Insulin Suppresses the Induction of CYP2B1 and CYP2B2 Gene Expression by Phenobarbital in Adult Rat Cultured Hepatocytes

doi:10.1006/bbrc.1996.1777

Biochemical and Biophysical Research Communications

Volume 229, Issue 1, 4 December 1996, Pages 182-188

https://doi.org/10.1006/bbrc.1996.1777 Get rights and content

Abstract

The effect of insulin on the phenobarbital (PB)-induced gene expression of CYP2B1 and CYP2B2 (CYP2B1/2B2) in adult rat hepatocytes was investigated. Insulin, which has been regarded as an essential hormone for primary hepatocytes, was found to strongly suppress the induction of CYP2B1/2B2 gene expression in hepatocytes cultured on EHS-gel. Although the induction by PB was not seen in monolayer hepatocytes cultured on type I collagen under standard culture conditions, the induced expression of the CYP2B1/2B2 gene was observed in monolayer hepatocytes by removing insulin from the medium. Further, we succeeded in maintaining the prolonged induction of CYP2B1/2B2 by PB in monolayer hepatocytes by using a medium containing dexamethasone but not insulin. Since the PB-induced UDP-glucuronosyltransferase gene expression was not reduced by insulin, the suppressive effect of insulin was considered to be specific to the CYP2B1/2B2 gene. These results demonstrate that insulin in media masks the PB-induced expression of the CYP2B1/2B2 gene in conventional monolayer hepatocytes and that the use of insulin-free media with primary hepatocytes provides a useful tool for investigating the molecular mechanism of CYP2B1/2B2 gene expression.

References (0)

Cited by (35)

Nuclear receptor phosphorylation in xenobiotic signal transduction
2020, Journal of Biological Chemistry
Citation Excerpt :
In addition to EGFR, PB can also bind the insulin receptor to repress ERK1/2 activation (71). Insulin has long been known to repress PB induction of CYP2B, and, conversely, diabetic livers increased CYP expressions (72–75). This PB-insulin interaction can now be understood by its competitive antagonism via the insulin receptor.
Nuclear pregnane X receptor (PXR, NR1I2) and constitutive active/androstane receptor (CAR, NR1I3) are nuclear receptors characterized in 1998 by their capability to respond to xenobiotics and activate cytochrome P450 (CYP) genes. An anti-epileptic drug, phenobarbital (PB), activates CAR and its target CYP2B genes, whereas PXR is activated by drugs such as rifampicin and statins for the CYP3A genes. Inevitably, both nuclear receptors have been investigated as ligand-activated nuclear receptors by identifying and characterizing xenobiotics and therapeutics that directly bind CAR and/or PXR to activate them. However, PB, which does not bind CAR directly, presented an alternative research avenue for an indirect ligand-mediated nuclear receptor activation mechanism: phosphorylation-mediated signal regulation. This review summarizes phosphorylation-based mechanisms utilized by xenobiotics to elicit cell signaling. First, the review presents how PB activates CAR (and other nuclear receptors) through a conserved phosphorylation motif located between two zinc fingers within its DNA-binding domain. PB-regulated phosphorylation at this motif enables nuclear receptors to form communication networks, integrating their functions. Next, the review discusses xenobiotic-induced PXR activation in the absence of the conserved DNA-binding domain phosphorylation motif. In this case, phosphorylation occurs at a motif located within the ligand-binding domain to transduce cell signaling that regulates hepatic energy metabolism. Finally, the review delves into the implications of xenobiotic-induced signaling through phosphorylation in disease development and progression.
Phenobarbital reduces blood glucose and gluconeogenesis through down-regulation of phosphoenolpyruvate carboxykinase (GTP) gene expression in rats
2015, Biochemical and Biophysical Research Communications
Citation Excerpt :
However, according to the proposed mechanism by Miao et al., there are some discrepancies found in acute effect of PB on PEPCK gene expression and in specificity of PB action to PEPCK gene in the present study. Acute treatment of PB did not show any inhibitory effect on PEPCK gene expression, even CYP2B gene expression was induced by activated CAR by PB (data not shown) [13]. It has been known that turnover rate of PEPCK mRNA was very rapid [24,25].
The regulatory mechanism of phosphoenolpyruvate carboykinase (GTP) (EC 4.1.1.32) (PEPCK) gene expression and gluconeogenesis by phenobarbital (PB), which is known to induce drug-metabolizing enzymes, was investigated. Higher level of PEPCK mRNA was observed in spherical rat primary hepatocytes on EHS-gel than monolayer hepatocytes on TIC (type I collagen). We found that PB directly suppressed PEPCK gene expression in spherical hepatocytes on EHS-gel, but not in those on TIC. PB strongly suppressed cAMP-dependent induction of PEPCK gene expression. Tyrosine aminotransferase (TAT), another gluconeogenic enzyme, was induced by cAMP, but not suppressed by PB. Chronic administration of PB reduced hepatic PEPCK mRNA in streptozotocin-induced diabetic and nondiabetic rats, and PB reduced blood glucose level in diabetic rats. Increased TAT mRNA in diabetic rats was not suppressed by PB. These results indicated that PB-dependent reduction is specific to PEPCK. From pyrvate challenge test, PB suppressed the increased gluconeogenesis in diabetic rats. PEPCK gene promoter activity was suppressed by PB in HepG2 cells. In conclusion, we found that spherical hepatocytes cultured on EHS-gel are capable to respond to PB to suppress PEPCK gene expression. Moreover, our results indicate that hypoglycemic action of PB result from transcriptional repression of PEPCK gene and subsequent suppression of gluconeogenesis.
Lipid components prepared from a freshwater Clam (Corbicula fluminea) extract ameliorate hypercholesterolaemia in rats fed high-cholesterol diet
2013, Food Chemistry
Citation Excerpt :
Expression was calculated using the standard curve method. Because the apo E mRNA level was not affected by any treatment used in the present study (data not shown), we used it as the normalisation standard (Oda, Nozawa, Hitomi, & Kakinuma,1995; Yoshida, Kimura, Oda, & Kakinuma, 1996). Previous studies demonstrated that the amounts of the CYP7A1 and ABCG5 mRNA have a good correlation with the activities or the protein levels (Noshiro, Mishimoto, & Okuda, 1990; Yu et al., 2005).
To explore the hypocholesterolaemic components in the fat fraction of freshwater clam extract (FCE), we further fractionated the fat fraction by silica gel column chromatography into nine fat subfractions. In the present study, we used exogenous hypercholesterolaemic rats induced by feeding a high-cholesterol diet; the doses of the added fat subfractions were equivalent to those in 30% FCE. Two (FF1, FF2) out of the nine fat subfractions strongly reduced serum cholesterol levels in the rats fed a high-cholesterol diet. Both FF1 and FF2 up-regulated the hepatic gene expression of cholesterol 7α-hydroxylase, a rate-limiting enzyme of bile acid biosynthesis. Thin-layer chromatography showed that FF1 primarily contained sphingolipids, while FF2 mainly contained triacylglycerols and sterol esters. These results indicate that fractions containing sphingolipids, triacylglycerols, and sterol esters are possibly responsible for the hypocholesterolaemic action in a novel manner through the up-regulation of the hepatic biosynthesis of bile acids.
Cell shape, cell-cell contact, cell-extracellular matrix contact and cell polarity are all required for the maximum induction of CYP2B1 and CYP2B2 gene expression by phenobarbital in adult rat cultured hepatocytes
2008, Biochemical Pharmacology
Citation Excerpt :
Various efforts have been made so far to observe sufficient induction of CYP2B1/2B2 in cultured hepatocytes [3,6–9], and the most successful methods have revolved around the manipulation of extracellular matrices. Several studies have established a method whereby primary hepatocytes can be reproducibly cultured on EHS gel (a reconstituted extracellular basement membrane gel from mouse Engelbreth-Holm-Swarm sarcoma) [6–12]. EHS gel is comprised of laminin, type IV collagen (TIVC), proteoglycan, and so on, and permits cells to be spherical.
The effect of cell shape, cell density, contact with extracellular matrix and cell polarity on the phenobarbital (PB)-induced gene expression of CYP2B1 and CYP2B2 (CYP2B1/2B2) in adult rat hepatocytes was investigated. High cell density enhanced the induction of CYP2B1/2B2 gene expression by PB. Hepatocytes cultured on EHS gel showed a spherical cell shape and highly enhanced the induction of CYP2B1/2B2 gene expression by PB. Although monolayer hepatocytes cultured on type I collagen (TIC) and type IV collagen exhibited poor induction of CYP2B1/2B2 gene expression by PB, monolayer cells on laminin showed substantial induction. The addition of soluble laminin to media did not show any effect on induction in monolayer hepatocytes cultured on TIC. Dishes coated with different concentrations of immovable laminin demonstrated complicated effects. Coating with higher concentrations of laminin resulted in greater induction of CYP2B1/2B2 gene expression by PB. On the other hand, when hepatocytes were cultured on dishes coated with lower concentrations of laminin, they became round and greater induction of CYP2B1/2B2 gene expression by PB was observed. Spherical hepatocytes cultured on low concentrations of TIC also showed highly enhanced induction of CYP2B1/2B2 gene expression by PB. EHS gel overlay to hepatocytes cultured on TIC and collagen sandwich configurations that are known to induce cell polarity enhanced the induction by PB. The induction of CYP2B1/2B2 gene expression needed cytoskeleton organization, such as actin filament, microtubule filament and intermediate filament. These results demonstrate that cell shape, cell density, contact with extracellular matrix and cell polarity all play critical roles in the induction of CYP2B1/2B2 gene expression by PB.
AMP-activated protein kinase mediates phenobarbital induction of CYP2B gene expression in hepatocytes and a newly derived human hepatoma cell line
2005, Journal of Biological Chemistry
Phenobarbital (PB) administration is known to trigger pleiotropic responses, including liver hypertrophy, tumor promotion, and induction of genes encoding drug-metabolizing enzymes. The induction of human CYP2B6 and the rat (CYP2B1) and mouse (Cyp2b10) homologues by PB is mediated by the nuclear receptor constitutive androstane receptor (CAR). The study of CYP2B gene regulation and CAR activity by PB has been difficult due to the lack of a cellular model. In this study, we describe a novel differentiated human hepatoma cell line (WGA), derived from HepG2, which expresses CYP2B6 and CAR. WGA cells represent a powerful system to study the regulation of CYP2B6 gene expression by PB. There is evidence that CAR activity is regulated by phosphorylation and that regulation of some CYP genes depends on the nutritional status of cells. The AMP-activated protein kinase (AMPK) functions as an energy sensor and is activated when cells experience energy-depleting stresses. In this report, we show that addition of 5-amino-imidazole carboxamide riboside, an AMPK activator, to WGA and human hepatocytes induces CYP2B6 gene expression. Expression of a constitutively active form of AMPK mimics the PB induction of CYP2B6 and CYP2B1 gene expression. Conversely, the expression of a dominant negative form of AMPK inhibits the induction of these genes by PB. Finally, we demonstrate, for the first time, that AMPK activity increases in cells cultured with PB. Our data strongly support a role for AMPK in the PB induction of CYP2B gene expression and provide new insights into the regulation of gene expression by barbiturate drugs.
Apolipoprotein A-I gene expression is upregulated by polychlorinated biphenyls in rat liver
2000, Journal of Nutritional Biochemistry
Xenobiotics such as polychlorinated biphenyls (PCB) increase serum cholesterol level (especially high density lipoprotein cholesterol) and apolipoprotein A-I (apo A-I) level in rats. The effect of PCB on serum apo A-I and hepatic apo A-I gene expression and the relationship between apo A-I and drug-metabolizing enzymes in rats were investigated. Serum levels of cholesterol and apo A-I were increased by dietary addition of PCB in a dose-dependent manner (0–500 mg/kg diet). Hepatic apo A-I mRNA level was also elevated by PCB in a similar fashion. Serum level of cholesterol gradually increased during feeding period of PCB (200 mg/kg diet, 105 days) and reached a two-fold higher level in PCB group than in controls. The levels of serum apo A-I and hepatic apo A-I mRNA linearly elevated during feeding period of PCB and were increased 3- or 4-fold, respectively, compared to controls. Although acute administration (16 hr) of PCB, 3-methylcholanthrene, and phenobarbital induced cytochrome P-450 gene expression in the liver, hepatic apo A-I gene expression was not increased by these xenobiotics. These results indicated that the serum levels of cholesterol and apo A-I had positive correlation with hepatic level of apo A-I mRNA in rats fed PCB, and that hepatic apo A-I gene expression was dependent upon intake of PCB but was not directly related to the induction of drug-metabolizing enzymes. This study demonstrated that xenobiotic-induced hyper-alpha-cholesterolemia would be caused by the increased apo A-I gene expression and cholesterol synthesis in the liver, coordinately.

View all citing articles on Scopus

^☆: Abbreviations: apo, apolipoprotein; ANOVA, analysis of variance; CYP, cytochrome P-450; Dex, dexamethasone; EHS, Engelbreth-Holm-Swarm; IRS, insulin responsive sequence; IRS1, insulin receptor substrate 1; PB, phenobarbital; PEPCK, phosphoenolpyruvate carboxykinase; PI-3 kinase, phosphatidylinositol-3 kinase; TAT, tyrosine aminotransferase; TIC, type I collagen; UGT, UDP-glucuronosyltransferase.

¹: To whom correspondence and reprint requests should be addressed. Fax: +81-52-789-5050. E-mail: hirooda@ agr.nagoya-u.ac.jp.

View full text

Regular ArticleInsulin Suppresses the Induction of CYP2B1 and CYP2B2 Gene Expression by Phenobarbital in Adult Rat Cultured Hepatocytes☆

Abstract

Regular Article
Insulin Suppresses the Induction of CYP2B1 and CYP2B2 Gene Expression by Phenobarbital in Adult Rat Cultured Hepatocytes☆