Ji et al. BMC Genomics 2012, 13:441 http://www.biomedcentral.com/1471-2164/13/441 Page 3 of 16 Figure 1 Venn diagram of overlapping and unique effects of fasting and insulin neutralization on gene expression. (A) A total of 2016 unique genes were differentially expressed (FDR adjusted p-value <0.05) between one or more pairwise treatment comparisons; (B) A total of 1401 genes with absolute fold change ≥1.5 among the differentially expressed genes. were attributable to fasting, with 917 up-regulated and 863 down-regulated genes in fasted vs. fed adipose tis- sue. Insulin neutralization altered expression of 92 genes, 72 of which were also differentially expressed with fasting (Figure 1A). All genes that were affected by both treatments changed in the same direction (i.e., up- or down-regulated in both groups). Real-time RT-PCR was employed to validate differential expression based on the microarray data. Eleven genes were selected based on fold-change or biological functions of interest (Table 1). Differential expression under fasting versus fed conditions was validated for all genes except pre-B-cell leukemia homeobox 3 (PBX3). Ten of the eleven genes were also differentially expressed in insulin neutralized compared to fed birds based on QPCR. Genes that were differentially expressed in at least one pairwise comparison were clustered to visualize the si- milarities between groups and to determine if insulin- neutralized expression profiles were more similar to fasted or to fed status. As shown in Figure 2A, samples within each of the three experimental groups clustered together. The dendrogram also showed that the fasting group was distant from fed and insulin-neutralized groups, which were closer to each other. To further visualize relationships between treatments with regard to gene expression, distinct clusters of genes were extracted and submitted to gene set enrichment analysis to identify GO terms and pathways that were significantly overrepresented among genes contained in these clusters. Seven clusters repre- sented four general patterns of similarities between treat- ments (Figure 2B). Clusters 1, 3 and 4 consisted of genes with higher expression in fasting compared to both insulin- neutralized and fed conditions, with insulin-neutralized intermediate between fasted and fed. This set of genes was significantly enriched in GO terms related to protein and lipid catabolism and to cell signaling, including regulation of the stress-sensitive NFκB cascade (Table 2). These three clusters were also enriched in members of the KEGG path- ways ubiquitin mediated proteolysis, sphingolipid meta- bolism, PPAR signaling, fatty acid metabolism and the peroxisome. The rate-limiting genes for fatty acid oxidation (ACOX1 and CPT1A), along with fatty acid binding pro- teins 5 and 6, are contained in these three clusters. Clusters 5 and 7 also contained genes with higher levels in fasted vs. the other two groups, but with comparable expression levels between insulin-neutralized and fed, and thus no clear effect of insulin loss. These two clusters were signifi- cantly enriched in pathways related to signaling and metab- olism, including enzyme linked receptor protein signaling pathway (p = 0.0097) and in the KEGG pathways for glycer- olipid metabolism and PPAR. Genes responsible for the lat- ter enrichment include PPARΔ, which was recently shown to increase total oxidative metabolism in white adipose tis- sue [21]. Clusters 2 and 6 contained genes expressed at lowest levels in fasted chickens. Genes in cluster 2 were expressed at intermediate levels in the insulin-neutralized group relative to fed and fasted. This set of genes was sig- nificantly enriched in GO annotations related to monosac- charide catabolic process and glucose metabolism, and in genes comprising the KEGG pathways for carbohydrate metabolism, TCA cycle and glycolysis (Table 2). Finally,