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• W2048363513 abstract "Many proinflammatory cytokines and hormones have been demonstrated to be involved in insulin resistance. However, the molecular mechanisms whereby these cytokines and hormones inhibit insulin signaling are not completely understood. We observed that several cytokines and hormones that induce insulin resistance also stimulate SOCS3 expression in 3T3-L1 adipocytes and that SOCS3 mRNA is increased in adipose tissue of obese/diabetic mice. We then hypothesized that SOCS3 may mediate cytokine- and hormone-induced insulin resistance. By using SOCS3-deficient adipocytes differentiated from mouse embryonic fibroblasts, we found that SOCS3 deficiency increases insulin-stimulated IRS1 and IRS2 phosphorylation, IRS-associated phosphatidylinositol 3-kinase activity, and insulin-stimulated glucose uptake. Moreover, lack of SOCS3 substantially limits the inhibitory effects of tumor necrosis factor-α to suppress IRS1 and IRS2 tyrosine phosphorylation, phosphatidylinositol 3-kinase activity, and glucose uptake in adipocytes. The ameliorated insulin signaling in SOCS3-deficient adipocytes is mainly due to the suppression of tumor necrosis factor-α-induced IRS1 and IRS2 protein degradation. Therefore, our data suggest that endogenous SOCS3 expression is a key determinant of basal insulin signaling and is an important molecular mediator of cytokine-induced insulin resistance in adipocytes. We conclude that SOCS3 plays an important role in mediating insulin resistance and may be an excellent target for therapeutic intervention in insulin resistance and type II diabetes. Many proinflammatory cytokines and hormones have been demonstrated to be involved in insulin resistance. However, the molecular mechanisms whereby these cytokines and hormones inhibit insulin signaling are not completely understood. We observed that several cytokines and hormones that induce insulin resistance also stimulate SOCS3 expression in 3T3-L1 adipocytes and that SOCS3 mRNA is increased in adipose tissue of obese/diabetic mice. We then hypothesized that SOCS3 may mediate cytokine- and hormone-induced insulin resistance. By using SOCS3-deficient adipocytes differentiated from mouse embryonic fibroblasts, we found that SOCS3 deficiency increases insulin-stimulated IRS1 and IRS2 phosphorylation, IRS-associated phosphatidylinositol 3-kinase activity, and insulin-stimulated glucose uptake. Moreover, lack of SOCS3 substantially limits the inhibitory effects of tumor necrosis factor-α to suppress IRS1 and IRS2 tyrosine phosphorylation, phosphatidylinositol 3-kinase activity, and glucose uptake in adipocytes. The ameliorated insulin signaling in SOCS3-deficient adipocytes is mainly due to the suppression of tumor necrosis factor-α-induced IRS1 and IRS2 protein degradation. Therefore, our data suggest that endogenous SOCS3 expression is a key determinant of basal insulin signaling and is an important molecular mediator of cytokine-induced insulin resistance in adipocytes. We conclude that SOCS3 plays an important role in mediating insulin resistance and may be an excellent target for therapeutic intervention in insulin resistance and type II diabetes. Insulin resistance is a fundamental aspect of the etiology of type II diabetes, a prevalent and serious metabolic disorder worldwide (1Kahn B.B. Flier J.S. J. Clin. Investig. 2000; 106: 473-481Crossref PubMed Scopus (2411) Google Scholar). Insulin resistance is also commonly seen with obesity (1Kahn B.B. Flier J.S. J. Clin. Investig. 2000; 106: 473-481Crossref PubMed Scopus (2411) Google Scholar). One of the major links between the two disorders is proinflammatory cytokines (1Kahn B.B. Flier J.S. J. Clin. Investig. 2000; 106: 473-481Crossref PubMed Scopus (2411) Google Scholar). Increased production of proinflammatory cytokines in obesity has been causally linked to insulin resistance (2Hotamisligil G.S. Int. J. Obes. 2003; 27: S53-S55Crossref PubMed Scopus (556) Google Scholar). TNFα 1The abbreviations used are: TNFα, tumor necrosis factor-α; SH, Src homology; MEFs, mouse embryonic fibroblasts; PI, phosphatidylinositol; Wt, wild type; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; RT, reverse transcriptase; DIO, diet-induced obesity; GH, growth hormone; IFNγ, interferon-γ; AT II, angiotensin II; IL, interleukin; SOCS, suppressor of cytokine signaling 1The abbreviations used are: TNFα, tumor necrosis factor-α; SH, Src homology; MEFs, mouse embryonic fibroblasts; PI, phosphatidylinositol; Wt, wild type; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; RT, reverse transcriptase; DIO, diet-induced obesity; GH, growth hormone; IFNγ, interferon-γ; AT II, angiotensin II; IL, interleukin; SOCS, suppressor of cytokine signaling is a major cytokine linked to insulin resistance in obesity (3Uysal K.T. Wiesbrok S.M. Marino M.W. Hotamisligil G.S. Nature. 1997; 389: 610-614Crossref PubMed Scopus (1882) Google Scholar). Increased production of TNFα within adipose tissue has been reported in obese humans and animal models, and this has been linked to insulin resistance (4Hotamisligil G.S. Arner P. Caro J.F. Atkinson R.L. Spiegelman B.M. J. Clin. Investig. 1995; 95: 2409-2415Crossref PubMed Scopus (2940) Google Scholar, 5Yamakawa T. Tanaka S. Yamakawa Y. Kiuchi Y. Isoda F. Kawamoto S. Okuda K. Sekihara H. Clin. Immunol. Immunopathol. 1995; 75: 51-56Crossref PubMed Scopus (76) Google Scholar). Although the role of proinflammatory cytokines in mediating insulin resistance has drawn extensive attention, the molecular mechanisms whereby most cytokines inhibit insulin signaling are incompletely understood.Many cytokines stimulate the tissue-specific expression of suppressor of cytokine signaling proteins (SOCSs), a group of signaling proteins characterized by their ability to down-regulate cytokine signaling (6Krebs D. Hilton D.J. Stem Cells. 2001; 19: 378-387Crossref PubMed Scopus (650) Google Scholar). The SOCS protein family includes eight members (CIS and SOCS1 to SOCS7), and each member contains a central SH2 domain and a conserved C-terminal SOCS box (7Endo T.A. Masuhara M. Yokouchi M. Suzuki R. Sakamoto H. Mitsui K. Matsumoto A. Tanimura S. Ohtsubo M. Misawa H. Miyazaki T. Leonor N. Taniguchi T. Fujita T. Kanakura Y. Komiya S. Yushimura A. Nature. 1997; 387: 921-924Crossref PubMed Scopus (1221) Google Scholar, 8Naka T. Narazaki M. Hirata M. Matsumoto T. Minamoto S. Aono A. Nishimoto N. Kajita T. Taga T. Yoshizaki K. Akira S. Kishimoto T. Nature. 1997; 387: 924-929Crossref PubMed Scopus (1128) Google Scholar, 9Starr R. Willson T.A. Viney E.M. Murray L.J. Rayner J.R. Jenkins B.J. Gonda T.J. Alexander W.S. Metcalf D. Nicola N.A. Hilton D.J. Nature. 1997; 387: 917-921Crossref PubMed Scopus (1793) Google Scholar). Cytokine binding to its receptor activates the JAK-STAT signaling pathway, leading to induction of SOCS mRNA and protein. Induced SOCS proteins in turn inhibit cytokine signaling (10Krebs D.L. Hilton D.J. J. Cell Sci. 2000; 113: 2813-2819Crossref PubMed Google Scholar). SOCS proteins bind directly via their SH2 domains to tyrosine-phosphorylated JAK or activated cytokine receptors to suppress cytokine signaling (10Krebs D.L. Hilton D.J. J. Cell Sci. 2000; 113: 2813-2819Crossref PubMed Google Scholar). An additional mechanism by which SOCS proteins inhibit signaling involves targeted proteasomal degradation of signaling proteins, via a SOCS box-mediated ubiquitination complex (11Zhang J.G. Farley A. Nicholson S.E. Willson T.A. Zugaro L.M. Simpson R.J. Moritz R.L. Cary D. Richardson R. Hausmann G. Kile B.J. Kent S.B.H. Alexander W.S. Metcalf D. Hilton D.J. Nicola N.A. Baca M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2071-2076Crossref PubMed Scopus (519) Google Scholar, 12Kamizono S. Hanada T. Yasukawa H. Minoguchi S. Kato R. Minoguchi M. Hattori K. Hatakeyama S. Yada M. Morita S. Kitamura T. Kato H. Nakayama K. Yoshimura A. J. Biol. Chem. 2001; 276: 12530-12538Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar).Previous studies from our laboratory have established SOCS3 as a negative regulator of leptin signaling. Leptin induces SOCS3, which in turn inhibits leptin signaling (13Bjørbæk C. El-Haschimi K. Frantz J.D. Flier J.S. J. Biol. Chem. 1999; 274: 30059-30065Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar, 14Bjørbæk C. Elmquist J.K. Frantz J.D. Shoelson S.E. Flier J.S. Mol. Cell. 1998; 1: 619-625Abstract Full Text Full Text PDF PubMed Scopus (842) Google Scholar), suggesting that SOCS3 may play a potential role in regulating leptin sensitivity. Recent reports have also demonstrated that SOCS3 is capable of blocking insulin signaling (15Emanuelli B. Peraldi P. Filloux C. Sawka-Verhelle D. Hilton D.J. Van Obberghen E. J. Biol. Chem. 2000; 275: 15985-15991Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, 16Emanuelli B. Peraldi P. Filloux C. Chavey C. Freidinger K. Hilton D.J. Hotamisligil G.S. Van Obberghen E. J. Biol. Chem. 2001; 276: 47944-47949Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar, 17Peraldi P. Filloux C. Emanuelli B. Hilton D.J. Van Obberghen E. J. Biol. Chem. 2001; 276: 24614-24620Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), at least partially through SOCS3-mediated proteasomal degradation of IRS1 and IRS2 (18Rui L. Yuan M. Frantz D. Shoelson S. White M.F. J. Biol. Chem. 2002; 277: 42394-42398Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar).Prior studies demonstrating SOCS3 inhibition of insulin signaling employed forced SOCS3 expression using transfection or adenoviral vectors, so little is known regarding the physiological role of SOCS3 in insulin signaling in insulin-sensitive tissues, such as adipocytes. Accordingly, this study was designed to determine the role of endogenous SOCS3 in setting the level of insulin signaling and mediating adipocyte insulin resistance in response to external factors. In this study, we established an Socs3-deficient adipocyte model, using adipocytes differentiated from mouse embryonic fibroblasts (MEFs) from wild-type or Socs3-deficient embryos. We found that SOCS3 deficiency increases insulin-stimulated IRS1 and IRS2 phosphorylation, enhances downstream PI 3-kinase activity, and leads to increased insulin-stimulated glucose uptake. Moreover, SOCS3 deficiency blocks TNFα-induced inhibition of insulin signaling, and this is largely attributed to the suppression of TNFα-induced IRS1 and IRS2 protein degradation. Taken together, these data demonstrate that the level of SOCS3 expression is a determinant of insulin signaling and is a mediator of cytokine-induced insulin resistance in adipocytes. Because SOCS3 plays an important role in mediating both leptin and insulin resistance, it could be a unique mediator of both obesity and the associated metabolic syndrome.EXPERIMENTAL PROCEDURESAntibodies and Reagents—The rabbit SOCS3 antiserum was generated as described previously (13Bjørbæk C. El-Haschimi K. Frantz J.D. Flier J.S. J. Biol. Chem. 1999; 274: 30059-30065Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar), and goat polyclonal anti-SOCS3 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-IR, IRS-1, and IRS2 antibodies were kindly provided by Dr. Ronald Kahn or purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Protein A-agarose, rabbit polyclonal anti-peroxisome proliferator-activated receptor-γ antibody, and goat polyclonal anti-SCD1 antibody were purchased from Santa Cruz Biotechnology. Rabbit polyclonal anti-GLUT4 antibody was purchased from Chemicon International (Temecula, CA). Mouse monoclonal anti-phosphotyrosine 4G10, rabbit polyclonal anti-p85, rabbit polyclonal anti-Akt1, and rabbit monoclonal anti-phospho-Akt1 (Ser-473) antibodies were obtained from Upstate Biotechnology, Inc. TNFα, IFNγ, interleukin-6, growth hormone, 3-isobutyl-1-methylxanthine, dexamethasone, insulin, and lactacystin were all purchased from Sigma. 2-Deoxy-3H-glucose was from Amersham Biosciences.Cell Culture—Murine 3T3-L1 preadipocytes were propagated in DMEM (Invitrogen) containing 10% FBS (growth medium). Confluent preadipocytes were induced to differentiate with DMEM containing 10% FBS supplemented with 0.5 mm 3-isobutyl-1-methylxanthine, 1 μm dexamethasone, and 200 nm insulin (differentiation medium). Cells were incubated in this differentiation medium for 2 days and then cultured in growth medium containing 200 nm insulin for another 2 days. Subsequently, cells were maintained in growth medium throughout the adipocyte stage and cultures were refed every 3 days.Primary MEFs for three Socs3 genotypes (Socs +/+, +/-, and -/-) were generated from 10- to 12-day embryos by crossing heterozygous Socs3 mice, according to a modified 3T3 protocol (19Todaro G.T. Green H. J. Cell Biol. 1963; 17: 299-313Crossref PubMed Scopus (1994) Google Scholar). Briefly, embryos were dissected, and the heads were removed and used for genotyping. The remaining bodies whose visceral organs had been removed were minced and digested in trypsin-EDTA buffer (2.5 g of trypsin, 0.4 g of EDTA, 7 g of NaCl, 0.3 g of Na2HPO4, 0.24 g of KH2PO4, 0.37 g of KCl, 1 g of d-glucose, 3.0 g of Tris-base, 1 ml of phenol red, and H2O added to 1 liter, pH 7.6) at 37 °C with occasional shaking for 30 min. Disassociated cells from individual embryos were collected by centrifugation and cultured in DMEM containing 10% FBS in T75 flasks. Forty eight h later, adherent cells were split into 100-mm plates at a density of 1.2 × 106/plate. Cells were then cultured in DMEM containing 10% FBS supplemented with 10% preconditioned medium (1st passage medium saved) (growth medium) and split every 3 days at the density indicated above. Cells were frozen at low passages (<5) for future experiments. For differentiation, confluent monolayers of MEFs were induced to differentiate with a differentiation medium (DMEM) containing 10% FBS supplemented with 0.5 mm 3-isobutyl-1-methylxanthine, 1 μm dexamethasone, 1.7 μm insulin, and 10 μm troglitazone. Cells were renewed in this medium every 3 days. On day 9, cells were switched to the above medium in which 3-isobutyl-1-methylxanthine and troglitazone were withdrawn and were then maintained in this medium until the appearance of cytoplasmic lipid accumulation. Cells were then changed to growth medium (DMEM) containing 10% FBS only and were ready for use. Five independent lineages from each genotype were generated and evaluated for differentiation efficiency.Total RNA Extraction and Quantitative RT-PCR—Adipocyte total RNA was extracted by using the RNeasy Mini Kit (Qiagen, Valencia, CA) and underwent DNase I treatment (Qiagen), according to the manufacturer's instructions. Mouse adipocyte SOCS3 mRNA was quantitatively measured using a Stratagene Mx 4000 Multiplex Quantitative PCR system (Stratagene, Cedar Creek, TX) with a Brilliant Single-Step Quantitative RT-PCR kit (Stratagene), as described previously (20Shi H. Norman A.W. Ojamura W.H. Sen A. Zemel M.B. FASEB J. 2002; 16: 1808-1810Crossref PubMed Google Scholar). The primers and probe for the mouse Socs3 (GenBank™ accession number 88328.1) measurement are as follows: forward primer, 5′-gcgggcacctttcttatcc-3′; reverse primer, 5′-tccccgactgggtcttgac-3′; and probe, 5′-FAM-tcggaccagcgccacttcttcac-BHQ-1–3′. A pooled mouse adipocyte total RNA was serially diluted in the range of 0.1–100 ng and was used to established an SOCS3 standard curve; total RNA for unknown samples was also diluted in this range. Reactions of real time RT-PCR for standards and unknown samples were performed according to the instructions for the Stratagene Mx 4000 quantitative RT-PCR system and single-step quantitative RT-PCT kit (Stratagene). The SOCS3 mRNA quantitation was further normalized by the corresponding cyclophilin mRNA measurement as follows: forward primer, 5′-ggtggagagcaccaagacaga-3′; reverse primer, 5′-gccggagtcgacaatgatg-3′; and probe, 5′-HEX-agccgggacaagccactgaaggat-BHQ-1–3′.Immunoprecipitation and Immunoblotting—Adipocytes were harvested and homogenized in a modified radioimmunoprecipitation assay (RIPA) lysis buffer containing 50 mm Tris-HCl, 1 mm EDTA, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 200 μm Na3VO3, 1% protease inhibitor mixture (Sigma), and 1% phosphatase inhibitor mixture (Sigma). Cell homogenates were incubated on ice for 45 min to solubilize all proteins, and insoluble portions were removed by centrifugation at 14,000 × g at 4 °C for 15 min. Two mg of cell lysate was incubated overnight with the appropriate antibodies and protein A/G-agarose (Santa Cruz Biotechnology) at 4 °C with constant gentle mixing. Agarose beads were collected by centrifugation, washed with ice-cold RIPA lysis buffer three times and phosphate-buffered saline two times, and then boiled in 2× Laemmli sample buffer for denaturation of proteins.For immunoblotting, proteins from immunoprecipitates or whole cell lysates were separated by SDS-PAGE. Proteins on the gels were transferred to Hybond ECL nitrocellulose membrane (Amersham Biosciences). The transferred membranes were blocked, washed, and incubated with various primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies. Visualization was detected with chemiluminescence reagent, using the ECL Western blotting analysis system (Amersham Biosciences). The blots were quantified by using densitometry (Amersham Biosciences).PI 3-Kinase Assay—Two mg of cell lysate was subjected to immunoprecipitation with anti-IRS1 antibody. The immunoprecipitates were used to determine the IRS1-associated PI 3-kinase activity, using a competitive enzyme-linked immunosorbent assay kit according to the company's instructions (Echelon, Salt Lake City, UT).Glucose Transport Assay—MEFs were grown and differentiated in 12-well plates. At least 1 week before the assay, insulin was withdrawn from the medium; 72 h prior to the assay, the glucose concentration was reduced to 5 mm in the medium. MEF adipocytes were incubated overnight in serum-free DMEM with or without 1 nm TNFα. Cells were then incubated in KRH buffer (129 mm NaCl, 4.8 mm KCl, 1.2 mm MgSO4, 1.8 mm CaCl2, and 20 mm HEPES, pH 7.4) with or without 100 nm insulin for 20 min. To start the glucose transport, 1 μCi of 2-deoxy-[3H]glucose was added to each well and incubated for 10 min. To stop the transport, cells were rinsed with ice-cold KRH buffer three times and lysed with 0.1% SDS. Cell lysates were added to scintillation vials with 5 ml of scintillation liquid and counted. One well of cells from each treatment group included 50 μm cytochalasin B to determine nonspecific transport, which was subtracted from each sample.Statistical Analysis—All data are expressed as mean ± S.E. Data were evaluated for statistical significance by one-way analysis of variance, and significantly different group means were then separated by the least significant difference test using SPSS (Chicago). p < 0.05 is considered as significant.RESULTSSOCS3 mRNA Is Elevated in Fat Depots of Obese/Diabetic and Diet-induced Obesity (DIO) Mice—To assess the potential relevance of SOCS3 in mediating insulin resistance, we quantitated SOCS3 mRNA in fat depots of obese/diabetic and DIO mice. Fig. 1a shows that SOCS3 mRNA was increased in subcutaneous, epididymal, and mesenteric fat pads of ob/ob and db/db mice, compared with WT control. Similar results were observed in DIO mice (Fig. 1b). These data suggest that SOCS3 mRNA expression is increased in adipose tissue of mice with genetic and acquired obesity.SOCS3 mRNA Expression Is Increased by Hormones and Cytokines in 3T3-L1 Adipocytes—We tested the effects of several hormones and cytokines, which have been shown to be involved in insulin resistance, on SOCS3 mRNA expression in 3T3-L1 adipocytes. Fig. 2, a and b, shows that several hormones, including insulin, growth hormone (GH), angiotensin II (AT II), and cytokines including TNFα, IL6, and IFNγ, did indeed stimulate SOCS3 mRNA expression in 3T3-L1 adipocytes. Dexamethasone and leptin had no effect (data not shown). Although TNFα and insulin caused a slow but relatively sustained stimulation of SOCS3 expression, IL6, IFNγ, AT II, and GH stimulated a more rapid increase of SOCS3 mRNA that was less sustained. These data suggest that up-regulation of SOCS3 may contribute to the insulin resistance caused by inflammatory cytokines and hormones.Fig. 2Regulation of SOCS3 mRNA in 3T3-L1 adipocytes by cytokines and hormones. 3T3-L1 cells were cultured and differentiated as described under “Experimental Procedures.” 3T3-L1 adipocytes were serum-free for 12 h and then treated with (b) cytokines including TNFα (10 ng/ml), IL6 (5 ng/ml), and IFNγ (100 ng/ml) and (a) hormones including insulin (100 nm), GH (100 nm), and AT II (1 μm) in a time course indicated in the figures. Adipose tissue total RNA was isolated, and SOCS3 mRNA was measured using real time RT-PCR as described under “Experimental Procedures.” All data are expressed as mean ± S.E. (n = 5).View Large Image Figure ViewerDownload (PPT)Establishment of a SOCS3-deficient Adipocyte Model—Because deletion of the Socs3 gene in mice results in embryonic lethality at day 13 (21Marine J.C. McKay C. Wang D. Tophem D.J. Parganas E. Nakajima H. Pendaeville H. Yasukawa H. Sasaki A. Yoshimura A. Ihle J.N. Cell. 1999; 98: 617-627Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar), we utilized MEFs as a cell model to explore further the role of SOCS3 in adipocyte insulin signaling. MEFs can be differentiated at 60–70% as shown by Oil Red O staining (Fig. 3a). Although MEFs of three genotypes (+/+, +/-, and -/-) underwent similar differentiation, as revealed by lipid accumulation and by the equal expression of adipocyte-phenotypic genes (Fig. 3b), Wt, Het, and Ko MEFs exhibited differential SOCS3 expression (Fig. 3c). Real time RT-PCR measurement showed that SOCS3 mRNA was not detectable in Ko MEFs, whereas SOCS3 expression in Het MEFs was decreased by 50%, compared with that in Wt cells (Fig. 3c, lower panel). Similar results were observed when SOCS3 protein levels were measured by immunoprecipitation followed by Western blot (Fig. 3c, upper panel). Moreover, Het MEFs exhibited less SOCS3 mRNA expression in response to TNFα treatment, compared with Wt MEFs. TNFα treatment caused a 10-fold increase of SOCS3 mRNA in Wt MEFs, whereas TNFα stimulated less SOCS3 expression in Het MEFs, which showed a 5-fold increase (Fig. 3d). Therefore, these MEFs are a useful cell model for studying the role of endogenous SOCS3 in insulin signaling in adipocytes.Fig. 3Establishment of an SOCS3-deficient adipocyte model.a, Oil Red O staining of differentiated Wt (+/+), Het (+/-), and Ko (-/-) MEFs. MEFs were generated and differentiated as described under “Experimental Procedures.” b, equal expression of adipocyte genes in differentiated Wt, Het, and Ko MEFs. Cell lysates were used for immunoblotting with the indicated antibodies as described under “Experimental Procedures.” c, differential expression of SOCS3 in differentiated Wt, Het, and Ko MEFs. SOCS3 protein from lysates was immunoprecipitated and immunoblotted with SOCS3 antibody. Adipocyte total RNA was isolated, and SOCS3 mRNA was measured using real time RT-PCR. Data are expressed as mean ± S.E. (n = 6); *, p < 0.05 versus Het. d, Het adipocytes express less SOCS3 mRNA in response to TNFα stimulation. Adipocytes were serum-free for 12 h and then treated with TNFα (1 nm) for 12 h. Adipocyte total RNA was isolated, and SOCS3 mRNA was measured using real time RT-PCR. Data are expressed as mean ± S.E. (n = 5); *, p < 0.001 versus Wt control and Het TNFα, **, p < 0.05 versus Het control.View Large Image Figure ViewerDownload (PPT)SOCS3 Deficiency Increases Insulin-stimulated Glucose Uptake in Adipocytes—To study the physiological consequence of SOCS3 deficiency for insulin signaling in adipocytes, we measured insulin-stimulated glucose uptake in differentiated MEFs. In Wt adipocytes, insulin stimulated 2-deoxy-[3H]glucose uptake by 3-fold, whereas insulin stimulated a 4-fold increase of glucose uptake in Ko adipocytes (Fig. 4). Moreover, lack of SOCS3 attenuated TNFα-induced inhibition of insulin-stimulated glucose uptake. Pretreatment of Wt adipocytes with TNFα for 12 h caused a 50% reduction of insulin-stimulated glucose uptake, whereas TNFα only exerted a 25% inhibition of glucose uptake in Ko adipocytes (Fig. 4). Het adipocytes exhibited an intermediate increase in insulin-stimulated glucose uptake and attenuation on inhibition of TNFα on glucose uptake (Fig. 4). These data demonstrate a gene dosage effect for the relationship between SOCS3 expression and insulin-stimulated glucose uptake in both the presence and absence of TNFα.Fig. 4SOCS3 deficiency increases insulin-stimulated 2-deoxy-[3H]glucose uptake. MEFs were generated and differentiated as described under “Experimental Procedures.” Adipocytes were pretreated with TNFα (1 nm) for 12 h, and glucose transport assay was conducted as described under “Experimental Procedures.” Data are expressed as mean ± S.E. (n = 6); *, p < 0.001 versus Wt and Het insulin alone, **, p < 0.001 versus Wt and Het insulin plus TNFα; ***, p < 0.001 versus Wt insulin plus TNFα. Con, control.View Large Image Figure ViewerDownload (PPT)Endogenous SOCS3 Levels Regulate Insulin-stimulated IRS1 Phosphorylation—We then investigated how endogenous SOCS3 levels affect insulin signaling in adipocytes. Fig. 5a shows that insulin-stimulated phosphorylation of IR remains at the same level among three genotypes, demonstrating that SOCS3 deficiency does not alter IR phosphorylation in these cells. However, insulin-stimulated phosphorylation of IRS1 was increased in Ko and Het MEFs by 50 and 20%, respectively, compared with Wt adipocytes (Fig. 5b). This suggests that IRS might be a major signaling molecule through which SOCS3 targets insulin signaling in adipocytes.Fig. 5SOCS3 deficiency increases insulin-stimulated phosphorylation of IRS1.a, SOCS3 deficiency does not alter insulin-stimulated phosphorylation of IR. Adipocytes were serum-free for 16 h and stimulated with 100 nm insulin for 5 min. IR protein was immunoprecipitated (IP) and immunoblotted (IB) with IR and phosphotyrosine antibodies. b, SOCS3 deficiency increases insulin-stimulated phosphorylation of IRS1. The experiments were performed as described in a except using IRS1 antibody. The blot is a representative of three similar experiments. Blots were quantified using a densitometry. Data are expressed as mean ± S.E. (n = 3); *, p < 0.05 versus Wt insulin.View Large Image Figure ViewerDownload (PPT)SOCS3 Is Required for the Action of TNFα to Inhibit Insulin Signaling in Adipocytes—Fig. 6a (left panel) shows that pretreatment of Wt adipocytes with TNFα for 12 h inhibited insulin-stimulated IRS1 phosphorylation by 80%. This was substantially attenuated in Ko and Het adipocytes, which showed approximately a 40% inhibition. In the TNFα treatment groups, in fact, the absolute levels of insulin-stimulated IRS1 phosphorylation in Het and Ko MEFs were increased by 2–3-fold, compared with Wt adipocytes. As shown in Fig. 6a (right panel), TNFα treatment reduced the IRS1 protein level by 80% in Wt adipocytes, whereas TNFα exerted less inhibitory effects on IRS1 protein levels in Ko and Het adipocytes, with approximately a 40% inhibition. These data indicate that IRS1 phosphorylation is well matched with IRS1 protein levels in the TNFα treatment study, suggesting that the protection of TNFα-induced IRS1 protein degradation by SOCS3 deficiency may account for the attenuation of inhibition by TNFα of IRS1 phosphorylation in Socs3-deficient cells. To confirm further this protection of SOCS3 deficiency on IRS1 protein degradation, we treated the Ko and Wt adipocytes with TNFα in a time course study. Fig. 6b shows that TNFα treatment caused IRS1 protein degradation from 9 to 48 h in Wt adipocytes, with the lowest levels at 12 and 24 h. In contrast, TNFα was unable to exert this action on IRS1 protein in Ko adipocytes. This effect of TNFα on IRS1 was specific, as TNFα was without effect on the degradation of p85 (Fig. 6b, lower panel). Moreover, pretreatment of adipocytes with lactacystin, a proteasomal inhibitor, completely blocks the effect of TNFα on IRS1 degradation (Fig. 6b, upper left). Similar results were observed on the effect of SOCS3 deficiency on IRS2 phosphorylation and protein levels (Fig. 6c). These data suggest that SOCS3 is required for TNFα-induced IRS1 and -2 protein degradation and subsequent hypophosphorylation, and may therefore be required for TNFα-caused impairment of insulin signaling.Fig. 6SOCS3 deficiency substantially blocks the ability of TNFα to inhibit insulin signaling.a, SOCS3 deficiency substantially blocks TNFα inhibition of IRS1 phosphorylation. Adipocytes were pretreated with TNFα (1 nm) for 12 h and then stimulated with 100 nm insulin for 5 min. IRS1 protein was immunoprecipitated (IP) and immunoblotted (IB) with IRS1 (upper right) and phosphotyrosine (upper left) antibodies. The blot is a representative of three similar experiments. Blots were quantified by densitometry. Lower left panel, data are expressed as mean ± S.E. (n = 3); *, p < 0.05 versus Wt insulin alone; **, p < 0.01 versus Wt insulin plus TNFα. Lower right panel, data are expressed as mean ± S.E. (n = 3); *, p < 0.01 versus Wt insulin plus TNFα. b, SOCS3 deficiency prevents TNFα-induced degradation of IRS1. Adipocytes were serum-free for 12 h, then pretreated with or" @default.
• W2048363513 created "2016-06-24" @default.
• W2048363513 creator A5002600582 @default.
• W2048363513 creator A5017534508 @default.
• W2048363513 creator A5028162489 @default.
• W2048363513 creator A5065083532 @default.
• W2048363513 date "2004-08-01" @default.
• W2048363513 modified "2023-10-16" @default.
• W2048363513 title "Suppressor of Cytokine Signaling 3 Is a Physiological Regulator of Adipocyte Insulin Signaling" @default.
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