<30355F D D DBFF8C0FA2DC0B1C5C2BDC2B9DAB1D9B1D428B0A3C7E0C0A7BFF8BCF6C1A4295FC0FAC0DAB1B3C1A4B9DDBFB528C8E6B9E9292E687770>

Size: px

Start display at page:

Download "<30355F D D DBFF8C0FA2DC0B1C5C2BDC2B9DAB1D9B1D428B0A3C7E0C0A7BFF8BCF6C1A4295FC0FAC0DAB1B3C1A4B9DDBFB528C8E6B9E9292E687770>"

수인 송
6 years ago
Views:

1 대한내분비학회지 : 제 23권제 1 호 2008 원 저 /jkes 알파 - 리포산이 HepG2 간세포에서 SREBP-1c 발현에미치는효과 계명대학교의과대학내과학교실, 경북대학교의과대학내과학교실 1 윤태승 민애경 김남경 김미경 조호찬 김혜순 황재석 류성열 박근규 이인규 1 Effects of Alpha-lipoic Acid on SREBP-1c Expression in HepG2 Cells Tae Sung Yun, Ae Kyung Min, Nam Kyung Kim, Mi-Kyung Kim, Ho Chan Cho, Hye Soon Kim, Jae-Seok Hwang, Seong-Yeol Ryu, Keun-Gyu Park, In-Kyu Lee 1 Department of Internal Medicine, Keimyung University School of Medicine; and Department of Internal Medicine, Kyungpook National University School of Medicine 1 ABSTRACT Background: Non-alcoholic fatty liver disease is common in patients with insulin resistance. Sterol regulatory element binding protein-1c (SREBP-1c) is a member of a family of transcription factors that have been recognized as key regulators for lipid accumulation in the liver that activate enzymes involved in the fatty acid biosynthetic pathway. This study was designed to evaluate whether alpha-lipoic acid (ALA) inhibits insulin-stimulated SREBP-1c expression. Methods: We investigated the effects of ALA on insulin-stimulated SREBP-1c expression in a human hepatoma cell line (HepG2 cells) using Northern and Western blot analysis. We also examined the effect of ALA on the promoter activity of the SREBP-1c gene to examine whether ALA can affect SREBP-1c expression at the transcriptional level. To discern the mechanism by which ALA inhibits SREBP-1c expression, we examined the role of AMP-activated protein kinase (AMPK). Results: Insulin increased the expression of SREBP-1c mrna and protein in HepG2 cells in a dose depended manner. Co-treatment with ALA inhibited the insulin increased SREBP-1c expression in a dose-dependent manner. ALA also inhibited insulin-stimulated activation of the SREBP-1c promoter activity, indicating that ALA inhibited SREBP-1c expression at the transcriptional level. ALA increased phosphorylation of AMPK in HepG2 cells. Inhibition of the AMPK activity by compound C markedly reversed the inhibitory effects of ALA for insulin-stimulated SREBP-1c expression. These results suggest that ALA-induced suppression of SREBP-1c expression is at least in part mediated via AMPK activation. Conclusion: The present study suggests that ALA has an inhibitory effect on insulin-stimulated SREBP-1c expression. Therefore, further studies on the effects of ALA on hepatic steatosis in an animal model need to be performed. (J Kor Endocr Soc 23:27~34, 2008) ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ Key Words: alpha-lipoic acid, insulin resistance, non-alcoholic fatty liver disease, SREBP-1c 1) 접수일자 : 2007년 8월 2일통과일자 : 2007년 11월 13일책임저자 : 박근규, 계명대학교의과대학내과학교실 * 본연구는대한내분비학회연구지원 (2006년) 으로수행되었고 2006년도정부재원 ( 교육인적자원부학술연구조성사업비 ) 으로한국학술진흥재단의지원을받아연구되었음 (KRF E00133)

2 대한내분비학회지 : 제 23 권제 1 호 2008 서론비알콜성지방간질환은알코올섭취력은거의없지만알코올성지방간과유사한간조직소견을가지는질환으로 [1] 지방간에서부터지방간염, 간경변증에이르기까지다양한병변을통칭하는표현이다 [2]. 비알콜성지방간질환은최근여러연구를통하여비만, 당뇨병, 고혈압, 지질대사이상등을포함하는대사증후군과관련이있음이입증되었고, 그중에서도비만이중요한원인인자로알려져있다 [3]. 비만으로인한고인슐린혈증은비알콜성지방간질환의병태생리학적발생기전의원인으로생각되고비알콜성지방간질환을간에서일어나는대사증후군의한형태로보는견해가우세하다 [4,5]. 비알콜성지방간질환의발병시작은중성지방합성의증가로인해세포내중성지방이증가하는것이특징으로인슐린은간의지방생합성에중요한호르몬중의하나다. 고인슐린혈증으로인한간의지방산합성은지방산생합성경로에중요한효소인, acetyl-coa carboxylase (ACC) 와 fatty acid synthase (FAS) 유전자의전사활성이증가하여일어나며 [6] 이과정에 sterol regulatory element binding protein (SREBPs) 가인슐린의역할을매개하는중요한전사인자로알려져있다 [7,8]. SREBPs는지방산과콜레스테롤의생합성경로에관련되는효소를활성화하여간에서지방산과콜레스테롤합성을조절하는중요한전사활성인자이다. SREBP에는 1a, 1c 및 2의세가지종류가있으며, SREBP-1a와 SREBP-1c는주로지방산및중성지방의합성에관여하고 SREBP-2는콜레스테롤대사에관여하는것으로알려져있다 [9]. 간조직에서는 SREBP-1c의발현이우세하며 [10] 간세포에서의중성지방합성과관련된 FAS, ACC, stearoyl-cap desaturase, ATP citrate lyase, malic enzyme과같은유전자의발현을조절한다 [11]. 인슐린저항성으로인한고인슐린혈증은간의 SREBP-1c 의발현을증가시킴으로써지방산의생합성을증가시키고결과적으로간조직에중성지방의축적을유발한다 [12,13]. 이러한인슐린저항성과간에서중성지방의축적에대한 SREBP-1c의역할은동물실험을통해서증명되었다. 고도비만과인슐린저항성의특징인 ob/ob mice에서지방간병변이관찰되고 [14] ob/ob mice의 Srebp-1 유전자를비활성화시켰을때간조직에중성지방의축적이약 50% 정도감소함을보고한연구를 [15] 통해 SREBP-1c가인슐린저항성동물모델에서지방간의발생에중요한역할을담당함을알수있다. 알파- 리포산 (alpha-lipoic acid, ALA) 은 metal chelating, 활성산소제거, 내인성항산화제재생, 산화적손상복구의기능을가지고있는강력한항산화물질로서 [16] 미토콘드리아내에서호흡효소의보조인자로서작용한다 [17]. 최근 Lee 등의연구에서알파 -리포산이 Otuska Long Evans Tokushima Fatty (OLETF) 쥐의골격근에서 AMP-activated protein kinase (AMPK) 를활성화하여인슐린감수성을증가시켰고중성지방축적을감소시켰다고보고하였다 [18]. 따라서, 본연구자들은알파 -리포산이간조직에서도 AMPK를활성화시키는지, 그리고알파 -리포산이지방세포에서 SREBP-1c 의발현과활성에어떠한미치는지영향을살펴보고자본연구를수행하였다. 대상및방법 1. 재료 SREBP-1c 항체는 BD Bioscience (San Jose, CA) 에서구입하였고 phospho-ampk 항체는 Cell signaling Technology (Danvers, MA), actin 항체는 Sigma (Saint Louis, MD) 에서구입하였다. [α- 32 P]dCTP는 Amersham Biosciences (Little Chalfont, UK) 에서구입하였다. 실험에사용된인슐린은 Novo nordisk (Bagsvrd, Denmark) 로부터, 알파- 리포산은 Viatris GmbH & Co. KG (Frankfurt, Germany) 로부터제공받았다. 2. 세포배양사람간암세포주 (human hepatoma cell line) 인 HepG2 세포는 minimum essential medium (MEM) 배지에항생제 (antibiotics) 와 fetal bovine serum (FBS) 을첨가하여배양하였다. 세포는일정한습도를유지하는 37 항온기에서공기 (95%) 와 CO 2 (5%) 의혼합기체를공급하면서배양하고, 3~4일마다계대배양하였다. HepG2 세포는인슐린처리하기전 0.5% FBS가첨가된 MEM 배지에서 24시간동안배양하여세포들을휴지기에들어가게하였다. 그리고인슐린은 6시간동안농도별로처리하였고, 인슐린을처리하기 18 시간전에알파 -리포산을농도별로처리하고각실험에필요한조건을주어 SREBP-1c의발현을확인하였다. 알파- 리포산에의한 p-ampk의발현은농도별로확인하기위해서알파- 리포산을 0.5, 1, 2 mmol/l로 24시간동안처리하였다. 3. 노던블롯 (Northern Blot) 분석노던블롯에사용된 SREBP-1c에대한방사선표지소식자는 [α- 32 P]2 -deoxycytidine 5 -triphosphate (dctp) 를이용한임의프라이머라벨방법 (random primer DNA labelling kit system, Amersham, Arlington Heights, IL) 으로제작하였다. 방사선표지소식자는 NAP-5 Column (Pharmacia, Uppsala, Sweden) 으로정제하였다. 이후, 20 μg의 RNA를 1% 포름알데하이드-아가로스겔에전기영동을시행한후나일론막으로이동시켰다. RNA가이동된나일론막은 UV cross-linker를사용하여 RNA를나일론막에고정하였다. 이후나일론막을방사선표지소식자와함께 Express

0, 150 mm NaCl, 5 mm EDTA, 1% NP-40, 100 μm PMSF), 1 μg/ml 단백질분해효소억제제 (Luepetin, Aprotinin), 1 mm DTT를처리하여단백질을추출하였다. 각시료를시료완충액과섞어 5분간끓인후얼음위에서식혔다.

3 - 윤태승외 9 인 : 알파 - 리포산이 HepG2 간세포에서 SREBP-1c 발현에미치는효과 - Hyb TM 용액에서 24시간동안 65 에서보합결합 (hybridization) 시킨후여러차례정해진규칙에따라세정을시행하였다. 세정이끝난나일론막은 -70 에서 24~48시간동안 X-ray 필름에노출시킨후 mrna 발현을밀도계측기 (densitometer) 를이용하여분석하였다. 4. 웨스턴블롯 (Western Blot) 분석 Hep G2 세포에 IPH 완충액 (50 mm Tris-HCl, ph 8.0, 150 mm NaCl, 5 mm EDTA, 1% NP-40, 100 μm PMSF), 1 μg/ml 단백질분해효소억제제 (Luepetin, Aprotinin), 1 mm DTT를처리하여단백질을추출하였다. 각시료를시료완충액과섞어 5분간끓인후얼음위에서식혔다. Sodium dodecyl sulfate-polyacrylamide 겔에서전기영동하여단백질은크기별로분리한후 Immobilon-P transfermembrane (Milipore, Billerica) 으로이동시켰다. 차단완충액 (blocking buffer) 으로차단하고 SREBP-1c 항체및 phospho-ampk 항체로 4 에서 16시간반응시킨후, horseradish peroxidase 가표지된이차항체로상온에서 2시간반응시켰다. ECL plus (Amersham Biosciences) 를사용하여단백질발현을확인하였다. Membrane을 actin 항체와다시반응시켜일정한양의단백질을사용하였는지확인하였다. 5. Luciferase 활성측정인슐린과알파-리포산이 SREBP-1c 전사활성에미치는영향을알아보고자 Lipofectamin (Invitrogen, Carlsbad, CA) 을이용하여 HepG2 세포에 SREBP-1c promoter construct (300 ng/well) 를과발현시켰고형질도입의효율을보정하기위하여 pcmv-β-gal plasmid (200 ng/well) 를 co-transfection 하였다. Human SREBP-1c promoter ( ) 는 Dr. Tarling으로부터제공받아사용하였다 [19]. 형질도입 5시간후에 0.5% FBS가들어있는배지로교체를해준후, 24시간더배양하였다. 이후세포를 PBS로두번세척하고 100 μl의 reporter lysis buffer (Promega, WI, USA) 를이용하여분해하였다. 4, 1200 rpm에서 10분동 안원심분리후 20 ul의상등액을발광분석기 (SIRUS Luminometer; Berthold, Pforzheim, Germany) 를사용하여전사활성을측정하였다. β-galactosidase 활성은상등액 20 ul를 ELISA reader를이용하여흡광도를 420 nm에서측정하고이값을 luciferase 활성수치를보정하는데사용하였다. 5. 통계학적인처리결과들은평균 ± 표준오차로표시하고, 변수의분석들은 Duncan's test를사용하였다. P값이 0.05 미만인경우에통계적으로유의하다는판정을하였으며, 모든실험은독립적으로 3회이상실시하여통계처리를하였다. 결과 1. 인슐린이 SREBP-1c 발현에미치는영향인슐린이 SREBP-1c mrna 발현에미치는영향을알아보기위해 HepG2 세포에인슐린을용량별로처리하고노던블롯을시행하였다. SREBP-1c mrna의발현은인슐린의용량을 50 nmol/l에서 200 nmol/l로증가시킴에따라현저히증가하였다 (Fig. 1A). 인슐린투여가 SREBP-1c 단백질발현과활성형 SREBP-1c로전환에미치는효과를알아보기위해인슐린을용량별로처리하고 SREBP 전구형과활성형의양을웨스턴블롯을이용하여측정하였다. 인슐린은 HepG2세포에서전구형및활성형 SREBP-1c 단백질발현을용량의존적으로증가시켰다 (Fig. 1B). 2. 알파-리포산이 SREBP-1c 발현에미치는효과알파- 리포산이인슐린에의해증가된 SREBP-1c의발현에미치는효과를알아보기위해 HepG2 세포에알파- 리포산을농도별로 18시간전처치후 100 nm 인슐린을처리하고 SREBP-1c 단백질발현변화를웨스턴블롯을통해확인하였다. 알파- 리포산을 0.5, 1, 2 및 4 mmol/l의농도로처리하였을경우인슐린에의해증가된전구형 SREBP-1c의단백질발현이용량 -의존적으로감소하였고, 활성형 SREBP-1c A B Fig. 1. Effects of insulin on SREBP-1c mrna and protein expressions in HepG2 cells. A. Northern blot analysis of the effect of insulin on SREBP-1c mrna expression in HepG2 cell. B. Western blot analysis of the effect of insulin on SREBP-1c protein expression in HepG2 cells. Cells were treated for 24 hours with 50, 100, 200 nmol/l of insulin. The protein levels were normalized by β-actin levels

대한내분비학회지 : 제 23 권제 1 호 2008 A B Fig. 2. Effect of ALA on insulin-stimulated SREBP-1c protein expression in HepG2 cells. A. Western blot analysis of the effect of ALA on insulin-stimulated SREBP-1c protein expression.

Quantification of data expressed as mean ± SEM of three separate measurements. Statistical significance was determined as *P < 0.01, and **P < 0.001 compared with insulin alone. Fig. 3.

HepG2 cells were transfected with a SREBP-1c-promoter-luciferase construct (300 ng/well) and then stimulated with 100 nmol/l insulin for 6 h, with or without pretreatment with the indicated dosages

4 대한내분비학회지 : 제 23 권제 1 호 2008 A B Fig. 2. Effect of ALA on insulin-stimulated SREBP-1c protein expression in HepG2 cells. A. Western blot analysis of the effect of ALA on insulin-stimulated SREBP-1c protein expression. HepG2 cells were treated with 100 nmol/l insulin for 6 h, with or without pretreatment with the indicated dosages of ALA for 24 h. The protein levels were normalized by β-actin levels. B. Quantification of data expressed as mean ± SEM of three separate measurements. Statistical significance was determined as *P < 0.01, and **P < compared with insulin alone. Fig. 3. Effect of ALA on insulin-stimulated SREBP-1c promotor activity. HepG2 cells were transfected with a SREBP-1c-promoter-luciferase construct (300 ng/well) and then stimulated with 100 nmol/l insulin for 6 h, with or without pretreatment with the indicated dosages of ALA for 24 h. Data are presented as the mean ± SEM of three separate measurements. Statistical significance was determined as *P < compared with control, **P < 0.05, #P < 0.01, ##P < compared with insulin alone. 단백질의발현도알파 -리포산에의해용량의존적으로감소하였다 (Fig. 2). 3. 알파 - 리포산이 SREBP-1c 전사활성에미치는효과 알파- 리포산이 SREBP-1c의발현을억제하는효과가전사수준에서일어나는지를알아보기위하여 luciferase assay 를통해 HepG2세포에 human SREBP-1c 전사조절부위 (promoter) 를발현시키고인슐린단독혹은인슐린과알파- 리포산의동시처리가 SREBP-1c촉진자의활성도에미치는효과를측정하였다. 인슐린 (100 nmol/l) 은 SREBP-1c 촉진자의활성을현저히증가시켰고, 알파- 리포산을 0.5, 1, 2, 및 4 mmol/l로처리할경우인슐린에의하여증가된 SREBP-1c 전사활성이용량의존적으로억제되었다 (Fig. 3). 4. 알파 - 리포산이 AMPK 활성에미치는효과 다음으로알파- 리포산이간세포에서 AMPK를활성화시키는지를알아보기위하여 HepG2 세포에알파-리포산을용량별로처리하고 AMPK의인산화를측정하였다. 알파- 리포산처리에의해전체 AMPK 발현은변화가없었으나, AMPK의인산화는용량의존적으로증가하였다 (Fig. 4). 5. AMPK 억제제가알파 - 리포산에의한 SREBP-1c 억제효과에미치는효과 마지막으로인슐린에의해증가된 SREBP-1c 단백질발현을억제하는알파 -리포산에대한효과가 AMPK를통해이루어지는지를알아보기위해 AMPK 억제제인 compound C 를처리하여 SREBP-1c 단백질발현의변화를웨스턴블롯으로측정하였다. 알파-리포산은인슐린에의해증가된

- 윤태승외 9 인 : 알파 - 리포산이 HepG2 간세포에서 SREBP-1c 발현에미치는효과 - A B Fig. 4. Effect of ALA on phosphorylation of AMPK in HepG2 cells. A. Western blot analysis of the effect of ALA on AMPK phosphorylation.

Quantification of data expressed as mean ± SEM of three separate measurements. Statistical significance was determined as *P < 0.05, **P < 0.01 and #P < 0.001 compared with basal expression.

HepG2 cells were treated with 100 nmol/l insulin for 6 h, without or with 5, 10 um of compound C. B. Quantification of data expressed as mean ± SEM of three separate measurements.

SREBP-1c 단백질발현을억제하였고 compound C를 5, 10 um 처리하였을때알파- 리포산에의해억제된 SREBP-1c 단백질발현이약 50% 정도회복되었다 (Fig. 5). 고찰비알코올성지방간질환은간에서발현되는대사증후군의한형태이다 [3].

5 - 윤태승외 9 인 : 알파 - 리포산이 HepG2 간세포에서 SREBP-1c 발현에미치는효과 - A B Fig. 4. Effect of ALA on phosphorylation of AMPK in HepG2 cells. A. Western blot analysis of the effect of ALA on AMPK phosphorylation. HepG2 cells were treated with 0.5, 1, 2 mmol/l ALA. The levels of AMPK phosphorylation were normalized by total AMPK protein levels. B. Quantification of data expressed as mean ± SEM of three separate measurements. Statistical significance was determined as *P < 0.05, **P < 0.01 and #P < compared with basal expression. A B Fig 5. Effect of Compound C on ALA inhibition of insulin-stimulated SREBP-1c protein expression. A. Western blot analysis of the effect of compound C on ALA inhibition of insulin-stimulated SREBP-1c protein expression. HepG2 cells were treated with 100 nmol/l insulin for 6 h, without or with 5, 10 um of compound C. B. Quantification of data expressed as mean ± SEM of three separate measurements. Statistical significance was determined as *P < compared with control, **P < 0.001, #P < 0.01 compared with insulin alone. SREBP-1c 단백질발현을억제하였고 compound C를 5, 10 um 처리하였을때알파- 리포산에의해억제된 SREBP-1c 단백질발현이약 50% 정도회복되었다 (Fig. 5). 고찰비알코올성지방간질환은간에서발현되는대사증후군의한형태이다 [3]. 인슐린저항성에의한고인슐린혈증은간조직에서 SREBP-1c를활성화시키고지방합성에중요효소인 FAS, ACC 및 SCD 등의발현을증가시켜지방축적을유발한다 [11]. 최근여러연구에서세포내에너지항상성을조절하는 AMPK가간조직에서의지방대사에중요한역할을수행함이보고되었다 [20]. AMPK는세포의에너지수준을감지하는역할을하는이질이합체로세포내 AMP가증가되면활성화되어지방산의 β-산화를자극하고지방생성을억제한다 [21]. AMPK가인산화되면지방합성기전에관 여하는 ACC와같은효소들이비활성화되어간의지방산합성이감소하게된다. 또한인산화된 AMPK는 SREBP-1c의발현을억제하여 SREBP-1c의표적유전자로알려진 FAS, L-type pyruvate kinase (LPK) 등의발현을감소시킨다. 경구용혈당강하제인 metformin과 thiazolidinedione계열의약물인 pioglitazone과 rosigitazone이간세포에서 AMPK를활성화시켜 SREBP-1c 발현을감소시킬뿐만아니라, SREBP-1c의표적유전자를감소시켜간에서의지방합성을억제함이보고되었다 [22~25]. 따라서본연구자들은간세포에서알파-리포산이 AMPK를활성시키는지와 AMPK 활성을통해 SREBP-1c의발현을억제시키는지를알아보았다. 알파- 리포산은자연적으로발생하는 short-chain 지방산으로두개의 sulfur 분자를가지고있으며강력한항산화효과가있다. 또한, 알파- 리포산은미토콘드리아의호흡효소의중요한보조인자로작용함으로써미토콘드리아의기능을향상시킨다 [16,17]. 최근 Lee 등은비만쥐의혈관내피

6 대한내분비학회지 : 제 23 권제 1 호 2008 세포에서 AMPK 활성의감소가혈관내피세포의기능이상을발생시키는데중요한역할을하며알파- 리포산을처리하였을때혈관내피세포의 AMPK를활성화시킴으로써혈관기능이상을개선시킬수있다고보고하였다 [26]. 또한근육세포에서알파- 리포산이 AMPK를활성화시켜인슐린감수성을개선시키고, 중성지방축적을감소시킨다고보고하였다 [18]. 본연구결과사람의간암세포주인 HepG2 세포에알파- 리포산을처리할경우용량의존적으로 AMPK의인산화가증가됨을확인함으로써알파- 리포산이간조직에서도 AMPK의활성화시킬수있음을알수있었다. 또한 compound C로 AMPK의활성을억제하였을경우알파- 리포산에의해억제된 SREBP-1c의발현이의미있게회복되어알파-리포산이인슐린에의해증가된 SREBP-1c 발현을억제하는기전에 AMPK의활성화가관여함을알수있었다. SREBP family는구조적으로 basic helix-loop-helix (bhlh) 형태를가진지방분화에관여하는전사인자로서, 이들중 SREBP-1c 단백질은소포체 (endoplasmic reticulum) 의막에비활성화된상태로결합되어있다가콜레스테롤의결핍이나인슐린에의해 SREBP cleavage-activation protein (SCAP) 이활성화되면 SREBP-1c는골지체로이동하게되며, 이후에 Site 1 Protease (S1P) 와 Site 2 Protease (S2P) 에의해분해되어활성형이핵으로이동하여 SREBP-1c의표적유전자의발현을유도한다 [11]. 스트렙토조토신 (steroptozotocin) 으로당뇨병을유발한쥐모델에서 SREBP-1c mrna가감소하고인슐린을주입하였을때증가한다고보고되었고 [27] 이러한생리학적변화는일차배양한쥐의간세포에서인슐린이 SREBP-1c의유전자의전사활성의증가를통하여일어남이증명되었다 [13,28,29]. 본연구자는 HepG2 세포에인슐린을처리한후 SREBP-1c의발현을관찰하였는데, 인슐린에의해 SREBP-1c 전사조절부위의활성과 SREBP-1c mrna, 전구형및활성형단백질의발현이모두증가함을관찰하였다. 또한알파 -리포산은인슐린에의해증가된 SREBP-1c의발현을억제하였고전구형 SREBP-1c 단백질에서활성형 SREBP-1c 단백질로의전환을억제함을관찰하였다. 알파- 리포산은인슐린에의해증가된 SREBP-1c 전사조절부위의활성을억제하였는데이러한결과는알파 -리포산의 SREBP-1c 발현억제효과는 SREBP-1c의전사활성을억제함으로써일어남을보여준다. 이상의결과를요약하면, 인슐린은 HepG2세포에서 SREBP-1c의 mrna와전구형단백질및활성형단백질발현을증가시켰고, 알파- 리포산은인슐린에의하여증가된 SREBP-1c 전사촉진자활성과총단백질및활성형단백질의발현을억제하였다. 알파- 리포산이 SREBP-1c의발현억제기전에는 AMPK의활성화가부분적으로관여할것으로사료된다. 따라서, 알파- 리포산이 SREBP-1의발현억제기 전에 AMPK 활성이외의다른기전이있는지에대한실험이필요할것으로생각되며또한동물실험을통해알파- 리포산이지방간의발생을예방할수있는지를알아보고나아가인슐린저항성을가진지방간환자를대상으로알파- 리포산이지방간의치료에유용한지를알아보는임상연구가필요할것으로생각된다. 요약연구배경 : 비알콜성지방간은인슐린저항성이있는환자에서흔히발생하는간질환이다. SREBP-1c는지방산의생합성에관여하는효소를활성화시켜서간내지방축적을일으키는중요한전사조절인자이다. 본연구는알파-리포산이간세포에서인슐린에의해증가된 SREBP-1c 발현에미치는영향을알아보고자하였다. 방법 : 간세포로 human hepatoma cell line인 HepG2 세포를사용하였다. 인슐린과알파 -리포산이 SREBP-1c 발현에미치는영향을노던블롯과웨스턴블롯으로확인하였다. SREBP-1c 전사조절부위 (promoter) 활성분석을통해알파- 리포산이 SREBP-1c의전사조절에관여하는지알아보았다. 알파-리포산이 AMPK 활성에미치는효과는 AMPK의인산화로평가하였으며 AMPK 억제제인 compound C를이용하여알파-리포산의억제효과가 AMPK를통해이루어지는지알아보았다. 결과 : 인슐린은용량- 의존적으로 HepG2 세포에서 SREBP-1c mrna 및단백질발현을증가시켰다. 알파- 리포산은인슐린에의해증가된 SREBP-1c의발현을억제하였다. 알파- 리포산은인슐린에의해증가된 SREBP-1c 전사촉진자의활성을억제하였다. 알파- 리포산은 HepG2 세포에서 AMPK의인산화를증가시켰다. AMPK 활성억제제인 compound C는알파 -리포산에의한 SREBP-1c 발현억제효과를의미있게회복시켰다. 결론 : 알파- 리포산은 HepG2 세포에서인슐린에의해증가된 SREBP-1c의발현을효과적으로억제함을알수있었다. 향후알파 -리포산이고인슐린혈증에의한지방간의발생에대한효과가있는지에대한동물및임상실험이추가적으로필요할것으로사료된다. 참고문헌 1. Clark JM, Brancati FL, Diehl AM: Nonalcoholic fatty liver disease. Gastroenterology 122: , Angulo P: Nonalcoholic Fatty liver disease. N Eng J Med 346: , Marceau P, Biron S, Hould FS, Marceau S, Simard S, Thung SN, Kral JG: Liver pathology and the

7 - 윤태승외 9 인 : 알파 - 리포산이 HepG2 간세포에서 SREBP-1c 발현에미치는효과 - metabolic syndrome X in severe obesity. J Clin Endocrinol Metab 84: , Kim TH, Yoo K: Obesity and Fatty liver disease. Korean J Intern Med 68: , Marchesini G, Brizi M, Bianchi G, Tomassetti S, Bugianesi E, Lenzi M, McCullough AJ, Natale S, Forlain G, Melchionda N: Nonalcholic fatty liver disease; a feature of the metabolic syndrome. Diabetes 50: , Hillgartner FB, Salati LM, Goodridge AG: Physiological and molecular mechanisms involved in nutritional regulation of fatty acid synthase. Physiol Rev 75:47-76, Horton JD, Bashmakov Y, Shimomura I, Shimano H: Regulation of sterol regulatory element binding proteins in livers of fasted and refed mice. Proc Natl Acad Sci USA 95: , Foretz M, Pacot C, Dugail I, Lemarchand P, Guichard C, Lie`pvre XL, Berthelier-Lubrano C, Spiegelman B, Kim JB, Ferre P, Foufelle F: ADD1/SREBP-1c is required in the activation of hepatic lipogenic gene expression by glucse. Mol Cell Biol 19: , Brown MS, Goldstein JL: The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 89: , Shimomura I, Shimano H, Horton JD, Goldstein JL, Brown MS: Differential expression of exons 1a and 1c in mrnas for sterol regulatory element binding protein-1 in human and mouse organs and cultured cells. J Clin Invst 99: , Horton JD, Goldstein JL, Brown MS: SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest 109: , Shimomura I, Bashmakov Y, Horton JD: Increased levels of nuclear SREBP-1c associated with fatty livers in two mouse models of diabetes mellitus. J Biol Chem 274: , Foretz M, Guichard C, Ferre P, Foufelle F: Sterol regulatory element binding protein-1c is a major mediator of insulin action on the hepatic expression of glucokinase and lipogenesis-related genes. Proc Natl Acad Sci USA 96: , Halaas, JL.Gajiwala KS, Maffei M, Cohen SL, Chait BT, Rabinowitz D, Lallone RL, Burley SK, Friedman JM: Weight-reducing effects of the plasma protein encoded by the obese gene. Science 269: , Yahagi N, Shimano H, Hasty AH, Matsuzaka T, Ide T, Yoshikawa T, Amemiya-Kudo M, Tomita S, Okazaki H, Tamura Y, Lizuka Y, Ohashi K, Osuga J, Harada K, Gotoda T, Nagai R, Ishibashi S, Yamada N: Absence of sterol regulatory element-binding protein-1 (SREBP-1) ameliorates fatty livers but not obesity or insulin resistance in Lepob/Lepob mice. J Biol Chem 277: , Biewenga GP, Haenen SR, Bast A: The pharmacology of the antioxidant lipoic acid. Gen pharmacol 29: , Packer K, Kraemer K, Rimbach G: Molecular aspects of lipoic acid in the prevention of diabetes complications. Nutrition 17: , Lee WJ, Song KH, Koh EH, Won JC, Kim HS, Park HS, Kim MS, Kim SW, Lee KU, Park JY: Alpha-lipoic acid increases insulin sensitivity by activating AMPK in skeletal muscle. Biochem Biophys Res Commun 332: , Tarling E, Salter A, Bennett A: Transcriptional regulation of human SREBP-1c(sterol-regulataroy-element-binding protein -1c) : a key regulator of lipogenesis. Biochem Soc Trans 32: , You M, Matsumoto M, Pacold CM, Cho WK, Crabb DW: The role of AMP-activated protein kinase in the action of ethanol in the liver. Gastroenterology 127: , Hardie DG: Minireview. The AMP-activated protein kinase cascade: the key sensor of cellular energy status. Endocrinology 144: , Zhou G, Myers R, Li Y, Chen Y, Shen X, Fenyk -Melody J, Wu M, Ventre J, Doebber T, Fujii N, Musi N, Hirshman MF, Goodyear LJ, Moller DE: Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest 108: , Fryer LG, Parbu-Patel A, Carling D: The Anti- diabetic drugs rosiglitazone and metformin stimulate AMP -activated protein kinase through distinct signaling pathways. J Biol Chem 277: , Saha AK: Pioglitazone treatment activates AMP-activated protein kinase through distinct signaling pathways. J

8 대한내분비학회지 : 제 23 권제 1 호 2008 Biol Chem 277: , Neuschwander-Tetri BA, Brunt EM, Wehmeier KR, Oliver D, Bacon BR: Improved nonalcoholic steatohepatitis after 48 weeks of treatment with the PPAR-gamma ligand rosiglitazone. Hepatology 38: , Lee WJ, Lee IK, Kim HS, Kim YM, Koh EH, Won JC, Han SM, Kim MS, Jo I, Oh GT, Park IS, Youn JH, Park SW, Lee KU, Park JY: alpha-lipoic acid prevents endothelial dysfunction in obese rats via activation of AMP-activated protein kinase. Arterioscler Thromb Vasc Biol 25: , Shimomura I, bashmakov Y, Ikemoto S, Horton JD, Brown MS, Goldstein JL: Insulin selectively increases SREBP-1c mrna in the livers of rats with streptozotocin-induced diabetes. Proc Natl Acad Sci USA 96: , Azzout-Marniche D, Becard D, Guichard C, Foretz M, Ferre P, Foufelle F: Insulin effects on sterol regulatory element binding protein 1c (SREBP-1c) transcriptional activity in rat hepatocytes. Biochem J 350: , Deng X, Cagen LM, Wilcox HG, Park EA, Raghow R, Elam MB: Regulation of the rat SREBP-1c promoter in primary rat hepatocytes. Biochem Biophy Res Commun 290: ,

Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ

Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee University College of Medicine & Hospital E mail : ycell2@yahoo.co.kr Abstract