Abstract

The regulation of cytochrome P450 (CYP) 2E1, the ethanol-inducible isoform, is particularly complex. The level is affected by a variety of other foreign compounds, by insulin (as studied in several laboratories), and by triiodothyronine (T₃), which has not been previously examined at the molecular level. In the present investigation, a stably transfected HepG2 cell line harboring a rabbitCYP2E1 minigene containing the coding sequence together with 1.6 kilobases of the 5′ flanking region and the untranslated region (UTR), as well as 0.5 kilobases of the 3′ UTR, was established. Western blot analysis showed that 1 μm insulin decreased the CYP2E1 protein level in a dose- and time-dependent manner, whereas 1 μm T₃ increased the level 2-fold in 1 day and 8-fold in 5 days. Similarly, steady state CYP2E1 mRNA levels were decreased by insulin but were increased by T₃. Neither hormone affected the transcription rate of the CYP2E1 5′ flanking region with an UTR/luciferase fusion gene, indicating that the regulation is post-transcriptional in this system under our experimental conditions. When the CYP2E1 3′ UTR was removed from the minigene, CYP2E1 mRNA and protein were up-regulated by insulin but were not affected by T₃. These findings, including mRNA half-life determinations, indicate that the effects of insulin and T₃ are a result of altered mRNA stability and that the 3′ UTR of CYP2E1 contains regulatory information for these hormone-mediated effects.