Diurnal regulation of Acyl-CoA synthetase 3 (ACSF3) governs rhythmic mitochondrial lysine-malonylation and daily hepatic metabolism

Hepatic daily rhythms are coordinated by feeding and the molecular circadian clock, ensuring metabolic homeostasis. Disrupted feeding schedules promote circadian misalignment and metabolic diseases but the underlying mechanisms remain scarce. Post-translational modifications have emerged as key regulators of circadian metabolic outputs. Here, we show that the mitochondrial enzyme Acyl-CoA synthetase family member 3 (ACSF3) oscillates in phase with different feeding schedules to drive rhythmic lysine-malonylation and coordinate daily hepatic metabolism. Hepatic Acsf3 knockdown drastically affected lysine-malonylation rhythms, decreased fasting glycemia, insulin sensitivity and AKT phosphorylation indicative of perturbed glucose homeostasis. Concomitantly, Acsf3 knockdown shifted lipid oxidation from mitochondria to peroxisomes, enhanced lipogenesis and triglyceride synthesis, while increasing diurnal autophagy. Multi-omics profiling uncovered specific lysine-malonylation targets in glycolysis, the TCA cycle, fatty-acid oxidation and autophagy. Our findings uncover hepatic ACSF3 as a pivotal molecular nexus that integrates feeding time with dynamic protein lysine-malonylation and orchestrates the diurnal rhythm of liver metabolism.