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Expression and Cellular Localization of Glucose Transporters (GLUT1, GLUT3, GLUT4) During Differentiation of Myogenic Cells Isolated from Rat Fïtuses

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Expression and Cellular Localization of Glucose Transporters (GLUT1, GLUT3, GLUT4) During Differentiation of Myogenic Cells Isolated from Rat Fïtuses Journal of Cell Science 107, 487-496 (1994) 487 Printed in Great Britain © The Company of Biologists Limited 1994 Expression and cellular localization of glucose transporters (GLUT1, GLUT3, GLUT4) during differentiation of myogenic cells isolated from rat fœtuses Isabelle Guillet-Deniau*, Armelle Leturque and Jean Girard Centre de Recherche sur l’Endocrinologie Moléculaire et le Développement, 9 rue Jules Hetzel, 92 190 Meudon Bellevue, France *Author for correspondence SUMMARY Skeletal muscle regeneration is mediated by the prolifera- cose, was 2-fold higher in myotubes than in myoblasts. tion of myoblasts from stem cells located beneath the basal Glucose deprivation increased the basal rate of glucose lamina of myofibres, the muscle satellite cells. They are transport by 2-fold in myoblasts, and 4-fold in myotubes. functionally indistinguishable from embryonic myoblasts. The cellular localization of the glucose transporters was The myogenic process includes the fusion of myoblasts into directly examined by immunofluorescence staining. multinucleated myotubes, the biosynthesis of proteins GLUT1 was located on the plasma membrane of myoblasts specific for skeletal muscle and proteins that regulates and myotubes. GLUT3 was located intracellularly in glucose metabolism, the glucose transporters. We find that myoblasts and appeared also on the plasma membrane in three isoforms of glucose transporter are expressed during myotubes. Insulin or IGF-I were unable to target GLUT3 fœtal myoblast differentiation: GLUT1, GLUT3 and to the plasma membrane. GLUT4, the insulin-regulatable GLUT4; their relative expression being dependent upon glucose transporter isoform, appeared only in contracting the stage of differentiation of the cells. GLUT1 mRNA and myotubes in small intracellular vesicles. It was translocated protein were abundant only in myoblasts from 19-day-old to the plasma membrane after a short exposure to insulin, rat fœtuses or from adult muscles. GLUT3 mRNA and as it is in skeletal muscle in vivo. These results show that protein, detectable in both cell types, increased markedly there is a switch in glucose transporter isoform expression during cell fusion, but decreased in contracting myotubes. during myogenic differentiation, dependent upon the GLUT4 mRNA and protein were not expressed in energy required by the different stages of the process. myoblasts. They appeared only in spontaneously contract- GLUT3 seemed to play a role during cell fusion, and could ing myotubes cultured on an extracellular matrix. Insulin be a marker for the muscle’s ability to regenerate. or IGF-I had no effect on the expression of the three glucose transporter isoforms, even in the absence of glucose. The Key words: glucose transporter, localization, fœtal myoblast, rate of glucose transport, assessed using 2-[3H]deoxyglu- differentiation INTRODUCTION actin, myosin heavy chain, acetylcholine receptor and creatine phosphokinase (Davis et al., 1987; Shih et al., 1990; Pinset et A specific phenotype for skeletal muscle requires the prolifer- al., 1991). ation of myoblasts from multipotential stem cells, and their In adult skeletal muscle, glucose transport is a facilitative subsequent morphological and biochemical differentiation. diffusion process mediated by GLUT1 and GLUT4 glucose Myogenic cells can be isolated from fœtal or adult muscle. In transporters. They belong to a family of proteins that differ adult muscle, the mononucleated satellite cells are quiescent functionally in terms of tissue specificity, affinity for hexoses myoblasts that lie beneath the external basal lamina of and hormonal regulation: six isoforms, designated GLUT1 to myofibres; they are able to recapitulate the normal embryonic GLUT7, and a pseudo-gene (GLUT6) have been described development of skeletal muscle through proliferation and (Bell et al., 1990; Kasanicki and Pilch, 1990; Waddell et al., fusion to give rise to cross-striated, contractile myofibres, in 1992). The GLUT1 gene is expressed in a wide range of the case of muscle injury (Mauro, 1961). Therefore, on a func- tissues and cultured cells; GLUT2 mainly in liver and pan- tional basis, satellite cells are developmentally indistinguish- creatic β cells; GLUT4, the insulin-regulatable isoform, essen- able from embryonic myoblasts in that both serve as myogenic tially in muscle and adipose tissue; GLUT5 in intestine; and precursors. The steps involved in the myogenic process GLUT7 probably in liver microsomes. GLUT3 seems to be include: (1) the expression of muscle-specific activating the major glucose transporter of neuronal processes (Maher et factors, such as MyoD1 and myogenin (Thayer et al., 1989); al., 1992) and grey-matter regions of the brain (Haber et al., (2) the fusion of myoblasts into multinucleated myotubes and 1993). Using cDNA probes to the human GLUT3 sequence, the biosynthesis of muscle-specific proteins, such as muscle GLUT3 mRNA was detected in many tissues from human, 488 I. Guillet-Deniau, A. Leturque and J. Girard rabbit, monkey, rat and mouse, especially in brain (Yano et fibroblasts. Non-attached cells were recovered and plated at a density al., 1991). GLUT3 has been found to be weakly expressed in of 1.5×104 cells/ml onto gelatin-coated flasks (TPP), in MEM/199 human placenta (Shepherd et al., 1992) and, recently, in medium containing 10% horse serum, 5 mM glucose, and antibiotics. human testis and spermatozoa (Haber et al., 1993). Several In some experiments, the cells were cultured on an extracellular isoforms may be expressed simultaneously or successively in matrix (Matrigel, Beckton-Dickinson). The cells were fed fresh the same tissue: GLUT1 and GLUT3 are both expressed in the medium the day after plating. At day 4, the medium was replaced by MEM/199 medium without serum; the cells were allowed to fuse and brain (Maher et al., 1992; Pardridge et al., 1990; Gerhart et differentiate for 6 to 11 days at 37°C, in a 7% CO2 incubator. At day al., 1992), whereas GLUT1, very abundant in fœtal skeletal 8, 90% of the cells were multinucleated spontaneously contracting muscle, is replaced by GLUT4 in adult skeletal muscle (San- myotubes. Myoblasts were harvested after 48 hours of culture in talucia et al., 1992). MEM/199 medium containing 10% horse serum. The concentration The GLUT3 facilitative glucose transporter (a 496 amino of glucose was <0.25 mM in glucose-deprived medium containing acid isoform) was first cloned from a human skeletal muscle horse serum. cDNA library (Kayano et al., 1988) and found to have 64% Adult satellite cells were extracted from hind-limbs of 1-month-old and 58% amino acid identity with the GLUT1 and GLUT4 rats; muscles were finely sliced and dissociated in an enzyme mixture isoforms, respectively (Kayano et al., 1988; Bell et al., 1990). containing one part of 0.25% trypsin (Gibco) and two parts of colla- genase (type II, Worthington, 131 units/mg) in phosphate-buffered More recently, a mouse GLUT3 cDNA was cloned from a × β saline (PBS, pH 7.4), by agitation (3 15 minutes) at 37°C. Every 15 TC-3 murine cell-line and a mouse brain cDNA library. This minutes, the muscles were disrupted by trituration with a wide-bore mouse cDNA encodes for a 493 amino acid peptide that has pipette. After neutralization of the enzyme activity with an equal 83% amino acid identity with the human GLUT3 protein volume of DMEM containing 20% fœtal calf serum, the cell suspen- (Nagamatsu et al., 1992). The main difference resides in the sion was centrifuged for 10 minutes at 700 g and filtered to discard carboxy termini of mouse and human GLUT3 proteins (Maher muscle fibre debris. The cells were plated onto gelatin-coated flasks et al., 1992; Nagamatsu et al., 1992). Several antisera has been and cultured in DMEM containing 20% fœtal calf serum for myoblast raised against carboxy-terminal amino acids of the mouse proliferation. Adult myoblasts were harvested 2 days after plating. GLUT3 protein (Maher et al., 1992; Bilan et al., 1992; Gould Hexose uptake et al., 1992). They can detect GLUT3 protein in intact neuronal cells of the rat (Maher et al., 1991) and in plasma membranes Cells, grown on 13 mm gelatin-coated Thermanox coverslips (Nunc), were placed in serum-free medium for 4 hours before measurement of rat L6 myogenic cell line (Bilan et al., 1992). of hexose uptake. Coverslips were washed 4 times in Krebs-Ringer The kinetic parameters of the GLUT3 glucose transporter phosphate buffer without glucose, containing 0.5% BSA (130 mM were determined in Xenopus oocytes injected with mRNA NaCl, 5 mM KCl, 1.3 mM CaCl2, 1.3 mM MgSO4, 10 mM Na2HPO4, encoding for human GLUT3. This transporter was found to be pH 7.4), and incubated at 37°C in 1 ml buffer containing 1 mM 2- a D-glucose, D-mannose and D-xylose transporter (Gould et deoxy-D-glucose and 3 mCi/ml 2-deoxy-D-[1-3H]glucose al., 1991; Colville et al., 1993). The relative Km values of the (Amersham, Bucks, UK; 13.9 Ci/mmole), or 1 mM D-xylose + 0.1 transporters for 2-deoxyglucose are: GLUT3 (1.8 mM) < mCi/ml D-[U-14C]xylose (CEA, Saclay, France, 210 mCi/mmole). GLUT4 (4.6 mM) < GLUT1 (6.9 mM) < GLUT2 (17.1 mM) The uptake was linear for at least 15 minutes, and 10 minutes was (Burant and Bell, 1992). The expression of GLUT3, with a low chosen as the time for the assay. The coverslips were rinsed five times K for hexoses, may be required under conditions of high with cold PBS and immediately immersed in scintillant and counted. m In each experiment, 4 coverslips were used at each time point. Non- glucose demand or hypoglycæmia to utilize low concentrations specific uptake was measured by incubating cells in buffer contain- of blood glucose efficiently. ing 0.3 mCi/ml L-[1-3H(N)]glucose (New England Nuclear, UK, 20 Since the GLUT3 cDNA was first cloned in human fœtal Ci/mmole).
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