Mechanisms of milk lipid secretion

乳脂分泌机制

• 批准号: 7683687
• 负责人: James Lewis McManaman
• 金额: $ 31.84万
• 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 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7683687
• 关键词: Adenovirus Vector; Adipose tissue; Alveolar; Animals; Apical; Binding; Binding Proteins; Biochemical; Biogenesis; C-terminal; Calories; Capsid Proteins; Cell Culture Techniques; Cell membrane; Cells; Cellular Structures; Child; Cholesterol; Cholesterol Esters; Data; Defect; Dissection; Endoplasmic Reticulum; Epithelial Cells; Essential Fatty Acids; Eukaryotic Cell; Exhibits; Failure; Family; Fatty Liver; Functional disorder; Funding; Future; Gene Expression; Goals; Growth; Health; Human; Immunofluorescence Immunologic; Indium; Knockout Mice; Lactation; Lipase; Lipids; Lipolysis; Lipoproteins; Liver; Mammalian Cell; Mammary gland; Mediating; Membrane; Metabolic Diseases; Metabolism; Milk; Modeling; Molecular; Molecular Abnormality; Molecular Structure; Mus; Muscle Cells; Mutagenesis; Mutate; Mutation; N-terminal; National Institute of Child Health and Human Development; Neonatal; Non-Insulin-Dependent Diabetes Mellitus; Nonesterified Fatty Acids; Nutritional; Obesity; Organ; Physiological; Pregnancy; Process; Proteins; Rodent; Role; Serum; Source; Structure; System; Techniques; Tertiary Protein Structure; Testing; Tissues; Transgenic Mice; Transgenic Organisms; Triglycerides; Variant; Wild Type Mouse; adipophilin; apical membrane; base; cell type; gain of function; in vivo; in vivo Model; insight; lipid metabolism; lipid transport; loss of function; member; membrane synthesis; milk fat globule; mutant; non-alcoholic; particle; perilipin; pregnant; prevent; promoter; public health relevance; pup; research study

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.

描述（申请人提供）：。脂质合成是在乳腺分化为分泌器官的过程中被诱导的；在人类和啮齿类动物的哺乳期，乳腺被认为是体内最活跃的脂肪生成器官之一。乳脂分泌是一个受到严格调控的过程，需要含有甘油三酯和胆固醇酯的液滴（胞质脂滴-CLD）合成、组装和运输到乳腺上皮细胞的顶膜，并通过独特的膜出芽机制分泌。本研究的长期目标是阐明CLD形成、转运和分泌的分子和细胞机制。我们推测亲脂蛋白（ADPH）是脂滴结合蛋白家族中的一员，是CLD形成和分泌所必需的，从而在哺乳期整合脂的合成和分泌。我们建议通过使用转基因和腺病毒方法，确定选择性破坏小鼠乳腺中ADPH和/或密切相关的PAT蛋白TIP47的表达的功能后果，来验证这一假设。将使用表达N端和c端截断形式ADPH的转基因小鼠来确定特定ADPH结构域对乳脂形成和分泌的影响。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域的突变，以鉴定分子决定因素，这些决定因素将挽救由缺乏该蛋白质引起的缺陷。ADPH和TIP47都被假设在许多哺乳动物细胞类型的脂质积累中起作用。由于乳腺中的脂质合成是稳健的，并且受到定义良好的启动子系统的发育调节，并且可以通过转基因和腺病毒技术操纵乳腺上皮细胞，因此所提出的研究为了解许多细胞和组织中脂质储存的分子相互作用和结构-功能关系提供了极好的机会。NICHD与健康的关系：CLD是乳脂的来源，这是新生儿生长所必需的，也是膜合成所需的必需脂肪酸和胆固醇的来源，特别是在中枢神经系统。然而，越来越多的人认识到CLDs在真核细胞中脂质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；而甘油三酯和胆固醇酯的来源是由肝脏分泌的血清脂蛋白颗粒，以及由乳腺上皮细胞分泌的乳脂球。CLDs是细胞和组织脂质代谢中重要的稳态元素，可能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性疾病的一个突出病理特征，如肥胖、ii型糖尿病和非酒精性肝性脂肪变性，这些疾病正在增加儿童的健康问题。在本应用中提出的调节CLD正常形成和代谢的基本分子和细胞机制的特征，对于理解导致脂质代谢紊乱的病理生理的分子异常至关重要。公共卫生相关性：本提案的长期目标是阐明CLD形成、运输和分泌的分子和细胞机制。我们推测亲脂蛋白（ADPH）是脂滴结合蛋白家族中的一员，是CLD形成和分泌所必需的，从而在哺乳期整合脂的合成和分泌。我们建议通过使用转基因和腺病毒方法，确定选择性破坏小鼠乳腺中ADPH和/或密切相关的PAT蛋白TIP47的表达的功能后果，来验证这一假设。表达ADPH突变形式的转基因小鼠将用于确定特定ADPH结构域对乳脂形成和分泌的影响。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，或这些蛋白的特定功能域的突变，以确定分子决定因素，这些决定因素将拯救由ADPH缺失引起的缺陷。