Effects of Training on Monocarboxylate Transporters (MCT1, MCT2 and MCT4) in Vastus Lateralis Muscle from Standardbred Horses

During intense exercise, lactic-acid accumulates in skeletal muscle and protons build up which causes a decrease in pH and leads to inhibition of muscle function and eventually fatigue. A family of Monocarboxylate Transporters (MCTs) have been identified in equine skeletal muscle but its function during training has not yet been fully elucidated. We hypothesize an important function of MCT1, 2 and 4 in equine muscle. Six Standardbred geldings were trained for a total of 22 weeks in 2 phases (endurance phase and high intensity phase). Exercise intensity during the training sessions were based on fixed percentages of the peak heart frequency (HRpeak) determined during a Standard Exercise Test (SET). At the beginning and end of each phase a SET was performed. Venous blood was drawn from the jugular vein before the test (t=0 minutes), after the warming up (t=9 minutes), and every 5 minutes during the SET (t=14, 19, 24, 29, 34). Samples were kept on ice until whole blood lactic acid had been analysed. Muscle biopsies were taken approximately 60 minutes before each SET using a modified Bergström biopsy needle. Blood and fat tissue were removed from the biopsy and the biopsy was there after immediately frozen in liquid nitrogen for biochemical analysis and Western-blotting for MCT expression. Training (SET1 vs SET2) resulted in an increase in the total plasma lactic acid accumulation during exercise as measured as the area under the curve during the 20-minute endurance run in the SET. Semi quantative densitometric analysis of the blots showed that MCT1 and MCT4 remain unaltered upon training. MCT2 expression was non-significantly increased by more than 60%. We report the expression of MCT1, MCT2 and MCT4 proteins in skeletal muscle of Standardbred trotters during a physical training regimen. The relative high expression of MCT2 was surprising but fits well with the gastrointestinal function of the horse. The high expression of MCT2 suggests an important function in the absorption of short chain fatty acids although such function needs further investigation.


Introduction
Exercise and training have a major impact on muscle metabolic function. During high intensity exercise or during the later stages of endurance exercise, lactic acid accumulates. At physiological pH, in the muscle cell (between 6 and 8), lactic acid is dissociated in a lactic acid anion (La -) and a proton. Accumulation of the Laand the protons during exercise in the muscle cell lead to inhibition of metabolic enzymes and the development of fatigue [1]. A better understanding in the relation between lactic acid accumulation and fatigue will not only lead to designing better training programs for athletes (horses and humans) but will also be beneficial for patient groups (e.g., cancer patients) in which fatigue during rest or physical activity is hampering Activities of Daily Living (ADL). In addition, more fundamental knowledge might also lead to the development of new targeted drugs or nutritional supplements that counteract the effects of lactic acid in muscle cells. The La -, however, cannot diffuse out of the cell because of the electric charge of the molecule. Removal of lactic acid from the exercising muscle cells is facilitated by a family of Monocarboxylate Transporters (MCT's) [2][3][4][5]. MCT's mediate membrane transport with an obligatory 1:1 coupling between monocarboxylates (among which the La -) and a proton [6]. Therefore, MCT's have an important function in the maintenance of intracellular pH homeostasis. To date, a family of 14 MCT isoforms have been identified that are involved in the transport of lactic acid, pyruvate, ketone bodies, and branched-chain ketoacids [2,3]. Of all the isoforms discovered so far, MCT1, MCT2 and MCT4 are most abundantly expressed in skeletal muscle [7][8][9][10]. MCT1 is predominantly found in the oxidative fibre types, and has a high correlation with lactic acid uptake and oxidative metabolism [11,12]. The expression of MCT1 is therefore associated with the uptake of lactic acid for oxidation. MCT4 on the other hand, is mainly expressed in glycolytic muscle fibres and is, despite a relative low affinity for lactic acid, associated with lactic acid efflux from the muscle cell.
The properties of MCT2 have been described in less detail. The protein has been shown to be expressed in sarcolemma of slow oxidative muscle fibres [8,9] and its expression during developmental stages in rat brain suggest a function in the influx of monocarboxylates in oxidative tissue [13]. Interestingly, it has been shown that in humans and rats MCT1 and MCT4 expression are increased in response to either endurance or high intensity training [14][15][16] but remarkably not intermittent training [17]. Additionally, plasma lactic acid and Lactic Acid Dehydrogenase (LDH) activity and LDH-isoenzyme expression have been shown to correlate with MCT proteins [18][19][20]. MCT proteins have been found in equine skeletal muscle and red blood cells [5] but its role during training has not yet been fully elucidated. Since plasma lactic acid accumulation during exhaustive exercise is a well-known phenomenon in horses [21] we hypothesize an important role in equine skeletal muscle of MCT1, 2 and 4 as well.
An 18-week training program was applied to investigate the effects of training on MCT protein expression in skeletal muscle. Finally, plasma lactic acid accumulation during a sub-maximal exercise test and LDH activity and LDH-isoenzyme expression were measured.

Horses
In this study, 6 Standardbred geldings were used. Horses were aged 20 ± 2 months in this study and were owned by the Faculty of Veterinary Medicine of the University of Utrecht, The Netherlands. The horses were individually housed and their diet consisted of grass silage supplemented with concentrate feed and vitamin supplements and met nutrient requirements for maintenance and performance (58 MJ NE (range 54-66). Salt blocks and water were available ad libitum. All procedures were approved by the Institutional Animal Care and Medical Ethical Committee of the Utrecht University, and complied with the principles of laboratory animal care.

Training
The training period consisted of a total of 22 weeks divided in 2 phases. The exercise intensity during the training sessions were based on fixed percentages of the peak Heart Frequency (HR peak ), as obtained in a previous study with Standardbred trotters [22]. Heart Frequency (HF) was measured with a Polar heart rate monitor (Polar S610i, Polar Electro, Kempele, Finland) during the training sessions and speed and inclination of the treadmill were adjusted on a weekly basis in order to achieve the desired HF.
The horses received an initial training of 4 weeks to get accustomed to trotting on a high-speed treadmill (Mustang 2000, Kagra, Graber HG, Switzerland). Each training session was preceded by 30 min warming-up at the walking machine followed by 8 min warming-up at the treadmill, which consisted of 4 min at 1.6 m/s and 4 min at 3.0-4.0 m/s, no incline. The training program during phase 1 consisted of endurance training: week 1, 30% HR peak for 20 min 3/week; week 2, 30% HR peak for 25 min 4/week; week 3, 40% HR peak for 30 min 4/week; and week 4, 50% HR peak for 35 min 4/week. Each training session ended with a cooling down consisted of a 5 min walk at the treadmill followed by 30 min walk at the walking machine.
The training phase (18 weeks) consisted of two types of exercise, endurance running and interval running. The days of interval running were alternated with days of endurance running. Each training session was preceded by 30 min warming-up at the walking machine followed by 8 min warming-up at the treadmill, which consisted of 4 min at 1.6 m/s and 4 min at 4.5 m/s, no incline. The endurance running included 20-24 min of continuous level running at 60% HR peak or 16-18 min at 75% HR peak . The interval training included three 3-min bouts at 80-90% HR peak or four 2-min bouts at 80-90% HR peak and interspersed with 3-min or 2-min periods at 60% HR peak . Each training session ended with a cooling down consisted of a 5 min walk at the treadmill followed by 30 min walk at the walking machine. The horses exercised 4 days/wk throughout the entire training period of phase 2. On the resting days, the horses walked for 60 minutes at the walking machine. . 03 . a 4-min warming up period of walking at 1.5m/s followed by 4 minutes of trot at 4.5m/s. Next, after 1 minute of additional walking at 1.5m/s horses trotted for 20 min at approximately 80% HR peak . Finally, horses were allowed to cool down for 5 min at 1.5 m/s. Heart rate was measured using a Polar S610i heart rate meter and continuous ECG monitoring (Cardio Perfect Stress 4.0; Cardio Perfect Inc, Atlanta, GA, USA). Venous blood was drawn from the jugular vein before the test (t=0 minutes), after the warming up (t=9 minutes), and every 5 minutes during the SET (t=14, 19,24,29,34). Samples were kept on ice until whole blood lactic acid and pH had been analysed (ABL-605 Radiometer Copenhagen, Westlake, Ohio).

Exercise test
Muscle biopsies were taken approximately 60 minutes before the SET. A 5-cm deep biopsy of the M. Vastus Lateralis (VL) taken under local anaesthesia (lidocaine hydro chlorine (2%) without adrenalin) using a modified Bergström biopsy needle (Maastricht instruments, Maastricht, The Netherlands) with a diameter of 7 mm. Blood and fat tissue were removed from the biopsy and the biopsy was there after immediately frozen in liquid nitrogen for biochemical analysis. Frozen muscle tissue was stored at -80ºC.

Biochemical analysis
For enzymatic analysis 50 mg of muscle tissue was homogenised in 1 ml SET buffer (250 mm sucrose; 2 mm EDTA; 10 mm Tris-HCL) with an Ultra-turrax homogeniser. Homogenates were subsequently sonificated 3 times and centrifuged at 15.000g for 10 min. Supernatants were stored at -80ºC until analysed. All chemicals used were of analytical grade.
Total LDH in muscle homogenates was determined using a commercially available assay (ABX diagnostics; Radiometer Nederland BV, Zoetermeer, The Netherlands). Distribution of different LDH iso-enzymes in muscle homogenates was analysed with a commercially available kit (Sebia Benelux S.A., Brussels, Belgium).

Western blotting of MCT1, 2 and 4 proteins
Approximately 50 mg of muscle tissue was homogenized with potter tubes in ice-cold buffer containing 210 mm Sucrose, 30 mm HEPES, 5 mm EDTA, 2 mm EGTA and 1 minitablet protease inhibitors (Roche Applied Science, Almere, and The Netherlands). The sample was diluted with a buffer containing 1.17 M KCl, 58.3 mm Na Pyrophosphate and 1 mm DTT. Samples were centrifuged at 150,000g for 90 minutes and the supernatant (cytosol fraction) was stored at -80ºC. The pellet was suspended in 50 µl buffer containing 10 mm Tris, 1 mm EDTA and 0.1% Triton X-100 (membrane fraction), and stored at -80ºC until blotting procedures.
Polyacrylamide Sodium Dodecysulphate (SDS) gel electrophoresis was performed according to Laemmli [23]. In short, 50 µl homogenate was boiled 5 minutes in an equal volume of SDS sample buffer containing 2.3% SDS and 5% β-mercapto-ethanol and subsequently centrifuged for 5 minutes. Equal amounts of protein (25 µg) were loaded on 10% polyacrylamide gels and electrophorized at 200V for 55 minutes. After electrophoresis protein was transferred to a nitrocellulose membrane by blotting for 60 minutes at 100V.
For detection of MCT protein nitrocellulose sheets were pre-treated with Odysey blocking buffer (Licor Biosciences; Westburg b.v., Leusden The Netherlands) diluted 1:1 in PBS for 60 minutes. Incubation with MCT1, 2 and 4 antibodies (Santa Cruz Biotechnology; Tebu-bio, Heerhugowaard, The Netherlands), diluted 1:10000 in blocking buffer, was carried out overnight (16 hours) at room temperature with gentle shaking. After three washing steps with 0.1% Tween20 in PBS blots were incubated for 60 minutes with fluorescent conjugated donkey anti goat secondary antibody (Rockland; Tebu-bio, Heerhugowaard, The Netherlands). Blots were scanned with an odyssey IR scanner (Licor Biosciences; Westburg b.v., Leusden The Netherlands) and results were expressed as integral intensities and as relative intensities to a positive control sample obtained from rat gastrocnemius muscle.

MCT expression
A representative Western blot showing MCT1, MCT2 and MCT4 is shown in the figures 1.1-1.3. Semi quantative densitometric analysis of the blots showed that MCT1 and MCT4 remain unaltered upon training. MCT2 expression was non-significantly increased by more than 60%. MCT2 showed a large inter-individual variation ( Figure 2).  . 04 . Figure 2(A,B) show representative blots of MCT1, 2 and 4. Lane M represents protein weight marker, R represents rat gastrocnemius control muscle sample, E represents equine vastus lateralis muscle and +P represents an example of pre-incubation with control peptide.
Also, no significant differences due to 18 weeks of training could be observed in total LDH activity expressed as µmol/gram muscle/minute (223.9 ± 58.8 vs 187.1 ± 36.7 for TT1 and TT2, respectively) ( Figure 4).

Discussion
We report the expression of lactic acid transport proteins MCT1, MCT2 and MCT4 in skeletal muscle of Standardbred trotters under a physical training regimen. The relative high expression of MCT2 was not anticipated, but fits well with the gastrointestinal function of the horse. The expression of high levels of MCT2 in the rumen and small intestine of reindeer suggests their involvement in the absorption of Short Chain Fatty Acids (SCFA) . 05 . [24]. It is well known that a major difference between humans and horses is the large availability of volatile Short Chain Fatty Acids (SCFA) derived from hindgut fermentation in horses. This is a particularly interesting and probably underestimated energy source of horse skeletal muscle. The SCFA acetate, propionate, and butyrate are monocarboxylates which represent the most abundant anions in the colonic lumen. Stein et al., [25] showed that the uptake of short-chain fatty acids uptake into the CaCo2 cell line is initiated by a pH-dependent and carrier mediated transport mechanism involving the MCT's. These findings are in line with the findings of Gill et al., who reported an imported role for MCT's in the uptake of short-chain fatty acids [26]. Furthermore, Koho et al., reported MCT2 and MCT1 as isoforms with the greatest affinity for SCFA in reindeer liver tissue [24]. Therefore, we propose that MCT2 functions as a monocarboxylate transporter with special emphasis on SCFA in skeletal muscle. Based on similarities in muscle fibre type distribution, enzymatic activity and protein expression in horses and humans, we propose similar roles for MCT proteins in equine skeletal muscle (e.g., transport of lactic acid to the exterior of the cells).
Antibodies used to identify MCT proteins in this study, were raised against human (MCT1 and 4) and mouse (MCT2) MCT proteins, and the question remains whether these antibodies cross-react with equine MCT. The function and structure of MCT proteins seems to be highly conserved among species [2]. Nevertheless, in our Western blotting multiple bands were visible, also in a positive control sample of rat gastrocnemius muscle. Pre-incubation with control peptide showed selective diminishment of one specific band in the region of interest however. This band could therefore be designated as the specific MCT protein.
Plasma lactic acid concentrations were significantly higher after a period of training. This result seems to contrast to other reports indicating an increased lactic acid clearance post training [21,27,28]. Although an explanation for the higher plasma lactic acid concentration observed in the trained horses in the present study is not easy to explain it could be related to the exercise intensity of the SET. Both SETs were performed at the same relative workload. Therefore, energy expenditure was increased during SET2 probably leading to increases in muscular lactic acid production, clearance from the muscle and hence and increased plasma concentration [29]. Evidence for this assumption comes from the heart frequencies as recorded during the second test. These were also increased which is indicative for an increased workload. Another explanation could be the underestimated load capacity of the horses in the first SET and therefore was of too moderate intensity to produce significant amounts of lactic acid. An increase in plasma lactic acid can also be the result of a decreased plasma lactic acid clearance once lactic acid is present in the circulation. In horses, however, there is no substantial support for the concept of clearance from the blood. Finally, it must be mentioned that equines have the unique ability of increasing red blood cell volume during exercise. It has previously been shown that red blood cells express MCT proteins with a high affinity in equines and could have a large part in lactic acid clear-ance from plasma, especially during exercise [30,31].
Previous studies show that endurance training results in a significant upregulation of the expression of both MCT1 and MCT4 [15] in human skeletal muscle, or selectively MCT1 [32] in rodents. In this study, we could not observe significant changes in the expression of MCT1 or MCT4 after training. Recently, similar results have been found by Millet et al in human cyclists after a period of intermittent anaerobic training [17]. It has been suggested that adaptations in expression of MCT1 due to exercise and training are intensity dependent. In line with this hypothesis, Baker et al., (1998) showed no increase in rat muscle MCT1 expression after moderate intensity training but did find an increase in some muscles after high intensity training [14]. Pilegaard et al., showed increase in MCT1 and MCT4 during high intensity training [16]. In contrast, Juel et al. and Bickham et al., were not able to detect increases in MCT4 protein expression after sprint training in human subjects [33,34]. Compared to these studies, the training load in our study were relatively low. This could be one explanation for the absence of an increase in the expression of MCT1 and MCT4 in equine skeletal muscle.
To the best of our knowledge, the effect of training on the expression of MCT2 has not been demonstrated, yet. We found an increase in MCT2 protein expression after 18 weeks of training. Although this increase was not significant due to large individual differences, it is plausible to suggest that this may be the result of an increase in SCFA metabolism after training. In contrast to humans and rodents, SCFA are a major energy substrate in horses. As MCT2 may transport monocarboxylates with a special emphasis on SCFA, as proposed in this paper, the metabolic potential of these substrates may be underestimated during sub-maximal exercise.
The activity of LDH showed a small, non-significant, decrease that was also observed in other studies [15]. Parallel to the decrease in total LDH activity, the relative expression of LDH-5 isoenzyme was also decreased (NS). Decreases in LDH activity have been observed previously after training in horses [21,35] and are considered an adaptation to endurance training and associated with concomitant increases in fat oxidation.

Conclusion
We report, for the first time, that MCT1, MCT2 and MCT4 proteins are abundantly expressed in skeletal muscle of exercising physically trained Standardbred trotters. This implicates that lactic acid homeostasis during exercise in equines is regulated in a similar pattern as in other mammals. The relative high abundance of MCT2 might implicate a regulatory role in short chain fatty acid transport in equine skeletal muscle. Upon training MCT2 expression showed a non-significant increase, and may reflect an increase in SCFA metabolism after training. This is supported by the observation of a (also non-significant) decrease in LDH activity and decrease in relative LDH-5 isoenzyme expression. We suggest that hindgut formation and the subsequent