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

Abstract

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 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 intensified hepatic PERK activation to asparaginase, yet surprisingly, mRNA levels of key ISR gene targets such as and 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 () 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 expression, whereas hydrogen sulfide production is promoted via GCN2-ATF4 pathway.

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

“…This suppressive role of GCN2 was first observed by us in mice fed a leucine devoid diet to Gcn2 -/-mice (10), then confirmed and further detailed using mice treated with asparaginase (8,9,19,25). This work also strongly supports the findings of Averous et al who used a model of leucine deprivation in mouse embryonic fibroblasts (MEFs) (20) to conclude that GCN2 and eIF2 phosphorylation, but not ATF4, are required for continuous suppression of mTORC1 during leucine or arginine deprivation.…”

“…-/-mice demonstrate PERK activation and eIF2 phosphorylation alongside highly activated mTORC1 (8). Thus, phosphorylation of eIF2 by PERK failed to prevent mTORC1 activation in obese mice.…”

“…In all the cases, these increases in mRNA levels were observed no earlier than 3 h following the injection of asparaginase, again confirming that acute inhibition of mTORC1 does not involve an ATF4-driven transcriptional program. Interestingly, our previous studies show that induction of Trib3 mRNA by asparaginase requires GCN2, but not ATF4, as Atf4 -/-animals treated with asparaginase demonstrate 10-fold greater increases in Trib3 mRNA levels to asparaginase than wild type animals, whereas no induction whatsoever is observed in Gcn2 -/-animals (8,32). This confirms that Trib3 gene is controlled by other transcriptional factors in addition to ATF4 (18), but whether TRIB3 represents the major effector of GCN2 pathway that contributes to sustained inhibition of hepatic mTORC1 in WT, as well as in Atf4 -/-animals requires further investigation.…”

“…Inappropriate activation of mTORC1 during cytoplasmic or proteotoxic stress has deleterious effects for both cells (7) and organisms (8)(9)(10)(11)(12). Our lab was the first to identify the existence of coordinated crosstalk between the ISR and mTORC1 pathway in mammals following amino acid deprivation (10).…”

“…Our lab was the first to identify the existence of coordinated crosstalk between the ISR and mTORC1 pathway in mammals following amino acid deprivation (10). Since then, multiple labs have explored the basis for this bi-directional relationship in tissues and in normal and cancer (8,(13)(14)(15)(16)(17)(18)(19)(20)(21). Despite these efforts, the core factor(s) coordinating ISR-mTORC1 communication during amino acid stress remain mysterious.…”