Insulin Metabolism and Protein Degradation by HEPG2 Hepatocytes Treated with HIV-Protease Inhibitors

Abstract

Objectives: To explore the effects of human immunodeficiency virus protease inhibitors (HPI) on insulin metabolism and protein degradation in HepG2 hepatocytes in vitro. Methods: To see if HIV-protease inhibitors affect insulin degradation in a dose-dependent manner, HepG2 cells were incubated with various concentrations of tipranavir, indinavir, or atazanavir. After 125I-insulin was added, its degradation was measured by precipitation with trichloroacetic acid (TCA). To see the effect of HPIs on protein degradation, HepG2 cells labeled overnight with 3H-leucine were incubated with 50 mM of an HPI, followed by another HPI incubation including concentrations of insulin ranging from 10-12 to 10-6 M. Cells were solubilized and proteins were precipitated using TCA. Degradation was quantified as percent TCA soluble, normalized, plotted, and then compared using student’s t-test or one- way ANOVA. Results: Cellular insulin degradation was inhibited only by tipranavir at the highest concentrations of 75 and 100 mM (12.06 ± 1.07%, p=0.047 and 9.35 ± 0.44%, p=0.024, respectively) when compared to the control (17.01 ± 1.37%; n=3). None of the concentrations of indinavir or atazanavir decreased insulin degradation significantly. From the protein degradation experiments, the log EC50 of the control (no HPI) insulin dose-response curve was not statistically different compared to those of the individual HPIs. Conclusions: Except for high concentrations of tipranavir, it appears that HPI does not inhibit the cellular degradation of insulin. HPIs do not appear to inhibit the role of insulin in the inhibition of protein degradation significantly.