Role of the Toll-like Receptor 4/NF- B Pathway in Saturated Fatty Acid-Induced Inflammatory Changes in the Interaction Between Adipocytes and Macrophages

【摘要】  Objective- Previous studies demonstrated that obese adipose tissue is characterized by increased infiltration of macrophages, suggesting that they might represent an important source of inflammation. Using an in vitro coculture system composed of 3T3-L1 adipocytes and RAW264 macrophages, we previously demonstrated that saturated fatty acids (FAs) and tumor necrosis factor (TNF)- derived from adipocytes and macrophages, respectively, play a major role in the coculture-induced inflammatory changes.

Methods and Results- Coculture of adipocytes and macrophages resulted in the activation of nuclear factor- B (NF- B), a primary regulator of inflammatory responses, in both cell types. Pharmacological inhibition of NF- B markedly suppressed the coculture-induced production of proinflammatory cytokines and adipocyte lipolysis. Peritoneal macrophages obtained from Toll-like receptor 4 (TLR4) mutant mice exhibited marked attenuation of TNF production in response to saturated FAs. Notably, coculture of hypertrophied adipocytes and TLR4-mutant macrophages resulted in marked inhibition of proinflammatory cytokine production and adipocyte lipolysis. We also observed that endogenous FAs, which are released from adipocytes via the ß 3 -adrenergic stimulation, resulted in the activation of the TLR4/NF- B pathway.

Conclusion- These findings suggest that saturated FAs, which are released in large quantities from hypertrophied adipocytes via the macrophage-induced adipocyte lipolysis, serve as a naturally occurring ligand for TLR4, thereby inducing the inflammatory changes in both adipocytes and macrophages through NF- B activation.

Using an in vitro coculture system composed of adipocytes and macrophages, we demonstrate that saturated FAs, which are released in large quantities from hypertrophied adipocytes via the macrophage-induced adipocyte lipolysis, serve as a naturally occurring ligand for TLR4, thereby inducing the inflammatory changes in both adipocytes and macrophages through NF- B activation.

【关键词】  adipocytes fatty acids macrophages nuclear factor B Tolllike receptor

Introduction

The metabolic syndrome is a constellation of visceral fat obesity, impaired glucose metabolism, atherogenic dyslipidemia, and blood pressure elevation, which all increase independently a risk of atherosclerotic diseases. 1,2 There is considerable evidence that visceral fat obesity is a key etiologic factor in the development of the metabolic syndrome. 3,4 Recent studies have suggested a role for fat-derived biologically active substances (collectively termed adipocytokines) as pathogenic contributors. 3,4 Indeed, in obese adipose tissue, the production of proinflammatory and antiinflammatory cytokines is dysregulated, which may play an important role in the development of obesity-related sequelae. 5,6 Thus, obesity may be viewed as a metabolic as well as a chronic low-grade inflammatory disease.

Nuclear factor- B (NF- B), a primary regulator of inflammatory responses, plays a critical role in a variety of physiological and pathologic processes. 7 In several models of obesity in rodents, the NF- B signaling pathway is activated in insulin-responsive tissues such as the skeletal muscle and liver, where it may be involved in the development of inflammatory changes and insulin resistance. 8-10 Although adipocytes also express components of the NF- B pathway, 11 its pathophysiologic role in the adipose tissue is poorly understood. Lappas et al have recently reported that inhibition of NF- B suppresses the release of proinflammatory cytokines from the human adipose tissue ex vivo, 12 suggesting the possible role of NF- B in the adipose tissue. However, the molecular mechanism underlying activation of the NF- B pathway in obese adipose tissue is largely unknown.

Previous studies have demonstrated that obese adipose tissue is characterized by increased infiltration of macrophages, suggesting that they might represent an important source of inflammation in the adipose tissue. 13,14 We have recently developed an in vitro coculture system composed of adipocytes and macrophages and demonstrated that a paracrine loop involving free fatty acids (FFAs) and tumor necrosis factor- (TNF- ) derived from adipocytes and macrophages, respectively, establishes a vicious cycle that augments the inflammatory changes in both adipocytes and macrophages; ie, marked upregulation of proinflammatory cytokines such as monocyte chemoattractant protein-1 (MCP-1) and TNF- and downregulation of antiinflammatory cytokine adiponectin. 15 These findings also led us to speculate that macrophages, when infiltrated into obese adipose tissue, induce the release of FFAs from adipocytes via lipolysis, which, in turn, may serve as a proinflammatory cytokine locally in the adipose tissue. However, the functional significance of NF- B in obese adipose tissue remains to be elucidated.

FFAs represent an important energy source mobilized from triglycerides stored in the adipose tissue, particularly during periods of starvation, but recent evidence has suggested the pathophysiologic roles other than the supply of nutrients in times of fasting or increased energy demand. There are a couple of reports demonstrating that exogenous administration of saturated fatty acids (FAs) exerts the proinflammatory effects in certain cells. 15-17 Lee et al demonstrated previously that exogenous administration of saturated FAs induces inflammatory markers in macrophages through the activation of one of Toll-like receptors (TLRs) or TLR4, an essential receptor for the recognition of lipopolysaccharide (LPS). 18 It has been recognized that the NF- B pathway plays an important role in the TLR4-mediated immunomodulatory effect. 19 These findings, taken together, suggest that saturated FAs act as a naturally occurring ligand for TLR4. However, the physiological and pathophysiologic implication of the proinflammatory properties of saturated FAs through TLR4 in vitro as well as in vivo remains to be elucidated. Moreover, whether FFAs released from adipocytes via lipolysis can induce the inflammatory changes has not been addressed.

Using the coculture of adipocytes and macrophages, 15 we examined the role of NF- B in each cell type and the functional significance of saturated FAs as a paracrine mediator of inflammation in the interaction between adipocytes and macrophages. This study provides evidence that saturated FAs, the release of which is activated by macrophages via adipocyte lipolysis, play a major role in the activation of NF- B in macrophages through TLR4. Our data suggest that the TLR4/NF- B pathway plays an important role in the development of inflammatory changes in obese adipose tissue.

Materials and Methods

Materials and Antibodies

Details are described in online Materials and Methods (available athttp://atvb.ahajournals.org.).

Cell Culture

RAW264 macrophage cell line (RIKEN BioResource Center, Tsukuba, Japan) and 3T3-L1 preadipocytes (American Type Culture Collection, Manassas, Va) were maintained in Dulbecco?s modified Eagle?s medium (Nacalai Tesque) containing 10% fetal bovine serum. Differentiation of 3T3-L1 preadipocytes to mature adipocytes was performed as previously described 15 and used as differentiated 3T3-L1 adipocytes at day 8 to 10 after the induction of differentiation. Hypertrophied 3T3-L1 adipocytes with larger lipid droplets, when cultured up to day 21 to 23, were also used. 15 Murine peritoneal macrophages were prepared as described 15 using C3H/HeJ mice which have defective LPS signaling due to a missense mutation in the TLR4 gene 20 and control C3H/HeN mice (CLEA Japan, Tokyo, Japan) at 8 to 10 weeks of age. All animal experiments were conducted in accordance to the guidelines of Tokyo Medical and Dental University Committee on Animal Research (No. 0060026).

Coculture of Adipocytes and Macrophages

Coculture of adipocytes and macrophages was performed as described 15 with some modifications (supplemental Figure IA). In the contact system, serum starved differentiated 3T3-L1 adipocytes ( 0.5 x 10 6 cells) were cultured in a 35-mm dish and macrophages (1.0 x 10 5 cells of RAW264 macrophages or peritoneal macrophages) were plated onto 3T3-L1 adipocytes. The cells were cultured for 24 hour with contact each other and harvested. As a control, adipocytes and macrophages, the numbers of which were equal to those in the coculture, were cultured separately and mixed after harvest. Some experiments were performed with the transwell system, in which cells were cocultured using transwell inserts with a 0.4 µm porous membrane (Corning) to separate adipocytes from macrophages. After incubation for 24 hours, the adipocytes in the lower dish were harvested.

Quantitative Real-Time Polymerase Chain Reaction

Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen) and quantitative real-time PCR was performed with an ABI Prism 7000 Sequence Detection System using PCR Master Mix Reagent (Applied Biosystems). 15,21 Primers used in this study were described elsewhere. 15,21 Levels of mRNA were normalized to those of 36B4 mRNA.

Measurement of MCP-1 and TNF Levels in Culture Media

The MCP-1 and TNF levels in culture supernatants were determined by commercially available enzyme-linked immunosorbent assay kit (R&D Systems). 15

Lipolysis Assay

Differentiated 3T3-L1 adipocytes were cocultured with macrophages in the medium containing 2% bovine serum albumin (Sigma) for 24 hours. The concentration of FFAs in the medium was measured using an acyl-coenzyme A (CoA) oxidase-based colorimetric assay kit (NEFA-C; WAKO Pure Chemicals). 15

Transient Transfection and Luciferase Assay

The luciferase reporter construct for NF- B activity ( B Luc) was described elsewhere. 22 The luciferase reporter construct without any cis -acting DNA elements was used as a negative control. A luciferase reporter assay was performed as previously described. 23,24 In brief, differentiated 3T3-L1 adipocytes and RAW264 macrophages were transiently transfected by electroporation (Nucleofector system; Amaxa) with a luciferase reporter vector and pRL-TK vector (Promega) as an internal control for transfection efficiency. After incubation for 24 hours, cells were serum starved for another 24 hours and used for the experiments. The luciferase activity was determined using a Dual-Luciferase Reporter Assay System (Promega).

Coculture of Adipocytes and Ba/F3 Cells

Interleukin (IL)-3-dependent lymphocyte-like Ba/F3 cells were cultured in RPMI 1640 (Nacalai Tesque) supplemented with 10% FBS, 100 µmol/L 2-mercaptoethanol, and IL-3. 25 Ba/F3 cells stably expressing an NF- B reporter with or without the TLR4 complex (TLR4, MD-2, and CD14) were described elsewhere. 25 The TLR4 mRNA expression level was 50-fold higher in the Ba/F3 cells expressing the TLR4 complex and an NF- B reporter than in those expressing an NF- B reporter alone (data not shown). The Ba/F3 cells were cocultured with 3T3-L1 adipocytes in the medium containing 2% bovine serum albumin for 24 hours in the presence or absence of ß 3 adrenergic receptor agonist CL346243 (supplemental Figure IB). In this study, mRNA expression of ß3 adrenergic receptor was negligible in Ba/F3 cells relative to 3T3-L1 adipocytes (data not shown).

Statistical Analysis

Data were expressed as the mean±SE. Statistical analysis was performed using analysis of variance followed by Scheffe test unless otherwise described. P <0.05 was considered to be statistically significant.

Results

Role of NF- B in the Coculture-Induced Inflammatory Changes

To explore the role of NF- B in the interaction between adipocytes and macrophages, we examined the effect of NF- B inhibitors on the inflammatory changes in the coculture system composed of 3T3-L1 adipocytes and RAW264 macrophages (supplemental Figure IA). Treatment with two different NF- B inhibitors, BAY11-7085 and calpain inhibitor-I, effectively inhibited the coculture-induced upregulation of MCP-1 and TNF mRNA expression in the contact system ( P <0.05, Figure 1 A). We also observed that blockade of NF- B activity markedly suppressed the contact coculture-induced MCP-1 and TNF secretion ( P <0.05, Figure 1 B). Similar results were obtained using the transwell coculture system. Pharmacological inhibition of NF- B with BAY11-7085 markedly reduced the coculture-induced MCP-1 mRNA in 3T3-L1 adipocytes ( P <0.01, Figure 1 C). Furthermore, BAY11-7085 also completely inhibited the coculture-induced FFA release in the contact system ( P <0.01, Figure 1 D).

Figure 1. Role of NF- B in the induction of inflammatory changes by the coculture. Differentiated 3T3-L1 adipocytes were cocultured with RAW264 macrophages (1 x 10 5 cells/35-mm dish) for 24 hours. A and B, Effects of NF- B inhibitors on the coculture-induced mRNA expression (A) and secretion (B) of proinflammatory cytokines in the contact system. C, Effect of BAY11-7085 on MCP-1 mRNA expression in the transwell system. D, Effect of BAY11-7085 on the coculture-induced FFA release in the contact system. E and F, Activation of NF- B in 3T3-L1 adipocytes (E) and RAW264 macrophages (F) by the coculture in the contact system. An NF- B reporter plasmid ( B Luc) was transiently transfected into 3T3-L1 adipocytes or RAW264 macrophages. After 48-hour incubation, the coculture of 3T3-L1 adipocytes and RAW264 macrophages was performed and the luciferase activity was determined. ct indicates control culture; co, coculture; BAY, BAY11-7085 10 µmol/L; Cal, calpain inhibitor-I 10 µmol/L, RLA, relative luciferase activity. * P <0.05, ** P <0.01 vs Veh/control, # P <0.05, ## P <0.01 vs Veh/coculture. n=6.

Using an NF- B reporter assay, we observed significant activation of NF- B in 3T3-L1 adipocytes cocultured with RAW264 macrophages ( P <0.01, Figure 1 E), which was suppressed by treatment with BAY11-7085 (data not shown). Moreover, in the coculture system, NF- B was also significantly activated in RAW264 macrophages ( P <0.01, Figure 1 F). These observations, taken together, indicate that NF- B is activated in both adipocytes and macrophages in the coculture system, which plays a critical role in the coculture-induced increase in proinflammatory cytokines and adipocyte lipolysis.

Role of NF- B in the Induction of Inflammatory Changes in Adipocytes and Macrophages

To elucidate precisely the relative contribution of adipocytes and macrophages to the coculture-induced inflammatory changes, we examined mRNA expression in adipocyte and macrophage fractions separated by the magnetic cell sorting system (supplemental Figure IIA). Adiponectin and F4/80, which are known to be expressed specifically in adipocytes and macrophages, 13-15 respectively, are expressed exclusively in the respective fractions (supplemental Figure IIB), indicating the purity of each fraction. In this study, MCP-1 mRNA was significantly up-regulated in both fractions by the coculture and the extent was more marked in the adipocyte fraction ( P <0.01, supplemental Figure IIC). By contrast, TNF mRNA was mostly expressed in the macrophage fraction, which was markedly increased by the coculture ( P <0.01, supplemental Figure IIC). These observations are consistent with our previous observation that MCP-1 and TNF are mainly derived from adipocytes and macrophages, respectively, in the coculture system. 15

We previously demonstrated that TNF is a major macrophage-derived paracrine mediator of inflammation in adipocytes in the coculture system. 15 We therefore examined the effect of TNF on NF- B activation and its role in TNF -induced proinflammatory cytokine production and lipolysis in 3T3-L1 adipocytes. The data demonstrate that NF- B is activated in adipocytes treated with recombinant TNF ( P <0.01, Figure 2 A). Moreover, BAY11-7085 and calpain inhibitor-I significantly suppressed the TNF -induced MCP-1 mRNA expression and secretion in adipocytes ( P <0.05, Figure 2B and 2 C). Interestingly, there was no significant change in the TNF -induced FFA release, when treated with BAY11-7085 ( Figure 2 D).

Figure 2. Role of NF- B in the TNF -induced inflammatory changes in adipocytes. A, Activation of NF- B in differentiated 3T3-L1 adipocytes by treatment with recombinant TNF. An NF- B reporter plasmid ( B Luc) was transiently transfected into 3T3-L1 adipocytes. After 48-hour incubation, 3T3-L1 adipocytes were treated with recombinant TNF for 10 hours in the presence or absence of BAY11-7085 and the luciferase activity was determined. B and C, Effects of BAY11-7085 and calpain inhibitor-I on the TNF -induced mRNA expression (B) and secretion (C) of MCP-1 in 3T3-L1 adipocytes. D, Effect of BAY11-7085 on FFA release by treatment with recombinant TNF. RLA indicates relative luciferase activity; BAY, BAY11-7085 10 µmol/L; Cal, calpain inhibitor-I 10 µmol/L; TNF, recombinant TNF 1 ng/mL. ** P <0.01 vs Veh; # P <0.05, ## P <0.01 vs TNF/Veh. n=6.

Because saturated FAs are an important adipocyte-derived paracrine mediator of inflammation in macrophages in the coculture system, 15 we examined the effect of palmitate, a saturated FA produced most abundantly in 3T3-L1 adipocytes, 26 using RAW264 macrophages. An NF- B reporter assay revealed significant activation of NF- B in RAW264 macrophages treated with palmitate as well as LPS ( P <0.01, Figure 3 A). Moreover, NF- B inhibitors significantly suppressed the palmitate-induced TNF mRNA expression and secretion in RAW264 macrophages ( P <0.01, Figure 3B and 3 C). These observations, taken together, indicate that NF- B is activated in both adipocytes and macrophages in the coculture system and that activation of NF- B plays a critical role in the coculture-induced inflammatory changes in both cell types.

Figure 3. Role of NF- B in the saturated FA-induced inflammatory changes in macrophages. A, Activation of NF- B in RAW264 macrophages by treatment with palmitate. An NF- B reporter plasmid ( B Luc) was transiently transfected into RAW264 macrophages. After 48-hour incubation, RAW264 macrophages were treated with palmitate (Pal, 100 to 200 µmol/L) or LPS (0.3 to 10 ng/mL) for 10 hours and the luciferase activity was determined. B and C, Effects of BAY11-7085 and calpain inhibitor-I on the palmitate (200 µmol/L)-induced mRNA expression (B) and secretion (C) of TNF in RAW264 macrophages. RLA indicates relative luciferase activity; BAY, BAY11-7085 10 µmol/L; Cal, calpain inhibitor-I 10 µmol/L. * P <0.05, ** P <0.01 vs Veh, ## P <0.01 vs Veh/Pal. n=6.

Role of TLR4 in the Saturated FA-Induced Inflammatory Changes in Macrophages

To explore the molecular mechanism underlying the saturated FA-induced inflammatory changes in macrophages, we performed DNA microarray analysis of RAW264 macrophages treated with palmitate and LPS. Forty-seven genes were commonly up-regulated by either treatment (supplemental Figure IIIA). They include proinflammatory cytokines/growth factors (TNF, IL-1ß, vascular endothelial growth factor, and platelet-derived growth factor-B) and chemokines (Cxcl2, Ccl3, and Ccl4), which are implicated in the pathophysiology of chronic inflammatory diseases. We also analyzed the transcription factors regulating the extracted 47 genes using Transcription Regulatory Element Database (supplemental Figure IIIB). The data reveal that the upregulated genes are known to be dependent on NF- B family (NF- B1 [p50], RelA [p65], and c-Rel). These observations, taken together, suggest that palmitate and LPS signals through common pathways.

We next investigated the role of TLR4 in the saturated FA-induced inflammatory changes in RAW264 macrophages. Peritoneal macrophages obtained from TLR4-mutant C3H/HeJ mice exhibited marked attenuation of TNF mRNA expression, when treated with LPS or palmitate ( P <0.01, Figure 4 A), indicating that TLR4 is involved in the palmitate-induced inflammatory changes in macrophages. We also examined whether endogenous FFAs released from adipocytes are capable of activating the TLR4/NF- B signaling pathway (supplemental Figure IB). The Ba/F3 cells stably expressing the TLR4 complex and an NF- B reporter, when cocultured with 3T3-L1 adipocytes, exhibited significant NF- B activation by the treatment with ß 3 adrenergic receptor agonist CL346243 ( P <0.01, Figure 4 B). We observed that FFA levels in the media were markedly increased by the treatment with CL346243 ( P <0.01, Figure 4 C). In this setting, there was no appreciable increase in the activity of NF- B in the Ba/F3 cells expressing an NF- B reporter alone (data not shown). In addition, treatment with CL346243 did not change the activity of NF- B in the Ba/F3 cells expressing an NF- B reporter (data not shown). These observations, taken together, suggest that endogenous FFAs released from adipocytes can activate the TLR4/NF- B pathway.

Figure 4. Role of TLR4 in the saturated FA-induced inflammatory changes in macrophages. A, Marked attenuated response to palmitate as well as LPS in peritoneal macrophages from TLR4 mutant C3H/HeJ mice (HeJ) relative to those from wild-type C3H/HeN mice (HeN). LPS 100 ng/mL; Pal 200 µmol/L. B and C, Activation of the TLR4/NF- B pathway by endogenous FFAs released from 3T3-L1 adipocytes. The Ba/F3 cells stably expressing the TLR4 complex and an NF- B reporter were cocultured with 3T3-L1 adipocytes for 24 hours in the presence or absence of ß 3 adrenergic receptor agonist CL346243, and the luciferase activity (B) and FFA levels in the media (C) were determined. D, Effect of endotoxin-binding antibiotics polymyxin B (Poly, 10 µg/mL) on LPS- and palmitate-induced TNF mRNA expression in RAW264 macrophages. LPS 10 ng/mL; Pal 200 µmol/L. E, Time course of LPS- and palmitate-induced TNF mRNA expression in RAW264 macrophages. LPS 100 ng/mL; Pal, 200 µmol/L. * P <0.05, ** P <0.01 vs each Veh; ## P <0.01. n=4 to 8.

We next compared the proinflammatory properties of palmitate and LPS. LPS-binding antibiotics polymyxin B did not inhibit the palmitate-induced TNF mRNA expression in RAW264 macrophages, whereas it almost completely inhibited the LPS-induced up-regulation of TNF ( Figure 4 D). In addition, the time course of TNF mRNA expression induced by palmitate was obviously different from that by LPS ( Figure 4 E). In this study, TNF mRNA expression was up-regulated in RAW264 macrophages as early as 1 hour after the treatment with LPS and declined gradually 8 hours after the treatment. By contrast, TNF mRNA expression remained unchanged 3 hours after the treatment with palmitate and thereafter increased up to 24 hours after the treatment. These observations suggest both palmitate and LPS signals, although differently, through the TLR4/NF- B pathway.

We next examined the role of TLR4 in adipocytes in the interaction between adipocytes and macrophages because recent reports have shown that 3T3-L1 adipocytes and the mature adipocyte fraction obtained from the adipose tissue express TLR4 mRNA. 27,28 Western blot analysis confirmed TLR4 protein expression in differentiated 3T3-L1 adipocytes, but the levels were much lower than RAW264 macrophages (supplemental Figure IVA). We also observed that treatment with LPS significantly upregulated MCP-1 mRNA expression but palmitate did not (supplemental Figure IVB). These observations suggest that palmitate, a much less potent TLR4 ligand than LPS, does not play a major role in the induction of inflammatory changes in adipocytes.

Role of TLR4 in the Coculture-Induced Inflammatory Changes and FFA Release

Using the coculture system composed of adipocytes and macrophages, we also examined the functional significance of TLR4 in the interaction between adipocytes and macrophages. We cocultured normal-sized differentiated 3T3-L1 adipocytes (8 to 10 days after differentiation) with peritoneal macrophages obtained from C3H/HeJ and C3H/HeN mice. TNF and IL-1ß mRNA expression was significantly attenuated, when cocultured with C3H/HeJ macrophages in the contact system ( P <0.05, Figure 5 A and data not shown), but there was rather significant increase in MCP-1 mRNA expression ( Figure 5 A). This was an unexpected result and the reason is currently unknown. In C3H/HeJ macrophages, when cocultured with 3T3-L1 adipocytes, other proinflammatory cytokines may be upregulated in compensation for the decrease in TNF and IL-1ß, thus inducing MCP-1 mRNA expression in 3T3-L1 adipocytes. We also used hypertrophied 3T3-L1 adipocytes (21 to 23 days after differentiation) in the contact system, which exhibited more marked inflammatory changes and FFA release than normal-sized 3T3-L1 adipocytes. 15 Expression of both MCP-1 and TNF mRNAs was significantly downregulated in the coculture of hypertrophied 3T3-L1 adipocytes with C3H/HeJ macrophages relative to that with C3H/HeN macrophages ( P <0.05, Figure 5 B). These results were also confirmed in the transwell system ( P <0.01, Figure 5 C). In this study, FFA levels in the culture media were significantly decreased in the contact coculture of hypertrophied 3T3-L1 adipocytes with C3H/HeJ macrophages relative to that with C3H/HeN macrophages ( P <0.05, Figure 5 D). These observations, taken together, indicate that TLR4 expressed in macrophages plays an important role in the coculture-induced inflammatory changes and adipocyte lipolysis.

Figure 5. Role of TLR4 in the coculture-induced inflammatory changes and FFA release. Normal-sized and hypertrophied 3T3-L1 adipocytes were cocultured with peritoneal macrophages (1 x 10 5 cells/35-mm dish) from C3H/HeN (HeN) and C3H/HeJ (HeJ) for 24 hours. A and B, Role of TLR4 in the inflammatory changes induced by the contact coculture with normal-sized 3T3-L1 adipocytes (A) and with hypertrophied 3T3-L1 adipocytes (B). C, Role of TLR4 in the inflammatory changes induced by the transwell coculture with hypertrophied 3T3-L1 adipocytes. D, Role of TLR4 in the adipocyte lipolysis by the contact coculture with hypertrophied 3T3-L1 adipocytes. ct indicates control culture; co, coculture. ** P <0.01 vs each control culture; # P <0.05, ## P <0.01. n=4 to 6.

Discussion

This study demonstrates for the first time that coculture of adipocytes and macrophages results in the activation of NF- B relative to the control culture. Importantly, this activation occurs in both adipocytes and macrophages. These observations suggest that the NF- B pathway is activated in obese adipose tissue, in which there may be an intimate crosstalk between adipocytes and macrophages. We also found that pharmacological suppression of NF- B activity results in the inhibition of the coculture-induced proinflammatory cytokine production in adipocytes, suggesting the pathophysiologic role of the NF- B pathway in the interaction between adipocytes and macrophages. This is consistent with a recent report by Lappas et al that the NF- B pathway is involved in the release of proinflammatory cytokines from the human adipose tissue ex vivo. 12 We previously reported that coculture of adipocytes and macrophages results in the activation of mitogen-activated protein kinases (MAP kinases) such as extracellular signal regulated kinase (ERK) and c-Jun NH 2 -terminal kinase (JNK), which are involved in the induction of inflammatory changes. 15 These observations, taken together, suggest that in addition to the MAP kinase pathway, the NF- B pathway plays a role in the regulation of inflammatory changes in the interaction between adipocytes and macrophages.

In this study, we demonstrated that pharmacological suppression of NF- B activity results in inhibition of the coculture-induced lipolysis in adipocytes, suggesting the role of the NF- B pathway in the regulation of FFA release from obese adipose tissue. This discussion is supported by the previous in vivo study that treatment with high doses of aspirin, an inhibitor of the NF- B pathway, markedly decreases serum FFA levels in ob/ob mice. 29 Although TNF is a major macrophage-derived mediator of inflammation and lipolysis in adipocytes, 15 pharmacological suppression of NF- B activity did not inhibit the TNF -induced lipolysis, while it inhibited the TNF -induced proinflammatory cytokine production in adipocytes ( Figure 2 ), suggesting the differential roles of the NF- B pathway in the TNF -induced inflammation and lipolysis in adipocytes. Because we and others have shown that NF- B is involved in the induction of TNF production ( Figure 3 ), 8 it is conceivable that in the coculture of adipocytes and macrophages, activation of the NF- B pathway in macrophages increases TNF production in macrophages, which in turn induces adipocyte lipolysis. Because there are a couple of lines of evidence demonstrating that TNF increases adipocyte lipolysis through the activation of MAP kinases, 30,31 the data of this study suggest that TNF plays a role in the regulation of adipocyte lipolysis independent of the NF- B pathway in adipocytes. Indeed, we found that pharmacological inhibition of the ERK and JNK pathways significantly reduces FFA concentrations in the media in the coculture (Suganami et al, unpublished data, 2006). Collectively, we speculate that macrophages, when infiltrated into obese adipose tissue, play a critical role in the regulation of lipolysis and thus circulating FFA levels probably through the interaction between adipocytes and macrophages.

We previously demonstrated that FFAs are a major proinflammatory mediator in macrophages in the coculture system. 15 Lee et al also reported that exogenous administration of saturated FAs are capable of inducing cyclooxygenase-2 expression activating the TLR4/NF- B pathway in cultured macrophages. 18 However, the pathophysiologic role of TLR4 in the interaction between adipocytes and macrophages has not been addressed so far. The microarray analysis of RAW264 macrophages treated with either palmitate or LPS revealed similar expression profiles of inflammation-related genes, suggesting that palmitate and LPS signal at least partly through common NF- B-related pathways. The data herein provide the genetic evidence that palmitate induces the inflammatory changes through TLR4; exogenous administration of palmitate increases TNF production in wild-type macrophages but not in TLR4-mutant macrophages. In this study, Ba/F3 cells expressing the TLR4 complex and an NF- B reporter exhibit significant NF- B activation, when the FFA levels in the media are increased by ß 3 -adrenergic stimulation-induced adipocyte lipolysis ( Figure 4 ). These observations suggest that endogenous FFAs are capable of activating NF- B in macrophages. In this context, we have recently observed that 3T3-L1 adipocytes, when cocultured with RAW264 macrophages, release many kinds of saturated FAs in the culture media, of which palmitate is a major component (T. Suganami et al, unpublished data, 2006). Furthermore, coculture of hypertrophied adipocytes with TLR4-mutant macrophages results in marked inhibition of proinflammatory cytokine production and lipolysis relative to that with wild-type macrophages ( Figure 5 ). These observations, taken together, suggest that saturated FAs such as palmitate, when released in larger quantities from hypertrophied adipocytes cocultured with macrophages, serve as a naturally occurring ligand for TLR4 expressed in macrophages, where they play a pathophysiologic role in the inflammatory changes in the interaction between adipocytes and macrophages.

Although both saturated FAs and LPS signal through the TLR4/NF- B pathway, there might be marked differences between the inflammatory changes. For instance, the time course of TNF mRNA expression induced by palmitate is obviously different from that induced by LPS ( Figure 4 ). In this study, palmitate is also capable of inducing proinflammatory cytokine production with less potency than LPS. The potential endogenous TLR4 ligands other than saturated FAs include heat shock protein HSP60 and extracellular matrix components such as fibronectin and hyaluronic acid, 32-34 the pathophysiologic role of which remains to be elucidated so far. Unlike LPS, the palmitate-induced TNF mRNA expression is resistant to the treatment with polymyxin B, which is similar to the report that fibronectin activates the TLR4/NF- B pathway, which is unaffected by polymyxin B. 33 These observations suggest that endogenous TLR4 ligands such as saturated FAs and fibronectin can activate the TLR4/NF- B pathway differently from a well-defined exogenous TLR4 ligand or LPS. This discussion may explain the chronic (or long-term) low-grade versus acute (or short-term) high-grade inflammatory changes elicited by endogenous versus exogenous ligands, respectively.

A previous study showed that apolipoprotein E-deficient mice lacking TLR4 exhibit reduced atherosclerosis that is associated with reduction in circulating levels of proinflammatory cytokines and numbers of macrophages in atheromatous plaques, suggesting the potential therapeutic target of TLR4 in atherosclerosis. 35 Evidence has accumulated indicating that elevated levels of circulating FFAs, especially saturated FFAs derived from dietary animal fat or adipocyte lipolysis, in obesity potentially contribute to the development of the inflammatory changes in the vascular wall. 17,36 It is, therefore, likely that saturated FAs, which are released in large quantities from hypertrophied adipocytes via the macrophage-induced adipocyte lipolysis, induce the inflammatory changes locally in obese adipose tissue and systemically in circulating monocytes and/or macrophages infiltrated into atheromatous plaques through the TLR4/NF- B pathway. The above discussion supports the concept that antagonism of the TLR4/NF- B pathway offers a novel therapeutic strategy to prevent or treat obesity-induced inflammation and thus the metabolic syndrome associated with excess adiposity.

In conclusion, this study provides in vitro evidence that the TLR4/NF- B pathway plays a critical role in the saturated FA-induced inflammatory changes in the interaction between adipocytes and macrophages (supplemental Figure V). To the best of our knowledge, there have been no studies on the susceptibility/resistance of mice with TLR4 deficiency to diet-induced disturbances in glucose and lipid metabolism. The results of this study should motivate future studies using the TLR4 mutant mice to test whether they have different metabolic responses to high-fat/high carbohydrate diets than wild-type mice. The data of this study will also help to elucidate the molecular mechanisms underlying the inflammatory changes in obese adipose tissue and identify the therapeutic targets that may reduce obesity-induced inflammation.

Acknowledgments

We thank Miyako Tanaka, Tae Mieda, and Maya Yashima for technical and secretarial assistance, and the members of the Ogawa laboratory for helpful discussions.

Sources of Funding

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 Astellas Foundation for Research on Metabolic Disorders, Japan Diabetes Foundation, Mishima Kaiun Memorial Foundation, The Novo Nordisk Insulin Study Award, Cell Science Research Foundation, Smoking Research Foundation, Japan Heart Foundation/Novartis Grant for Research Award on Molecular and Cellular Cardiology, and Chiyoda Mutual Life Foundation.

Disclosures

None.

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作者单位：Department of Molecular Medicine and Metabolism (T.S., K.T.-K., J.N., M.I., X.Y., Y.K., Y.O.) and the Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstitution of Tooth and Bone (Y.O.), Medical Research Institute, Tokyo Medical and Dental University; the Tsukuba