VDAC in Ethanol and Aldehyde-Induced Mitochondrial Dysfunction

描述(申请人提供)：酒精性肝病(ALD)、非酒精性脂肪性肝炎(NASH)和各种形式的毒物相关性脂肪性肝炎(TASH)的肝脏病理无法区分。ALD的发病机制可能与有毒乙醛的生成有关，而NASH和TASH则与脂质过氧化和/或毒物代谢生成丙二醛(MDA)、4-羟基壬烯醛(HNE)、氯乙醛(ClAld)等有关。除氧和短链脂肪酸外，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(MOM)。这个项目的中心假设是乙醇和醛关闭了VDAC，降低了MoM的通透性，并抑制了正常的线粒体功能。这种VDAC关闭允许有毒醛的选择性和更快的线粒体氧化，有毒醛自由地渗透到线粒体中。虽然VDAC封闭术在促进乙醛脱毒方面是适应的，但在促进脂肪变性和脂毒性方面可能不适应。因此，我们建议：1)表征乙醇和醛对培养肝细胞尿失禁的影响，因为尿失禁是一个主要的耗能过程，依赖于代谢产物在线粒体膜上的交换。我们将表征乙醇、乙酸乙酯、丙二醛、HNE、氯丙二醛和其他醛对尿液呼吸和外膜通透性的影响，以期外源性乙醛和乙醇代谢形成的乙酸乙酯会引起剂量依赖性的尿失禁抑制和对低分子量(≤3 kDa)溶质的MOM通透性降低。后续实验确定了介导VDAC关闭的激酶通路。2)利用VDAC亚型的单硫化siRNA和双敲除/敲除，确定三种单独的VDAC亚型在乙醇和醛抑制肝细胞尿失禁中的作用。3)评价醛、激酶和VDAC在体内外脂肪变性和脂毒性中的作用。在初步实验中，乙醛促进肝细胞与Intralipid孵育后依赖c-jun氨基末端激酶的脂肪变性，并敏化肿瘤坏死因子-α(肿瘤坏死因子-α)依赖的细胞凋亡。我们将评估这种细胞杀伤的潜在机制，以及特定的VDAC亚型在促进脂肪变性和细胞死亡中的作用。ALD和NASH在美国是广泛流行的疾病，治疗很大程度上无效。缺乏有效的治疗反映了我们对潜在病因的忽视。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封闭为这些疾病的潜在病理生理提供了一个共同的机制，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.