Training does not protect against exhaustive exercise-induced lactate transport capacity alterations

Nicolas Eydoux, Guillaume Py, Karen Lambert, Hervé Dubouchaud, Christian Préfaut, and Jacques Mercier

Laboratoire de Physiologie des Interactions, Service Central de Physiologie Clinique, Hôpital Arnaud de Villeneuve, 34295 Montpellier, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The effects of endurance training on lactate transport capacity remain controversial. This study examined whether endurance training 1) alters lactate transport capacity, 2) can protect against exhaustive exercise-induced lactate transport alteration, and 3) can modify heart and oxidative muscle monocarboxylate transporter 1 (MCT1) content. Forty male Wistar rats were divided into control (C), trained (T), exhaustively exercised (E), and trained and exercised (TE) groups. Rats in the T and TE groups ran on a treadmill (1 h/day, 5 days/wk at 25 m/min, 10% incline) for 5 wk; C and E were familiarized with the exercise task for 5 min/day. Before being killed, E and TE rats underwent exhaustive exercise (25 m/min, 10% grade), which lasted 80 and 204 min, respectively (P < 0.05). Although lactate transport measurements (zero-trans) did not differ between groups C and T, both E and TE groups presented an apparent loss of protein saturation properties. In the trained groups, MCT1 content increased in soleus (+28% for T and +26% for TE; P < 0.05) and heart muscle (+36% for T and +33% for TE; P < 0.05). Moreover, despite the metabolic adaptations typically observed after endurance training, we also noted increased lipid peroxidation byproducts after exhaustive exercise. We concluded that 1) endurance training does not alter lactate transport capacity, 2) exhaustive exercise-induced lactate transport alteration is not prevented by training despite increased MCT1 content, and 3) exercise-induced oxidative stress may enhance the passive diffusion responsible for the apparent loss of saturation properties, possibly masking lactate transport regulation.

carrier-mediated transport; MCT1 content; physical training


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE IMPACT OF TRAINING ON skeletal muscle lactate transport capacity has been studied, but the conclusions remain controversial because of different methodological approaches and experimental procedures. Some authors (27, 30) reported an increase in lactate transport in sarcolemmal vesicles after either repeated bouts of moderate-intensity running or a progressive endurance training program. Their results, however, were not comparable, because they chose different L-lactate concentrations and methodologies, i.e., zero-trans conditions vs. equilibrium exchange experiments. In contrast, Roth and Brooks (34) demonstrated that neither sprint-trained nor endurance-trained rats presented different apparent maximal transport velocity (Vm) and Michaelis-Menten constant (Km) of sarcolemmal lactate transport rate compared with sedentary rats. Furthermore, using soleus muscle strips and after 3 wk of moderate- or high-intensity treadmill endurance training in rats, Baker et al. (2) showed that lactate uptake was increased only at the higher training intensity.

A concordant relationship was also found in this last study (2) between the first monocarboxylate transporter isoform (MCT1) to be cloned and sequenced and lactate uptake in oxidative skeletal muscles. The proton-linked MCT1 correlates well with muscle fiber oxidative capacity and heart-form lactate dehydrogenase (LDH). It is highly correlated to citrate synthase activity and thus is only slightly expressed and detected in white muscle (25, 39). Because lactate represents a major metabolic end-product for glycolytic fibers, the extrusion of lactic acid implies the presence of another sarcolemmal protein. The MCT4 isoform (formerly called MCT3) fulfils this role because it is present in all muscle fibers except those that are totally oxidative (39). The main difference between MCT1 and MCT4 seems to lie in their regulation rather than in their intrinsic properties, because Wilson et al. (39) found that MCT1 and MCT4 responded differently to chronic electrical stimulation and denervation.

It is well documented that a bout of aerobic physical exercise markedly increases O2 uptake and consumption due to the increased skeletal muscle energy requirement. This increased O2 consumption further augments the generation of the reactive oxygen species (ROS) when the scavenging capacity of both nonenzymatic and enzymatic defense mechanisms are overwhelmed (8). This is especially the case during an acute bout of exhaustive exercise (19). ROS have been reported to cause modifications in cellular biochemical components such as protein, lipid, and DNA (1, 35). Furthermore, ROS, like lactate anion and proton, are suggested to be implicated in oxidative skeletal muscle fatigue (15, 35, 36). Indeed, Sen's review (35), among others, reported that ROS alter such transport systems as the potassium transport and finally contribute to the onset of fatigue. Polyunsaturated fatty acids are another ROS target, and their peroxidation may lead to fluidity and permeability alterations (8, 35). Moreover, lactate anion, independently from proton and thus pH modifications, may decrease muscle force production by inhibiting Ca2+ release from the sarcoplasmic reticulum and/or by changing ionic strength (15, 36).

In our laboratory, we found a decrease in lactate transport capacity at low physiological lactate concentration (1 mM) and a loss of the protein saturation properties at concentrations above the Km after an acute bout of exhaustive exercise (9). We obtained the same pattern of response with a submaximal nonexhaustive exercise (11). Although the mechanisms responsible for this are not clear, we speculated that free radical-induced lipid peroxidation could be involved; also, we cannot exclude the possible implication of MCT isoforms (yet unidentified) other than MCT1 and MCT4 to explain our findings.

The threefold purpose of the present study was 1) to investigate whether endurance training alters lactate/H+ transport capacity, 2) to determine whether such training limits or prevents the acute exercise-induced lactate transport alteration, and 3) to examine whether MCT1 expression is modified after a bout of exhaustive exercise following a training program compared with no training.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. All experiments were performed in accordance with the Helsinki accords for humane treatment of laboratory animals. Forty male Wistar rats weighing 170-200 g were individually housed in a temperature-controlled room and maintained with food and drink ad libitum in a 12:12-h light-dark cycle (lights on at 7 PM), thus allowing exercise during their active phase. The body weight of the rats was monitored throughout the experimental period. They were randomly divided into the following groups: control (C, n = 10), exercised (E, n = 10), trained (T, n = 10), and trained and exercised (TE, n = 10). All animals were familiarized with a motor-driven treadmill for 3-4 days, 5 min/day, on a 10% grade. The running speed was initially 15 m/min and was gradually increased to 25 m/min within 2 days. The C and E rats were maintained at this speed, grade, and running time for the duration of the training program (5 wk). Endurance-trained animals (T and TE) were made to run 5 days each wk for 1 h/day for 5 wk at 25 m/min on a 10% incline. Twenty-four hours after the final training or handling session, venous blood from E and TE rats was obtained by tail snipping of the caudal vein before and just after they were exercised to exhaustion (running parameters as above). A 50-µl aliquot of the pre- and postexercise blood samples was immediately mixed with 200 µl of ice-cold 7% perchloric acid and centrifuged at 1,500 g for 10 min at 4°C. The supernatant was analyzed enzymatically for lactate content according to the method of Gutmann and Wahlefeld (13). All rats were killed by cervical dislocation either at rest (C and T) or at the point of exhaustion (E and TE), scheduled at the same time of the day to eliminate diurnal effects and ~4 h after their last meal (fed state). The intensity of the acute bout of exhaustive exercise (25 m/min and 10% grade) was selected to represent a relative workload of ~70-75% of their maximal oxygen uptake (6, 19). Exhaustion was defined as the inability of the rats to run on the treadmill despite electric shocks and to upright themselves when placed on their back.

Reagents. Reagents of the highest quality available were purchased from Sigma Chemicals, unless otherwise stated.

Tissue preparation. After cervical dislocation, hindlimb muscles and the heart (Hrt) were rapidly removed. Portions of red and white gastrocnemius (RG and WG, respectively), soleus (Sol), and Hrt were quickly frozen in liquid N2 and stored at -80°C until used for biochemical analysis and Western blotting. The remaining hindlimb muscles (~18-20 g) were used for sarcolemmal isolation.

Biochemical analysis. Malondialdehyde (MDA) content was measured by the thiobarbituric acid method (29). Weighed portions (RG) were homogenized (10% wt/vol) with a glass tissue homogenizer in 8.1% SDS, acetate buffer (20% acetic acid solution adjusted to pH 3.5 with NaOH), and 0.8% aqueous solution of thiobarbituric acid. After heating at 95°C for 60 min, the red pigment produced was extracted with n-butanol-pyridine mixture, and the absorbance of the organic layer was measured at 532 nm. Tetramethoxypropane was used as an external standard, and results were expressed in nanomoles per milligram of protein in tissue homogenate.

Glutathione peroxidase (GPX) enzyme activity was determined in Sol, WG, and RG by use of the procedure of Flohé and Günzler (11) with t-butyl hydroperoxide as substrate. Briefly, GPX activity determination was based on a coupled reaction where the glutathione reductase enzyme oxidized the NADPH to NADP. GPX activity is directly related to the decrease in NADPH absorbance at 340 nm and is expressed as nanomoles of NADPH oxidized per minute per milligram of protein. We chose the GPX antioxidant enzyme because of the reported tight coupling between oxidative capacity and its activity and because GPX is the only antioxidant enzyme to be upregulated in muscles from exercise-trained rats (23).

Citrate synthase (CS) enzyme activity was assayed in RG according to Srere (37). Changes in absorbance were recorded over 3 min at 412 nm, and the results were expressed in micromoles per minute per gram of protein.

Muscle glycogen measurements were made according to the method of Lo et al. (24) on portions (30-50 mg) of predominantly RG. Muscles were boiled in 30% KOH saturated with Na2SO4 until homogenization (usually 30 min). Homogenates were kept on ice, and glycogen was precipitated by addition of a 1.2 volume of 95% ethanol. Samples were centrifuged for 30 min at 840 g, and pellets were resuspended in H2O. Assays were conducted on aliquots against appropriate blanks at 490 nm. Results were determined from a standard curve generated at the same time and are expressed in milligrams of glycogen per gram of tissue.

Sample preparation for Western blotting. Proteins were isolated from muscles for Western blotting, as previously described by McCullagh et al. (25), with slight modifications. Briefly, portions of Sol, WG, and Hrt muscle (~60 mg) were homogenized in 2 ml of buffer A (210 mM sucrose, 2 mM EGTA, 40 mM NaCl, 30 mM HEPES, 5 mM EDTA, and 2 mM phenylmethylsulfonyl fluoride, pH 7.4) for ten passes in a glass tissue homogenizer. Homogenates were transferred to ultracentrifuge tubes, and 2 ml of buffer A, used to rinse the glass homogenizer, were added to these tubes. An aliquot of 200 µl was partitioned and stored at -80°C for enzymatic activity measurements. Then, 2.85 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 base, 1 mM EDTA, pH 7.4). The pellet was resuspended in 570 µl of buffer C and homogenized for two interrupted 10-s bursts with an Ultra-Turrax T25 (Labo Moderne, Paris, France) set at 50% maximal power. Then, 3.33 µl of 16% SDS were added per milligram of muscle, and the 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 immunoblot detection of MCT1. Muscle protein concentrations were determined in triplicate by the bicinchoninic acid assay (Pierce, Interchim, Montluçon, France) with the use of BSA as a standard.

Western blotting of MCT1. A polyclonal antipeptide antibody (gift from Dr. G. A. Brooks, Department of Integrative Biology, University of California, Berkeley, CA) directed against the carboxy terminus of rat MCT1 was produced by immunizing New Zealand White rabbits with the synthetic peptide corresponding to amino acids 478-494 (PLQNSSGDPAEEESPV) (18). The polyclonal antibody yielded a single band on a Western blot that corresponded to 43 kDa, consistent with the molecular mass reported for MCT1 (12, 25, 39). In preliminary work, when the immunizing peptide was present at a concentration of 5 µg/ml, antibody binding was inhibited (data not shown).

Protein (20 µg) samples of the muscles, hearts, and prestained molecular mass markers (Bio-Rad, Ivry-sur-Seine, France) were separated on 12% SDS-polyacrylamide gels (150 V for ~90 min). Proteins were then transferred from the gels to polyvinylidene difluoride membranes (100 V for 60 min). Membranes were incubated on a shaker overnight at room temperature in buffer D [20 mM Tris base, 137 mM NaCl, 0.1 M HCl, adjusted to pH 7.5, 0.1% (vol/vol) Tween 20, and 5% (wt/vol) nonfat dried milk]. Membranes were then incubated with diluted carboxy-terminal MCT1 antibody (1:3,000) in buffer D for 2 h, followed by three washes in buffer E (buffer D without dried milk, one 15-min wash and two 5-min washes), followed by incubation for 1 h with goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (1:1,000; BI 2407, BioSys, Compiègne, France) in buffer E. Membranes were washed as before in buffer E, and MCT1 was detected with the use of an enhanced chemiluminescence detection method by exposing the membranes to film (Biomax MR, Kodak) at room temperature, according to the instructions of the manufacturer. Film was developed and fixed by means of a Hyperprocessor, RNP 1700 (Amersham, Les Ulis, France). MCT1 protein band densities were obtained by scanning the blots on a scanner connected to a Mac G3/300-MHz computer with appropriate software.

Sarcolemmal isolation and characterization. Sarcolemmal vesicles were purified from hindlimb muscles with a procedure routinely used in our laboratory (9, 10). All subsequent steps were carried out at 4°C. After elimination of fatty, nervous, and connective tissues, muscles (usually 15-20 g) were homogenized in ice-cold 250 mM sucrose, 1 mM EDTA, and 20 mM HEPES at pH 7.4 with two bursts (2 × 5 s) of the Ultra-Turrax T25 at 80% of maximal power. The homogenate was centrifuged twice at 900 g in a Sorvall RC-28S (Du Pont de Nemours, Germany) with an SA-600 rotor. Supernatants were filtered, and a 1-ml aliquot was partitioned and saved at 4°C for subsequent analysis. The remaining crude homogenate (CH) was diluted with a volume of KCl medium (3 M KCl, 250 mM sodium pyrophosphate, pH 7.4) equal to 10% of the CH volume and pelleted by ultracentrifugation in a Beckman 60Ti rotor (200,000 g for 45 min at 4°C). Pellets were resuspended with the use of Teflon pestle homogenization in 30 ml of sucrose medium. The suspension was centrifuged twice at 280 g, and supernatants were then collected and centrifuged (200,000 g, 45 min at 4°C). Pellets were homogenized with a glass tissue homogenizer in 7-8 ml of 40% (wt/vol) sucrose. A discontinuous density gradient was constructed by addition of 8 ml of each of the sucrose solutions, 38, 32, 27, and 12%, in layers. After an overnight centrifugation at 130,000 g at 4°C in an SW27 rotor, the 27% sucrose band (corresponding to sarcolemmal vesicles and termed F2) was harvested and diluted with a Krebs-Ringer-HEPES (KRH) buffer (118 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 50 mM HEPES, pH 7.5) and washed free of sucrose in a 60Ti rotor (200,000 g for 80 min). The vesicles were resuspended in KRH buffer up to 4 mg/ml and stored at -80°C until used for the transport experiments. Proteins were determined according to the procedure of Bradford (5), with bovine gamma globulin as a standard.

Sarcolemmal characterization was achieved with K+-stimulated p-nitrophenyl phosphatase (K+-pNPPase) assay, as described previously (9). Its total activity was measured in 40 mM HEPES, 0.8 mM EGTA, 4 mM MgCl2, 20 mM KCl, and 5 mM pNPPase, pH 7.4. The absorbance of the p-nitrophenol formed was read at 410 nm. Nonspecific K+-pNPPase activity was determined in a KCl-free medium, which, when subtracted from the total activity, gave the specific K+-pNPPase activity expressed in micromoles per hour per milligram. The purification index (PI) was defined as the ratio of the specific activity from the F2 fraction to the specific activity measured in CH. Skeletal muscle sarcolemmal yield was the ratio of milligrams of sarcolemmal protein obtained in F2 to the muscle weight in grams (wet weight).

Lactate transport studies. All measurements were performed in zero-trans conditions in duplicate. L-[U-14C]lactate (specific activity 155 mCi/mmol; Amersham) was diluted in 280 mM sucrose and 50 mM HEPES, pH 7.4, and different unlabeled L(+)-lactate concentrations. Reciprocal decreases in sucrose were used to maintain the same total isosmotic buffer strength. Reactions were initiated by delivering 50 µg of sarcolemmal vesicles in tracer-containing medium and stopped at appropriate time intervals by vacuum filtration on nitrocellulose filters (Whatman WCN, average pore size of 0.45 µm; Bio-Rad). Filters were then rinsed three times with an ice-cold isosmotic medium consisting of KRH buffer with 3 mM HgCl2, pH 7.4, dissolved with ethylene glycol monomethyl ether, and the radioactivity was counted in a scintillation analyzer (Packard 2200). Nonspecific transport activities were determined by preincubation of vesicles in tracer-containing medium with KRH buffer containing 3 mM HgCl2, which was used to fix the time 0 points. Results were expressed in nanomoles per milligram of protein. Measurements of initial lactate uptake were done for 1, 10, 30, 50, and 100 mM external lactate concentrations at 0 and 10 s. Slopes gave initial rates of lactate uptake expressed in nanomoles of lactate per minute per milligram of protein.

Statistical analysis. All results are expressed as means ± SE. Statistical significance was assessed by Student's t-test for nonrelated samples. Mean values of MCT1 content in each group were compared by one-way analysis of variance. Throughout the study, a probability level of P < 0.05 was used.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Body weight and endurance time. The mean body weights of the four groups of animals before training were 180-200 g (n = 40). After 5 wk of treadmill training, the body weights of the T and TE rats were 340 ± 9 and 340 ± 2 g, respectively, and the C and E rats weighed 374 ± 7 and 360 ± 12 g, respectively (Table 1). The net weight gains of the trained groups (T and TE) were significantly lower compared with those of the untrained groups (C and E) (P < 0.05). The mean endurance time of running to exhaustion was 80 ± 9 min for the E group and 204 ± 11 min for the TE group. Thus our 5-wk training program dramatically increased the endurance capacity of the rats.

                              
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Table 1.   Morphological and biochemical parameters of groups C, E, T, and TE

Blood lactate. Blood lactate concentration at the point of exhaustion was significantly elevated above rested values (preexercise, 1.47 ± 0.12 and 1.30 ± 0.06 mM; postexercise, 1.96 ± 0.20 and 1.51 ± 0.05 mM in the E and TE rats, respectively). Moreover, TE presented a lower increase in blood lactate postexercise compared with E (P < 0.05; see Fig. 1).


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Fig. 1.   Blood lactate concentration before and after exhaustive exercise in exercised (E, open bars) and trained exercised (TE, hatched bars) groups; n = 10. Values are means ± SE and expressed in mM. * Significantly different between groups, P < 0.05.

Muscle glycogen and MDA contents, CS, and GPX activities. As reported in Table 2, the results show a dramatic decrease in RG glycogen concentration, with 62 and 57% reductions in the E and TE groups compared with the C and T groups, respectively (P < 0.01). Although the decrease in muscle glycogen was lower after running to exhaustion when rats were trained, we did not observe a significant difference in glycogen content between the E and TE groups. Training also caused an increase in the resting activities of RG CS (+59%) and Sol and RG GPX (+93 and +159%, respectively). Whereas CS activity remained unaffected by the run to exhaustion, GPX activity decreased in the TE group compared with the T group for Sol and RG skeletal muscles (-36 and -38%, respectively). Changes in WG GPX activity were observed only after the single bout of exhaustive exercise, not after training (P < 0.01). MDA content in RG muscle was significantly increased at the point of exhaustion compared with rest (P < 0.05) in rats from both E and TE groups, which indicated that the extent of peroxidative reactions was enhanced.

                              
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Table 2.   Glycogen and MDA contents and CS activity in RG muscle, and GPX activity in Sol and RG and WG muscles from C, E, T, and TE rats

Sarcolemmal isolation and characterization. Using gradient density fractionation and the activity of the sarcolemmal membrane marker K+-pNPPase, we determined a purification index for each sarcolemmal vesicle preparation, defined as the ratio of the activity of the CH to the specific activity of the sarcolemmal fraction. As reported in Table 1, we obtained similar biochemical characteristics in the four groups of rats with regard to crude and purified membrane protein yield and K+-pNPPase activity. Hence, there were no significant differences in the purification index of each group.

Lactate transport capacity and MCT1 expression. Lactate uptake into sarcolemmal vesicles did not differ in the C and T groups, regardless of the concentration tested (Fig. 2; P > 0.05). Initial rates displayed saturation kinetics with a Vm of 355 vs. 410 nmol · min-1 · mg protein-1 and a Km of 28 vs. 32 mM in the C vs. T groups, respectively. In contrast, we observed a similar marked increase (P < 0.05) in the initial rate of 100 mM lactate uptake in the two exhaustively exercised groups (E and TE) compared with that in the unexercised groups (C and T; Fig. 2). Moreover, this increase in lactate transport capacity was associated with an apparent loss in the Michaelis-Menten saturation properties, because the uptake increased linearly with external lactate concentrations (Fig. 2).


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Fig. 2.   Kinetics of initial rate of lactate uptake into sarcolemmal vesicles from control (C), trained (T), E, and TE rats at various external lactate concentrations. Assays were performed in duplicate at pH 7.4 on 5-7 different membrane preparations, and results are shown as means ± SE. * Significantly different between C and E groups, P < 0.05; # significantly different between T and TE groups, P < 0.05.

Representative Western blots showing the expression of MCT1 in Hrt and Sol in the four groups are given in Fig. 3. In the two trained groups, MCT1 content was increased in both Sol (+28% for T and +26% for TE; P < 0.05) and Hrt (+36% for T and +33% for TE; P < 0.05). Moreover, running to exhaustion did not further increase the expression of sarcolemmal and cardiac MCT1 (group TE vs. group T; P > 0.05), as shown in Fig. 3.


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Fig. 3.   Representative Western blots of monocarboxylate transporter 1 (MCT1) protein and comparison of MCT1 content in heart (A) and in soleus muscle (B) from C, T, E, and TE rats. MCT1 content values are means ± SE; n = 5 in each group. Heart and soleus muscle MCT1 contents are expressed as a percentage of control group, where MCT1 content was set at 100%. * Significantly different, P < 0.05.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This is the first study to show that rodent lactate transport protein saturation properties were altered after an acute bout of exhaustive exercise irrespective of the trained state. These observations parallel those of a previous work, in which we observed a similar loss in the saturation kinetics after a single run to exhaustion (9). These changes were associated with an increased expression of muscle MCT1 isoform in both heart and soleus muscle after training. We also found that our running program elicited typical metabolic adaptations that are widely encountered in the literature, i.e., increases in citrate synthase and glutathione peroxidase activities and a lower blood lactate increase postexercise (4, 16, 23, 34). These modifications were concomitant with an elevation in lipid peroxidation byproducts, as indicated by the malondialdehyde content after the acute exercise run.

Discrepancies exist in the literature regarding the effect of training on lactate transport capacity. Roth and Brooks (34) observed no modifications after endurance and sprint training, whereas two other studies reported an increase (27, 32). In the present work, the absence of change in initial rates of lactate uptake between the control and trained rats confirms the former findings (34). The Michaelis-Menten Km constant and the maximal transport velocity, i.e., the Vm value of the two unexercised groups (C and T), were similar. Moreover, this absence of difference is in agreement with results in rats trained at a low 50% maximum oxygen uptake (VO2 max) intensity (32) and in noncompetitive trained subjects (30). The discrepancy concerning training effect seems to be due mostly to different experimental approaches and treadmill programs, and it appears that the muscle lactate transport system, in terms of protein affinity and velocity, requires minimal training intensity for adaptation. With a treadmill speed demanding ~78% VO2 max, McDermott and Bonen (27) concluded that the increase in lactate transport capacity was caused by a lowered posttraining Km, i.e., a higher affinity. Using giant sarcolemmal vesicles, others suggested both an increase in the amount of lactate carrier and a higher affinity for its substrate (32), but at present, and surprisingly, the well accepted notion that training increases lactate transport capacity has been based on observations of only one to three different lactate concentrations (27, 32). In addition, these studies present discordant findings for concentrations above the Km. Although Roth and Brooks had their own methodology and used female rats, one of the strengths of their study was that they performed transport assays at 10 different concentrations, at all of which they failed to obtain any change in lactate uptake. Given the different results reported, we chose five lactate concentrations ranging from 1 to 100 mM of lactate to surround the Km value of ~40 mM (34) and to determine kinetic parameters by plotting initial rates of uptake as a function of lactate concentrations.

The more recent studies showing a relationship between increases in MCT1 and lactate uptake in muscle or citrate synthase-related oxidative capacity also used different procedures or populations (2, 25, 31). Although we used a training program similar to those studied by other authors in terms of treadmill speed and grade (27, 34), we demonstrated a greater heart and soleus MCT1 content after training independent of any lactate uptake alteration. As already reported in experiments with moderate endurance training (2, 4), our findings confirm that MCT1 can be increased with training. In fact, we chose an average training intensity between the two values used by Baker et al. (2), and the data we obtained are also intermediate. This suggests that training intensity and duration seem to be important factors involved in the MCT1 expression level. Nevertheless, this alteration in MCT1 content in the trained group was not associated with a concomitant higher muscle lactate uptake compared with the control group. This indicates a minor role of MCT1 in the whole hindlimb lactate transport capacity. This in turn lends credence to the results of Price et al. (33), which demonstrate that MCT4 (formerly called mammalian MCT3) is the major lactate transporter isoform in muscle tissue, a role that the low MCT1 expression cannot fulfill (22). Moreover, in line with previous studies, the increase in MCT1 content occurs when the oxidative metabolism is enhanced, suggesting that the MCT1 isoform may be coordinately expressed with enzymes such as citrate synthase (2, 25).

We also showed for the first time that an acute run to exhaustion after a training period did not further alter sarcolemmal MCT1 expression, as opposed to what occurs for plasma membrane glucose transporters (14). The trained group exercised to exhaustion, and the untrained exhausted group shared the same pattern of response in terms of rates of lactate uptake. Despite different MCT1 contents, however, acute regulation of skeletal muscle lactate transport seems to rely on at least one other MCT isoform (25, 33). Indeed, we have previously shown that an acute regulation occurs independently of any MCT1 alteration and oxidative stress (11). Thus our findings contradict a previous study (26), in which a 38% increase in MCT1 in one T-tubule fraction of subcellular plasma membranes was reported after an acute exercise. In another study (4), the same authors stated that translocation of this transport protein was not likely; however, they gave no explanation for the T-tubule MCT1 increase. Similarly, Bonen and McCullagh (3) demonstrated that an acute bout of exercise slightly enhanced lactate transport. The authors suspected that this result was due to the novel experimental design they used. Until such reports are confirmed, our observations suggest that the MCT1-mediated lactate transport is regulated quite differently from that of the glucose transport system, which involves translocation (14, 28). Moreover, we hypothesize that MCT1 could represent only a minor part of the total protein involved in muscle lactate transport regulation during a single bout of exercise.

We observed a loss of the protein saturation properties in the two groups of exhaustively exercised rats. This confirmed our previous observations (9). Moreover, it is noteworthy that training did not protect against the effects of a single run to exhaustion. In fact, strenuous physical exercise-induced oxidative damage has received considerable attention in recent years, and the studies on oxidative stress are numerous (35). In organs such as skeletal muscle, antioxidant defenses act to cope with the reactive oxygen species produced during exercise (8) before they can attack biomembranes, leading to lipid peroxidation (8, 35). In the present investigation, we found a significantly increased concentration in malondialdehyde, a commonly used index for lipid peroxidation, in the exercised and trained exercised rats. As lipid peroxidation has been suggested to induce membrane selective permeability, i.e., loss of structural integrity (8), this increased permeability may explain the absence of the Michaelis-Menten saturation curve in the two exercised groups. In fact, under the physiological 1 mM lactate concentration, the carrier system accounts for 90% of the total flux. At higher concentrations, the relative part of passive diffusion increases, reaching 30% at 50 mM (21). Thus we can assume that an increase in membrane permeability caused by the exercise-induced oxidative stress would permit a higher lactate uptake into sarcolemmal vesicles by passive diffusion. Furthermore, because the protein yields and purification indexes of our membrane preparations were similar in the four groups, it is unlikely that exhaustive exercise interacted with the sarcolemmal purification procedure. Another relevant finding of our study was that the training sessions were sufficient to trigger a marked increase in GPX activity in active oxidative muscle fibers, as frequently reported (17, 20, 35, 38). Nevertheless, the training-induced increase in antioxidant defenses would confer only a limited ability to prevent generation of reactive oxygen species under an acute exhaustive exercise, as indicated by the 40% enhancement of MDA in the trained exercised rats. This was also evident in the study of Venditti and Di Meo (38), in which they noted a similar loss of sarcoplasmic and endoplasmic reticulum integrity as well, after completion of an exhaustive exercise. Consequently, we can assume that the short-term regulation reported elsewhere (11) could be masked in part by the deleterious effect of the exhaustive exercise-induced oxidative stress, especially at the higher lactate concentrations.

In summary, this study reports an apparent loss in the saturation properties of the sarcolemmal lactate transporter after an acute exhaustive exercise, irrespective of the training status. The present findings also demonstrate no training effect on lactate transport capacity and confirm that the observed alterations were independent of an increase in muscle MCT1 content. Because the MDA-lipid peroxidation marker increased at the point of exhaustion, we suspect that the exhaustive exercise-induced oxidative stress is a potent factor of membrane fragility and permeability, which in turn would explain the saturation loss by an increase in membrane passive diffusion. Because MCT1 seems to be coordinately expressed with enzymes of oxidative metabolism (i.e., citrate synthase), lactate transport regulation during a single bout of exercise may be mediated by one or more isoforms of a muscle lactate transport protein in addition to MCT1.


    ACKNOWLEDGEMENTS

The authors thank G. A. Brooks (Department of Integrative Biology, University of California, Berkeley) for providing us with the MCT1 antibody, M. Rossi (Laboratoire de Médecine Nucléaire, CHU Lapeyronie, Montpellier, France) for advice and technical assistance regarding the handling of the radiochemical materials, P. Boulanger and Dr. G. Roizès (Institut de Biologie, Montpellier) for their technical support, and A. Fabre and B. Aimetti (Institut de Biologie, Département de Physiologie) for their help in familiarizing the rats with the exercise task.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: N. Eydoux, Laboratoire de Physiologie des Interactions, Service Central de Physiologie Clinique, Hôpital A. de Villeneuve, 371 Avenue du Doyen G. Giraud, 34295 Montpellier cedex 5, France (E-mail: physio34{at}aol.com).

Received 12 August 1999; accepted in final form 13 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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