Diabetes Publish Ahead of Print, published online April 28, 2008 PPARβ/δ prevents NF-κB activation in adipocytes Activation of Peroxisome Proliferator-Activated Receptor β/δ (PPARβ/δ) Inhibits LPS-induced Cytokine Production in Adipocytes by Lowering NF-κB Activity via ERK1/2 Ricardo Rodríguez-Calvo1, Lucía Serrano1, Teresa Coll1, Norman Moullan2, Rosa M. Sánchez1, Manuel Merlos1, Xavier Palomer1, Juan C. Laguna1, Liliane Michalik2, Walter Wahli2 and Manuel Vázquez-Carrera1. 1Pharmacology Unit, Department of Pharmacology and Therapeutic Chemistry, Faculty of Pharmacy, University of Barcelona, IBUB (Institut de Biomedicina de la UB), and CIBERDEM-Instituto de Salud Carlos III, Diagonal 643, E-08028 Barcelona, Spain and 2Center for Integrative Genomics, National Research Center Frontiers in Genetics, University of Lausanne, CH-1015 Lausanne, Switzerland. Corresponding author: Manuel Vázquez-Carrera Unitat de Farmacologia. Facultat de Farmàcia. Diagonal 643. E-08028 Barcelona. Spain E-mail: [email protected] Received for publication 07 February 2008 and accepted in revised form 21 April 2008. Copyright American Diabetes Association, Inc., 2008 PPARβ/δ prevents NF-κB activation in adipocytes Objective: Chronic activation of the nuclear factor (NF)-κB in white adipose tissue leads to increased production of pro-inflammatory cytokines, which are involved in the development of insulin resistance. It is presently unknown whether Peroxisome Proliferator-Activated Receptor (PPAR)β/δ activation prevents inflammation in adipocytes. Research Design and Methods and Results: Firstly, we examined whether the PPARβ/δ agonist GW501516 prevents LPS-induced cytokine production in differentiated 3T3-L1 adipocytes. Treatment with GW501516 blocked LPS-induced IL-6 expression and secretion by adipocytes and the subsequent activation of the STAT3-SOCS3 pathway. This effect was associated with the capacity of GW501516 to impede LPS- induced NF-κB activation. Secondly, in in vivo studies, white adipose tissue from Zucker Diabetic Fatty (ZDF) rats, compared to that of lean rats, showed reduced PPARβ/δ expression and PPAR DNA-binding activity, which was accompanied by enhanced IL-6 expression and NF-κB DNA-binding activity. Furthermore, IL-6 expression and NF-κB DNA-binding activity was higher in white adipose tissue from PPARβ/δ-null mice than in wild-type mice. Since mitogen-activated protein kinase (MAPK)–extracellular signal– related kinase (ERK)1/2 (MEK1/2) is involved in LPS-induced NF-κB activation in adipocytes, we explored whether PPARβ/δ prevented NF-κB activation by inhibiting this pathway. Interestingly, GW501516 prevented ERK1/2-phosphorylation by LPS. Further, white adipose tissue from animal showing constitutively increased NF-κB activity, such as ZDF rats and PPARβ/δ-null mice, also showed enhanced phospho-ERK1/2 levels. Conclusions: These findings indicate that activation of PPARβ/δ inhibits enhanced cytokine production in adipocytes by preventing NF-κB activation via ERK1/2, an effect that may contribute to prevent insulin resistance. 2 PPARβ/δ prevents NF-κB activation in adipocytes ccumulating evidence implicates a and MCP-1). Of note, NF-κB activation by low-grade chronic systemic LPS requires mitogen-activated protein inflammatory response to nutrient kinase (MAPK)–extracellular signal– A excess as a key mechanism that links related kinase (ERK)1/2 (MEK1/2) obesity to metabolic disorders, including activation, since inhibition of this pathway insulin resistance and cardiovascular reduces LPS-induced cytokine production disease (1). Thus, models of diet-induced in adipocytes (9). and genetic obesity show increased Recent evidence suggests that adipose tissue expression and content of inflammatory processes induced by pro-inflammatory cytokines (such as obesity and high-fat diet cause systemic tumor necrosis factor α [TNFα], insulin resistance via a mechanism interleukin [IL] 1, monocyte chemo- involving TLR4 (10). For instance, attractant protein-1 [MCP-1] and IL-6) (2- saturated free fatty acids (FFA) activate 4). Of these cytokines, IL-6 correlates TLR4-mediated inflammatory signaling in most strongly with insulin resistance and adipocytes and macrophages and this type 2 diabetes (5-7); its plasma levels effect is blunted in the absence of this are increased 2-3 fold in patients with receptor (10). These observations obesity and type 2 diabetes compared indicate that enhanced adipose tissue with lean control subjects (6). At the lipolysis observed in insulin-resistant cellular level, insulin resistance and states may release the endogenous enhanced expression of these cytokines ligand for TLR4 to induce inflammation by adipose tissue during obesity, and also (11). In addition, it has been under a high-fat diet have been linked to demonstrated that high-fat diets augment activation of the pro-inflammatory plasma LPS to a concentration sufficient transcription factor NF-κB (4). This to increase body weight, fasting glycemia nuclear factor is activated by surface and inflammation (12). Furthermore, LPS proteins that recognize foreign receptor-deleted mice (CD14 mutants) substances, the so-called pattern are hypersensitive to insulin, and the recognition receptors, such as toll-like development of insulin resistance, obesity receptor-4 (TLR4). This receptor is and diabetes in this animal model is expressed on virtually all human cells and delayed in response to a high-fat diet binds a wide spectrum of exogenous and (12). endogenous ligands, including bacterial In recent years Peroxisome LPS (8). In the presence of LPS, the Proliferator-Activated Receptor β/δ TLR4 complex (including CD-14 and an (PPARβ/δ) activation has been proposed accessory protein, MD-2), recruits the as a potential treatment for insulin adaptor protein, myeloid differentiation resistance (13). PPARs are members of factor-88 (MyD88), which in turn recruits the nuclear receptor superfamily of interleukin-1 receptor-associated kinase ligand-inducible transcription factors. (IRAK), leading to NF-κB activation and They form heterodimers with retinoid X enhanced expression of several receptors (RXRs) and bind to consensus inflammatory mediators (including IL-6 DNA sites composed of direct repeats PPARβ/δ prevents NF-κB activation in adipocytes (DRs) of hexameric DNA sequences activity, and enhanced IL-6 expression separated by 1 bp (DR1) (14). Ligand and NF-κB DNA-binding activity in white binding induces a conformational change adipose tissue. Likewise, IL-6 expression in PPAR-RXR complexes, thereby and NF-κB DNA-binding activity was releasing co-repressors in exchange for higher in this tissue in PPARβ/δ-null mice co-activators, which leads to the than in wild-type mice. Since MAPK– recruitment of the basal transcription ERK1/2 (MEK1/2) is involved in NF-κB machinery and enhanced gene activation in adipocytes (9), we explored expression. In addition, PPARs may whether PPARβ/δ blocked NF-κB suppress inflammation through diverse activation by inhibiting this pathway. In mechanisms, such as reduced release of agreement with this possibility, inflammatory factors or stabilization of GW501516 prevented ERK1/2- repressive complexes at inflammatory phosphorylation by LPS. In contrast, gene promoters (15-18). Of the three animal models showing increased NF-κB PPAR isotypes found in mammals, activity in white adipose tissue, the ZDF PPARα (NR1C1) (19) and rat and the PPARβ/δ-null mice, showed PPARγ(NR1C3) are the targets for enhanced phospho-ERK1/2 levels. hypolipidemic (fibrates) and anti-diabetic Overall, on the basis of our findings, we (thiazolidinediones) drugs, respectively. propose that PPARβ/δ activation be Finally, activation of the third isotype, considered a molecular target to prevent PPARβ/δ (NR1C2), by high-affinity inflammation of adipose tissue and the ligands (including GW501516) enhances metabolic alterations associated with this fatty acid catabolism in adipose tissue process, such as insulin resistance. and skeletal muscle, thereby delaying weight gain (for review see (13)). RESEARCH DESIGN AND METHODS However, there is no information Materials. The PPARβ/δ ligand available on whether PPARβ/δ ligands GW501516 was from Biomol Research prevent inflammation in adipocytes. Here Labs Inc. (Plymouth Meeting, PA). Other we examined whether PPARβ/δ activation chemicals were from Sigma (St. Louis, by GW501516 prevents LPS-induced MO). inflammation in adipocytes. We found that this drug prevented LPS-induced IL-6 Cell culture. 3T3-L1 preadipocytes expression and secretion by adipocytes. (ATCC) were grown to confluence in This effect was associated with the Dulbecco’s Modified Eagle’s Medium capacity of the PPARβ/δ ligand to prevent (DMEM) supplemented with 10% bovine LPS-induced NF-κB activation. calf serum. Two 2 days after confluence Consistent with the role of PPARβ/δ in (day 0), differentiation of the 3T3-L1 cells blocking NF-κB-induced IL-6 expression, was induced in DMEM containing 10% a genetic model of obesity and insulin fetal bovine serum, resistance, the ZDF rat, showed reduced methylisobutylxanthine (500 µM), PPARβ/δ expression and DNA-binding dexamethasone (0.25 µM), and insulin 2 PPARβ/δ prevents NF-κB activation in adipocytes (10 µg/ml) for 48 h. The cells were then The generation of PPARβ/δ null mice incubated in 10% FBS/DMEM with insulin was described previously (20). for 8 days. Medium was changed every 2 Measurements of mRNA. Levels of days. Fat droplets were observed in more mRNA were assessed by the reverse than 90% of cells after day