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Browsing by Author "Wang, Yongping"
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Item Dietary Methionine Restriction Regulates Liver Protein Synthesis and Gene Expression Independently of Eukaryotic Initiation Factor 2 Phosphorylation in Mice(Oxford University Press, 2017-06) Pettit, Ashley P.; Jonsson, William O.; Bargoud, Albert R.; Mirek, Emily T.; Peelor, Frederick F., III; Wang, Yongping; Gettys, Thomas W.; Kimball, Scot R.; Miller, Benjamin F.; Hamilton, Karyn L.; Wek, Ronald C.; Anthony, Tracy G.; Biochemistry and Molecular Biology, School of MedicineBackground: 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.Item General control nonderepressible 2 deletion predisposes to asparaginase-associated pancreatitis in mice(American Physiological Society, 2016-06-01) Phillipson-Weiner, Lindsey; Mirek, Emily T.; Wang, Yongping; McAuliffe, W. Geoffrey; Wek, Ronald C.; Anthony, Tracy G.; Biochemistry and Molecular Biology, School of MedicineTreatment 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.Item Obesity challenges the hepatoprotective function of the integrated stress response to asparaginase exposure in mice(American Society for Biochemistry and Molecular Biology, 2017-04-21) Nikonorova, Inna A.; Al-Baghdadi, Rana J. T.; Mirek, Emily T.; Wang, Yongping; Goudie, Michael P.; Wetstein, Berish B.; Dixon, Joseph L.; Hine, Christopher; Mitchell, James R.; Adams, Christopher M.; Wek, Ronald C.; Anthony, Tracy G.; Biochemistry and Molecular Biology, School of MedicineObesity 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 versus 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 Gcn2 intensified hepatic PERK activation to asparaginase, yet surprisingly, mRNA levels of key ISR gene targets such as Atf5 and Trib3 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 (Atf4) 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 Atf4 expression, whereas hydrogen sulfide production is promoted via GCN2-ATF4 pathway.Item Role of activating transcription factor 4 in the hepatic response to amino acid depletion by asparaginase(SpringerNature, 2017-04-28) Al-Baghdadi, Rana J. T.; Nikonorova, Inna A.; Mirek, Emily T.; Wang, Yongping; Park, Jinhee; Belden, William J.; Wek, Ronald C.; Anthony, Tracy G.; Department of Biochemistry and Molecular Biology, School of MedicineThe 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.Item Transcription factor ATF4 directs basal and stress-induced gene expression in the unfolded protein response and cholesterol metabolism in the liver(American Society for Cell Biology, 2016-05-01) Fusakio, Michael E.; Willy, Jeffrey A.; Wang, Yongping; Mirek, Emily T.; Al Baghdadi, Rana J. T.; Adams, Christopher M.; Anthony, Tracy G.; Wek, Ronald C.; Department of Biochemistry & Molecular Biology, IU School of MedicineDisturbances in protein folding and membrane compositions in the endoplasmic reticulum (ER) elicit the unfolded protein response (UPR). Each of three UPR sensory proteins-PERK (PEK/EIF2AK3), IRE1, and ATF6-is activated by ER stress. PERK phosphorylation of eIF2 represses global protein synthesis, lowering influx of nascent polypeptides into the stressed ER, coincident with preferential translation of ATF4 (CREB2). In cultured cells, ATF4 induces transcriptional expression of genes directed by the PERK arm of the UPR, including genes involved in amino acid metabolism, resistance to oxidative stress, and the proapoptotic transcription factor CHOP (GADD153/DDIT3). In this study, we characterize whole-body and tissue-specific ATF4-knockout mice and show in liver exposed to ER stress that ATF4 is not required for CHOP expression, but instead ATF6 is a primary inducer. RNA-Seq analysis indicates that ATF4 is responsible for a small portion of the PERK-dependent UPR genes and reveals a requirement for expression of ATF4 for expression of genes involved in oxidative stress response basally and cholesterol metabolism both basally and under stress. Consistent with this pattern of gene expression, loss of ATF4 resulted in enhanced oxidative damage, and increased free cholesterol in liver under stress accompanied by lowered cholesterol in sera.