Background Dietary sulfur amino acid restriction (SAAR) improves body composition and metabolic health across several model organisms in part through induction of the integrated stress response (ISR). Objective We investigate the hypothesis that activating transcription factor 4 (ATF4) acts as a converging point in the ISR during SAAR. Methods Using liver-specific or global gene ablation strategies, in both female and male mice, we address the role of ATF4 during dietary SAAR. Results We show that ATF4 is dispensable in the chronic induction of the hepatokine fibroblast growth factor 21 while being essential for the sustained production of endogenous hydrogen sulfide. We also affirm that biological sex, independent of ATF4 status, is a determinant of the response to dietary SAAR. Conclusions Our results suggest that auxiliary components of the ISR, which are independent of ATF4, are critical for SAAR-mediated improvements in metabolic health in mice.

Stearoyl‐CoA desaturases are ER‐resident enzymes that convert saturated fatty acids into monounsaturated fatty acids and have been studied using whole body, liver, skin, and adipose knockout models, but their specific role in the intestine is not well understood. Stearoyl‐CoA desaturase 1 (SCD1) gene and protein expression increases along the length of the intestine and was found to be induced by fasting followed by refeeding a high sucrose very low‐fat diet, similar to what has been established in liver. In addition to SCD1, stearoyl‐CoA desaturase 2 (SCD2) was also found to be expressed and regulated in a similar manner to SCD1 in the intestine. Intestine specific SCD1 knockout (iKO) mice were generated by crossing SCD1 floxed mice with mice expressing the Cre‐recombinase gene under the villin promoter. These mice have longer intestines and increased lipid absorption compared to floxed counterparts. In addition, iKO mice have increased bile acids, a more hydrophilic bile acid pool, and have increased plasma GLP‐1 and brown adipose tissue (BAT) Iodothyronine Deiodinase 2 (Dio2) gene expression. When placed on a low‐fat diet (10% fat) or a high‐fat diet (45% fat), the iKO mice had increased food intake and energy expenditure while maintaining similar or leaner body weights as their floxed counterparts. The iKO mice showed a trend of improved glucose tolerance. Overall, these results show that stearoyl‐CoA desaturases are expressed and regulated in the intestine. Knocking out SCD1 in an intestine‐specific manner leads to increased bile acids and these increased bile acids may contribute to the increased GLP‐1 and bile acid‐induced thermogenesis in BAT. When fed a high fat diet, iKO mice demonstrated an increased basal metabolic rate, increased food intake, and improved glucose tolerance.

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.