[Evidence for the internalization and degradation of insulin in cultured Bri-7K human lymphocytes].
We have studied the internalization of insulin and the mechanism of insulin degradation in cultured Bri-7K human lymphocytes. The internalization of 125I-insulin was observed by using an acid extraction technique by Heigler's method which removed surface-bound insulin, leaving only intracellular insulin associated with cells. Insulin binding to Bri-7K cells reached a peak at 15 min and showed a gradual fall thereafter at 37 degrees C. Internalization of insulin rose with a peak in 30 min, and its rate was nearly 25% of the total specific cell-associated radioactivity. Insulin degrading activities in Bri-7K cells were examined as to precipitability with trichloroacetic acid (TCA method), Sephadex G-50 column chromatography (gel chromatography method) and the ability to bind to specific receptors on Bri-7K cells (rebinding method). Under conditions demonstrating that the insulin degradation didn't take place in the medium but was cell-mediated, the insulin degradation was not demonstrated by the TCA method. Chromatographic studies showed that the extractable radioactivity consisted almost entirely of intact insulin, whereas the supernatant and the nonextractable radioactivities consisted of large molecular weight material and intact insulin. Thus, the apparent degradation of insulin was not detectable by the TCA and gel chromatography methods, however, using the rebinding method, we could obtain results demonstrating that fair amounts of insulin in the supernatant were degraded. These results suggest that the products of insulin degradation are peptides with a molecular size similar to insulin itself but with little or no receptor binding activity. In conclusion, insulin internalizes into Bri-7K cells at physiological temperature, and the mechanism of insulin degradation in Bri-7K cells seems to be a limiting proteolysis of insulin by specific enzyme(s).
D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D003470:Culture Media; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D008214:Lymphocytes / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism; D013696:Temperature
D003470:Culture Media; D007328:Insulin; D011972:Receptor, Insulin