Skip to main content
SpringerLink
Log in

Insulin-Degrading Enzyme in a Human Colon Adenocarcinoma Cell Line (Caco-2)

  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

Abstract

The activity of insulin-degrading enzyme (IDE), a thiol metalloprotease degrading insulin in many insulin target cells, was determined in human colon adenocarcinoma (Caco-2) cells. Insulin-degrading activity was localized in the cytosol of Caco-2 cells, accounting for 88% of total activity. Western blots and immunoprecipitation showed that IDE was present in the cytosol of Caco-2 cells and contributed to more than 93% cytosolic insulin-degrading activity. Cytosolic insulin degradation was strongly inhibited by IDE inhibitors, including N-ethylmaleimide, 1,10-phenanthroline, p-chloromericuribenzoate, and EDTA, but was not significantly or not as extensively inhibited by strong inhibitors of proteasome, i.e., chymostatin, soybean trypsin inhibitor, leupeptin, and Dip-F. These results suggest that IDE is present in Caco-2 cells, that Caco-2 IDE has properties similar to those of its counterparts in insulin-target tissues, and that it significantly contributes to intracellular insulin degradation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

REFERENCES

  1. A short note. Pharm. J. 252:564 (1994).

    Google Scholar 

  2. W. D. Vlahos, T. A. Seemayer and J. F. Yale. Diabetes prevention in BB rats by inhibition of endogenous insulin secretion. Metab. 40, 825–829 (1991).

    Google Scholar 

  3. Z. Shao, Y. Li, R. Krishnamoorthy, T. Chermak and A. K. Mitra. Differential effects of anionic, cationic, nonionic, and physiologic surfactant on the dissociation, α-chymotryptic degradation, and enteral absorption of insulin hexamers. Pharm. Res. 10, 243–250 (1993).

    Google Scholar 

  4. S. Fujii, T. Yokoyama, T, K. Ikegaya, F. Sato and N. Yokoo. Promoting effect of the new chymotrypsin inhibitor FK-448 on the intestinal absorption of insulin in rats and dogs. J. Pharm. Pharmacol. 37, 545–549 (1985).

    Google Scholar 

  5. R. J. Schilling and A. K. Mitra. Degradation of insulin by trypsin and alpha-chymotrypsin. Pharm. Res. 8, 721–727 (1991).

    Google Scholar 

  6. A. Yamamoto, E. Hayakawa and V. H. L. Lee. Insulin and proinsulin proteolysis in mucosal homogenates of the rabbit: implications in peptide delivery from nonoral routes. Life Sci. 47, 2465–2474 (1990).

    Google Scholar 

  7. W. C. Duckworth. Insulin degradation:mechanisms, products, and significance, Endocr. Rev. 9, 319–345 (1988).

    Google Scholar 

  8. W. C. Duckworth. Insulin degrading enzyme, In (P. Cuatrecasas and S. Jacobs) Insulin, Springer-Verlag, Berlin Heilelberg, Germany (1990).

    Google Scholar 

  9. W. L. Kuo, A. G. Montag and M. R. Rosner. Insulin-degrading enzyme is differentially expressed and developmentally regulated in various rat tissues. Endocrinol. 132, 604–611 (1993).

    Google Scholar 

  10. A. R. Hilgers, R. A. Conradi and P. S. Burton. Caco-2 cell monolayers as a model for drug transport across the intestinal mucosa. Pharm. Res. 7, 902–910 (1990).

    Google Scholar 

  11. S. Howell, A. J. Kenny and A. J. Turner. A survey of membrane peptidases in two human colonic cell lines, Caco-2 and HT-29, Biochem. J. 284, 595–601 (1992).

    Google Scholar 

  12. A. H. Dantzig, D. C. Duckworth and L. B. Tabas. Transport mechanisms responsible for the absorption of loracarbef, cefixime, and cefuroxime axetil into human intestinal Caco-2 cells, Biochimica et Biophysica Acta 1191, 7–13 (1994).

    Google Scholar 

  13. R. S. MacDonald, W. H. Thornton, Jr. and T. L. Bean. Insulin and IGF-1 receptors in a human intestinal adenocarcinoma cell line (Caco-2): regulation of Na+ glucose transport across the brush-border, J. Receptor Res. 13, 1093–1113 (1993).

    Google Scholar 

  14. J. P. F., Bai, M. Hu, P., Subramanian, H. I. Mosberg, and G. L. Amidon. Utilization of peptide carrier system to improve the intestinal absorption: Targeting prolidase as a prodrug converting enzyme. J. Pharm. Sci. 81, 113–116 (1992).

    Google Scholar 

  15. M. M. Bradford. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 (1976).

    Google Scholar 

  16. W. C. Duckworth, M. A. Heinemann and A. Kitabchi. Purification of insulin-specific protease by affinity chromatography, Proc. Nat. Acad. Sci. 69, 3698–3702 (1972).

    Google Scholar 

  17. K. Yokono, Y. Imamura, K. Shii, H. Sakai and S. Baba. Purification and characterization of insulin-degrading enzyme from pig skeletal muscle, Endocrinol. 108, 1527–1532 (1981).

    Google Scholar 

  18. W. C. Duckworth, F. G. Hamel, J. Liepnieks, D. Peavy, B. Frank and R. Rabkin. Insulin degradation products from perfused rat kidney. Am. J. Physiol. 256, E208–E214 (1989).

    Google Scholar 

  19. K. Shii, K. Yokono, S. Bara and R. A. Roth. Purification and characterization of insulin-degrading enzyme from human erythrocytes. Diabetes 35, 675–683 (1986).

    Google Scholar 

  20. A. Tsuji and K. Kurachi. Isolation and characterization of a novel large protease accumulated in mammalian cells in the presence of inhibitors. J. Biol. Chem. 264, 16093–16099 (1989).

    Google Scholar 

  21. A. P. Arrigo, K. Tanaka, A. L. Goldberg and W. J. Welch. Identity of the 19S ‘prosome’ particle with the large multifunctional protease complex of mammalian cells (the proteasone). Nature 331, 192–194 (1988).

    Google Scholar 

  22. K. Yokono, Y. Imamura, K. Shii, H. Sakai and S. Baba. Purification and characterization of insulin-degrading enzyme from pig skeletal muscle, Endocrinol. 108, 1527–1532 (1981).

    Google Scholar 

  23. K. Shii and R. A. Roth. Inhibition of insulin degradation by hepatoma cells after microinjection of monoclonal antibodies to a specific cytosolic protease, Proc. Natl. Acad. Sci. 83, 4147–4151 (1986).

    Google Scholar 

  24. K. Shii, A. Baba, K. Yokono, and R. A. Roth. Covalent linkage of 1251-insulin to a cytosolic insulin-degrading enzyme, J. Biol. Chem. 260, 6503–6506 (1985).

    Google Scholar 

  25. J. J. Golstein and J. N. Livingston. An evaluation of the importance of lysosomal and neutral cytosol proteases in insulin degradation by adipocytes. Endocrinol. 108, 953–960 (1981).

    Google Scholar 

  26. W. L. Kuo, B. D. Gehm, M. R. Rosner, W. Li and G. Keller, Inducible expression and cellular localization of insulin-degrading enzyme in a stably transfected cell line. J. Biol. Chem. 269, 22599–22606 (1994).

    Google Scholar 

  27. A. Dorn, H. G. Bernstein, M. Reiser, A. Rinne, S. Ansorge. Degradation of insulin and glucagon in developing rat kidney: immunolocalization of insulin-glucagon-specific protease and quantitive estimation. J. Histochem. Cytochem. 34, 411–417 (1986).

    Google Scholar 

  28. S. Harada, R. M. Smith, J. A. Smith, L. Jarett, Inhibition of insulin-degrading enzyme increases translocation of insulin to the nucleus in H35 rat hepatoma cells: evidence of a cytosolic pathway, Endocrinol. 132, 2293–2298 (1993).

    Google Scholar 

  29. H. Kirschke and A. J. Barrett. Chemistry of lysosomal proteases in (F. J. Ballard ed) “From lysosomes: Their role in protein breakdown”. pp: 193–238, Academic Press, London (1987).

    Google Scholar 

  30. R. A. Roth, M. L. Mesirow, D. J. Cassell, K. Yokono and Baba S. Characterization of an insulin-degrading enzyme from cul tured human lymphocytes. Diabetes Res. Clin. Pract. 1, 31–39 (1985).

    Google Scholar 

  31. W. C. Duckworth, F. G. Hamel, R. Bennett, M. P. Ryan and R. A. Roth. Human red blood cell insulin-degrading enzyme and rat skeletal muscle insulin protease share antigenic sites and generate identical products from insulin, J. Biol. Chem. 265, 2984–2987 (1990).

    Google Scholar 

Download references

Authors
  1. Jane P. F. Bai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Matt J. P. Hsu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. W. Thomas Shier
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bai, J.P.F., Hsu, M.J.P. & Shier, W.T. Insulin-Degrading Enzyme in a Human Colon Adenocarcinoma Cell Line (Caco-2). Pharm Res 12, 513–517 (1995). https://doi.org/10.1023/A:1016241610649

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1016241610649

Search

Navigation