| Amino acids are the main substrates used for synthesis of milk protein. Except amino acids, small peptides also participate in the synthesis of milk protein. This study was conducted to evaluate effects of different amino acids patterns and small peptides on synthesis of milk protein in cultured lactating bovine mammary tissues/epithelial cells. The following series of experiments were performed:(1) Effects of amino acids profiles and small peptides on synthesis of milk protein (Expt 1); (2) Uptake of small peptides in bovine mammary gland (Expt 2); (3) Functions of small peptides in promoting synthesis of milk protein (Expt 3).Expt 1 was to investigate effects of ratio of threonine (Thr) to phenylalanine (Phe) and small peptides on asl casein gene expression and synthesis of milk protein in cultured bovine mammary tissues. Mammary tissues was obtained from Holstein dairy cow and incubated in DMEM/F12. Concentration of lactogenic hormones, ratio of Thr to Phe (117μg/mL) (0.7,0.8,0.9,1.1, and 1.2) or concentration of peptide-bound Phe or Lys (0%,5%,10%,15%, and 20%) in the experimental medium were adjusted. After incubated with experimental medium, tissues were collected for gene expression examination and medium was collected for milk protein determination. The results showed that combination addition of prolactin, insulin and hydrocortisone at 500 ng/mL,5000 ng/mL and 1000 ng/mL most enhanced the synthesis of milk protein (P<0.05). Different ratios of Thr to Phe markedly affected the asl casein gene expression and synthesis of milk protein, with the optimal ratio at 1:1. Additional small peptides increased gene expression of asl casein. The highest abundance of asl casein mRNA was observed when 10% Phe-Phe,10% Phe-Thr,10% Thr-Phe-Phe, 15% Lys-His,10% Lys-Phe,5% Lys-Lys,5% Lys-Leu and 5% Lys-Thr was respectively added (P<0.05). And synthesis of milk protein was enhanced by adding all small peptides. The results also demonstrated that small peptides consisting of amino acids residues which is hydrophobic and bulky had higher enhancement of synthesis of milk protein than small peptides with hydrophilic and charged amino acids residues. All these results demonstrated that appropriate lactogenic hormones and ratio of amino acids promote synthesis of milk protein; Peptide-bound Phe and Lys can be used for synthesis of milk protein by mammary gland, and they have higher utilization efficiency than free amino acids. Expt 2 was carried out to investigate expression of oligopeptide transporter (PepT) and its potential function in bovine mammary gland. To detect expression of PepT in bovine mammary epithelial cells, methods of real time PCR, immunohistochemistry and immunocytochemistry were used. After that, effects of lactogenic hormones (prolactin, hydrocortisone, or insulin) and the substrates (peptide-bound Phe) on PepT were investigated. And then, functions of PepT2 was inhibated using DEPC to investigate potential role of PepT2 in small peptides transportation. The results showed that there was no PepTl gene expression in bovine mammary gland, and the PepT2 mRNA and protein were successfully detected in bovine mammary epithelial cells. PepT2 gene expression was enhanced by addition of 50 ng/mL,500 ng/mL, and 5000 ng/mL prolactin,10 ng/mL and 100 ng/mL hydrocortisone, and 50 ng/mL,500 ng/mL,5000 ng/mL, and 50000 ng/mL insulin (P<0.05). Peptide-bound Phe promoted gene expression of PepT2. Compared with control, addition of Phe-Phe at 5% and 10% and Thr-Phe-Phe at 5%,10%, and 15% most enhanced the PepT2 mRNA level (P<0.05). When addition of Phe-Thr was 10%, gene expression of PepT2 trended to be increased (P=0.064). Because of comprising different amino acids residues, Phe-Phe and Thr-Phe-Phe induced higher PepT2 mRNA level than equivalent Phe-Thr (P<0.05). Responses of PepT2 to lactogenic hormones and small peptides allow concluding that it may play an important role in bovine mammary gland. Addition of Phe-Phe at 10% significantly increased mRNA abundance of asl casein and PepT2 (P<0.05), and had no effect on dipeptidase 2 (DPEP2) gene expression. However, inhibition of PepT2 function (DEPC,0.5 mM) decreased asi casein gene mRNA level and synthesis of milk protein promoted by Phe-Phe (P<0.05). These results showed that there is PepT2 expression in bovine mammary gland, and it can be regulated by lactogenic hormones and small peptides; Inhibition of PepT2 function decreased synthesis of milk protein promoted by small peptides, it plays an important role in uptake of small peptides by bovine mammary gland.Expt 3 was conducted to investigate the molecular mechanism of synthesis regulation of milk protein by small peptides. Concentrations of peptide-bound Phe or Lys in the experimental medium were adjusted. After incubated with experimental medium, tissues and medium were collected for gene expression examination and amino acids determination. When 10% of free Phe was replaced by Phe-Phe, concentration of Lys, Phe, leucine (Leu), isoleucine (Ile), and valine (Val) in culture medium markedly decreased (P<0.05). Addition of Phe-Phe significantly increased mRNA abundance of asl casein, ATB0+ and y+CAT2 (P<0.05), and had no effect on TAT1 and ASCT1 gene expression. Except for Lys-Phe, addition of Lys-His, Lys-Leu, Lys-Thr and Lys-Lys all enhanced ATB0+mRNA level (P<0.05). Lys-His and Lys-Lys significantly enhanced all gene expression of mTOR,4E-BP1 and S6K1 (P<0.05). It can be inferred that uptake of small peptides can decrease competitive inhibition among amino acids and promotes uptake of amino acids by mammary gland; Besides this, small peptides also participates in synthesis of milk protein as the signaling molecule.In summary, small peptides could be efficiently used for synthesis of milk protein in bovine mammary gland, and PepT2 may play an important role in uptake of small peptides by bovine mammary gland; Besides as substrates, small peptides also can promote uptake of amino acids and induce signal transduction in bovine mammary gland.
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