Very high-dose vitamin D₃ supplementation reduces the expression of genes and proteins engaged in β-oxidation in healthy pigs

Vitamin D plays a multifaceted role in the body, influencing a wide range of physiological processes. While its benefits in deficiency states are well recognized, the effects of high-dose vitamin D₃ supplementation in vitamin D-sufficient individuals remain poorly understood. In this study, we applied an integrative transcriptomic and proteomic approach to assess the dose-dependent effects of long-term dietary vitamin D₃ supplementation (5000 and 10,000 IU/kg feed) in healthy pigs. Despite the absence of phenotypic alterations in fattening characteristics, we observed significant molecular changes in liver tissue, particularly in pathways related to fatty acid β-oxidation, amino acid catabolism, and oxidative stress response. High-dose vitamin D₃ supplementation led to consistent downregulation of key genes and proteins involved in mitochondrial and peroxisomal β-oxidation, including ACSL5, ACADVL, HADHA, ACAA1 (gene expression), and ACADM, ECHDC1, and ECHDC2 (protein level). These findings suggest a reduced hepatic capacity for activating and degrading long-, very long-, and medium-chain fatty acids, potentially resulting in the accumulation of lipid intermediates and a shift toward alternative metabolic pathways. Our findings indicate that very high-dose vitamin D₃ supplementation in non-deficient states may lead to adverse metabolic shifts in the liver, including lipid accumulation and compromised energy metabolism. These effects appear to be dose-dependent, and while they may not manifest phenotypically in short-lived species, they offer important insights into non-classical toxicological effects of high-dose vitamin D₃ supplementation. Importantly, this study highlights the context-dependent nature of vitamin D’s effects and provides a new direction for research focused on its metabolic roles beyond classical pathways.