Homocysteine, ER stress and alcoholic liver injury

描述(由申请人提供)：酒精性肝病(ALD)的发病机制仍然不确定，尽管最近取得了相当大的进展。我们提出了一个新的假说，建立在酒精对同型半胱氨酸代谢的已知影响的基础上，将其与内质网应激反应以及随后的脂肪肝、坏死和细胞凋亡联系起来，这是我们在灌胃酒精喂养的小鼠模型中观察到的。我们的初步结果表明，喂食甜菜碱可逆转酒精所致的高同型半胱氨酸血症、内质网应激和肝脏病理改变。在此假设和初步结果的基础上，本文提出了五个目标。首先，我们将对乙醇诱导的内质网应激进行详细的描述，包括酒精引起的小鼠肝脏的细胞和区域区划，乙醇对内质网应激敏感性和细胞凋亡信号的影响，以及内质网应激对肿瘤坏死因子敏感性的影响。其次，我们将试图通过利用各种具有ER应激反应的特定成分的基因缺失的小鼠(例如SREBP-1、CHOP、caspase 12)或其他调节SREBP的信号机制(LXRa缺失小鼠)来确定ER应激反应的关键成分在酒精性肝病中的作用。第三，我们将通过评估底物池大小、同型半胱氨酸代谢酶的表达和示踪动力学来确定乙醇引起的高同型半胱氨酸血症的机制。这些研究将使我们能够确定肝脏是同型半胱氨酸的主要来源，以及是否甲基化和/或硫化减少是导致同型半胱氨酸过剩的主要原因。第四，我们将确定同型半胱氨酸在乙醇诱导的内质网应激中的作用。我们将采用几种不同的策略来加强同型半胱氨酸与内质网对乙醇的应激反应之间联系的证据。我们将确定是否通过同时增加同型半胱氨酸与过量的饮食蛋氨酸或乙酸胍或使用胱硫氨酸合成酶+/-小鼠来加强酒精性肝损伤。此外，我们将评估相同喂养对同型半胱氨酸、内质网应激和肝损伤的影响，并通过评估乙醇喂养对肿瘤坏死因子-R1缺失小鼠的同型半胱氨酸和内质网应激反应来确定肿瘤坏死因子是否作用于同型半胱氨酸的上游。第五，我们将通过将甜菜碱与牛磺酸、二甲基磺酸盐进行比较，并通过确定甜菜碱是否对MAT1A基因缺失的小鼠具有保护作用，来确定甜菜碱喂养对乙醇保护的作用机制。此外，我们将评估甜菜碱喂养对肝脏基因表达的整体影响，以寻找甜菜碱替代效应的线索，这可能也有助于其保护作用。我们期待我们将获得重要的新知识，这些知识可以应用于ALD的预防和治疗

DESCRIPTION (provided by applicant): The pathogenesis of alcoholic liver disease (ALD) remains uncertain despite considerable recent progress. We present a new hypothesis which builds upon the known effect of alcohol on homocysteine metabolism by linking this to the ER stress response and consequent fatty liver, necrosis, and apoptosis which we have observed in the intragastric ethanol fed mouse model. Our preliminary results indicate that feeding betaine reverses the hyperhomocysteinemia, ER stress and liver pathology in response to alcohol. Five aims are proposed which build on this hypothesis and preliminary results. First, we will complete a detailed characterization of ethanol-induced ER stress including the cellular and zonal compartmentation alcoholic mouse liver, the effect of ethanol on the susceptibility to ER stress and apoptotic signaling, and the effect of ER stress on susceptibility to TNF. Second, we will attempt to ascertain the contribution of key components of the ER stress response to alcoholic liver disease by exploiting various mice which have gene deletions of specific components of the ER stress response (e.g. SREBP-1, CHOP, caspase 12) or other signaling mechanisms for regulating SREBP (LXRa null mice). Third, we will determine the mechanism of hyperhomocysteinemia in response to ethanol by assessing substrate pool sizes, homocysteine metabolizing enzyme expression and tracer kinetics. These studies will allow us to determine that the liver is the major source of homocysteine and whether decreased remethylation and/or transulfuration are primarily responsible for excess homocysteine. Fourth, we will determine the role of homocysteine in ethanol-induced ER stress. We will employ several different strategies to strengthen the evidence for linkage of homocysteine and the ER stress response to ethanol. We will determine if alcoholic liver injury is potentiated by concomitantly raising homocysteine with either excess dietary methionine or guanidinoacetate or use of cystathionine ¿- synthase +/- mice. In addition, we will assess the effect of SAMe feeding on homocysteine, ER stress and liver injury and determine if TNF acts upstream of homocysteine by assessing homocysteine and ER stress in response to ethanol feeding in TNF-R1 null mice. Fifth, we will determine the mechanism of action of betaine feeding in protecting against ethanol by comparing it to taurine, dimethlysulfoniopropriate, and by determining if betaine protects in MAT1A null mice. In addition, we will assess the global effects of betaine feeding on hepatic gene expression to look for clues of alternative effects of betaine which might also contribute to its protective action. We anticipate that we will obtain important new knowledge which can be applied to the prevention and treatment of ALD