Ji et al. BMC Genomics 2012, 13:441 http://www.biomedcentral.com/1471-2164/13/441 (presumably derived largely from pancreas) was elevated comparably in both treatment groups [18], while GCG expression in adipose tissue was only up-regulated by in- sulin neutralization. Tissue metabolomic analysis highlighted effects of in- sulin neutralization that were divergent from fasting and not readily apparent from microarray data. Most of the tissue amino acids that were measured were higher with insulin-neutralization but lower with fasting when each group was compared to ad libitum fed controls. This pattern parallels the levels of αNH2NPN levels in blood [18]. Low levels in fasted adipose tissue were most likely due to oxidation of the carbon skeletons for cellular en- ergy through the tricarboxylic acid cycle (TCA) cycle and/or for glyceroneogenesis, in the absence of dietary glucose. Increased amino acid catabolism was reflected in the differential expression profiles of the fasted vs. fed comparison (Figure 3; Additional file 1). In the insulin neutralized group, however, glucose supply from food was maintained and preferentially oxidized for energy. Elevated amino acids in the insulin neutralized group may also reflect reduced utilization due to the lack of insulin’s anabolic effects, particularly on the proliferating cell population within adipose tissue. The metabolomics approach used here measured only metabolite pool sizes at the time that tissues were harvested, rather than the effect of fasting or insulin neutralization on the rates of metabolism through glycolysis and the TCA cycle. The latter would be much more informative with respect to the dynamic impact of treatment, but requires the use of isotopic labeling (e.g., by feeding 13C-labelled glucose) which was not performed in this study. Nonetheless, we were able to demonstrate significant effects on some metabolites that inform the parallel changes in gene ex- pression, particularly in relation to amino acid metabol- ism. Combined clustering of metabolomic and gene expression together identified a set of genes correlated with many amino acid levels, including PIK3R1, ME and MCD. Conclusions In summary, we determined that adipose tissue metabol- ism in the chicken is regulated by energy status and, to a lesser extent by insulin. Although adipose tissue is not a primary site of lipogenesis in chicken, the rate-limiting genes for fatty acid synthesis were suppressed by fasting. Likewise, fasting appeared to increase aspects of insulin sensitivity based on expression profiles, despite the view that chicken adipose tissue is relatively insensitive to insu- lin. Consistent with this paradigm, insulin neutralization significantly altered the expression of only a few genes related to glucose and lipid metabolism. Nonetheless, a considerable number of genes were altered by insulin neutralization, many of which thus far have unclear roles Page 13 of 16 in adipose biology. Expression profiles suggest that even short term fasting alters fat storage in broilers by enhan- cing the oxidation of fatty acids. The initiating events that trigger upregulation of the corresponding genes are un- clear, but there is considerable evidence for activation of PPARa, LXRa, and potentially other transcription factors that are activated by fatty acid ligands. Further studies are warranted to identify these triggers because of their poten- tial impact on fat storage. Our data also suggest that broiler chicks may be an informative model organism in which to investigate dietary effects on adipose develop- ment in light of what appears to be a relationship between energy intake and adipogenesis. The results of this study thus have dual benefit for both the poultry industry and for studies of obesity in humans. Methods Animals Male broiler chicks (ISA 915, Institut de Sélection Animale, Saint Brieuc, France) from which samples were collected for this study were hatched and raised under standard conditions, as originally described by Dupont [18] and in accordance with the guidelines for Care and Use of Agricul- tural Animals in Agricultural Research and Teaching. Briefly, at 16–17 days of age, chicks of similar body weights were either allowed to continue feeding (ad libitum fed controls), fasted for five hours, or fed but injected at 0, 2 and 4 hours with porcine anti-insulin serum (insulin neu- tralized). Both the fed and fasted groups received injections of normal porcine serum as a vehicle control. Abdominal adipose tissue samples were harvested and rapidly snap- frozen in liquid nitrogen, pulverized, then stored at -80°C until analysis. Adipose samples from five birds in each group were used for both microarray and metabolomic analyses. Gene expression Total RNA was isolated from chicken adipose samples using the RNeasy Lipid kit and incorporating an on- column DNase treated with the RNase-free DNase Set according to the manufacturer's protocol (Qiagen.com). RNA quality and concentration were measured using the Experion System (Bio-Rad.com); only RNA passing recom- mended standards of quality was used for further studies. Transcriptome profiling was performed by Genome Quebec (Montreal, Canada) using the Affymetrix Gene- Chip Chicken Genome Array (San Diego, CA). Microarray data from this study are deposited in the Gene Expression Omnibus (GEO) under the accession number GSE35581. For real time RT-PCR validation, cDNA was synthesized using the iScript cDNA Synthesis kit (Bio-Rad.com). Com- mercially designed and validated primer sets (QuantiTect) and the associated SYBR Green master mix (Qiagen.com) were used to assay gene expression on a CFX96 real-time