Involvement of resveratrol in crosstalk between adipokine adiponectin and hepatokine fetuin-A in vivo and in vitro

    Involvement of resveratrol in crosstalk between adipokine adiponectin and hepatokine fetuin-A in vivo and in vitro Hee Jae Lee, Yunsook Lim, Soo Jin Yang PII: DOI: Reference:

S0955-2863(15)00148-5 doi: 10.1016/j.jnutbio.2015.06.001 JNB 7376

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The Journal of Nutritional Biochemistry

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18 February 2015 15 May 2015 3 June 2015

Please cite this article as: Lee Hee Jae, Lim Yunsook, Yang Soo Jin, Involvement of resveratrol in crosstalk between adipokine adiponectin and hepatokine fetuin-A in vivo and in vitro, The Journal of Nutritional Biochemistry (2015), doi: 10.1016/j.jnutbio.2015.06.001

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Involvement of resveratrol in crosstalk between adipokine adiponectin and hepatokine

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fetuin-A in vivo and in vitro Hee Jae Leea, Yunsook Limb, Soo Jin Yangc,*

Division of Food and Nutrition and Human Ecology Research Institute, Chonnam National

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a

Department of Food and Nutrition, Seoul Women’s University, Seoul, Korea

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c

Department of Food and Nutrition, Kyung Hee University, Seoul, Korea

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b

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University, Gwangju, Korea

Corresponding author: Soo Jin Yang, Ph.D.*

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Address: Department of Food and Nutrition, Seoul Women’s University, Seoul, 139-774, Korea

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Telephone: 82-2-970-5643 / Fax: 82-2-976-4049 / E-mail: [email protected]

Conflict of interest

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Running title: resveratrol and adipose tissue-liver crosstalk

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There are no conflicts of interest. Funding sources

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF 2014R1A1A2A16055328) and Chonnam National University (2012). The funder had no role in study design, data collection, and analysis and interpretation, decision to publish, or preparation of the manuscript. Keywords: adiponectin, crosstalk, fetuin-A, resveratrol

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Abstract

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Metabolic homeostasis is maintained by the coordinated regulation of several physiological processes and organ crosstalk. Especially, the interaction between adipose tissue and liver is

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critical for the regulation of glucose and lipid metabolism. This study investigated the

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involvement of resveratrol (RSV) in the crosstalk between adipokine adiponectin and hepatokine

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fetuin-A. Adipocytes-hepatocytes co-culture system and a high-fat (HF) diet-induced obesity (DIO) mouse model were utilized. Protein levels of adiponectin and fetuin-A were analyzed in

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adipocytes and hepatocytes with the knockdown of adiponectin and fetuin-A, respectively. After six weeks of the HF diet, RSV was delivered via an osmotic pump for four weeks. The

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experimental groups were lean control fed with a standard diet, HF diet-induced obese control,

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and HF_RSV (8 mg/kg/day). After 4 weeks of each treatment, blood and tissues were collected, and the levels of adiponectin and fetuin-A were analyzed. RNA interference during co-culture of

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adipocytes and hepatocytes demonstrated the existence of crosstalk between adiponectin and

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fetuin-A. The four-week RSV treatment resulted in increased serum adiponectin and decreased serum fetuin-A in DIO mice. The serum levels of adiponectin and fetuin-A were inversely related. In epididymal fat depots, RSV increased adiponectin, peroxisome proliferator-activated receptor (PPAR) alpha, PPAR gamma, sirtuin1, and AMP-activated protein kinase (AMPK). RSV lowered fetuin-A and NF-κB, and increased liver AMPK. These results demonstrate the crosstalk between adiponectin and fetuin-A, and suggest that RSV may be involved in adipose tissue and liver crosstalk through the interaction between adiponectin and fetuin-A.

Keywords: Adiponectin; Crosstalk; Fetuin-A; Resveratrol

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ACCEPTED MANUSCRIPT 1. Introduction

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The interaction between the adipose tissue and other metabolic organs is critical for the maintenance of body homeostasis, which is regulated by the coordination of several

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physiological processes and organ crosstalk. Among major metabolic organs, adipose tissue is

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considered as a central endocrine organ in the secretion of hormones and cytokines in response to cellular signals, and in the modulation of organ crosstalk in adipose tissue, skeletal muscle,

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cardiovascular system, and pancreas through autocrine/paracrine and endocrine crosstalk [1-3].

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Especially, adipose tissue and liver play a central role in the glucose and lipid metabolism, and excessive secretion of adipokines and other related pro-inflammatory cytokines is closely

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associated with liver diseases including fatty liver and non-alcoholic fatty liver diseases (NAFLD)

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[4-5]. Representative bioactive factors affecting NAFLD are adiponectin, resistin, visfatin, retinol-binding protein 4 (RBP4), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, IL-1,

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IL-18, and fetuin-A [6-8]. These factors act in various ways by regulating insulin signaling and modulating the secretion and activation of inflammatory cytokines/pathways [6,9]. Adiponectin

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sensitizes insulin signaling [10,11] and acts as an anti-inflammatory cytokine by down-regulating the pro-inflammatory cytokines TNF-α and IL-6 in adipose tissue and liver [12]. Other adipokines, such as resistin, visfatin, and RBP4, could also be involved in the regulation of insulin signaling and may alter inflammatory responses in a similar way to adiponectin [13-15]. The concept that adipose tissue-derived factors are involved in insulin signaling and inflammation in liver suggests that these factors are critical in adipose tissue-liver crosstalk. Among adipokines, the direct action of adiponectin and adiponectin-mediated inflammation is a key link between insulin resistance and inflammation through autocrine/paracrine and endocrine crosstalk. Especially, liver-related action has been suggested to be closely related to the 3

ACCEPTED MANUSCRIPT modulation of hepatokine fetuin-A [16]. Fetuin-A is a glycoprotein that is extensively expressed in liver, kidney and brain, and which secreted mainly from the liver [17] and, to a lesser extent,

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from adipose tissue. Circulating levels of fetuin-A are elevated in several pathological conditions

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including obesity, type 2 diabetes, and NAFLD [17]. Because elevated levels of fetuin-A are positively correlated with hepatic fat contents, it has been suggested as a marker of simple

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hepatic steatosis [18,19]. Lower levels of adiponectin and higher levels of fetuin-A may mediate

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the pathophysiological processes of metabolic diseases, with fetuin-A having an inverse relationship with adiponectin [20,21]. However, the direct relationship between adiponectin and

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fetuin-A has not yet been thoroughly investigated.

Resveratrol (RSV) is a functional polyphenol compound present mainly in the skin of grapes

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and mulberry, and in red wine [22]. It has beneficial effects on cardiovascular diseases, cancer, Alzheimer's disease, and other aging-related chronic diseases [23-25]. In addition, RSV improves glucose control and hepatic metaflammation in a diet-induced obesity (DIO) mouse model [26]

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and in humans [27]. RSV administration increases serum adiponectin levels [26,27]. But, effects

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of RSV on fetuin-A or its possible involvement in the organ crosstalk have not yet been investigated. These results suggest the potential role of adiponectin and the possible involvement of adiponectin-fetuin crosstalk in the RSV-mediated improvements. We hypothesized that RSV is involved in the crosstalk between the adipokine adiponectin and the hepatokine fetuin-A. To test this hypothesis, RSV-mediated alterations of adiponectin and fetuin-A were analyzed in co-cultured adipocytes and hepatocytes with the knockdown of adiponectin and fetuin-A, respectively. Also, whether RSV administration alters adiponectin, fetuin-A, and related factors in epididymal fat and liver tissues was analyzed in a high fat (HF)fed DIO mouse model. 4

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2. Methods and materials

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2.1 Animal care

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Male, 6-week-old C57BL/6J mice were provided by Central Lab Animal Inc. (Seoul, Korea).

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The mice were maintained in a temperature- and humidity-controlled room on a 12 h light/dark cycle and fed standard irradiated rodent chow (10% kcal from fat; Research Diets, New

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Brunswick, NJ, USA) with unlimited access to food and water. After a 1-week acclimation

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period, 30 mice were fed with the standard chow diet or HF diet (45% kcal from fat; Research Diets). DIO was induced in a subset of mice using the HF diet. After 6 weeks of feeding the

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standard chow diet or HF diet, vehicle (50% dimethyl sulfoxide) or RSV (Cayman Chemical,

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Ann Arbor, MI, USA) was administered using an Alzet Model 1004 osmotic pump (Durect Corp., Palo Alto, CA, USA) with the assigned diet for another 4 weeks. The osmotic pump was

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implanted subcutaneously in the back. The experimental groups (n = 10 per group) were lean control (CON), HF diet-induced obese control (HF) and HF diet administered RSV (8 mg/kg/day)

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treatment (HF_RSV). This study protocol conformed to the specifications outlined in the National Institutes of Health Guiding Principles for the Care and Use of Laboratory Animals and was approved by the Institutional Animal Care and Use Committee of Chonnam National University (Approved protocol Number CNU IACUC-YB-2012-15).

2.2 Blood and tissue collection After 4 weeks of treatment, overnight fasted mice were anesthetized with an intraperitoneal injection of Zoletil/Rompun, and blood was collected from the abdominal aorta. Tissues were harvested, weighted and stored at -80°C until further analysis. 5

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2.3 Cells and culture conditions

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3T3-L1 fibroblasts (American Type Culture Collection, Manassas, VA, USA) were cultured to

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confluence in Dulbecco’s modified Eagle medium (DMEM; GIBCO, Grand Island, NY, USA) supplemented with 10% (v/v) bovine calf serum (BCS; GIBCO) and 1% penicillin-streptomycin

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(GIBCO) in a CO2 incubator at 37°C. On day 2 post-confluence (designated as differentiation

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day 0), cells were induced to differentiate with DMEM containing 10% fetal bovine serum (FBS; GIBCO), 5 μg/ml insulin (Sigma-Aldrich, St. Louis, MO, USA), 1 μmol/L dexamethasone

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(Sigma-Aldrich) and 0.05 mmol/L 3-isobutyl-1-methylxanthine (IBMX; Sigma-Aldrich). After 2 days, the medium was replaced with DMEM supplemented with 10% FBS and 5 μg/ml insulin.

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The cells were subsequently re-fed every 48 h with DMEM containing 10% FBS. AML12 mouse hepatocytes (American Type Culture Collection) were cultured in DMEM/F-12 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS, antibiotics (100 units/ml

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penicillin and 100 μg/ml streptomycin), 0.1 μM dexamethasone and a mixture of insulin,

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transferrin and selenium (Invitrogen).

2.4 RNA interference (RNAi) for the knock-down of adiponectin and fetuin-A RNAi-mediated gene silencing was performed according to the manufacturer’s instructions. Cells were transfected with negative control siRNA (Stealth RNAi negative control duplexes; Invitrogen) or Stealth RNAi siRNA targeting adiponectin (for adipocytes) or fetuin-A (for hepatocytes) using Lipofectamine (Invitrogen). After a 24 h transfection, co-culture of differentiated adipocytes (differentiation day 7) and AML12 mouse hepatocytes was conducted. Subsets of cells were incubated with RSV (50 μΜ; Cayman Chemical) for 24 h. 6

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2.5 Adipocyte-hepatocyte co-culture conditions

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Co-culture of differentiated adipocytes (differentiation day 7) and AML12 mouse hepatocytes

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was conducted according to a published protocol [28]. Briefly, 3T3-L1 cells were seeded in 12well plates and induced to differentiate. The hepatocytes were plated onto membrane inserts (BD

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Biosciences, Franklin Lakes, NJ, USA) and transferred to plate wells incubating adipocytes on

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day 7 of differentiation. This resulted in an assembly of the two cells types sharing the culture medium but being separated by the membrane of the insert. Distance from the bottom of the

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culture dish to the membrane was 0.9 mm. Cells were maintained in DMEM containing 10% FBS and co-cultures were conducted for 24 h. Integrity of both cell types was routinely checked

2.6 Metabolic parameters

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by light microscopy.

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Concentration of free fatty acids (FFA) was measured with a commercial kit (Wako Pure

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Chemical Industries, Osaka, Japan). Triglyceride (TG) contents were measured with enzymatic assays (Sigma-Aldrich). Activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were analyzed by assay kits (Abcam, Cambridge, UK). Commercially available ELISA kits were used to measure adiponectin (AdipoGen, Incheon, Korea) and fetuinA (R&D Systems, Minneapolis, MN, USA).

2.7 Isolation of total RNA and quantitative reverse transcription-polymerase chain reaction (RT-PCR)

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ACCEPTED MANUSCRIPT Total RNA was isolated from cells and tissues with a PureLink RNA Mini Kit (Invitrogen). Reverse transcription was performed using a SuperScript III First-Strand Synthesis System

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(Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions. mRNA

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expression was quantified by real-time PCR (StepOnePlus; Life Technologies, Carlsbad, CA, USA). Synthesized cDNA was mixed with Power SYBR Green PCR Master Mix (Applied

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Biosystems) and a gene-specific primer (Bioneer, Daegeon, Korea). Individual reactions for

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target and 18S were carried out separately with negative controls lacking cDNA. The conditions used were 95°C for 10 min, followed by 40 cycles of denaturation (95°C for 10 s), annealing

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(Tm (°C) for 15 s), and extension (72°C for 60 s). The cycle number for threshold of detection was determined by StepOne Software (Life Technologies). mRNA expression of each target was

2.8 Statistical analysis

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normalized to that of 18S gene and expressed as fold change relative to controls.

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Statistical analysis was performed using SPSS Statistics 21 (SPSS, Chicago, IL, USA). Data

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are expressed as mean ± SEM. One-way ANOVA was performed to compare the groups. Statistical significance was defined as P < .05.

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ACCEPTED MANUSCRIPT 3. Results

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3.1 Body and tissue weights

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To investigate the modulatory effects of exogenous RSV administration, mice were subjected to 4 weeks of RSV treatment using an osmotic pump (8 mg/kg/day). Chronically RSV-treated

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mice were indistinguishable from vehicle-treated mice in terms of weight gain and tissue weights

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of epididymal and subcutaneous adipose depots (Table 1). HF-fed mice had higher levels of body weight changes and tissue weights of epididymal and subcutaneous adipose depots as well

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as livers compared with controls. However, RSV treatment significantly reduced liver weights

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expressed as percentage of body weight to a similar extent as controls.

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3.2 RSV improves serum parameters of lipid profile and liver function Serum concentrations of FFA and TG were elevated in HF-fed DIO mice compared with

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controls (Table 2). Also, two representative markers of liver function (ALT and AST) were high

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in HF-fed mice showing HF diet-induced liver toxicity. In contrast, RSV-treated DIO mice showed a significant reduction in serum lipid profiles (FFA and TG) and liver function markers (ALT and AST).

3.3 RSV is involved in the crosstalk of adiponectin and fetuin-A in the co-culture system of adipocytes and hepatocytes To test our hypothesis that RSV is involved in crosstalk between adipokine adiponectin and hepatokine fetuin-A, RSV-mediated alterations of adiponectin and fetuin-A were analyzed in the adipocytes-hepatocytes co-culture system with the knockdown of adiponectin and fetuin-A, respectively. After 3T3-L1 adipocyte differentiation for 6 days, siRNA targeting adiponectin was 9

ACCEPTED MANUSCRIPT transfected into the subsets of differentiated adipocytes. Also, the subsets of AML12 mouse hepatocytes were transfected with siRNA targeting fetuin-A. After 24 h transfection, co-culture

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of differentiated adipocytes (differentiation day 7) and hepatocytes was conducted for additional

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24 hours in the absence or presence of RSV (50 μΜ). Detailed co-culture set-up is presented in Fig. 1A. During siRNA transfection and co-culture, cell morphology and cell viability were

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monitored, and were not affected by treatments. Notably, adiponectin knockdown in adipocytes

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elevated fetuin-A in AML12 hepatocytes in the co-culture system, which was significantly reduced with RSV treatment (Fig. 1B). Moreover, fetuin-A knockdown in hepatocytes increased

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adiponectin levels in adipocytes in the co-culture system (Fig. 1C). RSV administration for 24 h resulted in the evidently increased adiponectin levels in adipocytes co-cultured with hepatocytes

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with fetuin-A knockdown. These findings demonstrate the existence of the crosstalk between adipose factor adipokine and hepatic factor fetuin-A.

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3.4 RSV-mediated alteration of the systemic and tissue levels of adiponectin and fetuin-A in HF-fed DIO mouse model

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To investigate whether RSV mediates the alteration of adiponectin and fetuin-A in the HF-fed DIO mouse model, RSV was administered for 4 weeks and the systemic and tissue levels of these bioactive factors were analyzed. Systemic concentrations of adiponectin were lower in HFDIO mice compared with the controls, which was restored by RSV treatment (Fig. 2A). In contrast, serum fetuin-A was higher in HF-DIO mice compared with control mice (Fig. 2B). RSV-treated DIO mice had significantly reduced serum fetuin-A. Correlation analysis showed an inverse relationship between serum adiponectin and serum fetuin-A (r = -0.869, P < .001) suggesting the possibility of the interaction between adiponectin and fetuin-A (Fig. 2C).

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ACCEPTED MANUSCRIPT In epididymal adipose depots, gene expression and protein levels of adiponectin were lower in HF-DIO mice, which was restored with RSV treatment (Figs. 3A and B). Also, the known

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downstream targets of adiponectin and its related factors, peroxisome proliferator-activated

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receptor (PPAR)-α, PPAR-γ, Sirt1 and AMP-activated protein kinase (AMPK) were reduced in epididymal adipose depots from HF-DIO mice. RSV treatment significantly increased the

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expression levels of PPAR-α, PPAR γ, and sirtuin1 (Sirt1) (Figs. 3C-E). In addition, AMPK

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expression showed increased tendency by RSV (P = .051; Fig. 3F).

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Consistent with serum results, hepatic fetuin-A was increased in HF-DIO mice and RSV administration led to the marked reduction in hepatic fetuin-A (Figs. 4A and B). Moreover,

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reduced levels of AMPK (a nutrient and energy sensor) and elevated nuclear factor kappa-light-

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chain-enhancer of activated B cells (NF-κB), a downstream target of fetuin-A, in HF-DIO mice were altered by RSV administration (Figs. 4C and D). Thus, the marked alterations of

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adiponectin and fetuin-A, as well as related factors imply a coordinated regulation of cell- and

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organ-crosstalk by these two factors in RSV-treated co-culture system and DIO mice.

4. Discussion Here we show that crosstalk between adiponectin and fetuin-A exists and RSV has substantial metabolic benefits in a DIO mouse model relating to the adipokine-hepatokine crosstalk. RSV protects against diet-induced increase in liver tissue weight, which is accompanied by improvements in serum lipids and liver functions. Chronic HF feeding-induced hepatic steatosis is often characterized by increased accumulation of lipid droplets, hepatomegaly, impaired liver function, and hepatic metaflammation [26,29]. Presently, RSV alleviated HF-induced 11

ACCEPTED MANUSCRIPT impairments in liver weight, activities of liver function-related enzymes (ALT and AST), and lipid profiles. These improvements seem to be mediated in part by a change in cytokines and

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other bioactive factors affecting NAFLD [4,5].

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Various products of the adipose tissue and liver have been characterized including cytokines such as adiponectin, leptin, resistin, visfatin, RBP4, TNF-α, IL-6, IL-1, IL-18, and fetuin-A [16].

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Adiponectin is one of key adipokines regulating insulin signaling, inflammation, as well as

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glucose and lipid metabolism in adipose tissue and liver [30,31]. Adiponectin is negatively associated with the pro-inflammatory cytokines TNF-α and IL-6 [32,33], and down-regulates

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these cytokines by suppressing the activation of Kupffer cells and hepatic stellate cells as well as by attenuating the translocation of NF-κB to the nucleus [34]. Whereas previous reports suggest

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that adiponectin is a key player in the adipose tissue-liver crosstalk [16,35,36], the existing evidences are limited in demonstrating the correlation of adiponectin and various indices of liver diseases without showing the direct interaction between two organs. Previous reports regarding

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the association and interaction between adiponectin and fetuin-A have demonstrated equivocal

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results. The initial efforts to investigate the relationship between adiponectin and fetuin-A suggested an inverse correlation among serum levels of these factors in overweight/obese subjects with myelodysplastic syndrome [20]. In addition, fetuin-A administration induced increased secretion of pro-inflammatory cytokines and decreased adiponectin levels in serum and adipose tissue in mice [21]. However, a recent study that analyzed two cohorts (the European Prospective Investigation into Cancer and Nutrition-Potsdam study cohort, and the Nurses’ Health study cohort) reported that adiponectin and fetuin-A are independently associated with diabetes risk and suggested that each factor may be involved in the development of type 2

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ACCEPTED MANUSCRIPT diabetes by different mechanisms [37]. So far, the direct relationship between adiponectin and fetuin-A has not yet been investigated.

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This study uncovered the direct interaction between adiponectin and fetuin-A utilizing an

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adipocyte-hepatocyte co-culture system. Fetuin-A, a main hepatokine, stimulates the production of pro-inflammatory cytokines, and interferes with insulin signaling in part by acting as an

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endogenous ligand of the Toll-like receptor [38]. Fetuin-A is considered as a biomarker of

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chronic inflammatory diseases and hepatic fat contents [18,19,39]. Fetuin-A overexpression is accompanied by the increased levels and activation of NF-κB [40]. In this study, the subsets of

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differentiated adipocytes and hepatocytes were transfected with siRNAs targeting adiponectin or fetuin-A, respectively. Then, vehicle or RSV was treated for additional 24 h in an adipocyte-

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hepatocyte co-culture system. Interestingly, adiponectin knockdown in adipocytes elevated fetuin-A in AML12 hepatocytes, and fetuin-A knockdown in hepatocytes increased adiponectin levels in adipocytes in a co-culture system, which suggests the existence of the crosstalk between

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adipose factor adipokine and hepatic factor fetuin-A. Moreover, RSV altered the interaction

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between the two cytokines as well as between the two cell types. This is the first evidence showing the direct interaction of adiponectin and fetuin-A as well as the RSV-mediated alterations on these factors in a co-culture system. Findings from our in vivo experiments support the concept that RSV may be involved in the crosstalk between adiponectin and fetuin-A. An inverse relationship was clearly observed between systemic levels of adiponectin and fetuin-A, and RSV increased adiponectin in epididymal adipose tissues, which is consistent with previous findings that RSV increases adiponectin in adipose tissue explants incubated with IL-1β [41]. Also, RSV restored hepatic fetuin-A levels to a similar extent as controls, probably in part from the increased adiponectin 13

ACCEPTED MANUSCRIPT production from adipose tissues. In addition to the alterations in adiponectin and fetuin-A, downstream targets and related

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factors of the two cytokines were affected by the high fat feeding and RSV treatment. High fat feeding suppressed the gene expression levels of PPAR-α, PPAR-γ, Sirt1 and AMPK in

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epididymal adipose depots, which were restored by RSV. AMPK reduces NF-κB activity, and

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NF-κB up-regulates fetuin-A in liver (17). Excess influx of glucose and fatty acids into liver

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reduces AMPK, which subsequently increases NF-κB activity and fetuin-A (17). RSV-mediated alterations in AMPK and NF-κB relating to fetuin-A implicate that RSV may improve HF diet-

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induced hepatic steatosis in part by AMPK-NF-κB-fetuin-A axis in the present study. Direct interaction of adiponectin and fetuin-A, as well as the RSV-mediated alterations on

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these factors, have not yet reported, except for this present interesting finding. Thus, the present findings offer valuable evidence to extend our understanding on the adipose tissue-liver organ crosstalk and RSV-mediated beneficial functions. Although the current findings are interesting

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and meaningful, this study has several limitations. First, this study did not examine the direct/in-

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direct mechanisms about the feedback regulation between adiponectin and fetuin-A. Second, the possibility that other cytokines secreted from liver and adipose tissues may involve in the crosstalk was not considered. In the future, it would be interesting to examine the detailed mechanisms underlying the adipose tissue-liver crosstalk. For example, the treatment of adiponectin and/or fetuin-A as well as the overexpression of these cytokines in a co-culture setting or in vivo tissue-specific manipulation would be valuable approaches to elucidate the mechanisms. We have shown here that adiponectin and fetuin-A interact each other in a co-culture system and possibly in vivo. Moreover, the results indicate a potential role of RSV in the crosstalk of 14

ACCEPTED MANUSCRIPT two cytokines. These findings further suggest that RSV exerts beneficial effects on metabolic

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regulation in part through the interaction between adiponectin and fetuin-A

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Acknowledgements

This research was supported by Basic Science Research Program through the National

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Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF

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2014R1A1A2A16055328) and Chonnam National University (2012). The funder had no role in study design, data collection, and analysis and interpretation, decision to publish, or preparation

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of the manuscript.

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ACCEPTED MANUSCRIPT [27] Chen S, Zhao X, Ran L, Wan J, Wang X, Qin Y, et al. Resveratrol improves insulin resistance, glucose and lipid metabolism in patients with non-alcoholic fatty liver disease:

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A randomized controlled trial. Dig Liver Dis 2015;47:226-32.

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[29] Buettner R, Scholmerich J, Bollheimer LC. High-fat diets: modeling the metabolic disorders of human obesity in rodents. Obesity (Silver Spring) 2007;15:798-808.

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immunity. Nat Rev Immunol 2006;6:772-83. [32] Bruun JM, Lihn AS, Verdich C, Pedersen SB, Toubro S, Astrup A, et al. Regulation of

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humans. Am J Physiol Endocrinol Metab 2003;285:E527-33. [33] Kern PA, Di Gregorio GB, Lu T, Rassouli N, Ranganathan G. Adiponectin expression from human adipose tissue: relation to obesity, insulin resistance, and tumor necrosis factoralpha expression. Diabetes 2003;52:1779-85. [34] Wulster-Radcliffe MC, Ajuwon KM, Wang J, Christian JA, Spurlock ME. Adiponectin differentially regulates cytokines in porcine macrophages. Biochem Biophys Res Commun 2004;316:924-9.

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[40] Dasgupta S, Bhattacharya S, Biswas A, Majumdar SS, Mukhopadhyay S, Ray S, et al. NFkappaB mediates lipid-induced fetuin-A expression in hepatocytes that impairs adipocyte function effecting insulin resistance. Biochem J 2010;429:451-62. [41] Olholm J, Paulsen SK, Cullberg KB, Richelsen B, Pedersen SB. Anti-inflammatory effect of resveratrol on adipokine expression and secretion in human adipose tissue explants. Int J Obes (Lond) 2010;34:1546-53.

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Fig. 1. Crosstalk between adiponectin (AdipoQ) and fetuin-A in an adipocytes-hepatocytes co-

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culture system. (A) Diagram of co-culture system, (B) fetuin-A levels in AML12 hepatocytes cocultured with adipocytes with AdipoQ knockdown (KD) in the absence or the presence of

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resveratrol (RSV), and (C) AdipoQ levels in 3T3-L1 adipocytes co-cultured with hepatocytes

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with fetuin-A KD in the absence or the presence of RSV. Protein levels of each cytokine were expressed as relative percentage of that of control. Data are expressed as mean ± SEM (n = 6 per

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group). Different letters within a variable are significantly different at P < .05. CON, control.

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Fig. 2. (A) Serum adiponectin, (B) serum fetuin-A, and (C) correlation between serum

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adiponectin and fetuin-A. Data are expressed as mean ± SEM (n = 10~13 per group). Different

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resveratrol.

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letters within a variable are significantly different at P < .05. CON, control; HF, high fat; RSV,

Fig. 3. Resveratrol (RSV) alters (A) gene expression and (B) protein levels of adiponectin in epididymal adipose depots of high-fat (HF) fed mice. In addition, gene expression of (C) peroxisome proliferator-activated receptor (PPAR) alpha, (D) PPAR gamma, (E) sirtuin1, and (F) AMP-activated protein kinase (AMPK) was analyzed. Gene expression of each target was normalized to that of 18S. Data are expressed as mean ± SEM (n = 10~13 per group). Different letters within a variable are significantly different at P < .05. CON, control.

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ACCEPTED MANUSCRIPT Fig. 4. Resveratrol (RSV) alters (A) gene expression and (B) protein levels of fetuin-A in livers of high-fat (HF) fed mice. In addition, gene expression of (C) AMP-activated protein kinase

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(AMPK) and (D) nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) was

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analyzed. Gene expression of each target was normalized to that of 18S. Data are expressed as mean ± SEM (n = 10~13 per group). Different letters within a variable are significantly different

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at P < .05. CON, control.

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ACCEPTED MANUSCRIPT Table 1. The effects of resveratrol (RSV) on body weight (BW) change, liver weight, and food intake HF

(n = 10)

(n = 12)

30.8 ± 0.6a

Body weight change (g)

11.3 ± 0.6a

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Tissue weight

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Post-treatment (g)

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19.5 ± 0.4

(n = 13)

20.1 ± 0.3

20.3 ± 0.3

39.5 ± 0.8b

38.1 ± 0.8b

18.2 ± 0.7b

17.9 ± 1.0b

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Body weight Baseline (g)

HF_RSV

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CON

3.0 ± 0.2a

4.9 ± 0.2b

5.0 ± 0.3b

Subcutaneous fat (% BW)

2.7 ± 0.2a

4.5 ± 0.3b

4.8 ± 0.5b

3.5 ± 0.3a

4.1 ± 0.1b

3.5 ± 0.1a

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Liver weight (% BW)

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Epididymal fat (% BW)

Data are expressed as mean ± SEM (n = 10~13 per group). Different letters within a variable are

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significantly different at P < .05. CON, control; HF, high fat.

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ACCEPTED MANUSCRIPT Table 2. Serum metabolic parameters in lean control mice and high-fat (HF) fed mice treated with or without resveratrol (RSV). HF

HF_RSV

(n = 10)

(n = 12)

(n = 13)

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787.8 ± 13.3a

955.8 ± 25.5b

892.5 ± 13.4c

Serum TG (mmol/L)

2.5 ± 0.2a

3.8 ± 0.2b

2.9 ± 0.1c

Serum ALT (U/L)

10.9 ± 0.9a

25.1 ± 1.1b

12.5 ± 1.2a

Serum AST (U/L)

17.2 ± 1.1a

24.0 ± 1.6b

22.5 ± 0.9b

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Serum FFA (mg/L)

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aminotransferase; CON, control; FFA, free fatty acids; HF, high fat; TG, triglyceride.

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Figure 4

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