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DETECTION L6 MUSCLE

OF THE GLUT3 FACILITATIVE CELLS: REGULATION

GLUCOSE

BY CELLULAR

AND INSULIN-LIKE

GROWTH

TRANSPORTER

DIFFERENTIATION,

1129-1137

IN RAT INSULIN

FACTOR-I

Philip J. Bilana, Yasuhide Mitsumoto a, Frances Maherb, Ian A.Simpsonb and Amira Klipa* aDivision of Cell Biology, The Hospital for Sick Children, 555 University Toronto, Ontario, CANADA M5G 1X8

Avenue,

bExperimenta1 Diabetes, Metabolism and Nutrition Section, Diabetes Branch NIDDK, National Institutes of Health, Bethesda, Maryland 20892

Received

June

19,

1992

The GLUT3 facilitative glucose transporter protein was found to be expressedin rat L6 musclecells. It was detected at both the myoblast and myotube stage. GLUT3 protein content per mg of total membrane protein increased significantly during L6 cell differentiation. Subcellular fractionation demonstratedthat the GLUT3 protein was predominantly localized in plasmamembrane-enrichedfractions of either myoblastsor myotubes. Short-term exposureof L6 myotubes to IGF-I or insulin causeda redistribution of GLUT3 protein from an intracellular membranefraction to the plasmamembrane,without affecting total membraneGLUT3 protein content. Long-term exposure of L6 myotubesto IGF-I produced an increaseof GLUT3 protein in total membranesand all subcellularmembranefractions, especiallythe plasmamembrane. We proposethat the GLUT3 glucosetransportermay play an important role in glucosemetabolismin developing muscle. 0 I.992Academic Press,Inc.

Facilitative glucosetransportis mediatedby tissue-specificglucosetransporterproteins. Five isoforms have been described, designated GLUT1 to GLUTS. Glucose transporter protein biosynthesisand protein subcellular localization and their regulation by hormones,metabolite levels, enviromental signalsand diseasedstateshave been extensively studied for the GLUTl, GLUT2 and GLUT4 isoforms. However, considerably lessis known about the regulation of the GLUT3 and GLUT5 isoforms (l-3). The presentstudy focuseson the detection of the GLUT3 glucose transporter protein in a muscle cell line and regulation of its levels by hormonesand cellular differentiation. The GLUT3 facilitative glucose transporter was first cloned from a human fetal muscle cDNA library (4) and found to have 64.4% and 57.5% amino acid identity with the GLUT1 and the GLUT4 isoforms, respectively (1, 4). The GLUT3 protein hasglucose transport function since microinjection of synthetic GLUT3 mRNA into Xenopus oocytes increased2-deoxy-D-

*To whom correspondenceshouldbe addressed. 0006-291X/92

1129

$4.00

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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glucose uptake compared to control oocytes.

Furthermore, cytochalasin B, an inhibitor of facilitative glucose transport, blocked this elevated transport rate (5). In humans, GLUT3 mRNA is detectable in a variety of tissues but most abundantly in the brain (1,4). In human fetal skeletal muscle, GLUT3 mRNA levels were predicted to be relatively low compared to brain, dropping to very low levels in adult muscle (4). The human GLUT3 cDNA has been used successfully

to

detect GLUT3 mRNA expression in brain from monkey, rabbit, rat and mouse (6). Recently, a GLUT3 cDNA was cloned from a PTC-3 murine cell-line and a mouse brain cDNA library and found to encode for a polypeptide that has an 83% amino acid identity with the human GLUT3 protein (7). The rat and mouse GLUT3 proteins must be closely related since antiserum raised against carboxy-terminal

amino acids 478-492 of the mouse GLUT3 protein can

detect rat GLUT3 protein by immunoblotting

or immunofluorescence

in intact cells (8, 9). In

contrast, there is limited amino acid identity between the mouse and the human carboxy-termini the GLUT3 protein (7, 8). In rat brain immunofluorescence

of

studies detected GLUT3 protein

expression solely on neuronal cell plasma membranes and not in astroglia (9). In humans, the GLUT3 glucose transporter may play a role in fetal skeletal muscle glucose transport since GLUT3 mRNA is present at this stage of muscle development (4). Because the rat L6 cell line is a model of developing skeletal muscle, we hypothesized that L6 cells may express the GLUT3 gene product. The L6 cell line was derived from neonatal rat thigh skeletal muscle cells and retains many morphological, differentiate

biochemical

spontaneously

and metabolic properties of skeletal muscle (10-13).

L6 cells

after confluency from single cell myoblasts into elongated multi-

nucleated myotubes when cultured in low concentrations

of serum (13). L6 cells express the

GLUT1 and GLUT4 glucose transporter gene products, detected at the protein and mRNA levels (14-16). Myoblasts express high levels of GLUT1 protein. As L6 cells differentiate the GLUT1 protein content per mg protein decreases (17). In contrast, expression of the GLUT4 protein is essentially turned on as the cells begin to differentiate into myotubes (17). Moreover, glucose transport

becomes more responsive

to insulin

(17) and IGF-I

(Bilan, P. J. and Klip, A.,

unpublished) as L6 myoblasts differentiate into myotubes. Recently we have demonstrated that both the GLUT1 and GLUT4 glucose transporter isofotms are recruited to the plasma membrane by acute exposure of L6 myotubes to IGF-I or insulin (18, 19). In the present study, we demonstrate that L6 myoblasts and myotubes express the GLUT3 protein; that the protein is primarily

localized to the plasma membrane in both myoblasts and

myotubes; and that the membrane distribution hormonal regulation by insulin and IGF-I.

and biosynthesis

of GLUT3

protein is under

EXPERIMENTAL Materials. a-MEM was obtained from the University of Toronto Tissue Culture Facility. Fetal bovine serum and antibiotics were obtained from Gibco/BRL. Insulin, gradient grade sucrose, bovine serum albumin (BSA) and fatty acid free BSA, Hepes, Tris base and protease inhibitors were purchased from Sigma. Human recombinant IGF-I was a gift from Dr. M. Vranic, University of Toronto. Ultracentrifugation products were supplied by Beckman. Electrophoresis/Immunoblotting reagents were purchased from BioRad. Electrophoresis equipment and precast 10% SDS-polyacrylamide Tris/glycine gels were supplied by Novex . 1130

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Anti-GLUT1 and anti-GLUT4 sera (RaGLUTRANS and RaIRGT, respectively) were purchased from East Acres Biologicals. Rabbit anti-mouse GLUT3 serum, a kind gift from Hoffman LaRoche was raised against the synthetic peptide GPAGVELNSMQPVKETPGNA, corresponding to C-terminal amino acids 474-493 (8,9). al-Na+/K+ ATPase was detected with hybridoma culture supernatant containing the isozyme-specific monoclonal antibody McKl, a kind gift from Dr. K.F. Sweadner, Harvard University (20). Rabbit anti-rat liver S-nucleotidase serum raised against a monoclonal antibody immunoadsorbed enzyme (21) was a kind gift from Dr. J.P. Luzio, Cambridge University. [ t*5I]Sheep anti-mouse IgG and [3H]Cytochalasin B were purchased from Amersham. [l*51]-Protein A was from ICN. Cell cultures. A clonally selected line of L6 muscle cells (selected for high fusion potential) was grown in a-MEM culture medium containing 2% fetal bovine serum and allowed to fuse and differentiate, as reported earlier (13, 14). Myoblast cultures were studied at aproximately 90% confluence before any myotube differentiation. Myotubes were studied when greater than 90% fusion was attained. Porcine insulin or human recombinant IGF-I were added at the indicated concentrations in serum-free a-MEM culture medium containing 25 mM glucose and 5 mg/ml fatty acid free BSA for the indicated times at 37oC. L6 myotubes incubated for short times with hormone were first incubated for at least 5 hours in the above supplemented a-MEM medium before addition of the hormone. For long-term incubations (8 hours) cultures were not depleted of serum prior to addition of hormone in supplemented a-MEM medium. Membrane isolation. Total membranes from L6 cells were prepared as described earlier (17). Plasma membranes and intracellular light microsomes were isolated as previously reported (18). Briefly, a post-nuclear supernatant yields a crude plasma membrane pellet and light microsome supernatant after a 1 hour, 3 1,OOOxgcentrifugation. Light microsomes were collected by a 190,OOOxg centrifugation step. Plasma membranes were further purified by discontinuous sucrose gradient centrifugation in a Beckman SW 41 swinging bucket rotor at 210,OOOxg for 2 hours 15 min. This step yielded three membrane fractions that sedimented at the interfaces of the 8%/32%, 32%/40% and 40%/50% (w/w) sucrose layers. Only the 8%/32% sucrose fraction was enriched in plasma membrane markers ( see RESULTS AND DISCUSSION). Immunoblotting. Glucose transporter isoforms were detected by immunoblot analysis essentially as described earlier (19) using C-terminus specific antibodies to each isoform. All antibodies and Protein A were diluted in 3% w/v BSA; 100 mM NaCl; 0.04% w/v NP40; 50 mM Tris-HCl pH 7.5 at the following dilutions: Anti-GLUT1 serum: 1:2500; anti-GLUT4 serum: 1: 1000; anti-GLUT3 serum: I :300; M&l: 1: 100; anti-5’-nucleotidase serum: 1:200. Polyclonal antisera were detected by 0.1 uCi/ml [r*sI]Protein A and McKl by 0.1 uCi/ml [t*5I]Sheep antimouse IgG. Autoradiographs were densitometrically scanned with a Discovery Series DNA 35 gel scanner (Protein Databases Inc.) Statistical analysis was performed by Student’s t-test. RESULTS

AND

DISCUSSION

Membrane characterization. The membranesbandingat the 8%/32% sucroseinterface were designatedas plasmamembranesbecausethey were enriched in the at-subunit of the Na+/K+-

ATPase, 5’-nucleotidaseand the GLUT1 glucosetransporter relative to all other fractions (fig 1A). The abundanceof these proteins in plasma membranesrelative to total membranesare shown in Table I. The proteins in this plasmamembranefraction were highly labeled when isolatedfrom intact cells incubatedwith the impermeantNHS-LC-Biotin (Pierce) protein-reactive biotinylated-probe (not shown). Although the membranefractions banding atop 40% and 50% sucrosealso contained surface-labeledproteins, data from several experiments indicated they were not as enriched in the al-subunit of the Na+/K+ ATPase, 5’-nucleotidaseor GLUT1 asthe plasma membranefraction (fig 1A). The light microsomeswere not enriched in the plasma membranemarkersal-Na+/K+ ATPase and 5’-nucleotidase(fig 1A and Table I). However, the GLUT1 protein was somewhat enriched in the light microsome fraction relative to total membranes(fig 1A and Table I). This indicatesthat GLUT1 protein is distributed to an internal membrane store in addition to the plasma membrane. Importantly, the GLUT4 glucose transporter was largely localized to the light microsomesand by a lesserextent to the plasma 1131

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A PMLMTM4050

al-Na/K

ATPase

4lbm~

- 106 B (-) peptide

5’.Nucleotidase

(+) peptide - 106-so-

GLUT1

-

49.5

GLUT3+ GLUT4

-49.5-

^i -

49.5

PM LM TM 40 50

PM LM TM 40 50

Figure 1. Characterization of subcellular membrane fractions from L6 myotubes. A. Plasma membranes (PM), light microsomes (LM), total membranes (TM), the 40% sucrose fraction (40) and the 50% sucrose fraction (50) were isolated from untreated L6 myotubes. Protein samples (15 ug ) were subjected to SDS-PAGE and immunoblotted with antibodies specific for the atNa+/K+ ATPase, 5’-nucleotidase, GLUT1 and GLUT4 as described in EXPERIMENTAL. Data are representative of at least 3 separate experiments. The nearest molecular weight marker is indicated on the right (x10-3). B. Subcellular membrane fractions were subjected to SDS-PAGE and immunoblotted as described in A. The filter was probed with rabbit anti-mouse GLUT3 serum without (-) or with (+) 10 &ml of GLUT3 C-terminal synthetic peptide, followed by [t*sI]Protein A and autoradiography. Molecular weight marker positions (x10-s) are indicated.

membranes(fig 1A and Table I). The GLUT4 glucose transporter is typically localized to the internal membranesof insulin-responsivecells (22-24). Thus, the membranesof the 8%/32% sucroseinterface and the light microsomesrepresent L6 myotube plasma membranesand an internal membranepool containing glucosetransporters,respectively. Subcellular distribution of GLUT3 protein. Figure 1B showsan immunoblot of L6 myotube membranefractions probed with the mouse-specificanti-C-terminal GLUT3 glucosetransporter antiserum. The antiserum reacts strongly with a broad protein band of approximately 45,000 relative molecular weight (Mr) in the plasmamembranefraction. This band was also detected with less intensity in the light microsomes, the 40% sucrose, the 50% sucrose and total membranefractions. The specificity of the antiserumwas determined by competition with 10 p@rnl synthetic peptide correspondingto amino acids474-493 of the mouseGLUT3 protein (fig 1B). This portion of the immunoblot (+ peptide) also showsthat several nonspecific proteins were detected by the anti-GLUT 3 serum. There were at leasttwo prominent nonspecific protein

TABLE Fraction TM

PM LM

I. Relative membrane marker and glucose transporter contents in subcellular membrane fractions from L6 myotubes al -Na+/K+ ATPase 1.00 1.55 0.71

S-nuclmtidase

1 .oo 1.73 1.09

GLUT1

GLUT4

GLUT3

1.00 1.86 1.27

1.00 1.23 1.92

1.00 1.54 0.82

Optical density/mg protein values were obtained by laser scanning densitometry from autoradiographs like those in figure 1 A and B for plasma membranes (PM) and light microsomes (LM) from untreated L6 myotubes. Results are expressed relative to the value in total membranes (TM).

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70,000 and 60,000 Mr. The 70,000 Mr band appeared in all membrane

fractions but the 60,000 M, band was detected only in total membranes and in the 40% and 50% sucrose fractions.

The 45,000 Mr protein was the only band that was not detected by the

antiserum in the presence of the synthetic C-terminal peptide (fig 1B). Therefore by this criterion the 45,000 Mr protein band is thought to be the GLUT3 glucose transporter. As stated above, GLUT3 protein was detected in all the isolated membrane fractions but particularly in the plasma membrane fraction.

The GLUT3

protein content per mg protein was higher in the plasma

membranes than in the light microsomes or total membranes (fig. 1B and Table I). GLUT3 protein during rnyogenesis. We have previously shown that the levels of expression of the GLUT1 and GLUT4 glucose transporters

are regulated during differentiation

of L6 cells

from myoblasts to myotubes (17). In the present study we observed that the GLUT3 protein content per mg protein in the total membranes increased by 34 +_7% (n=S, p

Detection of the GLUT3 facilitative glucose transporter in rat L6 muscle cells: regulation by cellular differentiation, insulin and insulin-like growth factor-I.

The GLUT3 facilitative glucose transporter protein was found to be expressed in rat L6 muscle cells. It was detected at both the myoblast and myotube ...
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