Ji et al. BMC Genomics 2012, 13:441 http://www.biomedcentral.com/1471-2164/13/441 Page 9 of 16 Figure 3 KEGG pathway analysis of genes differentially expressed in fasting vs. fed. Genes differentially expressed in fasting vs. fed were matched to KEGG pathway membership using ClueGO. The percentage and #genes/term indicates the percentage and number of the genes in the pathway that are contained in the set of genes altered by fasting. p < 0.1 * p < 0.05 **, p < 0.01, based on Benjamini-corrected p-value. neutralized tissue. The two clusters differed with respect to the insulin neutralized group: cluster 3 contained ana- lytes at intermediate levels between fasted and fed, while cluster 2 contained those at levels comparable to or greater than fed. Twelve of the 17 metabolites with sta- tistically suggestive or significant effects of treatment, in- cluding all of the amino acids and amino acid derivatives (Additional file 4), were present in cluster 2 along with a set of genes that included the p85α regulatory subunit of PI3 kinase (PIK3R1), as well as ME, malonyl CoA de- carboxylase and ELOVL6. Cluster 3 contained several metabolites including both NAD + and NADPH and was significantly enriched in GO annotations related to carbohydrate metabolism and in the KEGG pathways TCA cycle, glycolysis/gluconeogenesis, pyruvate metabol- ism and steroid biosynthesis (p < 0.05; Additional file 4). Clusters 7 and 8 consisted of genes and metabolites with higher levels in fasted than in the other two treatment groups. These clusters were significantly enriched in GO categories PPAR signaling and negative regulation of cellu- lar biosynthesis and also contained citrate and pyruvate (p < 0.05; Additional file 4). Discussion Despite roles as both a domestic food animal of worldwide economic importance and a widely used model organism with relevance for human obesity and insulin resistance, few studies have examined regulation of gene expression in chicken adipose tissue. To our knowledge, no studies of nutritional regulation of chicken adipose tissue at the gen- omic level have been reported in the published literature. Likewise, although insulin is the most well-defined hormo- nal mediator of metabolism in mammalian adipose tissue, its role in chicken remains to be clarified. Therefore the current study addressed two objectives: 1) characterize the transcriptomic and metabolomic response to energy ma- nipulation as a step toward enhanced understanding of adipose biology in chicken; and 2) identify the effects of insulin on chicken adipose tissue by including a group of birds in which insulin action was blocked by immunoneu- tralization with an anti-insulin antibody. We sought to both identify potential new targets for genetic selection or management strategies to reduce fat accumulation in commercial broilers and to further develop chicken as a model organism for studies of human obesity. Although intrinsic lipogenic activity is low in chicken adi- pose tissue, genes involved in fatty acid synthesis and stor- age were suppressed and those in fatty acid mobilization and oxidation were up-regulated by fasting. The 40 down- regulated genes with fold changes greater than three were significantly enriched for the GO annotation lipid biosyn- thetic process (FDR <0.05), including genes that control triglyceride synthesis (DGAT2 and AGPAT9) and fatty acid synthesis (ACACA, ACLY and ME), elongation (ELOVL6), and desaturation (FADS1). AGPAT9 and DGAT2 catalyze the initial and final steps, respectively, of de novo triglycer- ide synthesis. ACLY is the main enzyme for synthesis of cytosolic acetyl-CoA, which is carboxylated to malonyl- CoA by ACACA, the rate-limiting step in fatty acid synthe- sis. Reducing equivalents for the conversion of malonyl- CoA to palmitate are supplied by malic enzyme (ME). ELOVL6 catalyzes elongation of palmitate to stearate and appears to play a key role in insulin sensitivity [30,31]. Finally, FADS1 is rate-limiting for polyunsaturated fatty acids (PUFA) biosynthesis and was recently implicated in control of fasting glucose homeostasis in humans [32]. Genes altered by fasting in adipose tissue in this study over- lapped with those shown to be differentially expressed in chicken liver after 16 or 48 hours of fasting, including ACLY, ACOX1, BCAT1 and PDK4 [33]. These authors used a different array platform than ours, which precludes precise quantitative comparisons. However, among the genes changed in both studies, the fold changes observed in adipose tissue were consistently greater than those in liver, despite the longer duration of fasting in that study. For ex- ample, PDK4 expression was up-regulated ~ 18-fold by a five hour fast in adipose tissue, but only ~ 1.5-fold after a 16 hour fast in liver. While differences in sensitivity