Down-regulated genes Fold Gene Gene title change symbol 2.91 LOC425670 glucagon receptor precursor 2.08 1.98 1.84 1.79 BAK1 CCT3 SETD7 ST13 AHSA1 TOE1 AZIN1 BCL2-antagonist/killer 1 chaperonin containing TCP1, subunit 3 (gamma) SET domain containing (lysine methyltransferase) 7 suppression of tumorigenicity 13 (colon carcinoma) 1.62 (Hsp70 interacting protein) AHA1, activator of heat shock 90 kDa protein ATPase homolog 1 (yeast) target of EGR1, member 1 (nuclear) antizyme inhibitor 1 Page 8 of 16 Fold change 2.77 1.83 1.62 1.62 1.62 1.59 1.56 Up-regulated genes Gene Gene title symbol GCG Glucagon TCP11L2 t-complex 11 (mouse)-like 2 LOC416916 hypothetical LOC416916 MAGI1 SEPT10 LSM14A membrane associated guanylate kinase, WW and PDZ domain containing 1 septin 10 LSM14A, SCD6 homolog A (S. cerevisiae); similar 1.71 to LSM14 homolog A (SCD6, S. cerevisiae) FDR p-value <0.05. treatment groups based on p < 0.05 and an additional five were suggestive of significance (p < 0.10; Table 5). Tissue levels of amino acids were consistently lower in fasted vs. fed tissue, with statistically significant reductions in aspara- gine and glutamine (p < 0.01). Presumably, these effects were due to a change in the balance of protein synthesis/ proteolysis and to the catabolism of carbon skeletons for energy in response to energy restriction, which is con- sistent with up-regulated expression of genes involved in amino acid catabolism (Figure 3). They may also re- flect a decrease in plasma amino acid supply as Table 5 Fold change of fasting and insulin neutralization on adipose tissue metabolites D-glucono-1,5-lactone-6-phosphate Metabolite adenosine alanine arginine asparagine glutamine glycerol-3-phosphate histidine hypoxanthine N-acetyl-glutamate N-acetyl-L-serine ornithine proline serine threonine tyrosine a hexose-phosphate * p < 0.10, ** p < 0.05, *** p < 0.01, **** p < 0.001. Fasting/ Fed 0.57 0.66 0.75 0.47*** 0.86 0.48*** 0.23** 0.82 0.64 1.31 0.64 0.77 0.63 0.67 0.77 0.74 1.05 Ins-Neu/ Fed 1.21 1.57 1.34 1.00 0.75* 1.64* 0.72* 1.38 0.80 1.34 1.70 1.59 1.50 1.61 1.38 1.30 1.32** Ins-Neu/ Fasting 2.12* 2.38*** 1.78** 2.15*** 0.87 3.43**** 0.87 2.17*** 0.61** 2.09* 7.30*** 2.05* 2.39*** 2.39*** 1.80** 1.76** 1.26* Ji et al. BMC Genomics 2012, 13:441 http://www.biomedcentral.com/1471-2164/13/441 Table 4 Unique effects of insulin neutralization on differential gene expression suggested by the decrease in total plasma amino acid levels (evaluated by the content in total α-NH2-non- protein nitrogen (αNH2NPN), i.e., mostly total amino acids), as compared to fed controls [18]. In contrast to fasting, tissue amino acid levels tended to be increased in insulin-neutralized vs. fed, although only glutamine showed a statistically significant response. Comparison of insulin-neutralized vs. fasted chickens highlights the divergent effects of treatments on amino acids (Figure 4). Alanine, arginine, asparagine, glutamine, histidine, proline, serine, threonine and tyrosine levels were all significantly higher in insulin-neutralized vs. fasted, with differences ranging from 1.7- to 3.4-fold. Two metabolites related to glucose metabolism, D-glucono-1,5-lactone-6-phosphate and glycerol-3-phosphate, were lower in both fasted and insulin-neutralized treatments vs. fed, with the latter com- parison nearing statistical significance (p < 0.1). D-glu- cono-1,5-lactone-6-phosphate is a product of glucose-6- phosphate dehydrogenase (G6PDH), an enzyme that, in mammals is insulin-sensitive and rate-limiting for pentose phosphate pathway activity and production of cellular NADPH, an important cofactor for lipid metabolism [28]. However, pentose phosphate pathway activity is intrinsic- ally low in chicken and is not stimulated when lipogenesis is high; the production of cellular NADPH is more closely related to malic enzyme activity [29]. Glycerol-3- phosphate is a product of both glucose and pyruvate me- tabolism and is used in triacylglycerol synthesis. Lower levels with both treatments may reflect glycerol demand for fatty acid reesterification in light of the apparent in- crease in lipolysis in both treatment groups [18]. Correlated patterns of gene expression and metabolite abundance were extracted using hierarchical clustering to interconnect treatment effects on transcripts and metabolites. Clusters 2 and 3 contained genes and meta- bolites with lower abundance in fasted vs. fed or insulin