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 fÏtuses

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 fÏtal 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 fÏtuses 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, fÏtal 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 fÏtal 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 fÏtal 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% fÏtal 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% fÏtal 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). Proteins were measured by the method of Bradford (Bio- skeletal muscle, we put forward the hypothesis that myoblasts Rad, Richmond, CA) on 8 coverslips on the same day as the transport may express the GLUT3 glucose transporter in primary assay. culture. Our work was performed on 19-day fÏtal muscle myoblasts, which are much more abundant than adult muscle Northern blotting satellite cells. The aim of this study was to determine the quan- Total RNA was extracted from cells by the guanidine thiocyanate µ titative expression of glucose transporter isoforms during myo- method (Chomczynski and Sacchi, 1987). Total RNA (15 g/lane) genesis. was electrophoresed in 1% agarose/0.66 M formaldehyde gels, and transferred to a nylon membrane (Hybond-N, Amersham), with 20× SSC (3 M sodium chloride, 0.3 M sodium citrate). Prehybridization was performed in 50% formamide, 8× Denhardt’s solution, 0.5 M MATERIALS AND METHODS NaCl, 1% SDS, 7.5% dextran sulphate, 200 mg/ml salmon sperm DNA and 40 mM Tris-HCl (pH 7.5), at 42¡C for 6 hours. Hybridiz- Cell culture ation was performed overnight in the same buffer containing 106 Primary cultures of myoblasts from 19-day-old rat fÏtuses were cpm/ml of [32P]cDNA probes, labelled with [32P]dCTP using the Mul- grown as follows: hind-limbs were removed from fÏtuses, rinsed four tiprime System kit (Amersham). GLUT1 was detected with a 1700 bp times in Hanks’ solution (Gibco) and maintained in a 2:1 mixture of cDNA probe to human GLUT1, and GLUT3 with a 600 bp cDNA to MEM/199 media (Gibco) without serum, for 1 hour at 4¡C. mouse GLUT3 (provided by Dr Graeme Bell, Howard Hughes The skin and fingers were removed under a stereoscopic micro- Institute, Chicago, Illinois). GLUT4 was detected with a 2086 bp scope. Muscles were thoroughly minced in MEM/199 medium con- cDNA probe to rat insulin-regulatable glucose transporter (a gift from taining 10% horse serum. Myoblasts were separated from muscle Dr David James, Washington University, St Louis, Missouri). After fibres by mechanical disruption (40 pipettings with a wide-bore stripping of the glucose transporter probes, the blots were rehy- pipette), and filtered through a 70 µm nylon filter. After centrifuga- bridized with a 300 bp probe to the non-coding region of rat skeletal tion (700 g, 2 minutes) to pellet cellular debris, cells were plated onto α-actin (a gift from C. Pinset, Institut Pasteur, Paris; Minty et al., 150 cm2 culture flasks (TPP-Switzerland) for 1 hour, to remove 1981). The amount of total RNA loaded was checked with a synthetic Expression and localization of glucose transporters 489 anti-sense oligonucleotide probe (Bioprobe System, Paris, France) gift from Dr G. W. Gould, University of Glasgow, Scotland; Gould labelled with [γ-32P]ATP by the bacteriophage T4 polymerase kinase, et al., 1992). representing the sequence 1047 to 1070 of the 18 S rat rRNA (Chan Filters were exposed to Hyperfilms MP (Amersham) for 1 to 60 et al., 1984). minutes for ECL detection, and overnight for 125I-Protein A detection. Autoradiographs of northern blots were quantified by scanning den- Quantification of western blots was performed by scanning densito- sitometry (Hoefer Scientific, San Francisco). metry.

Western blotting Immunofluorescence Cells were rinsed 3 times with cold PBS, placed in hypotonic buffer Cells were grown on gelatin or Matrigel-coated Permanox 2 chambers (10 mM Hepes, 0.1 mM MgCl2, pH 7.4) at 4¡C for 30 minutes and slides (Lab-Tek, Nunc, Denmark) for 2 to 11 days. They were washed disrupted in a Dounce homogenizer (40 strokes). The lysate was cen- 3 times with PBS and fixed with 3% paraformaldehyde for 20 trifugated at 900 g for 5 minutes to pellet unbroken cells and nuclei. minutes, rewashed extensively with PBS and quenched for 10 minutes The supernatant was centrifuged at 140,000 g for 1 hour at 4¡C. The with 50 mM NH4Cl. Before internal staining, cells were permeabi- pellet, containing crude membranes, was resuspended in membrane lized with PBS/0.1% Triton X-100 for 4 minutes. The insulin-regu- buffer (250 mM sucrose, 1 mM EDTA, 1 mM phenylmethylsulfonyl latable glucose transporter was detected by a monoclonal antiserum fluoride, 5 mM Na2HPO4, 5 mM NaH2PO4, pH 7.4). raised against a cytoplasmic portion of the rat GLUT4 (Genzyme, Samples of 40 µg proteins were fractionated on 12% SDS-poly- Boston, MA). Antisera were diluted in PBS/0.2% gelatin (dilution acrylamide gels with a Mini-protean II apparatus (Bio-Rad) and elec- 1/1000 for anti-GLUT3, 1/5000 for anti-GLUT1 and anti-GLUT4). trically transferred to reinforced nitrocellulose (Schleicher and Cells were incubated with diluted primary antisera overnight at 4¡C, Schuell, Germany). Filters were first incubated in Tris-buffered washed 3 times with PBS, and treated for 20 minutes with fluorescein saline/0.1% Tween-20 (TBS/T) containing 5% non-fat dried milk for (FITC)-conjugated goat anti-rabbit IgG (dil:1/160) for GLUT3 4 hours, as a blocking step, then shaked overnight with antisera detection, rhodamine (TRITC)-conjugated goat anti-rabbit IgG diluted in TBS/T+1% milk at room temperature. Next day, the (dil:1/100) for GLUT1 detection, and FITC-conjugated sheep anti- antibody solution was removed and the blots were washed 3 times mouse IgG (dil:1/128) for GLUT4 detection. The nuclei were stained with TBS/T and developed with the Amersham enhanced chemilu- with Hoechst 33258 (Hoechst, Germany). The slides were mounted minescence system (ECL), or with 125I-Protein A. The antisera used in glycerol containing Mowiol 4-88 (Hoechst) and observed with an were rabbit polyclonal antisera to the intracellular C terminus of rat Olympus IMT 2 fluorescence microscope. brain GLUT1 (amino acids 479-492, 1/500 dilution, East-Acres Bio- Fluorescein- and rhodamine-conjugated secondary antibodies, and logicals, Southbridge, MA), or to the C-terminal 13 amino acid other analytical grade reagents were purchased from Sigma Chemical sequence of mouse GLUT3 (amino acids 481-493, 1/100 dilution, a Co. (St Louis, MO)

Fig. 1. Phase-contrast photomicrographs of fÏtal myoblast living cultures. Multiplicating myoblasts 2 days after plating (A). Myoblasts beginning to line up 3 days after plating (B). Fusion of myoblasts into multinucleated myotubes at days 4 and 5 (C and D). The cross-striation of the contractile apparatus was detectable at day 6 (E). The network of myoﬁbres contracted spontaneously after 8 days of culture on Matrigel (F). A,C,D,E, ×200; B, ×300; F, ×100. 490 I. Guillet-Deniau, A. Leturque and J. Girard

RESULTS when 10−6 M dexamethasone was added for 48 hours to the culture medium (Fig. 2B). Time course of fÏtal myoblasts differentiation The evolution of the different glucose transporter mRNAs Photomicrographs of fÏtal myoblast living cultures are shown expressed during fÏtal myoblasts differentiation is summa- in Fig. 1. On day 2 after plating, the myoblasts looked like rized in Fig. 3. − − small multiplicating spindle-shaped cells (Fig. 1A). On day 3, The effects of addition of 10 7 M insulin or 10 8 M IGF-I, the cells began to line up (Fig. 1B). Cell fusion occurred on or glucose deprivation, on the expression of glucose transporter days 4 and 5, and multinucleated myotubes appeared (Fig. 1C mRNAs in 6-day-cultured fÏtal myotubes are shown in Fig. 4. and D). Branched myotubes were formed on day 6, and the These treatments had no effect on the state of differentiation cross-striation of the contractile apparatus was detectable (Fig. of the cells, as assessed by the α-actin mRNA level. GLUT3 1E). On day 8 after plating, the myotubes formed a network of mRNA was quite unaffected by a 24 hour treatment with spontaneously contracting fibres, more abundant in the insulin or IGF-I, or glucose deprivation; in the same way, tri- presence of Matrigel (Fig. 1F). iodothyronine (0.1 µM), dexamethasone (1 µM) or di-butyryl cAMP (100 µM) were inefficient (data not shown). Moreover, Expression of glucose transporters in differentiating GLUT1 mRNA, weakly expressed in fÏtal myotubes, was fÏtal myoblast increased by 3-fold after a 24 hour glucose deprivation, Glucose transporter mRNA expression whereas insulin or IGF-I had no effect. The evolution of the glucose transporters mRNA levels during fÏtal myoblasts differentiation is shown in Fig. 2. Glucose transporter proteins The state of cell differentiation was assessed by a muscle- GLUT1 protein was detected by western blotting in fÏtal specific marker, skeletal α-actin; α-actin mRNA appeared 3 myoblasts and in 6-day-cultured myotubes as a band of about days after plating, when the cells began to line up and fuse, as 45 kDa (Fig. 5B). GLUT3 protein appeared in 3-day-cultured a 1.5 kb mRNA; it increased up to 11 days, in parallel with myoblasts as 2 major bands of 50 kDa and 55 kDa, fainter non- morphological differentiation (Fig. 2A and B). specific bands appearing between 60 and 80 kDa when GLUT1, expressed as a single 2.8 kb mRNA, was abundant detection was performed with ECL system (Gould et al., 1992). in fÏtal myoblasts; it decreased quickly during cell fusion (day GLUT3 protein increased by 6-fold in 5-day-cultured 4), and remained constant thereafter (Fig. 2A). myotubes, and decreased strongly in 8-day-cultured myotubes A 4 kb GLUT3 mRNA was detectable in adult and fÏtal (Fig. 5A). myoblasts as well; it increased transiently by 10-fold at day 5, Fig. 5B shows the hormonal regulation of GLUT1 and in myotubes (Fig. 2A). GLUT3 mRNA was no more detectable GLUT3 proteins in 6-day myotubes cultured in the presence or in contracting myotubes cultured on Matrigel for 9 to 11 days in the absence of glucose (24 hours). Insulin and IGF-I had no (Fig. 2B). effect on GLUT1; the only factor capable of increasing GLUT1 GLUT4 mRNA was not expressed in fÏtal myoblasts, or in by 3-fold was glucose deprivation. Neither hormones (insulin, fœtal myotubes cultured on gelatin-coated flasks; it appeared IGF-I) nor glucose deprivation induced a change in GLUT3 as a 2.8 kb mRNA only in terminally differentiated contract- protein in 6-day-cultured myotubes. ing myotubes forming networks after 9 to 11 days of culture Culture of fÏtal myoblasts in medium containing horse on Matrigel. GLUT4 mRNA increased by 2-fold in these cells, serum and either 5 mM or 20 mM glucose did not alter the time

A B

Fig. 2. Expression of glucose transporter and α-actin mRNAs during differentiation of rat cultured myoblasts. Cells were extracted from adult or fÏtal hind-limb muscles and cultured during 8 days on gelatin (A), or 11 days on Matrigel (B), in the absence or presence of 1 µM dexamethasone (Dex) for 24 hours. A 15 µg sample of total RNA, extracted as described in Materials and Methods, was analyzed by northern blotting. The nylon membranes were hybridized with 32P-labelled cDNA from GLUT1, GLUT3 or α-actin (A), and GLUT3, GLUT4 or α-actin (B). The amount of total RNA loaded was checked with an oligonucleotide probe raised against rat 18 S rRNA. This northern blot is representative of 4 independent experiments. Expression and localization of glucose transporters 491

myoblasts myotubes contracting myotubes

100 % GLUT1 GLUT3 80 GLUT4

40 mRNA (arbitrary units) 20 Fig. 3. Time course of GLUT1, GLUT3 and GLUT4 glucose transporter mRNA expression during differentiation of fÏtal myoblasts. Glucose 0 transporters mRNA were extracted from cells 2 4 6 8 10 12 Days between 2 days and 11 days after plating.

Fig. 4. Regulation of glucose transporter and α-actin mRNA expression by insulin, IGF-I or glucose deprivation in 6-day-cultured Fig. 5. Western blot analysis of glucose transporter proteins in fÏtal myotubes. Total RNA (15 µg) extracted from fÏtal myotubes myoblasts and myotubes. GLUT1 and GLUT3 proteins were cultured for 6 days on gelatin was analyzed by northern blotting, as analyzed by Western blotting in fÏtal muscle cells cultured on described in Materials and Methods. Control cells (C). Cells cultured gelatin. Samples of 40 µg protein from crude membranes were for 24 hours in the presence of: 100 nM insulin (I), 10 nM IGF-I fractionated by electrophoresis, according to the protocol described (IGF-I), absence of glucose (−G). Total RNA amounts were checked in Materials and Methods. The evolution of GLUT3 protein during with an oligonucleotide probe raised against rat 18 S rRNA. This fusion of myoblasts into myotubes is shown from day 3 to day 8 ((A) northern blot is representative of 5 independent experiments. detection by ECL system). The effect of a 24 hour treatment with 100 nM insulin (I) or 10 nM IGF-I (IGF-I) on GLUT1 and GLUT3 proteins was compared with myoblasts, or with control (C) in 6-day- course or magnitude of GLUT1 or GLUT3 expression (results cultured myotubes, in the presence of 5 mM glucose, or in the not shown). absence of glucose ((B) GLUT1 detected by ECL system; GLUT3 by 125I-Protein A). These western blots are representative of 3 Hexose transport rate in cultured fÏtal myoblasts independent experiments. The rate of hexose transport was assessed in myoblasts 2 days after plating, and in myotubes 6 days after plating (Fig. 6). To 2-[3H]deoxyglucose increased from 1.62±0.14 pmol/min per determine if GLUT3 was functional in these cells, we also µg protein in myoblasts to 3.61±0.15 pmol/min per µg protein measured D-xylose uptake, since GLUT3 has been reported to in myotubes, showing that the rate of glucose transport is be a D-xylose transporter (Gould et al., 1991). The uptake of dependent upon the stage of differentiation. D-U-[14C]xylose 492 I. Guillet-Deniau, A. Leturque and J. Girard pmol/min/µg protein * Fig. 6. Comparison between hexose 16 transport rates in fÏtal myoblasts and myotubes. 2-[3H]deoxy-D-glucose and D-[14C]xylose transport was * assayed in fÏtal myoblasts (white 14 Myoblasts Myoblasts columns) and 6-day-cultured myotubes (grey columns), as Myotubes Myotubes described in Materials and Methods. A 45 minute effect of 100 nM insulin 12 (I) or 10 nM IGFI-I (IGF-I), and a 24 hour effect of 1 µM dexamethasone (Dex) or glucose deprivation (−G) were studied on hexose uptake in 4 fÏtal myoblasts and 6-day-cultured myotubes. Values are means ± s.e.m. x x from 3 to 5 independent experiments performed in quadruplicate. Statistical 2 signiﬁcance of differences was + + + + assessed in a paired Student’s t-test. The asterisks indicate difference that is statistically signiﬁcant at P<0.001 0 when compared with: (+) myoblasts, CI -G -G+I CI IGF-I Dex - G - G+I for D-xylose transport; control myoblasts (x), or control myotubes (*) D-[14 C] XYLOSE uptake 2-[ 3 H] DEOXY-D-GLUCOSE uptake for 2-deoxy-D-glucose transport.

transport was negligible in myoblasts, and reached 0.98±0.15 clearly localized on the plasma membrane (Fig. 7F), as it was pmol/min per µg protein in myotubes. The hormonal control in myoblasts. Some GLUT3 staining appeared on the plasma of 2-deoxyglucose or xylose uptake was performed on membrane, in 6-day-cultured myotubes (Fig. 7D); neverthe- myoblasts and myotubes: a 24 hour treatment with 1 µM dex- less, at 8 days after plating, fluorescence was still observed amethasone was inefficient. A 45 minute treatment with 100 around the nuclei, even in the presence of insulin (Fig. 7E). nM insulin or 10 nM IGF-I did not affect the 2-deoxyglucose GLUT3 was no longer detectable in myotubes cultured on or xylose uptake in either cell type. These results suggest that Matrigel for 11 days (result not shown). the glucose transporters expressed in 6-day-cultured myotubes GLUT4 was immunodetected in 11-day myotubes cultured are insensitive to hormonal regulation by insulin, IGF-I and on Matrigel: the fluorescence was weakly observed on the dexamethasone. plasma membrane, and strongly in the perinuclear area (Fig. A 24 hour glucose deprivation induced a 2-fold increase in 7G). When the cells were treated for 45 minutes with 100 nM 2-deoxyglucose uptake (2.99±0.24 pmol/min per µg protein) insulin before being fixed, the staining appeared mainly on the in myoblasts, and a 4-fold increase (14.82±1.0 pmol/min per plasma membrane (Fig. 7H). The same experiment was µg protein) in myotubes, in accordance with the enhancement performed on cells treated with 10−6 M dexamethasone for 48 observed in GLUT1 mRNA and protein. This result was inde- hours before fixation: GLUT4 was much more abundant on the pendent of the presence of insulin. Glucose deprivation had no effect on D-xylose uptake. Cellular localization of glucose transporters in Fig. 7. Cellular localization of glucose transporter proteins by cultured fÏtal myoblasts immunofluorescence staining. Fœtal myoblasts and myotubes were probed with polyclonal anti-GLUT1, polyclonal anti-GLUT3, and The contamination by fibroblasts and other cell types was less monoclonal anti-GLUT4. Localization of bound antibodies was than 10% in our primary cultures of myoblasts. Therefore, the visualized with TRITC-conjugated secondary antibody for GLUT1, cultures were directly examined by immunofluorescence and with FITC-conjugated secondary antibodies for GLUT3 and staining to determine the cellular localization of glucose trans- GLUT4. All the nuclei were stained with HÏchst 33258 (A-I, lower porters. Specificity of staining was controled by the use of panels). Control of GLUT3 staining was performed with the rabbit preimmune serum, for GLUT3, and by the use of non- preimmune serum (A). GLUT3 was detected around the nuclei in permeabilized cells, for GLUT1 and GLUT4, since the anti- myoblasts (B), on the plasma membrane and in the perinuclear area bodies recognized intracellular epitopes of the proteins. in 6-day cultured myotubes (D), as well as in 8-day-cultured In 2-day-cultured myoblasts, specific GLUT1 immunoreac- myotubes after a 45 minute treatment with 100 nM insulin (E). tivity was detected at the periphery of the cell body, this pattern GLUT1 was visualized on the plasma membrane in myoblasts (C) and myotubes (F). GLUT4 protein was only detected in myotubes being consistent with a plasma membrane localization (Fig. cultured for 11 days on Matrigel (G-I). It was intracellular in the 7C). In contrast, specific GLUT3 staining was mainly intra- absence of insulin (G) and translocated to the plasma membrane after cellular: GLUT3 appeared as small vesicles located in the per- a 45 minute treatment with 100 nM insulin (H). In the presence of inuclear area (Fig. 7B). No staining was observed with rabbit insulin, GLUT4 protein was more abundant in myotubes cultured for preimmune serum (Fig. 7A). 48 hours with 1 µM dexamethasone (I). A,D,E,F,G,H,I, × 400; B,C, In 6-day-cultured myotubes, GLUT1 immunoreactivity was ×600. Expression and localization of glucose transporters 493 plasma membrane in the presence of insulin in these cells, but porter expressed in undifferentiated fÏtal myoblasts, and a lot of fluorescence still remained in the perinuclear area (Fig. GLUT3 the predominant isoform of the fusioning cells, 7I). whereas GLUT4 appeared only in spontaneously contracting These results show that GLUT1 was the major glucose trans- myotubes. 494 I. Guillet-Deniau, A. Leturque and J. Girard

DISCUSSION vation, or to changes in glucose concentration, stimuli that upregulated GLUT1 in myoblasts and myotubes. We studied the expression of three glucose transporters To date, there is only one variant of the L6 cell line known isoforms, GLUT1, GLUT3 and GLUT4, in rat fÏtal myoblasts to express a significant amount of GLUT4 in vitro (Koivisto et during differentiation. al., 1991). In our study, GLUT4 mRNA was undetectable when Despite the fact that GLUT3 was first cloned from a library the cells were cultured for 8 days on gelatin-coated flasks, but of human fÏtal skeletal muscle (Kayano et al., 1988), GLUT3 was expressed when they were cultured for 11 days on mRNA was undetectable in vivo in skeletal muscle, both in Matrigel, mainly in the presence of dexamethasone. In 11-day- adult (Haber et al., 1993; Yano et al., 1991; Shepherd et al., cultured myotubes, the GLUT4 mRNA level corresponded to 1992; Kayano et al., 1988; Gould et al., 1992; Mantych et al., the GLUT4 mRNA level in 15-day-old newborn rat (Postic et 1992), and in fÏtal rats (result not shown). Recently, the al., 1994) (data not shown). This result suggested that presence of GLUT3 mRNA and protein was reported in myotubes derived from fÏtal myoblasts could provide a better myoblasts and myotubes derived from the rat L6 cell line model system than cell lines for the study of the appearance of (Bilan et al., 1992). This apparent discrepancy with the above GLUT4 in developing skeletal muscle. On Matrigel, cells were mentioned data could be due to: (1) the difference in species multiplicating and differentiating quickly, and reached a (rat versus human); (2) the dilution of the GLUT3 signal terminal state of differentiation, in terms of frequency of con- (whole muscle versus myogenic cells); (3) the use of a cell line tractions. After 8 days of culture on gelatin, the contractions (L6 versus skeletal muscle in vivo). In order to determine if released the cells from the layer, preventing them from further the GLUT3 glucose transporter was present during myogenic differentiation and leading to cell loss. The relative abundance differentiation, we studied its expression in a primary culture of GLUT4 in cells cultured on Matrigel might be related to the of myoblasts extracted from rat fÏtal skeletal muscle. In our enhanced frequency of contraction observed, since the con- work, GLUT3 mRNA and protein were expressed in myoblasts tractile activity was known to increase GLUT4 expression in and 6-day myotubes, but were no longer present in contracting adult rat skeletal muscle (Ploug et al., 1990; Rodnick et al., myotubes. The presence of GLUT3 mRNA was not specific to 1990). Nevertheless, the presence of growth factors, cytokines fÏtal cells, since it was also detected in adult satellite cells. and hormones in Matrigel rendered difficult the study of During cell differentiation, GLUT3 mRNA increased dras- hormonal regulation of GLUT4 expression and rate of glucose tically when the cells were fusing. The transient rise in GLUT3 transport. mRNA was concomitant with an increase in GLUT3 protein, As assessed by immunofluorescence, the GLUT4 protein suggesting a role for this protein during cell fusion. The pro- expressed in the contracting myotubes was translocated to the gressive disappearance of GLUT3 mRNA and protein between plasma membrane under the short-term action of insulin. This days 8 to 11, observed by western blotting and confirmed by acute effect of insulin on the cellular localization of glucose immuno-staining, seemed to indicate that the GLUT3 half-life transporters (Wardzala and Jeanrenaud, 1981; Hirshman et al., did not exceed 3 days. It was not due to progressive cell 1993) is specific for the GLUT4 isoform in skeletal muscle damage, since the cells were still contracting together on day (Douen et al., 1990). The increase in GLUT4 protein observed 11. in myotubes treated with 10−6M dexamethasone for 48 hours The cellular localization of GLUT3, determined by immuno- could be explained by the stimulating effect of dexamethasone fluorescence, showed that GLUT3 protein was intracellular in on muscle cell differentiation, as assessed by enhanced α-actin myoblasts. In myotubes, the protein was located, as expected, mRNA expression in these cells. on the plasma membrane, but also in small intracellular Finally, GLUT1 mRNA, very abundant in myoblasts, vesicles around the nucleus. Such a localization was also decreased by 80% when the cells began to fuse, and remained described for GLUT1 in a primary culture of rat cerebellar quite constant thereafter. GLUT1 protein followed the same granule neurons, without any physiological explanation pattern. Despite the decrease in GLUT1 protein, the glucose (Maher et al., 1991). The intracellular localization of GLUT3 transport was 2-fold higher in myotubes than in myoblasts, protein also faced us with the problem of the role of GLUT3: suggesting that GLUT1 was not the only glucose transporter it was not consistent with this isoform making an important involved; GLUT3 made a contribution. In fact, by immuno- contribution to glucose transport in myoblasts. On the contrary, fluorescence, GLUT1 protein was always found on the plasma GLUT3 was functional in myotubes, since D-xylose was trans- membrane, whereas GLUT3 was found on the plasma ported. Nevertheless, the affinity of GLUT3 for 2-deoxyglu- membrane only in myotubes. The 2- to 4-fold increase cose and D-xylose being very different (Burant and Bell, observed in 2-deoxyglucose transport, in glucose-deprived 1992), the percentage of GLUT3 glucose transporter present cells, might be related to the increase in GLUT1 protein. This on the plasma membrane could not be extrapolated from this result is in agreement with previous observations on 3T3C2 result. (Haspel et al., 1986), L6 myocytes (Walker et al., 1989) and To be functional, a glucose transporter should be located on L8 myocytes (Wertheimer et al., 1991). In our experimental the plasma membrane (Wardzala and Jeanrenaud, 1981). We conditions, hexose transport was not affected by a short-term were unable to translocate GLUT3 from the cytoplasm to the exposure to insulin or IGF-I. Again, this agrees with the known plasma membrane in response to insulin or IGF-I. Our results regulation of glucose transport ensured by GLUT1. are not in agreement with those of Bilan et al. (1992), who Satellite cell activation is part of a continuum spanning reported a redistribution of GLUT3 protein from an intracel- muscle regulation, from early growth through regeneration, lular membrane fraction to the plasma membrane of L6 cells, including hypertrophy. The presence of GLUT3 in fÏtal by short-term exposure to insulin or IGF-I. We showed that myoblasts and adult satellite cells seems to indicate that this GLUT3 mRNA and protein were insensitive to glucose depri- isoform could be a good marker of the muscle’s ability for Expression and localization of glucose transporters 495 regeneration. It could be of particular interest to check if distribution of the human GLUT3 glucose transporter. Endocrinology 132, GLUT3 is expressed in muscle satellite cells extracted from 2538-2543. mdx mice, a strain that presents striking similarities to human Haspel, H. C., Wilk, E. W., Birnbaum, M. J., Cushman, S. W. and Rosen, O. (1986). Glucose deprivation and hexose transporter polypeptides of muscular dystrophy. It is well known that these cells are able murine fibroblasts. J. Biol. Chem. 261, 6778-6789. to multiply, but not to fuse at the same rate as normal satellite Hirshman, M.F., Goodyear, L.J., Horton, E.D., Warzdzala, L.J. and cells (Di Mario and Strohman, 1988). An increase in GLUT3 Horton, E.S. (1993). Exercise training increases GLUT4 protein in rat might reflect their capacity to fuse. This could be very adipose cells. Amer. J. Physiol. 264, E882-E889. important, since the abrupt onset of muscle fibre degeneration Kasanicki, M. A. and Pilch, P. F. (1990). Regulation of glucose transporter function. Diabetes 13, 219-227. observed at about 6 weeks in these mice is followed by regen- Kayano, T., Kuumoto, H., Eddy, T., Fan, Y.-S., Byers, M. G., Shows, T. B. erative cycles that restore a fairly normal muscle histological and Bell, G. I. (1988). Evidence for a family of human glucose transporter picture, as well as a muscle function (Torre and Duchen, like proteins. Sequence and gene loalization of a protein expressed in fetal 1987). skeletal muscle and other tissues. J. Biol. Chem. 263, 12245-12248. In conclusion, we have shown that there is a shift in glucose Koivisto, U.-M., Martinez-Valdez, H., Bilan, P., Burdett, E., Ramlal, T. and Klip, A. (1991). Differential regulation of the GLUT1 and GLUT4 transporter isoform expression during myogenic differen- glucose transport systems by glucose and insulin in L6 muscles cells in tiation. This might be consistent with the specific amount of culture. J. Biol. 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