Surgical stress resistance induced by single amino acid deprivation requires Gcn2 in mice

Abstract

Dietary restriction, or reduced food intake without malnutrition, increases life span, health span, and acute stress resistance in model organisms from yeast to nonhuman primates. Although dietary restriction is beneficial for human health, this treatment is not widely used in the clinic. Here, we show that short-term, ad libitum feeding of diets lacking essential nutrients increased resistance to surgical stress in a mouse model of ischemia reperfusion injury. Dietary preconditioning by 6 to 14 days of total protein deprivation, or removal of the single essential amino acid tryptophan, protected against renal and hepatic ischemic injury, resulting in reduced inflammation and preserved organ function. Pharmacological treatment with halofuginone, which activated the amino acid starvation response within 3 days by mimicking proline deprivation, was also beneficial. Both dietary and pharmacological interventions required the amino acid sensor and eIF2α (eukaryotic translation initiation factor 2α) kinase Gcn2 (general control nonderepressible 2), implicating the amino acid starvation response and translational control in stress protection. Thus, short-term dietary or pharmacological interventions that modulate amino acid sensing can confer stress resistance in models of surgical ischemia reperfusion injury.

Fig. 1

Protection against renal IR by dietary protein deficiency in the absence of reduced calorie intake.(A to H) Wild-type male B6D2F1 mice were given ad libitum (AL) access to complete or protein (Prot)-free chow, or pair-fed (PF) to the protein-free chow group with complete chow, for 14 days (A to D) or 6 days (E to H) before induction of renal IR injury. (A and E) Body weight of mice expressed as percent initial body weight: (A) n = 9 to 14 per group; (E) n = 12 per group. (B and F) Daily food intake expressed as weight of food eaten per total weight of animals in the cage: (B) n = 2cages per group; (F) n = 3 to 5 cages per group. (C and G) Serum urea on the indicated day before (day 0) or 1 or 2 days after renal IR: (C) n = 7 to 9 per group; (G) n = 7 to 8 per group. (D) Serum creatinine on day 2 after renal IR (n = 3 to 4 per group). (H) Kaplan-Meier survival curves of the indicated groups over 7 days after renal IR (n = 7 to 8 per group). Survival in the protein-free group was significantly improved over both complete diet groups (log-rank test: P = 0.0022 versus AL; P = 0.0001 versus PF). Error bars indicate SEM. Asterisks indicate the significance of the difference between the indicated groups within a given day after IR according to a one-way ANOVA followed by Tukey's multiple comparison test comparing all pairs of values. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 2

Protection against renal IR by isolated EAA deprivation. (A) Total food intake over 6-day preoperative period expressed as weight of food eaten per total weight of animals in the cage (n = 2 cages per group). (B) Body weights of mice fed complete, Trp– or Leu– chow ad libitum (AL) or pair-fed (PF) to Trp– or Leu– animals with complete chow over the 6-day preoperative period (n = 8 per group) expressed as percent initial weight. (C and D) Kidney function as measured by serum urea (C) and creatinine (D) 1 day after 25 min of renal IR. (E) Effect of isocaloric protein- (Prot–) and tryptophan-deficient diets on kidney function, as measured by serum urea. Mice were preconditioned for 1 week on the indicated diet restricted daily to 0.28 kcal per gram of initial weight (~35% DR) before and up to 3 days after 30 min of renal ischemia (n = 5 per group). Inset: Area under the curve (AUC) analysis. Error bars indicate SEM. Asterisks indicate the significance of the difference between the indicated group and the complete diet group according to a one-way ANOVA followed by Dunnett's multiple comparison test, or between the Trp– or Leu– groups and their respective pair-fed controls as indicated with Bonferroni'smultiple comparison test. *P <0.05; **P < 0.01; ***P < 0.001.

Fig. 3

Requirement for Gcn2 in tryptophan deficiency-mediated protection against renal and hepatic IR. (A to D) Gcn2-mediated protection by tryptophan deficiency against renal IR. (A) Body weights of male mice (n = 12per group) fed the indicated diet for 6 days expressed as percent initial weight. (B) Renal function as indicated by serum creatinine 1 day after renal IR (n = 10 per group) or mock ischemia (n = 2 per group). (C) Kidney damage as indicated by mRNA abundance of kidney damage marker Kim1 mRNA as detected by quantitative PCR (qPCR) in kidney samples harvested 1 day after renal IR (n = 9 to 10 per group) or mock ischemia (n = 2 per group) and expressed relative to the wild-type (WT) complete IR group. (D) Representative images of PAS-stained kidney sections 1 day after reperfusion showing the corticomedullary junction, where most tubular damage occurs. (E to G) Gcn2-mediated protection by tryptophan deficiency against hepatic IR. (E) Body weights of female mice (n = 12 to 13 per group)fed the indicated diet for 6 days expressed as percent initial weight. (F) Serum ALT before (0 hours) or 3 hours after 45 min of hepatic ischemia (n = 5 to 10 per group) or mock IR (n = 2 to 3 per group). (G) mRNA abundance of proinflammatory markers P-selectin (Psel) and IL-6 as detected by qPCR in liver samples harvested 3 hours after hepatic ischemia (n = 4 to 5 per group) expressed relative to the WT mock-treated group. Error bars indicate SEM. Asterisks indicate the significance of the difference between the indicated groups by Student's t test for effect of diet within the same genotype. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4

Organ-specific and systemic activation of the AASR by tryptophan deficiency. (A) Gene expression as determined by qPCR on material prepared from liver (top) and kidney (bottom) from wild-type (WT) male B6D2F1 mice preconditioned for 6 days with ad libitum access to tryptophan-deficient (Trp–) chow or pair-fed on complete chow (n = 4 to 5 per group). Gene expression is presented relative to complete diet treatment group (dashed line). (B) Gene expression in liver (top) and kidney (bottom) from WT or Gcn2^−/−C57BL/6 male mice (n = 4 to 5 per group) on the indicated diet for 6 days. Gene expression is presented relative to the WT complete diet treatment group (dashed line). (C) Numbers of peripheral neutrophils in whole blood prepared from mice as in (B). (D) Expression of growth hormone receptor (Ghr) and insulin-like growth factor 1 (Igf1) mRNAs in liver as determined by qPCR (n = 4 to 5 per group) normalized to the WT complete diet treatment group. (E) Serum Igf1 protein levels after 6 days on the indicated diet (n = 16 per group). Error bars indicate SEM. Asterisks indicate the significance of the difference between the indicated groups by Student's t test for effect of diet within the same genotype. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5

Protection against IR by pharmacological activation of the AASR. Preconditioning with HF for 3 to 6 days protected against renal IR. (A) Serum creatinine before (0 hours) or 8 to 24 hours after 25 min of bilateral renal IR (n = 5 per group). (B) Gene expression changes in liver (top) and kidney (bottom) upon 3 days of HF treatment as measured by qPCR and presented relative to the vehicle-treated group (n = 4 to 5 per group). (C)Complete blood cell counts upon 3 days of HF treatment expressed as a percentage of the vehicle-treated (n = 5 per group). Error bars indicate SEM. Asterisks indicate the significance of the difference between the indicated group and the vehicle-treated group by one-way ANOVA followed by Dunnett's multiple comparison test (A) or by Student's t test for effect of HF treatment (B and C). **P < 0.01; ***P < 0.001.

Fig. 6

Block of HF activity by excess proline in vitro and requirementfor Gcn2 in vivo. (A) Cytostatic/cytotoxic effects of increasing concentrations of HF added to proliferating MEF cultures and blockade by addition of excess proline to the medium as measured by the MTT assay and expressed as a percentage of cell number in the vehicle-treated group with no additional proline. (B) Phospho-eIF2α and Atf4 immunoblots of extracts prepared from MEFs treated with HF (0, 5, or 10 nM) or tunicamycin (Tun) (0, 1, or 5 μg/ml) for 3 hours in complete medium with or without proline (Pro) supplementation. eIF4E was used as a loading control. (C) Protection by HF against renal IR required Gcn2. Serum urea (top) and creatinine (bottom) before (day 0) and 1 day after 25 min of bilateral renal IR. Error bars indicate SEM. Asterisks indicate the significance of the difference between the indicated groups by Student's t test for effect of diet within the same genotype. *P < 0.05; **P < 0.01. (D) Representative images of PAS-stained kidney sections 1 day after reperfusion showing the corticomedullary junction, where most tubular damage occurs.