Franny Jongbloed

90 CHAPTER 4 P< 0.01) and 3-days of protein-free diet (-1.7; P< 0.001) diets, while MTORC1 (-1.6; P< 0.01) and mTOR (-0.6; P< 0.001) were downregulated after three days of fasting. Both non- protective diets 3-day CHO-free (+0.8; P< 0.01) and 3-day 30% DR (+1.3; P< 0.001) showed an upregulation of mTOR. mTOR activity was examined at protein level by examining ribosomal protein S6 phosphorylation through immunoblotting in kidney extracts from all intervention and control groups. S6 is a down-stream target of mTOR through S6 kinase 25 . Table 3. List of upstream transcription factors after meta-analysis with corresponding z-scores in the different dietary interventions Transcription Factor Dietary intervention Meta- analysis 3-days fasting 2 weeks 30% DR Protein- free CHO- free 3 days 30% DR SMAD7 – SMAD family member 7 +2.890 +0.718 -0.128 +2.466 N/A N/A * FOXO3 - Forkhead box O3 +2.729 +3.303 +1.042 +1.701 -0.314 +1.893 FOXO1 - Forkhead box O1 +2.692 +1.599 -0.109 +0.269 -1.850 +0.360 * HNF4A – Hepatocyte nuclear factor 4 alpha +2.462 +2.122 +1.179 +2.089 +0.109 +1.327 MYCN – v-myc avian myelocytomatosis viral oncogene +2.414 +1.982 N/A +1.245 -2.464 +1.091 CLOCK – Circadian Locomotor Output Cycles Kaput +2.373 +1.980 +1.119 N/A +1.940 +1.925 * HMGA1 – High mobility group AT-hook 1 +2.051 +1.432 +2.177 +0.356 N/A +0.785 MED1 – Mediator complex subunit 1 +2.008 +2.058 N/A N/A +0.102 N/A SPDEF – SAM pointed domain containing ETS transcription factor +2.000 +2.170 N/A +1.000 N/A N/A LYL1 – Lymphoblastic leukemia associated hematopoiesis regulator 1 +2.000 +2.000 N/A N/A N/A +1.982 SIM1 – Single-minded homolog 1 +2.000 +0.557 N/A N/A N/A N/A ARNT2 – Aryl hydrocarbon receptor nuclear translocator 2 +2.000 +0.557 N/A N/A N/A N/A GLI1 – GLI family zinc finger 1 -2.183 N/A N/A +0.057 N/A N/A * HSF1 – Heat shock factor protein 1 -2.697 -1.659 -2.653 -2.376 +0.462 -0.451 SREBF2 - Sterol regulatory element- binding transcription factor 2 -2.923 -3.420 N/A -0.243 +0.660 N/A SREBF1 - Sterol regulatory element- binding transcription factor 1 -3.889 -2.794 N/A -1.304 +1.229 -0.779 Upstream regulator analysis of the DEPS found in common after 3-days of fasting, two weeks 30% DR and 3-days of protein- free diet (meta-analysis) revealed 16 transcription factors (TFs) significantly regulated, of which 12 were activated and four were inhibited. Highest activated TFs were SMAD7 and FOXO3. *: TFs of interest, that are not or oppositely regulated in the non-protective CHO-free diet.