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1 ORIGINAL ARTICLES OLETF 쥐에서 Rosiglitazone에의한 AMPK의활성화및비알콜성지방간질환의호전효과 울산대학교의과대학서울아산병원내분비내과 조은희 이기업 Rosiglitazone Activates AMPK and Improves Non-Alcoholic Fatty Liver Disease in OLETF Rats Eun-Hee Cho, Ki-Up Lee Division of Endocrinology and Metabolism, Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine Abstract Background: Insulin resistance is very common in patients with nonalcoholic fatty liver disease (NAFLD). Glitazones improve insulin sensitivity by acting as a selective agonist of the peroxisome proliferators -activated receptor gamma (PPAR γ), and were shown to activate AMP-activated protein kinase (AMPK) in skeletal muscle and the liver. Glitazones were also shown to reduce hepatic lipogenesis. The aim of this study was to investigate whether the protective mechanism of rosiglitazone on NAFLD is associated with AMPK activation. Methods: Twelve OLETF rats were divided into 2 groups (control, treatment, n = 6 each). LETO rats served as controls. At 35 weeks of age, treatment group received rosiglitazone 4 mg/kg daily for 3 days. Fasting plasma glucose, insulin, free fatty acid, lactate and triglycerides were measured. Liver tissues from each group were processed for histological and hepatic triglyceride content analysis and western blotting. Results: Fasting plasma glucose, insulin and triglycerides levels were significantly lower in treatment group than in control group. Histologic examination disclosed decreased hepatic steatosis in treatment group. Hepatic triglyceride content was also decreased in treatment group. Sterol regulatory binding protein-1c (SREBP-1c) and fatty acid synthase (FAS) expression were increased and AMPK phosphorylation was reduced in OLETF rats compared with LETO rats, and these changes were reversed by rosiglitazone treatment. Conclusion: Rosiglitazone reduced hepatic steatosis in OLETF rats, and activated AMPK in the liver. These results suggest the role of AMPK activation in the protective action of rosiglitazone on NAFLD. (KOREAN DIABETES J 32:141~148, 2008) Key Words: AMP-activated protein kinase, Nonalcoholic fatty liver disease, Rosiglitazone, Triglyceride 서 론 비알콜성지방간질환 (nonalcoholic fatty liver disease, NAFLD) 은단순한지방간으로부터간경화를일으키는비알콜성지방간염 (nonalcoholic steatohepatitis, NASH) 에이르는다양한스펙트럼의비알콜성간질환을대표한다 1). 비 접수일자 : 2008 년 4 월 15 일, 통과일자 : 2008 년 4 월 22 일, 책임저자 : 이기업, 울산대학교의과대학서울아산병원내분비내과 141

2 알콜성지방간질환의발병기전에대해서는이제까지많은연구가있었으며대사증후군이나당뇨병환자에서그발생빈도가증가하는것으로보아인슐린저항성이중요한역할을하리라생각된다 2). 글리타존계통의약물은 peroxisome proliferators-activated receptor gamma (PPAR γ) 에선택적으로작용하는혈당강하제로인슐린감수성을증가시키는것으로알려져있다. 이약물중의하나인 rosiglitazone은 NAFLD에서염증을줄이고, 조직학적인지방축적을감소시킨다고보고되었다 3-6). AMP-activated protein kinase (AMPK) 는 serine/threonine kinase의일종으로세포내에너지 (ATP) 가결핍될때활성화되어세포내에너지생산을증가시키는효소이다 7). 활성화된 AMPK는세포내에서 acetyl-coa carboxylase (ACC) 활성을억제하여 malonyl-coa농도를감소시킨다. Malonyl- CoA는지방산이미토콘드리아로유입하는데있어 rate limiting enzyme인 carnitine palmitoyltransferase-1 (CPT-1) 을억제하는물질로 AMPK 활성화를통해 malonyl-coa의농도가감소하면 CPT-1에대한억제효과가감소되어지방산산화가증가하게된다. 공복상태에서 AMPK의활성화는간에서중성지방의합성에관여하는유전자를주로조절하는 sterol regulatory binding protein-1c (SREBP-1c) 를억제함으로지방합성을감소시킨다 8). 이전의연구에서글리타존이나 metformin같은약물의혈당강하효과가 AMPK활성을통한다는학설이제시된바있다 9). Rosiglitazone은골격근에서 AMPK를활성화시키고, 지방산산화를촉진하며 10,11), pioglitazone은간조직에서 AMPK를활성화시킴이보고되었다 12). 한편 metformin과글리타존은간에서 SREBP-1을감소시키고, fatty acid synthase (FAS) 를감소시킨다 13,14). AMPK활성화가간조직에서도지방산의산화를촉진하기때문에 15), rosiglitazone의 NAFLD 호전효과가 AMPK에의할가능성이많으나아직이에대한연구보고는없는상태이다. 이에본연구에서는 rosiglitazone투여가비만동물에서나타나는 NAFLD 발생과 AMPK 활성도에미치는영향을조사하였다. 대상및방법 1. 실험동물실험동물로는당뇨병, 비만모델인 Otsuka Long Evans Tokushima Fatty (OLETF) 쥐와그대조군으로 Long-Evans Tokushima Otsuka (LETO) 쥐를오츠카제약회사 (Tokushima, Japan) 에서제공받아사용하였다. 이들동물은아산생명과학 연구소에서 SPF (specific pathogen free) 상태로양육되었으며, 일반쥐사료와물을자유롭게섭취하도록하였다. 생후 35주된 6마리의 LETO쥐와총 12마리의 OLETF쥐를무작위로두군으로나누어실험을진행하였다. Rosiglitazone 투여전 OLETF 양군간의체중은비슷하였으며 ( 치료군 OLETF, 672 ± 20 g; 대조군, 660 ± 18 g), LETO쥐군과 OLETF 대조군은일반쥐사료를투여하였고, OLETF 치료군은 rosiglitazone (GlaxoSmithKline Pharmaceuticals, Philadelphia, PA) 을하루 4 mg/kg 추가한일반쥐사료를 3 일간투여하였다. 모든동물을케타민으로마취한후혈액채취를하였고, 이어서희생시킨다음간조직을적출하였다. 2. 혈중포도당, 인슐린, 유산, 중성지방, 유리지방산의측정혈중포도당및유산농도는 YSI glucose and lactate analyzer (YSI 2300: Yellow Springs Instruments, Yellow Springs, OH) 를이용하여효소법에의해측정하였다. 혈장인슐린은면역효소법 (Linco, St. Louis, MO) 을이용하였고, 중성지방및유리지방산은효소처리법 (Sigma triglyceride GPO-trinder, St. Louis, MO; Wako Chemical, Osaka, Japan) 으로측정하였다. 3. 간조직의중성지방함량의측정간조직의중성지방측정은 Sigma Triglyceride (GPO -Trinder) kit를이용하여측정하였다. 4. 조직학적분석적출된간조직은 4% 포르말린으로고정시킨다음파라핀에포매시켰다. 절편은 hematoxylin and eosin (H & E) 염색을시행하여광학현미경으로분석하였다. 또한간조직내지질은 oil red염색을시행하여 Olympus BX60 camera (Tokyo, Japan) 를이용해사진을얻었다. 5. Western Blot 분석간조직을 lysis buffer (20 mm Tris, 1 mm Na 3vo 4, 1 mm phenylmethylsufonyl fluoride, 50 mm NaF) 에서 30분간용해시킨뒤단백질정량을시행하였다. 이중 50 ug 단백질을 8% SDS PAGE gel에서전기영동한뒤 nitrocellulose membrane에 2시간동안전이시켰다. Blocking buffer에 membrane을 1시간동안담가두었다가 phosphoampk (Thr172, Cell Signalling, Beverly, MA) 나 total AMPK에대한일차항체와 4 에서밤동안반응시켰다. 수차례의세척 142

3 조은희외 1 인 : OLETF 쥐에서 Rosiglitazone 에의한 AMPK 의활성화및비알콜성지방간질환의호전효과 과정을거친후 Horseradish peroxidase가붙은 anti-rabbit 항체 (GIBCOBRL, Gaithersburg, MD) 와 chemiluminescent detection reagents (New England Biolabs, Beverly, MA) 에차례로반응시켜반응을유발하였다. SREBP-1c 항체는 BD Biosciences (San Jose, CA) 에서, actin에대한항체는 Sigma에서, 그리고 phospho-ampk과 total AMPK항체는 Cell Signaling Technology (Danvers, MA) 를이용하였다. 6. 통계분석모든자료는평균 ± 표준오차로표시하였으며, 통계분석은 Statistical Package for Social Science (SPSS), version 15.0 (SPSS Inc, Chicago, IL) computer program을사용하였다. 두군간의비교는 Mann-Whitney test를시행하였고, 세군간의비교는 one-way ANOVA (analysis of variance) 및 Duncan's multiple rage test를사용하였고, P < 0.05인경우를통계적으로유의한것으로간주하였다. (Table 1). 2. OLETF쥐에서 Rosiglitazone 투여에따른간조직의중성지방함량에대한효과 OLETF쥐는 LETO군에비해간조직의중성지방이통계적으로유의하게높았으며 (LETO, 27.6 ± 13.6 mg/dl; OLETF, ± 65.2 mg/dl, P < 0.05), rosiglitazone을투여한 OLETF쥐에서대조군인 OLETF쥐에비해간조직의중성지방함량이통계학적으로유의하게감소하였다 ( 치료군, 51.7 ± 31.4 mg/dl; 대조군, ± 65.2 mg/dl, P < 0.05) (Fig. 1). 3. OLETF쥐에서 Rosiglitazone투여에따른간의조직학적변화분석 OLETF쥐의간조직을 LETO 쥐와비교시현저한지방의축적이관찰되며, rosiglitazone을투여시 OLETF쥐의간조직내지방의축적이유의하게감소함이관찰되었다 (Fig. 2). 결 과 1. OLETF쥐에서 Rosiglitazone투여에따른혈중포도당, 인슐린, 유산, 중성지방에대한효과 OLETF쥐는 LETO쥐에비해통계적으로유의한체중, 혈당, 인슐린과중성지방의증가를보였으나, 유산과유리지방산은통계적으로유의한차이는없었다. Rosiglitazone 치료군에서혈중포도당은대조군 OLETF 쥐에비해유의하게감소하였고 ( 치료군, ± 27.8 mg/dl; 대조군, ± 77.2 mg/dl, P < 0.05), 혈중중성지방도대조군인 OLETF쥐에비해유의하게감소하였으나 ( 치료군, 50.9 ± 12.3 mg/dl; 대조군, ± 40.8 mg/dl, P < 0.05), 유산과유리지방산의농도는두군간유의한변화가없었다 Fig. 1. Effects of rosiglitazone (RGZ) on hepatic triglyceride (TG) content. * P < 0.05 vs. LETO rat. P < 0.05 vs. OLETF rat. Table 1. Effects of rosiglitazone on plasma glucose, lactate, free fatty acid and triglyceride levels LETO Untreated OLETF OLETF + RGZ Mean food intake (g/day) 25 ± ± ± 0.4 Body weight (g) 530 ± ± 21 * 667 ± 18 * Plasma glucose (mg/dl) ± ± 77.2 * ± 27.8 Plasma lactate (mg/dl) 16.8 ± ± ± 20.1 Plasma insulin (ng/ml) 64.8 ± ± 10.7 * 79.6 ± 15.5 Free fatty acid (μe/l) ± ± ± Triglycerides (mg/dl) 26.5 ± ± 40.8 * 50.9 ± 12.8 All data are expressed as means ± SEM; RGZ, rosiglitazone. * P < 0.05 vs. LETO rat. P < 0.05 vs. untreated OLETF rat. 143

and oil-red staining (D, E, F), respectively. Fig. 3.

4 LETO OLETF OLETF+RGZ Fig. 2. Effects of rosiglitazone (RGZ) on liver histology of OLETF rats. Liver histology of LETO rats (A, D), untreated OLETF rats (B, E), and OLETF rats treated with rosiglitazone (C, F), stained by hematoxylin-eosin staining ( 400; A, B, C). and oil-red staining (D, E, F), respectively. Fig. 3. Effects of rosiglitazone (RGZ) treatment on sterol regulatory binding protein-1c and fatty acid synthase expression in the liver tissue of OLETF rat. * P < 0.05 vs. LETO rat; P < 0.05 vs. OLETF rat. 4. OLETF쥐에서 Rosiglitazone 투여에따른 SREBP -1c, FAS, AMPK 활성도변화 OLETF쥐는 LETO쥐에비해유의한 SREBP-1c과 FAS 의발현의증가를보였고, AMPK 활성의지표인 AMPK인산화가유의하게감소하였다. Rosiglitazone을투여시 OLETF쥐의간조직내지방합성에관여하는 SREBP-1c과 FAS의억제가관찰되었고 (Fig. 3), AMPK의활성화지표인 AMPK인산화가유의하게증가함이관찰되었다 (Fig. 4). 고찰이미보고된바와같이당뇨병및비만쥐모델인 OLETF쥐에서 LETO쥐에비해혈중포도당과중성지방농도및간조직내중성지방의함량이증가하였으며지방간이발생하였다 16,17). Rosiglitazone 투여군에서는이들모든지표의개선이확인되었다 18). Rosiglitazone에의한지방간호전의기전을파악하기위해간조직의 AMPK 활성화를조사하였을때, 치료군에서 AMPK 활성화의지표인 144

조은희외 1 인 : OLETF 쥐에서 Rosiglitazone 에의한 AMPK 의활성화및비알콜성지방간질환의호전효과 Fig. 4. Effects of rosiglitazone (RGZ) treatment on AMPK phosphorylation in the liver tissue of OLETF rat. * P < 0.05 vs.

5 조은희외 1 인 : OLETF 쥐에서 Rosiglitazone 에의한 AMPK 의활성화및비알콜성지방간질환의호전효과 Fig. 4. Effects of rosiglitazone (RGZ) treatment on AMPK phosphorylation in the liver tissue of OLETF rat. * P < 0.05 vs. LETO rat; P < 0.05 vs. OLETF rat. AMPK 인산화가유의하게증가됨을알수있었다. 본연구에서는이전의연구들 17,18,40) 과는달리 rosiglitazone 을비교적단기간인 3일간투여하였다. 본연구의결과와는달리 Hockings 등 19) 은 Zucker fatty rat에서 3일간 rosiglitazone을투여했을때지방간의호전을보이지않았다고보고하였다. 이와같은차이가난이유는현재명확하지않다. 비알콜성지방간질환은알콜성간질환과임상적으로유사하나과다한알코올섭취없이발생한다. 비만과제2형당뇨병은비알콜성간질환의가장흔한위험인자이다 20). AMPK는세포내에너지상태를감지하는효소로서 ATP 에비하여 AMP가증가하는스트레스상황에서활성화한다. 최근연구들은비만이나제2형당뇨병에서조직내 AMPK 활성도가감소됨을보고하였고 11,21), rosiglitazone, metformin, alpha-lipoic acid와같은약물이나 adiponectin, leptin과같은호르몬들이 AMPK를활성화시킴을보고하였다 22-24). 간에서의 AMPK활성은 ACC를불활성화시키고 25), malonyl CoA의합성을감소시켜지방산산화를증가시킨다. 또한 AMPK는 SREBP-1c를감소시켜지방간발생을억제하고 26), phosphoenolpyruvate carboxykinase (PEPCK) 의전사를억제하여포도당의신합성을저해한다 27). 이외에도 AMPK 활성은 HMG CoA reductase를억제함으로써콜레스테롤생산을저하한다 28). 글리타존은 PPAR γ에선택적으로작용하여인슐린감수성을증가시키는약제로, PPAR γ는주로지방조직과췌장의베타세포, 혈관의내피세포, 대식세포그리고간의 stellate 세포에많이존재한다. 글리타존은주로지방조직에 서지방산의섭취와저장을증가시켜혈액내유리지방산농도를낮추고결과적으로간이나골격근으로유리지방산유입을감소시키는것으로알려져왔다 29). 그러나최근의연구들은글리타존이골격근 AMPK를활성화하고 10), 골격근에서의지방산산화를직접증가시킴을보고하고있다 10,30). 또한글리타존은지방조직에서 adiponectin의합성증가를통해간접적으로인슐린감수성을개선시킨다 31). Adiponectin은간에서 AMPK를활성화시킴이보고되어있기때문에 32) 본연구에서관찰한글리타존의 AMPK 활성이직접적인효과인지간접적인효과인지는확실치않다. 여러임상연구에서글리타존이조직학적으로확진된 NASH환자에서인슐린저항성을개선시키고, ALT수치를호전시키며, 간의조직학적소견을개선시킴을보고하고있다 6,33,34). 그러나이와같은기전은아직까지확정되지않았다. 서등 18) 은 rosiglitazone을 28주간투여한 OLETF쥐에서지방산산화에관련된효소인 fatty-acid transport protein (FATP), medium-chain acyl-coa dehydrogenase (MCAD) 의발현이증가하고, TNF-α가감소함을보고하였다. 최근의연구들이 mitochondia biogenesis에 AMPK가관여함을보고 35-38) 하는것과같이생각할때이연구결과는본연구결과와상응하는결과라생각된다. 한편 NASH 환자에서간세포내의미토콘드리아 DNA량이감소하거나미토콘드리아와연관된여러단백질의발현이감소되는등간세포의미토콘드리아의기능이상이 NASH의발생에관여한다는보고가있었으나 39), Caldwell 등 40) 은 rosiglitazone 투여가간에서미토콘드리아의크리스탈봉입체 (crystalline inclusions) 을증가시키는것외에다른미토콘드리아와관련된변화를유발하지못함을보고하였다. 저자들은글리타존이에너지센서인 AMPK의활성화를통해비알콜성간질환을개선시킴을보이고자하였으며, OLETF 쥐에 rosiglitazone을투여했을때 AMPK 활성화가증가함을보였고, 간의조직학적소견및간의지방함량이개선됨을밝혀냈다. 그러나 rosiglitazone 투여후간이나지방에서 PPAR-γ 발현, adiponectin의혈중농도및발현, adiponectin 수용체의발현의변화나인슐린감수성의변화등을측정하지못한제한점이있다. 또한, 본연구에서는 rosiglitazone에의한지방간질환의호전과 AMPK활성화가나타난다는것만을밝혀낸것으로 rosiglitazone에의한 AMPK 활성이지방간호전을가져왔다는것을확정적으로밝히기위해서는향후 AMPK에대한 dominant-negative나 sirna를이용해 AMPK 활성을억제했을때이와같은효과가상쇄되는지에대한추가연구가필요할것으로생각된다. 145

6 요약연구배경 : 인슐린저항성은비알콜성지방간질환환자의대부분에서나타나며비알콜성지방간질환의발생에중요한역할을한다. 글리타존은 peroxisome proliferators-activated receptor gamma (PPAR γ) 에선택적으로작용하여인슐린감수성을증가시키는약물로비알콜성지방간질환의치료제로주목받고있다. AMPK는세포내에너지상태를감지하는효소로서 ATP에비하여 AMP가증가하는스트레스상황에서활성화된다. 최근의다수의연구들은 AMPK가제 2형당뇨병과비만의병인에관여함을제시하고있고, adiponectin이나 metformin 등이간에서의 AMPK를활성화함을보고하였다. 본연구는 rosiglitazone이간조직에서 AMPK를활성화시키는지를규명하기위하여시행되었다. 방법 : 생후 35주된 6마리의 LETO쥐와각각 6마리의대조군및치료군 (rosiglitazone을하루에 4 mg/kg 3일간투여 ) OLETF쥐를대상으로혈액검사와간조직검사를실시하였다. 또한대조군및치료군의간조직내 SREBP-1c와 FAS의발현, 그리고 AMPK 인산화를 western blotting으로측정하였다. 결과 : Rosiglitazone을투여한 OLETF쥐군은대조군 OLETF 쥐에비해공복혈당과인슐린그리고중성지방농도가유의하게낮았으며, 조직학적검사상지방간의소견이현저히호전되었고, 간내중성지방의함량또한유의하게감소하였다. Rosiglitazone 치료군에서대조군에비해간조직내 SREBP-1과 FAS의발현이유의하게감소하였고, AMPK 활성의지표인 AMPK 인산화는유의하게증가하였다. 결론 : Rosiglitazon 투여는 OLETF쥐에서비알콜성지방간질환의조직학적호전을보였으며, AMPK 활성화를유의하게증가시킴이관찰되었다. 향후 rosiglitazone의지방간질환의호전에대한더구체적인기전연구가요구된다. 참고문헌 1. Matteoni CA, Younossi ZM, Gramlich T, Boparai N, Liu YC, McCullough AJ: Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity. Gastroenterology 116:1413-9, Chitturi S, Abeygunasekera S, Farrell GC, Holmes-Walker J, Hui JM, Fung C, Karim R, Lin R, Samarasinghe D, Liddle C, Weltman M, George J: NASH and insulin resistance: Insulin hypersecretion and specific association with the insulin resistance syndrome. Hepatology 35:373-9, Akyuz F, Demir K, Ozdil S, Aksoy N, Poturoglu S, Ibrisim D, Kaymakoglu S, Besisik F, Boztas G, Cakaloglu Y, Mungan Z, Cevikbas U, Okten A: The effects of rosiglitazone, metformin, and diet with exercise in nonalcoholic fatty liver disease. Dig Dis Sci 52: , Khashab M, Chalasani N: Use of insulin sensitizers in NASH. Endocrinol Metab Clin North Am 36: , Tahan V, Eren F, Avsar E, Yavuz D, Yuksel M, Emekli E, Imeryuz N, Celikel C, Uzun H, Haklar G, Tozun N: Rosiglitazone attenuates liver inflammation in a rat model of nonalcoholic steatohepatitis. Dig Dis Sci 52: , 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: , Winder WW, Hardie DG: AMP-activated protein kinase, a metabolic master switch: possible roles in type 2 diabetes. Am J Physiol 277:E1-10, Foretz M, Ancellin N, Andreelli F, Saintillan Y, Grondin P, Kahn A, Thorens B, Vaulont S, Viollet B: Short-term overexpression of a constitutively active form of AMP-activated protein kinase in the liver leads to mild hypoglycemia and fatty liver. Diabetes 54:1331-9, 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: , Bandyopadhyay GK, Yu JG, Ofrecio J, Olefsky JM: Increased malonyl-coa levels in muscle from obese and type 2 diabetic subjects lead to decreased fatty acid oxidation and increased lipogenesis; thiazolidinedione treatment reverses these defects. Diabetes 55: , Lessard SJ, Chen ZP, Watt MJ, Hashem M, Reid JJ, 146

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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