Franny Jongbloed

84 CHAPTER 4 As the three days of fasting on the one hand and two weeks 30% DR and 3-day 30% DR on the other hand were each performed as individual cohorts using their own control group, we had three different control groups for all interventions combined. No significant differences in gene expression profiles were observed between the different control diets (data not shown). The highest number of significantly differentially expressed probe sets (DEPS) was found after three days of fasting, namely 2,604, of which 1,268 were up- and 1,336 downregulated. Two weeks of 30% DR gave rise to a five times lower number: 492 DEPS, of which 265 were upregulated and 227 were downregulated. Three days of protein- free diet induced 391 DEPS, with 230 probe sets up- and 161 downregulated. Of the non- protective diets, the 3-day fat-free diet did not induce any DEPS when compared to control diet fed mice, while three days of a CHO-free diet induced 1,717 DEPS containing 613 up- and 1,104 downregulated sets. Three days of 30% DR resulted in 454 DEPS, of which 284 up- and 170 downregulated. Several analytic approaches were performed to compare the transcriptomic response between all diets. First, a comparison was made based on the number of overlapping DEPS and the corresponding significance of the overlap (Table 1). Based on the maximum relative overlap, the 3-day protein-free diet and 3-day fasting demonstrated the most resemblance to each other with 222 DEPS in common, corresponding to 53.3% relative overlap. Two- weeks 30% DR showed 45.9% relative overlap with three days of fasting, with 247 DEPS in common. To compare the significance of these overlapping fractions, the enrichment factors (EF) with corresponding P- values were calculated. Comparison of the two identically modified diets differing only in intervention time, namely 3-days 30% DR and two weeks 30%DR, resulted in the highest EF with a 39 times higher number of DEPS in common than would have been expected by chance (Table 1). Comparison of the 3-day protein-free diet with 3-day 30% DR resulted in an EF of 29. All enrichments were significant, with values lower than 8.66E-48, suggesting a significant common transcriptomic response between dietary interventions with the exception of a 3-day fat-free diet. As directionality of the DEPS was not accounted for in this comparison, we explored dendographic heat maps. Since the 3-day fasting dataset was hybridized on a different date, the date of hybridization caused a stronger effect than the biological signal (Figure S2). It was therefore not possible to integrate all complete datasets in one informative heat map analysis. As a solution, we assumed 3-day fasting to represent the widest transcriptomic protective response, and all data sets were limited to its highest number of 2,604 DEPS. The resulting heat map (Figure 2A) shows that the majority of the DEPS have similar directionalities, with the lowest expression levels in the fat-free diet. The horizontal dendogram, based on the (dis) similarity between expression data for probe sets, shows that three days of fasting portrays the largest differences with the other groups, however, this was an assumption, since the heat map is based on the probe sets differentially regulated after three days of fasting.