Methionyl-Methionine Promotes alpha-s1 Casein Synthesis in Bovine Mammary Gland Explants by Enhancing Intracellular Substrate Availability and Activating JAK2-STAT5 and mTOR-Mediated Signaling Pathways

Background: Interest is increasing in the role of peptide-bound amino acids (AAs) in milk protein synthesis because studies have found that the uptake of some essential AAs by the mammary gland cannot meet the requirements for milk protein synthesis. Although the role of dipeptides in milk protein synthesis is clearly established, little is known about the underlying mechanisms. Objective: The objective of this study was to determine whether small peptides can be taken up intact by the peptide transporters in mammary tissue explants and the underlying mechanisms of the effects of methionyl-methionine (Met-Met) supplementation on milk protein synthesis. Methods: Mammary tissue explants were cultured in conditional medium and then treated with different concentrations of Met-Met that replaced 0%, 5%, 10%, 15%, 20%, and 25% of free Met for another 24 h. In some experiments, explants were cultured with an optimal dose of Met-Met with or without the inhibitors of peptide transporter 2 [PepT2; diethylpyrocarbonate (DEPC), 0.1 mmol/L] and aminopeptidase N (APN; bestatin, 20 mu mol/L) for 24 h. Results: The substitutions of 15% free Met with Met-Met significantly promoted alpha-s1 casein (alpha(s1)-CN) expression in the mammary explants (P < 0.051. The inhibition of the PepT2 by DEPC or APN by bestatin significantly decreased the Met-Met stimulated increase of alpha(s1)-CN expression (P < 0.05). Compared with the control group (0% Met-Met), absorption of Val, Met, Leu, Phe, Lys, and His was improved, and mRNA abundance of the neutral and basic AA transporter was increased in the 15% Met-Met group (P < 0.05). In addition, the mRNA abundance of the mammalian target of rapamycin (mTOR), p70 ribosomal S6 kinase 1 gene, eukaryotic initiation factor 4E binding protein 1 gene, Janus kinase 2 (JAK2), and signal transducer and activator of transcription 5 (STAT5) was increased in the 15% Met-Met treated group IP < 0.051. Conclusion: Met-Met promoted alpha(s1)-CN synthesis in cultured bovine mammary gland explants, and this stimulation may be mediated by enhanced intracellular substrate availability and by activating JAK2-STAT5 and mTOR signaling pathways.