肝X受体的激活对小鼠糖尿病肝脏脂肪酸合成酶表达的调节

Activation of Hver X receptor regulates fatty acid synthase expression in diabetic liver

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T0901317（T0）（3mg·kg^-1·d^-1）或DMSO灌胃处理7d；T0（10μmol／L）刺激人肝癌细胞系HepG2细胞24h。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12h；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）mmol／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FASmRNA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FASmRNA表达升高约3．5倍（P〈0．05），可使db／db小鼠肝脏FASmRNA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。

Objective To investigate the effects of liver X receptor （LXR） 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 gavaging 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 premoter-luciferase reporter recombinants with or without pcDNA3. 1, LXR expression vector, or an active steml regulatory element binding pretein-1c （SREBP-1c） 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-1c 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 db/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 with 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 of the 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 HepG2 cells transfected with LXR and SREBP-1 c were 1.9 and 1.6 times those of the control ceils （botn P 〈 0.01 ）. Conclusion LXRE directly or indirect （ via SREBP-1c） upregulates the expression of FAS gene in the diabetic liver. LXR may mediate the hpid accumulation in hver of diabetes.