The role of LeuRS, an aminoacyl-tRNA synthetase, as an intracellular l-leucine sensor for the mTORC1 pathway has been the subject of much research recently. Despite this, the association between LeuRS and lactation in dairy cow mammary epithelial cells (DCMECs) remains unknown. In this study, we found that LeuRS expression in mammary gland tissue was significantly higher during lactation than pregnancy. Moreover, our data demonstrates that LeuRS is localized in the cytoplasm. Treatment with leucine increased DCMECs viability and proliferation, as well as mammalian target of rapamycin (mTOR), p-mTOR, ribosomal protein S6 kinase 1 (S6K1), p-S6K1, β-Casein, sterol regulatory element binding protein 1c (SREBP-1c), glucose transporter 1 (GLUT1), and Cyclin D1 mRNA and protein expression. Secretion of lactose and triglyceride were also increased. siRNA-mediated knockdown of LeuRS led to reduction in all of these processes. Based on these data, LeuRS up-regulates the mTOR pathway to promote proliferation and lactation of DCMECs in response to changes in the intracellular leucine concentration.

BackgroundLactose, as the primary osmotic component in milk, is the major determinant of milk volume. Glucose is the primary precursor of lactose. However, the effect of glucose on lactose synthesis in dairy cow mammary glands and the mechanism governing this process are poorly understood.ResultsHere we showed that glucose has the ability to induce lactose synthesis in dairy cow mammary epithelial cells, as well as increase cell viability and proliferation. A concentration of 12 mM glucose was the optimum concentration to induce cell growth and lactose synthesis in cultured dairy cow mammary epithelial cells. In vitro, 12 mM glucose enhanced lactose content, along with the expression of genes involved in glucose transportation and the lactose biosynthesis pathway, including GLUT1, SLC35A2, SLC35B1, HK2, β4GalT-I, and AKT1. In addition, we found that AKT1 knockdown inhibited cell growth and lactose synthesis as well as expression of GLUT1, SLC35A2, SLC35B1, HK2, and β4GalT-I.ConclusionsGlucose induces cell growth and lactose synthesis in dairy cow mammary epithelial cells. Protein kinase B alpha acts as a regulator of metabolism in dairy cow mammary gland to mediate the effects of glucose on lactose synthesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-016-0704-x) contains supplementary material, which is available to authorized users.

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.

Amino acids are required for the mammalian target of rapamycin (mTOR) signaling pathway and milk synthesis in bovine mammary epithelial cells (BMECs). However, the mechanism through which amino acids activate this pathway is largely unknown. Here we show that glycyl-tRNA synthetase (GlyRS) mediates amino acid-induced activation of the mTOR-S6K1/4EBP1 pathway, and milk protein and fat synthesis in BMECs. Among 19 aminoacyl-tRNA synthetases, only the mRNA expression of GlyRS and Leucyl-tRNA synthetase (LeuRS) were significantly increased by several amino acids including Met and Leu. We then observed that GlyRS knockdown abolished the stimulation of Met on milk protein and fat synthesis in BMECs, whereas GlyRS overexpression led to more significantly increased milk synthesis in cells treated with Met. By western blotting and qualitative real time-polymerase chain reaction analysis (qRT-PCR) analysis, we next revealed that GlyRS is required for amino acid-induced activation of the mTOR-S6K1/4EBP1 pathway. Thus, this study establishes that GlyRS mediates amino acid-induced activation of the mTOR pathway, thereby regulating milk protein and fat synthesis.

Spleen tyrosine kinase (SYK) is a nonreceptor tyrosine kinase that has been considered a hematopoietic cell-specific signal transducer involved in cell proliferation and differentiation. However, the role of SYK in normal mammary gland is still poorly understood. Here we show that SYK is expressed in mammary glands of dairy cows. Expression of SYK was higher in dry period mammary tissues than in lactating mammary tissues. Knockdown and overexpression of SYK affected dairy cow mammary epithelial cell proliferation as well as the expression of signal molecules involved in proliferation, including protein kinase B (PKB, also known as AKT1), p42/44 mitogen-activated protein kinase (MAPK), and signal transducer and activator of transcription 5 (STAT5). Dual-luciferase reporter assay showed that SYK increased the transcriptional activity of the AKT1 promoter, and cis-elements within the AKT1 promoter region from -439 to -84 bp mediated this regulation. These results suggest that SYK affects mammary epithelial cell proliferation by activating AKT1 at the transcriptional level in mammary glands of dairy cows, which is important for the mammary remodeling process in dry cows as well as for increasing persistency of lactation in lactating cows.