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

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(申请人提供)：乳脂是婴儿营养的重要来源，为人类婴儿提供40%的卡路里。干扰乳房上皮细胞(MECs)的脂质合成和/或分泌会导致哺乳异常和新生儿生长受阻。乳脂分泌是一个受到严格调控的腔上皮细胞特有的过程，需要合成、包装和运输含有甘油三酯和胆固醇酯的液滴(细胞质脂滴-CLDs)到顶端质膜，在那里它们由独特的膜包裹机制分泌。本研究的长期目标是阐明乳腺上皮细胞形成、运输和分泌CLDs的分子机制。我们推测，脂联素(ADPH)是一种重要的CLD表面相关蛋白，在CLDs的形成和分泌中都是必需的，因此在哺乳期乳腺中整合了脂质合成和脂质分泌。乳腺上皮细胞CLD的形成和分泌需要ADPH这一假设，这一假设将在缺乏ADPH基因的小鼠和Perilipin-A取代ADPH的转基因小鼠中得到验证。通过检测ADPH缺失型和Perilipin转基因动物的幼仔生长速度、乳汁中脂肪含量以及乳汁中CLD的形成、大小和亚细胞定位，以确定ADPH缺失对乳房功能、乳脂代谢和乳脂分泌的影响。腺病毒载体将被用来将ADPH的变异体及其功能域的突变体输送到ADPH缺失的小鼠的乳腺上皮细胞中，以确定其替代以何种分子形式修复因ADPH基因缺失而导致的缺陷。从ADPH缺失和Perilipin转基因动物分离的乳腺CLDs的蛋白质组学分析将被用来确定这些蛋白质对CLDs一般蛋白质组成的影响，并确定潜在的CLD形成和分泌的替代介质。ADPH是CLD相关蛋白PAT家族中的一员，被认为是许多哺乳动物细胞中甘油三酯储存和代谢的重要调节因子。乳腺上皮细胞，因为它可以被转基因和腺病毒技术操纵，提供了机会来了解适用于许多细胞和组织中脂肪储存的结构-功能关系。