INTRODUCTION

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CONSIDERABLE EVIDENCE for a lactate transport system has been demonstrated in a variety of tissues, including the perfused rat hindquarter (40) and perfused rat heart (16), isolated intact muscles (2, 15), highly purified plasma membranes (23, 30), giant sarcolemmal vesicles (14, 18), and isolated cardiac myocytes (26, 37) and vesicles (34, 35). The above-mentioned studies have shown that the lactate carrier system in the heart and skeletal muscle functions as a stereoselective proton symport facilitating the electroneutral cotransport of H⁺ and lactate (13, 22, 29, 37).

Recently a monocarboxylate transporter (MCT-1) was cloned independently by two groups (4, 8, 9). In rats, MCT-1 is present in skeletal muscle and the heart (20) as well as in many other tissues (9). In skeletal muscles there is a high correlation between MCT-1 and 1) lactate uptake, 2) heart-type lactate dehydrogenase content, and 3) citrate synthase activity (20). Overexpression of MCT-1 in Chinese hamster ovary cells (32) or in chronically stimulated skeletal muscles (19) increased lactate uptake. This indicates that MCT-1 is a critical transport protein for the movement of lactate across the sarcolemma.

Muscle activity patterns may be an important determinant for establishing the capacity of lactate transport in muscle. When muscle activity is reduced by hindlimb unweighting (6) or denervation (17), lactate transport is decreased. After chronic muscle stimulation (18) and exercise training (21, 25), lactate transport is increased. The increase in muscle lactate transport capacity is also related to the intensity of training (25). Presumably, these changes in lactate transport in muscle occur in response to concurrent changes in MCT-1. However, there are no studies available to show whether the training-induced increase in lactate transport in skeletal muscle is associated with an increase in MCT-1, whether such changes are more prevalent in oxidative and/or glycolytic types of skeletal muscles, and whether the exercise training intensity influences the magnitude of the changes that can occur. In addition, there are no studies that have examined the influence of exercise training on lactate uptake by the heart. In this tissue the lactate transport system is the major route for the elimination of protons from myocytes (36).

We have investigated the effects of training on 1) MCT-1 in oxidative and glycolytic rat hindlimb muscles and in the heart and 2) lactate uptake in isolated perfused hearts and in isolated soleus (Sol) muscles. Because the increase in muscle lactate transport capacity is also related to the intensity of training (25), we also examined 3) the effects of two training programs, with different running intensities, on heart and muscle MCT-1 and lactate uptake. Furthermore, because it appears that some transport proteins in muscle are increased within days of the onset of exercise training [e.g., glucose transporter protein isoforms GLUT-1 and GLUT-4 (24, 28)], we examined 4) the training-induced changes in MCT-1 every week in muscles and heart for 3 wk in each of the two training programs. We hypothesized that MCT-1 and lactate uptake in the heart would not be affected by training but that, in skeletal muscle, MCT-1 and lactate uptake would 1) be increased with training and 2) be increased more with more intense training and 3) that the increase would be greater in oxidative types of skeletal muscles.

	METHODS

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Animal care and training programs. Female Sprague-Dawley rats were housed in an air-conditioned room on a 12:12-h light-dark cycle. They were fed a diet of Purina rat chow and water ad libitum. Animals commenced training between 10 and 12 wk of age and were matched according to body weight as closely as possible. Rats were randomly assigned to a control or to one of two endurance-training groups. All rats were familiarized with a motor-driven treadmill at low speeds (~15-20 m/min, 8% grade) for ~5-10 min/day for 3-5 days. Control animals were either killed before initiation of training (day 0) or remained sedentary (limited to cage activity) for the duration of the training program (day 20), at which point they were killed. For training purposes the remaining rats were subdivided into either a moderate-intensity (21 m/min, 8% grade) or a high-intensity (31 m/min, 15% grade) running group. In both groups training occurred 5 days each week for 1 h/day for up to 3 wk. Because of the severity of treadmill running at 31 m/min up a 15% grade, the high-intensity trained group required periodic rests for the first 2 days of training. Thereafter, they ran continuously for 1 h each day. Each week some of the animals (n = 4-5) were killed, 24 h after the last training session, with an intraperitoneal injection of pentobarbital sodium (65 mg/100 g body wt). While animals were under anesthesia, before death occurred, heparin (500 IU/kg) was administered intravenously. Skeletal muscles [red (RG) and white gastrocnemius (WG), extensor digitorum longus (EDL), and Sol] were rapidly removed, frozen in liquid nitrogen, and stored at 80°C until analyzed for MCT-1 and citrate synthase activity. Lactate uptake was determined in fresh strips of isolated rat Sol muscles and in isolated, perfused rat hearts.

Lactate uptake by isolated perfused myocardium. Thirty seconds after the heparin was administered, hearts were rapidly excised and placed in perfusion buffer at 4°C. Isolated rat hearts were perfused in the Langendorff mode as described by Ferdinandy et al. (7). Briefly, each heart was immediately cannulated through the aorta and preperfused at 37°C, in a retrograde manner, at a constant perfusion pressure equivalent to 100 cm of water (10 kPa). The preperfusion medium consisted of a modified Krebs-Henseleit bicarbonate buffer (KHBB) containing (in mM) 118 NaCl, 4.3 KCl, 2.4 CaCl₂, 25 NaHCO₃, 1.2 KH₂PO₄, 1.2 MgSO₄, and 10 glucose, gassed continually with 95% O₂-5% CO₂ (pH 7.4 at 37°C). After 15 min, the hearts were then perfused with glucose-free KHBB containing 1 mM unlabeled lactate as substrate and 5 µCi of L-[U-¹⁴C]lactate and 5 µCi of D-[1-³H]sorbitol. All perfusate was filtered to remove any precipitated or particulate contaminants. In preliminary experiments we found that a 90-s perfusion period was sufficient to equilibrate the radiolabel in the perfused hearts while at the same time lactate uptake was still increasing linearly and oxidation was minimal (<10%). We did not take this small fraction of oxidized lactate into account in our studies. This has also been done in other studies when lactate oxidation was low [cf. Refs. 2, 15, 23 (text and references)]. At the end of the 90-s perfusion period, a small portion of the left ventricle (~50 mg) was obtained, blotted, and frozen in liquid nitrogen. The samples were stored at 80°C until analyzed for ¹⁴C and ³H in digested extracts (2, 17, 23). The remainder of the heart was dissected free of great vessels and atria and used for citrate synthase and MCT-1 determinations. All samples were stored at 80°C until analyzed.

Lactate uptake into intact skeletal muscle in vitro. Lactate uptake measurements were performed as previously described (2, 17, 23) in strips of Sol muscles (17). Briefly, the Sol muscle was separated from underlying connective tissue, and the lateral sections were stripped longitudinally into small strips from tendon to tendon by using a needle. Sol strips were then preincubated in 1.5 ml of gassed (95% O₂-5% CO₂) KHBB containing 10 mM glucose and 0.2% fatty acid-free bovine serum albumin (BSA) at 37°C for 15 min in a shaking water bath (80 cycles/min). The muscles were then transferred to new incubation vials that contained 1.5 ml of gassed KHBB, 0.2% BSA, 1 mM unlabeled lactate, 1 µCi L-[U-¹⁴C]lactate, and 1 µCi D-[1-³H]sorbitol. In preliminary time course experiments, an incubation time of 5 min provided the optimal measurement point for lactate uptake in Sol muscle strips because lactate oxidation was low (<15%), lactate uptake was in the linear range, and D-[1-³H]sorbitol had equilibrated with the extracellular compartment. To terminate lactate uptake, Sol strips were rapidly removed from the incubation medium, rinsed in 0.9% ice-cold saline, cut free of tendons, and snap-frozen in liquid nitrogen. The samples were stored at 80°C until analyzed for ¹⁴C and ³H in digested muscle extracts (2, 17, 23).

Sample preparation for Western blotting. Proteins were isolated from muscles (Sol, EDL, RG, and WG) and the heart for Western blotting as previously described (20). Briefly, muscles (~50 mg) were homogenized in 2 ml of buffer A [(in mM) 210 sucrose, 2 ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 40 NaCl, 30 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 5 EDTA, and 2 phenylmethylsulfonyl fluoride, pH 7.4] for two interrupted 15-s bursts with a Polytron homogenizer set at eight. Homogenates were transferred to centrifuge tubes, and 2 ml of buffer A, used to rinse the Polytron, were added to the tubes. Then, 3 ml of buffer B (1.167 M KCl, 58.3 mM tetrasodium pyrophosphate) were added, mixed briefly, and then set on ice for 15 min. After centrifugation at 230,000 g for 75 min at 4°C, the supernatant fluid was discarded and the pellet was washed thoroughly with 1-2 ml of buffer C [10 mM tris(hydroxymethyl)aminomethane (Tris) base and 1 mM EDTA, pH 7.4]. The pellet was resuspended in 600 µl of buffer C and homogenized for two interrupted 10-s bursts with a Polytron set at seven. Then, 200 µl of 16% sodium dodecyl sulfate were added, samples were removed from ice, vortex mixed, and centrifuged at 1,100 g for 20 min at room temperature. The supernatant was divided into aliquots and stored at 80°C for protein assay and immunoblot detection of MCT-1.

Western blotting of MCT-1. A polyclonal anti-peptide antibody similar to that used by Garcia et al. (9) directed against the COOH terminus of MCT-1 was produced by immunizing New Zealand White rabbits with a synthetic peptide corresponding to amino acids 478-494 of MCT-1. This antibody was raised and affinity purified as described elsewhere (4). The polyclonal antibody yielded a single band on a Western blot that corresponded to a molecular weight of ~43,000, consistent with the molecular weight reported for MCT-1 (8, 9). In preliminary work, MCT-1 detection was blocked by the synthetic peptide and MCT-1 was found to be abundant in rat red blood cell ghosts as expected (data not shown).

Citrate synthase determination. To monitor the mitochondrial adaptation to endurance training, the Sol, EDL, RG, WG, and the heart were assayed via a standard fluorometric method (5) for maximal citrate synthase activity after the third week of training at both intensities (i.e., moderate and high) and in control animals. Briefly, 5-10 mg of tissue were homogenized in 250 µl buffer [(in mM) 16 Na₂HPO₄, 4 KH₂PO₄, 5 2-mercaptoethanol, 0.5 mM EDTA as well as 0.02% BSA and 50% (vol/vol) glycerol, pH 7.4] by using a glass pestle. Homogenates were sonicated and stored frozen at 80°C. Homogenates were diluted 50-fold in 20 mM imidazole-HCl, 0.02% charcoal-filtered BSA. This diluted homogenate (10 µl) was used in the assay that was started by adding 100 µl of reagent 1 (50 mM Tris · HCl, 0.4 mM acetyl-CoA, 0.5 mM oxaloacetate, 0.25% BSA, pH 8.1) and allowed to react for 1 h at room temperature (this reaction is linear for more than 1 h). Ten microliters of 0.5 N NaOH was added, and the sample was heated to 95°C for 5 min. One milliliter of reagent 2 (100 mM Tris · HCl, 100 µM ZnCl₂, 0.01% BSA, 30 µM NADH, 0.003 U/ml citrate lyase, 3 U/ml malate dehydrogenase, pH 7.5) was added and left for 20 min, after which 60 µl of 1 N HCl were added, left for 10 min, followed by 100 µl of 6 N NaOH/imidazole, and heated to 60°C for 20 min. Citric acid standards were also included in the assay. The 6 N NaOH converts NAD⁺ to a highly fluorescent compound that was read on a fluorometer.

Statistical analysis. A one-way analysis of variance was used to analyze the effects of training on cardiac and individual skeletal muscle MCT-1 contents and citrate synthase activities. Lactate uptake data were assessed with a t-test. All data are reported as means ± SE.

	RESULTS

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The mean body weights of all the animals before training was 248 ± 3.0 g (n = 46). The moderately trained animals (21 m/min, 8% grade, 5 × 1 h/wk) gained ~20 g/wk over the 3-wk training period. This resulted in a 24% increase over their pretraining weights (P < 0.05), and this increase was similar to the weight gain during the 3-wk period in the control group (P > 0.05). In the high-intensity trained group (31 m/min, 15% grade, 5 × 1 h/wk), weight gains were more modest (5 g/wk), resulting in a net weight gain of 7% by the end of the third week of training (P < 0.05). This weight gain in the high-intensity trained group was lower than in the moderately trained and control groups (P < 0.05).

Heart and muscle citrate synthase activity. No changes were observed in citrate synthase activity in any of the skeletal muscles in the moderately trained group after 3 wk of training (P > 0.05). In the high-intensity trained group, training caused a significant increase in citrate synthase activity in Sol (+69%) and RG (+37%) (P < 0.05) but not in the EDL and the WG (P > 0.05) (Fig. 1). In the heart, citrate synthase activity was not increased in either the moderately trained or high-intensity trained groups (P > 0.05) (Fig. 1).

View larger version (37K): [in this window] [in a new window]   Fig. 1.   Citrate synthase activity in control muscles and heart and in muscles and hearts obtained from rats trained for 3 wk at a moderate running intensity (21 m/min, 8% grade, 1 h/day, 5 days/wk) and a high running intensity (31 m/min, 15% grade, 1 h/day, 5 days/wk). Values are means ± SE; n = 4-5 animals at each point. WG, white gastrocnemius; EDL, extensor digitorum longus; Sol, soleus; RG, red gastrocnemius; prot, protein. * P < 0.05 high-intensity trained vs. control animals.

MCT-1 in heart and skeletal muscles. With Western blotting we were able to detect a single band for MCT-1 in skeletal muscles and heart from trained and untrained animals (Fig. 2). This corresponds to observations in previous studies from our laboratory (19, 20). MCT-1 content was greatest in the heart (Fig. 3). There was a strong relationship between the mean values of citrate synthase activities and the MCT-1 content of the tissues (r = 0.98, Fig. 3).

View larger version (51K): [in this window] [in a new window]   Fig. 2.   Western blots of monocarboxylate transporter 1 (MCT-1) protein in Sol muscles before training (week 0) and at end of each week of training (21 m/min, 8% grade and 31 m/min, 15% grade) and in heart before training (week 0) and at end of each week of training (21 m/min, 8% grade). Note heart and Sol muscle data are not directly comparable because 10 µg protein/lane for heart and 30 µg protein/lane for Sol muscle were used.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Relationship between citrate synthase and MCT-1 in heart and selected muscles. Values are means ± SE; n = 4-5 animals at each point.

MCT-1 adaptations in metabolically heterogeneous skeletal muscles. In the moderately trained group, no changes were observed in MCT-1 content in Sol, RG, EDL, and WG muscles during the 3-wk training period (Fig. 4) (P > 0.05). With the higher intensity training, MCT-1 was increased by 70% in the Sol (P < 0.05) and 94% in the RG (P < 0.05) muscles at the end of the 3-wk training period (Fig. 4). This increase was already apparent after the first week of training in both muscles, with an increase of 30% in Sol MCT-1 content (P < 0.05) and an increase of 32% in the RG MCT-1 content (P < 0.05). In contrast, MCT-1 was not changed in either the EDL or WG with the high-intensity training (P > 0.05) (Fig. 4).

View larger version (14K): [in this window] [in a new window]   View larger version (12K): [in this window] [in a new window]   Fig. 4.   MCT-1 content in rat hindlimb skeletal muscles at weekly intervals during moderate- and high-intensity treadmill running (1 h/day). Values are means ± SE; n = 4-5 animals at each point. A-D: Sol, RG, EDL, and WG, respectively. For each muscle, MCT-1 content at onset of training (week 0) has been set at 100%. See Fig. 3 for differences in MCT-1 among muscles.

MCT-1 adaptations in the heart. MCT-1 content in the heart increased progressively with moderate training (Fig. 5, P < 0.05), attaining a 36% increase after 3 wk (i.e., 15 h) of running. When the training intensity was increased, a progressive MCT-1 increase was also observed in the heart (Fig. 5, P < 0.05), attaining a 44% increase after 3 wk (i.e., 15 h) of training (P < 0.05). Changes in MCT-1 in the heart between moderately trained and high-intensity trained animals did not differ (P > 0.05).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   MCT-1 in rat hearts at weekly intervals during moderate- and high-intensity treadmill running (1 h/day). Values are means ± SE; n = 4-5 at each point. For each muscle, MCT-1 content at onset of training (week 0) has been set at 100%.

Lactate uptake in skeletal muscles. Lactate uptake into incubated Sol muscle strips from sedentary control animals on days 0 and 20 did not differ (P > 0.05). Therefore, these data were pooled. At the end of the training period, lactate uptake in the moderately trained and control Sol muscle strips did not differ (Fig. 6, P > 0.05). In contrast, there was a marked increase (+79%) in lactate uptake by the Sol muscles from the high-intensity trained group (P < 0.05) (Fig. 6).

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Lactate uptake in Sol muscle and in rat hearts from control and 3-wk moderately trained animals (21 m/min, 8% grade, 1 h/day, 5 days/wk) and high-intensity (31 m/min, 15% grade, 1 h/day, 5 days/wk) trained animals. Values are means ± SE; n = 4-5 animals at each point. * P < 0.05, moderately or high-intensity trained vs. control animals. ** P < 0.05, high-intensity vs. moderately trained animals.

Lactate uptake in the heart. No changes in myocardial lactate uptake were observed in control animals between days 0 and 20 (P > 0.05). These data were therefore pooled. After 3 wk of training, lactate uptake was increased in both the moderately trained (+72%) and high-intensity trained groups (+173%) (P < 0.05) compared with in the control animals (Fig. 6). The increase in the high-intensity trained group was significantly greater than in the moderately trained group (P < 0.05) (Fig. 6).

	DISCUSSION

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This is the first study to show that MCT-1 can be increased in heart and skeletal muscles by exercise training. In skeletal muscles we found that 1) MCT-1 was increased only when the training intensity of training was high, 2) the MCT-1 increase occurred only in highly oxidative skeletal muscles (i.e., Sol and RG) not in glycolytic muscles (i.e., EDL and WG), and 3) in the Sol muscle, lactate uptake increased only when MCT-1 was also increased. In the heart we observed that 4) MCT-1 was increased at a lower training intensity than in skeletal muscles, 5) MCT-1 did not increase further when the training intensity was increased, 6) the increase in MCT-1 occurred independently of changes in the heart's oxidative capacity (as measured by citrate synthase activity), and 7) lactate uptake was increased much more after high-intensity training than after moderate-intensity training, despite similar increases in heart MCT-1 with these two training intensities.

MCT-1 and lactate uptake in control muscles and heart. In the present study the greater concentrations of MCT-1 in more oxidative types of muscles, the high correlation between MCT-1 and citrate synthase activity, and the greater MCT-1 content in the heart than in skeletal muscles confirm recent observations from this laboratory (20). On the basis of the greater concentrations of MCT-1 in oxidative muscles (present study, Refs. 19 and 20), and the correlations between MCT-1 and citrate synthase activity (present study, Ref. 20) and MCT-1 and heart-type lactate dehydrogenase content (20), we suggest that the primary role of MCT-1 is to facilitate the movement of lactate into the muscle cell and to dispose of this substrate via oxidative metabolism because the rate of glyconeogenesis in highly oxidative muscles is low (1, 3). Lactate uptake by the muscle cell can be from the the circulation and from nearby fibers in which lactate is produced. In the heart MCT-1 is primarily located on myocyte membranes facing each other, suggesting that MCT-1 facilitates the transfer of lactate and protons from myocyte to myocyte (12).

Effects of training on MCT-1. Training increased MCT-1 in the heart and only in some of the muscles. In the heart, MCT-1 increments occurred at the low training intensity (21 m/min, 8% grade, 1 h/day). Increasing the training intensity (31 m/min, 15% grade, 1 h/day) did not not result in a further increase in heart MCT-1 content. In contrast, skeletal muscle MCT-1 was increased only in the oxidative muscles (Sol and RG) and only with the higher running intensity (31 m/min, 15% grade, 1 h/day). Thus in Sol and RG there was clearly a critical level of running activity required before an increase in MCT-1 was observed. Presumably, at the higher training intensity all the muscles examined (Sol, RG, WG and EDL) were recruited, but it appears that the contractile activities of the WG and EDL were not sufficient to provoke changes in MCT-1. Although glycolytic muscle fibers contain little or no MCT-1 protein (20), MCT-1 content of these fibers can be increased because chronic electrical stimulation of the EDL elicited a large increase in MCT-1 (19). This lends credence to our argument that an adequate contraction stimulus is required, particularly in glycolytic muscles, before increases in MCT-1 will be observed.

Lactate uptake and MCT-1. It would be desirable to use a nonmetabolizable analog of lactate to examine the transport of this substrate. However, such an analog is not available. Therefore, in this study, as well as in others, we have used short incubation (2, 17) or perfusion periods (19, 20) to minimize the oxidation of lactate. In this manner, the lactate that accumulates in the tissue provides an index of lactate transport. Previously, it has been shown that lactate uptake in isolated Sol muscles and lactate transport into sarcolemmal vesicles are reasonably comparable (23). Therefore, we feel that the tissue lactate accumulation in this study provides a reasonable index of lactate transport.