NUTRIENT AND HORMONAL CONTROL OF A LIPID ABSORPTION GENE

流行病学研究已经证明了饮食与 胆固醇吸收效率与血浆胆固醇水平和风险 动脉粥样硬化 然而，目前只有很少的信息 可用于调节重要基因表达的机制 在这个吸收过程中。 该应用程序将解决可能的 特定营养素和肠激素对表达的作用 胰腺胆固醇酯酶，一种催化胆固醇的蛋白质 通过消化道吸收。 初步数据显示 胆固醇酯酶生物合成的急性期活化是 由肠道激素介导，导致胆固醇升高 酯酶mRNA翻译。 与此相反，长期喂养大鼠， 高脂肪/高胆固醇饮食增加胆固醇酯酶mRNA水平， 这是因为营养素激活了它的基因转录。 具体目标1是 旨在确定调节胆固醇的分子机制 酯酶mRNA翻译。 初步结果显示， 胆固醇酯酶特异性翻译抑制因子 在体外降低胆固醇酯酶mRNA翻译的细胞。 这 胍存在抑制性工厂 硫氰酸盐/苯酚/氯仿提取物，表明它是一种RNA。 计划进行实验以分离和表征这种特定的 翻译控制RNA（tcRNA）。 标准尺寸分级和凝胶 电泳技术将用于分离tcRNA， 序列分析 翻译调控的机制将是 通过确定tcRNA对胆固醇酯酶的作用来探索 多肽链起始和延伸。 第2章研究 脂肪和胆固醇增加胆固醇酯酶的机制 基因转录 特定类型的膳食脂肪对 胆固醇酯酶基因转录也将被探索。 的 胆固醇酯酶基因中的调节结构域将通过 各种序列赋予胆固醇反应性的能力， 转染的AR42J细胞中的报告基因。 可疑区域 假定的胆固醇响应元件将通过DNA 足迹法和凝胶阻滞测定。 数量有限的转基因 将用嵌合构建体进行小鼠实验以验证 基因转录和mRNA翻译的饮食反应性。 此外，研究结果将有助于我们对机理的理解 控制饮食中胆固醇的吸收

Epidemiology studies have documented a relationship between dietary cholesterol absorption efficiency with plasma cholesterol level and risk of atherosclerosis. However, very little information is currently available on the mechanism regulating the expression of genes important for this absorption process. This application will address the possible role of specific nutrients and intestinal hormones on the expression of pancreatic cholesterol esterase, a protein that catalyzes cholesterol absorption through the digestive tract. Preliminary data have indicated that acute phase activation of cholesterol esterase biosynthesis is mediated by intestinal hormones and results in increase cholesterol esterase mRNA translation. In contrast, chronic feeding of rats with a high fat/high cholesterol diet increases cholesterol esterase mRNA level, due to nutrient activation of its gene transcription. Specific Aim 1 is designed to determine the molecular mechanism regulating cholesterol esterase mRNA translation. Preliminary results have shown the presence of a cholesterol esterase-specific translation inhibitory factor in control cells that reduces cholesterol esterase mRNA translation in vitro. This inhibitory factory was present in guanadinium thiocyanate/phenol/chloroform extracts, suggesting that it is an RNA. Experiments are planned to isolate and characterize this specific translational control RNA (tcRNA). Standard size fractionation and gel electrophoresis techniques will be utilized to isolate the tcRNA for sequence analysis. The mechanism of translational regulation will be explored by determining the role of the tcRNA on cholesterol esterase polypeptide chain initiation and elongation. Specific Aim 2 will study the mechanism by which fat and cholesterol increase cholesterol esterase gene transcription. The role of specific types of dietary fat on cholesterol esterase gene transcription will also be explored. The regulatory domain in the cholesterol esterase gene will be identified by the ability of various sequences to confer cholesterol responsiveness of a reporter gene in transfected AR42J cells. Suspected regions for the putative cholesterol responsive elements will be evaluated by DNA footprinting and gel retardation assays. A limited number of transgenic mice experiments will be performed with the chimeric constructs to verify dietary responsiveness of gene transcription and translation of mRNA. Moreover, the results will contribute to our understanding on mechanism that controls dietary cholesterol absorption.