Autophagy is Disrupted in the Livers of Obese Mice Exposed to Asparaginase.

Amino acid insufficiency activates general control nonderepressible 2 (GCN2), resulting in preferential synthesis of Activating Transcription Factor 4 (ATF4) to promote cellular protection and recovery. Asparaginase is an enzyme which breaks down asparagine and glutamine and is used to treat acute lymphoblastic leukemia in children and adults. Patients who are overweight or obese carry greater risk of developing hepatotoxicity during treatment and we reported that diet-induced obesity predisposes mice to liver steatosis and dysfunction during asparaginase exposure. To understand the mechanism behind these risks, we subjected liver-specific ATF4 knock-out mice (ls-Atf4KO) and their wild type controls, to a high-fat diet (60% fat by kcal) or low fat chow (~18% fat by kcal) for 12 weeks before exposing them to 8 daily intraperitoneal injections of asparaginase (3 international units per gram body weight) or saline vehicle. Untargeted metabolomics were conducted on the livers of mice (n=6 per group; 3 male and 3 female). Asparaginase exposure decreased liver asparagine independent of diet or genotype. The liver metabolomes in lean mice were largely unaffected by asparaginase or loss of ATF4. In contrast, the liver metabolomes of obese mice exposed to asparaginase showed striking changes in sphingolipids that correlated with elevations in endoplasmic reticulum (ER) stress as indicated by the phosphorylation of protein kinase R-like ER-resident kinase (PERK). Increased synthesis of sphingolipids occurs during the induction of autophagy. Consistent with a sphingolipid signature of increased autophagosome formation, microtubule-associated protein 1 light chain 3 beta-II (LC3-II) was elevated in obese, ls-Atf4KO mice exposed to asparaginase. Additionally, ubiquitin-binding protein 62 (p62), a cargo protein which binds to toxic waste and is then incorporated into the autophagosome for degradation, was elevated in obese mice exposed to asparaginase. This increase in p62 levels indicates a block in fusion of the autophagosome with the lysosome. Levels of p62 were highest in obese ls-Atf4KO mice administered asparaginase. Overall, these data suggest that asparaginase minimally affects the liver metabolome in lean, wild-type mice. In contrast, asparaginase causes marked shifts in the liver metabolome of obese mice which coincides with ER stress and autophagy dysfunction. Loss of ATF4 exacerbates autophagy disruption in obese mice exposed to asparaginase, highlighting its role in remediating ER stress.