INSULIN AND ADIPOSE CELL COMMITMENT

脂肪细胞结果的测定和终末分化 在启动大量脂肪细胞的表达过程中- 特定基因，它们的mRNA水平协调上升，并增加 对胰岛素的反应性。这项拟议工作的总体目标 1)定义一种生脂激素的分子机制， 胰岛素，控制脂肪细胞的基因活性，以及2)理解 前脂肪细胞发育过程中发生的生化变化 对终末分化的脂肪细胞表型的承诺。这 参与脂肪细胞分化的一系列复杂事件将是 利用小鼠3T3-L1细胞系和编码该基因的 CCAAT/增强子结合蛋白(C/EBP)。3T3-L1细胞分化为 成纤维样前脂肪细胞成熟过程中适当的激素刺激 脂肪细胞。3T3-L1系统具有良好的脂肪特性 分化程序，对胰岛素高度敏感，使其 一种分析胰岛素作用和前脂肪细胞的优秀模型 承诺。C-EBP已被证明参与了 终末分化的脂肪细胞与脂肪细胞的调控 特定的基因表达。之前的研究表明，有一些 C-EBP基因5‘侧翼序列中的几个顺式元件 与3T3-L1核蛋白相互作用，并具有改变的DNaseI 对脂肪细胞分化的敏感性模式，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 胰岛素对3T3-L1脂肪细胞C/EBP基因表达的影响 细胞。对所涉及的调控途径中早期事件的分析 在前脂肪细胞到脂肪细胞的级联反应中 胰岛素对成脂调节因子C/EBP的影响将是 通过以下具体目标进行测试。首先，我们要测定荷尔蒙 对体内C/EBP基因表达水平的影响核连续转录分析 将用于检测C/EBP转录速率的变化 通过胰岛素、饮食和其他激素在脂肪细胞中表达基因。第二，我们将 利用CAT分析C/EBP基因活性的调控机制 含C/EBP启动子的报告基因载体的构建 3T3-L1细胞的表达分析。第三，由于C/EBP仅表示 在分化的3T3-L1脂肪细胞中，我们将鉴定前脂肪细胞 特异的脱氧核糖核酸结合蛋白(S)，一种假定的基因活性抑制因子， 它与C/EBP基因的5‘侧翼序列相互作用，使用 前脂肪细胞表达文库。这个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Zif268、C/EBP基因相互作用 产物和HLH蛋白。

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.