PHYTOTHERAPY RESEARCH Phytother. Res. 29: 398–406 (2015) Published online 2 December 2014 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.5264

Ellagic Acid Suppresses Lipid Accumulation by Suppressing Early Adipogenic Events and Cell Cycle Arrest Mi-Seon Woo,1† Hyeon-Son Choi,2† Min-Jung Seo,1 Hui-Jeon Jeon1 and Boo-Yong Lee3* 1

Department of Biomedical Science, CHA University, Kyonggi 463-836, South Korea Department of Food Science and Technology, Seoul Women’s University, Seoul 139-774, South Korea 3 Department of Food Science and Biotechnology, CHA University, Kyonggi 463-836, South Korea 2

Ellagic acid (EA) is a natural polyphenol found in various fruits and vegetables. In this study, we examined the inhibitory effect of EA on fat accumulation in 3T3-L1 cells during adipogenesis. Our data showed that EA reduced fat accumulation by down-regulating adipogenic markers such as peroxisome proliferator activated receptor γ (PPARγ) and the CCAAT/enhancer binding protein α (C/EBPα) at the mRNA and protein levels in a dosedependent manner. We found that the decrease in adipogenic markers resulted from reduced expression of some early adipogenic transcription factors such as KLF4, KLF5, Krox20, and C/EBPβ within 24 h. Also, these inhibitions were correlated with down-regulation of TG synthetic enzymes, causing inhibition of triglyceride (TG) levels in 3T3-L1 cells investigated by ORO staining and in zebrafish investigated by TG assay. Additionally, the cell cycle analysis showed that EA inhibited cell cycle progression by arresting cells at the G0/G1 phase. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: ellagic acid; adipogenesis; 3T3-L1 celles; adipogenic markers; zebrafish.

INTRODUCTION Phytochemicals, which are naturally found in plants, can be used to improve human health (Krishnaswamy and Raghuramulu, 1998). Ellagic acid (EA) is a phytochemical found in edible plants, as well as algae, mushroom, oak, nuts, and fruits. The most prevalent source of phytochemicals is fruits such as blackberries, cranberries, and pomegranates (Daniel et al., 1989). Phytochemicals typically exist in a tannin form called ellagitannin in plants. The acid–base status of the environment leads to hydrolysis of ellagitannin and spontaneous release of EA (Larrosa et al., 2006). In particular, ingested ellagitannin is hydrolyzed to release EA in the human gastrointestinal tract and metabolized by colon microbiota (Larrosa et al., 2006). Many studies have shown that EA possesses various biological effects. EA is known to inhibit cell proliferation of human peripheral blood mononuclear cells and modulate cytokine production (Anderson and Teuber, 2010). In addition, EA is known to inhibit the proliferation and metastasis of cancer cells by inducing apoptosis (Narayanan et al., 1999). Meanwhile, recent studies have shown that EA can alleviate metabolic syndrome induced by highfat diets (HFDs) in rats (Panchal et al., 2012). Also, recently, adipogenesis is inhibited by ellagic acid through coactivator-associated arginine methytransferase 1 (CARM1) mediated chromatin modification (Kang

* Correspondence to: Boo-Yong Lee, Department of Food Science and Biotechnology, CHA University, 222 Yatap, Bundang, Seongnam, Kyonggi 463-836, South Korea. E-mail: [email protected] † These authors contributed equally to this work.

Copyright © 2014 John Wiley & Sons, Ltd.

et al., 2014). However, how EA suppresses adipogenesis, as well as the lipid signaling and molecular events related to the inhibitory effect of EA, has not been explored. Obesity has become a global epidemic associated with affluence, resulting from excessive energy and a lack of physical activity (Itoi et al., 2012). This can result in metabolic complications such as type 2 diabetes, hypertension, and atherosclerosis (Das et al., 2010). Thus, controlling obesity is important to reduce the risk of many metabolic diseases (MacKenzie et al., 2012). Adipocytes, which are cells that store excessive energy, are commonly used to study lipid accumulation in white adipose tissue. Adipocyte development is accompanied by an increase in the size and number of adipocytes cells. The increase in size (hypertrophy) is usually achieved through triglyceride (TG) accumulation (Gonzales and Orlando, 2007). Also, the increase in cell number (hyperplasia) occurs through cell proliferation and differentiation (Mandenoff et al., 1982). These increases in adipose cells are activated by transcription factors such as peroxisome proliferatoractivated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) (Smas and Sul, 1997). Activation of these major adipogenic factors requires the expression of early adipogenic factors, including C/EBPβ, C/EBPδ, early growth response protein 2 (EGR2 or Krox20) (Chen et al., 2005), kruffel-like factors (KLF4 or 5) (Jiang et al., 2008), and reduced preadipocyte factor1 (pref1) (Smas and Sul, 1997). Recently, many studies have exploded anti-adipogenic effects of phytochemicals in 3T3-L1 cells through the adipogenic factor’s suppression (Seo et al., 2013; Woo et al., 2013). An investigation of lipogenesis-related factors during the adipocyte differentiation is important to define suppression of lipid accumulation, and this relationship has been well studied (Cooke and Naaz, 2005; Jung et al., 2013). Glyceride 3-phosphate Received 26 August 2014 Revised 22 October 2014 Accepted 09 November 2014

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Table 1. Primers used in this study Gene

Sence

Antisense

3T3-L1 cells GAPDH PPARγ aP2 C/EBPα C/EBPβ C/EBPδ Pref1 KLF4 KLF5 Krox20

5′-ACACATTGGGGGTAGGAACA-3′ 5′-CCAGAGTCTGCTGATCTGCG-3′ 5′-GACCTGGAAACTCGTCTCCA-3′ 5′-GGTGCGCAAGAGCCGAGATAAAG-3′ 5′-CAAGCTGAGCGACGAGTACA-3′ 5′-AGAAGCTGGTGGAGTTGTCG-3′ 5′-CTTTTCGTGGTGGTTTTCGT-3′ 5′-GCCTGTGGGTTCGCTATAAA-3′ 5′-CCGGAGACGATCTGAAACACG-3′ 5′-AGTTGGGTCTCCAGGTTGTG-3′

5′-AACTTTGGCATTGTGGAAGG-3′ 5′-GCCACCTCTTTGCTCTGATC-3′ 5′-CATGACACATTCCACCACCA-3′ 5′-AGTTCACGGCTCAGCTGTTCCAC-3′ 5′-AGCTGCTCCACCTTCTTCTG-3′ 5′-CGCAGGTCCCAAAGAAACTA-3′ 5′-GCAAGTCTCAGGAACCAAGC-3′ 5′-AAGGAATGGTCAGCCACATC-3′ 5′-GTTGATGCTGTAAGGTATGCCT-3′ 5′-GGAGATCCAGGGGTCTCTTC-3′

Zebrafish β-actin PPARγ aP2 SREBP-1

5′-CTCTTCCAGCCTTCCTTCCT-3′ 5′-CAGTTTGCAGAGAACAGCGT-3′ 5′-GAACTGAGCCTGGCATCTTC-3′ 5′-GAGCCTTCAGACACGTCCTC-3′

5′-CTTCTGCATACGGTCAGCAA-3′ 5′-GGCTCTTCTTGTGTATGCGG-3′ 5′-GGCAAACTTGTGCAGAAACA-3′ 5′-ACTCTTCTGGTGTGGCTGCT-3′

(G3P) which is a metabolite derived from glycolysis is converted by glycerol 3-phosphate acyltransferase to lysophosphatidic acid (LPA). LPA is altered to phosphatidic acid (PA) by lysophosphatidic acid acyltransferase theta (LPAATθ). PA, a precursor of acylglycerols, is catalyzed into diacylglycerol (DGA) by Lipin-1, and DGA is finally converted into TG by diglyceride acyltransferase (DGAT) (Colemen and Mashe, 2011). In addition, adipocyte proliferation is enhanced during cell cycle progression. In 3T3-L1 cell lines, the cell cycle is induced with a hormonal cocktail treatment to induce differentiation, and the cell cycle regulators such as cyclin A, B, D1, E, and cyclin-dependent kinases (CDKs) increased. Thus, suppressing the cell cycle in adipocytes is a target to inhibit adipocyte differentiation (Kim et al., 2011). Recent studies have examined zebrafish in vivo. As a type of vertebrate, zebrafish, scientifically known as Danio rerio, has a similar genetic background to other mammals (Rosania, 2012). Additionally, zebrafish are a good model to study embryology and genetics because of its transparent body. For adipogenesis and lipid accumulation, zebrafish are known to have similar adipogenic factors and signaling systems as mammals (Chu et al., 2012). A recent study showed that diet-induced obese zebrafish share common pathophysiological routes and adipogenic factors in lipid metabolism, such as sterol regulatory element-binding transcription factor 1 (SREBF1) and PPARγ, with mammals (Chu et al., 2012). In this study, we explored the role of EA on adipogenesis and cell cycle arrest in 3T3-L1 cells to find how EA prevents adipogenesis during the growth stages of adipocyte. In addition, we examined the anti-adipogenic effect of EA in high-fat dieted zebrafish model.

MATERIALS AND METHODS Reagents. Dulbecco’s modified Eagle’s medium (DMEM), bovine calf serum (BCS), fetal bovine serum (FBS), penicillin–streptomycin (P/S), phosphate-buffered saline (PBS), and trypsin–EDTA were obtained from Copyright © 2014 John Wiley & Sons, Ltd.

Gibco (Gaithersburg, MD). Dexamethasone (DEX), 3-isobutyl-1-methylxanthine (IBMX), insulin, Oil Red O, dimethyl sulfoxide (DMSO), and troglitazone were acquired from Sigma-Aldrich (St. Louis, MO). All other chemicals were obtained from Sigma-Aldrich. The ellagic acid (EA) used in this study was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell culture. 3T3-L1 preadipocytes (American Type Culture Collection CL-173; ATCC, Manassas, VA) were cultured, maintained, and differentiated as described below. Cells were plated and grown in DMEM with 3.7 g/L sodium bicarbonate, 1% P/S, and 10% BCS. For differentiation, postconfluent cells were treated with 10% FBS and a hormonal cocktail (MDI) consisting of 0.5 mM IBMX, 0.25 μM DEX, and 4 μg/mL insulin for 2 days. Culture medium was replaced with DMEM supplemented only with 4 μg/mL insulin and 10% FBS. The EA is dissolved into DMSO to make 100 mM EA. 100 mM EA is diluted to 5, 25, and 50 mM EA. 3T3-L1 preadipocytes were cultured with culture medium consisting of 0.1% EA-DMSO solution to make 5, 25, and 50 μM EA. Also, the control medium is treated with 0.1% DMSO to clear the effect of DMSO on the 3T3-L1 preadipocytes. For treatment, 2-day postconfluent cells were differentiated with MDI in the presence of 0, 5, 25, or 50 μM EA. This medium was replenished every other day. Differentiated 3T3-L1 cells were harvested at days 6 and 8 to examine time- and dose-dependent effects of EA, respectively followed by Oil red O staining, RT-PCR, and western blotting.

Oil Red O staining. After differentiation, cells were fixed in 10% formaldehyde in PBS for 1 h, washed with distilled water, and dried completely. Cells were stained with 0.5% Oil Red O solution in 60:40 (v/v) isopropanol: water for 30 min at room temperature, washed four times in water, and dried. Differentiation was monitored using microscopy and quantified via elution with Phytother. Res. 29: 398–406 (2015)

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isopropanol; optical density (OD) measurements were obtained at 490 nm.

RNA extraction and semiquantitative reverse transcriptionpolymerase chain reaction (RT-PCR). Total RNA was extracted from 3T3-L1 adipocytes with TRIzol reagent (Invitrogen, Carlsbad, CA) as per the manufacturer’s protocol. RNA samples with OD260/OD280 ratios above 2.0 were used for semiquantitative RT-PCR. Total RNA (1 μg) was used to generate cDNA using a RT-PCR system. The primers which we used in this study are shown in Table 1. PCR products were electrophoresed on 1.5% (v/v) agarose gels, stained with ethidium bromide, and photographed. Expression levels were quantified using a gel documentation and analysis system (ImageJ; NIH, Bethesda, MD). Western blot analysis. Protein extracts (50 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes. The membranes were immunoblotted with primary antibodies. Secondary antibodies conjugated to horseradish peroxidase (1:1000) were then applied for 1 h. Bands were visualized using enhanced chemiluminescence (ECL), and proteins were detected using LAS image software (Fujifilm, New York, NY).

Cell cycle analysis. To investigate the effects of EA on cell cycle progression during 3T3-L1 adipocyte differentiation, a BD FACS Calibur™ Flow Cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ) was used. For this analysis, preadipocytes were differentiated for 18 h and 24 h with or without EA. Next, 3T3-L1 cells were fixed in 70% ethanol for at least 2 h and washed with PBS. Cells were stained with 300-μL propidium iodide (PI) solution (50 μg/mL) containing 20-μL RNase (10 mg/mL) and incubated at 37 °C for 30–45 min. After staining, the cell cycle was monitored using flow cytometry.

Zebrafish maintenance. Zebrafish eggs were obtained from Chungnam University (Daejeon, Korea) and raised in egg water (0.6 g/L sea salt) under 14 h light/10 h dark cycles at a constant temperature of 28 ± 0.5 °C. Fertilized embryos were plated in 100-mmdiameter Petri dishes (with 20 mL of egg water) through day 5 postfertilization (dpf) at 50 embryos per dish. After 5 dpf, embryos were transferred to 200 mL of egg water and fed hardboiled egg yolk as a HFD with 0, 25, or 50 μM of EA or 2.5 μM of curcumin until 20 dpf. Nile Red staining. Nile Red (Invitrogen N-1142) stock solution was made in acetone (1.25 mg/mL) and stored in the dark at 20 °C. Zebrafish were transferred to Nile Red at a final working concentration of 0.5 μg/mL egg water and incubated for 30 min in the dark at room temperature. Zebrafish were anesthetized in Tricain (Sigma MS-222) at 200 μL/5 mL and mounted in 3% methylcellulose. An Eclipse E600 (Nikon, Tokyo, Copyright © 2014 John Wiley & Sons, Ltd.

Japan) fluorescence microscope was used for green fluorescent imaging.

Triglyceride (TG) analysis. A TG assay kit (Zen-Bio, Durham, NC) was used to explore the effect of EA on TG accumulation in vivo, as per the manufacturer’s protocol. At 20 dpf, zebrafish were collected and washed with wash buffer (TG-5-RB) and then removed the buffer. Lysis buffer (150 μL) wAS added to each tube and incubated at 50 °C for 20 min. After adding 135-μL wash buffer, we performed syringe homogenization and then centrifugation. Supernatants (80 μL) from each tube were transferred to new tubes, and then 20-μL reagent B solution was added to the samples. Tubes are incubated at 37 °C for 2 h. Next, the samples were added with 100-μL reagent A and incubated at room temperature for 15 min, and the absorbance was determined at 540 nm.

Statistical analysis. All experiments were repeated three times. The data were statistically analyzed by ANOVA and Duncan’s multiple range tests which provides significance levels for the difference between any pair of means in the data. A *P-value of