Franny Jongbloed

129 5 ABSENCE OF PROTEIN AND AMINO ACIDS PROTECT AGAINST HEPATIC IRI Table 2. List of significantly up-or downregulated upstream transcription factors in the liver with corresponding z-scores regulated by all three amino-acid-free diets Methionine-free Tryptophan-free Leucine-free Upstream Regulator State Z-score P- value Z-score P- value Z-score P- value NFE2L2 Activated 4.545 6.28 E -13 2.975 3.12 E -11 2.919 3.92 E -07 SMARCB1 Activated 2.026 9.12 E -06 2.436 9.74 E -04 2.465 2.14 E -06 SREBF2 Inhibited -2.804 1.26 E -04 -5.540 8.96 E -26 -5.159 1.87 E -28 FOXM1 Inhibited -2.671 1.66 E -04 -3.096 1.76 E -05 -2.391 6.91 E -06 Upstream regulator analysis of the differentially regulated genes found in common after 3-day methionine-free, tryptophan- free and leucine-free diet revealed four significantly regulated transcription factors, of which two were activated and two were inhibited. DISCUSSION In this study we demonstrated that, in addition to a protein-free diet, a Met-free, a Leu- free and a Trp-free diet for three days induce a similar protection against hepatic IRI. We show that cellular stress-, nuclear receptor-, and cell cycle regulation pathways are regulated by all three EAA-free diets, and that transcription factors NRF2, FOXM1, SREBF2 and SMARCB1 may be implicated in orchestrating this response. Previously, we have shown that two weeks of 30% DR as well as three days of fasting are sufficient to induce resistance against hepatic and renal IRI 1,10,18 . Recently, we and others showed that absence of protein is the main contributor to these effects 17,19 . Here, we further dissected the role of essential amino acids, and demonstrated that the absence of one single essential amino acid is sufficient to induce the increased stress resistance associated with DR leading to protection against hepatic IRI. Mice fed EAA-free diets voluntarily restricted food intake by approximately 30%. Since we showed that three days of 30% DR without deprivation of EAA is insufficient to induce protection against hepatic IRI, the restricted food intake due to EAA-free diets is ruled out as a factor and the absence of EAA during three days is therefore responsible for inducing these beneficial effects. All three EAA-free diets induced a similar protective effect, indicative of a common denominator of induction of the beneficial response. To extract this denominator, we determined mRNA expression profiles of liver tissue following administration of the EAA- free diets and performed microarray analyses. These data show that EAA deprivation modifies the transcriptome at three different pathway levels.