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1Department of Sports Sciences, University of Tokyo, Meguro-ku, Tokyo 153-8902, Japan; and 2Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada N1G 2W1
Submitted 16 December 2003 ; accepted in final form 13 April 2004
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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lactate; plantaris; soleus; tibialis anterior; heart; distance; monocarboxylate transporter
In skeletal muscle, MCT1 and MCT4 are coexpressed. Glycolytic muscle fibers express considerable quantities of MCT4, whereas MCT1 is predominantly present in oxidative muscle fibers in both slow- and fast-twitch muscles (2, 5, 12, 16, 36, 42, 47). It has been suggested that this MCT distribution among different types of muscle fibers may indicate that MCT1 is primarily involved with taking lactate up into the myocyte, whereas MCT4 may be primarily involved with extruding lactate (2, 5, 33). A number of studies have shown that training (1, 3, 12, 41) and chronically increased muscle contraction (5, 37) can increase MCT1 and MCT4 protein expression, as well as increase lactate transport (3, 5, 35, 37). Along with the suggestion that MCT1 and MCT4 have different roles, it also appears that MCT1 and MCT4 are regulated independently as animals age (22) and by the hormone 3,5,3'-triiodothyronine (48). Some studies also indicate that the expression of MCT1 and MCT4 is regulated differently by contractile activity. For example, an increase in MCT1 was only observed when exercise intensity exceeded a threshold (1), and with more intense aerobic exercise the increments in MCT1 were greater than in MCT4 (12, 41). Thus, although exercise intensity appears to be a factor regulating MCT1 and MCT4 expression, in chronic muscle stimulation studies it appeared that MCT1 expression was increased in relation to the amount of contractile activity that had been performed (5).
Whether the quantity of exercise can influence the expression of MCT1 and MCT4 has not been examined. However, this can be examined using voluntary wheel running, which is a low-intensity model of exercise that can induce adaptations similar to that found after low-intensity endurance training (14, 25, 43). Although there is considerable interanimal variation in the amount of self-selected running activity (14, 25, 32, 43), this variation, however, provides an opportunity to determine whether differences in the amount of running are related to the overexpression of MCTs. Because it appears that MCT1 overexpression is more easily induced than that of MCT4 (5, 41), we hypothesized that, with low-intensity wheel running, MCT1 would be upregulated, whereas MCT4 would not be altered. Therefore, we compared the changes in MCT1 and MCT4 in relation to the amount of exercise performed after 1, 3, and 6 wk of voluntary wheel running. For these purposes, we examined the heart and selected hindlimb muscles to discern whether there were specific responses in muscles with differing fiber composition.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Male ICR mice (Clea Japan, Tokyo, Japan), 4 wk of age, were used. Food (Oriental Yeast, Tokyo, Japan) and water were provided ad libitum. The room temperature was maintained at 23 ± 1°C with a 12-h light (2100 to 0900) and 12-h dark (0900 to 2100) cycle. All mice were weighed each week at the same time of day. Ethical approval for this work was obtained from the Committee on Animal Care at the University of Tokyo.
Training
Mice were randomly divided into control group or voluntary running wheel group for 1, 3, or 6 wk. Wheel running mice were housed individually in a cage (22 x 9 x 8 cm) with a running wheel (0.2-m diameter; Clea Japan). Mice ran freely in the wheel. The numbers of wheel revolutions were counted regardless of the running direction of the mice. Running distance was determined daily from the number of daily revolutions. Control group mice were housed with three per cage and did not have access to a running wheel.
Standardized Exercise Test
After 1, 3, or 6 wk of training, all mice performed a brief, standardized bout of exercise. Before this treadmill exercise, all mice had been familiarized with treadmill exercise by having run on the treadmill for 3–5 min on two to three occasions. The standardized exercise bout consisted of short-duration, intense treadmill running (i.e., 2 min of warming up at 20 m/min, 2 min of rest, and 2 min of running at 40 m/min). Immediately after the exercise, mice were killed by cervical dislocation. Within 5 min, all blood and tissue samples were obtained; specifically, the chest cavity was opened and blood was obtained via cardiac puncture, followed immediately by the removal of the heart and selected lower limb hindlimb muscles [soleus (Sol), plantaris (Pl), and tibialis anterior (TA)]. Tissues were frozen in liquid nitrogen and stored at –80°C until analyzed for MCT1 and MCT4 content. Concentrations of circulating lactate were determined enzymatically (19).
To examine the influence of MCT1 and MCT4 content on running performance, one group of animals ran in the running wheel for only 1 day. They did not perform the standardized exercise test, but instead they were killed by cervical dislocation at the end of the dark-light cycle, when they had completed most of their wheel running. Muscles (Pl, TA) were removed, frozen in liquid nitrogen, and stored at –80°C until analyzed for MCT1 and MCT4 content.
MCT Proteins
For the analysis of MCT, proteins were isolated from muscles as our laboratory has described previously (4, 5, 16, 36, 47, 48). MCTs were detected with Western blotting. Antibodies for MCT1 and MCT4 were raised in rabbits against the oligopeptide corresponding to the COOH terminus regions of each MCT (Qiagen, Tokyo, Japan). The procedures for the detection of MCT1 and MCT4 have been described in detail by our laboratory previously (4, 5, 16, 36, 47, 48). MCT1 and MCT4 blots were quantified using a scanner (model GT-8700, Epson, Tokyo, Japan) with appropriate software (NIH Image 1.62, National Institutes of Health, Bethesda, MD).
Statistical Analyses
All results are expressed relative to age-matched mice housed without access to a running wheel. Data are expressed as means ± SE. The group data were analyzed with analyses of variance. Significance was determined with Scheffé's F-test. Correlational analyses were used to compare the effects of running distance on MCT1. Significance was established at P < 0.05.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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There was a large increase in average daily running distance during the first half of the 6-wk experimental period. The running distance of the mice increased significantly from 3,014 ± 414 m/day (average of 7 days) during the first week to 4,013 ± 394 m/day (average of 7 days) in the third week (P < 0.05). Thereafter, the average daily running had reached a plateau during the next 3 wk. In week 6, the animals ran 4,207 ± 1,022 m/day (average of 7 days), which did not differ from the average daily running in week 3 (P > 0.05). At the end of weeks 1, 3, and 6, mice had run total distances of 21.1 ± 2.9, 83.6 ± 8.3 and 176.7 ± 42.9 km, respectively. However, there were large variations in the amount of the accumulated running distances among the mice over the 6-wk period (Table 1).
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Body weights were significantly lower in the wheel running group (P < 0.05) (Fig. 1). Except for the Sol muscle in week 3, no significant differences in the weights of the sampled muscles (mg muscle/g body wt) were found (Table 2).
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MCT1 and MCT4 proteins
Compared with the control groups at weeks 1 and 3, no changes in MCT1 protein were observed after 1 and 3 wk of running (P > 0.05; Fig. 2). Significant increases were observed in MCT1 after 6 wk of running in heart (+50%), Pl muscle (+31%), and TA muscle (+60%), compared with the 6-wk control group (P < 0.05; Fig. 2). There was an unexpected reduction in MCT1 in the Sol after 6 wk of running, compared with the 6-wk control group (–20%; P < 0.05).
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Relationship Between Running Distance and MCTs
Despite the fact that significant changes in MCT1 were not evident on a group basis until week 6, there were individual animal variations in MCT1 and the amount of running performed in the study. Therefore, MCT1 expression was compared with the amount of running completed after either 1 day or 1, 3, and 6 wk of voluntary wheel running.
Day 1 and week 1. There was no relationship between the 1-day running distance and MCT1 protein in either the Pl or TA muscles, when examined individually or when data of the two muscles were combined (Fig. 3). However, after 1 wk, there was an inverse correlation (r = –0.70, P < 0.05) between running distance and MCT1 protein in the hindlimb muscles (Sol, Pl, TA) (Fig. 4). This inverse correlation was also evident in the individual muscles, Pl (r = –0.86, P < 0.01), Sol (r = –0.76, P = 0.06), and TA (r = –0.65, P = 0.11). No relationship was evident between heart MCT1 and running distance (r = –0.31, P > 0.05).
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Week 6. There was a positive relationship between running distance and MCT1 in week 6 in the TA muscle (r = +0.72, P < 0.05), but this was not observed in the other two muscles (Pl: r = +0.51, P = 0.24; Sol: r = +0.57, P = 0.13). Therefore, there was no relationship when the data from the muscles were combined and correlated with accumulated running distance (r = +0.13, P > 0.05; Fig. 4). No relationship was observed between MCT1 in the heart and accumulated running distance (r = +0.30, P > 0.05).
MCT4. MCT4 in Pl and TA muscles did not correlate with running distances in weeks 1 (r = –0.39), 3 (r = 0.49), and 6 (r = 0.28) (P > 0.05). In addition, there were no relationships between MCT1 and MCT4 in the same muscles in any of the weeks.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Group Adaptations to Wheel Running Exercise
The well-known reduction in postexercise blood lactate that occurs with endurance training was also observed in the present study. Similarly, the increase in MCT1 after 6 wk of exercise training parallels previous observations in training studies with rodents (1, 16) and humans (3, 12, 41). Although differential tissue responsiveness to exercise has been observed for MCT1, with heart MCT1 being more easily induced than muscle MCT1 (1), the group changes in MCT1 in the heart were comparable to those of the TA and greater than in the Pl after 6 wk. Although others have shown that MCT4 is also increased with training (12, 16, 41), it appears from a number of studies that MCT1 expression is more easily induced than MCT4 (5, 41). When muscle contraction is highly aerobic, such as occurs with chronic low-frequency muscle stimulation, then only an increase in MCT1 expression is observed, whereas MCT4 is not altered (5). Because previous studies have shown that wheel running represents a low-intensity type of exercise (25, 43), it is quite likely that the upregulation of only the MCT1, but not MCT4, in the present study reflects the low intensity of wheel running exercise.
Variations in Running and MCT1 in Muscle at Selected Periods of Wheel Running
Variations in wheel running. It was evident from the data that considerable variation occurred in the running activities among animals, as well as in the MCT1 content in the muscles under investigation. It is well known that there are large differences in self-selected, wheel running activity among animals (14, 25). The running performances of the mice in our studies compare well with those of others (14, 25, 32, 45). The coefficient of variation (CV = 100·SE/mean) in running in our study (CV range 14–28% over the 6-wk period) is similar to those in other studies (CV range 10–21%) (14, 25). Moreover, the average running distance per day in our study is also comparable to that observed in voluntary wheel running for mice (14). We have no data on the quality of running activity that was performed, but it has been shown previously that the number of sustained 1-min running bouts is increased over an 8-wk period of wheel running (45). Others have shown that maximal oxygen uptake (32, 45) and mitochondrial enzymes (25) are modestly increased in mice after 8 wk of voluntary wheel running. Thus self-selected wheel running induces metabolic adaptations that are qualitatively similar to those observed with more intense, forced treadmill exercise training.
Variations in MCT1 responses to wheel running. Some very unique and unanticipated results were observed in the present study. Despite the fact that no statistically significant differences in MCT1 expression were observed in the wheel running groups after 1 and 3 wk of exercise, there were nevertheless considerable variations in MCT1 in the muscles examined. This prompted us to compare the MCT1 concentrations in the muscles with the amount of wheel running completed after 1 day and 1, 3 and 6 wk. We opted to compare the MCT1 responses with the total running distance completed, because previously our laboratory (5) had observed a good relationship between the increase in MCT1 and the amount of contractile activity in chronically stimulated muscles. We established at the outset (i.e., day 1) that mice with a greater content of muscle MCT1 at the beginning of the exercise program (day 1) were not a priori predisposed to more running. Indeed, the mice in this group ran the same amount on the first day as the average daily running distance observed in the 1-wk group. We then determined whether the accumulated running distances, after 1, 3, and 6 wk, were related to the MCT1 responses in mouse muscles.
MCT Responses at Selected Weeks of Running
MCT1 responses after the first week of wheel running. Because the mean MCT1 content did not differ between the control and wheel running animals (P > 0.05) after 1 wk of running, despite the fact that there was a significant negative correlation between running distance and MCT1, it was evident that some muscles were already increasing their MCT1 content, whereas others were reducing their MCT1 content. Importantly, these changes occurred in relation to the amount of exercise performed over the entire week (r = –0.70). Interestingly, the key changes occurred when animals had run >20 km or <20 km in the first week. In the animals that had run 18.3 ± 0.3 km, there was an increase in MCT1 compared with the control group (+37%; P < 0.05), whereas in those mice that had run considerably further (26.2 ± 0.7 km; P < 0.05), there was a reduction in MCT1 relative to the control group (–16.1 ± 7.9%; P < 0.05). Thus the quantity of running appeared to be a key predictive factor in the changes observed in MCT1, albeit not necessarily a positive factor if running exceeded 20 km in the first week.
We are not aware of any studies that have reported a negative dose-dependent response to exercise training within the first week. This is not surprising because most studies, unlike self-selected wheel running, control the amount of exercise performed. However, there is some evidence that metabolic adaptations are not necessarily positive within the first few days of increased muscle activity. For example, after 3 days of chronic stimulation it appears that GLUT4 is reduced by 50% in rat hindlimb muscle (50) and that citrate synthase activity is also reduced (
20%) after 2 days of chronic stimulation (46). Thus there is evidence that the expected adaptive responses are at times counterintuitive within the first week of increased muscle activity (present study and Refs. 46, 50).
Even with long-term muscle stimulation, metabolic responses are not necessarily positive. For example, with long-term chronic stimulation, our laboratory (4) has first observed an increase in MCT1 after 7 days in white TA muscle, followed by a decrease in MCT1 with prolonged stimulation (21 days). This also parallels time-dependent increases and reductions in some enzymes after prolonged, chronic muscle stimulation (10, 24, 34). Although the reasons for the transient changes in muscle metabolic adaptation in the first week are not known, it has been proposed that muscle remodeling, which accompanies prolonged (i.e., weeks) muscle contraction, may interfere, in an unknown manner, with maintaining the levels of some enzymes (9, 34) and perhaps MCT1 (4). Analogously, when exercise training is begun, some metabolic remodeling is also initiated, and in the very early phases (
1 wk) some reductions in some, but not all, proteins may occur (46, 50). Whether muscle damage occurs with "excessive" wheel running exercise in the first week of training is not known, but such presumed damage could reduce MCT1. Eccentric exercise-induced muscle damage can within 2 days reduce the GLUT4 transcription rate, GLUT4 mRNA, and GLUT4 protein content in rat skeletal muscle (30). Clearly, the present study and others (46, 50) have shown that reductions in selected proteins can occur within the first week of training, whereas the present study and others (3) have also shown that an increase in protein expression (MCT1) can occur. It appears that for MCT1, if the metabolic insult (i.e., running distance) in this very early phase (week 1) is too great, MCT1 will be repressed, whereas when it is more modest MCT1 is increased.
MCT1 responses after 3 wk of wheel running.
As was observed after 1 wk, there were no mean differences in MCT1 content in muscles when the control and wheel running animals were compared. But, just as in the week 1 group, some animals increased muscle MCT1 with exercise, whereas others had a reduced muscle MCT1 content, relative to controls. In contrast to the relationship between running distance after the first week of running, there was a positive relationship between the accumulated running distance over 3 wk and the skeletal muscle MCT1 (r = 0.76). A threshold in MCT1 responses appeared to exist at 100 km of accumulated running over the 3 wk period. This became more evident when the animals were subdivided into those that had run >100 km (152.5 ± 9.1 km in 3 wk) and those that had run <100 km (76.6 ± 5.9 km in 3 wk; P < 0.05). Compared with the control animals, there was an increase in MCT1 (+21 ± 8.5%; P < 0.05) in animals that had run further, whereas there was a reduction in MCT1 in the animals that had run less (–25 ± 7.5%; P < 0.05). Rodnick et al. (43) have shown that, in freely exercising rats, there can be marked differences with self-selected run distances, such that in their study there were three identifiable groups of runners: group 1, 2–5 km/day; group 2, 6–9 km/day; and group 3, 11 km/day. The greatest changes in oxidative capacity occurred in the animals that ran the most, because running intensity was similar among the groups (43). Mice that were genetically predisposed for running (10 km/day), when compared with normally running mice (5 km/day), showed a greater increase in muscle oxidative enzyme activity (25). Our data indicate that only when the exercise accumulation has exceeded a certain limit [i.e., running >100 km over 3 wk (7.3 km/day)] is there an increase in MCT1. We have no obvious explanation as to why MCT1 was reduced in some of the animals, despite having run 76.6 km (3.6 km/day) in 3 wk. Although in some studies no changes in enzyme activities occurred with wheel running (25, 43), none has observed a decrease relative to control animals. If exercise intensity had been insufficient, we would have expected no change in MCT1 relative to the control group; our laboratory (22) has shown previously that MCT1 expression is quite stable in mature rat muscle.
Despite the unusual observation in the week 3 group, it should be noted that the positive relationship between MCT1 and accumulated running after 3 wk reported in this study is robust. Recently, as part of other ongoing studies, the 3-wk wheel running study was repeated, and similar results were again observed. Indeed, the data from that study and the present study fell on the same regression line (data not shown). Thus it appears that the 3 wk MCT1 data are not an anomaly, although it is not clear why there is an MCT1 reduction in some animals.
MCT1 responses after 6 wk of wheel running. After 6 wk, MCT1 was increased in all Pl and TA muscles compared with the control group, whereas there was an unexpected reduction in soleus MCT1 content, for which we have no obvious explanation. However, others (23) have also observed transient adaptive responses with wheel running. For example, there was an improved insulin-stimulated glucose uptake in soleus muscle after 1 wk of wheel running, but this effect was lost after 2 and 4 wk of wheel running (23).
After 6 wk of running, there was no correlation between running distance and MCT1 content, even when the Sol muscle data were removed. It would appear therefore that, in the Pl and TA muscles, the critical running distance required to upregulate MCT1 had been attained. Hence, any correlation between MCT1 and accumulated running distance was lost. This parallels observations in chronically stimulated muscles in which MCT1 was upregulated in proportion to ascertain the amount of contractile activity, but once a maximum had been attained no further increase in MCT1 occurred despite the maintenance of more muscle activity (5). Others have observed that, with exercise training, enzymatic adaptations plateau and further exercise only serves to maintain the increased enzyme activity (13). Thus it appears that, after 6 wk of wheel running, the full MCT1 adaptations had occurred for this mode of exercise, and therefore no relationship with accumulated running distance was observed.
Exercise Intensity
The model used in the present study is generally considered to be a low-intensity exercise model. This appeared to contribute to the lack of change in MCT4, because this protein is increased when exercise intensity is high (12, 41). A certain level of exercise intensity is also required to increase MCT1 (1). However, these observations are based on studies using controlled quantities of exercise within the experimental groups. It appears, based on the present study, that the quantity of exercise, when exercise intensity is low, is also important in increasing MCT1. These factors (exercise intensity and exercise quantity) are often inversely related. The relative importance of these factors for inducing MCT 1 or MCT4 expression is not known.
Summary
We have shown that voluntary wheel running can reduce blood lactate accumulation after a standardized bout of intense exercise and increase MCT1 expression after 6 wk. The self-selected, low-intensity wheel running failed to alter the MCT4 content of the muscles. There was no relationship between changes in MCT1 and MCT4 or between the MCTs and postexercise circulating lactate. Because of the variations in self-selected running, we were able to observe distinct phases in MCT1 responses at different points over the 6-wk training period. It appears that, within the first week of training, MCT1 is negatively correlated with the accumulated running distance. On further analysis, it appears that excessive running represses MCT1, whereas more modest amounts of running increase MCT1 in week 1. In the third week of running, a positive relationship is observed between MCT1 and running distance, although as after 1 wk there is a threshold that must be exceeded before an increase over the control animals occurs. Below this threshold, MCT1 is reduced. Finally, except for the Sol muscle, MCT1 was increased in the TA and Pl muscles after 6 wk of wheel running. The observed increases in MCT1 in week 6 were not correlated with running distances, suggesting that an optimal exercise-induced adaptation in MCT1 had occurred between 3 and 6 wk of running, beyond which MCT1 did not increase further.
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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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.
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REFERENCES |
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