Cellular And Animal Metabolic Responses To Indispensable Amino Acid Limitation
The current thesis was guided by three principal research objectives. The first objective was to identify target genes responsive to indispensable amino acid deprivation with the goal of identifying potential components of the Integrated Stress Response as well as possibly identifying some genes that are differentially expressed in response to amino acid deprivation but not other stress conditions. The second objective was to determine whether the previously identified targets and eIF2[alpha]-kinasemediated responses would be induced in animals fed a diet that was marginal in essential amino acid composition. The third objective was to elucidate the role of elevated 4E-BP1 expression in regulating translation initiation during nutrient deficiency. To assess the role of amino acid deprivation on eIF2[alpha] phosphorylation and downstream transcriptional responses, we conducted additional microarray studies for leucine-depleted HepG2/C3A cells and compared these with results for cysteinedepleted cells. The comparison of cells exposed to deficiencies of two different amino acids was done to facilitate selection of genes whose expression is more likely to be altered specifically in response to GCN2-induced eIF2[alpha] phosphorylation. In addition, we assessed the effects of both cysteine and leucine deprivation on the phosphorylation of eIF2[alpha] and on the protein expression patterns of ATF4 and other ISR-related proteins. We have compiled a list of 120 genes whose expression was differentially expressed in HepG2/C3A cells cultured in either cysteine- or leucinedeficient medium, and this list contains many of the genes known to respond to eIF2[alpha] kinase activation and to be components of the ISR. Clearly, amino acid deficiency, including cysteine deficiency in the absence of oxidative stress, induces eIF2[alpha] phosphorylation, presumably by activation of GCN2, leading to changes in expression of ATF4 and stress-related target genes. To answer whether eIF2[alpha]-kinase-mediated responses would be induced in animals fed a sulfur amino acid (SAA) deficient diet not as imbalanced as one in which a single essential amino acid is totally absent, we fed rats soy protein-based diets that were either adequate or limiting in SAA. Rats fed a SAA-deficient diet grew more slowly than rats fed the control diet. Analysis of liver from rats fed these diets for 7 days showed that the SAA-deficient rats had higher levels of eIF2[alpha] phosphorylation and higher levels of activating transcription factor (ATF) 4, ATF3, asparagine synthetase, solute carrier 7A11, cysteinyl-tRNA synthetase, and cystathionine [gamma]-lyase. On the other hand, components of the integrated stress response (ISR) known to promote apoptosis or translational recovery were not induced. These results indicate that rats fed the SAA-deficient diet had a prolonged activation of an eIF2[alpha] kinase that leads to upregulation of adaptive components of the ISR. To assess the role of elevated 4E-BP1 expression in regulating translation initiation during nutrient deficiency, we fed rats various protein/amino acid-deficient diets with a varying degree of deficiency and achieved amino acid deficiency and reduction of growth. In these experiments we consistently observed a significant induction of total 4E-BP1 protein levels that did not parallel a reduction in 4E-BP1 phosphorylation and was independent of energy restriction or feed intake. Further, the induction of total 4E-BP1 appeared independent of eIF2[alpha] phosphorylation.
Integrated Stress Response; mTOR; gcn2
Stipanuk, Martha Harney
Stover, Patrick J; Miller, Dennis Dean; Boisclair, Yves R
Ph.D. of Nutrition
Doctor of Philosophy
dissertation or thesis