Alcohol alters hepatic microtubule and membrane dynamics

描述(申请人提供)：每年有2万多人死于酒精性肝病，这是美国人的第七大死因。因为肝脏是酒精代谢的主要部位，所以它是最容易受到酒精损伤的器官。在疾病的早期阶段，脂肪肝发展，导致肝细胞坏死，肝纤维化，并最终发展为肝硬变。尽管疾病的进展在临床上已被很好地描述，但酒精性肝损伤的分子基础尚不清楚。我们的长期目标是了解导致酒精肝毒性的机制。我们最近的研究是在培养的WIF-B细胞上进行的。这些肝细胞是高度分化的，并在培养中保持肝脏特异性活动，包括有效代谢乙醇的能力。因此，这些细胞是检测酒精引起的肝脏毒性的极佳模型，并允许我们进行无法在动物身上完成的机械研究。最近，我们发现乙醇处理的细胞中微管更稳定，乙酰化程度是对照组的2-3倍。我们在酒精喂养的大鼠肝细胞中证实了这些结果，表明这些发现具有生理学意义。我们进一步确定WIF-B细胞中微管乙酰化的增加依赖于乙醇代谢。这项提议侧重于从这些最近的结果中出现的两个主要问题。首先，是什么机制导致：；乙醇处理细胞中的微管超乙酰化和稳定性增加，以及其他肝脏蛋白是否通过类似的机制超乙酰化？其次，我们将测试在蛋白质运输中观察到的酒精诱导的缺陷是否与微管的超乙酰化有关。我们在WIF-B细胞培养和使用方面的丰富专业知识以及我们在极化肝细胞蛋白运输方面的专业知识使我们非常适合进行这些探索性、机械性的实验。这些新的研究领域将为我们在未来的肝毒性研究中应用新的方法和假设打开大门。

DESCRIPTION (provided by applicant): More than 20,000 people each year die of alcoholic liver disease, the seventh largest cause of death in Americans. Because the liver is the major site of ethanol metabolism, it is the most susceptible organ to alcohol-induced injury. In the early stages of the disease, a fatty liver develops which leads to hepatocyte necrosis, liver fibrosis, and ultimately to cirrhosis. Although the disease progression is well described clinically, the molecular basis for alcohol-induced liver injury is not understood. Our long-term goal is to understand the mechanisms that lead to alcohol-induced hepatotoxicity. Our recent studies have been performed in cultured WIF-B cells. These hepatic cells are highly differentiated and maintain liver-specific activities in culture, including the ability to efficiently metabolize ethanol. Thus, these cells are an excellent model to examine alcohol-induced hepatotoxicity and allow us to perform mechanistic studies that cannot be done in animals. Recently, we found that microtubules are more stable and acetylated 2-3-fold more in ethanol-treated cells than in control. We confirmed these results in hepatocytes from ethanol-fed rats indicating the findings have physiologic importance. We further determined that increased microtubule acetylation in WIF-B cells is dependent on ethanol metabolism. This proposal focuses on two major questions emerging from these recent results. First, what is the mechanism that leads:; to microtubule hyperacetylation and increased stability in ethanol-treated cells and are other hepatic proteins hyperacetylated via similar mechanisms? Secondly, we will test whether microtubule hyperacetylation ;contributes to alcohol-induced defects observed in protein trafficking. Our considerable expertise in the culture and use of WIF-B cells and our expertise in polarized hepatocyte protein trafficking situate us perfectly to perform these exploratory, mechanistic experiments. These novel research areas will open the door to novel approaches and hypotheses that we will apply to our future studies of hepatotoxicity.