GCN2 drives diurnal patterns in the hepatic integrated stress response and maintains circadian rhythms in whole body metabolism during amino acid insufficiency.

Disruptions in circadian rhythms are associated with an increased risk of developing metabolic diseases. General control nonderepressible 2 (GCN2), a primary sensor of amino acid insufficiency and activator of the integrated stress response (ISR), has emerged as a conserved regulator of the circadian clock in multiple organisms. The objective of this study was to examine diurnal patterns in hepatic ISR activation in the liver and whole body rhythms in metabolism.

Off-target depletion of plasma tryptophan by allosteric inhibitors of BCKDK.

The activation of branched chain amino acid (BCAA) catabolism has garnered interest as a potential therapeutic approach to improve insulin sensitivity, enhance recovery from heart failure, and blunt tumor growth. Evidence for this interest relies in part on BT2, a small molecule that promotes BCAA oxidation and is protective in mouse models of these pathologies. BT2 and other analogs allosterically inhibit branched chain ketoacid dehydrogenase kinase (BCKDK) to promote BCAA oxidation, which is presumed to underlie the salutary effects of BT2.

GCN2 is required to maintain core body temperature in mice during acute cold.

Nonshivering thermogenesis in rodents requires macronutrients to fuel the generation of heat during hypothermic conditions. In this study, we examined the role of the nutrient sensing kinase, general control nonderepressible 2 (GCN2) in directing adaptive thermogenesis during acute cold exposure in mice. We hypothesized that GCN2 is required for adaptation to acute cold stress via activation of the integrated stress response (ISR) resulting in liver production of FGF21 and increased amino acid transport to support nonshivering thermogenesis.

Kinetic proteomics identifies targeted changes in liver metabolism and the ribo-interactome by dietary sulfur amino acid restriction.

Dietary sulfur amino acid restriction (SAAR) protects against diet-induced obesity, extends healthspan, and coincides with an overall reduction in hepatic protein synthesis. To explore the underpinnings of SAAR-induced slowed growth and its impact on liver metabolism and proteostasis, we resolved changes in hepatic mRNA and protein abundances and compared synthesis rates of individual liver proteins. To achieve this, adult male mice were provided deuterium-labeled drinking water while freely consuming either a regular-fat or high-fat diet that was SAA restricted.

GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis.

A stress adaptation pathway termed the integrated stress response has been suggested to be active in many cancers including prostate cancer (PCa). Here, we demonstrate that the eIF2 kinase GCN2 is required for sustained growth in androgen-sensitive and castration-resistant models of PCa both in vitro and in vivo, and is active in PCa patient samples. Using RNA-seq transcriptome analysis and a CRISPR-based phenotypic screen, GCN2 was shown to regulate expression of over 60 solute-carrier () genes, including those involved in amino acid transport and loss of GCN2 function reduces amino acid import and levels.

Rates of protein synthesis are maintained in brain but reduced in skeletal muscle during dietary sulfur amino acid restriction.

Dietary interventions such as sulfur amino acid restriction (SAAR) target multiple drivers of aging, and show promise for preventing or delaying the onset of chronic diseases. SAAR promotes metabolic health and longevity in laboratory animals. The effects of SAAR on proteostasis remain relatively unexplored.

Activation and execution of the hepatic integrated stress response by dietary essential amino acid deprivation is amino acid specific.

Dietary removal of an essential amino acid (EAA) triggers the integrated stress response (ISR) in liver. Herein, we explored the mechanisms that activate the ISR and execute changes in transcription and translation according to the missing EAA. Wild-type mice and mice lacking general control nonderepressible 2 (Gcn2) were fed an amino acid complete diet or a diet devoid of either leucine or sulfur amino acids (methionine and cysteine).

Circulating metabolite homeostasis achieved through mass action.

Homeostasis maintains serum metabolites within physiological ranges. For glucose, this requires insulin, which suppresses glucose production while accelerating its consumption. For other circulating metabolites, a comparable master regulator has yet to be discovered.

Discordant regulation of eIF2 kinase GCN2 and mTORC1 during nutrient stress.

Appropriate regulation of the Integrated stress response (ISR) and mTORC1 signaling are central for cell adaptation to starvation for amino acids. Halofuginone (HF) is a potent inhibitor of aminoacylation of tRNAPro with broad biomedical applications. Here, we show that in addition to translational control directed by activation of the ISR by general control nonderepressible 2 (GCN2), HF increased free amino acids and directed translation of genes involved in protein biogenesis via sustained mTORC1 signaling.

Physiologic Responses to Dietary Sulfur Amino Acid Restriction in Mice Are Influenced by Atf4 Status and Biological Sex.

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.

Metabolomic Response of Equine Skeletal Muscle to Acute Fatiguing Exercise and Training.

The athletic horse, despite being over 50% muscle mass, remains understudied with regard to the effects of exercise and training on skeletal muscle metabolism. To begin to address this knowledge gap, we employed an untargeted metabolomics approach to characterize the exercise-induced and fitness-related changes in the skeletal muscle of eight unconditioned Standardbred horses (four male, four female) before and after a 12-week training period. Before training, unconditioned horses showed a high degree of individual variation in the skeletal muscle metabolome, resulting in very few differences basally and at 3 and 24 h after acute fatiguing exercise.

Age modulates liver responses to asparaginase-induced amino acid stress in mice.

Asparaginase is an amino acid-depleting agent used to treat blood cancers. Metabolic complications due to asparaginase affect liver function in humans. To examine how the liver response to asparaginase changes during maturity to adulthood, here we treated juvenile (2-week), young adult (8-week), and mature adult (16-week) mice with drug or excipient for 1 week and conducted RNA-Seq and functional analyses.

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 prevented hepatic phosphorylation of eukaryotic initiation factor 2α to asparaginase and instead unleashed mTORC1 activity.

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

Unlabelled: 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.

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

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). We explored whether a Met-restricted (MR) diet activates the ISR to reduce body fat and regulate protein balance. Male and female mice aged 3-6 mo with either whole-body deletion of general control nonderepressible 2 () or liver-specific deletion of protein kinase R-like endoplasmic reticulum kinase () alongside wild-type or floxed control mice were fed an obesogenic diet sufficient in Met (0.

Obesity challenges the hepatoprotective function of the integrated stress response to asparaginase exposure in mice.

Obesity 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 alongside genetic disruption of the ISR, on mechanisms of liver protection during chronic asparaginase exposure in mice.

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.

Transcription factor ATF4 directs basal and stress-induced gene expression in the unfolded protein response and cholesterol metabolism in the liver.

Disturbances 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).

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.

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.

Stromal elements act to restrain, rather than support, pancreatic ductal adenocarcinoma.

Sonic hedgehog (Shh), a soluble ligand overexpressed by neoplastic cells in pancreatic ductal adenocarcinoma (PDAC), drives formation of a fibroblast-rich desmoplastic stroma. To better understand its role in malignant progression, we deleted Shh in a well-defined mouse model of PDAC. As predicted, Shh-deficient tumors had reduced stromal content.

EMT and dissemination precede pancreatic tumor formation.

Metastasis is the leading cause of cancer-associated death but has been difficult to study because it involves a series of rare, stochastic events. To capture these events, we developed a sensitive method to tag and track pancreatic epithelial cells in a mouse model of pancreatic cancer. Tagged cells invaded and entered the bloodstream unexpectedly early, before frank malignancy could be detected by rigorous histologic analysis; this behavior was widely associated with epithelial-to-mesenchymal transition (EMT).