Lipid Metabolism in the Aging Immune System

描述（由申请人提供）：脂质在老年人的许多主要疾病（动脉粥样硬化、癌症、关节炎）中以及在衰老本身中起着重要作用。具体而言，磷脂酸（PA）通过合成磷脂修饰膜结构并作为有效的细胞内信号传导分子，在细胞功能中发挥核心作用。最近，细胞内的脂质结合蛋白，酰基辅酶A结合蛋白（ACBP），已成为PA合成和细胞内信号转导的重要调控因子。T细胞中的磷脂改变与老年人免疫功能下降有关。问题：衰老导致T细胞中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）确定通过甘油-3-磷酸酰基转移酶（GPAT）减少的PA生物合成是否至少部分地导致T细胞增殖减少; 2）确定ACBP在老年T细胞功能障碍中PA生物合成中的作用; 3）评估ACBP在老年T细胞亚群功能障碍中的作用。方法：青年和老年大鼠自由进食。将从脾T细胞中分离内质网（ER）（微粒体）和线粒体，因为ER和线粒体是PA从头生物合成的主要位点。本工作将着重于分别用（GPAT）和溶血磷脂酸酰基转移酶将甘油-3-磷酸酰化为溶血磷脂酸，然后再酰化为PA。将设计实验以确定衰老对GPAT的影响在本质上是转录前（基因表达）还是转录后（磷酸化，蛋白质水平）。这是重要的，因为GPAT是PA生物合成中的限速酶。ACBP在介导随年龄降低的T细胞功能中的影响将通过检查过表达的ACBP是否改变T细胞增殖以及衰老对ACBP水平的影响来评估，所述影响通过分别通过蛋白质免疫印迹和RT-PCR分析蛋白质和基因表达来进行。ACBP对CD4+和CD8 + T细胞亚群和记忆/幼稚T细胞功能随年龄的影响将通过将ACBP转染到随着年龄表现出改变的ACBP表达的那些老年T细胞群中并通过流式细胞术检查细胞因子产生来确定。结果：这些实验将深入了解PA合成和ACBP表达或功能的改变是否是T细胞增殖的年龄依赖性损失的重要机制解释。其次，将深入了解ACBP是否在特定T细胞亚群的年龄依赖性功能障碍中发挥作用。优点：表征衰老对ACBP和PA合成的影响可能最终导致旨在维持最佳免疫功能的饮食和/或药理学策略的发展，从而改善老年人的生活质量。

DESCRIPTION (provided by applicant): Lipids play a fundamental role in many of the major diseases (atherosclerosis, cancer, arthritis) in the elderly as well as in aging itself. Specifically, phosphatidic acid (PA) plays a central role in cell function by modifying membrane structure via synthesis into phospholipids and serving as a potent intracellular signaling molecule. Recently, the intracellular lipid binding protein, acyl-CoA binding protein (ACBP), has emerged as an important regulator of PA synthesis and intracellular signaling. Phospholipid alterations in T-cells are associated with reduced immune function in the elderly. Problem: Aging results in significant changes in phospholipid mass and fatty acid composition in T-cells, however, the biochemical and molecular mechanism(s) causing these alterations is not known. Purpose: 1) Determine if reduced PA biosynthesis via glycerol-3-phosphate acyltransferase (GPAT) accounts, at least in part, for reduced T-cell proliferation; 2) Determine the role of ACBP in PA biosynthesis in aged T-cell dysfunction; 3) Assess the role of ACBP in aged T-cell subset dysfunction. Methods: Young and old rats fed ad libitum diets will bell used. Endoplasmic reticulum (ER) (microsomes) and mitochondria will be isolated from the splenic T-cells because the ER and mitochondria are the major sites of de novo PA biosynthesis. The work will focus on the acylation of glycerol-3-phosphate to lysophosphatidic acid then to PA by (GPAT) and lysophosphatidic acid acyltransfcrase respectively. Experiments will be designed to determine if the aging effects on GPAT are pre- (gene expression) or posttranscriptional (phosphorylation, protein levels) in nature. This is significant because GPAT is the rate-limiting enzyme in PA biosynthesis. The influence of ACBP in mediating reduced T-cell function with age will be assessed by examining whether over-expressed ACBP alters T-cell oroliferation and what impact aging has on ACBP levels by analyzing protein and gene expression by western immunoblotting_and RT-PCR, respectively. ACBP's influence on CD4+ and CD8+ T-cell sub sets and memory/naive-T-cell function with age will be determined by transfecting ACBP into those aged T-cell populations which exhibit altered ACBP expression with age and examining cytokine production by flow cytometry. Outcome: The experiments will give insight into whether alterations in PA synthesis and ACBP expression or function are an important mechanistic explanation for the age-dependent loss of T-cell proliferation. Secondly, insight will be gained into whether ACBP plays a role in the age-dependent dysfunction of specific T-cell subsets. Benefit: Characterizing the influence of aging on ACBP and PA synthesis may ultimately lead to the development of dietary and/or pharmacologic strategies aimed at maintaining optimal immune function hence improving the quality of life in the elderly.