Franny Jongbloed

123 5 ABSENCE OF PROTEIN AND AMINO ACIDS PROTECT AGAINST HEPATIC IRI In this unbiased PCA plot, PC1 explains 15% and PC2 explains 13% of the total variance of the probe sets. Examination of the PCA showed distinct separation of the mice in the control group and in the Met-free group, with uniformity within these groups along PC2. However, the Met-free cluster was grouped closely on PC1 with the cluster of control mice. In contrast, the Leu-free and Trp-free groups showed a high degree of overlap, differentiating as a whole from the former two groups on PC1 and with an intermediate response on PC2 (Figure 3A). A dendogram, based on the unbiased hierarchical clustering of all mice, showed two distinct clusters of the control group and the Met-free group, which were connected higher up the dendogram (Figure 3B). The Leu-free and Trp-free groups had a similar hierarchy, with no distinctive clustering between the two groups. The transcriptomic response was further analyzed by determining the differentially expressed probe sets (DEPS) after each dietary intervention compared to the control group. The Met-free diet induced the smallest number of DEPS, namely 572 with 348 upregulated and 224 downregulated DEPS. The Leu-free diet resulted in 933 DEPS, of which 476 were upregulated and 457 downregulated. The Trp-free diet induced 1,155 DEPS of which 579 were upregulated and 576 were downregulated. To analyze the overlap between the groups, a Venn diagram was made with these DEPS, and 137 genes overlapped between all three diets (Figure 3C). The DEPS of the Leu-free and Trp-free diets showed the most similarity, with 603 genes present in both diets, comprising 65% of the DEPS induced by the Leu-free diet and 52% by the Trp-free diet. Of all differentially regulated genes in common between the Leu-free and Trp-free diets, 99% had the same directionality (results not shown). The Met-free induced the highest percentage of unique DEPS, namely 334 out of the 573 (58%). However, the directionality pattern of all differentially regulated genes reached an 84% agreement between Met-free and Leu-free diet and an 86% agreement between Met-free and Trp-free. Despite the stronger response induced by the Leu-free and Trp-free diets, there is a high overlap in transcriptional responses between all three EAA-free diets. Essential amino acid-free diets regulate the cellular stress response, nuclear signaling and cell cycle Subsequent unbiased analysis of the gene expression profiles was performed via pathway analysis and upstream transcription factor analysis. Analyses of differentially regulated pathways by the EAA-free diets focused on canonical pathways with a positive or negative z-score, which indicates a significant activation or inhibition of the pathway, respectively. Three days of Met-free diet resulted in 11 regulated pathways, of which eight activated and three inhibited. The most prominent activated pathways were involved in cellular stress and injury, cell cycle regulation and growth signaling. Inhibition occurred mainly of the nuclear receptor signaling pathway (Table 1A). The Leu-free diet induced 29 differentially regulated pathways, of which 21 activated and eight inhibited (Table 1B). These pathways