Metabolic transition of milk triacylglycerol synthesis in response to varying levels of palmitate in porcine mammary epithelial cells

Milk in mammals is a key source of lipids for offspring, providing both critical energy and essential fatty acids. For lactating sows, palmitic acid is one of the most abundant fatty acids in milk, providing 10~12% of the suckling pig total dietary energy supply. However, the effects of exogenous palmitic acid on milk fat synthesis in sow mammary glands are not well-known. In this study, we investigated the effects of palmitic acid on lipogenic genes in porcine mammary epithelial cells (pMECs) to explore the role of exogenous palmitic acid in mediating milk triacylglycerols (TAG) synthesis.

Results from this study suggest that palmitate stimulated the cytosolic TAG accumulation in pMECs, probably by promoting lipogenic genes and proteins that are involved in lipid synthesis. However, addition of palmitate decreased the fatty acid de novo synthesis in pMECs.

Porcine mammary epithelial cells were cultured for 24 h in the presence of different concentrations of palmitate (0, 25, 50, 100, 200, 400, and 600 μM). The effect of palmitate on cell viability was tested via MTT assay. Intracellular lipid accumulation was measured through Oil Red O staining, and TAG levels were quantified by enzymatic colorimetric methods. Expression of genes and proteins involved in milk fat biosynthesis were assayed with quantitative real-time polymerase chain reaction (qPCR) and Western blotting, respectively.

Incubation with palmitate promoted cellular lipid synthesis in a dose-dependent manner, as reflected by the increased TAG content and enhanced formation of cytosolic lipid droplets. The increased lipid synthesis by palmitate was probably attributable to the upregulated mRNA expression of genes associated with milk fat biosynthesis, including long-chain fatty acid uptake (LPL, CD36), intracellular activation and transport (ACSL3, FABP3), TAG synthesis (GPAM, AGPAT6, DGAT1), lipid droplet formation (PLIN2), and regulation of transcription (PPARγ). Western blot analysis of CD36 and DGAT1 proteins confirmed the increased lipid synthesis with increasing incubation of palmitate. However, the genes involved in fatty acid de novo synthesis (ACACA, FASN), fatty acid desaturation (SCD), and regulation of transcription (SREBP1, INSIG1) were inversely affected by incubation with increasing concentrations of palmitate. Western blot analysis of ACACA protein confirmed this decrease associated with increasing levels of palmitate. Conclusions: Results from this study suggest that palmitate stimulated the cytosolic TAG accumulation in pMECs, probably by promoting lipogenic genes and proteins that are involved in lipid synthesis. However, addition of palmitate decreased the fatty acid de novo synthesis in pMECs.