Aging and Lipid Metaboilism

背景资料：脂质在老年人的许多主要疾病（动脉粥样硬化，癌症，关节炎）以及衰老本身中起着重要作用。具体而言，磷脂及其代谢产物通过修饰膜结构以及细胞间和细胞内信号传导在细胞功能中发挥核心作用。最近，细胞内脂质结合蛋白，酰基辅酶A结合蛋白（ACBP），已成为一个重要的调节脂质代谢和信号转导。问题：衰老导致磷脂质量和脂肪酸组成的显著变化，然而，引起这些变化的生物化学和分子机制尚不清楚。目的：1）评估老化对参与磷脂酸（PA）生物合成的主要酶活性的影响，PA生物合成是导致几乎所有磷脂合成的中心生化途径; 2）评估老化对这些酶的脂肪酸特异性的影响; 3）确定年龄对ACBP水平和PA生物合成诱导的影响。方法：将使用自由进食或热量限制饮食的年轻和老年（C57 BL/6 x DBA/2）小鼠。内质网（ER）和线粒体将从肝脏分离，因为ER和线粒体是从头磷脂生物合成的主要位点。本论文的工作主要集中在甘油-3-磷酸酰基转移酶和溶血磷脂酸酰基转移酶分别催化甘油-3-磷酸酰化为溶血磷脂酸和PA。还将进行关于通过酰基辅酶A合成酶和酰基辅酶A水解酶将脂肪酸活化为酰基辅酶A的实验。脂肪酸必须是酰基辅酶A形式，以便被酰基转移酶利用。将使用油酰辅酶A进行动力学研究，存在或不存在增加酰基转移酶活性的白蛋白或ACBP。将使用饱和、单不饱和和多不饱和酰基辅酶A进行其他研究。ACBP的水平将分别通过蛋白质印迹和RT-PCR分析蛋白质和基因水平来确定。结果：实验将揭示衰老是否通过改变ER膜结构或改变ACBP表达来影响酶活性。其次，将深入了解年龄是否会改变磷脂生物合成中特定脂肪酸的利用率。优点：表征衰老和热量限制对磷脂代谢的影响可能最终导致旨在维持最佳细胞功能的饮食和/或药理学策略的发展，从而改善老年人的生活质量。

Background: Lipids play a fundamental role in many of the major diseases (atherosclerosis, cancer, arthritis) in the elderly as well as in aging itself. Specifically, phospholipids and their metabolites play a central role in cell function by modifying membrane structure and inter- and intracellular signaling. Recently, the intracellular lipid binding protein, acyl-CoA binding protein (ACBP), has emerged as an important regulator of lipid metabolism and signaling. Problem: Aging results in significant changes in phospholipid mass and fatty acid composition, however, the biochemical and molecular mechanism(s) causing these alterations is not known. Purpose: 1) Assess the influence of aging on the activities of the major enzymes involved in phosphatidic acid (PA) biosynthesis which is the central biochemical pathway leading to the synthesis of almost all phospholipids; 2) Evaluate the effects of aging on the fatty acid specificities of these enzymes; 3) Determine the influence of age on ACBP levels and the induction of PA biosynthesis. Methods: Young and old (C57BL/6 x DBA/2) mice fed ad libitum or calorie restricted diets will be used. Endoplasmic reticulum (ER) and mitochondria will be isolated from the liver because the ER and mitochondria are the major sites of de novo phospholipid biosynthesis. The work will focus on the sequential acylation of glycerol-3-phosphate to lysophosphatidic acid then to PA by glycerol-3-phosphate acyltransferase and lysophosphatidic acid acyltransferase respectively. Experiments will also be performed on the activation of fatty acids to acyl-CoAs by acyl-CoA synthetase and acyl-CoA hydrolase. The fatty acids have to be in the acyl-CoA form in order to be utilized by the acyltransferases. Kinetic studies will be conducted using oleoyl-CoA with or without albumin or ACBP present which increases acyltransferase activity. Additional studies will be done using saturated, monounsaturated and polyunsaturated acyl-CoAs. Levels of ACBP will be determined by analyzing protein and gene levels by western blotting and RT-PCR, respectively. Outcome: The experiments will reveal if aging influences enzyme activity by altering ER membrane structure or by altering ACBP expression. Secondly, insight will be gained into whether age alters the utilization of specific fatty acids for phospholipid biosynthesis. Benefit: Characterizing the influence of aging and calorie restriction on phospholipid metabolism may ultimately lead to the development of dietary and/or pharmacologic strategies aimed at maintaining optimal cellular function hence improving the quality of life in the elderly.