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Inhibitory effect of naringenin chalcone on inflammatory changes in the interaction between adipocytes and macrophages
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Life Sciences 81 (2007) 1272 – 1279 www.elsevier.com/locate/lifescie
Inhibitory effect of naringenin chalcone on inflammatory changes in the interaction between adipocytes and macrophages Shizuka Hirai a , Young-II Kim a , Tsuyoshi Goto a , Min-Sook Kang a , Mineka Yoshimura b , Akio Obata b , Rina Yu c , Teruo Kawada a,⁎ a
Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan b Research & Development Division, Kikkoman Corporation, Noda, Chiba 278-0037, Japan c Department of Food Science and Nutrition, University of Ulsan, Ulsan 680-749, South Korea Received 13 July 2007; accepted 4 September 2007
Abstract Obese adipose tissue is characterized by an enhanced infiltration of macrophages. It is considered that the paracrine loop involving monocyte chemoattractant protein (MCP)-1 and tumor necrosis factor (TNF)-α between adipocytes and macrophages establishes a vicious cycle that augments the inflammatory changes and insulin resistance in obese adipose tissue. Polyphenols, which are widely distributed in fruit and vegetables, can act as antioxidants and some of them are also reported to have anti-inflammatory properties. Tomato is one of the most popular and extensively consumed vegetable crops worldwide, which also contains many flavonoids, mainly naringenin chalcone. We investigated the effect of flavonoids, including naringenin chalcone, on the production of proinflammatory mediators in lipopolysaccharide (LPS)-stimulated macrophages and in the interaction between adipocytes and macrophages. Naringenin chalcone inhibited the production of TNF-α, MCP-1, and nitric oxide (NO) by LPS-stimulated RAW 264 macrophages in a dose-dependent manner. Coculture of 3T3-L1 adipocytes and RAW 264 macrophages markedly enhanced the production of TNF-α, MCP-1, and NO compared with the control cultures; however, treatment with naringenin chalcone dose-dependently inhibited the production of these proinflammatory mediators. These results indicate that naringenin chalcone exhibits antiinflammatory properties by inhibiting the production of proinflammatory cytokines in the interaction between adipocytes and macrophages. Naringenin chalcone may be useful for ameliorating the inflammatory changes in obese adipose tissue. © 2007 Elsevier Inc. All rights reserved. Keywords: Naringenin chalcone; MCP-1; TNF-α; NO; Adipocyte; Macrophage
Introduction Obesity is recognized as a risk factor for insulin resistance, cardiovascular diseases, and type 2 diabetes (Miranda et al., 2005). Recent studies indicate that obesity is associated with chronic, low-grade inflammation, suggesting that inflammation may be a potential mechanism by which obesity leads to insulin resistance (Fernandez and Ricart, 2003; Lyon et al., 2003; Dandona et al., 2004). Adipose tissue is composed of various cell types; mature adipocytes (30%–60%) (Klaus and Keijer, 2004; Klaus, 2004), ⁎ Corresponding author. Tel.: +81 774 38 3751; fax: +81 774 38 3752. E-mail address: [email protected] (T. Kawada). 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.09.001
preadipocytes, fibroblasts, endothelial cells, vascular cells, and macrophages. This implies paracrine interactions between adipocytes and nonadipocyte cells. Recent studies have demonstrated that obese adipose tissue is characterized by an enhanced infiltration of macrophages (Weisberg et al., 2003; Xu et al., 2003). Macrophages produce various inflammatory proteins including tumor necrosis factor (TNF)-α and nitric oxide (NO), and it has been shown that TNF-α and inducible nitric oxide synthase (iNOS) expression levels increase in obese adipose tissues (Hotamisligil et al., 1993; Perreault and Marette, 2001; Engeli et al., 2007). Chemokines, such as monocyte chemoattractant protein (MCP)-1, a member of the CC chemokine superfamily, play a pivotal role in monocyte/macrophage trafficking and activation. The major source of MCP-1 is immune cells such as monocytes
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and macrophages, but endothelial cells and adipocytes are also reported to produce MCP-1 (Xu et al., 2003; Kanda et al., 2006). Yu et al. (2006) reported that MCP-1 expression level in adipose tissue is higher in obese animals than in nonobese animals and mesenteric adipose tissue produced the highest level of MCP-1 among several different fat pads. These results suggest that MCP-1 plays a crucial role in adipose tissue inflammatory responses by activating and inducing the infiltration of macrophages into adipose tissues. Suganami et al. (2005) reported that the pacrine loop involving adipocytederived free fatty acid (FFA) and macrophage-derived TNF-α establishes a vicious cycle that augments the inflammatory changes and insulin resistance in obese adipose tissue. Therefore, to prevent obesity-related inflammation, it is important to decrease the production of MCP-1, which induces the migration of macrophages, and other proinflammatory factors, including TNF-α and FFA, in adipose tissue. Tomato is one of the most popular and extensively consumed vegetable crops in the world. There is evidence that regular tomato consumption decreases the incidence of chronic degenerative diseases such as cardiovascular diseases (Pandey et al., 1995) and platelet aggregation in type 2 diabetes (Sheryl and Lazarus, 2004). These beneficial effects of tomato are generally attributed to the different antioxidant molecules such as carotenoids, vitamins, and flavonoids. Flavonoids, which are widely distributed in fruit and vegetables, are also reported to have anti-inflammatory properties. The main flavonoids found in tomato are naringenin chalcone, naringenin, and rutin, which accumulate almost exclusively in peel. Flavanone naringenin, also abundant in citrus fruit, exerts antioxidant (van Acker et al., 2000), antiproliferative (So et al., 1996), and anti-inflammatory (Lyu and Park, 2005) effects. Rutin, a flavonol glycoside also found in citrus fruit peels is one of the metabolites of naringenin. Naringenin chalcone is an intermediate in the biosynthesis of flavonols and is converted into naringenin by chalcone isomerase (Muir et al., 2001). Some chalcones have been reported to have antiallergic, antioxidative, and anti-inflammatory effects (Lee et al., 2004, 2006; Hatziieremia et al., 2006). Naringenin chalcone accumulated in tomato peels is also reported to suppress allergic reactions through the inhibition of histamine release (Yamamoto et al., 2004); however, the effect of naringenin chalcone on inflammation has been unclear. Here, we investigated the effect of naringenin chalcone, comparing to the effect of naringenin and rutin, on the production of proinflammatory mediators in lipopolysaccharide (LPS)stimulated macrophages and in the interaction between adipocytes and macrophages. Materials and methods
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from naringenin, and separated from unconverted naringenin by preparative HPLC. All the components were dissolved in dimethyl sulfoxide to prepare stock solutions (200 mM). Cell culture The RAW 264 macrophage cell line, obtained from RIKEN BioResource Center (Tsukuba, Japan), was cultured in Dulbecco's modified Eagle's medium (DMEM) (Sigma) with 10% fetal bovine serum (FBS) (JRH Bioscience, Kansas, USA) and 100 U/ ml penicillin/100 μg/ml streptomycin (Gibco BRL, NY, USA) at 37 °C under a humidified 5% CO2 atmosphere. The cells were seeded in 12-well plates (1 × 106 cells/ml), and treated with 5 μg/ ml LPS (Sigma) and various concentrations of naringenin chalcone, naringenin, or rutin in serum-free medium for 24 h. 3T3-L1 preadipocytes (American Type Culture Collection, Manassas, VA, USA) were subcultured in DMEM with 10% FBS (Biological Industries, Kibbutz Beit Haemek, Israel), 100 U/ml penicillin/100 μg/ml streptomycin at 37 °C under a humidified 5% CO2 atmosphere. Differentiation of 3T3-L1 preadipocytes was induced by adipogenic agents (0.5 mM 3isobutyl-1-methylxanthine (Nacalai Tesque, Kyoto, Japan), 0.25 μM dexamethasone (Sigma), and 10 μg/ml insulin (Sigma)) in DMEM containing 10% FBS for 2 days after the cells reached confluence (day 0). Then, the medium was replaced with DMEM containing 10% FBS and 5 μg/ml insulin, and was changed every two days. Twenty days after induction of differentiation, the cells that accumulated large lipid droplets were used as hypertrophied 3T3-L1 adipocytes. Coculture of adipocytes and macrophages Adipocytes and macrophages were cocultured in a contact system as previously described (Suganami et al., 2005). Briefly, RAW 264 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. RAW 264 and 3T3L1 cells of equal numbers to those in the coculture were cultured separately as control cultures. Naringenin chalcone, naringenin, or rutin was added to the coculture at various concentrations as shown in each figure. After 24 h of treatment, culture supernatants were collected and stored at −20 °C until measurements. Measurement of MCP-1 and TNF-α production The concentrations of MCP-1 and TNF-α in the culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). ELISA was conducted using a READY-SET-GO! Mouse MCP-1 and TNF-α (eBioscience, CA, USA), according to the manufacturer's protocol.
Chemical reagent Measurement of nitric oxide release Naringenin and rutin were purchased from Sigma (MO, USA) and Wako Chemicals (Osaka, Japan), respectively. Naringenin chalcone was prepared by the method described by Miranda et al. (2000) with some modification. Briefly, naringenin chalcone was obtained by isomerization with 5% NaOH at 60 °C for 15 min
The amount of nitrite in cell-free culture supernatants was measured using Griess reagent (Granger et al., 1996). Briefly, 100 μl of supernatant was mixed with an equivalent volume of Griess reagent [1:1 (v/v) of 0.1% N-1-naphthyl-ethylenediamine
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Cell viability assay Cells were seeded in 96-well plates (1 × 105 cells/well), and treated with various concentrations of naringenin chalcone, naringenin, or rutin in the presence or absence of LPS for 24 h. The viability of RAW 264 macrophages was measured by CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega). Statistical analyses Data were expressed as means ± SE, and were statistically analyzed using one-way ANOVA and Turkey's multiple comparison tests. Statistical significance was set at P b 0.01.
Fig. 1. Chemical structures of naringenin chalcone, naringenin, and rutin.
in distilled water and 1% sulfanilamide in 5% phosphoric acid] on a 96-well flat bottom plate. After 10 min, absorbance at 570 nm was measured, and the amount of nitrite was calculated from the NaNO2 standard curve. Western blotting RAW 264 cells were carefully washed twice with ice-cold phosphate buffered saline (PBS) and placed immediately in lysis buffer containing 20 mM Tris HCl (pH 7.5), 15 mM NaCl, 1% Triton × 100 (Nacalai Tesque), and protease inhibitor cocktail (Nacalai Tesque). Lysate was centrifuged at 15,000 rpm for 5 min, and the supernatant was stored for subsequent analysis. Protein concentration of cell lysate was determined using DC protein assay reagents (BioRad Laboratories, CA, USA) on the basis of the method of Lowry et al. (1951). Fifteen micrograms of protein was subjected to 10% SDS-PAGE and separated products were transferred to an Immobilon-P membrane (Millipore, MA, USA). After blocking with 3% skim milk in PBS/0.1% Tween 20, the membrane was incubated with monoclonal anti-iNOS antibody (BD Transduction Laboratories, CA, USA) or anti-IκB-α antibody (Santa Cruz Biotechnology, CA, USA) overnight, and then with a secondary antibody conjugated to horseradish peroxidase (anti-mouse IgG (Upstate, NY, USA) for iNOS, or anti-rabbit IgG (Promega, WC, USA) for IκB-α (1/2000) ) for 1 h. Secondary antibody was visualized using chemiluminescence with an ECL Western blotting detection reagent (Amersham Biosciences, NJ, USA).
Fig. 2. Effects of naringenin chalcone and its metabolites on the secretion of MCP-1 by LPS-stimulated RAW 264 macrophages. RAW 264 cells were stimulated with LPS (5 μg/ml) and incubated with naringenin chalcone (A), naringenin (B), or rutin (C) for 24 h. MCP-1 level in the culture medium was measured by ELISA. Values are means ± SE of four replicants. ⁎P b 0.01 and ⁎⁎P b 0.001 vs culture treated with LPS alone, as analyzed by one-way ANOVA and Turkey's multiple comparison tests.
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Results Effects of naringenin chalcone and its metabolites on MCP-1 and TNF-α production by RAW 264 macrophages Regulation of proinflammatory chemokines and cytokines induced by inflammatory stimuli serves as a key mechanism of inflammatory control. To determine whether naringenin chalcone (Fig. 1A) suppresses MCP-1 and TNF-α production in macrophages, RAW 264 macrophages were stimulated with LPS in the presence or absence of naringenin chalcone or its metabolites, naringenin (Fig. 1B) or rutin (Fig. 1C). None of the tested flavonoids affected the viability of RAW 264 cells (data not shown). Naringenin chalcone significantly inhibited LPSinduced MCP-1 production by RAW 264 cells in a dose-
Fig. 4. Effects of naringenin chalcone and its metabolites on the expression of nitric oxide and inducible nitric oxide synthase (iNOS) by LPS-stimulated RAW 264 macrophages. RAW 264 cells were stimulated with LPS (5 μg/ml) and incubated with naringenin chalcone (A), naringenin (B), or rutin (C) for 24 h. Secretion level of nitric oxide was measured. iNOS protein level was analyzed by Western blotting. Values are means ± SE of four replicants. ⁎P b 0.01 and ⁎⁎P b 0.001 vs culture treated with LPS alone, as analyzed by one-way ANOVA and Turkey's multiple comparison tests.
Fig. 3. Effects of naringenin chalcone and its metabolites on the secretion of TNF-α by LPS-stimulated RAW 264 macrophages. RAW 264 cells were stimulated with LPS (5 μg/ml) and incubated with naringenin chalcone (A), naringenin (B), or rutin (C) for 24 h. The level of TNF-α in the culture medium was measured by ELISA. Values are means ± SE of four replicants. ⁎P b 0.01 and ⁎⁎P b 0.001 vs culture treated with LPS alone, as analyzed by one-way ANOVA and Turkey's multiple comparison tests.
dependent manner (Fig. 2A). Naringenin also dose-dependently inhibited MCP-1 production by LPS-stimulated RAW cells (Fig. 2B). On the other hand, the inhibitory effect of rutin on the production of MCP-1 was moderate and showed no dose dependence (Fig. 2C). Naringenin chalcone and naringenin also inhibited TNF-α production by LPS-stimulated RAW 264 cells (Fig. 3A, B); however, rutin did not affect the production of TNF-α (Fig. 3C).
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a marked increase in MCP-1 level (Fig. 6A). Naringenin chalcone or naringenin treatment in this coculture significantly decreased MCP-1 secretion level (Fig. 6A). The inhibitory effect of rutin on the secretion of MCP-1 was moderate and showed no dose dependence (Fig. 6A). The coculture of differentiated 3T3-L1 adipocytes and RAW 264 cells showed an enhanced TNF-α secretion about threefold that of the control culture (Fig. 6B). Naringenin chalcone and naringenin treatment significantly decreased TNF-α secretion level even at the lowest concentration (50 μM) (Fig. 6B). Rutin at 50 μM did not effect the production of TNF-α, however, higher concentrations of rutin significantly suppressed the secretion of TNF-α (Fig. 6 similar effects of naringenin chalcone and its metabolites on NO
Fig. 5. Effects of naringenin chalcone and its metabolites on IκB-α degradation in LPS-stimulated RAW 264 macrophages. RAW 264 cells were stimulated with LPS (5 μg/ml) and incubated with naringenin chalcone (A), naringenin (B), or rutin (C) for 30 min. Total cell lysates were extracted from cultured RAW 264 cells, and IκB-α protein expression was analyzed by Western blotting.
Effects of naringenin chalcone and its metabolites on the nitrite oxide production and iNOS expression level Naringenin chalcone and naringenin significantly decreased NO production in a dose-dependent manner (Fig. 4 A, B), but the inhibitory effect of rutin on the production of NO was moderate and showed no dose dependence (Fig. 4C). The level of iNOS expression by LPS-stimulated RAW 264 cells was dose-dependently decreased by naringenin chalcone or naringenin treatment (Fig. 4A, B); however, rutin did not affect iNOS protein expression level (Fig. 4C). Effects of naringenin chalcone and its metabolites on the degradation of IκB-α NF-κB is one of the major transcription factors that regulate the induction of MCP-1, TNF-α, and iNOS. To clarify the molecular mechanism underlying the anti-inflammatory action of naringenin chalcone, we investigated whether naringenin chalcone and its metabolites inhibit IκB-α degradation in LPSstimulated RAW 264 cells, which leads to the activation of NFκB. LPS treatment for 30 min markedly promoted IκB-α degradation (Fig. 5). The treatment with naringenin chalcone (Fig. 5A) or naringenin (Fig. 5B) suppressed the degradation of IκB-α, although dose-dependent effects were not observed. Rutin showed no effect or only exerted a moderate inhibition of IκB-α degradation in LPS-stimulated RAW 264 cells (Fig. 5C). Effects of naringenin chalcone and its metabolites on inflammatory changes by coculture of adipocytes and macrophages Secretion of MCP-1 by differentiated 3T3-L1 cells or RAW 264 cells was very low when they were cultured separately; however, coculture of these cells in the contact system revealed
Fig. 6. Effects of naringenin chalcone and its metabolites on inflammatory changes induced by the coculture of 3T3-L1 adipocytes and RAW 264 macrophages. Differentiated 3T3-L1 adipocytes were cocultured with RAW 264 macrophages (1 × 105 cells/well) for 24 h. MCP-1 (A) and TNF-α (B) levels in the coculture medium were measured by ELISA. Secretion level of nitric oxide (C) was also measured. Values are means ± SE of four replicants. ⁎P b 0.01 and ⁎⁎P b 0.001 vs non-treated coculture, as analyzed by one-way ANOVA and Turkey's multiple comparison tests.
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secretion were also observed; naringenin chalcone and naringenin dose-dependently suppressed coculture-induced NO secretion, but the inhibitory effect of rutin on the secretion of NO was moderate and showed no dose dependence (Fig. 6C). Discussion In the present study, we demonstrated that naringenin chalcone not only suppresses the production of proinflammatory cytokines in activated macrophages, but also the inflammatory changes in the interaction between adipocytes and macrophages. This implies that naringenin chalcone can inhibit inflammatory changes in obese adipose tissue. Obesity is associated with chronic, low-grade inflammation. Recent studies have demonstrated that obese adipose tissue is characterized by an enhanced infiltration of macrophages, suggesting that they are an important source of inflammation in adipose tissue (Weisberg et al., 2003; Xu et al., 2003). Therefore, it is important to inhibit the secretion of chemokines and proinflammatory factors from adipose tissue to prevent the migration of macrophages and subsequent chronic inflammation. Many flavonoids have been shown to have biological activities, including anti-inflammatory properties. Naringenin chalcone, which is abundant in tomato peels, is an intermediate in the biosynthesis of flavonols and is converted into naringenin by chalcone isomerase (Muir et al., 2001). Naringenin chalcone is reported to have antiallergic activities via modulation of immune cell activities (Yamamoto et al., 2004). Flavanone naringenin, also abundant in tomato and citrus fruit, is reported to suppress NO production and TNF-α secretion from RAW 264.7 macrophages (Lyu and Park, 2005). Our data showed that naringenin chalcone and naringenin significantly inhibited proinflammatory mediators such as MCP-1, TNF-α, and NO in LPS-stimulated RAW 264 macrophages. Furthermore, the inhibitory effect of naringenin chalcone was stronger than that of naringenin, indicating that naringenin chalcone induces strong anti-inflammatory activities. Because adipose tissue macrophages play a crucial role in augmenting inflammatory responses in obese adipose tissue, naringenin chalcone may be used for suppressing macrophageinduced inflammation in adipose tissue. On the other hand, the effects of rutin on the production of these proinflammatory mediators were not observed or were very weak, which is consistent with previous reports (Lyu and Park, 2005; Shen et al., 2002). Rutin, a flavonol glycoside commonly found in tomato and citrus fruit peels is one of the metabolites of naringenin. Rutin glycoside is too polar for cells to take up in vitro (Shen et al., 2002), although rutin can be absorbed when rutinose located at the C3 site of rutin is hydrolyzed in the small intestine by βglycosidase to quercetin (Morand et al., 2000), and exerts its antiinflammatory activities in vivo (Afana'seva et al., 2001; Shen et al., 2002). This may explain the ineffectiveness of rutin to induce macrophage responses. In various cell types, NO is produced by iNOS in inflammation (Nathan and Xie, 1994; Xie and Nathan, 1994). Naringenin is reported to inhibit NO production by reducing iNOS expression in RAW 264 cells (Tsai et al., 1999). Our data showed that naringenin chalcone, as well as naringenin, inhibited iNOS
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expression in LPS-stimulated RAW 264 cells, indicating that the suppressive effect of naringenin chalcone on the secretion of NO is also associated with decreased iNOS expression level. NF-κB is one of the major transcription factors that regulate the induction of MCP-1 (Wang et al., 2000), TNF-α (Tsai et al., 2000; Vallejo et al., 2000), and iNOS (Xie et al., 1994). In the unstimulated form, NF-κB is present in the cytosol bound to the inhibitory protein IκB. After the stimulation of cells by various agents, IκB becomes phosphorylated and this triggers a proteolytic degradation of IκB, leading to the activation of NF-κB (Yamamoto and Gaynor, 2001). Our data revealed that naringenin chalcone and naringenin inhibited the LPS-induced degradation of IκB-α in activated RAW 264 macrophages; however, this inhibitory effect was weak and LPS-induced degradation of IκB-α was not completely rescued even after the treatment with naringenin chalcone or naringenin at the highest concentration. Hatziieremia et al. (2006) reported that cardamonin, a chalcone derivative, inhibited LPS-induced NF-κB DNA binding without affecting the degradation of IκB-α or phosphorylation of NF-κB, suggesting a direct effect of chalcone on the transcription factor binding to DNA. Interestingly, some flavonoids including cardamonin (Hatziieremia et al., 2006), anthocyanidins (Hou et al., 2005), and green tea polyphenol (Hou et al., 2007) are reported to inhibit other transcription factors such as activator protein 1 (AP-1) and CCAAT/enhancer-binding protein (C/EBP) δ, which are identified to bind the cis-acting elements in the promoter of MCP-1 (Sekine et al., 2002), TNF-α (Tsai et al., 2000; Vallejo et al., 2000), and iNOS (Xie et al., 1994). Therefore, the antiinflammatory action of naringenin chalcone and naringenin may involve the inhibitory action of those transcription factors. Recently, it was reported that activation of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) which catalyzes the conversion of cortisone to active cortisol in adipocytes and macrophages is critically involved in dysfunction of adipose tissue associated with inflammation in obese patients (Ishii et al., 2007). Naringenin and some bioflavonoids are reported to inhibit 11βHSD in guinea pig kidney (Zhang et al., 1994; Zhang and Wang, 1997), thus in the present study, naringenin might inhibit the production of proinflammatory mediators via suppressing 11βHSD in RAW 264 macrophages. Further studies are required to clarify the exact mechanisms. It has been shown that paracrine interactions between adipocytes and nonadipocyte cells such as macrophages augment the inflammatory response of adipose tissue in obesity (Weisberg et al., 2003; Xu et al., 2003). Our data showed that the coculture of hypertrophied 3T3-L1 adipocytes and RAW 264 macrophages resulted in a marked upregulation of the expression of proinflammatory mediators, namely, MCP-1, TNF-α, and NO, which is consistent with a previous report (Suganami et al., 2005). Moreover, naringenin chalcone and naringenin significantly suppressed the coculture-induced upregulation of MCP-1, TNFα, and NO, although the suppressive effect of rutin on MCP-1 and NO production was moderate and showed no dose dependence. These results indicate that naringenin chalcone and naringenin can directly suppress chronic inflammatory responses in obese adipose tissue. In addition, adipocyte-derived FFA is another important mediator of the establishment of a vicious cycle that
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augments the inflammatory changes in obese adipose tissue (Suganami et al., 2005). Because naringenin has no effect on the lipolysis of 3T3-L1 adipocytes (Harmon and Harp, 2001), the inhibitory actions of naringenin chalcone and naringenin on the production of proinflammatory mediators such as MCP-1, TNFα, and NO may be more important than their action in the suppression of FFA release from adipocytes, which in turn contribute to the suppression of chronic inflammatory responses in obese adipose tissue. In obese adipose tissue, it is considered that both adipocytes and macrophages can be the sources of MCP1 (Bruun et al., 2005; Fain and Madan, 2005; Yu et al., 2006; Suganami et al., 2005), although the level of MCP-1 produced by the separately cultured 3T3-L1 adipocytes (15 ng/ml) and RAW 264 macrophages (6.8 ng/ml) was very low compared with that by cocultured cells (1156 ng/ml). On the other hand, we could not detect TNF-α in the culture medium of 3T3-L1 adipocytes, thus TNF-α is mainly derived from macrophages (Suganami et al., 2005). In this study, naringenin chalcone and naringenin inhibited MCP-1 and/or TNF-α production by RAW 264 macrophages; thus, naringenin chalcone and naringenin may inhibit the cocultureinduced MCP-1 production by both 3T3-L1 cells and RAW 264 cells, as well as TNF-α production mainly by RAW 264 cells. MCP-1 plays a crucial role in the augmentation of inflammatory responses in obesity by enhancing the migration of monocytes into adipose tissue and activation into macrophages (Yu et al., 2006). The coculture system we used in the present study could not reveal whether naringenin chalcone or naringenin affected the migration of RAW 264 cells to adipocytes. However, these flavonoids may prevent the recruitment of monocytes into obese adipose tissue by reducing the production of MCP-1 by adipocytes and macrophages in vivo. Further study is needed to clarify the effect of naringenin chalcone on the migration of monocytes. In summary, the results presented here showed that naringenin chalcone suppressed the production of proinflammatory mediators by activated macrophages and/or adipocytes cocultured with macrophages. Naringenin chalcone may be a useful phytochemical for suppressing the vicious cycle of chronic inflammation in obese adipose tissue, and improve obesity-related insulin resistance. Acknowledgements This work was supported by the Research and Development Program for New Bio-industry Initiatives and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sport, Science and Technology of Japan (15081205, 19380074 and 19780096). RY was supported by a grant from Korean Science and Engineering Foundation (KOSEF R01-2005-000-10408-0). References Afanas'eva, I.B., Ostrakhovitch, E.A., Mikhal'chik, E.V., Ibragimova, G.A., Korkina, L.G., 2001. Enhancement of antioxidant and anti-inflammatory activities of bioflavonoid rutin by complexation with transition metals. Biochemical Pharmacology 61 (6), 677–684. Bruun, J.M., Lihn, A.S., Pedersen, S.B., Richelsen, B., 2005. Monocyte chemoattractant protein-1 release is higher in visceral than subcutaneous
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Life Sciences 81 (2007) 1272 – 1279 www.elsevier.com/locate/lifescie
Inhibitory effect of naringenin chalcone on inflammatory changes in the interaction between adipocytes and macrophages Shizuka Hirai a , Young-II Kim a , Tsuyoshi Goto a , Min-Sook Kang a , Mineka Yoshimura b , Akio Obata b , Rina Yu c , Teruo Kawada a,⁎ a
Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan b Research & Development Division, Kikkoman Corporation, Noda, Chiba 278-0037, Japan c Department of Food Science and Nutrition, University of Ulsan, Ulsan 680-749, South Korea Received 13 July 2007; accepted 4 September 2007
Abstract Obese adipose tissue is characterized by an enhanced infiltration of macrophages. It is considered that the paracrine loop involving monocyte chemoattractant protein (MCP)-1 and tumor necrosis factor (TNF)-α between adipocytes and macrophages establishes a vicious cycle that augments the inflammatory changes and insulin resistance in obese adipose tissue. Polyphenols, which are widely distributed in fruit and vegetables, can act as antioxidants and some of them are also reported to have anti-inflammatory properties. Tomato is one of the most popular and extensively consumed vegetable crops worldwide, which also contains many flavonoids, mainly naringenin chalcone. We investigated the effect of flavonoids, including naringenin chalcone, on the production of proinflammatory mediators in lipopolysaccharide (LPS)-stimulated macrophages and in the interaction between adipocytes and macrophages. Naringenin chalcone inhibited the production of TNF-α, MCP-1, and nitric oxide (NO) by LPS-stimulated RAW 264 macrophages in a dose-dependent manner. Coculture of 3T3-L1 adipocytes and RAW 264 macrophages markedly enhanced the production of TNF-α, MCP-1, and NO compared with the control cultures; however, treatment with naringenin chalcone dose-dependently inhibited the production of these proinflammatory mediators. These results indicate that naringenin chalcone exhibits antiinflammatory properties by inhibiting the production of proinflammatory cytokines in the interaction between adipocytes and macrophages. Naringenin chalcone may be useful for ameliorating the inflammatory changes in obese adipose tissue. © 2007 Elsevier Inc. All rights reserved. Keywords: Naringenin chalcone; MCP-1; TNF-α; NO; Adipocyte; Macrophage
Introduction Obesity is recognized as a risk factor for insulin resistance, cardiovascular diseases, and type 2 diabetes (Miranda et al., 2005). Recent studies indicate that obesity is associated with chronic, low-grade inflammation, suggesting that inflammation may be a potential mechanism by which obesity leads to insulin resistance (Fernandez and Ricart, 2003; Lyon et al., 2003; Dandona et al., 2004). Adipose tissue is composed of various cell types; mature adipocytes (30%–60%) (Klaus and Keijer, 2004; Klaus, 2004), ⁎ Corresponding author. Tel.: +81 774 38 3751; fax: +81 774 38 3752. E-mail address: [email protected] (T. Kawada). 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.09.001
preadipocytes, fibroblasts, endothelial cells, vascular cells, and macrophages. This implies paracrine interactions between adipocytes and nonadipocyte cells. Recent studies have demonstrated that obese adipose tissue is characterized by an enhanced infiltration of macrophages (Weisberg et al., 2003; Xu et al., 2003). Macrophages produce various inflammatory proteins including tumor necrosis factor (TNF)-α and nitric oxide (NO), and it has been shown that TNF-α and inducible nitric oxide synthase (iNOS) expression levels increase in obese adipose tissues (Hotamisligil et al., 1993; Perreault and Marette, 2001; Engeli et al., 2007). Chemokines, such as monocyte chemoattractant protein (MCP)-1, a member of the CC chemokine superfamily, play a pivotal role in monocyte/macrophage trafficking and activation. The major source of MCP-1 is immune cells such as monocytes
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and macrophages, but endothelial cells and adipocytes are also reported to produce MCP-1 (Xu et al., 2003; Kanda et al., 2006). Yu et al. (2006) reported that MCP-1 expression level in adipose tissue is higher in obese animals than in nonobese animals and mesenteric adipose tissue produced the highest level of MCP-1 among several different fat pads. These results suggest that MCP-1 plays a crucial role in adipose tissue inflammatory responses by activating and inducing the infiltration of macrophages into adipose tissues. Suganami et al. (2005) reported that the pacrine loop involving adipocytederived free fatty acid (FFA) and macrophage-derived TNF-α establishes a vicious cycle that augments the inflammatory changes and insulin resistance in obese adipose tissue. Therefore, to prevent obesity-related inflammation, it is important to decrease the production of MCP-1, which induces the migration of macrophages, and other proinflammatory factors, including TNF-α and FFA, in adipose tissue. Tomato is one of the most popular and extensively consumed vegetable crops in the world. There is evidence that regular tomato consumption decreases the incidence of chronic degenerative diseases such as cardiovascular diseases (Pandey et al., 1995) and platelet aggregation in type 2 diabetes (Sheryl and Lazarus, 2004). These beneficial effects of tomato are generally attributed to the different antioxidant molecules such as carotenoids, vitamins, and flavonoids. Flavonoids, which are widely distributed in fruit and vegetables, are also reported to have anti-inflammatory properties. The main flavonoids found in tomato are naringenin chalcone, naringenin, and rutin, which accumulate almost exclusively in peel. Flavanone naringenin, also abundant in citrus fruit, exerts antioxidant (van Acker et al., 2000), antiproliferative (So et al., 1996), and anti-inflammatory (Lyu and Park, 2005) effects. Rutin, a flavonol glycoside also found in citrus fruit peels is one of the metabolites of naringenin. Naringenin chalcone is an intermediate in the biosynthesis of flavonols and is converted into naringenin by chalcone isomerase (Muir et al., 2001). Some chalcones have been reported to have antiallergic, antioxidative, and anti-inflammatory effects (Lee et al., 2004, 2006; Hatziieremia et al., 2006). Naringenin chalcone accumulated in tomato peels is also reported to suppress allergic reactions through the inhibition of histamine release (Yamamoto et al., 2004); however, the effect of naringenin chalcone on inflammation has been unclear. Here, we investigated the effect of naringenin chalcone, comparing to the effect of naringenin and rutin, on the production of proinflammatory mediators in lipopolysaccharide (LPS)stimulated macrophages and in the interaction between adipocytes and macrophages. Materials and methods
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from naringenin, and separated from unconverted naringenin by preparative HPLC. All the components were dissolved in dimethyl sulfoxide to prepare stock solutions (200 mM). Cell culture The RAW 264 macrophage cell line, obtained from RIKEN BioResource Center (Tsukuba, Japan), was cultured in Dulbecco's modified Eagle's medium (DMEM) (Sigma) with 10% fetal bovine serum (FBS) (JRH Bioscience, Kansas, USA) and 100 U/ ml penicillin/100 μg/ml streptomycin (Gibco BRL, NY, USA) at 37 °C under a humidified 5% CO2 atmosphere. The cells were seeded in 12-well plates (1 × 106 cells/ml), and treated with 5 μg/ ml LPS (Sigma) and various concentrations of naringenin chalcone, naringenin, or rutin in serum-free medium for 24 h. 3T3-L1 preadipocytes (American Type Culture Collection, Manassas, VA, USA) were subcultured in DMEM with 10% FBS (Biological Industries, Kibbutz Beit Haemek, Israel), 100 U/ml penicillin/100 μg/ml streptomycin at 37 °C under a humidified 5% CO2 atmosphere. Differentiation of 3T3-L1 preadipocytes was induced by adipogenic agents (0.5 mM 3isobutyl-1-methylxanthine (Nacalai Tesque, Kyoto, Japan), 0.25 μM dexamethasone (Sigma), and 10 μg/ml insulin (Sigma)) in DMEM containing 10% FBS for 2 days after the cells reached confluence (day 0). Then, the medium was replaced with DMEM containing 10% FBS and 5 μg/ml insulin, and was changed every two days. Twenty days after induction of differentiation, the cells that accumulated large lipid droplets were used as hypertrophied 3T3-L1 adipocytes. Coculture of adipocytes and macrophages Adipocytes and macrophages were cocultured in a contact system as previously described (Suganami et al., 2005). Briefly, RAW 264 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. RAW 264 and 3T3L1 cells of equal numbers to those in the coculture were cultured separately as control cultures. Naringenin chalcone, naringenin, or rutin was added to the coculture at various concentrations as shown in each figure. After 24 h of treatment, culture supernatants were collected and stored at −20 °C until measurements. Measurement of MCP-1 and TNF-α production The concentrations of MCP-1 and TNF-α in the culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). ELISA was conducted using a READY-SET-GO! Mouse MCP-1 and TNF-α (eBioscience, CA, USA), according to the manufacturer's protocol.
Chemical reagent Measurement of nitric oxide release Naringenin and rutin were purchased from Sigma (MO, USA) and Wako Chemicals (Osaka, Japan), respectively. Naringenin chalcone was prepared by the method described by Miranda et al. (2000) with some modification. Briefly, naringenin chalcone was obtained by isomerization with 5% NaOH at 60 °C for 15 min
The amount of nitrite in cell-free culture supernatants was measured using Griess reagent (Granger et al., 1996). Briefly, 100 μl of supernatant was mixed with an equivalent volume of Griess reagent [1:1 (v/v) of 0.1% N-1-naphthyl-ethylenediamine
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Cell viability assay Cells were seeded in 96-well plates (1 × 105 cells/well), and treated with various concentrations of naringenin chalcone, naringenin, or rutin in the presence or absence of LPS for 24 h. The viability of RAW 264 macrophages was measured by CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega). Statistical analyses Data were expressed as means ± SE, and were statistically analyzed using one-way ANOVA and Turkey's multiple comparison tests. Statistical significance was set at P b 0.01.
Fig. 1. Chemical structures of naringenin chalcone, naringenin, and rutin.
in distilled water and 1% sulfanilamide in 5% phosphoric acid] on a 96-well flat bottom plate. After 10 min, absorbance at 570 nm was measured, and the amount of nitrite was calculated from the NaNO2 standard curve. Western blotting RAW 264 cells were carefully washed twice with ice-cold phosphate buffered saline (PBS) and placed immediately in lysis buffer containing 20 mM Tris HCl (pH 7.5), 15 mM NaCl, 1% Triton × 100 (Nacalai Tesque), and protease inhibitor cocktail (Nacalai Tesque). Lysate was centrifuged at 15,000 rpm for 5 min, and the supernatant was stored for subsequent analysis. Protein concentration of cell lysate was determined using DC protein assay reagents (BioRad Laboratories, CA, USA) on the basis of the method of Lowry et al. (1951). Fifteen micrograms of protein was subjected to 10% SDS-PAGE and separated products were transferred to an Immobilon-P membrane (Millipore, MA, USA). After blocking with 3% skim milk in PBS/0.1% Tween 20, the membrane was incubated with monoclonal anti-iNOS antibody (BD Transduction Laboratories, CA, USA) or anti-IκB-α antibody (Santa Cruz Biotechnology, CA, USA) overnight, and then with a secondary antibody conjugated to horseradish peroxidase (anti-mouse IgG (Upstate, NY, USA) for iNOS, or anti-rabbit IgG (Promega, WC, USA) for IκB-α (1/2000) ) for 1 h. Secondary antibody was visualized using chemiluminescence with an ECL Western blotting detection reagent (Amersham Biosciences, NJ, USA).
Fig. 2. Effects of naringenin chalcone and its metabolites on the secretion of MCP-1 by LPS-stimulated RAW 264 macrophages. RAW 264 cells were stimulated with LPS (5 μg/ml) and incubated with naringenin chalcone (A), naringenin (B), or rutin (C) for 24 h. MCP-1 level in the culture medium was measured by ELISA. Values are means ± SE of four replicants. ⁎P b 0.01 and ⁎⁎P b 0.001 vs culture treated with LPS alone, as analyzed by one-way ANOVA and Turkey's multiple comparison tests.
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Results Effects of naringenin chalcone and its metabolites on MCP-1 and TNF-α production by RAW 264 macrophages Regulation of proinflammatory chemokines and cytokines induced by inflammatory stimuli serves as a key mechanism of inflammatory control. To determine whether naringenin chalcone (Fig. 1A) suppresses MCP-1 and TNF-α production in macrophages, RAW 264 macrophages were stimulated with LPS in the presence or absence of naringenin chalcone or its metabolites, naringenin (Fig. 1B) or rutin (Fig. 1C). None of the tested flavonoids affected the viability of RAW 264 cells (data not shown). Naringenin chalcone significantly inhibited LPSinduced MCP-1 production by RAW 264 cells in a dose-
Fig. 4. Effects of naringenin chalcone and its metabolites on the expression of nitric oxide and inducible nitric oxide synthase (iNOS) by LPS-stimulated RAW 264 macrophages. RAW 264 cells were stimulated with LPS (5 μg/ml) and incubated with naringenin chalcone (A), naringenin (B), or rutin (C) for 24 h. Secretion level of nitric oxide was measured. iNOS protein level was analyzed by Western blotting. Values are means ± SE of four replicants. ⁎P b 0.01 and ⁎⁎P b 0.001 vs culture treated with LPS alone, as analyzed by one-way ANOVA and Turkey's multiple comparison tests.
Fig. 3. Effects of naringenin chalcone and its metabolites on the secretion of TNF-α by LPS-stimulated RAW 264 macrophages. RAW 264 cells were stimulated with LPS (5 μg/ml) and incubated with naringenin chalcone (A), naringenin (B), or rutin (C) for 24 h. The level of TNF-α in the culture medium was measured by ELISA. Values are means ± SE of four replicants. ⁎P b 0.01 and ⁎⁎P b 0.001 vs culture treated with LPS alone, as analyzed by one-way ANOVA and Turkey's multiple comparison tests.
dependent manner (Fig. 2A). Naringenin also dose-dependently inhibited MCP-1 production by LPS-stimulated RAW cells (Fig. 2B). On the other hand, the inhibitory effect of rutin on the production of MCP-1 was moderate and showed no dose dependence (Fig. 2C). Naringenin chalcone and naringenin also inhibited TNF-α production by LPS-stimulated RAW 264 cells (Fig. 3A, B); however, rutin did not affect the production of TNF-α (Fig. 3C).
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a marked increase in MCP-1 level (Fig. 6A). Naringenin chalcone or naringenin treatment in this coculture significantly decreased MCP-1 secretion level (Fig. 6A). The inhibitory effect of rutin on the secretion of MCP-1 was moderate and showed no dose dependence (Fig. 6A). The coculture of differentiated 3T3-L1 adipocytes and RAW 264 cells showed an enhanced TNF-α secretion about threefold that of the control culture (Fig. 6B). Naringenin chalcone and naringenin treatment significantly decreased TNF-α secretion level even at the lowest concentration (50 μM) (Fig. 6B). Rutin at 50 μM did not effect the production of TNF-α, however, higher concentrations of rutin significantly suppressed the secretion of TNF-α (Fig. 6 similar effects of naringenin chalcone and its metabolites on NO
Fig. 5. Effects of naringenin chalcone and its metabolites on IκB-α degradation in LPS-stimulated RAW 264 macrophages. RAW 264 cells were stimulated with LPS (5 μg/ml) and incubated with naringenin chalcone (A), naringenin (B), or rutin (C) for 30 min. Total cell lysates were extracted from cultured RAW 264 cells, and IκB-α protein expression was analyzed by Western blotting.
Effects of naringenin chalcone and its metabolites on the nitrite oxide production and iNOS expression level Naringenin chalcone and naringenin significantly decreased NO production in a dose-dependent manner (Fig. 4 A, B), but the inhibitory effect of rutin on the production of NO was moderate and showed no dose dependence (Fig. 4C). The level of iNOS expression by LPS-stimulated RAW 264 cells was dose-dependently decreased by naringenin chalcone or naringenin treatment (Fig. 4A, B); however, rutin did not affect iNOS protein expression level (Fig. 4C). Effects of naringenin chalcone and its metabolites on the degradation of IκB-α NF-κB is one of the major transcription factors that regulate the induction of MCP-1, TNF-α, and iNOS. To clarify the molecular mechanism underlying the anti-inflammatory action of naringenin chalcone, we investigated whether naringenin chalcone and its metabolites inhibit IκB-α degradation in LPSstimulated RAW 264 cells, which leads to the activation of NFκB. LPS treatment for 30 min markedly promoted IκB-α degradation (Fig. 5). The treatment with naringenin chalcone (Fig. 5A) or naringenin (Fig. 5B) suppressed the degradation of IκB-α, although dose-dependent effects were not observed. Rutin showed no effect or only exerted a moderate inhibition of IκB-α degradation in LPS-stimulated RAW 264 cells (Fig. 5C). Effects of naringenin chalcone and its metabolites on inflammatory changes by coculture of adipocytes and macrophages Secretion of MCP-1 by differentiated 3T3-L1 cells or RAW 264 cells was very low when they were cultured separately; however, coculture of these cells in the contact system revealed
Fig. 6. Effects of naringenin chalcone and its metabolites on inflammatory changes induced by the coculture of 3T3-L1 adipocytes and RAW 264 macrophages. Differentiated 3T3-L1 adipocytes were cocultured with RAW 264 macrophages (1 × 105 cells/well) for 24 h. MCP-1 (A) and TNF-α (B) levels in the coculture medium were measured by ELISA. Secretion level of nitric oxide (C) was also measured. Values are means ± SE of four replicants. ⁎P b 0.01 and ⁎⁎P b 0.001 vs non-treated coculture, as analyzed by one-way ANOVA and Turkey's multiple comparison tests.
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secretion were also observed; naringenin chalcone and naringenin dose-dependently suppressed coculture-induced NO secretion, but the inhibitory effect of rutin on the secretion of NO was moderate and showed no dose dependence (Fig. 6C). Discussion In the present study, we demonstrated that naringenin chalcone not only suppresses the production of proinflammatory cytokines in activated macrophages, but also the inflammatory changes in the interaction between adipocytes and macrophages. This implies that naringenin chalcone can inhibit inflammatory changes in obese adipose tissue. Obesity is associated with chronic, low-grade inflammation. Recent studies have demonstrated that obese adipose tissue is characterized by an enhanced infiltration of macrophages, suggesting that they are an important source of inflammation in adipose tissue (Weisberg et al., 2003; Xu et al., 2003). Therefore, it is important to inhibit the secretion of chemokines and proinflammatory factors from adipose tissue to prevent the migration of macrophages and subsequent chronic inflammation. Many flavonoids have been shown to have biological activities, including anti-inflammatory properties. Naringenin chalcone, which is abundant in tomato peels, is an intermediate in the biosynthesis of flavonols and is converted into naringenin by chalcone isomerase (Muir et al., 2001). Naringenin chalcone is reported to have antiallergic activities via modulation of immune cell activities (Yamamoto et al., 2004). Flavanone naringenin, also abundant in tomato and citrus fruit, is reported to suppress NO production and TNF-α secretion from RAW 264.7 macrophages (Lyu and Park, 2005). Our data showed that naringenin chalcone and naringenin significantly inhibited proinflammatory mediators such as MCP-1, TNF-α, and NO in LPS-stimulated RAW 264 macrophages. Furthermore, the inhibitory effect of naringenin chalcone was stronger than that of naringenin, indicating that naringenin chalcone induces strong anti-inflammatory activities. Because adipose tissue macrophages play a crucial role in augmenting inflammatory responses in obese adipose tissue, naringenin chalcone may be used for suppressing macrophageinduced inflammation in adipose tissue. On the other hand, the effects of rutin on the production of these proinflammatory mediators were not observed or were very weak, which is consistent with previous reports (Lyu and Park, 2005; Shen et al., 2002). Rutin, a flavonol glycoside commonly found in tomato and citrus fruit peels is one of the metabolites of naringenin. Rutin glycoside is too polar for cells to take up in vitro (Shen et al., 2002), although rutin can be absorbed when rutinose located at the C3 site of rutin is hydrolyzed in the small intestine by βglycosidase to quercetin (Morand et al., 2000), and exerts its antiinflammatory activities in vivo (Afana'seva et al., 2001; Shen et al., 2002). This may explain the ineffectiveness of rutin to induce macrophage responses. In various cell types, NO is produced by iNOS in inflammation (Nathan and Xie, 1994; Xie and Nathan, 1994). Naringenin is reported to inhibit NO production by reducing iNOS expression in RAW 264 cells (Tsai et al., 1999). Our data showed that naringenin chalcone, as well as naringenin, inhibited iNOS
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expression in LPS-stimulated RAW 264 cells, indicating that the suppressive effect of naringenin chalcone on the secretion of NO is also associated with decreased iNOS expression level. NF-κB is one of the major transcription factors that regulate the induction of MCP-1 (Wang et al., 2000), TNF-α (Tsai et al., 2000; Vallejo et al., 2000), and iNOS (Xie et al., 1994). In the unstimulated form, NF-κB is present in the cytosol bound to the inhibitory protein IκB. After the stimulation of cells by various agents, IκB becomes phosphorylated and this triggers a proteolytic degradation of IκB, leading to the activation of NF-κB (Yamamoto and Gaynor, 2001). Our data revealed that naringenin chalcone and naringenin inhibited the LPS-induced degradation of IκB-α in activated RAW 264 macrophages; however, this inhibitory effect was weak and LPS-induced degradation of IκB-α was not completely rescued even after the treatment with naringenin chalcone or naringenin at the highest concentration. Hatziieremia et al. (2006) reported that cardamonin, a chalcone derivative, inhibited LPS-induced NF-κB DNA binding without affecting the degradation of IκB-α or phosphorylation of NF-κB, suggesting a direct effect of chalcone on the transcription factor binding to DNA. Interestingly, some flavonoids including cardamonin (Hatziieremia et al., 2006), anthocyanidins (Hou et al., 2005), and green tea polyphenol (Hou et al., 2007) are reported to inhibit other transcription factors such as activator protein 1 (AP-1) and CCAAT/enhancer-binding protein (C/EBP) δ, which are identified to bind the cis-acting elements in the promoter of MCP-1 (Sekine et al., 2002), TNF-α (Tsai et al., 2000; Vallejo et al., 2000), and iNOS (Xie et al., 1994). Therefore, the antiinflammatory action of naringenin chalcone and naringenin may involve the inhibitory action of those transcription factors. Recently, it was reported that activation of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) which catalyzes the conversion of cortisone to active cortisol in adipocytes and macrophages is critically involved in dysfunction of adipose tissue associated with inflammation in obese patients (Ishii et al., 2007). Naringenin and some bioflavonoids are reported to inhibit 11βHSD in guinea pig kidney (Zhang et al., 1994; Zhang and Wang, 1997), thus in the present study, naringenin might inhibit the production of proinflammatory mediators via suppressing 11βHSD in RAW 264 macrophages. Further studies are required to clarify the exact mechanisms. It has been shown that paracrine interactions between adipocytes and nonadipocyte cells such as macrophages augment the inflammatory response of adipose tissue in obesity (Weisberg et al., 2003; Xu et al., 2003). Our data showed that the coculture of hypertrophied 3T3-L1 adipocytes and RAW 264 macrophages resulted in a marked upregulation of the expression of proinflammatory mediators, namely, MCP-1, TNF-α, and NO, which is consistent with a previous report (Suganami et al., 2005). Moreover, naringenin chalcone and naringenin significantly suppressed the coculture-induced upregulation of MCP-1, TNFα, and NO, although the suppressive effect of rutin on MCP-1 and NO production was moderate and showed no dose dependence. These results indicate that naringenin chalcone and naringenin can directly suppress chronic inflammatory responses in obese adipose tissue. In addition, adipocyte-derived FFA is another important mediator of the establishment of a vicious cycle that
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augments the inflammatory changes in obese adipose tissue (Suganami et al., 2005). Because naringenin has no effect on the lipolysis of 3T3-L1 adipocytes (Harmon and Harp, 2001), the inhibitory actions of naringenin chalcone and naringenin on the production of proinflammatory mediators such as MCP-1, TNFα, and NO may be more important than their action in the suppression of FFA release from adipocytes, which in turn contribute to the suppression of chronic inflammatory responses in obese adipose tissue. In obese adipose tissue, it is considered that both adipocytes and macrophages can be the sources of MCP1 (Bruun et al., 2005; Fain and Madan, 2005; Yu et al., 2006; Suganami et al., 2005), although the level of MCP-1 produced by the separately cultured 3T3-L1 adipocytes (15 ng/ml) and RAW 264 macrophages (6.8 ng/ml) was very low compared with that by cocultured cells (1156 ng/ml). On the other hand, we could not detect TNF-α in the culture medium of 3T3-L1 adipocytes, thus TNF-α is mainly derived from macrophages (Suganami et al., 2005). In this study, naringenin chalcone and naringenin inhibited MCP-1 and/or TNF-α production by RAW 264 macrophages; thus, naringenin chalcone and naringenin may inhibit the cocultureinduced MCP-1 production by both 3T3-L1 cells and RAW 264 cells, as well as TNF-α production mainly by RAW 264 cells. MCP-1 plays a crucial role in the augmentation of inflammatory responses in obesity by enhancing the migration of monocytes into adipose tissue and activation into macrophages (Yu et al., 2006). The coculture system we used in the present study could not reveal whether naringenin chalcone or naringenin affected the migration of RAW 264 cells to adipocytes. However, these flavonoids may prevent the recruitment of monocytes into obese adipose tissue by reducing the production of MCP-1 by adipocytes and macrophages in vivo. Further study is needed to clarify the effect of naringenin chalcone on the migration of monocytes. In summary, the results presented here showed that naringenin chalcone suppressed the production of proinflammatory mediators by activated macrophages and/or adipocytes cocultured with macrophages. Naringenin chalcone may be a useful phytochemical for suppressing the vicious cycle of chronic inflammation in obese adipose tissue, and improve obesity-related insulin resistance. Acknowledgements This work was supported by the Research and Development Program for New Bio-industry Initiatives and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sport, Science and Technology of Japan (15081205, 19380074 and 19780096). RY was supported by a grant from Korean Science and Engineering Foundation (KOSEF R01-2005-000-10408-0). References Afanas'eva, I.B., Ostrakhovitch, E.A., Mikhal'chik, E.V., Ibragimova, G.A., Korkina, L.G., 2001. Enhancement of antioxidant and anti-inflammatory activities of bioflavonoid rutin by complexation with transition metals. Biochemical Pharmacology 61 (6), 677–684. Bruun, J.M., Lihn, A.S., Pedersen, S.B., Richelsen, B., 2005. Monocyte chemoattractant protein-1 release is higher in visceral than subcutaneous
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