Adipose tissue is no longer viewed as a passive repository for triacylglycerol storage and a source of free fatty acids but as an active endocrine and paracrine organ secreting an ever increasing number of cytokines that participate in diverse metabolic processes including food intake, regulation of energy balance, insulin action, glucose and lipid metabolism, angiogenesis and vascular remodeling, regulation of blood pressure, and coagulation [20-21]. Excess adipose tissue leads to obesity and metabolic syndrome, such as insulin resistance, type 2 diabetes, heart disease, atherosclerosis and hypertension . Exploring the molecular mechanism of adipose development and fat deposition is helpful for the therapy of obesity and related metabolic diseases. Our fat and lean broilers have similar body weight but acquire a divergent abdominal fat content. So, it is the ideal animal model to study the molecular basis of fat deposition. In the current study, we combined RNA-Seq and iTRAQ techniques on abdominal adipose tissues from 7-week-old FL and LL broilers, and identified a number of key DEGs and DEPs that may affect fat deposition (Table 1). These genes are mainly involved in lipid metabolism associated processes, such as long-chain fatty acids uptake, in situ lipogenesis (fatty acid and cholesterol synthesis), and lipid droplets accumulation.
Long-chain fatty acid uptake
In poultry, fatty acids are taken up by the adipose tissue, which mainly come from triglycerides in plasma lipoproteins (such as VLDL) synthesized and packaged by the liver, and also from triglycerides in portomicrons (PM) assembled by long-chain fatty acids in dietary fat . The triglycerides contained in VLDL and PM are hydrolyzed by lipoprotein lipase (LPL) located in adipose tissue-lined endothelial cells to produce free fatty acids, which can be taken up by adipocytes and then re-esterified and stored in lipid droplet as triglycerides . Previous studies suggested that increased uptake of fatty acids in abdominal adipose tissue is a major cause of obesity in chickens . In general, most cells show less ability in long-chain fatty acid uptake, whereas adipocytes and cardiomyocytes can efficiently and specifically absorb long-chain fatty acids . In the present study, DEG [caveolin 1 (CAV1), ACSL1, and solute carrier family 27 member 6 (SLC27A6)] and DEPs [LPL, CAV1, and ACSL1] were implicated in long-chain fatty acid uptake. So, we speculate that long-chain fatty acid uptake may play an important role in chicken adiposity.
LPL is considered to be a rate-limiting enzyme in fat accretion in chicken adipose tissue , responsible for decomposing triglycerides in VLDL or PM to release free fatty acids. CAV1 was identified as the main plasma membrane fatty acid binding protein in adipocytes that can bind long-chain fatty acids with high affinity . Lack of CAV1 results in the loss of caveolae and defects in long-chain fatty acid uptake in adipocytes . In addition, CAV1 can bind to the long chain fatty acids on the inner leaflet of the lipid bilayer, and transport fatty acids to the subcellular membrane compartment through vesicle-mediated transport . ACSL1 is an acyl-CoA synthetase, and functions as long-chain fatty acid transport protein in adipocyte . The first step in using long-chain fatty acids in cells is their esterification reaction with CoA, and this reaction is catalyzed by acyl-CoA synthetase. In humans, there are two related long-chain fatty acid activation-related protein families: fatty acid transporters (FATP) and long-chain acyl-CoA synthetase (ACSLs). ACSL1 was found to co-localize with FATP1 in a small number of 3T3-L1 cells . Furthermore, ACSL1 can promote fatty acid uptake into cells depending on their expression levels [33-34]. SLC27A6, also named FATP6, is a kind of FATP that can enhance the uptake of long-chain and very-long-chain fatty acids into cells . In the present study, the expression levels of LPL, ACSL1, CAV1 and SLC27A6 were significantly higher in the FL adipose tissue, indicating the adipose tissues of the fat broilers have stronger long-chain fatty acid uptake ability to synthesize more triglycerides.
Fatty acids synthesis
The liver is widely considered to be the main site of de novo lipid synthesis in avian species, with more than 70% of de novo fatty acid synthesis occurring in liver tissue , contradictory to the findings in the present study that a large number of lipogenic genes expressed in chicken abdominal fat tissue. Recent studies have also shown that the lipid synthesis ability of avian adipose tissue may be underestimated. Resnyk et al.  performed microarray analysis on 9-week-old chicken abdominal fat tissue and found many genes associated with lipogenesis were highly expressed in fat chicken. Similarly, one RNA-Seq analysis on 7-week-old broilers showed a large number of lipogenic genes were also up-regulated in abdominal adipose tissues from fat chicken . Another RNA sequencing analysis showed that the 7-week-old fast growth chickens (fatter than slow growth chickens) over-express numerous lipogenic genes in adipose tissue, which should enhance in situ lipogenesis and ultimately adiposity . Intriguingly, in the present study, we also found several key genes associated with fatty acids synthesis, including DEGs [ACACA, fatty acid desaturase 2 (FADS2), SCD, 3-hydroxyacyl-CoA dehydratase 1 (HACD1), HACD2, FASN, ACSL1, acyl-CoA synthetase bubblegum family member 1 (ACSBG1), and 3-oxoacyl-ACP synthase, mitochondrial (OXSM)] and DEPs [ACACA, FASN, SCD, ACSL1, acyl-CoA oxidase 1 (ACOX1), and hydroxysteroid 17-beta dehydrogenase 4 (HSD17B4)]. KEGG analysis showed that ACACA, OXSM, FASN, ACSBG1 and ACSL1 were enriched in fatty acid biosynthesis pathway, and FADS2, SCD, HACD1, HACD2, HSD17B4 and ACOX1 were enriched in the biosynthesis of unsaturated fatty acids pathway (Fig.3b and Table 1). It is worth noting that two proteins (ACACA and SCD) can work as critical enzymes to synthesize fatty acids. ACACA is the rate-limiting enzyme in fatty acid biosynthesis, which can catalyze the synthesis of malonyl-CoA from two molecules of acetyl-CoA, and produce fatty acids under the action of fatty acid synthase . SCD is a rate-limiting enzyme that catalyzes the formation of monounsaturated fatty acids from saturated fatty acids .
Thus, we found that the expression levels of genes related to fatty acid synthesis were significantly higher in the fat line, suggesting the adipose tissues in fat birds have stronger ability of triglycerides synthesis in adipocytes.
At the cellular level, the deposition of adipose tissue is the result of the increase of the number of adipocytes (adipogenesis) and the size of single fat cells (triglyceride and cholesterol accumulation in lipid droplets) [42-43].
Adipose tissue is the major site for the storage of cholesterol, containing both free and esterified forms of cholesterol . In the current study, some critical DEGs [epoxide hydrolase 2, cytoplasmic (EPHX2), cytochrome p450 oxidoreductase (POR), and hydroxysteroid 17-beta dehydrogenase 7 (HSD17B7)] and DEPs [acetyl-CoA acetyltransferase 1 (ACAT1), EPHX2, POR, and HSD17B7] were related to cholesterol synthesis. ACAT1 is an acetyl-coenzyme A acetyltransferase, which can catalyze the formation of cholesteryl esters from cholesterol and long-chain fatty acyl-CoAs . EPHX2 is a member of the epoxide hydrolase family, and the N-terminal activity of EPHX2 can increase the cell’s cholesterol level [46-47]. POR is a microsomal membrane-associated protein of two types: type I and type II, of which type II is responsible for cholesterol synthesis . Lanosterol-14α-demethylase and squalene monooxygenase can participate in cholesterol biosynthesis and require POR as the electron donor [49-50]. HSD17B7 belongs to the 17β-hydroxysteroid dehydrogenase (17β-HSD) family that catalyze the conversion of the keto group on the 17th carbon in steroids to their 17β-hydroxy forms, and function as the 3-ketosteroid reductase of cholesterol biosynthesis . In present study, ACAT1, EPHX2, POR and HSD17B7 were all up-regulated in abdominal adipose tissue of fat line in the current study, suggesting the fat broilers could accumulate more cholesterol to expand the size of adipocytes.
Lipid droplet accumulation
Lipid droplets are dynamic organelles involved in intracellular lipid metabolism in almost all eukaryotic cells, and in white adipocytes, the large unique lipid droplet occupies most of the cell space and volume .
In the present study, perilipin1 (PLIN1), PLIN4 and CAV1 associated with lipid droplet accumulation were up-regulated in FL compared with LL. PLINs are proteins that coat lipid droplets in adipocytes, which control the lipolysis of stored neutral lipids by cytosolic lipases. PLIN1 is the most abundant lipid droplet coat protein, and plays a crucial role in restricting adipose lipolysis under basal or fed conditions . PLIN4 mainly exists in white adipose tissue and is associated with tiny nascent lipid droplets. As a lipid droplet coat protein, PLIN4 can quickly package newly synthesized triacylglycerol, and store energy to the greatest extent during excessive nutrition . Another lipid droplet coat protein is CAV1, which is an essential component for the assembly of caveola organelles in highly differentiated cells, such as adipocytes. CAV1 usually plays a key structural role in the accumulation of lipid droplets in adipocytes, since the deletion of CAV1 can reduce lipid accumulation, which leads to progressive atrophy of white adipose tissue . PLIN1, PLIN4 and CAV1 were up-regulated in the adipose tissue of fat line, indicating that fat birds may accumulate more lipid droplets in adipocytes than the lean birds.
For LL chickens, there are numerous down-regulated DEGs [solute carrier family 11 member 1 (SLC11A1), lipopolysaccharide induced TNF factor (LITAF), lysosomal protein transmembrane 5 (LAPTM5), hexosaminidase subunit beta (HEXB), GM2 ganglioside activator (GM2A), deoxyribonuclease 2 beta (DNASE2B), cathepsin V (CTSV), cathepsin S (CTSS), cathepsin H (CTSH), cathepsin C (CTSC), clathrin light chain A (CLTA), ATPase H+ transporting V0 subunit d2 (ATP6V0D2), arylsulfatase B (ARSB), and acid phosphatase 5, tartrate resistant (ACP5)] and DEPs [palmitoyl-protein thioesterase 1 (PPT1), alpha-N-acetylgalactosaminidase (NAGA), glucosamine (N-acetyl)-6-sulfatase (GNS), cathepsin D (CTSD), cathepsin S (CTSS), and galactosidase alpha (GLA)] significantly enriched in “lysosome” by KEGG analysis. Lysosomes are small organelles (100-500 nm diameter) that contained proteasomes, lipases and nucleases . Autophagy is closely related to lysosomes because it targets defective organelles to lysosomes for degradation . Researchers reported that autophagy plays a complex role in adipose deposition. Zhao et al.  revealed that the activation of autophagy can suppress the adipogenesis of human adipose derived stem cells. Singh et al.  first reported the similarities in regulation and function between lipolysis and autophagy in hepatocytes such as autophagy is required for lipid droplet breakdown, and inhibition of autophagy will increase lipid storage. Another study shown inhibition of autophagy by Bisphenol A exposure will result in decreased lipid droplet degradation and increased ROS levels . Other studies also shown that autophagy plays essential roles in lipolysis, which could eliminate fat [61-62], although many researches have shown that autophagy plays a positive role in adipocyte differentiation [63-64].
In the present study, numerous key DEGs and DEPs related to lysosome pathway were up-regulated in lean line, speculating that the autophagy-lysosome pathway was activated in the abdominal adipose tissue of lean birds, which promote lipolysis in adipocytes and reduce lipid droplets accumulation. Herein, through the joint analysis of transcriptome and proteome, we found many key genes that may affect chicken fat deposition (Table 1). The differential expression and molecular function of these genes likely lead to the differential accumulation of abdominal fat content, although some of them have not been reported to be directly related to adiposity, such as amino acid metabolism-related genes (ADP ribosylation factor like GTPase 6 interacting protein 5 and glutathione S-transferase theta 1-like 1) and oxidation-reduction-related genes (steroid 5 alpha-reductase 3, glycerol-3-phosphate dehydrogenase 2, mitochondrial, retinol saturase and vesicle amine transport 1). Functions of these genes in adipose tissue development and fat deposition awaits further investigation.