Effects of methionine-containing dipeptides on α s 1 casein expression in bovine mammary epithelial cells

Effects of methionine-containing dipetides on αs1 casein gene expression were investigated in cultured bovine mammary epithelial cells. αs1Casein gene expression was higher in the culture added with methionylmethionine than in that with methionine. Addition of methionyllysine enhanced αs1 casein gene expression, compared with methionine plus lysine. These results indicated that mammary epithelial cells can utilize methionine-containing dipeptides for milk protein synthesis with a higher effi ciency than the respective amino acid.


INTRODUCTION
Mammary cells are able to utilize amino acids bound to peptides for tissue or milk protein synthesis (Backwell et al., 1996).Supplementation of rumen protected methionine (Met) can improve the effi ciency of milk protein synthesis (Nichols et al., 1998;Xu et al., 1998).However it is still uncertain if the Metcontaining dipeptides affect the milk protein synthesis.This study was conducted to investigate the effects of methionylmethionine (Met-Met) and methionyllysine (Met-Lys) on α s1 casein gene expression in cultured bovine mammary epithelial cells.

Reagents
DMEM/F12 was purchased from Gibco BRL Life Technologies.Prolactin, insulin, transferrin, trypsin were supplied by Sigma; foetal calf serum, and hydrocortisone were obtained from Sangon (Shanghai).Reagents for reverse transcriptase (RT)-PCR were obtained from Invitrogen.Methionine and lysine were obtained from AMRESCO; Met-Met and Met-Lys (purity>99.8%) were supplied by Zhongtai Biochemistry (Hangzhou).All other reagents were of the highest purity commercially available.

Preparation and culture of cells
Mammary tissues were obtained from two slaughtered Holstein dairy cows at the middle stage of lactation.Tissues were cut into 1 mm 3 pieces and then plated in collagen-treated 6-well culture plates and incubated at 37 o C under 95% air and 5% CO 2. The procedures of dispersion and culture of mammary epithelial cells were carried out.Briefl y, when cells covered 80% of the plates, the tissues were digested with 0.15% trypsin and 0.02% EDTA.The dispersed cells were fi ltrated through a 105 μm mesh and then seeded at a density of 5×10 4 cells/ml in DMEM/ F12 medium.The basal medium was replenished with 2 μg/ml hydrocortisone, 5 μg/ml transferrin, 10 μg/ml insulin, 1% glutamine, 1% penicillin and streptomycin, and 10% foetal calf serum.

Treatments
The optimal concentration of adding methionine was at 60 μg/ml for the expression of α s1 casein gene in cultured mammary epithelial cells (Yang et al., 2007).Lysine was also usually considered as a limiting amino acid.The proportion of lysine in amino acids of α s1 casein is triple of methionine (GENE BANK, M38641).In order to investigate effects of methionine-containing dipeptides on α s1 casein gene expression, the basal culture mediums were added with 60 μg/ml Met-Met in comparison to 60 μg/ml methionine, and with 120 μg/ml Met-Lys in comparison to 60 μg/ml methionine plus 60 μg/ml lysine, respectively.

Methods
Total cellular RNA was extracted by Trizol (Invitrogen).The RNA purity was determined by optical density (OD260 nm/OD280 nm absorption ratio >1.80).The RT-PCR was used to amplify the mRNA of α s1 casein and GAPDH in the system, and the procedures of RT-PCR were according to the previous reports (Wu et al., 2004).PCR products were analysed by electrophoresis on 1.2% agarose gel.The net intensities of individual bands were measured using Image Master VDS Software (Pharmacia Biotech, Sweden).The average level of three repeats was used for statistical analysis.

Statistical analysis
Statistical analyses were undertaken using the GLM procedure of SAS (2000).P<0.01 was considered as signifi cantly different.

RESULTS
This study adopted an in vitro model of mammary epithelial cells to evaluate the effects of methionine-containing dipeptides on α s1 casein expression.Addition  of 60 μg/ml Met-Met promoted the α s1 casein expression in cultured mammary epithelial cells as compared with 60 μg/ml methionine-supplemented group (Figure 1; P<0.01).Similarly, α s1 casein expression on 120 μg/ml of Met-Lys was more abundant than that on 60 μg/ml of methionine plus lysine (Figure 2; P<0.01).It is suggested that mammary epithelial cells can utilize dipeptides to synthesis milk protein.Furthermore, the effi ciency of utilization of methionine-containing dipeptides is higher than free amino acids.

DISCUSSION
In the current study, addition of Met-containing dipeptides as source of methionine and lysine promoted the α s1 casein expression in cultured mammary epithelial cells as compared with respective free amino acids.The results is consistent with the fi ndings that histidine-containing dipeptide enhanced milk protein production compared with free histidine (Backwell et al., 1996).The mechanism for utilization of dipeptides is complicated and still unclear.It has been reported previously that cultured mammary epithelial cell can utilize peptides containing methionine as a substitute for free methionine supplied for mammary tissue and secretory protein synthesis (Pan et al., 1996).In the experiment with rats in vivo by Shennan et al. (1998), it is observed that dipeptides were hydrolysed extracellularly followed by uptake of the constituent amino acids.However, it is hard to explain the signifi cantly increased effi ciency of gene expression when the dipeptide containing methionine was used as supplement of methionine, as the expression of α s1 casein would not increase when the concentration of free methionine was higher than 60 μg/ml in cultured mammary epithelial cell (Yang et al., 2007).
The methionine transporters may be saturated at 60 μg/ml, but intact dipeptides may be absorbed through differential dipeptide transporter located at the membrane of bovine mammary epithelial cells (Groneberg et al., 2002).The peptide transporter has been found in mammary gland of rats and rabbits.Although the differences between species and methods should be taken into consideration, there should exist different dipeptide transporter on the membrane of bovine mammary epithelial cells.α s1 casein contained Met-Lys sequence (GENE BANK, M38641), thus the dipeptides absorbed by epithelial cells may be directly used to synthesize casein.

CONCLUSIONS
Methionine-containing dipeptides can be utilized by mammary epithelial cells, and the effi ciency of utilization was higher than that of equivalent free amino acids.The increased effi ciency of casein αs1 may have resulted from both enhanced α s1 casein gene expression and direct use of these dipeptides by bovine mammary epithelial cells.

Figure 1 .
Figure 1.Comparison of the effect of methionine (Met) and methionylmethionine (Met-Met) on α s1 casein gene expression in bovine mammary epithelial cells.A and B -agarose gel electrophoresis.C -means of optical density ratio of electrophoretic α s1 casein to GAPDH.a,b P<0.01

Figure 2 .
Figure 2. Comparison of the effect of methionine (Met) plus lysine (Lys) and methionyllysine (Met-Lys) on α s1 casein gene expression in bovine mammary epithelial cells.A -agarose gel electrophoresis.B -means of optical density ratio of the electrophoretic α s1 casein to GAPDH.a,b P<0.01