title: Chymotrypsin substrate analogues inhibit endocytosis of insulin and insulin receptors in adipocytes.
abstract: To explore the possible role of proteolytic step(s) in receptor-mediated endocytosis of insulin, the effects of inhibitors of various classes of proteases on the internalization process were studied in isolated rat adipocytes. Intracellular accumulation of receptor-bound 125I-insulin at 37 degrees C was quantitated after rapidly dissociating surface-bound insulin with an acidic buffer (pH 3.0). Of the 23 protease inhibitors tested, only chymotrypsin substrate analogues inhibited insulin internalization. Internalization was decreased 62-90% by five different chymotrypsin substrate analogues: N-acetyl-Tyr ethyl ester, N-acetyl-Phe ethyl ester, N-acetyl-Trp ethyl ester, benzoyl-Tyr ethyl ester, and benzoyl-Tyr amide. The effect of the substrate analogues in inhibiting insulin internalization was dose-dependent, reversible, and required the full structural complement of a chymotrypsin substrate analogue. Cell surface receptor number was unaltered at 12 degrees C. However, concomitant with their inhibition of insulin internalization at 37 degrees C, the chymotrypsin substrate analogues caused a marked increase (160-380%) in surface-bound insulin, indicating trapping of insulin-receptor complexes on the cell surface. Additionally, 1 mM N-acetyl-Tyr ethyl ester decreased overall insulin degradation by 15-20% and also prevented the chloroquine-mediated increase in intracellular insulin, further indicating that surface-bound insulin was prevented from reaching intracellular chloroquine-sensitive degradation sites. The internalization of insulin receptors that were photoaffinity labeled on the cell surface with B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin was also inhibited 70-90% by the five chymotrypsin substrate analogues, as determined by the effects of the analogues on the accumulation of trypsin-insensitive (intracellular) 440-kD intact labeled receptors. In summary, these results show that chymotrypsin substrate analogues efficiently inhibit the internalization of insulin and insulin receptors in adipocytes and implicate a possible role for endogenous chymotrypsin-like enzyme(s) or related substances in receptor-mediated endocytosis of insulin.
mesh_terms: D000273:Adipose Tissue / Q000378:metabolism*; D000345:Affinity Labels; D000626:Aminopeptidases / Q000037:antagonists & inhibitors; D000818:Animals; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors; D002738:Chloroquine / Q000494:pharmacology; D002918:Chymotrypsin / Q000037:antagonists & inhibitors*; D004705:Endocytosis / Q000187:drug effects*; D007328:Insulin / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D011480:Protease Inhibitors / Q000494:pharmacology*; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism*; D013329:Structure-Activity Relationship; D011503:Transglutaminases / Q000037:antagonists & inhibitors; D014443:Tyrosine / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D000345:Affinity Labels; D007328:Insulin; D011480:Protease Inhibitors; D014443:Tyrosine; C007651:ethyl N-alpha-acetyl-tyrosinate; D002738:Chloroquine; D011503:Transglutaminases; D011972:Receptor, Insulin; D002268:Carboxypeptidases; D000626:Aminopeptidases; D002918:Chymotrypsin