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

Abstract

Amino acid availability is known to regulate diverse cell processes including the activation of p70 S6 kinase, initiation factors involved in mRNA translation, gene expression and cellular amino acid uptake. Essential amino acids, in particular the branched-chain amino acids (e.g. leucine), have been shown to be the dominant players in mediating these effects, although the precise nature by which they regulate these processes remain poorly understood. In this study we have investigated the mechanisms involved in the leucine-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-deprived for 1 h with L-leucine (2 mM) led to a rapid (>2-fold) activation of p70 S6 kinase, which was suppressed by both wortmannin and rapamycin. Consistent with this finding, addition of leucine caused a rapid ( approximately 5-fold) but transient stimulation of phosphatidylinositol 3-kinase (PI3K). PI3K activation was inhibited by wortmannin and was not dependent upon insulin receptor substrate-1 activation. Unlike stimulation by insulin, activation 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 (GSK-3). Leucine enhanced System A transport by approximately 50%. We have shown previously that this stimulation is protein-synthesis-dependent and in the current study we show that it was blocked by both wortmannin and rapamycin. Our findings indicate that PI3K and the mammalian target of rapamycin are components of a nutrient signalling pathway that regulates the activation of p70 S6 kinase and induction of System A in L6 cells. The activation of this pathway by leucine is also responsible for the inactivation of GSK-3, and this is likely to have important regulatory implications for translation initiation.

Protein Kinase C Phosphorylates the System N Glutamine Transporter SN1 (Slc38a3) and Regulates Its Membrane Trafficking and Degradation

“…Classical biochemical experiments early demonstrated adaptive, hormonal, and osmotic regulation of system A and N activities as measured by functional transport assays (36–38). A nutrition signaling cascade that includes activation of phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) has been shown to be important for upregulation of system A (39, 40). For SAT2, an amino acid response element regulating the promoter activity has been demonstrated (41).…”

Stimulation of mTORC1 with L-leucine Rescues Defects Associated with Roberts Syndrome

“…L-leucine (L-Leu) has been reported to enhance translation initiation and protein synthesis [56] via induction of the mTOR pathway [57], [58], [59]. We supplemented the culture medium with 10 mM L-Leu or D-leucine (D-Leu) up to 8 days, and cell proliferation was quantified using a bright-line hemocytometer with trypan blue staining to discriminate viable and dead cells.…”

The Effect of Leucine Restriction on Akt/mTOR Signaling in Breast Cancer Cell Lines In Vitro and In Vivo

“…Branched chain amino acids mediate mTOR activity (50) and their reduced concentrations can regulate apoptosis through mTOR (51–53). Another AA involved in regulation of mTOR pathway is glutamine, which leads to uptake of leucine and phosphorylation of S6K1 (54).…”