Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2000 Sep 1;350(Pt 2):361–368.

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.

K Peyrollier 1, E Hajduch 1, A S Blair 1, R Hyde 1, H S Hundal 1
PMCID: PMC1221262  PMID: 10947949

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.

Full Text

The Full Text of this article is available as a PDF (193.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alberta J. A., Auger K. R., Batt D., Iannarelli P., Hwang G., Elliott H. L., Duke R., Roberts T. M., Stiles C. D. Platelet-derived growth factor stimulation of monocyte chemoattractant protein-1 gene expression is mediated by transient activation of the phosphoinositide 3-kinase signal transduction pathway. J Biol Chem. 1999 Oct 22;274(43):31062–31067. doi: 10.1074/jbc.274.43.31062. [DOI] [PubMed] [Google Scholar]
  2. Alessi D. R., Cohen P. Mechanism of activation and function of protein kinase B. Curr Opin Genet Dev. 1998 Feb;8(1):55–62. doi: 10.1016/s0959-437x(98)80062-2. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Cross D. A., Alessi D. R., Cohen P., Andjelkovich M., Hemmings B. A. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature. 1995 Dec 21;378(6559):785–789. doi: 10.1038/378785a0. [DOI] [PubMed] [Google Scholar]
  5. Cross D. A., Alessi D. R., Vandenheede J. R., McDowell H. E., Hundal H. S., Cohen P. The inhibition of glycogen synthase kinase-3 by insulin or insulin-like growth factor 1 in the rat skeletal muscle cell line L6 is blocked by wortmannin, but not by rapamycin: evidence that wortmannin blocks activation of the mitogen-activated protein kinase pathway in L6 cells between Ras and Raf. Biochem J. 1994 Oct 1;303(Pt 1):21–26. doi: 10.1042/bj3030021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dufner A., Thomas G. Ribosomal S6 kinase signaling and the control of translation. Exp Cell Res. 1999 Nov 25;253(1):100–109. doi: 10.1006/excr.1999.4683. [DOI] [PubMed] [Google Scholar]
  7. Flakoll P. J., Wentzel L. S., Rice D. E., Hill J. O., Abumrad N. N. Short-term regulation of insulin-mediated glucose utilization in four-day fasted human volunteers: role of amino acid availability. Diabetologia. 1992 Apr;35(4):357–366. doi: 10.1007/BF00401203. [DOI] [PubMed] [Google Scholar]
  8. Fox H. L., Kimball S. R., Jefferson L. S., Lynch C. J. Amino acids stimulate phosphorylation of p70S6k and organization of rat adipocytes into multicellular clusters. Am J Physiol. 1998 Jan;274(1 Pt 1):C206–C213. doi: 10.1152/ajpcell.1998.274.1.C206. [DOI] [PubMed] [Google Scholar]
  9. Fox H. L., Pham P. T., Kimball S. R., Jefferson L. S., Lynch C. J. Amino acid effects on translational repressor 4E-BP1 are mediated primarily by L-leucine in isolated adipocytes. Am J Physiol. 1998 Nov;275(5 Pt 1):C1232–C1238. doi: 10.1152/ajpcell.1998.275.5.C1232. [DOI] [PubMed] [Google Scholar]
  10. Hajduch E., Alessi D. R., Hemmings B. A., Hundal H. S. Constitutive activation of protein kinase B alpha by membrane targeting promotes glucose and system A amino acid transport, protein synthesis, and inactivation of glycogen synthase kinase 3 in L6 muscle cells. Diabetes. 1998 Jul;47(7):1006–1013. doi: 10.2337/diabetes.47.7.1006. [DOI] [PubMed] [Google Scholar]
  11. Hundal H. S., Bilan P. J., Tsakiridis T., Marette A., Klip A. Structural disruption of the trans-Golgi network does not interfere with the acute stimulation of glucose and amino acid uptake by insulin-like growth factor I in muscle cells. Biochem J. 1994 Jan 15;297(Pt 2):289–295. doi: 10.1042/bj2970289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hundal H. S., Rennie M. J., Watt P. W. Characteristics of acidic, basic and neutral amino acid transport in the perfused rat hindlimb. J Physiol. 1989 Jan;408:93–114. doi: 10.1113/jphysiol.1989.sp017449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Iiboshi Y., Papst P. J., Kawasome H., Hosoi H., Abraham R. T., Houghton P. J., Terada N. Amino acid-dependent control of p70(s6k). Involvement of tRNA aminoacylation in the regulation. J Biol Chem. 1999 Jan 8;274(2):1092–1099. doi: 10.1074/jbc.274.2.1092. [DOI] [PubMed] [Google Scholar]
  14. Kimball S. R., Shantz L. M., Horetsky R. L., Jefferson L. S. Leucine regulates translation of specific mRNAs in L6 myoblasts through mTOR-mediated changes in availability of eIF4E and phosphorylation of ribosomal protein S6. J Biol Chem. 1999 Apr 23;274(17):11647–11652. doi: 10.1074/jbc.274.17.11647. [DOI] [PubMed] [Google Scholar]
  15. Laine R. O., Hutson R. G., Kilberg M. S. Eukaryotic gene expression: metabolite control by amino acids. Prog Nucleic Acid Res Mol Biol. 1996;53:219–248. doi: 10.1016/s0079-6603(08)60146-4. [DOI] [PubMed] [Google Scholar]
  16. Louard R. J., Barrett E. J., Gelfand R. A. Overnight branched-chain amino acid infusion causes sustained suppression of muscle proteolysis. Metabolism. 1995 Apr;44(4):424–429. doi: 10.1016/0026-0495(95)90047-0. [DOI] [PubMed] [Google Scholar]
  17. May M. E., Buse M. G. Effects of branched-chain amino acids on protein turnover. Diabetes Metab Rev. 1989 May;5(3):227–245. doi: 10.1002/dmr.5610050303. [DOI] [PubMed] [Google Scholar]
  18. McDowell H. E., Christie G. R., Stenhouse G., Hundal H. S. Leucine activates system A amino acid transport in L6 rat skeletal muscle cells. Am J Physiol. 1995 Nov;269(5 Pt 1):C1287–C1294. doi: 10.1152/ajpcell.1995.269.5.C1287. [DOI] [PubMed] [Google Scholar]
  19. McDowell H. E., Walker T., Hajduch E., Christie G., Batty I. H., Downes C. P., Hundal H. S. Inositol phospholipid 3-kinase is activated by cellular stress but is not required for the stress-induced activation of glucose transport in L6 rat skeletal muscle cells. Eur J Biochem. 1997 Jul 1;247(1):306–313. doi: 10.1111/j.1432-1033.1997.00306.x. [DOI] [PubMed] [Google Scholar]
  20. Navé B. T., Ouwens M., Withers D. J., Alessi D. R., Shepherd P. R. Mammalian target of rapamycin is a direct target for protein kinase B: identification of a convergence point for opposing effects of insulin and amino-acid deficiency on protein translation. Biochem J. 1999 Dec 1;344(Pt 2):427–431. [PMC free article] [PubMed] [Google Scholar]
  21. Patti M. E., Brambilla E., Luzi L., Landaker E. J., Kahn C. R. Bidirectional modulation of insulin action by amino acids. J Clin Invest. 1998 Apr 1;101(7):1519–1529. doi: 10.1172/JCI1326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Proud C. G., Denton R. M. Molecular mechanisms for the control of translation by insulin. Biochem J. 1997 Dec 1;328(Pt 2):329–341. doi: 10.1042/bj3280329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Scott P. H., Brunn G. J., Kohn A. D., Roth R. A., Lawrence J. C., Jr Evidence of insulin-stimulated phosphorylation and activation of the mammalian target of rapamycin mediated by a protein kinase B signaling pathway. Proc Natl Acad Sci U S A. 1998 Jun 23;95(13):7772–7777. doi: 10.1073/pnas.95.13.7772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Shigemitsu K., Tsujishita Y., Hara K., Nanahoshi M., Avruch J., Yonezawa K. Regulation of translational effectors by amino acid and mammalian target of rapamycin signaling pathways. Possible involvement of autophagy in cultured hepatoma cells. J Biol Chem. 1999 Jan 8;274(2):1058–1065. doi: 10.1074/jbc.274.2.1058. [DOI] [PubMed] [Google Scholar]
  25. Tsakiridis T., McDowell H. E., Walker T., Downes C. P., Hundal H. S., Vranic M., Klip A. Multiple roles of phosphatidylinositol 3-kinase in regulation of glucose transport, amino acid transport, and glucose transporters in L6 skeletal muscle cells. Endocrinology. 1995 Oct;136(10):4315–4322. doi: 10.1210/endo.136.10.7664650. [DOI] [PubMed] [Google Scholar]
  26. Van der Kaay J., Beck M., Gray A., Downes C. P. Distinct phosphatidylinositol 3-kinase lipid products accumulate upon oxidative and osmotic stress and lead to different cellular responses. J Biol Chem. 1999 Dec 10;274(50):35963–35968. doi: 10.1074/jbc.274.50.35963. [DOI] [PubMed] [Google Scholar]
  27. Wang X., Campbell L. E., Miller C. M., Proud C. G. Amino acid availability regulates p70 S6 kinase and multiple translation factors. Biochem J. 1998 Aug 15;334(Pt 1):261–267. doi: 10.1042/bj3340261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Xu G., Kwon G., Marshall C. A., Lin T. A., Lawrence J. C., Jr, McDaniel M. L. Branched-chain amino acids are essential in the regulation of PHAS-I and p70 S6 kinase by pancreatic beta-cells. A possible role in protein translation and mitogenic signaling. J Biol Chem. 1998 Oct 23;273(43):28178–28184. doi: 10.1074/jbc.273.43.28178. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES