Receptor-linked degradation of 125I-insulin is mediated by internalization in isolated rat hepatocytes.
When hepatocytes were freshly isolated from rat liver and incubated for various periods of time at 37 degrees C, the media from the incubation, when completely separated from the cells, actively degraded 125I-insulin. THis soluble protease activity was strongly inhibited by bacitracin but was unaffected by the lysosomatropic agent ammonium chloride (NH4Cl). When hepatocytes were incubated with 125I-insulin at 37 degrees C in the presence or absence of 8 mM NH4Cl the ligand initially bound to the plasma membrane and was subsequently internalized as a function of time. When hepatocytes were incubated at 37 degrees C for 30 minutes with 125I-insulin in the presence of bacitracin and NH4Cl or bacitracin alone and the cells were washed, diluted, and the cell-bound radioactivity allowed to dissociate, the percent intact 125I-insulin in the cell pellet and in the incubation media was greater in the presence of NH4Cl at each time point of incubation. Under these same conditions a higher proportion of the cell-associated radioactivity was internalized and a higher proportion was associated with lysosomes. The data suggest that receptor-mediated internalization is required for insulin degradation by the cell, and that this process, at least in part, involves lysosomal enzymes. Furthermore, the data demonstrate that internalization is not blocked by the presence of bacitracin or NH4Cl in the incubation media, but that degradation is inhibited.
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D001345:Autoradiography; D001414:Bacitracin / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism*; D007457:Iodine Radioisotopes; D008099:Liver / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism*
D007328:Insulin; D007457:Iodine Radioisotopes; D000643:Ammonium Chloride; D001414:Bacitracin; D011972:Receptor, Insulin