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1 J Korean Soc Food Sci Nutr 한국식품영양과학회지 45(8), 1107~1113(2016) 지방세포의분화에미치는 Acacia catechu 추출물의항비만효과 김동규 1 강민정 1 서화진 2 권오운 2 신정혜 1 1 ( 재 ) 남해마늘연구소 2 ( 재 ) 경북천연염색산업연구원 Effect of Acacia catechu Extract on 3T3-L1 Preadipocyte Differentiation Dong-Gyu Kim 1, Min Jung Kang 1, Hwa Jin Suh 2, Oh Oun Kwon 2, and Jung Hye Shin 1 1 Namhae Garlic Research Institute 2 Gyeongbuk Natural Color Institute ABSTRACT The purpose of this study was to investigate the effects of catechu water extract on adipogenesis in 3T3-L1 adipocytes. 3T3-L1 preadipocytes were differentiated with adipogenic regents by incubation for 9 days in the absence or presence of catechu extract ranging from μg/ml. The effect of catechu extracts on cell proliferation of 3T3-L1 preadipocytes was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The effect of catechu extracts on 3T3-L1 differentiation was examined by measuring intracellular lipid droplet and triglyceride contents. These results were obtained from preadipocyte proliferation and adipocyte differentiation of 3T3-L1. Catechu extracts inhibited lipid accumulation and remarkably decreased triglyceride contents in 3T3-L1 preadipocytes at a concentration showing no cytotoxicity. The anti-adipogenic effects of catechu appeared to be mediated by significant down-regulation of expression of peroxisome proliferator-activated receptor γ, CCAAT/enhancer-binding protein α, and sterol regulatory element-binding protein 1c proteins apart from expression of hormone-sensitive lipase. We suggest that catechu extracts significantly inhibit adipogenesis and can be used for regulation of obesity. Key words: adipogenesis, triglyceride, PPARγ, C/EBPα, SREBP-1 서 비만은현대사회에서인간의건강을위협하는가장큰적으로인식되고있다. 비만으로인한질병의발생은전세계적으로증가추세이며, 세계보건기구 (WHO) 의보고서에따르면비만은사망률및질병유발위험인자의증가와정비례관계가있다고알려져있다 (1). 질병유발위험인자로는유전적 ( 선천성유전질환, 세포사멸유전자 ), 환경적 ( 식습관, 운동, 사회적요인, 화학물질등 ) 요인들이포함된다 (2). 비만은신체활동으로소비할수있는에너지수준을넘어서는과잉의음식물섭취에의한에너지밀도와지방량의증가때문에발생한다 (3). 이는제2형당뇨병, 고혈압, 암, 관상동맥, 심장질환등과같은대표적성인질환을유발하는것으로알려져있다 (4). 비만의개선및예방과관련한 in vitro 실험에는형태학적으로그리고생화학적으로뚜렷한변화를나타내는지방전구세포인 3T3-L1 세포가주로사용되는데, 3T3-L1 지방전구세포는분화유도시성장이정지되고지방세포로의 Received 25 April 2016; Accepted 10 June 2016 Corresponding author: Jung Hye Shin, Namhae Garlic Research Institute, Gyeongnam 52430, Korea whanbee@hanmail.net, Phone: 론 분화가유도되는데이러한과정을 adipogenesis라고한다 (5). 지방전구세포의분화에는 insulin, dexamethasone, 3-isobutyl-1-methylxanthine(IBMX) 등의분화유도인자가필요하며 (6-8), 이러한유도체들의처리로세포의형태가변하기시작하고얼마간의세포분열이동반되며 5~7일이내에지방세포의분화가확인된다 (9). 지방세포의분화에는다양한전사인자 (transcription factors) 들과조절매개체 (coregulator) 가관여한다 (10-12). 특히전사인자인 peroxisome proliferator-activated receptor γ(pparγ), CCAAT/enhancer-binding protein α (C/EBPα) 그리고 sterol regulatory element-binding protein 1c(SREBP-1c) 는지방형성의주조절자로잘알려져있다 (13). C/EBP는현재까지 6종류가보고되어있는데, 특히 C/EBPα, β 그리고 δ 형태는지방형성과밀접하게관련된것으로알려져있다 (14). C/EBPβ와 δ는분화초기에발현되어지방형성을조절하는데, C/EBPδ는분화진행과정중에발현량이감소하고 C/EBPβ는 C/EBPα와 PPARγ의유전자발현을유도하여지방형성을진행하는데관여한다고알려져있다 (11). C/EBPα와 PPARγ는상호작용을통해발현이증가하고지방세포특이적유전자인 fatty acid synthase(fas) 를발현시킨다고보고되어있다 (15). 따라서 C/ EBPα와 PPARγ의발현은지방형성과정을진행함에있어

2 1108 김동규 강민정 서화진 권오운 신정혜 매우중요한전사인자임을알수있다. 천연식물소재들은페놀화합물을포함한다양한생리활성물질들을지니고있어최근들어그활성을연구하고규명하여식품소재로활용하기위한연구들이다양하게진행되고있다. 또한, 이들식물소재들은고유의색을지니고있어색소소재로도활용되며, 이들색소성분들은구조적특성이나색에따라기능이다르지만항산화작용및암의발생원인의하나인활성산소발생억제의기능을공통으로가지고있어생리활성소재로관심이증대되고있다 (16). 천연색소시료중하나인아선약 (Acacia catechu) 은콩과 (Leguminosae) 식물에속하며아시아와인도에서전통약제로써간질환, 저혈당, 해열, 소화제, 항산화, 항염증, 흉부통증등다양한증상과질환에널리이용되어왔다 (17-23). 아선약의주성분으로는아민류, 타닌산, 폴리페놀, 알칼로이드그리고플라보노이드등이있으며 (24,25), 그중탄닌은수렴작용을할뿐만아니라지사제로사용되고항균활성이뛰어난것으로알려져있다 (26). 기존의연구에따르면아선약추출물은높은총페놀함량을가진다는보고가있다 (27). 이처럼아선약과그성분에대한다양한생리학적활성이보고되어있지만항비만활성에대한분자생물학적작용기전에관한체계적인연구는전무한실정이다. 본연구에서는아선약추출물을이용하여 3T3-L1 세포에서지방축적및트리글리세라이드 (triglyceride) 의축적억제활성을평가하였고, 지방생성과정과연관이있는주요전사인자의발현에미치는영향을분석하였다. 또한, 지방전구세포에서지방세포로분화되는세포의증식에미치는아선약의효과를확인함으로써비만예방및개선을위한소재활용의기초자료를확보하고자하였다. 재료및방법실험재료및추출물의제조천연색소소재인아선약은원산지가인도네시아로 ( 주 ) 대명제약 (Seoul, Korea) 에서의약품용으로수입한것을구입하여시료로사용하였다. 기존연구 (27) 의아선약추출물이높은항산화능을보였기때문에동일한천연색소추출법을토대로하여아선약건조시료 1 kg에 catalase(biotouchr CAT200, AB Enzymes, Rajamäki, Finland) 및 alcalase (AlcalaseR, Novozymes, Dittingen, Switzerland) 를 1% 농도로첨가하고 10 L의물을넣어 45 C에서 10시간동안반응시킨후 80 C로조절된추출기 (Cosmos660, Kyungseo, Incheon, Korea) 를이용하여 2시간동안추출하였다. 그후추출물을여과하여농축기 (Rotavapor R-220, Buchi, Flawil, Switzerland) 로농축한다음분무건조기를이용해분말화하여실험용시료로사용하였다. 총폴리페놀및총플라보노이드함량측정총폴리페놀함량은 Dewanto 등 (28) 의방법에비색법으 로정량하였다. 추출물 100 ml에 2% Na 2CO 3 용액 2 ml를가한후 3분간방치하고, 50% Folin-Ciocalteu reagent 100 μl를가해 30분반응한다음 700 nm에서흡광도를측정하였다. 표준곡선은 gallic acid(sigma-aldrich Co., St. Louis, MO, USA) 를이용하여작성하였다. 총플라보노이드함량은 Davis 변법 (29) 을이용하였다. 시료용액 1 ml에 diethylene glycol 10 ml 및 1 N NaOH 1 ml를가하여잘혼합한후 30 C에서 1시간반응시킨다음 420 nm에서흡광도를측정하였다. 이때검량곡선은 quercetin(sigma-aldrich Co.) 을이용하여작성하였다. 세포배양실험에사용된 3T3-L1 전지방세포는 American Type Culture Collection(ATCC, Rockville, MD, USA) 에서분양받아실험에사용하였다. 3T3-L1 세포는 10% bovine calf serum(bcs, Gibco, Grand Island, NY, USA), 1% penicillin/streptomycin(sigma-aldrich Co.) 을첨가한 Dulbecco modified Eagle medium(dmem, Gibco) 배지를사용하여 37 C, 5% CO 2 조건하에서배양하였다. 세포가 confluent 한상태에서 0.05% trypsin ethylenediaminetetraacetic acid(edta, Gibco) 를사용하여세포를떼어내어 3,000 rpm, 3분에서원심분리한후현탁하여사용하였다. 지방세포분화유도및약물처리 3T3-L1 세포주를 10% BCS가첨가된 DMEM 배지에서배양후 confluent 한상태가되면 10% fetal bovine serum (FBS, Gibco), 1 μm dexamethasone(sigma-aldrich Co.), 0.5 mm 3-isobutyl-1-methylxanthine(IBMX, Sigma- Aldrich Co.) 과 10 μg/ml의인슐린 (Gibco) 을함유한분화유도배지로교환한후 3일간배양하였다. 그후 10% FBS, 1% penicillin/streptomycin 및 10 μg/ml의인슐린이포함된 DMEM 배지로 2일간격으로배지를교환하였다. 시료추출물역시 20 μg/ml의농도로 2일간격으로처리하였다. 세포독성평가 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(mtt, Sigma-Aldrich Co.) 방법을이용하여아선약추출물이 3T3-L1 세포의생존에미치는영향을측정하였다. 96-well plates에 cells/well 농도로 3T3-L1 세포를분주한뒤 24시간동안배양한후지방세포분화를유도하여농도별시료를처리한다음 8일동안배양하였다. 이후기존배지를제거한다음 MTT 용액 (5 μg/ml) 10 μl 를포함하는 DMEM 배지 100 μl를각 well에넣어 37 C, 5% CO 2 조건하에서 2~3시간반응시켰다. 다시반응배지를제거하고 dimethyl sulfoxide(sigma-aldrich Co.) 를 100 μl 넣어 10분간교반한다음 ELISA reader(epoch, Bioteck, Winooski, VT, USA) 로 570 nm에서흡광도를측정하여 control에대한백분율로세포독성을평가하였다.

3 지방세포의분화에미치는 Acacia catechu 추출물의항비만효과 1109 Oil red-o 염색및지방축적률측정 3T3-L1 세포에서지방축적률을측정하기위하여지방전구세포분화시생성되는 triglyceride를특정적으로염색시키는 Oil red-o(sigma-aldrich Co.) 염색법을이용하여분화억제여부를확인하였다. 3T3-L1 전지방세포는분화를유도한후 1~200 μg/ml의아선약추출물을 3, 5, 7일에처리하였으며염색은마지막날실시하였다. 먼저배지를제거하고 PBS로세척한후 10% formalin을가해실온에서 5분간반응시킨다음 formalin을제거하였다. 다시여기에 formalin을가해 1시간반응시켜세포를고정하였다. Formalin을제거한다음 60% isopropanol로세척한후각각의 well이완전히마를때까지방치하였다. 그후각각의 well 에 Oil red-o solution을첨가하여 20분간실온에서염색후멸균증류수를이용하여잔여 Oil red-o solution을세척하였다. 100% isopropanol을첨가하여실온에서 10분간반응하여용출된 Oil red-o를 ELISA reader로 520 nm에서흡광도를측정하였다. 지방축적률은시료추출물대신세포생육배지를처리하여같은방법으로실험한 control에대한백분율로나타내었다. Triglyceride 함량측정분화유도된 3T3-L1 지방세포에축적된 triglyceride는 triglyceride colorimetric assay kit(cayman, Ann Arbor, MI, USA) 을이용하여확인하였다. 상기와동일한과정에의해시료가처리되고분화가유도된세포를 PBS(pH 7.4) 로 3회세척한후 lysis buffer(1% Triton X-100 in PBS) 를첨가하여스크래퍼로세포를모아 4 C에서 30초간 sonication 하여 10,000 rpm(hanil, Seoul, Korea) 에서 10분간원심분리한다음상층액을분리하였다. 상층액 10 μl에 150 μl의 enzyme buffer solution을첨가하여혼합한후실온에서 15분간반응시킨다음 540 nm에서흡광도를측정하였다. 시료대신배지를처리한대조군의 triglyceride 양대비시료처리군의 triglyceride 양의백분율로중성지방축적률을나타내었다. Western blot 분석 3T3-L1 지방세포에 protein extraction solution(pro- PREPTM, intron Biotechnology Inc., Seongnam, Korea) 을포함한 50 mm Tris-Cl(pH 7.5), 150 mm NaCl, 1 mm dithiothreitol, 0.5% nomylphenoxylpolyethoxylethanol, 1% triton X-100, 10% deoxycholate, 0.1% sodium dodecyl sulfate(sds), 1 mm EDTA, 1X protease inhibitor(sigma-aldrich Co.) 를첨가하여용해한후 4 C 에서 30분간방치하여 13,000 rpm(hanil) 에서 20분간원심분리한다음상층액을분리하였다. Bovine serum albumin protein assay kit(thermo, Waltham, MA, USA) 을이용하여 50 μl의동일한단백질시료를 SDS sample buffer에서용해시켰다. 단백질을 4~12% SDS PAGE(Invitrogen, Carlsbad, CA, USA) 를이용하여전기영동한후 PVDF membrane에 transfer 하였다. 3% skim milk buffer에 blocking 한다음 C/EBPα, PPARγ 및 SREBP-1의 1차항체 (Cell Signaling Technology, Danvers, MN, USA) 를 1:1,000으로희석하여 4시간반응시킨후 tris-buffered saline Tween-20(TBST) 으로 3회세척하였다. 2차항체인 peroxidase-conjugated anti-rabbit 및 anti-mouse 항체 (1:2,000) 로 1시간반응시킨후다시 TBST로 3회세척한다음 ECL detection reagents(ecl plus kit; ELPIS, Daejeon, Korea) 를이용하여단백질의발현정도를확인하였다. 상대적인단백질발현정도는 β-actin을이용하여계산하였다. 통계처리데이터는평균 ± 표준오차로표현하였으며, 데이터의통계처리는 Statistical Package for Social Science(Version 10, SPSS, IBM, Armonk, NY, USA) 를이용하여분석하였다. Student's t-test 방법에의하여각각의실험군과대조군간의유의성차이를검증하였다 ( P<0.05, P<0.01, P< 0.001). 결과및고찰 총폴리페놀과총플라보노이드함량분석녹차를포함한녹황색채소의경우폴리페놀과플라보노이드계열의화합물이많이함유되어있어만성질환을예방하거나산화적스트레스를개선하는데활용되고있다 (30). 이러한항산화및항염증활성이좋은소재는식품뿐만아니라약용식물로부터다양하게섭취해야할필요성이있다 (31). 아선약추출물에대한총폴리페놀및플라보노이드함량측정결과는 Table 1에서보는바와같다. 폴리페놀의정량을위해 gallic acid를이용하여검량선을작성하고이에따라총폴리페놀의함량을계산한결과아선약추출물에는 ±1.36 mg/g이함유되어있었다. 총플라보노이드함량은 quercetin을이용하여검량선을작성하여산출하였으며그결과 4.11±0.59 mg/g의함량을확인할수있었다. 이처럼아선약추출물은총폴리페놀의함량이매우높고총플라보노이드함량도높으므로항산화, 항염증효능을통해비만발생요인을중화하는작용을할것으로생각한다. Table 1. Total polyphenol (TPC) and total flavonoid contents (TFC) from catechu Sample TPC (mg/g) 1) TFC (mg/g) 2) Catechu ± ±0.59 1) Results represent mean value±standard error mg gallic acid equivalents per 1 g of sample. 2) Results represent mean value±standard error mg quercetin equivalents per 1 g of sample.

1110 김동규 강민정 서화진 권오운 신정혜 Cell viability (%). 120 100 80 60 40 20 0 200 (μg/ml) Fig. 1. The effect of catechu extract on cell viability in 3T3-L1 adipocytes.

Data are expressed as the percent growth rate of cells cultured in the presence of samples compared with untreated control cells.

4 1110 김동규 강민정 서화진 권오운 신정혜 Cell viability (%) (μg/ml) Fig. 1. The effect of catechu extract on cell viability in 3T3-L1 adipocytes. Differentiated 3T3-L1 cells were treated with various concentrations of catechu extracts. Data are expressed as the percent growth rate of cells cultured in the presence of samples compared with untreated control cells. Values are mean±standard error of 8 replicates and asterisks above the bar are significantly different from control ( P<0.001). 3T3-L1 지방세포에서아선약추출물의세포독성 MTT는환원형화합물또는효소에의해쉽게환원되어발색을일으키는시약으로미토콘드리아내의산화환원효소 (mitochondrial dehydrogenase) 와반응하여보라색의불용성 formazan을형성한다 (32). MTT 분석법은살아있는세포생존율을확인하는방법으로잘알려져있다 (33). 세포분화에사용되는인슐린은체내에서다양한기능을수행하는호르몬으로에너지저장과이화작용을매개하기위하여식후에는그생성량이증가하고, 공복시에는지방세포에서에너지를방출시키고포도당분해를유도하기위해감소한다 (34). 주요발현부위는지방조직, 근육, 간으로이러한조직들은인슐린수용체를통해신호를받아들여서탄수화물, 지질, 단백질의물질대사와관련하여조직특이적으로영향을주는것으로알려져있다 (34). Hwang 등 (35) 은천연색소시료인정향, 감, 오배자, 황벽, 치자, 홍화황및아토나추출물의세포생존율을측정한결과 100 μg/ml 농도에 서아토나와오배자추출물이 70.43% 와 71.92% 로낮았으며나머지 5가지시료들은 90% 대의생존율을나타내는것으로확인하였다. 이러한결과를바탕으로세포독성실험을위한시료의농도범위를설정하였다. 3T3-L1 전구지방세포에분화를유도하는인슐린과함께아선약추출물을 1~200 μg/ml 농도로처리한결과 1, 10, 50 그리고 100 μg/ml 농도를처리한군에서는대조군과비교하여세포의형태학적변화가관찰되지않았으며, 또한세포생존율도유의성있는변화를보이지않았다. 반면 200 μg/ml 농도로처리하였을때는 55.32±2.36% 로세포생존율이유의적으로감소하였다 (Fig. 1). 이러한결과를토대로항비만활성을평가하기위한아선약추출물의처리유효농도는 1, 10, 50, 100 μg/ml로결정하였다. 3T3-L1 지방전구세포의 lipid droplet 생성에미치는아선약추출물의영향 Lipid droplet은 phospholipid monolayer에의하여둘러싸여진중성지방으로구성된비활성소낭으로서 precursor fibroblast에서부터지방세포로의분화과정에서나타나며, PPARγ와같은중요한 adipogenic transcription factor들에의해서조절되는것으로알려져있다 (36). 3T3-L1 전구지방세포가지방세포로의분화과정에나타나는 lipid droplet 생성에아선약추출물이어떠한영향을미치는지를확인하기위해 Oil-red O 염색법을통해 3T3- L1 지방세포내축적된지방의양을측정하였다. Oil-red O 염색시약은세포내지질과결합하며, 염색된세포의붉은색이강할수록세포내중성지질축적이많이이루어졌음을의미한다 (37). 위상차현미경을통해염색된지방세포의수를이미지로확인한결과 lipid droplet의형성이아선약추출물처리농도에의존적으로억제되는것을확인하였다 (Fig. 2A). 앞선이미지결과를통계처리한결과대조군보다 1, 10, 50 및 100 μg/ml 처리군에서각기 23%, 45%, A CTL 1 μg/ml 10 μg/ml B μg/ml 100 μg/ml Lipid accumulation (%) Concentration (μg/ml) Fig. 2. Inhibitory effects of catechu on the lipid accumulation in 3T3-L1 adipocyte. (A) Lipid droplet formation in mature adipocytes, mature adipocyte on control, and on 1, 10, 50, 100 μg/ml of catechu extracts. (B) Values are mean±standard error of 3 replicates and asterisks above the bars are significantly different from control ( P<0.05, P<0.01).

지방세포의분화에미치는 Acacia catechu 추출물의항비만효과 1111 61%, 82% 의유의적인지방축적률의감소를나타내었다 (Fig. 2B). 이결과를통해아선약추출물이 3T3-L1 지방세포에서지방축적억제효과를가지는것이확인되었다.

5 지방세포의분화에미치는 Acacia catechu 추출물의항비만효과 %, 82% 의유의적인지방축적률의감소를나타내었다 (Fig. 2B). 이결과를통해아선약추출물이 3T3-L1 지방세포에서지방축적억제효과를가지는것이확인되었다. 아선약추출물의 triglyceride 축적억제효과 Lipid droplet은 triglyceride와 cholesterol ester가소포체의이중층사이에축적될때생기며, 지방을저장하는기능을한다 (38). 특히이들중 triglyceride는중요에너지원으로사용되지만비만의주요원인으로도작용한다 (39, 40). 배양된세포에분화유도제와함께아선약추출물을 1~100 μg/ml의농도로처리한다음분화과정이끝난후분화된지방세포내에존재하는순수한 triglyceride 축적을관찰하였다 (Fig. 3). 아선약추출물을처리한실험군에서는농도의존적으로유의한 triglyceride 축적의감소효과가나타났다. 특히 100 μg/ml 농도로처리하였을때 62.42± 1.87% 의높은 triglyceride 축적억제효과를확인할수있었다. 따라서아선약추출물은 3T3-L1 지방세포의분화유도과정에서지방생성을억제하여지방축적을억제한다는것을확인할수있었다. 천연물추출물을이용한기존의연구에따르면 Kim 등 (41) 은분화가유도된지방세포에후박추출물을 100 μg/ ml로처리하였을때 triglyceride가 35% 감소하는것으로보고하였으며, Cha 등 (42) 은수세미오이추출물을 600 μg/ ml로처리하였을때대조군과비교하여 triglyceride가 Triglyceride content (%) (μg/ml) Fig. 3. Effect of catechu on triglyceride contents in 3T3-L1 adipocytes. Values are mean±standard error of 3 replicates and asterisks above the bars are significantly different from control ( P<0.05, P<0.01). 13.5% 감소하는것으로확인되었다. 이러한대부분의천연식물추출물들은지방세포로분화되는동안에특징적으로발현되는 transcription factor들 (C/EBP family, PPARγ, SREBP-1c 등 ) 에의해조절되는것으로알려져있다 (43). 지방세포분화관련전사인자들의발현에미치는아선약추출물의영향지방전구세포에서지방세포로분화되는 adipogenesis 과정은많은종류의 adipogenic transcription factor들의 A B 2.5 (μg/ml) 2.0 C/EBPα PPARγ SREBP-1 PPARγ/β-actin β-actin 0.0 (μg/ml) C 2.5 D SREBP-1/β-actin C/EBP α/β-actin (μg/ml) 0.0 (μg/ml) Fig. 4. Effects of catechu on the major adipogenic factor expression in 3T3-L1 adipocyte. PPARγ, SREBP-1, and C/EBPα expression levels in 3T3-L1 adipocytes were analyzed by western blot. β-actin was used as a control. Values are mean±standard error of 3 replicates and asterisks above the bars are significantly different from control ( P<0.05).

6 1112 김동규 강민정 서화진 권오운 신정혜 단계적인조절에의하여유발되는것으로알려져있고, 분화과정에서중추적인역할을하는인자들로는 PPARγ와 SREBF-1 그리고 C/EBPα가보고되어있으며 (11,43), 이들세가지전사인자는지방세포분화과정중각기다른시점에서발현이유도되며상호작용을통하여여러지방세포특이유전자들의발현을조절하고, 지방대사의활성화와지방세포분화를점진적으로유도해나간다 (44). C/EBPα는지방전구세포에서발현이관찰되지않지만분화과정의후반기에발현이촉진되어지방세포분화관련유전자의발현을조절하는역할을하고, PPARγ 전사인자의경우 ligand 특이성, cofactor recruitment 등에의해다양한기능을수행한다 (11). PPARγ 유전자는서로다른전사시작점과 alternative splicing을이용하여 PPARγ1과 PPARγ2 두종류의 isoform을형성하는데, 특히 PPARγ2가지방세포에서선택적으로발현하며지방세포분화와지방산대사조절에서중요한기능을담당한다 (11). SREBP-1의 isotype 중하나인 SREBP-1c는다양한조직에서발현되고있는 SREBP-2 와는달리지방세포에특이적으로높은발현을나타내며 (45), 콜레스테롤대사와지방산대사에관여하는수많은유전자의발현을조절함으로써지질생합성에직접관여한다 (13). 아선약추출물이분화관련기작과직접적인연관성을가진주요조절인자들의발현량에어떠한영향을미치는지를확인하고자분화유도제만처리한대조군과분화유도제와아선약을병용처리한실험군을각각분화시킨후 western blot을통해확인하였다 (Fig. 4A). 단독으로분화유도제만처리한대조군과비교하여 PPARγ와 SREBP-1의발현은모두농도의존적으로유의하게감소였으며, 또한 C/EBPα 의발현도 100 μg/ml 농도에서유의성있는감소효과를나타냈다 (Fig. 4B~D). 아선약추출물은 preadipocyte의분화초기에는내생적으로높은발현을보이는 SREBP-1c의발현을조절하여지방형성을억제시키고, 분화후기에는 PPARγ와 C/EBPα의발현을감소시켜지방세포로의분화를저해함으로써항비만활성을나타낼수있을것으로판단된다. 요약지방전구세포가지방세포로분화하는과정에서아선약추출물의영향을확인하고자분화유도제를처리하여 3T3-L1 세포에서아선약의세포독성, 지방축적억제효과, triglyceride 억제효능, 지방분화관련단백질들의발현을확인하였다. 아선약추출물을최대 200 µg/ml 농도까지세포에처리하여세포독성을측정한결과아선약추출물은 100 µg/ml 이하의농도에서는세포에독성을유발하지않는것으로확인되었다. Oil red-o 염색을통해지방축적률을확인한결과아선약추출물에의한농도의존적인지방축적의감소효과가있었다. Triglyceride 생성량역시농도의존적 인감소가확인되었다. 아선약에의한지방분화의감소가어떠한기작에의해조절되는지확인하고자관련지표단백질인 PPARγ와 SREBF-1 그리고 C/EBPα의발현량변화를확인해본결과 100 µg/ml 농도에서유의성있게감소하였다. 이상의결과를종합해볼때아선약추출물은지방세포의분화억제와분화된지방세포의지방축적을저해함으로써항비만효과를유도할것으로기대된다. 감사의글 본논문은산업통상자원부지역특화기술융복합연구지원사업 ( 과제번호 : R ) 성과의일부이며, 지원에감사드립니다. REFERENCES 1. Malterud K, Tonstad S Preventing obesity: Challenges and pitfalls for health promotion. Patient Educ Couns 76: Dewulf EM, Cani PD, Neyrinck AM, Possemiers S, Van Holle A, Muccioli GG, Deldicque L, Bindels LB, Pachikian BD, Sohet FM, Mignolet E, Francaux M, Larondelle Y, Delzenne NM Inulin-type fructans with prebiotic properties counteract GPR43 overexpression and PPARγrelated adipogenesis in the white adipose tissue of high-fat diet-fed mice. J Nutr Biochem 22: Hosogai N, Fukuhara A, Oshima K, Miyata Y, Tanaka S, Segawa K, Furukawa S, Tochino Y, Komuro R, Matsuda M, Shimomura I Adipose tissue hypoxia in obesity and its impact on adipocytokine dysregulation. Diabetes 56: Bays H, Blonde L, Rosenson R Adiposopathy: how do diet, exercise and weight loss drug therapies improve metabolic disease in overweight patients?. Expert Rev Cardiovasc Ther 4: Ailhaud G, Grimaldi P, Négrel R Cellular and molecular aspects of adipose tissue development. Annu Rev Nutr 12: Haugen F, Zahid N, Dalen KT, Hollung K, Nebb HI, Drevon CA Resistin expression in 3T3-L1 adipocytes is reduced by arachidonic acid. J Lipid Res 46: Tenney R, Stansfield K, Pekala PH Interleukin 11 signaling in 3T3-L1 adipocytes. J Cell Physiol 202: Guo X, Liao K Analysis of gene expression profile during 3T3-L1 preadipocyte differentiation. Gene 251: MacDougald OA, Hwang CS, Fan H, Lane MD Regulated expression of the obese gene product (leptin) in white adipose tissue and 3T3-L1 adipocytes. Proc Natl Acad Sci U S A 92: Wang Q, Li YC, Wang J, Kong J, Qi Y, Quigg RJ, Li X mir cluster accelerates adipocyte differentiation by negatively regulating tumor-suppressor Rb2/p130. Proc Natl Acad Sci U S A 105: Farmer SR Transcriptional control of adipocyte formation. Cell Metab 4: Rosen ED, MacDougald OA Adipocyte differentiation from the inside out. Nat Rev Mol Cell Biol 7:

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Journal of Life Science 2011, Vol. 21. No μ μ

Journal of Life Science 2011 Vol. 21. No. 8. 1120~1126 ISSN : 1225-9918 DOI : http://dx.doi.org/10.5352/jls.2011.21.8.1120 μ μ μ α β Journal of Life Science 2011, Vol. 21. No. 8 1121 μ μ 1122 생명과학회지 2011,