Retinoid Metabolism and Alcohol Induced Disease

说明（由申请人提供）：世界卫生组织2004年公布的对长期饮酒造成的死亡率的估计表明，每年约有180万人死于酒精。饮酒会使人上瘾，并损害身体的几乎每个器官。导致酒精相关疾病的分子事件是复杂的，尚未完全了解。类维生素A（维生素A及其代谢物）是有效的转录调节因子，介导正常细胞增殖、分化和凋亡。酒精的一个不良反应是通过损害类视黄醇代谢和肝脏（人体70%的类视黄醇储存在肝脏中）和外周组织中的作用来促进组织和器官的损伤。酗酒者通常肝脏和组织的类维生素a水平都非常低。这导致维持正常细胞增殖和分化的类维甲酸可用性降低，使细胞/组织更容易受到酒精诱导的损伤。文献中提出的解释为什么类维生素a稳态在酗酒者中受损的原因有：1。酒精通过竞争醇（视黄醇）脱氢酶和醛（视网膜）脱氢酶催化维甲酸合成，抑制转录活性类视黄酸的合成；2. 酒精加速类视黄醇氧化，涉及乙醇诱导的细胞色素，如Cyp2E1；和3。酒精增加了储存的类维生素a从肝脏向其他组织的动员。目前，在类视黄醇代谢领域工作的研究人员认为，调节细胞/组织内类视黄醇稳态的关键代谢事件是视黄醇酯合成（涉及卵磷脂：视黄醇酰基转移酶或LRAT）和视黄酸氧化（由细胞色素酶催化）。进一步认为，肝脏类视黄醇代谢通过视黄醇结合蛋白（retinol-binding protein， RBP）介导的视黄醇在器官间的快速转移与外周组织的代谢相结合。我们的研究将集中在肝脏类视黄醇储存和类视黄醇动员/再分配，以及这些重要的调节过程如何受到慢性酒精摄入的影响。我们将使用Lrat-/-和Rbp-/-小鼠以及仅在骨骼肌中表达脂蛋白脂肪酶（LpL）的小鼠（MCK-LpL0小鼠）来研究这些关系。我们已经研究并发表了这些小鼠在类维生素a储存、代谢和运输方面的特征描述，现在建议使用这些小鼠模型来研究酒精诱导的器官损伤。LRAT是组织/细胞类视黄醇稳态的中心调节器，控制视黄醇在视黄酸合成中的可用性。Lrat-/-小鼠在任何组织中储存的类维生素a很少，肝脏中没有。RBP主要由肝脏合成，是循环中视黄醇的唯一转运蛋白，占空腹循环中类视黄醇的95%。Rbp-/-小鼠正常地在肝脏中积累饮食中的类维甲酸，当维持类维甲酸充足的饮食时，其表型正常。然而，Rbp-/-小鼠不能将视黄醇从肝脏调动/重新分配到周围。LpL催化视黄醇酯的水解，其表达在活化的肝星状细胞（HSCs）中升高30倍以上，肝星状细胞是肝中类视黄醇储存的细胞部位。有人提出，LpL促进了在HSC激活时从类视黄酮储存中动员类视黄酮。健康MCK-LpL0小鼠的肝脏类视黄醇储存和动员是正常的，因为这些小鼠的血清和肝脏类视黄醇水平与匹配的野生型（WT）小鼠相比没有差异。因此，在活化的hsc中不能表达LpL的MCK-LpL0小鼠中，我们将能够确定LpL在酒精诱导的肝脏疾病中的作用。该项目包括两个目标。在目标1中，我们将问：缺乏肝类视黄醇储存如何影响酒精诱导的肝脏疾病的发展和肝脏再生？这些研究将包括使用Lrat-/-小鼠来探索肝类视黄醇储存和Lrat在酒精性肝病发展中的作用。在目标2中，我们将问：动员/重新分配肝类视黄醇储存到周围的能力是否有助于酒精诱导的组织/器官损伤的发展？在这里，我们将使用Rbp-/-小鼠，它们不能动员肝类视黄醇储存，以及MCK-LpL0小鼠，它们不能在肝脏中表达LpL，在肝脏损伤中，LpL被认为在动员肝视黄醇酯储存中起作用。我们还将在WT、Rbp-/-和MCK-LpL0小鼠中探讨动员/重新分配肝类视黄醇储存的能力是否有助于周围器官损伤的发展，特别是酒精性心肌病。

DESCRIPTION (provided by applicant): Estimates of death rates caused by chronic alcohol consumption, published in 2004 by the World Health Organization, indicate that alcohol accounts for approximately 1.8 million deaths per year. Alcohol consumption leads to addiction and damage to almost every organ in the body. The molecular events that underlie alcohol-associated disease are complex and not completely understood. Retinoids (vitamin A and its metabolites) are potent transcriptional regulators that are needed for mediating normal cell proliferation, differentiation and apoptosis. One adverse action of alcohol involves promotion of tissue and organ damage by impairing retinoid metabolism and actions in liver (where 70% of the body's retinoid is stored) and in peripheral tissues. Alcoholics generally have very reduced hepatic and tissue retinoid levels. This results in decreased retinoid availability for maintaining normal cell proliferation and differentiation, rendering cells/tissues more susceptible to alcohol-induced injury. Reasons proposed in the literature to explain why retinoid homeostasis is impaired in alcoholics are: 1. alcohol inhibits the synthesis of the transcriptionally active retinoid, retinoic acid, by competing for alcohol (retinol) dehydrogenases and aldheyde (retinal) dehydrogenases catalyzing retinoic acid synthesis; 2. alcohol accelerates retinoid oxidation, involving ethanol-inducible cytochromes like Cyp2E1; and 3. alcohol increases mobilization of stored retinoid from the liver to other tissues. It is currently believed by investigators working in the area of retinoid metabolism that the key metabolic events regulating retinoid homeostasis within cells/tissues are retinyl ester synthesis (involving lecithin:retinol acyltransferase or LRAT) and retinoic acid oxidation (catalyzed by cytochrome enzymes). It is further thought that hepatic retinoid metabolism is integrated with that of peripheral tissues, through rapid interorgan transfer of retinol mediate via retinol-binding protein (RBP). Our investigations will focus on hepatic retinoid storage and retinoid mobilization/redistribution from the liver and how these important regulatory processes are influenced by chronic alcohol intake. We will employ Lrat-/- and Rbp-/- mice and mice expressing lipoprotein lipase (LpL) solely in skeletal muscle (MCK-LpL0 mice) to investigate these relationships. We have studied and published descriptions of the characteristics of these mice with regards to retinoid storage, metabolism and transport and now propose to employ these mouse models to study alcohol-induced organ injury. LRAT is a central regulator of tissue/cellular retinoid homeostasis, controlling retinol availability for retinoic acid synthesis. Lrat-/- mice possess very little stored retinoid in any tissue, and none in liver. RBP is synthesized primarily by the liver and is the sole transport protein for retinol in the circulation, accounting for > 95% of the retinoid present in the fasting circulation. Rbp-/- mice accumulate dietary retinoid normally in liver and are phenotypically normal when maintained on a retinoid-sufficient diet. However Rbp-/- mice are unable to mobilize/redistribute retinol from the liver to the periphery. LpL catalyzes the hydrolysis of retinyl esters and its expression is elevated over 30-fold in activated hepatic stellate cells (HSCs), the cellular site of retinoid storage in the liver. It has been proposed that LpL facilitates retinoid mobilization from HSC retinoid stores upon HSC activation. Hepatic retinoid storage and mobilization are normal in healthy MCK-LpL0 mice since serum and hepatic retinoid levels are not different for these mice compared to matched chow fed wild type (WT) mice. Thus, using MCK-LpL0 mice which are unable to express LpL in activated HSCs, we will be able to define a role for LpL in alcohol-induced liver disease. The project consists of 2 Aims. In Aim 1 we will ask: How does the absence of hepatic retinoid stores influence alcohol-induced liver disease development and liver regeneration? These investigations will involve the use of Lrat-/- mice to explore the role that hepatic retinoid stores and LRAT have in the development of alcoholic liver disease. In Aim 2 we will ask: Does the ability to mobilize/redistribute hepatic retinoid stores to the periphery contribute to the development of alcohol-induced tissue/organ injury? Here we will employ Rbp-/- mice, which are unable to mobilize hepatic retinoid stores, and MCK-LpL0 mice which can not express LpL in liver, an organ where it is proposed LpL plays a role in mobilizing hepatic retinyl ester stores upon hepatic injury. We will also explore in WT, Rbp-/- and MCK-LpL0 mice whether the ability to mobilize/redistribute hepatic retinoid stores contributes to the development of peripheral organ injury, specifically to alcoholic cardiomyopathy. PUBLIC HEALTH RELEVANCE: Chronic alcohol consumption is a major public health problem, leading to addiction and damage of almost every organ in the body. There is compelling evidence that alcohol impairs vitamin A metabolism in tissues, especially liver, where 70% of the vitamin A present in the body is stored. This results in lessened vitamin A availability for maintaining normal cellular proliferation, differentiation and apoptosis in liver and other organs. We are proposing studies that will provide a more complete understanding of relationships between alcohol consumption, vitamin A metabolism and actions, and alcoholic organ damage.