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• W2052878956 abstract "Glucose homeostasis is controlled by insulin in part through the stimulation of glucose transport in muscle and fat cells. This insulin signaling pathway requires phosphatidylinositol (PI) 3-kinase-mediated 3′-polyphosphoinositide generation and activation of Akt/protein kinase B. Previous experiments using dominant negative constructs and gene ablation in mice suggested that two phosphoinositide phosphatases, SH2 domain-containing inositol 5′-phosphatase 2 (SHIP2) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) negatively regulate this insulin signaling pathway. Here we directly tested this hypothesis by selectively inhibiting the expression of SHIP2 or PTEN in intact cultured 3T3-L1 adipocytes through the use of short interfering RNA (siRNA). Attenuation of PTEN expression by RNAi markedly enhanced insulin-stimulated Akt and glycogen synthase kinase 3α (GSK-3α) phosphorylation, as well as deoxyglucose transport in 3T3-L1 adipocytes. In contrast, depletion of SHIP2 protein by about 90% surprisingly failed to modulate these insulin-regulated events under identical assay conditions. In control studies, no diminution of insulin signaling to the mitogen-activated protein kinases Erk1 and Erk2 was observed when either PTEN or SHIP2 were depleted. Taken together, these results demonstrate that endogenous PTEN functions as a suppressor of insulin signaling to glucose transport through the PI 3-kinase pathway in cultured 3T3-L1 adipocytes. Glucose homeostasis is controlled by insulin in part through the stimulation of glucose transport in muscle and fat cells. This insulin signaling pathway requires phosphatidylinositol (PI) 3-kinase-mediated 3′-polyphosphoinositide generation and activation of Akt/protein kinase B. Previous experiments using dominant negative constructs and gene ablation in mice suggested that two phosphoinositide phosphatases, SH2 domain-containing inositol 5′-phosphatase 2 (SHIP2) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) negatively regulate this insulin signaling pathway. Here we directly tested this hypothesis by selectively inhibiting the expression of SHIP2 or PTEN in intact cultured 3T3-L1 adipocytes through the use of short interfering RNA (siRNA). Attenuation of PTEN expression by RNAi markedly enhanced insulin-stimulated Akt and glycogen synthase kinase 3α (GSK-3α) phosphorylation, as well as deoxyglucose transport in 3T3-L1 adipocytes. In contrast, depletion of SHIP2 protein by about 90% surprisingly failed to modulate these insulin-regulated events under identical assay conditions. In control studies, no diminution of insulin signaling to the mitogen-activated protein kinases Erk1 and Erk2 was observed when either PTEN or SHIP2 were depleted. Taken together, these results demonstrate that endogenous PTEN functions as a suppressor of insulin signaling to glucose transport through the PI 3-kinase pathway in cultured 3T3-L1 adipocytes. Insulin is the primary hormone that regulates glucose homeostasis, and impairment of insulin action and secretion plays a critical role in the pathogenesis of diabetes mellitus (1Saltiel A.R. Cell. 2001; 104: 517-529Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar, 2Virkamaki A. Ueki K. Kahn C.R. J. Clin. Invest. 1999; 103: 931-943Crossref PubMed Scopus (721) Google Scholar). One of the primary metabolic responses mediated by insulin is the stimulation of glucose transport and glycogen synthesis in muscle and adipose tissue (3Czech M.P. Corvera S. J. Biol. Chem. 1999; 274: 1865-1868Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, 4Pessin J.E. Thurmond D.C. Elmendorf J.S. Coker K.J. Okada S. J. Biol. Chem. 1999; 274: 2593-2596Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar). It is now established that translocation of glucose transporter 4 (GLUT4) 1The abbreviations used are: GLUT4, glucose transporter 4; IR, insulin receptor; IRS, insulin receptor substrate; PI, phosphatidylinositol; PI(3,4,5)P3 (or PIP3), phosphatidylinositol 3,4,5-trisphosphate; PDK1, phosphoinositide-dependent protein kinase 1; SHIP, Src homology 2-containing inositol 5′-phosphatase; PTEN, phosphatase and tensin homolog deleted on chromosome 10; GSK-3, glycogen synthase kinase 3; siRNA, short interfering RNA; RNAi, RNA interference; DMEM, Dulbecco's modified Eagle's medium; MAPK, mitogen-activated protein kinase; SKIP, skeletal muscle- and kidney-enriched inositol phosphatase; PH, pleckstrin homology. 1The abbreviations used are: GLUT4, glucose transporter 4; IR, insulin receptor; IRS, insulin receptor substrate; PI, phosphatidylinositol; PI(3,4,5)P3 (or PIP3), phosphatidylinositol 3,4,5-trisphosphate; PDK1, phosphoinositide-dependent protein kinase 1; SHIP, Src homology 2-containing inositol 5′-phosphatase; PTEN, phosphatase and tensin homolog deleted on chromosome 10; GSK-3, glycogen synthase kinase 3; siRNA, short interfering RNA; RNAi, RNA interference; DMEM, Dulbecco's modified Eagle's medium; MAPK, mitogen-activated protein kinase; SKIP, skeletal muscle- and kidney-enriched inositol phosphatase; PH, pleckstrin homology. from intracellular compartments to the plasma membrane is mainly responsible for the insulin-stimulated increase in glucose uptake in these tissues (5Cushman S.W. Wardzala L.J. J. Biol. Chem. 1980; 255: 4758-4762Abstract Full Text PDF PubMed Google Scholar, 6Holman G.D. Kasuga M. Diabetologia. 1997; 40: 991-1003Crossref PubMed Scopus (188) Google Scholar, 7Rea S. James D.E. Diabetes. 1997; 46: 1667-1677Crossref PubMed Google Scholar, 8Bryant N.J. Govers R. James D.E. Nat. Rev. Mol. Cell. Biol. 2002; 3: 267-277Crossref PubMed Scopus (930) Google Scholar). This process is initiated when insulin binds and activates its receptor tyrosine kinase at the cell surface. The activated insulin receptor phosphorylates the insulin receptor substrate (IRS) family of proteins on tyrosine residues. IRS proteins propagate insulin signaling to the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase, which activates the p110 catalytic subunit (3Czech M.P. Corvera S. J. Biol. Chem. 1999; 274: 1865-1868Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, 4Pessin J.E. Thurmond D.C. Elmendorf J.S. Coker K.J. Okada S. J. Biol. Chem. 1999; 274: 2593-2596Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar). A growing number of experiments indicate that insulin-induced PI 3-kinase activation is critical for insulin-induced metabolic actions, including glucose uptake and glycogen synthesis (9White M.F. Am. J. Physiol. 2002; 283: E413-E422Crossref PubMed Scopus (44) Google Scholar, 10Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (837) Google Scholar, 11Saltiel A.R. Am. J. Physiol. 1996; 270: E375-E385PubMed Google Scholar). Thus, phosphorylation of phosphatidylinositol 4,5-bisphosphate by activated PI 3-kinase increases levels of PI(3,4,5)P3 at cellular membranes. This phosphorylated lipid is a potent modulator of the protein kinases PDK1, atypical protein kinase C λ/ζ, and Akt/Protein kinase B (12Stephens L. Anderson K. Stokoe D. Erdjument-Bromage H. Painter G.F. Holmes A.B. Gaffney P.R. Reese C.B. McCormick F. Tempst P. Coadwell J. Hawkins P.T. Science. 1998; 279: 710-714Crossref PubMed Scopus (911) Google Scholar, 13Stokoe D. Stephens L.R. Copeland T. Gaffney P.R. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Science. 1997; 277: 567-570Crossref PubMed Scopus (1046) Google Scholar). Blockade of the PI 3-kinase signaling pathway through the use of specific inhibitors or by the expression of a dominant negative p85 regulatory subunit disrupts the insulin regulation of glucose transport and glycogen synthesis (14Cheatham B. Vlahos C.J. Cheatham L. Wang L. Blenis J. Kahn C.R. Mol. Cell. Biol. 1994; 14: 4902-4911Crossref PubMed Scopus (1000) Google Scholar, 15Quon 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). Moreover, a recent study evaluating insulin signaling in cultured adipocytes depleted of Akt2 indicates a requirement of Akt2 activation in insulin-regulated glucose transport and GSK-3 phosphorylation (16Jiang Z.Y. Zhou Q.L. Coleman K.A. Chouinard M. Boese Q. Czech M.P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7569-7574Crossref PubMed Scopus (308) Google Scholar, 17Katome T. Obata T. Matsushima R. Masuyama N. Cantley L.C. Gotoh Y. Kishi K. Shiota H. Ebina Y. J. Biol. Chem. 2003; 278: 28312-28323Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Consistent with a role of Akt as a positive regulator of glucose homeostasis, mice deficient in Akt2 demonstrate a strong attenuation of insulin action on liver gluconeogenesis (18Cho H. Mu J. Kim J.K. Thorvaldsen J.L. Chu Q. Crenshaw 3rd, E.B. Kaestner K.H. Bartolomei M.S. Shulman G.I. Birnbaum M.J. Science. 2001; 292: 1728-1731Crossref PubMed Scopus (1545) Google Scholar). Thus, the PI 3-kinase/Akt pathway is an obligatory step in the control of glucose uptake in muscle and fat and the reduction of hepatic glucose output regulated by insulin. PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a dual-function lipid and protein phosphatase that was originally identified as a tumor suppressor gene frequently mutated or deleted in a variety human cancers (19Steck P.A. Pershouse M.A. Jasser S.A. Yung W.K. Lin H. Ligon A.H. Langford L.A. Baumgard M.L. Hattier T. Davis T. Frye C. Hu R. Swedlund B. Teng D.H. Tavtigian S.V. Nat. Genet. 1997; 15: 356-362Crossref PubMed Scopus (2512) Google Scholar, 20Li J. Yen C. Liaw D. Podsypanina K. Bose S. Wang S.I. Puc J. Miliaresis C. Rodgers L. McCombie R. Bigner S.H. Giovanella B.C. Ittmann M. Tycko B. Hibshoosh H. Wigler M.H. Parsons R. Science. 1997; 275: 1943-1947Crossref PubMed Scopus (4267) Google Scholar, 21Di Cristofano A. Pandolfi P.P. Cell. 2000; 100: 387-390Abstract Full Text Full Text PDF PubMed Scopus (1031) Google Scholar). PTEN has been shown to dephosphorylate PI(3,4,5)P3 at the 3′-phosphate, leading to decreased levels of this phospholipid and simultaneous reduction of Akt activity (22Maehama T. Dixon J.E. J. Biol. Chem. 1998; 273: 13375-13378Abstract Full Text Full Text PDF PubMed Scopus (2590) Google Scholar, 23Cantley L.C. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4240-4245Crossref PubMed Scopus (1745) Google Scholar). In Caenorhabditis elegans, the PTEN homolog, DAF-18, acts in insulin receptor-like pathway and regulates longevity and dauer larva development (24Ogg S. Ruvkun G. Mol. Cell. 1998; 2: 887-893Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 25Gil E.B. Malone Link E. Liu L.X. Johnson C.D. Lees J.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2925-2930Crossref PubMed Scopus (123) Google Scholar, 26Rouault J.P. Kuwabara P.E. Sinilnikova O.M. Duret L. Thierry-Mieg D. Billaud M. Curr. Biol. 1999; 9: 329-332Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 27Mihaylova V.T. Borland C.Z. Manjarrez L. Stern M.J. Sun H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7427-7432Crossref PubMed Scopus (147) Google Scholar). Drosophila melangaster devoid of PTEN have abnormally high concentrations of PI(3,4,5)P3 and are viable only if the affinity of the Akt ortholog for PI(3,4,5)P3 is decreased by a mutation in the pleckstrin homology (PH) domain (28Stocker H. Andjelkovic M. Oldham S. Laffargue M. Wymann M.P. Hemmings B.A. Hafen E. Science. 2002; 295: 2088-2091Crossref PubMed Scopus (169) Google Scholar). The sufficiency of an Akt PH domain mutant to rescue PTEN-deficient Drosophila indicates that Akt is the primary target activated by increased PI(3,4,5)P3 levels. Hyperactivation of the PI 3-kinase/Akt pathway also appears to be a main result of PTEN deletion in mammalian systems (29Stiles B. Gilman V. Khanzenzon N. Lesche R. Li A. Qiao R. Liu X. Wu H. Mol. Cell. Biol. 2002; 22: 3842-3851Crossref PubMed Scopus (119) Google Scholar, 30Stiles B. Wang Y. Stahl A. Bassilian S. Lee W.P. Kim Y.J. Sherwin R. Devaskar S. Lesche R. Magnuson M.A. Wu H. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2082-2087Crossref PubMed Scopus (342) Google Scholar, 31Hyun T. Yam A. Pece S. Xie X. Zhang J. Miki T. Gutkind J.S. Li W. Blood. 2000; 96: 3560-3568Crossref PubMed Google Scholar). A number of studies have been published that support a role for PTEN in the negative regulation of insulin-induced metabolic actions in 3T3-L1 adipocytes (32Ono H. Katagiri H. Funaki M. Anai M. Inukai K. Fukushima Y. Sakoda H. Ogihara T. Onishi Y. Fujishiro M. Kikuchi M. Oka Y. Asano T. Mol. Endocrinol. 2001; 15: 1411-1422Crossref PubMed Scopus (69) Google Scholar, 33Nakashima N. Sharma P.M. Imamura T. Bookstein R. Olefsky J.M. J. Biol. Chem. 2000; 275: 12889-12895Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 34Mosser V.A. Li Y. Quon M.J. Biochem. Biophys. Res. Commun. 2001; 288: 1011-1017Crossref PubMed Scopus (15) Google Scholar). In these studies, high expression of full-length PTEN suppressed insulin-stimulated Akt activation and glucose transport. Conversely, expression of dominant negative PTEN enhanced Akt activation (32Ono H. Katagiri H. Funaki M. Anai M. Inukai K. Fukushima Y. Sakoda H. Ogihara T. Onishi Y. Fujishiro M. Kikuchi M. Oka Y. Asano T. Mol. Endocrinol. 2001; 15: 1411-1422Crossref PubMed Scopus (69) Google Scholar). However, the role of PTEN is controversial as recent results derived from the expression of a dominant inhibitory PTEN mutant indicate that endogenous PTEN may not modulate the metabolic function of insulin in fat cells (34Mosser V.A. Li Y. Quon M.J. Biochem. Biophys. Res. Commun. 2001; 288: 1011-1017Crossref PubMed Scopus (15) Google Scholar). SHIP2 (Src homology 2-containing inositol 5′-phosphatase 2) is a recently identified lipid phosphatase that hydrolyzes the 5′-phosphate of the inositol ring from PI(3,4,5)P3. SHIP2 is more broadly detected than the isoform SHIP1, which is mainly expressed in hematopoietic cells (35Ishihara H. Sasaoka T. Hori H. Wada T. Hirai H. Haruta T. Langlois W.J. Kobayashi M. Biochem. Biophys. Res. Commun. 1999; 260: 265-272Crossref PubMed Scopus (116) Google Scholar, 36Pesesse X. Deleu S. De Smedt F. Drayer L. Erneux C. Biochem. Biophys. Res. Commun. 1997; 239: 697-700Crossref PubMed Scopus (199) Google Scholar, 37Krystal G. Semin. Immunol. 2000; 12: 397-403Crossref PubMed Scopus (120) Google Scholar). Numerous studies have shown that expression of dominant negative SHIP2 inactivates insulin signaling by blocking the PI 3-kinase/Akt pathway in 3T3-L1 adipocytes (38Wada T. Sasaoka T. Funaki M. Hori H. Murakami S. Ishiki M. Haruta T. Asano T. Ogawa W. Ishihara H. Kobayashi M. Mol. Cell. Biol. 2001; 21: 1633-1646Crossref PubMed Scopus (152) Google Scholar, 39Sasaoka T. Wada T. Fukui K. Murakami S. Ishihara H. Suzuki R. Tobe K. Kadowaki T. Kobayashi M. J. Biol. Chem. 2004; 279: 14835-14843Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 40Sasaoka T. Hori H. Wada T. Ishiki M. Haruta T. Ishihara H. Kobayashi M. Diabetologia. 2001; 44: 1258-1267Crossref PubMed Scopus (80) Google Scholar). Consistent with a role for SHIP2 as a negative regulator of insulin signaling, homozygous disruption of SHIP2 causes severe hypoglycemia and death within a few hours after birth (41Clement S. Krause U. Desmedt F. Tanti J.F. Behrends J. Pesesse X. Sasaki T. Penninger J. Doherty M. Malaisse W. Dumont J.E. Le Marchand-Brustel Y. Erneux C. Hue L. Schurmans S. Nature. 2001; 409: 92-97Crossref PubMed Scopus (317) Google Scholar). Heterozygous disruption of this gene leads to insulin hypersensitivity as demonstrated by an increase in GLUT4 translocation and glycogen synthesis in skeletal muscles in response to insulin. Injection of d-glucose resulted in a more rapid glucose clearance in SHIP2(+/-) mice than in wild-type mice (41Clement S. Krause U. Desmedt F. Tanti J.F. Behrends J. Pesesse X. Sasaki T. Penninger J. Doherty M. Malaisse W. Dumont J.E. Le Marchand-Brustel Y. Erneux C. Hue L. Schurmans S. Nature. 2001; 409: 92-97Crossref PubMed Scopus (317) Google Scholar). However, no information on insulin-induced Akt activation or on metabolic regulation by insulin in fat cells from heterozygous mice was provided in these studies. Furthermore, a recent study suggested that the phenotype of the SHIP2 knock-out mouse generated by Clement et al. (41Clement S. Krause U. Desmedt F. Tanti J.F. Behrends J. Pesesse X. Sasaki T. Penninger J. Doherty M. Malaisse W. Dumont J.E. Le Marchand-Brustel Y. Erneux C. Hue L. Schurmans S. Nature. 2001; 409: 92-97Crossref PubMed Scopus (317) Google Scholar) may have been due to an incomplete knock-out of SHIP2, as a separately generated SHIP2 knock-out mouse was normoglycemic and resistant to high fat diet-induced obesity (42Sleeman M.W. Wortley K.E. Lai K.M. Gowen L.C. Kintner J. Kline W.O. Garcia K. Stitt T.N. Yancopoulos G.D. Wiegand S.J. Glass D.J. Nat. Med. 2005; 11: 199-205Crossref PubMed Scopus (201) Google Scholar). Thus the role of SHIP2 as a negative regulator of insulin signaling in white adipose tissue remains unclear. Based on the above review of the literature, definitive experiments testing the role of PTEN or SHIP2 in insulin signaling in adipocytes through depletion of these proteins have not yet been conducted. In this present investigation, we have tested the relative importance of endogenous SHIP2 and PTEN in the control of insulin induced Akt activation and 2-deoxyglucose transport in 3T3-L1 adipocytes by RNAi-based gene silencing. Depletion of PTEN markedly enhanced insulin-stimulated Akt and GSK-3α phosphorylation, as well as insulin-stimulated deoxyglucose transport in 3T3-L1 adipocytes. Surprisingly, almost complete loss of SHIP2 had no detectable effect on these insulin-regulated events. Taken as a whole, these results reveal the surprising result that PTEN, but not SHIP2, functions as a suppressor of insulin signaling to glucose transport through the PI 3-kinase → Akt pathway in cultured adipocytes. Materials and Chemicals—Human insulin was obtained from Lilly. Rabbit polyclonal anti-PTEN, anti-IRS-1, and mouse monoclonal antiphosphotyrosine (clone 4G10) antibodies were from Upstate Biotechnology, Inc. Goat polyclonal anti-SHIP2 (I-20, sc-14502), mouse monoclonal anti-GSK-3α/β (0011-A, sc-7291), and anti-insulin receptor β-subunit (29B4, sc-09) were from Santa Cruz Biotechnology. Rabbit polyclonal antibodies against phospho-Akt (Ser-473), phospho-GSK-3α/β (Ser-21/9), phospho-Erk1/2, anti-Akt, and anti-Erk1/2 were from Cell Signaling Technology (Beverly, MA). Mouse anti-actin monoclonal antibody was from Sigma. Mouse anti-PIP3 monoclonal antibody and the PIP3 strips were from Echelon Biosciences Inc. The horseradish peroxidase-conjugated goat anti-rabbit IgG and rabbit anti-goat IgG were from Chemicon International. Cell Culture and Electroporation of 3T3-L1 Adipocytes—3T3-L1 fibroblasts were grown in DMEM supplemented with 10% fetal bovine serum, 50 μg/ml streptomycin, and 50 units/ml penicillin and differentiated into adipocytes as described (16Jiang Z.Y. Zhou Q.L. Coleman K.A. Chouinard M. Boese Q. Czech M.P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7569-7574Crossref PubMed Scopus (308) Google Scholar, 43Guilherme A. Soriano N.A. Bose S. Holik J. Bose A. Pomerleau D.P. Furcinitti P. Leszyk J. Corvera S. Czech M.P. J. Biol. Chem. 2004; 279: 10593-10605Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The 3T3-L1 adipocytes were transfected with siRNA duplexes by electroporation. Briefly, the adipocytes, on the 4th or 5th day of differentiation, were detached from culture dishes with 0.25% trypsin and 0.5 mg of collagenase/ml in phosphate-buffered saline, washed twice, and resuspended in phosphate-buffered saline. Half of the cells from one 150-mm dish were then mixed with the indicated siRNA duplexes, which were delivered to the cells by a pulse of electroporation with a Bio-Rad gene pulser II system at the setting of 0.18 kV and 950 microfarads capacitance. After electroporation, cells were immediately mixed with fresh medium before reseeding into multiple-well plates, designated for Western blotting or deoxyglucose uptake assay. siRNA-induced Degradation of PTEN and SHIP2—The siRNA species purchased from Dharmacon were designed to target the following cDNA sequences: scrambled, 5′-CAGTCGCGTTTGCGACTGG-3′; PTEN siRNA, 5′-GTATAGAGCGTGCAGATAA-3′; SHIP2 siRNA, 5′-GTGAGGAGGAGATATCTTT-3′. Either 30 nmol of scrambled siRNA, 30 nmol of PTEN siRNA, or 20 nmol of SHIP2 siRNA species were electroporated into 3T3-L1 adipocytes as described previously (16Jiang Z.Y. Zhou Q.L. Coleman K.A. Chouinard M. Boese Q. Czech M.P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7569-7574Crossref PubMed Scopus (308) Google Scholar, 43Guilherme A. Soriano N.A. Bose S. Holik J. Bose A. Pomerleau D.P. Furcinitti P. Leszyk J. Corvera S. Czech M.P. J. Biol. Chem. 2004; 279: 10593-10605Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). After 72 h, cells were harvested, and equal amounts of protein from different lysates resolved by SDS-PAGE and analyses by immunoblot with indicated antibodies. Immunoblotting—3T3-L1 adipocytes electroporated with indicated siRNA were starved overnight in serum-free DMEM medium. Cells were then incubated without or with the indicated insulin concentrations for 30 min and then harvested with lysis buffer containing 1% SDS. Equal amounts of protein from lysates were resolved by SDS-PAGE and electrotransfered to nitrocellulose membranes, which were incubated with the indicated antibodies overnight at 4 °C and then with horseradish peroxidase-linked secondary antibodies for 45 min at room temperature. Proteins were then detected with an enhanced chemiluminescence kit. Detection of Phosphatidylinositol 3,4,5-Trisphosphate (PIP3)— 3T3-L1 adipocytes electroporated with the indicated siRNA were starved overnight in serum-free DMEM medium. Cells were incubated without or with the indicated insulin concentrations for 10 min, and then phospholipids were extracted with 1:1 (v/v) chloroform:methanol, as described (44Lawe D.C. Sitouah N. Hayes S. Chawla A. Virbasius J.V. Tuft R. Fogarty K. Lifshitz L. Lambright D. Corvera S. Mol. Biol. Cell. 2003; 14: 2935-2945Crossref PubMed Google Scholar, 45Serunian L.A. Auger K.R. Cantley L.C. Methods Enzymol. 1991; 198: 78-87Crossref PubMed Scopus (130) Google Scholar). The lower phase was collected and washed twice with 1:1 (v/v) methanol:1 n HCl. Aliquots of the resulting organic phase were applied on activated (110 °C, 1 h) TLC plates. The phospholipids were separated by the chromatographic solvent system chloroform: methanol:acetone:acetic acid:water (80:26:30:24:16) with the PIP3 standard, run in parallel. The areas corresponding to PIP3 were eluted from the plate using chloroform:methanol (1:1). The organic phase was then dried, resuspended in a small volume, and spotted onto Hybond nitrocellulose for probing with anti-PIP3 monoclonal antibody (Echelon) to specifically detect PIP3. 2-Deoxyglucose Uptake Assay—Insulin-stimulated glucose transport in 3T3-L1 adipocytes was estimated by measuring 2-deoxyglucose uptake as described (16Jiang Z.Y. Zhou Q.L. Coleman K.A. Chouinard M. Boese Q. Czech M.P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7569-7574Crossref PubMed Scopus (308) Google Scholar). Briefly, siRNA-transfected cells were reseeded on 24-well plates and cultured for 72 h and then washed twice and starved for 2 h with Krebs-Ringer's Hepes buffer (130 mm NaCl, 5 mm KCl, 1.3 mm CaCl2, 1.3 mm MgSO4, 25 mm Hepes, pH 7.4) supplemented with 0.5% bovine serum albumin and 2 mm sodium pyruvate. Cells were then stimulated with insulin for 30 min at 37 °C. Glucose uptake was initiated by addition of [1,2-3H]2-deoxy-d-glucose to a final assay concentration of 100 μm for 5 min at 37 °C. Assays were terminated by three washes with ice-cold Krebs-Ringer's Hepes buffer, and the cells were solubilized with 0.4 ml of 1% SDS, and 3H uptake was quantitated by scintillation counting. Nonspecific deoxyglucose uptake was measured in the presence of 20 μm cytochalasin B and subtracted from each determination to obtain specific uptake. Insulin-stimulated Deoxyglucose Transport Is Enhanced by siRNA-based Depletion of Endogenous PTEN but Not by Depletion of SHIP2 in 3T3-L1 Adipocytes—It has been reported that both PTEN and SHIP2 phosphatases are expressed in 3T3-L1 adipocytes (32Ono H. Katagiri H. Funaki M. Anai M. Inukai K. Fukushima Y. Sakoda H. Ogihara T. Onishi Y. Fujishiro M. Kikuchi M. Oka Y. Asano T. Mol. Endocrinol. 2001; 15: 1411-1422Crossref PubMed Scopus (69) Google Scholar, 38Wada T. Sasaoka T. Funaki M. Hori H. Murakami S. Ishiki M. Haruta T. Asano T. Ogawa W. Ishihara H. Kobayashi M. Mol. Cell. Biol. 2001; 21: 1633-1646Crossref PubMed Scopus (152) Google Scholar). Studies that have invoked high expression of full-length or dominant negative constructs of SHIP2 or PTEN have concluded that both phosphatases are involved in PI(3,4,5)P3 hydrolysis and function as suppressors of the insulin-stimulated PI 3-kinase/Akt pathway in these cells (32Ono H. Katagiri H. Funaki M. Anai M. Inukai K. Fukushima Y. Sakoda H. Ogihara T. Onishi Y. Fujishiro M. Kikuchi M. Oka Y. Asano T. Mol. Endocrinol. 2001; 15: 1411-1422Crossref PubMed Scopus (69) Google Scholar, 33Nakashima N. Sharma P.M. Imamura T. Bookstein R. Olefsky J.M. J. Biol. Chem. 2000; 275: 12889-12895Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 34Mosser V.A. Li Y. Quon M.J. Biochem. Biophys. Res. Commun. 2001; 288: 1011-1017Crossref PubMed Scopus (15) Google Scholar, 38Wada T. Sasaoka T. Funaki M. Hori H. Murakami S. Ishiki M. Haruta T. Asano T. Ogawa W. Ishihara H. Kobayashi M. Mol. Cell. Biol. 2001; 21: 1633-1646Crossref PubMed Scopus (152) Google Scholar, 39Sasaoka T. Wada T. Fukui K. Murakami S. Ishihara H. Suzuki R. Tobe K. Kadowaki T. Kobayashi M. J. Biol. Chem. 2004; 279: 14835-14843Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 40Sasaoka T. Hori H. Wada T. Ishiki M. Haruta T. Ishihara H. Kobayashi M. Diabetologia. 2001; 44: 1258-1267Crossref PubMed Scopus (80) Google Scholar). Both PTEN and SHIP2 are also expected to be negative regulators of insulin-stimulated glucose transport, based on experiments in which these proteins were expressed at high levels in cultured adipocytes (32Ono H. Katagiri H. Funaki M. Anai M. Inukai K. Fukushima Y. Sakoda H. Ogihara T. Onishi Y. Fujishiro M. Kikuchi M. Oka Y. Asano T. Mol. Endocrinol. 2001; 15: 1411-1422Crossref PubMed Scopus (69) Google Scholar, 33Nakashima N. Sharma P.M. Imamura T. Bookstein R. Olefsky J.M. J. Biol. Chem. 2000; 275: 12889-12895Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 34Mosser V.A. Li Y. Quon M.J. Biochem. Biophys. Res. Commun. 2001; 288: 1011-1017Crossref PubMed Scopus (15) Google Scholar, 38Wada T. Sasaoka T. Funaki M. Hori H. Murakami S. Ishiki M. Haruta T. Asano T. Ogawa W. Ishihara H. Kobayashi M. Mol. Cell. Biol. 2001; 21: 1633-1646Crossref PubMed Scopus (152) Google Scholar, 39Sasaoka T. Wada T. Fukui K. Murakami S. Ishihara H. Suzuki R. Tobe K. Kadowaki T. Kobayashi M. J. Biol. Chem. 2004; 279: 14835-14843Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 40Sasaoka T. Hori H. Wada T. Ishiki M. Haruta T. Ishihara H. Kobayashi M. Diabetologia. 2001; 44: 1258-1267Crossref PubMed Scopus (80) Google Scholar). In addition, it has been shown that SHIP2(+/-) mice exhibit hypersensitivity to insulin and more rapid glucose clearance than wild-type mice (41Clement S. Krause U. Desmedt F. Tanti J.F. Behrends J. Pesesse X. Sasaki T. Penninger J. Doherty M. Malaisse W. Dumont J.E. Le Marchand-Brustel Y. Erneux C. Hue L. Schurmans S. Nature. 2001; 409: 92-97Crossref PubMed Scopus (317) Google Scholar), suggesting that SHIP2 may be a negative regulator for glucose transport. However, more definitive approaches, such as depletion of the endogenous phosphatases, have not been used to address the function of SHIP2 and PTEN phosphatases in insulin signaling in either primary or cultured adipocytes. Thus, our first set of experiments was designed to selectively deplete endogenous SHIP2 versus PTEN proteins in cultured 3T3-L1 adipocytes. Assays were then performed to examine how the absence of each one of these phosphatases affects insulin-stimulated signaling. To specifically decrease SHIP2 or PTEN expression levels in 3T3-L1 adipocytes, we used a siRNA-mediated gene silencing technique recently applied successfully to these cells (16Jiang Z.Y. Zhou Q.L. Coleman K.A. Chouinard M. Boese Q. Czech M.P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7569-7574Crossref PubMed Scopus (308) Google Scholar, 46Bose A. Guilherme A. Robida S.I. Nicoloro S.M. Zhou Q.L. Jiang Z.Y. Pomerleau D.P. Czech M.P. Nature. 2002; 420: 821-824Crossref PubMed Scopus (215) Google Scholar). 3T3-L1 adipocytes were electroporated with scrambled siRNA as a control or with siRNA targeted against mouse PTEN or SHIP2 mRNAs. After 72 h, the cells were harvested, and PTEN and SHIP2 protein expression levels were assessed by immunoblotting. As depicted in Fig. 1, the protein expression level of PTEN was reduced by ∼50% in 3T3-L1 adipocytes electroporated with PTEN-directed siRNA but not with scrambled or SHIP2-directed siRNA (Fig. 1). Expression of SHIP2 protein was unaffected by treatment of the cultured adipocytes with PTEN-directed siRNA (Fig. 2). However, electroporation of 3T3-L1 adipocytes with SHIP2-directed siRNA caused a marked reduction of SHIP2 protein expression (about 90% depletion, Fig. 2), without a detectable change in PTEN protein level (Fig. 1). Thus, selective depletion of both PTEN and SHIP2 phosphatases can be achieved by siRNA-mediated gene silencing in cultured adipocytes.Fig. 2Depletion of SHIP" @default.
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• W2052878956 title "PTEN, but Not SHIP2, Suppresses Insulin Signaling through the Phosphatidylinositol 3-Kinase/Akt Pathway in 3T3-L1 Adipocytes" @default.
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