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 [18]. 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 [19]. Previous studies suggested that increased uptake of fatty acids in abdominal adipose tissue is a major cause of obesity in chickens [20]. 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 [21]. In the present study, DEG (CAV1) 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 [22], 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 [23]. Lack of CAV1 results in the loss of caveolae and defects in long-chain fatty acid uptake in adipocytes [24]. 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 [25]. ACSL1 is an acyl-CoA synthetase, and functions as long-chain fatty acid transport protein in adipocyte [26]. 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 (ACS). 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 [27]. Furthermore, ACSL1 can promote fatty acid uptake into cells depending on their expression levels [28–29]. In the present study, the expression levels of LPL, ACSL1, and CAV1 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 [30], 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. [31] 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 [32]. 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 [33]. Intriguingly, in the present study, we also found several key genes associated with fatty acids synthesis, including DEGs (ACACA, OXSM, FADS2, SCD, PECR, and HACD2) and DEPs (ACACA, FASN, SCD ACSL1, and ACOX1). KEGG analysis showed that ACACA, OXSM, FASN, and ACSL1 were enriched in fatty acid biosynthesis pathway, and FADS2, SCD, PECR, HACD2, and ACOX1 were enriched in the biosynthesis of unsaturated fatty acids pathway. 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 [34]. SCD is a rate-limiting enzyme that catalyzes the formation of monounsaturated fatty acids from saturated fatty acids [35].
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.
Cholesterol synthesis
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) [36–37].
Adipose tissue is the major site for the storage of cholesterol, containing both free and esterified forms of cholesterol [38]. In the current study, some critical DEGs (EPHX2 and POR) and DEPs (ACAT1, EPHX2, and POR) 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 [39]. EPHX2 is a member of the epoxide hydrolase family, and the N-terminal activity of EPHX2 can increase the cell’s cholesterol level [40–41]. POR is a microsomal membrane-associated protein of two types: type I and type II, of which type II is responsible for cholesterol synthesis [42]. Lanosterol-14α-demethylase and squalene monooxygenase can participate in cholesterol biosynthesis and require POR as the electron donor [43–44]. EPHX2, POR and ACAT1 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 [45]. Perilipin1 (PLIN1), PLIN2, PLIN4 and CAV1 associated with lipid droplet accumulation were also discovered in the present study. 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 [46]. However, PLIN2 has minimal control over lipolysis, and may affect lipid droplets accumulation by a different mode. PLIN2 deficient mice can increase triglycerides accumulation in the heart by altered lipophagy [47]. 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 [48]. 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 [49]. PLIN1, PLIN4 and CAV1 were up-regulated and PLIN2 was down-regulated in the adipose tissue of fat line, indicating that fat birds may accumulate more lipid droplets in adipocytes than the lean birds.
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 (ARL6IP5 and GSTTL1) and oxidation-reduction-related genes (SRD5A3, GPD2, RETSAT and VAT1). Functions of these genes in adipose tissue development and fat deposition awaits further investigation.