Ethanol-induced steatosis involves impairment of lipophagy, associated with reduced Dynamin2 activity.
Ethanol-induced steatosis involves impairment of lipophagy, associated with reduced Dynamin2 activity.
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DOI：
10.1002/hep4.1063
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发表时间：
2017-08
影响因子：
5.1
通讯作者：
Casey CA
中科院分区：
文献类型：
--
作者：
Rasineni K;Donohue TM Jr;Thomes PG;Yang L;Tuma DJ;McNiven MA;Casey CA
脂滴 (LD) 是酒精性脂肪变性的核心细胞器，可被脂肪吞噬（自噬的一种特殊形式）分解。在这里，我们假设乙醇给药通过下调动力蛋白 2 (Dyn2) 来延缓脂肪吞噬，动力蛋白 2 是一种促进溶酶体重组的蛋白质，导致肝细胞脂肪变性。从饲喂 Lieber-DeCarli 对照或乙醇 (EtOH) 液体饮食 6-8 周的雄性 Wistar 大鼠中分离出原代肝细胞。肝细胞在含有或不含 Dyn2 抑制剂 dynasore 或 Src 抑制剂 SU6656 的完全培养基（补食）或无营养培养基（禁食）中孵育。使用蛋白质印迹分析对 Src 和 Dyn2 的磷酸化（活性）形式以及自噬标记物进行定量。使用共聚焦显微镜确定 LD 与自噬机制的共定位。与完全培养基中的肝细胞相比，配对喂养大鼠的肝细胞中，LD 分解在禁食期间加速，这是通过更小的 LD 和更低的甘油三酯 (TG) 含量来判断的。 SU6656 或 Dynasore 显着阻断了对照肝细胞中禁食诱导的 TG 损失。与对照组相比，EtOH 喂养大鼠的肝细胞中磷酸化 Src (pSrc) 和磷酸化 Dyn2 (pDyn2) 的含量分别降低了 66% 和 40%，并且禁食引起的 TG 损失率也较低。在与 Dynasore 共孵育的细胞中，禁食引起的 TG 损失速度较慢被阻断。对喂食 EtOH 的大鼠肝细胞进行显微镜检查，发现自噬体标记物 LC3 在 LD 上的共定位增加，同时溶酶体标记物 LAMP1 减少。与对照组相比，EtOH 喂养大鼠的整个肝脏和 LD 部分的 LC3II 和 p62 同时增加，表明脂肪吞噬受到阻碍。结论：长期服用乙醇会减慢肝细胞的自噬速率，部分原因是可激活其底物 Dyn2 的磷酸化 Src 激酶水平较低，从而导致溶酶体耗竭，从而导致 LD 分解。 （肝病学通讯 2017 年；1：501–512）
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 ethanol (EtOH) liquid diets for 6‐8 weeks. 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 using western blot analysis. Colocalization of LDs with autophagic machinery was determined using confocal microscopy. In hepatocytes from pair‐fed rats, LD breakdown was accelerated during fasting, as judged by smaller LDs and lower triglyceride (TG) content when compared with hepatocytes in complete media. Fasting‐induced TG loss in control hepatocytes was significantly blocked by either SU6656 or Dynasore. Compared with controls, hepatocytes from EtOH‐fed rats had 66% and 40% lower content of phosphorylated Src (pSrc) and phosphorylated Dyn2 (pDyn2), respectively, coupled with a lower rate of fasting‐induced TG loss. This slower rate of fasting‐induced TG loss was blocked in cells coincubated with Dynasore. Microscopic examination of EtOH‐fed rat hepatocytes revealed increased colocalization 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. Conclusion: 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. (Hepatology Communications 2017;1:501–512)
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影响因子：
64.8
作者：
Lee, Jae Man;Wagner, Martin;Xiao, Rui;Kim, Kang Ho;Feng, Dan;Lazar, Mitchell A.;Moore, David D.
通讯作者：
Moore, David D.
影响因子：
4.8
作者：
Ahn, S;Maudsley, S;Daaka, Y
通讯作者：
Daaka, Y
影响因子：
3.7
作者：
Kockx, Maaike;Karunakaran, Denuja;Kritharides, Leonard
通讯作者：
Kritharides, Leonard
影响因子：
4.8
作者：
Cao, WH;Luttrell, LM;Collins, S
通讯作者：
Collins, S
影响因子：
4
作者：
Guo, Yi;Cordes, Kimberly R.;Walther, Tobias C.
通讯作者：
Walther, Tobias C.