An Evaluation of the Importance of Lysosomal and Neutral Cytosol Proteases in Insulin Degradation by Adipocytes*

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Abstract
The properties of insulin degradation by lysosomal and cytosolic proteases from adipocytes were studied and compared to the insulin-degrading characteristics of intact cells. Previous studies have shown that the cytosol contains 99% of the total adipocyte insulin-degrading capacity at neutral pH; less than 0.1% is provided by the plasma membranes. As quantified by immunoprecipitation, total lysosomal degradation of [I25I]- iodoinsulin (0.5 nM) was approximately equal to neutral cytosol insulin degradation. Gel filtration of the insulin fragments demonstrated the formation of different products that were characteristic of each activity. The lysosomal and neutral insulin-degrading systems also demonstrated differential sensitivity to inhibitors. Bacitracin (1 mg/ml), N-ethyl maleimide (3 DIM), and unlabeled insulin (2 μM) inhibited neutral cytosol insulin degradation by 93%, 99%, and 89%, respectively. Lysosomal insulin degradation was inhibited 20% or less by these agents. In intact adipocytes, insulin degradation was inhibited by N-ethyl maleimide and bacitracin by 99% and 69% respectively, suggesting that the neutral cytosol activity constitutes the predominant route of physiological insulin degradation. The neutral cytosol insulin-degrading activity was further studied by fractionation of adipocyte cytosol. Gel filtration demonstrated a single peak of activity with a molecular weight of 190,000 ± 20,000. The neutral protease activity was purified 26-fold from the adipocyte homogenate by ultracentrifugation, gel filtration, and ion exchange chromatography. This activity exhibited a neutral pH optimum (6.0–8.0), was inhibited by N-ethyl maleimide and bacitracin, and generated the same labeled products from [125I]iodoinsulin as the unfractionated cytosol. The purified protease activity had linear Lineweaver-Burk kinetics, with a Km for insulin of 0.13 μM. Using insulin as substrate, the Ki values for desoctapeptide insulin and proinsulin were 0.06 and 0.24 μM, respectively. The activity was also inhibited by glucagon, PRL, and GH with Kj values of 5.3, 6.5, and 2.0 μM, respectively. The neutral cytosol degrading activity was resolved into two species with discontinuous polyacrylamide gel electrophoresis. Both proteolytic activities have very similar kinetic properties and generate identical fragments from [l25I]iodoinsulin. Therefore, although lysosomal insulin degradation is important in subcellular adipocyte systems, inhibitors that block insulin degradation in intact cells only affect the neutral cytosol activity. This neutral activity has a demonstrated specificity for insulin and appears to play a role in insulin degradation by adipocytes.