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Luteolin, a food-derived flavonoid, suppresses adipocyte-dependent activation of macrophages by inhibiting JNK activation
FEBS Letters 583 (2009) 3649–3654
journal homepage: www.FEBSLetters.org
Luteolin, a food-derived flavonoid, suppresses adipocyte-dependent activation of macrophages by inhibiting JNK activation Chieko Ando a,1, Nobuyuki Takahashi a,1, Shizuka Hirai a,1, Kanako Nishimura a, Shan Lin a, Taku Uemura a, Tsuyoshi Goto a, Rina Yu b, Joji Nakagami c, Shigeru Murakami c, Teruo Kawada a,* a b c
Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan Department of Food Science and Nutrition, University of Ulsan, Ulsan 680-749, South Korea Taisho Pharmaceutical Co. Ltd., Toshima-ku, Tokyo 170-8633, Japan
a r t i c l e
i n f o
Article history: Received 31 August 2009 Revised 28 September 2009 Accepted 16 October 2009 Available online 23 October 2009 Edited by Laszlo Nagy Keywords: Luteolin Macrophage Adipocyte JNK Inflammation
a b s t r a c t Interaction between adipocytes and macrophages contributes to the development of insulin resistance in obese adipose tissues. In this study, we examined whether luteolin, food-derived flavonoid, could suppress the production of inflammatory mediators of the interaction between adipocytes and macrophages. Experiments using a coculture system of adipocytes and macrophages showed that luteolin suppressed the production of inflammatory mediators. In addition, activated macrophages were targets for the suppressive effect of luteolin. Luteolin inhibited the phosphorylation of JNK and suppressed the production of inflammatory mediators in the activated macrophages. The findings indicate that luteolin can inhibit the interaction between adipocytes and macrophages to suppress the production of inflammatory mediators, suggesting that luteolin is a valuable foodderived compound for the treatment of metabolic syndrome. Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Obesity is often accompanied by hyperglycemia, hypertension, and hyperlipidemia, which together are called metabolic syndrome [1]. Recent studies have elucidated that obesity is characterized by low-grade chronic inflammation of adipose tissues and that such inflammation is one of the potential mechanisms underlying insulin resistance [2–4]. It has been indicated that adipose tissues contain various cells in addition to adipocytes, in which there are paracrine interactions between adipocytes and such non-adipocytes. Infiltration of macrophages is observed in obese adipose tissues [5,6]. The infiltrating macrophages are crucial contributors to the interactions to induce inflammation of obese adipose tissues. Generally, the nuclear factor-jB (NF-jB) pathway and mitogenactivated protein kinase (MAPK) pathway play indispensable roles in inflammation [7]. In obese adipose tissues, both pathways are involved in the production of inflammatory mediators from the activated macrophages and/or adipocytes [8,9]. Thus, the degradation of I-jB and activation of MAPK are valuable markers for the detection of inflammation of adipose tissues.
* Corresponding author. Fax: +81 774 38 3752. E-mail address: [email protected] (T. Kawada). 1 These authors equally contributed to this work.
Suganami and colleagues have reported that adipocyte-derived free fatty acid (FFA), monocyte chemoattractant protein (MCP)-1, and macrophage-derived tumor necrosis factor-a (TNFa) establish a vicious cycle enhancing inflammation and leading to insulin resistance in obese adipose tissue [8,9]. Matured adipocyte-derived MCP-1 enhances the infiltration of macrophages, which are activated by adipocyte-derived FFA to stimulate the release of TNFa from the infiltrating macrophages. The released TNFa in turn stimulates lipolysis in adipocytes to induce overproduction of FFA. Therefore, anti-inflammatory compounds that suppress such an inflammatory vicious cycle may contribute to the reduction of obesity-related insulin resistance. Foods contain various bioactive compounds. Among them, flavonoids are considered to be particularly valuable compounds, with multifunction including antioxidative, anticancer, and antiinflammatory effects [10–12]. Luteolin is a food-derived flavonoid, which is present in medicinal plants and in some vegetables and spices. Luteolin exhibits specific anti-inflammatory effects at micromolar concentrations, which are only partly explained by its antioxidant capacities. A recent review has provided the pharmacological data on the antioxidant, anti-inflammatory, and antiallergy functions of luteolin [12]. However, the effects of luteolin on low-grade chronic inflammation of obese adipose tissues remain to be clarified. Thus, the purpose of this study is to examine
0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2009.10.045
C. Ando et al. / FEBS Letters 583 (2009) 3649–3654
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2. Materials and methods 2.1. Chemicals and cell cultures Luteolin was purchased from Wako Chemicals (Osaka, Japan) and dissolved in DMSO to prepare a stock solution. All other chemicals were purchased from Sigma (MO, USA) and Nacalai Tesque (Kyoto, Japan). A RAW264 macrophage cell line (RIKEN BioResource Center, Tsukuba, Japan) was cultured in DMEM with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 lg/ml streptomycin (Gibco BRL, NY, USA) at 37 °C under a humidified 5% CO2 atmosphere as previously described [13–15]. The serum-free medium of RAW264 macrophages cultured for 24 h was collected as a RAW264 conditioned medium and stored at 20 °C until use. The cells were seeded in 12-well plates (5 105 cells/ml) and treated with various concentrations of luteolin in serum-free medium for 24 h. 3T3-L1 preadipocytes (American Type Culture Collection, VA, USA) were subcultured in DMEM with 10% FBS, 100 U/ml penicillin, and 100 lg/ml streptomycin at 37 °C under a humidified 5% CO2 atmosphere. Differentiation of 3T3-L1 preadipocytes was induced by treatment with adipogenic agents (0.5 mM 3-isobutyl1-methylxanthine, 0.25 lM dexamethasone, and 10 lg/ml insulin) in DMEM containing 10% FBS for 2 days after the cells reached confluence (day 0), as previously described [13,14]. Then, the medium was replaced with DMEM containing 10% FBS and 5 lg/ml insulin and was replaced every 2 days. Twenty days after the differentiation induction, the cells that accumulated large lipid droplets were used as hypertrophied 3T3-L1 adipocytes. The serum-free medium of hypertrophied 3T3-L1 adipocytes cultured for 12 h was collected as a 3T3-L1 conditioned medium and stored at 20 °C until use. RAW264 cells were seeded in 96-well plates and treated with various concentrations of luteolin for 24 h. The viability of RAW264 macrophages was measured by CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, MO, USA). 3T3-L1 cells were seeded in 96-well plates and induced to differentiate in DMEM containing various concentrations of luteolin. The medium was replaced every 2 days and cell viability was measured in the same procedure.
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the anti-inflammatory effects of luteolin on the interaction between adipocytes and macrophages to elucidate whether luteolin is effective for reducing inflammation in obese adipose tissues.
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Fig. 1. Luteolin inhibited inflammation in contact coculture system. Protein secretion levels of inflammatory mediators, TNFa (A) and MCP-1 (B), and release of NO (C). Luteolin was added to the contact coculture system of RAW264 macrophages and 3T3-L1 adipocytes. After 24 h, TNFa and MCP-1 secretion levels in the cultured medium were measured by ELISA. The secretion level of NO was measured using Griess reagent. The values are mean ± S.E.M. of 3–4 tests. *P < 0.05 and **P < 0.01 compared with a vehicle control.
2.2. Stimulation of macrophages and adipocytes Adipocytes and macrophages were cocultured in a contact system, as previously described [14,15]. Briefly, RAW264 cells (1 105 cells/ml) were plated onto dishes with serum-starved and hypertrophied 3T3-L1 cells, and the coculture was incubated in serum-free DMEM for 24 h. RAW264 and 3T3-L1 cells of equal concentrations to those in the coculture were cultured separately as control cultures. Luteolin was added to the coculture at various concentrations, as described in each figure legend. After 24 h of treatment, culture supernatants were collected and stored at 20 °C until use. 2.3. Measurement of inflammatory mediators The concentrations of TNFa and MCP-1 in the culture supernatants were determined by ELISA, which was conducted using Ready-Set-Go Mouse TNFa and MCP-1 kits (eBioscience, CA, USA), in according with the manufacturer’s protocols. NO release was measured using Griess reagent, as described previously
[14,15]. Briefly, 100 ll of supernatant was mixed with an equivalent volume of Griess reagent [1:1 (v/v) of 0.1% N-1-naphthyl-ethylenediamine in distilled water and 1% sulfanilamide in 5% phosphoric acid] on 96-well flat-bottom plates. After 10 min, the absorbance at 570 nm was measured and the amount of nitrite was calculated from the NaNO2 standard curve. Total RNA preparation and quantification of gene expression were performed as previously described [13,14]. The cells were washed with PBS and total RNA was prepared using Sepasol (R)-RNA Super (Nacalai Tesque) in according with the manufacturer’s protocol. Aliquots of total RNA were reverse-transcribed using M-MLV reverse transcriptase (Invitrogen Corp., CA, USA) and a thermal cycler (Takara PCR Thermal Cycler SP: Takara Shuzo Co., Kyoto, Japan). To quantify mRNA expression, real-time RTPCR was performed with a LightCycler System (Roche Diagnostics, Mannheim, Germany) using SYBR Green fluorescence signals. The oligonucleotide primers of inflammatory mediator genes were designed using a PCR primer selection program shown on the
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Fig. 2. Luteolin did not suppress adipocyte inflammation induced by macrophage-derived conditioned medium. Protein secretion and mRNA expression levels of TNFa (A, B) and MCP-1 (C, D). Hypertrophied 3T3-L1 adipocytes were incubated with the conditioned medium of RAW264 macrophages and various concentrations of luteolin for 24 h. The TNFa and MCP-1 secretions in the cultured medium were measured by ELISA. The mRNA expression levels of these mediators were measured by real-time RT-PCR. The values are mean ± S.E.M. of 3–4 tests.
website of the Virtual Genomic Center from the GenBank database. All PCR primers were previously described [13,14]. To compare mRNA expression levels among the samples, the copy numbers of all transcripts were divided by that of mouse 36B4 showing a constant expression level. All mRNA expression levels are presented as a ratio relative to that of a control in each experiment. 2.4. Western blotting Western blotting was performed as previously described [14,16]. In brief, RAW264 cells were washed with PBS and placed immediately in lysis buffer containing 20 mM Tris–HCl (pH 7.5), 15 mM NaCl, 1% Triton X-100 (Nacalai Tesque), and a protease inhibitor cocktail (Nacalai Tesque). The lysate was centrifuged at 15 000 rpm for 5 min, and the supernatant was stored for subsequent analysis. Protein concentration was determined using DC protein assay reagents (BioRad Laboratories, CA, USA) in accordance with the method of Lowry et al. [21]. Fifteen micrograms of protein was separated by 10% SDS–PAGE, and transferred to an Immobilon-P membrane (Millipore, MA, USA). After blocking, the membrane was incubated with an anti-I-jB-a (Santa Cruz Biotechnology, CA, USA), anti-JNK (Cell Signaling Technology, MA, USA), anti-pJNK (Cell Signaling Technology), or b-actin (Cell Signaling Technology) antibody, and then with a secondary antibody conjugated to horseradish peroxidase [anti-rabbit IgG: 1/2000 (Promega, WC, USA) ] for 1 h. The secondary antibody staining was visualized by chemiluminescence immunoassay using a chemiluminescent HRP substrate (Millipore). 2.5. Statistical analyses The data are presented as mean ± S.E.M. and were statistically analyzed using one-way ANOVA when their variances were heterogeneous and unpaired t-test. Differences were considered significant at P < 0.05.
3. Results 3.1. Effects of luteolin on inflammation in coculture of adipocytes and macrophages To examine the effects of luteolin on the interaction between adipocytes and macrophages, we used a coculture system using 3T3-L1 adipocytes and RAW264 macrophages, in which these cells were in direct contact. The coculture revealed marked increases in secretion levels of inflammatory mediators such as TNFa, MCP-1, and NO (Fig. 1A–C). Treatment with luteolin in the coculture system significantly decreased the secretion levels of the inflammatory mediators in a dose-dependent manner. In particular, the secretion of TNFa was markedly suppressed to a control level at a higher concentration of luteolin (20 lM). The concentrations of luteolin used in this experiment did not affect the viability of either RAW264 or 3T3-L1 cells (data not shown). These results indicate that luteolin has anti-inflammatory effects in the contact coculture system of adipocytes and macrophages. 3.2. Effects of luteolin on macrophage activation induced by adipocytederived conditioned medium Next, to clarify the anti-inflammatory effects of luteolin, we conducted medium change experiments. First, to examine the effect of luteolin on 3T3-L1 adipocytes, a conditioned medium of RAW264 macrophages was added to a hypertrophied 3T3-L1 adipocyte culture. The addition of the RAW264 conditioned medium significantly increased the levels of TNFa and MCP-1 protein secretion from 3T3-L1 cells (Fig. 2A and C). mRNA expression levels of the mediators were also increased by the RAW264 conditioned medium (Fig. 2B and D). However, the treatment with luteolin did not suppress the TNFa and MCP-1 expression at both protein secretion and mRNA expression levels. Thus, these results indicate that 3T3-L1 adipocytes are not a target of luteolin.
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Fig. 3. Luteolin suppressed macrophage activation induced by adipocyte-derived conditioned medium. RAW264 macrophages were incubated with the conditioned medium derived from the hypertrophied 3T3-L1 adipocytes (L1 medium) and various concentrations of luteolin for 24 h. The TNFa (A) and MCP-1 (C) protein levels were measured by ELISA. The secretion level of NO (E) was also measured using Griess reagent. The mRNA expression levels of TNFa (B), MCP-1 (D), and iNOS (F) were measured by real-time RTPCR. The values are mean ± S.E.M. of 3–4 tests. *P < 0.05 and **P < 0.01 compared with a vehicle control.
Second, to investigate the anti-inflammatory effect of luteolin on RAW264 macrophages, a conditioned medium of hypertrophied 3T3-L1 adipocytes was added to a RAW264 macrophage culture. The addition of the 3T3-L1 conditioned medium increased the level of TNFa secretion from RAW264 macrophages (Fig. 3A). Treatment with luteolin significantly decreased TNFa secretion level in a dose-dependent manner. The treatment with luteolin also suppressed TNFa mRNA expression (Fig. 3B). In addition to TNFa, the levels of secretion of MCP-1 and release of NO from RAW264 macrophages were increased by the 3T3-L1 conditioned medium (Fig. 3C and E). The increase was reduced to a control level by 20 lM luteolin (Fig. 3C and E). The addition of luteolin also significantly decreased the mRNA expression levels of MCP-1 and inducible-NO synthase (iNOS) (Fig. 3D and F). These anti-inflammatory effects of luteolin were observed in LPS-stimulated productions of inflammatory mediators. LPS (5 lg/ml) resulted in 414-, 15.1-, and 9.37-fold increases in productions of TNFa, MCP-1, and NO, respectively, in RAW264 macrophages. Luteolin (10 lM) inhibited the LPS-dependent productions in TNFa, MCP-1, and NO at 66.5%, 38.2%, and 56.2%, respectively. These effects of luteolin on the LPS-induced inflammation were a dose-dependent manner (data not shown). These results indicate that luteolin has anti-inflammatory effects on macrophages in terms of the interaction between adipocytes and macrophages.
3.3. Effects of luteolin on degradation of I-jB and activation of MAPK NF-jB is one of the major transcription factors that regulate the induction of various inflammatory mediators including TNFa, MCP-1, and iNOS [7]. To clarify the molecular mechanism underlying the anti-inflammatory effects of luteolin, we investigated whether luteolin inhibits I-jB degradation, which activates NFjB, in RAW264 macrophages. I-jB degradation was observed in RAW264 macrophages stimulated by the 3T3-L1 adipocyte-derived conditioned medium (Fig. 4A). However, the pretreatment with luteolin did not inhibit the degradation of I-jB. These results show that luteolin has no effect on I-jB degradation in RAW264 macrophages stimulated by the 3T3-L1 adipocyte-derived conditioned medium, although the conditioned medium promoted the degradation of I-jB. Next, we examined the effects of luteolin on the MAPK pathway. To determine which MAPK subtypes regulate the production of the inflammatory mediators, we used three different MAPK inhibitors to block the activation of each MAPK. A specific JNK inhibitor, SP600125 (10 lM), significantly inhibited the production of all the inflammatory mediators (Fig. 4B). However, an ERK inhibitor, PD98059 (10 lM), or a p38 inhibitor, SB239063 (10 lM), only partially inhibited the production. These results indicate that JNK regulates the production of all inflammatory mediators examined
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Fig. 4. Luteolin affected not I-jB degradation but JNK activation in RAW264 macrophages stimulated by 3T3-L1 adipocyte-derived conditioned medium. (A) RAW264 macrophages were pretreated with luteolin (5, 10 lM) for 1 h and then incubated with the hypertrophied 3T3-L1 adipocyte-derived conditioned medium (L1 medium) and luteolin for 30 min. Total cell lysates were extracted and then protein expression levels of I-jB-a and b-actin were analyzed by immunoblotting. (B) Secretion levels of inflammatory mediators: TNFa (left), MCP-1 (middle), and NO (right). RAW264 macrophages were incubated with the conditioned medium derived from the 3T3-L1 adipocytes (L1 medium) and SP600125 (a JNK inhibitor; 10 lM), PD98059 (an ERK inhibitor; 10 lM) or SB239063 (a p38 inhibitor; 10 lM) for 24 h. Protein secretion levels of TNFa and MCP-1 were measured by ELISA. Release of NO was measured using Griess reagent. The values are mean ± S.E.M. of 3–4 tests. *P < 0.05 and **P < 0.01 compared with a vehicle control. (C) RAW264 macrophages were pretreated with luteolin (5, 10 lM) for 1 h and then incubated with the 3T3-L1 adipocyte-derived conditioned medium and luteolin for 30 min. Total cell lysates were extracted and then the expression of phospho-JNK and total-JNK proteins was analyzed by immunoblotting.
in this study from the RAW264 macrophages activated by 3T3-L1 adipocytes. Finally, we investigated the effects of luteolin on the JNK activation. The stimulation of RAW264 with the 3T3-L1 adipocyte-derived conditioned medium resulted in an enhanced phosphorylation of JNK (Fig. 4C). The pretreatment of RAW264 macrophages with 5 and 20 lM luteolin inhibited JNK phosphorylation. These results indicate that luteolin inhibits the activation of JNK to suppress the inflammatory mediators in the contact coculture system, suggesting that a target of the anti-inflammatory effects of luteolin is the MAPK pathway in activated macrophages. 4. Discussion In this study, luteolin suppressed the production of proinflammatory mediators such as TNFa, MCP-1, and NO in a coculture system using 3T3-L1 adipocytes and RAW264 macrophages. Many reports have indicated that obesity is related to obese adipose tissue inflammation, which causes insulin resistance in the liver and adipose tissues [7,22–24]. Although the causes of inflammation related to obesity have not been completely elucidated, macrophages in filtrating into obese adipose tissues seem to play an indispensable role in the inflammation [5,6]. Macrophages infiltrate into obese adipose tissues as induced by MCP1 and then by FFA, both of which are derived from matured adipocytes. The infiltrating and activated macrophages in adipose tissues secrete TNFa to induce additional secretion of MCP-1 and FFA from the matured adipocytes. The cytokine paracrine loop establishes a vicious cycle that aggravates the inflammation and insulin resistance in obese adipose tissues [8,9]. Thus, blockade of this loop is an important target for the treatment of adipose tissue inflammation and insulin resistance. Several studies have already shown that luteolin has anti-inflammatory effects
on LPS-activated macrophages [25]. However, it was unclear whether luteolin can suppress the vicious cycle in adipose tissues to improve insulin resistance. Thus, we have examined the antiinflammatory effects of luteolin on the interaction between adipocytes and macrophages. Our used concentrations were 0.1– 20 lM, which is consistent to the previous studies (3–25 lM). These concentrations were used to LPS-stimulated macrophages in the previous studies. Luteolin showed more effective in our coculture experiments than in the LPS-used ones. This might be because the coculture system is weaker than the LPS system in term of the macrophage activation. Moreover, in our findings, the anti-inflammatory effects of luteolin were more effective in the coculture system than the adipocyte-derived conditioned medium (Figs. 1 and 3). This is because luteolin also suppressed lipolysis in 3T3-L1 adipocytes, which causes additional activation in the coculture system (data not shown), although we should perform further investigation of luteolin’s effects in adipocytes. The results in this study indicate that luteolin inhibits the production of proinflammatory mediators from macrophages, suggesting a possibility that luteolin is a valuable food-derived compound for improving inflammation-related insulin resistance. However, to elucidate whether luteolin can block the cytokine paracrine loop in obese adipose tissues, animal experiments using high fat diet-fed mice are indispensable in the future. In the LPS-induced inflammation, the Toll-like receptor-4 (TLR4) signaling pathway is very important, as it activates both NF-jB and MAPK pathways [26,27]. LPS binds to and activates TLR-4 to induce NF-jB- and AP-1-dependent transcription through I-jB degradation and MAP kinase activation, respectively. NF-jB and AP-1 coordinately stimulate mRNA induction of inflammatory mediators including TNFa, MCP-1, and iNOS. Thus, both transcriptional factors are indispensable for the production of proinflammatory mediators. In this study, luteolin inhibited the JNK activation
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but not I-jB degradation in the activated macrophages. The results are consistent with those of a previous study using LPS-stimulated microglia, in which IL-6 production was suppressed by luteolin through inhibition of JNK activation not I-jB degradation [28]. Moreover, it has been reported that JNK plays a significant role of the productions of inflammatory mediators in the coculture system [8,9]. Although there is a possibility that other MAP kinases (p38 and ERK) are involved in the mechanism underlying the antiinflammatory effect of luteolin, the inhibition of JNK activation seems to be a particular target of the anti-inflammatory effects of luteolin. This is because only inhibition of JNK could suppress the release of all inflammatory mediators derived from the activated macrophages. Various naturally occurring compounds exhibit positive effects on metabolic syndrome. We have already reported several foodderived factors that attenuate insulin resistance, obesity, and atherosclerosis [13–20]. For example, abietic acid and dehydroabietic acid (a derivative of abietic acid) exhibit anti-inflammatory effects similar to those of luteolin [13,15], although the mechanism underlying the effects is completely different from that of luteolin. Abietic acid and dehydroabietic acid serve as agonists of peroxisome proliferator-activated receptor-c (PPARc), which shows anti-inflammatory effects through its activation in macrophages [13,15]. Luteolin did not serve as the agonist in a luciferase reporter assay (data not shown). Recently, it has been reported that a combination of bioactive compounds is very effective in vivo [29]. In particular, a combination of compounds exhibiting different mechanisms by which anti-inflammatory effects are exerted seems to be most efficient. Therefore, luteolin may be suitable for the treatment of metabolic syndrome with PPARc agonists such as abietic acid and dehydroabietic acid, although additional experiments are required. In conclusion, luteolin, a food-derived flavonoid, suppressed the production of proinflammatory mediators such as TNFa, MCP-1, and NO from macrophages in a coculture system using 3T3-L1 adipocytes and RAW264 macrophages. In addition, the target of luteolin was macrophages not adipocytes, because luteolin inhibited the production of proinflammatory mediators from only the macrophages activated by the adipocyte-derived conditioned medium. In the activated macrophages, luteolin inhibited JNK activation to suppress mRNA expression of the mediators. The results indicate that luteolin suppresses the adipocyte-dependent activation of macrophages, suggesting a possibility that luteolin attenuates insulin resistance in obese adipose tissues with low-grade chronic inflammation. Acknowledgements The authors thank Yukiko Tada for technical assistance. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (15081205, 19380074, and 19780096). R.Y. was supported by a grant from the Korean Science and Engineering Foundation (KOSEF R01-2005-000-10408-0). References [1] Miranda, P.J., DeFronzo, R.A., Califf, R.M. and Guyton, J.R. (2005) Metabolic syndrome: definition, pathophysiology, and mechanisms. Am. Heart J. 149, 33–45. [2] Fernández-Real, J.M. and Ricart, W. (2003) Insulin resistance and chronic cardiovascular inflammatory syndrome. Endocr. Rev. 24, 278–301. [3] Lyon, C.J., Law, R.E. and Hsueh, W.A. (2003) Minireview: adiposity, inflammation, and atherogenesis. Endocrinology 144, 2195–2200. [4] Dandona, P., Aljada, A. and Bandyopadhyay, A. (2004) Inflammation: the link between insulin resistance, obesity and diabetes. Trend Immunol. 25, 4–7.
[5] Weisberg, S.P., McCann, D., Desai, M., Rosenbaum, M., Leibel, R.L. and Ferrante, A.W. (2003) Obesity is associated with macrophage accumulation in adipose tissue. J. Clin. Invest. 112, 1796–1808. [6] Xu, H., Barnes, G.T., Yang, Q., Tan, G., Yang, D., Chou, C.J., Sole, J., Nichols, A., Ross, J.S., Tartaglia, L.A. and Chen, H. (2003) Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance. J. Clin. Invest. 12, 1821–1830. [7] Guha, M. and Mackman, N. (2001) LPS induction of gene expression in human monocytes. Cell Signal 13, 85–94. [8] Suganami, T., Nishida, J. and Ogawa, Y. (2005) A paracrine loop between adipocytes and macrophages aggravates inflammatory changes: role of free fatty acids and tumor necrosis factor alpha. Arterioscler. Thromb. Vasc. Biol. 25, 2062–2068. [9] Suganami, T., Tanimoto-Koyama, K., Nishida, J., Itoh, M., Yuan, X., Mizuarai, S., Kotani, H., Yamaoka, S., Miyake, K., Aoe, S., Kamei, Y. and Ogawa, Y. (2007) Role of the Toll-like receptor 4/NF-kappaB pathway in saturated fatty acid-induced inflammatory changes in the interaction between adipocytes and macrophages. Arterioscler. Thromb. Vasc. Biol. 27, 84–91. [10] Teillet, F., Boumendjel, A., Boutonnat, J. and Ronot, X. (2008) Flavonoids as RTK inhibitors and potential anticancer agents. Med. Res. Rev. 28, 715–745. [11] Talhouk, R.S., Karam, C., Fostok, S., El-Jouni, W. and Barbour, E.K. (2007) Antiinflammatory bioactivities in plant extracts. J. Med. Food. 10, 1–10. [12] Middleton, E., Kandaswami, C. and Theoharides, T.C. (2000) The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol. Rev. 52, 673–751. [13] Takahashi, N., Kawada, T., Goto, T., Kim, C.S., Taimatsu, A., Egawa, K., Yamamoto, T., Jisaka, M., Nishimura, K., Yokota, K., Yu, R. and Fushiki, T. (2003) Abietic acid activates PPAR-gamma in RAW264.7 macrophages and 3T3-L1 adipocytes to regulate gene expression involved in inflammation and lipid metabolism. FEBS Lett. 550, 190–194. [14] Hirai, S., Kim, Y.I., Goto, T., Kang, M.S., Yoshimura, M., Obata, A., Yu, R. and Kawada, T. (2007) Inhibitory effect of naringenin chalcone on inflammatory changes in the interaction between adipocytes and macrophages. Life Sci. 81, 1272–1279. [15] Kang, M.S., Hirai, S., Goto, T., Kuroyanagi, K., Ezaki, Y., Takahashi, N. and Kawada, T. (2008) Dehydroabietic acid, herbal terpenoid, acts as ligands for PPARs in macrophages to regulate inflammation. Biochem. Biophys. Res. Commun. 369, 333–338. [16] Takahashi, N., Kawada, T., Yamamoto, T., Goto, T., Taimatsu, A., Aoki, N., Kawasaki, H., Taira, K., Yokoyama, K.K., Kamei, Y. and Fushiki, T. (2002) Overexpression and ribozyme-mediated targeting of transcriptional coactivators, CREB binding protein (CBP) and p300, revealed their indispensable roles in adipocyte differentiation through the regulation of PPAR-gamma. J. Biol. Chem. 277, 16906–16912. [17] Takahashi, N., Kawada, T., Goto, T., Yamamoto, T., Taimatsu, A., Kimura, K., Saitoh, M., Hosokawa, T., Miyashita, K. and Fushiki, T. (2002) Dual action of isoprenols to activate both PPAR-gamma and PPAR-alpha in 3T3-L1 adipocytes and HepG2 hepatocytes. FEBS Lett. 514, 315–322. [18] Goto, T., Takahashi, N., Kato, S., Egawa, K., Ebisu, S., Moriyama, T., Fushiki, T. and Kawada, T. (2005) Phytol, a phytochemical compound, activates PPARalpha and regulates lipid metabolism in PPAR-alpha-expressing cells. Biochem. Biophys. Res. Commun. 337, 440–445. [19] Kuroyanagi, K., Kang, M.S., Goto, T., Kusudo, T., Hirai, S., Yu, R., Yano, M., Sasaki, T., Takahashi, N. and Kawada, T. (2008) Citrus auraptene acts as an agonist for PPARs and enhances adiponectin production and MCP-1 reduction in 3T3-L1 adipocytes. Biochem. Biophys. Res. Commun. 366, 219–225. [20] Takahashi, N., Kang, M.S., Kuroyanagi, K., Goto, T., Kusudo, T., Hirai, S., Yu, R., Yano, M., Sasaki, T., Murakami, S. and Kawada, T. (2008) Auraptene, a citrus fruit compound, regulates gene expression as a PPAR-alpha agonist in HepG2 hepatocytes. BioFactors 33, 25–32. [21] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. [22] Hotamisligil, G.S. (1999) The role of TNFa and TNF receptors in obesity and insulin resistance. J. Int. Med. 245, 621–625. [23] Muoio, D.M. and Newgard, C.B. (2006) Obesity-related derangements in metabolic regulation. Annu. Rev. Biochem. 75, 367–401. [24] de Ferranti, S. and Mozaffarian, D. (2008) The perfect storm: obesity, adipocyte dysfunction, and metabolic consequences. Clin. Chem. 54, 945–955. [25] Xagorari, A., Papapetropoulos, A., Mauromatis, A., Economou, M., Fotsis, T. and Roussos, C. (2001) Luteolin inhibits an endotoxin-stimulated phosphorylation cascade and proinflammatory cytokine production in macrophages. J. Pharmacol. Exp. Ther. 296, 181–187. [26] Lee, J.Y., Sohn, K.H., Rhee, S.H. and Hwang, D. (2001) Saturated fatty acids, but not unsaturated fatty acids, induce the expression of cyclooxygenase-2 mediated through Toll-like receptor 4. J. Biol. Chem. 276, 16683–16689. [27] Shi, H., Kokoeva, M.V., Inouye, K., Tzameli, I., Yin, H. and Flier, J.S. (2006) TLR4 links innate immunity and fatty acid-induced insulin resistance. J. Clin. Invest. 116, 3015–3025. [28] Jang, S., Kelley, K.W. and Johnson, R.W. (2008) Luteolin reduces IL-6 production in microglia by inhibiting JNK phosphorylation and activation of AP-1. Proc. Natl. Acad. Sci. USA 105, 7534–7539. [29] Kiokias, S. and Gordon, M.H. 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Luteolin, a food-derived flavonoid, suppresses adipocyte-dependent activation of macrophages by inhibiting JNK activation Chieko Ando a,1, Nobuyuki Takahashi a,1, Shizuka Hirai a,1, Kanako Nishimura a, Shan Lin a, Taku Uemura a, Tsuyoshi Goto a, Rina Yu b, Joji Nakagami c, Shigeru Murakami c, Teruo Kawada a,* a b c
Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan Department of Food Science and Nutrition, University of Ulsan, Ulsan 680-749, South Korea Taisho Pharmaceutical Co. Ltd., Toshima-ku, Tokyo 170-8633, Japan
a r t i c l e
i n f o
Article history: Received 31 August 2009 Revised 28 September 2009 Accepted 16 October 2009 Available online 23 October 2009 Edited by Laszlo Nagy Keywords: Luteolin Macrophage Adipocyte JNK Inflammation
a b s t r a c t Interaction between adipocytes and macrophages contributes to the development of insulin resistance in obese adipose tissues. In this study, we examined whether luteolin, food-derived flavonoid, could suppress the production of inflammatory mediators of the interaction between adipocytes and macrophages. Experiments using a coculture system of adipocytes and macrophages showed that luteolin suppressed the production of inflammatory mediators. In addition, activated macrophages were targets for the suppressive effect of luteolin. Luteolin inhibited the phosphorylation of JNK and suppressed the production of inflammatory mediators in the activated macrophages. The findings indicate that luteolin can inhibit the interaction between adipocytes and macrophages to suppress the production of inflammatory mediators, suggesting that luteolin is a valuable foodderived compound for the treatment of metabolic syndrome. Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Obesity is often accompanied by hyperglycemia, hypertension, and hyperlipidemia, which together are called metabolic syndrome [1]. Recent studies have elucidated that obesity is characterized by low-grade chronic inflammation of adipose tissues and that such inflammation is one of the potential mechanisms underlying insulin resistance [2–4]. It has been indicated that adipose tissues contain various cells in addition to adipocytes, in which there are paracrine interactions between adipocytes and such non-adipocytes. Infiltration of macrophages is observed in obese adipose tissues [5,6]. The infiltrating macrophages are crucial contributors to the interactions to induce inflammation of obese adipose tissues. Generally, the nuclear factor-jB (NF-jB) pathway and mitogenactivated protein kinase (MAPK) pathway play indispensable roles in inflammation [7]. In obese adipose tissues, both pathways are involved in the production of inflammatory mediators from the activated macrophages and/or adipocytes [8,9]. Thus, the degradation of I-jB and activation of MAPK are valuable markers for the detection of inflammation of adipose tissues.
* Corresponding author. Fax: +81 774 38 3752. E-mail address: [email protected] (T. Kawada). 1 These authors equally contributed to this work.
Suganami and colleagues have reported that adipocyte-derived free fatty acid (FFA), monocyte chemoattractant protein (MCP)-1, and macrophage-derived tumor necrosis factor-a (TNFa) establish a vicious cycle enhancing inflammation and leading to insulin resistance in obese adipose tissue [8,9]. Matured adipocyte-derived MCP-1 enhances the infiltration of macrophages, which are activated by adipocyte-derived FFA to stimulate the release of TNFa from the infiltrating macrophages. The released TNFa in turn stimulates lipolysis in adipocytes to induce overproduction of FFA. Therefore, anti-inflammatory compounds that suppress such an inflammatory vicious cycle may contribute to the reduction of obesity-related insulin resistance. Foods contain various bioactive compounds. Among them, flavonoids are considered to be particularly valuable compounds, with multifunction including antioxidative, anticancer, and antiinflammatory effects [10–12]. Luteolin is a food-derived flavonoid, which is present in medicinal plants and in some vegetables and spices. Luteolin exhibits specific anti-inflammatory effects at micromolar concentrations, which are only partly explained by its antioxidant capacities. A recent review has provided the pharmacological data on the antioxidant, anti-inflammatory, and antiallergy functions of luteolin [12]. However, the effects of luteolin on low-grade chronic inflammation of obese adipose tissues remain to be clarified. Thus, the purpose of this study is to examine
0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2009.10.045
C. Ando et al. / FEBS Letters 583 (2009) 3649–3654
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2. Materials and methods 2.1. Chemicals and cell cultures Luteolin was purchased from Wako Chemicals (Osaka, Japan) and dissolved in DMSO to prepare a stock solution. All other chemicals were purchased from Sigma (MO, USA) and Nacalai Tesque (Kyoto, Japan). A RAW264 macrophage cell line (RIKEN BioResource Center, Tsukuba, Japan) was cultured in DMEM with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 lg/ml streptomycin (Gibco BRL, NY, USA) at 37 °C under a humidified 5% CO2 atmosphere as previously described [13–15]. The serum-free medium of RAW264 macrophages cultured for 24 h was collected as a RAW264 conditioned medium and stored at 20 °C until use. The cells were seeded in 12-well plates (5 105 cells/ml) and treated with various concentrations of luteolin in serum-free medium for 24 h. 3T3-L1 preadipocytes (American Type Culture Collection, VA, USA) were subcultured in DMEM with 10% FBS, 100 U/ml penicillin, and 100 lg/ml streptomycin at 37 °C under a humidified 5% CO2 atmosphere. Differentiation of 3T3-L1 preadipocytes was induced by treatment with adipogenic agents (0.5 mM 3-isobutyl1-methylxanthine, 0.25 lM dexamethasone, and 10 lg/ml insulin) in DMEM containing 10% FBS for 2 days after the cells reached confluence (day 0), as previously described [13,14]. Then, the medium was replaced with DMEM containing 10% FBS and 5 lg/ml insulin and was replaced every 2 days. Twenty days after the differentiation induction, the cells that accumulated large lipid droplets were used as hypertrophied 3T3-L1 adipocytes. The serum-free medium of hypertrophied 3T3-L1 adipocytes cultured for 12 h was collected as a 3T3-L1 conditioned medium and stored at 20 °C until use. RAW264 cells were seeded in 96-well plates and treated with various concentrations of luteolin for 24 h. The viability of RAW264 macrophages was measured by CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, MO, USA). 3T3-L1 cells were seeded in 96-well plates and induced to differentiate in DMEM containing various concentrations of luteolin. The medium was replaced every 2 days and cell viability was measured in the same procedure.
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the anti-inflammatory effects of luteolin on the interaction between adipocytes and macrophages to elucidate whether luteolin is effective for reducing inflammation in obese adipose tissues.
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Fig. 1. Luteolin inhibited inflammation in contact coculture system. Protein secretion levels of inflammatory mediators, TNFa (A) and MCP-1 (B), and release of NO (C). Luteolin was added to the contact coculture system of RAW264 macrophages and 3T3-L1 adipocytes. After 24 h, TNFa and MCP-1 secretion levels in the cultured medium were measured by ELISA. The secretion level of NO was measured using Griess reagent. The values are mean ± S.E.M. of 3–4 tests. *P < 0.05 and **P < 0.01 compared with a vehicle control.
2.2. Stimulation of macrophages and adipocytes Adipocytes and macrophages were cocultured in a contact system, as previously described [14,15]. Briefly, RAW264 cells (1 105 cells/ml) were plated onto dishes with serum-starved and hypertrophied 3T3-L1 cells, and the coculture was incubated in serum-free DMEM for 24 h. RAW264 and 3T3-L1 cells of equal concentrations to those in the coculture were cultured separately as control cultures. Luteolin was added to the coculture at various concentrations, as described in each figure legend. After 24 h of treatment, culture supernatants were collected and stored at 20 °C until use. 2.3. Measurement of inflammatory mediators The concentrations of TNFa and MCP-1 in the culture supernatants were determined by ELISA, which was conducted using Ready-Set-Go Mouse TNFa and MCP-1 kits (eBioscience, CA, USA), in according with the manufacturer’s protocols. NO release was measured using Griess reagent, as described previously
[14,15]. Briefly, 100 ll of supernatant was mixed with an equivalent volume of Griess reagent [1:1 (v/v) of 0.1% N-1-naphthyl-ethylenediamine in distilled water and 1% sulfanilamide in 5% phosphoric acid] on 96-well flat-bottom plates. After 10 min, the absorbance at 570 nm was measured and the amount of nitrite was calculated from the NaNO2 standard curve. Total RNA preparation and quantification of gene expression were performed as previously described [13,14]. The cells were washed with PBS and total RNA was prepared using Sepasol (R)-RNA Super (Nacalai Tesque) in according with the manufacturer’s protocol. Aliquots of total RNA were reverse-transcribed using M-MLV reverse transcriptase (Invitrogen Corp., CA, USA) and a thermal cycler (Takara PCR Thermal Cycler SP: Takara Shuzo Co., Kyoto, Japan). To quantify mRNA expression, real-time RTPCR was performed with a LightCycler System (Roche Diagnostics, Mannheim, Germany) using SYBR Green fluorescence signals. The oligonucleotide primers of inflammatory mediator genes were designed using a PCR primer selection program shown on the
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Fig. 2. Luteolin did not suppress adipocyte inflammation induced by macrophage-derived conditioned medium. Protein secretion and mRNA expression levels of TNFa (A, B) and MCP-1 (C, D). Hypertrophied 3T3-L1 adipocytes were incubated with the conditioned medium of RAW264 macrophages and various concentrations of luteolin for 24 h. The TNFa and MCP-1 secretions in the cultured medium were measured by ELISA. The mRNA expression levels of these mediators were measured by real-time RT-PCR. The values are mean ± S.E.M. of 3–4 tests.
website of the Virtual Genomic Center from the GenBank database. All PCR primers were previously described [13,14]. To compare mRNA expression levels among the samples, the copy numbers of all transcripts were divided by that of mouse 36B4 showing a constant expression level. All mRNA expression levels are presented as a ratio relative to that of a control in each experiment. 2.4. Western blotting Western blotting was performed as previously described [14,16]. In brief, RAW264 cells were washed with PBS and placed immediately in lysis buffer containing 20 mM Tris–HCl (pH 7.5), 15 mM NaCl, 1% Triton X-100 (Nacalai Tesque), and a protease inhibitor cocktail (Nacalai Tesque). The lysate was centrifuged at 15 000 rpm for 5 min, and the supernatant was stored for subsequent analysis. Protein concentration was determined using DC protein assay reagents (BioRad Laboratories, CA, USA) in accordance with the method of Lowry et al. [21]. Fifteen micrograms of protein was separated by 10% SDS–PAGE, and transferred to an Immobilon-P membrane (Millipore, MA, USA). After blocking, the membrane was incubated with an anti-I-jB-a (Santa Cruz Biotechnology, CA, USA), anti-JNK (Cell Signaling Technology, MA, USA), anti-pJNK (Cell Signaling Technology), or b-actin (Cell Signaling Technology) antibody, and then with a secondary antibody conjugated to horseradish peroxidase [anti-rabbit IgG: 1/2000 (Promega, WC, USA) ] for 1 h. The secondary antibody staining was visualized by chemiluminescence immunoassay using a chemiluminescent HRP substrate (Millipore). 2.5. Statistical analyses The data are presented as mean ± S.E.M. and were statistically analyzed using one-way ANOVA when their variances were heterogeneous and unpaired t-test. Differences were considered significant at P < 0.05.
3. Results 3.1. Effects of luteolin on inflammation in coculture of adipocytes and macrophages To examine the effects of luteolin on the interaction between adipocytes and macrophages, we used a coculture system using 3T3-L1 adipocytes and RAW264 macrophages, in which these cells were in direct contact. The coculture revealed marked increases in secretion levels of inflammatory mediators such as TNFa, MCP-1, and NO (Fig. 1A–C). Treatment with luteolin in the coculture system significantly decreased the secretion levels of the inflammatory mediators in a dose-dependent manner. In particular, the secretion of TNFa was markedly suppressed to a control level at a higher concentration of luteolin (20 lM). The concentrations of luteolin used in this experiment did not affect the viability of either RAW264 or 3T3-L1 cells (data not shown). These results indicate that luteolin has anti-inflammatory effects in the contact coculture system of adipocytes and macrophages. 3.2. Effects of luteolin on macrophage activation induced by adipocytederived conditioned medium Next, to clarify the anti-inflammatory effects of luteolin, we conducted medium change experiments. First, to examine the effect of luteolin on 3T3-L1 adipocytes, a conditioned medium of RAW264 macrophages was added to a hypertrophied 3T3-L1 adipocyte culture. The addition of the RAW264 conditioned medium significantly increased the levels of TNFa and MCP-1 protein secretion from 3T3-L1 cells (Fig. 2A and C). mRNA expression levels of the mediators were also increased by the RAW264 conditioned medium (Fig. 2B and D). However, the treatment with luteolin did not suppress the TNFa and MCP-1 expression at both protein secretion and mRNA expression levels. Thus, these results indicate that 3T3-L1 adipocytes are not a target of luteolin.
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Fig. 3. Luteolin suppressed macrophage activation induced by adipocyte-derived conditioned medium. RAW264 macrophages were incubated with the conditioned medium derived from the hypertrophied 3T3-L1 adipocytes (L1 medium) and various concentrations of luteolin for 24 h. The TNFa (A) and MCP-1 (C) protein levels were measured by ELISA. The secretion level of NO (E) was also measured using Griess reagent. The mRNA expression levels of TNFa (B), MCP-1 (D), and iNOS (F) were measured by real-time RTPCR. The values are mean ± S.E.M. of 3–4 tests. *P < 0.05 and **P < 0.01 compared with a vehicle control.
Second, to investigate the anti-inflammatory effect of luteolin on RAW264 macrophages, a conditioned medium of hypertrophied 3T3-L1 adipocytes was added to a RAW264 macrophage culture. The addition of the 3T3-L1 conditioned medium increased the level of TNFa secretion from RAW264 macrophages (Fig. 3A). Treatment with luteolin significantly decreased TNFa secretion level in a dose-dependent manner. The treatment with luteolin also suppressed TNFa mRNA expression (Fig. 3B). In addition to TNFa, the levels of secretion of MCP-1 and release of NO from RAW264 macrophages were increased by the 3T3-L1 conditioned medium (Fig. 3C and E). The increase was reduced to a control level by 20 lM luteolin (Fig. 3C and E). The addition of luteolin also significantly decreased the mRNA expression levels of MCP-1 and inducible-NO synthase (iNOS) (Fig. 3D and F). These anti-inflammatory effects of luteolin were observed in LPS-stimulated productions of inflammatory mediators. LPS (5 lg/ml) resulted in 414-, 15.1-, and 9.37-fold increases in productions of TNFa, MCP-1, and NO, respectively, in RAW264 macrophages. Luteolin (10 lM) inhibited the LPS-dependent productions in TNFa, MCP-1, and NO at 66.5%, 38.2%, and 56.2%, respectively. These effects of luteolin on the LPS-induced inflammation were a dose-dependent manner (data not shown). These results indicate that luteolin has anti-inflammatory effects on macrophages in terms of the interaction between adipocytes and macrophages.
3.3. Effects of luteolin on degradation of I-jB and activation of MAPK NF-jB is one of the major transcription factors that regulate the induction of various inflammatory mediators including TNFa, MCP-1, and iNOS [7]. To clarify the molecular mechanism underlying the anti-inflammatory effects of luteolin, we investigated whether luteolin inhibits I-jB degradation, which activates NFjB, in RAW264 macrophages. I-jB degradation was observed in RAW264 macrophages stimulated by the 3T3-L1 adipocyte-derived conditioned medium (Fig. 4A). However, the pretreatment with luteolin did not inhibit the degradation of I-jB. These results show that luteolin has no effect on I-jB degradation in RAW264 macrophages stimulated by the 3T3-L1 adipocyte-derived conditioned medium, although the conditioned medium promoted the degradation of I-jB. Next, we examined the effects of luteolin on the MAPK pathway. To determine which MAPK subtypes regulate the production of the inflammatory mediators, we used three different MAPK inhibitors to block the activation of each MAPK. A specific JNK inhibitor, SP600125 (10 lM), significantly inhibited the production of all the inflammatory mediators (Fig. 4B). However, an ERK inhibitor, PD98059 (10 lM), or a p38 inhibitor, SB239063 (10 lM), only partially inhibited the production. These results indicate that JNK regulates the production of all inflammatory mediators examined
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Fig. 4. Luteolin affected not I-jB degradation but JNK activation in RAW264 macrophages stimulated by 3T3-L1 adipocyte-derived conditioned medium. (A) RAW264 macrophages were pretreated with luteolin (5, 10 lM) for 1 h and then incubated with the hypertrophied 3T3-L1 adipocyte-derived conditioned medium (L1 medium) and luteolin for 30 min. Total cell lysates were extracted and then protein expression levels of I-jB-a and b-actin were analyzed by immunoblotting. (B) Secretion levels of inflammatory mediators: TNFa (left), MCP-1 (middle), and NO (right). RAW264 macrophages were incubated with the conditioned medium derived from the 3T3-L1 adipocytes (L1 medium) and SP600125 (a JNK inhibitor; 10 lM), PD98059 (an ERK inhibitor; 10 lM) or SB239063 (a p38 inhibitor; 10 lM) for 24 h. Protein secretion levels of TNFa and MCP-1 were measured by ELISA. Release of NO was measured using Griess reagent. The values are mean ± S.E.M. of 3–4 tests. *P < 0.05 and **P < 0.01 compared with a vehicle control. (C) RAW264 macrophages were pretreated with luteolin (5, 10 lM) for 1 h and then incubated with the 3T3-L1 adipocyte-derived conditioned medium and luteolin for 30 min. Total cell lysates were extracted and then the expression of phospho-JNK and total-JNK proteins was analyzed by immunoblotting.
in this study from the RAW264 macrophages activated by 3T3-L1 adipocytes. Finally, we investigated the effects of luteolin on the JNK activation. The stimulation of RAW264 with the 3T3-L1 adipocyte-derived conditioned medium resulted in an enhanced phosphorylation of JNK (Fig. 4C). The pretreatment of RAW264 macrophages with 5 and 20 lM luteolin inhibited JNK phosphorylation. These results indicate that luteolin inhibits the activation of JNK to suppress the inflammatory mediators in the contact coculture system, suggesting that a target of the anti-inflammatory effects of luteolin is the MAPK pathway in activated macrophages. 4. Discussion In this study, luteolin suppressed the production of proinflammatory mediators such as TNFa, MCP-1, and NO in a coculture system using 3T3-L1 adipocytes and RAW264 macrophages. Many reports have indicated that obesity is related to obese adipose tissue inflammation, which causes insulin resistance in the liver and adipose tissues [7,22–24]. Although the causes of inflammation related to obesity have not been completely elucidated, macrophages in filtrating into obese adipose tissues seem to play an indispensable role in the inflammation [5,6]. Macrophages infiltrate into obese adipose tissues as induced by MCP1 and then by FFA, both of which are derived from matured adipocytes. The infiltrating and activated macrophages in adipose tissues secrete TNFa to induce additional secretion of MCP-1 and FFA from the matured adipocytes. The cytokine paracrine loop establishes a vicious cycle that aggravates the inflammation and insulin resistance in obese adipose tissues [8,9]. Thus, blockade of this loop is an important target for the treatment of adipose tissue inflammation and insulin resistance. Several studies have already shown that luteolin has anti-inflammatory effects
on LPS-activated macrophages [25]. However, it was unclear whether luteolin can suppress the vicious cycle in adipose tissues to improve insulin resistance. Thus, we have examined the antiinflammatory effects of luteolin on the interaction between adipocytes and macrophages. Our used concentrations were 0.1– 20 lM, which is consistent to the previous studies (3–25 lM). These concentrations were used to LPS-stimulated macrophages in the previous studies. Luteolin showed more effective in our coculture experiments than in the LPS-used ones. This might be because the coculture system is weaker than the LPS system in term of the macrophage activation. Moreover, in our findings, the anti-inflammatory effects of luteolin were more effective in the coculture system than the adipocyte-derived conditioned medium (Figs. 1 and 3). This is because luteolin also suppressed lipolysis in 3T3-L1 adipocytes, which causes additional activation in the coculture system (data not shown), although we should perform further investigation of luteolin’s effects in adipocytes. The results in this study indicate that luteolin inhibits the production of proinflammatory mediators from macrophages, suggesting a possibility that luteolin is a valuable food-derived compound for improving inflammation-related insulin resistance. However, to elucidate whether luteolin can block the cytokine paracrine loop in obese adipose tissues, animal experiments using high fat diet-fed mice are indispensable in the future. In the LPS-induced inflammation, the Toll-like receptor-4 (TLR4) signaling pathway is very important, as it activates both NF-jB and MAPK pathways [26,27]. LPS binds to and activates TLR-4 to induce NF-jB- and AP-1-dependent transcription through I-jB degradation and MAP kinase activation, respectively. NF-jB and AP-1 coordinately stimulate mRNA induction of inflammatory mediators including TNFa, MCP-1, and iNOS. Thus, both transcriptional factors are indispensable for the production of proinflammatory mediators. In this study, luteolin inhibited the JNK activation
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but not I-jB degradation in the activated macrophages. The results are consistent with those of a previous study using LPS-stimulated microglia, in which IL-6 production was suppressed by luteolin through inhibition of JNK activation not I-jB degradation [28]. Moreover, it has been reported that JNK plays a significant role of the productions of inflammatory mediators in the coculture system [8,9]. Although there is a possibility that other MAP kinases (p38 and ERK) are involved in the mechanism underlying the antiinflammatory effect of luteolin, the inhibition of JNK activation seems to be a particular target of the anti-inflammatory effects of luteolin. This is because only inhibition of JNK could suppress the release of all inflammatory mediators derived from the activated macrophages. Various naturally occurring compounds exhibit positive effects on metabolic syndrome. We have already reported several foodderived factors that attenuate insulin resistance, obesity, and atherosclerosis [13–20]. For example, abietic acid and dehydroabietic acid (a derivative of abietic acid) exhibit anti-inflammatory effects similar to those of luteolin [13,15], although the mechanism underlying the effects is completely different from that of luteolin. Abietic acid and dehydroabietic acid serve as agonists of peroxisome proliferator-activated receptor-c (PPARc), which shows anti-inflammatory effects through its activation in macrophages [13,15]. Luteolin did not serve as the agonist in a luciferase reporter assay (data not shown). Recently, it has been reported that a combination of bioactive compounds is very effective in vivo [29]. In particular, a combination of compounds exhibiting different mechanisms by which anti-inflammatory effects are exerted seems to be most efficient. Therefore, luteolin may be suitable for the treatment of metabolic syndrome with PPARc agonists such as abietic acid and dehydroabietic acid, although additional experiments are required. In conclusion, luteolin, a food-derived flavonoid, suppressed the production of proinflammatory mediators such as TNFa, MCP-1, and NO from macrophages in a coculture system using 3T3-L1 adipocytes and RAW264 macrophages. In addition, the target of luteolin was macrophages not adipocytes, because luteolin inhibited the production of proinflammatory mediators from only the macrophages activated by the adipocyte-derived conditioned medium. In the activated macrophages, luteolin inhibited JNK activation to suppress mRNA expression of the mediators. The results indicate that luteolin suppresses the adipocyte-dependent activation of macrophages, suggesting a possibility that luteolin attenuates insulin resistance in obese adipose tissues with low-grade chronic inflammation. Acknowledgements The authors thank Yukiko Tada for technical assistance. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (15081205, 19380074, and 19780096). R.Y. was supported by a grant from the Korean Science and Engineering Foundation (KOSEF R01-2005-000-10408-0). References [1] Miranda, P.J., DeFronzo, R.A., Califf, R.M. and Guyton, J.R. (2005) Metabolic syndrome: definition, pathophysiology, and mechanisms. Am. Heart J. 149, 33–45. [2] Fernández-Real, J.M. and Ricart, W. (2003) Insulin resistance and chronic cardiovascular inflammatory syndrome. Endocr. Rev. 24, 278–301. [3] Lyon, C.J., Law, R.E. and Hsueh, W.A. (2003) Minireview: adiposity, inflammation, and atherogenesis. Endocrinology 144, 2195–2200. [4] Dandona, P., Aljada, A. and Bandyopadhyay, A. (2004) Inflammation: the link between insulin resistance, obesity and diabetes. Trend Immunol. 25, 4–7.
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