INSULIN AND ADIPOSE CELL COMMITMENT

脂肪细胞的测定和终末分化结果 在大量脂肪细胞表达的起始阶段， 特定的基因，其mRNA水平的协调上升， 对胰岛素的反应。 本拟议工作的总体目标 是：1）定义脂肪生成激素的分子机制， 胰岛素，控制脂肪细胞基因活性，和2）了解 前脂肪细胞分化过程中发生的生化变化 终末分化脂肪细胞表型的定型。 这 涉及脂肪细胞分化的一系列复杂事件将是 使用鼠3 T3-L1细胞系和编码 CCAAT/增强子结合蛋白（C/EBP）。 3 T3-L1细胞分化为 适当的激素刺激，从成纤维细胞样前脂肪细胞成熟 脂肪细胞 3 T3-L1系统具有良好表征的脂肪 分化程序，并高度响应胰岛素，使其 胰岛素作用和前脂肪细胞的良好分析模型 承诺. C-EBP已被证明参与了 终末分化的脂肪细胞及其调控 特异性基因表达 以前的研究表明， C-EBP基因5 ′侧翼序列中的几个顺式元件， 与3 T3-L1核蛋白相互作用，并具有改变的DNaseI 脂肪细胞分化后的敏感性模式，这可能是 在C-EBP基因调控中起重要作用。 我们实验室的最新数据显示， 显示胰岛素改变3 T3-L1脂肪细胞中C/EBP mRNA水平 细胞 分析涉及的调节途径中的早期事件 在前脂肪细胞向脂肪细胞定向的级联反应中， 胰岛素对脂肪生成调节因子C/EBP的影响， 通过以下具体目标进行测试。 首先，我们将确定荷尔蒙 对体内C/EBP mRNA水平的影响。 核连续转录测定 将用于测定C/EBP转录速率的改变， 基因在脂肪细胞中的作用。 二是 用CAT分析了C/EBP基因活性的调控机制 转染中含有C/EBP启动子的报告基因构建体 在3 T3-L1细胞中的表达测定。 第三，由于C/EBP仅表示 在分化的3 T3-L1脂肪细胞中，我们将鉴定前脂肪细胞 特异性DNA结合蛋白，一种假定的基因活性阻遏物， 与C/EBP基因的5'侧翼序列相互作用， 前脂肪细胞表达文库。 该项目的第四个目标是 分析C/EBP基因5 ′侧翼区的元件， 与即刻早期基因产物Zif 268、C/EBP基因相互作用 产品和HLH蛋白质。

The determination and terminal differentiation of adipose cells results in the initiation of the expression of a large number of adipocyte- specific genes, a coordinate rise in their mRNA levels and an increased responsiveness to insulin. The overall objective of this proposed work are: 1) to define the molecular mechanism by which a lipogenic hormone, insulin, controls adipocyte gene activity, and 2) to understand the biochemical changes that occur during the process of preadipocyte cell commitment to the terminally differentiated adipocyte phenotype. This complex series of events involved in adipocyte differentiation will be analyzed using the murine 3T3-L1 cell line and the gene encoding the CCAAT/Enhancer Binding Protein (C/EBP). 3T3-L1 cells differentiate with proper hormonal stimuli from fibroblast-like preadipocyte cells to mature adipocytes. the 3T3-L1 system has a well characterized adipose differentiation program and are highly responsive to insulin, making it an excellent model for analysis of insulin action and preadipose cell commitment. C-EBP has been shown to be involved in the development of the terminally differentiated adipocyte and the regulation of adipocyte- specific gene expression. Previous studies have shown that there are several cis-elements in the 5' flanking sequence of the C-EBP gene that interact with 3T3-L1 nuclear proteins, and have an altered DNaseI sensitivity pattern upon adipocyte differentiation, which may be important in C-EBP gene regulation. Recent data from our laboratory has shown that C/EBP mRNA levels are altered by insulin in 3T3-L1 adipocyte cells. Analysis of earlier events in the regulatory pathways involved in the cascade of preadipocyte to adipocyte cell commitment and the effects of insulin on the lipogenic regulatory factor, C/EBP, will be tested by the following specific aims. First, we will determine hormonal effects on C/EBP mRNA levels in vivo. Nuclear run-on transcription assay will be used to assay alterations in rates of transcription of the C/EBP gene in adipocytes by insulin, diet and other hormones. Second, we will analyze the mechanisms that control C/EBP gene activity using CAT reporter constructs containing the C/EBP promoter in transfection expression assays in 3T3-L1 cells. Third, since C/EBP is expressed only in the differentiated 3T3-L1 adipocytes we will identify the preadipocyte specific DNA-binding protein(s), a putative repressor of gene activity, that interacts with the 5' flanking sequence of the C/EBP gene, using a preadipocyte expression library. The fourth aim of this project is to analyze the elements in the 5' flanking region of the C/EBP gene that interact with the immediate early gene product Zif268, C/EBP gene product, and HLH proteins.