General Control Non-Derepressible 2 is a Homeostatic Regulator of Thermogenesis and Biological Rhythms
Creators: name: Levy, Jordan L.
Publisher: name: No Publisher Supplied
Year: 2024

description: Mammals adapt to changes in their environment such as temperature, light exposure and food availability by engaging a variety of homeostatic mechanisms. The integrated stress response (ISR) is an intracellular signaling network that functions to maintain homeostatic control of the proteome. The ISR activates adaptation following a variety of environmental threats such as nutrient scarcity, misfolded protein, viral infection and oxygen deprivation and serves as a key survival mechanism for cells to adapt changes in their intracellular environment. Activation of the ISR triggers phosphorylation of the translation factor, eukaryotic initiation factor 2 (eIF2) on its alpha subunit by a family of four protein kinases. Phosphorylated eIF2 slows cap-dependent translation while at the same time promotes the preferential synthesis of ATF4 and other stress-responsive DNA binding proteins function to realign the transcriptome towards survival and adaptation.The eIF2 kinase, general control non-derepressible 2 (GCN2) is a primary sensor of amino acid insufficiency. However, evidence for GCN2 activation in response to other forms of stress such as cold stress adaptation and the maintenance of the circadian clock are present in the literature. At this time, these insights are limited to either invitro models or have only been demonstrated in a single tissue. The objective of this dissertation was twofold: 1) to understand the functional role of GCN2 in response to acute cold stress 2) explore how activation of GCN2 by amino acid insufficiency impacts diurnal rhythms in the liver. The first thesis objective was addressed with an experimental design that examined the role of GCN2 in maintaining core body temperature under acute cold exposure. The results showed that cold exposure increases GCN2 and eIF2 phosphorylation in the BAT of male mice and corresponded with an increase in the transcript abundance of SLC transmembrane transporters and intracellular amino acid levels. Loss of GCN2 activity impaired ISR activation and SLC transporter expression in brown adipose resulting in acute cold intolerance independent of sex. The second thesis objective was addressed using two different experimental designs. In the first study, I examined the role of GCN2 and dietary leucine intake on the maintenance of circadian rhythms in whole body metabolism and peripheral tissues. In WT mice, hepatic GCN2 phosphorylation displayed a diurnal pattern which strongly correlated with diurnal changes in intracellular branched chain amino acid levels. Although global loss of Gcn2 did not alter circadian rhythms in whole body calorimetry measures, diurnal changes in hepatic ISR signaling, transcriptome and triglyceride content were lost. Similarly, in wildtype (WT) mice, leucine devoid diet (Leu D) feeding did not alter circadian rhythms in whole body calorimetry measures but altered diurnal rhythms in ISR activation and transcriptional execution in a time dependent manner. Lastly, global loss of Gcn2 under Leu D feeding impaired circadian rhythms in whole body metabolism and altered the transcripts in the liver changing over a diurnal cycle. Taken together, the results demonstrate a role for GCN2 in the maintenance of diurnal changes in ISR transcriptional execution in peripheral tissues which is likely driven by changes in intracellular branched chain amino acid levels. A second study examined how the ISR and mechanistic target of rapamycin complex 1 (mTORC1) signaling pathways are coordinated over a feeding-fasting cycle and the impact of acute dietary amino acid insufficiency (AAI) on diurnal rhythms in these signaling pathways and the circadian clock in the liver. Control-fed mice displayed diurnal rhythms in both ISR and mTORC1 signaling, with ISR activity antiphase to mTORC1. TrpD feeding disrupted these rhythms, and lead to significant changes in the identity and the temporal profile of the diurnal hepatic transcriptome, particularly in genes involved in amino acid metabolism and stress responses. Furthermore, TrpD feeding impacted the diurnal expression and ribosome association of clock genes. These findings demonstrate that acute changes in dietary amino acid intake significantly alters diurnal rhythms in hepatic ISR and mTORC1 signaling, affecting transcriptional and translational regulation. This study illustrates the diurnal relationship between GCN2 and mTORC1 in the liver and provides insights into the interplay between nutrient sensing and circadian regulation.The data within this dissertation expands our understanding of the physiological role of GCN2 in relaying changes in the intracellular environment to physiological outputs in the BAT and liver. The insights provided within this work highlight new functional outcomes of ISR signaling which hold broader implications for how to target this signaling pathway therapeutically can be leveraged to combat obesity and metabolic disorders.