Ethanol-Induced VDAC Closure in Hepatocytes

乙醇诱导肝细胞中的 VDAC 关闭

• 批准号: 7143363
• 负责人: EKHSON Lukmanovich HOLMUHAMEDOV
• 金额: $ 20.99万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7143363
• 关键词: acetaldehyde; adenosine triphosphate; adenylate cyclase; alcohol dehydrogenase; anions; biological transport; cellular respiration; confocal scanning microscopy; cytochrome P450; digitonin; enzyme activity; enzyme inhibitors; ethanol; fatty acids; fatty liver; free radical oxygen; ion channel blocker; laboratory rat; liver cells; membrane permeability; mitochondrial disease /disorder; mitochondrial membrane; nicotinamide adenine dinucleotide; oxidation; polarography; voltage gated channel

DESCRIPTION (provided by applicant): Ethanol ingestion produces rapid changes in hepatic mitochondrial metabolism. These changes include a swift increase in alcohol metabolism (SIAM) by alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ACDH), a near doubling of respiration, decreased ATP, activated glycolysis, glycogen depletion, inhibition of mitochondrial oxidation of medium and long chain fatty acids, and fatty liver changes (steatosis). The unifying hypothesis of this proposal is that closure of mitochondrial voltage dependent anion channels (VDAC) is a critical event leading to these acute alterations of mitochondrial metabolism after ethanol. VDAC is located in the mitochondrial outer membrane and conducts solutes of molecular weight less than 5 kDa into and out of the mitochondria. Open VDAC is essential for movement of respiratory substrates (e.g., medium and long chain fatty acids, pyruvate), ATP, ADP, Pi and other polar metabolites into and out of mitochondria. We specifically hypothesize that VDAC closure after ethanol blocks these exchanges. After VDAC closure, mitochondria still oxidize membrane permeant respiratory substrates, particularly acetaldehyde and short chain fatty acids. Inhibition of ATP release and the high redox potential of acetaldehyde/acetate then produce mitochondrial hyperpolarization, leading to non-ohmic proton leaks in the hperpolarized inner membrane and respiratory stimulation. Activation of uncoupling proteins and the weak uncoupling effect of acetate may also contribute to increased respiratory flux. Overall, accelerated respiration and selective oxidation of acetaldehyde have the physiological function of removing and detoxifying ethanol. Heretofore, examination of VDAC conductivity in situ has been very difficult. In our preliminary studies, we developed a novel confocal microscopic technique utilizing the distribution of a 3 kDa rhodamine-conjugated dextran in the mitochondria of permeabilized hepatocytes to assess the open/closed status of VDAC. These pilot experiments support the hypothesis that acute ethanol exposure induces VDAC closure. Accordingly,our Specific Aims are 1) to characterize changes of VDAC permeability in rat hepatocytes exposed to ethanol from measurements of respiration and RhoDex retention by permeabilized hepatocytes and isolated mitochondria; and 2). to determine which metabolites of ethanol oxidation (NADH, ROS, acetaldehyde,acetate) are responsible for ethanol-induced VDAC closure or whether closure is a direct effect of ethanol. Overall, this proposal addresses the high risk, high return hypothesis that VDAC closure after ethanol exposure causes mitochondrial dysfunction and promotes steatosis. If our preliminary data supporting this hypothesis can be extended, this new role of VDAC will represent a paradigm shift in our understanding of the effects of alcohol on the liver and the regulation of mitochondrial metabolism.

描述（由申请人提供）：乙醇摄入会导致肝线粒体代谢发生快速变化。这些变化包括乙醇脱氢酶 (ADH) 和乙醛脱氢酶 (ACDH) 导致的酒精代谢 (SIAM) 迅速增加、呼吸作用几乎加倍、ATP 减少、糖酵解激活、糖原消耗、中长链脂肪线粒体氧化的抑制酸和脂肪肝变化（脂肪变性）。该提议的统一假设是，线粒体电压依赖性阴离子通道（VDAC）的关闭是导致乙醇后线粒体代谢急剧改变的关键事件。 VDAC 位于线粒体外膜，将分子量小于 5 kDa 的溶质导入和导出线粒体。开放式 VDAC 对于呼吸底物（例如中链和长链脂肪酸、丙酮酸）、ATP、ADP、Pi 和其他极性代谢物进出线粒体的运动至关重要。我们特别假设乙醇后 VDAC 关闭会阻止这些交换。 VDAC 关闭后，线粒体仍然氧化膜渗透呼吸底物，特别是乙醛和短链脂肪酸。抑制 ATP 释放和高氧化还原电位 乙醛/乙酸盐随后产生线粒体超极化，导致超极化内膜中的非欧姆质子泄漏和呼吸刺激。解偶联蛋白的激活和乙酸盐的弱解偶联作用也可能导致呼吸通量增加。总体而言，乙醛的加速呼吸和选择性氧化具有清除乙醇、解毒的生理功能。 迄今为止，原位检查 VDAC 电导率一直非常困难。在我们的初步研究中，我们开发了一种新型共焦显微技术，利用 3 kDa 罗丹明缀合葡聚糖在透化肝细胞线粒体中的分布来评估 VDAC 的打开/关闭状态。这些初步实验支持了急性乙醇暴露会导致 VDAC 关闭的假设。因此，我们的具体目标是 1) 通过测量透化肝细胞和分离的线粒体的呼吸和 RhoDex 保留来表征暴露于乙醇的大鼠肝细胞中 VDAC 通透性的变化；和2）。确定乙醇氧化的哪些代谢物（NADH、ROS、乙醛、乙酸）负责乙醇诱导的 VDAC 关闭，或者关闭是否是乙醇的直接影响。总体而言，该提案解决了高风险、高回报的假设，即乙醇暴露后 VDAC 关闭会导致线粒体功能障碍并促进脂肪变性。如果我们支持这一假设的初步数据可以得到扩展，那么 VDAC 的这一新作用将代表我们理解酒精对肝脏的影响和线粒体代谢调节的范式转变。