Together, our data are consistent with the notion that ad libitum feeding of a nutritionally complete diet before surgical ischemia is a risk factor for inflammatory injury in preclinical models of IR injury. On the other hand, reduced intake of a complete diet, or ad libitum feeding of a diet deficient in protein or EAAs for as little as 6 days, provided significant protection. Pharmacological activation of the AASR with HF also provided protection. The protective effects of tryptophan deficiency and HF pretreatment were both dependent on the amino acid deprivation sensor Gcn2, indicating a role for translational control in AASR-induced stress resistance.
DR can be beneficial in humans and experimental organisms as measured by a number of endpoints, including acute stress resistance and longevity. In humans, these benefits include clinically relevant metabolic parameters such as blood pressure, glucose homeostasis, and blood lipid profiles (16
). Clinical applications of DR against acute stress are practically nonexistent, perhaps because of the difficulty of self-imposed food restriction and an assumption that DR benefits take a long time to accrue. Contrary to this notion, we and others have shown rapid onset of benefits of dietary preconditioning, including DR and fasting, in models of renal and hepatic ischemia (15
), and therapy with chemotherapeutic agents (58
). Here, we have shown that the benefits of reduced total food/calorie intake and reduced protein/EAAs are separable. Thus, reduced food intake per se is not required for the benefits of dietary preconditioning, obviating the difficulty of adherence to reduced food intake. Nevertheless, our data also suggest that reduced nutrient intake combined with reduced calorie intake may together be the most efficacious preconditioning regimen.
Our results are also consistent with a body of literature from lower, nonmammalian organisms, indicating that nutrient restriction rather than calorie restriction per se is a key variable in the benefits of DR (19
). Investigations of this issue in mammals have yielded equivocal results (59
), likely a result of the reliance on life span as an experimental endpoint, which constraints the experimental diets to those compatible with long-term survival. Here, we separated the effects of nutrient deprivation from calorie intake using clinically relevant, acute stress endpoints (instead of longevity) and short-term interventions compatible with incomplete diets. We conclude that nutrient restriction can contribute to DR benefits in mammals as in flies, consistent with evolutionary conservation of the nutritional basis of DR. Our data also support the idea that coordinately regulated reductions in food intake and body size are common both to DR (imposed through reduced food availability) and amino acid restriction (likely through initial aversion to food intake).
We also identified a role for Gcn2
in mediating the beneficial effects of amino acid starvation on stress resistance. Our data indicate that the response to dietary amino acid starvation is tissue-specific and does not involve transcriptional up-regulation of ARE-containing genes such as Nqo1
. Instead, we revealed a requirement for Gcn2
in the systemic response to dietary tryptophan deficiency, which included reduced serum Igf1 and reduced numbers of peripheral granulocytes. Reduced insulin/IGF-1 signaling is synonymous with extended longevity and increased stress resistance (62
). In lower organisms, these effects depend on activation of Foxo transcription factors and presumably increased expression of Foxo target genes, such as those involved in oxidative stress resistance. Our data showed no significant increase in expression of candidate antioxidant genes in the liver after tryptophan deficiency. However, in mammals, Igf1 also plays a prominent role in regulation of the immune system as a growth factor for myeloid lineages (64
). Igf1 can also contribute to inflammation by enhancing expression of endothelial cell adhesion molecules (65
), priming granulocytes for activation (66
), and suppressing granulocyte apoptosis (67
). We observed fewer circulating neutrophils after tryptophan deficiency and reduced expression of proinflammatory and cell adhesion markers after ischemic insult. Thus, reduced Igf1 caused by tryptophan deficiency may have a greater effect on inflammatory capacity than on oxidative stress resistance. Although there is precedent for Gcn2
-dependent immunosuppression of the adaptive immune system [for example, in Ido-mediated T cell anergy(68
) and asparaginase-based immunosuppression (69
)], direct effects on granulocytes have not been previously reported. T cells are also implicated in ischemic injury, because severe combined immunodeficient (SCID) or Rag−/−
mice lacking functional B or T lymphocytes are partially protected from damage (70
). HF inhibits T cell–mediated proinflammatory cytokine secretion by suppression of nuclear factor κB(NF-κB) activity (71
) and prevents in vitro differentiation of proinflammatory TH
17 cells (46
). IL-17 has been implicated in renal IR injury, although neutrophils rather than T cells appear to be the major source (72
). Future studies will be required to clarify in which tissues and cells GCN2 activation is required for adaptive stress resistance.
The observation that the lack of Gcn2
on its own, in the absence of any dietary intervention, led to protection against IR is paradoxical because activation of Gcn2 by either tryptophan deficiency or HF also led to protection. This could be a result of different roles of Gcn2 before and after acute stress. Although Gcn2 activation before stress resulted in potentially beneficial systemic changes (reduced Igf1, reduced circulating leukocytes), the lack of Gcn2 may be beneficial after stress for different reasons. In the case of renal damage by the ER stress activator tunicamycin, the lack of the proapoptotic transcription factor Chop, a downstream target of Atf4 activation, is protective (73
). Thus, activation of Gcn2 after stress may actually be detrimental if it increases apoptotic signaling and results in more cell death. Another nonmutually exclusive possibility is that the lack of Gcn2 results in compensatory activation of aspects of Gcn2 signal transduction through a different eIF2α kinase, for example, PERK. Recent studies indicate that Gcn2 may function as a repressor of mTOR (74
). De-repression of mTOR in the absence of Gcn2 could potentially increase protein synthesis, increase ER stress, and activate the unfolded protein response, resulting in the activation of PERK and stabilization of Atf4.
A useful strategy for harnessing the health benefits of DR could be pharmacological activation of DR target pathways by DR mimetics, abrogating the need for dietary changes. Known DR mimetics such as resveratrol and metformin are thought to work by activating pathways that regulate the response to reduced energy, including the NAD-dependent sirtuins and adenosine monophosphate (AMP)–activated protein kinase. Here, we identified a pathway involved in dietary preconditioning, the AASR, through manipulation of diet and then tested compounds already known to activate this response in the absence of dietary intervention. Our data suggest that HF may act as a dietary preconditioning mimetic. In addition to its ability to precondition against renal IR, we demonstrated the dependence of its activity on Gcn2
and the ability of l
-proline, but not d
-proline or any of 16 other amino acids, to competitively inhibit its biological activity. Consistent with our finding, dietary l
-proline can mitigate the skin-weakening effects of HF in chickens (75
). Further studies will be required to clarify the role of proline in the activation of AASR by HF.
Alternate day calorie restriction for 2 months is well tolerated and is highly effective in reducing levels of oxidative stress and inflammation in human subjects (76
). Short-term, preoperative dietary interventions are feasible and safe in patients participating in living organ donation for transplantation (78
). However, dietary recommendations are not a routine component of medical management around the time of surgery (79
), with the exception of preoperative overnight fasting, which serves a different purpose (80
). What is still required is a demonstration that short-term dietary preconditioning can reduce surgical stress in humans. For this test, vascular procedures with high risk of ischemic injury such as carotid endarterectomy may be appropriate. In addition, we need to know what diet works best and for how long to apply it before surgery. Our data indicate that isolated protein or amino acid deficiency can modulate stress resistance independent of DR; however, the benefits of energy and nutrient restriction may be additive or even synergistic. Finally, our studies were performed in young, healthy rodents; it will be necessary to determine whether this approach will work when needed most, such as in elderly or obese individuals. For example, in some experimental models of ischemic preconditioning, cardioprotection is lost as a function of age (81
In conclusion, we report that dietary activation of an evolutionarily conserved response to amino acid starvation protected mice against IR injury. This protection was mediated by the Gcn2 kinase, implicating translational control in dietary preconditioning. We also showed that HF, a drug already in clinical trials as a chemotherapeutic agent, can act as a dietary preconditioning mimetic against surgical stress in preclinical models. Modulation of the AASR by brief dietary or pharmacological interventions may thus be a promising approach for mitigation of surgical stress, including IR injury.