Time-resolved analysis of amino acid stress identifies eIF2 phosphorylation as necessary to inhibit mTORC1 activity in liver.

Amino acid availability is sensed by GCN2 (general control nonderepressible 2) and mechanistic target of rapamycin complex 1 (mTORC1), but how these two sensors coordinate their respective signal transduction events remains mysterious. In this study we utilized mouse genetic models to investigate the role of GCN2 in hepatic mTORC1 regulation upon amino acid stress induced by a single injection of asparaginase. We found that deletion of Gcn2 prevented hepatic phosphorylation of eukaryotic initiation factor 2α to asparaginase and instead unleashed mTORC1 activity. This change in intracellular signaling occurred within minutes and resulted in increased 5'-terminal oligopyrimidine mRNA translation instead of activating transcription factor 4 synthesis. Asparaginase also promoted hepatic mRNA levels of several genes which function as mTORC1 inhibitors, and these genes were blunted or blocked in the absence of Gcn2, but their timing could not explain the early discordant effects in mTORC1 signaling. Preconditioning mice with a chemical endoplasmic reticulum stress agent before amino acid stress rescued normal mTORC1 repression in the liver of Gcn2-/- mice but not in livers with both Gcn2 and the endoplasmic reticulum stress kinase, Perk, deleted. Furthermore, treating wildtype and Gcn2-/- mice with ISRIB, an inhibitor of PERK signaling, also failed to alter hepatic mTORC1 responses to asparaginase, although administration of ISRIB alone had an inhibitory GCN2-independent effect on mTORC1 activity. Taken together, the data show that activating transcription factor 4 is not required, but eukaryotic initiation factor 2α phosphorylation is necessary to prevent mTORC1 activation during amino acid stress.

Obesity challenges the hepatoprotective function of the integrated stress response to asparaginase exposure in mice.

Obesity increases risk for liver toxicity by the anti-leukemic agent asparaginase, but the mechanism is unknown. Asparaginase activates the integrated stress response (ISR) via sensing amino acid depletion by the eukaryotic initiation factor 2 (eIF2) kinase GCN2. The goal of this work was to discern the impact of obesity, alone versus alongside genetic disruption of the ISR, on mechanisms of liver protection during chronic asparaginase exposure in mice. Following diet-induced obesity, biochemical analysis of livers revealed that asparaginase provoked hepatic steatosis that coincided with activation of another eIF2 kinase PKR-like endoplasmic reticulum kinase (PERK), a major ISR transducer to ER stress. Genetic loss of Gcn2 intensified hepatic PERK activation to asparaginase, yet surprisingly, mRNA levels of key ISR gene targets such as Atf5 and Trib3 failed to increase. Instead, mechanistic target of rapamycin complex 1 (mTORC1) signal transduction was unleashed, and this coincided with liver dysfunction reflected by a failure to maintain hydrogen sulfide production or apolipoprotein B100 (ApoB100) expression. In contrast, obese mice lacking hepatic activating transcription factor 4 (Atf4) showed an exaggerated ISR and greater loss of endogenous hydrogen sulfide but normal inhibition of mTORC1 and maintenance of ApoB100 during asparaginase exposure. In both genetic mouse models, expression and phosphorylation of Sestrin2, an ATF4 gene target, was increased by asparaginase, suggesting mTORC1 inhibition during asparaginase exposure is not driven via eIF2-ATF4-Sestrin2. In conclusion, obesity promotes a maladaptive ISR during asparaginase exposure. GCN2 functions to repress mTORC1 activity and maintain ApoB100 protein levels independently of Atf4 expression, whereas hydrogen sulfide production is promoted via GCN2-ATF4 pathway.