MECHANISMS OF MILK LIPID SECRETION

描述（由申请方提供）：乳脂是婴儿营养的重要来源，为人类婴儿提供40%的热量。乳腺上皮细胞（MEC）中脂质合成和/或分泌的干扰导致泌乳异常和新生儿生长受损。乳脂质分泌是一个严格调节的腔上皮细胞特异性过程，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-CLD）至顶端质膜，在那里它们通过独特的膜转运机制分泌。本研究的长期目标是阐明乳腺上皮细胞形成、转运和分泌CLD的分子机制。我们推测，adipophilin（ADPH），一个突出的CLD表面相关蛋白，是所需的CLD的形成和分泌，从而整合在泌乳乳腺的脂质分泌与脂质合成。将在缺乏ADPH基因的小鼠和周脂蛋白A取代ADPH的转基因小鼠中测试ADPH是乳腺上皮细胞中CLD形成和分泌所需的假设。将在ADPH缺失和周脂蛋白转基因动物的组织中检查幼仔生长速率、乳汁的脂质含量和CLD形成、大小和亚细胞定位，以确定ADPH缺失对乳腺功能、CLD代谢和乳汁脂质分泌的影响。腺病毒载体将用于将ADPH的变体及其功能结构域的突变体递送至ADPH缺失小鼠的乳腺上皮细胞，以确定其替代物以何种分子形式拯救由ADPH基因缺失引起的缺陷。将使用来自ADPH无效和周脂蛋白转基因动物的分离的乳腺CLD的蛋白质组学分析来确定这些蛋白质对CLD的一般蛋白质组成的影响，并鉴定CLD形成和分泌的潜在替代介质。ADPH是CLD相关蛋白PAT家族的成员，其被认为是许多哺乳动物细胞类型中甘油三酯储存和代谢的重要调节剂。乳腺上皮细胞，因为它可以操纵的转基因和腺病毒技术，提供了机会，了解适用于许多细胞和组织中的脂质储存的结构-功能关系。

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.