MECHANISMS OF MILK LIPID SECRETION

乳脂分泌机制

• 批准号: 7094207
• 负责人: James Lewis McManaman
• 金额: $ 33.28万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-06 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7094207
• 关键词: Adenoviridae; cell line; dietary lipid; gene deletion mutation; genetically modified animals; human milk; immunofluorescence technique; laboratory mouse; lactation; lipid biosynthesis; mammary epithelium; molecular biology; peptides; protein biosynthesis; protein localization; protein sequence; protein structure function; proteomics; secretion; transfection /expression vector; triglycerides; Adenoviridae; cell line; dietary lipid; gene deletion mutation; genetically modified animals; human milk; immunofluorescence technique; laboratory mouse; lactation; lipid biosynthesis; mammary epithelium; molecular biology; peptides; protein biosynthesis; protein localization; protein sequence; protein structure function; proteomics; secretion; transfection /expression vector; triglycerides

DESCRIPTION (provided by applicant): Milk lipids are an important source of infant nutrition, providing 40% of calories to human infants. Interference with lipid synthesis and/or secretion in mammary epithelial cells (MECs) leads to abnormalities in lactation and impaired neonatal growth. Milk lipid secretion is a tightly regulated, luminal epithelial cell specific, process, requiring synthesis, packaging and transport of triglyceride and cholesterol ester containing droplets (cytoplasmic lipid droplets - CLDs) to the apical plasma membrane where they are secreted by a unique membrane envelopment mechanism. The long-term objective of this study is to elucidate the molecular mechanisms underlying the formation, transport and secretion of CLDs by mammary epithelial cells. We hypothesize that adipophilin (ADPH), a prominent CLD surface associated protein, is required for both formation and secretion of CLDs and thus integrates lipid synthesis with lipid secretion in the lactating mammary gland. The hypothesis that ADPH is required for CLD formation and secretion in mammary epithelial cells will be tested in mice lacking the ADPH gene and in transgenic mice in which perilipin-A has displaced ADPH. Pup growth rate, lipid content of milk and CLD formation, size and subcellullar localization will be examined in tissues of ADPH-null and perilipin transgenic animals to establish the effects of ADPH deletion on mammary function, CLD metabolism and milk lipid secretion. Adenoviral vectors will be used to deliver variants of ADPH and mutants of its functional domains to mammary epithelial cells of ADPH-null mice to determine in what molecular forms its replacement rescues defects induced by the absence of the ADPH gene. Proteomic analysis of isolated mammary CLDs from ADPH-null and perilipin transgenic animals will be used to establish effects of these proteins on the general protein composition of CLDs and to identify potential alternative mediators of CLD formation and secretion. ADPH is a member of the PAT family of CLD associated proteins that are thought to be important regulators of triglyceride storage and metabolism in many mammalian cell types. The mammary epithelial cell, because it can be manipulated by both transgenic and adenoviral technology, offers the opportunity to understand structure-function relations applicable to lipid storage in many cells and tissues.

描述（由申请人提供）：牛奶脂质是婴儿营养的重要来源，为人类婴儿提供40％的卡路里。乳腺上皮细胞（MEC）中脂质合成和/或分泌的干扰会导致泌乳异常和新生儿生长受损。牛奶脂质分泌是一种严格调节的，腔内上皮细胞的特异性，过程，需要甘油三酸酯和含甘油三酸酯和胆固醇酯的合成，包装和运输，含有液滴（细胞质脂质液滴 - CLDS- CLDS），向根尖质膜膜上，它们被独特的膜膜膜造型机构分泌出来。这项研究的长期目的是阐明乳腺上皮细胞对CLD的形成和分泌的分子机制。我们假设脂肪蛋白（ADPH）是一种显着的CLD表面蛋白，需要CLD的形成和分泌，因此将脂质合成与泌乳乳腺中的脂质分泌相结合。将在缺乏ADPH基因的小鼠和紫脂蛋白A替代ADPH的小鼠中测试乳腺上皮细胞中CLD形成和分泌所必需的ADPH的假设。幼崽的生长速率，牛奶和CLD形成的脂质含量，大小和亚皮拉尔定位将在ADPH-NULL和PERIPIN转基因动物的组织中进行检查，以确定ADPH缺失对乳腺功能，CLD代谢和牛奶脂质分泌的影响。腺病毒载体将用于将ADPH的ADPH及其功能结构域的突变体传递到ADPH无效小鼠的乳腺上皮细胞中，以确定由于缺乏ADPH基因引起的分子形成其替代救援缺陷。对来自ADPH-NULL和PERIPIN转基因动物分离的乳腺CLD的蛋白质组学分析将用于确定这些蛋白质对CLDS的一般蛋白质组成的作用，并确定CLD形成和分泌的潜在替代介质。 ADPH是PAT CLD相关蛋白质家族的成员，被认为是许多哺乳动物细胞类型中甘油三酸酯储存和代谢的重要调节剂。乳腺上皮细胞可以通过转基因和腺病毒技术来操纵乳腺细胞，它提供了理解适用于许多细胞和组织中脂质储存的结构功能关系的机会。