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. 1996 Aug 15;318(Pt 1):203–205. doi: 10.1042/bj3180203

Phorbol esters stimulate phosphatidylinositol 3,4,5-trisphosphate production in 3T3-L1 adipocytes: implications for stimulation of glucose transport.

B T Navé 1, K Siddle 1, P R Shepherd 1
PMCID: PMC1217608  PMID: 8761472

Abstract

The effects of insulin and phorbol 12-myristate 13-acetate (PMA) on the levels of cellular phosphoinositides were investigated in 3T3-L1 adipocytes. Stimulation for 4 min with PMA (1 microM) or insulin (10 nM) increased levels of PtdIns(3,4,5)P3 approx. 2-fold and 6-fold respectively. PMA also had a small effect on the cellular levels of PtdIns4P, whereas insulin had no effect on PtdIns4P levels; levels of PtdIns(4,5)P2 and PtdIns3P were not significantly affected by either agent. Insulin increased the levels of the p85 alpha subunit of phosphoinositide (PI) 3-kinase associated with membranes, whereas PMA decreased levels of membrane-associated p85 alpha. PMA did not increase PI 3-kinase activity in anti-phosphotyrosine or anti-p85 immunoprecipitates. The stimulation of glucose transport by insulin or PMA was blocked by 100 nM wortmannin or 10 ng/ml LY294002, indicating that PI 3-kinase is essential for stimulation by both agents. In summary, these results demonstrate: (1) that PMA and insulin stimulate PtdIns(3,4,5)P3 production by distinct mechanisms in 3T3-L1 adipocytes, and (2) that stimulation of PtdIns(3,4,5)P3 production by PMA is likely to be important in signalling pathways leading from PMA stimulation to end-point responses such as glucose transport.

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Selected References

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  1. Abrams C. S., Wu H., Zhao W., Belmonte E., White D., Brass L. F. Pleckstrin inhibits phosphoinositide hydrolysis initiated by G-protein-coupled and growth factor receptors. A role for pleckstrin's PH domains. J Biol Chem. 1995 Jun 16;270(24):14485–14492. doi: 10.1074/jbc.270.24.14485. [DOI] [PubMed] [Google Scholar]
  2. Apgar J. R. Activation of protein kinase C in rat basophilic leukemia cells stimulates increased production of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate: correlation with actin polymerization. Mol Biol Cell. 1995 Jan;6(1):97–108. doi: 10.1091/mbc.6.1.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arcaro A., Wymann M. P. Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses. Biochem J. 1993 Dec 1;296(Pt 2):297–301. doi: 10.1042/bj2960297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berhanu P., Olefsky J. M. Effects of insulin and insulin-like agents on the glucose transport system of cultured human fibroblasts. Diabetes. 1981 Jun;30(6):523–529. doi: 10.2337/diab.30.6.523. [DOI] [PubMed] [Google Scholar]
  5. Blackshear P. J., Witters L. A., Girard P. R., Kuo J. F., Quamo S. N. Growth factor-stimulated protein phosphorylation in 3T3-L1 cells. Evidence for protein kinase C-dependent and -independent pathways. J Biol Chem. 1985 Oct 25;260(24):13304–13315. [PubMed] [Google Scholar]
  6. Buchner K. Protein kinase C in the transduction of signals toward and within the cell nucleus. Eur J Biochem. 1995 Mar 1;228(2):211–221. [PubMed] [Google Scholar]
  7. Cheatham B., Vlahos C. J., Cheatham L., Wang L., Blenis J., Kahn C. R. Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation. Mol Cell Biol. 1994 Jul;14(7):4902–4911. doi: 10.1128/mcb.14.7.4902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clarke J. F., Young P. W., Yonezawa K., Kasuga M., Holman G. D. Inhibition of the translocation of GLUT1 and GLUT4 in 3T3-L1 cells by the phosphatidylinositol 3-kinase inhibitor, wortmannin. Biochem J. 1994 Jun 15;300(Pt 3):631–635. doi: 10.1042/bj3000631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Exton J. H. Some thoughts on the mechanism of action of insulin. Diabetes. 1991 May;40(5):521–526. doi: 10.2337/diab.40.5.521. [DOI] [PubMed] [Google Scholar]
  10. Gibbs E. M., Calderhead D. M., Holman G. D., Gould G. W. Phorbol ester only partially mimics the effects of insulin on glucose transport and glucose-transporter distribution in 3T3-L1 adipocytes. Biochem J. 1991 Apr 1;275(Pt 1):145–150. doi: 10.1042/bj2750145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hara K., Yonezawa K., Sakaue H., Ando A., Kotani K., Kitamura T., Kitamura Y., Ueda H., Stephens L., Jackson T. R. 1-Phosphatidylinositol 3-kinase activity is required for insulin-stimulated glucose transport but not for RAS activation in CHO cells. Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7415–7419. doi: 10.1073/pnas.91.16.7415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jackson T. R., Stephens L. R., Hawkins P. T. Receptor specificity of growth factor-stimulated synthesis of 3-phosphorylated inositol lipids in Swiss 3T3 cells. J Biol Chem. 1992 Aug 15;267(23):16627–16636. [PubMed] [Google Scholar]
  13. Keller S. R., Lienhard G. E. Insulin signalling: the role of insulin receptor substrate 1. Trends Cell Biol. 1994 Apr;4(4):115–119. doi: 10.1016/0962-8924(94)90065-5. [DOI] [PubMed] [Google Scholar]
  14. Kelly K. L., Ruderman N. B. Insulin-stimulated phosphatidylinositol 3-kinase. Association with a 185-kDa tyrosine-phosphorylated protein (IRS-1) and localization in a low density membrane vesicle. J Biol Chem. 1993 Feb 25;268(6):4391–4398. [PubMed] [Google Scholar]
  15. Klip A., Ramlal T. Cytoplasmic Ca2+ during differentiation of 3T3-L1 adipocytes. Effect of insulin and relation to glucose transport. J Biol Chem. 1987 Jul 5;262(19):9141–9146. [PubMed] [Google Scholar]
  16. Kotani K., Yonezawa K., Hara K., Ueda H., Kitamura Y., Sakaue H., Ando A., Chavanieu A., Calas B., Grigorescu F. Involvement of phosphoinositide 3-kinase in insulin- or IGF-1-induced membrane ruffling. EMBO J. 1994 May 15;13(10):2313–2321. doi: 10.1002/j.1460-2075.1994.tb06515.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Merrall N. W., Wakelam M. J., Plevin R., Gould G. W. Insulin and platelet-derived growth factor acutely stimulate glucose transport in 3T3-L1 fibroblasts independently of protein kinase C. Biochim Biophys Acta. 1993 Jun 6;1177(2):191–198. doi: 10.1016/0167-4889(93)90040-v. [DOI] [PubMed] [Google Scholar]
  18. Petritsch C., Woscholski R., Edelmann H. M., Parker P. J., Ballou L. M. Selective inhibition of p70 S6 kinase activation by phosphatidylinositol 3-kinase inhibitors. Eur J Biochem. 1995 Jun 1;230(2):431–438. doi: 10.1111/j.1432-1033.1995.0431h.x. [DOI] [PubMed] [Google Scholar]
  19. Rodriguez-Viciana P., Warne P. H., Dhand R., Vanhaesebroeck B., Gout I., Fry M. J., Waterfield M. D., Downward J. Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature. 1994 Aug 18;370(6490):527–532. doi: 10.1038/370527a0. [DOI] [PubMed] [Google Scholar]
  20. Stephens L., Eguinoa A., Corey S., Jackson T., Hawkins P. T. Receptor stimulated accumulation of phosphatidylinositol (3,4,5)-trisphosphate by G-protein mediated pathways in human myeloid derived cells. EMBO J. 1993 Jun;12(6):2265–2273. doi: 10.1002/j.1460-2075.1993.tb05880.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Stephens L., Smrcka A., Cooke F. T., Jackson T. R., Sternweis P. C., Hawkins P. T. A novel phosphoinositide 3 kinase activity in myeloid-derived cells is activated by G protein beta gamma subunits. Cell. 1994 Apr 8;77(1):83–93. doi: 10.1016/0092-8674(94)90237-2. [DOI] [PubMed] [Google Scholar]
  22. Stoyanov B., Volinia S., Hanck T., Rubio I., Loubtchenkov M., Malek D., Stoyanova S., Vanhaesebroeck B., Dhand R., Nürnberg B. Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase. Science. 1995 Aug 4;269(5224):690–693. doi: 10.1126/science.7624799. [DOI] [PubMed] [Google Scholar]
  23. Tanaka C., Nishizuka Y. The protein kinase C family for neuronal signaling. Annu Rev Neurosci. 1994;17:551–567. doi: 10.1146/annurev.ne.17.030194.003003. [DOI] [PubMed] [Google Scholar]
  24. Tanti J. F., Rochet N., Grémeaux T., Van Obberghen E., Le Marchand-Brustel Y. Insulin-stimulated glucose transport in muscle. Evidence for a protein-kinase-C-dependent component which is unaltered in insulin-resistant mice. Biochem J. 1989 Feb 15;258(1):141–146. doi: 10.1042/bj2580141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Turinsky J., Bayly B. P., O'Sullivan D. M. 1,2-Diacylglycerol and ceramide levels in rat skeletal muscle and liver in vivo. Studies with insulin, exercise, muscle denervation, and vasopressin. J Biol Chem. 1990 May 15;265(14):7933–7938. [PubMed] [Google Scholar]
  26. Vlahos C. J., Matter W. F., Hui K. Y., Brown R. F. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem. 1994 Feb 18;269(7):5241–5248. [PubMed] [Google Scholar]
  27. Vogt B., Mushack J., Seffer E., Häring H. U. The translocation of the glucose transporter sub-types GLUT1 and GLUT4 in isolated fat cells is differently regulated by phorbol esters. Biochem J. 1991 May 1;275(Pt 3):597–600. doi: 10.1042/bj2750597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Woscholski R., Waterfield M. D., Parker P. J. Purification and biochemical characterization of a mammalian phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase. J Biol Chem. 1995 Dec 29;270(52):31001–31007. doi: 10.1074/jbc.270.52.31001. [DOI] [PubMed] [Google Scholar]
  29. Yamamoto K., Lapetina E. G. Protein kinase C-mediated formation of phosphatidylinositol 3,4-bisphosphate in human platelets. Biochem Biophys Res Commun. 1990 Apr 30;168(2):466–472. doi: 10.1016/0006-291x(90)92344-y. [DOI] [PubMed] [Google Scholar]
  30. Yang J., Clarke J. F., Ester C. J., Young P. W., Kasuga M., Holman G. D. Phosphatidylinositol 3-kinase acts at an intracellular membrane site to enhance GLUT4 exocytosis in 3T3-L1 cells. Biochem J. 1996 Jan 1;313(Pt 1):125–131. doi: 10.1042/bj3130125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Zvelebil M. J., MacDougall L., Leevers S., Volinia S., Vanhaesebroeck B., Gout I., Panayotou G., Domin J., Stein R., Pages F. Structural and functional diversity of phosphoinositide 3-kinases. Philos Trans R Soc Lond B Biol Sci. 1996 Feb 29;351(1336):217–223. doi: 10.1098/rstb.1996.0019. [DOI] [PubMed] [Google Scholar]

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