e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 6 ( 2 0 1 3 ) 1253–1260

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Dieckol, a phlorotannin isolated from a brown seaweed, Ecklonia cava, inhibits adipogenesis through AMP-activated protein kinase (AMPK) activation in 3T3-L1 preadipocytes Seok-Chun Ko a , Myoungsook Lee b , Ji-Hyeok Lee a , Seung-Hong Lee c , Yunsook Lim d , You-Jin Jeon a,e,∗ a

Department of Marine Life Sciences, Jeju National University, Jeju 690-756, Republic of Korea Department of Food and Nutrition, Sungshin Women’s University, Seoul 136-742, Republic of Korea c Division of Food Bioscience, Konkuk University, Chungju, Chungbuk 380-701, Republic of Korea d Department of Food and Nutrition, Kyung Hee University, Seoul 130-701, Republic of Korea e Aqua Green Technology Co. Ltd., Jeju Bio-industry Center, Jeju 690-121, Republic of Korea b

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Article history:

In this study, we assessed the potential inhibitory effect of 5 species of brown seaweeds

Received 13 May 2013

on adipogenesis the differentiation of 3T3-L1 preadipocytes into mature adipocytes by

Received in revised form

measuring Oil-Red O staining. The Ecklonia cava extract tested herein evidenced profound

9 October 2013

adipogenesis inhibitory effect, compared to that exhibited by the other four brown sea-

Accepted 13 October 2013

weed extracts. Thus, E. cava was selected for isolation of active compounds and finally

Available online 23 October 2013

the three polyphenol compounds of phlorotannins were obtained and their inhibitory effect on adipogenesis was observed. Among the phlorotannins, dieckol exhibited great-

Keywords:

est potential adipogenesis inhibition and down-regulated the expression of peroxisome

Diekol

proliferator-activated receptor-␥ (PPAR␥), CCAAT/enhancer-binding proteins (C/EBP␣), sterol

Ecklonia cava

regulatory element-binding protein 1 (SREBP1) and fatty acid binding protein 4 (FABP4) in

Adipogenesis

a dose-dependent manner. The specific mechanism mediating the effects of dieckol was

3T3-L1 preadipocytes

confirmed by AMP-activated protein kinase (AMPK) activation. These results demonstrate

AMPK

inhibitory effect of dieckol compound on adipogenesis through the activation of the AMPK signal pathway. © 2013 Elsevier B.V. All rights reserved.

1.

Introduction

Currently, obesity has become one of the most common metabolic syndromes, and has come to pose a major worldwide threat to human health (Kim et al., 2011). Obesity is a serious chronic health problem, because it is a high risk factor for type 2 diabetes, hypertension

and cardiovascular disease, including atherosclerosis, hyperlipidemia and myocardial infarction (Giri et al., 2006; Kim and Kong, 2010). Cellular and molecular studies show that the development of obesity is characterized by increases in the number and size of mature adipocytes in the body produced by differentiation and mitogenesis (Herberg et al., 1974; Kim and Kong, 2010; Kim et al., 2010).

∗ Corresponding author at: Department of Marine Life Sciences, Jeju National University, Jeju 690-756, Republic of Korea. Tel.: +82 64 754 3475; fax: +82 64 756 3493. E-mail address: [email protected] (Y.-J. Jeon). 1382-6689/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.etap.2013.10.011

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e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 6 ( 2 0 1 3 ) 1253–1260

The 3T3-L1 preadipocyte cell line is model for investigating the differentiation of adipocytes and several stages related to obesity (Guo and Liao, 2000; Kim and Kong, 2010). The differentiation of preadipocytes into adipocytes is accompanied by sequential expression and activation of transcription factors governing expression of adipocyte-specific markers, such as CCAAT/enhancer binding proteins ␣ (C/EBP␣), peroxisome proliferator-activated receptor-␥ (PPAR␥) and basic helix–loop–helix family (ADD1/SREBP-1) (Ha et al., 2010; Kim et al., 2010). These factors play an important role in the regulation of adipogenesis by modulating the expression of their target genes in a coordinated fashion (Kang et al., 2011). Moreover, AMP-activated protein kinase (AMPK) is one of the well characterized and an important target for the prevention and treatment of obesity (Kim and Kong, 2010; Lee et al., 2009). AMPK complex is a heterorimer, which functions as a cellular energy sensor and has been shown to positively correlated with glucose and lipid homeostasis in adipocytes (Unger, 2004; Winder and Thomson, 2007). Seaweeds are known to provide an abundance of bioactive compounds with valuable biomedical and pharmaceutical potential (Lee et al., 2010). Among the seaweeds, brown seaweeds possess a variety of biological compounds, including fucoxanthin, fucoidan, phycocolloids and polyphenolcontaining phlorotannins (Halliwell and Gutteridge, 1999). Many reports on phlorotannins of brown seaweeds have pointed out a variety of biological effects, including antidiabetes (Lee et al., 2010), anti-oxidant (Ahn et al., 2007), anti-hypertensive (Wijesinghe et al., 2011), anti-plasmin inhibitor (Nakayama et al., 1989), anti-allergic (Sugiura et al., 2006) and hepatoprotective (Kang et al., 2012) effects. However, there currently is insufficient evidence for the effects of phlorotannins from brown seaweeds on adipogenesis related to obesity. Also, marine natural products provide a rich source of chemical diversity that can be used to design and develop new, potentially useful therapeutic agents. The objectives of the current study were to isolate phlorotannins from Ecklonia cava based on the results of analytical data, and to evaluate its inhibitory effects of adiogenesis and the expression levels of adipocyte marker proteins and genes in 3T3-L1 preadipocytes. In order to screen a proper sample having higher effect, we measured the differentiation inhibitory effect of extracts from the five species of brown seaweeds; E. cava was selected for further experiments, owing to its higher differentiation inhibitory effect in 3T3-L1 preadipocytes.

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), bovine serum (BS), Phosphate-buffered saline (pH 7.4; PBS) and penicillin–streptomycin (PS) were purchased from Gibco BRL (Grand Island, NY, USA). Antibodies to PPAR␥, C/EBP␣, FABP4, phospho-AMPK (Thr172) and phosphoACC were purchased were from Cell Signaling Technology (Bedford, MA, USA). Antibody to SREBP-1 and GAPDH were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 3-Isobutyl-1-methylxanthine (IBMX), dexamethasone, insulin, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents were of the highest grade available commercially.

2.2. Extraction procedure of extracts from brown seaweeds The brown seaweeds powder (2 g) was extracted three times with 80% methanol (100 ml) and filtered. After filtration, methanolic extracts were evaporated to dryness under vacuum and dissolved in DMSO, and then used for experiments, adjusting the final concentration of DMSO in the culture medium to

Dieckol, a phlorotannin isolated from a brown seaweed, Ecklonia cava, inhibits adipogenesis through AMP-activated protein kinase (AMPK) activation in 3T3-L1 preadipocytes.

In this study, we assessed the potential inhibitory effect of 5 species of brown seaweeds on adipogenesis the differentiation of 3T3-L1 preadipocytes ...
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