European Journal of Pharmacology 738 (2014) 40–48

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Molecular and cellular pharmacology

Inhibition of adipocyte inflammation and macrophage chemotaxis by butein Zheng Wang 1, Youngyi Lee 1, Jae Soon Eun, Eun Ju Bae n College of Pharmacy, Woosuk University, Wanju-gun, Jeollabuk-do, Republic of Korea

art ic l e i nf o

a b s t r a c t

Article history: Received 3 March 2014 Received in revised form 1 May 2014 Accepted 14 May 2014 Available online 27 May 2014

Adipose tissue inflammation has been proposed as a therapeutic target for the treatment of obesity and metabolic disorders such as insulin resistance and type 2 diabetes. Butein, a polyphenol of vegetal origin, exhibits anti-inflammatory effects in macrophages but it was not reported whether butein prevents adipocyte inflammation. Here, we investigated the effects of butein on adipocyte inflammation in 3T3-L1 cells and performed functional macrophage migration assays. Butein opposed the stimulation of inducible nitric oxide synthase (iNOS) protein expression and of nitric oxide production by simultaneous treatment of adipocytes with tumor necrosis factor alpha (TNFα), lipopolysaccharide (LPS), and interferon gamma (TLI). In addition, butein inhibited mRNA expression of pro-inflammatory genes and chemokines in adipocytes stimulated with TLI or conditioned medium from RAW 264.7 macrophages treated with LPS. These effects were associated with suppression of inhibitor of kappa B alpha degradation induced by TNFα and with nuclear factor-kappa B (NF-κB) p65 phosphorylation and acetylation. Moreover, butein prevented phosphorylation of extracellular signal-regulated kinases, c-Jun N-terminal kinase, and the mitogen-activated protein kinase (MAPK) p38. These results suggest that butein suppresses adipocyte inflammation by inhibiting NF-κB/MAPK-dependent transcriptional activity. Furthermore, conditioned media from adipocytes stimulated macrophage chemotaxis, whereas media from adipocytes treated with butein blocked macrophage migration, an effect that was consistent with suppression of MCP-1 secretion by adipocytes treated with butein. In addition, macrophages treated with butein exhibited a reduced ability to migrate toward adipocyte CM. In conclusion, butein may represent a therapeutic agent to prevent adipose tissue inflammation and the obesity-linked insulin resistance. & 2014 Elsevier B.V. All rights reserved.

Keywords: Butein Adipocytes Inflammation Chemokines Macrophage chemotaxis Chemical compounds studied in this article: Butein (PubChem CID: 5281222) TNF alpha (PubChem SID:57288613) LPS (PubChem CID: 11970143) IFN gamma (PubChem SID:57288557)

1. Introduction Obesity and insulin resistance or type 2 diabetes mellitus (T2DM) are associated with chronic low-grade inflammation states (Glass and Olefsky, 2012). In obese individuals, a surplus of calories increases fat mass, and under these conditions, adipocytes produce and release large amounts of cytokines/adipokines and

Abbreviations: T2DM, type 2 diabetes mellitus; CM, conditioned medium; ATM, adipose tissue macrophage; TNFα, tumor necrosis factor alpha; LPS, lipopolysaccharide; IFNγ, interferon gamma; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2; MCP-1, monocyte chemoattractant protein-1; CCL, C–C motif ligand; CXCL, C-X-C motif ligand; IL-6, interleukin-6; IκBα, inhibitor of kappa B alpha; NF-κB, nuclear factor-kappa B; ERK, extracellular signal-regulated kinases; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; QPCR, quantitative realtime RT-PCR; SIRT-1, sirtuin-1; PPARγ, peroxisome proliferatoractivated receptor gamma n Corresponding author at: College of Pharmacy, Woosuk University, 443 Samnye-ro, Wanju-gun, Jeollabuk-do 565-701, Republic of Korea. E-mail addresses: [email protected], [email protected] (E.J. Bae). 1 These two authors equally contributed to the work. http://dx.doi.org/10.1016/j.ejphar.2014.05.031 0014-2999/& 2014 Elsevier B.V. All rights reserved.

particular chemoattractants, termed chemokines, which recruit inflammatory cells into the adipose tissue. Macrophages infiltrating the adipose tissue amplify the inflammatory response in concert with adipocytes, aggravating systemic insulin resistance. Several studies demonstrated that insulin resistance and T2DM are associated closely with adipose tissue inflammation (Solinas et al., 2007; Patsouris et al., 2008), indicating that prevention of adipocyte inflammation and subsequent macrophage infiltration could be beneficial for individuals affected by T2DM (Osborn and Olefsky, 2012; Romeo et al., 2012). The role of nitric oxide (NO), a free-radical messenger molecule, and its synthase enzyme inducible NO synthase (iNOS) has been extensively studied in a wide variety of pathological settings, including inflammatory diseases. In response to inflammatory stimuli, iNOS is synthesized de novo to regulate the host defense. In the adipose tissue of rats administered lipopolysaccharide (LPS), iNOS expression is increased mainly in adipocytes (Ribiere et al., 1996) and iNOS and NO secretions mediate lipolysis and regulate the metabolism of energy substrates (Fiorucci et al., 2004). Interleukins (ILs) such as IL-6 and IL-1β affect adipocyte function and

Z. Wang et al. / European Journal of Pharmacology 738 (2014) 40–48

contribute to obesity-induced insulin resistance. IL-6 is expressed abundantly by adipose tissue and a negative correlation exists between plasma levels of IL-6 and systemic insulin sensitivity in humans (Kern et al., 2001). Chemokines are a family of proteins comprising more than 50 members and can be classified into two major subfamilies in accordance with the amino acid sequence: the C–C motif ligand (CCL) and the C-X-C motif ligand (CXCL) subfamilies. Among the chemokines derived from adipocytes, CCL2, also termed monocyte chemoattractant protein (MCP)-1, has been investigated extensively. MCP-1 expression is increased in the adipose tissue of obese individuals and the overexpression of MCP-1 in murine adipose tissue leads to macrophage recruitment and insulin resistance (Kamei et al., 2006; Kanda et al., 2006; McLaughlin et al., 2008; Tateya et al., 2010). The polyphenol butein is the biologically active component of Toxicodendron vernicifluum and has been shown to have pleiotropic effects such as induction of apoptosis and anti-cancer (Pandey et al., 2007), anti-fibrogenic (Lee et al., 2003), and antiinflammatory activity (Lee et al., 2004). Furthermore, butein inhibits the migration and the invasion of cancer cells by targeting the extracellular signal-regulated kinases (ERK) and nuclear factorkappa B (NF-κB) signaling pathways (Zhang et al., 2008). However, the effect of butein in adipose tissue inflammation associated with obesity remains to be established. In this study, the effects of butein on the LPS/cytokine-induced inflammatory responses and the mechanisms involved were investigated in 3T3-L1 adipocytes. In addition, the effects of butein on macrophage chemotaxis in response to adipocyte-derived chemoattractants were determined by functional macrophage migration assays.

2. Materials and methods 2.1. Reagents Tumor necrosis factor alpha (TNFα) and LPS were purchased from Sigma-Aldrich (St. Louis, MO) and interferon gamma (IFNγ) from R&D Systems (Minneapolis, MN). Butein was purchased from Sigma-Aldrich. The concentrations of reagents used in the experiments were: TNFα 10 ng/ml; LPS 10 ng/ml; IFNγ 250 U/ml. 2.2. Cell culture Murine 3T3-L1 preadipocyte cells were maintained in the growth medium containing Dulbecco's modified Eagle's medium (DMEM), 10% fetal bovine serum (FBS), 50 U/ml penicillin and 50 μg/ml streptomycin at 37 1C in a humidified atmosphere with 10% CO2, and induced to differentiate as previously described (Oh et al., 2010). Briefly, 2 day-post-confluent preadipocytes (day 0) were cultured in the growth medium containing 0.5 μM 3-isobutyl-1-1methylxanthine, 1 μM dexamethasone and 1 μg/ml insulin for 3 days. The cells were further incubated in the growth medium containing 1 μg/ml insulin for additional 3 days, and thereafter medium was replaced with fresh growth media every other days. Mature adipocytes at day 8 or more were used in the experiments. Murine macrophage RAW 264.7 cells were maintained in 10% FBS/ DMEM. 2.3. Cell viability Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenylthetrazolium bromide (MTT) assay. Cells in 96-well culture plates were incubated with butein at different concentrations for 24 h. After incubation, MTT (0.5 mg/ml in PBS) 20 μl was added to each well and the cells were further incubated for 3 h at

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37 1C. The formation of violet precipitate formazan was monitored at a wavelength of 595 nm with spectrophotometer. 2.4. NO measurement The production of NO was estimated by measuring the amount of nitrite, a stable metabolite of NO, using the Griess reagent as previously described (Lee et al., 2004). Briefly, 3T3-L1 adipocytes were pretreated with the indicated concentrations of butein for 1 h prior to the addition of TLI in 12-well plates. After 6 h, 100 μl aliquots of culture supernatants were mixed with an equal volume of a modified Griess reagent of a 1:1 mixture of 1% sulfanilamide in 30% acetic acid and 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in 60% acetic acid, at room temperature for 5 min, and the absorbance at 540 nm was measured using a spectrophotometer. 2.5. Cell lysis and Western blot analysis Preparation of whole cell lysates and Western blot analysis were performed as described previously (Oh et al., 2010). Briefly, the cells were lysed in the buffer containing 10 mM Tris–HCl (pH 7.1), 100 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Triton X-100, 0.5% Nonidet P-40, 1 mM dithiothreitol and 0.5 mM phenylmethylsulfonyl fluoride, supplemented with inhibitors of proteinase and phosphatase. The protein concentrations in the cell lysates were determined by using a Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). The aliquots of lysates were eletrophoresed in 6–10% sodium dodecyl sulfate-polyacrylamide gels (20 μg of protein/lane). The separated proteins were transferred to nitrocellulose membranes (GE Healthcare). The membranes were blocked with 0.4% skim milk in TBS-1% Tween 20 and incubated with the primary antibodies, followed by incubation with secondary antibodies. Immunoreactive protein was visualized by ECL chemiluminescence detection kit (Amersham Biosciences, Buckinghamshire, UK). Image was obtained using ChemiDoc™ XRS þ System (Bio-rad, Hercules, CA) or conventional developing method using Kodak film. Primary antibodies used were: iNOS from BD Biosciences (Palo Alto, CA), β-tubulin (#PA1-16947) from Thermo scientific (Waltham, MA), and acetylated-p65 (Lys310, #D0018) from Assay Biotech (Sunnyvale, CA), COX-2 (sc-1745) and IκBα (sc371) from Santa Cruz Biotechnology (Santa Cruz, CA), p-p65 (Ser536, ab76302) and p-p65 (Ser311, ab51059) from Abcam (Cambridge, MA), p-JNK (#92515), p-p38 (#4631) and p-ERK (#9101) from Cell Signaling Technology (Beverly, MA). 2.6. RNA isolation, semi-quantitative RT-PCR and quantitative realtime RT-PCR (QPCR) Total RNA was extracted from cells with TRIzol reagent (Invitrogen Carlsbad, CA). The total RNA (2 μg) was reverse-transcribed using random primer (Promega, Madison, WI). The following primer sequences were used: Nos2, sense (50 -AATCTTGGAGCGAGTTGTGG-30 ) and antisense (50 -CAGGAAGTAGGTGAGGGCTTG-30 ); Il6, sense (50 -CCAGAGATACAAAGAAATGATGG-30 ) and antisense (50 -ACTCCAGAAGACCAGAGGAAAT-30 ); Mcp1, sense (50 -TCTGGACCCATTCCTTCTTG-30 ) and antisense (50 -AGGTCCCTGTCATGCTTCTG30 ); Cxcl1, sense (50 -AATGAGCTGCGCTGTCAGTG-30 ) and antisense (50 -TGAGGGCAACACCTTCAAGC-30 ); Cxcl10, sense (50 -GACGGTCCGCTGCAACTG-30 ) and antisense (50 -GCTTCCCTATGGCCCTCATT-30 ); Tnfα, sense (50 -GCCACCACGCTCTTCTGCCT-30 ) and antisense (50 GGCTGATGGTGTGGGTGAGG-30 ); Pparg, sense (50 -TTGCTGAACGTGAAGCCCATCGAGG-30 ) and antisense (50 -GTCCTTGTAGATCTCCTGGAGCAG-30 ); Rps3, sense (50 -ATCAGAGAGTTGACCGCAGTTG-30 ) and antisense (50 -AATGAACCGAAGCACACCATAG-30 ). Semi-quantitative PCR products were separated on a 2.5% agarose gel. QPCR

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was performed as previously described (Oh et al., 2010) using an ABI 7000 and Stratagene 3000 MXP PCR cycler with the Sybr Green detection system 6. The mRNA expression of all genes tested is normalized to the Rps3 expression. 2.7. In vitro chemotaxis assay For the preparation of adipocyte conditioned medium (CM), mature 3T3-L1 adipocytes, more than 90% of cells showing large lipid droplets when observed under microscope, were used. At day 10 of differentiation, culture media were changed and further incubated with migration media (serum free, 0.2% endotoxin- and free fatty acid-free bovine serum albumin in DMEM) for 48 h. Treatment of cells with the different reagents was performed in the migration media. When the adipocytes were treated with TLI or TLI þbutein, cells were treated with the compounds for 3 h, then switched to the fresh migration media and further incubated for 48 h until CM harvest. Collected media were first centrifuged at 15,000g for 10 min to remove the cell debris and the collected supernatants were kept aliquot and frozen at  70 1C until a chemotaxis assay was performed. For the migration per se, 5  105 RAW264.7 cells suspended in migration media were placed in the upper chamber of an 8 μm pore size polycarbonate filter (24-transwell format; Corning, Lowell, MA), whereas adipocyte CM was placed in the lower chamber. After 3 h of migration, cells were fixed in formalin and stained with 0.1% crystal violet. Macrophages that had remained in the upper chamber were removed by swiping the filters with cotton tips. Macrophages found on the bottom of filter were counted as cells that had performed chemotaxis and quantified by purple color with imaging software. Cells were quantified from 5 fields/condition; each condition was performed in triplicate. 2.8. Statistical analysis The values presented are expressed as the mean 7S.E.M. The statistical significance of the differences between treatment groups was determined by one-way ANOVA using GraphPad Prism 4.0 (San Diego, CA). The P o0.05 was considered statistically significant.

et al., 2001), we first determined the range of non-cytotoxic concentration of butein in 3T3-L1 adipocytes. Increasing concentrations of butein did not affect adipocyte viability and all the subsequent experiments were conducted in the presence of 3–30 μM of butein (Fig. 1). Although the induction of iNOS by TNFα in various tissues, including adipose tissue, is well established, the extent of iNOS expression in 3T3-L1 adipocytes stimulated with cytokines/LPS appears to be variable (Lien et al., 2009; Merial-Kieny et al., 2003). Therefore, we examined whether 3T3-L1 adipocytes express iNOS when stimulated with TNFα. However, iNOS protein was not detected in adipocytes treated with TNFα alone or with TNFα in conjunction with LPS (Fig. 2A). In contrast, combined treatment of adipocytes with TNFα, LPS, and IFNγ (TLI) induced iNOS expression. Moreover, TLI treatment did not induce expression of the pro-inflammatory protein cyclooxygenase (COX)-2, implying that independent signaling pathways regulate the expression of iNOS and COX-2 in adipocytes. We next examined iNOS expression in adipocytes exposed to butein for different lengths of time. Butein pretreatment at 30 μM for 1 h suppressed iNOS induction from 3 h to 24 h after TLI stimulation, and the effect was concentrationdependent (Fig. 2B and C). Consistently, nitrite production in response to TLI treatment was significantly inhibited by pretreatment with butein (Fig. 2D). It has been reported that butein inhibits the tyrosine-specific protein kinase activity of epidermal growth factor receptor (IC50 ¼65 μM) in a cell-free system (Yang et al., 1998), implying that butein could act at the level of membrane receptor and there is a possibility that butein interferes with the binding between TLI and their receptors to oppose TLI-stimulated inflammation. To exclude the possibility that butein just interferes with the binding of TLI to their receptors and to test whether butein enters the cell to exert its pharmacological effects, we determined the effects of butein with or without washout of drug before TLI treatment. In agreement with the inhibitory effects of butein on iNOS induction by TLI as shown in Fig. 2B and C, butein pretreatment for 1 h without washout suppressed the iNOS expression induced by 6 h exposure of TLI (the third lane in Fig. 2E). Similarly, butein pretreatment for 7 h with subsequent drug washout before TLI treatment for 6 h resulted in inhibition of iNOS expression, indicating that butein may enter the cell to exert its pharmacological effects.

3. Results 3.1. Butein inhibits iNOS expression in 3T3-L1 adipocytes

3.2. Cytokine/chemokine secretion and mRNA transcription is suppressed by butein

Since butein was previously reported to have pro-apoptotic or anti-proliferative effects in cancer cells (Khan et al., 2012; Kim

To confirm the anti-inflammatory effect of butein in adipocytes, we measured IL-6 and MCP-1 levels in the culture media of

Butein Fig. 1. (A) Chemical structure of butein. (B) Effect of butein on the cell viability in 3T3-L1 adipocytes. Mature adipocytes were treated with butein for 24 h at the indicated concentrations in the presence of serum and then subjected to MTT assay. The results are expressed as the mean 7 S.E.M.

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TLI 3

iNOS

43

TLI + Butein

6

24

3

6

24 (h)

iNOS

β-tubulin

TLI (6 h) -

3

10

30 Butein (μM)

iNOS

Nitrite (μM)

β-tubulin

COX-2

β-tubulin TLI + Butein Relative Intensity

TLI (6 h) without washout -

1

with washout 1

3

7

Butein pretreatment (h)

iNOS β-tubulin TLI + Butein

Fig. 2. Butein inhibited iNOS protein expression in 3T3-L1 adipocytes. (A) Effect of cytokines/LPS on iNOS expression. Cells were treated with TNFα (10 ng/ml) alone or in combination with LPS (10 ng/ml) or with LPS þ IFNγ (250 U/ml) for 24 h. For COX-2 examination, the cell lysate obtained from the RAW 264.7 cells treated with LPS (10 ng/ ml) for 24 h was included as a positive control. β-tubulin was used as a loading control. (B) Time- and (C) concentration-dependent changes of iNOS expression by butein treatment. Cells were treated with the combination of TNFα, LPS and IFNγ (TLI) in the absence or the presence of butein at 30 μM for the indicated time periods (B) or at 3, 10 and 30 μM for 6 h (C). The relative levels of iNOS expression analyzed by densitometry were shown in the lower bar graph. (D) Nitrite production. The supernatants obtained from the cells which were treated as described in (C) were used to measure NO production. (E) Effect of butein treatment on the iNOS expression with or without washout of drug before TLI exposure. Cells were treated with TLI for 6 h either in the continuing presence of butein (1 h pretreatment, without washout) or after butein washout following drug treatment for 1, 3 or 7 h (with washout). *Po 0.05 versus Control, #Po 0.05 versus TLI.

adipocytes. Enzyme-linked immunosorbent assays revealed that TLI treatment for 24 h increased secretion of IL-6 and MCP-1, and this effect was opposed by pretreatment with butein for 1 h (Fig. 3A). In addition, semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis suggested that butein inhibited the stimulatory effects of TLI on mRNA expression of Nos2, Il6, and of the chemokine-encoding genes Mcp1, Cxcl1, and Cxcl10 (Fig. 3B). Conversely, the mRNA expression of the adipocytic gene Pparg was decreased by TLI treatment, confirming the role of cytokines and LPS in promoting adipocyte dedifferentiation. Butein reversed the suppression of Pparg by TLI, implying that butein may exhibit anti-dedifferentiating or anti-lipolytic effects in adipocytes. To further confirm the inhibitory effects of butein on the expression of gene markers of inflammation in adipocytes, inflammation was induced with an alternate approach. RAW 264.7 cells were incubated with LPS for 3 h, transferred to fresh media for 24 h, and CM, containing the mixture of cytokines and hormones secreted by macrophages was collected. Subsequently, we investigated the effect of butein on macrophage CM-induced inflammation gene expression in adipocytes. QPCR analysis demonstrated that expression of the pro-inflammatory genes Nos2, Mcp1, Il6, and Cxcl10 was increased by incubation with CM, but these events were suppressed by pretreatment with butein at concentrations of 10 μM and 30 μM,

confirming the anti-inflammatory effect of butein in adipocytes (Fig. 3C).

3.3. Butein suppresses NF-κB/MAPK signaling activated by TNFα in 3T3-L1 adipocytes Several studies reported that butein inhibits inflammatory responses mediated by NF-κB/MAPK signaling (Pandey et al., 2007). To elucidate the anti-inflammatory mechanisms of butein in 3T3-L1 adipocytes, we examined the effect of butein on signaling pathways activated by TNFα. Butein pretreatment resulted in complete blockade of IκBα degradation induced by TNFα (Fig. 4A). Since activation of the NF-κB component RelA/p65 requires phosphorylation and acetylation, we next examined the post-translational modifications of p65. Phosphorylation of p65 at Ser311 and Ser536 was enhanced by TNFα treatment but butein pretreatment prevented this effect. Similarly, acetylation of p65 at Lys310 was induced by TNFα treatment for 30 min and 60 min but was decreased by pretreatment with butein, although the effect was less pronounced than inhibition of phosphorylation. Moreover, ERK, JNK, and p38 MAPK phosphorylation was inhibited by butein pretreatment (Fig. 4B), suggesting suppression of these signaling pathways by butein. In summary, these findings indicate that butein inhibits

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IL-6

*

#

#

Nos2 Mcp1 Cxcl1

MCP-1

Cxcl10

* #

Il6 Pparg Rps3

*

*

#

# #

#

*

*

#

# #

#

Fig. 3. Butein inhibited the cytokine/chemokine secretion and mRNA expression of inflammatory genes in adipocytes. (A) Cytokine/chemokine levels in the media from the cells treated with TLI for 24 h with or without butein. (B) Adipocytes were treated with TLI for 3 h with or without butein and were subjected to semi-quantitative RT-PCR. Pparg and Rps3 were used as an adipocyte marker gene and a housekeeping gene, respectively. (C) Adipocytes were treated with macrophage CM (MΦ-CM) for 6 h with or without butein and QPCR was performed. MΦ-CM was collected from the RAW 264.7 cells which were treated with LPS 10 ng/ml for 3 h followed by washout with fresh media for further 24 h incubation. *Po 0.05, **Po 0.01 versus MΦ-CM.

activation of both the NF-κB and MAPK signaling pathways in adipocytes, leading to the abrogation of adipocyte inflammation. 3.4. Butein inhibits macrophage chemotaxis To investigate the functional consequences of the inhibitory activity of butein on adipocyte inflammation, we performed an

in vitro chemotaxis assay (Oh et al., 2010; Patsouris et al., 2009; Yu et al., 2006). RAW 264.7 macrophages were seeded onto the upper insert well of the chemotaxis chamber and incubated for 3 h in the presence of DMEM or adipocyte CM in the lower chamber. A 3-h migration was chosen since it provides optimal results in the macrophage chemotaxis assay, as described previously (Patsouris et al., 2009). Adipocyte CM significantly stimulated macrophage

Z. Wang et al. / European Journal of Pharmacology 738 (2014) 40–48

TNFα 10 30 60

10

60 (min)

IκBα p-p65 (Ser311) p-p65 (Ser536)

TNFα+Butein

TNFα

TNFα+Butein 10 30

45

30

60

10

30

60 (min)

p-ERK ERK p-JNK JNK

Ac-p65 (Lys310)

p-p38

β-tubulin

p38

Fig. 4. Effect of butein treatment on the TNFα-stimulated cell signaling in adipocytes. Immunoblot analysis for IκBα, phosphorylated- and acetylated-p65 (A) and the phosphorylations of ERK, JNK and p38 MAPK (B). Cells were pretreated with butein (30 μM for 0.5 h) and subsequently stimulated with TNFα for the time periods indicated.

Fig. 5. Macrophage chemotaxis was markedly inhibited by butein treatment. (A) Macrophage migration assays using DMEM (migration media) or CM from adipocytes treated with vehicle, TLI or TLI þbutein (30 μM) for 48 h. RAW 264.7 macrophages onto transwell in the presence of different media in the lower well were incubated for 3 h for migration. (B) Macrophage migration assays using adipocyte CM after vehicle or butein treatment to macrophages. RAW 264.7 cells were treated with vehicle or butein 30 μM for 3 h and the detached cells were used for migration assay in the presence of DMEM or CM. *P o0.05 versus DMEM, #Po 0.05 versus TLI in (A) or CM (B), respectively.

chemotaxis in comparison to DMEM and a more pronounced effect was observed with CM from adipocytes treated with TLI (Fig. 5A). However, macrophage migration was suppressed when cells were exposed to the CM obtained from adipocytes treated with TLI and butein, indicating that butein may inhibit the ability of adipocytes to secrete chemoattractants. This finding is consistent with the observation that butein treatment prevents expression of chemokine mRNA expression and secretion of the encoded proteins. Furthermore, exposure of macrophages, but not of adipocytes, to butein abrogated cell migration in response to adipocyte CM (Fig. 5B). These observations suggest that butein interferes with macrophage chemotaxis in response to adipocytederived chemoattractants, and this effect occurs when either adipocytes or macrophages are exposed to butein.

3.5. Butein has an anti-inflammatory effect in macrophages Adipose tissue comprises mainly adipocytes and infiltrated immune cells, largely macrophages. Given the inhibitory effect of butein on macrophage migration in response to adipocyte CM and on adipocyte inflammation, we next examined the effect of butein on the expression of inflammation genes in macrophages. RAW 264.7 cells were treated with 10 ng/ml LPS in the absence or presence of butein. Butein inhibited iNOS expression in a concentration-dependent manner (Fig. 6A and B), a finding that is consistent with previous results (Lee et al., 2004). COX-2 expression induced by LPS was suppressed by butein only at a concentration of 30 μM (Fig. 6B). Induction of Mcp1, Nos2, and Tnfα mRNA expression by LPS was suppressed by butein

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LPS

iNOS

Butein -

1

3

10

30 (μM)

iNOS COX-2 β-tubulin

LPS

COX-2

Butein -

10

30

(μM)

Mcp1 Nos2 Tnfα Rps3 Fig. 6. Butein blocked the expression of inflammatory genes/chemokine in macrophages. (A) RAW 264.7 cells were treated with LPS 10 ng/ml with or without butein for 24 h and then subjected to immunoblot analysis for iNOS and COX-2. (B) Quantification results of (A). n ¼5/group, *Po 0.05 versus control, #Po 0.05 versus LPS alone. (C) Semiquantitative RT-PCR results for MCP-1, iNOS and TNFα. Cells were pretreated with butein 30 min prior to LPS exposure for 6 h.

pretreatment (Fig. 6C). Taken together, these findings indicate that the anti-inflammatory responses and the suppression of chemokine expression by butein in adipocytes and macrophages lead to the inhibition of macrophage chemotaxis.

4. Discussion This study confirms that combined treatment of 3T3-L1 adipocytes with TNFα, LPS, and IFNγ increased iNOS expression, whereas neither TNFα alone nor TNFα in conjunction with LPS induced iNOS expression, suggesting the presence of a complex network of interactions between inflammatory cytokines and LPS in 3T3-L1 adipocytes (Kapur et al., 1999). iNOS plays a key role as a mediator of inflammation in obesity-linked insulin resistance as well as in endotoxemia (Carvalho-Filho et al., 2006; Kapur et al., 1999; Perreault and Marette, 2001). Indeed, iNOS expression is increased in adipose tissue as well as in the skeletal muscle and liver in obese subjects (Elizalde et al., 2000; Fujimoto et al., 2005; Pilon et al., 2000; Sugita et al., 2005). In the present work, we observed that butein prevented induction of iNOS by TLI or LPS in 3T3-L1 adipocytes and RAW 264.7 macrophages, respectively. Furthermore, butein suppressed both the induction of proinflammatory genes and chemokines in adipocytes by TLI and macrophage CM. Although the anti-inflammatory effects of butein are known, this is the first report demonstrating that butein inhibits inflammation in adipocytes, indicating that butein is a bona fide anti-inflammatory molecule. Suppression of NF-κB and MAPK signaling pathways is the molecular mechanism by which butein opposes inflammation in macrophages. NF-κB-dependent gene expression occurs in response to IκBα degradation induced by NF-κB activating signals, such as LPS or TNFα. Furthermore, the transcriptional activity of

NF-κB is modulated by post-translational modifications such as phosphorylation and acetylation, and these mechanisms are independent from IκBα degradation (Schmitz et al., 2001). In the current study, we confirmed that butein prevents degradation of IκBα stimulated by TNFα in adipocytes, and that butein completely inhibited the phosphorylation of p65 NF-κB at both Ser311 and Ser536, an event required for the transactivating activity of NF-κB (Duran et al., 2003; Jiang et al., 2003; Sanchez-Valdepenas et al., 2007). Acetylation of NF-κB at Lys310 is required for the transcriptional activity of RelA/p65 (Chen et al., 2002). In this study, we found that butein reduced the acetylation of NF-κB at Lys310, confirming the stimulatory role of butein on the activity of the NAD þ -dependent histone deacetylase sirtuin (SIRT)1, as proposed previously (Yang et al., 2009). Together, the current results suggest that butein may affect TNFα signaling at the level of IκB kinases, protein kinase (PK) Cζ and PKA, which are responsible for phosphorylation of Ser536 and Ser311 of p65 and regulate proinflammatory gene expression (Sakurai et al., 1999). Concurrently, activation of SIRT1 by butein and the subsequent deacetylation of NF-κB may support its anti-inflammatory function. In addition to the reduction of NF-κB signaling, we report that the activity of the MAPKs ERK, JNK, and p38 MAPK was decreased by butein treatment in adipocytes. Activation of these MAPKs in macrophages is linked with induction of pro-inflammatory genes, including iNOS (Chan et al., 1999; Chan and Riches, 2001). Our study revealed that the global inhibition of MAPK phosphorylation in conjunction with the impairment of NF-κB signaling may contribute to the potent anti-inflammatory action of butein in adipocytes. The current finding is in agreement with a previous report indicating that butein inhibits MAPK activation in macrophages to prevent inflammation (Lee et al., 2004). We also observed that suppression of Pparg mRNA expression by TLI was prevented by butein pretreatment. Peroxisome

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proliferator-activated receptor (PPAR) γ is a key molecule that preserves healthy adipose tissue function not only by regulating adipogenesis but also due to its anti-inflammatory role in macrophages and adipocytes (Nguyen et al., 2012). Previous studies showed that chemical activation of PPARγ or its overexpression opposed inflammation in vitro and in vivo, leading to increased insulin sensitivity, whereas the transactivating function of PPARγ is suppressed by inflammatory cytokines such as TNFα and ILs (Guilherme et al., 2009; van Asseldonk et al., 2010). In the present study, butein exhibited reversed suppression of PPARγ expression by TLI, an effect that, in combination with the blockade of NF-κB/MAPK signaling pathways, mediates the anti-inflammatory function of butein in adipocytes. In summary, these findings raise the possibility that butein treatment could have important therapeutic effects by reducing tissue inflammation and promoting insulin sensitivity. The present study is the first report of butein's ability to block macrophage migration in response to adipocyte CM. Adipose tissue expansion with obesity is associated with adipose tissue inflammation due to infiltration and of accumulation adipose tissue macrophage (ATM). The crucial role of monocyte/macrophage infiltration into adipose tissue for systemic insulin resistance is demonstrated, and prevention of ATM infiltration can ameliorate obesity-linked insulin resistance, providing a therapeutic target for metabolic syndrome. Herein, to assess the ability of macrophage to migrate toward adipocyte-derived chemotactic factors, we employed an in vitro chemotaxis assay with adipocyte CM. The adipocyte CM contains a variety of cytokines and chemokines and among adipocyte-derived chemokines, MCP-1 is known to possess the most strong chemoattractive activity and has been studied extensively. In adipose tissue, both adipocytes and the infiltrated immune cells are responsible for secretion of MCP-1. The important role of MCP-1 in adipose inflammation and systemic insulin resistance has been demonstrated in gene overexpression or deletion studies in adipocytes (Kamei et al., 2006; Kanda et al., 2006). In addition to MCP-1, multiple chemokines are derived from adipocytes and connect obesity to insulin resistance (Meijer et al., 2011). Chemokines from the CXCL subfamily, such as CXCL-1 and CXCL-10, play an essential role in chemotaxis and adipose inflammation (Pedersen et al., 2012). We report that butein treatment in adipocytes results in reduced expression of CXCLs as well as MCP-1 and these effects may contribute to inhibition of macrophage chemotaxis by butein. Furthermore, a 3-h-long exposure of macrophages to butein was sufficient to block macrophage chemotaxis. Previous studies demonstrated that MCP-1 induces macrophage migration largely via MAPK signaling (Huang et al., 2004), suggesting the possibility that butein acutely impairs the ability of macrophages to migrate in response to MCP-1 by inhibiting MAPK activity. Although the role of MCP-1 in ATM recruitment remains controversial (Kirk et al., 2008), we observed that butein significantly suppresses MCP-1 expression both in adipocytes and macrophages, implying that butein has the potential to block ATM recruitment in the adipose tissue of obese individuals. In conclusion, we provide evidence that butein exerts antiinflammatory effects in macrophages and adipocytes, possibly by inhibiting the NF-κB and MAPK signaling pathways. The current findings also demonstrate that butein inhibits chemokine production in adipocytes, therefore preventing macrophage chemotaxis. Further investigations of the effects of butein in vivo will determine whether butein has therapeutic potential for obesity-linked inflammatory diseases and insulin resistance.

Acknowledgments We thank Dr. Byung-Hyun Park at Medical School and Diabetes Research Center, Chonbuk National University for the generous

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provision of facilities for QPCR and chemotaxis assay and especially thank Dr. Sun-O Ka for all the technical help. This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A1014527).

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