Mechanisms of milk lipid secretion

描述(由申请人提供)：脂肪合成是在乳腺分化为分泌器官的过程中诱导的；在人类和啮齿动物的哺乳期，乳腺被认为是体内最活跃的造脂器官之一。乳脂分泌是一个严格调控的过程，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)到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。这项建议的长期目标是阐明CLD形成、运输和分泌的分子和细胞机制。我们推测脂磷脂(ADPH)是脂滴结合蛋白(PAT)家族中的一员，它是CLD形成和分泌所必需的，从而整合了哺乳过程中的脂肪合成和分泌。我们建议通过使用转基因和腺病毒方法来确定选择性干扰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检验这一假设。表达N-和C-末端截短型ADPH的转基因小鼠将被用来确定特定的ADPH结构域对乳脂形成和分泌的影响。腺病毒载体将被用来在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域的突变，以确定将修复由于缺乏该蛋白质而导致的缺陷的分子决定因素。ADPH和TIP47都被认为在许多哺乳动物细胞中具有脂质积累的功能。由于乳腺中的脂肪合成是强大的，并受到明确的启动子系统的发育调节，而且乳腺上皮细胞可以通过转基因和腺病毒技术进行操纵，因此本研究为了解许多细胞和组织中脂肪储存的分子相互作用和结构-功能关系提供了一个很好的机会。NICHD与健康相关：CLD是新生儿生长所需的乳脂的来源，以及膜合成所需的必需脂肪酸和胆固醇，特别是在中枢神经系统。然而，人们越来越多地认识到CLD在真核细胞中储存和细胞内脂类运输的普遍重要性。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，也是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是细胞和组织脂肪代谢中重要的动态平衡元素，可能会限制潜在有毒游离脂肪酸的可获得性。此外，非脂肪组织中CLD积聚增加是许多人类代谢性疾病的显著病理特征，如肥胖、II型糖尿病和非酒精性肝脂肪变性，这些疾病正日益增加对儿童健康的担忧。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，对于理解导致脂代谢紊乱的分子异常是至关重要的。与公共卫生相关：这项提案的长期目标是阐明CLD的形成、运输和分泌的分子和细胞机制。我们推测脂磷脂(ADPH)是脂滴结合蛋白(PAT)家族中的一员，它是CLD形成和分泌所必需的，从而整合了哺乳过程中的脂肪合成和分泌。我们建议通过使用转基因和腺病毒方法来确定选择性干扰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检验这一假设。表达突变形式的ADPH的转基因小鼠将被用于确定特定的ADPH结构域对乳脂形成和分泌的影响。腺病毒载体将被用来在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域的突变，以确定将修复因它们缺失而导致的缺陷的分子决定因素。

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.