Actu Phpiol Scand 1992, 144, 469471
Lactate transport during differentiation of skeletal muscle cells: evidence for a specific carrier in L6 myotubes K. E L ABIDA, A. D U V A L L E T , L. T H I E U L A R T , M. R I E U and M. BEAUDRY Laboratoire d e physiologie des adaptations, CHU Cochin, 24 rue du Faubourg Saint Jacques, 75014 Paris, France A., THIEULART, L., RIEU,M. & BEAUDRY, M. 1992. Lactate EL ABIDA, K., DUVALLET, transport during differentiation of skeletal muscle cells: evidence for a specific carrier in L6 myotubes. Acta Physiol Scand 144, 469471. Received 12 June 1991, accepted 8 October 1991. ISSN 0001-6772. Laboratoire de physiologie des adaptations, CIIU Cochin, 24 rue du Faubourg Saint Jacques, 75104 Paris, France. Cellular uptake of lactate involves a carrier in numerous types of mammalian cells. In cultured rat L6 myoblasts, lactate diffuses freely. We show that during myogenesis a carrier appears at the myotube stage. The degree of cellular differentiation was determined by microscopic observation and by measurement of a marker of biochemical differentiation : the increase in specific activity of creatine kinases. Key words : lactate transport, myogenesis, myotubes.
I t has been clearly demonstrated in numerous types of mammalian cells (Halestrap 1974, Connett 1986), that facilitated lactate transport occurs across the cell membrane. However, in contrast to literature reports we have found that lactate diffuses freely in cultured rat L6 myoblasts (Beaudry 1991). This may be because the L6 muscle cell is at an early stage of development. T h e aim of the present study was to determine the mode of lactate transport in cultured L6 cells at a later stage of development, when cellular fusion of myoblasts produces myotubes.
MATERIALS AND METHODS Radioactive ~-[U-'~C]lacticacid (171 mCi mmol-') was purchased from Amersham (Amersham, UK). All other chemicals were from Sigma (St Louis, MO, USA). Cell culture. Undifferentiated muscle cells of the L6 line were used as described previously (Beaudry 1991). The cells were cultured until myotubes were Correspondence : M. Beaudry, Laboratoire de physiologie des adaptations, CHU Cochin, 24 rue du Faubourg Saint Jacques, 75014 Paris, France.
obtained. When confluence is reached, the cells begin to fuse to form multinucleated myotubes, which increase in size and number until most of the nuclei of the mononucleated cells are incorporated into the multinucleated cells. Determination of' lactate uptake rates. Cell monolayers were washed three times in phosphate buffer saline (PBS) pH 7.4. In standard experiments ( n = 16), cells were incubated for 10 s in /-['4C]lactate (0.2 mCi ml-') and unlabelled lactate (1-20 mM) and were layered in p€I 7.4 PBS buffered with 1-5 mM HEPES at 0 "C. Some experiments were performed using L-lactate uptake inhibitor. Uptake was stopped when required by addition of 1 ml of cold PBS. Cells were promptly washed three times in cold PBS containing 1 ml of 1 mM phloretin, which immediately stops lactate efflux, and were then harvested in 0.5 ml 0.1 N NaOH and radioactivity was counted in Ready Safe liquid scintillation cocktail (Beckman Instruments, Fullerton, CA, USA). Measurement of creutine kinase activity. After washing, the cells were harvested by scraping with a rubber policeman. The cellular creatine kinase activity was measured by spectrophotometry using the CKNAC-activated UV system kit from Boehringer (Mannheim, Germany). This assay was performed at
469
A'. EI 14hir/rr e t al.
470
diKerercnt stages of culture so as to detine changes in specitic activity during cellular development. 'T'hc cellular protein content was estimated using the method of Imvr! et i t / . (1951) after dissolution of the cells in 200 //I of 0.1 s SaOI3.
KESuT Ts 1
T h e specific actility of creatine kinase was very low in the m p b l a s t cultures (Fig. 1). I t began to rise when m!-otubes formed, and had increased
Fig. 1. 1,actate uptakc in L A m!-otubes metabolic content is expressed as nmol mg-' protein. Each value is the mean of the d u e s obtained from I6 separate experiments, and the standard error does not exceed 10 ()
Lactate transport during differentiation of skeletal muscle cells: evidence for a specific carrier in L6 myotubes K. E L ABIDA, A. D U V A L L E T , L. T H I E U L A R T , M. R I E U and M. BEAUDRY Laboratoire d e physiologie des adaptations, CHU Cochin, 24 rue du Faubourg Saint Jacques, 75014 Paris, France A., THIEULART, L., RIEU,M. & BEAUDRY, M. 1992. Lactate EL ABIDA, K., DUVALLET, transport during differentiation of skeletal muscle cells: evidence for a specific carrier in L6 myotubes. Acta Physiol Scand 144, 469471. Received 12 June 1991, accepted 8 October 1991. ISSN 0001-6772. Laboratoire de physiologie des adaptations, CIIU Cochin, 24 rue du Faubourg Saint Jacques, 75104 Paris, France. Cellular uptake of lactate involves a carrier in numerous types of mammalian cells. In cultured rat L6 myoblasts, lactate diffuses freely. We show that during myogenesis a carrier appears at the myotube stage. The degree of cellular differentiation was determined by microscopic observation and by measurement of a marker of biochemical differentiation : the increase in specific activity of creatine kinases. Key words : lactate transport, myogenesis, myotubes.
I t has been clearly demonstrated in numerous types of mammalian cells (Halestrap 1974, Connett 1986), that facilitated lactate transport occurs across the cell membrane. However, in contrast to literature reports we have found that lactate diffuses freely in cultured rat L6 myoblasts (Beaudry 1991). This may be because the L6 muscle cell is at an early stage of development. T h e aim of the present study was to determine the mode of lactate transport in cultured L6 cells at a later stage of development, when cellular fusion of myoblasts produces myotubes.
MATERIALS AND METHODS Radioactive ~-[U-'~C]lacticacid (171 mCi mmol-') was purchased from Amersham (Amersham, UK). All other chemicals were from Sigma (St Louis, MO, USA). Cell culture. Undifferentiated muscle cells of the L6 line were used as described previously (Beaudry 1991). The cells were cultured until myotubes were Correspondence : M. Beaudry, Laboratoire de physiologie des adaptations, CHU Cochin, 24 rue du Faubourg Saint Jacques, 75014 Paris, France.
obtained. When confluence is reached, the cells begin to fuse to form multinucleated myotubes, which increase in size and number until most of the nuclei of the mononucleated cells are incorporated into the multinucleated cells. Determination of' lactate uptake rates. Cell monolayers were washed three times in phosphate buffer saline (PBS) pH 7.4. In standard experiments ( n = 16), cells were incubated for 10 s in /-['4C]lactate (0.2 mCi ml-') and unlabelled lactate (1-20 mM) and were layered in p€I 7.4 PBS buffered with 1-5 mM HEPES at 0 "C. Some experiments were performed using L-lactate uptake inhibitor. Uptake was stopped when required by addition of 1 ml of cold PBS. Cells were promptly washed three times in cold PBS containing 1 ml of 1 mM phloretin, which immediately stops lactate efflux, and were then harvested in 0.5 ml 0.1 N NaOH and radioactivity was counted in Ready Safe liquid scintillation cocktail (Beckman Instruments, Fullerton, CA, USA). Measurement of creutine kinase activity. After washing, the cells were harvested by scraping with a rubber policeman. The cellular creatine kinase activity was measured by spectrophotometry using the CKNAC-activated UV system kit from Boehringer (Mannheim, Germany). This assay was performed at
469
A'. EI 14hir/rr e t al.
470
diKerercnt stages of culture so as to detine changes in specitic activity during cellular development. 'T'hc cellular protein content was estimated using the method of Imvr! et i t / . (1951) after dissolution of the cells in 200 //I of 0.1 s SaOI3.
KESuT Ts 1
T h e specific actility of creatine kinase was very low in the m p b l a s t cultures (Fig. 1). I t began to rise when m!-otubes formed, and had increased
Fig. 1. 1,actate uptakc in L A m!-otubes metabolic content is expressed as nmol mg-' protein. Each value is the mean of the d u e s obtained from I6 separate experiments, and the standard error does not exceed 10 ()
