Designed Inhibitors of Insulin-Degrading Enzyme Regulate the Catabolism and Activity of Insulin
Figure 5
Effects of IDE inhibitors on insulin catabolism and signaling in cells.
A, Dose-response curve of Ii1-mediated inhibition of insulin degradation by CHO-IR cells. Note that the potency of Ii1 in this context is comparable to that obtained in vitro (c.f., Fig. 3A). B, Progress curve of insulin degradation by CHO-IR cells in the absence or presence of Ii1 (10 µM). Note that insulin catabolism is completely inhibited by Ii1. C–F, Effects of Ii1 on insulin catabolism in live cells. C, Representative images of live CHO-IR cells pre-loaded with FITC-ins and imaged at various time points in the presence or absence of Ii1 (10 µM). D,E, Quantitative analysis of intracellular (D) and extracellular (E) FITC fluorescence from 5 replicate experiments. Data are mean ± SEM. Note that these experiments were conducted at 22°C. F, Effects of Ii1 on insulin signaling in CHO-IR cells cold-loaded with insulin. Graph shows IR autophosphorylation (phospho-IR) determined by ELISA in response to 5-min incubation at 37°C in the presence of Ii1 (10 µM), ML3-XF (10 µM) or vehicle (DMSO). Data are mean ± SEM of 6 independent experiments, where data are normalized to IR autophosphorylation obtained for insulin and vehicle alone. *p<0.01. Western analysis of a representative experiment (lower panels) providing biochemical confirmation of the results obtained by ELISA. G, Preloaded insulin is rapidly catabolized by CHO-IR cells and blocked by IDE inhibitors. Graph shows amounts of 125I present in CHO-IR cells cold-loaded with 125I-insulin then incubated for 5 min at 37°C in the presence of IDE inhibitors (10 µM) or vehicle then washed to remove insulin catabolites. Data are mean ± SEM of 4 experiments expressed as a percentage of 125I present in control cells maintained at 4°C.