Mechanisms of milk lipid secretion

乳脂分泌机制

• 批准号: 8114111
• 负责人: James Lewis McManaman
• 金额: $ 30.17万
• 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/8114111
• 关键词: Adenovirus Vector; Adipocytes; 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; Hepatocyte; 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; 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（由申请人提供）：。在乳腺分化为分泌器官时诱导脂质合成。在人类和啮齿动物的泌乳过程中，乳腺估计是体内最活跃的脂肪生物器官之一。牛奶脂质分泌是一个严格调节的过程，需要甘油三酸酯和胆固醇和胆固醇酯的合成和运输，其中含有液滴（细胞质脂质液滴-CLD）与乳腺上皮细胞的顶层膜，并由独特的膜出现机制分泌。该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。我们假设脂蛋白（ADPH）是脂质液滴结合蛋白的钙蛋白（PAT）家族的成员，是CLD形成和分泌所必需的，因此在泌乳过程中整合了脂质合成和分泌。我们建议使用转基因和腺病毒方法在小鼠乳腺中选择性破坏ADPH和/或密切相关的PAT蛋白TIP47的表达的功能后果来检验这一假设。表达N-和C末端截短的ADPH形式的转基因小鼠将使用特定ADPH结构域对牛奶脂质形成和分泌的影响。腺病毒载体将用于表达ADPH和/或TIP47的变体，或这些蛋白质的特定功能结构域的突变，在ADPH-NULL小鼠的乳腺中，以鉴定该蛋白质缺乏诱导的缺陷的分子决定因素。假设ADPH和TIP47都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可靠的，并且在发育方面受到良好的启动子系统的调节，并且可以通过转基因和腺病毒技术来操纵乳腺上皮细胞，因此拟议的研究为许多细胞和组织中脂质储存的分子相互作用和结构储存关系提供了极好的机会。 NICHD健康相关性：CLD是牛奶脂质的来源，牛奶脂质是新生儿生长所需的，以及膜合成所需的必需脂肪酸和胆固醇，尤其是在CNS中。然而，人们对CLD在真核细胞中脂质的储存和细胞内运输中的一般重要性越来越多。这些细胞结构是肝脏，脂肪和肌肉细胞中甘油三酸酯的主要储存库。以及由肝脏分泌的血清脂蛋白颗粒中的甘油三酸酯和胆固醇酯的来源，以及由乳腺上皮细胞分泌的牛奶脂肪球。 CLD是细胞和组织脂质代谢中重要的稳态元素，可以限制潜在有毒的游离脂肪酸的可用性。此外，非脂肪组织中的CLD积累升高是许多人类代谢疾病的重要病理特征，例如肥胖症，II型糖尿病和非酒精性肝脂肪变性，这些病的健康问题正在增加儿童的健康问题。该应用中提出的调节正常形成和CLD代谢的基本分子和细胞机制的表征对于理解促进脂质代谢疾病病理生理的分子异常至关重要。公共卫生相关性：该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。我们假设脂蛋白（ADPH）是脂质液滴结合蛋白的钙蛋白（PAT）家族的成员，是CLD形成和分泌所必需的，因此在泌乳过程中整合了脂质合成和分泌。我们建议使用转基因和腺病毒方法在小鼠乳腺中选择性破坏ADPH和/或密切相关的PAT蛋白TIP47的表达的功能后果来检验这一假设。表达ADPH突变形式的转基因小鼠将用于定义特定ADPH结构域对牛奶脂质形成和分泌的影响。腺病毒载体将用于表达ADPH和/或TIP47的变体，或这些蛋白质的特定功能结构域的突变，在ADPH-NULL小鼠的乳腺中，以鉴定分子确定因素，这些分子决定因素将挽救因其缺失而诱导的缺陷。