Mechanisms of milk lipid secretion

描述（由申请人提供）：在乳腺分化为分泌器官的过程中诱导脂质合成;在人类和啮齿动物的哺乳期间，乳腺估计是体内最活跃的产脂器官之一。乳脂质分泌是一个严格调节的过程，需要合成、组装和运输含有甘油三酯和胆固醇酯的液滴（细胞质脂滴-CLD）至乳腺上皮细胞的顶膜，在那里它们通过独特的膜出芽机制分泌。本提案的长期目标是阐明CLD形成、转运和分泌的分子和细胞机制。我们推测，脂滴结合蛋白的围脂蛋白（PAT）家族的一员，亲脂蛋白（ADPH），需要CLD的形成和分泌，从而整合在哺乳期的脂质合成和分泌。我们建议通过使用转基因和腺病毒方法确定选择性破坏ADPH和/或密切相关的PAT蛋白TIP47在小鼠乳腺中表达的功能后果来测试这一假设。将使用表达N-和C-末端截短形式的ADPH的转基因小鼠来确定特定ADPH结构域对乳脂质形成和分泌的影响。腺病毒载体将用于在ADPH缺失小鼠的乳腺中表达ADPH和/或TIP47的变体，或这些蛋白质的特定功能结构域的突变，以鉴定将挽救由于缺乏该蛋白质而诱导的缺陷的分子决定簇。ADPH和TIP47都被假设在许多哺乳动物细胞类型中的脂质积累中起作用。由于乳腺中的脂质合成是稳健的，并且通过明确的启动子系统进行发育调控，并且乳腺上皮细胞可以通过转基因和腺病毒技术进行操作，因此拟议的研究提供了一个极好的机会来了解许多细胞和组织中脂质储存的分子相互作用和结构-功能关系。NICHD健康相关性：CLD是乳脂质的来源，乳脂质是新生儿生长所需的，并且是膜合成所需的必需脂肪酸和胆固醇，特别是在CNS中。然而，人们越来越认识到CLD在真核细胞中脂质的储存和细胞内运输中的普遍重要性。这些细胞结构是肝脏、脂肪和肌肉细胞中甘油三酯的主要储存库;以及肝脏分泌的血清脂蛋白颗粒和乳腺上皮细胞分泌的乳脂球中甘油三酯和胆固醇酯的来源。CLD是细胞和组织脂质代谢中的重要稳态元素，并且可用于限制潜在毒性游离脂肪酸的可用性。此外，非脂肪组织中CLD积累升高是许多人类代谢疾病的突出病理学特征，例如肥胖症、II型糖尿病和非酒精性肝脂肪变性，这些疾病正在增加儿童的健康问题。本申请中提出的调节CLD正常形成和代谢的基本分子和细胞机制的表征对于理解导致脂质代谢紊乱的病理生理学的分子异常是必不可少的。公共卫生相关性：本提案的长期目标是阐明CLD形成、转运和分泌的分子和细胞机制。我们推测，脂滴结合蛋白的围脂蛋白（PAT）家族的一员，亲脂蛋白（ADPH），需要CLD的形成和分泌，从而整合在哺乳期的脂质合成和分泌。我们建议通过使用转基因和腺病毒方法确定选择性破坏ADPH和/或密切相关的PAT蛋白TIP47在小鼠乳腺中表达的功能后果来测试这一假设。表达突变形式的ADPH的转基因小鼠将用于确定特定ADPH结构域对乳脂质形成和分泌的影响。腺病毒载体将用于在ADPH缺失小鼠的乳腺中表达ADPH和/或TIP47的变体，或这些蛋白质的特定功能结构域的突变，以鉴定将挽救由其缺失诱导的缺陷的分子决定簇。

DESCRIPTION (provided by applicant):. Lipid synthesis is induced during differentiation of the mammary gland into a secretory organ; and during lactation in humans and rodents the mammary gland is estimated to be among the most active lipogenic organs in the body. Milk lipid secretion is a tightly regulated process requiring synthesis, assembly and transport of triglyceride and cholesterol ester containing droplets (cytoplasmic lipid droplets -CLD) to the apical membrane of mammary epithelial cells where they are secreted by unique membrane budding mechanism. The long-term objectives of this proposal are to elucidate the molecular and cellular mechanisms underlying formation, transport, and secretion of CLD. We hypothesize that adipophilin (ADPH), a member of the perilipin (PAT) family of lipid droplet binding proteins, is required for CLD formation and secretion, thus integrating lipid synthesis and secretion during lactation. We propose to test this hypothesis by determining the functional consequences of selectively disrupting expression of ADPH and/or the closely related PAT protein, TIP47, in the mouse mammary gland using transgenic and adenoviral approaches. Transgenic mice expressing N- and C-terminally truncated forms of ADPH will be used to define effects of specific ADPH domains on milk lipid formation and secretion. Adenoviral vectors will be used to express variants of ADPH and/or TIP47, or mutations of specific functional domains of these proteins, in mammary glands of ADPH-null mice to identify molecular determinants that will rescue defects induced by absence of this protein. ADPH and TIP47 are both hypothesized to function in lipid accumulation in many mammalian cell types. Because lipid synthesis in the mammary gland is robust and developmentally regulated by well defined promoter systems, and mammary epithelial cells can be manipulated by transgenic and adenoviral techniques, the proposed studies offer an excellent opportunity to understand molecular interactions and structure-function relations of lipid storage in many cells and tissues. NICHD Health Relatedness: CLD are the source of milk lipids, which are required neonatal growth, and essential fatty acids and cholesterol needed for membrane synthesis, particularly in the CNS. However, there is increasing recognition of the general importance of CLDs in storage and intracellular trafficking of lipids in eukaryotic cells. These cellular structures are the primary storage depots for triglycerides in liver, adipose and muscle cells; and the source of triglycerides and cholesterol esters in serum lipoprotein particles secreted by the liver, and milk fat globules secreted by mammary epithelial cells. CLDs are important homeostatic elements in cellular and tissue lipid metabolism and may serve to limit the availability of potentially toxic free fatty acids. Furthermore, elevated CLD accumulation in non-adipose tissue is a prominent pathological feature of many human metabolic diseases, such as obesity, type-II diabetes and non-alcoholic hepatic steatosis, which are increasing health concerns for children. Characterization of the fundamental molecular and cellular mechanisms regulating normal formation and metabolism of CLD as proposed in this application is essential to understanding the molecular abnormalities contributing the pathophysiology of lipid metabolism disorders. PUBLIC HEALTH RELEVANCE: The long-term objectives of this proposal are to elucidate the molecular and cellular mechanisms underlying formation, transport, and secretion of CLD. We hypothesize that adipophilin (ADPH), a member of the perilipin (PAT) family of lipid droplet binding proteins, is required for CLD formation and secretion, thus integrating lipid synthesis and secretion during lactation. We propose to test this hypothesis by determining the functional consequences of selectively disrupting expression of ADPH and/or the closely related PAT protein, TIP47, in the mouse mammary gland using transgenic and adenoviral approaches. Transgenic mice expressing mutated forms of ADPH will be used to define effects of specific ADPH domains on milk lipid formation and secretion. Adenoviral vectors will be used to express variants of ADPH and/or TIP47, or mutations of specific functional domains of these proteins, in mammary glands of ADPH-null mice to identify molecular determinants that will rescue defects induced by their absence.