Tracy G. Anthony

The eIF2 kinase PERK and the integrated stress response facilitate activation of ATF6 during endoplasmic reticulum stress.

This study shows that the eIF2 kinase PERK is required not only for translational control but also for activation of ATF6 and its target genes in the unfolded protein response. The PERK pathway facilitates both the synthesis of ATF6 and trafficking of ATF6 from the endoplasmic reticulum to the Golgi for intramembrane proteolysis and activation of ATF6.

Regulation of adipose branched-chain amino acid catabolism enzyme expression and cross-adipose amino acid flux in human obesity

Elevated blood branched-chain amino acids (BCAA) are often associated with insulin resistance and type 2 diabetes, which might result from a reduced cellular utilization and/or incomplete BCAA oxidation. White adipose tissue (WAT) has become appreciated as a potential player in whole body BCAA metabolism. We tested if expression of the mitochondrial BCAA oxidation checkpoint, branched-chain α-ketoacid dehydrogenase (BCKD) complex, is reduced in obese WAT and regulated by metabolic signals. WAT BCKD protein (E1α subunit) was significantly reduced by 35-50% in various obesity models (fa/fa rats, db/db mice, diet-induced obese mice), and BCKD component transcripts significantly lower in subcutaneous (SC) adipocytes from obese vs. lean Pima Indians. Treatment of 3T3-L1 adipocytes or mice with peroxisome proliferator-activated receptor-γ agonists increased WAT BCAA catabolism enzyme mRNAs, whereas the nonmetabolizable glucose analog 2-deoxy-d-glucose had the opposite effect. The results support the hypothesis that suboptimal insulin action and/or perturbed metabolic signals in WAT, as would be seen with insulin resistance/type 2 diabetes, could impair WAT BCAA utilization. However, cross-tissue flux studies comparing lean vs. insulin-sensitive or insulin-resistant obese subjects revealed an unexpected negligible uptake of BCAA from human abdominal SC WAT. This suggests that SC WAT may not be an important contributor to blood BCAA phenotypes associated with insulin resistance in the overnight-fasted state. mRNA abundances for BCAA catabolic enzymes were markedly reduced in omental (but not SC) WAT of obese persons with metabolic syndrome compared with weight-matched healthy obese subjects, raising the possibility that visceral WAT contributes to the BCAA metabolic phenotype of metabolically compromised individuals.

The Leucine Content of a Complete Meal Directs Peak Activation but Not Duration of Skeletal Muscle Protein Synthesis and Mammalian Target of Rapamycin Signaling in Rats

This study examined the impact of leucine (Leu) derived from complete meals on stimulation of skeletal muscle protein synthesis (MPS). Expt. 1 examined time course changes in translation initiation and MPS after a meal. Male rats ( approximately 300 g) were trained for 5 d to eat 3 meals/d providing 20, 50, and 30% of energy from whey protein, carbohydrates, and fats, respectively. Plasma and skeletal muscle were collected at time 0 (baseline) after 12 h of food deprivation and then at 45, 90, 135, 180, and 300 min after a 4-g meal. Plasma Leu increased at 45 min and remained elevated through 180 min. MPS peaked at 45-90 min and returned to baseline by 180 min. Plasma Leu correlated with phosphorylation of ribosomal protein p70 S6 kinase (r = 0.723; P < 0.05), eukaryotic initiation factor 4E binding protein-1 (r = 0.773; P < 0.05), and MPS (r = 0.608; P < 0.05) over time. Expt. 2 examined 3 levels of protein intake (10, 20, and 30% of energy) from 2 sources (wheat and whey) with different Leu contents ( approximately 6.8 and approximately 10.9%, respectively) on stimulation of initiation and MPS. Rats were trained to eat 3 meals/d providing 14, 56, and 30% of energy from protein, carbohydrates, and fats. On d 6, MPS was evaluated at 90 min after rats consumed 1 of the 6 test meals. Whey protein stimulated initiation and MPS more than wheat and the differential response related to greater plasma Leu responses in the whey groups. These studies demonstrate that peak activation but not duration of MPS is proportional to the Leu content of a meal.

GCN2 Protein Kinase Is Required to Activate Amino Acid Deprivation Responses in Mice Treated with the Anti-cancer Agent l-Asparaginase

Asparaginase depletes circulating asparagine and glutamine, activating amino acid deprivation responses (AADR) such as phosphorylation of eukaryotic initiation factor 2 (p-eIF2) leading to increased mRNA levels of asparagine synthetase and CCAAT/enhancer-binding protein β homologous protein (CHOP) and decreased mammalian target of rapamycin complex 1 (mTORC1) signaling. The objectives of this study were to assess the role of the eIF2 kinases and protein kinase R-like endoplasmic reticulum resident kinase (PERK) in controlling AADR to asparaginase and to compare the effects of asparaginase on mTORC1 to that of rapamycin. In experiment 1, asparaginase increased hepatic p-eIF2 in wild-type mice and mice with a liver-specific PERK deletion but not in GCN2 null mice nor in GCN2-PERK double null livers. In experiment 2, wild-type and GCN2 null mice were treated with asparaginase (3 IU per g of body weight), rapamycin (2 mg per kg of body weight), or both. In wild-type mice, asparaginase but not rapamycin increased p-eIF2, p-ERK1/2, p-Akt, and mRNA levels of asparagine synthetase and CHOP in liver. Asparaginase and rapamycin each inhibited mTORC1 signaling in liver and pancreas but maximally together. In GCN2 null livers, all responses to asparaginase were precluded except CHOP mRNA expression, which remained partially elevated. Interestingly, rapamycin blocked CHOP induction by asparaginase in both wild-type and GCN2 null livers. These results indicate that GCN2 is required for activation of AADR to asparaginase in liver. Rapamycin modifies the hepatic AADR to asparaginase by preventing CHOP induction while maximizing inhibition of mTORC1.

Role of glutamine depletion in directing tissue-specific nutrient stress responses to L-asparaginase

l-Asparaginase is important in the induction regimen for treating acute lymphoblastic leukemia. Cytotoxic complications are clinically significant problems lacking mechanistic insight. To reveal tissue-specific molecular responses to this drug, mice were administered asparaginase from either Escherichia coli (clinically used) or Wolinella succinogenes (novel, glutaminase-free form). Both enzymes abolished serum asparagine, but only the E. coli form reduced circulating glutamine. E. coli asparaginase reduced protein synthesis in liver and spleen but not pancreas via increased phosphorylation of the translation factor eIF2. In contrast, treatment with Wolinella caused no untoward changes in protein synthesis in any tissue examined. Treating mice deleted for the eIF2 kinase, GCN2, with the E. coli enzyme showed eIF2 phosphorylation to be GCN2-dependent, but only initially. Furthermore, although eIF2 phosphorylation was not increased in the pancreas or by Wolinella asparaginase, expression of the amino acid stress response genes, asparagine synthetase and CHOP/GADD153, increased as a result of both enzymes, even in tissues demonstrating no change in eIF2 phosphorylation. Finally, signaling downstream of the mammalian target of rapamycin kinase was repressed in liver and pancreas by E. coli but not Wolinella asparaginase. These data demonstrate that the nutrient stress response to asparaginase is tissue-specific and exacerbated by glutamine depletion. Importantly, increased expression of asparagine synthetase and CHOP does not require eIF2 phosphorylation, signifying alternate or auxiliary means of inducing gene expression under conditions of amino acid depletion in the whole animal.

Defining meal requirements for protein to optimize metabolic roles of amino acids

Dietary protein provides essential amino acids (EAAs) for the synthesis of new proteins plus an array of other metabolic functions; many of these functions are sensitive to postprandial plasma and intracellular amino acid concentrations. Recent research has focused on amino acids as metabolic signals that influence the rate of protein synthesis, inflammation responses, mitochondrial activity, and satiety, exerting their influence through signaling systems including mammalian/mechanistic target of rapamycin complex 1 (mTORC1), general control nonrepressed 2 (GCN2), glucagon-like peptide 1 (GLP-1), peptide YY (PYY), serotonin, and insulin. These signals represent meal-based responses to dietary protein. The best characterized of these signals is the leucine-induced activation of mTORC1, which leads to the stimulation of skeletal muscle protein synthesis after ingestion of a meal that contains protein. The response of this metabolic pathway to dietary protein (i.e., meal threshold) declines with advancing age or reduced physical activity. Current dietary recommendations for protein are focused on total daily intake of 0.8 g/kg body weight, but new research suggests daily needs for older adults of ≥1.0 g/kg and identifies anabolic and metabolic benefits to consuming at least 20–30 g protein at a given meal. Resistance exercise appears to increase the efficiency of EAA use for muscle anabolism and to lower the meal threshold for stimulation of protein synthesis. Applying this information to a typical 3-meal-a-day dietary plan results in protein intakes that are well within the guidelines of the Dietary Reference Intakes for acceptable macronutrient intakes. The meal threshold concept for dietary protein emphasizes a need for redistribution of dietary protein for optimum metabolic health.

Selective mRNA translation during eIF2 phosphorylation induces expression of IBTKα

This study measured changes in global mRNA translation in response to ER stress. The analysis suggests that translation of a majority of gene transcripts is either repressed or resistant, whereas select key regulators are subject to preferential translation. From this last group, IBTKα is identified as a novel target of the UPR central to cell fate.

The eIF2 kinase GCN2 is essential for the murine immune system to adapt to amino acid deprivation by asparaginase.

Amino acid starvation by asparaginase (ASNase) enhances phosphorylation of eukaryotic initiation factor 2 (eIF2) by general control nonderepressible 2 (GCN2) kinase, leading to reduced global mRNA translation rates. This conserves energy and allows cells time to reprogram stress-related gene expression to alleviate cell injury. This study addressed the importance of GCN2 for the immune system to adapt to amino acid starvation by ASNase. GCN2(+/+) and GCN2(-/-) mice were injected once daily with ASNase or saline for up to 7 d. In both thymus and spleen, activation of amino acid stress response genes to ASNase, such as asparagine synthetase and CAAT enhancer binding protein homologous protein, required GCN2. ASNase reduced food intake and body weight in both genotypes, but spleen and thymus wet weights and total cell numbers in thymus, spleen, bone marrow, and mesenteric lymph nodes were less in GCN2(-/-) mice treated with ASNase (genotype x ASNase, P < 0.05). In the thymus, GCN2(-/-) mice treated with ASNase demonstrated enhanced apoptosis and fewer cells in all subpopulations examined (CD3+, CD4-8-, CD4+8+, CD4+8-, CD4-8+) compared with GCN2(+/+) mice treated with ASNase (genotype x ASNase, P < 0.05). In the spleen, GCN2 deletion magnified ASNase-induced reductions in CD4+ T cells, CD8+ T cells, CD19+ B cells, and CD11b+ leukocytes (genotype x ASNase, P < 0.05). These results indicate that loss of GCN2 enhances immunosuppression by ASNase and that this eIF2 kinase is broadly required for amino acid stress management in the immune system.

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

Whole-body and liver-specific ATF4–knockout mice are used to evaluate the role of ATF4 transcription factor in the unfolded protein response (UPR). ATF4 directs a small subset of UPR gene expression, and deletion of ATF4 in the liver triggers enhanced oxidative stress, disrupts cholesterol metabolism, and enhances cell death.

Role of GCN2-Independent Signaling Through a Noncanonical PERK/NRF2 Pathway in the Physiological Responses to Dietary Methionine Restriction.

Restricting availability of essential amino acids (EAAs) limits aminoacylation of tRNAs by their cognate EAAs and activates the nutrient-sensing kinase, general control nonderepressible 2 (GCN2). Activated GCN2 phosphorylates eukaryotic initiation factor 2 (eIF2), altering gene-specific translation and initiating a transcriptional program collectively described as the integrated stress response (ISR). Central GCN2 activation by EAA deprivation is also linked to an acute aversive feeding response. Dietary methionine restriction (MR) produces a well-documented series of physiological responses (increased energy intake and expenditure, decreased adiposity, and increased insulin sensitivity), but the role of GCN2 in mediating them is unknown. Using Gcn2−/− mice, we found that the absence of GCN2 had no effect on the ability of MR to reduce body weight or adiposity, increase energy intake and expenditure, increase hepatic transcription and release of fibroblast growth factor 21, or improve insulin sensitivity. Interestingly, hepatic eIF2 phosphorylation by MR was uncompromised in Gcn2−/− mice. Instead, protein kinase R–like endoplasmic reticulum (ER) kinase (PERK) was activated in both intact and Gcn2−/− mice. PERK activation corresponded with induction of the ISR and the nuclear respiratory factor 2 antioxidant program but not ER stress. These data uncover a novel glutathione-sensing mechanism that functions independently of GCN2 to link dietary MR to its metabolic phenotype.