Department of Gastroenterology, Justus Liebig University Giessen, Giessen, Germany.
Institute of Medical Virology, National Reference Centre for Hepatitis B Viruses and Hepatitis D Viruses, Justus Liebig University, Giessen, Germany.
Lab Invest. 2020 Nov;100(11):1411-1424. doi: 10.1038/s41374-020-0457-9. Epub 2020 Jul 1.
Clinical studies demonstrated that nonalcoholic steatohepatitis is associated with liver-related outcomes in chronic hepatitis B. Furthermore, primary biliary fibrosis and biliary atresia occurred in patients with HBV infection. Interestingly, hepatitis B virus surface protein (HBs) transgenic mice spontaneously develop hepatic steatosis. Our aim is to investigate the effect of Abcb4 knockout-induced cholestasis on liver steatosis in HBs transgenic mice. Hybrids of HBs transgenic and Abcb4 mice were bred on the BALB/c genetic background. Lipid synthesis, storage, and catabolism as well as proteins and genes that control lipid metabolism were analyzed using HPTLC, qPCR, western blot, electrophoretic mobility shift assay (EMSA), lipid staining, and immunohistochemistry. Hepatic neutral lipid depots were increased in HBs transgenic mice and remarkably reduced in Abcb4 and HBs/Abcb4 mice. Similarly, HPTLC-based quantification analyses of total hepatic lipid extracts revealed a significant reduction in the amount of triacylglycerols (TAG), while the amount of free fatty acids (FFA) was increased in Abcb4 and HBs/Abcb4 in comparison to wild-type and HBs mice. PLIN2, a lipid droplet-associated protein, was less expressed in Abcb4 and HBs/Abcb4. The expression of genes-encoding proteins involved in TAG synthesis and de novo lipogenesis (Agpat1, Gpat1, Mgat1, Dgat1, Dgat2, Fasn, Hmgcs1, Acc1, Srebp1-c, and Pparγ) was suppressed, and AMPK and CREB were activated in Abcb4 and HBs/Abcb4 compared to wild-type and HBs mice. Simulating cholestatic conditions in cell culture resulted in AMPK and CREB activation while FASN and PLIN2 were reduced. A concurrent inhibition of AMPK signaling revealed normal expression level of FASN and PLIN2, suggesting that activation of AMPK-CREB signaling regulates hepatic lipid metabolism, i.e. synthesis and storage, under cholestatic condition. In conclusions, in vivo and mechanistic in vitro data suggest that cholestasis reduces hepatic lipid storage via AMPK and CREB signaling. The results of the current study could be the basis for novel therapeutic strategies as NASH is a crucial factor that can aggravate chronic liver diseases.
临床研究表明，非酒精性脂肪性肝炎与慢性乙型肝炎的肝脏相关结局有关。此外，原发性胆汁性纤维化和胆道闭锁发生在乙型肝炎病毒感染患者中。有趣的是，乙型肝炎病毒表面蛋白（HBs）转基因小鼠自发发展为肝脂肪变性。我们的目的是研究 Abcb4 敲除诱导的胆汁淤积对 HBs 转基因小鼠肝脂肪变性的影响。HBs 转基因和 Abcb4 小鼠的杂交在 BALB/c 遗传背景下繁殖。使用 HPTLC、qPCR、western blot、电泳迁移率变动分析（EMSA）、脂质染色和免疫组织化学分析来分析脂质合成、储存和分解以及控制脂质代谢的蛋白质和基因。HBs 转基因小鼠的肝中性脂质库增加，而 Abcb4 和 HBs/Abcb4 小鼠的肝中性脂质库显著减少。同样，基于 HPTLC 的总肝脂质提取物定量分析显示，三酰甘油（TAG）的量显著减少，而游离脂肪酸（FFA）的量在 Abcb4 和 HBs/Abcb4 中增加与野生型和 HBs 小鼠相比。PLIN2，一种与脂滴相关的蛋白质，在 Abcb4 和 HBs/Abcb4 中的表达减少。参与 TAG 合成和从头脂肪生成的蛋白质编码基因的表达（Agpat1、Gpat1、Mgat1、Dgat1、Dgat2、Fasn、Hmgcs1、Acc1、Srebp1-c 和 Pparγ）受到抑制，而 AMPK 和 CREB 在 Abcb4 和 HBs/Abcb4 中被激活与野生型和 HBs 小鼠相比。在细胞培养中模拟胆汁淤积条件会导致 AMPK 和 CREB 的激活，同时 FASN 和 PLIN2 的减少。同时抑制 AMPK 信号通路显示 FASN 和 PLIN2 的表达水平正常，表明在胆汁淤积条件下，AMPK-CREB 信号通路的激活调节肝脂质代谢，即合成和储存。总之，体内和体外机制数据表明，胆汁淤积通过 AMPK 和 CREB 信号通路减少肝脂质储存。本研究的结果可为新型治疗策略提供基础，因为 NASH 是可加重慢性肝病的关键因素。
