Integration of Feeding Behaviour by the Liver Circadian Clock Reveals Network Dependency of Metabolic Rhythms [RNA-seq]

The mammalian circadian clock, expressed throughout the brain and body, controls daily metabolic homeostasis. Clock function in peripheral tissues is required, but not sufficient, for this task. Due to lack of specialized animal models, it is unclear how tissue clocks interact with extrinsic signals to drive molecular oscillations. Here, we present a model in which the interaction between feeding and the liver clock can be isolated in vivo by reconstituting Bmal1 exclusively in hepatocytes (Liver-RE) in otherwise clock-less mice. We found that the cooperative action of BMAL1 and the transcription factor CEBPB regulates daily liver metabolic transcriptional programs. Functionally, the liver clock and feeding rhythm are sufficient to drive temporal glucose homeostasis. By contrast, liver rhythms tied to redox and lipid metabolism required communication with the skeletal muscle clock, demonstrating peripheral clock cross-talk. Our results highlight how the inner workings of the clock system rely on communicating signals to maintain daily metabolism. Overall design: To study how CEBPB affects circadian transcription we generated transient knock-down (KD) cells by using a siRNA targeting Cebpb. Cells were synchronized with dexamethasone and RNA extracted at specific circadian time points (CT12 and CT24). To study the muscle clock-liver connection, we collected serum from WT and muscle specific BMAL1 knock-out mice (mKO) at zeitgeber time (ZT16). Cultured primary hepatocytes were treated with the collected serum for 24 hours.

遍布大脑与全身表达的哺乳动物昼夜节律时钟（circadian clock），调控每日代谢稳态。外周组织中的时钟功能是完成该调控任务的必要而非充分条件。由于缺乏专用动物模型，目前尚不明确组织时钟如何与外源性信号相互作用以驱动分子振荡。本研究构建了一种可在体内分离进食与肝脏时钟之间相互作用的模型：在完全丧失时钟功能的小鼠体内，仅在肝细胞中重构Bmal1基因（命名为Liver-RE小鼠）。研究发现，BMAL1与转录因子CEBPB的协同作用，调控肝脏每日代谢相关的转录程序。功能层面上，肝脏时钟与进食节律足以驱动节律性葡萄糖稳态的维持。与之相反，与氧化还原及脂质代谢相关的肝脏节律，需要与骨骼肌时钟进行信号交流，这证实了外周时钟间的交叉对话。本研究结果阐明了时钟系统的内在运作如何依赖信号交流以维持每日代谢稳态。
实验总体设计：为探究CEBPB对昼夜节律转录的调控作用，本研究通过靶向Cebpb的小干扰RNA（siRNA）构建了瞬时敲低（KD）细胞系。用地塞米松对细胞进行同步化处理，并在特定昼夜节律时间点（CT12与CT24）提取RNA。为探究肌肉时钟与肝脏时钟的互作关系，本研究在授时因子时间（ZT16）下，从野生型（WT）及骨骼肌特异性BMAL1敲除小鼠（mKO）体内采集血清。将采集的血清处理培养原代肝细胞，持续24小时。