Effect of Peroxisome Proliferator-activated Receptor-α Ligands in the Interaction Between Adipocytes and Macrophages in Obese Adipose Tissue

Authors

  • Takuya Toyoda,

    1. Department of Molecular Medicine and Metabolism, Medical Research Institute, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan
    2. Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstitution of Tooth and Bone, Medical Research Institute, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan
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  • Yasutomi Kamei,

    1. Department of Molecular Medicine and Metabolism, Medical Research Institute, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan
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  • Hirotsugu Kato,

    1. Department of Molecular Medicine and Metabolism, Medical Research Institute, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan
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  • Satoshi Sugita,

    1. Department of Molecular Medicine and Metabolism, Medical Research Institute, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan
    2. Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstitution of Tooth and Bone, Medical Research Institute, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan
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  • Motohiro Takeya,

    1. Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, Honjo, Kumamoto, Japan
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  • Takayoshi Suganami,

    1. Department of Molecular Medicine and Metabolism, Medical Research Institute, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan
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  • Yoshihiro Ogawa

    Corresponding author
    1. Department of Molecular Medicine and Metabolism, Medical Research Institute, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan
    2. Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstitution of Tooth and Bone, Medical Research Institute, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan
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(ogawa.mmm@mri.tmd.ac.jp)

Abstract

Objective: This study was designed to examine the effect of peroxisome proliferator-activated receptor-α (PPAR-α) ligands on the inflammatory changes induced by the interaction between adipocytes and macrophages in obese adipose tissue.

Methods and Procedures: PPAR-α ligands (Wy-14,643 and fenofibrate) were added to 3T3-L1 adipocytes, RAW264 macrophages, or co-culture of 3T3-L1 adipocytes and RAW264 macrophages in vitro, and monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) mRNA expression and secretion were examined. PPAR-α ligands were administered to genetically obese ob/ob mice for 2 weeks. Moreover, the effect of PPAR-α ligands was also evaluated in the adipose tissue explants and peritoneal macrophages obtained from PPAR-α-deficient mice.

Results: In the co-culture of 3T3-L1 adipocytes and RAW264 macrophages, PPAR-α ligands reduced MCP-1 and TNF-α mRNA expression and secretion in vitro relative to vehicle-treated group. The anti-inflammatory effect of Wy-14,643 was observed in adipocytes treated with macrophage-conditioned media or mouse recombinant TNF-α and in macrophages treated with adipocyte-conditioned media or palmitate. Systemic administration of PPAR-α ligands inhibited the inflammatory changes in adipose tissue from ob/ob mice. Wy-14,643 also exerted an anti-inflammatory effect in the adipose tissue explants but not in peritoneal macrophages obtained from PPAR-α-deficient mice.

Discussion: This study provides evidence for the anti-inflammatory effect of PPAR-α ligands in the interaction between adipocytes and macrophages in obese adipose tissue, thereby improving the dysregulation of adipocytokine production and obesity-related metabolic syndrome.

Introduction

Obesity is often accompanied by several lifestyle-related disorders, such as diabetes, hyperlipidemia, and hypertension, thereby conferring a higher risk of atherosclerosis (1,2). There is considerable evidence that obesity is a state of chronic low-grade inflammation (3,4). Obese adipose tissue exhibits the inflammatory changes characterized by the upregulation of proinflammatory adipocytokines, such as monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) and downregulation of an anti-inflammatory adipocytokine adiponectin (5,6,7,8,9). Weisberg et al. and Xu et al. independently reported increased infiltration of macrophages in obese adipose tissue and suggested that they were involved in the inflammatory changes in adipose tissue (10,11). These findings also led us to speculate that the dysregulation of adipocytokine production in obese adipose tissue results from the interaction between adipocytes and macrophages.

Recently, we have developed an in vitro co-culture system composed of adipocytes and macrophages and demonstrated that a paracrine loop involving saturated fatty acids and TNF-α, which are derived from adipocytes and macrophages, respectively, establishes a vicious cycle that aggravates the inflammatory changes (12). Furthermore, a previous study showed that macrophages in obese adipose tissue are localized to dead adipocytes, where they fuse to scavenge the residual lipid droplet to form multinucleate giant cells (13). These findings are reminiscent of the inflammatory changes found in atherosclerotic vascular walls (termed vascular remodeling), which are characterized by complex interactions among vascular endothelial and smooth muscle cells and monocyte-derived macrophages (14).

The peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily. Three receptor subtypes (PPAR-α, γ, δ) are expressed in mammals including humans (15,16,17,18). PPAR-α is expressed mainly in tissues and organs in which fatty acid oxidation is active, such as liver, kidney, and skeletal muscle (19,20,21,22), and its expression is also observed in adipose tissue (23,24). Previous studies including those with PPAR-α-deficient mice showed that PPAR-α-regulated genes participate in the regulation of not only lipid metabolism but also inflammation (25,26,27,28). For instance, GW7647, a PPAR-α ligand, has strongly inhibited atherosclerosis and attenuated macrophage-related inflammation in atherosclerotic foam cells (29). Given the anti-atherogenic effect of PPAR-α ligands, it is tempting to speculate that PPAR-α ligands are capable of reducing the inflammatory changes in obese adipose tissue, thereby improving the obesity-related metabolic syndrome. Tsuchida et al. previously reported that PPAR-α activation reduces obesity-related adipose tissue inflammation (23). However, how PPAR-α ligands inhibit the inflammatory changes induced by the interaction between adipocytes and macrophages has not been fully elucidated.

In this study, using an in vitro co-culture of adipocytes and macrophages, we examined the effect of PPAR-α ligands on the inflammatory changes induced by the interaction between adipocytes and macrophages. We also studied the in vivo effect of PPAR-α ligands on the inflammatory changes in adipose tissue from genetically obese ob/ob mice. Moreover, we studied the anti-inflammatory effect of PPAR-α ligands using the adipose tissue explants and peritoneal macrophages obtained from PPAR-α-deficient mice.

Methods and Procedures

Reagents

Wy-14,643 was purchased from Cayman Chemical (Ann Arbor, MI). Fenofibrate was purchased from Sigma-Aldrich (St Louis, MO). Mouse recombinant TNF-α was purchased from R&D Systems (Minneapolis, MN). Palmitate was purchased from Sigma-Aldrich, solubilized in ethanol, conjugated with fatty acids- and immunoglobulin-free bovine serum albumin (Sigma-Aldrich) in low serum medium as described (12). Preparation and characterization of anti-F4/80 antibody was reported previously (30). All other reagents were purchased from Sigma-Aldrich.

Animals

Five-week-old male C57B/6 ob/ob mice and wild-type mice were obtained from Charles River Japan (Tsukuba, Ibaraki, Japan). The PPAR-α-deficient mice (B6.129S4-Pparatm1Gonz) were purchased from Taconic (Germantown, NY). The animals were housed in individual cages in a temperature-, humidity-, and light-controlled room (12-h light and 12-h dark cycle) and allowed free access to water and standard chow (Oriental MF; 362 kcal/100 g, 5.4% energy as fat) (Oriental Yeast, Tokyo, Japan). All animal experiments were conducted in accordance with the guidelines of Tokyo Medical and Dental University Committee on Animal Research (No. 0060028).

Cell culture

3T3-L1 preadipocytes (American Type Culture Collection, Manassas, VA) and RAW264 macrophages (RIKEN BioResource Center, Tsukuba, Japan) were maintained in Dulbecco's modified Eagle's medium (Nacalai Tesque, Kyoto, Japan) containing 10% fetal bovine serum (BioWest, Miami, FL). 3T3-L1 preadipocytes were differentiated to mature adipocytes as described (12) and used as differentiated adipocytes 8 days after the induction of differentiation. Serum-starved 3T3-L1 adipocytes were treated with Wy-14,643 at doses of 10 and 100 μmol/l for 4 h, and thereafter incubated with the conditioned media from RAW264 macrophages or the media containing 1 ng/ml mouse recombinant TNF-α in the presence of Wy-14,643. Cells were harvested after 24 h of incubation. Serum-starved RAW264 macrophages were treated with Wy-14,643 at doses of 10 and 100 μmol/l for 4 h, and thereafter incubated with the conditioned media from 3T3-L1 adipocytes or the media containing 200 μmol/l palmitate in the presence of Wy-14,643.

Mouse peritoneal macrophages were isolated from 6- to 10-week-old male C57BL/6N mice or PPAR-α-deficient mice intraperitoneally injected with 3% thioglycollate medium (Nihon Seiyaku, Tokyo, Japan). Cells were plated onto 100-mm dishes in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and allowed to attach for 4 h. Then 1.0 × 106 attached cells were plated onto 60-mm dishes in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Serum-starved peritoneal macrophages were treated with Wy-14,643 at doses of 100 μmol/l for 4 h, and thereafter incubated with the media containing 500 μmol/l palmitate in the presence of Wy-14,643. Cells were harvested after 24 h of incubation.

Co-culture of adipocytes and macrophages

Co-culture of adipocytes and macrophages was performed as described (12). In brief, serum-starved differentiated 3T3-L1 adipocytes (∼1.5 × 106 cells) were cultured in 60-mm dish and RAW264 macrophages (1.0 × 105 cells) were plated onto 3T3-L1 adipocytes. Cells were cultured with contact each other in the presence or the absence of 10 and 100 μmol/l Wy-14,643 or fenofibrate for 24 h and harvested. As a control, adipocytes and macrophages, the numbers of which were equal to those used in the co-culture, were cultured separately and mixed after harvest.

Quantitative real-time PCR

Total RNA was extracted from cultured cells or mouse epididymal white adipose tissue (WAT) using the acid guanidium-phenol-chloroform method. Quantitative real-time PCR was performed with an ABI Prism 7000 Sequence Detection System using SYBR Green PCR Master Mix Reagent Kit (Applied Biosystems, Foster City, CA) as described (31). Primers used in this study were reported elsewhere (12). Levels of mRNA were normalized to those of 36B4 mRNA.

Measurement of MCP-1 and TNF-α levels in culture media

MCP-1 and TNF-α levels in culture supernatants were determined using commercially available enzyme-linked immunosorbent assay kits (R&D systems, Minneapolis, MN).

Lipolysis assay

Differentiated 3T3-L1 was co-cultured with RAW264 macrophages in the medium containing 1% fatty acids- and immunoglobulin-free bovine serum albumin for 24 h. The concentration of free fatty acids (FFAs) in the medium was measured using an acyl-CoA oxidase-based colorimetric assay kit (NEFA-C; WAKO Pure Chemicals, Osaka, Japan) (12).

Administration of PPAR-α ligands to ob/ob mice

Seven-week-old male ob/ob mice and wild-type mice were orally treated with Wy-14,643 or fenofibrate for 2 weeks. Control ob/ob mice and wild-type mice were treated with the vehicle (0.5% methylcellulose). The amount of Wy-14,643 administered was 20 mg/kg of body weight per day. Fenofibrate was administered at 300 mg/kg of body weight per day. In the pair-feeding experiment, a group of ob/ob mice was provided with the same amount of food as that consumed on the previous day by those treated with fenofibrate. Daily food intake and body weight were measured during the experiments. After a 2-week administration, blood parameters were measured, and epididymal WAT was dissected to be used for RNA analysis and histological examination.

Histological analysis and morphometry

The epididymal WAT was fixed with neutral-buffered formalin and embedded in paraffin. Five-micrometer sections were stained with hematoxylin and eosin and studied under ×200 magnification to compare the adipocyte cell size. The adipocyte diameter was measured using the software Win Roof (Mitani, Chiba, Japan). For macrophage staining, de-waxed 5-μm sections were treated with 0.05 mol/l proteinase K in 0.015 mol/l Tris-HCl, pH7.5 (Dako, Kyoto, Japan) for 5 min for antigen retrieval followed by 0.3% hydrogen peroxide in methanol to inactivate endogenous peroxidases. After treatment with normal goat serum to reduce nonspecific staining, sections were incubated overnight with an anti-mouse macrophage antibody, F4/80 (30). Normal rat serum was used as negative control. Simple stain mouse MAX-PO (Rat) (Nichirei, Tokyo, Japan) was used as a peroxidase-conjugated second antibody. Histochemical reaction was performed using 3,3′-diaminobenzidine (Dojindo, Kumamoto, Japan) as substrate. Sections were counterstained with hematoxylin. The number of F4/80-positive cells was counted in a blinded fashion under the microscope with a ×400 objective and an eyepiece grid indicating a 0.0625 mm2 field of view. More than 50 serial fields were examined, and the data were expressed as cells per square millimeter of adipose tissue examined.

Adipose tissue explants

The epididymal WAT was dissected out from male ob/ob mice at the age of 8–12 weeks and cut into small pieces. Incubation of adipose tissue fragments (each ∼0.2 g) was performed in six-well culture plates. Sterile condition was maintained during the removal, mincing and allocation of adipose tissue fragments, and the incubation with antibiotics. Each well contained 2 ml of Dulbecco's modified Eagle's medium containing 0.5% fetal bovine serum, to which the WAT fragments were added. After 12 h of quiescent incubation, the adipose tissue explants were treated with Wy-14,643 at a dose of 100 μmol/l for 12 h and subjected to RNA analysis. The stromal vascular fraction (SVF) from the adipose tissue explants were prepared by collagenase digestion (12).

Statistical analysis

Data are expressed as the mean ± s.e. Statistical analysis was performed using Student's t-test or ANOVA followed by Scheffe's test. P < 0.05 was considered to be statistically significant.

Results

Effect of PPAR-α ligands in the co-culture of adipocytes and macrophages

Co-culture of 3T3-L1 adipocytes and RAW264 macrophages resulted in marked increases of mRNA expression and secretion of proinflammatory cytokines, such as MCP-1 and TNF-α relative to the control culture (P < 0.01) (Figure 1a,b). It also increased significantly FFA release from adipocytes (P < 0.01) (Figure 1c). These data are consistent with our previous study (12).

Figure 1.

Anti-inflammatory effect of PPAR-α ligands in the co-culture system. (a) Effect of PPAR-α ligands on the co-culture-induced MCP-1 and TNF-α mRNA expression. (b) Effect of PPAR-α ligands on the co-culture-induced MCP-1 and TNF-α secretion in the culture media. (c) Effect of PPAR-α ligands on the co-culture-induced increase in FFA release. veh, vehicle; Wy, Wy-14,643 at doses of 10, 100 μmol/l; feno, fenofibrate at doses of 10, 100 μmol/l. **P < 0.01 vs. co-culture (−),P < 0.05, ††P < 0.01 vs. co-culture (+) alone. n = 4.

Using the above co-culture system, we examined the anti-inflammatory effect of PPAR-α ligands, Wy-14,643, and fenofibrate. In the control culture, MCP-1 and TNF-α mRNA expression were significantly reduced by PPAR-α ligands (MCP-1, P < 0.01, TNF-α, P < 0.05 for fenofibrate) (Figure 1a). The co-culture-induced increases in MCP-1 and TNF-α mRNA expression and secretion were significantly decreased by both Wy-14,643 and fenofibrate in a dose-dependent manner (P < 0.01) (Figure 1a,b). However, there was no significant change in adipocyte lipolysis when treated with either Wy-14,643 or fenofibrate (Figure 1c).

Effect of Wy-14,643 in adipocytes and macrophages

To examine whether PPAR-α ligands act directly on adipocytes, we examined the effect of Wy-14,643 on MCP-1 mRNA expression in adipocytes. Wy-14,643 significantly reduced basal MCP-1 mRNA expression in 3T3-L1 adipocytes in a dose-dependent manner (P < 0.01) (Figure 2a). Treatment of 3T3-L1 adipocytes with the conditioned media from RAW264 macrophages resulted in a significant induction of MCP-1 mRNA expression (P < 0.01). The increase in MCP-1 mRNA expression was inhibited significantly by Wy-14,643 in a dose-dependent manner (P < 0.01) (Figure 2a). Because TNF-α is a major paracrine mediator of inflammation in adipocytes in the co-culture system (12), we also examined the effect of Wy-14,643 on the TNF-α-induced MCP-1 expression. Treatment with mouse recombinant TNF-α resulted in a significant induction of MCP-1 mRNA expression and secretion (P < 0.01). Treatment with Wy-14,643 suppressed the TNF-α-induced MCP-1 mRNA expression and secretion in a dose-dependent manner (P < 0.01) (Figure 2b,c).

Figure 2.

Anti-inflammatory effect of Wy-14,643 in adipocytes. (a) Effect of Wy-14,643 on MCP-1 mRNA expression in 3T3-L1 adipocytes treated with macrophage-conditioned media. (b, c) Effect of Wy-14,643 on MCP-1 mRNA expression (b) and secretion (c) in 3T3-L1 adipocytes treated with mouse recombinant TNF-α (1 ng/ml). Wy, Wy-14,643 at doses of 10, 100 μmol/l; Mφ Med, RAW264 macrophage-conditioned media; veh, vehicle. **P < 0.01 vs. Mφ Med (−) alone or TNF-α (−) alone, P < 0.05, ††P < 0.01 vs. Mφ Med (+) alone or TNF-α (+) alone. n = 4.

We also examined the direct effect of Wy-14,643 on TNF-α mRNA expression in macrophages. RAW264 macrophages treated with the conditioned media from 3T3-L1 adipocytes showed a significant induction of TNF-α mRNA expression (P < 0.01), which was inhibited by Wy-14,643 in a dose-dependent manner (P < 0.01) (Figure 3a). Because saturated fatty acids are an important adipocyte-derived paracrine mediator of inflammation in macrophages in the co-culture system (12), we examined the effect of Wy-14,643 ligand on the TNF-α mRNA expression in macrophages treated with palmitate, a major saturated fatty acids released from 3T3-L1 adipocytes (32). Treatment with palmitate resulted in a significant induction of TNF-α mRNA expression and secretion in RAW264 macrophages (P < 0.01). In this study, Wy-14,643 also suppressed dose-dependently the palmitate-induced increase in TNF-α mRNA expression and secretion (P < 0.01) (Figure 3b,c).

Figure 3.

Anti-inflammatory effect of Wy-14,643 in macrophages. (a) Effect of Wy-14,643 on TNF-α mRNA expression in RAW264 macrophages treated with adipocyte-conditioned media. (b, c) Effect of Wy-14,643 on TNF-α mRNA expression (b) and secretion (c) in RAW264 macrophages treated with palmitate (200 μmol/l). Wy, Wy-14,643 at doses of 10, 100 μmol/l; L1 Med, 3T3-L1 adipocyte-conditioned media; veh, vehicle. **P < 0.01 vs. L1 Med (−) alone or palmitate(−) alone, P < 0.05, ††P < 0.01 vs. L1 Med (+) alone or palmitate (+) alone. n = 4.

Effect of PPAR-α ligands in ob/ob mice

To investigate the anti-inflammatory effect of PPAR-α ligands on obese adipose tissue in vivo, we examined the effect of systemic administration of PPAR-α ligands on the inflammatory changes in adipose tissue from genetically obese ob/ob mice. Table 1 summarizes the changes in the metabolic parameters observed in ob/ob mice and age-matched wild-type mice treated with PPAR-α ligands (Wy-14,643 or fenofibrate) or vehicle for 2 weeks. In this study, ob/ob mice that received systemic administration of PPAR-α ligands exhibited a slight decrease in body weight relative to vehicle-treated group. Blood glucose and serum concentrations of FFA and triglyceride were reduced significantly by administering PPAR-α ligands in ob/ob mice (P < 0.01) (Table 1). The food intake was decreased in PPAR-α ligand-treated mice. No adverse effect on health was noted throughout this study.

Table 1.  Metabolic parameters in ob/ob mice treated with PPAR-α ligands
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The liver weight was significantly increased in mice treated with PPAR-α ligands (P < 0.01). In this study, the weights of epididymal and subcutaneous WATs were decreased significantly in ob/ob mice treated with PPAR-α ligands (epididymal WAT, P < 0.05; subcutaneous WAT, P < 0.01 for fenofibrate) (Table 1). Histological examination of the epididymal WAT revealed no appreciable difference between PPAR-α ligand- and vehicle-treated groups. However, the size of mature adipocytes was significantly smaller in PPAR-α ligand-treated groups than in the vehicle-treated group (P < 0.01) (Figure 4a,b). There was no significant difference in the number of F4/80-positive macrophages in adipose tissue between PPAR-α ligand- and vehicle-treated ob/ob mice (Figure 4c).

Figure 4.

Anti-inflammatory effect of PPAR-α ligands in ob/ob mice. (a) Hematoxylin and eosin (HE) staining and immunohistochemical staining using anti-F4/80 antibody (F4/80) of the epididymal WAT. Original magnification, ×200. Bar = 100 μm. (b) Adipocyte size of epididymal WAT. (c) Number of F4/80-positive cells in epididymal WAT. n.d., not detected. n = 4. (d) Expression of MCP-1 and TNF-α mRNA in the epididymal WAT. WT, wild-type mice; veh, vehicle-treated mice; Wy, Wy-14,643-treated mice at 20 mg/kg of body weight per day; feno, fenofibrate-treated mice at 300 mg/kg of body weight per day. **P < 0.01 vs. vehicle-treated wild-type mice, P < 0.05, ††P < 0.01 vs. vehicle-treated ob/ob mice.n = 8–16.

Quantitative real-time PCR analysis revealed that expression of MCP-1 and TNF-α mRNA was increased significantly in the epididymal WAT from ob/ob mice relative to age-matched wild-type mice (P < 0.01) (Figure 4d). Systemic administration of PPAR-α ligands significantly reduced MCP-1 and TNF-α mRNA expression in adipose tissue from ob/ob mice (Figure 4d) (MCP-1, P < 0.01, TNF-α, P < 0.05 for Wy-14,643 and P < 0.01 for fenofibrate). In the pair-feeding experiment, significant decreases in body weight and the liver and the subcutaneous WAT weights were observed in pair-fed vehicle-treated ob/ob mice relative to ad lib-fed vehicle-treated ob/ob mice (Table 2). Histological examination of the epididymal WAT showed no appreciable difference between fenofibrate-treated and vehicle-treated groups in the pair-feeding experiment (data not shown). Although there was no significant difference in MCP-1 and TNF-α mRNA levels in the epididymal WAT between ad lib-fed vehicle-treated and pair-fed vehicle-treated ob/ob mice, they were reduced significantly in pair-fed fenofibrate-treated ob/ob mice relative to pair-fed vehicle-treated ob/ob mice (P < 0.01) (Figure 5).

Table 2.  Metabolic parameters in pair-fed fenofibrate-treated ob/ob mice
inline image
Figure 5.

Anti-inflammatory effect of fenofibrate in pair-fed ob/ob mice. Effect of fenofibrate on MCP-1 and TNF-α mRNA expression in the epididymal WAT from pair-fed ob/ob mice. veh, vehicle-treated ob/ob mice; feno, fenofibrate-treated ob/ob mice at 300 mg/kg of body weight per day; n.s., not significant. *P < 0.05 vs. ad lib-fed vehicle treatedob/ob mice, ††P < 0.01 vs. pair-fed vehicle treated ob/ob mice. n = 8.

Effect of Wy-14,643 on the adipose tissue explants obtained from ob/ob mice

To elucidate whether PPAR-α ligands affect directly on adipose tissue in vivo, we examined the effect of Wy-14,643 on MCP-1 and TNF-α expression levels in the adipose tissue explants ex vivo, which were obtained from ob/ob mice as described in Research Methods and Procedures. Treatment with Wy-14,643 significantly suppressed MCP-1 and TNF-α mRNA expression relative to vehicle-treated group (MCP-1, P < 0.05 and TNF-α, P < 0.01) (Figure 6a). Wy-14,643 also reduced significantly TNF-α mRNA expression in SVF obtained from the adipose tissue explants (P < 0.05) (Figure 6b).

Figure 6.

Anti-inflammatory effect of Wy-14,643 on the adipose tissue explants from ob/ob mice. (a) Effect of Wy-14,643 on MCP-1 and TNF-α mRNA expression in the adipose tissue explants from ob/ob mice. (b) Effect of Wy-14,643 on TNF-α mRNA expression in stromal vascular fraction (SVF) separated from ob/ob adipose tissue explants. Wy, Wy-14,643 at a dose of 100 μmol/l. *P < 0.05, **P < 0.01 vs. Wy (−). n = 6.

Effect of Wy-14,643 on the adipose tissue explants and peritoneal macrophages obtained from PPAR-α-deficient mice

To evaluate whether PPAR-α ligands act on adipose tissue via PPAR-α, we also examined the effect of Wy-14,643 on the adipose tissue explants and peritoneal macrophages obtained from non-obese PPAR-α-deficient mice. Significant elevation of MCP-1 mRNA expression level was observed in the adipose tissue explants from PPAR-α-deficient mice relative to those from wild-type mice (P < 0.01) (Figure 7a). Treatment of the adipose tissue explants from wild-type mice with recombinant TNF-α (1 ng/ml) as well as 3T3-L1 adipocytes in Figure 2b resulted in significant induction of MCP-1 mRNA expression (P < 0.01). In this study, the increase in MCP-1 mRNA expression was inhibited significantly by Wy-14,643 (P < 0.01) (Figure 7a). Treatment with TNF-α induced MCP-1 mRNA expression significantly in the adipose tissue explants from PPAR-α-deficient mice (P < 0.01). TNF-α-induced MCP-1 mRNA expression in PPAR-α-deficient mice adipose tissue explants was also suppressed by Wy-14,643 (P < 0.05) (Figure 7a). As observed in RAW264 macrophages (Figure 3b), treatment with palmitate resulted in significant induction of TNF-α mRNA expression in peritoneal macrophages from wild-type mice (P < 0.05) and PPAR-α-deficient mice (P < 0.01) (Figure 7b). The palmitate-induced TNF-α mRNA expression in peritoneal macrophages from wild-type mice was reduced in the presence of Wy-14,643 (P < 0.05). However, reduction of TNF-α mRNA expression was not observed in palmitate-treated peritoneal macrophages from PPAR-α-deficient mice (Figure 7b).

Figure 7.

Effect of Wy-14,643 on the adipose tissue explants and peritoneal macrophages obtained from PPAR-α-deficient mice. (a) Effect of Wy-14,643 on MCP-1 mRNA expression in the adipose tissue explants from PPAR-α-deficient mice. (b) Effect of Wy-14,643 on TNF-α mRNA expression in peritoneal macrophages from PPAR-α-deficient mice. Wy, Wy-14,643 at a dose of 100 μmol/l. *P < 0.05, **P < 0.01. n = 4–6.

Discussion

There is considerable evidence that macrophage infiltration is increased in obese adipose tissue (10,11,33,34,35), which is reminiscent of the inflammatory changes found in atherosclerotic vascular walls (or vascular remodeling) characterized by complex interactions among vascular endothelial cells, smooth muscle cells, monocyte-derived infiltrated macrophages, etc. (14). Given the antiatherogenic effect of PPAR-α ligands (29), it is likely that PPAR-α ligands are capable of reducing the inflammatory changes in obese adipose tissue, thereby improving obesity-related metabolic syndrome. Recently, we have developed an in vitro co-culture system composed of 3T3-L1 adipocytes and RAW264 macrophages and demonstrated that co-culture of adipocytes and macrophages may provide the unique in vitro experimental system with which to assess anti-inflammatory drugs in the interaction between adipocytes and macrophages in obese adipose tissue (12). This study was designed to elucidate how PPAR-α ligands exert the anti-inflammatory effect in the interaction between adipocytes and macrophages.

Using the co-culture of adipocytes and macrophages, we demonstrated that PPAR-α ligands attenuate the co-culture-induced increase in proinflammatory adipocytokine MCP-1 and TNF-α mRNA expression and secretion relative to vehicle-treated group. These observations indicate that PPAR-α ligands exert the anti-inflammatory effect in the interaction between adipocytes and macrophages in vitro. Because PPAR-α is expressed in both mature adipocytes and SVF from obese adipose tissue (23), it is important to know whether PPAR-α ligands can act directly on adipocytes or macrophages, or both. The data of this study showed that Wy-14,643 reduces MCP-1 mRNA expression in adipocytes treated with macrophage-conditioned media or those treated with recombinant TNF-α. It also reduced significantly TNF-α mRNA expression in macrophages treated with adipocyte-conditioned media or those treated with palmitate. These observations, taken together, suggest that PPAR-α ligands act directly on both adipocytes and macrophages.

In this study, administration of PPAR-α ligands inhibited MCP-1 and TNF-α mRNA expression in adipose tissue from ob/ob mice. Although food intake of ob/ob mice treated with PPAR-α ligands was reduced significantly relative to vehicle-treated group as reported previously (36), pair-feeding ob/ob mice to the amount of food consumed by those treated with PPAR-α ligands did not improve MCP-1 and TNF-α mRNA expression in adipose tissue (Figure 5), suggesting that the anti-inflammatory effect of PPAR-α ligands is not due to the decreased food intake. This is consistent with a previous observation that PPAR-α ligands increase AdipoR1 and AdipoR2 mRNA expression independently of the decreased food intake (23). In this study, we observed no appreciable reduction in macrophage infiltration into adipose tissue from ob/ob mice treated with Wy-14,643 for 2 weeks (Figure 4c), suggesting that Wy-14,643 can suppress proinflammatory gene expression with no change in macrophage infiltration as it does in the co-culture experiments. In this regard, Tsuchida et al. previously reported that macrophage infiltration is inhibited in KKAy mice fed a high-fat diet, when they were treated with Wy-14,643 for 8 weeks (23). This may be due to the differences in the experimental protocols such as mouse strains (ob/ob mice vs. KKAy mice), the doses of Wy-14,643 administered, the treatment duration, and high-fat vs. standard diets.

We also found that expression of MCP-1 and TNF-α mRNAs is reduced significantly in the adipose tissue explants prepared from ob/ob mice when treated with Wy-14,643 (Figure 6a), thus supporting the direct effect of PPAR-α ligands on obese adipose tissue. Furthermore, Wy-14,643 inhibited TNF-α mRNA expression in SVF obtained from ob/ob adipose tissue explants (Figure 6b), suggesting that it acts directly on SVF (possibly macrophages) in the adipose tissue explants. In this study, we could not examine the direct effect of Wy-14,643 on mature adipocytes because the mature adipocyte fraction, when separated from the adipose tissue explants, might be contaminated to a large extent with SVF; it expressed a substantial amount of F4/80 mRNA level (data not shown).

A previous study showed that PPAR-α ligands, such as oleoylethanolamide and Wy-14,643, are able to promote lipolysis in isolated mouse adipocytes under basal conditions (37), which we reproduced in separate experiments (unpublished data). In this study, PPAR-α ligands did not affect the co-culture-induced increase in FFA release, although they suppressed TNF-α secretion (Figure 1b,c). This may be because PPAR-α ligands suppress the co-culture-induced TNF-α production, thereby reducing the otherwise increased adipocyte lipolysis. It is conceivable that systemic administration of PPAR-α ligands increases adipose lipolysis locally in adipose tissue from ob/ob mice. However, they might induce fatty acid oxidation in liver, thereby leading to the reduced level of serum FFAs (Table 1).

In the adipose tissue explants from non-obese PPAR-α-deficient mice, which may not be massively infiltrated with macrophages, the basal expression level of MCP-1 mRNA was higher than in those from wild-type mice, suggesting that PPAR-α exerts an anti-inflammatory effect in adipocytes. Interestingly, Wy-14,643 suppressed TNF-α-induced MCP-1 mRNA expression in adipose tissue from PPAR-α-deficient mice (Figure 7a). It has been known that Wy-14,643 at high concentrations (>100 μmol/l) can cross activate PPAR-γ (38). PPAR-γ is expressed in both adipocytes and macrophages, and its expression is much abundant in adipocytes (39). Thus, the effect of Wy-14,643 observed in the adipose tissue explants may be partially mediated by PPAR-γ expressed in adipocytes. Consistently, a PPAR-α ligand bezafibrate tended to elevate plasma adiponectin concentrations in PPAR-α-deficient mice and adiponectin secretion in the adipocyte cultured media from PPAR-α-deficient mice, which might be attributed to the partial PPAR-γ activation induced by bezafibrate (24).

The molecular mechanism for the anti-inflammatory effect of PPAR-α ligands has not been fully elucidated. Recently, we demonstrated that co-culture of adipocytes and macrophages results in the activation of the nuclear factor-κB (NF-κB) pathway, which is involved in the co-culture-induced inflammatory changes (40,41). Because PPAR-α regulates intracellular signaling pathways, such as the NF-κB and activator protein-1 pathways, negatively through direct protein-protein interaction (42,43,44,45), it is conceivable that the anti-inflammatory effect of PPAR-α ligands observed in this study is mediated at least in part through the inhibition of the NF-κB pathway.

PPAR-α is expressed abundantly in tissues with a high capacity of mitochondrial and peroxisomal fatty acid β-oxidation, such as liver, skeletal muscle, and kidney, which are responsible for most of the known metabolic effect of PPAR-α ligands (17,19,20,21,22). For instance, PPAR-α ligands increase fatty acid uptake and fatty acid oxidation, and improve very low density lipoprotein production in liver. PPAR-α is also expressed in vascular cells, such as endothelial cells, smooth muscle cells, and monocytes/macrophages (43,46,47,48), where it reduces vascular inflammatory changes and thus atherosclerosis. This study demonstrates that PPAR-α ligands are capable of improving the inflammatory changes in obese adipose tissue. It is, therefore, likely that improvement of the inflammatory changes in obese adipose tissue may contribute to the metabolic effect of PPAR-α ligands. Further investigations using mice with adipocyte-specific PPAR-α deficiency and those with macrophage-specific PPAR-α deficiency are required to elucidate how the anti-inflammatory effect of PPAR-α ligands in obese adipose tissue are involved in their metabolic effect.

Because our in vitro co-culture system reproduces the dysregulation of adipocytokine production in obese adipose tissue in vivo (12), it is useful to screen and evaluate chemicals that can reduce the inflammatory changes in obese adipose tissue (41). This study demonstrates that PPAR-α ligands exert an anti-inflammatory effect in the interaction between adipocytes and macrophages in vitro and that they also reduce the inflammatory changes in obese adipose tissue in vivo, which reinforce the usefulness of our co-culture system as a unique screening system for developing a new therapeutic strategy to reduce the obesity-induced adipose tissue inflammation.

This study demonstrates the anti-inflammatory effect of PPAR-α ligands in the interaction between adipocytes and macrophages in obese adipose tissue. The results of this study suggest that besides the well-known lipid-lowering effect, PPAR-α ligands exert an anti-inflammatory effect in obese adipose tissue, which may contribute to the improvement of obesity-related metabolic syndrome.

Acknowledgment

We thank Ms. M. Yashima for secretarial assistance and the members of the Ogawa laboratory for helpful discussion. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and Ministry of Health, Labour and Welfare of Japan, and research grants from Japan Research Foundation for Clinical Pharmacology, Novo Nordisk Insulin Study Award, The Kato Memorial Trust for Nambyo Research, the Kanae Foundation for Life and Socio-Medical Science, the Mitsui Sumitomo Insurance Welfare Foundation, Japan Diabetes Foundation, and The Takeda Science Foundation.

Disclosure

The authors declared no conflict of interest.