Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1989 Nov;86(21):8368–8372. doi: 10.1073/pnas.86.21.8368

Isoproterenol stimulates phosphorylation of the insulin-regulatable glucose transporter in rat adipocytes.

D E James 1, J Hiken 1, J C Lawrence Jr 1
PMCID: PMC298282  PMID: 2554313

Abstract

We have examined the acute effects of insulin and isoproterenol on the phosphorylation state of the insulin-regulatable glucose transporter (IRGT) in rat adipocytes. The IRGT was immunoprecipitated from either detergent-solubilized whole-cell homogenates or subcellular fractions of 32P-labeled fat cells and subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The 32P-labeled IRGT was detected by autoradiography as a species of apparent Mr 46,000. Insulin stimulated translocation of the IRGT from low-density microsomes to the plasma membrane but did not affect phosphorylation of the transporter in either fraction. Isoproterenol inhibited insulin-stimulated glucose transport by 40% but was without effect on the subcellular distribution of the transporter in either the presence or absence of insulin. Isoproterenol stimulated phosphorylation of the IRGT 2-fold. Incubating cells with dibutyryl-cAMP and 8-bromo-cAMP also stimulated phosphorylation 2-fold, and the transporter was phosphorylated in vitro when IRGT-enriched vesicles were incubated with cAMP-dependent protein kinase and [gamma-32P]ATP. These results suggest that isoproterenol stimulates phosphorylation of the IRGT via a cAMP-dependent pathway and that phosphorylation of the transporter may modulate its ability to transport glucose.

Full text

PDF
8368

Images in this article

8370Image
on p.8370
8371Image
on p.8371
8371Image
on p.8371

Selected References

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

  1. Avruch J., Alexander M. C., Palmer J. L., Pierce M. W., Nemenoff R. A., Blackshear P. J., Tipper J. P., Witters L. A. Role of insulin-stimulated protein phosphorylation in insulin action. Fed Proc. 1982 Aug;41(10):2629–2633. [PubMed] [Google Scholar]
  2. Birnbaum M. J., Haspel H. C., Rosen O. M. Cloning and characterization of a cDNA encoding the rat brain glucose-transporter protein. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5784–5788. doi: 10.1073/pnas.83.16.5784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blok J., Gibbs E. M., Lienhard G. E., Slot J. W., Geuze H. J. Insulin-induced translocation of glucose transporters from post-Golgi compartments to the plasma membrane of 3T3-L1 adipocytes. J Cell Biol. 1988 Jan;106(1):69–76. doi: 10.1083/jcb.106.1.69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chamberlain J. P. Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal Biochem. 1979 Sep 15;98(1):132–135. doi: 10.1016/0003-2697(79)90716-4. [DOI] [PubMed] [Google Scholar]
  5. Cohen P. The role of protein phosphorylation in the hormonal control of enzyme activity. Eur J Biochem. 1985 Sep 16;151(3):439–448. doi: 10.1111/j.1432-1033.1985.tb09121.x. [DOI] [PubMed] [Google Scholar]
  6. Cushman S. W., Wardzala L. J. Potential mechanism of insulin action on glucose transport in the isolated rat adipose cell. Apparent translocation of intracellular transport systems to the plasma membrane. J Biol Chem. 1980 May 25;255(10):4758–4762. [PubMed] [Google Scholar]
  7. Denton R. M. Early events in insulin actions. Adv Cyclic Nucleotide Protein Phosphorylation Res. 1986;20:293–341. [PubMed] [Google Scholar]
  8. Elbrink J., Bihler I. Membrane transport: its relation to cellular metabolic rates. Science. 1975 Jun 20;188(4194):1177–1184. doi: 10.1126/science.1096301. [DOI] [PubMed] [Google Scholar]
  9. Flier J. S., Mueckler M., McCall A. L., Lodish H. F. Distribution of glucose transporter messenger RNA transcripts in tissues of rat and man. J Clin Invest. 1987 Feb;79(2):657–661. doi: 10.1172/JCI112864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gibbs E. M., Allard W. J., Lienhard G. E. The glucose transporter in 3T3-L1 adipocytes is phosphorylated in response to phorbol ester but not in response to insulin. J Biol Chem. 1986 Dec 15;261(35):16597–16603. [PubMed] [Google Scholar]
  11. Haystead T. A., Sim A. T., Carling D., Honnor R. C., Tsukitani Y., Cohen P., Hardie D. G. Effects of the tumour promoter okadaic acid on intracellular protein phosphorylation and metabolism. Nature. 1989 Jan 5;337(6202):78–81. doi: 10.1038/337078a0. [DOI] [PubMed] [Google Scholar]
  12. James D. E., Brown R., Navarro J., Pilch P. F. Insulin-regulatable tissues express a unique insulin-sensitive glucose transport protein. Nature. 1988 May 12;333(6169):183–185. doi: 10.1038/333183a0. [DOI] [PubMed] [Google Scholar]
  13. James D. E., Burleigh K. M., Kraegen E. W. In vivo glucose metabolism in individual tissues of the rat. Interaction between epinephrine and insulin. J Biol Chem. 1986 May 15;261(14):6366–6374. [PubMed] [Google Scholar]
  14. James D. E., Lederman L., Pilch P. F. Purification of insulin-dependent exocytic vesicles containing the glucose transporter. J Biol Chem. 1987 Aug 25;262(24):11817–11824. [PubMed] [Google Scholar]
  15. James D. E., Strube M., Mueckler M. Molecular cloning and characterization of an insulin-regulatable glucose transporter. Nature. 1989 Mar 2;338(6210):83–87. doi: 10.1038/338083a0. [DOI] [PubMed] [Google Scholar]
  16. Joost H. G., Weber T. M., Cushman S. W. Qualitative and quantitative comparison of glucose transport activity and glucose transporter concentration in plasma membranes from basal and insulin-stimulated rat adipose cells. Biochem J. 1988 Jan 1;249(1):155–161. doi: 10.1042/bj2490155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Joost H. G., Weber T. M., Cushman S. W., Simpson I. A. Activity and phosphorylation state of glucose transporters in plasma membranes from insulin-, isoproterenol-, and phorbol ester-treated rat adipose cells. J Biol Chem. 1987 Aug 15;262(23):11261–11267. [PubMed] [Google Scholar]
  18. Kashiwagi A., Huecksteadt T. P., Foley J. E. The regulation of glucose transport by cAMP stimulators via three different mechanisms in rat and human adipocytes. J Biol Chem. 1983 Nov 25;258(22):13685–13692. [PubMed] [Google Scholar]
  19. Krebs E. G., Beavo J. A. Phosphorylation-dephosphorylation of enzymes. Annu Rev Biochem. 1979;48:923–959. doi: 10.1146/annurev.bi.48.070179.004423. [DOI] [PubMed] [Google Scholar]
  20. Kuroda M., Honnor R. C., Cushman S. W., Londos C., Simpson I. A. Regulation of insulin-stimulated glucose transport in the isolated rat adipocyte. cAMP-independent effects of lipolytic and antilipolytic agents. J Biol Chem. 1987 Jan 5;262(1):245–253. [PubMed] [Google Scholar]
  21. 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]
  22. Lawrence J. C., Jr, Hiken J. F., Inkster M., Scott C. W., Mumby M. C. Insulin stimulates the generation of an adipocyte phosphoprotein that is isolated with a monoclonal antibody against the regulatory subunit of bovine heart cAMP-dependent protein kinase. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3649–3653. doi: 10.1073/pnas.83.11.3649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lawrence J. C., Jr, James C. Activation of glycogen synthase by insulin in rat adipocytes. Evidence of hormonal stimulation of multisite dephosphorylation by glucose transport-dependent and -independent pathways. J Biol Chem. 1984 Jun 25;259(12):7975–7982. [PubMed] [Google Scholar]
  24. Lienhard G. E., Kim H. H., Ransome K. J., Gorga J. C. Immunological identification of an insulin-responsive glucose transporter. Biochem Biophys Res Commun. 1982 Apr 14;105(3):1150–1156. doi: 10.1016/0006-291x(82)91090-7. [DOI] [PubMed] [Google Scholar]
  25. Mueckler M., Caruso C., Baldwin S. A., Panico M., Blench I., Morris H. R., Allard W. J., Lienhard G. E., Lodish H. F. Sequence and structure of a human glucose transporter. Science. 1985 Sep 6;229(4717):941–945. doi: 10.1126/science.3839598. [DOI] [PubMed] [Google Scholar]
  26. Oka Y., Czech M. P. Photoaffinity labeling of insulin-sensitive hexose transporters in intact rat adipocytes. Direct evidence that latent transporters become exposed to the extracellular space in response to insulin. J Biol Chem. 1984 Jul 10;259(13):8125–8133. [PubMed] [Google Scholar]
  27. RODBELL M. METABOLISM OF ISOLATED FAT CELLS. I. EFFECTS OF HORMONES ON GLUCOSE METABOLISM AND LIPOLYSIS. J Biol Chem. 1964 Feb;239:375–380. [PubMed] [Google Scholar]
  28. Simpson I. A., Cushman S. W. Hormonal regulation of mammalian glucose transport. Annu Rev Biochem. 1986;55:1059–1089. doi: 10.1146/annurev.bi.55.070186.005211. [DOI] [PubMed] [Google Scholar]
  29. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  30. Smith U., Kuroda M., Simpson I. A. Counter-regulation of insulin-stimulated glucose transport by catecholamines in the isolated rat adipose cell. J Biol Chem. 1984 Jul 25;259(14):8758–8763. [PubMed] [Google Scholar]
  31. Suzuki K., Kono T. Evidence that insulin causes translocation of glucose transport activity to the plasma membrane from an intracellular storage site. Proc Natl Acad Sci U S A. 1980 May;77(5):2542–2545. doi: 10.1073/pnas.77.5.2542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Thorens B., Sarkar H. K., Kaback H. R., Lodish H. F. Cloning and functional expression in bacteria of a novel glucose transporter present in liver, intestine, kidney, and beta-pancreatic islet cells. Cell. 1988 Oct 21;55(2):281–290. doi: 10.1016/0092-8674(88)90051-7. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES