DEVELOPMENTAL REGULATION OF CYTOPLASMIC LIPID DROPLET SYNTHESIS

在乳腺上皮细胞中，形成并分泌到乳汁中的细胞质脂滴为膜合成提供必需的脂肪酸和胆固醇，并提供很大比例的新生儿卡路里。由于乳腺是身体中最活跃的脂肪生成器官之一，它提供了一个理想的模型系统，以了解参与脂滴形成的细胞和分子过程。这个项目的总体目标是确定脂质， 在内质网中合成的甘油三酯和胆固醇酯被包装到细胞质脂滴中。脂滴相关蛋白和小窝蛋白的PAT家族（Perilipin、Adipophilin、TIP47）已显示在其它组织中脂滴的形成、稳定和代谢中起重要作用。在乳腺中，这一过程是由妊娠激素协调和调节的。我们推测乳腺肺泡上皮细胞的内质网发生了结构改变 在妊娠期间，导致形成特定的脂滴组装结构域，其含有脂质合成的酶以及脂滴组装所必需的蛋白质。在具体目标1中，我们使用形态学技术来定义脂质滴形成过程中内质网结构的时间变化。这些变化将与特定目的2中脂质合成酶的表达和脂质合成相关。候选基因的表达水平将在特定目标3中改变，以开发参与脂滴形成的蛋白质相互作用的精确分子模型。这些实验旨在确定参与脂滴形成的结构和生化过程，并为理解激素在此过程中的作用提供框架。这个提议的独特之处在于，CLD在包含所有合成机器的ER中的专门领域内形成的假设。如果这一假设是正确的，它将大大改变我们目前对CLD形成起始所涉及的细胞生物学机制的理解。

In mammary epithelial cells cytoplasmic lipid droplets that are formed and secreted into milk provide essential fatty acids and cholesterol for membrane synthesis and a large percentage of neonatal calories. Since the mammary gland is one of the most active lipogenic organs of the body it provides an ideal model system to understand the cellular and molecular processes involved in lipid droplet formation. The overall goal of this project is to define how lipids, triglycerides and cholesterol esters, synthesized in the endoplasmic reticulum, are packaged into cytoplasmic lipid droplets. The PAT family (Perilipin, Adipophilin, TIP47) of lipid droplet associated proteins and caveolins have been shown to play an essential role in the formation, stabilization and metabolism of lipid droplets in other tissues. In the mammary gland this process is developmentally coordinated and regulated by the hormones of pregnancy. We hypothesize that the endoplasmic reticulum of mammary alveolar epithelial cells undergoes structural alterations during pregnancy resulting in the formation of specific lipid droplet assembly domains that contain the enzymes of lipid synthesis as well as the proteins necessary for lipid droplet assembly. In Specific Aim 1 we use morphological techniques to define the temporal alterations in the structure of the endoplasmic reticulum during lipid droplet formation. These changes will be correlated with expression of lipid synthetic enzymes and lipid synthesis in Specific Aim 2. The expression levels of candidate genes will be altered in Specific Aim 3 to develop a precise molecular model for the interactions of proteins involved in formation of lipid droplets. These experiments are designed to identify the structural and biochemical processes involved in formation of lipid droplets and to provide a framework for understanding the role of hormones in this process. The unique aspect of this proposal is the hypothesis that CLD form within specialized domains in the ER containing all the synthetic machinery. If this hypothesis is correct it will substantially alter our current understanding of the cell biological mechanisms involved in the initiation of CLD formation.