The activation of signaling pathways and the regulation of heme-oxygenase 1 and cytochrome P450 2E1 by ketone bodies in primary cultured rat hepatocytes.

摘要

Diabetes is characterized by elevated levels of ketone body acetoacetate (AA) and 3-hydroxybutyrate (3HB). High levels of ketone bodies have been implicated in generation of cellular oxidative stress. Diabetes is also characterized by elevated levels of cytochrome P450 2E1 (CYP2E1), which has been implicated in cellular oxidative stress. Ketone body activation of cellular signaling pathways associated with oxidative stress, the adaptive response by the cells to this oxidative stress, and the role of ketone bodies in the regulation of CYP2E1 have not been established. Hence, the effects of AA and 3HB on CYP2E1 gene expression, activation of intracellular signaling pathways and components, and oxidative stress have been examined in our laboratory. AA, but not 3HB, activates Erk1/2 and p38 MAPK, but not JNK. Maximal activation occurred at 12 h. AA treatment resulted in the generation of reactive oxygen species and the depletion of cellular glutathione levels, which was ameliorated by the antioxidants NAC and Trolox. NAC and Trolox also ameliorated AA-mediated Erk1/2 and p38 MAK activation, suggesting that this activation is associated with oxidative stress. Also, AA, but not 3HB, increased the inducible HO-1 protein expression, a stress response protein, which was ameliorated by the p38 MAPK inhibitor SB203580, but not by the Mek inhibitor PD98059, and the antioxidants NAC and Trolox. These results suggest that p38 MAPK signaling pathway maybe the link between oxidative stress and the upregulation of HO-1 induced by AA. Also, AA in the absence of insulin or in the presence of diabetic insulin levels decreases CYP2E1 mRNA expression in a concentration- and time-dependent manner. AA activates p70S6K and PKC significantly following 6 h treatment. Inhibition of PI 3-K with wortmannin and LY294002, or mTOR with rapamycin, or PKC with bisindolylmaleimide, ameliorated the AA-mediated downregulation of CYP2E1 mRNA expression. The AA-mediated inhibition of CYP2E1 mRNA is through the inhibition of gene transcription as evident by heterogenous nuclear RNA analysis. Interestingly, AA, but not 3HB, increased CYP2E1 protein levels significantly at 24 h and 48 h. This increase was due to increased translational efficiency as evident by polysomal distribution studies and increased protein half life as evident by degradation studies. These studies illustrate the complex mechanisms that regulate CYP2E1 gene expression in response to ketone bodies.