Signalling mechanisms involved in the regulation of mammary protein synthesis by amino acids
The aim of this study was to develop an in vitro mammary model, based on rat mammary explants, which could be used to examine the effects of amino acid profile and concentration in the media on protein synthesis. Secondly, to ascertain whether these responses to amino acids, in particular leucine and α ketoisocaproic acid, were transmitted through the mTOR/p70 S6 kinase signalling pathway. Mammary explant protein synthesis was found to be stimulated up to twofold in response to graded levels of a complete mixture of amino acids (2 x and 4 x, normal rat plasma concentrations). The acute (1 h) stimulation of protein synthesis was at the level of translation. Inhibition of mTOR by rapamycin did not block the stimulation of protein synthesis by amino acids. In fact, when total amino acid concentrations were increased 0.5 to 4-fold, p70 S6 kinase activity decreased, despite the fact that protein synthesis was elevated up to 2.5 fold. When explants were incubated with either leucine or its transamination product α ketoisocaproic acid at 4 x normal levels in the presence of other amino acids (1 x), p70 S6 kinase activity was increased. There was a tendency for p70 S6 kinase activity to be blocked when transamination was inhibited. The failure to decrease protein synthesis by inhibition of transamination, despite the fact that p70 S6 kinase activity was inhibited, suggests that other translation factors may be more important in regulating mammary protein synthesis. This Phd thesis demonstrates a novel role for amino acids in mammary protein synthesis, whereby amino acids modulate the activity of the translation regulator p70 S6 kinase. In particular leucine and its transamination are important in the regulation of p70 S6 kinase activity. This provides the starting point for future studies exploring the role of translation factors in the regulation of mammary protein synthesis.