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 genomic level have been reported in the published literature. Likewise, although insulin is the most well-defined hormonal 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 manipulation 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 immunoneutralization 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 adipose tissue, genes involved in fatty acid synthesis and storage 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 biosynthetic 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 triglyceride 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 synthesis. 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
. Genes altered by fasting in adipose tissue in this study overlapped with those shown to be differentially expressed in chicken liver after 16 or 48 hours of fasting, including ACLY, ACOX1, BCAT1 and PDK4
. 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 example, 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 between the two array platforms must be kept in mind, these data suggest that adipose tissue metabolism in chicken is at least as sensitive to energy status as hepatic metabolism. Our results indicate that both fatty acid synthesis and storage are dynamically regulated by energy status in chicken adipose tissue, despite its modest (~ 15%) contribution to the amount of stored fatty acids.
Both fasted and insulin-neutralized birds exhibited significant increases in plasma glucagon. Parallel elevations in plasma NEFA suggested that this resulted in significant lipolysis of stored triacylglycerol in both treatment groups. During fasting, a considerable percentage of the liberated fatty acids are re-esterified in adipocytes, and only a small fraction traditionally have been thought to be oxidized in the mitochondria of adipocytes through beta oxidation
. However, recent studies in mice and in human adipose tissue demonstrate that in some conditions fatty acid oxidation in white adipose tissue is considerable and may be an important determinant of obesity
[35–37]. Consistent with this concept, we found significant increases in a number of key enzymes that mediate mobilization of fatty acids and their oxidation, including the rate-limiting enzymes in both mitochondrial and peroxisomal fatty acid oxidation (CPT1A and ACOX1, respectively). We measured tissue levels of beta-hydroxybutyrate, a ketone product of beta oxidation, to confirm that changes in gene expression had functional consequences and found them to be significantly elevated in adipose tissue of fasted vs. fed chickens. Levels were numerically but not statistically higher in insulin-neutralized adipose tissue (data not shown). Qualitatively, fasting-induced changes in gene expression resemble those induced by the fibrate class of drugs, which activate PPARα and promote fatty acid oxidation in white adipose tissue and are used clinically to treat hyperlipidemia
[35, 37–40]. These data suggest that dietary activation of PPARα, for example through supplementation with fatty acids that preferentially bind and activate this member of the PPAR family
, may be a means to attenuate fat deposition in commercial broilers. Such action may underlie the reduced abdominal fat mass reported in broilers that were fed diets rich in n-3 PUFA
Both fasting and insulin neutralization elicited marked upregulation of PDK4. PDK4 is a nutrient sensing fuel switch that phosphorylates and inactivates pyruvate dehydrogenase, which shifts fuel use from glucose to fatty acids and spares glucose for the brain during periods of fasting
[23, 43–45]. PDK4 also enhances glycerol synthesis in white adipose tissue by shunting pyruvate into glyceroneogenesis, at least in the fed state
. Hepatic and skeletal muscle expression of PDK4 is increased by fatty acids, acetyl CoA, NADH and the diabetic state and decreased by insulin and pyruvate (rev. in
[23, 47]). Little is known about PDK4 in chicken, but a recent study suggests it acts as a glycogen sensor in muscle and thus plays comparable roles to those in mammals
. In mouse white adipose tissue, PDK4 expression was shown to be induced by activation of p38MAPK
, which we found to be significantly up-regulated with fasting and, to a lesser extent, with insulin neutralization
. Although PDK4 was up-regulated in both treatment groups, and both groups showed evidence of increased lipolysis
, only fasted chickens presented a gene expression signature and tissue beta-hydroxybutyrate levels that were clearly indicative of fatty acid oxidation. Although we did not measure malonyl-CoA levels, we predict that they were reduced with fasting, but not insulin neutralization, based on reduced expression of ACACA. Malonyl-CoA allosterically binds and inhibits CPT1A, minimizing fatty acid transport and subsequent oxidation in mitochondria
. With insulin neutralization, increased PDK4 may thus be more aligned with the demand for glycerol needed to re-esterify fatty acids liberated by lipolysis
. Additional experiments are needed to confirm that manipulation of PDK4 alters fatty acid oxidation in chicken adipose tissue and to delineate its relative contributions to fatty acid oxidation and glyceroneogenesis under varying metabolic states. If manipulation of PDK4 does alter fatty acid oxidation, our results highlight this pathway as a potential target for reducing fatness, which has relevance for both poultry and humans.
Microarray data indicate that the effects of fasting in chicken adipose tissue extend beyond metabolism. GO analysis highlighted pathways such as cell cycle and cytokine-cytokine receptor interaction that are most likely related to changes in the stromal vascular fraction, which contains proliferating preadipocytes and cells of the immune system. In particular, a number of genes that regulate multiple steps in adipogenesis were significantly altered by fasting. Chickens rapidly accumulate abdominal fat after hatch, and until approximately 7 weeks of age this is due more to formation of new adipocytes than to adipocyte hypertrophy
. Adipocytes arise from mesenchymal stem cells in a two stage process of lineage commitment to an adipocyte fate, followed by differentiation of fibroblast-like preadipocytes into mature fat-storing cells
. Members of both the Wnt (MSC lineage commitment) and TGFβ/BMP (MSC lineage commitment and preadipocyte competence) signaling pathways were significantly regulated by fasting. Fasting down-regulated expression of CEBPα and PPARγ, two transcription factors that orchestrate the cascade of gene expression changes that lead to terminal adipocyte differentiation
. Expression of other adipogenic mediators including fibroblast growth factor 2 (FGF2), fibroblast growth factor receptor 1 (FGFR1)
, and nuclear receptor corepressor 1 (NCOR1)
 were also significantly regulated by fasting. Collectively, these changes suggest that adipocyte number in chickens is dynamically tied to energy status, at least in young chicks (such as those used herein) that are rapidly 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
. 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 nutritional state, chickens may thus be particularly valuable 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 mediate insulin signals that are associated with lipid rafts
, 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
. 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 potential of PI3K-independent effects of insulin on glucose utilization in chicken adipose tissue.
Insulin is not considered to be a key regulator of glucose metabolism in chicken adipose tissue, although it does induce glucose disposal in chicken liver and muscle
. 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 lipogenesis 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 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 metabolism 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 insulin deprivation. Interestingly, this cluster was significantly 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 hyperglucagonemia present in both treatment groups
. In vitro experiments using primary adipocytes or adipose explants will be useful to confirm specific effects of insulin 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 preproglucagon (fold change = 2.91), and down-regulation of the glucagon 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
). Adipose tissue expresses both transcripts, with PGA being slightly more abundant, and is the third highest preproglucagon expressing tissue in chicken, behind pancreas and the proventriculus
. 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 (presumably derived largely from pancreas) was elevated comparably in both treatment groups
, while GCG expression in adipose tissue was only up-regulated by insulin neutralization.
Tissue metabolomic analysis highlighted effects of insulin 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
. Low levels in fasted adipose tissue were most likely due to oxidation of the carbon skeletons for cellular energy 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 expression, particularly in relation to amino acid metabolism. Combined clustering of metabolomic and gene expression together identified a set of genes correlated with many amino acid levels, including PIK3R1, ME and MCD.