肝脏胰岛素受体急性敲除对载脂蛋白B和三酰甘油分泌的影响

Effects of acute knockdown of hepatic insulin receptor on secretion of apolipoprotein B and triglyceride inmice

目的分析肝脏组织特异性的胰岛素信号急性缺失对载脂蛋白B和三酰甘油分泌的影响。方法应用Cre-LoxP系统原理，在重组酶基因的上游插入肝组织特异性白蛋白基因启动子，以此构建组织特异性表达的腺病毒。扩增纯化后的腺病毒经胰岛素受体Floxed小鼠尾静脉注射，分别在注射2d、4d和6d后收集小鼠血浆，测定新生载脂蛋白B和极低密度脂蛋白三酰甘油分泌速率，抽提肝脏脂质并用酶法检测脂质含量，并用免疫印迹法分析胰岛素受体和脂代谢相关蛋白在肝脏内的表达。结果小鼠在注射含重组酶的腺病毒2d、4d和6d后，胰岛素受体表达依次降低30．50％（P〈0．05）、60．12％（P〈0．01）和99．54％（P〈0．001），极低密度脂蛋白三酰甘油的分泌速率分别降低20．43％（P〈0．05）、33．63％（P〈0．05）和44．21％（P〈0．01）；下调了固醇调控结合蛋白1和脂肪酸合成酶以及极低密度脂蛋白形成相关蛋白的表达，但不影响新生载脂蛋白B100的分泌和肝脏脂质含量；而在含重组酶的腺病毒注射6d后新生载脂蛋白B48升高约35．07％（P〈0．05）。结论肝脏胰岛素信号急性缺失降低极低密度脂蛋白中三酰甘油的分泌速率并呈现时间依赖性，揭示小鼠肝脏分泌较小的极低密度脂蛋白颗粒，这种变化可能和肝脏内脂肪酸的合成速度下降有关。

Objective To analyze the effects of acute depletion of liver specific insulin signaling on secretion of apolipoprotein B （apoB） and triglyceride （TG）. Methods Based on Cre LoxP principle, a promoter of hepatic tissue specific albumin gene was inserted into upstream of the cre recombinase gene. Albumin-Cre adenovirus （Ad-CRE） and GFP adenovirus （Ad-GFP） were amplified in 293A cells and purified before intravenous administration to mice. After adenovirus infection for 2 days, 4 days and 6 days, blood samples from mice were collected and hepatic tissues were frozen. The secretion rates of hepatic newly synthesized apoB and very low density lipoprotein （VLDL） TG were determined by injection of Triton WR 1339. The levels of plasma cholesterol （TC） and TG were measured. The expressions of insulin receptor and other lipoprotein metabolism related proteins in hepatic tissues were analyzed by Western blot. Results After 2 d, 4 d and 6 d of the Ad CRE injection into mice, insulin receptor expression was reduced by 30.50% （P〈0. 05）, 60. 12%（P〈0.01） and 99.54% （P〈0. 001）, and VLDL-TG secretion rate was decreased by 20.43% （P〈0.05）, 33.63% （P〈0.05） and 44.21% （P〈0.01）, respectively. Expressions of sterol regulatory binding proteins 1, fatty acid synthase, and the related proteins of VLDL-formation were decreased, but there were no changes in hepatic secretion of apoB100 and hepatic lipids. The hepatic secretion of apoB48 was increased by 35.07% （P〈0.05） 6 d after Ad-CRE injection. Conclusions Acute depletion ofhepatic insulin receptor might reduce VLDL-TG secretion in manner of time dependent, and increase the assembly and secretion of smaller apoB-containing lipoproteins in mice liver, which is probably associated with decreased lipogenesis.