AIM-Dependent Lipolysis Induces Macrophage Migration.

We then tested whether AIM itself attracts macrophages. However, AIM showed no chemoattractive activity in a macrophage migration assay using RAW264.1 mouse macrophage cells (Fig. 2A, Left). In contrast, conditioned medium from 3T3-L1 adipocytes that had been challenged with rAIM for 72 h (AIM CM) efficiently attracted macrophage cells (Fig. 2A, Left). A comparable effect was observed with conditioned medium from cells treated with C75, a specific FAS inhibitor that also induces lipolysis (14). AIM CM also attracted J774.1 mouse monocyte cells (Fig. S3A). Furthermore, 3T3-L1 adipocytes were treated with rAIM in the presence of a CD36-neutralizing antibody (mouse IgA), which inhibits AIM-dependent lipolysis by disturbing the endocytosis of AIM into adipocytes (14), and the conditioned medium (AIM+αCD36 CM) was assessed in the macrophage migration assay. The AIM+αCD36 CM did not efficiently attract macrophages (Fig. 2A, Right), suggesting that AIM-induced lipolysis in adipocytes appears to be responsible for macrophage recruitment. The CD36-neutralizing antibody itself had no direct effect on the macrophage migration (Fig. S3B).

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Fig. 2.

AIM-dependent lipolysis induces chemokine production in adipocytes via TLR4 stimulation. (A) Chemotaxis of RAW 264.1 cells in response to specified stimulant. Attractants: rAIM (25 μg/mL), C75 (25 μM), AIM CM/C75 CM: conditioned medium from 3T3-L1 adipocytes treated for 3 d with rAIM (25 μg/mL) or C75 (25 μM), respectively; AIM+αCD36 CM/AIM+IgA CM: conditioned medium from 3T3-L1 adipocytes treated for 3 d with rAIM (25 μg/mL) in the presence of anti-CD36 Ab or mouse IgA (10 μg/mL each), respectively; none CM, control CM: treated without rAIM or C75; and FM: fresh DMEM culture medium containing 10% FBS. Averages from n = 3 ± SEM. MCP-1 (100 ng/mL) was used as a positive control. (B) Degradation of IkBα in 3T3-L1 adipoctytes in response to specified stimulant in the absence (−) or presence (+) of a TIRAP inhibitor (100 μM). LPS (100 ng/mL) was used as a positive control. Representative immunoblotting results are presented. The density of the signal was quantified using National Institutes of Health Image J image analysis software and presented as values relative to those of prestimulation (Lower two panels). n = 3. Error bar: SEM. *, versus the value at prestimulation (0 min). (C) QPCR analysis of mRNA levels for MCP-1, CCL5/RANTES, MCP-2, and MCP-3 using RNA isolated from 3T3-L1 adipocytes treated with specified stimulant for 24 h in the absence (white bars) or presence (black bars) of a TIRAP inhibitor. Values were presented as relative expression to those without stimulation (none). n = 3 for each. Error bar: SEM. (D and E) No degradation of IkBα or expression induction of mRNA for chemokine genes in 3T3-L1 adipoctytes in response to rAIM alone (25 μg/mL) (D) or AIM+αCD36 CM (E).

Fatty Acids Effluxed from Adipocytes in Response to AIM-Dependent Lipolysis Stimulated TLR Signaling Pathway and Induced Chemokine Production in Adipocytes.

Accumulating evidence has demonstrated that saturated fatty acids activate TLR signaling cascade and that this response is tightly associated with obesity-induced inflammation (21–25). Thus, it is plausible that an increase in blood AIM may induce vigorous lipolysis in obese adipose tissues, and saturated fatty acids effluxed from adipocytes as a result of lipolysis might activate chemokine production in adipocytes via the stimulation of TLR(s) in a paracrine/autocrine fashion (26–28). Indeed, palmitic and stearic acids, the major fatty acids comprising triglyceride droplets (29) and well known as stimulators of TLR4 and TLR2 (21, 25, 30, 31), were identified as the components released by adipocytes in response to lipolysis induced by AIM or C75 when the profile of fatty acids in AIM CM and C75 CM was evaluated by gas-chromatography mass-spectrometry analysis.

Consistent with this result, both AIM CM and C75 CM efficiently stimulated the TLR signaling cascade and chemokine production in 3T3-L1 adipocytes, as assessed by degradation of IkBα (Fig. 2B) and mRNA expression of chemokines such as MCP-1, CCL5/RANTES, MCP-2, and MCP-3, which affects macrophages (Fig. 2C). AIM CM induced substantial levels of protein of these chemokines as assessed by ELISA (Fig. S4A). These responses diminished when adipocytes were treated with AIM CM or C75 CM in the presence of a toll–interleukin-1 receptor domain containing adapter protein (TIRAP) inhibitor, which specifically interferes with the interaction of TLR4 (as well as TLR2) and the adapter protein TIRAP/Mal, resulting in attenuation of TLR signaling (Fig. 2 B and C) (32). Furthermore, we confirmed that similar effects of TLR activation and chemokine production were observed when 3T3-L1 adipocytes were treated with palmitic acid (PA) or stearic acid (SA) and that the responses induced by each fatty acid were reduced when subjected to the TIRAP inhibitor (Fig. S5). Consistent with the results from macrophage migration assay presented in Fig. 2A, neither rAIM alone (Fig. 2D and Fig. S4B) nor AIM+αCD36 CM (Fig. 2E and Fig. S4C) stimulated IkBα degradation or chemokine mRNA and protein expression in adipocytes. These findings clearly indicate the necessity of the lipolytic process in the overall activation of TLR signaling cascade by AIM.

Involvement of TLR4.

As TIRAP is downstream of not only TLR4 but also other TLRs, including TLR2 (32), the precise involvement of TLR4 in macrophage recruitment was further verified. We first suppressed TLR4 expression by siRNA in 3T3-L1 adipocytes and assessed the induction of MCP-1 by AIM CM. As shown in Fig. S6 A–C, induction of both mRNA and protein of MCP-1 by AIM CM was significantly reduced in cells transfected with siRNA for TLR4. In addition, we injected rAIM i.v. into wild-type and TLR4^−/− mice and thereafter assessed the state of lipolysis and chemokine production in epididymal adipose tissue. In both types of mice, the mRNA levels of FSP27 (also called Cidec), Perilipin, and Adipophilin, coating elements for lipid droplets, were decreased after challenging with rAIM (Fig. 3A), a finding consistent with the progression of lipolysis reported previously (17, 33, 34). Similarly, the increase in blood FFA and glycerol levels was equivalent in TLR4^−/− and wild-type mice (Fig. 3B). In contrast, induction of mRNA for chemokines by rAIM injection was significantly less efficient in TLR4^−/− than in wild-type mice (Fig. 3C). In line with this, phosphorylation levels of c-Jun N-terminal kinases (JNKs) in epididymal fat, which represent the state of TLR activation, were up-regulated in wild-type mice but not in TLR4^−/− mice (Fig. 3D). Furthermore, the rAIM injection increased mRNA levels for M1 macrophage markers in epididymal adipose tissue of wild-type but not TLR4^−/− mice, demonstrating that AIM-induced lipolysis could not recruit inflammatory macrophages into adipose tissue in the absence of TLR4 (Fig. 3E). There was no significant change in mRNA levels for M2 macrophage markers in both TLR4^−/− and wild-type mice (Fig. 3E). Histological analysis revealed the presence of IL-6 expressing M1 macrophages after the rAIM injection in epididymal adipose tissue of wild-type mice but not of TLR4^−/− mice (Fig. S6D).

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Fig. 3.

Involvement of TLR4 in adipose tissue macrophage recruitment by AIM in vivo. (A–E) TLR4^−/− and wild-type littermate mice (B6 background) were i.v. injected with rAIM or BSA three times every other day (400 μg in 200 μL PBS per injection). The day after the third injection (day 8 from the first injection), mice were killed, and lipolysis, chemokine expression, and adipose tissue macrophage accumulation were analyzed. n = 5 for each. (A) mRNA levels for FSP27, Perilipin, and Adipophilin were assessed by QPCR using RNA isolated from epididymal fat. Values were presented as relative expression to those of fat tissue injected with BSA. Error bar: SEM. (B) Serum levels for FFA and glycerol. (C) mRNA levels for chemokines. (D) Immunoblotting for total and phosphorylated JNK in epididymal fat. Immunoblot for β-actin is also presented. Results from three mice for each group are presented. Note that comparable results were obtained in five independent mice in each group. (E) mRNA levels for F4/80 pan-macrophage marker, M1 and M2 macrophage markers to assess macrophage recruitment. (F) Immunoblotting for total and phosphorylated JNK using lysates obtained from epididymal fats of AIM^+/+ and AIM^−/− mice fed a HFD for 12 wk (n = 4–6). Relative values of phosphorylated JNK signals to total JNK are also presented (Lower graph). (G) QPCR analysis of mRNA levels for chemokine genes in epididymal fat tissue and (H) serum MCP-1 concentration in AIM^+/+ and AIM^−/− mice fed a HFD for 0 (lean) or 12 wk (obese); n = 6–8.

Consistent results were obtained in obese AIM^+/+ and AIM^−/− mice after 12 wk on a HFD. In epididymal fat, phosphorylation levels of JNKs were decreased in AIM^−/− mice compared with AIM^+/+ mice (Fig. 3F). In addition, chemokine mRNA levels were also lower in AIM^−/− than in AIM^+/+ adipose tissue (Fig. 3G). Moreover, the serum level of MCP-1 was lower in AIM^−/− than in AIM^+/+ mice (Fig. 3H).

It is possible that fatty acids effluxed from adipocytes may stimulate TLR4 expressed not only on adipocytes but also on resident M2 macrophages within adipose tissue in a paracrine fashion and may induce chemokine expression in macrophages. To assess this possibility, we stained epididymal fat from wild-type AIM^+/+ mice fed a HFD for 6 wk for MR, a M2 macrophage marker, and MCP-1. As shown in Fig. S7, both adipocytes and M2 macrophages stained positive for MCP-1. As expected, in AIM^−/− mice, neither adipocytes nor resident macrophages showed obvious MCP-1 expression. Thus, in summary, AIM-induced lipolysis provoked the efflux of saturated fatty acids, including palmitic and stearic acids, from adipocytes, and these fatty acids stimulated chemokine production in both adipocytes and resident macrophages via TLR4 activation, resulting in M1 macrophage migration.

We thank Genostaff Inc. for technical assistance in histology. This work was supported by Grants-in-Aid for Scientific Research (B) (Japan Society for the Promotion of Science), the Global Centers of Excellence (COE) Program (T.M.), Kanae Foundation for the Promotion of Medical Science, the Astellas Foundation for Research on Metabolic Disorders, and the Ono Medical Research Foundation (S.A.).