Transepithelial transport of insulin: I. Insulin degradation by insulin-degrading enzyme in small intestinal epithelium.
OBJECTIVE
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.
D000818:Animals; D015153:Blotting, Western; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism*; D007339:Insulysin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D007421:Intestine, Small / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D008297:Male; D011233:Precipitin Tests; D051381:Rats; D017207:Rats, Sprague-Dawley; D013347:Subcellular Fractions / Q000187:drug effects / Q000201:enzymology
D004791:Enzyme Inhibitors; D007328:Insulin; D007339:Insulysin