The Trimeric GTP-binding Protein (Gq/G11) α Subunit Is Required for Insulin-stimulated GLUT4 Translocation in 3T3L1 Adipocytes
2000; Elsevier BV; Volume: 275; Issue: 10 Linguagem: Inglês
10.1074/jbc.275.10.7167
ISSN1083-351X
AutoresMakoto Kanzaki, Robert T. Watson, Nikolai O. Artemyev, Jeffrey E. Pessin,
Tópico(s)PI3K/AKT/mTOR signaling in cancer
ResumoTo investigate the potential role of trimeric GTP-binding proteins regulating GLUT4 translocation in adipocytes, wild type and constitutively active Gq(Gq/Q209L), Gi (Gi/Q205L), and Gs (Gs/Q227L) α subunit mutants were expressed in 3T3L1 adipocytes. Although expression of neither the wild type nor Gi/Q205L and Gs/Q227L α subunit mutants had any effect on the basal or insulin-stimulated translocation of a co-expressed GLUT4-enhanced green fluorescent protein (EGFP) fusion protein, expression of Gq/Q209L resulted in GLUT4-EGFP translocation in the absence of insulin. In contrast, microinjection of an inhibitory Gq/G11 α subunit-specific antibody but not a Gi or Gsα subunit antibody prevented insulin-stimulated endogenous GLUT4 translocation. Consistent with a required role for GTP-bound Gq/G11, expression of the regulators of G protein signaling (RGS4 and RGS16) also attenuated insulin-stimulated GLUT4-EGFP translocation. To assess the relationship between Gq/G11 function with the phosphatidylinositol 3-kinase dependent pathway, expression of a dominant-interfering p85 regulatory subunit, as well as wortmannin treatment inhibited insulin-stimulated but not Gq/Q209L-stimulated GLUT4-EGFP translocation. Furthermore, Gq/Q209L did not induce thein vivo accumulation of phosphatidylinositol-3,4,5-trisphosphate (PIP3), whereas expression of the RGS proteins did not prevent the insulin-stimulated accumulation of PIP3. Together, these data demonstrate that insulin stimulation of GLUT4 translocation requires at least two independent signal transduction pathways, one mediated through the phosphatidylinositol 3-kinase and another through the trimeric GTP-binding proteins Gq and/or G11. To investigate the potential role of trimeric GTP-binding proteins regulating GLUT4 translocation in adipocytes, wild type and constitutively active Gq(Gq/Q209L), Gi (Gi/Q205L), and Gs (Gs/Q227L) α subunit mutants were expressed in 3T3L1 adipocytes. Although expression of neither the wild type nor Gi/Q205L and Gs/Q227L α subunit mutants had any effect on the basal or insulin-stimulated translocation of a co-expressed GLUT4-enhanced green fluorescent protein (EGFP) fusion protein, expression of Gq/Q209L resulted in GLUT4-EGFP translocation in the absence of insulin. In contrast, microinjection of an inhibitory Gq/G11 α subunit-specific antibody but not a Gi or Gsα subunit antibody prevented insulin-stimulated endogenous GLUT4 translocation. Consistent with a required role for GTP-bound Gq/G11, expression of the regulators of G protein signaling (RGS4 and RGS16) also attenuated insulin-stimulated GLUT4-EGFP translocation. To assess the relationship between Gq/G11 function with the phosphatidylinositol 3-kinase dependent pathway, expression of a dominant-interfering p85 regulatory subunit, as well as wortmannin treatment inhibited insulin-stimulated but not Gq/Q209L-stimulated GLUT4-EGFP translocation. Furthermore, Gq/Q209L did not induce thein vivo accumulation of phosphatidylinositol-3,4,5-trisphosphate (PIP3), whereas expression of the RGS proteins did not prevent the insulin-stimulated accumulation of PIP3. Together, these data demonstrate that insulin stimulation of GLUT4 translocation requires at least two independent signal transduction pathways, one mediated through the phosphatidylinositol 3-kinase and another through the trimeric GTP-binding proteins Gq and/or G11. insulin receptor substrate pleckstrin homology enhanced green fluorescent protein cation-independent mannose-6-phosphate receptor transferrin receptor phosphatidylinositol phospholipase C regulators of G protein signaling phosphatidylinositol (3.Rea S. James D.E. Diabetes. 1997; 46: 1667-1677Crossref PubMed Google Scholar, 4.Kandror K.V. Pilch P.F. Am. J. Physiol. 1996; 271: E1-E14Crossref PubMed Google Scholar, 5.Cheatham B. Kahn C.R. Endocr. 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More recently, endothelin-1 (Gq/G11-coupled receptor agonist) has been observed to stimulate glucose transport and GLUT4 translocation in 3T3L1 adipocytes (56.Kishi K. Muromoto N. Nakaya Y. Miyata I. Hagi A. Hayashi H. Ebina Y. Diabetes. 1998; 47: 550-558Crossref PubMed Scopus (150) Google Scholar). Although these data provide circumstantial evidence that the trimeric GTP-binding protein Gq and/or G11 can couple to GLUT4 translocation, the relationship between insulin signaling and adrenergic activation on GLUT4 translocation remains unclear. Furthermore, whether receptor agonists that induce GLUT4 translocation directly function through Gq/G11 activation has not yet been determined. To address these issues, we have observed that expression of a constitutively active α subunit mutant of Gq, but not Gs or Gi, is sufficient to induce GLUT4 translocation in 3T3L1 adipocytes. In addition, this Gq signaling pathway displays the same characteristics as osmotic shock and GTPγS stimulation, in that it is PI 3-kinase-independent but requires tyrosine kinase activation. Furthermore, inhibition of Gq/G11 function prevented the insulin-stimulated translocation of GLUT4. Together, these data strongly suggest that insulin must utilize at least two independent pathways (PI 3-kinase-dependent and Gq/G11-dependent) that functionally integrate to induce GLUT4 translocation in 3T3L1 adipocytes. A mouse monoclonal anti-GLUT4 antibody (RDI-GLUT4abmX) was purchased from Research Diagnostics, Inc. (Flanders, NJ). The cDNA for the cation-independent mannose-6-phosphate receptor (CI/MPR) was kindly provided by Dr. Richard Roth (Stanford University, CA). Texas Red-conjugated transferrin was obtained from Molecular Probes (Eugene, OR). Rabbit polyclonal Gq/G11α, Gαi1/2 and Gsα subunit (carboxyl-terminal) antibodies were purchased from Calbiochem (La Jolla, CA). A sheep polyclonal anti-maltose-binding protein was generously gifted by Dr. Morris Birnbaum (University of Pennsylvania). Cy5-conjugated Donkey anti-mouse IgG and fluorescien isothiocyanate-conjugated Donkey anti-sheep IgG were purchased from Jackson Immunoresearch Laboratories (West Grove, PA). Vectashield was obtained from Vector Laboratories (Burlingame, CA). Mini-prep DNA kit was purchased from Invitrogen (Carlsbad, CA). The GLUT4-enhanced green fluorescent protein (EGFP) fusion construct was prepared as described previously and cloned into the mammalian expression plasmid pcDNA3 (57.Thurmond D.C. Ceresa B.P. Okada S. Elmendorf J.S. Coker K. Pessin J.E. J. Biol. Chem. 1998; 273: 33876-33883Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Similarly, a CI/MPR-EGFP fusion protein was generated by cloning EGFP at the carboxyl-terminal domain of CI/MPR. The Gq/WT and constitutively active Gq/Q209L α subunits were obtained from Dr. John Exton (Vanderbilt University) in the mammalian expression plasmid pCMV. The Gs/WT α subunit in the mammalian expression plasmid pCR3.1 was provided by Dr. Mario Ascoli (Department of Pharmacology, The University of Iowa). Gi2/WT and constitutively active mutants Gi2/Q205L and Gs/Q227L in the mammalian expression plasmid pcDNA1 were purchased from the American Type Tissue Culture (Manassas, VA). The cDNAs for RGS4, RGS16, and the inactive RGS16 mutant RGS16/N131A were subcloned into pcDNA3 using theBamHI/XhoI sites (for RGS4) and theEcoRI/XhoI sites (for RGS16 and RGS16/N131A). The wild type and dominant-interfering mutants of the p85 regulatory subunit of the type I PI 3-kinase p85/WT and Δp85 were kindly provided by Dr. Wataru Ogawa (Kobe University, Kobe, Japan) and were subcloned into pcDNA3 using the BamHI/EcoRI sites. The GRP cDNA was generously provided by Dr. Michael Czech (University of Massachusetts). The PH domain was then subcloned carboxyl-terminal to EGFP generating the EGFP-GRP/PH fusion construct. 3T3L1 preadipocytes were cultured in DMEM containing 25 mm glucose, 10% calf serum at 37 °C in a 8% CO2 atmosphere and induced to differentiate into an adipocyte phenotype as described previously (58.Olson A.L. Knight J.B. Pessin J.E. Mol. Cell. Biol. 1997; 17: 2425-2435Crossref PubMed Scopus (209) Google Scholar). Confluent cultures were induced to differentiate by incubation of the cells with DMEM containing 25 mm glucose, 10% fetal bovine serum, 1 μg/ml insulin, 1 mm dexamethasone, and 0.5 mm isobutyl-1-methylxanthine. After 4 days, the medium was changed to DMEM, 25 mm glucose, 10% fetal bovine serum, and 1 μg/ml insulin for an additional 4 days. The medium was then changed to DMEM containing 25 mm glucose and 10% fetal bovine serum. Under these conditions, greater than 95% of the cell population morphologically differentiated into adipocytes. The adipocytes were maintained for an additional 4–8 days prior to use. The differentiated 3T3L1 adipocytes were transfected by electroporation as described previously (59.Min J. Okada S. Coker K. Ceresa B.P. Elmendorf J.S. Syu L.-J. Noda Y. Saltiel A.R. Pessin J.E. Mol. Cell. 1999; 3: 751-760Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Briefly, the adipocytes were put into suspension by mild trypsinization and electroporated with plasmids under low voltage conditions (160 V, 950 microfarad). The cells were then allowed to adhere to tissue culture dishes for 36 h, and the adipocytes were then serum-starved for 2 h prior to experiments. In some experiments, the electroporated adipocytes were seeded on cover slips. 3T3L1 adipocytes were grown on 35-mm tissue culture dishes, and prior to microinjection, the medium was changed to Lebovitz's L-15 medium containing 0.1% bovine serum albumin. Differentiated 3T3L1 adipocytes were impaled using an Eppendorf model 5171 micromanipulator and injected with 2–4 mg/ml antibodies plus 4 mg/ml MBP-Ras in 100 mm KCl, 5 mm Na2PO4, pH 7.2, directly into the cell cytoplasm with an Eppendorf model 5246 transjector. Preparation of plasma membrane sheets from the microinjected 3T3L1 adipocytes was performed as described previously (59.Min J. Okada S. Coker K. Ceresa B.P. Elmendorf J.S. Syu L.-J. Noda Y. Saltiel A.R. Pessin J.E. Mol. Cell. 1999; 3: 751-760Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Briefly, the cells were rinsed once in ice-cold phosphate-buffered saline (PBS) and incubated with 0.5 mg/ml poly-d-lysine (Sigma) for 1 min. Cells were then swollen in a hypotonic buffer (23 mm KCl, 10 mm HEPES, 2 mm MgCl2, 1 mm EGTA, pH 7.5) by three successive rinses. The swollen cells were sonicated for 3 s at power setting 5 with a Fisher Sonic Dismembrator model 550 fitted with a 5-mm microtip set 0.7 cm above the surface of the cell monolayer in 10 ml of sonication buffer (70 mm KCl, 30 mmHEPES, 6 mm MgCl2, 3 mm EGTA, 1 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, pH 7.5). The bound plasma membranes sheets were washed twice in sonication buffer and subjected to immunofluorescence confocal microscopy as described below. The isolated plasma membrane sheets were fixed with sonication buffer containing 2% paraformaldehyde for 30 min, and the reaction was quenched by incubation with PBS containing 100 mm glycine for 20 min. After three rinses in PBS, the sheets were blocked overnight at 4 °C in 5% donkey serum in PBS. The blocked sheets were incubated at room temperature for 1 h with a 1:100 dilution of the monoclonal GLUT4 antibody in combination with a 1:1000 dilution of polyclonal sheep MBP antiserum. The plasma membrane sheets were then washed three times with PBS and incubated for 1 h with a 1:100 dilution of fluorescien isothiocyanate-conjugated donkey anti-sheep IgG in combination with a 1:100 dilution of Cy5-conjugated donkey anti-mouse IgG. Following incubation with the secondary antibodies, the membrane sheets were rinsed three times in PBS and mounted for microscopic analysis using Vectashield mounting medium. Confocal images were obtained on a Bio-Rad MRC 600 laser confocal microscope (The University of Iowa Central Microscopy Research Facility). Differentiated 3T3L1 adipocytes were electroporated and allowed to recover as described above. The cells were then placed in serum-free DMEM for 2 h and incubated with 10 μg/ml of Texas Red-conjugated transferrin at 4 °C for 30 min. The cells were then extensively washed in serum-free DMEM to remove all the unbound Texas Red-cojugated transferrin, and the cells were warmed to 37 °C for 30 min. The cells were then fixed with 4% paraformaldehyde in PBS for 20 min and visualized by confocal fluorescent microscopy. The in vivo production of PIP3 was determined by a modification of the ARNO/PH domain GFP fusion protein assay (60.Venkateswarlu K. Oatey P.B. Tavare J.M. Cullen P.J. Curr. Biol. 1998; 8: 463-466Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). Briefly, 3T3L1 adipocytes were transfected with a cDNA encoding for the GRP/PH domain fused to EGFP (EGFP-GRP/PH). The GRP/PH domain has high affinity for PIP3 with substantially lower affinity for other phosphatidylinositol phosphates (60.Venkateswarlu K. Oatey P.B. Tavare J.M. Cullen P.J. Curr. Biol. 1998; 8: 463-466Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 61.Klarlund J.K. Guilherme A. Holik J.J. Virbasius J.V. Chawla A. Czech M.P. Science. 1997; 275: 1927-1930Crossref PubMed Scopus (372) Google Scholar). Because the insulin stimulation of PIP3 formation occurs at the plasma membrane, the plasma membrane localization of the EGFP-GRP/PH fusion protein directly reflects the accumulation of PIP3. Recently several studies have shown that receptor agonists coupled to Gi and Gq/G11 can result in GLUT4 translocation in both transfected fibroblasts and 3T3L1 adipocytes (50.Kishi K. Muromoto N. Nakaya Y. Miyata I. Hagi A. Hayashi H. Ebina Y. Diabetes. 1998; 47: 550-558Crossref PubMed Scopus (147) Google Scholar, 55.Kishi K. Hayashi H. Wang L. Kamohara S. Tamaoka K. Shimizu T. Ushikubi F. Narumiya S. Ebina Y. J. Biol. Chem. 1996; 271: 26561-26568Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 56.Kishi K. Muromoto N. Nakaya Y. Miyata I. Hagi A. Hayashi H. Ebina Y. Diabetes. 1998; 47: 550-558Crossref PubMed Scopus (150) Google Scholar). To determine the potential involvement of the Gq/G11 α subunit as an intermediate in a potential signal transduction cascade leading to GLUT4 translocation, we examined the effect of Gq/WT and constitutively active Gq/Q209L α subunit expression (Fig.1). In these experiments we also co-transfected the cells with the GLUT4-EGFP fusion protein to easily assess only the transfected cell population. As typically observed for the endogenous GLUT4 protein, in the basal state GLUT4-EGFP was localized to the perinuclear region and small vesicles scattered throughout the cytoplasm (data not shown). This distribution was identical to cells co-expressing GLUT4-EGFP and the Gq/WT α subunit (Fig. 1 A, panel a). As expected, insulin stimulation resulted in the translocation of the GLUT4-EGFP protein to the plasma membrane detected as a continuous rim of cell surface fluorescence in both GLUT4-EGFP transfected and Gq/WT co-transfected cells (Fig. 1 A, panel b). In contrast, cells expressing Gq/Q209L resulted in the constitutive association of the GLUT4-EGFP protein with the plasma membrane in the absence of insulin (Fig. 1 A, panel c). The subsequent insulin stimulation had no further effect on the extent but did slightly increase the number of cells displaying GLUT4-EGFP cell surface rim fluorescence (Fig. 1 A,panel d). In control experiments, expression of wild type Gs and Gi2 (Gs/WT, Gi2/WT) or constitutively active Gs and Gi2 (Gs/Q227L, Gi/Q205L) had no effect on either the basal or insulin-stimulated translocation of GLUT4-EGFP (Fig. 1 A, panels e–l). Quantitation of these data by scoring the transfected cells for GLUT4-EGFP plasma membrane rim fluorescence is presented in Fig. 1 B. Together, these data suggest that expression of constitutively active of Gαq is sufficient to induce GLUT4 translocation, which is not mimicked by the expression of constitutively active Gsor Gi2. Although the stimulation of GLUT4-EGFP translocation by Gq/Q209L appeared to be specific for this family of trimeric GTP-binding proteins, the overexpressed proteins can potentially impinge upon signaling pathways not necessarily utilized by the endogenous protein. Thus, to examine the role of the endogenous Gq/G11 α subunit, we microinjected 3T3L1 adipocytes with several specific antibodies and examined the translocation of the endogenous GLUT4 proteins by single cell immunofluorescence in isolated plasma membrane sheets (Fig.2). In these experiments, all the antibodies were prepared against the carboxyl-terminal region of the individual α subunits, which inhibits the coupling between the trimeric GTP-binding protein with their respective receptors (62.Williams C.J. Mehlmann L.M. Jaffe L.A. Kop
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