Abstract：
The organization of a circadian system includes an endogenous pacemaker system, input pathways for environmental synchronizing (entraining) stimuli, and output pathways through which the clock regulates physiological and behavioral processes, for example, the glucose-sensing mechanism in the liver. The liver is the central regulator of metabolism and one of our peripherals clocks. In mammals, central to this pacemaker are the transcription factors Circadian Locomotor Output Cycles Kaput (CLOCK) and BMAL1 (Brain and Muscle ARNT-Like 1). BMAL1 dimerizes with CLOCK, and this heterodimer then binds to the E-box promoter elements (CACGTG) present in clock and clock-controlled genes (CCGs). However, we are just beginning to understand how output pathways and regulatory mechanisms of CCGs are involved in rhythmic physiological processes. Glucokinase (GCK) is a fundamental enzyme in glucose homeostasis, catalyzing the high Km phosphorylation of glucose and allowing its storage. Moreover, gck is a dependent circadian gene. This study aims to determine the contribution of clock genes to hepatic gck expression and to define the specific role of E-box sequences on the circadian regulation of hepatic gck. Results showed that gck expression follows a circadian rhythm in rat hepatocytes in vitro. Accordingly, bmal1 expression induces the glucokinase circadian rhythmic expression in hepatocytes and the analysis of human and rat gck promoters, indicating the presence of E-box regions. Moreover, the basal activity of gck promoter was increased by clock/bmal1 co-transfection but inhibited by Period1/Period2 (per1/per2) co-transfection. Thus, the data suggest that the clock proteins tightly regulate the transcriptional activity of the gck promoter.
摘要：
昼夜节律系统的组织包括内源性起搏器系统，环境同步(夹带)刺激的输入途径，以及时钟调节生理和行为过程的输出途径，例如,肝脏中的葡萄糖感应机制。肝脏是新陈代谢的中央调节器，也是我们的外围时钟之一。在哺乳动物中,该起搏器的核心是转录因子昼夜节律运动输出周期Kaput（CLOCK）和BMAL1（脑和肌肉ARNT样1）。BMAL1与时钟二聚化，然后，该异二聚体与时钟和时钟控制基因（CCG）中存在的E-box启动子元件（CACGTG）结合。然而,我们才刚刚开始了解CCG的输出途径和调节机制是如何参与有节奏的生理过程的。葡萄糖激酶（GCK）是葡萄糖稳态的基本酶，催化葡萄糖的高Km磷酸化并允许其储存。此外，gck是一个依赖的昼夜节律基因。本研究旨在确定时钟基因对肝脏gck表达的贡献，并确定E-box序列在肝脏gck昼夜节律调节中的特定作用。结果表明，体外大鼠肝细胞中gck的表达遵循昼夜节律。因此，bmal1表达诱导肝细胞葡萄糖激酶昼夜节律表达和人和大鼠gck启动子的分析，表明E-box区域的存在。此外，gck启动子的基础活性通过clock/bmal1共转染增加，但被Period1/Period2（per1/per2）共转染抑制。因此，数据表明，时钟蛋白紧密调节gck启动子的转录活性。
