Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1995 Nov 1;311(Pt 3):787–795. doi: 10.1042/bj3110787

Chloroquine augments the binding of insulin to its receptor.

A P Bevan 1, J R Christensen 1, J Tikerpae 1, G D Smith 1
PMCID: PMC1136071  PMID: 7487933

Abstract

The effect of chloroquine on the interaction of insulin with its receptor has been investigated under both equilibrium and non-equilibrium conditions. Chloroquine was found to augment insulin binding in a pH-dependent manner between pH 6.0 and pH 8.5, with the maximum occurring at approximately pH 7.0. Analysis of the equilibrium binding data in terms of independent binding sites gave equivocal results but suggested an increase in the high-affinity component. Analysis using the negative co-operativity binding model of De Meyts, Bianco and Roth [J. Biol. Chem. (1976) 251, 1877-1888] suggested that the affinity at both high and low occupancy was increased equally. The kinetics of association of insulin with the plasma-membrane receptor indicated that, although the net rate of association increased in the presence of chloroquine, this was due to a reduction in the dissociation rate rather than an increase in the association rate. This was confirmed by direct measurement of the rates of dissociation. Dissociation was found to be distinctly biphasic, with fast and slow components. Curve fitting suggested that the decrease in dissociation rate in the presence of chloroquine was not due to a decrease in either of the two dissociation rate constants, but rather to an increase in the amount of insulin dissociating by the slow component. It was also found that the increase in dissociation rate in the presence of excess insulin, ascribed to negative co-operativity, could be accounted for by an increase in the amount of insulin dissociating by the faster pathway, rather than by an increase in the dissociation rate constant. Thus chloroquine appears to have the opposite effect to excess insulin, and evidence was found for the induction of positive co-operativity in the insulin-receptor interaction at high chloroquine concentrations. Evidence was also found for the presence of low-affinity chloroquine binding sites with binding parameters similar to the concentration dependence of the chloroquine-induced augmentation of insulin binding.

Full text

PDF
787

Selected References

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

  1. Avruch J., Wallach D. F. Preparation and properties of plasma membrane and endoplasmic reticulum fragments from isolated rat fat cells. Biochim Biophys Acta. 1971 Apr 13;233(2):334–347. doi: 10.1016/0005-2736(71)90331-2. [DOI] [PubMed] [Google Scholar]
  2. Backer J. M., Kahn C. R., White M. F. The dissociation and degradation of internalized insulin occur in the endosomes of rat hepatoma cells. J Biol Chem. 1990 Sep 5;265(25):14828–14835. [PubMed] [Google Scholar]
  3. Backer J. M., Shoelson S. E., Haring E., White M. F. Insulin receptors internalize by a rapid, saturable pathway requiring receptor autophosphorylation and an intact juxtamembrane region. J Cell Biol. 1991 Dec;115(6):1535–1545. doi: 10.1083/jcb.115.6.1535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Balage M., Grizard J., Sornet C., Simon J., Dardevet D., Manin M. Insulin binding and receptor tyrosine kinase activity in rat liver and skeletal muscle: effect of starvation. Metabolism. 1990 Apr;39(4):366–373. doi: 10.1016/0026-0495(90)90250-g. [DOI] [PubMed] [Google Scholar]
  5. Ballotti R., Kowalski A., White M. F., Le Marchand-Brustel Y., Van Obberghen E. Insulin stimulates tyrosine phosphorylation of its receptor beta-subunit in intact rat hepatocytes. Biochem J. 1987 Jan 1;241(1):99–104. doi: 10.1042/bj2410099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blazar B. R., Whitley C. B., Kitabchi A. E., Tsai M. Y., Santiago J., White N., Stentz F. B., Brown D. M. In vivo chloroquine-induced inhibition of insulin degradation in a diabetic patient with severe insulin resistance. Diabetes. 1984 Dec;33(12):1133–1137. doi: 10.2337/diab.33.12.1133. [DOI] [PubMed] [Google Scholar]
  7. Christensen J. R., Smith G. D., Peters T. J. Characterization of insulin uptake into subcellular fractions of perfused rat liver using two different iodinated tracers. Cell Biochem Funct. 1985 Jan;3(1):13–19. doi: 10.1002/cbf.290030105. [DOI] [PubMed] [Google Scholar]
  8. De Meyts P., Roth J. Cooperativity in ligand binding: a new graphic analysis. Biochem Biophys Res Commun. 1975 Oct 27;66(4):1118–1126. doi: 10.1016/0006-291x(75)90473-8. [DOI] [PubMed] [Google Scholar]
  9. De Meyts P., Roth J. Cooperativity in ligand binding: a new graphic analysis. Biochem Biophys Res Commun. 1975 Oct 27;66(4):1118–1126. doi: 10.1016/0006-291x(75)90473-8. [DOI] [PubMed] [Google Scholar]
  10. DeMeyts P., Bainco A. R., Roth J. Site-site interactions among insulin receptors. Characterization of the negative cooperativity. J Biol Chem. 1976 Apr 10;251(7):1877–1888. [PubMed] [Google Scholar]
  11. Deger A., Krämer H., Rapp R., Koch R., Weber U. The nonclassical insulin binding of insulin receptors from rat liver is due to the presence of two interacting alpha-subunits in the receptor complex. Biochem Biophys Res Commun. 1986 Mar 13;135(2):458–464. doi: 10.1016/0006-291x(86)90016-1. [DOI] [PubMed] [Google Scholar]
  12. Dennis P. A., Aronson N. N., Jr The effects of low temperature and chloroquine on 125I-insulin degradation by the perfused rat liver. Arch Biochem Biophys. 1981 Nov;212(1):170–176. doi: 10.1016/0003-9861(81)90356-8. [DOI] [PubMed] [Google Scholar]
  13. Doherty J. J., 2nd, Kay D. G., Lai W. H., Posner B. I., Bergeron J. J. Selective degradation of insulin within rat liver endosomes. J Cell Biol. 1990 Jan;110(1):35–42. doi: 10.1083/jcb.110.1.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dorling P. R., Le Page R. N. A rapid high yield method for the preparation of rat liver cell plasma membranes. Biochim Biophys Acta. 1973 Aug 9;318(1):33–40. doi: 10.1016/0005-2736(73)90333-7. [DOI] [PubMed] [Google Scholar]
  15. Faure R., Baquiran G., Bergeron J. J., Posner B. I. The dephosphorylation of insulin and epidermal growth factor receptors. Role of endosome-associated phosphotyrosine phosphatase(s). J Biol Chem. 1992 Jun 5;267(16):11215–11221. [PubMed] [Google Scholar]
  16. Fujita-Yamaguchi Y., Choi S., Sakamoto Y., Itakura K. Purification of insulin receptor with full binding activity. J Biol Chem. 1983 Apr 25;258(8):5045–5049. [PubMed] [Google Scholar]
  17. Gammeltoft S. Insulin receptors: binding kinetics and structure-function relationship of insulin. Physiol Rev. 1984 Oct;64(4):1321–1378. doi: 10.1152/physrev.1984.64.4.1321. [DOI] [PubMed] [Google Scholar]
  18. Gu J. L., Goldfine I. D., Forsayeth J. R., De Meyts P. Reversal of insulin-induced negative cooperativity by monoclonal antibodies that stabilize the slowly dissociating ("Ksuper") state of the insulin receptor. Biochem Biophys Res Commun. 1988 Jan 29;150(2):694–701. doi: 10.1016/0006-291x(88)90447-0. [DOI] [PubMed] [Google Scholar]
  19. Hamel F. G., Mahoney M. J., Duckworth W. C. Degradation of intraendosomal insulin by insulin-degrading enzyme without acidification. Diabetes. 1991 Apr;40(4):436–443. doi: 10.2337/diab.40.4.436. [DOI] [PubMed] [Google Scholar]
  20. Jørgensen K. H., Larsen U. D. Homogeneous mono-(125)i-insulins. Preparation and characterization of mono-(125)i-(tyr a14)-and mono-(125)i-(tyr a19)-insulin. Diabetologia. 1980;19(6):546–554. doi: 10.1007/BF00253183. [DOI] [PubMed] [Google Scholar]
  21. Kahn C. R., Freychet P., Roth J., Neville D. M., Jr Quantitative aspects of the insulin-receptor interaction in liver plasma membranes. J Biol Chem. 1974 Apr 10;249(7):2249–2257. [PubMed] [Google Scholar]
  22. Khan M. N., Baquiran G., Brule C., Burgess J., Foster B., Bergeron J. J., Posner B. I. Internalization and activation of the rat liver insulin receptor kinase in vivo. J Biol Chem. 1989 Aug 5;264(22):12931–12940. [PubMed] [Google Scholar]
  23. Krupp M. N., Livingston J. N. Insulin binding to solubilized material from fat cell membranes: evidence for two binding species. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2593–2597. doi: 10.1073/pnas.75.6.2593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  25. Marsh J. W., Westley J., Steiner D. F. Insulin-receptor interactions. Presence of a positive cooperative effect. J Biol Chem. 1984 May 25;259(10):6641–6649. [PubMed] [Google Scholar]
  26. Maxfield F. R., Yamashiro D. J. Endosome acidification and the pathways of receptor-mediated endocytosis. Adv Exp Med Biol. 1987;225:189–198. doi: 10.1007/978-1-4684-5442-0_16. [DOI] [PubMed] [Google Scholar]
  27. Munson P. J., Rodbard D. Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem. 1980 Sep 1;107(1):220–239. doi: 10.1016/0003-2697(80)90515-1. [DOI] [PubMed] [Google Scholar]
  28. Murphy R. F., Powers S., Cantor C. R. Endosome pH measured in single cells by dual fluorescence flow cytometry: rapid acidification of insulin to pH 6. J Cell Biol. 1984 May;98(5):1757–1762. doi: 10.1083/jcb.98.5.1757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ohkuma S., Poole B. Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3327–3331. doi: 10.1073/pnas.75.7.3327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Olefsky J. M. The insulin receptor. A multifunctional protein. Diabetes. 1990 Sep;39(9):1009–1016. doi: 10.2337/diab.39.9.1009. [DOI] [PubMed] [Google Scholar]
  31. Pease R. J., Smith G. D., Peters T. J. Characterization of insulin degradation by rat-liver low-density vesicles. Eur J Biochem. 1987 Apr 1;164(1):251–257. doi: 10.1111/j.1432-1033.1987.tb11018.x. [DOI] [PubMed] [Google Scholar]
  32. Pease R. J., Smith G. D., Peters T. J. Degradation of endocytosed insulin in rat liver is mediated by low-density vesicles. Biochem J. 1985 May 15;228(1):137–146. doi: 10.1042/bj2280137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Powrie J. K., Smith G. D., Shojaee-Moradie F., Sönksen P. H., Jones R. H. Mode of action of chloroquine in patients with non-insulin-dependent diabetes mellitus. Am J Physiol. 1991 Jun;260(6 Pt 1):E897–E904. doi: 10.1152/ajpendo.1991.260.6.E897. [DOI] [PubMed] [Google Scholar]
  34. Schacterle G. R., Pollack R. L. A simplified method for the quantitative assay of small amounts of protein in biologic material. Anal Biochem. 1973 Feb;51(2):654–655. doi: 10.1016/0003-2697(73)90523-x. [DOI] [PubMed] [Google Scholar]
  35. Schäffer L. A model for insulin binding to the insulin receptor. Eur J Biochem. 1994 May 1;221(3):1127–1132. doi: 10.1111/j.1432-1033.1994.tb18833.x. [DOI] [PubMed] [Google Scholar]
  36. Seymour C. A., Peters T. J. Enzyme activities in human liver biopsies: assay methods and activities of some lysosomal and membrane-bound enzymes in control tissue and serum. Clin Sci Mol Med. 1977 Mar;52(3):229–239. doi: 10.1042/cs0520229. [DOI] [PubMed] [Google Scholar]
  37. Smith G. D., Amos T. A., Mahler R., Peters T. J. Effect of chloroquine on insulin and glucose homoeostasis in normal subjects and patients with non-insulin-dependent diabetes mellitus. Br Med J (Clin Res Ed) 1987 Feb 21;294(6570):465–467. doi: 10.1136/bmj.294.6570.465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Smith G. D., Christensen J. R., Rideout J. M., Peters T. J. Hepatic processing of insulin. Characterization of differential inhibition by weak bases. Eur J Biochem. 1989 May 1;181(2):287–294. doi: 10.1111/j.1432-1033.1989.tb14723.x. [DOI] [PubMed] [Google Scholar]
  39. Sorimachi K., Okayasu T., Yasumura Y. Increase in insulin binding affinity by chloroquine in cultured rat hepatoma cells. Endocr Res. 1987;13(1):49–60. doi: 10.1080/07435808709023662. [DOI] [PubMed] [Google Scholar]
  40. Trischitta V., Wong K. Y., Brunetti A., Scalisi R., Vigneri R., Goldfine I. D. Endocytosis, recycling, and degradation of the insulin receptor. Studies with monoclonal antireceptor antibodies that do not activate receptor kinase. J Biol Chem. 1989 Mar 25;264(9):5041–5046. [PubMed] [Google Scholar]
  41. Wang C. C., Goldfine I. D., Fujita-Yamaguchi Y., Gattner H. G., Brandenburg D., De Meyts P. Negative and positive site-site interactions, and their modulation by pH, insulin analogs, and monoclonal antibodies, are preserved in the purified insulin receptor. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8400–8404. doi: 10.1073/pnas.85.22.8400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. de Duve C., de Barsy T., Poole B., Trouet A., Tulkens P., Van Hoof F. Commentary. Lysosomotropic agents. Biochem Pharmacol. 1974 Sep 15;23(18):2495–2531. doi: 10.1016/0006-2952(74)90174-9. [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]
  44. de Vries C. P., Van Haeften T. W., Rao B. R., Van der Veen E. A. Effect of the insulin degradation inhibitors bacitracin and chloroquine on insulin binding data to H35 rat hepatoma cells. Diabete Metab. 1990 Mar-Apr;16(2):70–76. [PubMed] [Google Scholar]

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

RESOURCES