College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, P.R. China.
Center for Molecular and Translational Medicine, Georgia State University, Atlanta, GA 30303, USA.
Mol Med Rep. 2019 Oct;20(4):3224-3232. doi: 10.3892/mmr.2019.10549. Epub 2019 Aug 1.
Alcohol consumption causes liver steatosis in humans. Metabolic disorders of lipids are one of the factors that cause liver steatosis in hepatocytes. Hepatic Niemann‑Pick C1‑like 1 (NPC1L1) regulates lipid homeostasis in mammals. The relationship between NPC1L1 and autophagy in those with a history of alcohol abuse is unclear. The present study aimed to investigate the function of NPC1L1 in the activation of hepatic autophagy in a mouse model with a human (h)NPC1L1 transgene under alcohol feeding conditions. The mice expressing hNPC1L1 (Ad‑L1) or controls (Ad‑null) were created by retro‑orbital adenovirus injection. The Ad‑L1 and Ad‑null mice were fed with alcohol or a non‑alcoholic diet to mimic chronic alcohol consumption in humans. Hepatic autophagy was demonstrated in isolated primary hepatocytes by monitoring autophagic vacuoles under fluorescence microscopy, and by western blotting for autophagic makers. Isolated hepatocytes from the livers of Ad‑L1 mice were treated with different doses of ezetimibe to study the restoration of autophagy. Chronic alcohol feeding caused liver injury and steatosis, shown by significantly higher levels of plasma alanine transaminase and aspartate transaminase activity, and by hematoxylin and eosin staining in Ad‑L1 and Ad‑null mice. Compared to Ad‑null control mice, the microtubule‑associated proteins 1A/1B light chain 3 (LC3) particles in the isolated hepatocytes of Ad‑L1 mice were decreased, both under alcohol and non‑alcoholic feeding. The ratio of LC3II/LC3I was significantly decreased, and the level of p62/sequestosome‑1 protein was significantly increased in Ad‑L1 mice compared with Ad‑null mice after alcohol feeding. Levels of LC3II protein were statistically increased in hepatocytes isolated from Ad‑L1 mice with ezetimibe treatment. The increase in LC3II expression was dose dependent. Within the tested range, it reached its highest level at 40 µM. The livers of Ad‑L1 mice represent a more human‑like state for the study of hepatic autophagy. Hepatic expression of human NPC1L1 resulted in an inhibition of autophagy; it may contribute to alcoholic fatty liver disease in humans.
酒精摄入会导致人类肝脏脂肪变性。脂质代谢紊乱是导致肝细胞脂肪变性的因素之一。肝 Niemann-Pick C1 样 1（NPC1L1）在哺乳动物中调节脂质稳态。在有酗酒史的人群中，NPC1L1 与自噬之间的关系尚不清楚。本研究旨在探讨 NPC1L1 在酒精喂养条件下表达人 NPC1L1（h）NPC1L1 转基因的小鼠模型中肝自噬激活中的作用。通过 retro-orbital 腺病毒注射创建表达 hNPC1L1（Ad-L1）或对照（Ad-空）的小鼠。Ad-L1 和 Ad-空小鼠用酒精或非酒精饮食喂养，以模拟人类慢性酒精摄入。通过荧光显微镜观察自噬小泡，western blot 检测自噬标志物，在分离的原代肝细胞中检测肝自噬。用不同剂量的依泽替米贝处理来自 Ad-L1 小鼠肝脏的分离肝细胞，以研究自噬的恢复情况。慢性酒精喂养导致 Ad-L1 和 Ad-空小鼠的肝损伤和脂肪变性，表现为血浆丙氨酸转氨酶和天冬氨酸转氨酶活性显著升高，以及苏木精和伊红染色。与 Ad-空对照小鼠相比，在酒精和非酒精喂养下，Ad-L1 小鼠分离的肝细胞中的微管相关蛋白 1A/1B 轻链 3（LC3）颗粒减少。与 Ad-空小鼠相比，酒精喂养后，Ad-L1 小鼠的 LC3II/LC3I 比值显著降低，p62/自噬体-1 蛋白水平显著升高。用依泽替米贝处理后，Ad-L1 小鼠分离的肝细胞中 LC3II 蛋白水平统计学上增加。LC3II 表达增加呈剂量依赖性。在测试范围内，在 40μM 时达到最高水平。Ad-L1 小鼠的肝脏代表了更适合研究肝自噬的人类样状态。肝表达人 NPC1L1 导致自噬抑制；它可能导致人类酒精性脂肪肝疾病。
