Franny Jongbloed

245 9 SUMMARY AND DISCUSSION the following essential amino acids (EAA): methionine, leucine or tryptophan. These diets were followed by induction of acute stress in the form of liver IRI. Most literature about amino acids and DR studied a methionine restricted diet, and it showed that the results are slightly in favor of methionine being responsible for the beneficial effects on aging 13,16,17 . The extent to which tryptophan and leucine deprived diets have been examined is small, however do point towards a favorable role of essential amino acids deprivation on increased lifespan 5 . We showed that hepatic IRI, as a model for acute stress, was significantly reduced by all three EAA-free diets. These effects were already visible six hours after hepatic IRI in the leucine-free and tryptophan-free diets, while all EAA-free diets showed these effects at 24 hours post-IRI. We concluded that it is possible to provide robust protection against IRI with a diet as short as three days lacking only one essential amino acid and that these essential amino acids are able to produce this protection in a similar manner. In conclusion, by extending current DR regimens we showed that three days of fasting is able to induce protection against renal IRI in different animal models, including aged and overweight mice that more resemble the human population. In addition, by exploring novel DR regimens we could induce the protection against IRI with protein restriction and EAA-restriction as short as three days. Of interest, carbohydrate and fat deprivation were not able to induce protection and even aggravated the detrimental effects of IRI. Therefore, macronutrients are not equally responsible for the beneficial effects of DR and separation of total calorie restriction from protein restriction enabled us to develop a short-term DR regimen that is more specific and could be more suited to use in a clinical setting. Insight into the mechanisms underlying dietary restriction Parallel with the vastly increasing number of studies about DR is the amount of studies focusing on the mechanisms underlying DR 18-20 . Many genes and pathways have been proposed to play an important role in the beneficial effects of DR, however none of those has been validated extensively in different studies 19,21-23 . Despite the fact that the exact mechanisms underlying DR remain to be elucidated, several pathways and genes have been put forward by many authors and are therefore of particular interest. These pathways include the NRF2-mediated stress response and mTOR nutrient signaling 24,25 . With an unbiased approach, we collected and analyzed transcriptome profiles of kidney and liver after various short-term dietary interventions that were either protective or non-protective against renal and hepatic IRI in order to examine the responsible mechanisms for ourselves. Interestingly, aforementioned pathways were on our top list of pathways and transcription factors as well. Transcription factor nuclear factor-erythroid like 2 (NRF2) is known to be the initiator of the phase II response, which is a detoxifying response to for instance oxidative and xenobiotic metabolism 24 . The NRF2-pathway was activated by all our protective diets,