Insulin binding to erythrocytes incubated in vitro at physiological temperature.
We have investigated the previously described time-dependent increase in specific insulin binding to freshly isolated human erythrocytes incubated at 37 C. We found that at 37 C, specific insulin binding to erythrocytes rose to and remained at equilibrium for the first 90 min of incubation; thereafter, it rose in a rapid linear fashion, directly related to the increase in the degradation of unbound insulin and paralleling the intensity of visible hemolysis. The rise in specific binding was intensified by conditions in which hemolysis was enhanced and attenuated by conditions designed to limit hemolysis or by agents capable of inhibiting the degradation of insulin despite hemolysis. Thus, gentle handling of the cells prevents hemolysis, the degradation of insulin, and the increase in apparent insulin binding. Furthermore, 5% albumin, 2.5 mM N-ethylmaleimide, or excess unlabeled insulin (100 micrograms/ml) inhibited insulin degradation (even in the presence of hemolysis) and prevented the rise in insulin binding. The rise in cell-associated radioactivity after 90 min of incubation at 37 C was due to cellular uptake of products of insulin degradation, since degraded [125I]insulin rapidly associated with freshly prepared erythrocytes. Acid extraction studies suggested that about 60% of the cell-associated degraded material was intracellular, while the remaining approximately 40% was bound to the cell surface. The data suggest that the rise in binding in erythrocytes incubated at 37 C is a result of insulin degradation products which associate with the cells. The generation of degraded insulin is due to hemolysate released from leaky cells, and this phenomenon is unique to the in vitro situation.
D000328:Adult; D000818:Animals; D001831:Body Temperature; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D006461:Hemolysis; D006801:Humans; D066298:In Vitro Techniques; D007328:Insulin / Q000097:blood*; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D013997:Time Factors
D007328:Insulin; D005033:Ethylmaleimide