β-catenin signaling pathway in 3T3-L1 adipocytes

    Inhibitory effect 6-gingerol on adipogenesis through activation of the Wnt/βcatenin signaling pathway in 3T3-L1 adipocytes Chunbo Li, Lin Zhou PII: DOI: Reference:

S0887-2333(15)00243-X doi: 10.1016/j.tiv.2015.09.023 TIV 3638

To appear in: Received date: Revised date: Accepted date:

3 March 2015 19 September 2015 24 September 2015

Please cite this article as: Li, Chunbo, Zhou, Lin, Inhibitory effect 6-gingerol on adipogenesis through activation of the Wnt/β-catenin signaling pathway in 3T3-L1 adipocytes, (2015), doi: 10.1016/j.tiv.2015.09.023

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ACCEPTED MANUSCRIPT Title: Inhibitory effect 6-gingerol on adipogenesis through activation of the Wnt/β-catenin signaling pathway in 3T3-L1 adipocytes

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Author name: Chunbo Li

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Department of Gynaecology and Obstetrics, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai, China, 200040.

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Lin Zhou

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Department of urology, Zhongshan Hospital, Fudan university. Shanghai, China, 200032.

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Corresponding: Chunbo Li, Department of Gynaecology and Obstetrics, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, 536 Changle Road,

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Shanghai, China, 200040. Telephone: +86 15021803963. E-mail: [email protected] Disclosure of financial support: None

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Disclosure of funding: None

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ACCEPTED MANUSCRIPT Inhibitory effect of 6-gingerol on adipogenesis through activation of the Wnt/β-catenin signaling pathway in 3T3-L1 adipocytes

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Abstract

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6-Gingerol has been reported to inhibit adipogenesis and lipid content accumulation.

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However, the mechanism of its anti-adipogenic effect remains unclear. Our aim is to investigate the molecular mechanism of the anti-adipogenic effect of 6-gingerol. The lipid

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content in adipocytes was measured by Oil Red O staining and cells viability was

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analyzed by MTT assay. The extent of suppression of differentiation by 6-gingerol was characterized by measuring the triglyceride content and GPDH activity. The regulation of

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adipogenic markers and the components of the Wnt/β-catenin pathway were analyzed by

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real-time PCR and western blotting. The nuclear location of β-catenin was identified using immunofluorescence assay.Small interfering RNA transfection was conducted to

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elucidate the crucial role of β-catenin in anti-adipogenic effect of 6-gingerol. Our results showed that 6-gingerol inhibited the adipogenesis and lowered the mRNA expression levels of transcription factors and the key lipogenic enzymesin 3T3-L1 cells. The effect of 6-gingerol on adipogenic differentiation was accompanied by stimulating the activation of the Wnt/β-catenin signaling. In addition,we found 6-gingerol induced phosphorylations of glycogen synthase kinase-3β(GSK-3β), and promotedthe nuclear accumulation of β-catenin. Importantly, the inhibitory effect of 6-gingerol on adipogenic differentiationwasreversed after the siRNA knockdown of β-catenin was added. Our findings demonstrated that 6-gingerol inhibits the adipogenic differentiation of 3T3-L1 cells through activating the Wnt/β-catenin signaling pathway.

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ACCEPTED MANUSCRIPT Keywords 6-gingerol

naturel adipogenic differentiation WNT/β-catenin1.

Introduction

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Adipogenesis is an intricate differentiation process that is related topreadipocyte

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proliferation, differentiation, and intracellular lipid content accumulation(Gerriets & MacIver, 2014; A. Park, Kim, & Bae, 2014). During adipogenic differentiation,

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committed preadipocytes will withdraw from the cell cycle before undergoing adipogenic

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conversion(Cignarelli et al., 2012; A. Park et al., 2014). The appropriate combination of adipogenic and mitogenic signals participate in the subsequent process of the

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differentiation, which changes the morphological and biochemical characteristics of the mature adipocytes(Billon & Dani, 2012; Laudes, 2011). The differentiation process is

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mediated by various adipogenic transcription factors.Peroxisome proliferator-activated receptor-γ (PPAR-γ)，a member of the nuclear hormone receptor superfamily, is the main

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regulator of adipogenic differentiation, which mainlymaintains mainly adipocyte-specific function such as lipid storage(Cristancho & Lazar, 2011). CCAAT/enhancer binding protein-α (C/EBP-α) ， a member of the C/EBP family, plays important roles in preadipocyte differentiation(Cristancho & Lazar, 2011). The transcriptional factors cooperatively regulate the expression of the downstream lipid metabolizing enzymes such as Fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC)(Zamani & Brown, 2011). The Wnt/β-catenin signaling pathway is an endogenous inhibitory force and a key regulator of adipose biology(Laudes, 2011). Recently, the Wnt/β-catenin signaling pathway has been reported to be a pivotal negative regulator in the process of adipogenic differentiation(Beg et al., 2014; Kim, Song, Kim, & Hwang, 2014; Lee et al., 2011).

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ACCEPTED MANUSCRIPT Canonically, Wnt ligands (exogenous or autocrine) stimulation of the low density lipoprotein receptor-related protein (LRP) and Frizzled (Fz) co-receptors activates

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disheveled (Dvl) and promotes the disruption of the glycogen synthase kinase 3-AXIN-adenomatous polyposis coli (AXIN-GSK3β-APC) complex, which results in the

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further stabilization and nuclear translocation of β-catenin(Malinauskas & Jones, 2014;

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Prestwich & Macdougald, 2007). In the nucleus, β-catenin binds to a transcription factor

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named T-cell factors (TCFs)/lymphoid-enhancing factors (LEFs) and activates the transcription of cyclin D1 (CCND1) and PPAR-δ, which aresuppressors of PPAR-γand

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C/EBP-α(Prestwich & Macdougald, 2007). Recently, some studies have reported that traditional medicinal herbs such as Shikonin and Kirenolcan inhibit the differentiation

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and lipogenesis of 3T3-L1 cells through the activation of the Wnt/β-catenin signaling pathway(Kim et al., 2014; Lee et al., 2011).

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6-Gingerol, the rhizome of Zingiber officinale, is a globally important herb with a long medicinal history(Rani, Krishna, Padmakumari, Raghu, & Sundaresan, 2012; Yob et al., 2011).

Ginger

phytochemicals,

specifically

6-gingerol

(S-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-3-decanone), account forthe main pungent component of ginger and have been widely used as an antioxidant, anti-inflammation, and anti-tumor agent worldwide(Rani et al., 2012; Yob et al., 2011). Moreover, 6-gingerol has an inhibitory effect on xanthine oxidase, which is responsible for the generation of reactive oxygen species (ROS) such as superoxide anion(Caliceti, Nigro, Rizzo, & Ferrari, 2014). Recently, several studies demonstrated that 6-gingerol effectively suppresses body weight gain and body fat accumulation in rats that were fed a high-fat diet(Saravanan, Ponmurugan, Deepa, & Senthilkumar, 2014; Tzeng & Liu, 2013). However, the exact

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ACCEPTED MANUSCRIPT mechanism of its anti-obesity is still intricate. Inhibiting adipogenesis with therapeutic agents isbelieved to be an important therapeutic measurein preventing and treating

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obesity.A study by Tzeng et al demonstrated that 6-gingerol prevents adipogenesis and the accumulation of cytoplasmic lipid content in 3T3-L1 cells by attenuating the

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phosphorylation of GSK3β(Tzeng & Liu, 2013). GSK3β, an important downstream

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signaling protein in the Wnt/β-catenin signaling pathway, has been previously described

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to be involved in preadipocyte differentiation(Prestwich & Macdougald, 2007). However, it remains unclear whether the suppression of 6-gingerol in adipogenesis is related to the

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Wnt/β-catenin signaling pathway. The present study investigated the effects and partial underlying mechanisms of 6-gingerol in adipogenic differentiation in mouse 3T3-L1 cells.

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The results of our study provide direct evidence that 6-gingerol exerts anti-adipogenic

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2. Materials and method

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effects by activating the Wnt/β-catenin signaling pathway.

2.1. Chemicals and reagents

6-gingerol was purchased from Sigma (USA). Dulbecco’s modified Engle medium (DMEM), fetal bovine serum (FBS), and Trizol were purchased from Gibco (USA). 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), Insulin, Oil Red O, phosphate buffered saline (PBS) and MTT assay were purchased from Sigma (USA).Antibodies against phosphorylated GSK3β, GSK3β, CCND1, ACC, FAS, C/EBP-α, PPAR-γ and a-Tubulin were purchased from Cell Signaling Technology (USA). β-catenin small interfering RNA (siRNA) and control siRNA were purchased from Santa Cruz Biotechnology. Lipofectamine RNAiMAX transfection reagent was purchased from Invitrogen (USA).All other chemicals were purchased from Sigma (USA).

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ACCEPTED MANUSCRIPT 2.2. Cell culture and differentiation The 3T3-L1 cells were purchased from Cyagen Biosciences Inc (Guangzhou, China).The

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cells were cultured in DMEM supplemented with 10% FBS, and 100units/ml penicillin,

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and 100units/ml streptomycin, and they were incubated at 37℃in a humidified，5% CO2

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incubator. After thecells were cultured for 1 day, the growth medium was replaced with differentiation medium(DMEM with 500uMIBMX, 1uM dexamethasone, 1nM insulin,

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and 125nM indomethacin), which was changed every 2 days during adipogenic differentiation. To evaluate the effect of 6-gingerol on the adipogenesis, the cells were

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treated with 5, 10, and 15ug/ml of 6-gingerol. After the addition of 6-gingerol for1, 3 and 7 days, the 3T3-L1 cells were lysed for experimental analysis. Three independent

2.3. Oil Red O staining

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experiments were performed in quadruplicate.

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After the addition of varying concentrations of 6-gingerol in the adipogenic medium for 7 days, the plates were washed three times with PBS and fixed with 10% formaldehyde for 30 min at room temperature. After washing with distilled water, the cells were stained with filtered 3 mg/ml Oil Red O dissolved in 60% isopropanol for 10 min. Each well was then washed thoroughly with water, and images of the stained wells were recorded with a TE-2000U bright-field optical microscope (Nikon, Tokyo, Japan). For quantification, the Oil Red O was eluted by the addition of 100% isopropanol to each well and the absorbance was measured spectrophotometrically at 590 nm with a spectrophotometer. 2.4. Cell viability The3T3-L1 cells (1×103 cells/well) were cultured in 96-well plates and grown at 37℃ in

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ACCEPTED MANUSCRIPT a 5%CO2 incubator. After overnight incubation, the cells were treated with various concentrations of 6-gingerol (0, 5, 10 and 15 mM) in adipogenic medium for 1, 3 and 7

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days. MTT solution (10 ml) was then added to each well and incubated at 37℃ for 4

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hours to allow the formation of formazan crystals. The absorbance was then measured on

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an optical 96-well microplate reader at 570 nm. Further studies were performed with 6-gingerol at a concentration of15 ug/ml.

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2.5. GPDH activity assay

For the GPDH activity assay, differentiated cells were treated with various concentrations

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of 6-gingerol for 7 days. Cells were then washed twice with PBS, and 50 mM Tris-HCl (pH 7.5) containing 1 mM EDTA was then added to the cells. The harvested cells were

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sonicated for 5s at 20% amplitude. After centrifugation at 12,000g for 5 min, the supernatants were assayed for GPDH activity. GPDH activity was assessed by measuring

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NADH absorbance at 340nm using a microplate reader (VERSAmax, USA). GPDH activity was normalized to total DNA content determined using a Hoechst dye assay. 2.6. Quantification of triglycerides Differentiated cells were washed twice with PBS and 50 mM Tris-HCl (pH 7.5) containing 1 mM EDTA was then added to the cells. On day 7, the cells were measured for triglyceride content. The triglyceride content in the cell lysates was quantified using the Triglyceride E test WAKO (Wako Pure Chemical Industries, Osaka, Japan). The concentration was corrected using DNA as an internal standard. DNA was quantified using a Quant-iTTM dsDNA HS Assay kit (Invitrogen, Palsley, UK). 2.7. Real Time-PCR The total RNA was extracted from differentiated cells using Trizol reagent according to

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ACCEPTED MANUSCRIPT the manufacturer’s instructions (Invitrogen). For real-time PCR, approximately 2ug of total RNA was converted to single-stranded cDNA using a commercial cDNA synthesis

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kit (Promega Corporation), which was subsequently used for quantitative real-time PCR

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analysis in a Light Cycler 480 using SYBR Green master mix (Roche Diagnostics). The statistical analysis of the quantitative real-time PCR was obtained by using the

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comparative threshold-cycle (CT) method, which calculates the relative changes in gene

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expression level of the target gene normalized to the housekeeping gene (GAPDH)and relative to a calibrator that serves as the control group. These measurements were

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performed in duplicates for each sample using cells from three different cultures, and

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2.8. Western Blot analysis

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each experiment was repeated three times.

The protein expression of the key transcription regulatorβ-catenin, AKT, pAKT, CCND1,

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GSK3β, PPAR-γ, C/EBP-α, FAS, and ACC were evaluated by western blotting. Cultured and differentiated cells were harvested using a cell scraper and lysed with ice-cold RIPA buffer containing 25mM Tris-HCl, pH 7.6, 150mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS) and a protease inhibitor cocktail (Sigma-Aldrich) to obtain the total cell lysates. The total cell lysates were centrifuged at 12,000 rpm for 20 min at 4 °C to remove insoluble materials. The protein concentrations were determined using a BCA protein assay kit (Pierce, Rockford, IL, USA). Protein extract samples (20ug) were separated by 8-10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes at 150 mA for 1 h. The membranes were then blocked for 1h at room temperature with phosphate buffered saline (PBS) containing 5% skim milk and 0.1% Tween 20, and the

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ACCEPTED MANUSCRIPT membranes were then incubated with 1:1000 dilutions of primary antibodies overnight at 4°C and subsequently with a horseradish peroxidase-conjugated anti-rabbit secondary

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antibody for 1 h at room temperature. Proteins were detected with an enhanced

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chemiluminescence (ECL) detection system (Amersham Biosciences, Little Chalfont, UK) and visualized with the G:BOX EF imaging system and the Gene Snap programme

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(Syngene). The Western blotting results were confirmed twice by independent

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experiments. 2.9. Immunofluorescence analysis

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Differentiated cells were treated with various concentrations of 6-gingerol. On days 7, the cells were fixed in 4% paraformaldehyde and permeabilized in 0.25% triton X-100/PBS.

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An antibody against β-catenin (1: 50; Abgent, USA) was added to the cells overnight at

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4 °C. A DyLight 488 AffiniPure Goat Anti-Rabbit gG(H+L) (EarthOx, USA) was added

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for 1 h at room temperature in the dark, and then 0.1 μg/ml DAPI was then added for 1 min. Samples were viewed with a Leica confocal microscope (Leica TCS SP5, Germany).

2.10. β-catenin knockdown by siRNA transfection Two days after reaching confluence, 3T3-L1 cells were cultured in serum-free medium for 1 h and transfected with 60nM of β-catenin small interference RNA (siRNA) or a non-related control siRNA using the lipofectamine RNAiMAX transfection reagent. After 9h, the transfected cells were differentiated according to the differentiation protocol. Then the cells were then treated with 15 ug/ml 6-gingerol as study groups. The control group received no additional treatment. After 7 days, the total RNA and protein extracts were further prepared for real-time PCR and Western blotting, respectively.

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ACCEPTED MANUSCRIPT 2.11. Statistical analysis All experiments were repeated independently at least three times, and all results

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werepresented as mean ± standard deviation (SD). Statistical significances for comparisons between treated samples and corresponding untreated samples were

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evaluated using one-way ANOVA with a posthoc dunnett’s or students’s t-test. Statistical

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analysis was performed using SPSS 20.0 (SPSS, Chicago, IL, USA). P values <0.05 were

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considered statistically significant. 3. Result

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3.1 6-gingerol inhibits adipogenic differentiation of 3T3-L1 cells Examination of lipid contentformation indifferentiated 3T3-L1 adipocytes on day 7 by

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Oil Red O stainingdemonstrated that 6-gingerol reduced the accumulation of lipid content in a concentration-dependent manner (Figure.1A). The highest 6-gingerol concentration

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(15ug/ml) resulted in the largest reduction in intracellular lipid contentformation. On days 3 and 7, the isopropanol-extracted intracellular Oil Red O showed that 6-gingerol inhibited the differentiation of 3T3-L1 cells into adipocytes and prevented lipid contentaccumulation in comparison with control group (Figure.1B). However, no significant difference was observed between the experimental group and control group on day 1. The MTT assay showed that 6-gingerol (5, 10, and 15ug/ml) had no significant effect on cell viability (Figure.1B). To further characterize the suppression of differentiation

by

6-gingerol,

the

cellular

triglyceride

(TG)

content

and

glycerol-3-phosphate dehydrogenase (GPDH) activity were evaluated. Our results showed that 6-gingerol decreased the TG level and GPDH activity during adipogenic differentiation (Figure. 1C). The protein levels of adipogenic differentiation markers,

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ACCEPTED MANUSCRIPT such as PPAR-γ and C/EBP-α,were markedly decreased by 6-gingerol during adipogenesis (Figure.1D). The results were consistent with the mRNA expression

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levelsof PPAR-γ and C/EBP-α (Figure.1E). In addition, the mRNA expression levels of

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PPAR-γ and C/EBP-α downstream target genes, such as FAS and ACC, were also evaluated.The protein levels examined by western blotting showed a significant reduction

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in these protein levels compared with those of the control (Figure.1D). Notably, the

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expression of these genes was significantly down-regulated upon addition of 6-gingerol (Figure.1E). Taken together, these results suggested that 6-gingerol inhibitsadipogenic

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differentiation of 3T3-L1 cells through the down-regulation of PPAR-γ and C/EBP-α, which further inhibitsthe expression of FAS and ACC.

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3.2 6-gingerol activates the expression of the Wnt/β-catenin signaling pathway

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Activation of the canonical Wnt/β-catenin signaling pathway is responsible for the dephosphorylation and nuclear translocation of β-catenin.To confirm whether 6-gingerol activates thecanonical Wnt/β-catenin signaling pathway, we initially analysed the effect of 6-gingerol on β-catenin and CCND-1during adipogenic differentiation by Western blotting and real-time PCR. As shown in Fig.2A, 6-gingerol increased the protein expression levels of β-catenin and CCND-1 on days3 and 7 compared with those of the control. Consistent with the protein levels, the mRNA levels of β-catenin and CCND-1 also increased with 6-gingerol treatment(Fig.2B). However, no significant difference was observed between the experimental group and control group on day 1. The effect of 6-gingerol on the nuclear translocation of β-catenin was further confirmed by an immunofluorescence assay. Confocal microscopy showed that the level of nuclear

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ACCEPTED MANUSCRIPT β-catenin increasedafter 6-gingerol treatment during adipogenic differentiation,indicating that 6-gingerol could promotes the nuclear translocation of β-catenin (Fig. 2C). Our study

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also demonstrated that 6-gingerol increased the mRNA and protein levels of LRP6 and

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DVL2 compared with those of the control on days3 and 7. Similarly, no significant difference was found between experimental group and control group. This finding

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demonstrated that 6-gingerolup-regulates several components of the Wnt/β-catenin

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pathway, resulting in the reduced expression of adipogenesis-mediated markers and promoting the nuclear translocation of β-catenin.

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3.3 6-gingerol promotes GSK3βphosphorylation

GSK3β, an important down-stream signaling protein in the Wnt/β-catenin signaling

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pathway,phosphorylatesβ-catenin and prevents LEF/TCF-activated transcription activity. Thus, we next investigated if the activation of Wnt/β-catenin signaling due to

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6-gingerolis involvedin GSK3β phosphorylation during adipogenic differentiation. As shown in Fig.3, 6-gingerol enhanced the phosphorylation levels of GSK3β in a time-dependent manner. These data suggested that the activation of Wnt/β-catenin signaling induced by 6-gingerol is dependent on GSK3β activity. 3.4siRNA-mediated knockdown of β-catenin attenuates the anti-adipogenic effect of 6-gingerol To evaluate the involvementof β-catenin in the anti-adipogenic effects of 6-gingerol, a siRNA knockdown experiment was performed. ThemRNA and protein expression levels ofβ-catenin and its transcriptional product, CCND1,were decreasedin 3T3-L1 cells transfected with β-catenin siRNA compared with cells transfected with control siRNA (Fig.5A). Our results showed that pretreatment with β-catenin siRNA effectively

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ACCEPTED MANUSCRIPT decreased the inhibitory effect of 6-gingerol on adipogenic differentiation, as shown in Fig.4B. We next examined the effect of 6-gingerol on the adipogenic transcription factors

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such as PPAR-γ and C/EBP-α. Our results demonstrated that the PPAR-γ and C/EBP-α protein levels, which were down-regulated by 6-gingerol, were significantly restored by

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β-catenin siRNA(Fig.4C).Moreover, the decreases in the mRNA expression levels of

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PPAR-γand C/EBP-α due to 6-gingerol treatment wereeffectively attenuated by the

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addition of β-catenin siRNA(Fig. 4D). Our findingsstrongly confirmed that 6-gingerol inhibits adipogenic differentiation through the activation of the Wnt/β-catenin signaling

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pathway. 4. Discussion

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Obesity and its associated increased mortality and morbidity rates are rapidly becoming a worldwide health problem(Gerriets & MacIver, 2014; A. Park et al., 2014). Obesity due

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to excessive energy intake and low energy expenditure, initiates the over-accumulation of lipid content in adipocytes(A. Park et al., 2014). Theformation of new adipocytes from undifferentiated preadipocytes isreported to be an important factor for obesity(Billon & Dani, 2012; Cignarelli et al., 2012). Thus, a reduction in the number of adipocytes is becoming a new therapeutic target for adiposity. Several studies focusing on the discovery of safe and effective natural products, have highlighted the benefits ofregulating adipogenesis and fat accumulation in preadipocytes(Beg et al., 2014; Kim et al., 2014; Lee et al., 2011). An important finding of the present study showed that 6-gingerol haspotential suppressive impacts on the adipogenic differentiationvia activation ofthe Wnt/β-catenin signaling pathway. Previously, 6-gingerol has been reported to haveanti-cancer, anti-angiogenic and

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ACCEPTED MANUSCRIPT anti-inflammatory activities(Hathaichanok Impheng, 2015; Saha, Das, & Chaudhuri, 2013). A study by Lin et al showed that 6-gingerol at a relatively low concentration of

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50uM could inhibits the growth of colon cancer cells through the induction of cell cycle

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arrest(Lin, Lin, & Tsay, 2012). However, Lee et al demonstrated that treatment with concentration greater than 300 uM 6-gingerol induces apoptosis in HCT-116, SW480,

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HT-29, LoVo and Caco-2 cells(Seong-Ho Lee, 2008). It has also been reported that

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60uM 6-gingerol decreases the induction of apoptosis of COLO-205 cells(Zeng et al., 2015). Tzeng et al showed a15ug/ml (50uM)dose of 6-gingerol significantly inhibited in

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vitro adipogenesis by attenuating the Akt/GSK3β pathway(Tzeng & Liu, 2013). These results support the hypothesis that 6-gingerol exertsdifferent effects depending on the

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tested dose. A low concentration of 6-gingerol tends to inhibit the growth of cells through the induction of cell cycle arrest instead of apoptosis. However, a higherconcentration of

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6-gingerol is required to induceapoptosis. Thus, a 6-gingeroldose of 15ug/ml may be utilized to evaluate the inhibitory effect of 6-gingerol on adipogenic differentiation in 3T3-L1 cells.

The 3T3-L1 cell line is one of the most well-characterized and reliable adipocyte differentiation model systemss for researching the molecular mechanisms of adipogenesis(James M. Ntambi, 2000). To evaluate the effects of 6-gingerol on the differentiation of preadipocytes into adipocytes, confluent 3T3-L1 preadipocytes were treated with various concentrations of 6-gingerol during differentiation. Adipogenesis, which occurs in two major (adipocyte determination and terminal differentiation, is the developmentalprocess of preadipocyte differentiation into mature adipocytes(Newton et al., 2011). PPAR-γand C/EBP-αare two direct adipogenic transcription activators that

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ACCEPTED MANUSCRIPT play key roles inadipogenicdifferentiation, and their expression patterns determine adipose differentiation(Newton et al., 2011). Generally, the activation of C/EBP-α gene

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expression promotes the adipogenic programme of preadipocytes. Next, the expression and activation of PPAR-γinitiatesthe adipogenesis of cells that have not been committed

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to an adipocyte lineage(Shen et al., 2014; Wang et al., 2013). PPAR-γ and C/EBP-αexert

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theirpositive feedback regulation to induce their own expression and to activate

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downstream target genes. Although both factors have critical cooperative and synergistic roles in the late stages of adipogenic differentiation, neither factors is expressed at high

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levels in preadipocytes(White & Stephens, 2010). Previous studies have also demonstrated that the factors are not both involved in the early stages of adipogenic

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differentiation. In the present study, 6-gingerol decreased lipid contentaccumulation, as measured by Oil Red O staining, and down-regulatedboth the protein and mRNA levels PPAR-γand

C/EBP-αin

a

concentration-dependent

manner

on

day 7

of

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of

differentiation.Moreover, the expressions levels of adipocyte-specific protein enzymes, including FAS and ACC, which cooperatively determine the later stages of adipogenic differentiation and biosynthesis of fatty acids and triacylglycerols(Min, Lee, Song, Han, & Chung, 2014), were significantly decreased by 6-gingerol in 3T3-L1 cells. Our findings indicated that 6-gingerol reduces lipid accumulation and fatty acid utilization by controlling these biosynthesis-related genes during adipogenic differentiation. Previous studies have demonstrated that the Wnt/β-catenin signaling pathway inhibitsthe adipogenic differentiation of preadipocytesby blocking the induction of PPAR-γ and C/EBP-α signaling(Prestwich & Macdougald, 2007; Shen et al., 2014; Wang et al., 2013). In addition, the Wnt/β-catenin signaling pathway is activated when the secreted

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ACCEPTED MANUSCRIPT glycoprotein Wnt binds to a cell surface receptor, such as the frizzled receptor and LRP5/6 co-receptors(Laudes, 2011). LRP6is a key receptor that plays a pivotal role in

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initiating a series of signal transduction events and its deficiency spontaneously leads to adipogenic differentiation(Malinauskas & Jones, 2014). DVL2 is the only activated

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member of the DVL family, and regulates the disruption of the Axin–GSK3β–APC

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complex by promoting the phosphorylation of GSK3β(Prestwich & Macdougald, 2007).

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In the present study, 6-gingerol significantly increased the mRNA and protein expression levels of LRP6 and DVL2 in differentiated 3T3-L1 adipocytes on days 3 and 7.However,

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no significant difference on day 1 was observed between the experimental and control group.It was reported that the expression levels of compounds of the Wnt/β-catenin

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signaling increases significantly after the natural herbs such as shikonin, kirenol, or phytoestrogenic molecule desmethylaritin was added for two days(Kim et al., 2014; Lee

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et al., 2011; Wang et al., 2013). Wang et al concluded that the inhibitory of the Wnt/β-catenin signaling does not influence the induction of the early adipogenic differentiation(Wang et al., 2013). In addition, our findings demonstrated that 6-gingerol activates the Wnt/β-catenin signaling in a time-dependent manner. GSK3β is a key negative regulator of β-catenin in the Wnt/β-catenin signaling pathway, that promotes the degradation of β-catenin in quiescent cells(Zamani & Brown, 2011). In addition, the phosphorylation of GSK3β reportedly results in the inhibition of GSK3β, thus leading to the stimulation of the Wnt/β-catenin signaling pathway(Pan et al., 2014). In the present study, we found 6-gingerol increased the phosphorylation level of GSK3β, suggesting that 6-gingerol may inhibit β-catenin degradation via GSK3β phosphorylation. In addition, our findings indicated that 6-gingerol did not influence the phosphorylation

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ACCEPTED MANUSCRIPT level of GSK3β on day 1, which was similar with a study by Tzeng et al that demonstrated the 6-gingeorl regulated the phosphorylation level of GSK3βon days

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4(Tzeng & Liu, 2013).

in

the

nucleus

and

plays

an

important

role

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β-catenin, a component of the cadherin cell adhesion complex, is predominantly localized in

tissue

development

and

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regeneration(Prestwich & Macdougald, 2007). Recently,β-catenin was shown to be a

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negative regulator of adipogenic differentiation, that may directly decrease the activity of PPAR-γ via an interaction between the TCF/LEF-binding domain of β-catenin and the

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catenin-binding domain of PPAR-γ(Malinauskas & Jones, 2014; Prestwich & Macdougald, 2007; Wang et al., 2013). In addition, β-catenin activates the transcription

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of target genes, such as CCND1 (a β-catenin-TCF/LEF target gene) and indirectly suppresses the activity of PPAR-γ and C/EBP-α(Prestwich & Macdougald, 2007). In the

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present study, we found 6-gingerol could up-regulated the mRNA expression levels of β-catenin and CCND1. Furthermore, western blot and immunofluoresence analyses confirmed that 6-gingerol increased the protein expression levels of β-catenin and CCND1 and promoted the nuclear translocation of β-catenin. Our study also showed that the expression levelsof β-catenin and CCND1 could be significantly lower following the siRNA-mediated knockdown of β-catenin, which further demonstrated the vital association between β-catenin and CCND1(Cristancho & Lazar, 2011). Moreover, the relatively lower expression levels of PPAR-γ and C/EBP-α due to 6-gingerol treatment were recovered expression by the siRNA-mediated knockdown of β-catenin, suggesting that 6-gingerol down-regulated PPAR-γ and C/EBP-α through β-catenin up-regulation. These results support the conclusion that 6-gingerol activates the Wnt/β-catenin signal

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ACCEPTED MANUSCRIPT pathway. Obesity is strongly associated with a number of chronic diseases, such as insulin

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resistance, atherosclerosis, type 2 diabetes and chronic inflammation(Gerriets & MacIver, 2014; A. Park et al., 2014). Recently, several studies have proposed potential links

and

metabolic

syndrome

presenthigher

circulating

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obesity

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between obesity, metabolic syndrome and cancer(Gallagher & LeRoith, 2010). Both concentrations

of

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inflammation-related molecules, such as leptin, IL-6, and TNF, which are related to tumor growth and metastasis(Crujeiras & Casanueva, 2015). It has been reported that

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these tumor-promoting adipokines are released from fat tissue(J. Park, Morley, Kim, Clegg, & Scherer, 2014). In addition, cancer cells, peritumoral adipocytes and

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preadipocytes are thought to interact with one another, resulting in more aggressive tumor

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behavior(Freese et al., 2015). 6-gingerol, as a traditional natural medicine, has been

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demonstrated to be effective in suppressing the growth and proliferation of cancer cells(Hathaichanok Impheng, 2015). The potential anti-adipogenic effects of 6-gingerol may provide

some

additional

protection

against

tumorigenesis

by reducing

tumor-promoting adipokines, angiogenic factors and inflammatory adipokines released from adipose tissue as well as by inhibiting the interaction between cancer cells and preadipocytes. These results suggest a possible links between the remarkable preventive and anti-cancer effects of 6-gingeorl and its anti-adipogenic activity found in the present study. According to our conclusions, our findings indicated 6-gingerol activatesthe cell surface receptors, followed by the cellular accumulation and nuclear translocation of β-catenin, ultimately leading to the inhibition of adipogenesis. We conclude that 6-gingerol inhibits

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ACCEPTED MANUSCRIPT adipocyte differentiation and lipid accumulation by regulating the expression of PPAR-γ and C/EBP-α and their downstream lipogenic enzymes such as FAS and ACC via

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activating the Wnt/β-catenin signaling pathway. Our findings warrant further study into

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the development of 6-gingerol as a natural anti-obesity agent for the prevention and treatment of obesity. Further studies are needed to elucidate the anti-obesity activity of

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6-gingerol in vivo and to characterize the underlying mechanisms in an obese mouse

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Conflict of interest: None declared

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model.

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Acknowledgements: We thank Mr Carlbring in Linköping University and Miss CM Xia

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in Fudan University for revising our manuscript.

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Figure legends:

Figure.1 6-gingerol inhibits adipogenic differentiation in 3T3-L1 cells.(A) Inhibitory effects of 6-gingerol on intracellular lipid content formation. Representative morphological change of adipocyte differentiation was stained by Oil Red O staining on days 7. (B) Lipid content were quantified in terms of the absorbance of Oil Red O and the cell viability was further assessed by MTT assay on days 1, 3 and 7 after adipogeic indcution. (C) Triglyceride (TG) content (per mg protein) and Glycerol-3-phosphate dehydrogenase (GPDH) activity (U/mg protein) were measured with a triglyceride

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ACCEPTED MANUSCRIPT estimation kit (Sigma) and a GPDH activity assay, respectively. (D-E) The protein level and mRNA level of transcription factor (PPAR-γ and C/EBP-α) and lipogenic enzymes

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(FAS and ACC) treated with different concentration of 6-gingerol was evaluated by

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western blotting and real-time RCR, respecitively. α-Tubulin and GAPDH was used as internal control. *p>0.05 or **p<0.05 compared with CON. All results are showed as the

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mean±SD of three independent experiment. CON: adipogenic induction without the

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addition of 6-gingerol (0 ug/ml).

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Figure.2 6-gingerol activates the Wnt/β-catenin signaling pathway in 3T3-L1 cells. (A)The protein level of LRP6, DVL2, β-catenin and CCND1 in 3T3-L1 cells on days 1, 3

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and 7 treated with 6-gingerol at 15ug/ml was evaluated by western blotting. (B)The

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mRNA level of LRP6, DVL2, β-catenin and CCND in 3T3-L1 cells on days 1, 3 and 7

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treated with 6-gingerol at 15 ug/ml was evaluated by real-time PCR. α-Tubulin and GAPDH was used as internal control. *p>0.05 or **p<0.05 compared with CON. All results are showed as the mean±SD of three independent experiment. CON: adipogenic induction without the addition of 6-gingerol (0 ug/ml). EXP: adipogenic induction with the addition of 6-gingerol (15ug/ml). (C) 6-gingerol induced β-catenin nuclear translocation during adipogenic differentiation. 3T3-L1 cells were treated with 6-Gingerol at 15ug/ml for 1, 3 and 7 days, and were measured by the immunofluorescence staining for β-catenin (green) using anti-dephosphorylated β-catenin. The nucleis were stained with DAPI (blue).

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ACCEPTED MANUSCRIPT Figure.3 Activation of the Wnt/β-catenin signaling pathway by 6-gingerol is responsible for phosphorylation of GSK3β. The phosphorylation of GSK3β and total GSK3β in

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3T3-L1 on days 1, 3 and 7 treated with 6-gingerol at 15ug/ml were evaluated by western

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blotting (upper panel). The intense of the protein bands were quantified and calculated as percentages of the control (lower panel). Values are expressed as the fold of increase to

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control culture. *p>0.05, **p<0.05.CON: adipogenic induction without the additon of

siRNA-mediated

6-gingerol-induced

knockdown

activation

of

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Figure.4

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6-gingerol (0 ug/ml). EXP: adipogenic induction with the adition of 6-gingerol (15ug/ml).

of

β-catenin

Wnt/β-catenin

significantly

signaling

blocked

pathway

and

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6-gingerol-induced inhibitory effect of adipogenic differentiation. (A) Effect of

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6-gingerol in the presence or absence of β-catenin siRNA on the protein and mRNA

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levels of β-catenin and CCND1 during adipogenesis of 3T3-L1 was evaluated by western blotting and real-time PCR. (B) β-catenin siRNA blocked the inhibitory effect of 6-gingerol on adipogenic differentiation, which was confirmed by Oil O staining. (C-D) Effect of 6-gingerol in the presence or absence of β-catenin siRNA on the protein and mRNA levels of PPAR-γ and C/EBP-α during adipogenesis of 3T3-L1 was evaluated by western blotting and real-time PCR. α-Tubulin and GAPDH was used as internal control. All results are showed as the mean±SD of three independent experiment. CON siRNA: transfection with empty plasmid. *p>0.05 or **p<0.05 compared with CON siRNA and 6-gingerol treatment. #p>0.05 or ##p<0.05 compared with CON siRNA alone.

Graphical abstract: Mechanism of 6-gingerol-induced inhibition of adipogenesis

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the up-regulation of β–catenin which inhibits the epression of PPAR-γ and C/EBP-α.

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ACCEPTED MANUSCRIPT Highlights 1. 6-gingerol inhibits adipogenic differentiation and lipid accumulation.

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2. 6-gingerol activates the Wnt/β-catenin signaling pathway during adipogenic

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differentiation.

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3.β-catenin plays a critical role in adipogenic differentiation.

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