Mar Biotechnol (2010) 12:299–307 DOI 10.1007/s10126-009-9224-z ORIGINAL ARTICLE 1-(3′,5′-dihydroxyphenoxy)-7-(2″,4″,6-trihydroxyphenoxy)2,4,9-trihydroxydibenzo-1,4-dioxin Inhibits Adipocyte Differentiation of 3T3-L1 Fibroblasts Chang-Suk Kong & Jung-Ae Kim & Byul-Nim Ahn & Thanh Sang Vo & Na-Young Yoon & Se-Kwon Kim Received: May 2009 / Accepted: 14 July 2009 / Published online: 13 August 2009 # Springer Science + Business Media, LLC 2009 Abstract In this study, we isolated the phloroglucinol derivative, 1-(3′,5′-dihydroxyphenoxy)-7-(2″,4″,6-trihydroxyphenoxy)-2,4,9-trihydroxydibenzo-1,4-dioxin (1), from Ecklonia cava and evaluated its potential inhibition on adipocyte differentiation in 3T3-L1 cells Lipid accumulation along with the expression of several genes associated with adipogenesis and lipolysis was examined at the end of differentiation Lipid accumulation level was examined by measuring triglyceride content and Oil-Red O staining The expression levels of several genes and proteins were examined using reverse-transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, and Western blot analysis Compound significantly reduced lipid accumulation and downregulated peroxisome proliferator-activated receptor-γ, sterol regulatory elementbinding protein 1c, and CCAAT/enhancer-binding proteins α in a dose-dependent manner Moreover, the presence of compound induced downregulation of adipogenic target genes such as fatty acid binding protein 4, fatty acid transport protein 1, fatty acid synthase, acyl-CoA synthetase 1, lipoprotein lipase, and leptin According to the lipolytic response, compound downregulated perilipin and hormone-sensitive lipase while upregulating tumor necrosis factor alpha Therefore, these results suggest that compound might decrease lipid accumulation during adipocyte differentiation by modulating adipogenesis and lipogenesis Furthermore, compound could be developed as a functional agent effective in improving obesity Keywords Adipocyte differentiation Lipid accumulation Adipogenesis 3T3-L1 Abbreviations ACS1 Acyl-CoA synthetase C/EBPα CCAAT/enhancer-binding proteins DMEM Dulbecco’s modified Eagle medium FABP Fatty acid binding protein FAS Fatty acid synthase FATP Fatty acid transport protein FBS Fetal bovine serum HSL Hormone-sensitive lipase LPL Lipoprotein lipase PBS Phosphate-buffered saline PPARγ Peroxisome proliferator-activated receptor-γ RT-PCR Reverse-transcription polymerase chain reaction SREBP1c Sterol regulatory element-binding protein 1c TNF-α Tumor necrosis factor alpha Introduction C.-S Kong : N.-Y Yoon : S.-K Kim Marine Bioprocess Research Center, Pukyong National University, Busan 608-737, South Korea J.-A Kim : B.-N Ahn : T S Vo : S.-K Kim (*) Department of Chemistry, Pukyong National University, Busan 608-737, South Korea e-mail: sknkim@pknu.ac.kr Obesity is defined as excessive body weight in the form of fat and is characterized by increases in the number and size of fat cells as well as their lipid stores (Matsuo et al 2001) Obesity is not only one of the serious public health problems but also predisposes a person to a variety of pathological disorders such as hyperglycemia, hypertension, cardiovascular disease, etc (Xavier and Sunyer 2002; Lee et al 2005; Giri et al 2006) Adipocytes play a central role in regulating adipose 300 mass and obesity, related not only to lipid homeostasis and energy balance but also to the secretion of various transcription factors (Kim 2007) The relationship between occurrence of obesity and adipocyte differentiation or fat accumulation has been previously reported (Jeon et al 2004) It is known that 3T3-L1 cells have served as a wellestablished and useful in vitro model for the assessment and facilitation of the cellular regulatory mechanisms of adipocyte differentiation (Cho et al 2008) 3T3-L1 cells can induce differentiation of preadipocytes to adipocytes in the presence of an adipogenic cocktail The programmed differentiation of preadipocytes involves several stages related to obesity (Tang et al 2003) For these reasons, many research efforts have been conducted in 3T3-L1 cells to search for new health benefit foods/agents for obesity Natural marine products include an abundant source of chemical diversity A number of clinical trials have been carried out widely for natural marine products from marine seaweeds or marine algae Even from ancient times, marine algae have been emerged as staple diet and as an alternative medicine in many Asian countries such as in Korea, Japan, and China due to their abundance of natural bioactive substances (Ali et al 2000) They are classified into three typical groups based on pigmentation: brown, red, and green algae, which are referred to as Phaeophyceae, Rhodophyceae, and Chlorophyceae, respectively Ecklonia cava is a brown alga (Laminariaceae), is abundantly distributed in seas all over the world, and is used as a seasoned vegetable in coastal areas This seaweed grows at a water depth of 2–25 m in the sublittoral zone along the coast of Korea (Maegawa et al 1987) In recent works, a wealth of evidence has demonstrated that E cava possesses a number of biological activities, including matrix metalloproteinase inhibitory activity, protease inhibitory activity, antioxidative activity, anti-inflammatory activity, anti-HIV1 activity, and antiallergic effects (Kim et al 2006, 2008; Artan et al 2008; Le et al 2009) However, there are no reports on the effect of components of E cava on adipocyte differentiation related to obesity In the present study, we isolated the phloroglucinol derivative, 1-(3′,5′-dihydroxyphenoxy)-7-(2″,4″,6-trihydroxyphenoxy)-2,4,9-trihydroxydibenzo-1,4-dioxin (1), from E cava and investigated its potential inhibitory effect on adipocyte differentiation in 3T3-L1 cells Its effect on lipid accumulation in cultured 3T3-L1 adipocytes was examined by directly measuring triglyceride levels and Oil-Red O staining To understand the mechanism by which lipid accumulation in adipocytes is decreased by the phloroglucinol derivative, the expression levels of several genes and proteins associated with adipogenesis and lipolysis were examined using reverse-transcription polymerase chain reaction (RT-PCR), quantitative real-time RT-PCR, and Western blot analysis Mar Biotechnol (2010) 12:299–307 Materials and Methods Plant Material Leafy thalli of Ecklonia cava were collected along Jeju Island coast of South Korea during the period from October 2004 to March 2005 A voucher specimen has been deposited in the author’s laboratory The collected sample was freeze-dried and kept at −25ºC until use Extraction and Isolation The lyophilized powder (4.0 kg) of E cava was percolated in hot EtOH (3×10 l) The crude extract (584.3 g) was partitioned with organic solvents to yield n-hexane (114.3 g), CH2Cl2 (40.6 g), EtOAc (55.0 g), and n-BuOH (96.5 g) fractions, as well as an H2O residue (277.9 g) The EtOAc fraction (55.0 g) of E cava was subjected to column chromatography over a silica gel with CH2Cl2:MeOH (30:1 to 1:1), yielding 16 subfractions (EF01 to EF16) 1-(3′,5′dihydroxyphenoxy)-7-(2″,4″,6-trihydroxyphenoxy)-2,4,9trihydroxydibenzo-1,4-dioxin (43.4 mg) was isolated from fraction 11 (EF11, 135 mg) with RP-18 (20% MeOH to 100% MeOH, gradient) and Sephadex LH-20 (100% MeOH) Its structural identity was verified by comparison with published spectral data (Fig 1; Okada et al 2004) 1-(3′,5′-dihydroxyphenoxy)-7-(2″,4″,6-trihydroxyphenoxy)2,4,9-trihydroxydibenzo-1,4-dioxin (1) H-nuclear magnetic resonance (NMR; 400 MHz, DMSO-d6) δ: 5.72 (2H, d, J=2.0 Hz, H-2′, 6′), 5.79 (1H, d, J=3.1 Hz, H-6), 5.80 (1H, t, J=2.0 Hz, H-4′), 5.86 (2H, s, H-3″, 5″), 6.01 (1H, d, J=3.1 Hz, H-8), 6.14 (1H, s, H-3), 9.0 (1H, s, H-4″), 9.12 (4H, d, J=6.3 Hz, 3′, 5′-OH, 2″, 6″-OH), 9.20 (1H, s, 2-OH), 9.40 (1H, s, 4-OH), 9.61 (1H, s, 9-OH); 13 C-NMR (100 MHz, DMSO-d6) δ: 160.3 (C-1′), 158.8 Fig Chemical structure of phloroglucinol derivative isolated from Ecklonia cava (1) 1-(3′,5′-dihydroxyphenoxy)-7-(2″,4″,6-trihydroxyphenoxy)-2,4,9-trihydroxydibenzo-1,4-dioxin Mar Biotechnol (2010) 12:299–307 (C-3′, 5′), 154.8 (C-4″), 154.5 (C-7), 151.2 (C-2″, 6″), 146.0 (C-9), 145.9 (C-2), 142.3 (C-5a), 141.8 (C-4), 137.1 (C-10a), 123.9 (C-9a), 123.1 (C-4a), 122.5 (C-1″), 122.2 (C-1), 98.9 (C-3), 98.3 (C-8), 96.2 (C-4′), 94.8 (C-3″, 5″), 93.6 (C-2′, 6′), 93.4 (C-6) Cell Culture and Adipocyte Differentiation Mouse 3T3-L1 preadipocytes were grown to confluence in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere of 5% CO2 At day postconfluence (designated “day 0”), cell differentiation was induced with a mixture of methylisobutylxanthine (0.5 mM), dexamethasone (0.25 μM), and insulin (5 μg/ml) in DMEM containing 10% FBS After 48 h (day 2), the induction medium was removed and replaced by DMEM containing 10% FBS supplemented with insulin (5 μg/ml) alone This medium was changed every days The compound was treated into culture medium of adipocytes at day After treatment with the compound for days, the adipose tissue was lysed for analysis Cytotoxicity of the compound was evaluated by MTT assay Any significant toxic effect was not observed on the cells treated with the compound up to a concentration of 100 μM (data were not shown) Therefore, the experiments were carried out up to a concentration of 50 μM Measurement of Triglyceride Content Cellular triglyceride contents were measured using a commercial triglyceride assay kit (Triglyzyme-V, Eiken Chemical, Tokyo, Japan) according to the manufacturer’s instructions Cells were treated with the compound with the concentration of 1, 5, 10, 20, and 50 μM in 12-well plates during the adipocyte differentiation for days (from day to day 7) The cells were washed twice with phosphate-buffered saline (PBS), scraped in 75 μl of homogenizing solution (154 mM KCl, mM EDTA, 50 mM Tris, pH 7.4), and sonicated to homogenize the cell suspension The residual cell lysate was centrifuged at 3,000×g for at 4°C to remove fat layer The supernatants were assayed for triglyceride content and protein content Triglyceride was normalized to protein concentration determined by the bovine serum albumin as a standard Results were expressed as milligrams of triglyceride per milligram of cellular protein Oil-Red O Staining For Oil-Red O staining (Havel 2000), cells were washed gently with PBS twice, fixed with 3.7% fresh formaldehyde 301 (Sigma, St Louis, MO, USA) in PBS for h at room temperature, and stained with filtered Oil-Red O solution (60% isopropanol and 40% water) for at least h After staining of lipid droplets with red, the Oil-Red O staining solution was removed, and the plates were rinsed with water and dried Images of lipid droplets of the 3T3-L1 adipocytes were collected using an Olympus microscope (Tokyo, Japan) Finally, the dye retained in the cells was eluted with isopropanol and quantified by measuring the optical absorbance at 500 nm using a microplate reader (Tecan Austria GmbH, Austria) RNA Extraction and Reverse-Transcription Polymerase Chain Reaction Total RNA was isolated from 3T3-L1 adipocytes using TRIzol reagent (Invitrogen Co., CA, USA) For synthesis of first-strand cDNA, μg of RNA was added to RNase-free water and oligo (dT), denaturated at 70°C for min, and cooled immediately RNA was reversetranscribed in a master mix containing 1× RT buffer, mM dNTPs, 500 ng oligo (dT), 140 U M-MLV reserve transcriptase, and 40 U RNase inhibitor at 42°C for 60 and at 72°C for using an automatic Whatman thermocycler (Biometra, UK) The target cDNA was amplified using the gene-specific primers (Table 1) The amplification cycles were carried out at 95°C for 45 s, 60°C for min, and 72°C for 45 s After 30 cycles, the PCR products were separated by electrophoresis on 1.5% agarose gel for 30 at 100 V Gels were then stained with mg/ml ethidium bromide visualized by UV light using AlphaEase® gel image analysis software (Alpha Innotech, CA, USA) Quantitative Real-Time RT-PCR Analysis One microliter of each RT reaction was amplified in a 25-μl PCR assay volume using MasterMix containing HotStarTaq Plus DNA Polymerase, QuantiFast SYBR PCR Buffer, dNTP Mix, SYBR Green I dye, and ROX dye (Qiagen, Germany) Quantitative SYBR Green realtime PCR was performed on Rotor gene 6000 (Corbett Life Science) using the following program: samples were incubated for an initial denaturation at 95°C for 10 min, followed by 40 PCR cycles Each cycle proceeded at 95°C for 15 s, 60°C for 30 s, and 72°C for 15 s Relative quantification was calculated using the 2ÀðΔΔCTÞÀmethod (Livak and Á Schmittgen 2001), where ΔΔCT ¼ C À C T;target T;actin treated sample À À CT;target ÀCT;actin Þcontrol sample To confirm amplification of specific transcripts, melting curve profiles (cooling the sample to 40°C and heating slowly to 95°C with continuous measurement of fluorescence) were produced at the end of each PCR 302 Table Gene-specific primers used for the RT-PCR and realtime RT-PCR analysis Mar Biotechnol (2010) 12:299–307 Gene Direction Sequence PPARγ Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward 5′-TTT TCA AGG GTG CCA GTT TC-3′ 5′-AAT CCT TGG CCC TCT GAG AT-3′ 5′-TGT TGG CAT CCT GCT ATC TG-3′ 5′-AGG GAA AGC TTT GGG GTC TA-3′ 5′-TTA CAA CAG GCC AGG TTT CC-3′ 5′-GGC TGG CGA CAT ACA GTA CA-3′ 5′-TCA CCT GGA AGA CAG CTC CT-3′ 5′-AAT CCC CAT TTA CGC TGA TG-3′ 5′-TGC CTC TGC CTT GAT CTT TT-3′ 5′-GGA ACC GTG GAT GAA CCT AA-3′ 5′-TTG CTG GCA CTA CAG AAT GC-3′ 5′-AAC AGC CTC AGA GCG ACA AT-3′ 5′-TCC AAG GAA GCC TTT GAG AA-3′ 5′-CCA TCC TCA GTC CCA GAA AA-3′ 5′-CAA CCC AGA ACC ATG GAA GT-3′ Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse 5′-CTG ACT GCA TGG AGA GGT CA-3′ 5′-GGA TCA GGT TTT GTG GTG CT-3′ 5′-TTG TGG CCC ATA AAG TCC TC-3′ 5′-AAG GAT CCT GCA CCT CAC AC-3′ 5′-CCT CTG CTG AAG GGT TAT CG-3′ 5′-GAG GGA CAC ACA CACACC TG-3′ 5′-CCC TTT CGC AGC AAC TTT AG-3′ 5′-AGG CCT TGT GTT GTG TTT CCA-3′ 5′-TGG GGG ACA GCT TCC TTC TT-3′ Forward Reverse 5′-CCA CAG CTG AGA GGG AAA TC-3′ 5′-AAG GAA GGC TGG AAA AGA GC-3′ SREBP1c C/EBPα FABP4 FATP1 FAS LPL ACS1 Leptin Perilipin HSL TNFα β-actin Western Blot Analysis Statistical Analysis Western blotting was performed according to standard procedures Briefly, cells were lysed in radioimmunoprecipitation assay buffer containing 50 mM Tris–HCl (pH 8.0), 0.4% Nonidet P-40, 120 mM NaCl, 1.5 mM MgCl2, mM phenylmethylsulfonyl fluoride, 80 μg/ml leupeptin, mM NaF, and mM dithiothreitol at 4°C for 30 Cell lysates (50 μg) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto a polyvinylidene fluoride membrane (Amersham Pharmacia Biotech., England, UK), blocked with 5% skim milk, and hybridized with primary antibodies (diluted 1:1,000, Santa Cruz Biotechnology, CA, USA) After incubation with horseradish-peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, CA, USA) at room temperature, immunoreactive proteins were detected using a chemiluminescent ECL assay kit (Amersham Pharmacia Biosciences, England, UK) according to the manufacturer's instructions Western blot bands were visualized using a LAS3000® Luminescent image analyzer (Fujifilm Life Science, Tokyo, Japan) Data were expressed as mean ± SE (n=3) Differences between the means of the individual groups were assessed by one-way ANOVA with Duncan's multiple-range tests Differences were considered significant at p