This study was conducted to determine the optimum ratio of lysine and methionine (Lys:Met) to enhance milk protein concentration in vitro, focusing on the regulation of genes related to the JAK2-STAT5 and the mammalian target of rapamycin (mTOR) signaling pathways. A preliminary dose response study revealed that casein concentration peaked (2.5-2.7 ppm) at a supplemental Lys concentration of 1.2 mM and Met at 0.5 mM. At the peak casein concentration cell proliferation rate also was higher. Furthermore, the expression of CSN1S1, CSN1S2, CSN2, CSN3, LALBA, JAK2, STAT5, and MTOR was upregulated with both Lys and Met compared with the control. A subsequent experiment was conducted as a 5 × 3 factorial design with supplemental Lys plus Met at different ratios. When the supplemental concentration of Lys was 1.2 mM and Met was 0.4 mM (∼3:1), the concentration of casein peaked. Therefore, we measured gene expression, mTOR protein expression, and phosphorylated mTOR (p-mTOR) in cultures incubated with 3:1 Lys:Met (Lys&Met). Expression of CSN1S1 and LALBA were the most highly expressed genes (P < 0.01). The upregulation of CSN2, CSN3, CSN1S2 isoforms (P < 0.01) and JAK2, ELF5, mTOR (P < 0.05) was also observed. Total mTOR protein expression was greater (P < 0.05) with Lys alone and also Lys&Met. However, Lys&Met resulted in the greatest (P < 0.05) p-mTOR. Results suggest that peak concentration of casein at a supplemental 3:1 Lys:Met is driven in part via upregulation of the mRNA expression of components of the JAK-STAT and mTOR pathways.
Keywords: amino acids; mammary cell; milk protein.