Aging and Lipid Metaboilism

背景：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。具体来说，磷脂及其代谢物通过改变膜结构和细胞间和细胞内信号传导在细胞功能中起着核心作用。近年来，细胞内脂质结合蛋白酰基辅酶a结合蛋白（ACBP）已成为脂质代谢和信号传导的重要调节因子。问题：衰老会导致磷脂质量和脂肪酸组成的显著变化，然而，引起这些变化的生化和分子机制尚不清楚。目的：1)评估衰老对参与磷脂酸（PA）生物合成的主要酶活性的影响，磷脂酸是导致几乎所有磷脂合成的主要生化途径；2)评价衰老对这些酶脂肪酸特异性的影响；3)确定年龄对ACBP水平及PA生物合成诱导的影响。方法：采用老年小鼠（C57BL/6 × DBA/2）和老年小鼠（C57BL/6 × DBA/2）随意饲喂或限热量饲料。内质网（ER）和线粒体将从肝脏中分离出来，因为内质网和线粒体是从头合成磷脂的主要部位。本研究的重点是分别通过甘油-3-磷酸酰基转移酶和溶血磷脂酸酰基转移酶将甘油-3-磷酸依次酰基化为溶血磷脂酸，然后依次酰基化为PA。实验还将进行脂肪酸对酰基辅酶a合成酶和酰基辅酶a水解酶的活化。脂肪酸必须是酰基辅酶a的形式才能被酰基转移酶利用。动力学研究将使用油酰辅酶a进行，有或没有白蛋白或ACBP存在，增加酰基转移酶活性。将使用饱和、单不饱和和多不饱和酰基辅酶a进行进一步的研究。ACBP水平将分别通过western blotting和RT-PCR分析蛋白和基因水平来确定。结果：实验将揭示衰老是否通过改变内质网膜结构或改变ACBP表达来影响酶活性。其次，了解年龄是否会改变特定脂肪酸对磷脂生物合成的利用。益处：描述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/或药物策略的发展，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.