Original Article Korean J Obes 2016 June;25(2): pissn X eissn PPARγ 에의해유도된지방세포생성에대한테

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1 Original Article Korean J Obes 216 June;25(2):68-76 pissn X eissn PPARγ 에의해유도된지방세포생성에대한테스토스테론의억제효과 정선효 목원대학교의생명 보건학부 he Inhibitory Effect of estosterone on PPARγ-induced Adipogenesis Sunhyo Jeong Division of biomedical Engineering & Health Science Management, Mokwon University, Daejeon, Korea Background: Peroxisome proliferator-activated receptor γ (PPARγ) plays a major role in adipocyte differentiation. estosterone is well known for inhibiting adipocyte metabolism in men. o investigate the inhibitory mechanism of testosterone on adipogenesis, this study evaluated the effects of testosterone on PPARγ expression and activity in adipocytes using in vitro approaches. Methods: After differentiated 33-L1 adipocytes were treated with PPARγ agonist troglitazone and sex hormone testosterone, the effects of testosterone on troglitazone-induced triglyceride accumulation and expression of genes involved in adipogenesis were investigated. We also investigated whether testosterone regulates troglitazone-induced PPARγ reporter activity in 33-L1 preadipocytes. Results: estosterone decreased triglyceride accumulation in differentiated 33-L1 cells compared with the vehicle treated control group. estosterone also decreased the expression of PPARγ mrna as well as PPARγ dependent adipocyte-specific genes, such as adipocyte fatty acid binding protein and tumor necrosis factor α. Moreover, testosterone treatment inhibited triglyceride accumulation, and the expression of PPARγ and adipocyte-specific genes caused by troglitazone in differentiated 33-L1 cells. estosterone decreased troglitazone-induced PPARγ reporter activity. Also, treatment with testosterone led to an inhibition of troglitazone-induced PPARγ reporter activity in PPARγ and androgen receptor (AR) expressed 33-L1 preadipocytes. Conclusion: hese results suggest that testosterone interferes with the actions of PPARγ on adipogensis by an AR-dependent component. In addition, this study may have provided valuable molecular and biological insights regarding testosterone therapy in obese hypogonadal men. Key words: estosterone, glitazone, PPARγ, Adipogenesis, 33-L1 adipocytes 서론 비만은대사성질환 ( 당뇨병, 심혈관계질환등 ) 의유병률과밀접한관련이있기때문에산업화된국가에서는널리퍼져있는건강문제로인식되고있다. 1 흡수된에너지와소비된에너지의불균형으로에너지의소비보다공급이많아질때과량의에너지는지방형태로지방조직에저장된다. 지방이지속적으로체내지방조직에축적되면지방조직이증가되어결국비만으로이어지게된다. 비만은식생활습관, 활동량부족, 스트레스및내분비계의변화등 여러요인에의해발생한다. 특히남성들은나이및성선기능저하증 (hypogonadism) 등에의해생식선성스테로이드호르몬인테스토스테론이감소되면서지방조직의무게가증가되고, 고인슐린증, 심혈관질환, 고지혈증및제2형당뇨병등을포함한대사증후군의발병률을증가시킨다. 2-5 그러나이러한남성들이테스토스테론을보충받으면제지방무게 (fat-free mass) 는증가되는반면, 지방무게는감소되고비만과관련된대사증후군의발병이감소되는경향을나타낸다. 6,7 따라서테스토스테론은남성에서지방조직대사를조절하는중요한인자로인식되고있다. 또한성스테로이드호르몬과성스테로이드호 Corresponding author Sunhyo Jeong Division of biomedical Engineering & Health Science Management, College of echno- Sciences, Mokwon University, 88 Doanbuk-ro, Seo-gu, Daejeon 35349, Korea el Fax jsh227@mokwon.ac.kr Received Nov. 19, 215 Reviewed Dec. 17, 215 Accepted Feb. 22, 216 Copyright 216 Korean Society for the Study of Obesity his is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 68

2 정선효 estosterone Inhibits PPARγ-induced Adipogenesis 르몬의수용체가지방조직에서높게발현되기때문에, 지방조직은성스테로이드호르몬의주요표적기관이된다. 8,9 지방조직증가는지방세포로의지방축적으로지방세포의크기가비대해지는과정과미분화된지방전구세포의증식과분화로성숙한지방세포의수가증가되는과정을통해일어난다. 1 이러한과정중지방전구세포가성숙한지방세포로분화되는지방세포생성 (adipogenesis) 는지방세포의형태적변화, 지질의축적및지방대사조절과관련된유전자발현의변화와관련이있으며 11, 지방세포생성에관한연구는 33-L1 지방전구세포를이용한 in vitro model이널리사용되고있다 L1 지방전구세포에 fetal calf serum과 3-isobutyl-1-methylxanthine (IBMX), dexamethasone 및 insulin이포함된지방세포분화배지인 MDI medium을처리하면지방세포로분화되는지방세포생성과정이일어난다. 이러한 MDI medium이처리된 33-L1 지방전구세포는세포속에중성지방이축적되고지방세포특이적유전자들이발현되면서성숙한지방세포로분화된다. 13 지방세포생성은 CCAA/enhancer binding proteins (C/EBPs), peroxisome proliferator-activated receptor γ (PPARγ) 등의 transcription factors 에의해조절된다. 특히 PPARγ 는지방세포생성과정을조절하는주요한 transcription factor 로알려져있으며지방세포생성과정에대한 PPARγ의중요성은지방전구세포를이용한 in vitro에서널리연구되고있다 PPARγ는지방세포생성의후기단계에서발현이증가되며지방세포특이적유전자의발현을촉진시킴으로써지방세포특이적기능을조절한다. 17 PPARγ는 ligand에의해활성화되는 transcription factor 로써지방세포특이적유전자의발현과지방세포분화에중요한역할을담당하며, PPARγ는백색지방과갈색지방에서높은수준으로발현된다. 18 리간드에의해활성화된 PPARγ는 retinoic X receptor와 heterodimer를이룬후, 지질대사와관련된지방세포특이적유전자들의 promoters/enhancers에존재하는 peroxisome proliferator response elements (PPREs) 에결합하여 PPARγ- 표적유전자의발현을조절한다. 19 hiazolidinediones (ZDs; troglitazone과 rosiglitazone) 은 PPARγ agonist로서지방세포생성과 lipid accumulation을촉진시키며지방세포생성동안 PPARγ-responsive genes의발현을유도한다. 2,21 지방조직에서대부분의 PPARγ- 표적유전자는 adipocyte fatty acid binding protein (ap2) 과 fatty acid synthase 등의 lipogenic pathway와직접적인관련이있는유전자들이다. 11,22 또한지방세포분화과정동안 PPARγ는지방세포에서생성되고분비되는 tumor necrosis factor α (NFα) 와 leptin 등의지방세포유전자들 (adipogenic genes) 을유도한다. 23,24 지방세포생성은 PPARγ뿐만아니라성스테로이드호르몬에의해서도영향을받는다. Dehydroepiandrosterone을비롯한남성성스 테로이드호르몬에의해지방전구세포가지방세포로분화되는과정이억제된다고보고되고있다 그러나남성성스테로이드호르몬이남성의지방조직대사에주요한요인이됨에도불구하고남성의지방조직대사에대한남성성스테로이드호르몬의작용기전이분자세포생물학적측면에서명확히규명되지않고있다. 따라서본연구는 33-L1 지방전구세포를이용한 in vitro 연구를통해남성호르몬인테스토스테론이 troglitazone에의해유도된 PPARγ 활성에미치는영향과그작용기전을규명함으로써, 테스토스테론에의한지방세포생성조절에대한정확한분자세포생물학적조절기전을규명하여남성호르몬생성의이상으로발생되는비만과관련된대사질환의예방및치료에유익한기초자료를제공하고자한다. 방법 1. 33L1 지방전구세포배양및지방세포로의분화유도 33-L1 지방전구세포 (ACC, Manassas, VA, USA) 는 6-well plates 에서 bovine calf serum (Invitrogen, Carlsbad, CA, USA) 이포함된 Dulbecco s modified Eagle s medium (DMEM) 배지 (Gibco-Brl, Grand Island, NY, USA) 로배양되었다. 2일후 1% fetal bovine serum (FBS) 이포함된 DMEM배지에.5 mmol/l IBMX, 1 μmol/l dexamethasone 및 1 μg/ml insulin이첨가된 MDI medium 으로교체하여 (day ) 2일동안지방세포분화를유도한후 (day 2), 2일마다 1% FBS가포함된 DMEM 배지로교체하여 6일동안배양하였다 (day 6). 배양 6일째에세포의 total RNA를추출하였고 Oil red O 염색액으로염색하였다. Vehicle, 테스토스테론및 troglitazone은 MDI medium을첨가한 2일동안처리하였다. 2. 분화된 33L1 세포에서의중성지방축적의분석세포를 phosphate buffered saline (PBS) 으로세척하고 1% formaldehyde로 1시간동안고정시킨후 PBS로세척하고 4% Oil red O 염색액으로 2시간동안염색하였다. 세포를 PBS로세척한후중성지방축적정도를광학현미경으로분석하였다. 3. arget genes 발현분석 otal RNA는 rizol (Invitrogen, Avenue Carlsbad, USA) 을사용하여추출하였으며, 역전사-중합효소연쇄반응 (reverse transcription-polymerase chain reaction, R-PCR) 을이용하여 mrna의발현양을측정하였다. Complementary DNA는 total RNA 2 μg과 reverse primer.5 μg을혼합하여최종약 14 μl를준비하여 75 C에서 15분동안열처리 (heating) 한후, 5분동안얼음속에보관하였다. 여기에 5X M-MLV reaction buffer, 1 mm dnp mixture, 2 units 69

3 정선효 estosterone Inhibits PPARγ induced Adipogenesis M-MLV R (Promega, Madison, WI, USA) 를첨가하여최종양이 25 μl가되게한후, 42 C에서 1시간동안반응시켰다. R reaction 5 μl에 1X reaction buffer (Mg 2 포함 ), 1 mm dnp, 5 unites aq polymerase (Solgent, Daejeon, South Korea), 그리고 1 μm primer 를첨가하여최종 5 μl가되게한후, RC- 1M Programmable hermal Controller (MJ Research, INC., Waltham, MA, USA) 를이 하여 transfection하였다. 반응 6시간후신선한배지 1 ml를첨가하고 vehicle, 테스토스테론및 troglitazone을처리하였다. 처리 24시간후배지를제거하고배양한세포를수확하였다. Luciferase와 β-galactosidase 활성은 Promega사 (Promega, Madison, WI, USA) 의 kit 인 Reporter Lysis Buffer가포함된 Luciferase Assay System과 β-galactosidase Enzyme Assay System 을사용하여측정하였다. 용하여 PCR 을실시하였다. able 1 은 primer sequences 와 PCR conditions 이다. 5. 통계분석 모든값은 mean ± standard deviation (SD) 으로표시하였다. 통계 4. ransient transfection 및 luciferase assay psg5-mpparγ gene 과 PPRE3-tk-luc reporter gene 의발현벡터 (expression vector) 는 Dr. Gonzalez (National Cancer Institute, NIH, Bethesda, MD) 로부터받은것이며, psg5-ar gene constructs 의발 현벡터 (expression vector) 는 Dr. Cynthia Heinlein (University of Rochester Medical Center, Rochester, NY) 로부터받은것이다. Murine preadipocyte cell line 33-L1 cells 는 6-well tissue culture plates (2 1 4 cells/well) 에서 1% fetal bovine serum (Gibco-Brl, Grand Island, NY, USA), penicillin G (1 U/mL), streptomycin sulfate (1 μg/ml), amphotericin B (.25 μg/ml) 및 2-mercaptoethanol (5 μ M) 이포함된 DMEM 배지로 24 시간동안배양한후 transfecton 하였 다. 모든 transfection 의경우 well 당각각 2 ng 의 plasmid 가사용되 었고 lipofectamine (Life technologies, Rockville, MD, USA) 을사용 able 1. Sequences of oligonucleotide primers and polymerase chain reaction conditions Genes Size (bp) Primer sequences A ( C) Cycle 분석은 ukey s multiple-comparison test에의한 one-way ANOVA 를실시하였다. 유의수준은 P<.5로설정하였다. 결과 L1 세포의분화에대한 troglitazone과테스토스테론의영향 33-L1 지방전구세포가지방세포로분화되었는지를조사하기위해서, 중성지방을염색하는 Oil red O 염색을이용하여 33-L1 세포에서중성지방의축적을측정하였다 (Fig. 1). 33-L1 지방전구세포는중성지방의축적이나타나지않았으며, 지방세포로분화된 33-L1 세포에서 vehicle 이처리된대조군세포와 troglitazone이처리된세포는중성지방의축적이확연히관찰되었다. 그러나테스토스테론이처리된세포는 vehicle 이처리된세포에비해중성지방의축적이현저히감소되었으며, 특히 troglitazone과테스토스테론이동시처리된세포는 troglitazone이처리된세포에비해중성지방의축적이감소되었다. PPARγ 34 F:5 -attctggcccaccaacttcgg-3 R:5 -tggaagcctgatgctttatcccca-3 ap2 417 F:5 -caaaatgtgtgatgcctttgtg-3 R:5 -ctcttcctttggctcatgcc-3 NFα 387 F:5 -ctcgagtgacaagcccgtag-3 R:5 -ttgacctcagcgctgagcag-3 β-actin 35 F:5 -tggaatcctgtggcatccatgaaa-3 R:5 -taaaacgcagctcagtaacagtcc-3 A, annealing temperature; bp, base pair 지방세포특이적인유전자의발현에대한 troglitazone과테스토스테론의영향 33-L1 세포에서 troglitazone 처리에의해증가된 triglyceride droplets 축적이테스토스테론에의해감소된것이 PPARγ 와 PPARγ- 의존적인지방세포특이적유전자의 mrna 발현의변화에의한것인지를조사하였다. glitazone 이처리된세포는 vehicle이처리된 Fig. 1. Differentiation of 33-L1 preadipocytes into adipocytes. Microphotographs were obtained on days and 6. Oil red O staining was performed to identify lipid droplet formation (magnification, 1). ; 1 μmol/l troglitazone; ; 1 μmol/l testosterone. 7

4 정선효 estosterone Inhibits PPARγ-induced Adipogenesis 대조군세포에비해 PPARγ mrna 발현이 16.8% 증가하였다 (P<.5) (Fig. 2). 그러나테스토스테론이처리된세포는 vehicle 이처리된대조군세포에비해 PPARγ mrna 발현이 15.3% 감소되었으며, troglitazone이처리된세포에비해 troglitazone과테스토스테론이동시처리된세포는 troglitazone에의해유도된 PPARγ mrna 발현이 38.2% 감소되었다 (P<.5) (Fig. 2). PPARγ- 의존적인지방세포특 이적유전자인 ap2와 NFα의 mrna 발현은대조군세포에비해 troglitazone이처리된세포에서각각 28.7% 와 25.1% 씩증가되었지만, 테스토스테론이처리된세포는대조군세포에비해각각 15.1% 와 18.7% 씩감소되었다 (P<.5) (Fig. 3). glitazone 에의해증가된 ap2와 NFα의 mrna 발현은 troglitazone과테스토스테론의동시처리에의해각각 36.8% 와 34.1% 씩감소되었다 (P<.5) (Fig. 3). A 1..8 PPARγ/β-actin mrna (R.D.U) B PPARγ β-actin Fig. 2. Effect of Co-treatment with troglitazone () and testosterone () on the mrna expression of PPARγ in differentiated 33-L1 adipoctyes. (A) Cells were treated with vehicle, 1 μmol/l troglitazone (), 1 μmol/l testosterone () or 1 μmol/l plus 1 μmol/l. otal cellular RNA was extracted from differentiated cells on day 6. All values are expressed as mean± SD of R.D.U. (relative density units) using β-actin. (B) Representative R-PCR photographs from an independent experiment. P<.5, as compared with control group, P<.5, as compared with group. A.8.8 ap2/β-actin mrna (R.D.U) NFα/β-actin mrna (R.D.U) B ap2 NF-α β-actin Fig. 3. Effect of Co-treatment with troglitazone () and testosterone () on the mrna expression of PPARγ target genes involved in adipogenesis in differentiated 33-L1 adipoctyes. (A) Cells were treated with vehicle, 1 μmol/l troglitazone (), 1 μmol/l testosterone () or 1 μmol/l plus 1 μmol/l. otal cellular RNA was extracted from differentiated cells on day 6. All values are expressed as mean± SD of R.D.U. (relative density units) using β-actin. (B) Representative R- PCR photographs from an independent experiment. P<.5, as compared with control group; P<.5, as compared with group. 71

5 정선효 estosterone Inhibits PPARγ induced Adipogenesis Relative luciferase activity ( 1 4 ) Relative luciferase activity ( 1 4 ) PPARγ (μm) Fig. 4. Effect of Co-treatment with troglitazone () and testosterone () on the PPARγ reporter gene expression in 33-L1 preadipoctyes with PPARγ. 33-L1 preadipocytes were transiently transfection with psg5-mpparγ gene and PPRE3-tkluc reporter gene. Cells were treated with a 1 μmol/l troglitazone (), or 1 μmol/l plus testosterone (). After incubation for 24 hours, cells were harvested, lysed and subsequently assays for luciferases and β-galactosidase activities. All values are expressed as mean± SD of relative luciferase units/β-galactosidase activity. P<.5, as compared with lane 1; P<.5, as compared with lane 2; P<.5, as compared with lane PPARγ report gene 의발현에대한 troglitazone 과 테스토스테론의영향 glitazone 에의해유도된 PPARγ mrna 발현과 PPARγ- 의존적 인지방세포특이적유전자 mrna 발현에대한테스토스테론의억 제적영향을이해하기위한분자세포생물학적조절기전을조사하기 위해, 33-L1 preadipocytes 를 PPARγ expression constructs, androgen receptor (AR) expression constructs 및 luciferase reporter gene construct (PPRE 3-tk-luc) 로 transient transfection 시켰다 (Figs. 4 and 5). PPARγ 로 transfection 시킨세포는 PPARγ 로 transfection 시키지 않은세포에비해 luciferase reporter activity 가증가되었다 (P<.5) (Fig. 4, lane 2 vs lane 1). PPARγ 로 transfection 시킨세포에서 troglitazone 처리는 PPARγ transfection 에의해유도된 luciferase reporter activity 를 86.6% 증가시켰다 (P<.5) (Fig. 4, lane 3 vs lane 2). 그러나 troglitazone 과테스토스테론의동시처리는 troglitazone 에의해유도 된 luciferase reporter activity 를테스토스테론처리농도에의존적으 로감소시겼으며 (P<.5) (Fig. 4, lane 4, 5, and 6 vs lane 3), 특히 troglitazone 과 1 μm 테스토스테론동시처리에의해 44.8% 감소시켰다 (P<.5) (Fig. 4, lane 6 vs lane 3). 따라서이러한결과는테스토스테 론이 troglitazone 에의해활성화된 PPARγ 의전사작용을억제시켰 PPARγ AR (μm) Fig. 5. Effect of Co-treatment with troglitazone () and testosterone () on the PPARγ reporter gene expression in 33-L1 preadipoctyes with PPARγ and AR. 33-L1 preadipocytes were transiently transfection with psg5-mpparγ gene, PPRE3-tk-luc reporter gene and psg5-ar gene. Cells were treated with a 1 μmol/ L troglitazone (), or 1 μmol/l plus testosterone (). After incubation for 24 hours, cells were harvested, lysed and subsequently assays for luciferases and β-galactosidase activities. All values are expressed as mean± SD of relative luciferase units/β-galactosidase activity. P<.5, as compared with lane 1; P<.5, as compared with lane 2; P<.5, as compared with lane 3; P<.5, as compared with lane 4. 음을보여주었다. 또한 PPARγ 와 AR 로동시에 transfection 시킨세포는 PPARγ 로 transfection 시킨세포에비해 luciferase reporter activity 가 48.2% 감 소되었다 (P<.5) (Fig. 5, lane 3 vs lane 2). PPARγ 와 AR 로동시에 transfection 시킨세포에서 troglitazone 처리는 vehicle 처리에비해 luciferase reporter activity 를 139.9% 증가시켰지만 (P<.5) (Fig. 5, lane 4 vs lane 3), troglitazone 처리에의해증가된 luciferase reporter activity 는 troglitazone 과테스토스테론의동시처리에의해감소되었 으며 (P<.5) (Fig. 5, lane 5, 6, and 7 vs lane 4), 특히 troglitazone 과 1 μm 테스토스테론동시처리에의해 3.5% 감소되었다 (P<.5) (Fig. 5, lane 7 vs lane 4). 고찰 본연구는지방세포로분화된 33-L1 세포를이용한 in vitro 연구 를통해 PPARγ 활성에의해유도된지방세포생성에대한남성호르몬 인테스토스테론의억제적작용을조사하였다. 테스토스테론은분화 된지방세포에서의중성지방축적을감소시켰으며 PPARγ 와 PPARγ- 72

6 정선효 estosterone Inhibits PPARγ-induced Adipogenesis 의존적인지방세포특이적유전자의 mrna 발현도감소시켰다. 또한 PPARγ agonist인 troglitazone 처리에의해지방세포에축적된중성지방이테스토스테론처리에의해감소되었고, troglitazone 처리에의해증가된 PPARγ와 PPARγ-의존적인지방세포특이적유전자의 mrna 발현도테스토스테론처리에의해감소되었다. 추가적으로 PPARγ를발현시킨 33-L1 지방전구세포뿐만아니라 PPARγ와 AR 을발현시킨 33-L1 지방전구세포에서도 troglitazone에의해유도된 PPARγ reporter activity 는테스토스테론처리에의해억제되었다. 비만은생활습관의영향뿐만아니라성스테로이드호르몬의변화와도밀접한관련이있다. 여성의경우여성호르몬인 estrogen의생성이감소되는폐경시기가되면급속도로복부비만이진행된다. 28,29 여성뿐만아니라남성도나이가들어감에따라남성호르몬의분비가점점감소되고, 이시기부터남성은근육과뼈의양이감소되는데비해체지방의양이증가된다. 2 남성성스테로이드호르몬인 androgens 는근육, 지방, 연골및뼈세포로분화가가능한 C3H 11/2 pluripotent cells가근육세포로분화되는것을촉진하였지만지방세포로의분화는억제하였다. 3 또한 epididymal preadipocytes 의 primary culture에서의 androgens 처리는지방분화의후기단계표지자 (marker) 인 glycerol 3-phosphate dehydrogenase activity를감소시켰다. 8 이러한연구결과는지방세포생성에대한테스토스테론의억제작용을보여준본연구결과를뒷받침해주고있으며남성호르몬이직 간접적인조절기전을통해지방세포분화를억제한다는것을시사하고있다. 본연구에서 PPARγ agonist인 troglitazone에의해증가된중성지방축적및 PPARγ mrna 발현과 PPARγ- 의존적인지방세포특이적인유전자들의 mrna 발현이 troglitazone과테스토스테론의동시처리에의해감소되었다. 이러한연구결과는테스토스테론이 troglitazone에의해활성화된 PPARγ의작용을억제시킴으로써지방세포생성과정을억제한다는것을시사하고있다. Androgens (; 테스토스테론, dihydrotestosterone) 의작용은 androgen receptor (AR) 와의결합을통해조절된다. 31 AR의리간드인 androgen과의결합으로활성화된 AR은표적유전자에존재하는 androgen response elements와결합하여표적유전자의전사를조절한다. 32 그러나테스토스테론은 AR에의존적인메커니즘뿐만아니라 AR에비의존적인메커니즘을통해서도작용한다. 33 Bourghardt 등 34 의연구에의하면, 테스토스테론처리는고환이절제된 mice의 wild mice와 AR knockout (ARKO) mice 모두에서동맥경화증을개선하였다. 또한콜레스테롤이포함된먹이를섭취한 AR mutant testicular feminized mice (fm) 에서테스토스테론처리는지방선조 (fatty streak) 형성을감소시켰다. 35 반면콜레스테롤을섭취한토끼에서 AR blocker 인 flutamide 처리는동맥경화성플라크영역 (atherosclerotic plaque area) 을감소시키는테스토스테론의영향을 억제하였다. 36 이러한연구결과들은동맥경화증에대한테스토스테론의생리학적작용이 AR-의존적또는 AR-비의존적조절기전에의해조절된다는것을시사하고있다. 그러나지방세포생성에대한테스토스테론의생리학적작용은 AR-의존적조절기전에의해조절된다. 11/2 cells에서 androgens (; 테스토스테론, dihydrotesotosterone) 처리에의해감소되었던지방세포의수와지방세포생성의주요조절인자인 C/EBPα 단백질발현이 AR antagonist 인 bicalutamide 처리에의해지방세포생성에대한 androgens 의이러한효과가차단됨으로써, 지방세포생성에대한테스토스테론의작용이 AR-의존적조절기전에의해조절된다는것이보고되고있다. 3 또한지방세포생성에관한연구에널리사용되고있는 33-L1 지방전구세포는 AR이낮은수준으로발현되고있으며, 33-L1 지방전구세포를지방세포로분화시키는동안 AR의발현량은증가된다. 37 따라서본연구에사용된 33-L1 지방전구세포에는낮은수준의 AR이발현되어있으므로, PPARγ를 transfection시킨 33-L1 지방전구세포에서 AR을 transfection시키지않은경우뿐만아니라 AR을 transfection시킨경우에도 troglitazone에의해유도된 PPARγ reporter activity가테스토스테론처리에의해억제된본연구결과들은, 테스토스테론이 AR-의존적조절기전에의해지방세포생성에대한 PPARγ의작용을억제조절한다는것을시사하고있다. AR과결합한테스토스테론이 troglitazone에의해유도된 PPARγ 의전사활성을억제시킨본연구결과에대한분자세포생물학적조절기전의가능성은발표된몇몇논문들에의해설명될수있다. PPARγ 와성호르몬수용체 (; estrogen receptor 또는 androgen receptor) 는리간드에의해활성화되는 transcription factor 이다. 이러한수용체들의전사작용의활성은 transcriptional coactivators에의해증가되며이러한작용은리간드의존재에의존적이다. 38,39 최근연구결과에의하면 AR의대표적인 coactivators인 ARA7은 AR뿐만아니라 PPARγ 의 coactivators 로서작용한다고보고되고있고, dihydro테스토스테론에의해활성화된 AR이 PPARγ-ARA7 transactivation을방해함으로써지방세포에서 PPARγ-mediated response와 AR-mediated response 간에 negative signaling cross-talk 가일어난다는것을제시하고있다. 4 또한 androgen은 Wnt signaling pathway 에관여하여지방세포분화를억제한다. Androgen이 AR에결합함으로써발생하는세포내의현상으로 AR과 β-catenin이결합한후이결합체가세포의핵속으로이동하여 Wnt signaling pathway 에관여해서 CF4/LEF 의활성을촉진시킴으로써지방분화의주요인자인 PPARγ, C/EBP-α 및 C/EBP-δ의억제와관련된지방세포생성를억제하였다. 41 따라서이러한연구결과들을바탕으로, 테스토스테론에의해활성화된 AR에의해 troglitazone에의해유도된 PPARγ reporter activity가억제된다는본연구결과는, 테스토스테론이 AR의직 간접적인 73

7 정선효 estosterone Inhibits PPARγ induced Adipogenesis 조절기전에의해 troglitazone에의해유도된 PPARγ activity의작용을억제조절한다는것을시사하고있다. 결론적으로본연구는테스토스테론이 AR-의존적조절기전에의해 troglitazone에의해유도된 PPARγ의작용을억제시킴으로써지방세포생성를억제한다는것을제시하고있다. 또한본연구는테스토스테론이남성의지방조직대사와비만조절에중요한요인임을시사하고있고, 더나아가테스토스테론감소로인해발생하는지방조직대사및비만과관련된대사증후군의치료및예방에대한분자세포생물학적조절기전을이해하는데기초자료를제공할것으로기대된다. 요약 배경 : Peroxisome proliferator-activated receptor γ (PPARγ) 는지질대사와지방세포분화에대해중요한역할을한다. 또한테스토스테론은남성의지방세포대사작용을억제하는것으로알려져있다. 따라서본연구는테스토스테론에의한지방세포생성의억제조절기전을규명하기위해, in vitro 연구를이용하여지방세포에서의 PPARγ expression과 activity에대한테스토스테론의영향을조사하였다. 방법 : 분화된 33-L1 지방세포에 troglitazone과테스토스테론을처리한후, troglitazone에의해유도된중성지방축적과지방세포생성과관련된유전자들의발현에대한테스토스테론의영향을조사하였다. 또한테스토스테론이 33-L1 지방전구세포에서 troglitazone에의해유도된 PPARγ reporter activity를조절하는지에대해서도조사하였다. 결과 : 테스토스테론은분화된 33-L1 지방세포에서중성지방축적을감소시켰다. 그리고테스토스테론은 PPARγ mrna의발현뿐만아니라 PPARγ에의존적인지방세포특이적유전자인 adipocyte fatty acid binding protein 과 tumor necrosis factor α의발현을억제하였다. 테스토스테론의처리는분화된 33-L1 지방세포에서 troglitazone에의해유도된중성지방축적뿐만아니라 PPARγ와지방세포특이적유전자들의발현을억제하였다. 또한 PPARγ와 androgen receptor (AR) 가발현되는 33-L1 지방전구세포에서테스토스테론은 troglitazone에의해유도된 PPARγ reporter activity도감소시켰다. 결론 : 본연구결과는테스토스테론이 AR-의존적조절기전에의해지방세포생성에대한 PPARγ의작용을방해한다는것을시사하고있으며, 남성의비만조절에대한테스토스테론치료요법의유익한분자생물학적정보를제공할것으로사료된다. 중심단어 : 테스토스테론, troglitazone, PPARγ, 지방세포생성, 33-L1 지방세포 Conflicts of Interest he researcher claims no conflicts of interest. References 1. Hursting SD, Dunlap SM. Obesity, metabolic dysregulation, and cancer: a growing concern and an inflammatory (and microenvironmental) issue. Ann N Y Acad Sci 212;1271: Blouin K, Després JP, Couillard C, remblay A, Prud homme D, Bouchard C, et al. Contribution of age and declining androgen levels to features of the metabolic syndrome in men. Metabolism 25;54: Kapoor D, Malkin CJ, Channer KS, Jones H. Androgens, insulin resistance and vascular disease in men. Clin Endocrinol (Oxf) 25;63: Martin AC. Osteoporosis in men: a review of endogenous sex hormones and testosterone replacement therapy. J Pharm Pract 211;24: Mori Y, Hoshino K, Yokota K, Yokose, ajima N. Increased visceral fat and impaired glucose tolerance predict the increased risk of metabolic syndrome in Japanese middle-aged men. Exp Clin Endocrinol Diabetes 25;113: enover JL. Experience with testosterone replacement in the elderly. Mayo Clin Proc 2;75:S Wang C, Swerdloff RS, Iranmanesh A, Dobs A, Snyder PJ, Cunningham G, et al. ransdermal testosterone gel improves sexual function, mood, muscle strength, and body composition parameters in hypogonadal men. J Clin Endocrinol Metab 2;85: Dieudonne MN, Pecquery R, Leneveu MC, Giudicelli Y. Opposite effects of androgens and estrogens on aipogenesis in rat preadipocytes: evidence for sex and site-related specificities and possible involvement of insulin-like growth factor 1 receptor and peroxisome proliferator-activated receptor gamma2. Endocrinology 2;141: Pedersen SB, Børglum JD, Eriksen EF, Richelsen B. Nuclear estradiol binding in rat adipocytes. Regional variations and regulatory influences of hormones. Biochim Biophys Acta 1991;193: Liu L, Li Y, ollefsbol O. Gene-environment interactions and epigenetic basis of human diseases. Curr Issues Mol Biol 28;1: 74

8 정선효 estosterone Inhibits PPARγ-induced Adipogenesis Rosen ED, Spiegelman BM. Molecular regulation of aipogenesis. Annu Rev Cell Dev Biol 2;16: Green H, Meuth M. An established pre-adipose cell line and its differentiation in culture. Cell 1974;3: Cornelius P, MacDougald OA, Lane MD. Regulation of adipocyte development. Annu Rev Nutr 1994;14: Butterwith SC. Molecular events in adipocyte development. Pharmacol her 1994;61: Hu E, ontonoz P, Spiegelman BM. ransdifferentiation of myoblasts by the adipogenic transcriprion factors PPARγ and C/EBPα. Proc Natl Acad Sci U S A 1995;92: MacDougald OA, Lane MD. ranscriptional regulation of gene expression during adipocyte differentiation. Annu Rev Biochem 1995;64: Ntambi JM, Young-Cheul K. Adipocyte differentiation and gene expression. J Nutr 2;13:3122S-6S. 18. Kliewer SA, Forman BM, Blumberg B, Ong ES, Borgmeyer U, Mangelsdorf DJ, et al. Differential expression and activation of a family of murine peroxisome proliferator-activated receptors. Proc Natl Acad Sci U S A 1994;91: Rosen ED, Walkey CJ, Puigserver P, Spiegelman BM. ranscriptional regulation of adipogenesis. Genes Dev 2;14: Gimble JM, Robinson CE, Wu X, Kelly KA, Rodriguez BR, Kliewer SA, et al. Peroxisome proliferator-activated receptor-gamma activation by thiazolidinediones induces adipogenesis in bone marrow stromal cells. Mol Pharmacol 1996;5: Sottile V, Seuwen K. Bone morphogenetic protein-2 stimulates adipogenic differentiation of mesenchymal precursor cells in synergy with BRL (rosiglitazone). FEBS Lett 2;475: Spiegelman BM, Choy L, Hotamisligil GS, Graves RA, ontonoz P. Regulation of adipocyte gene expression in differentiation and syndromes of obesity/diabetes. J Biol Chem 1993;268: Kern PA, Saghizadeh M, Ong JM, Bosch RJ, Deem R, Simsolo RB. he expression of tumor necrosis factor in human adipose tissue. Regulation by obesity, weight loss, and relationship to lipoprotein lipase. J Clin Invest 1995;95: Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature 1994;372: Gordon GB, Newitt JA, Shantz LM, Weng DE, alalay P. Inhibition of the conversion of 33 fibroblast clones to adipocytes by dehydroepiandrosterone and related anticarcinogenic steroids. Cancer Res 1986;46: Lea-Currie YR, Monroe D, Mcintosh MK. Dehydroepiandrosterone and related steroids alter 33-L1 preadipocyte proliferation and differentiation. Comp Biochem Physiol C Pharmacol oxicol Endocrinol 1999;123: McIntosh M, Hausman D, Martin R, Hausman G. Dehydroepiandrosterone attenuates preadipocyte growth in primary cultures of stromal-vascular cells. Am J Physiol 1998;275(2 Pt 1):E Fukunaga, Abe, Ishida Y, Kondoh M. Subcutaneous fat and muscle distribution patterns in middle and old aged Japanese. J herm Biol 1993;18: Jeong S, Han M, Lee H, Kim M, Kim J, Nicol CJ, et al. Effects of fenofibrate on high-fat diet-induced body weight gain and adiposity in female C57BL/6J mice. Metabolism 24;53: Singh R, Artaza JN, aylor WE, Gonzalez-Cadavid NF, Bhasin S. Androgens stimulate myogenic differentiation and inhibit aipogenesis in C3H 11/2 pluripotent cells through an androgen receptor-mediated pathway. Endocrinology 23;144: Dieudonne MN, Pecquery R, Boumediene A, Leneveu MC, Giudicelli Y. Androgen receptors in human preadipocytes and adipocytes: regional specificities and regulation by sex steroids. Am J Physiol 1998;274(6 Pt 1):C Claessens F, Denayer S, Van ilborgh N, Kerkhofs S, Helsen C, Haelens A. Diverse roles of androgen receptor (AR) domains in AR-mediated signaling. Nucl Recept Signal 28;6:e Liu PY, Death AK, Handelsman DJ. Androgens and cardiovascular disease. Endocr Rev 23;24: Bourghardt J, Wilhelmson AS, Alexanderson C, De Gendt K, Verhoeven G, Krettek A, et al. Androgen receptor-dependent and independent atheroprotection by testosterone in male mice. Endocrinology 21;151: Nettleship JE, Jones H, Channer KS, Jones RD. Physiological testosterone replacement therapy attenuates fatty streak formation and improves high-density lipoprotein cholesterol in the fm mouse: an effect that is independent of the classic androgen receptor. Circulation 27;116: Li S, Li X, Li Y. Regulation of atherosclerotic plaque growth and stability by testosterone and its receptor via influence of inflammatory reaction. Vascul Pharmacol 28;49:

9 정선효 estosterone Inhibits PPARγ induced Adipogenesis 37. Hartig SM, Feng Q, Ochsner SA, Xiao R, McKenna NJ, McGuire SE, et al. Androgen receptor agonism promotes an osteogenic gene program in preadipocytes. Biochem Biophys Res Commun 213;434: Arany Z, Sellers WR, Livingston DM, Eckner R. E1A-associated p3 and CREB-associated CBP belong to a conserved family of coactivators. Cell 1994;77: Yeh S, Chang C. Cloning and characterization of a specific coactivator, ARA7, for the androgen receptor in human prostate cells. Proc Natl Acad Sci U S A 1996;93: Heinlein CA, ing HJ, Yeh S, Chang C. Identification of ARA7 as a ligand-enhanced coactivator for the peroxisome proliferatoractivated receptor gamma. J Biol Chem 1999;274: Singh R, Artaza JN, aylor WE, Braga M, Yuan X, Gonzalez-Cadavid NF, et al. estosterone inhibits adipogenic differentiation in 33-L1 cells: nuclear translocation of androgen receptor complex with beta-catenin and -cell factor 4 may bypass canonical Wnt signaling to down-regulate adipogenic transcription factors. Endocrinology 26;147: