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• W2095024386 abstract "The protein-tyrosine phosphatases PTP1B and Syp have both been implicated as modulators of the mitogenic actions of insulin. However, the roles of these protein-tyrosine phosphatases in the metabolic actions of insulin are not well characterized. In this study, we directly assessed the ability of PTP1B and Syp to modulate insulin-stimulated translocation of the insulin-responsive glucose transporter GLUT4 in a physiologically relevant insulin target cell. Primary cultures of rat adipose cells were transiently transfected with either wild-type PTP1B (PTP1B-WT), wild-type Syp (Syp-WT), or the catalytically inactive mutants PTP1B-C/S or Syp-C/S. The effects of overexpression of these constructs on insulin-stimulated translocation of a co-transfected epitope-tagged GLUT4 were studied. Cells overexpressing either PTP1B-C/S or Syp-WT had insulin dose-response curves similar to those obtained with control cells expressing only epitope-tagged GLUT4. In contrast, for cells overexpressing PTP1B-WT the level of GLUT4 on the cell surface at each insulin dose (ranging from 0 to 60 nM) was significantly lower than that observed in the control cells. Interestingly, cells overexpressing the dominant inhibitory mutant Syp-C/S also had a small but statistically significant impairment in insulin responsiveness. At a maximally stimulating concentration of insulin (60 nM), cell surface epitope-tagged GLUT4 was approximately 20% less than that of the control cells. It is possible that effects from high level overexpression of Syp and PTP1B constructs may not reflect what occurs under physiological conditions. Nevertheless, our data raise the possibility that PTP1B may be a negative regulator of insulin-stimulated glucose transport, while Syp may have a small role as a positive mediator of the metabolic actions of insulin. The protein-tyrosine phosphatases PTP1B and Syp have both been implicated as modulators of the mitogenic actions of insulin. However, the roles of these protein-tyrosine phosphatases in the metabolic actions of insulin are not well characterized. In this study, we directly assessed the ability of PTP1B and Syp to modulate insulin-stimulated translocation of the insulin-responsive glucose transporter GLUT4 in a physiologically relevant insulin target cell. Primary cultures of rat adipose cells were transiently transfected with either wild-type PTP1B (PTP1B-WT), wild-type Syp (Syp-WT), or the catalytically inactive mutants PTP1B-C/S or Syp-C/S. The effects of overexpression of these constructs on insulin-stimulated translocation of a co-transfected epitope-tagged GLUT4 were studied. Cells overexpressing either PTP1B-C/S or Syp-WT had insulin dose-response curves similar to those obtained with control cells expressing only epitope-tagged GLUT4. In contrast, for cells overexpressing PTP1B-WT the level of GLUT4 on the cell surface at each insulin dose (ranging from 0 to 60 nM) was significantly lower than that observed in the control cells. Interestingly, cells overexpressing the dominant inhibitory mutant Syp-C/S also had a small but statistically significant impairment in insulin responsiveness. At a maximally stimulating concentration of insulin (60 nM), cell surface epitope-tagged GLUT4 was approximately 20% less than that of the control cells. It is possible that effects from high level overexpression of Syp and PTP1B constructs may not reflect what occurs under physiological conditions. Nevertheless, our data raise the possibility that PTP1B may be a negative regulator of insulin-stimulated glucose transport, while Syp may have a small role as a positive mediator of the metabolic actions of insulin. Insulin is an important regulator of growth and metabolism. The pleiotropic actions of insulin are initiated by the binding of insulin to its receptor and the resultant activation of intrinsic receptor tyrosine kinase activity (1Quon M.J. Butte A.J. Taylor S.I. Trends Endocrinol. Metab. 1994; 5: 369-376Abstract Full Text PDF PubMed Scopus (48) Google Scholar). Because tyrosine kinase activity is central to insulin signaling, protein-tyrosine phosphatases (PTPases) 1The abbreviations used are: PTPase(s)protein-tyrosine phosphataseIRS-1insulin receptor substrate-1pNPPp-nitrophenyl phosphateMES4-morpholineethanesulfonic acidWTwild-typeHAhemagglutinin may be important for modulating insulin signal transduction pathways (2Goldstein B.J. Receptor. 1993; 3: 1-15PubMed Google Scholar). Although there is good evidence that PTPases regulate mitogenic actions of insulin, the roles of various PTPases in metabolic actions of insulin are not well characterized. protein-tyrosine phosphatase insulin receptor substrate-1 p-nitrophenyl phosphate 4-morpholineethanesulfonic acid wild-type hemagglutinin The ubiquitously expressed prototype nontransmembrane PTPase PTP1B was among the first PTPases to be identified, cloned, and characterized (3Tonks N.K. Diltz C.D. Fischer E.H. J. Biol. Chem. 1988; 263: 6722-6730Abstract Full Text PDF PubMed Google Scholar, 4Tonks N.K. Diltz C.D. Fischer E.H. J. Biol. Chem. 1988; 263: 6731-6737Abstract Full Text PDF PubMed Google Scholar, 5Chernoff J. Schievella A.R. Jost C.A. Erikson R.L. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2735-2739Crossref PubMed Scopus (168) Google Scholar, 6Frangioni J.V. Beahm P.H. Shifrin V. Jost C.A. Neel B.G. Cell. 1992; 68: 545-560Abstract Full Text PDF PubMed Scopus (508) Google Scholar, 7Barford D. Keller J.C. Flint A.J. Tonks N.K. J. Mol. Biol. 1994; 239: 726-730Crossref PubMed Scopus (48) Google Scholar, 8Jia Z. Barford D. Flint A.J. Tonks N.K. Science. 1995; 268: 1754-1758Crossref PubMed Scopus (559) Google Scholar). PTP1B dephosphorylates the insulin receptor both in vitro and in intact cells (9Tappia P.S. Sharma R.P. Sale G.J. Biochem. J. 1991; 278: 69-74Crossref PubMed Scopus (17) Google Scholar, 10Hashimoto N. Zhang W.R. Goldstein B.J. Biochem. J. 1992; 284: 569-576Crossref PubMed Scopus (62) Google Scholar, 11Lammers R. Bossenmaier B. Cool D.E. Tonks N.K. Schlessinger J. Fischer E.H. Ullrich A. J. Biol. Chem. 1993; 268: 22456-22462Abstract Full Text PDF PubMed Google Scholar). In addition, PTP1B regulates the mitogenic actions of insulin (12Tonks N.K. Cicirelli M.F. Diltz C.D. Krebs E.G. Fischer E.H. Mol. Cell. Biol. 1990; 10: 458-463Crossref PubMed Scopus (88) Google Scholar, 13Ahmad F. Li P.M. Meyerovitch J. Goldstein B.J. J. Biol. Chem. 1995; 270: 20503-20508Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). Interestingly, in tissue culture models an increase in the level and activity of PTP1B has been associated with insulin resistance induced by exposure to high glucose levels. In addition, the level and activity of PTP1B in human skeletal muscle is positively correlated with in vivo measures of insulin sensitivity (14Ide R. Maegawa H. Kikkawa R. Shigeta Y. Kashiwagi A. Biochem. Biophys. Res. Commun. 1994; 201: 71-77Crossref PubMed Scopus (47) Google Scholar, 15Maegawa H. Ide R. Hasegawa M. Ugi S. Egawa K. Iwanishi M. Kikkawa R. Shigeta Y. Kashiwagi A. J. Biol. Chem. 1995; 270: 7724-7730Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 16Kusari J. Kenner K.A. Suh K.I. Hill D.E. Henry R.R. J. Clin. Invest. 1994; 93: 1156-1162Crossref PubMed Scopus (111) Google Scholar). Syp (also known as SH-PTP2, PTP1D, SHPTP3, or PTP2C) is a cytosolic PTPase containing two SH2 domains in addition to a catalytic phosphatase domain (17Feng G.S. Pawson T. Trends Genet. 1994; 10: 54-58Abstract Full Text PDF PubMed Scopus (169) Google Scholar). Binding of the SH2 domains of Syp to phosphotyrosine motifs on either the insulin receptor or insulin receptor substrate-1 (IRS-1) results in activation of Syp PTPase activity (18Sugimoto S. Wandless T.J. Shoelson S.E. Neel B.G. Walsh C.T. J. Biol. Chem. 1994; 269: 13614-13622Abstract Full Text PDF PubMed Google Scholar, 19Ugi S. Maegawa H. Olefsky J.M. Shigeta Y. Kashiwagi A. FEBS Lett. 1994; 340: 216-220Crossref PubMed Scopus (29) Google Scholar). Recently, a number of studies have shown that Syp participates in Ras and mitogen-activated protein kinase-dependent pathways as a positive mediator of mitogenic actions of insulin and other growth factors (20Yamauchi K. Milarski K.L. Saltiel A.R. Pessin J.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 664-668Crossref PubMed Scopus (268) Google Scholar, 21Xiao S. Rose D.W. Sasaoka T. Maegawa H. Burke Jr., T.R. Roller P.P. Shoelson S.E. Olefsky J.M. J. Biol. Chem. 1994; 269: 21244-21248Abstract Full Text PDF PubMed Google Scholar, 22Milarski K.L. Saltiel A.R. J. Biol. Chem. 1994; 269: 21239-21243Abstract Full Text PDF PubMed Google Scholar, 23Noguchi T. Matozaki T. Horita K. Fujioka Y. Kasuga M. Mol. Cell. Biol. 1994; 14: 6674-6682Crossref PubMed Scopus (350) Google Scholar). In addition, Hausdorff et al (24Hausdorff S.F. Bennett A.M. Neel B.G. Birnbaum M.J. J. Biol. Chem. 1995; 270: 12965-12968Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) have investigated the role of Syp in differentiated 3T3-L1 cells (tissue culture cells capable of differentiating into an adipocyte-like phenotype under appropriate conditions). They report that microinjection of either the SH2 domains of Syp or anti-Syp antibodies interfered with the mitogenic actions of insulin, but had no detectable effect on the insulin-stimulated translocation of the insulin-responsive glucose transporter GLUT4 (24Hausdorff S.F. Bennett A.M. Neel B.G. Birnbaum M.J. J. Biol. Chem. 1995; 270: 12965-12968Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). One of the most important metabolic actions of insulin is to increase glucose transport in tissues such as muscle and fat by recruiting GLUT4 to the cell surface. Previously, we used a transient transfection system for rat adipose cells in primary culture to demonstrate roles for the insulin receptor tyrosine kinase, IRS-1, and phosphatidylinositol 3-kinase in the insulin-stimulated translocation of GLUT4 (25Quon M.J. Zarnowski M.J. Guerre-Millo M. de la Luz Sierra M. Taylor S.I. Cushman S.W. Biochem. Biophys. Res. Commun. 1993; 194: 338-346Crossref PubMed Scopus (71) Google Scholar, 26Quon M.J. Guerre-Millo M. Zarnowski M.J. Butte A.J. Em M. Cushman S.W. Taylor S.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5587-5591Crossref PubMed Scopus (87) Google Scholar, 27Quon M.J. Butte A.J. Zarnowski M.J. Sesti G. Cushman S.W. Taylor S.I. J. Biol. Chem. 1994; 269: 27920-27924Abstract Full Text PDF PubMed Google Scholar, 28Quon M.J. Chen H. Ing B.L. Liu M.L. Zarnowski M.J. Yonezawa K. Kasuga M. Cushman S.W. Taylor S.I. Mol. Cell. Biol. 1995; 15: 5403-5411Crossref PubMed Scopus (143) Google Scholar). In the present study, we used a similar approach to overexpress wild-type or catalytically inactive mutant forms of PTP1B or Syp to directly test the roles of these PTPases in modulating insulin-stimulated translocation of GLUT4 in a physiologically relevant insulin target cell. Our data suggest that PTP1B may function as a negative regulator of the metabolic actions of insulin, while Syp may mediate a small positive effect on the ability of insulin to recruit GLUT4 to the cell surface. An expression vector (pCIS2) that generates high expression levels in transfected rat adipose cells (25Quon M.J. Zarnowski M.J. Guerre-Millo M. de la Luz Sierra M. Taylor S.I. Cushman S.W. Biochem. Biophys. Res. Commun. 1993; 194: 338-346Crossref PubMed Scopus (71) Google Scholar) was used as the parent vector for subsequent constructions. The cDNA coding for human GLUT4 with the influenza hemagglutinin epitope (HA1) inserted in the first exofacial loop of GLUT4 was subcloned into pCIS2 (GLUT4-HA). An XbaI/SmaI fragment containing the cDNA for human PTP1B (generous gift from Dr. Jonathan Chernoff) was ligated into XbaI/HpaI sites in the multiple cloning region of pCIS2 (PTP1B-WT). An XbaI/SmaI fragment containing the cDNA for a catalytically inactive mutant PTP1B with a cysteine to serine substitution at position 215 (generous gift from Dr. Jonathan Chernoff) was ligated into XbaI/HpaI sites in the multiple cloning region of pCIS2 (PTP1B-C/S). An XbaI/XhoI fragment containing the cDNA for human Syp (generous gift from Dr. Benjamin Neel) was ligated into the multiple cloning region of pCIS2 (Syp-WT). An XhoI/DraI fragment containing the cDNA for a catalytically inactive mutant Syp with a cysteine to serine substitution at position 459 (generous gift from Dr. Benjamin Neel) was ligated into XhoI/HpaI sites in the multiple cloning region of pCIS2 (Syp-C/S). Milligram quantities of the plasmid DNA vectors described above were obtained using a Magic Megaprep kit (Promega). The wild-type and mutant sequences in the catalytic domain of the respective PTP1B and Syp constructs were confirmed by direct sequencing. Isolated adipose cells were prepared from the epididymal fat pads of male rats (170-200 g, CD strain, Charles River Breeding Laboratories, Wilmington, MA) by collagenase digestion as described (25Quon M.J. Zarnowski M.J. Guerre-Millo M. de la Luz Sierra M. Taylor S.I. Cushman S.W. Biochem. Biophys. Res. Commun. 1993; 194: 338-346Crossref PubMed Scopus (71) Google Scholar, 29Karnieli E. Zarnowski M.J. Hissin P.J. Simpson I.A. Salans L.B. Cushman S.W. J. Biol. Chem. 1981; 256: 4772-4777Abstract Full Text PDF PubMed Google Scholar). Isolated adipose cells were transfected by electroporation as described (25Quon M.J. Zarnowski M.J. Guerre-Millo M. de la Luz Sierra M. Taylor S.I. Cushman S.W. Biochem. Biophys. Res. Commun. 1993; 194: 338-346Crossref PubMed Scopus (71) Google Scholar, 26Quon M.J. Guerre-Millo M. Zarnowski M.J. Butte A.J. Em M. Cushman S.W. Taylor S.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5587-5591Crossref PubMed Scopus (87) Google Scholar, 27Quon M.J. Butte A.J. Zarnowski M.J. Sesti G. Cushman S.W. Taylor S.I. J. Biol. Chem. 1994; 269: 27920-27924Abstract Full Text PDF PubMed Google Scholar, 28Quon M.J. Chen H. Ing B.L. Liu M.L. Zarnowski M.J. Yonezawa K. Kasuga M. Cushman S.W. Taylor S.I. Mol. Cell. Biol. 1995; 15: 5403-5411Crossref PubMed Scopus (143) Google Scholar). Cells from multiple cuvettes were pooled to obtain the necessary volume of cells for each experiment. Table I shows the combinations and concentrations of plasmid DNA as well as the number of cuvettes used for each of the insulin dose-response experiments.Table ITransfection of PTP1B and Syp constructs in rat adipose cellsGroupExperimental constructs versus controlNumber of cuvettesGLUT4-HAExperimentalpCIS2μg/cuvetteExperimental2024Control2024Nonspecific106 Open table in a new tab 20 h after electroporation, adipose cells were processed as described (26Quon M.J. Guerre-Millo M. Zarnowski M.J. Butte A.J. Em M. Cushman S.W. Taylor S.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5587-5591Crossref PubMed Scopus (87) Google Scholar, 27Quon M.J. Butte A.J. Zarnowski M.J. Sesti G. Cushman S.W. Taylor S.I. J. Biol. Chem. 1994; 269: 27920-27924Abstract Full Text PDF PubMed Google Scholar, 28Quon M.J. Chen H. Ing B.L. Liu M.L. Zarnowski M.J. Yonezawa K. Kasuga M. Cushman S.W. Taylor S.I. Mol. Cell. Biol. 1995; 15: 5403-5411Crossref PubMed Scopus (143) Google Scholar) and treated with insulin at final concentrations of 0, 0.024, 0.072, 0.3, or 60 nM at 37°C for 30 min. Cell surface epitope-tagged GLUT4 was determined by using the anti-HA1 mouse monoclonal antibody 12CA5 (Boehringer Mannheim) in conjunction with 125I-labeled sheep anti-mouse IgG as described (26Quon M.J. Guerre-Millo M. Zarnowski M.J. Butte A.J. Em M. Cushman S.W. Taylor S.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5587-5591Crossref PubMed Scopus (87) Google Scholar, 27Quon M.J. Butte A.J. Zarnowski M.J. Sesti G. Cushman S.W. Taylor S.I. J. Biol. Chem. 1994; 269: 27920-27924Abstract Full Text PDF PubMed Google Scholar, 28Quon M.J. Chen H. Ing B.L. Liu M.L. Zarnowski M.J. Yonezawa K. Kasuga M. Cushman S.W. Taylor S.I. Mol. Cell. Biol. 1995; 15: 5403-5411Crossref PubMed Scopus (143) Google Scholar). Cells transfected with the empty expression vector pCIS2 were used to determine nonspecific binding of the antibodies. Typically, the nonspecific binding was ∼30% of the total binding to cells transfected with GLUT4-HA and maximally stimulated with insulin (26Quon M.J. Guerre-Millo M. Zarnowski M.J. Butte A.J. Em M. Cushman S.W. Taylor S.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5587-5591Crossref PubMed Scopus (87) Google Scholar). The actual specific counts were comparable from experiment to experiment (see figure legends). The lipid weight from a 200-μl aliquot of cells was determined as described (30Cushman S.W. Salans L.B. J. Lipid Res. 1978; 19: 269-273Abstract Full Text PDF PubMed Google Scholar) and used to normalize the data for each sample. Expression of recombinant PTP1B, Syp, or GLUT4-HA was confirmed by immunoblotting extracts of cells that were prepared at the same time and had undergone transfection in parallel with the cells used for the translocation assay described above. Whole cell homogenates were prepared from cells co-transfected with GLUT4-HA (2 μg/cuvette) and either pCIS2, PTP1B-WT, PTP1B-C/S, Syp-WT, or Syp-C/S (4 μg/cuvette). Cells from 15 cuvettes were pooled for each group. After electroporation and overnight incubation, the cells were washed once and resuspended in 3 ml of TES buffer (20 mM Tris, 1 mM EDTA, 8.73% sucrose, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 10 μg/ml soybean trypsin inhibitor, 10 μg/ml bovine serum albumin, 1 mM phenylmethylsulfonyl fluoride, pH 7.4, 18°C). The cells were homogenized by being passed through a 25-gauge needle three times and then were centrifuged for 10 min at 400 × g, 4°C, to pellet nuclei. The fat cake and pellet were discarded. For detection of PTP1B and GLUT4-HA, the total membrane fraction was isolated from the whole cell homogenate by centrifuging for 30 min at 400,000 × g, 4°C. The pellet containing the total membrane fraction was resuspended in 600 μl of TES buffer and stored at -70°C until further processing. For immunodetection of PTP1B constructs, aliquots of the total membrane fractions from each group containing equal amounts of protein (500 μg) were solubilized in Laemmli sample buffer and subjected to SDS-polyacrylamide gel electrophoresis. The contents of the gel were transferred to nitrocellulose, and the PTP1B protein was detected with a polyclonal anti-PTP1B antibody (Upstate Biotechnology, Inc., Lake Placid, NY) and visualized using an antibody against rabbit IgG in conjunction with an enhanced chemiluminescent detection system (ECL, Amersham). For immunodetection of Syp constructs, EDTA and aprotinin (final concentrations of 2 mM and 1.3 μg/ml, respectively) were added to aliquots of whole cell homogenates containing equal amounts of protein (800 μg). The samples were centrifuged for 20 min at 17,000 × g, and the supernatant was precleared for 30 min at 4°C by incubating with 20 μl of protein A-agarose (Bio-Rad) that had been prewashed in lysis buffer (phosphate-buffered saline with 1% Nonidet P-40, 2 mM EDTA, and 1.3 μg/ml aprotinin). The samples were then immunoprecipitated by incubating for 45 min at 4°C with a human-specific polyclonal anti-Syp antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) followed by incubation with 20 μl of prewashed protein A-agarose for 45 min at 4°C. The immunoprecipitated samples were then washed three times in lysis buffer and eluted by adding 60 μl of Laemmli sample buffer and incubating at 98°C for 5 min. Twenty μl of each sample was loaded per lane and subjected to SDS-polyacrylamide gel electrophoresis. The contents of the gel were transferred to nitrocellulose, and Syp protein was detected with a monoclonal anti-Syp antibody (Transduction Laboratories, Lexington, KY) and visualized using an antibody against mouse IgG in conjunction with an enhanced chemiluminescent detection system (ECL, Amersham). To determine relative levels of GLUT4-HA in each group of transfected cells, total membrane fractions were prepared as above, and samples were immunoprecipitated with an anti-HA antibody followed by immunoblotting with an anti-GLUT4 antibody as described previously (28Quon M.J. Chen H. Ing B.L. Liu M.L. Zarnowski M.J. Yonezawa K. Kasuga M. Cushman S.W. Taylor S.I. Mol. Cell. Biol. 1995; 15: 5403-5411Crossref PubMed Scopus (143) Google Scholar). Cells were transfected with the various PTPase constructs as described above. After incubation overnight, cells were stimulated with insulin (60 nM) for 2 min, and cell extracts were prepared exactly as described above for the immunoblotting experiments (except 150 mM NaCl and 1 mM dithiothreitol were added to the TES buffer). PTPase activity in the cell extracts was determined by measuring the hydrolysis of p-nitrophenyl phosphate (pNPP). Equal aliquots of the cell extracts (80 μg of total protein in 50 μl of TES buffer) were added to 450 μl of a reaction mixture containing 50 mM MES, 150 mM NaCl, 2.5 mM EDTA, 0.1% bovine serum albumin, 2 mM dithiothreitol, and 50 mM pNPP and incubated for 10 min at 30°C. The reaction was stopped by the addition of 500 μl of 2 M KOH, and the amount of product (p-nitrophenyl) produced was measured by determining the absorbance at 405 nM in a spectrophotometer. Nonspecific absorbance was corrected for by subtracting the absorbance at 405 nM determined in the absence of cell extract. Insulin dose-response curves were compared using multivariate analysis of variance. Paired t tests were used to compare individual points where appropriate. p values of less than 0.05 were considered statistically significant. The insulin dose-response curves were fit to the equation y = a + b [x/(x + k)] using a Marquardt-Levenberg nonlinear least squares algorithm. When plotted on linear log axes, this equation gives a sigmoidal curve where the parameters are associated with the following properties: a = basal response, a + b = maximal response, k = half-maximal dose (ED50), and x = concentration of insulin. To directly evaluate the role of PTP1B in insulin-stimulated translocation of GLUT4, we overexpressed either wild-type or catalytically inactive mutant forms of human PTP1B in rat adipose cells. We confirmed that PTP1B-WT and PTP1B-C/S were overexpressed at comparable levels in our system by immunoblotting total membrane fractions isolated from transfected cells with an anti-PTP1B antibody (Fig. 1). In the lane containing cell extracts from control cells transfected with the empty expression vector pCIS2, there is a faint band representing endogenous rat PTP1B. The lanes containing extracts from groups of cells transfected with either PTP1B-WT or PTP1B-C/S show that the recombinant PTP1B constructs were expressed at levels that are much higher than the endogenous rat PTP1B levels. Since only ∼5% of the adipose cells that have undergone electroporation are actually transfected (26Quon M.J. Guerre-Millo M. Zarnowski M.J. Butte A.J. Em M. Cushman S.W. Taylor S.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5587-5591Crossref PubMed Scopus (87) Google Scholar), we estimate that there was at least a 100-fold overexpression of PTP1B-WT and PTP1B-C/S relative to endogenous PTP1B in the transfected cells. We also tested the PTPase activity of the recombinant PTP1B constructs by assessing the ability of cell extracts from insulin-stimulated transfected cells to hydrolyze the substrate pNPP. PTPase activity in extracts derived from the group of cells transfected with PTP1B-WT was approximately 3 times higher than that of the control cells transfected with the empty expression vector pCIS2, consistent with a high level of overexpression in the 5% of transfected cells. PTPase activity in extracts derived from the group of cells transfected with PTP1B-C/S was not significantly different from that of the control cells (data not shown). After confirming overexpression of the recombinant PTP1B constructs in transfected adipose cells, we next determined their effects on the ability of insulin to recruit a co-transfected epitope-tagged GLUT4 to the cell surface. The insulin dose-response curve for control cells co-transfected with the empty expression vector pCIS2 and GLUT4-HA showed a 2.5-fold increase in cell surface GLUT4-HA upon maximal insulin stimulation (60 nM) with an ED50 of 0.06 nM. In contrast, the insulin dose-response curve for cells overexpressing PTP1B-WT demonstrated both decreased sensitivity and decreased responsiveness to insulin (Fig. 2A). In the absence of insulin, the basal level of cell surface GLUT4 for cells overexpressing PTP1B-WT was approximately half of that seen in the basal state for the control cells. Furthermore, at every insulin dose the amount of GLUT4 at the cell surface of cells overexpressing PTP1B-WT was less than that of the control cells (ED50 = 0.18 nM). At 60 nM insulin, the maximal insulin response for cells overexpressing PTP1B-WT was approximately 80% of the maximal response for the control cells. Thus, the insulin sensitivity of cells overexpressing PTP1B-WT was decreased approximately 3-fold while the insulin responsiveness was decreased by about 20%. When analyzed by multivariate analysis of variance, the difference in the dose-response curve caused by overexpression of PTP1B-WT was highly significant (p < 1 × 10-10). In contrast to PTP1B-WT, we were unable to detect any significant difference in the insulin-stimulated translocation of GLUT4-HA in cells overexpressing the mutant PTP1B-C/S when compared with the control cells (Fig. 2B). It is possible that the lower level of cell surface GLUT4-HA we observed in cells overepxressing PTP1B-WT is due to an effect of PTP1B-WT on expression of GLUT4-HA rather than to an effect on insulin sensitivity or responsiveness. Therefore, we measured total GLUT4-HA in cells co-transfected with pCIS2, PTP1B-WT, or PTP1B-C/S by immunoprecipitating total membrane fractions with an anti-HA antibody followed by immunoblotting with an anti-GLUT4 antibody (Fig. 3). We observed comparable levels of GLUT4-HA in all groups. Taken together, our data suggest that overexpression of PTP1B-WT has an inhibitory effect on insulin signal transduction pathways related to GLUT4 translocation. We confirmed overexpression of Syp-WT and Syp-C/S by using a human-specific polyclonal anti-Syp antibody to immunoprecipitate whole cell homogenates of cells transfected with either the empty expression vector pCIS2, Syp-WT, or Syp-C/S in conjunction with immunoblotting with a monoclonal anti-Syp antibody (Fig. 4). The lanes containing extracts from cells transfected with Syp-WT or Syp-C/S show that the two recombinant Syp constructs are overexpressed at comparable levels. A specific band representing endogenous rat Syp is not seen in cells transfected with pCIS2 because the samples were immunoprecipitated with a human-specific antibody. We tested the PTPase activity of the recombinant Syp constructs by assessing the ability of cell extracts from insulin-stimulated transfected cells to hydrolyze the substrate pNPP. PTPase activity in extracts derived from the group of cells transfected with Syp-WT was approximately 2 times higher than that of the control cells transfected with the empty expression vector pCIS2, consistent with a high level of overexpression in the 5% of transfected cells. PTPase activity in extracts derived from the group of cells transfected with Syp-C/S was not significantly different from than that of the control cells (data not shown). To gain insight into the ability of Syp to modulate insulin-stimulated translocation of GLUT4, we tested the effects of overexpressing either Syp-WT or Syp-C/S on the ability of insulin to recruit co-transfected GLUT4-HA to the cell surface (Fig. 5). Control cells co-transfected with pCIS2 and GLUT4-HA had a 3-fold increase in cell surface GLUT-HA upon maximal insulin stimulation with an ED50 of 0.06 nM. Cells overexpressing Syp-WT had an insulin dose-response curve that was not significantly different from that of the control cells (p = 0.43) (Fig. 5A). In contrast, cells overexpressing Syp-C/S had decreased insulin responsiveness when compared with control cells (Fig. 5B). The level of cell surface GLUT4-HA for cells overexpressing Syp-C/S was approximately 20% lower than that of the control cells at the maximally stimulating dose of insulin (p < 0.02). However, there was no significant change in the estimated value of the half-maximal dose of insulin (ED50) for cells overexpressing Syp-C/S when compared with the control cells. To help rule out the possibility that the difference in the insulin dose-response curve for cells overexpressing Syp-C/S is due to an effect of the mutant PTPase on expression of GLUT4-HA, we evaluated total levels of GLUT4-HA in cells co-transfected with GLUT4-HA and either pCIS2, Syp-WT, or Syp-C/S. We immunoprecipitated total membrane fractions from each group of transfected cells with an anti-HA antibody, followed by immunoblotting with an anti-GLUT4 antibody (Fig. 3). The results of this experiment demonstrate that there is no detectable effect of overexpressing Syp-WT or Syp-C/S on the total level of GLUT4-HA in transfected cells. Thus, the difference in the insulin dose-response curve of cells overexpressing Syp-C/S is most likely due to an effect on signal transduction pathways related to GLUT4 translocation. Since Syp-C/S is known to behave in a dominant inhibitory manner, our results suggest that Syp may play a small positive role in mediating insulin-stimulated translocation of GLUT4. A large family of PTPases is thought to be involved in modulating signal transduction pathways initiated by receptor tyrosine kinases such as the insulin receptor (31Tonks N.K. Yang Q. Flint A.J. Gebbink M.F. Franza Jr., B.R. Hill D.E. Sun H. Brady-Kalnay S. Cold Spring Harbor Symp. Quant. Biol. 1992; 57: 87-94Crossref PubMed Scopus (28) Google Scholar, 32Neel B.G. Semin. Cell Biol." @default.
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