Degradation of insulin by isolated rat renal cortical endosomes

Abstract

It has been widely accepted that in kidney, degradation of insulin occurs in lysosomes. It is thought that after internalization into the cell, insulin dissociates from its receptor, which then recycles to the plasma membrane, while the hormone is transported in endosomes to the lysosomes, where it is degraded. However, earlier studies from this laboratory have suggested that insulin may also be degraded in an extralysosomal site, most likely endosomes. Indeed, studies in other tissues, most notably liver, have shown that insulin degradation does take place in endosomes. Since the intracellular processing of insulin differs between different tissues and cell types, and as the kidney is a major site of insulin degradation, we set out to determine directly whether endosomes degrade internalized insulin in the kidney. Rats were injected with [125I]monoiodoinsulin, labeled at either the A14 or B26 tyrosine. After killing, the kidney cortex was excised, and heavy endosomes were prepared by differential and isopycnic centrifugation. The isolated [125I]insulin-loaded endosomes were incubated for up to 60 min in intracellular medium, and degradation of [125I] insulin was estimated by means of precipitation in trichloroacetic acid. In the presence of ATP (10 mM), the percent degraded was increased over the control value (no ATP present), but under these circumstances, degradation was greater when the endosomes contained internalized 125I-labeled [B26]insulin than with A14-labeled [125I]insulin (26% vs. 13% degraded/h). In the absence of ATP, the percent degraded increased when the pH of the incubation medium was lowered. Radiolabeled material was extracted from endosomes, and Sephadex G-50 analysis revealed the presence of high mol wt, insulin-size, and low mol wt material. Reverse phase HPLC analysis of the insulin-size material revealed the presence of intact insulin and a number of degradation products. The elution profiles of some of these products were consistent with that reported to arise from the action of the insulin-degrading enzyme. Western blot analysis with the antiinsulin-degrading enzyme monoclonal antibody 9B12 confirmed the presence of the enzyme in endosomal preparations. We conclude that degradation of insulin does occur in kidney cortical endosomes, probably involves the insulin-degrading enzyme, and results in the formation of relatively large intermediate products as well as low mol wt products.