Ji et al. BMC Genomics 2012, 13:441 http://www.biomedcentral.com/1471-2164/13/441 Table 1 Fold change verification of gene expression by RT-PCR Page 4 of 16 Gene symbol FBXO8 DUSP5 BNIP3 PBX3 IL10RB EGR1 NAB1 PDK4 CTSL2 AGTR1 SESN1 Gene title F-box protein 8 Dual specificity phosphatase 5 BCL2/adenovirus E1B 19 kDa interacting protein 3 Pre-B-cell leukemia homeobox 3 Interleukin 10 receptor, beta Early growth response 1 NGFI-A binding protein 1 (EGR1 binding protein 1) Pyruvate dehydrogenase kinase, isozyme 4 Angiotensin II receptor, type 1 Cathepsin L2 Sestrin 1 FDR p-value: * p < 0.05, ** p < 0.005, *** p < 0.0005. cluster 6 consisted of genes that were also lowest in fasting but showed no clear effect of insulin loss, with similar ex- pression in fed and insulin-neutralized groups. This set of genes was significantly enriched for the KEGG pathways steroid biosynthesis (p = 0.0043), glyoxylate and dicarboxy- late metabolism (P = 0.012) and pyruvate metabolism (P = 0.033), along with a number of genes involved in lipid biosynthesis, which was the highest scoring GO category (p = 0.6). Cluster 8 was a distinct, small cluster with variable expression within group and no significant GO or KEGG annotations. Global biological responses to fasting and to insulin neutralization were further characterized using KEGG pathway matching, based on genes with statistically signifi- cant differential expression (FDR < 0.05) and absolute fold- change ≥1.5. Genes altered exclusively by fasting repre- sented a wide range of cellular pathways, indicating signifi- cant effects of even a five hour fast on adipose function and metabolism in chicken. Fasting exerted significant effects on pathways related to carbohydrate, amino acid and lipid metabolism and synthesis. Within the categories related to lipid metabolism, fasting up-regulated expres- sion of genes involved in fatty acid oxidation (e.g., acyl- CoA oxidase 1 (ACOX1), acetyl-CoA carboxylase beta (ACACB), carnitine palmitoyltransferase 1A (CPT1A)) and down-regulated expression of genes that control fatty acid, cholesterol and triacylglycerol synthesis (e.g., 1-acyl- glycerol-3-phosphate O-acyltransferase 9 (AGPAT9), ATP citrate lyase (ACLY), farnesyl diphosphate synthase (FDPS), acetyl-Coenzyme A carboxylase alpha (ACACA) and acetoacetyl-CoA synthetase (AACS)). Fasting also up- regulated expression of many genes involved in proteolysis and amino acid degradation. In addition to pathways high- lighted by KEGG analysis, fasting down-regulated a number of genes (e.g., TGFβ, BMP) that mediate mesenchymal stem Microarray fold change Fasting Ins-Neu vs. Fed vs. Fed 4.16*** 2.09** 9.43*** 2.98*** −1.62*** 1.74*** 2.43* 2.43*** 17.28*** 2.09*** 4.15 *** 1.86** 1.05 1.53 1.07 1.02 −1.58 1.06 7.06** 1.55* 2.05* 1.125 Ins-Neu vs. Fasting −1.99*** −8.98*** −1.95** 1.75*** −1.71** −3.86** −2.29*** −2.45** −1.35* −2.02** −1.65 Quantitative PCR fold change Fasting Ins-Neu Ins-Neu vs. Fed vs. Fed vs. Fasting 2.59*** 2.76*** 1.06 8.13*** 3.45*** −1.22 1.76*** 2.58*** 1.67** 18.33*** 2.96*** 3.52** 1.58* 1.69*** 2.26*** 1.83*** 1.42* −1.36 1.52** 4.01*** 2.053*** 1.72* 1.47* −4.82*** −1.52* 2.22*** −1.24 −3.52*** −1.10 −4.57*** −1.44 −2.05* 1.07 cell (MSC) commitment, an early step in the formation of new adipocytes (Additional file 1). Finally, a number of phosphodiesterases were up-regulated with fasting, pre- sumably in response to the increased plasma glucagon [18] and subsequent elevations in cyclic adenosine monopho- sphate (cAMP; Additional file 1). Collectively, these cat- egories indicate that chicken adipose tissue responds to a relatively short duration (five hour) fast with sweeping changes in gene expression that suppress synthesis and storage of lipids and other macromolecules and up-regulate mobilization and metabolism of fatty acids and proteins. Loss of insulin action also resulted in significant effects on adipose gene expression, the majority of which over- lapped with the response to fasting (Table 3; Additional file 2). Several genes central to energy metabolism were affected. Diacylglycerol O-acyltransferase homolog 2 (DGAT2), which catalyzes the final and only committed step in triacylglycerol synthesis, was down-regulated (10.5-and 6.1-fold, respectively, fasted and insulin neutra- lized) in both treatment groups relative to the fed group. Conversely, acyl-Coenzyme A binding domain containing 5 (ACBD5) and pyruvate dehydrogenase kinase 4 (PDK4) were significantly up-regulated in both treatments relative to fed controls. ACBD5 is one of a family of long chain fatty acyl CoA trafficking proteins that play roles in both triglyceride synthesis and beta-oxidation [22]. PDK4, which was up-regulated vs. fed by ~ 17-fold with fasting and 6-fold with insulin neutralization, acts as a fuel switch by phosphorylating and inactivating pyruvate dehydrogen- ase, shifting metabolism from glycolysis to fatty acid oxi- dation [23]. Fasting and insulin neutralization also up- regulated expression of the type I angiotensin II receptor (AGTR1). Angiotensin II alters adipocyte lipid metabolism and insulin signaling [24-26], and increased AGTR1 ex- pression in adipose tissue is associated with enhanced