Thoroughbreds are athletes with a high capacity for exercise. Oxidation of carbohydrates and fat provides the energy for their high level of performance. Equine skeletal muscle contains a high amount of energy stored as glycogen.1 This muscle glycogen is readily available during exercise and is thought to be the most important energy substrate for horses. Fat is stored primarily in adipose tissue and muscle. Although fatty acids cannot provide energy as rapidly as can carbohydrates and are less efficient per unit of oxygen than are carbohydrates, they provide much more energy per gram of wet weight than do carbohydrates. Therefore, the oxidative capacity of fatty acids may also be important for exercise performance in horses.
It is widely accepted that the transfer of fatty acids across the plasma membrane of muscle cells is primarily a protein-mediated process. Three types of membrane-associated proteins have been identified as potential fatty acid transporters: fatty acid binding protein, fatty acid transport protein, and FAT/CD36.2,3 Of these, most of the fatty acid uptake into the muscle tissue of mammals has been attributed to FAT/CD36.4,5 Similar to findings in the skeletal muscle of rodents and humans, FAT/CD36 protein has been detected in the skeletal muscle of horses.6 However, whether training results in an increase in FAT/CD36 content in equine muscles is not known.
Peroxisome proliferator-activated receptor-γ coactivator-1α is emerging as the master regulator of mitochondrial biogenesis and oxidative capacity in skeletal muscle.7 The PGC-1α protein content increases in humans and rodents as a result of acute exercise8,9 and endurance training.10,11 A single bout of exercise can increase PGC-1α mRNA in equine muscle.12 In another study13 conducted by our laboratory group, we reported that PGC-1α content increases with growth in equine muscle. However, whether PGC-1α protein content increases as a result of training is unknown. Because studies14,15 in humans have found that HIT effectively increases oxidative capacity, we considered it appropriate to use training of a higher intensity than that of conventional endurance training.
Metabolomics is an emerging tool used to gain insights into cellular and physiologic responses. Because increases in the maximal enzyme activities and protein contents of transporter proteins do not necessarily reflect energy metabolism during exercise, we considered it appropriate to use a metabolome differential display method based on CE-TOFMS described previously16,17 to measure muscle metabolites related to glycolysis substrates in muscle during maximal exercise.
The objective of the study reported here was to investigate changes in PGC-1α and FAT/CD36 protein contents in Thoroughbreds in response to training. We also sought to investigate changes in substrate concentrations related to carbohydrate metabolism, as determined via CE-TOFMS.
Materials and Methods
Animals—Twelve Thoroughbreds (3 to 4 years old; 6 males and 6 females) were included in the study. The pedigree of each horse was known. Horses had been raised in accordance with standard procedures of the Equine Research Institute of the Japan Racing Association for use in research investigations. All horses received concentrate and hay twice daily, in amounts in accordance with National Research Council feeding standards, and water was available ad libitum. The horses underwent surgery to translocate a carotid artery from the carotid sheath to a subcutaneous location to facilitate arterial catheterization. At least 1 month was allowed to elapse between surgery and any exercise experiments. The study protocols were reviewed and approved by the Animal Welfare and Ethics Committee of the Japan Racing Association Equine Research Institute.
Treadmill measurements—After translocation of the carotid artery, horses were acclimated to running on a treadmill.a Horses were subjected to IET on a treadmill at an incline of 6% to identify each horse's 
o2max and the speed required to attain it. Horses were fed 3 hours before IET. The IET protocol involved walking (1.7 m/s) for 2 minutes and trotting (3.5 m/s) for 5 minutes. The speed of the treadmill was then increased to 6, 8, and 10 m/s (in increments of 1 m/s at 1-minute intervals) until the horse was too exhausted to maintain its position at the front of the treadmill. During this procedure, each horse was fitted with an open-flow mask for o2max measurement. Briefly, oxygen and carbon dioxide concentrationsb and ambient temperature and relative humidityc were measured continuously. All instrument signals were stored on a computer with an analogue-to-digital converter, and calculations were performed with a software analysis package.d The mean o2 for the last 15 seconds of each treadmill speed was considered the o2 for that speed, and o2max was determined at a plateau point via linear regression analysis.
A catheter inserted in a jugular vein was used for collection of a venous blood sample (10 mL) during the IET; the samples were used for measurement of plasma lactate and glucose concentrations, as described elsewhere.18 Blood samples were centrifuged at 1,800 × g for 10 minutes at 4°C. Plasma was harvested, and plasma lactate concentrations were measured with an automated blood lactate analyzer.e Plasma free fatty acid concentration was measured via an enzymatic colorimetric technique by use of a kitf as described by the manufacturer. Lactate, glucose, and free fatty acid concentrations were measured in duplicate.
Training procedure—Horses were subjected to HIT for 18 weeks. During the initial 10 weeks, horses ran at 90% of o2max for 3 minutes. During weeks 11 to 18, horses ran at 110% of o2max for 3 minutes. The training program consisted of a warmup period (walking at 1.7 m/s for 1 minute and trotting at 3.5 m/s for 3 minutes), cantering at 90% (or 110%) of o2max for 3 minutes, and a cooldown period (walking at 1.7 m/s for 3 minutes). Training was conducted 5 d/wk on a treadmill at an incline of 6%. Horses were subjected to IET before and after the 18-week training period. The final IET was performed at least 24 hours after the last training exercise. Intensity of training was determined on the basis of the pretraining o2max. Veterinarians monitored health of the horses during the training period. All horses completed the 18 weeks of training and both IETs. On days horses did not train, they were kept in a pasture.
Muscle biopsy specimens—Muscle biopsy specimens were obtained at rest under local anaesthesia (2% lidocaineg) with a 2-mm-diameter needleh from all horses from the same portion of the gluteus medius muscle and at the same depth (5 cm from the skin surface) before and after the training period. Samples for CE-TOFMS were obtained immediately after both IETs. Specimens were frozen in melting isopentane cooled by liquid nitrogen and stored at −80°C until analyzed.
Western blotting—Muscle specimens were homogenized, and proteins were separated via SDS-PAGE as described elsewhere.13,19,20 The anti–PGC-1α antibody against amino acids 777 to 797 in the carboxyl terminus of the human sequencei was used as in a previous study.13 The anti–CD36 antibody against the human sequence near the N-terminus of CD36j was obtained; it was predicted that the anti–CD36 antibody would react with other mammalian species (ie, equine) because of sequence homology. Also, expression of FAT/CD36 protein in equine muscle has been detected with the human CD36 antibody.6 Equal quantities of protein were loaded into each lane, and a common standard was included in all blots. A portion of each sample was analyzed for total protein content via the Bradford assay,k with bovine serum albumin as the standard. Equal loading and blotting was confirmed by staining with a temporary protein stain.l Blots were developed via enhanced chemiluminescencem and subsequently quantified with a chemiluminescent imaging system.n Densitometric analyses of the recorded images were performed with commercially available software.o In a preliminary experiment, we confirmed there was a linear relationship between the amount of protein loaded (5 to 20 μg) for electrophoresis and the chemiluminescence of the band in the final spectrum, and single clear bands for PGC-1α and CD36 were observed.
Enzyme activities—Activities of PFK, CS, and β-HAD were measured via standard procedures. The activities were evaluated as described in other studies (PFK,21 CS,22 and β-HAD23).
CE-TOFMS—All CE-TOFMS experiments were performed with a capillary electrophoresis systemp by personnel at a metabolomics analysis lab.q The experiments were performed as previously reported.16,17 Briefly, muscle biopsy specimens (50 mg) were added to 500 μL of ice-cold methanol containing internal standards and homogenized at 4,000 × g for 60 seconds at 4°C. Extracts then were transferred to a separate tube, mixed with 200 μL of water and 500 μL of chloroform, and centrifuged at 2,300 × g for 5 minutes at 4°C. The aqueous layer was centrifugally filtered through a 5-kDa cutoff membrane to remove proteins. The filtrate was lyophilized, dissolved in 50 mL of water, and subjected to CE-TOFMS analysis. Separations were conducted in a fused silica capillary (inner diameter, 50 μm; length, 80 cm). Sample solutions were injected at 5 kPa for 25 seconds, and voltage of 30 kV was applied. Electrospray ionization–time-of-flight mass spectrometry was conducted in the negative ion mode; the capillary voltage was set at 3,500 V. The scan range was 50 to 1,000 m/z.
Statistical analysis—Data were expressed as mean ± SEM. Paired t tests were used to analyze the data. Values of P < 0.05 were considered significant.
Results
Mean ± SEM body weight was not altered by HIT (495.5 ± 9.8 kg before HIT and 500.9 ± 9.5 kg after HIT). Peak speed and o2max during IET increased significantly after HIT. The RER at 10 m/s during IET decreased significantly; however, at exhaustion during IET, the RER was not altered after HIT (Table 1). The plasma lactate concentration at 10 m/s during IET decreased significantly after HIT, but the concentration at exhaustion during IET was not altered after HIT. The plasma free fatty acid concentration after IET increased significantly after HIT, whereas the glucose concentration did not (Table 2). The fructose 1,6-diphosphate, phosphoenolpyruvate, and pyruvate concentrations in the gluteus medius muscle during IET decreased significantly after HIT. Fructose 6-phosphate and glucose 6-phosphate concentrations during IET decreased, but not significantly, after HIT (Table 3). The PGC-1α and FAT/CD36 protein contents increased significantly after HIT (Figure 1). Activities of CS and β-HAD in the gluteus medius muscle increased significantly after HIT, whereas the PFK activity remained unchanged (Figure 2).
Mean ± SEM values for body weight, o2max, peak speed, and RER during an IET in 12 Thoroughbreds before (untrained) and after HIT for 18 weeks (trained).
| Horses | Body weight (kg) | o2max (mL/kg/min) | Peak speed (m/s) | RER | |
|---|---|---|---|---|---|
| 10 m/s | Exhaustion | ||||
| Untrained | 495.5 ± 9.8 | 162.5 ± 3.5 | 11.1 ± 0.1 | 1.08 ± 0.04 | 1.21 ± 0.03 |
| Trained | 500.9 ± 9.5 | 180.5 ± 2.9* | 12.8 ± 0.2* | 0.99 ± 0.02* | 1.29 ± 0.02 |
Value differs significantly (P < 0.05) from the corresponding value for the untrained horses.
Mean ± SEM plasma concentrations of glucose, lactate, and free fatty acids during an IET in 12 Thoroughbreds before (untrained) and after HIT for 18 weeks (trained).
| Variable | Untrained | Trained | ||||
|---|---|---|---|---|---|---|
| Basal | 10 m/s | Exhaustion | Basal | 10 m/s | Exhaustion | |
| Glucose (mM) | 5.7 ± 0.1 | 6.2 ± 0.2 | 6.7 ± 0.3 | 5.7 ± 0.2 | 5.6 ± 0.3 | 6.5 ± 0.4 |
| Lactate (mM) | 0.80 ± 0.1 | 15.90 ± 2.5 | 22.30 ± 2.9 | 0.83 ± 0.1 | 6.60 ± 1.1* | 20.50 ± 2.3 |
| Free fatty acids (mM) | 0.24 ± 0.02 | ND | 0.29 ± 0.03 | 0.26 ± 0.04 | ND | 0.40 ± 0.03* |
ND = Not determined.
See Table 1 for remainder of key.
Mean ± SEM values for muscle metabolites measured via CE-TOFMS metabolomics in specimens obtained at exhaustion during an IET in 4 Thoroughbreds before (untrained) and after HIT for 18 weeks (trained).
| Metabolite | Untrained | Trained |
|---|---|---|
| Glucose 1-phosphate | 185 ± 22 | 108 ± 38 |
| Glucose 6-phosphate | 4,238 ± 462 | 2,299 ± 748 |
| Fructose 6-phosphate | 684 ± 70 | 359 ± 125 |
| Fructose 1,6-diphosphate | 1,652 ± 229 | 802 ± 196* |
| 3-phosphoglycerate | 343 ± 25 | 283 ± 44 |
| Phosphoenolpyruvate | 68.0 ± 3.2 | 50.0 ± 6.0* |
| Pyruvate | 192.0 ± 9.5 | 145.0 ± 9.5* |
| Lactate | 48,596 ± 11,046 | 31,337 ± 7,591 |
Values reported are nmol/g of muscle tissue.
See Table 1 for remainder of key.
Mean ± SEM PGC-1α (A) and FAT/CD36 protein (B) content in the gluteus medius muscle of 12 Thoroughbreds before (untrained) and after HIT for 18 weeks (trained). *Value differs significantly (P < 0.05) from the value for the untrained horses. AU = Arbitrary units.
Citation: American Journal of Veterinary Research 73, 11; 10.2460/ajvr.73.11.1813
Mean ± SEM activities of PFK (A), CS (B), and β-HAD (C) in the gluteus medius muscle of 12 Thoroughbreds before (untrained) and after HIT for 18 weeks (trained). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 73, 11; 10.2460/ajvr.73.11.1813
Discussion
In the study reported here, we determined that training can induce a shift from carbohydrate oxidation to fatty acid oxidation during IET in Thoroughbreds. The novel observations of this study are that after HIT for 18 weeks, skeletal muscle PGC-1α and FAT/CD36 protein expression increased; activities of CS and β-HAD increased, whereas PFK activity remained unchanged; and concentrations of muscle metabolites related to glycogenolysis (fructose 1,6-diphosphate, phosphoenolpyruvate, and pyruvate) measured via metabolomics analysis decreased at exhaustion during IET. Thus, these results suggested that training would induce a shift from carbohydrate oxidation to fatty acid oxidation with increases in FAT/CD36 protein content and β-HAD activity. Less glycogenolysis occurred during exercise, but maximal activity of PFK was not altered.
Training increases the skeletal muscle mitochondrial content and maximal capacity for carbohydrate and fat oxidation, and it permits the exercise at higher absolute power outputs.24,25 Endurance training in horses results in increases in Vo2max and the oxidative enzyme activity of muscles.26,27 The present study revealed that HIT for 18 weeks resulted in an 11% increase in Vo2max and 50% and 59% increases in the maximal activities of key mitochondrial enzymes (CS and β-HAD, respectively), but not a glycolytic enzyme (PFK), which was in accordance with results of earlier studies28,29 that used conventional race training in Thoroughbreds. Moreover, we found a significant increase in FAT/CD36 protein content after HIT in association with an increase in β-HAD activity in Thoroughbreds.
The capacity for increased fatty acid oxidation induced by exercise training is considered to be most closely associated with fatty acid uptake across the plasma membrane by FAT/CD36.5 Analysis of these data suggested that training increased the reliance on lipids as an energy source as a result of an increase in plasma free fatty acid uptake. Investigators in another study6 reported that FAT/CD36 protein expression is higher in red oxidative skeletal muscle than in white glycolytic skeletal muscle in horses. Because training induces a change in muscle fiber composition from fast glycolytic fibers to fast and slow oxidative fibers,30 the change in FAT/CD36 content in the present study may have been associated with a transition in fiber type. Regarding other metabolite transporters, investigators in a study31 reported an increase in glucose transporter 4 secondary to training in horses. In addition, in another study19 conducted by our laboratory group, we reported that monocarboxylate transporters 1 and 4, which are lactate transporters, increased after HIT. Thus, HIT increases the capacity for oxidation of both fat and carbohydrates (including lactate).
In the present study, we also found an increase in PGC-1α content in equine skeletal muscle after training. Recent studies8,32 have indicated that PGC-1α has a pivotal role in mitochondrial biogenesis in skeletal muscle. Combined with our previous result of increases in PGC-1α content with growth, the results for the present study suggest that PGC-1α plays a role as a master regulator in oxidative adaptation in horses, as has been reported in rodents8,10 and humans.11,33
In the present study, we used metabolomics analysis (CE-TOFMS) and found that although PFK activity was not altered, concentrations of muscle metabolites related to glycogenolysis (fructose 1,6-diphosphate, phosphoenolpyruvate, and pyruvate) decreased at exhaustion during IET after training. In addition, fructose 6-phosphate and glucose 6-phosphate concentrations decreased slightly. It must be acknowledged that these results for CE-TOFMS were based on a relatively small sample size. However, despite this, we were able to detect significant changes in muscle metabolites related to glycogenolysis.
Furthermore, we detected a decrease in plasma lactate concentration at submaximal exercise intensities. The RER decreases during both high- and moderate-intensity exercise because of an increase in oxidative capacity secondary to training.34,35 Similar to results in these reports, RER during submaximal exercise intensities decreased significantly after training in the present study. This decrease in RER may have been attributable to the decrease in lactate production in muscles because a portion of expired carbon dioxide is derived from the buffering of lactic acid. However, decreases in plasma lactate concentrations during exercise might be attributable to increased lactate oxidation and glycogenesis or gluconeogenesis from lactate in the liver.36,37 These results suggested that glycolysis during exhaustive exercise is suppressed by HIT, whereas fatty acid oxidation is increased. Similar to the results for the present study, it was reported in another study25 that after training in humans, there was less glycogenolysis during exercise at 90% of o2peak because the larger mitochondrial oxidative capacity after training afforded a tighter coupling between the supply of ATP and the demand for ATP during exercise. The muscle glycogen concentration decreases rapidly during maximal exercise, and muscle glycogen depletion is a major cause of exercise fatigue.38 Therefore, glycogen sparing after training as a result of an increase in oxidative capacity and a decrease in glycogenolysis may be a major factor in increases in exercise capacity.
Collectively, the present study indicated that there can be significant increases in the capacity for oxidation of fatty acids in Thoroughbreds after training. These changes may contribute to increases in exercise performance of Thoroughbreds.
ABBREVIATIONS
| β-HAD | β-3-hydroxyacyl CoA dehydrogenase |
| CE-TOFMS | Capillary electrophoresis–time-of-flight mass spectrometry |
| CS | Citrate synthase |
| FAT/CD36 | Fatty acid translocase |
| HIT | High-intensity training |
| IET | Incremental exercise testing |
| PFK | Phosphofructokinase |
| PGC-1α | Peroxisome proliferator-activated receptor-γ coactivator-1α |
| RER | Respiratory exchange ratio |
| o2max | Maximal oxygen consumption |
Mustang, Kagra AG, Fahrwangen, Switzerland.
METS-900, VISE Medical, Chiba, Japan.
HMI38, Vaisala, Vantaa, Finland.
DATAQ, Akron, Ohio.
YSI 2300 STAT Plus, Yellow Springs Instruments, Yellow Springs, Ohio.
NEFA C test kit, Wako Pure Chemicals, Osaka, Japan.
Lidocaine, Fujisawa Pharmaceutical Co, Osaka, Japan.
Disposable biopsy system, Sheen Man Co, Osaka, Japan.
Calbiochem, San Diego, Calif.
Santa Cruz Biotechnology, Santa Cruz, Calif.
Bradford protein assay, Bio-Rad, Hercules, Calif.
Ponceau S solution, Sigma-Aldrich, St Louis, Mo.
GE Healthcare, Amersham, Buckinghamshire, England.
ChemiDoc, Bio-Rad Laboratories, Hercules, Calif.
Bio-Rad Quantity One, Bio-Rad Laboratories, Hercules, Calif.
Agilent CE-TOFMS system, Agilent Technologies, Waldbronn, Germany.
Human Metabolome Technologies Inc, Tsuruoka, Japan.
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