Rasineni Karuna, Donohue Terrence M, Thomes Paul G, Yang Li, Tuma Dean J, McNiven Mark A, Casey Carol A
The Liver Study Unit, VA Nebraska-Western Iowa Health Care System (VA NWIHCS).
Department of Internal Medicine, University of Nebraska Medical Center.
Hepatol Commun. 2017 Aug;1(6):501-512. doi: 10.1002/hep4.1063. Epub 2017 Jul 10.
Lipid droplets (LDs), the organelles central to alcoholic steatosis, are broken down by lipophagy, a specialized form of autophagy. Here, we hypothesize that ethanol administration retards lipophagy by down-regulating Dynamin 2 (Dyn2), a protein that facilitates lysosome re-formation, contributing to hepatocellular steatosis.
Primary hepatocytes were isolated from male Wistar rats fed Lieber-DeCarli control or EtOH liquid diets for 6-8 wk. Hepatocytes were incubated in complete medium (fed) or nutrient-free medium (fasting) with or without the Dyn2 inhibitor Dynasore or the Src inhibitor SU6656. Phosphorylated (active) forms of Src and Dyn2, and markers of autophagy were quantified by Western Blot. Co-localization of LDs-with autophagic machinery was determined by confocal microscopy.
In hepatocytes from pair-fed rats, LD breakdown was accelerated during fasting, as judged by smaller LDs and lower TG content when compared to hepatocytes in complete media. Fasting-induced TG loss in control hepatocytes was significantly blocked by either SU6656 or Dynasore. Compared to controls, hepatocytes from EtOH-fed rats had 66% and 40% lower content of pSrc and pDyn2, respectively, coupled with lower rate of fasting-induced TG loss. This slower rate of fasting-induced TG loss was blocked in cells co-incubated with Dynasore. Microscopic examination of EtOH-fed rat hepatocytes revealed increased co-localization of the autophagosome marker LC3 on LDs with a concomitant decrease in lysosome marker LAMP1. Whole livers and LD fractions of EtOH-fed rats exhibited simultaneous increase in LC3II and p62 over that of controls, indicating a block in lipophagy.
Chronic ethanol administration slowed the rate of hepatocyte lipophagy, owing in part to lower levels of phosphorylated Src kinase available to activate its substrate, Dyn2, thereby causing depletion of lysosomes for LD breakdown.
脂滴（LDs）是酒精性脂肪变性的核心细胞器，通过脂噬（一种特殊形式的自噬）被分解。在此，我们假设乙醇给药通过下调发动蛋白2（Dyn2）来延缓脂噬，Dyn2是一种促进溶酶体重组的蛋白质，会导致肝细胞脂肪变性。
从喂食Lieber-DeCarli对照或乙醇液体饮食6 - 8周的雄性Wistar大鼠中分离原代肝细胞。肝细胞在完全培养基（喂食）或无营养培养基（禁食）中培养，添加或不添加Dyn2抑制剂Dynasore或Src抑制剂SU6656。通过蛋白质印迹法对Src和Dyn2的磷酸化（活性）形式以及自噬标志物进行定量。通过共聚焦显微镜确定脂滴与自噬机制的共定位。
在配对喂养大鼠的肝细胞中，与完全培养基中的肝细胞相比，禁食期间脂滴分解加速，表现为脂滴更小且甘油三酯含量更低。SU6656或Dynasore可显著阻断对照肝细胞中禁食诱导的甘油三酯损失。与对照组相比，乙醇喂养大鼠的肝细胞中磷酸化Src和磷酸化Dyn2的含量分别降低了66%和40%，同时禁食诱导的甘油三酯损失率也更低。与Dynasore共同孵育的细胞中，这种较慢的禁食诱导甘油三酯损失率被阻断。对乙醇喂养大鼠肝细胞的显微镜检查显示，自噬体标志物LC3在脂滴上的共定位增加，同时溶酶体标志物LAMP1减少。乙醇喂养大鼠的全肝和脂滴部分显示LC3II和p62比对照组同时增加，表明脂噬受阻。
长期给予乙醇会减缓肝细胞脂噬的速度，部分原因是可用于激活其底物Dyn2的磷酸化Src激酶水平较低，从而导致用于脂滴分解的溶酶体耗竭。
