Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1997 Jan;17(1):190–198. doi: 10.1128/mcb.17.1.190

Differential effects of constitutively active phosphatidylinositol 3-kinase on glucose transport, glycogen synthase activity, and DNA synthesis in 3T3-L1 adipocytes.

E U Frevert 1, B B Kahn 1
PMCID: PMC231743  PMID: 8972199

Abstract

Phosphatidylinositol 3-kinase (PI3K) activation is necessary for many insulin-induced metabolic and mitogenic responses. However, it is unclear whether PI3K activation is sufficient for any of these effects. To address this question we increased PI3K activity in differentiated 3T3-L1 adipocytes by adenovirus-mediated expression of both the inter-SH2 region of the regulatory p85 subunit of PI3K (iSH2) and the catalytic p110 alpha subunit (p110). Coexpression resulted in PI3K activity that exceeded insulin-stimulated activity by two- to fivefold in cytosol, total membranes, and the low density microsome (LDM) fraction, the site of greatest insulin stimulation. While insulin increased glucose transport 15-fold, coexpression of iSH2-p110 increased transport (5.2-) +/- 0.7-fold with a parallel increase in GLUT4 translocation to the plasma membrane. Constitutive activation of PI3K had no effect on maximally insulin-stimulated glucose transport. Neither basal nor insulin-stimulated activity of glycogen synthase or mitogen-activated protein kinase was altered by iSH2-p110 coexpression. DNA synthesis was increased twofold by insulin in control 3T3-L1 adipocytes transduced with beta-galactosidase-encoding recombinant adenovirus, while iSH2-p110 coexpression increased DNA synthesis fivefold. These data indicate that (i) increased PI3K activity is sufficient to activate some but not all metabolic responses to insulin, (ii) activation of PI3K to levels exceeding the effect of insulin in adipocyte LDM results in only a partial stimulation of glucose transport, and (iii) increased PI3K activity in the absence of growth factor or oncoprotein stimulation is a potent stimulus of DNA synthesis.

Full Text

The Full Text of this article is available as a PDF (445.3 KB).

Selected References

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

  1. Becker T. C., Noel R. J., Coats W. S., Gómez-Foix A. M., Alam T., Gerard R. D., Newgard C. B. Use of recombinant adenovirus for metabolic engineering of mammalian cells. Methods Cell Biol. 1994;43(Pt A):161–189. doi: 10.1016/s0091-679x(08)60603-2. [DOI] [PubMed] [Google Scholar]
  2. Cheatham B., Kahn C. R. Insulin action and the insulin signaling network. Endocr Rev. 1995 Apr;16(2):117–142. doi: 10.1210/edrv-16-2-117. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Dallner G., Siekevitz P., Palade G. E. Biogenesis of endoplasmic reticulum membranes. II. Synthesis of constitutive microsomal enzymes in developing rat hepatocyte. J Cell Biol. 1966 Jul;30(1):97–117. doi: 10.1083/jcb.30.1.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. DeFronzo R. A., Bonadonna R. C., Ferrannini E. Pathogenesis of NIDDM. A balanced overview. Diabetes Care. 1992 Mar;15(3):318–368. doi: 10.2337/diacare.15.3.318. [DOI] [PubMed] [Google Scholar]
  6. DePaolo D., Reusch J. E., Carel K., Bhuripanyo P., Leitner J. W., Draznin B. Functional interactions of phosphatidylinositol 3-kinase with GTPase-activating protein in 3T3-L1 adipocytes. Mol Cell Biol. 1996 Apr;16(4):1450–1457. doi: 10.1128/mcb.16.4.1450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Frost S. C., Lane M. D. Evidence for the involvement of vicinal sulfhydryl groups in insulin-activated hexose transport by 3T3-L1 adipocytes. J Biol Chem. 1985 Mar 10;260(5):2646–2652. [PubMed] [Google Scholar]
  8. 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]
  9. Hara K., Yonezawa K., Sakaue H., Kotani K., Kotani K., Kojima A., Waterfield M. D., Kasuga M. Normal activation of p70 S6 kinase by insulin in cells overexpressing dominant negative 85kD subunit of phosphoinositide 3-kinase. Biochem Biophys Res Commun. 1995 Mar 17;208(2):735–741. doi: 10.1006/bbrc.1995.1399. [DOI] [PubMed] [Google Scholar]
  10. Heller-Harrison R. A., Morin M., Guilherme A., Czech M. P. Insulin-mediated targeting of phosphatidylinositol 3-kinase to GLUT4-containing vesicles. J Biol Chem. 1996 Apr 26;271(17):10200–10204. doi: 10.1074/jbc.271.17.10200. [DOI] [PubMed] [Google Scholar]
  11. Herbst J. J., Andrews G. C., Contillo L. G., Singleton D. H., Genereux P. E., Gibbs E. M., Lienhard G. E. Effect of the activation of phosphatidylinositol 3-kinase by a thiophosphotyrosine peptide on glucose transport in 3T3-L1 adipocytes. J Biol Chem. 1995 Oct 27;270(43):26000–26005. doi: 10.1074/jbc.270.43.26000. [DOI] [PubMed] [Google Scholar]
  12. Hu Q., Klippel A., Muslin A. J., Fantl W. J., Williams L. T. Ras-dependent induction of cellular responses by constitutively active phosphatidylinositol-3 kinase. Science. 1995 Apr 7;268(5207):100–102. doi: 10.1126/science.7701328. [DOI] [PubMed] [Google Scholar]
  13. Isakoff S. J., Taha C., Rose E., Marcusohn J., Klip A., Skolnik E. Y. The inability of phosphatidylinositol 3-kinase activation to stimulate GLUT4 translocation indicates additional signaling pathways are required for insulin-stimulated glucose uptake. Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10247–10251. doi: 10.1073/pnas.92.22.10247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kahn B. B. Facilitative glucose transporters: regulatory mechanisms and dysregulation in diabetes. J Clin Invest. 1992 May;89(5):1367–1374. doi: 10.1172/JCI115724. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kahn B. B., Rosen A. S., Bak J. F., Andersen P. H., Damsbo P., Lund S., Pedersen O. Expression of GLUT1 and GLUT4 glucose transporters in skeletal muscle of humans with insulin-dependent diabetes mellitus: regulatory effects of metabolic factors. J Clin Endocrinol Metab. 1992 May;74(5):1101–1109. doi: 10.1210/jcem.74.5.1569156. [DOI] [PubMed] [Google Scholar]
  16. Kaplan D. R., Whitman M., Schaffhausen B., Pallas D. C., White M., Cantley L., Roberts T. M. Common elements in growth factor stimulation and oncogenic transformation: 85 kd phosphoprotein and phosphatidylinositol kinase activity. Cell. 1987 Sep 25;50(7):1021–1029. doi: 10.1016/0092-8674(87)90168-1. [DOI] [PubMed] [Google Scholar]
  17. Katagiri H., Asano T., Ishihara H., Inukai K., Shibasaki Y., Kikuchi M., Yazaki Y., Oka Y. Overexpression of catalytic subunit p110alpha of phosphatidylinositol 3-kinase increases glucose transport activity with translocation of glucose transporters in 3T3-L1 adipocytes. J Biol Chem. 1996 Jul 19;271(29):16987–16990. doi: 10.1074/jbc.271.29.16987. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Klippel A., Escobedo J. A., Hirano M., Williams L. T. The interaction of small domains between the subunits of phosphatidylinositol 3-kinase determines enzyme activity. Mol Cell Biol. 1994 Apr;14(4):2675–2685. doi: 10.1128/mcb.14.4.2675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Klippel A., Reinhard C., Kavanaugh W. M., Apell G., Escobedo M. A., Williams L. T. Membrane localization of phosphatidylinositol 3-kinase is sufficient to activate multiple signal-transducing kinase pathways. Mol Cell Biol. 1996 Aug;16(8):4117–4127. doi: 10.1128/mcb.16.8.4117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kotani K., Carozzi A. J., Sakaue H., Hara K., Robinson L. J., Clark S. F., Yonezawa K., James D. E., Kasuga M. Requirement for phosphoinositide 3-kinase in insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. Biochem Biophys Res Commun. 1995 Apr 6;209(1):343–348. doi: 10.1006/bbrc.1995.1509. [DOI] [PubMed] [Google Scholar]
  22. Labarca C., Paigen K. A simple, rapid, and sensitive DNA assay procedure. Anal Biochem. 1980 Mar 1;102(2):344–352. doi: 10.1016/0003-2697(80)90165-7. [DOI] [PubMed] [Google Scholar]
  23. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  24. Lamphere L., Carpenter C. L., Sheng Z. F., Kallen R. G., Lienhard G. E. Activation of PI 3-kinase in 3T3-L1 adipocytes by association with insulin receptor substrate-1. Am J Physiol. 1994 Mar;266(3 Pt 1):E486–E494. doi: 10.1152/ajpendo.1994.266.3.E486. [DOI] [PubMed] [Google Scholar]
  25. Navé B. T., Haigh R. J., Hayward A. C., Siddle K., Shepherd P. R. Compartment-specific regulation of phosphoinositide 3-kinase by platelet-derived growth factor and insulin in 3T3-L1 adipocytes. Biochem J. 1996 Aug 15;318(Pt 1):55–60. doi: 10.1042/bj3180055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Okada T., Kawano Y., Sakakibara T., Hazeki O., Ui M. Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes. Studies with a selective inhibitor wortmannin. J Biol Chem. 1994 Feb 4;269(5):3568–3573. [PubMed] [Google Scholar]
  27. Powis G., Phil D. Inhibitors of phosphatidylinositol signalling as antiproliferative agents. Cancer Metastasis Rev. 1994 Mar;13(1):91–103. doi: 10.1007/BF00690420. [DOI] [PubMed] [Google Scholar]
  28. Rameh L. E., Chen C. S., Cantley L. C. Phosphatidylinositol (3,4,5)P3 interacts with SH2 domains and modulates PI 3-kinase association with tyrosine-phosphorylated proteins. Cell. 1995 Dec 1;83(5):821–830. doi: 10.1016/0092-8674(95)90195-7. [DOI] [PubMed] [Google Scholar]
  29. Rodriguez-Viciana P., Warne P. H., Vanhaesebroeck B., Waterfield M. D., Downward J. Activation of phosphoinositide 3-kinase by interaction with Ras and by point mutation. EMBO J. 1996 May 15;15(10):2442–2451. [PMC free article] [PubMed] [Google Scholar]
  30. Ruderman N. B., Kapeller R., White M. F., Cantley L. C. Activation of phosphatidylinositol 3-kinase by insulin. Proc Natl Acad Sci U S A. 1990 Feb;87(4):1411–1415. doi: 10.1073/pnas.87.4.1411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Serunian L. A., Auger K. R., Roberts T. M., Cantley L. C. Production of novel polyphosphoinositides in vivo is linked to cell transformation by polyomavirus middle T antigen. J Virol. 1990 Oct;64(10):4718–4725. doi: 10.1128/jvi.64.10.4718-4725.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Shepherd P. R., Navé B. T., Siddle K. Insulin stimulation of glycogen synthesis and glycogen synthase activity is blocked by wortmannin and rapamycin in 3T3-L1 adipocytes: evidence for the involvement of phosphoinositide 3-kinase and p70 ribosomal protein-S6 kinase. Biochem J. 1995 Jan 1;305(Pt 1):25–28. doi: 10.1042/bj3050025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sjölander A., Yamamoto K., Huber B. E., Lapetina E. G. Association of p21ras with phosphatidylinositol 3-kinase. Proc Natl Acad Sci U S A. 1991 Sep 15;88(18):7908–7912. doi: 10.1073/pnas.88.18.7908. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Thomas J. A., Schlender K. K., Larner J. A rapid filter paper assay for UDPglucose-glycogen glucosyltransferase, including an improved biosynthesis of UDP-14C-glucose. Anal Biochem. 1968 Oct 24;25(1):486–499. doi: 10.1016/0003-2697(68)90127-9. [DOI] [PubMed] [Google Scholar]
  35. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Walsh J. P., Caldwell K. K., Majerus P. W. Formation of phosphatidylinositol 3-phosphate by isomerization from phosphatidylinositol 4-phosphate. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9184–9187. doi: 10.1073/pnas.88.20.9184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Weiland M., Bahr F., Höhne M., Schürmann A., Ziehm D., Joost H. G. The signaling potential of the receptors for insulin and insulin-like growth factor I (IGF-I) in 3T3-L1 adipocytes: comparison of glucose transport activity, induction of oncogene c-fos, glucose transporter mRNA, and DNA-synthesis. J Cell Physiol. 1991 Dec;149(3):428–435. doi: 10.1002/jcp.1041490311. [DOI] [PubMed] [Google Scholar]
  38. Welsh G. I., Foulstone E. J., Young S. W., Tavaré J. M., Proud C. G. Wortmannin inhibits the effects of insulin and serum on the activities of glycogen synthase kinase-3 and mitogen-activated protein kinase. Biochem J. 1994 Oct 1;303(Pt 1):15–20. doi: 10.1042/bj3030015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wiese R. J., Mastick C. C., Lazar D. F., Saltiel A. R. Activation of mitogen-activated protein kinase and phosphatidylinositol 3'-kinase is not sufficient for the hormonal stimulation of glucose uptake, lipogenesis, or glycogen synthesis in 3T3-L1 adipocytes. J Biol Chem. 1995 Feb 17;270(7):3442–3446. doi: 10.1074/jbc.270.7.3442. [DOI] [PubMed] [Google Scholar]
  40. Yamauchi K., Holt K., Pessin J. E. Phosphatidylinositol 3-kinase functions upstream of Ras and Raf in mediating insulin stimulation of c-fos transcription. J Biol Chem. 1993 Jul 15;268(20):14597–14600. [PubMed] [Google Scholar]
  41. 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]
  42. Yki-Järvinen H., Koivisto V. A. Natural course of insulin resistance in type I diabetes. N Engl J Med. 1986 Jul 24;315(4):224–230. doi: 10.1056/NEJM198607243150404. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES