1 3 5 dihydroxyphenoxy 7 2 4 6 trihy

3T3-L1 Abbreviations ACS1 Acyl-CoA synthetase 1 C/EBPα CCAAT/enhancer-binding proteins DMEM Dulbecco’s modified Eagle medium FABP Fatty acid binding protein FAS Fatty acid synthase FATP

ORIGINAL ARTICLE

′,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: 6 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-trihy-droxyphenoxy)-2,4,9-trihydroxydibenzo-1,4-dioxin (1),

from Ecklonia cava and evaluated its potential inhibition

on adipocyte differentiation in 3T3-L1 cells Lipid

accu-mulation 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 1 significantly

reduced lipid accumulation and downregulated peroxisome

proliferator-activated receptor-γ, sterol regulatory

element-binding protein 1c, and CCAAT/enhancer-element-binding proteins

α in a dose-dependent manner Moreover, the presence

of compound 1 induced downregulation of adipogenic

target genes such as fatty acid binding protein 4, fatty acid

transport protein 1, fatty acid synthase, acyl-CoA

synthe-tase 1, lipoprotein lipase, and leptin According to the

lipolytic response, compound 1 downregulated perilipin

and hormone-sensitive lipase while upregulating tumor

necrosis factor alpha Therefore, these results suggest that

compound 1 might decrease lipid accumulation during

adipocyte differentiation by modulating adipogenesis and lipogenesis Furthermore, compound 1 could be developed

as a functional agent effective in improving obesity Keywords Adipocyte differentiation Lipid accumulation Adipogenesis 3T3-L1

Abbreviations ACS1 Acyl-CoA synthetase 1 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 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

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

DOI 10.1007/s10126-009-9224-z

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

well-established and useful in vitro model for the assessment and

facilitation of the cellular regulatory mechanisms of

adipo-cyte 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

metal-loproteinase inhibitory activity, protease inhibitory activity,

antioxidative activity, anti-inflammatory activity,

anti-HIV-1 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-trihy-droxyphenoxy)-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

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)-2,4,9-trihydroxydibenzo-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)

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 1 Chemical structure of phloroglucinol derivative isolated from Ecklonia cava (1) 1-(3 ′,5′-dihydroxyphenoxy)-7-(2″,4″,6-trihydroxy-phenoxy)-2,4,9-trihydroxydibenzo-1,4-dioxin

(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

atmo-sphere of 5% CO2 At 1 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 2 days The compound 1 was treated

into culture medium of adipocytes at day 0 After

treatment with the compound 1 for 7 days, the adipose

tissue was lysed for analysis Cytotoxicity of the

com-pound 1 was evaluated by MTT assay Any significant

toxic effect was not observed on the cells treated with the

compound 1 up to a concentration of 100μM (data were

not shown) Therefore, the experiments were carried out

up to a concentration of 50μM

Measurement of Triglyceride Content

Cellular triglyceride contents were measured using a

commercial triglyceride assay kit (Triglyzyme-V, Eiken

Chemical, Tokyo, Japan) according to the

manufac-turer’s instructions Cells were treated with the

com-pound 1 with the concentration of 1, 5, 10, 20, and 50μM

in 12-well plates during the adipocyte differentiation for

7 days (from day 0 to day 7) The cells were washed

twice with phosphate-buffered saline (PBS), scraped in

75 μl of homogenizing solution (154 mM KCl, 1 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 5 min at 4°C to remove fat

layer The supernatants were assayed for triglyceride

content and protein content Triglyceride was

normal-ized 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

(Sigma, St Louis, MO, USA) in PBS for 1 h at room temperature, and stained with filtered Oil-Red O solution (60% isopropanol and 40% water) for at least 1 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 synthe-sis of first-strand cDNA, 2 μg of RNA was added to RNase-free water and oligo (dT), denaturated at 70°C for 5 min, and cooled immediately RNA was reverse-transcribed in a master mix containing 1× RT buffer,

1 mM dNTPs, 500 ng oligo (dT), 140 U M-MLV reserve transcriptase, and 40 U RNase inhibitor at 42°C for 60 min and at 72°C for 5 min 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 1 min, and 72°C for 45 s After 30 cycles, the PCR products were separated by electrophoresis

on 1.5% agarose gel for 30 min at 100 V Gels were then stained with 1 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 real-time PCR was performed on Rotor gene 6000 (Corbett Life Science) using the following program: samples were

incubat-ed for an initial denaturation at 95°C for 10 min, followincubat-ed 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 2
ΔΔCTð Þmethod (Livak and Schmittgen

2001), where ΔΔCT ¼ CT ;target
CT ;actin

treated sample

CT;target
CT;actinÞcontrol 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

Western Blot Analysis

Western blotting was performed according to standard

procedures Briefly, cells were lysed in

radioimmunopreci-pitation assay buffer containing 50 mM Tris–HCl (pH 8.0),

0.4% Nonidet P-40, 120 mM NaCl, 1.5 mM MgCl2, 2 mM

phenylmethylsulfonyl fluoride, 80μg/ml leupeptin, 3 mM

NaF, and 1 mM dithiothreitol at 4°C for 30 min 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

tempera-ture, 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)

Statistical Analysis 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<0.05 The statistical software package SAS v9.1 (SAS Institute Inc., Cary, NC, USA) was used for the analysis

Results Effect of Compound 1 on Intracellular Lipid Accumulation

in Adipocytes

To explore whether compound 1 affects differentiation of preadipocytes into adipocytes, its effect on the induction of terminal differentiation markers at the end of the differenti-ation period (day 7) was investigated (Fig 2) Lipid accu-mulation was quantified by directly measuring triglyceride levels and Oil-Red O staining Treatment with compound 1 reduced the triglyceride content of differentiated adipocyte

Table 1 Gene-specific primers

used for the RT-PCR and

real-time RT-PCR analysis

lysate in a dose-dependent manner (p<0.05) Triglycerides of fully differentiated adipocytes were stained with Oil-Red O staining solution In the absence of compound 1, fully differentiated cells had many lipid droplets, indicating lipid accumulation This observation was further supported with the quantitative analysis of neutral lipid content by measuring the absorbance at 500 nm The absorbance value of Oil-Red O eluted solution represents lipid droplet accumulation in the cytoplasm Lipid accumulation in cells was concentration-dependently inhibited in the presence of compound 1 (p< 0.05) The absorbance value of eluted dye was decreased according to the increased concentrations during adipocyte differentiation This was assessed by morphological changes based on Oil-Red O staining of cellular triglyceride contents Inhibition of Adipogenesis

To determine whether compound 1 affects the expression of transcription factors, RT-PCR and Western blotting analysis were conducted (Fig 3) Treatment with compound 1 reduced the size and intensity of the lytic zone for the regulation of peroxisome proliferator-activated receptor-γ (PPARγ), differentiation-dependent factor 1/sterol

regulato-ry element-binding protein 1c (SREBP1c), and CCAAT/ enhancer-binding proteins (C/EBPα) genes, compared to fully differentiated control adipocytes This inhibition pattern was in a dose-dependent manner Treatment with compound 1 also suppressed the protein expression of PPARγ, SREBP1c, and C/EBPα

We further studied whether compound 1 regulates the expression of adipogenic target genes such as adipocyte fatty acid binding protein 4 (FABP4), fatty acid transport protein 1 (FATP1), fatty acid synthase (FAS), lipoprotein lipase (LPL), acyl-CoA synthetase 1 (ACS1), and leptin Treatment with compound 1 during adipocyte differentiation induced significant downregulation of the FATP, FAS, LPL, ACS1, and leptin genes in a dose-dependent manner (Fig.4) Effect of Compound 1 on Lipolysis

To assess the lipolytic response during adipocyte differenti-ation, the gene expression levels of perilipin, hormone-sensitive lipase (HSL), and tumor necrosis factorα (TNF-α) were determined by using RT-PCR (Fig.5) Treatment with compound 1 downregulated perilipin and HSL genes expressions, while TNF-α expression was upregulated, compared to fully differentiated control adipocytes

Effect of Compound 1 on Genes Expression by Real-Time RT-PCR Analysis

The effect of compound 1 on gene expression during adipocyte differentiation was also confirmed by using

real-Fig 2 Effect of compound 1 on lipid accumulation in 3T3-L1

adipocytes Confluent 3T3-L1 preadipocytes were differentiated into

adipocytes in medium contained with or without different

concentra-tion of compound 1 for 7 days (from day 0 to day 7) The lipid

accumulation was measured by triglyceride assay (a) and Oil-Red O

staining (b and c) Representative image are presented a –d Means

with the different letters are significantly different (p<0.05) by

Duncan's multiple-range test Con: fully differentiated control

adipo-cytes (0.5 mM methylisobutylxanthine, 0.25 μM dexamethasone, and

5 μg/ml insulin)

time RT-PCR (Fig.6) Treatment with compound 1 induced

dose-dependent downregulation of PPARγ compared to

fully differentiated adipocytes without sample treatment

Moreover, the expression levels of the SREBP1c, C/EBPα,

FABP4, FATP1, FAS, LPL, ACS1, and leptin genes were

downregulated in the presence of compound 1 The

expression levels of the perilipin and HSL genes were

downregulated, while TNF-α gene was upregulated by

treatment with compound 1 These results correspond with

the results of the RT-PCR analysis

Discussion

Obesity is a serious socioeconomic health problem with

increasing prevalence, as it is associated with diseases such

as type 2 diabetes, hypertension, cancer, osteoarthritis, and

heart disease (Lee et al 2005) Obesity is defined as the heavy accumulation of fat in the body’s fat cells up to a serious degree Many studies have been conducted to search for new health benefit materials that can decrease obesity Possible mechanisms in reference to antiobesity actions include reducing incorporation of glucose and free fatty acids into triglyceride, increasing oxidation of glucose and/or fatty acids, or increasing lipolysis (Evans et al

2002) It is known that adipocyte differentiation as well

as the amount of lipid accumulation determines the occurrence and development of obesity (Jeon et al 2004) Adipocytes play a vital role in energy balance, specifically

in triglyceride storage and release of free fatty acids

In this study, effect of the phloroglucinol derivative, 1- (3′,5′-dihydroxyphenoxy)-7-(2″,4″,6-trihydroxyphenoxy)-2,4,9-trihydroxydibenzo-1,4-dioxin (1), from E cava on differentiation of preadipocytes into adipocytes was

inves-Fig 3 Effect of compound 1

on expression of PPAR γ,

SREBP1c, and C/EBP α

gene (a) and protein (b) in

3T3-L1 cells Confluent 3T3-L1

preadipocytes were differentiated

into adipocytes in medium

contained with or without

different concentration of

compound 1 for 7 days (from

day 0 to day 7) a–e Means

with the different letters are

significantly different (p<0.05)

by Duncan's multiple-range test.

Con: fully differentiated control

adipocytes (0.5 mM

methyliso-butylxanthine, 0.25 μM

dexa-methasone, and 5 μg/ml insulin)

Fig 4 Effect of compound 1 on expression of FABP4, FATP1, FAS,

LPL, ACS1, and leptin genes in 3T3-L1 cells Confluent 3T3-L1

preadipocytes were differentiated into adipocytes in medium contained

with or without different concentration of compound 1 for 7 days

(from day 0 to day 7) a –d Means with the different letters at each gene are significantly different (p<0.05) by Duncan's multiple-range test Con: fully differentiated control adipocytes (0.5 mM methyl-isobutylxanthine, 0.25 μM dexamethasone, and 5 μg/ml insulin)

tigated in 3T3-L1 cells Lipid accumulation and the

expression of several genes associated with adipogenesis

and lipolysis during differentiation were also examined at

the end of differentiation in cultured 3T3-L1 adipocytes

Triglyceride levels during adipocyte differentiation were

significantly reduced in the presence of compound 1 in a

concentration-dependent manner (p<0.05; Fig.2) Oil-Red

O staining showed that culture of 3T3-L1 under

differen-tiation conditions remarkably induced many lipid droplets,

indicating lipid accumulation The presence of compound 1

reduced the absorbance value of Oil-Red O eluted solution

in the cytoplasm of treated cells in a

concentration-dependent manner This result means that compound 1

inhibits adipogenesis under differentiation conditions by

reducing lipid accumulation Our study demonstrates the

effective inhibition of lipid formation by compound 1

Therefore, it would be interesting to evaluate the mechanism

action of compound 1 in 3T3-L1 adipocytes associated with adipogenesis or lipolysis Adipocyte differentiation leads to

a series of programmed changes in specific gene Adipo-genesis can be induced through the action of several enzymes such as FAS, ACC, acyl-CoA synthetase, and glycerol-3-phosphate acyltransferase under differentiation conditions Expression of these genes is regulated by transcription factors such as PPARγ, SREBP1c, and C/ EBPα These factors are known to be critical activators for adipogenesis and showed early changes in gene expression during adipocyte differentiation (Latasa et al.2000; Luong

et al.2000; Ericsson et al.1997) PPARγ and C/EBPα play central roles in adipocyte differentiation and coordinate expression of genes involved in creating or maintaining the phenotype of adipocytes (Rosen 2005) They are induced

as central transcriptional regulators prior to the transcrip-tional activation of many adipocyte-specific genes

Over-Fig 6 Effect of compound 1 on

the results by real-time RT-PCR

analysis Real-time RT-PCR

analysis was carried out for

PPAR γ, SREBP1c, and C/

EBP α (a), FABP4, FATP1, FAS,

LPL, ACS1, and leptin (b), and

perilipin, HSL, and TNF- α (c).

Confluent 3T3-L1 preadipocytes

were differentiated into

adipo-cytes in medium contained with

or without different concentration

of compound 1 for 7 days (from

day 0 to day 7) a –d Means

with the different letters at each

gene are significantly different

(p<0.05) by Duncan's

multiple-range test Con: fully

differenti-ated control adipocytes (0.5 mM

methylisobutylxanthine, 0.25 μM

dexamethasone, and 5 μg/ml

insulin)

Fig 5 Effect of compound 1 on expression of perilipin, HSL, and

TNF- α genes in 3T3-L1 cells Confluent 3T3-L1 preadipocytes were

differentiated into adipocytes in medium contained with or without

different concentration of compound 1 for 7 days (from day 0 to day

7) a –e Means with the different letters at each gene are significantly different (p < 0.05) by Duncan's multiple-range test Con: fully differentiated control adipocytes (0.5 mM methylisobutylxanthine, 0.25 μM dexamethasone, and 5 μg/ml insulin)

expression of these transcription factors can accelerate

differentiation of preadipocytes into adipocytes SREBP1c

can critically cross-activate a ligand-binding domain of

PPARγ and promote the production of an endogenous

PPARγ ligand (Bruce and Jeffery 2001) The effect of

compound 1 on the expression levels of PPARγ, SREBP1c,

and C/EBPα, as a major marker of adipogenesis, was

inves-tigated (Fig.3) Expression levels of PPARγ, SREBP1c, and

C/EBPα were remarkably induced in cultures of 3T3-L1

cells under differentiation conditions However, these

tran-scription factors were significantly downregulated by

com-pound 1 in a dose-dependent manner, compared to fully

differentiated adipocytes without compound 1 The presence

of compound 1 also downregulated the protein expression

levels of PPARγ, SREBP1c, and C/EBPα in a

dose-dependent manner

Downregulation of SREBP1c and C/EBPα by

com-pound 1 might reduce fatty acid synthesis as well as the

synthesis and activity of PPARγ, resulting in inhibition of

lipid accumulation by blocking adipogenesis PPARγ and

C/EBPα synergistically activate a number of

adipocyte-specific gene promoters (Gregoire et al.1998) Therefore,

we investigated the effect of compound 1 on regulation of

adipogenic target genes such as FABP4, FATP1, FAS, LPL,

ACS1, and leptin As a result, treatment with compound 1

under differentiation conditions induced downregulation of

the FABP4, FATP1, FAS, LPL, ACS1, and leptin genes

The FABP4 and FATP1 play important roles in the obesity

pathway linked to fatty acid metabolism (Salas et al.2007)

Cellular uptake of long-chain fatty acid can be facilitated by

fatty acid transporters such as FABP4 and FATP1

Therefore, decreased expression levels of FABP4 and

FATP1are in accordance with decreased fatty acid

utiliza-tion in cells and decreased transport of fatty acids into the

cells, respectively (Salas et al.2007) Traditionally, FAS has

been considered as a terminal marker of adipocyte

differentiation Activated PPARγ and SREBP1c are able

to induce expression of FAS and clearly cross-activate the

FAS promoter (Palmer et al 2002) LPL catalyzes the

hydrolysis reactions of triglycerides, in which plasma

triglycerides are metabolized to free fatty acids for

triglyceride synthesis in adipose cells (Yamaguchi et al

2002) The levels of LPL in adipose tissue are dependent

upon the triglyceride level in fat cells; therefore, elevated

LPL activity in adipocytes is closely linked with obesity

(Bullo et al.2002) Leptin is secreted exclusively in adipose

tissue in proportion to triglyceride stores and adipose cell

size The concentration of leptin in adipocytes is positively

correlated with adipose tissue mass (Maffei et al 1995)

Therefore, leptin secretions are known to be as indicative

markers of obesity Our results suggest that compound 1

might inhibit adipocyte differentiation and adipogenesis

through PPARγ-, SREBP1c-, and C/EBPα-mediated

adipo-genesis mechanism, associated with the downstream pro-moters of adipocyte-specific genes such as FABP4, FATP1, FAS, LPL, ACS1, and leptin

We also examined whether the reduction effect of lipid accumulation by compound 1 is associated with lipolysis Lipolysis includes some critical processes such as phos-phorylation of perilipin and HSL translocation into lipid droplets (Ardevol et al 2000) Perilipin is a protein that coats lipid droplets in adipocytes and acts as a protective coating from natural lipases such as HSL (Greenberg et al

1991; Londos et al 1999) Perilipin expression is elevated

in obese animals and humans HSL mediates hydrolysis of triglycerides into free fatty acids and glycerol for later use

in metabolism in a process called lipolysis (Londos et al

1999) Moreover, the cytokine TNF-α is an important mediator of lipid metabolism and plays a role in inducing lipolysis and apoptosis of adipocytes (Salas et al 2007; Zhang et al 2008) Furthermore, TNF-α can perturb the normal regulation of energy metabolism, and enhanced TNF-α expression can be achieved with the decrease of lipidic depots in white adipose tissue, the inhibition of insulin action, and the promotion of apoptosis (Salas et al

2007; Sethi and Hotamisligil1999) Therefore, the lipolytic response was evaluated by measuring the expression levels

of perilipin, HSL, and TNF-α during adipocyte differenti-ation Compound 1 downregulated perilipin and HSL levels while upregulating TNF-α level, compared to fully differ-entiated adipocytes

In conclusion, our results revealed that compound 1 is one of the active components of E cava capable of inhibiting adipocyte differentiation and adipogenesis in 3T3-L1 cells At the molecular level, compound 1 inhibited expression of PPARγ, SREBP1c, and C/EBPα through an adipogenesis mechanism related to the downstream pro-moters of adipocyte-specific genes, including FABP4, FATP1, FAS, LPL, ACS1, and leptin Therefore, our study suggests that compound 1 might decrease lipid accumula-tion during adipocyte differentiaaccumula-tion by modulating adipo-genesis and lipoadipo-genesis Although the exact molecular signaling mechanism of compound 1 remains to be elucidated, it holds promise as a functional agent in improving obesity

Acknowledgement This research was supported by a grant from Marine Bioprocess Research Center of the Marine Biotechnology Program funded by the Ministry of Land, Transport, and Maritime, Republic of Korea.

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