HORMONAL REGULATION OF ENZYME LEVELS IN MAMMALIAN TISSUE

肝脏和肾脏皮质中葡萄糖合成的激素控制 对于所有哺乳动物中葡萄糖稳态的调节至关重要。的 β-烯醇式丙酮酸盐是β-烯醇式丙酮酸盐的主要代谢途径 羧激酶（GTP）（PEPCK）。这种基因的表达水平 酶对各种饮食和激素信号敏感。我们有 定位了启动子中与转录结合的反应元件 因子，并确定了改变转录速率的信号 处于各种营养和荷尔蒙状态。PEPCK启动子（-460至460） +73）包含三个高度互动的区域， 基因的转录。区域1负责控制基础 基因的转录及其对cAMP的反应;区域2包含 肝和肾特异性表达所需的序列以及 甲状腺激素控制基因转录;区域3具有调节 参与基因对糖皮质激素反应的元件， 胰岛素解释PEPCK复杂调控模式的模型 基因转录是基于先前的研究而发展起来的。这 在此期间，将对该模型进行详细测试。我们建议 确定施加在每个调节细胞上的生理调节， 通过分析PEPCK启动子的三个区域内的元件， 在转基因小鼠中发挥作用。我们将确定负责的机制 用于抑制由饮食引起的PEPCK基因转录 碳水化合物，并描述胰岛素和/或 糖类。此外，特异性转录的相互作用 参与控制PEPCK基因转录的因素将是 研究，使用纯化的转录因子在体外结合 通过体内PEPCK启动子的紫外光足迹法测定， 通过使用酵母双杂交技术分析蛋白质-蛋白质相互作用， 系统在C/EBP α和HNF-1基因中具有“敲除”的小鼠将被称为“小鼠”。 分析以更好地了解这些蛋白质在控制 PEPCK基因在肝脏和肾脏中的组织特异性表达。 初步研究表明，C/EBPa可能是关键的 PEPCK基因在肝脏中的发展和维持 围产期的葡萄糖稳态。的相互作用 与区域1和2结合的蛋白质将通过“元件移位”来确定。 实验和双杂交系统的使用。这个目标 研究的目的是开发一个系统来解剖PEPCK的区域 通过体外分析进行详细的研究。的结果 研究应该提供对一种关键代谢酶的控制的深入了解 并允许制定一套代谢原则， 应用于代谢途径中关键点的其他酶。这 研究对于理解疾病的病因有明确的意义。 糖尿病和控制糖尿病的更基本的过程 哺乳动物的葡萄糖稳态。

The hormonal control of glucose synthesis in the liver and kidney cortex is critical for the regulation of glucose homeostasis in all mammals. The pace setting enzyme in the pathway of gluconeogenesis is P-enolpyruvate carboxykinase (GTP) (PEPCK). The level of expression of the gene for this enzyme is sensitive to a variety of dietary and hormonal signals. We have mapped the response elements in the promoter which bind transcription factors and determined the signals which alter the rate of transcription in various nutritional and hormonal states. The PEPCK promoter (-460 to +73) contains three highly interactive regions which coordinately control transcription of the gene. Region 1 is responsible for controlling basal transcription of the gene and its response to cAMP; Region 2 contains sequences required for the liver and kidney specific expression and for thyroid hormone control of gene transcription; Region 3 has regulatory elements involved in the response of the gene to glucocorticoids and insulin. A model to explain the complex pattern of regulation of PEPCK gene transcription has been developed based on previous research. This model will be tested in detail during this granting period. We propose to determine the physiological regulation exerted on each of the regulatory elements within the three regions of the PEPCK promoter by analysis of function in transgenic mice. We will determine the mechanism responsible for the inhibition of PEPCK gene transcription caused by dietary carbohydrate and delineate between an effect caused by insulin and/or carbohydrate. In addition, the interaction of specific transcription factors involved in the control of PEPCK gene transcription will be studied, using the purified transcription factors in an in vitro binding assay, by ultra violet photofootprinting of the PEPCK promoter in vivo and by analysis of protein-protein interactions employing the yeast two-hybrid system. Mice with "knock outs" in the genes for C/EBPa and HNF-l will be analyzed to better understand the role of these proteins in controlling the tissue specific expression of the PEPCK gene in the liver and kidney. Preliminary studies suggest that C/EBPa may be critical for the development of the PEPCK gene in the liver and for the maintenance of glucose homeostasis during the perinatal period. The interactions of proteins binding to Regions 1 and 2 will be determined by "element shift" experiments and by the use of the two hybrid system. The goal of this research is to develop a system to dissect the Regions of the PEPCK promoter for detailed study by in vitro analysis. The results of this research should provide insight into the control of a key metabolic enzyme and allow the formulation of a set of metabolic principles which can be applied to other enzymes at key points in metabolic pathways. This research has clear implications for the understanding of the etiology of diabetes and the more basic processes which underlie the control of glucose homeostasis in mammals.