GCN2 drives diurnal patterns in the hepatic integrated stress response and maintains circadian rhythms in whole body metabolism during amino acid insufficiency

Disruptions in circadian rhythms are associated with increased risk of developing metabolic diseases. General control nonderepressible 2 (GCN2), a primary sensor of amino acid insufficiency and activator of the integrated stress response (ISR), has emerged as a conserved regulator of the circadian clock in multiple organisms. The objective of this study was to examine diurnal patterns in hepatic ISR activation in the liver and whole-body rhythms in metabolism. We hypothesized that GCN2 activation cues hepatic ISR signaling over a natural 24 h feeding fasting cycle. To address our objective, wild type (WT) and whole body Gcn2 knockout (GCN2 KO) mice were housed in metabolic cages and provided free access to either a Control or leucine-devoid diet (LeuD) for 8-days in total darkness. On the last day, blood and livers were collected at circadian time (CT) 3 and CT15. In livers of WT mice, GCN2 phosphorylation followed a diurnal pattern that was guided by intracellular branched chain amino acid concentrations (r2=0.93). Feeding LeuD to WT mice increased hepatic ISR activation at CT15 only. Diurnal oscillation in hepatic ISR signaling, the hepatic transcriptome including lipid metabolic genes, and triglyceride concentrations were substantially reduced or absent in GCN2 KO mice. Further, mice lacking GCN2 were unable to maintain circadian rhythms in whole body energy expenditure, respiratory exchange ratio and physical activity when fed LeuD. In conclusion, GCN2 activation functions to maintain diurnal ISR activation in the liver and has a vital role in the mechanisms by which nutrient stress affects whole-body metabolism. For this study, liver tissue was collected from WT and whole body GCN2 KO mice fed a Control or LeuD diet and killed at CT03 or CT15. Our comparisons look at the impact of GCN2 deletion, LeuD feeding and their interaction on diurnal patterns in the hepatic transcriptome. For this experiment we compare the differential expression of transcripts between the two time points within our various treatment groups. In addition, to observe the time dependent impact of LeuD feeding on the hepatic transcriptome, we compare the differential gene expression of Control and LeuD fed WT mice at the CT03 and CT15 time points.昼夜节律（circadian rhythms）紊乱与代谢性疾病发病风险升高密切相关。一般性调控阻遏蛋白激酶2（General control nonderepressible 2, GCN2）是氨基酸不足的核心感受器，同时也是整合应激反应（integrated stress response, ISR）的激活因子，现已被证实为多种生物体内昼夜节律的保守调控因子。 本研究旨在探究肝脏内整合应激反应激活的昼夜模式，以及全身代谢的昼夜节律。我们提出假说：在自然24小时摄食-禁食周期中，GCN2的激活会介导肝脏整合应激信号通路的活性变化。 为达成研究目标，我们将野生型（wild type, WT）及Gcn2全身敲除（GCN2 KO）小鼠饲养于代谢笼中，在完全黑暗环境下自由摄食对照饮食或无亮氨酸饮食（leucine-devoid diet, LeuD），共计8天。实验最后一天，分别于昼夜时间（circadian time, CT）3点及CT15点采集血液与肝脏样本。 在野生型小鼠的肝脏中，GCN2磷酸化水平呈现昼夜节律模式，该模式由细胞内支链氨基酸浓度所调控（决定系数r²=0.93）。仅在CT15时点，给野生型小鼠饲喂无亮氨酸饮食可增强肝脏整合应激反应的激活。 GCN2敲除小鼠体内，肝脏整合应激信号通路的昼夜振荡、包含脂质代谢基因在内的肝脏转录组变化，以及肝脏甘油三酯浓度均显著降低甚至消失。 此外，在饲喂无亮氨酸饮食时，缺乏GCN2的小鼠无法维持全身能量消耗、呼吸交换率及运动活性的昼夜节律。 综上，GCN2的激活可维持肝脏内整合应激反应的昼夜激活模式，并在营养应激调控全身代谢的机制中发挥关键作用。 本研究的样本来自野生型及GCN2全身敲除小鼠的肝脏组织，这些小鼠分别饲喂对照饮食或无亮氨酸饮食，并于CT03点或CT15点被处死。 本次对比分析聚焦于GCN2敲除、无亮氨酸饮食及其交互作用对肝脏转录组昼夜模式的影响。 本实验还将比较各处理组内两个时间点的转录本差异表达情况。 此外，为观察无亮氨酸饮食对肝脏转录组的时间依赖性影响，我们还对比了在CT03点及CT15点时，饲喂对照饮食与无亮氨酸饮食的野生型小鼠的基因差异表达情况。