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. Overall design: 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.