l-Arginine regulates protein turnover in porcine mammary epithelial cells to enhance milk protein synthesis
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Milk is an important food for mammalian neonates, but its insufficient production is a nutritional problem for humans and other animals. Recent studies indicate that dietary supplementation with l-arginine (Arg) increases milk production in mammals, including sows, rabbits, and cows. However, the underlying molecular mechanisms remain largely unknown. The present study was conducted with porcine mammary epithelial cells (PMECs) to test the hypothesis that Arg enhances milk protein synthesis via activation of the mechanistic target of rapamycin (mTOR) cell signaling. PMECs were cultured for 4 days in Arg-free basal medium supplemented with 10, 50, 200, or 500 μmol/L Arg. Rates of protein synthesis and degradation in cells were determined with the use of l-[ring-2,4-3H]phenylalanine. Cell medium was analyzed for β-casein and α-lactalbumin, whereas cells were used for quantifying total and phosphorylated levels of mTOR, ribosomal protein S6 kinase (p70S6K), 4E-binding protein 1 (4EBP1), ubiquitin, and proteasome. Addition of 50–500 μmol/L Arg to culture medium increased (P < 0.05) the proliferation of PMECs and the synthesis of proteins (including β-casein and α-lactalbumin), while reducing the rates of proteolysis, in a dose-dependent manner. The phosphorylated levels of mTOR, p70S6K and 4EBP1 were elevated (P < 0.05), but the abundances of ubiquitin and proteasome were lower (P < 0.05), in PMECs supplemented with 200–500 μmol/L Arg, compared with 10–50 μmol/L Arg. These results provide a biochemical basis for the use of Arg to enhance milk production by sows and have important implications for improving lactation in other mammals (including humans and cows).
KeywordsArginine Mammary gland Milk protein mTOR signaling
Dulbecco’s modified eagle medium
4E-binding protein 1
Fetal bovine serum
Mechanistic target of rapamycin
Porcine mammary epithelial cells
Ribosomal protein S6 kinase
Tris–Tween buffered saline
Q. Ma was a recipient of a postdoctoral fellowship from the China Scholarship Council. This work was supported Agriculture and Food Research Initiative Competitive Grants (2014-67015-21770) from the USDA National Institute of Food and Agriculture, and Texas A&M AgriLife Research (H-8200). The authors thank our research assistants for technical assistance.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This study involved the cultures of an established cell line and did not require an animal use protocol.
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