Emily T. Mirek

GCN2 is required to increase fibroblast growth factor 21 and maintain hepatic triglyceride homeostasis during asparaginase treatment.

The antileukemic agent asparaginase triggers the amino acid response (AAR) in the liver by activating the eukaryotic initiation factor 2 (eIF2) kinase general control nonderepressible 2 (GCN2). To explore the mechanism by which AAR induction is necessary to mitigate hepatic lipid accumulation and prevent liver dysfunction during continued asparaginase treatment, wild-type and Gcn2 null mice were injected once daily with asparaginase or phosphate buffered saline for up to 14 days. Asparaginase induced mRNA expression of multiple AAR genes and greatly increased circulating concentrations of the metabolic hormone fibroblast growth factor 21 (FGF21) independent of food intake. Loss of Gcn2 precluded mRNA expression and circulating levels of FGF21 and blocked mRNA expression of multiple genes regulating lipid synthesis and metabolism including Fas, Ppara, Pparg, Acadm, and Scd1 in both liver and white adipose tissue. Furthermore, rates of triglyceride export and protein expression of apolipoproteinB-100 were significantly reduced in the livers of Gcn2 null mice treated with asparaginase, providing a mechanistic basis for the increase in hepatic lipid content. Loss of AAR-regulated antioxidant defenses in Gcn2 null livers was signified by reduced Gpx1 gene expression alongside increased lipid peroxidation. Substantial reductions in antithrombin III hepatic expression and activity in the blood of asparaginase-treated Gcn2 null mice indicated liver dysfunction. These results suggest that the ability of the liver to adapt to prolonged asparaginase treatment is influenced by GCN2-directed regulation of FGF21 and oxidative defenses, which, when lost, corresponds with maladaptive effects on lipid metabolism and hemostasis.

Dietary Methionine Restriction Regulates Liver Protein Synthesis and Gene Expression Independently of Eukaryotic Initiation Factor 2 Phosphorylation in Mice

Background: The phosphorylation of eukaryotic initiation factor 2 (p-eIF2) during dietary amino acid insufficiency reduces protein synthesis and alters gene expression via the integrated stress response (ISR).Objective: We explored whether a Met-restricted (MR) diet activates the ISR to reduce body fat and regulate protein balance.Methods: Male and female mice aged 3-6 mo with either whole-body deletion of general control nonderepressible 2 (Gcn2) or liver-specific deletion of protein kinase R-like endoplasmic reticulum kinase (Perk) alongside wild-type or floxed control mice were fed an obesogenic diet sufficient in Met (0.86%) or an MR (0.12% Met) diet for ≤5 wk. Ala enrichment with deuterium was measured to calculate protein synthesis rates. The guanine nucleotide exchange factor activity of eIF2B was measured alongside p-eIF2 and hepatic mRNA expression levels at 2 d and 5 wk. Metabolic phenotyping was conducted at 4 wk, and body composition was measured throughout. Results were evaluated with the use of ANOVA (P < 0.05).Results: Feeding an MR diet for 2 d did not increase hepatic p-eIF2 or reduce eIF2B activity in wild-type or Gcn2-/- mice, yet many genes transcriptionally regulated by the ISR were altered in both strains in the same direction and amplitude. Feeding an MR diet for 5 wk increased p-eIF2 and reduced eIF2B activity in wild-type but not Gcn2-/- mice, yet ISR-regulated genes altered in both strains similarly. Furthermore, the MR diet reduced mixed and cytosolic but not mitochondrial protein synthesis in both the liver and skeletal muscle regardless of Gcn2 status. Despite the similarities between strains, the MR diet did not increase energy expenditure or reduce body fat in Gcn2-/- mice. Finally, feeding the MR diet to mice with Perk deleted in the liver increased hepatic p-eIF2 and altered body composition similar to floxed controls.Conclusions: Hepatic activation of the ISR resulting from an MR diet does not require p-eIF2. Gcn2 status influences body fat loss but not protein balance when Met is restricted.

General control nonderepressible 2 deletion predisposes to asparaginase-associated pancreatitis in mice

Treatment with the antileukemic agent asparaginase can induce acute pancreatitis, but the pathophysiology remains obscure. In the liver of mice, eukaryotic initiation factor 2 (eIF2) kinase general control nonderepressible 2 (GCN2) is essential for mitigating metabolic stress caused by asparaginase. We determined the consequences of asparaginase treatment on the pancreata of wild-type (WT, GCN2-intact) and GCN2-deleted (ΔGcn2) mice. Mean pancreas weights in ΔGcn2 mice treated with asparaginase for 8 days were increased (P < 0.05) above all other groups. Histological examination revealed acinar cell swelling and altered staining of zymogen granules in ΔGcn2, but not WT, mice. Oil Red O staining and measurement of pancreas triglycerides excluded lipid accumulation as a contributor to acini appearance. Instead, transmission electron microscopy revealed dilatation of the endoplasmic reticulum (ER) and accumulation of autophagic vacuoles in the pancreas of ΔGcn2 mice treated with asparaginase. Consistent with the idea that loss of GCN2 in a pancreas exposed to asparaginase induced ER stress, phosphorylation of protein kinase R-like ER kinase (PERK) and its substrate eIF2 was increased in the pancreas of asparaginase-treated ΔGcn2 mice. In addition, mRNA expression of PERK target genes, activating transcription factors 4, 3, and 6 (Atf4, Atf3, and Atf6), fibroblast growth factor 21 (Fgf21), heat shock 70-kDa protein 5 (Hspa5), and spliced Xbp1 (sXbp1), as well as pancreas mass, was elevated in the pancreas of asparaginase-treated ΔGcn2 mice. Furthermore, genetic markers of oxidative stress [sirtuin (Sirt1)], inflammation [tumor necrosis factor-α (Tnfα)], and pancreatic injury [pancreatitis-associated protein (Pap)] were elevated in asparaginase-treated ΔGcn2, but not WT, mice. These data indicate that loss of GCN2 predisposes the exocrine pancreas to a maladaptive ER stress response and autophagy during asparaginase treatment and represent a genetic basis for development of asparaginase-associated pancreatitis.

Evaluating the effect of 20-hydroxyecdysone (20HE) on mechanistic target of rapamycin complex 1 (mTORC1) signaling in the skeletal muscle and liver of rats

Phytoecdysteroids such as 20-hydroxyecdysone (20HE) are nutritional supplements marketed as enhancers of lean body mass. In this study the impact of 20HE ingestion on protein kinase B/Akt-mechanistic target of rapamycin complex 1 signaling in the skeletal muscle and liver of male rats was found to be limited. Bioavailability of 20HE, whether consumed alone or with leucine, also remained low at all doses ingested. Additional work is necessary to clarify 20HE mechanism of action in vivo.

­­­­­­­­­­­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.

Role of activating transcription factor 4 in the hepatic response to amino acid depletion by asparaginase

The anti-leukemic agent asparaginase activates the integrated stress response (ISR) kinase GCN2 and inhibits signaling via mechanistic target of rapamycin complex 1 (mTORC1). The study objective was to investigate the protective role of activating transcription factor 4 (ATF4) in controlling the hepatic transcriptome and mediating GCN2-mTORC1 signaling during asparaginase. We compared global gene expression patterns in livers from wildtype, Gcn2−/−, and Atf4−/− mice treated with asparaginase or excipient and further explored selected responses in livers from Atf4+/− mice. Here, we show that ATF4 controls a hepatic gene expression profile that overlaps with GCN2 but is not required for downregulation of mTORC1 during asparaginase. Ingenuity pathway analysis indicates GCN2 independently influences inflammation-mediated hepatic processes whereas ATF4 uniquely associates with cholesterol metabolism and endoplasmic reticulum (ER) stress. Livers from Atf4−/− or Atf4+/− mice displayed an amplification of the amino acid response and ER stress response transcriptional signatures. In contrast, reduction in hepatic mTORC1 signaling was retained in Atf4−/− mice treated with asparaginase. Conclusions: GCN2 and ATF4 serve complementary roles in the hepatic response to asparaginase. GCN2 functions to limit inflammation and mTORC1 signaling whereas ATF4 serves to limit the amino acid response and prevent ER stress during amino acid depletion by asparaginase.