A Novel Role of Neuregulin in Skeletal Muscle
2001; Elsevier BV; Volume: 276; Issue: 21 Linguagem: Inglês
10.1074/jbc.m008100200
ISSN1083-351X
AutoresElisabeth Suárez, Daniel Q. Bach, Joan A. Cadefau, Manuel Palacı́n, António Zorzano, Anna Gumà,
Tópico(s)Ion channel regulation and function
ResumoNeuregulins regulate the expression of acetylcholine receptor genes and induce development of the neuromuscular junction in muscle. In studying whether neuregulins regulate glucose uptake in muscle, we analyzed the effect of a recombinant neuregulin,rheregulin-β1-(177–244) (HRG), on L6E9 muscle cells, which express the neuregulin receptors ErbB2 and ErbB3. L6E9 responded acutely to HRG by a time- and concentration-dependent stimulation of 2-deoxyglucose uptake. HRG-induced stimulation of glucose transport was additive to the effect of insulin. The acute stimulation of the glucose transport induced by HRG was a consequence of the translocation of GLUT4, GLUT1, and GLUT3 glucose carriers to the cell surface. The effect of HRG on glucose transport was dependent on phosphatidylinositol 3-kinase activity. HRG also stimulated glucose transport in the incubated soleus muscle and was additive to the effect of insulin. Chronic exposure of L6E9 cells to HRG potentiated myogenic differentiation, and under these conditions, glucose transport was also stimulated. The activation of glucose transport after chronic HRG exposure was due to enhanced cell content of GLUT1 and GLUT3 and to increased abundance of these carriers at the plasma membrane. However, under these conditions, GLUT4 expression was markedly down-regulated. Muscle denervation is associated with GLUT1 induction and GLUT4 repression. In this connection, muscle denervation caused a marked increase in the content of ErbB2 and ErbB3 receptors, which occurred in the absence of alterations in neuregulin mRNA levels. This fact suggests that neuregulins regulate glucose transporter expression in denervated muscle. We conclude that neuregulins regulate glucose uptake in L6E9 muscle cells by mechanisms involving the recruitment of glucose transporters to the cell surface and modulation of their expression. Neuregulins may also participate in the adaptations in glucose transport that take place in the muscle fiber after denervation. Neuregulins regulate the expression of acetylcholine receptor genes and induce development of the neuromuscular junction in muscle. In studying whether neuregulins regulate glucose uptake in muscle, we analyzed the effect of a recombinant neuregulin,rheregulin-β1-(177–244) (HRG), on L6E9 muscle cells, which express the neuregulin receptors ErbB2 and ErbB3. L6E9 responded acutely to HRG by a time- and concentration-dependent stimulation of 2-deoxyglucose uptake. HRG-induced stimulation of glucose transport was additive to the effect of insulin. The acute stimulation of the glucose transport induced by HRG was a consequence of the translocation of GLUT4, GLUT1, and GLUT3 glucose carriers to the cell surface. The effect of HRG on glucose transport was dependent on phosphatidylinositol 3-kinase activity. HRG also stimulated glucose transport in the incubated soleus muscle and was additive to the effect of insulin. Chronic exposure of L6E9 cells to HRG potentiated myogenic differentiation, and under these conditions, glucose transport was also stimulated. The activation of glucose transport after chronic HRG exposure was due to enhanced cell content of GLUT1 and GLUT3 and to increased abundance of these carriers at the plasma membrane. However, under these conditions, GLUT4 expression was markedly down-regulated. Muscle denervation is associated with GLUT1 induction and GLUT4 repression. In this connection, muscle denervation caused a marked increase in the content of ErbB2 and ErbB3 receptors, which occurred in the absence of alterations in neuregulin mRNA levels. This fact suggests that neuregulins regulate glucose transporter expression in denervated muscle. We conclude that neuregulins regulate glucose uptake in L6E9 muscle cells by mechanisms involving the recruitment of glucose transporters to the cell surface and modulation of their expression. Neuregulins may also participate in the adaptations in glucose transport that take place in the muscle fiber after denervation. epidermal growth factor rheregulin-β1-(177–244) insulin-responsive aminopeptidase secretory component-associated membrane proteins vesicle-associated membrane proteins fetal bovine serum phosphate-buffered saline supernatant plasma membrane low density vesicle membrane polymerase chain reaction Skeletal muscle is the main tissue that contributes to glucose disposal in absorptive conditions. A limiting step in this process is glucose transport, which is mediated by different glucose transporters; GLUT1 is responsible for basal transport and GLUT4 is responsible for insulin- or exercise-stimulated glucose transport through translocation to the plasma membrane (1Douen A.G. Ramlal T. Rastogi S. Bilan P.J. Cartee G.D. Vranic M. Holloszy J.O. Klip A. J. Biol. Chem. 1990; 265: 13427-13430Abstract Full Text PDF PubMed Google Scholar). GLUT1 is highly expressed in fetal muscle, but during perinatal life it is markedly repressed, whereas GLUT4 is induced (2Santalucı́a T. Camps M. Castelló A. Muñoz P. Nuel A. Testar X. Palacı́n M., Zorzano A. Endocrinology. 1992; 130: 837-846Crossref PubMed Scopus (0) Google Scholar). This effect is temporally coincident with the process of innervation of the muscle fiber (3Castelló A. Cadefau J. Cussó R. Testar X. Hesketh J.E. Palacı́n M. Zorzano A. J. Biol. Chem. 1993; 268: 14998-15003Abstract Full Text PDF PubMed Google Scholar). 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The first group of neuregulins contain a transmembrane domain and a cytosolic tail, reside as membrane proteins, and are released to the extracellular milieu after proteolytic cleavage. Most of the released forms contain an N-terminal Ig-like domain that binds to the glycosaminoglycan portion of proteoglycans in the extracellular matrix. Common to all isoforms, there is a C-terminal EGF1-like domain defined by six cysteine residues that fold the domain into compact, protease-resistant β-sheets by forming three disulfide bonds. The EGF-like domain is sufficient to elicit biological responses. After proteolysis, the EGF-like domain is released and binds to members of the ErbB family of tyrosine kinase receptors, ErbB3 (HER3) and ErbB4 (HER4). ErbB4 shows ligand-stimulated tyrosine kinase activity. ErbB3, however, is a tyrosine kinase-deficient receptor because an aspartate and a glutamate in the kinase domain that are critical for autophosphorylation are replaced by other residues. Neuregulin binding to ErbB3 signals through heterodimerization with ErbB2 (HER2, c-neu), which displays tyrosine kinase activity (20Carraway K.L. Cantley L.C. Cell. 1994; 78: 5-8Abstract Full Text PDF PubMed Scopus (582) Google Scholar, 21Sliwkowski M.X. Schaefer G. Akita R.W. Lofgren J.A. Fitzpatrick V.D. Nuijens A. Fendly B.M. Cerione R.A. Vandlen R.L. Carraway K.L. J. Biol. Chem. 1994; 269: 14661-14665Abstract Full Text PDF PubMed Google Scholar). Neuregulins have major effects on the growth and development of epithelial cells (22Peles E. Yarden Y. Bioessays. 1993; 15: 815-824Crossref PubMed Scopus (260) Google Scholar), and generation of knockout mice has demonstrated that they are essential for the development of cranial nerve, ganglia, and Schwann cell precursors along peripheral nerves in the trunk (23Meyer D. Birchmeier C. Nature. 1995; 378: 386-390Crossref PubMed Scopus (1037) Google Scholar). In the hearts of mutant embryos for neuregulin, ErbB2 and ErbB4, ventricular trabeculation does not occur, which results in developmental arrest and embryo death at embryonic day 10.5 (23Meyer D. Birchmeier C. Nature. 1995; 378: 386-390Crossref PubMed Scopus (1037) Google Scholar, 24Lee K.-F. Simon H. Chen H. Bates B. Hung M.-C. Hauser C. Nature. 1995; 378: 394-398Crossref PubMed Scopus (1077) Google Scholar, 25Gassman M. Casagranda F. Orioli D. Simon H. Lai C. Klein R. Lemke G. Nature. 1995; 378: 390-393Crossref PubMed Scopus (930) Google Scholar). Neuregulins also affect the biology of skeletal muscle; they are potent activators of the expression of acetylcholine receptors (26Altiok N. Bessereau J.-L. Changeux J.-P. EMBO J. 1995; 14: 4258-4266Crossref PubMed Scopus (130) Google Scholar, 27Jo S.A. Zhu X. Marchionni M.A. Burden S.J. Nature. 1995; 373: 158-161Crossref PubMed Scopus (241) Google Scholar, 28Chu G.C. Moscoso L.M. Sliwkowski M.X. Merlie J.P. Neuron. 1995; 14: 329-339Abstract Full Text PDF PubMed Scopus (126) Google Scholar). In addition, the neuregulins GGF2 and NRGα1 activate myogenic differentiation (29Florini J.R. Samuel D.S. Ewton D.Z. Kirk C. Sklar R.M. J. Biol. Chem. 1996; 271: 12699-12702Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 30Kim D. Chi S. Lee K.H. Rhee S. Kwon Y.K. Chung C.H. Kwon H. Kang M.-S. J. Biol. Chem. 1999; 274: 15395-15400Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). There is also evidence for the operation of a neuregulin-ErbB3 autocrine signaling pathway during an early stage of myoblast differentiation (30Kim D. Chi S. Lee K.H. Rhee S. Kwon Y.K. Chung C.H. Kwon H. Kang M.-S. J. Biol. Chem. 1999; 274: 15395-15400Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). In the mature muscle fiber, ErbB2 and ErbB3 are concentrated at the neuromuscular junction, and therefore it is thought that neuregulins regulate the protein composition and the functioning of the neuromuscular junction (26Altiok N. Bessereau J.-L. Changeux J.-P. EMBO J. 1995; 14: 4258-4266Crossref PubMed Scopus (130) Google Scholar, 27Jo S.A. Zhu X. Marchionni M.A. Burden S.J. Nature. 1995; 373: 158-161Crossref PubMed Scopus (241) Google Scholar, 31Zhu X. Lai C. Thomas S. Burden S.J. EMBO J. 1995; 14: 5842-5848Crossref PubMed Scopus (155) Google Scholar, 32Moscoso L.M. Chu G.C. Gautman M. Noakes P.G. Merlie J.P. Sanes J.R. Dev. Biol. 1995; 172: 158-169Crossref PubMed Scopus (160) Google Scholar, 33Rimer M. Cohen I. Lomo T. Burden S.J. McMahan U.J. Mol. Cell. Neurosci. 1998; 12: 1-15Crossref PubMed Scopus (67) Google Scholar). Here we examined the effects of the neuregulinrheregulin-β1-(177–244) on glucose uptake in L6E9 muscle cells and the mechanism involved. Our results indicate that HRG stimulates glucose transport and translocates glucose transporters to the cell surface in muscle cells. Additionally, chronic exposure to HRG also stimulates glucose transport and causes alteration of the expression pattern of glucose transporters in muscle cells. Our data are compatible with a model in which neuregulins regulate glucose disposal in or near the neuromuscular junction in innervated muscle fiber. In addition, neuregulin may also participate in the adaptations in glucose uptake that take place in the muscle fiber after denervation. The L6E9 rat skeletal muscle cell line was kindly provided by Dr. B. Nadal-Ginard (Harvard University, Boston, MA). Recombinant heregulin (rheregulin-β1-(177–244) (HRG)) was obtained from Genentech, Inc. (South San Francisco, CA). Dulbecco's modified Eagle's medium, fetal bovine serum (FBS), glutamine, and antibiotics were purchased from BioWhittaker (Walkersville, MD). Most commonly used chemicals and wortmannin were from Sigma. Pepstatin, leupeptin, and aprotinin were from ICN (Costa Mesa, CA). Immobilon polyvinylidene difluoride was obtained from Millipore Corp. (Bedford, MA). Purified porcine insulin was a kind gift from Lilly Co. ECL reagents were from Amersham Pharmacia Biotech UK (Little Chalfont, Buckinghamshire, UK). 2-Deoxy-d-[3H]glucose was obtained from American Radiolabeled Chemicals, Inc. (St. Louis, MO). 2-Deoxy-d-[14C]glucose,d-[3H]mannitol, and the tissue solubilizer Protosol were obtained from PerkinElmer Life Sciences. All chemicals were of the highest purity grade available. Bradford reagent and all electrophoresis agents and molecular weight markers were obtained from Bio-Rad (Hercules, CA). Polyclonal antibody OSCRX (raised against the 15 C-terminal amino acid residues from GLUT4) was produced in our laboratory by Dr. Conxi Mora. Anti-GLUT1 antibody was purchased from Diagnostic International (Karlsdorf, Germany). Anti-rat/mouse GLUT3 antibody was kindly provided by Dr. Gwyn W. Gould (University of Glasgow, UK). Anti-ErbB3 (C-17) and ErbB2 (Neu) (C-18) antibodies were purchased from Santa Cruz Biotechnologies, Inc. (Santa Cruz, CA). Monoclonal antibodies against myosin heavy chain (MF20) and against α1-Na+/K+-ATPase (α6F) were purchased from Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA). Anti-IRAP and anti-SCAMPs antibodies (3F8) were kindly provided by Dr. Paul Pilch (Boston University, Boston, MA). Anti-VAMP2 and anti-cellubrevin antibodies were kindly provided by Dr. Joan Blasi (Universitat de Barcelona). Tissue culture material was purchased from Corning Inc. L6E9 myoblasts were grown in monolayer culture in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) antibiotics (10,000 units/ml of penicillin G and 10 mg/ml streptomycin), 2 mm glutamine, and 25 mm Hepes (pH 7.4). Pre-confluent myoblast (80–90%) were induced to differentiate by lowering FBS to final concentration of 2% (v/v). To analyze the acute effect of HRG on glucose transport and transporters, fully differentiated myotubes were depleted of serum containing 0.2% bovine serum albumin for 4.5 h. 90 min before the end of this period, HRG was added (except in the time course experiments), and when insulin action was studied, it was added for the last 30 min of this period. For the chronic HRG treatment, HRG was added 24 h after the cells were changed to the low serum medium, and studies were carried out after 1, 2, or 3 days of HRG treatment. Cells were cultured on 6-well plates. Transport (34Kaliman P. Viñals F. Testar X. Palacı́n M. Zorzano A. Biochem. J. 1995; 312: 471-477Crossref PubMed Scopus (35) Google Scholar) was initiated by washing the cells twice in a transport solution (20 mm Hepes, 137 mm NaCl, 4.7 mm KCl, 1.2 mm MgSO4, 1.2 mmKH2PO4, 2.5 mm CaCl2, 2 mm pyruvate, pH 7.4). Cells were then incubated for 10 min with a transport solution that contained 0.1 mm2-deoxy-d-glucose and 1 μCi of 2-deoxy-d-[3H]glucose uptake (10 mCi/mmol). To determine background labeling, we incubated the cells for 10 min with ice-cold 50 mm glucose in phosphate-buffered saline buffer (PBS) containing the same specific activity of 2-deoxy-d-[3H]glucose. Uptake was stopped by the addition of 2 volumes of ice-cold 50 mm glucose in PBS. Cells were washed twice in the same solution and disrupted with 0.1m NaOH, 0.1% SDS. Radioactivity was determined by scintillation counting. Protein was determined by the Bradford method. Each condition was run in duplicate or triplicate. Glucose transport was linear during the period assayed (data not shown). Homogenates were obtained from cells cultured on 6-well plates at 2, 3, or 4 days of differentiation with one plate from each group. Cells were placed on ice, washed twice in ice-cold PBS, and scraped into 2 ml of PBS. Cells were pelleted at 3,000 rpm for 5 min and resuspended in 300 μl of lysis buffer (20 mmHepes, 350 mm NaCl, 20% (v/v) glycerol, 1% (v/v) Nonidet P-40, 1 mm MgCl2, 0.5 mm EDTA, 0.1 mm EGTA, pH 7.9) containing freshly added protease inhibitors (1 mm dithiothreitol, 0.1% (v/v) phenylmethylsulfonyl fluoride, 0.1% (v/v) aprotinin). The homogenate was shaken for 30 min at 4 °C and centrifuged at 1,000 rpm for 20 min, and the supernatant (SN) was collected and kept at −20 °C. For total membrane preparation (35Mitsumoto Y. Klip A. J. Biol. Chem. 1992; 267: 4957-4962Abstract Full Text PDF PubMed Google Scholar), 3–4 10-cm dishes were used for each experimental group. Cells were washed and scraped into 10 ml of PBS as described previously and pelleted at 1,000 rpm for 5 min. They were then resuspended in 3 ml/dish of cold homogenization buffer (250 mm sucrose, 2 mm EGTA, 5 mm sodium azide, 20 mm Hepes, pH 7.4) containing freshly added protease inhibitors (200 μm phenylmethylsulfonyl fluoride, 1 μm leupeptin, 1 μm pepstatin) and homogenized in a glass Dounce homogenizer (pestle A, 20 strokes). Homogenate was centrifuged at 2000 rpm for 5 min, and the supernatant (SN1) was kept. The pellet was resuspended in 1.5 ml of homogenization buffer/dish, re-homogenized, and centrifuged in the same way. The new pellet was discarded, and the supernatant (SN2) was pooled with SN1 and centrifuged at 190,000 × g for 1 h 10 min. The pellet (total membranes) was collected and resuspended in 250 μl of 20 mm Hepes, pH 7.4. When membrane fractionation was required (35Mitsumoto Y. Klip A. J. Biol. Chem. 1992; 267: 4957-4962Abstract Full Text PDF PubMed Google Scholar), cells were cultured on 2–4 15-cm dishes for each experimental group and treated initially as described above. The pooled SN1 and SN2 were centrifuged at 24,000 ×g for 1 h. The pellet, a partially purified plasma membrane (PM) fraction, was resuspended in 500 μl of 20 mm Hepes, pH 7.4, and the SN was further centrifuged at 190,000 × g for 1 h. The new pellet, a low density fraction (LDM) of intracellular origin, was resuspended in 200 μl of Hepes solution. Cells were cultured in 10-cm dishes. After treatments, cells were washed twice in cold PBS, and 1 mm ZnSO4 in 20% dimethyl sulfoxide was added at room temperature and incubated for 1 min. Cells were then washed gently in cold PBS and fixed with 2.5% glutaraldehyde in PBS for 2 min. Cells were then treated for 1 min with 50% ethanol and rinsed with PBS before staining with filtered 0.04% Giemsa in PBS, pH 6.8, overnight. Finally, cells were rinsed with tap water. Under an optical microscope, several randomly chosen fields were photographed, and the nuclei per cell were counted. For studies of incubated soleus muscle, male Wistar rats (250 g) were anesthetized with pentobarbital, 5–7 mg/100 g of body weight, and strips were isolated by a modification of the method of Crettaz et al. (36Crettaz M. Prentki M. Zaninetti D. Jeanrenaud B. Biochem. J. 1980; 186: 525-534Crossref PubMed Scopus (241) Google Scholar). For denervation studies, the peroneal nerve of anesthetized male rats (ketamine, 20 mg/kg of body weight) was severed unilaterally. Three days after denervation, tibialis anterior and extensor digitorum longus muscles were dissected and frozen in liquid nitrogen. All procedures were reviewed and approved by the local ethics committee. Muscles were processed to obtain total membranes as reported previously (37Gumà A. Zierath J.R. Wallberg-Henriksson H. Klip A. Am. J. Physiol. 1995; 268: E613-E622PubMed Google Scholar). Isolated strips of soleus muscles were incubated as reported (38Gumà A. Testar X. Palacı́n M. Zorzano A. Biochem. J. 1988; 253: 625-629Crossref PubMed Scopus (39) Google Scholar). HRG (3 nm, 120 min) was added after 30 min of muscle incubation, and 1 h later, insulin (100 nm, 60 min) was added to the medium. Previous to the uptake period, muscles were washed for 10 min with a glucose-depleted medium. Thereafter, muscles were incubated in the presence of 2-deoxy-d-[14C]glucose uptake (1 mCi/mmol) and d-[3H]mannitol (0.5 mCi/mmol), as an extracellular space marker, for 20 min, the time in which linear conditions are maintained. Muscles were then frozen and processed as reported (38Gumà A. Testar X. Palacı́n M. Zorzano A. Biochem. J. 1988; 253: 625-629Crossref PubMed Scopus (39) Google Scholar). SDS-polyacrylamide gel electrophoresis was performed on membrane protein. Proteins were transferred to Immobilon in buffer consisting of 20% methanol, 200 mm glycine, 25 mm Tris, pH 8.3. After transfer, the filters were blocked with 5% nonfat dry milk in Tris-buffered saline solution for 1 h at room temperature and then incubated overnight at 4 °C with antibodies directed against GLUT4 (1:800), GLUT1 (1: 1300), GLUT3 (1:500), ErbB3 (1:500), ErbB2 (1:500), myosin heavy chain (1:20), α1-subunit of the Na+/K+ ATPase (1:100), IRAP (1/1000), SCAMPs (1/3000), VAMP2 (1/1000), and cellubrevin (1/500), all diluted in 1% (w/v) bovine serum albumin, 0.067% (w/v) sodium azide in Tris-buffered saline, 0.09% (v/v) Tween 20. The immune complex was detected using an ECL chemiluminescence system. The autoradiograms were quantified using scanning densitometry. Immunoblots were performed in conditions in which autoradiographic detection was in the linear response range. cDNA was synthesized from 2 μg of total RNA at 20 μl of final reaction volume the using SuperScript II Rnase H− Reverse Transcriptase System (Life Technologies, Inc.). (dT)15 was used as the primer at 0.4 μm. Genomic contamination was monitored by enzyme-free controls. The resulting cDNA was diluted 1/10, and 1 μl of this dilution was amplified by PCR in an MJ Research PT-100 thermocycler at 25 μl final reaction volume. PCR primers 5′-TCAGAGCTTCGAATTAACAAAGC-′3 and 5′-GTGGTCATGGCTGATAGATAC CT-′3 (Life Technologies, Inc.), corresponding to 627–649 and 1608–1630 base pairs, respectively, of rat NDF (sequence with total identity to all neuregulin isoforms) were added at 0.4 μm and dNTPs at 0.2 mm. 1.25 units of Taq Expand High Fidelity and its corresponding buffer with Mg2+ were used (Roche Molecular Biochemicals). PCR was performed as follows: an initial step of 3 min at 94 °C; 35 cycles of 30 s at 94 °C, 30 s at 52 °C, and 2 min at 72 °C; and a final step of 5 min at 72 °C. Nonsaturating conditions were ensured by previous assays with the same cDNAs samples subjected to different number of PCR cycles (20 to 35) and in which the maintenance of linearity was determined (data not shown). For electrophoretic analysis, 5 μl of the final reaction volume was loaded in 1.5% agarose gel. As a first step, we assayed the level of neuregulin receptors ErbB2 and ErbB3 in total membrane extracts obtained from L6E9 myoblasts and cells after 2 and 3 days of differentiation induced by exposure to low serum conditions (Fig.1). The ErbB2 content was maximal in myoblasts, and expression was somewhat reduced during differentiation. In contrast, ErbB3 levels were low in myoblasts but rose during differentiation (a 2-fold increase on day 3 of differentiation) (Fig.1). Under these conditions, the abundance of the α1subunit of the Na+/K+-ATPase remained unaltered, indicating that the pattern of changes detected for neuregulin receptors was specific. Our results indicate the presence of neuregulin receptors in L6E9 muscle cells, which is consistent with reports in different muscle cell lines (25Gassman M. Casagranda F. Orioli D. Simon H. Lai C. Klein R. Lemke G. Nature. 1995; 378: 390-393Crossref PubMed Scopus (930) Google Scholar, 32Moscoso L.M. Chu G.C. Gautman M. Noakes P.G. Merlie J.P. Sanes J.R. Dev. Biol. 1995; 172: 158-169Crossref PubMed Scopus (160) Google Scholar). Fully differentiated L6E9 myotubes were incubated with different concentrations of HRG for 90 min (Fig. 2 A). HRG stimulated glucose transport with half-maximal effects detected at 2 nm HRG and maximal effects (2–2.5-fold stimulation of glucose transport) observed between 3 and 5 nm HRG, which is consistent with other reports on the effects of neuregulins (26Altiok N. Bessereau J.-L. Changeux J.-P. EMBO J. 1995; 14: 4258-4266Crossref PubMed Scopus (130) Google Scholar, 30Kim D. Chi S. Lee K.H. Rhee S. Kwon Y.K. Chung C.H. Kwon H. Kang M.-S. J. Biol. Chem. 1999; 274: 15395-15400Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar,39Si J. Miller D.S. Mei L. J. Biol. Chem. 1997; 272: 10367-10371Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Time course studies were also performed at maximal concentrations of HRG (3 nm) (Fig. 2 B). The maximal effect of HRG on glucose transport was reached between 60 and 90 min of incubation. The effect of a maximal concentration of HRG (3 nm for 90 min) on glucose transport was comparable with the effect caused by a supramaximal concentration of insulin (1 μm for 30 min) (Fig. 2 C). Furthermore, the combination of HRG and insulin caused an additive stimulation of glucose transport (Fig.2 C). To determine whether the effect of HRG on glucose transport required phosphatidylinositol 3-kinase activity, wortmannin (1 μm) (40Tsakiridis T. McDowell H. Walker T. Downes C.P. Hunda H.S. Vranic M. Klip A. Endocrinology. 1995; 136: 4315-4322Crossref PubMed Google Scholar) was added 30 min prior to HRG. Insulin-stimulated glucose transport was inhibited by wortmannin, and under these conditions wortmannin also blocked HRG-induced glucose transport (Fig.3). Incubation of L6E9 myotubes for 90 min in the presence of HRG did not alter the cell content of GLUT4, GLUT1, or GLUT3 (data not shown). In the next step, L6E9 myotubes were incubated in the absence or presence of 3 nm HRG for 90 min and then were further subjected to subcellular fractionation of membranes. This yielded two membrane fractions: PM, which were highly enriched in plasma membrane markers such as the α1 subunit of the Na+/K+-ATPase, and LDM, which are of intracellular origin. A typical experiment starting with four 15-cm dishes yielded ∼3–5 mg of membrane proteins in the PM fraction and 0.5–1.5 mg of membrane proteins in the LDM fraction. Incubation of cells for 90 min in the presence of HRG caused a 43% increase in the abundance of GLUT4 in PM fractions and a 50% decrease in GLUT4 in LDMs (Fig. 4, A and B), consistent with HRG-induced GLUT4 translocation. HRG also caused a 45 a
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