Pan Xiaoli, Wang Pei, Luo Jinzhuo, Wang Zhijun, Song Yuhu, Ye Jin, Hou Xiaohua
Department of Gastroenterology and Hepatology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, No. 1277 Jiefang Avenue, Wuhan, 430022, Hubei, China.
Endocrine. 2015 Apr;48(3):834-47. doi: 10.1007/s12020-014-0384-x. Epub 2014 Aug 20.
Non-alcoholic fatty liver disease (NAFLD) is characterized by steatosis associated with liver inflammation. As NAFLD progresses, triglycerides increase within hepatocytes, causing typical vacuoles that resemble adipocytes. However, whether these morphological changes in hepatocytes indicate potential functional changes is unclear. C57BL/6J mice were fed a high-fat diet (HFD) containing 42% fat. Markers for adipocytes in the liver were measured using real-time PCR, Western blot, and double immunofluorescent labeling. Cytokines in cell culture supernatants were quantified with ELISA. To determine the macrophage phenotype, hepatic classical M1 markers and alternative M2 markers were analyzed. After a 24-week feeding period, adipocyte markers aP2 and PPARγ increased at both the mRNA and protein level in the liver of HFD-fed mice. FITC-labeled aP2 and rhodamine-labeled albumin were both stained in the cytoplasm of steatotic hepatocytes as observed under confocal laser scanning microscopy. Cell membrane-bound E-cadherin and albumin expression were reduced in steatotic hepatocytes compared to controls. However, hepatic adiponectin and adiponectin receptor-2 expression decreased with upregulation of hepatic CD36, suggesting impaired adiponectin activity in livers of HFD-fed mice. Moreover, steatotic primary hepatocytes not only released pro-inflammatory cytokines such as TNFα, MCP-1, IL-6, and IL-18, but also could activate macrophages when co-cultured in vitro. In vivo, hepatic expression of M1 genes such as iNOS and TNFα was markedly increased in HFD-fed mice. In contrast, hepatic expression of M2 genes such as Arg1 and CD206 was significantly reduced. Specifically, the ratio of TNFα to CD206 in HFD-fed mice was notably upregulated. Overexpression of adipocyte-specific genes in hepatocytes and their secretory function and epithelial phenotype impairment in NAFLD cause functional changes in steatotic hepatocytes aside from morphological changes. This suggests that adipogenic changes in hepatocytes are involved in pathogenesis of NAFLD.
非酒精性脂肪性肝病（NAFLD）的特征是脂肪变性伴有肝脏炎症。随着NAFLD的进展，肝细胞内甘油三酯增加，形成类似脂肪细胞的典型空泡。然而，肝细胞的这些形态学变化是否表明潜在的功能变化尚不清楚。给C57BL/6J小鼠喂食含42%脂肪的高脂饮食（HFD）。使用实时PCR、蛋白质印迹和双重免疫荧光标记法检测肝脏中脂肪细胞的标志物。用酶联免疫吸附测定法对细胞培养上清液中的细胞因子进行定量。为了确定巨噬细胞表型，分析了肝脏经典M1标志物和替代性M2标志物。在24周的喂养期后，高脂饮食喂养小鼠肝脏中脂肪细胞标志物aP2和PPARγ在mRNA和蛋白质水平均升高。在共聚焦激光扫描显微镜下观察到，FITC标记的aP2和罗丹明标记的白蛋白均在脂肪变性肝细胞的细胞质中染色。与对照组相比，脂肪变性肝细胞中细胞膜结合的E-钙黏蛋白和白蛋白表达降低。然而，肝脏脂联素和脂联素受体-2表达降低，同时肝脏CD36上调，提示高脂饮食喂养小鼠肝脏中脂联素活性受损。此外，脂肪变性的原代肝细胞不仅释放促炎细胞因子如TNFα、MCP-1、IL-6和IL-18，而且在体外共培养时还能激活巨噬细胞。在体内，高脂饮食喂养小鼠肝脏中iNOS和TNFα等M1基因的表达明显增加。相反，Arg1和CD206等M2基因的肝脏表达显著降低。具体而言，高脂饮食喂养小鼠中TNFα与CD206的比值明显上调。肝细胞中脂肪细胞特异性基因的过表达及其分泌功能和上皮表型损伤在NAFLD中除形态学变化外还导致脂肪变性肝细胞的功能变化。这表明肝细胞的脂肪生成变化参与了NAFLD的发病机制。
