An integrative approach to assessing effects of a short-term Western diet on gene expression in rat liver.

Consumption of a diet rich in saturated fatty acids and carbohydrates contributes to the accumulation of fat in the liver and development of non-alcoholic steatohepatitis (NASH). Herein we investigated the hypothesis that short-term consumption of a high fat/sucrose Western diet (WD) alters the genomic and translatomic profile of the liver in association with changes in signaling through the protein kinase mTORC1, and that such alterations contribute to development of NAFLD. The results identify a plethora of mRNAs that exhibit altered expression and/or translation in the liver of rats consuming a WD compared to a CD. In particular, consumption of a WD altered the abundance and ribosome association of mRNAs involved in lipid and fatty acid metabolism, as well as those involved in glucose metabolism and insulin signaling. Hepatic mTORC1 signaling was enhanced when rats were fasted overnight and then refed in the morning; however, this effect was blunted in rats fed a WD as compared to a CD. Despite similar plasma insulin concentrations, fatty acid content was elevated in the liver of rats fed a WD as compared to a CD. We found that feeding had a significant positive effect on ribosome occupancy of 49 mRNAs associated with hepatic steatosis (e.g., LIPE, LPL), but this effect was blunted in the liver of rats fed a WD. In many cases, changes in ribosome association were independent of alterations in mRNA abundance, suggesting a critical role for diet-induced changes in mRNA translation in the expression of proteins encoded by those mRNAs. Overall, the findings demonstrate that short-term consumption of a WD impacts hepatic gene expression by altering the abundance of many mRNAs, but also causes wide-spread variation in mRNA translation that potentially contribute to development of hepatic steatosis.

ATF4-Mediated Upregulation of REDD1 and Sestrin2 Suppresses mTORC1 Activity during Prolonged Leucine Deprivation.

BACKGROUND: The protein kinase target of rapamycin (mTOR) in complex 1 (mTORC1) is activated by amino acids and in turn upregulates anabolic processes. Under nutrient-deficient conditions, e.g., amino acid insufficiency, mTORC1 activity is suppressed and autophagy is activated. Intralysosomal amino acids generated by autophagy reactivate mTORC1. However, sustained mTORC1 activation during periods of nutrient insufficiency would likely be detrimental to cellular homeostasis. Thus, mechanisms must exist to prevent amino acids released by autophagy from reactivating the kinase. OBJECTIVE: The objective of the present study was to test whether mTORC1 activity is inhibited during prolonged leucine deprivation through ATF4-dependent upregulation of the mTORC1 suppressors regulated in development and DNA damage response 1 (REDD1) and Sestrin2. METHODS: Mice (8 wk old; C57Bl/6 × 129SvEV) were food deprived (FD) overnight and one-half were refed the next morning. Mouse embryo fibroblasts (MEFs) deficient in ATF4, REDD1, and/or Sestrin2 were deprived of leucine for 0-16 h. mTORC1 activity and ATF4, REDD1, and Sestrin2 expression were assessed in liver and cell lysates. RESULTS: Refeeding FD mice resulted in activation of mTORC1 in association with suppressed expression of both REDD1 and Sestrin2 in the liver. In cells in culture, mTORC1 exhibited a triphasic response to leucine deprivation, with an initial suppression followed by a transient reactivation from 2 to 4 h and a subsequent resuppression after 8 h. Resuppression occurred concomitantly with upregulated expression of ATF4, REDD1, and Sestrin2. However, in cells lacking ATF4, neither REDD1 nor Sestrin2 expression was upregulated by leucine deprivation, and resuppression of mTORC1 was absent. Moreover, in cells lacking either REDD1 or Sestrin2, mTORC1 resuppression was attenuated, and in cells lacking both proteins resuppression was further blunted. CONCLUSIONS: The results suggest that leucine deprivation upregulates expression of both REDD1 and Sestrin2 in an ATF4-dependent manner, and that upregulated expression of both proteins is involved in resuppression of mTORC1 during prolonged leucine deprivation.

Suppression of p16 Induces mTORC1-Mediated Nucleotide Metabolic Reprogramming.

Reprogrammed metabolism and cell cycle dysregulation are two cancer hallmarks. p16 is a cell cycle inhibitor and tumor suppressor that is upregulated during oncogene-induced senescence (OIS). Loss of p16 allows for uninhibited cell cycle progression, bypass of OIS, and tumorigenesis. Whether p16 loss affects pro-tumorigenic metabolism is unclear. We report that suppression of p16 plays a central role in reprogramming metabolism by increasing nucleotide synthesis. This occurs by activation of mTORC1 signaling, which directly mediates increased translation of the mRNA encoding ribose-5-phosphate isomerase A (RPIA), a pentose phosphate pathway enzyme. p16 loss correlates with activation of the mTORC1-RPIA axis in multiple cancer types. Suppression of RPIA inhibits proliferation only in p16-low cells by inducing senescence both in vitro and in vivo. These data reveal the molecular basis whereby p16 loss modulates pro-tumorigenic metabolism through mTORC1-mediated upregulation of nucleotide synthesis and reveals a metabolic vulnerability of p16-null cancer cells.

Evidence for a role for Sestrin1 in mediating leucine-induced activation of mTORC1 in skeletal muscle.

Previous studies established that leucine stimulates protein synthesis in skeletal muscle to the same extent as a complete mixture of amino acids, and the effect occurs through activation of the mechanistic target of rapamycin in complex 1 (mTORC1). Recent studies using cells in culture showed that the Sestrins bind leucine and are required for leucine-dependent activation of mTORC1. However, the role they play in mediating leucine-dependent activation of the kinase in vivo has been questioned because the dissociation constant of Sestrin2 for leucine is well below circulating and intramuscular levels of the amino acid. The goal of the present study was to compare expression of the Sestrins in skeletal muscle to other tissues and to assess their relative role in mediating activation of mTORC1 by leucine. The results show that the relative expression of the Sestrin proteins varies widely among tissues and that in skeletal muscle Sestrin1 expression is higher than Sestrin3, whereas Sestrin2 expression is markedly lower. Analysis of the dissociation constants of the Sestrins for leucine as assessed by leucine-induced dissociation of the Sestrin·GAP activity toward Rags 2 (GATOR2) complex revealed that Sestrin1 has the highest affinity for leucine and that Sestrin3 has the lowest affinity. In agreement with the dissociation constants calculated using cells in culture, oral leucine administration promotes disassembly of the Sestrin1·GATOR2 complex but not the Sestrin2 or Sestrin3·GATOR2 complex. Overall, the results presented herein are consistent with a model in which leucine-induced activation of mTORC1 in skeletal muscle in vivo occurs primarily through release of Sestrin1 from GATOR2.