MECHANISMS OF ACTIONS OF ALCOHOL AND OMEGA 3-FATTY ACIDS

描述（改编自申请人的摘要）： 这种竞争性的持续应用是：乙醇暴露下调 α-2，6-唾液酸转移酶（2，6-ST）在分子水平上导致 载脂蛋白E（ApoE）糖基化缺陷及其与 HDL。乙醇也影响神经鞘磷脂（SPM）和其他磷脂（PL）。 高密度脂蛋白。这些缺陷导致胆固醇逆向转运（RCT）受损， 被低膳食欧米茄3脂肪酸（欧米茄3FA）逆转。本实验室曾 以下发表的（10篇出版物和1篇综述）和初步数据， 支持上述假设：1。乙醇损害唾液酸化， 转铁蛋白（Tf）和载脂蛋白E（ApoE）的表达。2.两 在两个尸检肝脏中，2，6-ST-mRNA和GAPDH mRNA水平似乎是完整的 来自非酒精对照的样品。3. 50和100 mM乙醇降低ST 在稳定表达Cyp 2 E的人HepG 2细胞（Cyp 2 E细胞）和高表达Cyp 2 E的人HepG 2细胞中的mRNA 乙醇脱氢酶（ADH细胞），但不是在野生型。4.然而，在这方面， 50 μ M浓度的乙醛（Ach）降低2，6-ST mRNA，即使在Wilde 类型. 5.乙醇使肝脏2，6-STmRNA不稳定，推测是通过顺式和反式- 作用因素6.去唾液酸化的ApoE对HDL具有低亲和力，导致其 人和大鼠RCT功能受损。7.两种慢性 酒精喂养的大鼠和人类酗酒者显示出有缺陷的RCT。8.极低膳食 ω 3FA恢复了乙醇介导的HDL RCT能力抑制。9.两 20：5和22：6 omega 3FA含量的HDL增加4-4.7倍，omega 3FA喂养 与对照组相比。10. HDL中鞘磷脂（SPM）含量为 乙醇组降低，同时伴随RCT抑制。11. HDL来自 与不饮酒者相比，慢性酗酒者的HDL SPM也较低。12. 缺乏SPM的HDL显示RCT功能受损。因此，以下 提出了具体的目标，以测试假设，以了解机制， 行动的酒精和欧米茄3FA：糖基化：具体目标1：是蛋白质 参与乙醇介导的肝脏2，6-ST mRNA不稳定的因素？ 乙醇如何影响2，6-ST蛋白及其mRNA的分布？做 酗酒者的肝脏也表现出2，6-ST蛋白及其mRNA的减少。 具体目标2：乙醇的体内效应能否在人肝脏中模拟 细胞系统？乙醇的活跃代谢是其在 这些细胞系统？顺式和反式因子是什么 去稳定化2，6-ST mRNA？Ach在临床上是否对这些效应负责 相关水平？功能后果：具体目标3：如何做欧米茄3脂肪酸 改变乙醇介导的HDL RCT功能缺陷？具体目标4： 乙醇影响HDL SPM和其他PL？人类酗酒者是否改变了SPM 和HDL中其他PL水平的关系乙醇引起的SPM和其他 PL？酗酒者是否改变了HDL中的SPM和其他PL水平？并 乙醇诱导的SPM和HDL的其他PL的损失影响其RCT功能？的 实验室将在人类和动物系统中使用分子 生物学、免疫化学和生物化学方法。

DESCRIPTION (Adapted from the applicant's abstract): The major hypotheses of this competing continuation application are: Ethanol exposure down-regulates alpha-2,6-sialyltransferase (2,6-ST) at the molecular level leading to defective glycosylation of apolipoprotein E (ApoE) and its association with HDL. Ethanol also affects sphingomyelin (SPM) and other phospholipids (PL) of HDL. These defects lead to impaired reverse cholesterol transport (RCT) that are reversed by low dietary omega 3-faty acids (omega 3FA). This laboratory has the following published (10 publication & 1 review) and preliminary data in support of the above hypotheses: 1. Ethanol impaired sialylation of both transferring (Tf) and ApoE by down-regulating 2,6-ST and its mRNA. 2. Both 2,6-ST-mRNA and GAPDH mRNA levels seemed to be intact in two autopsy liver samples from non-alcoholic controls. 3. Ethanol at 50 and 100mM decreased ST mRNA in human HepG2 cells stably expressing Cyp2E (Cyp2E cells) and high alcohol dehydrogenase (ADH cells), but not in Wild type. 4. However, acetaldehyde (Ach) at 50muM concentration decreased 2,6-ST mRNA even in Wilde type. 5. Ethanol destabilized liver 2,6-STmRNA, presumably via cis and trans- acting factors. 6. Desialylated ApoE had low affinity for HDL resulting in its impaired RCT function in both human and rat. 7. HDLs from both chronic ethanol-fed rats and human alcoholics showed defective RCT. 8. Very low dietary omega 3FA restored ethanol-mediated inhibition of RCT capacity of HDL. 9. Both 20:5 and 22:6 omega 3FA contents of HDL increased 4-4.7-fold in omega 3FA-fed group compared to the control group. 10. Sphingomyelin (SPM) content of HDL was decreased in ethanol group with concomitant inhibition of RCT. 11. HDLs from chronic human alcoholics also had lower HDL SPM compared to non-drinkers. 12. HDL devoid of SPM showed impaired RCT function. Therefore, the following specific aims are proposed to test hypotheses to understand the mechanisms of actions of alcohol and omega 3FA: Glycosylation: Specific Aim 1: Are protein factors involved in the ethanol-mediated destabilization of liver 2,6-ST mRNA? How does ethanol affect the distribution of 2,6-ST protein & its mRNA? Do livers from human alcoholics also exhibit decreased 2,6-ST protein & its mRNA? Specific Aim 2: Can the effects of ethanol in vivo be mimicked in human liver cell systems? Is active metabolism of ethanol a prerequisite for its effects in these cell systems? What are the cis and trans factors responsible for destabilizing 2,6-ST mRNA? Is Ach responsible for these effects at clinically relevant levels? Functional Consequences: Specific Aim 3: How do omega 3 FA alter ethanol-mediated defects in the RCT function of HDL? Specific Aim 4: Does ethanol affect the HDL SPM and other PL? Do human alcoholics have altered SPM and other PL levels in their HDL? Does ethanol-induced loss of SPM and other PL? Do human alcoholics have altered SPM and other PL levels in their HDL? Does ethanol-induced loss of SPM and other PL from HDL affect its RCT function? The laboratory will accomplish these in human and animal systems using molecular biology, immunochemical and biochemical approaches.