Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2000 May 15;348(Pt 1):83–91.

Chronic insulin effects on insulin signalling and GLUT4 endocytosis are reversed by metformin.

P R Pryor 1, S C Liu 1, A E Clark 1, J Yang 1, G D Holman 1, D Tosh 1
PMCID: PMC1221039  PMID: 10794717

Abstract

Decreases in insulin-responsive glucose transport and associated levels of cell surface GLUT4 occur in rat adipocytes maintained in culture for 20 h under hyperinsulinaemic and hyperglycaemic conditions. We have investigated whether this defect is due to reduced signalling from the insulin receptor, GLUT4 expression or impaired GLUT4 trafficking. The effects of chronic insulin treatment on glucose transport and GLUT4 trafficking were ameliorated by inclusion of metformin in the culture medium. In comparison with the ic insulin treatment attenuated changes in signalling processes leading to glucose transport. These included insulin receptor tyrosine phosphorylation, phosphoinositide 3-kinase activity and Akt activity, which were all reduced by 60-70%. Inclusion of metformin in the culture medium prevented the effects of the chronic insulin treatment on these signalling processes. In comparison with cells maintained in culture without insulin, the total expression of GLUT4 protein was not significantly altered by chronic insulin treatment, although the level of GLUT1 expression was increased. Trafficking rate constants for wortmannin-induced cell-surface loss of GLUT4 and GLUT1 were assessed by 2-N-4-(1-azi-2, 2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannose-4-yloxy)-2-propyla min e (ATB-BMPA) photolabelling. In comparison with cells acutely treated with insulin, chronic insulin treatment resulted in a doubling of the rate constants for GLUT4 endocytosis. These results suggest that the GLUT4 endocytosis process is very sensitive to the perturbations in signalling that occur under hyperinsulinaemic and hyperglycaemic conditions, and that the resulting elevation of endocytosis accounts for the reduced levels of net GLUT4 translocation observed.

Full Text

The Full Text of this article is available as a PDF (261.6 KB).

Selected References

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

  1. Björnholm M., Kawano Y., Lehtihet M., Zierath J. R. Insulin receptor substrate-1 phosphorylation and phosphatidylinositol 3-kinase activity in skeletal muscle from NIDDM subjects after in vivo insulin stimulation. Diabetes. 1997 Mar;46(3):524–527. doi: 10.2337/diab.46.3.524. [DOI] [PubMed] [Google Scholar]
  2. Calera M. R., Martinez C., Liu H., Jack A. K., Birnbaum M. J., Pilch P. F. Insulin increases the association of Akt-2 with Glut4-containing vesicles. J Biol Chem. 1998 Mar 27;273(13):7201–7204. doi: 10.1074/jbc.273.13.7201. [DOI] [PubMed] [Google Scholar]
  3. Cheatham B., Volchuk A., Kahn C. R., Wang L., Rhodes C. J., Klip A. Insulin-stimulated translocation of GLUT4 glucose transporters requires SNARE-complex proteins. Proc Natl Acad Sci U S A. 1996 Dec 24;93(26):15169–15173. doi: 10.1073/pnas.93.26.15169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cooksey R. C., Hebert L. F., Jr, Zhu J. H., Wofford P., Garvey W. T., McClain D. A. Mechanism of hexosamine-induced insulin resistance in transgenic mice overexpressing glutamine:fructose-6-phosphate amidotransferase: decreased glucose transporter GLUT4 translocation and reversal by treatment with thiazolidinedione. Endocrinology. 1999 Mar;140(3):1151–1157. doi: 10.1210/endo.140.3.6563. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Czech M. P., Corvera S. Signaling mechanisms that regulate glucose transport. J Biol Chem. 1999 Jan 22;274(4):1865–1868. doi: 10.1074/jbc.274.4.1865. [DOI] [PubMed] [Google Scholar]
  7. Davidson M. B., Peters A. L. An overview of metformin in the treatment of type 2 diabetes mellitus. Am J Med. 1997 Jan;102(1):99–110. doi: 10.1016/s0002-9343(96)00353-1. [DOI] [PubMed] [Google Scholar]
  8. Dominguez L. J., Davidoff A. J., Srinivas P. R., Standley P. R., Walsh M. F., Sowers J. R. Effects of metformin on tyrosine kinase activity, glucose transport, and intracellular calcium in rat vascular smooth muscle. Endocrinology. 1996 Jan;137(1):113–121. doi: 10.1210/endo.137.1.8536601. [DOI] [PubMed] [Google Scholar]
  9. Galuska D., Nolte L. A., Zierath J. R., Wallberg-Henriksson H. Effect of metformin on insulin-stimulated glucose transport in isolated skeletal muscle obtained from patients with NIDDM. Diabetologia. 1994 Aug;37(8):826–832. doi: 10.1007/BF00404340. [DOI] [PubMed] [Google Scholar]
  10. Garvey W. T., Olefsky J. M., Marshall S. Insulin receptor down-regulation is linked to an insulin-induced postreceptor defect in the glucose transport system in rat adipocytes. J Clin Invest. 1985 Jul;76(1):22–30. doi: 10.1172/JCI111950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Garvey W. T., Olefsky J. M., Matthaei S., Marshall S. Glucose and insulin co-regulate the glucose transport system in primary cultured adipocytes. A new mechanism of insulin resistance. J Biol Chem. 1987 Jan 5;262(1):189–197. [PubMed] [Google Scholar]
  12. Goodyear L. J., Giorgino F., Sherman L. A., Carey J., Smith R. J., Dohm G. L. Insulin receptor phosphorylation, insulin receptor substrate-1 phosphorylation, and phosphatidylinositol 3-kinase activity are decreased in intact skeletal muscle strips from obese subjects. J Clin Invest. 1995 May;95(5):2195–2204. doi: 10.1172/JCI117909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Handberg A., Vaag A., Vinten J., Beck-Nielsen H. Decreased tyrosine kinase activity in partially purified insulin receptors from muscle of young, non-obese first degree relatives of patients with type 2 (non-insulin-dependent) diabetes mellitus. Diabetologia. 1993 Jul;36(7):668–674. doi: 10.1007/BF00404079. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Holman G. D., Kasuga M. From receptor to transporter: insulin signalling to glucose transport. Diabetologia. 1997 Sep;40(9):991–1003. doi: 10.1007/s001250050780. [DOI] [PubMed] [Google Scholar]
  16. Holman G. D., Kozka I. J., Clark A. E., Flower C. J., Saltis J., Habberfield A. D., Simpson I. A., Cushman S. W. Cell surface labeling of glucose transporter isoform GLUT4 by bis-mannose photolabel. Correlation with stimulation of glucose transport in rat adipose cells by insulin and phorbol ester. J Biol Chem. 1990 Oct 25;265(30):18172–18179. [PubMed] [Google Scholar]
  17. Holman G. D., Lo Leggio L., Cushman S. W. Insulin-stimulated GLUT4 glucose transporter recycling. A problem in membrane protein subcellular trafficking through multiple pools. J Biol Chem. 1994 Jul 1;269(26):17516–17524. [PubMed] [Google Scholar]
  18. Hresko R. C., Heimberg H., Chi M. M., Mueckler M. Glucosamine-induced insulin resistance in 3T3-L1 adipocytes is caused by depletion of intracellular ATP. J Biol Chem. 1998 Aug 7;273(32):20658–20668. doi: 10.1074/jbc.273.32.20658. [DOI] [PubMed] [Google Scholar]
  19. Inoue G., Cheatham B., Kahn C. R. Different pathways of postreceptor desensitization following chronic insulin treatment and in cells overexpressing constitutively active insulin receptors. J Biol Chem. 1996 Nov 8;271(45):28206–28211. doi: 10.1074/jbc.271.45.28206. [DOI] [PubMed] [Google Scholar]
  20. Jhun B. H., Rampal A. L., Liu H., Lachaal M., Jung C. Y. Effects of insulin on steady state kinetics of GLUT4 subcellular distribution in rat adipocytes. Evidence of constitutive GLUT4 recycling. J Biol Chem. 1992 Sep 5;267(25):17710–17715. [PubMed] [Google Scholar]
  21. Kitamura T., Ogawa W., Sakaue H., Hino Y., Kuroda S., Takata M., Matsumoto M., Maeda T., Konishi H., Kikkawa U. Requirement for activation of the serine-threonine kinase Akt (protein kinase B) in insulin stimulation of protein synthesis but not of glucose transport. Mol Cell Biol. 1998 Jul;18(7):3708–3717. doi: 10.1128/mcb.18.7.3708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Klein H. H., Matthaei S., Drenkhan M., Ries W., Scriba P. C. The relationship between insulin binding, insulin activation of insulin-receptor tyrosine kinase, and insulin stimulation of glucose uptake in isolated rat adipocytes. Effects of isoprenaline. Biochem J. 1991 Mar 15;274(Pt 3):787–792. doi: 10.1042/bj2740787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kohn A. D., Summers S. A., Birnbaum M. J., Roth R. A. Expression of a constitutively active Akt Ser/Thr kinase in 3T3-L1 adipocytes stimulates glucose uptake and glucose transporter 4 translocation. J Biol Chem. 1996 Dec 6;271(49):31372–31378. doi: 10.1074/jbc.271.49.31372. [DOI] [PubMed] [Google Scholar]
  24. Kozka I. J., Clark A. E., Holman G. D. Chronic treatment with insulin selectively down-regulates cell-surface GLUT4 glucose transporters in 3T3-L1 adipocytes. J Biol Chem. 1991 Jun 25;266(18):11726–11731. [PubMed] [Google Scholar]
  25. Kozka I. J., Holman G. D. Metformin blocks downregulation of cell surface GLUT4 caused by chronic insulin treatment of rat adipocytes. Diabetes. 1993 Aug;42(8):1159–1165. doi: 10.2337/diab.42.8.1159. [DOI] [PubMed] [Google Scholar]
  26. Krook A., Roth R. A., Jiang X. J., Zierath J. R., Wallberg-Henriksson H. Insulin-stimulated Akt kinase activity is reduced in skeletal muscle from NIDDM subjects. Diabetes. 1998 Aug;47(8):1281–1286. doi: 10.2337/diab.47.8.1281. [DOI] [PubMed] [Google Scholar]
  27. Lima F. B., Thies R. S., Garvey W. T. Glucose and insulin regulate insulin sensitivity in primary cultured adipocytes without affecting insulin receptor kinase activity. Endocrinology. 1991 May;128(5):2415–2426. doi: 10.1210/endo-128-5-2415. [DOI] [PubMed] [Google Scholar]
  28. Matthaei S., Hamann A., Klein H. H., Benecke H., Kreymann G., Flier J. S., Greten H. Association of Metformin's effect to increase insulin-stimulated glucose transport with potentiation of insulin-induced translocation of glucose transporters from intracellular pool to plasma membrane in rat adipocytes. Diabetes. 1991 Jul;40(7):850–857. doi: 10.2337/diab.40.7.850. [DOI] [PubMed] [Google Scholar]
  29. Matthaei S., Reibold J. P., Hamann A., Benecke H., Häring H. U., Greten H., Klein H. H. In vivo metformin treatment ameliorates insulin resistance: evidence for potentiation of insulin-induced translocation and increased functional activity of glucose transporters in obese (fa/fa) Zucker rat adipocytes. Endocrinology. 1993 Jul;133(1):304–311. doi: 10.1210/endo.133.1.8391425. [DOI] [PubMed] [Google Scholar]
  30. Nolte L. A., Rincón J., Wahlström E. O., Craig B. W., Zierath J. R., Wallberg-Henriksson H. Hyperglycemia activates glucose transport in rat skeletal muscle via a Ca(2+)-dependent mechanism. Diabetes. 1995 Nov;44(11):1345–1348. doi: 10.2337/diab.44.11.1345. [DOI] [PubMed] [Google Scholar]
  31. Rossetti L., DeFronzo R. A., Gherzi R., Stein P., Andraghetti G., Falzetti G., Shulman G. I., Klein-Robbenhaar E., Cordera R. Effect of metformin treatment on insulin action in diabetic rats: in vivo and in vitro correlations. Metabolism. 1990 Apr;39(4):425–435. doi: 10.1016/0026-0495(90)90259-f. [DOI] [PubMed] [Google Scholar]
  32. Saad M. J., Maeda L., Brenelli S. L., Carvalho C. R., Paiva R. S., Velloso L. A. Defects in insulin signal transduction in liver and muscle of pregnant rats. Diabetologia. 1997 Feb;40(2):179–186. doi: 10.1007/s001250050660. [DOI] [PubMed] [Google Scholar]
  33. Santos R. F., Nomizo R., Wajhenberg B. L., Reaven G. M., Azhar S. Changes in insulin receptor tyrosine kinase activity associated with metformin treatment of type 2 diabetes. Diabete Metab. 1995 Oct;21(4):274–280. [PubMed] [Google Scholar]
  34. Satoh S., Nishimura H., Clark A. E., Kozka I. J., Vannucci S. J., Simpson I. A., Quon M. J., Cushman S. W., Holman G. D. Use of bismannose photolabel to elucidate insulin-regulated GLUT4 subcellular trafficking kinetics in rat adipose cells. Evidence that exocytosis is a critical site of hormone action. J Biol Chem. 1993 Aug 25;268(24):17820–17829. [PubMed] [Google Scholar]
  35. Simpson I. A., Yver D. R., Hissin P. J., Wardzala L. J., Karnieli E., Salans L. B., Cushman S. W. Insulin-stimulated translocation of glucose transporters in the isolated rat adipose cells: characterization of subcellular fractions. Biochim Biophys Acta. 1983 Dec 19;763(4):393–407. doi: 10.1016/0167-4889(83)90101-5. [DOI] [PubMed] [Google Scholar]
  36. Stith B. J., Goalstone M. L., Espinoza R., Mossel C., Roberts D., Wiernsperger N. The antidiabetic drug metformin elevates receptor tyrosine kinase activity and inositol 1,4,5-trisphosphate mass in Xenopus oocytes. Endocrinology. 1996 Jul;137(7):2990–2999. doi: 10.1210/endo.137.7.8770923. [DOI] [PubMed] [Google Scholar]
  37. Stith B. J., Woronoff K., Wiernsperger N. Stimulation of the intracellular portion of the human insulin receptor by the antidiabetic drug metformin. Biochem Pharmacol. 1998 Feb 15;55(4):533–536. doi: 10.1016/s0006-2952(97)00540-6. [DOI] [PubMed] [Google Scholar]
  38. Summers S. A., Garza L. A., Zhou H., Birnbaum M. J. Regulation of insulin-stimulated glucose transporter GLUT4 translocation and Akt kinase activity by ceramide. Mol Cell Biol. 1998 Sep;18(9):5457–5464. doi: 10.1128/mcb.18.9.5457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Taylor L. P., Holman G. D. Symmetrical kinetic parameters for 3-O-methyl-D-glucose transport in adipocytes in the presence and in the absence of insulin. Biochim Biophys Acta. 1981 Apr 6;642(2):325–335. doi: 10.1016/0005-2736(81)90449-1. [DOI] [PubMed] [Google Scholar]
  41. Thomas C. R., Turner S. L., Jefferson W. H., Bailey C. J. Prevention of dexamethasone-induced insulin resistance by metformin. Biochem Pharmacol. 1998 Nov 1;56(9):1145–1150. doi: 10.1016/s0006-2952(98)00151-8. [DOI] [PubMed] [Google Scholar]
  42. Thomson M. J., Williams M. G., Frost S. C. Development of insulin resistance in 3T3-L1 adipocytes. J Biol Chem. 1997 Mar 21;272(12):7759–7764. doi: 10.1074/jbc.272.12.7759. [DOI] [PubMed] [Google Scholar]
  43. Tordjman K. M., Leingang K. A., James D. E., Mueckler M. M. Differential regulation of two distinct glucose transporter species expressed in 3T3-L1 adipocytes: effect of chronic insulin and tolbutamide treatment. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7761–7765. doi: 10.1073/pnas.86.20.7761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Traxinger R. R., Marshall S. Coordinated regulation of glutamine:fructose-6-phosphate amidotransferase activity by insulin, glucose, and glutamine. Role of hexosamine biosynthesis in enzyme regulation. J Biol Chem. 1991 Jun 5;266(16):10148–10154. [PubMed] [Google Scholar]
  45. Traxinger R. R., Marshall S. Recovery of maximal insulin responsiveness and insulin sensitivity after induction of insulin resistance in primary cultured adipocytes. J Biol Chem. 1989 May 15;264(14):8156–8163. [PubMed] [Google Scholar]
  46. 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]
  47. 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]
  48. Yang J., Holman G. D. Comparison of GLUT4 and GLUT1 subcellular trafficking in basal and insulin-stimulated 3T3-L1 cells. J Biol Chem. 1993 Mar 5;268(7):4600–4603. [PubMed] [Google Scholar]
  49. Zierath J. R., He L., Gumà A., Odegoard Wahlström E., Klip A., Wallberg-Henriksson H. Insulin action on glucose transport and plasma membrane GLUT4 content in skeletal muscle from patients with NIDDM. Diabetologia. 1996 Oct;39(10):1180–1189. doi: 10.1007/BF02658504. [DOI] [PubMed] [Google Scholar]

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

RESOURCES