Applied Biosystems, Foster City, Calif.

h.

TRIZOL, Invitrogen Corp, Carlsbad, Calif.

i.

SuperScript VILO, Invitrogen Corp, Carlsbad, Calif.

j.

Operon Biotechnologies, Tokyo, Japan.

k.

GE Healthcare, Amersham, Buckinghamshire, England.

l.

ChemiDoc, Bio-Rad Laboratories, Hercules, Calif.

m.

Bio-Rad Quantity One, Bio-Rad Laboratories, Hercules, Calif.

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Appendix

Primer sequences used for real-time PCR assay.

GeneForward primerReverse primer
MCT15′-GATTCTTGGCGGCTGCTTGTCAGG-3′5′-TGCCAATCATGGTCAGAGCCGGA-3′
MCT45′-ATGGTGTCTGCGTCCTTCTGCGGA-3′5′-AGCGCCAAACCCAAGCCGGTAA-3′
GAPDH5′-GAGATCAAGAAGGTGGTGAAGC-3′5′-CATCGAAGGTGGAAGAGTGG-3′

GAPDH = Glyceraldehyde-3-phosphate dehydrogenase.

Contributor Notes

Supported by the Japan Racing Association. Dr. Kitaoka was supported in part by a Research Fellowship for Young Scientists from the Japan Society for the Promotion of Science.

Address correspondence to Dr. Hatta (hatta@idaten.c.u-tokyo.ac.jp).