VDAC in Ethanol and Aldehyde-Induced Mitochondrial Dysfunction

描述（由申请方提供）：酒精性肝病（ALD）、非酒精性脂肪性肝炎（NASH）和毒物相关性脂肪性肝炎（TASH）的肝脏病理学无法区分。ALD的发病机制可能与毒性乙醛（AcAld）的产生有关，而NASH和TASH与脂质过氧化和/或毒物代谢产生的丙二醛（MDA）、4-羟基壬烯醛（HNE）、氯乙醛（ClAcAld）等有关。除了氧和短链脂肪酸的主要例外，所有线粒体代谢物都通过开放的电压依赖性阴离子通道（VDAC）穿过线粒体外膜。该项目的中心假设是乙醇和醛类关闭VDAC，降低线粒体的通透性并抑制正常的线粒体功能。这种VDAC关闭允许自由渗透线粒体的有毒醛的选择性和更快速的线粒体氧化。虽然VDAC闭合在促进醛解毒方面是适应性的，但VDAC闭合在促进脂肪变性和脂毒性方面可能是不适应的。因此，我们提议：1）表征乙醇和醛对培养的肝细胞中的尿素生成的影响，因为尿素生成是依赖于代谢物跨线粒体膜的交换的主要能量消耗过程。我们将表征乙醇、AcAld、MDA、HNE、ClAcAld和其他醛类对尿素产生呼吸和外膜通透性的影响，期望外源醛类和乙醇代谢形成的AcAld将引起剂量依赖性的尿素产生抑制和对低分子量（≤ 3 kDa）溶质的膜通透性降低。后续实验鉴定了介导VDAC闭合的激酶途径。2)使用硫代磷酸酯siRNA单和双敲除/敲低VDAC亚型，确定三种单独的VDAC亚型对乙醇和醛抑制肝细胞中尿素生成的贡献。3)评价醛类、激酶和VDAC在体外和体内脂肪变性和脂毒性中的作用。在初步实验中，醛促进肝细胞与Intralidid孵育后依赖于c-Jun N-末端激酶（JNK）的脂肪变性，并对肿瘤坏死因子-α（TNFα）依赖性细胞凋亡敏感。我们将评估这种细胞杀伤的机制以及特定VDAC亚型在促进脂肪变性和细胞死亡中的作用。ALD和NASH是美国广泛流行的疾病，治疗在很大程度上无效。缺乏有效的治疗反映了我们对潜在病因的无知。特别是，ALD、NASH和TASH相同的组织病理学基础尚不清楚。醛依赖性VDAC闭合为这些疾病的潜在病理生理学提供了一种共同的机制，这可能会导致更好的治疗和预防策略。

DESCRIPTION (provided by applicant): Liver pathology in alcoholic liver disease (ALD), non-alcoholic steatohepatitis (NASH) and forms of toxicant-associated steatohepatitis (TASH) is indistinguishable. ALD pathogenesis may be related to generation of toxic acetaldehyde (AcAld), whereas NASH and TASH are associated with generation of malondialdehyde (MDA), 4-hydroxynonenal (HNE), chloracetaldehyde (ClAcAld) and others by lipid peroxidation and/or toxicant metabolism. With the major exceptions of oxygen and short chain fatty acids, all mitochondrial metabolites cross mitochondrial outer membranes (MOM) via open voltage dependent anion channels (VDAC). The central hypothesis of this project is that ethanol and aldehydes close VDAC, decrease permeability of MOM and suppress normal mitochondrial function. Such VDAC closure allows the selective and more rapid mitochondrial oxidation of toxic aldehydes, which permeate mitochondria freely. Although VDAC closure is adaptive in promoting aldehyde detoxification, VDAC closure may be maladaptive in promoting steatosis and lipotoxicity. Accordingly, we propose to: 1) Characterize the effects of ethanol and aldehydes on ureagenesis in cultured hepatocytes, since ureagenesis is a major energy-consuming process that is dependent on exchange of metabolites across mitochondrial membranes. We will characterize the effects of ethanol, AcAld, MDA, HNE, ClAcAld and other aldehydes on ureagenic respiration and outer membrane permeability with the expectation that exogenous aldehydes and AcAld formed by ethanol metabolism will cause dose-dependent inhibition of ureagenesis and a decrease of MOM permeability to low molecular weight (≤ 3 kDa) solutes. Follow-on experiments identify kinase pathways mediating VDAC closure. 2) Determine the contributions of the three individual VDAC isoforms to suppression of ureagenesis in hepatocytes by ethanol and aldehydes using phosphorothioate siRNA single and double knockout/knockdowns of the VDAC isoforms. 3) Evaluate the role of aldehydes, kinases and VDAC in steatosis and lipotoxicity in vitro and in vivo. In preliminary experiments, aldehyde promoted steatosis dependent on c-Jun N-terminal kinase (JNK) after incubation of hepatocytes with Intralipid and sensitized to tumor necrosis factor-α (TNFα)-dependent apoptosis. We will evaluate mechanisms underlying this cell killing and the role of specific VDAC isoforms in promoting both steatosis and cell death. ALD and NASH are widely prevalent diseases in the U.S. for which therapy is largely ineffective. Lack of effective therapy reflects our ignorance of the underlying etiologies. In particular, the basis for the identical histopathology of ALD, NASH and TASH is unknown. Aldehyde-dependent VDAC closure provides a shared mechanism for the underlying pathophysiology of these diseases, which will likely lead to better strategies for treatment and prevention.