Franny Jongbloed

131 5 ABSENCE OF PROTEIN AND AMINO ACIDS PROTECT AGAINST HEPATIC IRI Third, all three EAA-free diets induced a strong activation of the NRF2-mediated oxidative stress response , both on transcription factor and pathway level. NRF2 increases stress resistance 30 , and we have already implicated NRF2 in the protection against renal IRI after three days of fasting 1,17,18 . In response to environmental stress, including protein deprivation, NRF2 is activated by phosphorylation of eIF2α via Gcn2 30,34,35 and results in the transcription of genes involved in antioxidant defense, reduction of inflammation and cell survival. In NRF2 knock-out models, similar effects of DR on cellular stress injury as in NRF2 proficient models could not be induced 30,36 . This indicates that NRF2 is indeed a major player yet not the only player in the protection against IRI. Our results demonstrate that activation of nuclear receptors signaling, inhibition of cellular proliferation and activation of the NRF2- pathway might be the essential package required to induce the beneficial effects of hepatic IRI. Validation studies should further emphasize on how these regulators induce protection against oxidative stress induced by IRI. Taken together, we report that three essential amino acid-free diets given for three days protect against hepatic IRI in a mouse model possibly mediated via transcription factors NRF2, FOXM1, SREBF2 and SMARCB1. Further in depth analysis through functional and pharmacological modulation of these factors may provide mimetics for treatment against ROS-related injury. MATERIAL AND METHODS Animals Male C57BL/6 (approximately 25 grams) were obtained from Harlan (Horst, the Netherlands). Animals were kept under standard laboratory conditions, and were housed in individually ventilated cages (n = 3 animals/cage). All mice had ad libitum (AL) access to food and water (acidified with HCl). All experiments were performed with the approval of the appropriate local ethical board. Diets Before dietary intervention, all mice were acclimatized during seven days and fed the standard Special Diet Services (SDS) chow. All other diets used for dietary intervention were purchased from Research Diets, Inc. (New Brunswick, NJ, USA). The SDS chow was used as a control diet for the fasted and 30% dietary restricted mice 1 . As a control for the protein-free and EAA-free diet, a specific control diet was used since this diet has the same protein source as the EAA-free diets, including all EAA (Table S2). The protein source of the EAA-free diets is derived from a crystalline L-amino acid mixture leaving out one of the specific amino acids (Met-free, Leu-free or Trp-free). During the experiment, 3-day 30%