Effect of Bacitracin on Retroendocytosis and Degradation of Insulin in Cultured Kidney Epithelial Cell Line

Abstract

In an earlier study, we described the presence of a retroendocytotic pathway for insulin in a cultured kidney epithelial cell line. Derived from the opossum kidney (OK), these cells possess many features of proximal tubule epithelium, which is the major site of kidney insulin metabolism. We studied the interaction between the retroendocytotic and the degradative pathways with bacitracin as a pharmacological probe. Monolayers of OK cells were loaded with 125I-labeled insulin over 30 min, acid washed to remove membrane-bound insulin, then incubated in fresh medium for 60 min while the release of intracellular radioactivity was monitored. In experiments carried out in the presence of bacitracin (2 mM), there was a twothirds increase in intracellular radioactivity at the end of the loading phase. Measurements made during the subsequent release phase showed that bacitracin reduced the release of degradation products. Thus, although controls released 72.1 ± 8.1% of the internalized radioactivity as trichloroacetic acid (TCA)-soluble products, bacitracin-treated cells released 59.2 ± 9.4% (P < 0.02). In contrast, release of TCAprecipitable insulin increased from 15.2 ± 4.6% in controls to 25.8 ± 3.7% in bacitracin-treated cells (P < 0.01). In separate experiments analyzed by gelexclusion chromatography, 6.4 ± 0.6% of radioactivity released from preloaded control cells into medium over 60 min was insulin sized compared to 29.7 ± 1.4% in bacitracin-treated cells. High-performance liquid chromotography revealed that 61.5 ± 3.5% of this insulin-sized material released from control cells preloaded with A14-insulin eluted as intact insulin and the remainder as unidentified intermediate degradation products. In the bacitracin group, 48.0 ± 2.1% of the radioactivity eluted with insulin, and 18.5 ± 0.6% eluted under a series of peaks representing intermediate products. Included in this material was a doublet, which has been identified in previous experiments with OK cells, other cell types, and liver endosomes. Previous characterization of this doublet indicates that it results from cleavage of insulin by insulin protease or a similar enzyme. We conclude that, by inhibiting the initial steps in the degradation of insulin, bacitracin diverts intact insulin and early degradation products from the degradative to the retroendocytotic pathway. These findings suggest that insulin degradation commences in an extralysosomal site, probably recycling endosomes.