Effects of AICAR, Rapamycin, and Non-essential Amino Acids on Cell Signaling in Bovine Mammary Tissue
Studies in animal and cell culture models have revealed that essential amino acids (AA) play a role in regulating protein synthesis not only as substrates but also through direct signaling to the protein synthetic machinery. Essential AA affect the mammalian target of rapamycin (mTOR) signaling pathway in bovine mammary cells, which is associated with increased milk protein synthesis. Three experiments were conducted to study the effect of AICAR, an activator of AMP kinase, rapamycin, a mTOR complex 1 inhibitor, and non-essential AA on mTOR signaling in mammary tissue. Three Holstein cows in late lactation were slaughtered and mammary tissue was collected from uninfected rear quarters. Tissue slices (120 ± 30 mg) were incubated for 4 h in the respective treatment medium. In the first experiment, treatments consisted of complete Dulbeco Modified Eagle Medium (DMEM) or DMEM with essential AA (EAA) at 5% of regular concentrations, and AICAR at 0, 0.4, or 4.0 mM. The second experiment consisted of the same EAA treatments supplemented with rapamycin at 0, 0.5, and 10 µM. In the third experiment, tissue slices from 2 cows were incubated in Minimum Essential Medium supplemented with 4.5 g glucose,10 µg insulin, and per liter of media. The amino acids examined were Ala, Asx, Glx, Gly, Pro, and Ser where Asx represented Asn plus Asp, and Glx represented Glu plus Gln. Phosphorylated and total forms of mTOR, eukaryotic elongation factor 2, and ribosomal protein S6 were determined by Western blotting and the ratio of phosphorylated to total ratio was calculated. Essential AA had no effect on signaling proteins in either experiment. AICAR significantly reduced mTOR phosphorylation, but had no effect on phosphorylation of eEF2 or rpS6. Rapamycin did not affect phosphorylation of mTOR or eEF2. However, rapamycin significantly reduced rpS6 phosphorylation. NEAA had no effect on phosphorylation of signaling proteins in the mTOR pathway. There was no effect of EAA on protein synthesis rates, implying that there are other causes regulating synthesis rates, besides cell signaling.
Keywords: rapamycin, AICAR, mTOR