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Troglitazone Antagonizes Tumor Necrosis Factor-α-induced Reprogramming of Adipocyte Gene Expression by Inhibiting the Transcriptional Regulatory Functions of NF-κB

2003; Elsevier BV; Volume: 278; Issue: 30 Linguagem: Inglês

10.1074/jbc.m303141200

ISSN

1083-351X

Autores

Hong Ruan, Henry J. Pownall, Harvey F. Lodish,

Tópico(s)

Adipokines, Inflammation, and Metabolic Diseases

Resumo

Troglitazone (TGZ), a member of the thiazolidinedione class of anti-diabetic compounds and a peroxisome proliferator activator receptor-γ (PPAR-γ) agonist, restores systemic insulin sensitivity and improves the full insulin resistance syndrome in vivo. The mechanisms underlying its in vivo function are not understood. Here we investigated the potential functional interaction between PPAR-γ and NF-κB in adipocytes. We show that TGZ selectively blocked tumor necrosis factor-α-induced and NF-κB-dependent repression of multiple adipocyte-specific genes and induction of growth phase and other genes. This occurs without interfering with NF-κB expression, activation, nuclear translocation, or DNA binding and without suppressing NF-κB-dependent survival signals. Notably, the expressions of some tumor necrosis factor-α-induced genes in adipocytes were unaffected by PPAR-γ activation. In reporter gene assays in HeLa cells, ectopic expression of PPAR-γ abolished induction of a NF-κB-responsive reporter gene by the p65 subunit (RelA) of NF-κB, and the inhibition was further enhanced in the presence of TGZ. Conversely, overexpression of p65 inhibited induction of a PPAR-γ-responsive reporter gene by activated PPAR-γ in a dose-dependent manner. The inhibitory effect was independent of the presence of NF-κB-binding sites in the promoter region. Other NF-κB family members, p50 and c-Rel as well as the S276A mutant of p65, blocked PPAR-γ-mediated gene transcription less effectively. Thus, p65 antagonizes the transcriptional regulatory activity of PPAR-γ in adipocytes, and PPAR-γ activation can at least partially override the inhibitory effects of p65 on the expression of key adipocyte genes. Our data suggest that inhibition of NF-κB activity is a mechanism by which PPAR-γ agonists improve insulin sensitivity in vivo and that adipocyte NF-κB is a potential therapeutic target for obesity-linked type 2 diabetes. Troglitazone (TGZ), a member of the thiazolidinedione class of anti-diabetic compounds and a peroxisome proliferator activator receptor-γ (PPAR-γ) agonist, restores systemic insulin sensitivity and improves the full insulin resistance syndrome in vivo. The mechanisms underlying its in vivo function are not understood. Here we investigated the potential functional interaction between PPAR-γ and NF-κB in adipocytes. We show that TGZ selectively blocked tumor necrosis factor-α-induced and NF-κB-dependent repression of multiple adipocyte-specific genes and induction of growth phase and other genes. This occurs without interfering with NF-κB expression, activation, nuclear translocation, or DNA binding and without suppressing NF-κB-dependent survival signals. Notably, the expressions of some tumor necrosis factor-α-induced genes in adipocytes were unaffected by PPAR-γ activation. In reporter gene assays in HeLa cells, ectopic expression of PPAR-γ abolished induction of a NF-κB-responsive reporter gene by the p65 subunit (RelA) of NF-κB, and the inhibition was further enhanced in the presence of TGZ. Conversely, overexpression of p65 inhibited induction of a PPAR-γ-responsive reporter gene by activated PPAR-γ in a dose-dependent manner. The inhibitory effect was independent of the presence of NF-κB-binding sites in the promoter region. Other NF-κB family members, p50 and c-Rel as well as the S276A mutant of p65, blocked PPAR-γ-mediated gene transcription less effectively. Thus, p65 antagonizes the transcriptional regulatory activity of PPAR-γ in adipocytes, and PPAR-γ activation can at least partially override the inhibitory effects of p65 on the expression of key adipocyte genes. Our data suggest that inhibition of NF-κB activity is a mechanism by which PPAR-γ agonists improve insulin sensitivity in vivo and that adipocyte NF-κB is a potential therapeutic target for obesity-linked type 2 diabetes. Type 2 diabetes is characterized, in part, by elevated plasma levels of free fatty acids and glucose and is associated with a cluster of abnormalities such as central obesity, dyslipidemia, hyperinsulinemia, elevated plasma inflammatory markers, impaired fibrinolysis, vascular abnormalities, and hypertension (1Olefsky J.M. Nolan J.J. Am. J. Clin. Nutr. 1995; 61: 980-986Crossref PubMed Scopus (110) Google Scholar, 2Reaven G.M. Annu. Rev. Med. 1993; 44: 121-131Crossref PubMed Scopus (768) Google Scholar). These abnormalities are also referred to as metabolic or insulin resistance syndrome (2Reaven G.M. Annu. Rev. Med. 1993; 44: 121-131Crossref PubMed Scopus (768) Google Scholar, 3Olefsky J.M. Saltiel A.R. Trends Endocrinol. Metab. 2000; 11: 362-368Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 4Lebovitz H.E. Banerji M.A. Recent Prog. Horm. Res. 2001; 56: 265-294Crossref PubMed Scopus (164) Google Scholar) and are risk factors for cardiovascular and cerebrovascular diseases. In obesity-linked type 2 diabetes, decreased overall insulin sensitivity is a fundamental defect that precedes the development of the full insulin resistance syndrome and subsequent β cell failure (3Olefsky J.M. Saltiel A.R. Trends Endocrinol. Metab. 2000; 11: 362-368Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 4Lebovitz H.E. Banerji M.A. Recent Prog. Horm. Res. 2001; 56: 265-294Crossref PubMed Scopus (164) Google Scholar). Treatments of type 2 diabetes, such as correcting relative insulin deficiency, inhibiting hepatic glucose production, and delaying glucose absorption from the gastrointestinal tract, lower plasma glucose levels but do little to improve insulin sensitivity. In time, these interventions often fail to restore metabolic homeostasis and to prevent the development of most of the complications of type 2 diabetes. Thus, there remains a great need to restore insulin responsiveness in the clinical management of type 2 diabetes. Thiazolidinediones (TZD), 1The abbreviations used are: TZD, thiazolidinediones; TGZ, troglitazone; TNF-α, tumor necrosis factor-α; PPAR-γ, peroxisome proliferator activator receptor-γ; ELISA, enzyme-linked immunosorbent assay; PPRE-luc, The PPAR-γ-responsive luciferase reporter gene; PEPCK, phosphoenolpyruvate carboxykinase; HSL, hormone-sensitive lipase; IκBs, inhibitor of κB proteins; IL, interleukin; TG, triglyceride; FFA, free fatty acid.1The abbreviations used are: TZD, thiazolidinediones; TGZ, troglitazone; TNF-α, tumor necrosis factor-α; PPAR-γ, peroxisome proliferator activator receptor-γ; ELISA, enzyme-linked immunosorbent assay; PPRE-luc, The PPAR-γ-responsive luciferase reporter gene; PEPCK, phosphoenolpyruvate carboxykinase; HSL, hormone-sensitive lipase; IκBs, inhibitor of κB proteins; IL, interleukin; TG, triglyceride; FFA, free fatty acid. a class of anti-diabetic medications and synthetic ligands for PPARγ, decrease plasma free fatty acid concentrations as well as fasting and postprandial plasma glucose levels in patients with type 2 diabetes by improving insulin sensitivity in major insulin-target tissues. In addition, TZD reduce plasma triglyceride levels, improve the plasma lipoprotein profile, lower blood pressure in diabetic hypertensive patients, and correct the proinflammatory and procoagulant state (5Hauner H. Diabetes Metab. Res. Rev. 2002; 18: 10-15Crossref PubMed Scopus (298) Google Scholar). Taken together, TZD target insulin resistance and restore metabolic homeostasis while improving the cluster of abnormalities that occur in type 2 diabetes. However, the direct target tissue(s) of TZD and the molecular mechanism(s) by which TZD sensitize the major insulin-responsive tissues in vivo remain elusive. TZD are high affinity ligands for peroxisome proliferator receptor activator-γ (PPAR-γ). PPAR-γ has two protein isoforms, PPAR-γ1 and PPAR-γ2 (6Zhu Y. Qi C. Korenberg J.R. Chen X.N. Noya D. Rao M.S. Reddy J.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7921-7925Crossref PubMed Scopus (601) Google Scholar, 7Fajas L. Auboeuf D. Raspe E. Schoonjans K. Lefebvre A.M. Saladin R. Najib J. Laville M. Fruchart J.C. Deeb S. Vidal-Puig A. Flier J. Briggs M.R. Staels B. Vidal H. Auwerx J. J. Biol. Chem. 1997; 272: 18779-18789Abstract Full Text Full Text PDF PubMed Scopus (1083) Google Scholar). Whereas PPAR-γ1 is ubiquitously expressed at low levels in many tissues including muscle, PPAR-γ2 is most highly expressed in adipose tissue (8Braissant O. Foufelle F. Scotto C. Dauca M. Wahli W. Endocrinology. 1996; 137: 354-366Crossref PubMed Scopus (1737) Google Scholar, 9Tontonoz P. Hu E. Graves R.A. Budavari A.I. Spiegelman B.M. Genes Dev. 1994; 8: 1224-1234Crossref PubMed Scopus (1996) Google Scholar). Notably, adipose tissue is not only essential for maintaining the overall in vivo insulin sensitivity but is also a source of endocrine activity that modulates several physiological processes that include systemic energy metabolism, inflammation, blood coagulation, blood vessel tone, and reproduction. A number of adipocyte-derived factors have been implicated in insulin resistance in obesity and obesity-linked type 2 diabetes. Thus, it is likely that TZD could directly target molecular mediator(s) of insulin resistance in adipocytes and restore the metabolic function and endocrine signals of adipose tissue, and thereby contribute to the improved systemic insulin sensitivity. Tumor necrosis factor-α (TNF-α), an autocrine/paracrine factor that is highly expressed in adipose tissues of obese animals and human subjects, is a potential molecular mediator of insulin resistance that is of physiological importance. Although many factors may trigger the development of insulin resistance in humans (10Boden G. Endocrinol. Metab. Clin. 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We recently substantiated the critical role of TNF-α-regulated gene expression in adipocytes in the development of systemic insulin resistance by association of gene expression profiles in major insulin-responsive tissues with overall in vivo insulin sensitivity in rats infused with TNF-α (24Ruan H. Miles P.D.G. Ladd C.M. Ross K. Golub T.R. Olefsky J.M. Lodish H.F. Diabetes. 2002; 51: 3176-3188Crossref PubMed Scopus (222) Google Scholar). We also demonstrated that NF-κB activation by TNF-α is obligatory in the repression of key adipocyte genes and induction of many proinflammatory and acute phase proteins (22Ruan H. Hacohen N. Golub T.R. Van Parijs L. Lodish H.F. Diabetes. 2002; 51: 1319-1336Crossref PubMed Scopus (419) Google Scholar). These data assert the importance of TNF-α and TNF-α-induced NF-κB activation in the etiology of insulin resistance in adipocytes. It is well established that TZD counteract a number of effects of TNF-α on adipocytes, including adipocyte differentiation (25Ohsumi J. Sakakibara S. Yamaguchi J. Miyadai K. Yoshioka S. Fujiwara T. Horikoshi H. Serizawa N. Endocrinology. 1994; 135: 2279-2282Crossref PubMed Scopus (66) Google Scholar) and gene expression (17Stephens J.M. Pekala P.H. J. Biol. Chem. 1992; 267: 13580-13584Abstract Full Text PDF PubMed Google Scholar, 18Zhang B. Berger J. Hu E. Szalkowski D. White-Carrington S. Spiegelman B.M. Moller D.E. Mol. Endocrinol. 1996; 10: 1457-1466Crossref PubMed Scopus (310) Google Scholar, 22Ruan H. Hacohen N. Golub T.R. Van Parijs L. Lodish H.F. Diabetes. 2002; 51: 1319-1336Crossref PubMed Scopus (419) Google Scholar, 26Szalkowski D. White-Carrington S. Berger J. Zhang B. Endocrinology. 1995; 136: 1474-1481Crossref PubMed Google Scholar, 27Ribon V. Johnson J.H. Camp H.S. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14751-14756Crossref PubMed Scopus (179) Google Scholar), insulin signaling (28Peraldi P. Xu M. Spiegelman B.M. J. Clin. Invest. 1997; 100: 1863-1869Crossref PubMed Scopus (298) Google Scholar), insulin-stimulated glucose uptake (26Szalkowski D. White-Carrington S. Berger J. Zhang B. Endocrinology. 1995; 136: 1474-1481Crossref PubMed Google Scholar), lipogenesis (29Yamauchi T. Kamon J. Waki H. Murakami K. Motojima K. Komeda K. Ide T. Kubota N. Terauchi Y. Tobe K. Miki H. Tsuchida A. Akanuma Y. Nagai R. Kimura S. Kadowaki T. J. Biol. Chem. 2001; 276: 41245-41254Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar), and lipolysis (19Souza S.C. Yamamoto M.T. Franciosa M.D. Lien P. Greenberg A.S. Diabetes. 1998; 47: 691-695Crossref PubMed Scopus (153) Google Scholar). Yet the molecular mechanisms for these effects of TZD are not known. Interestingly, TZD-induced PPAR-γ activation in monocytes or macrophages suppresses the induction of many inflammatory and immune response mediators such as TNF-α, IL-1β, IL-6, metalloproteases, and inducible nitric-oxide synthase in part by inhibiting the activities of NF-κB, signal transducers and activators of transcription, and AP-1 (30Jiang C. Ting A.T. Seed B. Nature. 1998; 391: 82-86Crossref PubMed Scopus (538) Google Scholar, 31Ricote M. Li A.C. Willson T.M. Kelly C.J. Glass C.K. Nature. 1998; 391: 79-82Crossref PubMed Scopus (3252) Google Scholar). In addition, PPAR-α agonists inhibit cytokine-induced VCAM-1 and IL-6 expression in endothelial cells and vascular smooth muscle cells through interference with NF-κB and AP-1 action by protein-protein interactions and cofactor squelching (32Marx N. Sukhova G.K. Collins T. Libby P. Plutzky J. Circulation. 1999; 99: 3125-3131Crossref PubMed Scopus (546) Google Scholar, 33Delerive P. De Bosscher K. Besnard S. Vanden Berghe W. Peters J.M. Gonzalez F.J. Fruchart J.C. Tedgui A. Haegeman G. Staels B. J. Biol. Chem. 1999; 274: 32048-32054Abstract Full Text Full Text PDF PubMed Scopus (978) Google Scholar). However, experimental data demonstrating a functional interaction between NF-κB and PPAR-γ in the context of adipocytes are lacking. Herein we have used a number of complementary experimental approaches to determine whether ligand-induced PPAR-γ activation can override the transcription-repressive effects of NF-κB activation by TNF-α on key adipocyte genes, and whether NF-κB family members can directly inhibit the transcriptional regulatory activity of PPAR-γ. Oligonucleotide Microarray Data Collection and Analysis—3T3-L1 adipocytes were untreated or stimulated with TNF-α (1 nmol/liter), troglitazone (1 μmol/liter), or both for various times. Total RNA at each time point was isolated, converted to biotin-labeled cRNA targets, hybridized to MG74Av2 oligonucleotide arrays (Affymetrix, Santa Clara, CA), and scanned on Affymetrix scanners essentially as described previously (22Ruan H. Hacohen N. Golub T.R. Van Parijs L. Lodish H.F. Diabetes. 2002; 51: 1319-1336Crossref PubMed Scopus (419) Google Scholar, 34Golub T.R. Slonim D.K. Tamayo P. Huard C. Gaasenbeek M. Mesirov J.P. Coller H. Loh M.L. Downing J.R. Caligiuri M.A. Bloomfield C.D. Lander E.S. Science. 1999; 286: 531-537Crossref PubMed Scopus (9188) Google Scholar). Gene expression data were analyzed and presented using a set of web-based analysis tools developed in the Genome Center at the Whitehead Institute for Biomedical Research (Cambridge, MA) and the Cluster and TreeView software (35Eisen M.B. Spellman P.T. Brown P.O. Botstein D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14863-14868Crossref PubMed Scopus (13198) Google Scholar). Identification of adipocyte-abundant genes was determined by selecting those with 3-fold or higher expression levels in adipocytes than fibroblasts. We then excluded genes expressed at a very low level by setting an arbitrary threshold value for the array measurements (Average Differences, according to Affymetrix), and genes whose expression levels were below 150 at all time points and under all conditions were excluded. We also excluded genes whose differences in expression levels (maximal value-minimal value) between any two time points were less than 150. Thus, these filters allow us to include adipocyte-abundant genes that exhibit robust changes in steady state mRNA levels in response to at least one treatment. Next we used a modified score system, originally described by Hacohen and co-workers (36Huang Q. Liu D. Majewski P. Schulte L.C. Korn J.M. Young R.A. Lander E.S. Hacohen N. Science. 2001; 294: 870-875Crossref PubMed Scopus (666) Google Scholar), to identify genes regulated by TNF-α, troglitazone, or both. Briefly, let R i and C i be the steady state mRNA levels of treated samples and control samples, respectively, at the ith time point. Define μC to be the mean expression level of samples of the control time course and δC as the standard deviation of expression levels in the control samples. Then we can define a score, S i = (R i – μC)/δC, to measure the statistical significance of the changes in gene expression in the treated samples at each time point R i. Genes with low scores are a consequence of large variation in mRNA levels in the control time course (high noise) or small differences between the control and treated samples. By setting a threshold value for S i (see below), we can exclude genes whose expression levels were not significantly affected by a treatment or fluctuated over time under control conditions. Identification of up-regulated genes was determined by requiring one of the following: 1) S i ≥ 4 for at least 1 time point; 2) S i ≥ 1.4 for at least two consecutive time points. Down-regulated genes were selected by requiring S i ≤ –1.4 for at least three consecutive time points or S i ≤ –3 for at least one time point. We used this score system to identify TNF-α-regulated transcripts from the 175 adipocyte-abundant known genes, and we then compared the list of identified genes with our master list of 64 TNF-α-affected adipocyte-enriched genes that have been verified previously by Northern Blotting, semi-quantitative RT-PCR, or literature search (22Ruan H. Hacohen N. Golub T.R. Van Parijs L. Lodish H.F. Diabetes. 2002; 51: 1319-1336Crossref PubMed Scopus (419) Google Scholar, 24Ruan H. Miles P.D.G. Ladd C.M. Ross K. Golub T.R. Olefsky J.M. Lodish H.F. Diabetes. 2002; 51: 3176-3188Crossref PubMed Scopus (222) Google Scholar). The score system identified 102 TNF-α-affected genes (Table I) including 51 genes from the master list. The scores of the rest of the 13 master list genes are very close to but did not pass the score threshold and were excluded from the TNF-α-regulated gene list in this study. The 13 genes are as follows: CD 36; lactate dehydrogenase 2; apoE; sterol carrier protein 2; 11-β-hydroxysteroid dehydrogenase; cytochrome P450; carbonic anhydrase 3; catalase; adenylate kinase isozyme 3; complement component C3; amyloid β (A4) precursor-like protein 2; GADD 45; and Rho B. This indicates that the score system might give false-negative results. However, the inclusion of these 13 genes would not have changed our interpretation of the results, and we therefore used this score system to assess genes regulated by other treatments.Table IRegulation of adipocyte-abundant genes by troglitazone and TNF-αGBAGene name-Fold (Fat/FB)TNF-αTGZTNF-α + TGZEnergy metabolism (cholesterol and steroid)U37799Scavenger receptor class B16.1-2.51.4NCX8999817-β-Hydroxysteroid dehydrogenase type IV8.8-2.11.4-1.5X75926ATP-binding cassette 1521.8NC1.6Energy Metabolism (fatty acid, triglyceride, and lipoprotein)AJ223066FABP5, epidermal3-21.5NCX61431Diazepam-binding inhibitor5.7-2.51.5NCZ24722Apolipoprotein C2-linked35-5.6NC-1.9M63335Lipoprotein lipase23-1.7NC-2.1U15977Long chain fatty acyl-CoA synthetase52-2.51.5NCX95281Retinal short chain dehydrogenase4.5-3.81.7-1.5U07159Acetyl-CoA dehydrogenase, medium chain11-2.4NCNCAF030343Peroxisomal/mitochondrial dienoyl-CoA isomerase3.5-2.3NCNCU01170Carnitine palmitoyltransferase 24-1.71.3NCAF006688Peroxisomal acyl-CoA oxidase5.7-1.51.91.3X13135Fatty-acid synthase4.7-3.9NC-2.3M21285Steroyl-CoA desaturase 130-1.6-1.1-1.3J02652Malic enzyme4.2-2.2NCNCU67611Transaldolase5.7-31.2NCU11680Glycerol-3-phosphate acyltransferase4.1-2.2NC-1.7AF078752Diacylglycerol acyltransferase29-3.81.4NCM97957Pyruvate carboxylase homologous protein279-2.4NC-1.3AF009605Phosphoenolpyruvate carboxykinase4.2-4.33.51.8M25558Glycerolphosphate dehydrogenase 1103-4.6NCNCU69543Hormone-sensitive lipase166-3.81.51.3AF064748Plasma membrane associated S3-12 protein (perilipin family)42-2.22.2NCEnergy metabolism (glucose)M23383GLUT473-4.4NC-2.4Y11666Hexokinase II3.7-1.6-1.5-1.4U68564Isocitrate dehydrogenase 3 (NAD+), γ3.2-1.8-1.5-1.7X98848Fructose-2,6-bisphosphatase23-10.6NC-3.2AF020039NADP-dependent isocitrate dehydrogenase3-2.4NC-1.9Metabolism (phospholipid)U88624Calcium-independent phospholipase A212.6-5.9-1.4-2U25051Phosphatidylethanolamine N-methyltransferase4.9-3.3-1.8-1.7Metabolism (amino acid and nucleic acid)L47335Branched chain α-ketoacid decarboxylase E1α20-2.3NCNCAB020202Adenylate kinase isozyme 24.8-3-1.2NCAJ238636Nucleoside diphosphatase11-4NCNCL31783Uridine kinase7.2-2.6NCNCMetabolism (other)U35741Thiosulfate sulfurtransferase9.4-4.1-2-2.7M63245Aminolevulinate synthase5.5-2.2NCNCX13752δ-Aminolevulinate dehydratase4.6-4.2NC-2.4U27195Leukotriene C4 synthase11-1.4NC1.3M32032Selenium-binding protein 17.1-2.8-2.9-4.9U96401Aldehyde dehydrogenase-2-like31-2.4NCNCX51941Methylmalonyl-CoA mutase3.8-2.1NC-2.2AJ222660Nifs-like protein precursor3.2-1.61.3NCL11163Acety CoA dehydrogenase, short chain3.6-1.9NCNCU86108Nicotinamide N-methytransferase4-5.6NCNCStress responseL35528Manganese superoxide dismutase4.63.6-1.44.5Secreted protein (acute phase reactant)M27008Orosomucoid 1113-2.4-1.2-2.2Secreted protein (extracellular matrix)AF011450Procollagen, type XV51-2NC1.4U69176Laminin, α411-1.61.4NCX53929Decorin162NCNCSecreted protein (hormone and plasma protein)AA718169Resistin587-5.5NC-2.1U49915ACRP30 (AdipoQ)379-1.8-1.1-1.4AF045887Angiotensinogen135-4NCNCU49430Ceruloplasmin5.18.4NC5Secreted protein (immune and defense response and protease inhibitor)X04673Adipsin370-1.9NC-1.4M96827Haptoglobin10.55.3NC6.6M64086Spi 2 proteinase inhibitor6.72.7NC1.9Transcription factor and transcriptional regulationAB012273CEBP-γ4-3.4NCNCAF053062Receptor interacting protein 1407.7-5.9-1.7-3.3AF085745Nuclear orphan receptor LXR-α64-1.7NCNCM62362CEBP-α17-2.91.1NCU10374PPAR-γ14-2.5-2.3-5.9X95279Spot 14135-7.3NCNCY15001Iroquois homeobox protein 36-1.7-1.3-1.6U63387Chromobox homolog 44-4.1NC-4.1AF077659Homeodomain-interacting protein kinase 234-4.81.3-1.7AF077660Homeodomain-interacting protein kinase 34.6-3.31.91.5U06924STAT 15.61.721.7Cell cycle and proliferationAF011908Apoptosis-associated tyrosine kinase18-5.9NCNCD78382Tob10-2.2NCNCX95280G0S2-like protein86-8.7NCNCU19596Cdk4 and Cdk6 inhibitor p18 protein5.2-4.2-2.2-2.7ApoptosisM61737Adipocyte-specific mRNA858-9.61.91.5Cell adhesionAF078705Amine oxidase, copper containing 330-2.3-1.4-2.2X69902Integrin α68.3-3NCNCU89915Junctional adhesion molecule3.61.6NC2.8Protein degradationX81323Tripeptidyl peptidase II3.7-2.7NC-1.7AB024427Ring finger protein 115.8-2NC-1.9SignalingAB016080Calcium-binding protein Kip213-6.2NC-2.1AF009246Ras-related protein128-10.72-6.1AF093669Peroxisomal biogenesis factor6.6-2.5NCNCM13071Raf-related oncogene3-1.7NC-1.6U44940Quaking4.7-21.8NCU58883c-Cbl associated protein18-3.42.2NCU67187G protein signaling regulator 231-7.1-4.1-7.6X72862Adrenergic receptor, β343-13NC-10.4Y12577ADP-ribosylation like 412-52.8NCU28168Adenomatosis polyposis coli5.1-2.3NCNCTransportAF091390Phospholemman precursor6.6-1.8NC-1.8AB010100Aquaporin 75.8-2.31.5-1.4Z48670ATP-binding cassette, subfamily D, member 262-5NC-3.3AF098633GLUT4 vesicle protein5.3-1.5NCNCAF084575Adaptor protein complex-3 3A subunit4.4-2-1.2-2.1Immune response and MHC-relatedM28233Interferon-γ receptor9.61.3NC1.6U60091ATP-binding cassette transporter 24.74.2NC4.3AJ007970mGBP-2 protein3.815.8NC11M27134Histocompatibility 2, K region locus 24.72.5NC3.6M69069Histocompatibility 2, D region locus 13.43.1NC3.6X00246MHC class I antigen3.63.2NC3.6Y00629Histocompatibility 2, T region locus 238.63.5-2.12.7OtherL28835Peroxisome membrane protein29-3.3NC-2.2AL078630Genomic DNA sequence from chromosome 17, containing the genes for γ-aminobutyric acid B receptor 1, and five 7 transmembrane receptor (rhodopsin family)6.272.617 Open table in a new tab Fold changes in gene expression in response to a treatment relative to control were calculated as the following: F up = max{L 1, L 2,... L n-1} for genes up-regulated by a treatment, and F down = –1/min{L 1, L 2,... L n-1} for down-regulated genes, where L i = geomean{R i R i+1}/geomean{C i C i+1}. Oligonucleotide microarray data were also collected from wild-type 3T3-L1 adipocytes and adipocytes expressing a dominant negative inhibitor of NF-κB, IκB-α-DN, treated with TNF-α (1 nmol/liter) for 0, 0.5, 1, and 2 h (22Ruan H. Hacohen N. Golub T.R. Van Parijs L. Lodish H.F. Diabetes. 2002; 51: 1319-1336Crossref PubMed Scopus (419) Google Scholar). Identification of TNF-α-repressed genes was determined by requiring one of the following: 1) mRNA level decreases 30% or more for at least two consecutive time points; and 2) mRNA level decreases 50% or more at the end of the 2-h incubation. Tissue Culture—3T3-L1 cells were purchased from ATCC (Manassas, VA), maintained as fibroblasts, and differentiated into adipocytes as described previously (37Student A.K. Hsu R.Y. Lane M.D. J. Biol. Chem. 1980; 255: 4745-4750Abstract Full Text PDF PubMed Google Scholar). HeLa cells were provided by Dr. C. C. Zhang (Whitehead Institute, Cambridge, MA), and were propagated in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Western Blot Analysis—3T3-L1 adipocytes were incubated in growth media or media containing TNF-α (1 nmol/liter), troglitazone (1 μmol/liter), or both together for various times. Cell lysates were separated by SDS-PAGE and electroblotted onto a nitrocellulose membrane (Amersham Biosciences). The filter was incubated with various primary antibodies, washed, and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody. Bound antibodies were detected using the enhanced chemiluminescence Western blotting analysis system (Amersham Biosciences). Blots were stained with Ponceau S solution to visualize the amount of total protein in each lane. Plasmids—The reporter gene PEPCK-Luc was generated by subcloning the 2100-bp fragment of the PEPCK gene promoter (38Magnuson M.A. Quinn P.G. Granner D.K. J. Biol. Chem. 1987; 262: 14917-14920Abstract Full Text PDF PubMed Google Scholar) (provided by Dr. D. Granner, Vanderbilt, TN) into pTAL-Luc (Clontech, La Jolla, CA). The NF-κB-responsive luciferase reporter gene was from Clontech. The PPAR-γ-responsive luciferase reporter gene (PPRE-luc) was constructed by subcloning 6 tandem repeats of PPAR-γ-response elements into the pTAL-luc vector. The expression plasmids p65 (RelA), p65 (S276A) mutant, and p50 were provided by Dr. D. Granner (39Waltner-Law M. Daniels M.C. Sutherland C. Granner D.K. J. Biol. Chem. 2000; 275: 31847-31856Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar); c-Rel was a gift from Dr. W. Tam (Whitehead Institute, Cambridge, MA). Murine full-length PPAR-γ2 was subcloned into pIRES2-GFP expression vector (Clontech), and the sequence was verified by DNA sequencing. Transfection Assays—All transfe

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