Skip to main content
SpringerLink
Log in

Transepithelial Transport of Insulin: I. Insulin Degradation by Insulin-Degrading Enzyme in Small Intestinal Epithelium

  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

Abstract

Purpose. The purpose of this study was to determine the existence of insulin-degrading enzyme (EC 3.4.22.11) (IDE) in rat intestinal enterocytes.

Methods. Subcellular fractionation, biochemical characterization, immunoprecipitation, and western blots were employed.

Results. Insulin-degrading activity was localized in the cytosol, constituting 92% of total insulin-degrading activity. Cytosolic insulin-degrading activity had a pH optimum of 7.5, was almost completely inhibited by IDE inhibitors (N-ethylmaleimide, 1,10-phenanthroline, EDTA, p-chloromercuribenzoate, bacitracin), but was not or only weakly inhibited by others (aprotinin, chymostatin, leupeptin, and diisopropyl phosphofluoridate.) Further, cytosolic insulin-degrading activity had a Km of 78 nM, sharing a similar Km value with insulin-degrading enzyme in non-purified forms. Approximately, 87 ± 1.7% of cytosolic insulin-degrading activity was removed by the monoclonal antibody to IDE. On the SDS gel, the molecular weight of cytosolic IDE was 110 KD which is the same as that of human IDE.

Conclusions. IDE is the major enzyme which degrades insulin in enterocytes.

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.

Similar content being viewed by others

REFERENCES

  1. 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 

  2. 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 

  3. 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 

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

    Google Scholar 

  5. 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 

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

    Google Scholar 

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

    Google Scholar 

  8. 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 

  9. D. J. Pillion, V. Ganapathy, and F. H. Leibach. Identification of insulin receptors on the mucosal surface of colon epithelial cells. J. Biol. Chem. 260:5244–5247 (1985).

    Google Scholar 

  10. R. L. Gingerich, W. R. Gilbert, P. G. Comens, and J. R. Gavin III. Identification and characterization of insulin receptors in basolateral membranes of dog intestinal mucosa. Diabetes 36:1124–1129 (1987).

    Google Scholar 

  11. N. Gallo-Payet and J. S. Hugon. Insulin receptors in isolated adult mouse intestinal cells: Studies in vivo and in organ culture. Endocrinol. 114:1885–1892 (1984).

    Google Scholar 

  12. M. Bendayan, E. Ziv, R. Ben-Sasson, H. Bar-On, and M. Kidron. Morpho-cytochemical and biochemical evidence for insulin absorption by the rat ileal epithelium. Diabeteologia, 33:197–204 (1990).

    Google Scholar 

  13. M. Kessler, O. Acuto, C. Storelli, H. Murer, M. Muller, and G. Semenza. A modified procedure for the rapid preparation of efficiently transporting vesicles form small intestinal brush border membranes. Biochim. Biophys. Acta 506:136–154 (1978).

    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. J. P. F. Bai. Subcellular distribution of proteolytic activities degrading bioactive peptides and analogues in the small intestinal and colonic enterocytes. J. Pharm. Pharmacol. 46:671–675 (1994).

    Google Scholar 

  17. M. Igarashi and V. P. Hollander. Acid phosphatase from rat liver: Purification, crystallization, and properties. J. Biol. Chem. 243:6084–6089 (1968).

    Google Scholar 

  18. M. Reim, C. Bahrke, R. Kuchelkorn, and T. Kuwert. Investigation of enzyme activities in severe burns of the anterior eye segment, Graefe's Arch. Clin. Exp. Ophthalmol. 231:308–312 (1993).

    Google Scholar 

  19. 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 

  20. 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 

  21. 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 

  22. 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 

  23. 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 

  24. 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 proteasome). Nature 331:192–194 (1988).

    Google Scholar 

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

    Google Scholar 

  26. 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 

  27. 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 

  28. U. Derewenda, Z. S. Derewenda, G. G. Dodson, and R. E. Hubbard, Insulin structure. In P. Cuatrecasas and S. Jacobs (eds.), Insulin, Springer-Verlag, Berlin Heidelberg, Germany, 1990.

    Google Scholar 

  29. A. H. Pekar and B. H. Frank. Conformation of proinsulin. A comparison of insulin and proinsulin self-association of neutral pH. Biochem. 11:4013–4016 (1972).

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pharmaceutics, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota, 55455

    Jane P. F. Bai & L. L. Chang

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

    You can also search for this author in PubMed Google Scholar

  2. L. L. Chang
    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., Chang, L.L. Transepithelial Transport of Insulin: I. Insulin Degradation by Insulin-Degrading Enzyme in Small Intestinal Epithelium. Pharm Res 12, 1171–1175 (1995). https://doi.org/10.1023/A:1016263926946

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1016263926946

Search

Navigation