Loss of hepatic circadian organization in fatty liver syndrome identifies CLOCK as a regulator of lipid homeostasis in laying hens.

Abstract

Fatty liver syndrome (FLS) is a major metabolic disorder in laying hens, but how hepatic lipid dysregulation is linked to circadian disruption remains unclear. Here, we investigated the role of circadian regulation in FLS pathogenesis and focused on the core clock gene Clock Circadian Regulator (CLOCK). In a high-fat diet-induced FLS model in Lohmann pink layers, affected birds showed hepatomegaly, hepatic lipid accumulation, elevated serum total cholesterol, triglycerides, alanine aminotransferase, and aspartate aminotransferase, increased hepatic pro-inflammatory gene expression, and reduced interleukin-10 (IL-10) expression. FLS was also accompanied by reduced laying performance, delayed and dispersed oviposition, and altered temporal profiles of reproductive hormones. Time-series liver transcriptomics across six circadian time points showed extensive transcriptional remodeling in FLS, with dysregulated genes enriched mainly in lipid metabolic and circadian pathways. Pathway analyses indicated increased fatty acid biosynthesis together with reduced fatty acid degradation, glycerophospholipid metabolism, and ether lipid metabolism. Rhythmicity analysis further revealed a marked collapse of hepatic circadian organization: the number of rhythmically expressed genes decreased from 1,046 in controls to 81 in FLS, and core clock genes including CLOCK, cryptochrome 2 (CRY2), and basic helix-loop-helix family member e40 (BHLHE40) lost rhythmic expression. Series Test of Cluster analysis, weighted gene co-expression network analysis, and protein-protein interaction analysis consistently placed CLOCK at the center of the circadian regulatory network associated with hepatic metabolic disturbance. Functional assays in primary chicken hepatocytes showed that CLOCK overexpression reduced lipid accumulation, intracellular total cholesterol and triglyceride levels, and expression of lipogenic genes, while increasing peroxisome proliferation-activated receptor alpha (PPARα) expression and altering multiple circadian regulators. In an oleic acid-induced steatosis model, CLOCK overexpression similarly reduced lipid deposition and inflammatory gene expression, while increasing IL-10, nicotinamide phosphoribosyl transferase (NAMPT), and Sirtuin 1 (SIRT1) expression. This study provides mechanistic insight into the circadian basis of FLS and highlights CLOCK as a potential molecular target for the prevention and intervention of metabolic liver disorders in poultry.