Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1996 Apr 1;315(Pt 1):271–279. doi: 10.1042/bj3150271

Sustained signalling from the insulin receptor after stimulation with insulin analogues exhibiting increased mitogenic potency.

B F Hansen 1, G M Danielsen 1, K Drejer 1, A R Sørensen 1, F C Wiberg 1, H H Klein 1, A G Lundemose 1
PMCID: PMC1217182  PMID: 8670118

Abstract

The metabolic and mitogenic potencies of six different insulin analogues were determined by measuring glucose transport in primary adipocytes and DNA synthesis in CHO cells respectively. Three analogues showed a disproportionately high mitogenic potency compared with their metabolic potency, and were up to 7 times more mitogenically than metabolically potent when compared with human insulin. The mitogenic/metabolic potency ratio of the analogues was found to be inversely correlated with the insulin receptor dissociation rate constant (Kd) in an exponential fashion (r=0.99), with a disproportionately greater increase in mitogenic potential compared with metabolic potential for analogues with Kd values of less than 40% of that of human insulin. To investigate the molecular mechanisms behind the correlation between the increased half-life of the receptor-ligand complex (low Kd) and mitogenicity, 3 h time-course experiments were performed. Slow ligand dissociation from the insulin receptor induced a parallel sustained activation of the insulin receptor tyrosine kinase. A similar pattern was observed for insulin receptor autophosphorylation and Shc phosphorylation, whereas the duration of insulin receptor substrate-1 phosphorylation with low-Kd analogues and with insulin was similar Thus the increased half-life of the ligand-receptor complex induces sustained activation of the insulin receptor tyrosine kinase and sustained phosphorylation of Shc, which may be the cause of the disproportionately high mitogenic potency seen for some insulin analogues.

Full Text

The Full Text of this article is available as a PDF (561.1 KB).

Selected References

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

  1. Andersen A. S., Kjeldsen T., Wiberg F. C., Christensen P. M., Rasmussen J. S., Norris K., Møller K. B., Møller N. P. Changing the insulin receptor to possess insulin-like growth factor I ligand specificity. Biochemistry. 1990 Aug 14;29(32):7363–7366. doi: 10.1021/bi00484a002. [DOI] [PubMed] [Google Scholar]
  2. Avruch J., Zhang X. F., Kyriakis J. M. Raf meets Ras: completing the framework of a signal transduction pathway. Trends Biochem Sci. 1994 Jul;19(7):279–283. doi: 10.1016/0968-0004(94)90005-1. [DOI] [PubMed] [Google Scholar]
  3. Backer J. M., Myers M. G., Jr, Shoelson S. E., Chin D. J., Sun X. J., Miralpeix M., Hu P., Margolis B., Skolnik E. Y., Schlessinger J. Phosphatidylinositol 3'-kinase is activated by association with IRS-1 during insulin stimulation. EMBO J. 1992 Sep;11(9):3469–3479. doi: 10.1002/j.1460-2075.1992.tb05426.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brange J., Owens D. R., Kang S., Vølund A. Monomeric insulins and their experimental and clinical implications. Diabetes Care. 1990 Sep;13(9):923–954. doi: 10.2337/diacare.13.9.923. [DOI] [PubMed] [Google Scholar]
  5. De Meyts P. The structural basis of insulin and insulin-like growth factor-I receptor binding and negative co-operativity, and its relevance to mitogenic versus metabolic signalling. Diabetologia. 1994 Sep;37 (Suppl 2):S135–S148. doi: 10.1007/BF00400837. [DOI] [PubMed] [Google Scholar]
  6. Debant A., Clauser E., Ponzio G., Filloux C., Auzan C., Contreres J. O., Rossi B. Replacement of insulin receptor tyrosine residues 1162 and 1163 does not alter the mitogenic effect of the hormone. Proc Natl Acad Sci U S A. 1988 Nov;85(21):8032–8036. doi: 10.1073/pnas.85.21.8032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Di Guglielmo G. M., Baass P. C., Ou W. J., Posner B. I., Bergeron J. J. Compartmentalization of SHC, GRB2 and mSOS, and hyperphosphorylation of Raf-1 by EGF but not insulin in liver parenchyma. EMBO J. 1994 Sep 15;13(18):4269–4277. doi: 10.1002/j.1460-2075.1994.tb06747.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dikic I., Schlessinger J., Lax I. PC12 cells overexpressing the insulin receptor undergo insulin-dependent neuronal differentiation. Curr Biol. 1994 Aug 1;4(8):702–708. doi: 10.1016/s0960-9822(00)00155-x. [DOI] [PubMed] [Google Scholar]
  9. Drejer K., Kruse V., Larsen U. D., Hougaard P., Bjørn S., Gammeltoft S. Receptor binding and tyrosine kinase activation by insulin analogues with extreme affinities studied in human hepatoma HepG2 cells. Diabetes. 1991 Nov;40(11):1488–1495. doi: 10.2337/diab.40.11.1488. [DOI] [PubMed] [Google Scholar]
  10. Drejer K. The bioactivity of insulin analogues from in vitro receptor binding to in vivo glucose uptake. Diabetes Metab Rev. 1992 Oct;8(3):259–285. doi: 10.1002/dmr.5610080305. [DOI] [PubMed] [Google Scholar]
  11. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  12. Hosomi Y., Shii K., Ogawa W., Matsuba H., Yoshida M., Okada Y., Yokono K., Kasuga M., Baba S., Roth R. A. Characterization of a 60-kilodalton substrate of the insulin receptor kinase. J Biol Chem. 1994 Apr 15;269(15):11498–11502. [PubMed] [Google Scholar]
  13. Karnieli E., Zarnowski M. J., Hissin P. J., Simpson I. A., Salans L. B., Cushman S. W. Insulin-stimulated translocation of glucose transport systems in the isolated rat adipose cell. Time course, reversal, insulin concentration dependency, and relationship to glucose transport activity. J Biol Chem. 1981 May 25;256(10):4772–4777. [PubMed] [Google Scholar]
  14. Klein H. H., Kowalewski B., Drenckhan M., Neugebauer S., Matthaei S., Kotzke G. A microtiter well assay system to measure insulin activation of insulin receptor kinase in intact human mononuclear cells. Decreased insulin effect in cells from patients with NIDDM. Diabetes. 1993 Jun;42(6):883–890. doi: 10.2337/diab.42.6.883. [DOI] [PubMed] [Google Scholar]
  15. Kuhné M. R., Pawson T., Lienhard G. E., Feng G. S. The insulin receptor substrate 1 associates with the SH2-containing phosphotyrosine phosphatase Syp. J Biol Chem. 1993 Jun 5;268(16):11479–11481. [PubMed] [Google Scholar]
  16. Lee C. H., Li W., Nishimura R., Zhou M., Batzer A. G., Myers M. G., Jr, White M. F., Schlessinger J., Skolnik E. Y. Nck associates with the SH2 domain-docking protein IRS-1 in insulin-stimulated cells. Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11713–11717. doi: 10.1073/pnas.90.24.11713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Markussen J., Diers I., Hougaard P., Langkjaer L., Norris K., Snel L., Sørensen A. R., Sørensen E., Voigt H. O. Soluble, prolonged-acting insulin derivatives. III. Degree of protraction, crystallizability and chemical stability of insulins substituted in positions A21, B13, B23, B27 and B30. Protein Eng. 1988 Jul;2(2):157–166. doi: 10.1093/protein/2.2.157. [DOI] [PubMed] [Google Scholar]
  18. Marshall C. J. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell. 1995 Jan 27;80(2):179–185. doi: 10.1016/0092-8674(95)90401-8. [DOI] [PubMed] [Google Scholar]
  19. McPherson G. A. Analysis of radioligand binding experiments. A collection of computer programs for the IBM PC. J Pharmacol Methods. 1985 Nov;14(3):213–228. doi: 10.1016/0160-5402(85)90034-8. [DOI] [PubMed] [Google Scholar]
  20. Miralpeix M., Sun X. J., Backer J. M., Myers M. G., Jr, Araki E., White M. F. Insulin stimulates tyrosine phosphorylation of multiple high molecular weight substrates in Fao hepatoma cells. Biochemistry. 1992 Sep 22;31(37):9031–9039. doi: 10.1021/bi00152a046. [DOI] [PubMed] [Google Scholar]
  21. Pang L., Milarski K. L., Ohmichi M., Takata Y., Olefsky J. M., Saltiel A. R. Mutation of the two carboxyl-terminal tyrosines in the insulin receptor results in enhanced activation of mitogen-activated protein kinase. J Biol Chem. 1994 Apr 8;269(14):10604–10608. [PubMed] [Google Scholar]
  22. Ponzio G., Contreres J. O., Debant A., Baron V., Gautier N., Dolais-Kitabgi J., Rossi B. Use of an anti-insulin receptor antibody to discriminate between metabolic and mitogenic effects of insulin: correlation with receptor autophosphorylation. EMBO J. 1988 Dec 20;7(13):4111–4117. doi: 10.1002/j.1460-2075.1988.tb03305.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pronk G. J., McGlade J., Pelicci G., Pawson T., Bos J. L. Insulin-induced phosphorylation of the 46- and 52-kDa Shc proteins. J Biol Chem. 1993 Mar 15;268(8):5748–5753. [PubMed] [Google Scholar]
  24. Rolband G. C., Williams J. F., Webster N. J., Olefsky J. M. Deletion of the insulin receptor beta-subunit acidic domain results in enhanced metabolic signaling. Endocrinology. 1993 Sep;133(3):1437–1443. doi: 10.1210/endo.133.3.8396020. [DOI] [PubMed] [Google Scholar]
  25. Sadoul J. L., Peyron J. F., Ballotti R., Debant A., Fehlmann M., Van Obberghen E. Identification of a cellular 110 000-Da protein substrate for the insulin-receptor kinase. Biochem J. 1985 May 1;227(3):887–892. doi: 10.1042/bj2270887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sasaoka T., Draznin B., Leitner J. W., Langlois W. J., Olefsky J. M. Shc is the predominant signaling molecule coupling insulin receptors to activation of guanine nucleotide releasing factor and p21ras-GTP formation. J Biol Chem. 1994 Apr 8;269(14):10734–10738. [PubMed] [Google Scholar]
  27. Sasaoka T., Rose D. W., Jhun B. H., Saltiel A. R., Draznin B., Olefsky J. M. Evidence for a functional role of Shc proteins in mitogenic signaling induced by insulin, insulin-like growth factor-1, and epidermal growth factor. J Biol Chem. 1994 May 6;269(18):13689–13694. [PubMed] [Google Scholar]
  28. Scahill S. J., Devos R., Van der Heyden J., Fiers W. Expression and characterization of the product of a human immune interferon cDNA gene in Chinese hamster ovary cells. Proc Natl Acad Sci U S A. 1983 Aug;80(15):4654–4658. doi: 10.1073/pnas.80.15.4654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schäffer L., Larsen U. D., Linde S., Hejnaes K. R., Skriver L. Characterization of the three 125I-iodination isomers of human insulin-like growth factor I (IGF1). Biochim Biophys Acta. 1993 Dec 8;1203(2):205–209. doi: 10.1016/0167-4838(93)90084-5. [DOI] [PubMed] [Google Scholar]
  30. Skolnik E. Y., Batzer A., Li N., Lee C. H., Lowenstein E., Mohammadi M., Margolis B., Schlessinger J. The function of GRB2 in linking the insulin receptor to Ras signaling pathways. Science. 1993 Jun 25;260(5116):1953–1955. doi: 10.1126/science.8316835. [DOI] [PubMed] [Google Scholar]
  31. Sun X. J., Crimmins D. L., Myers M. G., Jr, Miralpeix M., White M. F. Pleiotropic insulin signals are engaged by multisite phosphorylation of IRS-1. Mol Cell Biol. 1993 Dec;13(12):7418–7428. doi: 10.1128/mcb.13.12.7418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sun X. J., Rothenberg P., Kahn C. R., Backer J. M., Araki E., Wilden P. A., Cahill D. A., Goldstein B. J., White M. F. Structure of the insulin receptor substrate IRS-1 defines a unique signal transduction protein. Nature. 1991 Jul 4;352(6330):73–77. doi: 10.1038/352073a0. [DOI] [PubMed] [Google Scholar]
  33. Takata Y., Webster N. J., Olefsky J. M. Intracellular signaling by a mutant human insulin receptor lacking the carboxyl-terminal tyrosine autophosphorylation sites. J Biol Chem. 1992 May 5;267(13):9065–9070. [PubMed] [Google Scholar]
  34. Takata Y., Webster N. J., Olefsky J. M. Mutation of the two carboxyl-terminal tyrosines results in an insulin receptor with normal metabolic signaling but enhanced mitogenic signaling properties. J Biol Chem. 1991 May 15;266(14):9135–9139. [PubMed] [Google Scholar]
  35. Thies R. S., Ullrich A., McClain D. A. Augmented mitogenesis and impaired metabolic signaling mediated by a truncated insulin receptor. J Biol Chem. 1989 Aug 5;264(22):12820–12825. [PubMed] [Google Scholar]
  36. Traverse S., Seedorf K., Paterson H., Marshall C. J., Cohen P., Ullrich A. EGF triggers neuronal differentiation of PC12 cells that overexpress the EGF receptor. Curr Biol. 1994 Aug 1;4(8):694–701. doi: 10.1016/s0960-9822(00)00154-8. [DOI] [PubMed] [Google Scholar]
  37. Uchida T., Matozaki T., Noguchi T., Yamao T., Horita K., Suzuki T., Fujioka Y., Sakamoto C., Kasuga M. Insulin stimulates the phosphorylation of Tyr538 and the catalytic activity of PTP1C, a protein tyrosine phosphatase with Src homology-2 domains. J Biol Chem. 1994 Apr 22;269(16):12220–12228. [PubMed] [Google Scholar]
  38. White M. F., Kahn C. R. The insulin signaling system. J Biol Chem. 1994 Jan 7;269(1):1–4. [PubMed] [Google Scholar]
  39. Whitesell R. R., Gliemann J. Kinetic parameters of transport of 3-O-methylglucose and glucose in adipocytes. J Biol Chem. 1979 Jun 25;254(12):5276–5283. [PubMed] [Google Scholar]
  40. Wilden P. A., Backer J. M., Kahn C. R., Cahill D. A., Schroeder G. J., White M. F. The insulin receptor with phenylalanine replacing tyrosine-1146 provides evidence for separate signals regulating cellular metabolism and growth. Proc Natl Acad Sci U S A. 1990 May;87(9):3358–3362. doi: 10.1073/pnas.87.9.3358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wilden P. A., Siddle K., Haring E., Backer J. M., White M. F., Kahn C. R. The role of insulin receptor kinase domain autophosphorylation in receptor-mediated activities. Analysis with insulin and anti-receptor antibodies. J Biol Chem. 1992 Jul 5;267(19):13719–13727. [PubMed] [Google Scholar]
  42. Zapf A., Hsu D., Olefsky J. M. Comparison of the intracellular itineraries of insulin-like growth factor-I and insulin and their receptors in Rat-1 fibroblasts. Endocrinology. 1994 Jun;134(6):2445–2452. doi: 10.1210/endo.134.6.8194471. [DOI] [PubMed] [Google Scholar]
  43. de Meyts P., Roth J., Neville D. M., Jr, Gavin J. R., 3rd, Lesniak M. A. Insulin interactions with its receptors: experimental evidence for negative cooperativity. Biochem Biophys Res Commun. 1973 Nov 1;55(1):154–161. doi: 10.1016/s0006-291x(73)80072-5. [DOI] [PubMed] [Google Scholar]

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

RESOURCES