Effect of nelfinavir on insulin metabolism, proteasome activity and protein degradation in HepG2 cells

HIV-1 protease inhibitors have revolutionized the treatment of HIV infection, but their use has been associated with lipodystrophy and insulin resistance. One suggestion for this has been the inhibition of insulin-degrading enzyme (IDE). We have previously demonstrated that insulin, through IDE, can inhibit the proteasome, thus decreasing cytosolic protein degradation. We examined whether the protease inhibitor nelfinavir inhibited IDE and its effect on protein degradation both in vitro and in whole cells. I-125-Insulin degradation was measured by trichloroacetic acid precipitation. Proteasome activities were measured using fluorogenic peptide substrates. Cellular protein degradation was measured by prelabelling cells with H-3-leucine and determining the release of TCA-soluble radioactivity. Nelfinavir inhibited IDE in a concentration-dependent manner with 50% inhibition at the maximal concentration tested, 100 mu m. Similarly, the chymotrypsin-like and trypsin-like activities of the proteasome were decreased with an IC50 of approximately 3 mu m. The ability of insulin to inhibit the proteasome was abrogated by nelfinavir. Treatment of HepG2 cells with 50 mu m nelfinavir decreased I-125-insulin degradation and increased cell-associated radioactivity. Insulin alone maximally decreased protein degradation by 15%. Addition of 50 mu m nelfinavir inhibited cellular protein degradation by 14% and blunted the effect of insulin. These data show that nelfinavir inhibits IDE, decreases insulin's ability to inhibit protein degradation via the proteasome and provides another possible mechanism for the insulin resistance seen in protease inhibitor-treated HIV patients.