Acta Physiol Scand 1991, 141, 379-381

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Lactate transport in skeletal muscle cells: uptake in L6 myoblasts M. BEAUDRY, A. D U V A L L E T , L. T H I E U L A R T , K. E L A B I D A and M. R I E U Laboratoire d e physiologie des adaptations, CHU Cochin, 24 rue d u Faubourg Saint Jacques, 75014 Paris, France. BEAUDRY, M., DUVALLET, A., THIEULART, L., EL ABIDA,K. & RIEU,M. 1991. Lactate transport in skeletal muscle cells: uptake in L6 myoblasts. Acta Physiol Scand 141, 379-381. Received 7 August 1990, accepted 11 October 1990. ISSN 00014772. Laboratoire de physiologie des adaptations, CHU Cochin, 24 rue du Faubourg Saint Jacques, 75014 Paris, France. During exercise, lactate is produced by degradation of glucose-6-phosphate during glycolysis in the contracting muscles. This lactate is metabolized during and after exercise in the muscle itself and also in the liver and other muscles, which can use it as an energy metabolite or can resynthetize glycogen. Lactate is transported in the blood, and the rate of muscular utilization may be limited by two factors: the rate of metabolic utilization by the muscle cell; and the rate of transport across the membrane regulating lactate transfer from the blood to the cell. We have studied lactate uptake in L6 muscle cells by incorporation of 14C-lactate. The uptake rates were linear for 20 seconds with 5 mM lactate and 10 seconds with 20 mM. The uptake during 10 seconds for physiological lactate concentrations (1-20 mM) gave a straight line passing through the origin. Lactate uptake was not altered by specific inhibitors of lactate transport (2.5 mM a cyano-4-hydroxycinnamic acid. 5 ,UM 4,4’-diisothiocyanostilbene-2,2’disulphonic acid) or by the stereospecific D-lactate inhibitor. The results suggest that L-lactate uptake in L6 cells occurs by passive diffusion. Key words: lactate uptake, myoblast, rat.

During and after exercise lactate produced by degradation of glucose-6-phosphate in contracting muscles is metabolized in the muscle, liver and other muscles. T h e transportation occurs via the blood, and two factors may intervene in the limitation of the rate of muscular utilization : the rate of transport across the membrane regulating transfer from the blood to the cell; and the rate of metabolic utilization by the muscle cell. Although the release from muscle cells has been studied quite extensively (Mason 1988, Juel 1989), its entry is not fully understood and contradictions remain, despite studies suggesting transmembrane transport (Koch 1981, Mason 1988). A variety of forms of transport have been

reported for other cell types from different species : transport by lactate/Na+ across the brush border of renal cells, by lactate/H+ exchange in human erythrocytes and rat hepatocytes, passive diffusion in toadfish hepatocytes and red blood cells of fish. Also it may be that this specificity does not depend on the state of cellular differentiation. T h e variation noted in lactate concentrations in the muscle and in the blood could be explained by differences in the mechanism of lactate uptake. I t is for this reason that we first studied transport of lactate between extra-intracellular media in undifferentiated cultured muscle cells. T h e L6 cell used here is comparable with the muscle satellite cell in terms of differentiation.

Correspondence : M. Beaudry, Laboratoire de physiologie des adaptations, CHU Cochin, 24 rue du Faubourg Saint Jacques, 75014 Paris, France

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MATERIALS AND METHODS Cell culture. Undifferentiated muscle cells of L6 line were used in this study. L6 is an established cell line obtained from rat muscles, immortalized by treatment with methylcholantrene. In culture, these mononucleated muscle cells go through a proliferative phase when they divide actively, When they achieve confluency the cells stop dividing and they align. These aligned cells can fuse to form multinucleated myotubes. Cells of L6 line were seeded at a concentration of 2 x lo3 cells/cm2 in 75 cm2 culture flasks. Cultures were grown in Dulbecco's Modified Eagle Medium (DMEM) cbntaining 5% foetal calf serum and 1% penicilin-streptomycin, in humidified air containing 7% CO,. Cell viability was greater than 95% as estimated by trypan blue exclusion. The standard medium was changed once every two days. Determination of lactate uptake rates. Subconfluent monolayer cultures were harvested by trypsin treatment (trypsin 0.25%, 10 min, 37 "C), and seeded in 2 cm2 tissue culture multiwell plate. Monolayers were washed three times in phosphate buffer saline CaZ+, Mg2+ free (PBS), pH = 7.4. Radioactive lactate was purchased from Amersham L [U14-C] lactic acid 172 mci/mMol. Other chemicals were purchased from Sigma. In standard experiments (n = 16) cells were incubated during various times in the C14 lactate and non labelled lactate (1-20 mM, 0.2 pCi ml-'), layered in PBS pH 7.4 buffered with hepes 15 mM at 20 "C. Some experiments (n = 16) were performed using L-lactate uptake inhibitors. Uptake was stopped when required by addition of 1 ml cold PBS. Cells were immediately washed three times in cold PBS containing 1 ml phloretin which stops lactate efflux, and were then harvested in 0.5 ml NaOH 0.1 N and counted in dynagel (J. T. Baker) for radioactivity. The cell protein content was estimated by use of the Lowry method.

I n our experimental conditions, specific inhibitors of lactate transport in mammalian cells have no significant effect on the rate of lactate uptake. Inhibitors and cells were preincubated 1 min before the uptake was performed. The uptake is not inhibited by 2,s mM a-cyano-4-hydroxycinnamate ( L U R = 0.25), a specific inhibitor of

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5 10 20 30 40 50 60 Time (in seconds) Fig. 1. Time course of lactate in intracellular water of L6 cells. The studies are performed at two physiological concentrations of lactate in extracellular water (A = 20 mM, B = 5 mM). The time scale represents the time of incorporation of C14and cold lactate in the myoblasts.

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RESULTS Lactate uptake rates (Fig. 1) were linear for about 20 s at 5 mM and 10 s at 20 mM L-lactate. L-lactate uptake rates a t 10 s were used in our experiments. After 30 s lactate uptake reached a plateau. In these conditions we assumed that lactate was not metabolized (Walsh 1987). When lactate uptake was performed at physiological concentrations of lactate (1-20 mM) (Fig. 2), saturation was not observed and the double reciprocal plot passed through the origin (Lactate uptake rate - LUR - with 5 mM LLactate = 0.278 nmol sec-' mg-' protein).

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Lactate (nmol* I-') Fig. 2. Relation between lactate concentration in extracellular and intracellular water. Metabolic content is expressed as nmol mg-' protein. Meansc! SEM are given. For each extracellular content, twelve measures are performed.

Lactate uptuke in L6 myoblusts lactate transportor by 5 ,LAM 4,4'-diisothiocyanostilbene-2,2'disulphonic acid (DIDS) (LUR = 0.259), which inhibited the mechanism of the general anion transporter, 5 mM D-lactate (LUR = 0.262), a stereospecific inhibitor of L-lactate uptake. These results show, that the uptake of Llactate by L 6 cells had no specificity. In addition, variations of the extracellular pH had no significant effect on L-lactate uptake (pH = 6, LUR = 0.282 - p H = 7.4, LUR = 0.278 - p H = 8, L U R = 0.301 nmol sec-' mg-' protein).

DISCUSSION Uptake of lactate by L 6 rat muscle cells was studied by measuring the initial rate of transport of labelled L-lactate at physiological concentrations and in the presence of inhibitors of nonspecific transport (DIDS) and specific transport (-cyano-4-hydroxycinnamate or D-lactate). It remained linear for at least 10 s at 20 mM and 20 s at 5 mM; so all data are expressed as the initial rate of uptake in nmol sec-' mg-' protein. Uptake is non-saturable and was not inhibited by two compounds classically used as inhibitors of lactate transport in mammalian cells. Furthermore, uptake is not stereospecific. These results are in agreement with those reported by Walsh (1987 - toadfish hepatocytes), and Moon (1986 - fish red blood cells) and suggest that transport of lactate involves simple diffusion. In contrast, Roth (1989) suggests that a specific transporter exists in the membrane of mature cells with a lactate/H+ cotransport, and a V,,, of 126.6 nmol mg-' protein minute-', and a K m of 40.6 mM. Indirectly, Jorfeldt calculated a maximal rate of lactate uptake approximately 0.08 mmoles min-' 100 ml-' muscle (1970 - men muscle). Extracellular changes in pH do not alter the rate of transport in rat L6 cells and toadfish hepatocytes, and hence p H does not seem to play an important role in diffusion. T h e L6 cell is an artificially modified, undifferentiated cell, which cannot synthesize

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certain specific proteins (myosin heavy chain, phosphorylase, phosphorylase kinase, phosphocreatine kinase). Similarly, certain membrane proteins such as acetylcholine receptors and catecholamine receptors differ between L6 muscle cells and mature cells (Wahrmann 1987). It is therefore possible that transport systems specific to lactate are absent. If they exist in mature mammalian cells, as Roth (1989) and Brooks (1989) have shown, lactate transporters may be produced during cellular differentiation, which modifies a large number of enzymes and enzymatic activities (creatine kinase and acetylcholinesterase) involved in bioenergetics, glycolysis in particular. The authors would particularly like to thank J. P. Wahrmann and D. Labie (INSERM U15) for ex-

perimental assistance and helpful suggestions. REFERENCES BROOKS, C.A. & ROTH,D.A. 1989. Characterization of the lactate transporter in muscle. Med Sci Sports Exerc, 21, S35. JORFELDT, L. 1970. Metabolism of L(+)Lactate in human skeletal muscle during exercise. Acta Physiol Scand, Suppl 338. JUEL,C. & WIBRAND, F. 1989. Lactate transport in isolate mouse muscles studied with a tracer technique - kinetics, stereospecificity, pH dependency and maximal capacity. Acta Physiol Scand 137, 33-39. KOCH,A., WEBSTER, B. & LOWELL, S. 1981. Cellular uptake of L-lactate in mouse diaphragm. Biophys 3 36, 775-796. MASON,M. J. & THOMAS, R. C. 1988. A microelectrode study of the mechanisms of L-lactate entry into and release from frog sartorius muscle. 3 Physiol400, 459-479. MOON,T.W. 1986. The utilization and uptake of L( +)-lactate by red blood cells of teleost fish. XXXth Intern Congress Physiol Sciences : Banff Satellite Symposium, July. M. & FAVARD-SERENO, WAHRMANN, J.P., RECOUVREUR, C. 1977. Development and regulation of the phosphorylase-glycogen complex in myogenic cells of the L6 line. 3 Cell Sci 26, 77-91. WALSH, P.J. 1987. Lactate uptake by toadfish hepatocytes : Passive diffusion is sufficient.3Exp Bid 130, 295-304.