Activation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) by xenobiotic inducers of cytochromes P450 is part of a pleiotropic response that includes liver hypertrophy, tumor promotion, effects on lipid homeostasis, and energy metabolism. Here, we describe an acute response to CAR and PXR activators that is associated with induction of Insig-1, a protein with antilipogenic properties. We first observed that activation of CAR and PXR in mouse liver results in activation of Insig-1 along with reduced protein levels of the active form of sterol regulatory element binding protein 1 (Srebp-1). Studies in mice deficient in CAR and PXR revealed that the effect on triglycerides involves these two nuclear receptors. Finally, we identified a functional binding site for CAR and PXR in the Insig-1 gene by in vivo, in vitro, and in silico genomic analysis. Our experiments suggest that activation of Insig-1 by drugs leads to reduced levels of active Srebp-1 and consequently to reduced target gene expression including the genes responsible for triglyceride synthesis. The reduction in nuclear Srebp-1 by drugs is not observed when Insig-1 expression is repressed by small interfering RNA. In addition, we observed that Insig-1 is also a target of AMP-activated kinase, the hepatic activity of which is increased by activators of CAR and PXR and is known to cause a reduction of triglycerides. The fact that drugs that serve as CAR or PXR ligands induce Insig-1 might have clinical consequences and explains alterations in lipid levels after drug therapy.
Previous studies have shown that glucose increases the glucose transporter (GLUT2) mRNA expression in the liver in vivo and in vitro. Here we report an analysis of the effects of glucose metabolism on GLUT2 gene expression. GLUT2 mRNA accumulation by glucose was not due to stabilization of its transcript but rather was a direct effect on gene transcription. A proximal fragment of the 5' regulatory region of the mouse GLUT2 gene linked to a reporter gene was transiently transfected into liver GLUT2-expressing cells. Glucose stimulated reporter gene expression in these cells, suggesting that glucose-responsive elements were included within the proximal region of the promoter. A dose-dependent effect of glucose on GLUT2 expression was observed over 10 mM glucose irrespective of the hexokinase isozyme (glucokinase K(m) 16 mM; hexokinase I K(m) 0.01 mM) present in the cell type used. This suggests that the correlation between extracellular glucose and GLUT2 mRNA concentrations is simply a reflection of an activation of glucose metabolism. The mediators and the mechanism responsible for this response remain to be determined. In conclusion, glucose metabolism is required for the proper induction of the GLUT2 gene in the liver and this effect is transcriptionally regulated.
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.
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