Acetoacetate Induces CYP2E1 Protein and Suppresses CYP2E1 mRNA in Primary Cultured Rat Hepatocytes

Abstract

The ketone body acetoacetate (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 in primary cultured rat hepatocytes. AA activates p70 ribosomal S6 kinase (p70S6K) and protein kinase C (PKC) by ϳ2-to 2.5-fold, respectively, following 6-h treatment. The AAmediated activation of p70S6K, but not PKC, was abolished by inhibition of PI 3-K with LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] or wortmannin, in agreement with p70S6K being downstream of phosphatidylinositol 3-kinase (PI 3-K). Inhibition of PI 3-K, mTOR with rapamycin, or PKC with bisindolylmaleimide ameliorated the AA-mediated down-regulation of CYP2E1 mRNA expression. Neither the mitogen-activated protein kinase kinase inhibitor PD98059 (2Ј-amino-3Ј-methoxyflavone) nor the p38 mitogen-activated protein kinase inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole] ameliorated the AA-mediated suppression of CYP2E1 mRNA expression. Heterogeneous nuclear RNA analysis revealed that AA suppressed CYP2E1 gene transcription by ϳ50% and that inhibition of PI 3-K and PKC diminished this AA-mediated effect on transcription. CYP2E1 mRNA half-life slightly increased from ϳ24 h in untreated hepatocytes to ϳ32 h in AA-treated cells. Interestingly, AA increased CYP2E1 protein levels by ϳ2-and 2.5-fold at 24 and 48 h, respectively. DL-␤-Hydroxybutyrate was without effect. Polysomal distribution studies revealed that AA increased the proportion of RNA associated with the actively translated polysomal fractions versus the 40S to 60S untranslated fractions by ϳ40%. CYP2E1 protein half-life increased from ϳ8 h in untreated hepatocytes to ϳ24 in AA-treated cells. These data show that AA decreases CYP2E1 mRNA expression through inhibition of gene transcription while simultaneously elevating CYP2E1 protein levels through increased translation and decreased protein degradation.Acetoacetate (AA) and DL-␤-hydroxybutyrate (3HB) comprise two major ketone bodies, which are produced primarily in the liver. Ketone bodies are considered an emergency source of glucose (Casazza et al., 1984). In humans, ketone bodies (AA plus 3HB) with plasma concentrations higher than 1 mM are considered hyperketonemic. Ketone body concentrations may be elevated up to 10 mM in severe ketosis, relative to concentrations less than 0.5 mM under normal physiologic conditions (Jain et al., 2003). Hyperketonemia is a very common feature in diabetes mellitus.AA and 3HB effects on CYP2E1 expression remain controversial. An increase in CYP2E1 protein and/or activity has been associated with hyperketonemia (Shimojo, 1994); however, neither AA nor 3HB elevated CYP2E1 mRNA levels in the presence of insulin in primary cultured rat hepatocytes (Zangar and Novak, 1997) or in vivo (Barnett et al., 1992). In addition, we have reported that AA, but not 3HB, in the absence of insulin substantially decreased CYP2E1 mRNA levels in primary cultured rat hepatocytes c...

The role of intracellular signaling in insulin-mediated regulation of drug metabolizing enzyme gene and protein expression

“…16). In addition, our results also suggest that mTOR and downstream p70S6 kinase may effect AA-mediated inhibition of CYP2E1 mRNA expression (Abdelmegeed et al, 2005). In contrast, neither p38 MAPK nor ERK signaling pathways appear to play an active role in mediating the downregulation of CYP2E1 mRNA levels by AA as shown by the use of the MEK inhibitor PD98059 and the p38 MAPK inhibitor SB202190.…”

“…Although AA decreased CYP2E1 mRNA, it significantly increased CYP2E1 protein levels which appears to be associated with increased mRNA translation and may reflect activation of eIF4E/4E-BP1 phosphorylation, as well as activation of the p70S6 kinase. This is supported in part, by the inhibitory effect of rapamycin, and inhibitor of mTOR, on AAmediated downregulation of CYP2E1 expression (Abdelmegeed et al, 2005).…”

“…Because hyperketonemia is a well established event in diabetes, our laboratory has also studied the effect of the two major ketone bodies acetoacetate (AA) and DL-beta-hydroxybutyrate on the expression of CYP2E1 mRNA and protein Abdelmegeed et al, 2005). We showed that AA significantly decreased CYP2E1 mRNA levels in primary cultured rat hepatocytes mainly through the inhibition of CYP2E1 gene transcription as monitored by the heterogeneous nuclear RNA analysis (Fig.…”

Opposite Effect of Diabetes Mellitus Induced by Streptozotocin on Oral and Intravenous Pharmacokinetics of Verapamil in Rats

“…Some hepatic cytochrome P450 isozymes of diabetic rats were reversed or partly reversed to the level of the controls after insulin treatment (Yamazoe et al, 1989). It has been reported that insulin and ketone bodies participated in the regulation of hepatic CYP2E1 in diabetic rats (Woodcroft et al, 2002;Abdelmegeed et al, 2005). However, the mechanisms involved in the regulation of CYP3A in liver and intestine by diabetes mellitus were not reported and need to be further investigated.…”

Decreased exposure of simvastatin and simvastatin acid in a rat model of type 2 diabetes

“…The activity and expression of hepatic Cyp3a in DM rats were associated with HOMA-IR, which suggests that insulin resistance or relative insufficiency of insulin may explain the affecting expression and activity of hepatic Cyp3a. Several reports have demonstrated that insulin and ketone bodies participating in the regulation of cytochrome P450 isozymes in the liver of DM rats and insulin treatment reversed or partially reversed alteration induced by diabetes [17,[37][38][39] . The elevated total serum cholesterol and triglyceride levels in DM rats were consistent with the increases in activity and expression of hepatic Cyp3a, suggesting that high cholesterol and triglyceride levels may explain inducing Cyp3a expression and activity.…”