The effects of L-type amino acid transporter 1 on milk protein synthesis in mammary glands of dairy cows

Abstract

The mammary gland requires the uptake of AA for milk protein synthesis during lactation. The L-type amino acid transporter 1 (LAT1, encoded by SLC7A5), found in many different types of mammalian cells, is indispensable as a transporter of essential AA to maintain cell growth and protein synthesis. However, the function of LAT1 in regulating milk protein synthesis in the mammary gland of the dairy cow remains largely unknown. For the current study, we characterized the relationship between LAT1 expression and milk protein synthesis in lactating dairy cows and investigated whether the mammalian target of rapamycin complex 1 (mTORC1) signaling controls the expression of LAT1 in their mammary glands. We found that LAT1 and the heavy chain of its chaperone, 4F2, were expressed in mammary tissues of lactating cows, with the expression levels of LAT1 and the 4F2 heavy chain being significantly greater in lactating mammary tissues with high-milk protein content (milk yield, 33.8 ± 2.1 kg/d; milk protein concentration >3%, wt/vol,; n = 3) than in tissues from cows with low-milk protein content (milk yield, 33.7 ± 0.5 kg/d; milk protein concentration <3%, wt/vol; n = 3). Immunofluorescence staining of sectioned mammary tissues from cows with high and low milk protein content showed that LAT1 was located on the whole plasma membrane of alveolar epithelial cells, suggesting that LAT1 provides essential AA to the mammary gland. In cultured mammary epithelial cells from the dairy cows with high-milk protein content, knockdown of LAT1 expression decreased cell viability and β-casein expression; in contrast, overexpression of LAT1 had the opposite effect. Inhibition of mTORC1 by rapamycin attenuated the phosphorylation of molecules related to mTORC1 signaling and caused a marked decrease in LAT1 expression in the cultured cells; expression of β-casein also decreased significantly. These results suggest that LAT1 is involved in milk protein synthesis in the mammary glands of lactating dairy cows and that the mTORC1 signaling pathway might be a control point for regulation of LAT1 expression, which could ultimately be used to alter milk protein synthesis.

U2AF65 enhances milk synthesis and growth of bovine mammary epithelial cells by positively regulating the mTOR‐SREBP‐1c signalling pathway

“…23 Moreover, recent studies have indicated that the regulation of protein translation and cell proliferation by amino acids and hormones via the mTOR signalling pathway is a crucial determinant of milk production in BMECs. 15,16,24 In accordance with these previous reports, and based on our data that U2AF65 regulates milk synthesis and cellular growth and controls the mRNA expression of mTOR and subsequent mTOR phosphorylation, we propose that U2AF65 regulates milk synthesis and cell growth via mTOR signalling. We observed that mTOR protein levels were relatively stable in cells overexpressing U2AF65 or transfected FIGURE 4 Effects of U2AF65 overexpression and knockdown on the mTOR and SREBP-1c signalling protein by splicing.…”

“…BMECs treated with amino acids and hormones showed higher U2AF65 expression than that of the control group ( Figure 1C and 1D). 15,16 The above findings suggested that expression of U2AF65 responded to extracellular stimuli, such as amino acids and hormones and might be involved in the regulation of milk synthesis in BMECs. 15,16 The above findings suggested that expression of U2AF65 responded to extracellular stimuli, such as amino acids and hormones and might be involved in the regulation of milk synthesis in BMECs.…”

“…15,16 The total proteins in milk comprise about 80% caseins, and β-casein represents about 35% of caseins, which is the most abundant milk protein. 15,16 The total proteins in milk comprise about 80% caseins, and β-casein represents about 35% of caseins, which is the most abundant milk protein.…”

“…15,16 The purity of BMECs was verified by immunofluorescence staining for the epithelial cell marker, CK18 ( Figure 1A). In response to the stimulation by hormones and nutrients, BMECs proliferate and synthesize milk components.…”

“…In addition, the mTOR-SREBP-1c pathway has been confirmed to be a central pathway that controls the synthesis of milk protein, milk fat, and lactose and regulates the growth of BMECs. 15,16 To date, it has not been reported whether U2AF65 can enhance the mTOR-SREBP-1c pathway.…”