VDAC in Ethanol and Aldehyde-Induced Mitochondrial Dysfunction

描述（由申请人提供）：酒精性肝病（ALD）、非酒精性脂肪性肝炎（NASH）和毒物相关脂肪性肝炎（TASH）的肝脏病理难以区分。ALD的发病机制可能与毒性乙醛（AcAld）的产生有关，而NASH和TASH则与脂质过氧化和/或毒性代谢产生丙二醛（MDA）、4-羟基壬烯醛（HNE）、氯乙醛（ClAcAld）等有关。除了氧和短链脂肪酸外，所有线粒体代谢物都通过开放电压依赖性阴离子通道（VDAC）穿过线粒体外膜（MOM）。该项目的中心假设是乙醇和醛类物质关闭VDAC，降低MOM的通透性，抑制正常线粒体功能。这样的VDAC关闭允许选择性和更快速的线粒体氧化有毒醛，这些醛可以自由地渗透线粒体。虽然VDAC关闭在促进醛解毒方面是适应性的，但VDAC关闭在促进脂肪变性和脂肪毒性方面可能是不适应性的。因此，我们建议：1)表征乙醇和醛类物质对培养肝细胞脲原作用的影响，因为脲原作用是一个主要的能量消耗过程，依赖于代谢物在线粒体膜上的交换。我们将表征乙醇、AcAld、MDA、HNE、ClAcAld等醛类对脲原呼吸和外膜通透性的影响，期望乙醇代谢形成的外源醛类和AcAld会引起剂量依赖性的脲原作用抑制，降低MOM对低分子量（≤3kda）溶质的通透性。后续实验确定了介导VDAC关闭的激酶途径。2)利用硫代siRNA单敲除和双敲除VDAC异构体，确定三种VDAC异构体对乙醇和醛类抑制肝细胞尿变性的贡献。3)评价醛、激酶和VDAC在体内外脂肪变性和脂肪毒性中的作用。在初步实验中，醛促进脂质内培养的肝细胞依赖于c-Jun n -末端激酶（JNK）的脂肪变性，并对肿瘤坏死因子-α (TNFα)依赖的细胞凋亡敏感。我们将评估这种细胞杀伤的机制以及特定VDAC异构体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，治疗基本上无效。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。醛依赖的VDAC关闭为这些疾病的潜在病理生理提供了一种共享机制，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.