NUTRITIONAL AND HORMONAL REGULATION OF HEPATIC GENES

肝脏是处理摄入的碳水化合物的主要器官， 哺乳动物，因此，t负责将过量的膳食 碳水化合物转化为甘油三酯。增加碳水化合物的摄入量导致t 增加肝脏脂肪生成，该过程部分受 参与形成的酶的生产变化 甘油三酯目前对这些机制知之甚少， 哪些营养因子控制着哺乳动物系统中的基因表达 本研究的长期目标是探索这一途径。 为此，我们将重点放在两个肝基因的表达 - 丙酮酸激酶和S14基因。两者的转录率 这些基因对碳水化合物喂养的反应迅速而显著。 这些变化可以在培养的原代肝细胞中模拟， 使我们能够识别对基因调控至关重要的DNA序列， 碳水化合物的表达。对于S14和丙酮酸激酶，DNA 来自5 '侧翼区的序列显示能够 使葡萄糖激活连接的报告基因。我们假设 一种常见的碳水化合物应答转录因子 在这些基因的5 '侧翼区的位点。或者说目标是 并表征顺式作用DNA序列和反式作用因子 负责控制这个过程。这将通过 (1)使用诱变技术， 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）鉴定 与这些相互作用的肝碳水化合物反应因子 DNA序列;和（3）纯化和克隆碳水化合物- 肝脏反应因子。这个克隆体将提供一个关键的工具 进行进一步的研究，以探索 肝细胞可以“感知”糖酵解增加，并通过改变 特异性基因表达在现代人中， 简单的糖在饮食中，这一途径及其控制可能是一个 导致过度转化为甘油三酯的因素和问题 与肥胖有关。 S14基因的表达也受到甲状腺激素的调节。 激素.这两种刺激以协同方式控制S14基因表达。 方式实验室最近的证据表明 在T3受体和碳水化合物途径的组成部分之间。 使用先前资助中获得的T3受体的cDNA克隆 期间，我们将测试各种模型的相互作用的受体 和碳水化合物反应因子。这些研究将提供 深入了解两种不同刺激可以作用于 协调控制基因表达。

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.