NUTRITIONAL AND HORMONAL REGULATION OF HEPATIC GENES

肝脏基因的营养和激素调节

• 批准号: 2137945
• 负责人: HOWARD C TOWLE
• 金额: $ 16.88万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-04-01 至 1996-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2137945
• 关键词: affinity chromatography; carbohydrate metabolism; dietary carbohydrates; enzyme induction /repression; gel mobility shift assay; gene expression; genetic library; genetic promoter element; genetic regulatory element; genetic transcription; genetically modified animals; hormone receptor; hormone regulation /control mechanism; laboratory mouse; laboratory rat; lipid biosynthesis; liver cells; molecular cloning; northern blottings; nucleic acid sequence; nutrient interaction; nutrition related tag; protein purification; protein sequence; pyruvate kinase; site directed mutagenesis; tissue /cell culture; transcription factor; triiodothyronine

The liver is the primary organ dealing with ingested carbohydrates in mammals and, as such, t is responsible for converting excess dietary carbohydrates to triglycerides. Increased intake of carbohydrate leads t increased hepatic lipogenesis and this process is regulated in part by changes in the production of enzymes involved in the formation of triglycerides. Little is currently known regarding the mechanisms by which nutritional factors control gene expression in mammalian systems and the long range goal of this research is to explore this pathway. For this purpose, we are focusing on the expression of two hepatic genes - pyruvate kinase and the S14 gene. The rates of transcription of both of these genes respond rapidly and markedly to carbohydrate feeding. These changes can be mimicked in cultured primary hepatocytes which allows us to identify DNA sequences critical for the regulation of gene expression by carbohydrate. For both S14 and pyruvate kinase, DNA sequences from the 5'-flanking region were shown to be capable of conferring glucose- activation to a linked reporter gene. We hypothesize that a common carbohydrate-responsive transcription factor is binding to sites in the 5'-flanking region of these genes. Or goal is to identify and characterize the cis-acting DNA sequences and trans-acting factor(s) responsible for controlling this process. This will be accomplished by (1) using mutagenesis techniques, essential DNA sequences for the carbohydrate response of the pyruvate kinase and S14 genes will be defined in transfected hepatocytes and transgenic mice; (2) identifying the hepatic carbohydrate-responsive factor(s) which interacts with these DNA sequences; and (3) purifying and cloning of the carbohydrate- responsive factor from liver. This clone will provide a critical tool for further studies to explore the signalling pathway by which the hepatocyte can 'sense' increased glycolysis and respond by changing specific gene expression. In modern man with the ready availability of simple sugars in the diet, this pathway and its control could be a factor leading to excess conversion to triglycerides and problems associated with obesity. The expression of the S14 gene is also regulated in response to thyroid hormone. The two stimuli control S14 gene expression in a synergistic manner. Recent evidence from the laboratory suggests an interaction between the T3 receptor and components of the carbohydrate pathway. Using CDNA clones to the T3 receptor obtained in the previous funding period, we will test various models for the interaction of the receptor and the carbohydrate-responsive factor. These studies should provide insight into the mechanism by which two distinct stimuli can act to coordinately control gene expression.

肝脏是处理摄入的碳水化合物的主要器官 哺乳动物及其t负责转化多余的饮食 碳水化合物到甘油三酸酯。碳水化合物铅的摄入量增加 肝脂肪生成增加，该过程部分受到 涉及形成的酶的生产的变化 甘油三酸酯。目前对有关机制的知之甚少 哪些营养因子控制哺乳动物系统中的基因表达 这项研究的远距离目标是探索这一途径。 为此，我们专注于两个肝基因的表达 - 丙酮酸激酶和S14基因。两者的转录率 这些基因在碳水化合物喂养方面迅速响应。 这些变化可以在培养的原发性肝细胞中模仿 允许我们识别对基因调节至关重要的DNA序列 通过碳水化合物表达。对于S14和丙酮酸激酶，DNA 显示5'FANKing区域的序列能够 将葡萄糖激活转换为链接的报告基因。我们假设 常见的碳水化合物反应转录因子与 这些基因的5'Fanking区域中的位置。或目标是确定 并表征顺式作用DNA序列和反式作用因子（S） 负责控制此过程。这将通过 （1）使用诱变技术，必需的DNA序列 丙酮酸激酶和S14基因的碳水化合物反应将是 在转染的肝细胞和转基因小鼠中定义； （2）识别 与这些相互作用的肝碳水化合物反应因子 DNA序列； （3）碳水化合物的纯化和克隆 肝脏的反应因素。该克隆将提供关键的工具 对于进一步的研究，以探索信号通路 肝细胞可以“感觉”增加糖酵解，并通过改变 特定基因表达。在现代人中有现成的可用性 饮食中的简单糖，这种途径及其控制可能是 导致过量转化为甘油三酸酯和问题的因素 与肥胖有关。 S14基因的表达也受到甲状腺的响应调节 激素。两个刺激控制S14基因表达在协同作用中 方式。实验室的最新证据表明相互作用 在T3受体和碳水化合物途径的成分之间。 将cDNA克隆用于先前资金中获得的T3受体 时期，我们将测试各种受体相互作用的模型 和碳水化合物反应因子。这些研究应提供 深入了解两个不同刺激可以采取的机制 协同控制基因表达。