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대한한방내과학회지제 35 권 1 호 (2014 년 3 월 ) Korean J. Orient. Int. Med. 2014:35(1)70-78 장수영, 정유선, 신현철대구한의대학교한의과대학내과학교실 The Effects of Hoechunyanggyeok-san on hyperglycemia and Dyslipidemia in db/db mice Soo-young Jang, Yu-sun Jung, Hyeon-cheol Shin Dept. of Internal Medicine of Korean Medicine, College of Korean Medicine, Dae-Gu Haany University ABSTRACT Objectives : Hoechunyanggyeok-san (HYS) is a traditional herbal medicine, which has been clinically used for treating febrile and inflammatory diseases. HYS has been reported to be a useful treatment for diabetes, atherosclerosis and hyperlipidemia in the type 1 diabetic model. However, the mechanism of the effects of HYS against hyperglycemia and hyperlipidemia is poorly understood. In the present study, we investigated the underlying mechanism of ameliorative effect of HYS on hyperglycemia and hyperlipidemia in vivo. Methods : HYS (10, 50 mg/kg/day, p.o.) was administered every day for 2 weeks to db/db mice and its effect was compared with vehicle-treated db/db mice. To confirm serum glucose and triglyceride (TG) changes, serological testing was performed. The levels of sterol regulatory element-binding protein-1 (SREBP-1) activity and Sirtuin1 (SIRT1), AMP-activated protein kinase (AMPK), and acetyl-coa carboxylase α (ACCα) expression were analyzed by western blot analysis. Results : The administration of HYS significantly decreased the elevated serum glucose and TG in db/db mice. HYS administration increased the levels of SIRT1 and AMPK expression compared with the vehicle-treated group. Moreover, HYS treatment significantly inhibited SREBP-1 activity and ACCα expression in the liver, while the vehicle-treated group exhibited their increase. Conclusions : In conclusion, HYS is suggested to have an improvement effect on hyperglycemia and hyperlipidemia by activating the SIRT1/AMPK signaling pathway and inhibiting SREBP-1. Key words : Hoechunyanggyeok-san, hyperlipidemia, SIRT1, SREBP-1, ACCα Ⅰ. 서론 최근고지방, 고탄수화물위주의식문화로인한 교신저자 : 신현철경북포항시남구새천년대로 411 대구한의대학교포항한방병원 3 내과 TEL: 054-281-0055 FAX: 054-281-7464 E-mail: ungaeshin@naver.com 이논문은 2014 년도대구한의대학대학원한의학박사학위논문임. 비만인구의급증이세계적인주요건강문제로대두되고있다. 이로인한합병증에는대부분관상동맥질환, 고혈압, 이상지질혈증등이있으며, 인슐린저항성을일으켜제 2형당뇨병발병에중요한역할을하는것으로여겨지고있다. 현재비만에대해서는약물요법및외과적수술등이시도되고있으나여러가지부작용으로인해아직까지효과적인치료법은부족한실정이다 1-4. 70

장수영 정유선 신현철 이에비만을포함한여러대사성질환에서한약의활용가능성에대한연구가활발히진행되고있다. 이중回春凉膈散은萬病回春 5 에收錄된方劑로三焦火盛, 口舌生瘡을주치증으로하고 6, 臨床에서는당뇨병에도활용되고있으며, 回春凉膈散에대한실험적연구로는홍 7 의 streptozotocin 투여백서의혈당량에미치는영향등이있으나탄수화물및지질대사의조절기전에관한연구는부족한형편이다. 효모균에존재하는 nicotinamide adenine dinucleotide (NAD + ) 의존적탈아세틸화효소인 Sir2의포유류 homologue 중 Sir2와가장유사한것으로알려진 Sirtuin1(SIRT1) 은당과지질대사, 미토콘드리아생합성, 염증, 면역, 세포사등다양한범위에서세포기능을조절하고있다 8,9. 또한, SIRT1 은세포내에너지항상성유지에센서역할을담당하는효소인 AMP-activated protein kinase(ampk) 의 upstream kinase중하나인 liver kinase B1(LKB1) 을탈아세틸화함으로써 AMPK 활동을촉진하고, 이는다시 NAD + 증가를초래하여 SIRT1 활성을증가시킴으로써 SIRT1 과 AMPK 는상호작용으로세포대사를조절하게된다 10-12. Sterol regulatory element-binding proteins(srebps) 는콜레스테롤과지방산합성에관여하는유전자들의발현을조절하는전사인자로, 주로간과지방조직에서발현된다 13,14. SREBP isoform 에는 SREBP-1a, 1c, 2의세가지가있는데, 이중 SREBP-1a 와 SREBP-1c 는지방산대사와중성지방의합성에주로관여하며 acetyl-coa carboxylase(acc), fatty acid synthase (FAS), stearoyl-acp desaturase, ATP citrate lyase, malic enzyme 과같은지방합성에관련된효소들의발현을증가시키는것으로알려져있다 13,15. 그중 ACC는 acetyl-coa 의비가역적카르복시화촉진을통해 malonyl-coa 를생성하는효소로, 미토콘드리아내에서지방산산화를억제하고지방산생합성에중요한역할을한다 16. 또한최근연구결과에따르면 SIRT1 과 AMPK 가각각 SREBP-1 을직접적으로탈아세틸화, 인산화시킴으로써전사활동을억제하는것으로보고된바있다 17,18. 이에본연구에서는 db/db 마우스에서回春凉膈散의당및지질대사장애개선효과를관찰하고, 그생화학적기전이 SIRT1, AMPK 및 SREBP-1 발현조절을중심으로함을확인한바이에보고하는바이다. Ⅱ. 재료및방법 1. 동물생후 12주령된수컷 db/db 마우스 (C57BL/KsJ) 18마리와 db/m 마우스 6마리를샘타코바이오코리아 ( 오산, 한국 ) 로부터공급받았다. NIH#3( 샘타코바이오코리아 ) 먹이와물을자유롭게섭취하도록하였으며, 2주간일정한온도 (23±1 ), 습도 ( 약 60%) 와밤낮주기 (12시간씩) 가유지되는환경에적응시킨후 18마리의 db/db 마우스는체중과혈당을측정하여각군의평균체중과혈당이거의동일하게 3군 (n=6) 으로나누어정상군 (db/m 마우스, n=6) 포함총 4군으로배정하였다. 정상군과대조군에게는생리식염수를, Hoechunyanggyeok-san(HYS) 투여군에는 HYS 추출물 10 또는 50 mg/kg body weight 을 2주간매일경구투여하였다. 투여종료후마취상태에서심장천자를통해모든마우스의혈액을채취하였고, 간은빠르게적출하여액체질소에냉동한뒤 -80 에서보관하였다. 2. 약재및추출본실험에사용한약재는휴먼허브 ( 대구, 한국 ) 에서구입하여사용하였다. 回春凉膈散 (Table 1) 1첩분량 35 g에물 600 ml를가하여 3시간동안전탕하였다. 전탕액을여과지 (Whatman NO.2, Maidstone, England) 로여과한뒤, Evaporator(BUCHI, Switzerland) 를이용하여감압농축한후, 동결건조하여얻은분말을생리식염수에희석하여실험동물에경구투여 71

하였다. Table 1. Composition of Hoechunyanggyeok-san. Herb name Scientific name Amounts (g) 連翹 Forsythia koreana (Rehder) Nakai 5 桔梗 Platycodon grandiflorum (Jacq.) A. DC. 3 枳殼 Citrus aurantium L. 3 生地黃 Rehmannia glutinosa (Gaetner) Libosch. 3 赤芍藥 Paeonia lactiflora Pallas 3 當歸 Angelica acutiloba (S. et Z.) Kitagawa 3 黃芩 Scutellaria baicalensis George 3 黃連 Coptis chinensis Franch. 3 梔子 Gardenia jasminoides Ellis 3 薄荷 Mentha arvensis L. var. piperascens Malinv. 3 甘草 Glycyrrhiza glabra Fisch. 3 Total 35 3. 시약 Polyvinylidene difluoride(pvdf) membranes는 Millipore Corporation(Bedford, MA, USA) 에서구입하였고, β-actin, histone, SREBP-1, ACCα, SIRT1, AMPK의항체는 Santa Cruz Biotechnology(Santa Cruz, CA, USA) 로부터구입하였다. 모든혈청분석에사용된 kit는신양화학 ( 부산, 한국 ) 에서구입하였고, 이외에모든시약은 Sigma Aldrich(St. Louis, MO, USA) 로부터구입하여사용하였다. 4. Cytosol extract 간조직 100 mg을 100 mm Tris-HCl(pH 7.4), 20 mm β-glycerophosphate, 20 mm NaF, 2 M Na 3 VO 4, 1 mm EDTA, 0.5 mm PMSF, 1 μm pepstatin, 80 mg/l trypsin inhibitor 가포함된 homogenate buffer 1ml(10 mm HEPES(pH 7.8), 10 mm KCl, 2 mm MgCl 2, 1 mm DTT, 0.1 mm EDTA, 0.1 mm PMSF) 와함께얼음으로차갑게하여조직파쇄기 (Bio Spec Product, USA) 로분쇄하였다. 미토콘드리아침전물을얻기위해 4 에서 12,000 rpm으로 15분간원심분리하였다. 5. Nuclear extract 간조직 100 mg을 500 μl hypotonic buffer A(10 mm HEPES(pH 7.8), 10 mm KCl, 2 mm MgCl 2, 1 mm DTT, 0.1 mm EDTA, 0.1 mm PMSF) 에넣어차가운상태에서조직파쇄기 (Bio Spec Product, USA) 로분쇄하였다. 10% NP-40 용액 62.5 μl을첨가하여 12,000 rpm으로 30초간원심분리하였다. 이렇게얻어진핵분획을 10% NP-40 가더해진 buffer A에한번헹구어 12,000 rpm으로원심분리하고, 100 μl의 buffer B(50 mm HEPES, 50 mm KCl, 0.3 mm NaCl, 0.1 mm EDTA, 1 mm DTT, 0.1 mm PMSF, 10% glycerol) 를첨가해재부유시킨뒤 4 에서 12,000 rpm으로 10분간원심분리하였다. 핵단백질을포함한상층액은수집되어 -80 에서냉동보관되었다. 6. 혈청분석심장에서채혈한혈액을원심분리하여혈청을얻었다. 혈청 glucose, triglyceride 는 glucose, triglyceride assay kit를사용하여제작사 ( 신양화학 ) 의 protocol 에따라측정하였다. 혈청샘플 5 μl에시약 200 μl 를첨가한후 37 에서 5분간 incubate 한뒤 540 nm에서흡광도를측정하였다. 7. Western blot analysis SIRT1, AMPK, SREBP-1, ACCα, histone, β- actin을확인하기위해각각의단백질을 8% SDSpolyacrylamide gel electrophoresis(sds-page) 를통해전기영동시켰다. 분리된단백질들을 nitrocellulose membrane 에서 90 V로 1시간 30분동안전기영동하였다. 5% 탈지분유가함유된 TBST(20 mm Tris, 500 mm NaCl, 0.05% Tween 20) 로 1시간동안 72

장수영 정유선 신현철 blocking 한뒤 TBST 로 washing 한후, 1차항체 (SIRT-1, AMPK, SREBP-1, ACCα, histone, β- actin) 를 5% 의탈지분유가들어있는 TBST에희석 (1:1,000) 하여 membrane을 4 에서 overnight 동안반응시켰다. 실온에서 10분씩 3번 TBST 로 membrane 을 washing 한후, 2차항체 (horseradish peroxidase -conjugated donkey anti-rabbit antibody; 1:5,000) 를 5% 의탈지분유가들어있는 TBST에희석 (1:2,000) 하여실온에서 1시간동안반응시켰다. TBST 로 washing 한후 enhanced chemiluminescence(amersham) 로 Hyperfilm(Amersham) 에노출시킨후단백질의발현정도를분석하였다. Band density는 ATTO Densitograph Software(ATTO Corporation, Tokyo, Japan) 로측정하여 histone 또는 β-actin과의비율로정량하였다. 8. 통계처리실험결과는평균과표준편차 (mean±sd) 로표기하였으며, SPSS 18.0 for Windows program을사용하여 one-way analysis of variance(anova) 를실시하여유의수준 p-value<0.05에서 Dunnett's t-test 의다중검정을하였다. Ⅲ. 결과 1. HYS이혈청 glucose 에미치는영향비만으로인해초래되는고혈당에대한 HYS의효과를확인하기위해혈청 glucose 를측정해본결과, 실험시작시 104.52±3.49 mg/dl에서 118.49±5.30 mg/dl로의변화를보인정상군에비해대조군에서는 475.05±39.51 mg/dl에서 596.97±10.66 mg/dl로현저한증가를보였다. 이에비해실험군의경우 HYS 10 mg 투여군은 473.13±42.64 mg/dl에서 525.49±22.57 mg/dl, 50 mg 투여군에서는 474.95±42.76 mg/dl에서 408.16±56.00 mg/dl로의변화를보여투여종료후대조군에비해유의하게낮은수준을보였고 HYS 50 mg 투여군은투여전에비해혈청 glucose 가현 저하게감소하였다 (Fig. 1). Fig. 1. Effects of HYS on serum glucose level in db/db mice. HYS50 : HYS 50 mg/kg treated db/db mice. Bars represent means±sd. Significantly different from vehicle-treated db/db mice values by Dunnett's t-test ( p<0.05, p<0.001). 2. HYS이혈청 triglyceride에미치는영향고혈당과함께비만으로인해초래되는고지혈증에대한 HYS의개선효과를확인하기위해 db/db 마우스에서혈청 triglyceride 를측정하였다. 정상군 (106.94±6.69 mg/dl) 과비교하여 182.67±18.70 mg/dl 로뚜렷한증가를보인대조군에비해실험군은 HYS 10 mg 투여군에서 125.00±10.56 mg/dl, 50 mg 투여군에서 109.17±9.39 mg/dl로통계적으로유의하게감소하였다 (Fig. 2). 특히 HYS 50 mg 투여군에서는정상군수준에가까운감소효과를보였다. 이상의결과를통해 HYS이비만으로유발되는고혈당및고지혈증을완화하는효능이있음을알수있었고, 특히지질수치를개선하는데더욱유의한효과가있음을확인할수있었다. 73

(B) Fig. 2. Effects of HYS on serum triglyceride level in db/db mice. HYS50 : HYS 50 mg/kg treated db/db mice. Bars represent means±sd. Significantly different from vehicle-treated db/db mice values by Dunnett's t-test ( p<0.05, p<0.01). 3. HYS이 SIRT1, AMPK 발현에미치는영향 HYS의당및지질대사조절기전을구체적으로탐구하기위해 SIRT1과상호촉진작용을통해세포대사를조절하는 AMPK의발현정도를 Western blotting을통해확인하였다. 그결과, 정상군에비해대조군에서 SIRT1 의발현이유의하게감소하였고, 실험군은 HYS 10, 50 mg 투여군모두에서정상군에가까운발현을보였다 (Fig. 3A). AMPK 의발현정도를확인해본결과에서는, 유의성은없었으나대조군에비해 HYS 투여군에서농도의존적으로발현이증가하는경향을보였다 (Fig. 3B). (A) Fig. 3. Effects of HYS on SIRT1 and AMPK expression in db/db mice liver. SIRT1 (A), AMPK (B) protein expressions in liver. HYS50 : HYS 50 mg/kg treated db/db mice. β- actin was used for loading control. Bars represent means±sd. Significantly different from vehicle-treated db/db mice values by Dunnett's t-test ( p<0.01). 4. HYS이 SREBP-1 활성에미치는영향 HYS 이혈중지질수치개선에더욱유의한효과를보인결과를토대로지질대사의조절기전을더구체적으로알아보고자콜레스테롤과지방산합성에관여하는유전자들의발현을조절하는전사인자인 SREBP-1 의활성에대한 HYS 의효과를확인해보았다. 그결과, 대조군에서 SREBP-1 활성이정상군에비해유의하게증가하였으며, 실험군은 HYS 50 mg 투여군에서유의성있게줄어든결과를보였다 (Fig. 4). 이로써 HYS은 SREBP-1 활성을억제해지질대사를조절함을알수있었으며, 아울러결과 3에서 SREBP-1 의활성을직접적으로억제하는 SIRT1 과 AMPK 의발현이촉진된결과와유관하다하겠다. 74

장수영 정유선 신현철 HYS50 : HYS 50 mg/kg treated db/db mice. β- actin was used for loading control. Bars represent means±sd. Significantly different from vehicle-treated db/db mice values by Dunnett's t-test ( p<0.05). Ⅳ. 고찰 Fig. 4. Effects of HYS on SREBP-1 activity in db/db mice liver. HYS50 : HYS 50 mg/kg treated db/db mice. Histone was used for loading control. Bars represent means ±SD. Significantly different from vehicle-treated db/db mice values by Dunnett's t-test ( p<0.05, p<0.01). 5. HYS이 ACCα 발현에미치는영향 SREBP-1 의하위표적효소인 ACCα 발현에대한 HYS의효과를확인해본결과에서는, SREBP-1 과마찬가지로대조군에서 ACCα 발현이정상군에비해유의하게증가하였다. 이에비해실험군은 HYS 50 mg 투여군에서정상군수준에가까운발현을보여유의한감소효과를확인하였다 (Fig. 5). 이로써 HYS 이 SREBP-1 활성억제를통해 ACCα 발현을낮춤으로써지질합성을효과적으로제어하고지방산산화를촉진함을알수있었다. Fig. 5. Effects of HYS on ACCα expression in db/db mice liver. 回春凉膈散은明代龔廷賢의萬病回春 5 에기재되어있으며, 連翹黃芩梔子桔梗黃連生地黃枳殼當歸赤芍藥薄荷甘草의 11 種藥物로構成되어있고, 三焦火盛, 口舌生瘡을주치증으로하는方劑이다. 이에三焦의火가盛하여나타나는口內糜爛 ( 口內炎 ), 頭瘡, 鼻血, 目赤, 脣裂등과, 胃熱로인한發斑頭瘡의黑陷, 癮炎등에주로활용되고있다 6. 현재까지回春凉膈散에대해서는주로항염증에대한효과를밝히는연구 19-21 위주로보고되어왔으나, 本方과관련하여臨床에서는上焦中焦積熱로因한煩燥多渴咽乾, 胸膈煩熱, 面赤脣焦, 口舌生瘡, 消渴等의적응증 22-24 으로당뇨병에도활용되고있지만, 이에대해서는 streptozotocin 투여백서의혈당량및혈중지질감소효과를확인한보고 7 외에혈당및혈중지질개선효능과관련한구체적인기전연구는부족한실정이다. 비만은에너지소모에비해칼로리섭취가지나칠경우에발생하며, 영양과잉으로인해생리적수준을넘어선활성산소의생성으로세포에스트레스상황을유도하고, 고혈압, 이상지질혈증, 제 2형당뇨병, 심혈관질환, 비알콜성지방간등의여러질병을일으키게된다 25,26. 현재비만에대한치료적접근으로는식이및운동을통한생활습관개선이나인지행동치료와같은정신과적치료, 수술요법, 약물요법등이있다. 수술요법에는루와이위우회술, 위조절밴드등이있으나수술후사망, 외과적합병증및잦은재수술등의위험부담이있고, 약물요법은음식물의섭취 흡수를감소시키거나에너지소비를증가시키는효과가있으나심혈관질환, 기분장애및약물남용등의여러부작용으로사용에제한이따르는실정이다 4. 75

이에비만및그와관련된여러합병증의예방과치료에이상의여러부작용들을최소화하면서당및지질대사를효율적으로조절할수있는치료제가필요한실정으로, 본연구에서는回春凉膈散의활용가능성을검토해보았다. 비만으로인해제 2형당뇨병이초래되는모델인 db/db 마우스에回春凉膈散을 2주간투여하여고혈당및고지혈증에대한개선효과를관찰할수있었고, 그중에서도특히혈중중성지방의감소효과가더욱유의함을확인하였다. 이로써回春凉膈散이당및지질대사를유의하게조절하는효과가있음을알수있었다. 回春凉膈散의고혈당및고지혈증완화효과에대한구체적기전을알아보기위해세포에너지대사과정에서중추적역할을하는 SIRT1과 AMPK 를중심으로그하위기전에해당하는지표들의발현변화를확인해보았다. 먼저 db/db 마우스에서 SIRT1과 AMPK의발현량을 Western blotting 을통해확인해본결과, 대조군에서 SIRT1 의발현이줄어든반면回春凉膈散투여군에서는정상군에가까운발현을보였고, AMPK의경우는유의성은없었으나回春凉膈散의투여농도가증가함에따라그발현량이늘어나는경향을보였다. 이로써 HYS은 AMPK 보다는 SIRT1 의발현활성을통한신호전달에더많은영향을미쳐당및지질대사에대한조절효능을가짐을알수있었다. 回春凉膈散이특히지질대사에강한조절력을보임에따라지질합성유전자의발현을촉진하는전사인자이면서 SIRT1 과 AMPK 로부터그활성이직접적으로억제되는 SREBP-1 의활성과지방산합성의주요효소인 ACCα의발현변화를관찰해보았다. 콜레스테롤과지방산합성에관여하는유전자들의발현을조절하는 SREBPs 는 helix-loop-helix-leucine zipper famaily 에속하는전사인자로, SREBP-1a, 1c, 2의세가지 isoform 으로존재한다 13,14. 이중지방산대사와중성지방의합성에주로관여하는 SREBP-1 은단백질로발현되어불활성화상태 (native form) 로소포체에존재하다가세포내 sterol 이고갈되면골지체로이동하여두단계의 cleavage 를통해활성화되고, 활성화된상태 (mature form) 의 SREBP-1 은핵안으로이동해표적유전자들의발현을증가시킨다 13,27-29. 이중에서특히지질대사효소인 ACC의발현을증가시키는데, ACC는미토콘드리아내에서지방산산화를억제하고지방산생합성에중요한역할을하며, ACCα는 lipogenic tissue에많이존재하고 ACCβ 는골격근과심장등 oxidative tissue 에많이분포하는것으로알려져있다 16,30. 본실험에서는 SREBP-1 의활성이정상군에비해유의하게증가되어지질합성이항진되어있는것으로보이는대조군과달리, 回春凉膈散투여군에서는 SREBP-1 활성과 ACCα 발현이모두줄어든결과를보였다. 이로써回春凉膈散은 SIRT1 과 AMPK 의촉진및그에따른 SERBP-1 활성과 ACCα 발현억제를통해동화작용에서이화작용으로의대사를활성화시킴으로써포도당및지방대사장애를개선하는데유효한효과가있고, 특히 SIRT1, SREBP-1, ACC를주축으로하는신호전달경로를통해지질대사를효과적으로조절함을알수있었다 (Fig. 6). Fig. 6. Possible action mechanisms of HYS on the lipid metabolism. HYS inhibited triglyceride synthesis through increasing SIRT1 and AMPK which regulate SREBP-1 and ACC in lipid metabolism. 76

장수영 정유선 신현철 비만으로인해유발된고혈당에대한回春凉膈散의저하효과는확인하였으나당대사와관련한실험은부족하였다. 따라서향후구체적인기전연구를통해回春凉膈散의고지질혈증, 고혈당조절효과에관한실험적결과를마련하여비만, 당뇨병및그로인한합병증치료에있어回春凉膈散의임상적활용에관한객관적인근거를확고히해야할것으로사료된다. Ⅴ. 결론비만으로인한당및지질대사이상에대해回春凉膈散의효과및대사조절기전을 db/db 마우스에서알아본결과, 고혈당과고중성지방혈증을개선 완화하는효과를관찰하였고, 당및지질대사를효율적으로조절하였으며, 특히지질대사에대한개선효과가더욱유의함을확인한바다음과같은결론을얻었다. 回春凉膈散은 SIRT1 과 AMPK의촉진및그에따른 SREBP-1과 ACCα의억제에의하여당및지질대사이상을개선함으로써혈당과혈중중성지방수치를감소시키는효과를보였다. 이로써回春凉膈散은비만에서의탄수화물및지질대사이상을교정함으로써, 비만으로초래되는대사성질환의치료및예방에응용될수있을것으로사료된다. 참고문헌 1. Klop B, Elte JWF, Cabezas MC. Dyslipidemia in obesity: mechanisms and potential targets. Nutrients 2013;5:1218-40. 2. Kim JH, Jo GJ, Choi GM, Han JH, Yoon DK, Kim SM. The relationship between plasma Visfatin level, obesity and metabolic syndrome in women without diabetes. Korean J Obes 2009;18(1):15-23. 3. Speigelman BM, Flier JS. Obesity and the regulation of energy balance. Cell 2001;104(4) :531-43. 4. Rodgers RJ, Tschop MH, Wilding JPH. Antiobesity drugs: past, present and future. Disease Models & Mechanisms 2012;5:621-6. 5. Gong JH. Man-byeong-hoe-chun. Seoul: Haeng-rim -seo-won; 1972, p. 16. 6. Hwang DY. Bang-yak-hap-pyeon. Seoul: Yeongrim-sa; 1985, p. 304-5. 7. Hong JS. Effect of Hoechounyanggeuksan on the glucose in diabetic rats induced by streptozotocin. J. of Oriental Medicine 1991;14:397-412. 8. Kitada M, Koya D. SIRT1 in type 2 diabetes: mechanisms and therapeutic pontential. Diabetes Metab J 2013;37:315-25. 9. Kitada M, Kume S, Takeda-Watanabe A, Kanasaki K, Koya K. Sirtuins and renal disease: relationship with aging and diabetic nephropathy. Clin Sci (Lond) 2013;124:153-64. 10. Ruderman N, Prentki M. AMP kinase and malonyl-coa: targets for therapy of the metabolic syndrome. Nat Rev Drug Discov 2004;3:340-51. 11. Lan F, Cacicedo JM., Ruderman N, Ido Y. SIRT1 modulation of the acetylation status, cytosolic localization and activity of LKB1. Possible role in AMP-activated protein kinase activation. J Biol Chem 2008;283(41):27628-35. 12. Canto C, Gerhart-Hines Z, Feige JN, Lagouge M, Milne JC., Elliott PJ et al. AMPK regulates energy expenditure by modulating NAD + metabolism and SIRT1 activity. Nature 2009;458(7241):1056-60. 13. Brown MS, Goldstein JL. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 1997;89:331-40. 14. Weber LW, Boll M, Stampfl A. Maintaining 77

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