Supplementing Different Forms of L-Methionine and Acetate Alters the Expression Patterns of mRNA, Proteins and Metabolites Related to Milk Protein Synthesis and Energy Metabolism in Bovine Mammary Cells

N-Acetyl-L-Methionine (NALM) can be used as a rumen-protected methionine, which is cleaved into L-Met and acetate in the small intestine or liver, with high bioavailability. This study was conducted to compare the efficacy of NALM and its digested forms on milk protein synthesis using immortalized bovine mammary epithelial cell line (MAC-T Cell); L-Methionine (L-Met), conjugated L-Met and acetate (NALM), and non-conjugated form (L-Met+Acetate; digested form of NALM). Then, the mechanism of milk protein synthesis is also elucidated through omics analysis. L-Met+Acetate group showed the highest milk protein concentration in the media (P < 0.05) as well as in the total protein (P < 0.05), and also showed the highest beta casein (P < 0.05) and S6K1 (P < 0.05) mRNA expression levels compared to L-Met and NALM. On the other hand, L-Met treated group showed the highest mTOR gene expression level than NALM and L-Met+Acetate groups (P < 0.05). A total of 39 upregulated and 77 downregulated proteins, 62 upregulated and 80 downregulated proteins, 50 upregulated and 81 downregulated proteins were observed in L-Met, NALM, and L-Met+Acetate treated groups, respectively. Interestingly, based on metabolic pathway analysis, L-Met+Acetate treated group stimulates the ATP synthesis, cell cycle, Ubiquitin proteasome and TGF-beta signaling pathways but not in the NALM treated group. On the contrary, the NALM and L-Met treated groups stimulate the PI3 kinase and pyruvate metabolism pathways but not in the L-Met+Acetate treated group, indicating that acetate group conjugated in NALM cannot be utilized as an energy source in MAC-T cell, and consequently resulting cells to produce energy via the pyruvate pathway. Metabolite analysis shows that NALM treated group resulted in the increase of 12 metabolites and the decrease of UMP (P < 0.05). On the other hand, L-Met+Acetate treated group showed the increase of 13 metabolites and the decrease of IMP and pantothenate (P < 0.05). In summary, L-Met+Acetate treated group showed better performance in the expression of the beta casein and S6K1 mRNA, the stimulation of pathways and metabolites involved in ATP synthesis, and the increase of other metabolites related to energy production. As a result, L-Met+Acetate treated group showed higher protein concentration (P < 0.05) in the MAC-T cells compared to other groups, possibly suggesting that digested forms of NALM (L-Met and Acetate) can be effectively utilized for the increase of milk protein synthesis in the mammary gland after absorption and digestion of NALM in small intestine or liver.