Ji et al. BMC Genomics 2012, 13:441 http://www.biomedcentral.com/1471-2164/13/441 chickens is dynamically tied to energy status, at least in young chicks (such as those used herein) that are rap- idly forming new adipocytes. An elegant study by Arner et al. concluded that adipocyte number in humans is a major determinant of adult fat mass and is determined during early childhood [54]. Less is known about this process in humans due to the limitations of sampling adipose tissue, particularly during development and from different abdominal depots. In light of what appears to be sensitive regulation of adipogenesis by nu- tritional state, chickens may thus be particularly valu- able models in which to elucidate mechanisms of adipocyte hyperplasia during development that would inform the study of human obesity. It is worth noting that, despite the uncertainty about insulin signaling in chicken adipose tissue, fasting altered the expression of several messengers encoding elements of the insulin signaling cascade. Expression of PIK3CB, which encodes the catalytic p110 subunit of PI3K, was up-regulated with fasting, while PIK3R1, which encodes the regulatory p85 subunit, was down-regulated. Such regulation could maintain some insulin signals despite a fall in plasma insulin level. CBLB and CRK, which medi- ate insulin signals that are associated with lipid rafts [55], were also up-regulated with fasting. In mammals, this pathway stimulates glucose uptake independently of PI3K activation, which may shed light on the apparent refractoriness of PI3K activity to insulin that was described in chicken skeletal muscle [18]. Therefore, the potential effects of insulin on lipid storage and energy utilization appear to be defended in the fasting state, when insulin levels fall, by enhanced insulin sensitivity at the post-receptor level. Additional studies are needed to confirm this effect and to further explore the poten- tial of PI3K-independent effects of insulin on glucose utilization in chicken adipose tissue. Insulin is not considered to be a key regulator of glu- cose metabolism in chicken adipose tissue, although it does induce glucose disposal in chicken liver and muscle [14]. It is therefore not surprising that the majority of genes significantly altered by both insulin neutralization and fasting are not related to glucose metabolism and lipid synthesis. The main exception is DGAT2, which catalyzes the final step in esterification of fatty acids into triglycerides. In fact, DGAT2 showed the most extreme down-regulation (6.1- and 10.5-fold, insulin-neutralized and fasted, respectively) in each treatment group, which is surprising considering that other genes related to lipo- genesis were only regulated by fasting. Suppression of DGAT2 expression may be due to feedback by lipolysis, which appeared to be increased in both treatment groups based on plasma NEFA levels. In general, our data indicate that insulin deprivation altered fatty acid and glucose metabolism in a manner comparable to Page 12 of 16 fasting but to a lesser extent, such that most genes involved in these pathways did not exhibit statistically significant changes in expression. For example, cluster analysis (Figure 2) revealed that some genes upregulated by fasting were also increased by insulin neutralization (clusters 1, 3 and 4); these three clusters were enriched with genes in the KEGG pathways for fatty acid metab- olism and PPAR signaling, including both ACOX1 and CPT1A, among others. Similarly, among genes that were downregulated by fasting, clustering discriminated a set of genes (Figure 2, cluster 2) with a trend to also be decreased (albeit to a lesser extent than in fasting) by in- sulin deprivation. Interestingly, this cluster was signifi- cantly enriched in functions related to carbohydrate metabolism, suggesting that insulin does play some role in chicken adipose glucose metabolism. Comparable trends appeared in the metabolomic data. For example, stearate and palmitate (the only fatty acids measured by our MS platform) were lower (although not significantly) in both fasted and insulin neutralized compared to fed birds (Additional file 3). While the purpose of our study design was to determine the specific effects of insulin on chicken adipose tissue, we cannot exclude the possibility that some of the overlapping changes in gene expression were secondary to systemic factors, such as hypergluca- gonemia present in both treatment groups [18]. In vitro experiments using primary adipocytes or adipose explants will be useful to confirm specific effects of insu- lin on genes identified herein. Of the 13 changes in expression that were unique to insulin neutralization, the most interesting responses were up-regulation of GCG, which encodes preprogluca- gon (fold change = 2.91), and down-regulation of the glu- cagon receptor (LOC425670, fold change = −2.77). The proglucagon system in avians is more complex than in mammals. The avian preproglucagon locus encodes two distinct precursor proteins that yield different peptides through alternative posttranslational processing: the class A transcript (PGA) yields glucagon and glucagon- like peptide-1 (GLP-1), while the class B transcript (PGB) additionally produces glucagon-like peptide-2 (GLP-2) and is more like the mammalian transcript (rev. in [56]). Adipose tissue expresses both transcripts, with PGA being slightly more abundant, and is the third highest preproglucagon expressing tissue in chicken, be- hind pancreas and the proventriculus [57]. We used transcript-specific QPCR to determine that only the PGB transcript was up-regulated by insulin neutralization (data not shown). Additional experiments are necessary to delineate which of the encoded peptides are up- regulated in parallel, but the coincident down-regulation of the glucagon receptor suggests a paracrine glucagon axis in chicken adipose tissue, and one that is regulated by insulin. In support of this concept, plasma glucagon