Franny Jongbloed

124 CHAPTER 5 represented mainly an activation of the cellular immune response and intracellular and secondmessenger signaling, in combination with an inhibition of nuclear receptor signaling and cellular stress and injury. Analysis of the Trp-free diet revealed 15 pathways, including eight activated and seven inhibited pathways. The activated pathways were mostly involved in cellular immune responses and cell cycle regulation, while inhibited pathways included nuclear receptor signaling and cancer pathways (Table 1C). Comparison of overlapping pathways revealed that five out of the 11 pathways regulated by the Met-free diet were also regulated by the Leu-free diet, and four out of the 11 pathways by the Trp-free diet, all with similar directionalities. These overlapping pathways were amongst the highest regulated pathways including activation of NRF2-mediated stress response and G2/MCheckpoint Regulation pathways, and inhibition of the LPS/IL-1 mediated inhibition of RXR Function as well as the LXR/RXR activation pathway. In addition to the pathways in common between the three EAA-free diets, the Leu-free and Trp-free diets had another four pathways in common of which two had the same directionality, namely Calcium- induced T-lymphocyte apoptosis and Sphingosine-1-phosphate signaling . Analysis of the upstream transcription factors (TFs) activated or inhibited by the EAA- free diets resulted in a list of factors depicted in Table S1. The highest activated TF by the Met-free diet was NFE2L2, i.e. NRF2, followed by ATF4 and SMARCB1. NFE2L2 and SMARCB1 were also significantly activated by the Leu-free and Trp-free diets, while ATF4 met a significant z-score in the Trp-free diet and showed a trend towards significance in the Leu-free diet (1.838). The strongest inhibited TF by the Met-free diet was SREBF2, followed by FOXM1 and MYBL2. Both SREBF2 and FOXM1 were also significantly inhibited by the Leu- and Trp-free diets, while MYBL2 only reached significance in the after the Leu-free (-2.000) and not in the Trp-free diet (-1.633) (Table 2). Of the 18 significantly regulated TFs by the Met-free diet, 14 and 13 TFs were also regulated by the Leu-free and Trp-free diet, respectively. All of these TFs showed the same directionality in all three EAA-free diets.