Novel insights into the circadian modulation of lipid metabolism in chicken livers revealed by RNA sequencing and weighted gene co-expression network analysis

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The circadian clock is crucial for maintaining lipid metabolism homeostasis in mammals. Despite the economic importance of fat content in poultry, research on the regulatory effects and molecular mechanisms of the circadian clock on avian hepatic lipid metabolism has been limited. In this study, we observed significant diurnal variations (P<0.05) in triglyceride (TG), free fatty acids (FFA), fatty acid synthase (FAS), and total cholesterol (TC) levels in the chicken embryonic liver under 12-h light/12-h dark incubation conditions, with TG, FFA, and TC concentrations showing significant cosine rhythmic oscillations (P<0.05). However, such rhythmic variations were not observed under complete darkness incubation conditions. Using transcriptome sequencing technology, we identified 157 genes significantly upregulated at night and 313 genes significantly upregulated during the 12-h light/12-h dark cycle. These circadian differential genes are involved in processes and pathways such as lipid catabolic process regulation, meiotic cell cycle, circadian rhythm regulation, positive regulation of the MAPK cascade, and glycerolipid metabolism. Weighted gene co-expression network analysis (WGCNA) revealed 3 modules-green, blue, and red-that significantly correlate with FFA, FAS, and TG, respectively. Genes within these modules were enriched in processes and pathways including the cell cycle, light stimulus response, circadian rhythm regulation, phosphorylation, positive regulation of the MAPK cascade, and lipid biosynthesis. Notably, we identified ten hub genes, including protein kinase C delta (PRKCD), polo like kinase 4 (PLK4), clock circadian regulator (CLOCK), steroid 5 alpha-reductase 3 (SRD5A3), BUB1 mitotic checkpoint serine/threonine kinase (BUB1B), shugoshin 1 (SGO1), NDC80 kinetochore complex component (NDC80), NIMA related kinase 2 (NEK2), minichromosome maintenance complex component 4 (MCM4), polo like kinase 1 (PLK1), potentially link circadian regulation with lipid metabolic homeostasis. These findings demonstrate the regulatory role of the circadian clock in chicken liver lipid metabolism homeostasis and provide a theoretical basis and molecular targets for optimizing the circadian clock to reduce excessive fat deposition in chickens, which is significant for the healthy development of the poultry industry.