Anti-inflammatory effect of resveratrol on TNF-α-induced MCP-1 expression in adipocytes

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Biochemical and Biophysical Research Communications 369 (2008) 471–477 www.elsevier.com/locate/ybbrc

Anti-inflammatory effect of resveratrol on TNF-a-induced MCP-1 expression in adipocytes Jian Zhu a,b, Wei Yong b, Xiaohong Wu a, Ying Yu b, Jinghuan lv b, Cuiping Liu a, Xiaodong Mao a, Yunxia Zhu b, Kuanfeng Xu a, Xiao Han b,*, Chao Liu a,* b

a Department of Endocrinology, First Affiliated Hospital, Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, China Key Laboratory of Human Functional Genomics of Jiangsu Province, School of Basic Medical Science, Jiangsu Province Diabetes Center, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, China

Received 31 January 2008 Available online 20 February 2008

Abstract Chronic low-grade inflammation characterized by adipose tissue macrophage accumulation and abnormal cytokine production is a key feature of obesity and type 2 diabetes. Adipose-tissue-derived monocyte chemoattractant protein (MCP)-1, induced by cytokines, has been shown to play an essential role in the early events during macrophage infiltration into adipose tissue. In this study we investigated the effects of resveratrol upon both tumor necrosis factor (TNF)-a-induced MCP-1 gene expression and its underlying signaling pathways in 3T3-L1 adipoctyes. Resveratrol was found to inhibit TNF-a-induced MCP-1 secretion and gene transcription, as well as promoter activity, which based on down-regulation of TNF-a-induced MCP-1 transcription. Nuclear factor (NF)-jB was determined to play a major role in the TNF-a-induced MCP-1 expression. Further analysis showed that resveratrol inhibited DNA binding activity of the NF-jB complex and subsequently suppressed NF-jB transcriptional activity in TNF-a-stimulated cells. Finally, the inhibition of MCP-1 may represent a novel mechanism of resveratrol in preventing obesity-related pathologies. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Resveratrol; NF-jB; MCP-1; Sirt1; 3T3-L1 adipoctye; Inflammation

Obesity is closely associated with a state of chronic, low-grade inflammation characterized by abnormal cytokine production and activation of inflammatory signaling pathways in adipose tissue [1]. Recent studies have indicated that adipose tissue macrophages accumulating during diet-induced obesity are not only an important source of adipose tissue inflammation but also alter insulin sensitivity and promote atherosclerosis [2,3]. Monocyte chemoattractant protein (MCP)-1 chemotactically recruits monocytes to sites of inflammation. This protein is traditionally thought to be expressed mainly by endo* Corresponding authors. Fax: +86 25 86862731 (X. Han), +86 25 83674006 (C. Liu). E-mail addresses: [email protected] (X. Han), liuchao@nfmcn. com (C. Liu).

0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.02.034

thelial cells and macrophages. However, it has recently been shown to be primarily expressed by adipose tissues. MCP-1 is expressed by cultured human primary adipocytes dissociated from other cell types present in fat pads [4]. In situ hybridization and laser microcapture experiments confirmed adipocytes as the major source of MCP-1 expression in adipose tissue [5]. Adipocytederived MCP-1 induces macrophage infiltration into adipose tissues and thus secretes inflammatory cytokines including tumor necrosis factor (TNF)-a, which in turn leads to the dysfunction of adipocytes [6]. In addition, MCP-1 inhibits insulin-dependent glucose uptake and the expression of adipogenic genes; thus, MCP-1deficient mice lack insulin resistance and are resistant to atherosclerosis [5]. These data suggest that the modulation of adipocyte-derived MCP-1 is a useful strategy

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for preventing not only obesity-induced inflammation but also the development of obesity-related pathologies. Resveratrol is a naturally occurring polyphenol present in red wine, berries, and peanuts. Numerous studies now support resveratrol as the major bioactive ingredient in red wine that contributes to its heart-protective effect [7]. Recently, resveratrol has been receiving increasing attention as a potent activator of Sirt1, which was evidenced by the findings that resveratrol can mimic caloric restriction and delay the aging process in a silent information regulator 2 (Sir2)-dependent manner [8]. More recently, oral administration of resveratrol was shown to improve insulin sensitivity of mice fed a high-fat diet [9]. Given that a highfat diet increases production of MCP-1 from adipose tissue and thus induces accumulation of adipose tissue macrophages, as well as changes the expression of adipocytokine [10,11]. We surmise that the beneficial roles of resveratrol in improving insulin sensitivity and preventing atherosclerosis are at least in part due to altering MCP-1 expression. Therefore, the present study was designed to determine whether resveratrol attenuates TNF-a-induced MCP-1 secretion and gene expression in 3T3-L1 adipocytes, and if so, to investigate the related cellular mechanism. Materials and methods Reagents. Recombinant TNF-a was purchased from R&D Systems (Minneapolis, MN, USA). Resveratrol, isobutylmethylxanthine (IBMX), dexamethasone (DEX), insulin, and BAY11-7082 were from Sigma– Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), sodium pyruvate were from Gibco-BRL (Rockville, MD, USA). Fetal bovine serum (FBS) was from PAA Laboratories (GmbH, Linz, Austria). c-32P ATP was purchased from Perkin–Furui Life Sciences (Perkin, CHN). The plasmids encoding Sirt1 were kindly provided by Dr. W. Bai (University of South Florida) [12]. pSV-b-galactosidase control vector was from Promega (Madison, USA). pNF-jB-luc construct was purchased from Clontech (Palo Alto, CA, USA). The monoclonal antiSirt1 was from Upstate Biotechnology (Temecula, CA, USA). Cell culture. The 3T3-L1 preadipocyteline was cultured in DMEM containing 25 mM glucose (DMEM-H), 1 mM sodium pyruvate, 100 U/ ml penicillin, 100 lg/ml streptomycin, and 10% FBS. For induction of differentiation, two days after postconfluence, the cells were stimulated to differentiate with DMEM containing 10% FBS, 167 nM insulin, 0.5 M IBMX, and 1 M DEX for 2 days. Cells were then maintained in 10% FBS/ DMEM medium with 167 nM insulin for another 2 days, followed by culturing with 10% FBS/DMEM medium for an additional 4 days, at which time 90% of cells were mature adipocytes with accumulated fat droplets. All stimulations were carried out after culturing the 3T3-L1 adipocytes in DMEM-H without any additions for the time periods indicated. Plamids construction. For construction of the MCP-1 promoter luciferase reporter plasmids, the 2.6 kb fragment containing the region from 2642 to +81 of the murine MCP-1 promoter was subcloned into pGL3 basic luciferase reporter gene vector to give the parental pMCP1-luc construct. Within this parental construct, the consensus sites (positions 2471 to 2461, 50 -GGGAACTTCC-30 was changed to 50 -ACGATATCAG-30 ) were inactivated by site-directed mutagenesis for transcription factor NF-jB (pMCP1 mut-NF-jB-luc) as described previously [13]. All plasmids were verified by DNA sequencing. Transient transfection and luciferase assays. HEK293 cells were plated at a density of 1.5  105 cells per well on 24-well plates for 24 h. Plasmids were transiently co-transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Relative

luciferase activity was measured with a luminometer (TD-20/20; Turner Designs, CA, USA) using luciferase assay system (Promega, Madison, WI, USA) according to the instructions. The b-galactosidase activity was detected to normalize the luciferase activity. Electrophoretic gel mobility shift assay. Nuclear extracts were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (Pierce, Woburn, MA, USA) as the manufacturer’s instruction. Nuclear extracts (5 lg) prepared from each group were incubated with 32P-labeled DNA probe (50 -AGTTGAGGGGACTTTCCCAGGC-30 ) in the binding buffer for 15 min at 37 °C, and the DNA-protein complex formed was separated from free oligonucleotide on 5% native polyacrylamide gels. A doublestranded mutated oligonucleotide, 50 -AGTTGAGGCGACTTTCCCA GGC-30 was used to examine the specificity of binding of NF-jB to the DNA. The specificity of binding was also examined by competition with the unlabeled oligonucleotide. After electrophoresis, the gel was dried and then exposed to X-ray film. The radioactive bands were quantitated by ImageQuant software. Real-time RT-PCR assay. The total RNA was isolated from 3T3-L1 adipocytes with TRIzol reagent (Invitrogen Life Technologies Inc., Carlsbad, CA, USA). A quantity of 1 lg RNA was reverse-transcribed using M-MLV reverse transcription system (Promega, Madison, WI, USA). Real time RT-PCR was performed by the ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA, USA) using SYBR Green fluorescence signals, as described previously [14]. The following primers were used: MCP-1, forward 50 -GCCCCACTCACCTG CTGCTACT-30 , reverse 50 -CCTGCTGCTGGTGATCCTCTTGT-30 ; IL6, forward 50 -AACGATGATGCACTTGCAG-30 , reverse 50 -GAGCATT GGAAATTGGGGTA-30 ; adiponectin, forward 50 -TGGTGAGAAGG GTGAGAA-30 , reverse 50 -AGATCTTGGTAAAGCGAATG-30 ; 36B4, forward 50 -AAGCGCGTCCTGGCATTGTCT-30 , reverse 50 -CCGCAG GGGCAGCAGTGGT-30 . Levels of mRNA, expressed as relative mRNA levels compared with control, were calculated after normalization to 36B4. Cytokine ELISA. The conditioned culture medium from 3T3-L1 adipocytes was collected from each sample. The concentrations of MCP-1 were assayed using a mouse MCP-1 ELISA kit (R&D Systems, Minneapolis, MN, USA). Each concentration was obtained from the standard curve and expressed as per mg of the total extractable cell protein. Immunoblotting. Cell lysates were separated by 10% SDS–PAGE horizontal gels and electrotransferred to polyvinylidene difluoride (PVDF) membrane and blocked in 5% milk. The membranes were probed with anti-Sirt1 and anti-b-actin antibodies diluted in blocking solution followed by horseradish peroxidase conjugated secondary antibody. Proteins were detected with an enhanced chemiluminescence detection system (ECLPlus, Amersham Pharmacia, Arlington, IL, USA), followed by exposure to X-ray films. Data Analysis. Results are expressed as mean ± SEM. Data were analyzed using unpaired Student’s t tests or one-way analysis of variance between groups (ANOVA) with Bonferroni post hoc test. Statistical analysis was performed with statistical analysis software SPSS 12.0 software. P < 0.05 was considered to have significant difference.

Results Resveratrol inhibits TNF-a-induced changes of MCP-1 secretion The 3T3-L1 adipocytes in normal state released 646 ± 24 pg/mg protein of MCP-1 to the culture media during incubation for 24 h, whereas the cells markedly increased MCP-1 production, up to 1446 ± 53 pg/mg protein, upon exposure to TNF-a alone. No significant difference in MCP-1 production was found between normal 3T3L1 adipocytes and the cells treated with resveratrol (50 lM) alone (Fig. 1A). However, resveratrol inhibited TNF-a-induced MCP-1 production in a dose-dependent

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Fig. 1. Effects of resveratrol treatment on TNF-a-induced changes of adipokines in 3T3-L1 adipocytes. (A) 3T3-L1 adipocytes were untreated or pretreated with various concentrations of resveratrol (RSV, lM) for 6 h and and then were stimulated for 24 h by the addition of TNF-a. The secreted protein MCP-1 in conditioned medium were measured as described in Materials and methods. Values were standardized by the total extractable protein, and expressed as means ± SEM (n = 4–6). (D) After exposure to various concentrations of resveratrol (RSV, lM) for 6 h, 3T3-L1 adipocytes were treated with TNF-a for 12 h. The mRNA expression levels were measured by quantitative RT-PCR. Values are normalized to 36b4 RNA expression levels and expressed relative to untreated control. Results were expressed as means ± SD and are representative of three independent experiments. #p < 0.01 vs. media alone-treated group, *p < 0.01 vs. with TNF-a only treated group. **p < 0.01 vs. media alone-treated group.

manner, corresponding to 38% inhibition at 10 lM, 70% at 25 lM and 84% at 50 lM. Resveratrol inhibits TNF-a-induced changes of MCP-1 expression To investigate whether resveratrol inhibited TNF-ainduced MCP-1 secretion by altering the gene expression, real time quantitative RT-PCR was carried out with total RNA extracted from 3T3-L1 adipocytes of each group. MCP-1 transcript markedly increased when the cells were stimulated with TNF-a alone compared to the control (Fig. 1B). Resveratrol attenuated TNF-a induced transcription of MCP-1 in a dose-dependent manner. The regulatory effect of resveratrol on MCP-1 transcription was further examined by promoter luciferase activity analysis. HEK293 cells, which have been used as a simplified cellular model for studying transcription regulation of adipocyte gene expression, were transfected with MCP-1 promoter that was fused to luciferase gene as a reporter. Upon exposure to TNF-a alone, the transfected cells increased luciferase expression up to 4.25-fold over the basal levels. However, resveratrol inhibited TNF-ainduced luciferase expression in a dose-dependent manner (Fig. 4B). No significant difference in the luciferase expres-

sion was found between transfected HEK293 cells and the cells treated with resveratrol (50 lM) alone. Furthermore, resveratrol inhibited TNF-a-induced increase in the production of IL-6 in a dose-dependent manner (Fig. 1C). Resveratrol itself alone increased the adiponectin gene expression, also effectively reversed the decrease in adiponectin production induced by TNF-a from 25 to 50 lM (Fig. 1D). NF-jB mediated TNF-a-induced MCP-1 expression and promoter activity We further tested which molecule implicated in TNF-asignaling was potential mediator of the positive effect of this cytokine on MCP-1 synthesis. 3T3-L1 adipocytes were pretreated with 5 lM BAY11-7082, a NF-jB inhibitor, for 2 h before the addition of 10 ng/mL TNF-a for 12 h. The total RNA was extracted from 3T3-L1 adipocytes of each group followed by real time quantitative RT-PCR analysis. The enhanced expression of MCP-1 mRNA by stimulation of TNF-a was suppressed by BAY11-7082 treatment. At a BAY11-7082 concentration of 5 lM, the MCP-1 mRNA expression was inhibited from 4.5-fold to 1.4-fold (Fig. 2A). To further confirm the role of NF-jB in the regulation of TNF-a-induced MCP-1 gene expression, HEK293 cells

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expression of IjBa caused a decrease of MCP-1 promoter activity from 4.6-fold to 1.42-fold, In addition, we inactivated the NF-jB DNA binding site of MCP-1 promoter by site-directed mutagenesis, and repeated the transient transfection study. The levels of inhibition were similar to the drop in fold-induction observed when IjBa was overexpressed (Fig. 2C), indicating NF-jB played a major role in TNF-a-induced expression of MCP-1. Resveratrol inhibited TNF-a-induced activation of NF-jB To determine whether the regulatory function of resveratrol was mediated by NF-jB, we investigated the effect of this compound on TNF-a-induced NF-jB transcriptional activity by using HEK293 cells harboring pNF-jBluc construct including four copies of the jB sequence that were fused to luciferase gene as a reporter. Upon exposure to TNF-a alone, the transfected cells increased luciferase expression up to 6.2-fold over the basal level, indicating that cellular NF-jB is transcriptionally functional. No significant difference in the luciferase expression was found between transfected HEK293 cells and the cells treated with resveratrol (50 lM) alone. However, resveratrol inhibited TNF-a-induced activition of NF-jB (Fig. 4D). The phenomenon of NF-jB transcriptional activity is preceded by DNA binding activity of NF-jB complex. We next determined whether resveratrol could affect DNA binding activity of the NF-jB complex. An NFjB-specific [32P]-labeled oligonucleotide was reacted with nuclear extracts of TNF-a-stimulated 3T3-L1 adipocytes, and then subjected to electrophoretic mobility shift assay. DNA binding activities of NF-jB were markedly increased compared with exposure to TNF-a alone. Resveratrol exhibited dose-dependent inhibitory effects on TNF-ainduced DNA binding activity of NF-jB complex (Fig. 3).

Fig. 2. NF-kB mediated TNF-a-induced MCP-1 expression and promoter activity. (A) 3T3-L1 adipocytes were untreated or pretreated with BAY11-7082 (BAY, 5 lM) or Sirtinol (10 lM) for 6 h and then added with TNF-a (T, 10 ng/ ml) stimulation for 12 h. Values are normalized to 36b4 RNA expression levels and expressed relative to TNF-a alone treated group. (B) HEK293 cells were transfected with pMCP1-luc reporter construct, pSV-bgal vector and IjBa expression vector or pCMV as control. (C) HEK293 cells were co-transfected with firefly luciferase reporter construct (pMCP1-luc or pMCP1/mut-jB-luc) and pSV-bgal plasmid. Twenty four hours after transfection, all cells were untreated or treated with 10 ng/ml TNF-a for 6 h. Luciferase expression as a reporter for MCP-1 promoter activity is represented as relative fold. Values are means ± SEM of three independent experiments. *p < 0.01 vs. TNF-a alonetreated group, **p < 0.01 vs. wild type promoter group.

were transiently transfected with wild type MCP-1 promoter reporter construct and IjBa protein expression vector followed by TNF-a stimulation. Treatment with TNF-a resulted in 4.25-fold increase in MCP-1 reporter gene expression, which was repressed by co-transfection of IjBa plasmid compared to control (Fig. 2B). When fold induction by TNF-a was determined, we found that over-

Overexpression of Sirt1 inhibited TNF-a-induced MCP-1 promoter and NF-jB activity Resveratrol was reported to be an activator of Sirt1 in vivo and in vitro. Thus, we next examined the effects of modulating Sirt1 on the TNF-a-induced MCP-1 promoter and NF-jB activity. As shown in Fig. 4A, HEK293 cells overexpressing of Sirt1 displayed a remarkable reduction in TNF-a-induced MCP-1 promoter activity and NF-jB activity relative to cells co-transfected with the control vector. However, sirtinol, an inhibitor of Sirt1, increased the basal and TNF-a-induced MCP-1 promoter activity and NF-jB activity (Fig. 4C and D). Furthermore, Sirtinol amplified the TNF-a-induced increased effects on MCP-1 mRNA transcription (Fig. 2A). We next explored whether the exogenous overexpression of Sirt1 protein affect DNA binding activity of NF-jB complex. No significant difference was detected between those cells transfected with Sirt1 plasmid and control vector (Fig. 4F). This result suggested that activation of Sirt1 could not alter the DNA

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Fig. 3. Resveratrol suppress TNF-induced NF-kB binding activity. 3T3L1 adipocytes were preincubated at 37 °C for 6 h with indicated different concentrations of resveratrol (RSV, lM), followed by 1 h incubation with 10 ng/ml TNF-a. After these treatments, nuclear extracts were prepared and then assayed for NF-jB activation.

binding activity of NF-jB but inhibited NF-jB dependent transcription activity induced by TNF-a. Discussion Among inflammatory molecules up-regulated in adipose tissues of obese animals and humans, MCP-1 has been viewed as an pivotal candidate adipocytokine, initiating macrophage infiltration of the adipose tissue and inducing systemic insulin resistance [15]. In the present study, we demonstrated for the first time that resveratrol effectively inhibited TNF-a-induced increase in the secretion of MCP-1 in 3T3-L1 adipocytes (Fig. 1A). Furthermore, resveratrol not only attenuated TNF-a-induced synthesis of MCP-1 but also inhibited TNF-a-induced MCP-1 promoter activity (Figs. 1B and 4B). These results indicated that resveratrol could regulate TNF-a-induced MCP-1 expression at the transcription level. In addition, resveratrol attenuated TNF-a-induced changes of mRNA expression of other adipocytokines, IL-6 and adiponectin, in dose-dependent manners (Fig. 1C and D). Transcription factor NF-jB has been evidenced to play a major role in the TNF-a-induced expression of adipocytokines including IL-6 and PAI-1 in adipocytes[16]. In the present study, we determined the key role of NF-jB in MCP-1 gene expression induced by TNF-a. First, BAY11-7082 abolished the TNF-a-induced MCP-1 gene expression in 3T3-L1 adipocytes (Fig. 2A). Second, EMSA analysis revealed that TNF-a stimulated NF-jB binding activity in 3T3-L1 adipocytes (Fig. 3). Third, overexpres-

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sion of IjB-a abolished the effects of TNF-a-stimulated MCP-1 promoter activity (Fig. 2B). At last, luciferase assay NF-jB mediated the activation of the TNF-a-responsive element of the MCP-1 gene (Fig. 2C). Recent studies have reported that the effect of resveratrol on NF-jB activation triggered by cytokine is cell-type specific. Resveratrol suppressed TNF-a-induced activation of NF-jB in U-937, HeLa and T-Jurkat cells, as well as coronary arterial endothelial cells [17]. However, treatment of glomerular mesangial cells with resveratrol significantly and dose-dependently enhanced NF-jB activation triggered by proinflammatory cytokines [18]. Our results showed that resveratrol attenuates TNF-ainduced NF-jB activity in 3T3-L1 cells (Fig. 3). These evidences indicated that resveratrol is a potent inhibitor of TNF-a-induced activation of NF-jB and thus inhibited TNF-a-induced changes of adipokines in 3T3-L1 adipocytes. It has been demonstrated that resveratrol induces a conformational change in Sirt1 protein, which lowers the Km for both the acetylated substrate and nicotinamide adenine dinucleotide (NAD), therefore enhancing the deacetylation of Sirt [19] and Sirt1-dependent cellular processes, such as axonal protection [20] and fat mobilization [21]. Recently, Yeung, et al. [22] reported that Sirt1 could physically interact with the RelA/p65 subunit of NF-jB and inhibits its transcription by deacetylating RelA/p65 at lysine 310. Taken together, these experiments raised the possibility that resveratrol could attenuate TNF-a-induced MCP-1 gene expression in Sirt1-dependent manner. Our results showed that overexpression of Sirt1 protein in HEK293 cells resulted in downregulation of TNF-a-induced MCP-1 promoter activity and NF-jB activity (Fig. 4A). Furthermore, treatment of the HEK293 cells with sirtinol, a Sirt1 inhibitor, increased the basal and TNF-a-induced promoter activity (Fig. 4C and D). Additionally, Sirt1 is traditionally thought to be located in the nucleus, however, it has recently been shown also located in the cytoplasm, such as in the pancreas islet beta cell [23], HEK293 cell [24]. Sirt1 protein directly interacts with IRS-2 protein which regulates insulin-induced IRS-2 tyrosine phosphorylation and its downstream Akt activation by deacetylating IRS-2 in cytoplasm [24]. In the present study, overexpression of Sirt1 protein did not alter the TNF-a-induced NF-jB binding activity (Fig. 4E). Based on these evidences described above, a possible explanation was that deacetylated NF-jB could still bind to the NF-jB consensus sequences of target genes, however, it was not able to initiate gene transcription. These results suggest activation of Sirt1 may be a cellular mechanism of resveratrol’s effect on MCP-1 expression. In conclusion, resveratrol can attenuate the TNF-a induced MCP-1 genes expression and secretion in 3T3-L1 adipocyte. The effects were mediated by directly repressing NF-jB binding activity, as well as indirectly inhibiting NFjB dependent transcription activity through activation of Sirt1. Our studies contribute to the understanding of the anti-inflammatory effect of resveratrol and provide a novel

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Fig. 4. Modulation of Sirt1 affects TNF-a induced MCP-1 promoter activity and NF-kB activation. (A) HEK293 cells were co-transfected with firefly luciferase reporter construct (pMCP1-luc or pNF-jB-luc) and pcDNA-Sirt1(Sirt1) or pcDNA3.1 vector (VC). TNF-a (10 ng/ml) was added the next day for 6 h before harvesting the cells. (B, C) HEK293 cells were transient co-transfected pMCP1-luc reporter construct and pSV-bgal plasmid. Cells were left untreated (Ctl) or treated with resveratrol (RSV, lM) (B) or 10 lM sirtinol (C) for 6 h and then stimulated by addition of 10 ng/ml TNF-a for 6 h. (D) HEK293 cells were transiently co-transfected with the pNF-jB-luc reporter construct and pSV-bgal plasmid. Cells were left untreated (Ctl) or treated with resveratrol (RSV, 50 lM) or Sirtinol (10 lM) for 6 h and then 10 ng/ml TNF-a was added for 6 h. The luciferase activities were measured as Fig. 2. Results are means ± SEM of three independent experiments. #p < 0.01 vs. media treated group. ##p < 0.01 vs. TNF-a treated group. *p < 0.01 vs. TNF-a treated group. (E) Western blot analysis demonstrates the protein expression of Sirt1 in HEK293. (F) HEK293 cells were transfected with pcDNA-Sirt1 (Sirt1) or pcDNA3.1 vector (VC). Twenty four hours after transfection, cells were treated with or without TNF-a (10 ng/ml) 1 h, nuclear extracts were prepared and then assayed for NF-jB activation by EMSA.

mechanism of resveratrol in preventing obesity-related pathologies. Acknowledgment This work was supported by grants from the Science Foundation of Jiangsu Province (BK2007251) and the Special Funds for Major State Basic Research Program of China (973 Program, 2006CB503908).

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