Degradation of insulin by isolated mouse pancreatic acini. Evidence for cell surface protease activity.
In the present study, we have used isolated mouse pancreatic acini to investigate the relationship between 125I-insulin binding and its degradation in order to probe the nature and cellular localization of the degradative process. In these cells, the proteolysis of 125I-insulin was dependent on time and cell concentration, and was saturated by unlabeled insulin with a Km of 290 nM. Since this value was much higher than the Kd for insulin binding to its receptor (1.1 nM), the data indicated that 125I-insulin degradation by acini occurred primarily via nonreceptor mechanisms. Several lines of evidence suggested that insulin was being degraded by the neutral thiol protease, insulin degrading enzyme (IDE). First, insulin degradation was inhibited by thiolreacting agents such as N-ethylmaleimide and p-chloromercuribenzoate. Second, the Km for degradation in acini was similar to the reported Km for IDE in other tissues. Third, the enzyme activity had a relative mol wt of approximately 130,000 by gel filtration, a value similar to that reported for purified IDE. Fourth, the degrading activity was removed with a specific antibody to IDE. Other lines of evidence suggested that enzymes located on the cell surface played a role in insulin degradation by acini. First, the nonpenetrating sulfhydryl reacting agent 5,5' dithiobis-2-nitrobenzoic acid blocked 125I-insulin degradation. Second, a specific antibody to IDE identified the presence of the enzyme on the cell surface. Third, chloroquine, leupeptin and antipain, agents that inhibit lysosomal function, did not influence 125I-insulin degradation. Fourth, highly purified pancreatic plasma membranes degraded 125I-insulin.
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002729:Chloromercuribenzoates / Q000494:pharmacology; D002850:Chromatography, Gel; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism*; D007457:Iodine Radioisotopes; D007515:Islets of Langerhans / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D010447:Peptide Hydrolases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000187:drug effects / Q000378:metabolism*
D002729:Chloromercuribenzoates; D007328:Insulin; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D011972:Receptor, Insulin; D010447:Peptide Hydrolases; D005033:Ethylmaleimide