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.

Our previous studies have suggested a role for AMP-activated protein kinase (AMPK) in the induction of CYP2B6 by phenobarbital (PB) in hepatoma-derived cells (Rencurel et al., 2005). In this study, we showed in primary human hepatocytes that: 1) 5Ј-phosphoribosyl-5-aminoimidazol-4-carboxamide 1-␤-D-ribofuranoside and the biguanide metformin, known activators of AMPK, dose-dependently increase the expression of CYP2B6 and CYP3A4 to an extent similar to that of PB. 2) PB, but not the human nuclear receptor constitutive active/androstane receptor (CAR) ligand 6-(4-chlorophenyl)imidazol[2,1-6][1,3]thiazole-5-carbaldehyde, dose-dependently increase AMPK activity. 3) Pharmacological inhibition of AMPK activity with compound C or dominant-negative forms of AMPK blunt the inductive response to phenobarbital. Furthermore, in transgenic mice with a liver-specific deletion of both the ␣1 and ␣2 AMPK catalytic subunits, basal levels of Cyp2b10 and Cyp3a11 mRNA were increased but not in primary culture of mouse hepatocytes. However, phenobarbital or 1,4 bis[2-(3,5-dichloropyridyloxy)]benzene, a mouse CAR ligand, failed to induce the expression of these genes in the liver or cultured hepatocytes from mice lacking hepatic expression of the ␣1 and ␣2 subunits of AMPK. The distribution of CAR between the nucleus and cytosol was not altered in hepatocytes from mice lacking both AMPK catalytic subunits. These data highlight the essential role of AMPK in the CAR-mediated signal transduction pathway.

Transcriptional activation of cytochrome P450 (CYP) genes and various drug metabolizing enzymes by the prototypical inducer phenobarbital (PB) and many other drugs and chemicals is an adaptive response of the organism to exposure to xenobiotics. The response to PB is mediated by the nuclear receptor constitutive androstane receptor (CAR), whereas the chicken xenobiotic receptor (CXR) has been characterized as the PB mediator in chicken hepatocytes. Our previous results suggested an involvement of AMP-activated protein kinase (AMPK) in the molecular mechanism of PB induction. Here, we show that the mechanism of AMPK activation is related to an effect of PB-type inducers on mitochondrial function with consequent formation of reactive oxygen species (ROS) and phosphorylation of AMPK by the upstream kinase LKB1. Gain-and loss-of-function experiments demonstrate that LKB1-activated AMPK is necessary in the mechanism of drug induction and that this is an evolutionary conserved pathway for detoxification of exogenous and endogenous chemicals. The activation of LKB1 adds a proximal target to the so far elusive sequence of events by which PB and other drugs induce the transcription of multiple genes.drug metabolism ͉ induction ͉ mitochondria ͉ reactive oxygen species E volution has provided organisms with an elaborate defense system against foreign compounds (xenobiotics). The liver of vertebrates contains numerous enzymes that can transform potentially toxic xenobiotics (e.g., nutrients or drugs) or endobiotics (e.g., bile acids) to inactive and excretable metabolites. The expression of these enzymes can be adapted to the needs for detoxification by a process called induction. Phenobarbital (PB) is the prototype of a number of drugs that induce their own and the metabolism of other xenobiotics. Induction of drug metabolism is part of a pleiotropic response of the liver to xenobiotic exposure, which includes liver hypertrophy, tumor promotion, and induction of numerous genes in addition to those encoding for drug-metabolizing enzymes and drug transporters (1). PB also was shown to decrease the transcription of gluconeogenic enzymes such as phosphoenolpyruvate carboxykinase 1 (PEPCK1) and glucose-6-phosphatase (G6P), and of several hepatic genes responsible for fatty acid metabolism (2). Moreover, PB increases the transcription of ␦-aminolevulinic acid synthase 1 (ALAS1), the rate-limiting enzyme in the synthesis of heme, the prosthetic group of cytochromes P450 (CYPs) (3, 4). The molecular details of the mechanisms by which PB causes these effects are incompletely understood.The transcriptional activation by PB of genes encoding drugmetabolizing enzymes, such as Cyp2b10 in mouse and CYP2B6 in human, is mediated by the nuclear receptor constitutive androstane receptor (CAR) (5). The interaction of PB with CAR is complex. PB apparently does not bind directly to CAR, but rather triggers its translocation from cytoplasm to the nucleus by as yet unknown mechanisms. In addition, phosphorylation and dephosphorylation events str...

Nuclear receptors (NRs) are transcription factors activated by a multitude of hormones, other endogenous substances, and exogenous molecules. These proteins modulate the regulation of target genes by contacting their promoter or enhancer sequences at specific recognition sites. The identification of these response elements is the first step toward detailed insight into the regulatory mechanisms affecting a gene. We have developed NUBIScan, a computer algorithm to predict DNA recognition sites for NRs in the regulatory regions of genes. The algorithm is based on weighted nucleotide distribution matrices and combines scores from both half-sites necessary for NR dimer binding. It provides more specific identification of functional sites than previous in silico approaches, as evidenced by scanning published regulatory regions of drug-inducible genes and comparing the obtained predictions with experimental results. In prospective analyses, NUBIScan consistently identified new functional NR binding sites in sets of large sequences, which had eluded previous analyses. This is exemplified by the detailed functional analysis of the flanking region of two genes. This approach therefore facilitates the selection of likely sites of gene regulation for subsequent experimental analysis.

Heme is an essential component in oxygen transport and metabolism in living systems. In non-erythropoietic cells, 5-aminolevulinate synthase (ALAS1) is the first and rate-limiting enzyme in the heme biosynthesis pathway. ALAS1 expression and heme levels are increased in vivo by drugs and other chemical inducers of cytochrome P450 hemoproteins through mechanisms that are poorly understood. In the present studies, a chicken genomic cosmid library was employed to isolate a major portion of the ALAS1 gene. Two drug-responsive enhancer sequences, 176 and 167 base pairs in length, were identified in the 5-flanking region of the gene in reporter gene assays in the hepatoma cell line LMH. The relative potency of inducers to activate these enhancers corresponds to induction of ALAS1 mRNA levels in LMH cells. Analysis of putative transcription factor binding sites within the enhancers revealed DR5 and DR4 type recognition sequences for nuclear receptors. Drug activation of the enhancer elements was reduced at least 60% after mutagenesis of individual nuclear receptor binding sites and was virtually eliminated following alteration of both recognition sites within the respective elements. Electrophoretic mobility shift assays and transactivation studies demonstrate direct interactions between the nuclear receptor binding sites and the recently described chicken xenobiotic-sensing receptor, (CXR) implicating drug activation mechanisms for ALAS1 similar to those found in inducible cytochrome(s) P450. This is the first report describing direct transcriptional activation of ALAS1 by drugs via drug-responsive enhancer sequences.