Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Release of Insulin Receptor Substrate Proteins from an Intracellular Complex Coincides with the Development of Insulin Resistance

2000; Elsevier BV; Volume: 275; Issue: 6 Linguagem: Inglês

10.1074/jbc.275.6.3819

ISSN

1083-351X

Autores

Sharon F. Clark, Juan-Carlos Molero, David E. James,

Tópico(s)

Adipokines, Inflammation, and Metabolic Diseases

Resumo

Insulin receptor substrate (IRS) proteins are major substrates of the insulin receptor (IR). IRS-1 associates with an insoluble multiprotein complex, possibly the cytoskeleton, in adipocytes. This localization may facilitate interaction with the IR at the cell surface. In the present study, we examined the hypothesis that the release of IRS proteins from this location may be a mechanism for insulin desensitization. We show that a second IRS protein, IRS-2, is associated with a multiprotein complex in adipocytes with similar characteristics to the IRS-1 complex. Insulin treatment (15–60 min) caused the release of IRS-1 and IRS-2 from this complex (high speed pellet; HSP) into the cytosol, whereas the level of tyrosyl-phosphorylated IRS proteins remained constant. Chronic insulin treatment resulted in a dramatic reduction in IRS-1 and IRS-2 in the HSP, eventually (>2 h) leading to IRS protein degradation and decreased levels of tyrosyl-phosphorylated IRS proteins. Okadaic acid, which rapidly induces insulin resistance in adipocytes independently of IR function, caused an almost quantitative release of IRS-1 into the cytosol commensurate with a significant reduction in tyrosyl-phosphorylated IRS proteins. Platelet-derived growth factor, a factor known to compromise insulin signaling, caused a more moderate release of IRS proteins from the HSP. Collectively, these results suggest that the assembly of IRS-1/IRS-2 into a multiprotein complex facilitates coupling to the IR and that the regulated release from this location may represent a novel mechanism of insulin resistance. Insulin receptor substrate (IRS) proteins are major substrates of the insulin receptor (IR). IRS-1 associates with an insoluble multiprotein complex, possibly the cytoskeleton, in adipocytes. This localization may facilitate interaction with the IR at the cell surface. In the present study, we examined the hypothesis that the release of IRS proteins from this location may be a mechanism for insulin desensitization. We show that a second IRS protein, IRS-2, is associated with a multiprotein complex in adipocytes with similar characteristics to the IRS-1 complex. Insulin treatment (15–60 min) caused the release of IRS-1 and IRS-2 from this complex (high speed pellet; HSP) into the cytosol, whereas the level of tyrosyl-phosphorylated IRS proteins remained constant. Chronic insulin treatment resulted in a dramatic reduction in IRS-1 and IRS-2 in the HSP, eventually (>2 h) leading to IRS protein degradation and decreased levels of tyrosyl-phosphorylated IRS proteins. Okadaic acid, which rapidly induces insulin resistance in adipocytes independently of IR function, caused an almost quantitative release of IRS-1 into the cytosol commensurate with a significant reduction in tyrosyl-phosphorylated IRS proteins. Platelet-derived growth factor, a factor known to compromise insulin signaling, caused a more moderate release of IRS proteins from the HSP. Collectively, these results suggest that the assembly of IRS-1/IRS-2 into a multiprotein complex facilitates coupling to the IR and that the regulated release from this location may represent a novel mechanism of insulin resistance. insulin receptor insulin receptor substrate phosphatidylinositide platelet-derived growth factor Chinese hamster ovary high speed pellet hemagglutinin polyacrylamide gel electrophoresis phosphate-buffered saline The insulin receptor (IR)1 is a member of the tyrosine kinase growth factor receptor family (1.White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar). One property that distinguishes the IR from other growth factor receptors is its ability to induce the tyrosine phosphorylation of a family of intracellular signaling molecules referred to as insulin receptor substrate (IRS) proteins (2.Lavan B.E. Lane W.S. Lienhard G.E. J. Biol. Chem. 1997; 272: 11439-11443Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 3.Lavan B.E. Fantin V.R. Chang E.T. Lane W.S. Keller S.R. Lienhard G.E. J. Biol. Chem. 1997; 272: 21403-21407Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 4.Sun X.J. Rothenberg P. Kahn C.R. Backer J.M. Araki E. Wilden P.A. Cahill D.A. Goldstein B.J. White M.F. Nature. 1991; 352: 73-77Crossref PubMed Scopus (1291) Google Scholar, 5.Sun X.J. Wang L.M. Zhang Y.T. Yenush L. Myers M.G. Glasheen E. Lane W.S. Pierce J.H. White M.F. Nature. 1995; 377: 173-177Crossref PubMed Scopus (767) Google Scholar). IRS proteins contain a pleckstrin homology domain and a phosphotyrosine binding domain, both of which are required for the efficient tyrosine phosphorylation of these proteins by the activated insulin receptor tyrosine kinase (6.Gustafson T.A. He W. Craparo A. Schaub C.D. O'Neill T.J. Mol. Cell. Biol. 1995; 15: 2500-2508Crossref PubMed Scopus (327) Google Scholar, 7.Yenush L. Makati K.J. Smith-Hall J. Ishibashi O. Myers M.G. White M.F. J. Biol. Chem. 1996; 271: 24300-24306Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). The first member of the IRS family to be identified, IRS-1, encodes a 160-kDa protein that is highly expressed in physiologically insulin-responsive tissues such as adipocytes and muscle cells and has been implicated in the control of a number of insulin-sensitive metabolic pathways including glucose transport, lipid deposition, and glycogen synthesis (8.Ogawa W. Matozaki T. Kasuga M. Mol. Cell. Biochem. 1998; 82: 13-22Crossref Scopus (102) Google Scholar). In response to insulin, the phosphorylation of multiple tyrosine residues within the C terminus of IRS-1 by the IR leads to the generation of highly specific binding sites for a number of Src homology 2 domain-containing downstream signaling molecules such as phosphatidylinositide (PI) 3-kinase, Syp, Nck, Fyn, and Grb-2 (8.Ogawa W. Matozaki T. Kasuga M. Mol. Cell. Biochem. 1998; 82: 13-22Crossref Scopus (102) Google Scholar, 9.Lavan B.E. Kuhne M.R. Garner C.W. Anderson D. Reedijk M. Pawson T. Lienhard G.E. J. Biol. Chem. 1992; 267: 11631-11636Abstract Full Text PDF PubMed Google Scholar, 10.Sun X.J. Crimmins D.L. Myers M.J. Miralpeix M. White M.F. Mol. Cell. Biol. 1993; 13: 7418-7428Crossref PubMed Google Scholar). PI 3-kinase appears to be a central insulin-signaling molecule, because inhibition of its activity by either pharmacological agents or dominant-negative mutants profoundly abrogates several biological responses to this hormone (11.Okada T. Kawano Y. Sakakibara T. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3568-3573Abstract Full Text PDF PubMed Google Scholar, 12.Cheatham B. Vlahos C.J. Cheatham L. Wang L. Blenis J. Kahn C.R. Mol. Cell. Biol. 1994; 14: 4902-4911Crossref PubMed Scopus (1002) Google Scholar, 13.Kotani K. Carozzi A.J. Sakaue H. Hara K. Robinson L.J. Clark S.F. Yonezawa K. James D.E. Kasuga M. Biochem. Biophys. Res. Commun. 1995; 209: 343-348Crossref PubMed Scopus (144) Google Scholar, 14.Quon 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; 5: 5403-5411Crossref Scopus (143) Google Scholar). In basal cells, IRS-1 is also highly phosphorylated on serine and threonine (Ser/Thr) residues, and insulin acutely stimulates a further increase in IRS-1 Ser/Thr phosphorylation (4.Sun X.J. Rothenberg P. Kahn C.R. Backer J.M. Araki E. Wilden P.A. Cahill D.A. Goldstein B.J. White M.F. Nature. 1991; 352: 73-77Crossref PubMed Scopus (1291) Google Scholar, 15.Keller S.R. Kitagawa K. Aebersold R. Lienhard G.E. Garner C.W. J. Biol. Chem. 1991; 266: 12817-12820Abstract Full Text PDF PubMed Google Scholar). The signaling function of IRS-1 Ser/Thr phosphorylation is unknown, although this may regulate the docking of other types of signaling molecules such as 14-3-3 proteins (16.Craparo A. Freund R. Gustafson T.A. J. Biol. Chem. 1997; 272: 11663-11669Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 17.Ogihara T. Isobe T. Ichimura T. Taoka M. Funaki M. Sakoda H. Onishi Y. Inukai K. Anai M. Fukushima Y. Kikuchi M. Yazaki Y. Oka Y. Asano T. J. Biol. Chem. 1997; 272: 25267-25274Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Defects within insulin signaling pathways comprise a major locus for the development of insulin resistance in disease states such as non-insulin-dependent diabetes mellitus (18.De Fronzo R.A. Bonadonna R.C. Ferrannini E. Diabetes Care. 1992; 15: 318-368Crossref PubMed Scopus (1903) Google Scholar). Certain forms of insulin resistance, such as that induced by tumor necrosis factor-α, okadaic acid, or chronic insulin treatment, may be due to uncoupling of the IR activity toward IRS-1 (19.Hotamisligil G.S. Peraldi P. Budavari A. Ellis R. White M.F. Spiegelman B.M. Science. 1996; 271: 665-668Crossref PubMed Scopus (2223) Google Scholar, 20.Tanti J.-F. Gremeaux T. Cormont M. Van Obberghen E. Le Marchand-Brustel Y. Am. J. Physiol. 1993; 64: E868-E873Google Scholar, 21.Kozka I.J. Clark A.E. Holman G.D. J. Biol. Chem. 1991; 266: 11726-11731Abstract Full Text PDF PubMed Google Scholar). While the molecular basis for this defect is unclear, a common observation in cells subjected to these conditions is that IRS-1 becomes hyperphosphorylated on serine and threonine residues and various intracellular Ser/Thr kinases, including glycogen synthase kinase-3, protein kinase C-α, mitogen-activated protein kinase, and protein kinase B/Akt, have been implicated in mediating this effect (22.Hotamisligil G.S. Murray D.L. Choy L.N. Spiegelman B.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4854-4858Crossref PubMed Scopus (1052) Google Scholar, 23.Tanti J.F. Gremeaux T. Van Obberghen E. Le Marchand-Brustel Y. J. Biol. Chem. 1994; 269: 6051-6057Abstract Full Text PDF PubMed Google Scholar, 24.Ricort J-M. Van Obberghen E. Le Marchand-Brustel Y. Diabetologia. 1995; 38: 1148-1156Crossref PubMed Scopus (76) Google Scholar, 25.Eldar-Finkelman H. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9660-9664Crossref PubMed Scopus (286) Google Scholar, 26.Chin J.E. Liu F. Roth R.A. Mol. Endocrinol. 1994; 8: 51-58Crossref PubMed Scopus (0) Google Scholar, 27.De Fea K. Roth R.A. J. Biol. Chem. 1997; 272: 31400-31406Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 28.Li J. DeFea K. Roth R.A. J. Biol. Chem. 1999; 274: 9351-9356Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). The decline in insulin sensitivity invoked by tumor necrosis factor-α has recently been attributed to a dominant negative effect exerted by hyperphosphorylated IRS-1 upon the IR intrinsic tyrosine kinase (19.Hotamisligil G.S. Peraldi P. Budavari A. Ellis R. White M.F. Spiegelman B.M. Science. 1996; 271: 665-668Crossref PubMed Scopus (2223) Google Scholar). However, neither okadaic acid nor chronic insulin treatment appear to disrupt IR tyrosine kinase activity in response to acute insulin activation (20.Tanti J.-F. Gremeaux T. Cormont M. Van Obberghen E. Le Marchand-Brustel Y. Am. J. Physiol. 1993; 64: E868-E873Google Scholar, 21.Kozka I.J. Clark A.E. Holman G.D. J. Biol. Chem. 1991; 266: 11726-11731Abstract Full Text PDF PubMed Google Scholar, 24.Ricort J-M. Van Obberghen E. Le Marchand-Brustel Y. Diabetologia. 1995; 38: 1148-1156Crossref PubMed Scopus (76) Google Scholar). These latter observations suggest that other mechanisms may operate to induce insulin resistance. We have recently provided evidence to suggest that IRS-1 is enriched in a cytoskeletal fraction in adipocytes that is insoluble in a range of nonionic detergents (29.Clark S.F. Martin S. Carozzi A.J. Hill M.M. James D.E. J. Cell Biol. 1998; 140: 1211-1225Crossref PubMed Scopus (159) Google Scholar). This accounts for early reports suggesting that IRS-1 is bound to membranes, because the cytoskeletal fraction co-fractionates with microsomal membranes during ultracentrifugation (30.Kelly K.L. Ruderman N.B. J. Biol. Chem. 1993; 268: 4391-4398Abstract Full Text PDF PubMed Google Scholar, 31.Heller-Harrison R.A. Morin M. Czech M.P. J. Biol. Chem. 1995; 270: 24442-24450Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The anchoring of IRS-1 to the cytoskeleton may be of particular importance to the efficacy of insulin signaling by providing a platform for localizing IRS-1 within proximity to the insulin receptor. This arrangement may also provide a robust link between IRS-1 and downstream signaling proteins such as PI 3-kinase, which also appears to associate with this insoluble fraction (29.Clark S.F. Martin S. Carozzi A.J. Hill M.M. James D.E. J. Cell Biol. 1998; 140: 1211-1225Crossref PubMed Scopus (159) Google Scholar). One functional consequence of this spatial localization is that it may create a unique site for the generation of specific signals required for insulin action. Consistent with the latter notion, platelet-derived growth factor (PDGF) also activates PI 3-kinase in adipocytes but has no significant effect on PI 3-kinase-dependent functions in these cells, including glucose transport and glycogen synthesis (32.Wiese R.J. Mastick C.C. Lazar D.F. Saltiel A.R. J. Biol. Chem. 1995; 270: 3442-3446Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). It has previously been reported that IRS-1 exists in at least two distinct pools in adipocytes: the cytoskeletal component and the cytosol (31.Heller-Harrison R.A. Morin M. Czech M.P. J. Biol. Chem. 1995; 270: 24442-24450Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 33.Kublaoui B. Lee J. Pilch P.F. J. Biol. Chem. 1995; 270: 59-65Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Furthermore, short term insulin treatment triggers the release of IRS-1 from the cytoskeletal fraction into the cytosol (31.Heller-Harrison R.A. Morin M. Czech M.P. J. Biol. Chem. 1995; 270: 24442-24450Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). It has been argued that the cytoskeletal component represents the functional pool of IRS-1, because most of the tyrosyl-phosphorylated IRS-1 is found in this pool, and there is a net increase in PI 3-kinase in this fraction in response to insulin (34.Inoue G. Cheatham B. Emkey R. Kahn C.R. J. Biol. Chem. 1998; 273: 11548-11555Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Hence, it may be postulated that the deactivation of IRS-1 corresponds to its translocation from this fraction into the cytosol. This model raises the possibility that the cytosolic pool of IRS-1 is nonfunctional presumably due to its inaccessibility to the IR. Therefore, inappropriate accumulation of IRS-1 in the cytosol may disengage this protein from the receptor, resulting in a state of insulin resistance. In the present study, we have tested the notion that the intracellular location of IRS-1 can be modified under conditions that normally cause insulin resistance and/or alter insulin signaling potential. In addition, we have extended this hypothesis to include the IRS-1 homologue, IRS-2, which is also expressed in 3T3-L1 adipocytes. Our results show that a significant proportion of IRS-1 and IRS-2 are found in a detergent-resistant insoluble fraction that has properties analogous to the cytoskeleton. Moreover, IRS proteins translocate from this location into the cytosol following exposure of adipocytes to chronic insulin treatment or okadaic acid or PDGF. These data suggest that the intracellular location of IRS proteins is regulated in a way that may influence insulin action. All tissue culture media was purchased from Life Technologies, Inc., except fetal calf serum, which was obtained from Trace Biosciences (Clayton, Australia). Insulin was obtained from Calbiochem, and PDGF.B was from Life Technologies, Inc. Bovine serum albumin was purchased from ICN (Costa Mesa, CA). Unless specified, all other reagents were from Sigma. The GLUT4 polyclonal antibody (R820) was raised against a synthetic peptide as described previously (35.James D.E. Brown R. Navarro J. Pilch P.F. Nature. 1988; 333: 183-185Crossref PubMed Scopus (472) Google Scholar). The anti-phosphotyrosine monoclonal antibody (4G10) was kindly provided by Dr. B. Druker (Oregon Health Sciences University, Portland, OR), and polyclonal antibodies raised against Akt-2 were provided by Dr. M. Birnbaum (Howard Hughes Medical Institute, Philadelphia, PA). All other antibodies used in this study were purchased from the following sources: anti-IRS-1 polyclonal antibody from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); anti-p85 and anti-IRS-2 polyclonal antibodies from Upstate Biotechnology Inc. (Lake Placid, NY); anti-hemagglutinin (HA) monoclonal antibody from Babco (Richmond, CA); peroxidase-coupled secondary antibodies from Amersham Pharmacia Biotech (Little Chalfont, United Kingdom). Full-length mouse IRS-1 cDNA in pBluescriptSK, generously provided by Drs. S. Keller and G. Lienhard (Dartmouth Medical School, Hanover, NH), was used as template in a polymerase chain reaction to generate a construct encoding IRS-1 tagged at the C terminus with the HA epitope (HA-IRS-1). In this reaction, the forward primer consisted of the pBluescript sequencing primer, T7, whereas the reverse primer was comprised of the following sequence: 5′-CTGCGGTCGACTAAGCGTAATCTGGAACATCGTATGGGTAAGCTTGACGATCCTCTGGCTGCTTCTGGAAGCTGATGCTGGC-3′ (Pacific Oligos, Lismore, Australia). A cDNA fragment encoding the entire sequence of HA-IRS-1 was then amplified using standard polymerase chain reaction protocols. The amplified product was isolated, digested with SalI, and subcloned intoSalI sites of a eukaryotic expression vector, pMEX (36.Benito M. Porras A. Nebrada A.R. Santos E. Science. 1991; 253: 565-568Crossref PubMed Scopus (141) Google Scholar). Clones carrying the insert in the desired orientation were verified by restriction mapping. 3T3-L1 fibroblasts were cultured and differentiated into adipocytes as described previously (37.Piper R.C. Hess L.J. James D.E. Am. J. Physiol. 1991; 260: C570-C580Crossref PubMed Google Scholar). Cells were serum-starved in Dulbecco's modified Eagle's medium supplemented with 0.1% bovine serum albumin and 2 mmglutamine for 2 h at 37 °C and subsequently incubated with insulin (1 μm) for 0.25 h (acute) or for 0.25, 1, 2, or 4 h (chronic) or with PDGF (50 ng/ml) for 1 h at 37 °C. Where indicated, cells were incubated with wortmannin (100 nm) for 15 min and then insulin (1 μm) and wortmannin for 1 h at 37 °C. Medium was replaced with medium containing fresh wortmannin (100 nm) and insulin (1 μm) 30 min prior to harvesting cells. In other experiments, cells were incubated with okadaic acid (2.5–5.0 μm) for 30 min, during which insulin (1 μm) was added to the incubation medium for the last 15 min. In glucose uptake assays, 3T3-L1 adipocytes were serum-starved in Krebs-Ringer phosphate buffer (2.5 mm HEPES (pH 7.4), 120 mmNaCl, 6 mm KCl, 1.2 mm MgSO4, 10 mm CaCl2, 0.4 mmNaH2PO4, 0.6 mmNa2HPO4), KRP, containing 0.1% bovine serum albumin and 3.0 mm sodium pyruvate, for 2 h at 37 °C. Cells were then incubated with PDGF (50 ng/ml) for 1 h and subsequently incubated with insulin (1 μm) for a further 15 min. CHO cells overexpressing the insulin receptor (CHO-IR) were kindly donated by Dr. M White (Harvard Medical School, Boston, MA) and were maintained in culture as described previously (29.Clark S.F. Martin S. Carozzi A.J. Hill M.M. James D.E. J. Cell Biol. 1998; 140: 1211-1225Crossref PubMed Scopus (159) Google Scholar). Prior to transfection, cells were seeded in 60-mm dishes and grown for a further 24 h to achieve 60–80% confluence. Transient transfections were performed by incubating cells in medium (Dulbecco's modified Eagle's medium, nonessential amino acids, 2 mml-glutamine) containing 6 μg of HA-IRS-1/pMEX and 30 μl of Lipofectamine reagent for 5 h at 37 °C. Transfection medium was then replaced with CHO cell culture media, and cells were grown to 100% confluence. Transfected cells were subsequently serum-starved and exposed to chronic insulin treatment as described above for adipocytes. In preliminary experiments, immunofluorescence studies of CHO-IR cells transfected with HA-IRS-1 cDNA showed that at least 20–30% of cells expressed full-length HA-IRS-1 protein. After incubation with the appropriate agents, 3T3-L1 adipocytes were washed three times with ice-cold HES buffer (20 mm HEPES (pH 7.4), 1 mm EDTA, 250 mm sucrose) and homogenized in the same buffer supplemented with phosphatase and protease inhibitors (2 mm sodium orthovanadate, 10 mm sodium fluoride, 1 mm tetra-sodium pyrophosphate, 1 mm ammonium molybdate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 250 μm phenylmethylsulfonyl fluoride). Subcellular fractions were isolated by differential centrifugation as previously detailed (37.Piper R.C. Hess L.J. James D.E. Am. J. Physiol. 1991; 260: C570-C580Crossref PubMed Google Scholar). All procedures were performed at 4 °C. Briefly, cell homogenates were centrifuged at 13,000 × g for 20 min. The resulting pellet was then resuspended in HES buffer and layered onto a 1.12 m sucrose cushion as described by Piperet al. (37.Piper R.C. Hess L.J. James D.E. Am. J. Physiol. 1991; 260: C570-C580Crossref PubMed Google Scholar). After centrifugation at 77,000 ×g for 1 h, the plasma membrane fraction was collected from the 1.12 m sucrose interface. The supernatant from the 13,000 × g centrifugation step was subjected to centrifugation at 30,000 × g for 30 min to pellet the high density microsomal fraction. The resultant supernatant was subjected to further centrifugation at 175,000 × g for 75 min to obtain the high speed pellet (HSP). The supernatant from this centrifugation step was designated the cytosol fraction. The membrane pellets were solubilized with 1% SDS in PBS. In some experiments, the HSP fraction from 3T3-L1 adipocytes was resuspended and incubated in HES containing 1% Triton X-100 (Pierce) for 1 h on ice. Insoluble material was collected by centrifugation at 175,000 ×g for 75 min at 4 °C, and the resultant pellet was solubilized in 1% SDS in PBS. CHO-IR cells overexpressing HA-IRS-1 were subjected to chronic insulin treatment, and the HSP from these cells was obtained as described previously (29.Clark S.F. Martin S. Carozzi A.J. Hill M.M. James D.E. J. Cell Biol. 1998; 140: 1211-1225Crossref PubMed Scopus (159) Google Scholar) with some modifications. All steps were performed at 4 °C. Cells were washed in ice-cold HES and then homogenized in HES buffer containing phosphatase and protease inhibitors by passage through a 22-gauge needle. The homogenate was subjected to centrifugation at 17,500 × g for 15 min to remove high density microsomes, plasma membranes, mitochondria/nuclei, and cell debris. The supernatant was then centrifuged at 175,000 ×g for 75 min. The resultant supernatant was designated the cytosol. The pellet (HSP), was solubilized in 1% SDS in PBS. The amount of protein present in all samples was determined using BCA reagent (Pierce). Samples were subjected to SDS-PAGE (38.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207522) Google Scholar) and transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA). Membranes were blocked with 3% bovine serum albumin in TBST buffer (20 mm Tris·HCl (pH 7.6), 150 mmNaCl, 0.05% Tween 20) (anti-phosphotyrosine antibody) or with 5% skim milk powder in PBS buffer (all other antibodies). The membranes were incubated with primary antibody, washed, and then incubated with the appropriate horseradish peroxidase-coupled secondary antibody for 30 min at room temperature. Immunoreactive proteins were visualized by autoradiography using enhanced chemiluminescence (Supersignal, Pierce, or ECL Plus, Amersham Pharmacia Biotech), according to the instructions from the manufacturer. The protein bands were quantified by densitometry (GS-700 Imaging densitometer, Bio-Rad) using nonsaturated exposed x-ray films. Statistical analysis of the data was performed using Microsoft Excel software. All data are presented as the mean value. 2-Deoxy-[3H]glucose uptake was measured as described previously (39.Robinson L.J. Razzack Z.F. Lawrence J.C. James D.E. J. Biol. Chem. 1993; 268: 26422-26427Abstract Full Text PDF PubMed Google Scholar). Briefly, 3T3-L1 adipocytes were treated in the absence or presence of growth factor in 950 μl of KRP containing 1% bovine serum albumin and 3.0 mm sodium pyruvate. The assay was initiated by adding 50 μl of 1 mm 2-deoxy-[3H]glucose (20 μCi/mmol)/KRP and, after 3 min, was terminated by washing cells rapidly three times with ice-cold PBS. Cells were subsequently solubilized in 1% Triton X-100, and 3H was quantitated by scintillation counting (Packard 1900CA liquid scintillation analyzer, Packard Instrument Co.). Glucose uptake was measured in duplicate in all treatments. Nonspecific uptake of 2-deoxy-[3H]glucose was determined by adding 50 μm cytochalasin B to the appropriate controls prior to the commencement of assays. In basal adipocytes, IRS-1 is enriched in a microsomal fraction, often referred to as the low density microsomes (31.Heller-Harrison R.A. Morin M. Czech M.P. J. Biol. Chem. 1995; 270: 24442-24450Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 33.Kublaoui B. Lee J. Pilch P.F. J. Biol. Chem. 1995; 270: 59-65Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 40.Ricort J-M. Tanti J.F. Van Obberghen E. Le Marchand-Brustel Y. Eur. J. Biochem. 1996; 239: 17-22Crossref PubMed Scopus (85) Google Scholar). In addition to intracellular membranes that contain the insulin-regulatable glucose transporter, GLUT4, the low density microsomes fraction also contains cytoskeleton and other large protein complexes. In fact, >60% of the protein in this fraction is insoluble in nonionic detergents, so we now refer to this fraction as the high speed pellet, or HSP (41.Hill M.M. Clark S.F. James D.E. Electrophoresis. 1997; 18: 2629-2637Crossref PubMed Scopus (15) Google Scholar). In contrast to membrane proteins such as GLUT4, IRS-1 remains insoluble following treatment of the HSP with nonionic detergents, and it does not have a buoyant density that enables it to float up through a 50% sucrose solution (29.Clark S.F. Martin S. Carozzi A.J. Hill M.M. James D.E. J. Cell Biol. 1998; 140: 1211-1225Crossref PubMed Scopus (159) Google Scholar). Based on these data, we proposed that IRS-1 is either attached to the cytoskeleton or found in a large protein complex that enables it to be pelleted during high speed centrifugation. In certain cellular contexts, IRS-2 may functionally substitute for IRS-1 in mediating insulin action (42.Zhou L. Chen L. Lin C.H. Cong L.-N. McGibbon M.A. Sciacchitano S. Lesniak M.A. Quon M.J. Taylor S.I. J. Biol. Chem. 1997; 272: 29829-29833Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). In 3T3-L1 adipocytes, the subcellular distribution of IRS-2 is indistinguishable from IRS-1 (Fig.1 A). Consistent with recent studies (34.Inoue G. Cheatham B. Emkey R. Kahn C.R. J. Biol. Chem. 1998; 273: 11548-11555Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 43.Anai O. Ono H. Funaki M. Fukushima Y. Inukai K. Ogihara T. Sakoda H. Onishi Y. Yazaki Y. Kikuchi M. Oka Y. Asano T. J. Biol. Chem. 1998; 273: 29686-29692Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), we observed a significant amount (60–80% of the total) of both IRS-1 and IRS-2 in the HSP fraction in basal adipocytes. Low levels were found in the plasma membrane and high density microsome fractions, possibly due to contamination of these fractions with HSP-derived material. Significant levels (20–40%) of both IRS-1 and IRS-2 were found in the cytosol under basal conditions. Consistent with previous studies (31.Heller-Harrison R.A. Morin M. Czech M.P. J. Biol. Chem. 1995; 270: 24442-24450Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 34.Inoue G. Cheatham B. Emkey R. Kahn C.R. J. Biol. Chem. 1998; 273: 11548-11555Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar), both IRS-1 and IRS-2 were released from the HSP into the cytosol upon acute treatment of the cells with insulin (Fig. 1 A). The HSP pool of IRS-2 exhibited similar biochemical properties to that of IRS-1, in that it remained insoluble following treatment with the nonionic detergent, Triton X-100 (Fig.1 B). In addition, flotation analysis of the HSP as described previously (29.Clark S.F. Martin S. Carozzi A.J. Hill M.M. James D.E. J. Cell Biol. 1998; 140: 1211-1225Crossref PubMed Scopus (159) Google Scholar) showed that, in contrast to membrane-associated proteins but identical to IRS-1, IRS-2 does not float up through a 50% sucrose solution (data not shown). These data are consistent with a model where the HSP pool of both IRS-1 and IRS-2 are not membrane-associated. As depicted in Fig. 1 Aand described in more recent studies (34.Inoue G. Cheatham B. Emkey R. Kahn C.R. J. Biol. Chem. 1998; 273: 11548-11555Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar), both IRS-1 and IRS-2 undergo acute insulin-stimulated release from the HSP into the cytosol. With extended insulin treatment (15–60 min), there was significant loss of immunoreactive IRS-1 and IRS-2 from the HSP (>80%), but the amount of tyrosine-phosphorylated IRS proteins remained fairly constant during this time (Fig. 2, A andB). This suggests that a relatively small component of the total IRS protein pool is tyrosine-phosphorylated in response to insulin at steady state and that this pool of IRS proteins is maintained despite a marked loss of immunoreactive protein from the HSP fraction following prolonged exposure to insulin. The amount of IRS proteins in the HSP continued to decline in response to long term exposure to insulin, and it was not until the cells had been incubated in the presence of insulin for up to 4 h before there was a significant decrease in tyrosine-phosphorylated IRS proteins in the HSP f

Referência(s)