Inhibitory Effect of Piceatannol on TNF-α-Mediated Inflammation and

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Inhibitory Effect of Piceatannol on TNF-α-Mediated Inflammation and Insulin Resistance in 3T3-L1 Adipocytes Yanfang Li,†,§ Puyu Yang,§ Qimeng Chang,# Jing Wang,† Jie Liu,*,† Yuan Lv,§ Thomas T. Y. Wang,⊗ Boyan Gao,⊥ Yaqiong Zhang,§ and Liangli Lucy Yu*,⊥

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Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology & Business University, Beijing 100048, China § Institute of Food and Nutraceutical Science, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China # Department of Surgery, Fudan University Minhang Hospital, Shanghai 201199, China ⊗ Diet, Genomics, and Immunology Laboratory, Agricultural Research Service (ARS), U.S. Department of Agriculture, Beltsville, Maryland 20705, United States ⊥ Department of Nutrition and Food Science, University of Maryland, College Park, Maryland 20742, United States S Supporting Information *

ABSTRACT: Piceatannol, a bioactive component in grape and blueberry, was examined for its potential in decreasing the inflammatory activities in adipocytes using a cocultured adipocyte and macrophage system, and suppressing tumor necrosis factor-α (TNF-α)-mediated inflammation and the related insulin resistance using a 3T3-L1 adipocyte model. Piceatannol at 10 μM significantly reduced the release of inflammatory cytokines of TNF-α and monocyte chemoattractant protein-1 (MCP-1) by 19 and 31% in the cocultured system, respectively. Pretreatment with piceatannol also inhibited TNF-α-induced expression of interleukin-6 (IL-6) and MCP-1 at both mRNA and protein levels in the 3T3-L1 adipocytes. Piceatannol also partially improved the malfunction of insulin-stimulated glucose uptake, which was reduced by TNF-α in 3T3-L1 adipocytes. Furthermore, the inhibitions were mediated by significant blocking of IκBα phosphorylation and nuclear factor-κB (NF-κB) activation through suppressing nuclear translocation of NF-κB p65 along with c-Jun N-terminal kinase (JNK)−mitogen activated protein kinase (MAPK) activation. In addition, the Akt-dependent forkhead box O1 (FoxO1) signaling pathway was involved in the restoration of insulin-stimulated glucose uptake through suppressing the down-regulation of phosphorylation of Akt and FoxO1 expressions. These results suggested the potential of piceatannol in improving chronic inflammatory condition and insulin sensitivity in obese adipose tissues. KEYWORDS: piceatannol, TNF-α, 3T3-L1 adipocytes, inflammation, insulin resistant

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adipocytes using a conditioned medium model.4 Piceatannol treatment at 30 μmol/L concentration was able to inhibit the expression of TNF-α, IL-6, and nitric oxide at both mRNA and protein levels. However, the possible molecular mechanism behind the anti-inflammation activity of piceatannol was not investigated. It was also interesting whether and how piceatannol may act in a cocultured adipocyte and macrophage system, which allows simultaneous functional interaction between the two cell types. In addition, it has been widely accepted that TNF-α secreted from macrophages might infiltrate into obese adipose tissue and increase the risk of obesity-associated insulin resistant.5−8 A cocultured adipocyte and macrophage system may mimic an inflammatory status in obese tissues and has been used as an experimental model for investigating the simultaneous functional interaction between the two cell types since it was developed by Suganami and others.9 This cocultured adipocyte and

INTRODUCTION Piceatannol (3,3′,4,5′-trans-tetrahydroxystilbene) is an analogue and a metabolite of resveratrol that presents in many fruits including grapes and blueberries.1 Use of resveratrol in dietary intervention to reduce the risk of chronic human diseases is limited due to its poor bioavailability and rapid metabolism. Hydroxylated resveratrol derivatives, including piceatannol, are possible alternatives to resveratrol for health food applications. Increasing evidence has indicated the potential benefits of piceatannol in immunomodulatory, antiadipogenesis, antiproliferative, and anti-inflammatory properties.1−4 Piceatannol suppressed tumor necrosis factor-α (TNF-α) activation of nuclear factor-κB NF-κB)-dependent reporter gene expression in myeloid cells, lymphocytes, and epithelial cells.2 TNF-α is the primary activator of pro-inflammatory signaling cascades that regulate the pro-inflammatory gene transcriptions such as interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and resistin gene transcriptions, leading to a selffeeding inflammatory cycle. Recently, Takayuki and others observed that piceatannol suppressed the pro-inflammation gene expression in RAW 264.7 macrophages induced by lipopolysaccharide (LPS) and the culture medium of 3T3-L1 © 2017 American Chemical Society

Received: Revised: Accepted: Published: 4634

April 5, 2017 May 17, 2017 May 23, 2017 May 23, 2017 DOI: 10.1021/acs.jafc.7b01567 J. Agric. Food Chem. 2017, 65, 4634−4641

Article

Journal of Agricultural and Food Chemistry

PCR were 50 °C for 2 min, 95 °C for 10 min, and 40 cycles of amplification at 95 °C for 15 s and 60 °C for 1 min. Quantifications were performed in triplicate, and the experiments were carried out independently three times. Changes in the expression of target genes were expressed as relative mRNA levels and normalized to a housekeeping gene, β-actin; data were analyzed using the 2−ΔΔCt method. The primer sequences for each gene were performed with the following primers: MCP-1, forward, 5′-TCTGGACCCATTCCTTCTTG-3′ and reverse, 5′-AGGTCCCTGTCATGCTTCTG-3′; IL-6, forward, 5′-CACGGCCTTCCCTACTTCAC-3′ and reverse, 5′-TGCAAGTGCATCATCGTTGT-3′; β-actin, forward, 5′-TGTCCACCTTCCAGCAGATGT-3′ and reverse, 5′-AGCTCAGTAACAGTCCGCCTAGA-3′. Glucose Uptake. The glucose uptake assay was performed using a commercial kit (BioVision, Milpitas, CA, USA) according to the manufacturer’s protocol with minor modification. 2-Deoxyglucose (2-DG) has been widely used because of its structural similarity to glucose. 2-DG can be taken up by glucose transporters and metabolized to 2-DG-6-phosphate (2-DG6P) and thus accumulated in the cells. The accumulated 2-DG6P is directly proportional of 2-DG (or glucose) uptake by cells. In brief, cells were incubated in Krebs Ringer phosphate Hepes buffer (KRPH, containing 121 mM NaCl, 4.9 mM KCl, 1.2 mM MgSO4, 0.33 mM CaCl2, and 12 mM Hepes, pH 7.4) for 4 h. The cells were pretreated with 10 μM piceatannol for 24 h followed by cotreatment with 10 ng/mL TNF-α for 24 h. The culture medium was discarded and replaced with 900 μL of KRPH with or without 10 nM insulin for 60 min at 37 °C. After 30 min of incubation in the KRPH with 10 mM 2-DG at 37 °C, cells were immediately washed with ice-cold KRPH three times and lysed by extraction buffer for 10 min; reaction mix substrate was added. Accumulated 2-DG6P was oxidized to generate NADPH, which can be determined by measuring the fluorescence at Ex/Em = 535/587 nm using a Tecan PRO M200microplate reader (Mannedorf, Switzerland). Western Blotting. Cells were carefully washed twice with ice-cold PBS, harvested by scraping, and immediately mixed with 200 μL of icecold radio-immunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktail (Sigma) and phosphatase inhibitors (Roche Diagnostics, Mannheim, Germany) for each well. To remove the insoluble materials, lysates of the whole cells were centrifuged at 10,000g for 20 min at 4 °C. The protein concentrations in the lysates were determined using a BCA protein assay kit (Pierce, Rockford, IL, USA). For the nucleus p65 protein analysis, nuclear proteins were obtained using a Beyotime Nuclear and Cytoplasmic Extraction Kit (Beyotime Inc., Nantong, Jiangsu, China) according to the manufacturer’s instruction. The protein concentration for each sample was adjusted to an equal amount with different volumes of loading buffer and denatured in boiled water for 5 min. To separate different proteins, equal aliquots (40 μg) of protein samples were subjected to 12% polyacrylamide gel electrophoresis and then electrotransferred to PVDF membranes at 25 V and 1.5 A for 60 min. The membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 2 h. After being washed with TBST three times, the membranes were incubated with specific target protein antibodies overnight at 4 °C, followed by incubation with secondary antibodies with conjugated horseradish peroxidase (HRP) for 1.5 h at ambient temperature. Peroxidase activity was visualized using the chemiluminescence method with an ECL kit (Bio-Rad, Hercules, CA, USA) with ChemiDoc XRS+ (Bio-Rad), with β-actin as an internal control. Films were scanned, and the protein levels of the specific bands were quantified by detecting the densitometry using the Image Lab software and calculated according to the internal β-actin control. Statistical Analysis. Each experiment was performed in triplicate. Data were expressed as the mean ± SD. The statistical significance of difference was analyzed using one-way ANOVA, followed by Tukey’s test. All statistical analyses were performed using SPSS 21.0 software. P < 0.05 was considered to be statistically significant. Graph was created on GraphPad Prism (version 6.00, Graphpad Software Inc., San Diego, CA, USA).

macrophage system also provides an excellent opportunity for investigating whether and how piceatannol may improve insulin sensitivity in the adipocytes under inflammatory status, which has not been reported to the best of our knowledge.10 Therefore, this research was conducted to investigate whether and how piceatannol might attenuate TNF-α mediated inflammation and insulin resistance in adipocytes using a cocultured adipocyte and macrophage model. The information obtained from this research might be used to promote the utilization of piceatannol or grape and other fruits to reduce the risk of chronic inflammation in obese adipose tissue and improve obesity-related insulin resistance.

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MATERIALS AND METHODS

Chemicals. Piceatannol with a purity of >98% (HPLC), insulin, 3-isobutyl-1-methylxanthine (IBMX), and dexamethasone were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum (FCS), and fetal bovine serum (FBS) were obtained from Life Technologies (New York, NY, USA). Antibodies against IκBα, p-IκBα, p65, JNK, p-JNK, ERK1/2, p-ERK1/2, p38, p-p38, Akt, p-Akt, GSK3β, p-GSK3β, FoxO1, p-FoxO1, and β-actin and the secondary antibodies were purchased from the Cell Signaling Technology (Beverly, MA, USA). All other chemicals were purchased from Sigma (St. Louis, MO, USA) with the highest quality available and used without further purification. Cell Culture and Differentiation of 3T3-L1 Preadipocytes. The 3T3-L1 preadipocytes (American Type Culture Collection, ATCC, Manassas, VA, USA) were cultured and differentiated as previously described following a laboratory protocol.11 Briefly, 3T3-L1 preadipocytes were cultured in DMEM supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% FCS at 37 °C, in a humidified atmosphere of 95% air and 5% CO2. The cells were cultured in six-well cell culture plates at a density of 1 × 105 cells/mL and allowed to grow overnight to achieve adherence. The culture medium was renewed every 2 days until cells reached confluence and then cultured for another 2 days; the cells were then cultured in a differentiation medium containing 10% FBS−DMEM, 1 μg/mL insulin, 0.25 μM dexamethasone, and 0.5 mM IBMX for 48 h. The cells were then cultured in 10% FBS−DMEM supplemented with 1 μg/mL insulin for another 48 h. The culture medium was changed to 10% FBS−DMEM medium every 2 days until cells achieved full adipocyte morphology. The 3T3-L1 adipocytes were treated with different concentrations of piceatannol and subsequently treated with 10 ng/mL of TNF-α for 15 min or 24 h. Coculture of 3T3-L1 Adipocytes and RAW 264.7 Macrophages. RAW 264.7 macrophages were purchased from the ATCC and cocultured with differentiated 3T3-L1 adipocytes in a Trans-well system (Corning Inc., Acton, MA, USA). 3T3-L1 preadipocytes were seeded in 12-well plates and differentiated as described above. RAW 264.7 macrophage cells (6 × 104 cells/mL) were seeded in Trans-well inserts with a 0.4 μm porous membrane and cultured in DMEM supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% FBS at 37 °C, in a humidified atmosphere of 95% air and 5% CO2. 3T3-L1 adipocytes were cocultured with RAW 264.7 cells and treated with 5% BSA−DMEM containing 5 and 10 μM piceatannol for 24 h. Subsequently, cells and coculture medium were collected and stored at −80 °C until analysis. MCP-1, TNF-α, and IL-6 Productions. The concentration of MCP-1, TNF-α, and IL-6 in the coculture media were determined with three enzyme-linked immunosorbent assay kits (eBioscience, San Diego, CA, USA), according to the manufacturer’s protocol. Real-Time PCR. Total RNA from vehicle or piceatannol-treated differentiated 3T3-L1 adipocytes was extracted with the TrizoL reagent (Invitrogen, Carlsbad, CA, USA). An equal aliquot (0.4 μg) of total RNA was used for the synthesis of first-stand cDNA with an IScript reverse transcriptase kit (Bio-Rad) according to the manufacturer’s protocol. The quantitative real-time PCR amplification and detection were performed subsequently using the cDNA on an ABI 7900 HT (Applied Biosystems, Carlsbad, CA, USA). The cycling parameters for 4635

DOI: 10.1021/acs.jafc.7b01567 J. Agric. Food Chem. 2017, 65, 4634−4641

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Journal of Agricultural and Food Chemistry

Figure 1. Effects of piceatannol on inflammatory changes. (A) Effects of piceatannol on the secretion of inflammatory mediators in Trans-well coculture system. Differentiated 3T3-L1 adipocytes were cocultured with RAW 264.7 macrophages for 24 h in the absence or presence of 5 and 10 μM piceatannol. Released protein levels of (B) TNF-α, (C) MCP-1, and (D) IL-6 in the coculture medium were measured by ELISA, and those from 3T3-L1 adipocytes or RAW 264.7 macrophages alone were used as controls. Effects of piceatannol on TNF-α induced relative mRNA expressions of (E) IL-6 and (F) MCP-1 were measured by RT-PCR. Effects of piceatannol on TNF-α induced protein expressions of (G) IL-6 and (H) MCP-1 were measured by ELISA. Adipocytes were pre-incubated with 5 and 10 μM piceatannol for 24 h and then treated with 10 ng/mL TNF-α for 24 h before cells were harvested. Values are the mean ± SD of three independent experiments carried out in triplicate. Different letters above each bar indicate significant differences between means (P < 0.05).

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RESULTS AND DISCUSSION Piceatannol Significantly Reduced TNF-α and MCP-1 Secretion in a Cocultured System of 3T3-L1 Adipocytes and RAW 264.7 Macrophages. In obese adipose tissue,

TNF-α from the infiltrated macrophages plays an important role in developing obesity-related insulin resistance.6 The effect of piceatannol on the inflammatory changes in adipose tissue was examined in the cocultured 3T3-L1 adipocytes and RAW 4636

DOI: 10.1021/acs.jafc.7b01567 J. Agric. Food Chem. 2017, 65, 4634−4641

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Journal of Agricultural and Food Chemistry

in the cocultured system (Figure 1D). It was interesting whether TNF-α might play a role in the overexpression of MCP-1 and IL-6 in the cocultured adipocytes and whether piceatannol may reduce their expressions. As shown in Figure 1E,F, TNF-α significantly increased the mRNA expression of MCP-1 and IL-6, and pretreatment with 5 and 10 μM piceatannol was able to significantly reverse the TNF-α-induced MCP-1 and IL-6 mRNA overexpression in the 3T3-L1 adipocytes. The inhibition of TNF-α-induced MCP-1 mRNA expression was in a dose-dependent manner (Figure 1E). Piceatannol was also able to suppress the TNF-α-induced MCP-1 and IL-6 expressions at the protein level (Figure 1G,H). The suppression of MCP-1 protein production was also dosedependent (Figure 1G). It needs to be pointed out that the treatment concentrations for all of these tests had no adverse effects on 3T3-L1 adipocytes and RAW 264.7 macrophages growth according to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Figures S1 and S2). TNF-α has been known to up-regulate the production of several important inflammatory cytokines and chemokines such as MCP-1and IL-6 in adipocytes, leading to down-regulation of glucose uptake in the adipocytes.8,9 The data in Figure 1 indicate a potential of piceatannol in suppressing TNF-αinduced production of inflammatory cytokines and chemokines in adipocytes, and it became interesting whether and how piceatannol might be able to modulate TNF-α-induced insulin resistance in adipocytes. Piceatannol Improved TNF-α-Mediated Insulin Resistance in 3T3-L1 Adipocytes. Glucose uptake was determined by 2-DG6P uptake in the 3T3-L1 adipocytes treated with TNF-α with or without piceatannol pretreatment in the absence or presence of insulin. Treatment with TNF-α for 24 h significantly

Figure 2. Effects of piceatannol and TNF-α on glucose uptake in 3T3-L1 adipocytes. Fully differentiated 3T3-L1 adipocytes were all treated with 10 ng/mL TNF-α alone or pretreated with 10 μM piceatannol in the absence or presence of insulin for 24 h. 2-DG6P uptake was determined. Values are the mean ± SD of three independent experiments carried out in triplicate. Different letters above each bar indicate significant difference (P < 0.05).

264.7 macrophages at 5 and 10 μM concentrations using a Transwell system. The expressions of TNF-α, MCP-1, and IL-6 were significantly greater in the cocultured system as compared with that in only 3T3-L1 adipocytes or RAW 264.7 macrophages (Figure 1A−D). This overexpression of TNF-α and MCP-1 in the cocultured system was dose dependently reversed by preincubation with 5 and 10 μM piceatannol (Figure 1B,C), whereas piceatannol was not able to significantly reduce IL-6 expression

Figure 3. Effects of piceatannol on NF-κB pathway relevant protein expressions induced by TNF-α with different concentrations of piceatannol in 3T3-L1 adipocytes. 3T3-L1 adipocytes were pretreated with 5 and 10 μM piceatannol for 24 h, followed by cotreatment with 10 ng/mL TNF-α for another 15 min. The protein levels of (A) phosphor (p)-IκBα and total (t)-IκBα and (B) total (t) p65 and total β-actin in the nucleus were determined by Western blotting. β-Actin was used as a loading control. The protein levels of the bands were quantified by densitometry. The results were reported as the ratio of phosphorylated/total protein contents and expressed in amounts relative to the control values. Values are the mean ± SD of three independent experiments. Different letters indicate significant difference between means (P < 0.05). 4637

DOI: 10.1021/acs.jafc.7b01567 J. Agric. Food Chem. 2017, 65, 4634−4641

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Figure 4. Effects of piceatannol on MAPK pathway relevant protein expressions induced by TNF-α with different levels of piceatannol in 3T3-L1 adipocytes. 3T3-L1 adipocytes were pretreated with 5 and 10 μM piceatannol for 24 h, followed by cotreatment with 10 ng/mL TNF-α for another 15 min. The protein levels of (A) phosphor (p) or total (t) ERK1/2, JNK, and p38 were determined by Western blotting. β-Actin was used as a loading control. The protein levels of the bands were quantified by densitometry. The results were reported as the ratio of phosphorylated/total protein contents: (B) p-JNK/JNK, (C) p-ERK/ERK, and (D) p-p38/p38 contents were expressed in amounts relative to the control values. Values are the mean ± SD of three independent experiments. Different letters above each bar indicate significant difference (P < 0.05).

decreased both basal and insulin-stimulated 2-DG6P uptake in the 3T3-L1 adipocytes (Figure 2), supporting the conclusion that pretreatment with 10 μM piceatannol significantly improved TNF-α-mediated reduction of 2-DG6P uptake regardless of insulin presence (Figure 2). Pretreatment with 10 μM piceatannol could significantly alleviate TNF-α-induced insulin resistance by improving 21.7% 2-DG6P uptake in the presence of insulin under the experimental conditions. These results suggested the potential health benefits of fruits and other foods rich in piceatannol. Piceatannol Attenuated TNF-α-Induced Adipocyte Inflammation through the NF-κB and MAPK/JNK Pathway. To further understand how piceatannol could suppress the TNF-α-induced adipocyte inflammation and insulin resistance, the effect of piceatannol on nuclear factor-κB (NF-κB) and three mitogen-activated protein kinases (MAPKs) signaling pathways were investigated. Piceatannol significantly suppressed TNF-αinduced phosphorylation and degradation of IκBα in a dosedependent manner (Figure 3A). Piceatennol also was able to does-dependently inhibit the TNF-α induced nuclear translocation of p65 protein (Figure 3B). Together, these data suggested that the NF-κB signaling pathway might be involved in TNF-α-induced inflammatory reactions and glucose uptake in adipocytes, as well as the effects of piceatannol in reducing the risk of inflammation and insulin resistance in adipose tissues. The NF-κB pathway has been

recognized for its role in adipocyte lipolysis, inflammation, and insulin resistance.6 NF-κB may be activated by TNF-α through phosphorylation and proteasomal degradation of IκBα, a part of the NF-κB complex, stimulating the translocation of RelA (p65)/p50 complexes to nucleus and consequently inducing the production of enzymes and cytokines important for inflammation and insulin resistance, such as MCP-1 and IL-6. However, how piceatannol might block the phosphorylation of IκBα and inhibit the p65 nuclear translocation has not been fully understood. A previous study has shown that TNF-α-induced NF-κB activation might be through a canonical activation pathway.12 Briefly, TNF-α might activate tumor necrosis factor receptor (TNFR) and stimulate other signal transducing adaptor proteins and kinases, leading to the activation of IKKβ, which results in the phosphorylation and degradation of IκBα, nuclear translation of p65, and activation of target gene transcription.12−15 Piceatannol has been found to block NF-κB activation through direct modification of IKKβ at the cysteine 179 residue16 and inhibition of p65 phosphorylation.2 Taken together with the results shown in Figure 3 that piceatannol reduced TNFαinduced phosphorylation of IκBα and nuclear translation of p65, IKKβ would be an interesting key target for future studies to fully understand how piceatannol and other stilbenoids might suppress TNF-α-induced phosphorylation of IkBα and p65. The potential role of piceatannol in TNF-α induced phosphorylation of c-Jun N-terminal kinase (JNK), extracellular 4638

DOI: 10.1021/acs.jafc.7b01567 J. Agric. Food Chem. 2017, 65, 4634−4641

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Journal of Agricultural and Food Chemistry

Figure 5. Effects of piceatannol on TNF-α-mediated suppression of the insulin signaling cascades in 3T3-L1 adipocytes. Cells were pretreated with blank or with 10 μM piceatannol for 24 h, followed by treatment with or without 10 ng/mL TNF-α for 24 h. Then 10 nM insulin was added at the last 30 min. (A) Cultures were then harvested to determine the protein expression of phosphor (p) or total (t) Akt, GSK3β, and FoxO1 using Western blotting. β-Actin was used as a loading control. The protein levels of the bands were quantified by densitometry, and results were expressed as the ratio of (B) p-Akt/Akt, (C) p-GSK3β/GSK3β, and (D) p-FoxO1/FoxO1 protein contents and reported relative to the control values. Values are the mean ± SD of three independent experiments. Different letters above each bar indicate significant difference (P < 0.05).

examine whether and how piceatannol might reduce the risk of TNF-α-induced insulin resistance in adipocytes through the insulin signaling pathway. Piceatannol Enhanced the Insulin Signaling through Improving Phosphorylation of Akt, GSK3β, and FoxO1. Insulin signaling inhibition is a possible mechanism for insulin resistance. To further elucidate how piceatannol could alleviate the TNF-α-induced adipocyte insulin resistance, the effect of piceatannol on insulin signal cascades, including Akt, glycogen synthase kinase 3β (GSK3β), and Forkhead box O1 (FoxO1), were examined. Pretreatment of piceatannol was able to significantly repair the TNF-α-induced phosphorylation suppression of Akt in the adipocytes (Figure 5A,B), and the effect required the presence of insulin (Figure 5B). Piceatannol had no significant effect on GSK3β phosphorylation regardless of the insulin presence (Figure 5C). In addition, piceatannol pretreatment was able to improve the phosphorylation of FoxO1 with or without the presence of insulin (Figure 5A,D). These results are consistent with that on glucose uptake in Figure 2. The findings from this study agreed with the observation by Tsai and others that carnosic acid was able to attenuate TNF-α-induced 3T3-L1 adipocyte inflammation via suppressing NF-κB and enhancing insulin sensitivity via the Akt-dependent FoxO1 signaling pathway.21 Akt, GSK3β. and FoxO1 play an important role in insulin-regulated glucose uptake,22−24 and food factors capable of enhancing their phosphorylation might potentially improve insulin resistance, including enhancing glucose uptake.25−29 The results from this study suggested that piceatannol might

signal-regulated kinase (ERK), and p38 was investigated to understand its possible involvement in the MAPK pathway (Figure 4). Pretreatment with piceatannol was able to attenuate TNF-α-mediated activation of JNK in a dose-dependent manner (Figure 4A,B), with no significant effect on the phosphorylation of ERK (Figure 4C) or p38 (Figure 4D). JNK, ERK, and p38 are three important MAPK family kinases. These kinases may be activated by inflammatory stimuli and oxidative stress and are contributors to obesity-associated inflammation and insulin resistance. JNK-dependent MAPK pathway could interact with the PI3K/Akt pathway, important for insulin actions such as glucose uptake.6,17 Taken together, the data suggested that the NF-κB pathway and JNK phosphorylation might be the possible primary targets for piceatannol to alleviate the TNF-α-induced inflammation and insulin resistance in adipocytes under the experimental conditions. The findings were supported by an earlier report from Sakamoto and others that dietary daidzein, an isoflavone compound common in soybean, could down-regulate proinflammatory gene expression by inhibiting the JNK pathway in a coculture of adipocytes and macrophages.18 The findings from this study were also supported by Yang and others’ observation that bitter melon could reduce high-fat diet induced insulin resistance and diabetes in OLETF rats through suppressing the NF-κB and JNK pathways.19 NF-κB and MAPK/JNK pathways may induce specific insulin receptor substrate 1 (IRS-1) serine phosphorylation, resulting in an impaired downstream insulin receptor signaling.19,20 It was interesting to further 4639

DOI: 10.1021/acs.jafc.7b01567 J. Agric. Food Chem. 2017, 65, 4634−4641

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Journal of Agricultural and Food Chemistry

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restore the TNF-α-induced insulin resistance in 3T3-L1 adipocytes through the Akt-dependent FoxO1 signaling pathway and potentially improve insulin resistance. In summary, this study observed for the first time that piceatannol significantly reduced TNF-α and MCP-1 secretions in a cocultured system of 3T3-L1 adipocytes and RAW 264.7 macrophages and further demonstrated that it could attenuate TNF-α-induced adipocyte inflammation possibly through the NF-κB and MAPK/JNK pathways. The results from the present study also suggested that piceatannol could improve the TNF-αmediated insulin resistance in 3T3-L1 adipocytes through activating the Akt−FoxO1 insulin cascade signal pathway to enhance insulin signaling. These findings suggested that piceatannol might be used to improve obesity-associated glucose uptake disorder through its attenuation of chronic inflammatory condition and improvement in insulin sensitivity in obese adipose tissues. Additional research is needed to confirm the observations and better understand the role of piceatannol in insulin resistance.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b01567. Cytotoxicity of piceatannol in 3T3-L1 adipocytes and in RAW 264.7 macrophages (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(L.Y.) Phone: (301) 0405-0761. Fax: (301) 314-3313. E-mail: [email protected]. *(J.L.) E-mail: [email protected]. ORCID

Liangli Lucy Yu: 0000-0001-6497-0864 Funding

This research was supported by grants from the National High Technology Research and Development Program of China (Grants 2013AA102202 and 2013AA102207), a grant from the Foundation for Young Scientist of Beijing Technology & Business University (Grant QNJJ2017-07), and funding from the Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology & Business University (BTBU) and the Beijing Excellent Talents Funding for the Youth Scientist Innovation Team. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED 2-DG6P, 2-deoxyglucose-6-phosphate; ERK, extracellular signalregulated kinase; FoxO1, forkhead box O1; GSK3β, glycogen synthase kinase 3β; IL-6, interleukin-6; JNK, c-Jun N-terminal kinase; KRPH, Krebs Ringer phosphate Hepes buffer; MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemoattractant protein-1; NF-κB, nuclear factor-κB; PI3K, phosphatidylinositol 3-kinase; TNF-α, tumor necrosis factor-α

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