Evidence of the lack of receptor-mediated insulin degradation in human cultured lymphocytes (RPMI-1788 line).
We studied insulin degradation in human cultured lymphocytes (RPMI-1788 line) with a small but significant number of lysosomes under the electron microscope. Insulin degradation determined by the TCA solubility method was 64.6 +/- 1.2% (mean +/- SEM) at a trace concentration after the incubation with 2.0 x 10(7) cells (4.0 x 10(7) cells/ml) for 60 min at 37 degrees C. Because insulin degradation was 54.6 +/- 7.0% in the cell-free buffer in which 2.0 x 10(7) cells were previously incubated, most of the insulin was degraded outside of the cells. Gel filtration of the radioactive materials also revealed that most of the labeled insulin in the medium was degraded, and the main peak of the cell-associated radioactivities was intact labeled insulin. Chloroquine, a lysosomotropic agent, failed not only to increase insulin binding but also to decrease the insulin degradation. Other lysosomal protease inhibitors, antipain and leupeptin had also no effect on insulin degradation. In contrast, bacitracin (500 micrograms/ml) significantly decreased the insulin degradation analyzed by TCA solubility, receptor-rebinding, and the gel filtration method. These results suggest that insulin molecules are degraded by the enzymes leaked from the cells. The non-receptor mediated process, which is the bacitracin sensitive pathway, might be a general mechanism of insulin degradation in human cultured lymphocytes in vitro.
D001414:Bacitracin / Q000494:pharmacology; D002460:Cell Line; D002738:Chloroquine / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D008214:Lymphocytes / Q000378:metabolism* / Q000648:ultrastructure; D011480:Protease Inhibitors / Q000494:pharmacology; D011972:Receptor, Insulin / Q000502:physiology*
D007328:Insulin; D011480:Protease Inhibitors; D001414:Bacitracin; D002738:Chloroquine; D011972:Receptor, Insulin