Processing of insulin by bovine endothelial cells in culture. Internalization without degradation.
Insulin binding and processing was studied in monolayer cultures of bovine aortic endothelial cells. Specific 125I-insulin binding was both time and temperature dependent. Maximum binding at 37 degrees C occurred at 90 min, and was 3.8%/mg protein and, at 15 degrees C, 7%/mg protein at 4 h. 125I-insulin was crosslinked to its receptor using disuccinimidyl suberate (DSS), and the structure of the receptor complex was identified by SDS-polyacrylamide gel electrophoresis and autoradiography; a major band with Mr = 145,000 was identified, which corresponds to the alpha-subunit of the insulin receptor reported in other tissues. Receptor-bound insulin was internalized, and both the rate and the amount of internalization were temperature dependent. The rate of internalization was slowest at 4 degrees C, and fastest at 37 degrees C, and the maximum amount of 125I-insulin internalized in 120 min was 16% at 4 degrees C, 45% at 15 degrees C, and 81% at 37 degrees C. Despite the high rate of internalization, endothelial cells do not appear to degrade insulin significantly, as determined by gel chromatography and TCA solubility (7% at 4 h) of media-associated radioactivity. In addition, the majority of internalized insulin (75%) was released by 60 min, largely as intact insulin. Chloroquine treatment at high concentration did not exert any major effect on insulin binding or degradation within the first 60 min, but thereafter produced a marked increase in cell-associated radioactivity.(ABSTRACT TRUNCATED AT 250 WORDS)
D000345:Affinity Labels; D000818:Animals; D001011:Aorta / Q000166:cytology / Q000378:metabolism; D001345:Autoradiography; D002417:Cattle; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology; D002850:Chromatography, Gel; D004727:Endothelium / Q000166:cytology / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism*; D013696:Temperature
D000345:Affinity Labels; D007328:Insulin; D002738:Chloroquine; D011972:Receptor, Insulin