Lactate is not only the end product of glycolysis but also an oxidizable substrate in skeletal muscles.1,2 Lactate is primarily produced in fast-twitch muscle fibers and oxidized in slow-twitch muscle fibers. Lactate is transported across the plasma membrane via proton-linked MCTs.3,4 Among the 14 MCT proteins, MCT1 and MCT4 appear to be the most important in skeletal muscles.5,6 Monocarboxylate transporter 1 is involved in the uptake of lactate by myocytes, and its expression is highly correlated with muscle oxidative capacity.7 On the other hand, MCT4 is involved in the release of lactate from myocytes, and its expression is associated with muscle glycolytic capacity.8
It is known that lactate transport and the levels of MCT protein expression in muscles change in accordance with metabolic demands in mammals. For example, the expression levels of MCT1 and MCT4 in the skeletal muscle increase after endurance and sprint training in rats and humans.9,10 Although changes in expression levels of MCTs during training have been evaluated, few studies11–14 have investigated the changes in MCTs in horses during development, despite the fact that growth is known to cause various alterations in muscle metabolism. In a cross-sectional study,15 we have shown that during development in rats, the expressions of MCT1 in the heart and soleus muscles increase but that of MCT4 in those muscles decreases with age. However, to our knowledge, longitudinal studies of the changes in amounts of MCTs in muscle have not been conducted hitherto.
Horses are suitable animals for use in a longitudinal study because muscle samples can be repeatedly collected from the same site. Results of a previous study13 indicated that the body weight of Thoroughbreds at the age of 24 months is almost equivalent to that of adults, and that the proportion of type IIA fibers in the GMM increases from the age of 2 months to 24 months because of the transition of type IIX fibers to type IIA fibers during the growth period. Also, it is known that in horses, the activities of CS and 3-hydroxyacyl-coenzyme A dehydrogenase increase with age during the growth phase.11,13 Moreover, it has been indicated that the maximal oxygen uptake in horses increases from 18 to 24 months of age.12 These data suggest that horses acquire muscle oxidative capacity as they grow. Peroxisome proliferator-activated receptor-γ coactivator-1α appears to be a key regulator of energy metabolism on the basis of the fact that it is a transcription coactivator that interacts with a broad range of transcription factors.16,17 In mouse cardiac myocytes, the transient expression of the PGC-1α gene induces an increase in mitochondrial gene expressions.18 In other studies,19,20 the overexpression of muscle-specific PGC-1α in transgenic mice led to enhanced mitochondrial biogenesis and the formation of slow-twitch fibers. Furthermore, because PGC-1α is associated with MCT1 expression, PGC-1α may influence lactate metabolism.21 However, the effect of growth on the expression of PGC-1α in horses is also unknown. The purpose of the study reported here was to undertake longitudinal analysis of the changes in MCT1 and MCT4 content and in indicators of energy metabolism in the GMM of Thoroughbreds during growth.
Materials and Methods
Animals—Six Thoroughbreds (3 males and 3 females) were selected for the study. All the horses received concentrate twice daily, and water and hay were available ad libitum. From birth, the horses received pasture exercise in an area of 1,000 m2 for 4 h/d with the remainder of the day spent in a stall. Ethical approval for the study was obtained from the Japan Racing Association. From all horses, muscle samples from the left hind limb were collected at the ages of 2, 6, 12, and 24 months; each sample was obtained at rest from the same portion of the GMM and at the same depth (5 cm deep to the skin surface) by use of a previously described method.13,22 Four muscle pieces from each horse at each time point were prepared for western blotting and measurements of 3 enzyme activities. The samples were frozen in liquid nitrogen and stored at −80°C until analysis.
Western blotting—The muscle proteins in each sample were separated via SDS-PAGE, followed by western blotting according to the protocol described in previous reports.15,21,23,24 Rabbit antibodiesa against the oligopeptide corresponding to the C-terminus regions of human MCT1 (CQKDTEGGPKEEESPV) and MCT4 (CEPEKNGEVVHTPETSV) were used.15,23,24 The specificity of these antibodies for the respective horse MCTs was verified via peptide blocking. For peptide blocking, the antibody was preincubated with the synthetic peptide (1 μg/mL) for 60 minutes at room temperature (approx 22°C) prior to use in immunoblotting. The anti-PGC-1α antibodyb had been used in previous studies in rats21 and human skeletal muscles25 and was predicted to react with horse muscles because of sequence homology between humans and horses. In a preliminary experiment, we confirmed a linear relation between the amount of protein (5 to 20 μg) loaded for electrophoresis and the chemiluminescence of the band in the final spectrum. Mouse skeletal muscles were used as positive controls because MCT1, MCT4, and PGC-1α were previously reported to be abundant in mouse skeletal muscles.3,21
The procedures for preparation of horse muscle samples for western blotting have been described in detail previously.26 Briefly, each GMM sample (15 to 20 mg) was homogenized in a buffer (210mM sucrose, 2mM ethylene glycol-bis-[β-aminoethyl ether-N,N,N′,N′]-tetra acetic acid, 5mM EDTA, and dimethylsulfoxide; pH, 7.5). Then, 3 mL of another buffer (1.167M KCl and 58.3mM tetrasodium pyrophosphate) was added, mixed briefly, and placed on ice for 15 minutes. After centrifugation at 160,000 × g for 75 minutes at 4°C, the supernatant was discarded, and the pellet was washed thoroughly with a third buffer (10mM 2-amino- 2- hydroxymethyl-1, 3-propanediol, and 1mM EDTA; pH, 7.4). The pellet was then homogenized in 400 μL of the third buffer. After 200 μL of 16% SDS was mixed with the pellet, the samples were removed from ice, vortex-mixed, and centrifuged at 1,100 × g for 20 minutes at room temperature. The supernatant was divided into aliquots and stored at −80°C for the protein assay. Following detergent solubilization, a detergent-compatible colorimetric assayc was used to measure the protein concentrations in each sample.
The muscle protein samples (10 μg) and prestained molecular-weight markersd were separated on 12% SDS polyacrylamide gels for 1 hour at 150 V. The proteins were then transferred from the gel to hydrophobic polyvinylidene difluoride transfer membranese for 90 minutes at 100 V. Membranes were incubated on a shaker for 2 hours in another buffer (20mM Tris base, 137mM saline [0.9% NaCl] solution, 0.1M HCl, 0.1% Tween 20, and 3% bovine serum albumin; pH, 7.5) at room temperature. Membranes were then incubated with primary antibody (1:4,000) in the buffer for 2 hours, followed by 3 washes for 15 minutes twice in a different buffer (20mM Tris base, 137mM saline solution, 0.1M HCl, and 0.1% Tween 20; pH, 7.5) and by incubation for 1 hour with secondary antibody (1:4,000) in the wash buffer. Membranes were washed as before with the wash buffer; antibody binding was detected and assessed by use of an enhanced chemiluminescence systemf and an imaging system.g The densitometric analyses of the captured images were performed by use of software.h
Enzyme analyses—The activities of PFK and CS in the GMM samples were analyzed to determine the muscle's glycolytic and oxidative capacities. The PFK activity was determined by use of the standard procedures described by Shonk and Boxer,27 and the CS activity was determined by use of procedures described by Serere.28 The amounts of each of 5 LDH isoenzymes were determined by use of an LDH isoenzyme electrophoresis kit.24,i Lactate dehydrogenase isoenzymes in whole muscle homogenates (1 μg of protein) were separated by means of agarose gel electrophoresis for 40 minutes at 100 V, according to the manufacturer's instructions. After electrophoresis, the gels were covered with a blotting membrane saturated with substrate (208 mmol lithium lactate, 5.6 mmol nicotinamide adenine dinucleotide, 2.4 mmol p-nitro blue tetrazolium chloride, and 0.33 mmol phenazine methosulfate) and incubated for 20 minutes at 45°C. The gels were fixed in 5% acetic acid and dried. The LDH isoenzyme bands were scanned and quantified by use of an imaging system.g
Statistical analysis—Data are expressed as mean ± SE. Data for MCT1, MCT4, and PGC-1α were normalized to the results obtained at 2 months of age for analysis. Data for LDH were represented as relative amounts of each of 5 LDH isoenzymes. A 1-way repeated-measures ANOVA was used to test for the difference across all time points. A Tukey post hoc test was used to identify individual differences. Significance was established at a value of P < 0.05.
Results
A sample of GMM was successfully collected from each of the 6 Thoroughbreds at each time point. There were no noticeable adverse effects associated with sample collection in any horse.
Body weight—The mean weight of the 6 horses increased progressively during the study period. Mean ± SE weights of horses at 2, 6, 12, and 24 months of age were 139.3 ± 5.6 kg, 256.6 ± 6.5 kg, 384.0 ± 6.8 kg, and 476.6 ± 8.7 kg, respectively.
Assessments of MCT1, MCT4, and PGC-1α contents—Via western blotting, a single clear band for MCT1, MCT4, and PGC-1α each was observed. The data obtained in this study were normalized to the results obtained at 2 months of age (designated as 100%). The MCT1 content increased with growth during the study period and was significantly (P < 0.05) different at 24 months (86% increase), compared with the findings at 2 months (Figure 1). The PGC-1α content also changed during the study period and was significantly (P < 0.05) increased at 24 months (124% increase), compared with the findings at 2 months (Figure 2). In contrast, the MCT4 content remained unchanged as horses aged from 2 to 24 months.
Representative results of western blotting and mean ± SE MCT1 (A) and MCT4 (B) content in samples of GMM collected from 6 Thoroughbreds at the ages of 2, 6, 12, and 24 months. The bands represent the respective MCTat 2, 6, 12, and 24 months from left to right. In the graphs, MCT1 or MCT4 content is expressed as a percentage of the respective content at 2 months of age. *At this time point, MCT1 content is significantly (P < 0.05) different from that at 2 months of age.
Citation: American Journal of Veterinary Research 72, 8; 10.2460/ajvr.72.8.1107
Representative results of western blotting and mean ± SE PGC-1α content in samples of GMM collected from 6 Thoroughbreds at the ages of 2, 6, 12, and 24 months. The bands represent PCG-α at 2, 6, 12, and 24 months from left to right. In the graph, PGC-1α content is expressed as a percentage of the content at 2 months of age. *At this time point, PGC-1α content is significantly (P < 0.05) different from all other values.
Citation: American Journal of Veterinary Research 72, 8; 10.2460/ajvr.72.8.1107
Assessments of CS, PFK, and LDH activities—In the study horses, GMM CS activity significantly (P < 0.05) changed with growth; the activity at 24 months of age was increased by 36% from the activity at 2 months of age (Figure 3). During the study period, the PFK activity remained unaltered. Changes in the proportions of 5 isoenzymes of LDH in GMM samples were detected as the horses aged from 2 to 24 months (Figure 4). Compared with findings at 2 months of age, the percentages of the LDH1 and LDH2 isoenzymes (with respect to the total amount of all 5 isoenzymes) at 12 (52% and 43%, respectively; P < 0.05) and 24 months (41% and 32%, respectively; P < 0.05) were higher.
Mean ± SE activity of CS (A) and PFK (B) in samples of GMM collected from 6 Thoroughbreds at the ages of 2, 6, 12, and 24 months. Activity of CS or PFK is expressed as a percentage of the respective activity at 2 months of age. *At this time point, CS activity is significantly (P < 0.05) different from values at 2 and 6 months of age.
Citation: American Journal of Veterinary Research 72, 8; 10.2460/ajvr.72.8.1107
Relative amounts of each of 5 LDH isoenzymes (as a percentage of total LDH content) in samples of GMM collected from 6 Thoroughbreds at the ages of 2, 6, 12, and 24 months. *At this time point, value for LDH1 is significantly (P < 0.05) different from the value at 2 months of age. †At this time point, value for LDH2 is significantly (P < 0.05) different from the value at 2 months of age.
Citation: American Journal of Veterinary Research 72, 8; 10.2460/ajvr.72.8.1107
Discussion
Thoroughbreds were used in the present study on the basis of the hypotheses that growth-related changes in the expression levels of MCTs may be different in the muscles of animals, such as horses, that can sustain high-intensity exercises for a few minutes. Because muscle and plasma lactate concentrations in Thoroughbreds are high after strenuous exercise,29,30 lactate metabolism can be considered as one of the key factors responsible for their exercise capacity. There are sparse data regarding MCT expression in horses. Koho et al31 reported that MCT1 and MCT4 were present in the muscles of Standardbreds, and we previously reported the effects of training on MCT1 and MCT4 in muscles of Thoroughbreds.26 However, to our knowledge, studies have not been performed to investigate the changes in MCTs attributable to development in Thoroughbreds.
At 12 months of age, horses attain approximately 60% to 70% of adult body weight and 90% of adult height, and at 24 months of age, horses weigh almost the same as adult horses,14 as was evident in the present study. Full-scale training for racing usually begins when horses are 18 to 24 months old. However, the horses used in the present study did not undergo any training except voluntary exercise during pasturage in an area of approximately 1,000 m2 for 4 h/d. Ohmura et al32 reported that mean daily activity (distance moved) of untrained 18-month-old horses pastured in 2 hectares for 7 h/d was 2.9 ± 0.7 km/d in winter and 6.9 ± 0.4 km/d in summer. They also reported that most of the young horses' activity was walking. On the basis of data from studies9,33 in humans and rodents, it is known that a high intensity of training is necessary to increase MCT1 and MCT4 contents in muscles; thus, the results of the present study mainly appear to represent the effects of growth on lactate metabolism.
In the horses of the present study, the increase in the amount of MCT1 with increasing age was considered indicative of an increase in the level of lactate influx into the skeletal muscle. Moreover, the percentages of LDH1 and LDH2 isoenzymes (which facilitate lactate oxidation) increased with growth, although LDH5 is the dominant isoenzyme in skeletal muscles. Citrate synthase activity was also significantly increased during growth from 2 to 24 months. Therefore, the muscular capacity to acquire and use lactate, accompanied by an increase in oxidation capacity, develops in growing Thoroughbreds. This finding is consistent with results of a previous study,13 which indicated that the proportion of GMM type IIA fibers—fibers that have high oxidative and glycolytic capacities—is greater in 24-month-old Thoroughbreds than that in 2-month-old Thoroughbreds. Peroxisome proliferator-activated receptor-γ coactivator-1α induces mitochondrial biogenesis and slow-twitch fiber formation,18–20 and the data obtained in the present study indicated that the PGC-1α content of the GMM in Thoroughbreds increases with growth. This suggests that PGC-1α may influence muscular oxidative capacity in horses not only during intensive training but also during growth. However, further studies should be performed to investigate the relationship between PGC-1α expression and growth-related changes in equine skeletal muscles.
On the basis of the data obtained for GMM MCT4 content and PFK activity in the present study, it appears that, in Thoroughbreds, the capacity to produce and release lactate during high-intensity exercise such as racing is similarly present during growth. The findings regarding the PFK activity observed in the present study are consistent with those reported in previous studies,34,35 which indicated that skeletal muscles of horses essentially have high glycolytic capacity during the early developmental phases but that capacity does not increase during growth. In contrast to Thoroughbreds, humans are known to acquire glycolytic capacity during growth. The PFK activity in the muscles of boys (age, 11 to 13 years) is reportedly lower than that of adults.36 The increase in the glycolytic capacity in humans after puberty is partly because of an increase in the number of fast-type fibers in skeletal muscle (hypertrophy). Two-month-old Thoroughbreds already have a high glycolytic capacity, indicating that they can or possibly have to move swiftly and suddenly to survive even during the early developmental phases. Because lactate is produced and used in skeletal muscles during exercise and acquirement of glycolytic and oxidative capacities is essential for strenuous exercise, Thoroughbreds can continue high-intensity exercises for longer duration by acquiring the capacity to use lactate in their skeletal muscles as an energy resource during growth.26,31
The results of the present study have indicated that developmental changes related to lactate metabolism occur in the GMMs of Thoroughbreds. The MCT1 protein content, the oxidative enzyme activity, and the percentage of LDH1 and LDH2 isoenzymes (in relation to the total amount of all 5 isoenzymes) in the GMM increase during development from 2 to 24 months of age, suggesting an increase in lactate oxidation capacity during growth. However, the MCT4 content and glycolytic enzyme activity in the GMM remain unchanged during that growth period.
ABBREVIATIONS
| CS | Citrate synthase |
| GMM | Gluteus medius muscle LDH Lactate dehydrogenase |
| MCT | Monocarboxylate transporter PGC-1α Peroxisome proliferator-activated receptor-γ coactivator-1α |
| PFK | Phosphofructokinase |
Operon, Tokyo, Japan.
Calbiochem, San Diego, Calif.
DC Protein Assay, Bio-Rad, Hercules, Calif.
BioDynamics Laboratory Inc, Tokyo, Japan.
Hybond-P PVDF transfer membranes, Amersham Biosciences, Piscataway, NJ.
Amersham Biosciences, Piscataway, NJ.
Chemidoc system, Bio-Rad, Hercules, Calif.
Quantity One software, version 4.6.1, Bio-Rad, Hercules, Calif.
Beckman Paragon LD, Beckman Instruments Inc, La Brea, Calif.
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