L-leucine availability regulates phosphatidylinositol 3-kinase, p70 S6 kinase and glycogen synthase kinase-3 activity in L6 muscle cells: evidence for the involvement of the mammalian target of rapamycin (mTOR) pathway in the L-leucine-induced up-regulation of System A amino acid transport

Amino acid availability is known to regulate diverse cell processes includi ng the activation of p70 S6 kinase, initiation factors involved in mRNA tra nslation, gene expression and cellular amino acid uptake. Essential amino a cids, in particular the branched-chain amino acids (e.g. leucine), have bee n shown to be the dominant players in mediating these effects, although the precise nature by which they regulate these processes remain poorly unders tood. In this study we have investigated the mechanisms involved in the leu cine-induced modulation of p70 S6 kinase and addressed whether this kinase participates in the up-regulation of the System A amino acid transporter in L6 muscle cells. Incubation of muscle cells that had been amino acid-depri ved for 1 h with L-leucine (2 mM) led to a rapid(> 2-fold) activation of p7 0 S6 kinase, which was suppressed by both wortmannin and rapamycin. Consist ent with this finding, addition of leucine caused a rapid (approximate to 5 -fold) but transient stimulation of phosphatidylinositol 3-kinase (PI3K). P I3K activation was inhibited by wortmannin and was not dependent upon insul in receptor substrate-1 activation. Unlike stimulation by insulin, activati on of neither protein kinase B nor p42/p44 mitogen-activated protein kinase accompanied the leucine-induced stimulation of PI3K. However, the leucine- induced activation of PI3K and p70 S6 kinase did result in the concomitant inactivation of glycogen synthase kinase-3 (G5K-3). Leucine enhanced System A transport by approximate to 50%. We have shown previously that this stim ulation is protein-synthesis-dependent and in the current study we show tha t it was blocked by both wortmannin and rapamycin, Our findings indicate th at PI3K and the mammalian target of rapamycin are components of a nutrient signalling pathway that regulates the activation of p70 S6 kinase and induc tion of System A in L6 cells. The activation of this pathway by leucine is also responsible for the inactivation of G5K-3, and this is likely to have important regulatory implications for translation initiation.