Ethanol and retinoid metabolism/function

描述（由申请人提供）：本项目旨在确定乙醇对正常维生素A（视黄醇）营养期间体内atRA（全反式维甲酸）浓度的影响，并确定乙醇对atRA稳态影响的机制和功能后果。我们开发了一种LC/MS/MS分析与飞摩尔灵敏度的小生物样品中的atRA的具体定量，并使用它来生成atRA及其异构体在血清和多种组织的几个品系的小鼠的参考值。该测定法用于确定乙醇对正常维生素A营养期间稳态atRA浓度的影响。与预期相反，乙醇喂养（Lieber-DeCarli液体饮食，36%的热量，1个月）没有改变atRA浓度在肝脏，肾脏，嗅球，纹状体，丘脑和小脑的C57 BL/6成年小鼠，但引起2至50倍的atRA浓度增加在海马，皮质，睾丸和血清。初步数据表明，乙醇，喂养同样的方式，以母鼠从e13通过e18，增加atRA在海马和皮质的e19胚胎的比例，母鼠BAC。0.08%的母鼠BAC分别导致海马和皮质atRA增加4倍和30倍。我们还发现，海马星形胶质细胞生物合成atRA，和atRA诱导树突状生长在原代小鼠海马神经元。atRA通过定位于树突状RNA颗粒并刺激翻译的RAR 1形式刺激树突状生长。我们试图应用这些技术和洞察力，严格确定的后果，大坝乙醇摄入对atRA浓度在海马的e19胚胎，并了解乙醇的影响的生物后果。要测试的假设是：乙醇摄入的母鼠增加内源性浓度的atRA在海马体的胚胎，这些atRA增加海马体发育过程中的乙醇毒性和/或致畸。具体目的是确定慢性乙醇对以下方面的影响：1）海马发育早期（e13-e18）胚胎中的内源性atRA; 2）发育中海马中类维生素A调节的基因表达（转录和翻译）; 3）发育中海马的神经发生; 4）海马星形胶质细胞合成和分泌atRA的机制; 5）海马神经元中atRA的功能。这项工作的目的是提供新的见解维甲酸代谢和生物学，以及维生素A和乙醇之间的相互作用，使用新技术和新的见解最近产生的维甲酸在海马体中的功能。研究结果可能会影响治疗酗酒者的方法。公共卫生相关性：维生素A是脊椎动物生命所必需的，因为它是胚胎发育所必需的，并调节中枢神经系统的功能，以及许多其他功能。酒精（乙醇）摄入会导致肝脏中维生素A储存的严重消耗，并被认为会抑制维生素A活化成其激素形式，全反式视黄酸（atRA）。我们有新的数据使用最近开发的技术，乙醇增加atRA在特定区域的大脑，包括海马体。本项目旨在确认观察到的慢性乙醇摄入的大坝增加了胚胎的海马体中的atRA的浓度，其大坝在海马体发育过程中摄入乙醇，并确定乙醇对atRA功能在海马体中的影响。

DESCRIPTION (provided by applicant): This project seeks to determine the impact of ethanol on atRA (all-trans-retinoic acid) concentrations in vivo during normal vitamin A (retinol) nutriture, and to identify mechanisms and functional consequences of ethanol effects on atRA homeostasis. We developed a LC/MS/MS assay with femtomol sensitivity for specific quantitation of atRA in small biological samples, and used it to generate reference values for atRA and its isomers in serum and multiple tissues of several strains of mice. The assay was applied to determine the impact of ethanol on steady-state atRA concentrations during normal vitamin A nutriture. Contrary to expectations, ethanol feeding (Lieber-DeCarli liquid diet, 36% of calories, 1 month) did not alter atRA concentrations in liver, kidney, olfactory bulb, striatum, thalamus and cerebellum of C57BL/6 adult mice, but caused 2 to 50-fold increases in atRA concentrations in hippocampus, cortex, testis and serum. Preliminary data showed that ethanol, fed the same way to dams from e13 through e18, increased atRA in the hippocampus and cortex of e19 embryos in proportion to dam BAC. A dam BAC of 0.08% caused 4 and 30- fold increases, respectively, in hippocampus and cortex atRA. We also showed that hippocampus astrocytes biosynthesize atRA, and atRA induces dendritic growth in primary mouse hippocampus neurons. atRA stimulates dendritic growth through a form of RAR1 that localizes to dendritic RNA granules and stimulates translation. We seek to apply these techniques and insight to determine rigorously the consequences of dam ethanol ingestion on atRA concentrations in hippocampus of e19 embryos, and to understand the biological consequences of the ethanol effects. The hypothesis to be tested is: ethanol ingestion by dams increases endogenous concentrations of atRA in the hippocampus of their embryos; these atRA increases during hippocampus development contribute to ethanol toxicity and/or teratology. The specific aims are to determine the effects of chronic ethanol on: 1) endogenous atRA in the embryo during early hippocampus development (e13-e18); 2) retinoid-regulated gene expression (transcription and translation) in the developing hippocampus; 3) neurogenesis in the developing hippocampus; 4) mechanisms of atRA biosynthesis and secretion by hippocampus astrocytes; 5) atRA functions in hippocampus neurons. This work aims to provide new insight into retinoid metabolism and biology, and the interactions between vitamin A and ethanol, using new technology and new insights generated recently about retinoid function in the hippocampus. The results could influence approaches to treating alcoholics. PUBLIC HEALTH RELEVANCE: Vitamin A is essential for vertebrate life, because it is necessary for embryonic development, and regulates function of the central nervous system, among many other functions. Alcohol (ethanol) ingestion causes severe depletion of vitamin A storage in the liver, and has been postulated to inhibit activation of vitamin A into its hormonal form, all-trans-retinoic acid (atRA). We have new data using recently developed techniques that ethanol increases atRA in specific areas of the brain, including the hippocampus. This project seeks to affirm the observation that chronic ethanol ingestion by dams increases the concentrations of atRA in the hippocampus of embryos whose dams have ingested ethanol during hippocampus development, and to determine the effects of ethanol on atRA function in the hippocampus.