INSULIN AND ADIPOSE CELL COMMITMENT

胰岛素和脂肪细胞的承诺

• 批准号: 2145545
• 负责人: ROBERT J CHRISTY
• 金额: $ 10.15万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-05-01 至 1998-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2145545
• 关键词: 3T3 cells; DNA binding protein; adipocytes; affinity chromatography; cell differentiation; cyclic AMP; diet; dietary carbohydrates; dietary lipid; fasting; gel mobility shift assay; gene expression; genetic transcription; glucocorticoids; hormone receptor; insulin; laboratory mouse; messenger RNA; molecular cloning; northern blottings; nuclear runoff assay; nucleic acid sequence; nucleoproteins; nutrition related tag; protein purification; regulatory gene; transfection; triiodothyronine; western blottings

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.

脂肪细胞的测定和终端分化结果 在启动大量脂肪细胞的表达时 特定基因，其mRNA水平的协调升高，增加 对胰岛素的反应。 这项拟议工作的总体目标 是：1）定义脂肪激素的分子机制， 胰岛素，控制脂肪细胞基因活性，2）了解 在前细胞细胞过程中发生的生化变化 对最终分化的脂肪细胞表型的承诺。 这 脂肪细胞分化涉及的复杂事件将是 使用鼠3T3-L1细胞系和编码的基因分析 CCAAT/增强子结合蛋白（C/EBP）。 3T3-L1细胞与 从成纤维细胞样细胞细胞到成熟的适当的激素刺激 脂肪细胞。 3T3-L1系统具有良好的脂肪特征 差异化计划，对胰岛素有很高的反应，使得 分析胰岛素作用和前脂蛋白细胞的绝佳模型 承诺。 C-EBP已被证明参与了 末端分化的脂肪细胞和脂肪细胞的调节 特定基因表达。 以前的研究表明有 C-EBP基因的5'侧翼序列中的几个顺式元素 与3T3-L1核蛋白相互作用，并改变了DNasei 脂肪细胞分化后的敏感性模式，这可能是 在C-EBP基因调控中很重要。 我们实验室的最新数据 表明C/EBP mRNA水平通过3T3-L1脂肪细胞中的胰岛素改变 细胞。 分析涉及的调节途径中的早期事件 在脂肪细胞级别的脂肪细胞级联和脂肪细胞的承诺中 胰岛素对脂肪生成调节因子C/EBP的影响将是 通过以下特定目的测试。 首先，我们将确定荷尔蒙 对体内C/EBP mRNA水平的影响。 核跑步转录测定法 将用于测定C/EBP转录率的改变 胰岛素，饮食和其他激素中的脂肪细胞基因。 第二，我们会的 分析使用CAT控制C/EBP基因活性的机制 转染中包含C/EBP启动子的记者结构 3T3-L1细胞中的表达测定。 第三，因为C/EBP仅表示 在差异化的3T3-L1脂肪细胞中，我们将识别前脂肪细胞 特定的DNA结合蛋白（S），一种基因活性的推定阻遏物， 与C/EBP基因的5'侧翼序列相互作用，使用A 前脂肪细胞表达式库。 该项目的第四个目标是 分析C/EBP基因5'侧翼区域的元素 与立即的早期基因产物ZIF268，C/EBP基因相互作用 产品和HLH蛋白。