Identification of a 16-Nucleotide Sequence That Mediates Post-transcriptional Regulation of Rat CYP2E1 by Insulin

Abstract

Insulin directly down-regulates the gene expression of the rat CYP2E1 by altering its mRNA stability (De Waziers, I., Garlatti, M., Bouguet, J., Beaune, P. H., and Barouki, R. (1995) Mol. Pharmacol. 47, 474 -479). Because the regulation of CYP mRNA stability was poorly understood, the molecular mechanisms involved in this regulation in the rat hepatoma H4IIEC3 cell line were studied. By using RNase T1 protection methods, the formation of a major CYP2E1 RNA-protein complex was observed. By competition experiments, the binding site of this complex was located on a 16-nucleotide sequence in the 5-proximal region of the CYP2E1-coding sequence. Insulin did not modify the binding pattern of proteins to this sequence. and transfections of expression vectors or antisense oligonucleotides were undertaken to demonstrate the actual functionality of the 16-mer sequence. The insertion of this sequence in a luciferase gene was sufficient to render the chimeric mRNA sensitive to insulin. Furthermore, transfection of H4IIEC3 cells with antisense oligonucleotide complementary to this sequence blocked the insulin effect on the CYP2E1 mRNA expression, i.e. its rapid degradation. All these results demonstrate that this 16-nucleotide sequence is implicated in the CYP2E1 post-transcriptional regulation by insulin.

Stable Expression, Activity, and Inducibility of Cytochromes P450 in Differentiated HepaRG Cells

“…The cause of the loss of CYP2E1 activity was unclear. Its inhibition by insulin has been reported in a rat hepatoma cell line (Moncion et al, 2002). However, medium depletion or lower concentrations of insulin did not prevent CYP2E1 activity loss in HepaRG cells (data not shown).…”

D2-receptor-linked signaling pathways regulate the expression of hepatic CYP2E1

“…Mean ± SE; ⁎ P b 0.025; ⁎⁎ P b 0.005; ⁎⁎⁎ P b 0.001. group of diverse xenobiotics may increase the expression of CYP2E1 by increasing its transcription or translation, pathophysiological conditions, such as disturbances in carbohydrate metabolism, may up-regulate the gene by both transcriptional and post-transcriptional mechanisms (Dong et al, 1988;Thummel and Schenkman, 1990;Novak and Woodcroft, 2000;Moncion et al, 2002). Insulin can reverse the up-regulation of CYP2E1 in diabetes suggesting that this endogenous signaling pathway is involved in the regulation (Moncion et al, 2002). The data of this study add to the complexity of the CYP2E1 regulation by demonstrating that another endogenous signaling pathway, a D 2 -dopaminergic receptor pathway, is also involved.…”

“…Increasing evidence suggests that endogenous processes and signaling pathways may play a role in the regulation of CYP2E1. For example, disturbances in glucose metabolism and an insulindependent signaling pathway may influence the hepatic levels of CYP2E1 (Woodcroft et al, 2002;Moncion et al, 2002). Recent evidence also showed that increased noradrenergic neurotransmission was associated with increased CYP2E1-catalyzed pnitrophenol hydroxylation (PNP), suggesting the involvement of catecholamines in the regulation of this isozyme (Konstandi et al, 1999).…”

Ethanol increases mitochondrial cytochrome P450 2E1 in mouse liver and rat hepatocytes

“…Cytochrome P450 2E1 (CYP2E1) metabolises many endogenous or exogenous small molecules such as acetone, glycerol, ethanol, acetaminophen, carbon tetrachloride, halothane or nitrosamines [1][2][3]. Hepatic expression of the CYP2E1 mRNA and/or expression of the CYP2E1 protein are increased in different physiological or pathological conditions, such as fasting, a high fat diet, diabetes, obesity or ethanol intoxication [1,4].…”