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Hydrolysis of vitamin A esters in liver

维生素A酯在肝脏中的水解

基本信息

批准号：
7289291
负责人：
EARL Howard HARRISON
金额：
$ 33.55万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-09-30 至 2011-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7289291
关键词：
1,2-diacylglycerol Accounting Acids Air All-Trans-Retinol Bile Acids and Salts Binding Biological Assay Carboxylic Ester Hydrolases Cell Line Cell Separation Cells Cholesterol Esters Chylomicrons Complementary DNA Complex Coupled Cultured Cells Diglycerides Drug or chemical Tissue Distribution Emulsions Enzymatic Biochemistry Enzyme Kinetics Enzymes Esters Facility Construction Funding Category Goals Hepatic Hepatic Stellate Cell Hepatocyte Hydrolysis In Vitro Incubated Kidney Kinetics Knowledge Lipids Lipoproteins Liposomes Liver Liver Microsomes Localized Lysophospholipids Messenger RNA Metabolic Metabolism Mono-S Mus Peripheral Pharmaceutical Preparations Phenotype Phospholipids Physiological Plasma Play Polymerase Chain Reaction Primary carcinoma of the liver cells Protein Secretion Proteins RNA Interference Rate Rattus Relative (related person)Retinoids Retinol Binding Proteins Role Sequence Analysis Serum Site Small Interfering RNA Smooth Muscle Myocytes Substrate Specificity System Testing Testis Time Tissues Transfection Triglycerides Vitamin A Water bile salts carboxylesterase cell type chylomicron remnant enzyme substrate hepatoma cell in vivo laser capture microdissection macrophage monolayer retinyl esterase stellate cell uptake

项目摘要

DESCRIPTION (provided by applicant): Hydrolysis of retinyl esters (RE) in the liver plays a key role in the body's metabolism of vitamin A. Dietary vitamin A enters the liver as RE in chylomicron remnants, which undergo hydrolysis and reesterification for storage. Prior to mobilization, RE are hydrolyzed and free retinol binds to retinol-binding protein for secretion into the plasma. Neutral and acid retinyl ester hydrolases (REHs) have been purified from rat liver and shown by sequence analysis to be carboxylesterases, ES-2 and ES-10. The goal of this project is to define the role of ES-2, ES-10, and the related ES-3 and ES-4, in the metabolism of vitamin A. Carboxylesterases have been studied with other substrates, but it is not known if they are important in vitamin A metabolism. Their efficiencies in catalyzing the hydrolysis of RE compared with other esters are also unknown. These gaps in knowledge will be explored with the following specific aims: AIM 1: Enzymoloqy of REHs. Enzyme kinetics will be used to study the substrate specificity of the carboxylesterases and to test the hypothesis that these enzymes differ in their intrinsic ability to catalyze the hydrolysis of RE and other lipid esters. AIM 2: Tissue distribution of carboxylesterases. Real-time PCR (for mRNA), immunodetection (for protein) and enzyme assay will be used to define the levels and distributions of enzymes in various tissues and to test the hypothesis that the carboxylesterases are present in peripheral tissues as well as in liver. AIM 3: Cell type distribution in liver. Laser Capture Microdissection and cell separations coupled with real-time PCR, immunodetection, and enzyme assays will be used to define the distribution of carboxylesterases between stellate cells & hepatocytes and to test the hypotheses that carboxylesterase ES-2 is localized exclusively in hepatocytes and that carboxylesterase ES-10 is localized in hepatocytes and stellate cells. AIM 4: Physiologic roles of the carboxylesterases in the metabolism of RE in cultured cells, cDNA transfection and RNA interference will be used to modulate carboxylesterase enzyme levels to test the hypothesis that the various carboxylesterases play a metabolic role in the hepatic uptake and/or metabolism of chylomicron RE, and to test the hypothesis that the carboxylesterases play a metabolic role in the turnover of intracellular RE and cholesteryl esters in cells that store them.

描述（由申请人提供）：视黄酯（RE）在肝脏中的水解在维生素A的体内代谢中起关键作用。膳食维生素A进入肝脏作为RE在乳糜微粒残余物，经历水解和再酯化储存。在动员之前，RE被水解，游离视黄醇与视黄醇结合蛋白结合，分泌到血浆中。中性和酸性视黄酯水解酶（REH）已从大鼠肝脏中纯化，并通过序列分析显示为羧酸酯酶，ES-2和ES-10。本项目的目标是确定ES-2、ES-10以及相关的ES-3和ES-4在维生素A代谢中的作用。羧酸酯酶已与其他底物一起研究，但不知道它们是否在维生素A代谢中重要。与其他酯相比，它们催化RE水解的效率也是未知的。这些知识的差距将与以下具体目标进行探讨：目的1：REH的酶学。酶动力学将用于研究羧酸酯酶的底物特异性，并测试这些酶在催化RE和其他脂质酯水解的内在能力方面不同的假设。目的2：羧酸酯酶的组织分布。实时PCR（mRNA）、免疫检测（蛋白质）和酶测定将用于确定各种组织中酶的水平和分布，并检验羧酸酯酶存在于外周组织和肝脏中的假设。目的3：肝细胞类型分布.激光捕获显微切割和细胞分离结合实时PCR、免疫检测和酶测定将用于确定羧酸酯酶在星状细胞和肝细胞之间的分布，并测试羧酸酯酶ES-2仅位于肝细胞中以及羧酸酯酶ES-10位于肝细胞和星状细胞中的假设。目标4：羧酸酯酶在培养细胞中RE代谢中的生理作用、cDNA转染和RNA干扰将用于调节羧酸酯酶水平以检验各种羧酸酯酶在乳糜微粒RE的肝摄取和/或代谢中起代谢作用的假设，并检验羧酸酯酶在储存它们的细胞内RE和胆固醇酯的周转中起代谢作用的假设。