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DOI 10.1002/mnfr.201500021

Mol. Nutr. Food Res. 2015, 59, 1458–1471

RESEARCH ARTICLE

Dieckol, a major phlorotannin in Ecklonia cava, suppresses lipid accumulation in the adipocytes of high-fat diet-fed zebrafish and mice: Inhibition of early adipogenesis via cell-cycle arrest and AMPK␣ activation Hyeon-Son Choi1∗ , Hui-Jeon Jeon2∗ , Ok-Hwan Lee3 and Boo-Yong Lee2 1

Department of Food Science and Technology, Seoul Women’s University, Hwarang, Nowon, Seoul, South Korea Department of Food Science and Biotechnology, CHA University, Seongnam, Gyeonggi, South Korea 3 Department of Food Science and Biotechnology, Kangwon National University, Chuncheon, South Korea 2

Scope: Dieckol is a major polyphenol of Ecklonia cava. This study demonstrates a mechanistic role for dieckol in the suppression of lipid accumulation using three models. Methods and results: Mice were split into four experimental groups (n = 10 per group): normal diet, high-fat diet (HFD), and dieckol-supplemented diets. Dieckol-supplemented mice groups showed a significant decrease of body weight gain (38%) as well as fats of organs including epididymal (45%) compared with a HFD-fed group. LDL cholesterol level was reduced by 55% in dieckol-supplemented group. Adipogenic factors and lipid synthetic enzymes were analyzed via real-time PCR or immunoblotting. Dieckol regulated mRNA expressions of early adipogenic genes in 3T3-L1 cells. These results were reflected in downregulation of late adipogenic factors, resulting in a decrease in triacylglycerol content. These data were also verified in zebrafish and mouse models. Dieckol activated AMP-activated protein kinase ␣ (AMPK␣) signaling to inhibit lipid synthesis in 3T3-L1 and mouse model. Dieckol was also shown to inhibit mitotic clonal expansion via cell-cycle arrest. Conclusion: Our data demonstrate that dieckol inhibits lipid accumulation via activation of AMPK␣ signaling and cell-cycle arrest.

Received: January 19, 2015 Revised: March 19, 2015 Accepted: April 9, 2015

Keywords: 3T3-L1 / Adipogenesis / Dieckol / Mice / Zebrafish

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Additional supporting information may be found in the online version of this article at the publisher’s web-site

Introduction

Ecklonia cava is an edible brown alga native to Korea and Japan. It contains many polyphenol compounds, primarily phlorotannins [1]. Extract of Ecklonia cava has been demonstrated to have numerous biological activities, includ-

Correspondence: Dr. Boo-Yong Lee E-mail: [email protected] Abbreviations: ACC, acetyl-CoA carboxylase; AICAR, 5Aminoimidazole-4-carboxamide ribonucleotide; AMPK, AMPactivated protein kinase; BCS, bovine calf serum; DGAT, diacylglycerol acyltransferase; DEX, dexamethasone; DKLD, low dose of dieckol; DKHD, high dose of dieckol; FBS, fetal bovine serum; HFD, high fat diet; IBMX, 3-isobutyl-1-methylxanthine; MCE, mitotic clonal expansion  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

ing anti-inflammatory, anti-oxidative, anti-adipogenetic, antihyperlipidemic, and anti-aging effects [2–4]. This seaweed extract has been commercialized in the form of supplemental remedies. The product Seapolynol consists of a mixture of polyphenols, including the phlorotannin compounds dieckol, bieckol derivatives, pholorofurofucoeckol-A, eckol, and phloroeckol [5]. Dieckol is known to be a major phlorotannin of both Seapolynoland Ecklonia cava itself [5]. Various biological studies of dieckol to date have been reported [6–8]. Lee et al. reported that dieckol protects human umbilical vein endothelial cells from oxidative stress [9]. Dieckol is also known to improve memory in mice [10], and to inhibit adopogenesis in adipocytes [11]. ∗ These authors contributed equally to this work. Colour Online: See the article online to view Figs. 1, 2, 5, and 6 in colour.

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Obesity is caused by excessive accumulation of body fat as a result of an energy imbalance, leading to complex metabolic diseases, such as diabetes and atherosclerosis [12]. Formation of body fat is dependent upon the differentiation status of adipocytes (fat cells). Therefore, regulation of adipocyte differentiation is acknowledged to be a key factor in the control of obesity at the cellular level. Hormonal and nutritional regulation of adipocyte differentiation are generally well understood [13, 14]. In cell-based research, adipocyte differentiation can be initiated by supplementing growth-arrested confluent cell cultures with a hormonal cocktail composed of insulin, dexamethasone, and 3-isobutyl-1-methylxanthine (IBMX). Differentiation is then achieved with the expression of various transcription factors. Expression of early adipogenic factors, such as C/EBP␤, C/EBP␦, KLF4, and KLF5 increases initially, followed by late adipogenic factors, including C/EBP␣ and PPAR␥ [15,16]. In particular, activation of late adipogenic factors is associated with the synthesis of triglyceride lipids. Triglyceride synthesis is regulated by triglyceride synthetic enzymes, such as lipin 1 and diacylglycerol acyltransferase (DGAT); these promote lipid accumulation in the terminal adipogenic stage of adipocyte differentiation [17–19]. In addition, AMP-activated kinase (AMPK) plays an important role as an energy sensor that regulates lipid metabolism [20]. Activation of AMPK suppresses fatty acid synthesis via deactivation of acetyl-CoA carboxylase (ACC) [21]. The mouse is a well-established animal model for adipogenic research that has been instrumental in generating an enormous amount of data. In recent years, the zebrafish has also been recognized as a useful animal with which to model various diseases. The advantages of the zebrafish model include a shorter generation time and lower costs compared to other animal models [22]. In addition, the zebrafish provides abundant embryos, and has a transparent body, enabling direct observation of phenotypes [22]. Notably, the mechanisms of lipid accumulation of zebrafish are known to be similar to those of other mammals [23]. As a result, research into metabolic diseases, including obesity, has been performed using the zebrafish model [24]. Dieckol is the most abundant polyphenol in Ecklonia cava, an edible seaweed known to have physiological activity. The objective of this study is to examine the effects of dieckol on adipogenesis and lipid accumulation, and demonstrate their mechanisms. In this study, we investigated the inhibitory effect of dieckol on lipid accumulation in 3T3-L1 cells, focusing on early adipogenesis, the cell cycle, and cell signaling. The anti-adipogenic effect of dieckol was further evaluated in both zebrafish and mouse models.

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Materials and methods

2.1 Materials Dieckol ( 90% purity) was obtained from Botamedi, Inc, (Jeju, Korea). DMEM, bovine calf serum (BCS), fetal bovine  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

serum (FBS), penicillin-Gibco (Gaithersburg, MD, USA). Dexamethasone (DEX), IBMX, insulin, Oil red O, and nitroblue tetrazolium were purchased from Sigma (St. Louis, MO, USA). Antibodies against PPAR␥, C/EBP␣, FABP4(aP2), C/EBP␤ Akt, p-Akt, ERK, p-ERK, AMPK␣, p-AMPK␣, ACC, p-ACC, and ␣-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against LPAAT ␾, LIPIN1, and DGAT1 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Aprotinin, leupeptin, benzamidine, pepstatin, sodium orthovanadate, phenylmethylsulfonyl fluoride, and phosphatase inhibitor cocktails I and II were purchased from Sigma. Dorsomorphin dihydrochloride (compound C) and aminoimidazole-4carboxamide riboside(AICAR) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). All other chemicals were purchased from Sigma.

2.2 Cell culture and harvesting 3T3-L1 preadipocytes were plated and cultured in DMEM medium containing 3.7 g/L sodium bicarbonate, 1% P/S, and 10% BCS at 37⬚C in 5% CO2 . Two-day postconfluent cells were induce to differentiate via treatment with 10% FBS and MDI hormone cocktail (0.5 mM IBMX, 1.0 ␮M DEX, and 1.67 ␮M insulin; day 0). The medium was replaced with DMEM containing 1.67 ␮M insulin and 10% FBS for a further 2 days (day 2). This medium was then replaced with fresh DMEM containing 10% FBS every other day until the indicated time point (days 6–12). Dieckol (25, 50, and 100 ␮M) were dissolved in DMSO. The final concentration of DMSO in medium was 0.1% in all experiments.

2.3 Trypan blue assay Postconfluent 3T3-L1 cells were induced to differentiate by treatment for 2 days with MDI cocktail with or without dieckol. Cells were collected after two washes with PBS. Trypan blue dye (0.5%, Sigma-Aldrich) was added to the cultures to enable counting of viable cells via microscopic observation.

2.4 Oil red O staining and lipid quantification Differentiated 3T3-L1 cells or liver tissue samples, which were washed with PBS, were fixed with 4% formaldehyde at 4⬚C for 1 h. After washing with PBS, the fixed cells were stained with 0.5% Oil red O in 60:40 v/v isopropanol/H2 O for 2 h at room temperature, and then washed three times with water. The extent of lipid accumulation was observed microscopically and photographed. A 100% isopropanol was used to elute Oil red O dye, and the absorbance at 490 nm was determined. www.mnf-journal.com

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2.5 RNA extraction and semiquantitative RT-PCR Total RNA was extracted from differentiated (day 6 or 8) or undifferentiated cells, zebrafish, and mouse liver tissues using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. cDNA was produced from 1 ␮g of total RNA using a Maxime RT PreMix Kit (iNtRON Biotechnology, Inc). cDNA was amplified using SYBR Green 2X matermix Kit (m.biotech., Inc). Mixture was synthesized by CFX96 Real-Time system and c1000 thermal cycler (BioRad, Inc) and subjected to PCR analysis at 40 cycles. Quantification analysis generated using the CFX ManagerTM software (BioRad, Inc). The specific primers listed in Supporting Information Table 1.

2.6 Western blotting and protein estimation Proteins (25 or 50 ␮g) extracted from differentiated or undifferentiated cells and mouse liver tissues were performed by SDS-PAGE and immunoblot analysis with indicated antibodies. Bands were visualized by enhanced chemiluminescence, and detected using LAS imaging software (Fuji, New York, NY, USA).

2.7 Quantification of triglycerides Accumulated triglycerides were measured using a Total Triglyceride Assay Kit (Zen-Bio, Inc.).

2.8 Analysis of cell-cycle progression Cell-cycle progression of cultured cells was assayed by flow cytometry. Postconfluent preadipocytes were treated with MDI in the presence or absence of dieckol (50 or 100 ␮M) for the indicated periods of time. Harvested cells were fixed with 70% ethanol for 2 h at 4⬚C, washed with PBS, and centrifuged. For staining of DNA, the resulting pellet was resuspended in 40 ␮g/mL propidium iodine solution containing 1 mg/mL RNase A at 37⬚C for 30 min. The cell-cycle progression of samples (10 000 cells per experiment) was analyzed using a BD FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA), according to the manufacturer’s instructions.

2.9 Animal care and experimental protocol All experimental mice were housed in a specific pathogen-free facility at CHA University, Seongnam, Korea. The project was approved by the Institutional Animal Care and Use Committee of CHA University (IACUC140001). Forty male Imprinting Control Region mice (5 weeks old), purchased from Joong-Ang Experimental Animal Co., Seoul, Korea were housed under the following conditions: 21 ± 2.0⬚C in  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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temperature, 50% ± 5% relative humidity, 12 h light/12 h dark cycle. All of the mice were fed rodent chow and tap water ad libitum for 1 week prior to their partition into the following experimental groups (n = 10 per group): normal diet, high-fat diet (HFD) and dieckol-supplemented diets (DKLD; 15 mg/kgBW/day and DKHD; 60 mg/kgBW/day). The normal diet was a purified diet based on the Purina Laboratory Rodent Diet 38057 (Dyets Inc., Bethlehem, PA, USA). The HFD was identical to the normal diet, but supplemented with 350 g fat/kg (300 g lard plus 50 g corn oil) and 1% cholesterol (Research Diets Inc., Bethlehem, NJ, USA). The dieckolsupplemented diets were identical to the HFD, but supplemented with 15 mg/kgBW/day (DKLD) or 60 mg/kgBW/day (DKHD) of dieckol. At the end of the experimental period (11 week), the animals were anesthetized with ether, following a 12-h fast. To analyze plasma lipid levels, blood was drawn from the abdominal aorta into an EDTA-coated tube. The plasma was isolated by centrifuging the blood at 2000 × g for 15 min at 4⬚C. The epididymal, mesenteric, and inguinal fat pads were removed, rinsed with PBS, and weighed. The plasma, liver, and visceral fat pad samples were collected and stored at −80⬚C. All zebrafish experiments were approved by the internal Animal Ethics Committee at CHA University. Embryos and larvae of zebrafish (Danio rerio) were initially obtained from Chungnam National University (Daejeon, South Korea). Larvae were maintained in a 100-mm plate at a density of 20 larvae per 100 mL, and fed ad libitum with hardboiled egg yolk as a high-fat diet (HFD) once per day, in the presence or absence of the indicated compounds (17–20 dpf) or vehicle (dimethyl sulfoxide, DMSO) for 12–15 days (17–20 dpf). Dieckol and DMSO were treated as a concentration of 0.1% v/v in each group plates. Two doses of dieckol (1 and 4 ␮M were examined, and CCM (curcumin, 2.5 ␮M) was used as a positive control. Zebrafish larvae used for imaging analysis were starved for 24 h prior to Nile red staining, to ensure that their digestive tracts were empty.

2.10 Nile red staining, fluorescence imaging, and quantification A stock solution (1.25 mg/mL) of Nile red (Invitrogen N-1142) was prepared in acetone and stored in the dark at –20⬚C. For staining of zebrafish, the water in which the fish were maintained was supplemented with Nile red stock solution to reach a final concentration of 50 ng/mL. Fish were incubated in this solution for 5 min at 28⬚C in the dark, before washing with distilled water three times. Anesthesia was induced with a few drops of a tricaine (Sigma) stock solution (4 mg/mL, pH 7). The fish were mounted in 3% methylcellulose, and Nile red staining was imaged under a fluorescence dissecting microscope (TE300, Nikon) equipped with a green fluorescent protein long-pass filter. Fluorescence images were acquired with a Pixera Penguin 600CL digital camera and the InStudio software (Pixera). www.mnf-journal.com

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2.11 Biochemical analysis Plasma concentrations of total cholesterol, HDL-cholesterol, LDL-cholesterol, triglyceride, albumin, glucose, and insulin were determined enzymatically using commercial kits (Asan Pharmaceutical, Gyeonggi, South Korea).

2.12 Statistical analysis All data are expressed as means ± standard error of the mean of triplicate determinants [25]. All statistical analyses were performed using the Statistical Package for Social Sciences version 12.0 (SPSS, Chicago, IL, USA). A one-way analysis of variance was used for comparisons among groups. Significant differences between the mean values were assessed using Duncan’s test. p values less than 0.05 were considered to indicate statistical significance.

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Results

3.1 Effects of dieckol on lipid accumulation, adipogenic factors, and triglyceride synthetic enzymes during adipocyte differentiation Dieckol inhibited lipid accumulation during adipocyte differentiation in a dose-dependent manner (Fig. 1B). A 25 ␮M of dieckol showed just a slight decrease of lipid accumulation and a clear reduction (47%) was observed from 50 ␮M. In particular, 100 ␮M dosage of dieckol led to a remarkable (63%) reduction in lipid accumulation, compared with the control (Fig. 1B). the effect of dieckol mRNA levels of adipogenic factors also decreased in a dose-dependent manner (S.2A). The dose-dependent downregulation of adipogenic genes by dieckol was reflected in the protein levels (S.2C). Furthermore, dieckol inhibited triglyceride accumulation during adipocyte differentiation in a dose-dependent manner (Fig. 1C). The inhibitory effect of dieckol was associated with the downregulation of triglyceride synthetic enzymes, including LPAAT␾, LIPIN1, and DGAT1 (Fig. 1D and E). This result indicates that dieckol inhibits triglyceride synthesis by suppressing the triglyceride synthetic pathway and expression of adipogenic factors during adipocyte differentiation.

lipid accumulation, and no inhibitory effect was evident when treatment was initiated after day 4 (Fig. 2B and C). This result indicates that dieckol-induced inhibition of lipid accumulation mostly occurs in the early adipogenic stages. In the early adipogenic period, the number of adipocytes increases greatly, in a process known as mitotic clonal expansion (MCE) [25]. A Trypan blue assay demonstrated that dieckol inhibited cell proliferation in a dose-dependent manner. The untreated control group showed a great increase (ca. twofold) in cell numbers after 2 days. Dieckol treatment suppressed this increase in cell numbers by 80% (Fig. 2D). This result indicates that dieckol inhibits adipocyte differentiation in MCE stage.

3.3 Effect of dieckol on cell-cycle progression We therefore examined the effect of dieckol on cell-cycle progression during adipocyte differentiation. Induction of differentiation induced normal cell-cycle progression from the G0 /G1 phase to the S and G2 /M phases, within 24 h (Fig. 3A and B). The cell population in the G0 /G1 phase decreased to 44%, whereas the cell population in the S phase increased from 9 to 21% (Fig. 3B), compared with undifferentiated postconfluent cells. However, dieckol treatment inhibited cell-cycle progression in a dose-dependent manner (Fig. 3A). Dieckol treatment reduced the S-phase cell population to 18 and 10% following 50 and 100 ␮M doses, respectively. Moreover, the proportion of G1 phase cells was increased to 53% by 50 ␮M dieckol, and 63% by 100 ␮M dieckol, suggesting that dieckol arrests the cell cycle. For protein levels of cell cycle regulators, cyclin D and p-Rb, which are G1 phase regulators, were dose-dependently decreased by dieckol treatment, consistent with dieckol-induced cell-cycle arrest (Fig. 3B). In addition, cyclin A, a major cell-cycle regulators in S phase, and its partner, cyclin-dependent kinase 2 (CDK2) were also downregulated by dieckol treatment, correlated with the reduction in the S-phase cell population (Fig. 3B–D). However, expression of p27, a negative regulator, was greatly increased by dieckol treatment (Fig. 3C and D). Our data suggest that dieckol inhibits cell proliferation by regulating cyclins and cyclin-dependent kinases during adipocyte differentiation.

3.4 Effect of dieckol on the expression of early adipogenic factors, and lipid metabolic signaling 3.2 Effect of dieckol on mitotic clonal expansion We investigated the adipogenic stage that is inhibited by dieckol. Treatment with dieckol (100 ␮M) during the early adipogenic stage (days 0–2 and 0–4) resulted in significant inhibition of lipid accumulation (Fig. 2B and C), compared with the control. Dieckol treatment during days 0–4 resulted in an inhibition of lipid accumulation by 40%, compared with the control (Fig. 2B and C). Treatment with dieckol after day 2 resulted in only a weak inhibitory effect (20%) on  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Genetic regulation of the early adipogenic stage is a key process in adipocyte differentiation [26]. We therefore examined the effect of dieckol on the mRNA expression of transcription factors associated with early adipogenesis. The mRNA expression of the early adipogenic factors C/EBP␤, C/EBP␦, KLF4, KLF5, and ETS2 was significantly inhibited by dieckol treatment. mRNA expressions of KLF 4 and 5 decreased by around 40%, compared to the control condition (Fig. 4A). A significant reduction of C/EBP␤, C/EBP␦, and ETS2 mRNA www.mnf-journal.com

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Figure 1. The effect of dieckol on lipid accumulation and expression of adipogenic factors and triglyceride synthetic enzymes during adipocyte differentiation. Differentiation was induced by incubation with a hormone cocktail in the presence or absence of dieckol for 8 days. (A) Chemical structure of dieckol. (B) After 8 days, the cells were stained with Oil red O, which was subsequently eluted in isopropanol, and its optical density was measured at 490 nm. (C) Triglyceride (TG) accumulation was determined using a TG assay kit (Zen Bio, Inc.). (D) Differentiated or undifferentiated cells were harvested at day 6 for extraction of protein, which was subjected to Western blotting, and (E) levels of triglyceride synthetic enzymes were quantified by Image J software. CON: fully differentiated group (control), ND: not differentiated. Data are presented as means ± SEM from three replicates. Means not designated by a common superscript are different (p < 0.05).

expression levels was observed in 100 ␮M rather than in 50 ␮M. KLF4 and KLF5 mRNA levels was shown to be reduced from 50 ␮M in a dose dependent way. The protein level of C/EBP␤ was also significantly reduced by dieckol (Fig. 4C and D). Conversely, the mRNA expressions of KLF2, a negative regulator of adipocyte differentiation, and preadipocyte factor-1 (pref-1) were increased by over threefold compared to control cells in 100 ␮M (Fig. 4B). However, low dosage (50 ␮M) of dieckol did not lead to any difference in mRNA levels. Our data indicates that dieckol inhibits adipocyte differentiation via regulation of early adipogenic factors. We next determined the effects of dieckol on AKT and ERK, both of which are associated with cell proliferation. Dieckol inhibited the activation of ERK and AKT in a dose-dependent manner by suppressing its phosphorylation (Fig. 4E). This result indicates that dieckol inhibits cell proliferation and lipid accumulation in adipocytes by suppressing AKT and ERK signaling.  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Furthermore, dieckol treatment resulted in activation of AMPK␣ a key energy sensor in cell metabolism, by increasing its phosphorylation level (Fig. 4E). These data suggest that dieckol inhibits lipid accumulation by activating AMPK␣ signaling. To determine whether inhibition of dieckol on lipid synthetic signalings is mediated through AMPK␣ activation, we examine the effect of AMPK␣ inhibitor (or activator) on dieckol-induced phosphorylation of AMPK␣ and its target protein, ACC. As expected, the basal phosphorylation of AMPK␣ and ACC was effectively suppressed by compound C, a specific AMPK␣ inhibitor, and dieckol significantly increased their phosphorylations (Fig. 4G and H). Compound C significantly suppressed the ability of dieckol to activate AMPK␣, and weakened the dieckol-induced phosphorylation of ACC. These results indicate that effect of dieckol on ACC, which is responsible for the lipid synthesis, was mediated through AMPK␣ activation. In addition, AICAR, an AMPK␣ www.mnf-journal.com

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Figure 2. The effect of dieckol on mitotic clonal expansion during the early stage of adipogenesis. (A) Differentiation was induced by incubation with a hormone cocktail in the presence or absence of dieckol on days 0–2, 0–4, 0–6, 2–4, 2–6, or 4–6. (B) After 6 days, the cells were stained with Oil red O, which was subsequently eluted in isopropanol and visualized at 490 nm. (C) Lipid accumulation at each stage was expressed as a percentage relative to the control (100%). (D) After differentiation for 24 or 48 h, the number of cells stained with Trypan blue was determined using a hemocytometer. INS: insulin, GM: growth medium, MDI: hormonal cocktail, FBS: differentiation medium, DMSO: dimethyl sulfoxide. CON: fully differentiated group (control), ND: not differentiated. Data are presented as means ± SEM from three replicate experiments. Means not designated by a common superscript are different (p < 0.05).

activator, effectively increased AMPK␣ and ACC phosphorylation, and such AICAR-induced increase was maximized by cotreatment with dieckol (Fig. 4I and J). These results suggest that dieckol deactivates ACC via AMPK␣ activation to inhibit lipid synthesis.

3.5 Effect of dieckol on lipid accumulation in zebrafish The effect of dieckol on lipid accumulation in zebrafish was determined by Nile red staining. Fluorescence microscopy revealed that dieckol reduced the levels of body lipids in zebrafish in a dose-dependent manner, compared with the control (Fig. 5A). Strong Nile red staining was observed in the pericardial, peri-orbital, and pectoral fin plate regions of the control group, and such fluorescence was significantly reduced in the dieckol treatment groups (Fig. 5A). Notably, fish treated with curcumin, a known anti-adipogenic compound [27], displayed a marked reduction in Nile red staining. Quantitative fluorescence analysis showed that treatment of fish with 2 and 4 ␮M dieckol resulted in a significant decrease (40 and 60%, respectively) in Nile red staining, compared with the control group (Fig. 5B). This result correlated with the results of the triglyceride assay, which indicated a decrease in  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

triglyceride content by approximately 40% in the dieckol (4 ␮M) group (Fig. 5C). As in the cell line study, dieckol effectively decreased the mRNA expression of the adipogenic factors PPAR␥, C/EBP␣, fatty acid-binding protein 11a (FABP11a), and sterol regulatory element binding factor-1 (SREBF-1) in 4 ␮M of dieckol (Fig. 5D). These data suggest that dieckol inhibits triglyceride accumulation in zebrafish by downregulating adipogenic factors.

3.6 Effect of dieckol on lipid accumulation in the mouse Mice fed a HFD supplemented with dieckol showed reductions of final body weight (35%) and body weight gain (38%) in high dose of dieckol (DKHD, 60 mg/kgBW/day), without a significant change in food intake, compared with the control group (Fig. 6A–C). Low dosage of dicekol (DKLD, 15 mg/kgBW/day) was shown to have a little reduction in body weight, but no significant difference was observed. The food efficiency ratio of dieckol (60 mg)-fed mice was reduced by 45% compared with that of HFD-fed mice (Fig. 6D). This result is consistent with the reduction (3844%) of epididymal, mesentric, and inguinal fats which are observed in dieckolfed mice of 60 mg/kgBW/day rather than 15 mg/kgBW/day www.mnf-journal.com

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Figure 3. The effect of dieckol on the cell-cycle progression of 3T3-L1 cells during differentiation. 3T3-L1 cells, in which differentiation was induced by treatment with a hormone cocktail with or without dieckol for 24 h, were harvested, fixed with 70% ethanol and stained with propidium iodide. (A) Stained DNA was analyzed using a flow cytometer. (B) The cell population in each stage of the cell cycle was determined using BD CellQuest Pro software (Becton Dickinson, San Jose, CA). (C) After 24 h of differentiation, cells were lysed for analysis of cyclins and cdk2 by Western blotting, and (D) each protein levels were quantified using ImageJ software. CON: fully differentiated group (control), ND: not differentiated. Data are expressed as means ± SEM from three replicates. Means not designated by a common superscript are different (p < 0.05).

(Fig. 6E). Dieckol significantly decreased the elevation of plasma triglycerides, total cholesterol, and LDL cholesterol that is induced by HFD (Supporting Information Table 2). In particular, the LDL cholesterol level was 55% lower than in the HFD-fed group, and similar to the normal diet group. However, the plasma glucose and insulin levels were not significantly affected by dieckol supplementation. In addition, Oil Red O staining indicated that there was a significant difference in liver status among the treatment groups. The HFD resulted in a fatty liver with a white color, while normal diet-fed mice had relatively healthy, dark-pink livers (Fig. 6F). A high dose of dieckol (60 mg) significantly blocked the elevation of liver fat induced by a HFD, resulting in similar ORO staining and liver coloration as the normal diet-fed group. This result indicates that dieckol prevents the development of a fatty liver in response to a HFD. In addition, the protein levels of major adipogenic factors, such as PPAR␥ and C/EBP␣, were effectively inhibited by dieckol supplementation (Fig. 6G and I), and TG synthetic enzymes were downregulated in dieckol-suplemented mice (DKHD) (Fig. 6H and K). Conversely, the energy sensor AMPK␣ was activated by dieckol supplementation. Activation of AMPK␣ increased phosphorylation of its target protein (ACC) (Fig. 6G and J). This dieckol-induced effects were obvious in high  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

dose of dieckol (60 mg/kgBW/day) rather than in low dosase (15 mg/kgBW/day). Generally, these results are consistent with the results of the 3T3-L1 study (Fig. 4). Our data suggest that dieckol-mediated inhibition of lipid accumulation in mice is associated with the downregulation of adipogenic factors and lipid synthetic enzyme as well as activation of AMPK␣ signaling.

4

Discussion

In this study, we demonstrated that dieckol suppresses lipid accumulation in 3T3-L1 cells, high-fat diet-fed zebrafish, and mice. Furthermore, we revealed that the anti-adipogenic effect of dieckol was mediated via inhibition of early adipogenic responses, including cell-cycle progression, and activation of AMPK␣ signaling. Previous studies have reported inhibitory effects of Ecklonia cava polyphenols on adipogenesis. Phloroeckol and dioxinodehydroeckol were shown to have anti-adipogenic effects in adipocytes [3,28]. Polyphenols from Ecklonia cava were also shown to be beneficial in clinical trials involving overweight human [29]. In particular, Dieckol is known to be the most abundant polyphenol in Ecklonia cava extract [1, 5]. Yogogawa et al. used HPLC analysis to show www.mnf-journal.com

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Figure 4. The effect of dieckol on the expression of early adipogenic factors and signaling pathways. Differentiation of 3T3L1 preadipocytes was induced by incubation with a hormone cocktail in the presence or absence of dieckol. After 1–4 h, cells were harvested for mRNA or protein analysis. (A, B) mRNA expression of early adipogenic factors was evaluated by qRT-PCR. These values were normalized to b-actin mRNA according to the comparative threshold method (⌬CT ). (C, D) C/EBP␤ protein levels were determined by Western blotting (C), and quantified using ImageJ software (D). (E) Expression levels of signaling factors were analyzed in lysates harvested after 1 or 2 h of differentiation, measured by immunoblotting, and (F) quantified by Image J software. (G–J) Inhibitor (Compound C, 10 ␮M) and activator (AICAR, 10 ␮M) of AMPKs signaling pathway were treated to measure the dependent effect of dieckol (DK, 100 ␮M) on the AMPK␣ signaling pathway. CON: fully differentiated group (control), ND: not differentiated. Data are presented as means ± SEM from three replicate experiments. Means not designated by a common superscript are different (p < 0.05).

that dieckol comprises 8.2% of the polyphenol content of a commercially available Ecklonia cava extract (SeapolynolTM , Botamedi, Inc, Jeju, Korea), making it the most common polyphenol compound in the samples tested [5]. Recent studies have demonstrated the biological activities of dieckol. Lee et al. showed that dieckol protects human endothelial cells from high-glucose-induced oxidative stress [9]. Hwang et al. reported a suppressive effect of dieckol on UVB-induced skin cancer [7]. Interestingly, a recent study showed an inhibitory effect of diekcol on adipogenesis of 3T3-L1 via AMPK␣ activation [25]. However, an anti-adipogenic mechanism of dieckol still needs to be investigated via animal study. Current study identified the anti-adipogenic effect of dieckol in various animal models with early adipogenic and signaling mechanisms, supporting previous studies.  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Our data demonstrated that dieckol inhibits early adipogenesis by regulating early adipogenic factors, such as KLF2 and C/EBP␣ (Fig. 4). Dieckol-mediated inhibition of early adipogenesis is associated with the inhibition of ERK and AKT signaling and arrest of cell cycle progression (Fig. 4E). A recent study reported phytochemical-induced inhibition of MCE via cell cycle arrest, consistent with our data [30], which is one of the main mechanisms for inhibition of adipocyte differentiation and lipid accumulation in 3T3-L1. In addition, dieckol activates AMPK␣, which is known to play a critical role in energy regulation, in both 3T3-L1 cells and a mouse model (Fig 4E and 6G). AMPK␣ deactivates ACC, a fatty acid synthetic enzyme, via phosphorylation, which can contribute to the suppression of TG accumulation [21]. Our data on inhibitor or activator of AMPK␣ showed that dieckol www.mnf-journal.com

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Figure 4. Continued.

deactivates ACC via AMPKa activation (Fig. 4G–J). Interestingly, AICAR, which has been widely used as an activator of AMPK␣, has been known to have anti-adipogenic effects [31, 32], suggesting that dieckol and AICAR may have similar mechanisms to inhibit lipid accumulation through AMPK␣ activation. Dieckol-induced activation of AMPK␣, a negative regulator of biosynthetic metabolism, appears to be a major signaling mechanism for reduction of lipid accumulation, as seen in 3T3-L1 cells and in mice. Thus, Dieckol is thought to have multiple mechanisms including suppression of MCE via cell-cycle arrest, inhibition of AKT/ERK signaling, and deactivation of a lipid synthetic pathway. Dieckol supplementation significantly improves the plasma lipid status, with respect to triglyceride and cholesterol levels, of mice fed a HFD. This result is supported by the observation that adipogenic factors are downregulated by dieckol. However, the current study did not clearly address how dieckol supplementation reduced cholesterol synthesis. One of the master genes for cholesterol synthesis is SREBP. SREBPs are major transcription factors involved in the synthesis of two major lipid building blocks: fatty acids and cholesterol [33]. SREBP-2, in particular, is a critical transcription factor that is responsible for cholesterol synthesis pathways. Although dieckol inhibited zebrafish SREBF-1 mRNA expression, a detailed cholesterol– related mechanistic action of dieckol should, therefore, be ex C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

tensively addressed in a following study. Because AMPK␣ activation is known to negatively regulate transcriptional activity of SREBP [33], dieckol is also thought to inhibit SREBP transcription activity in mice model. Furthermore, we never overlook the possibility of increased energy expenditure as a cause of the reduction of bodyweight gain in dieckol-supplemented mice. AMPKa also activates NAD-dependent protein deacetylase sirtuin 1 (SIRT1), resulting in activation of SIRT1 targets including peroxisome proliferator-activated receptor-␥ coactivator 1␣ (PGC1a) [34]. These signalings are known to promote thermogenic responses, in which energy expenditure is increased via heat production with the decrease of fat accumulation [35]. In this state, gene expressions of uncoupling proteins (UCP1 and UCP3) are known to be upregulated [35,36]. Choi et al. reported that indole-3-carbinol, a vegetable compound, binds to SIRT1 and enhances its activity in 3T3L1 cells [37]. Thus, Dieckol is expected to positively regulate these thermogenic signaling associated with energy expenditure in mice. The analysis of energy expenditure-related genes would be performed in the next study. We adopted the zebrafish as another animal model with which to demonstrate dieckol-mediated inhibition of lipid accumulation. Zebrafish studies present fewer ethical issues relative to animal studies involving mammals. This model also features similar genetic backgrounds and phenotypes www.mnf-journal.com

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Figure 5. The effect of dieckol on lipid accumulation in zebrafish. Larvae were grown to 20 dpf in the presence or absence of the indicated compounds, stained with Nile red (Invitrogen) for 5 min in the dark, and anesthetized with tricaine (Sigma). (A) Live larvae were visualized under a fluorescence microscope (upper), and the image color was inversed (bottom). (B) Photographic images were subjected to quantification using the ImageJ software. (C) Triglyceride levels in zebrafish were determined using a TG Assay Kit (Zen Bio, Inc.). (D) RNA expression of adipogenic factors in zebrafish was quantified by qRT-PCR. CON: hardboiled egg yolk treated group, CCM: curcumin (2.5 ␮M) treated group. Data are presented as means ± SEM of 10 zebrafish. Means not designated by a common superscript are different (p < 0.05).

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Figure 6. The effect of dieckol on lipid accumulation of mice. (A–G) HFD-fed mice were assayed for body weight gain (A, B), food intake (C), food efficiency ratio (D), liver (E), epididymal white adipocyte tissue (eWAT), mesenteric white adipose tissue (mWAT), and inguinal white adipose tissue (iWAT) (F), and protein expressions of adipogenic factors, AMPK␣ ACC (G), and lipogenic proteins (H) were analyzed by immunoblotting. Protein levels were quantified using Image J software (I, J, K). PPAR␥, C/EBP␣, DGAT1, LIPIN1, and LPAAT1 levels were expressed as the ratio to ␤-actin. Data are presented as means ± SEM of eight mice. Means not designated by a common superscript are different (p < 0.05).

to mammals, in relation to various diseases conditions [22]. Particularly, we used zebrafish as a model to see the effect of dieckol on early stage of lipid accumulation, because zebrafish fat was known to be detectable from 15 dpf [38]. We used zebrafish, which were grown for 17–20 dpf, for the analysis of early stage of fat accumulation by Nile-red staining. Dieckol was shown to inhibit fat accumulation of zebrafish from early stage (Fig. 5). However, further studies, including early adipogenic factors, should be executed. Finally, our data demonstrated that dieckol effectively suppressed HFD-induced lipid accumulation in zebrafish, consistent with the cell line and mouse model studies (Fig. 5). We, therefore, conclude that dieckol inhibits lipid accumulation in all three models. However, downsides of inhibition of adipogenesis in high fat diet conditions should be also considered. Adipocytes importantly function as a storage site for the excessive energy, a mechanical insulator, and an endocrine organ in the body [39]. Excessive adipogenic inhibition even in the presence of high fat diet could block normal adipogenesis which is necessary for the function of body. This condition could result in the occurrence of lipodystrophy, in which fat is accumulated in wrong places such as muscle and liver, providing another metabolic  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

disorders like insulin resistance and hepatosteatosis [39]. Besides, the functions as mechanical insulation against external forces could be diminished. Moreover, weakened endocrine functions could lead to the disturbance in the release of hormones such as estrogen and leptin [40, 41]. Therefore, these possible side effects derived from dieckol-induced adipogenic inhibition might have to be checked in the use of dieckol as the obesity medication. Ecklonia cava contains various polyphenols other than dieckol, including many eckol derivatives and phlorotannins. Phloroeckol and dioxinodehydroeckol have been shown to have anti-adipogenic activity in adipocytes, but the other Ecklonia cava–derived polyphenols are yet to be investigated. It would be informative to compare the anti-adipogenic activities of the different polyphenols, to establish which compound is the most potent. We tested the effect of eckol monomer, which is also found in Ecklonia cava, on lipid accumulation during 3T3-L1 adipogenesis, but it did not have an inhibitory effect at the same doses as dieckol (Supporting Information Fig. 3). In the current study, we showed that dieckol inhibits lipid accumulation in various models, including the zebrafish. Our www.mnf-journal.com

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Figure 6. Continued.

findings suggest that dieckol inhibits early adipogenic events by suppressing cell cycle progression, and plays important roles in regulating AMPK␣, ERK, and AKT signaling to inhibit lipid accumulation. Our data will be useful in developing a dieckol-based anti-obesity agent.

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This research was a part of the project entitled “Studies on adipogenic signaling pathways regulated by phytochemicals from edible agricultural and marine products.” funded by the Ministry of Education and Science Technology, Korea, to Boo-Yong Lee and “2013 RIA1A2064024” funded by National Research Foundation of Korea to Boo-Yong Lee. www.mnf-journal.com

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The authors have declared no conflicts of interest.

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Dieckol, a major phlorotannin in Ecklonia cava, suppresses lipid accumulation in the adipocytes of high-fat diet-fed zebrafish and mice: Inhibition of early adipogenesis via cell-cycle arrest and AMPKα activation.

Dieckol is a major polyphenol of Ecklonia cava. This study demonstrates a mechanistic role for dieckol in the suppression of lipid accumulation using ...
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