Franny Jongbloed

246 CHAPTER 9 including three days of fasting in the aged-overweight mice in chapter 2 , the protein-free diet used against renal IRI in chapter 4 , and all EAA-free diets given prior to hepatic IRI in chapter 5 . Previous gene expression datasets of three days of fasting in young-lean male mice and two weeks of 30% DR demonstrated the activation of NRF2 as well 1 . Despite that the importance of NRF2 in the protection against DR has been described in multiple studies as well as our studies, a Nrf2 knock-out mice model submitted to DR showed similar increase in lifespan as control mice. Therefore, the exclusive role for NRF2 could not be determined and other factors are of essence in the protection as well 26 . Another one of those factors could be the nutrient-sensing pathway of mammalian target of Rapamycine (mTOR). MTOR has extensively been linked to an increase of lifespan and stress resistance and several studies that investigated the effects of a mTOR-inhibitor, rapamycin, show promising results 25,27-29 . In our renal IRI model, the protective diets all activated the transcription factor hepatocyte nuclear receptor factor alpha (HNF4A). HNF4A results in downregulation of metabolic sensors, in particular the mTOR pathway 30,31 . Using Western blot analysis, we examined S6 phosphorylation as a down-stream target of mTOR through immunoblotting. We showed a significant decrease in S6 phosphorylation after three days of fasting, indicating downregulation of mTOR 25 . The EAA-free diets downregulated mTOR as well, although not significantly in all diets. Since the mTOR pathway came up downregulated in both kidney and liver, this demonstrates its importance in the overall induction of stress resistance by DR. Moreover, renal and hepatic gene expression profiles showed multiple common features even though different DR regimens were used. The nuclear receptor signaling was proposed to be upregulated via the activation of retinoid acids (RAs), for instance the PPAR-pathway as seen in the aged-overweight fasting mice studied in chapter 2 , and FOXO3, which is the transcription factor that was activated the most after DR in the kidney in chapter 4 . Both RAs and FOXO3 are linked to increased stress resistance and protection against various forms of ischemia 32-34 . The stress resistance pathway mediated by all diets was NRF2, as mentioned before, but also the eukaryotic translation initiation factor 2a (EIF2a). Via activation of general control nonderepressible 2 ( Gcn2 ), the EIF2a signaling response activates and mediates genes involved in antioxidant defense, reduction of inflammation and cell survival 35 . Previously, Gcn2 was thought to be required for inducing stress resistance in mice, although a more recent study showed that Gcn2 -deprivation was not sufficient to indeed provide protection against hepatic IRI and other factors might be required 12,36 . One of these additional factors might be the inhibition of cellular proliferation via inhibition of transcription factors FOXM1 and SMARCB1 24,37 as seen after the EAA-free diets in chapter 5 , thereby inducing the adaptive response that shifts resources from growth to maintenance as seen by DR 38 . We concluded that it is possible to provide robust protection against IRI with a diet as short as three days lacking protein or only one essential amino acid. We propose a mechanism-of-action model, based