CAJ | 학술논문

目的探讨肝X受体(LXR)对糖尿病肝脏脂肪酸合成酶(FAS)表达的影响及机制.方法将16周龄、雄性、C57BL/6背景下瘦素受体基因缺陷的db/db小鼠和对照的db/m小鼠,分别予以LXR激动剂TO901317(TO)(3 mg·kg-1·d-1)或DMSO灌胃处理7 d;TO(10 μmol/L)刺激人肝癌细胞系HepG2细胞24 h.此外,HepG2细胞转染小鼠FAS启动子报告基因表达质粒,同时转染pcDNA3.1表达载体或LXR或活化型固醇调节元件结合蛋白(SREBP-1c)表达质粒12 h;采用免疫组织化学方法检测FAS蛋白在肝脏上的表达,实时荧光定量PCR和蛋白印迹方法分别在mRNA和蛋白水平检测FAS和SREBP-1的表达及TO对其表达的影响,荧光素酶报告基因方法检测LXR激动剂和SREBP-1c对小鼠FAS启动子活性的影响.结果免疫组化结果显示FAS蛋白在肝脏广泛表达,主要位于肝细胞胞质内.TO可显著降低db/db小鼠空腹血糖水平[由(12.00±1.06)mmol/L下降到(7.73±0.69)mmool/L,P＜0.01]和糖化血红蛋白水平(由5.67%±0.10%下降到4.87%±0.08%,P＜0.01).db/db小鼠肝脏FAS mRNA水平明显高于db/m小鼠,约为5.5倍(P＜0.01);在蛋白水平,db/db小鼠肝脏上的FAS也明显高于db/m小鼠.TO处理可使db/m小鼠肝脏FAS mRNA表达升高约3.5倍(P＜0.05),可使db/db小鼠肝脏FAS mRNA升高约1.7倍(P＜0.05);TO处理可使db/m小鼠肝脏SREBP-1 mRNA升高约2.4倍(P＜0.05),使db/db小鼠肝脏SREBP-1 mRNA升高约2.1倍(P＜0.01).TO能够上调HepG2细胞FAS的mRNA表达水平,升高约1.9倍(P＜0.05);LXR的激活还能显著增加HepG2细胞中FAS基因启动子活性,约为对照组的1.5倍;过表达LXR或SREBP-1c也能增加HepG2细胞FAS基因启动子活性,分别为对照组的1.9倍和1.6倍(均P＜0.01).结论 LXR可能通过其本身的直接作用和SREBP-1C的间接作用上调肝脏FAS的表达,LXR可能介导了糖尿病肝脏的脂质生成过程.
목적 탐토간X수체(LXR)대당뇨병간장지방산합성매(FAS)표체적영향급궤제.방법 장16주령、웅성、C57BL/6배경하수소수체기인결함적db/db소서화대조적db/m소서,분별여이LXR격동제TO901317(TO)(3 mg·kg-1·d-1)혹DMSO관위처리7 d;TO(10 μmol/L)자격인간암세포계HepG2세포24 h.차외,HepG2세포전염소서FAS계동자보고기인표체질립,동시전염pcDNA3.1표체재체혹LXR혹활화형고순조절원건결합단백(SREBP-1c)표체질립12 h;채용면역조직화학방법검측FAS단백재간장상적표체,실시형광정량PCR화단백인적방법분별재mRNA화단백수평검측FAS화SREBP-1적표체급TO대기표체적영향,형광소매보고기인방법검측LXR격동제화SREBP-1c대소서FAS계동자활성적영향.결과 면역조화결과현시FAS단백재간장엄범표체,주요위우간세포포질내.TO가현저강저db/db소서공복혈당수평[유(12.00±1.06)mmol/L하강도(7.73±0.69)mmool/L,P＜0.01]화당화혈홍단백수평(유5.67%±0.10%하강도4.87%±0.08%,P＜0.01).db/db소서간장FAS mRNA수평명현고우db/m소서,약위5.5배(P＜0.01);재단백수평,db/db소서간장상적FAS야명현고우db/m소서.TO처리가사db/m소서간장FAS mRNA표체승고약3.5배(P＜0.05),가사db/db소서간장FAS mRNA승고약1.7배(P＜0.05);TO처리가사db/m소서간장SREBP-1 mRNA승고약2.4배(P＜0.05),사db/db소서간장SREBP-1 mRNA승고약2.1배(P＜0.01).TO능구상조HepG2세포FAS적mRNA표체수평,승고약1.9배(P＜0.05);LXR적격활환능현저증가HepG2세포중FAS기인계동자활성,약위대조조적1.5배;과표체LXR혹SREBP-1c야능증가HepG2세포FAS기인계동자활성,분별위대조조적1.9배화1.6배(균P＜0.01).결론 LXR가능통과기본신적직접작용화SREBP-1C적간접작용상조간장FAS적표체,LXR가능개도료당뇨병간장적지질생성과정.
Objective To investigate the effects of liver X receptor(L)(R)on the expression of fatty acid synthase(FAS)in diabetic liver.Methods Sixteen-week-old male db/db mice with C57BL/6 background were administered via garaging of TO901317(TO),a LXR synthetic agonist,at the dose of 3 mg·kg-1·d-1 or dimethyl sulfide(DMSO),a vehicle alone for 7 days.Then the mice were killed with their livers taken out to undergo immunohistochemistry to observe the distribution of FAS protein.Human hepatocellular liver carcinoma cell of the line HepG2 were cultured with TO(10 μmol/L)or DMSO for 24 hours.Another HepG2 cells were transfected with mouse FAS promoter-luciferase reporter recombinants with or without pcDNA3.1,LxR expression vector.or an active sterol regulatory element binding protein-1 c(SREBP-1 c)expression vector for 12 hours.Real-time PCR and Western blotting were used to detect the levels of mRNA and protein of FAS and SREBP-1 c respectively.Luciferase reporter assay was utilized to examine the activity of mouse FAS promoter.Results FAS was abundantly expressed in the mouse livers,especially in the cytoplasm of liver cells.The FAS mRNA levels of the livers of the dh/db mice was about 5.5 times as high as that of the db/m mice(P＜0.01).The FAS protein levels in the livers of db/db and db/m mice treated witll TO were 1.7 and 3.5 times higher than those of the control mice(both P＜0.05).The SREBP-1 mRNA levels in the liver ofthe db/m and db/db mice treated with TO were 2.4 and 2.1 times higher compared with the control mice(P＜0.05,P＜0.01).Luciferase test showed that the FAS promoter activity of the HepG2 cells treated with TO Was 1.5 times that of the control cells(P＜0.01).The FAS promoter activities of the HeDG2 cells transfected with LXR and SREBP-1 c were 1.9 and 1.6 times those of the control cells(botn P＜0.01).Conclusion LXRE directly or indirect(via SREBP-1 c)upregulates the expression of FAS gene in the diabetic liver.LXR may mediate the 1ipid accumulation in liver of diabetes.