Inhibitory Effect of Piceatannol on TNF-Î±-Mediated Inflammation and

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Inhibitory effect of piceatannol on TNF-# 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 J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 23 May 2017 Downloaded from http://pubs.acs.org on May 24, 2017

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Journal of Agricultural and Food Chemistry

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Inhibitory Effect of Piceatannol on TNF-α Mediated

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Inflammation and Insulin Resistance in 3T3-L1 Adipocytes

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Yanfang Li,a,b Puyu Yang,b Qimeng Chang,c Jing Wang,a Jie Liu,a* Yuan Lv,b

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Thomas T Y Wang,d Boyan Gao,e Yaqiong Zhang,b Liangli (Lucy) Yue*

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a

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Technology & Business University, Beijing 100048, China

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b

Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing

Institute of Food and Nutraceutical Science, School of Agriculture and Biology,

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Shanghai Jiao Tong University, Shanghai 200240, China

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c

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China

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d

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USDA, Beltsville, MD 20705

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e

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MD 20742, USA

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Short title: Inhibitory Effect of Piceatannol on Inflammation and Insulin Resistance

Department of Surgery, Fudan University Minhang Hospital, Shanghai 201199,

Diet, Genomics, and Immunology Laboratory, Agricultural Research Service (ARS),

Department of Nutrition and Food Science, University of Maryland, College Park,

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* Contact Information of the Corresponding Author:

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Liangli (Lucy) Yu at Tel: 301-0405-0761, Fax: 301-314-3313, and Email:

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[email protected]; correspondence may also be directed to Jie Liu, Email:

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[email protected]

ACS Paragon Plus Environment


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ABSTRACT

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Piceatannol, a bioactive component in grapes and blueberry, was examined for its

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potential in decreasing the inflammatory activities in adipocytes using a co-cultured

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adipocytes and macrophages system, and suppressing TNF-α mediated inflammation

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and the related insulin resistance using a 3T3-L1 adipocytes model. Piceatannol at 10

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µM significantly reduced the release of inflammatory cytokines of TNF-α and MCP-1

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by 19% and 31% in the co-cultured system, respectively. The pretreatment with

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piceatannol also inhibited TNF-α induced expression of IL-6 and MCP-1 at both

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mRNA and protein levels in the 3T3-L1 adipocytes. Piceatannol also partially

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improved the malfunction of insulin-stimulated glucose uptake, which was reduced by

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TNF-α in 3T3-L1 adipocytes. Furthermore, the inhibitions were mediated by

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significant blocking of IκBα phosphorylation and NF-κB activation through

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suppressing nuclear translocation of NF-κB p65 along with JNK-MAPK activation. In

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addition, Akt-dependent FoxO1 signaling pathway was involved in the restoration of

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insulin-stimulated glucose uptake through suppressing the down-regulation of

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phosphorylation of Akt and FoxO1 expressions. These results suggested the potential

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of piceatannol in improving chronic inflammatory condition and insulin sensitivity in

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obese adipose tissues.

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KEYWORDS: Piceatannol, TNF-α, 3T3-L1 adipocytes, inflammation, insulin

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resistant


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INTRODUCTION

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Piceatannol (3,3’,4,5’-trans-trihydroxystilbene) is an analog and a metabolite of

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resveratrol that presents in many fruits including grapes and blueberries.1 Use of

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resveratrol in dietary intervention to reduce the risk of chronic human diseases is

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limited due to its poor bioavailability and rapid metabolism. Hydroxylated resveratrol

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derivatives, including piceatannol, is a possible alternative to resveratrol for health

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food applications. Increasing evidence indicated the potential benefits of piceatannol

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in immunomodulatory, anti-adipogenesis, anti-proliferative and anti-inflammatory

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properties.1-4 Piceatannol suppressed tumor necrosis factor-α (TNF-α) activation of

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NF-κB-dependent reporter gene expression in myeloid cells, lymphocytes and

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epithelial cells.2 TNF-α is the primary activator of pro-inflammatory signaling

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cascades, that regulate the pro-inflammatory gene transcriptions such as interleukin-6

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(IL-6), monocyte chemoattractant protein-1 (MCP-1) and resistin gene transcriptions,

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leading to a self-feeding inflammatory cycle. Recently, Takayuki and others observed

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that piceatannol suppressed the proinflammation gene expression in RAW264.7

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macrophages induced by LPS- and the culture-medium of 3T3-L1 adipocytes using a

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conditioned medium model.4 Piceatannol treatment at 30 µmol/L concentration was

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able to inhibit the expression of TNF-α, IL-6 and nitric oxide at both mRNA and

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protein levels. However, the possible molecular mechanism behind the anti-

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inflammation activity of piceatannol was not investigated. It was also interesting

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whether and how piceatannol may act in a co-cultured adipocytes and macrophages

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system, which allows simultaneous functional interaction between the two cell types.



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In addition, it has been widely accepted that TNF-α secreted from macrophages might

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infiltrate into obese adipose tissue and increase the risk of obesity-associated insulin

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resistant.5-8 A co-cultured adipocytes and macrophages system may mimic an

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inflammatory status in obese tissues, and has been used as an experimental model for

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investigating the simultaneous functional interaction between the two cell types since

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it was developed by Suganami and others.9 This co-cultured adipocytes and

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macrophages system also provides an excellent opportunity for investigating whether

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and how piceatannol may improve insulin sensitivity in the adipocytes under

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inflammatory status, which has not been reported based on our best knowledge.10

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Therefore, this research was conducted to investigate whether and how piceatannol

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might attenuate TNF-α mediated inflammation and insulin resistance in adipocytes

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using a co-cultured adipocytes and macrophages model. The information obtained

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from this research might be used to promote the utilization of piceatannol or grape

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and other fruits to reduce the risk of chronic inflammation in obese adipose tissue, and

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improve obesity-related insulin resistance.

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MATERIALS AND METHODS

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Chemicals. Piceatannol with a purity > 98% (HPLC), insulin, 3-isobutyl-1-

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methylxanthine (IBMX) and dexamethasone were purchased from Sigma-Aldrich

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(St.Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fatal calf

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serum (FCS) and fatal bovine serum (FBS) were obtained from Life Technologies

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(New York, NY, USA). Antibodies against IκBα, p-IκBα, p65, JNK, p-JNK, ERK1/2,


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p-ERK1/2, p38, p-p38, Akt, p-Akt, GSK3β, p-GSK3β, FoxO1, p-FoxO1 and β-actin,

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and the secondary antibodies were purchased from the Cell Signaling Technology

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(Beverly, MA, USA). All other chemicals were purchased from Sigma (St. Louis,

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MO, USA) with the highest quality available and used without further purification.

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Cell culture and differentiation of 3T3-L1 pre-adipocytes. The 3T3-L1 pre-

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adipocytes (American Type Culture Collection, ATCC, Manassas, VA, USA) were

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cultured and differentiated as previously described following a laboratory protocol.11

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Briefly, 3T3-L1 pre-adipocytes were cultured in DMEM supplemented with 100

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U/mL penicillin, 100 μg/mL streptomycin and 10% FCS at 37 °C, in a humidified

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atmosphere of 95% air and 5% CO2. The cells were cultured in six-well cell culture

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plates at a density of 1 × 105 cells/mL, and allowed to grow overnight to achieve

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adherence. After the culture medium was renewed every two days until cells reached

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confluence and cultured for another two days, the cells were cultured in a

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differentiation medium containing 10% FBS-DMEM, 1 μg/mL insulin, 0.25 μM

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dexamethasone and 0.5 mM IBMX for 48 h. The cells were then cultured in 10%

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FBS-DMEM supplemented with 1 μg/mL insulin for another 48 h. The culture

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medium was changed to 10% FBS-DMEM medium every two days till cells achieved

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full adipocytes morphology. The 3T3-L1 adipocytes were treated with different

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concentrations of piceatannol, and subsequently treated with 10 ng/mL of TNF-α for

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15 min or 24 h.



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Co-culture of 3T3-L1 adipocytes and RAW264.7 macrophages. RAW264.7

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macrophages were purchased from the American Type Culture Collection (ATCC,

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Manassas, VA, USA) and co-cultured with differentiated 3T3-L1 adipocytes in a

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Trans-well system (Corning Inc., Acton, MA, USA). 3T3-L1 pre-adipocytes were

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seeded in twelve-well plates, and differentiated as described above. RAW 264.7

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macrophage cells (6 × 104 cells/mL) were seeded in Trans-well inserts with a 0.4 μm

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porous membrane and cultured in DMEM supplemented with 100 U/mL penicillin,

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100 μg/mL streptomycin and 10% FBS at 37 °C, in a humidified atmosphere of 95%

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air and 5% CO2. 3T3-L1 adipocytes were co-cultured with RAW 264.7 cells, and

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treated with 5% BSA-DMEM medium containing 5 and 10 μM piceatannol for 24 h.

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Subsequently, cells and co-culture medium were collected and stored at -80 °C until

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analysis.

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MCP-1, TNF-α and IL-6 productions. The concentration of MCP-1, TNF-α and

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IL-6 in the co-culture media were determined with three enzyme-linked

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immunosorbent assay kits (eBioscience, San Diego, CA, USA), according to the

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manufacturer protocol.

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Real-time PCR. Total RNA from vehicle or piceatannol-treated differentiated

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3T3-L1 adipocytes was extracted with the TrizoL reagent (Invitrogen, Carlsbad, CA,

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USA). Equal aliquot (0.4 μg) of total RNA was used for the synthesis of first-stand

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cDNA with an IScript reverse transcriptase kit (Bio-Rad) according to the

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manufacturer protocol. The quantitative real time PCR amplification and detection


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were performed subsequently using the cDNA on an ABI 7900 HT (Applied

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Biosystems, Carlsbad, CA, USA). The cycling parameters for PCR were 50 °C for 2

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min, 95 °C for 10 min, and 40 cycles of amplification at 95 °C for 15 s and 60 °C for

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1 min. Quantifications were performed in triplicate, and the experiments were carried

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out independently for three times. Changes in the expression of target genes were

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expressed as relative mRNA levels and normalized to a housekeeping gene β-actin

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and data were analyzed using the 2-ΔΔCt method. The primer sequences for each gene

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were performed with the following primers: MCP-1, Forward: 5’-

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TCTGGACCCATTCCTTCTTG-3’ and Reverse: 5’-

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AGGTCCCTGTCATGCTTCTG-3’ IL-6, Forward: 5’-

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CACGGCCTTCCCTACTTCAC-3’ and Reverse: 5’-

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TGCAAGTGCATCATCGTTGT-3’; β-actin, Forward: 5’-

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TGTCCACCTTCCAGCAGATGT-3’ and Reverse: 5’-

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AGCTCAGTAACAGTCCGCCTAGA-3’.

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Glucose uptake. Glucose uptake assay was performed using a commercial kit

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(BioVision, Milpitas, CA, USA) according to the manufacture protocol with minor

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modification. 2-deoxyglucose (2-DG) has been widely used because of its structural

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similarity to glucose. 2-DG can be taken up by glucose transporters and metabolized

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to 2-DG-6-phosphate (2-DG6P) and thus accumulated in the cells. The accumulated

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2-DG6P is directly proportional of 2-DG (or glucose) uptake by cells. In brief, cells

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were incubated in Krebs ringer phosphate hepes buffer (KRPH, containing121 mM



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NaCl, 4.9 mM KCl, 1.2 mM MgSO4, 0.33 mM CaCl2 and 12 mM HEPES, pH 7.4) for

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4 h. The cells were pretreated with 10 μM piceatannol for 24 h followed by co-

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treatment with 10 ng/mL TNF-α for 24 h. The culture medium was discarded and

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replaced with 900 μL KRPH with or without 10 nM insulin for 60 min at 37 °C. After

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30 min incubation in the KRPH with 10 mM 2-DG at 37 °C, cells were immediately

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washed with ice-cold KRPH for three times, lysed by extraction buffer for 10 min and

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added reaction mix substrate. Accumulated 2-DG6P was oxidized to generate

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NADPH, which can be determined by measuring the fluorescence at Ex/Em =

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535/587 nm using a Tecan PRO M200microplate reader (Mannedorf, Switzerland).

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Western-blotting. Cells were carefully washed twice with ice-cold PBS, harvested

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by scraping, and immediately mixed with 200 μL of ice-cold radio

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immunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktail

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(Sigma, St. Louis, MO, USA) and phosphatase inhibitors (Roche Diagnostics,

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Mannheim, Germany) for each well. To remove the insoluble materials, lysates of the

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whole cells were centrifuged at 10,000g for 20 min at 4 °C. The protein

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concentrations in the lysates were determined using a BCA protein assay kit (Pierce,

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Rockford, IL, USA). For the nucleus p65 protein analysis, nuclear proteins were

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obtained using a Beyotime Nuclear and Cytoplasmic Extraction Kit (Beyotime Inc.,

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Nantong, Jiangsu, China) according to the manufacturer’s instruction. The protein

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concentration for each sample was adjusted to an equal amount with different volume

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of loading buffer and denatured in boiled water for 5 min. To separate different


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proteins, equal aliquot (40 μg) of protein samples were subjected to 12%

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polyacrylamide gel electrophoresis and then electro-transferred to PVDF membranes

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at 25 V, 1.5A for 60 min. The membranes were blocked with 5% non-fat milk in Tris

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buffered saline containing 0.1% Tween-20 (TBST) for 2 h. After being washed with

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TBST for three times, the membranes were incubated with specific target protein

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antibodies overnight at 4 °C, followed by incubating with secondary antibodies with

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conjugated horseradish peroxidase (HRP) for 1.5 h at an ambient temperature.

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Peroxidase activity was visualized using the chemiluminescence method with an ECL

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kit (Bio-Rad, Hercules, CA, USA) with ChemiDocTM XRS+ (Bio-Rad), with β-actin

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as an internal control. Films were scanned, and the protein levels of the specific bands

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were quantified by detecting the densitometry using the Image Lab software and

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calculated according to the internal β-actin control.

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Statistical analysis. Each experiment was performed in triplicate. Data were

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expressed as mean ± SD. The statistical significance of difference was analyzed using

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one-way ANOVA, followed by Tukey’s test. All statistical analyses were performed

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using SPSS 21.0 software. P < 0.05 was considered to be statistically significant.

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Graph was created on GraphPad Prism (Version 6.00, Graphpad Software Inc., San

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Diego, CA, USA).

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RESULTS AND DISCUSSION Piceatannol significantly reduced TNF-α and MCP-1 secretion in a co-

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cultured system of 3T3-L1 adipocytes and RAW 264.7 macrophages. In obese

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adipose tissue, TNF-α from the infiltrated macrophages play an important role in

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developing obesity-related insulin resistance.6 The effect of piceatannol on the

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inflammatory changes in adipose tissue was examined in the co-cultured 3T3-L1

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adipocytes and RAW 264.7 macrophages at 5 and 10 μM concentrations using a

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trans-well system. The expressions of TNF-α, MCP-1 and IL-6 were significantly

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greater in the co-cultured system as compared with that in only 3T3-L1 adipocytes or

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RAW 264.7 macrophages (Figure 1A-D). This overexpression of TNF-α and MCP-1

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in the co-cultured system was dose dependently reversed by pre-incubation with 5 and

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10 μM piceatannol (Figure 1B and 1C), whereas piceatannol was not able to

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significantly reduce the IL-6 expression in the co-cultured system (Figure 1D). It was

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interesting whether TNF-α might play a role in the overexpression of MCP-1 and IL-6

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in the co-cultured adipocytes, and whether piceatannol may reduce their expressions.

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As shown in Figure 1E and 1F, TNF-α significantly increased the mRNA

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expression of MCP-1 and IL-6, and pre-treatment with 5 and 10 μM piceatannol was

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able to significantly reverse the TNF-α induced MCP-1 and IL-6 mRNA over-

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expression in the 3T3-L1 adipocytes. The inhibition of TNF-α induced MCP-1

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mRNA expression was in a dose-dependent matter (Figure 1E). Piceatannol was also

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able to suppress the TNF-α induced MCP-1 and IL-6 expressions at the protein level


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(Figure 1G and 1H). The suppression on MCP-1 protein production was also dose-

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dependent (Figure 1G). It needs to be pointed out that the treatment concentrations

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for all these tests had no adverse effects on 3T3-L1 adipocytes and RAW 264.7

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macrophages growth according to 3-(4,5-dimethylthiazol-2-yl)-2,5-

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diphenyltetrazolium bromide (MTT) assay (Figures S1 and S2).

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TNF-α has been known for up-regulating the production of several important

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inflammatory cytokines and chemokines such as MCP-1and IL-6 in adipocytes,

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leading to down-regulation of glucose-uptake in the adipocytes.8,9 The data in Figure

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1 indicated a potential of piceatannol in suppressing TNF-α induced production of

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inflammatory cytokines and chemokines in adipocytes, and it became interesting

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whether and how piceatannol might be able to modulate TNF-α induced insulin

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resistance in adipocytes.

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Piceatannol improved TNF-α mediated insulin resistance in 3T3-L1

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adipocytes. The glucose uptake was determined by 2-deoxyglucose-6-phosphate (2-

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DG6P) uptake in the 3T3-L1 adipocytes treated with TNF-α with or without

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piceatannol pretreatment in the absence or presence of insulin. Treatment with TNF-α

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for 24 h significantly decreased both basal and insulin-stimulated 2-DG6P uptake in

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the 3T3-L1 adipocytes (Figure 2), supporting the conclusion that pre-treatment with

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10 μM piceatannol significantly improved TNF-α mediated reduction of 2-DG6P

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uptake regardless of insulin presence (Figure 2). Pre-treatment with 10 μM

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piceatannol could significantly alleviate TNF-α induced insulin resistant by



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improving 21.7% of 2-DG6P uptake in the presence of insulin under the experimental

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conditions. These results suggested the potential health benefits of fruits and other

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foods rich in piceatannol.

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Piceatannol attenuated TNF-α induced adipocytes inflammation through the

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NF-κB and MAPK/JNK pathway. To further understand how piceatannol could

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suppress the TNF-α induced adipocytes inflammation and insulin resistance, the

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effect of piceatannol on nuclear factor-κB (NF-κB) and three mitogen activated

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protein kinases (MAPKs) signaling pathways were investigated. Piceatannol

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significantly suppressed TNF-α induced phosphorylation and degradation of IκBα in a

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dose-dependent manner (Figure 3A). Piceatennol also was able to does-dependently

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inhibit the TNF-α induced nuclear translocation of p65 protein (Figure 3B).

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Together, these data suggested that NF-κB signaling pathway might be involved in

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TNF-α induced inflammatory reactions and glucose-uptake in adipocytes, as well as

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the effects of piceatannol in reducing the risk of inflammation and insulin resistance

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in the adipose tissues. NF-κB pathway has been recognized for its role on adipocytes

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lipolysis, inflammation and insulin resistant.6 NF-κB may be activated by TNF-α

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through phosphorylation and proteasomal degradation of IκBα, a part of the NF-κB

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complex, stimulating to the translocation of RelA (p65)/p50 complexes to nucleus and

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consequently induce the production of enzymes and cytokines important for

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inflammation and insulin resistance, such as MCP-1 and IL-6. However, how

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piceatannol might block the phosphorylation of IκBα and inhibit the p65 nuclear


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translocation has not been fully understood. A previous study has shown that TNF-α-

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induced NF-κB activation might be through a canonical activation pathway.12 Briefly,

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TNF-α might activate tumor necrosis factor receptor (TNFR) and stimulating other

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signal transducing adaptor proteins and kinases leading to the activation of IKKβ,

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which results in the phosphorylation and degradation of IκBα, nuclear translation of

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p65, and activation of target gene transcription.12-15 Piceatannol has been found to

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block NF-κB activation through direct modification of IKKβ at the cysteine 179

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residue16 and inhibition of p65 phosphorylation.2 Taking together with the results

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shown in Figure 3A and 3B that piceatannol reduced TNFα-induced phosphorylation

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of IκBα and nuclear translation of p65, IKKβ would be an interesting key target for

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future studies to fully understand how piceatannol and other stilbenoids might

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suppress TNF-α-induced phosphorylation of IkBα and p65.

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The potential role of piceatannol on TNF-α induced phosphorylation of c-Jun N-

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terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38 were

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investigated to understand its possible involvement in the MARK pathway (Figure 4).

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Pre-treatment with piceatannol was able to attenuate TNF-α mediated activation of

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JNK in a dose-dependent manner (Figure 4A and 4B), with no significant effect on

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the phosphorylation of ERK (Figure 4C) or p38 (Figure 4D). JNK, ERK and p38 are

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important three MAPK family kinases. These kinases may be activated by

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inflammatory stimuli and oxidative stress, and are contributors to obesity-associated



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inflammation and insulin resistance. JNK-dependent MAPK pathway could interact

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with the PI3K/Akt pathway, important for insulin actions such as glucose uptake.6,17

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Taking together, the data suggested that NF-κB pathway and JNK phosphorylation

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might be the possible primary targets for piceatannol to alleviate the TNF-α induced

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inflammation and insulin resistance in adipocytes under the experimental conditions.

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The findings were supported by an earlier report from Yuri Sakamoto and others that

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dietary daidzein, an isoflavone compound common in soybean, could down-regulate

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pro-inflammatory gene expression by inhibiting the JNK pathway in a co-culture of

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adipocytes and macrophages.18 The findings from this study were also supported by

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Yang and others’ observation that bitter melon could reduce high-fat diet induced

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insulin resistance and diabetes in OLETF rats through suppressing NF-κB and JNK

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pathways.19 NF-κB and MAPK/JNK pathways may induce specific insulin receptor

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substrate 1 (IRS-1) serine phosphorylation, resulting in an impaired downstream

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insulin receptor signaling.19,20 It was interesting to further examine whether and how

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piceatannol might reduce the risk of TNF-α induced insulin resistance in adipocytes

289

though insulin signaling pathway.

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Piceatannol enhanced the insulin signaling through improving

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phosphorylation of Akt, GSK3β and FoxO1. Insulin signaling inhibition is a

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possible mechanism for insulin resistant. To further elucidate how piceatannol could

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alleviate the TNF-α induced adipocyte insulin resistance, the effect of piceatannol on

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insulin signal cascades, including Akt, glycogen synthase kinase 3β (GSK3β) and


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Forkhead box O1 (FoxO1) were examined. Pretreatment of piceatannol was able to

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significantly repair the TNF-α induced phosphorylation suppression of Akt in the

297

adipocytes (Figure 5A and 5B), and the effect required the presence of insulin (figure

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5B). Piceatannol had no significant effect on GSK3β phosphorylation regardless of

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the insulin presence (Figure 5C). In addition, piceatannol pre-treatment was able to

300

improve the phosphorylation of FoxO1 with or without the presence of insulin (Figure

301

5A and 5D). These results are in consistent with that on glucose-uptake in the Figure

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2. The findings from this study agreed with the observation by Tsai and others that

303

carnosic acid was able to attenuate TNF-α induced 3T3-L1 adipocyte inflammation

304

via suppressing NF-κB and enhancing insulin sensitivity via Akt-dependent FoxO1

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signaling pathway.21 Akt, GSK3β and FoxO1 play an important role in insulin-

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regulated glucose uptake22-24 and food factors capable of enhancing their

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phosphorylation might potentially improve insulin resistant including enhancing

308

glucose uptake.25-29 The results from this study suggested that piceatannol might

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restore the TNF-α induced insulin resistance in 3T3-L1 adipocytes through Akt-

310

dependent FoxO1 signaling pathway, and potentially improve insulin resistant.

311

In summary, this study observed for the first time that piceatannol significantly

312

reduced TNF-α and MCP-1 secretions in a co-cultured system of 3T3-L1 adipocytes

313

and RAW 264.7 macrophages, and further demonstrated that it could attenuate TNF-α

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induced adipocyte inflammation possibly through the NF-κB and MAPK/JNK

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pathways. The results from the present study also suggested that piceatannol could



316

improve the TNF-α mediated insulin resistance in 3T3-L1 adipocytes through

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activating Akt-FoxO1 insulin cascade signal pathway to enhance the insulin signaling.

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These findings suggested that piceatannol might be used to improve the obesity-

319

associated glucose uptake disorder through its attenuation of chronic inflammatory

320

condition and improvement in insulin sensitivity in the obese adipose tissues.

321

Additional research is needed to confirm the observations and better understand the

322

role of piceatannol in insulin resistance.

323 324

ACKNOWLEDGEMENTS

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This research was supported by grants from the National High Technology Research

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and Development Program of China (Grant Nos. 2013AA102202; 2013AA102207), a

327

grant from the Foundation for Young Scientist of Beijing Technology & Business

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University (Grant No. QNJJ2017-07), and funding from the Beijing Advanced

329

Innovation Center for Food Nutrition and Human Health, Beijing Technology &

330

Business University (BTBU) and the Beijing Excellent Talents Funding for the Youth

331

Scientist Innovation Team.


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ABBREVIATIONS

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2-DG6P, 2-deoxyglucose-6-phosphate (2-DG6P); ERK, extracellular signal-regulated

334

kinase; FoxO1, forkhead box O1; GSK3β, glycogen synthase kinase 3β; IL-6,

335

interleukin-6; JNK, c-Jun N-terminal kinase; KRPH, krebsringer phosphatehepes

336

buffer; MAPK, mitogen activated protein kinase; MCP-1, monocyte chemoattractant

337

protein-1; NF-κB, nuclear factor-κB; PI3K, phosphatidylinositol 3-kinase; TNF-α,

338

tumor necrosis factor-α.

339

Conflict of interest

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The authors declare that there are no conflicts of interest.



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REFERENCES

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(1) Piotrowska, H.; Kucinska, M.; Murias, M. Biological activity of piceatannol:

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tumor necrosis factor alpha-mediated insulin resistance and lipolytic acceleration in

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3T3-L1 adipocytes. Planta Med. 2014, 80, 880-886.

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necrosis factor alpha. Arterioscler. Thromb. Vasc. Biol. 2005, 25, 2062-2068.

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stilbene, in metabolic diseases: A Review. J Med Food. 2017, 20, 1–12.

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Gynostemma pentaphyllum saponin and its adipogenesis inhibitory effect through

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modulating Wnt/β-catenin pathway and cell cycle in mitotic clonal expansion. J Funct

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catechol-type polyphenol, inhibits phorbol ester-induced NF-κB activation and

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437

FIGURE CAPTIONS

438

Figure 1. Effects of piceatannol on inflammatory changes. A) Effects of piceatannol

439

on the secretion of inflammatory mediators in Transwell co-culture system.

440

Differentiated 3T3-L1 adipocytes were co-cultured with RAW 264.7 macrophages for

441

24 h in the absence or presence of 5 and 10 μM piceatannol. Released protein levels

442

of B) TNF-α; C) MCP-1; and D) IL-6 in the co-culture medium were measured by

443

ELISA and those from 3T3-L1 adipocytes or RAW 264.7 macrophages alone were

444

used as controls. Effects of piceatannol on TNF-α induced relative mRNA

445

expressions of E) IL-6 and F) MCP-1 were measured by RT-PCR. Effects of

446

piceatannol on TNF-α induced protein expressions of G) IL-6 and H) MCP-1 were

447

measured by ELISA. Adipocytes were pre-incubated with 5 and 10 μM piceatannol

448

for 24 h and then treated with 10 ng/mL TNF-α for 24 h before cells were harvest.

449

Values are mean ± SD of three independent experiments carried out in triplicate.

450

Different letters indicate significant differences between means (P < 0.05).

451

Figure 2. Effects of piceatannol and TNF-α on glucose uptake in 3T3-L1 adipocytes.

452

Fully differentiated 3T3-L1 adipocytes were all treated with 10 ng/mL TNF-α alone

453

or pretreatment with 10 μM piceatannol in the absent or present of insulin for 24 h.

454

The 2-DG6P uptake was determined. Values are mean ± SD of three independent

455

experiments carried out in triplicate. Different letters for each column mean

456

significantly different (P < 0.05).



457

Figure 3. Effects of piceatannol on NF-κB pathway relevant protein expressions

458

induced by TNF-α with different concentrations of piceatannol in 3T3-L1 adipocytes.

459

3T3-L1 adipocytes were pre-treated with 5 and 10 μM piceatannol for 24 h, followed

460

by co-treatment with 10 ng/mL TNF-α for another 15 min. The protein levels of A)

461

phosphor(p)-IκBα and total(t)-IκBα; B) total(t) p65 and total β-actin in nuclear were

462

determined by Western blotting. β-actin was used as a loading control. The protein

463

levels of the bands were quantified by densitometry. The results were reported as the

464

ratio of phosphorylated/total protein contents and expressed in amounts relative to the

465

control values. Values are the mean ± SD of three independent experiments. Different

466

letters indicate significantly difference between means (P < 0.05).

467

Figure 4. Effects of piceatannol on MAPK pathway relevant protein expressions

468

induced by TNF-α with different levels of piceatannol in 3T3-L1 adipocytes. 3T3-L1

469

adipocytes were pre-treated with 5 and 10 μM piceatannol for 24 h, followed by co-

470

treatment with 10 ng/mL TNF-α for another 15 min. The protein levels of A)

471

phosphor (p) or total (t) ERK1/2, JNK and p38 were determined by Western blotting.

472

β-actin was used as a loading control. The protein levels of the bands were quantified

473

by densitometry. The results were reported as the ratio of phosphorylated/total protein

474

contents: B) p-JNK/JNK; C) p-ERK/ERK; and D) p-p38/p38 contents were expressed

475

in amounts relative to the control values. Values are the mean ± SD of three

476

independent experiments. Different letters mean significantly different (P< 0.05).


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477

Figure 5. Effects of piceatannol on TNF-α-mediated suppression of the insulin

478

signaling cascades in 3T3-L1 adipocytes. Cells were pretreated with Blank or with 10

479

μM piceatannol for 24 h, and followed by treatment with or without 10 ng/mL TNF-α

480

for 24 h. Then 10nM insulin was added at the last 30 min. A) Cultures were then

481

harvested to determine the protein expression of phosphor (p) or total (t) Akt, GSK3β

482

and FoxO1 using western blotting. β-actin was used as a loading control. The protein

483

levels of the bands were quantified by densitometry, and results were expressed as the

484

ratio of B) p-Akt/Akt; C) p-GSK3β/GSK3β; and D) p- FoxO1/FoxO1 protein contents

485

and reported relative to the control values. Values are the mean ± SD of three

486

independent experiments. Different letters mean significantly different (P< 0.05).



Figure 1.


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Figure 2.



Figure 3.


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Figure 4.



Figure 5.


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TOC graphic Food Materials Grape

Passion fruit

Akt

+ Piceatannol

+

TNF-α MCP-1 IL-6 Low-grade Chronic Inflammation

FoxO1

GSK-3β

FoxO1

P

P

+

P

Obesity Macrophages

+

GSK-3β

+ NF-κB

P

Akt

Adipocytes


Glycogen and Protein Synthesis

Inhibitory Effect of Piceatannol on TNF-Î±-Mediated Inflammation and

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