L‐arginine stimulates the proliferation of mouse mammary epithelial cells and the development of mammary gland in pubertal mice by activating the GPRC6A/PI3K/AKT/mTOR signalling pathway

Amino acids have been shown to affect the development of mammary gland (MG). However, it is unclear whether L-arginine promotes the development of pubertal MG. Therefore, our study aims to explore the effect of L-arginine on the development of MG in pubertal mice. To investigate its internal mechanism of action, we will use mouse mammary epithelial cells (mMECs) line. Whole-mount staining showed that L-arginine can promote the extension of MG duct. In vitro, 0.4 mM L-arginine could activate the G protein-coupled receptor family C, group 6, subtype A (GPRC6A)/phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signalling pathway and increase the phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4EBP1) to promote the synthesis of cell cycle regulatory protein D1 (Cyclin D1), leading to the dissociation of the retinoblastoma tumour suppressor protein (Rb)-E2F1 transcription factor (E2F1) complex in mMECs and releasing E2F1 to promote cell proliferation. Furthermore, GPRC6A was knocked down or inhibition of the PI3K/AKT/mTOR signalling pathway with corresponding inhibitors completely abolished the arginine-induced promotion of mMECs proliferation. In vivo, it was further confirmed that 0.1% L-arginine can activate the PI3K/AKT/mTOR signalling pathway in the MG of pubertal mice. These results were able to indicate that L-arginine stimulates the development of MG in pubertal mice through the GPRC6A/PI3K/AKT/mTOR signalling pathway.