Yellow feather broiler chickens are widely consumed in China, and chicken meat production has increased in recent years. IMF and AF contents strongly affect the quality of chicken meat. Although the mechanisms underlying fat deposition in broiler chickens are well known, differences in the rate of fat deposition in BM and AF tissues have not been determined. A large number of DEGs can be identified by RNA-seq analysis; however, the characterization of gene expression patterns and correlation with phenotypes are challenging, underscoring the need to perform weighted co-expression network analyses. In the study, DEGs were identified in BM and AF by RNA-seq analysis, and joint analysis with WGCNA to identify specifically expressed genes and signaling pathways for IMF and AF deposition. The molecular regulatory mechanisms of fat deposition in different tissues at the same age were elucidated in chicken.
Based on the differentially expressed genes, the results of KEGG signaling pathway analysis showed that DEGs from BM and AF tissues were jointly enriched in the AGE-RAGE signaling pathway, amino acid biosynthesis, cytokine-cytokine receptor interactions, local adhesion, GnRH signaling pathway, MAPK signaling pathway, metabolic pathway, in diabetic complications. tight junctions and other signaling pathways. Studies have shown that MAPK signaling pathway is involved in lipid deposition [13, 14], which can regulate PPAR pathway. Moreover, PPAR signaling pathway also has the function of regulating lipid metabolism [16, 17].
In contrast, DEGs in BM tissue were specifically enriched in pathways related to gluconeogenesis (glycolysis/gluconeogenesis, starch, sucrose metabolism, fructose, mannose metabolism, pentose, and glucuronide). LDHA and LDHB in the glycolysis/gluconeogenesis signaling pathway are involved in pyruvate metabolism and tricarboxylic acid cycle, which can facilitate the glycolytic process by converting pyruvate to lactate . In addition, GBE1 is essential in starch and glycogen formation metabolism, and there is a strong link between the hexokinase family genes HK1, HK2, HK3 in this pathway and the metabolic process of glucose. These data suggest that IMF deposition in BM tissue is mainly dependent on glucose metabolism, with some energy metabolism involved in the process.
DEGs in BM tissue were specifically enriched in pathways related to fatty acid syntheses (PPAR signaling, glycerolipid metabolism, glycerophospholipid metabolism, and unsaturated fatty acid biosynthesis). Sixteen genes were enriched in the PPAR signaling pathway, including HMGCS2 (catalyze ketogenesis)[19, 20], ACOX1 (lipid degradation), ADIPOQ (adipocyte differentiation), APOA1 and ME3 (cholesterol metabolism), SCD (fatty acid transporter proteins), PLIN1 and PLIN2 (lipid droplet protection)[25–27], ACSL1, FABP7, FABP1, FADS2. The formation of intracellular lipid droplets is a highly conserved process, including fatty acid transport and activation, neutral lipid synthesis, and lipid droplet formation, which is regulated by many factors and pathways. These results suggest that AF deposition is dependent on fatty acid synthesis and transport and lipid droplet formation.
WGCNA was performed using RNA-seq data to identify expressed genes in different modules and predict the role of genes in lipid deposition. Several modules were significantly associated with IMF/AFW/AFP, lipid composition, and fatty acids metabolism in BM and AF, and the expressed genes significantly associated with the phenotypes in the modules were aggregated together and analyzed jointly with DEGs, which could further identify the functional genes that play important roles in the IMF and AF deposition.
We identified 114 expressed genes associated with Metabolic pathways, Starch and sucrose metabolism, Intestinal immune network for IgA production, Adrenergic signaling in cardiomyocytes, and Glycolysis/Gluconeogenesis in BM. These pathways are regulated by genes such as ALDH5A1, LDHA, GAPDH, GBE1, and GPX1 and may play roles in IMF deposition. A total of 1229 expressed genes associated with 41 signaling pathways were identified in AF, of which 20 pathways that may play roles in AF deposition via PPAR signaling pathway, amino acid biosynthesis, oxidative phosphorylation, may be controlled by several genes, including ADIPOQ, ELOVL6, HMGCS2, ME3, DGKE, AOX1, UBB, SCD, APOC3, and FABP1. The results revealed that IMF deposition in BM tissue was regulated by gluconeogenesis-related pathways (glycolysis/gluconeogenesis, starch and, sucrose metabolism signaling pathways), and by several genes, including GAPDH, LDHA, GPX1, ALDH5A1, and GBE1, among which, the key differential gene ALDH5A1 can catalyze a step in the degradation of the inhibitory neurotransmitter γ-aminobutyric acid . T The protein encoded by LDHA is involved in pyruvate metabolism by catalyzing the conversion of lactate to pyruvate in anaerobic glycolysis. GAPDH plays an important role in glycerol metabolism. These data further suggest that IMF deposition in BM is dependent on gluconeogenesis and energy metabolism.
While expressed genes in AF tissue are enriched to PPAR signaling pathway, oxidative phosphorylation, amino acid biosynthesis, and other signaling pathways, acting through genes such as FABP1, ELOVL6, SCD, and ADIPOQ. PPAR signaling pathway is involved in lipid droplets formation and mitochondrial metabolism, and fatty acid oxidation and lipid synthesis are necessary for cellular signaling . FABP1 plays a crucial role in the PPAR pathway, PPARG expression, as well as fatty acid uptake, transport, and metabolism in vivo. In addition, PPARϒ can be regulated by modulating SCD1 expression to control fatty acid synthesis in adipocytes . ELOVLs encode long-chain and extra-long-chain fatty acid elongases that play an important role in the fatty acid synthesis and can limit fatty acid elongation, and ELOVL6 is involved in the synthesis of fatty acid enzymes in vivo, promoting fatty acid elongation. ACAT2 encodes acetyl-coenzyme A acetyltransferase 2, which is involved in acetyl-coenzyme A metabolism. These results suggest that AF deposition may result from changes in fatty acid synthesis through mitochondrial activity.
The above results suggest that different signaling pathways regulate fat deposition in BM and AF tissues. IMF deposition in BM was associated with pyruvate and citrate metabolism through GAPDH, LDHA, GPX1, GBE1, and other genes, whereas AF deposition was related to acetyl-coenzyme A and glycerophospholipid metabolism through FABP1, ELOVL6, SCD, and ADIPOQ. The transcriptional regulation of genes in network modules associated with traits and metabolic pathway analysis can provide new insights into the genetic mechanisms governing fat deposition in broiler chickens.