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Tumor Necrosis Factor- Suppresses Adipocyte-Specific Genes and Activates Expression of Preadipocyte Genes in 3T3-L1 Adipocytes N

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Tumor Necrosis Factor- Suppresses Adipocyte-Specific Genes and Activates Expression of Preadipocyte Genes in 3T3-L1 Adipocytes N Tumor Necrosis Factor-␣ Suppresses Adipocyte-Specific Genes and Activates Expression of Preadipocyte Genes in 3T3-L1 Adipocytes Nuclear Factor-␬B Activation by TNF-␣ Is Obligatory Hong Ruan,1 Nir Hacohen,1 Todd R. Golub,1,2 Luk Van Parijs,3,4 and Harvey F. Lodish1,3 ␣ ␣ expression, and insulin responses. However, absence of Tumor necrosis factor- (TNF- ) is a contributing ␬ cause of the insulin resistance seen in obesity and NF- B activation abolished suppression of >98% of the genes normally suppressed by TNF-␣ and induction of obesity-linked type 2 diabetes, but the mechanism(s) by ␣ which TNF-␣ induces insulin resistance is not under- 60–70% of the genes normally induced by TNF- . More- over, extensive cell death occurred in I␬B␣-DN؊ stood. By using 3T3-L1 adipocytes and oligonucleotide ␣ microarrays, we identified 142 known genes reproduc- expressing adipocytes after2hofTNF- treatment. ibly upregulated by at least threefold after 4 h and/or Thus the changes in adipocyte gene expression induced ␣ by TNF-␣ could lead to insulin resistance. Further, 24 h of TNF- treatment, and 78 known genes down- ␬ ␣ regulated by at least twofold after 24 h of TNF-␣ NF- B is an obligatory mediator of most of these TNF- incubation. TNF-␣؊induced genes include transcription responses. Diabetes 51:1319–1336, 2002 factors implicated in preadipocyte gene expression or NF-␬B activation, cytokines and cytokine-induced pro- teins, growth factors, enzymes, and signaling molecules. Importantly, a number of adipocyte-abundant genes, nsulin resistance is a major defect frequently seen in including GLUT4, hormone sensitive lipase, long-chain obesity and obesity-linked type 2 diabetes (1–3). fatty acyl-CoA synthase, adipocyte complement-related Tumor necrosis factor-␣ (TNF-␣) has been impli- protein of 30 kDa, and transcription factors CCAAT/ cated as a contributing cause of insulin resistance enhancer binding protein-␣, receptor retinoid X recep- I associated with the above diseases by its expression tor-␣, and peroxisome profilerator؊activated receptor ␥ were significantly downregulated by TNF-␣ treatment. pattern as well as by provocative in vitro and in vivo Correspondingly, 24-h exposure of 3T3-L1 adipocytes to models (4–7). TNF-␣ is expressed in adipose tissue, and its TNF-␣ resulted in reduced protein levels of GLUT4 and mRNA and protein levels are significantly increased in several insulin signaling proteins, including the insulin adipose tissue of obese animals (8–10) and humans (11,12). receptor, insulin receptor substrate 1 (IRS-1), and pro- Long-term exposure of cultured cells (1–5 days) (13) or tein kinase B (AKT). Nuclear factor-␬B (NF-␬B) was animals (14) to TNF-␣ induces insulin resistance, whereas activated within 15 min of TNF-␣ addition. 3T3-L1 neutralization of TNF-␣ in fa/fa Zucker rats for 3 days adipocytes expressing I␬B␣-DN, a nondegradable NF-␬B increases insulin sensitivity, reduces hyperinsulinemia, inhibitor, exhibited normal morphology, global gene and increases insulin receptor (IR) tyrosine kinase activity in adipose tissue (5). Moreover, data from gene knockout animals indicate that the absence of TNF-␣ or TNF recep- From the 1Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, Massachusetts; the 2Dana-Farber Cancer tors significantly improves insulin sensitivity in genetic Institute and Department of Pediatrics, Harvard Medical School, Boston, (ob/ob) and dietary (high-fat diet) models of rodent obesity Massachusetts; the 3Department of Biology, Massachusetts Institute of Tech- (15,16). Thus, TNF-␣ clearly contributes to the develop- nology, Cambridge, Massachusetts; and the 4Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts. ment of insulin resistance in the above settings. Address correspondence and reprint requests to Professor Harvey F. Although the effects of TNF-␣ are well known, the Lodish, Whitehead Institute for Biomedical Research, 9 Cambridge Center, ␣ Room 601, Cambridge, MA 02142. E-mail: [email protected]. mechanisms by which TNF- induces insulin resistance Received for publication 27 December 2001 and accepted in revised form are not fully understood. Because the tyrosine kinase 18 February 2002. activity of the IR is critical for insulin action (17), the ACRP30, adipocyte complement-related protein of 30 kDa; Adm, adreno- ␣ medullin; AKT, protein kinase B; AP-1, activator protein 1; Bcl-3, B-cell effects of TNF- on insulin-induced tyrosine phosphoryla- leukemia/lymphoma-3; CDK, cyclin-dependent kinase; CEBP-␣, CCAAT/ tion of the IR and IR substrate 1 (IRS-1) have been the enhancer binding protein-␣; EST, expressed sequence tag; Fra-1, Fos-related focus of intensive studies. TNF-␣ treatment of a number of antigen-1; GATA-6, GATA-binding protein 6; GFP, green fluorescent protein; HMGP-1C, high mobility group protein-1 isoform C; HSL, hormone-sensitive cell lines, including primary human adipocytes, rat hepa- lipase; IKK, I␬B kinase; IKKi, inducible IKK; IL-6, interleukin 6; IR, insulin toma Fao cells, human embryonic kidney 293 cells, and receptor; IRS-1, IR substrate 1; LPSBP, lipopolysaccharide binding protein; LTR, long terminal repeat; MAPKKK, mitogen-activated protein kinase kinase NIH-3T3 fibroblasts, has been shown to result in immedi- kinase; MMP-3, matrix metalloproteinase 3; NF-␬B, nuclear factor-␬B; PAI-1, ate inhibition of insulin-induced tyrosine phosphorylation plasminogen activator inhibitor 1; pMIG, pMSCV-IRES-GFP; pI␬B␣-DN, of IR and IRS-1 (18–20). In contrast, short-term TNF-␣ pMSCV-I␬B␣__DN-IRES-GFP; PPAR-␥, peroxisome profileratorϪactivated re- ceptor ␥; RXR, receptor retinoid X receptor-␣; Spi2-1, serine protease inhibitor treatment (15 min to 2 h) of 3T3-L1 adipocytes promotes 2-1; TNF, tumor necrosis factor-␣; VCAM-1, vascular cell adhesion molecule 1. insulin-mediated tyrosine phosphorylation of IRS-1 (21), DIABETES, VOL. 51, MAY 2002 1319 TNF-␣ AND GENE EXPRESSION IN ADIPOCYTES whereas long-term TNF-␣ exposure (6 h to 5 days) dimin- inducing kinase (a MAPKKK) induces responses typical of ishes tyrosine phosphorylation of both IR and IRS-1 impaired insulin signaling, such as attenuated insulin (22,23). Thus, the effects of TNF-␣ on insulin signaling activation of the IR, IRS-2, and AKT. Conversely, inhibi- appear to be related to the cell type and duration of TNF-␣ tion of IKK␤ increases systemic insulin sensitivity in ob/ob treatment. mice or during high-fat feeding (32). Taken together, these Chronic (6 h to 5 days) TNF-␣ treatment of 3T3-L1 data implicate NF-␬B as an obligatory mediator of TNF- adipocytes leads to decreased tyrosine phosphorylation of ␣ induction of insulin resistance in 3T3-L1 adipocytes, and IRS-1, with a concomitant increase in serine phosphoryla- suggest that adipocyte NF-␬B may be a reasonable target tion (13,22,23). The increased serine phosphorylation of for drug development. IRS-1 inhibits the tyrosine kinase activity of the IR and has been postulated as a mechanism by which long-term RESEARCH DESIGN AND METHODS TNF-␣ treatment impairs insulin action (23,24). Because Cell culture. 3T3-L1 cells were purchased from American Type Culture phosphorylation and dephosphorylation are rapid events Collection (Rockville, MD), maintained as fibroblasts, and differentiated into adipocytes, as previously described (25). TNF-␣ and insulin treatments were that take place in seconds to minutes, an increase in serine performed as described in figure legends. phosphorylation and a decrease in tyrosine phosphoryla- Stable cell lines. The retroviral expression vector pMSCV-IRES-GFP (green tion of IRS-1 after a 5-day exposure to TNF-␣ seems fluorescent protein, pMIG), as well as the construct pMSCV-I␬B␣__DN-IRES- unlikely to be caused by a direct effect of TNF-␣. Support- GFP (pI␬B␣-DN) encoding the dominant negative I␬B␣ (a nondegradable mutant of I␬B␣) and GFP were gifts from L. Van Parijs (Massachusetts ing this notion, Stephens et al. (25) showed that insulin Institute of Technology, Cambridge, MA). To generate retroviruses, Phoenix resistance induced by chronic (4-day) TNF-␣ treatment is packaging cells were transfected with 8 ␮g pMIG or pI␬B␣-DN plasmid cDNA associated with a loss of IRS-1 and GLUT4 proteins rather using Polyfect (Qiagen, Chatsworth, CA). The supernatant containing the than a defect in IR signaling. Thus, the effects of TNF-␣ on retroviruses was collected 48 h after transfection and used to infect 3T3-L1 preadipocytes in the presence of 4 ␮g/ml polybrene. Then, 48 h after infection, tyrosine and/or serine phosphorylation of IRS-1, and its cells were selected for GFP expression by flow cytometry. Because both GFP subsequent impact on insulin signaling and induction of and I␬B␣-DN are under control of the retroviral long terminal repeat (LTR) insulin resistance have not been established. promoter, protein levels of GFP are therefore indicative of I␬B␣-DN expres- TNF-␣ is a well-known transcriptional regulator, which, sion. GFP-positive cells were amplified once and were sorted again by flow ␬ ␬ cytometry to select cells with the top 1% of GFP expression. through the activation of nuclear factor- B (NF- B), acti- Northern blot analysis. Blots containing 20 ␮g total RNA per lane isolated vator protein 1 (AP-1), and possibly other DNA binding from indicated cells were hybridized with 32P-labeled cDNAs encoding mouse proteins, regulates the expression of diverse immune and plasminogen activator inhibitor-1 (PAI-1), adipocyte complement-related pro- inflammatory response genes in many cell
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