Effects of essential amino acids availability on amino acids uptake, metabolism and casein synthesis in bovine mammary epithelial cells.

Under current dairy cow feeding conditions, the conversion efficiency of dietary nitrogen into milk protein is less than 30%. A thorough understanding of amino acids (AA) uptake and utilization in mammary in response to AA supply is essential for accurately modeling nutritional requirements. This study utilized bovine mammary epithelial cells (BMEC) as a model to investigate the effects of increasing total essential amino acids (EAA) concentrations (0.00, 0.97, 1.94, 3.88, and 7.76 mM) on AA uptake, metabolism, and casein synthesis. The control group (0.00 mM) contained the basal medium without any additional EAA supplementation. The EAA mixture consisted of Thr, Lys, Met, His, Arg, Leu, Ile, Val, and Phe, maintained at fixed relative proportions that matched those found in the mammary artery of dairy cows at peak lactation. Nine cell culture plates were used per treatment, with 6 plates for casein expression analysis and 3 plates for proteomics analysis. After 24 h of incubation with the EAA, cells were harvested for the respective analyses. The culture medium from 6 plates was collected for individual AA content measurement. Data were analyzed using SAS (version 9.4). Orthogonal polynomial contrasts were used to determine the linear, quadratic, and cubic effects of treatments. Significance was declared at P†<†0.05. As EAA concentrations in the BMEC medium increased, protein expression of αS1-casein, β-casein, and κ-casein, as well as total EAA, total AA, and each individual EAA uptake (except for Lys) increased quadratically (Pquadratic < 0.01). Meanwhile, the uptake efficiency of total EAA, Thr, Met, Ile, Phe, and His decreased linearly (Plinear < 0.01). Moreover, with increasing total EAA concentration from 0.97 to 7.76 mM, decreases in the uptake efficiency of Thr, Ile, and Phe were greater than for other EAA. Lysine uptake efficiency showed no difference among EAA-treated groups (P†>†0.05). The protein expression of key enzymes involved in Met catabolism exhibited significant decreases with increasing EAA concentration (Plinear < 0.01). In contrast, The protein expression of key enzymes associated with Lys (Plinear†=†0.008), branched-chain amino acids (Pquadratic†<†0.05), and Arg (Plinear†<†0.001 or Pquadratic†<†0.001) catabolism demonstrated opposite trends, showing significant increases with increasing EAA concentration. These findings suggest that when EAA supply exceeds BMEC metabolic requirements, further increases in EAA supply reduce EAA utilization efficiency without enhancing casein synthesis. Furthermore, individual EAA demonstrate distinct uptake patterns and catabolic responses to changes in total EAA concentration.