Citrulline Modulates Milk Fat Metabolism via the RTN3-FABP5 Axis in Bovine Mammary Epithelial Cells

Milk fat critically determines the nutritional value, flavor, and processing characteristics of dairy products. Citrulline, a nonproteinogenic amino acid enriched in cucurbitaceous fruits, has been implicated in lipid metabolism and metabolic health. However, its function in milk fat synthesis within bovine mammary epithelial cells (BMECs) and the associated molecular mechanisms remain undefined. Therefore, the study sought to determine Citrulline's impact on milk fat synthesis and clarify its mechanism. We initially examined reticulon 3 (RTN3) expression in the mammary glands of dairy cows with high and low milk fat contents. Functional assays demonstrated that RTN3 overexpression in BMECs markedly elevated milk fat synthesis, whereas RTN3 knockdown suppressed it. Mechanistically, RTN3 directly interacted with fatty acid-binding protein 5 (FABP5) and enhanced its stability by limiting ubiquitin-mediated proteasomal degradation. Screening further identified citrulline as a natural compound that upregulates RTN3, reduces FABP5 ubiquitination, and promotes milk-fat synthesis in BMECs. Collectively, our findings demonstrate that food-derived citrulline promotes milk-fat biosynthesis through the RTN3/FABP5 axis, supporting amino-acid-based nutritional strategies to optimize milk lipid composition.