Robert D Finn

Colin J Henderson

Claire L Scott

C Roland

C Roland Wolf
email: c.r.wolf@dundee.ac.uk

Unsaturated Fatty Acid Regulation of Cytochrome P450 Expression via a CAR-Dependent pathway

Keywords: Constitutive androstane receptor, Cytochrome P450, Linoleic acid, P450 Oxidoreductase, Pregnane X receptor, Steatosis

The liver is responsible for key metabolic functions, including control of normal homeostasis in response to diet and xenobiotic metabolism/detoxification. We have previously shown that inactivation of the hepatic cytochrome P450 system through conditional deletion of P450 oxidoreductase (POR) induces hepatic steatosis, liver growth and P450 expression. We have exploited a new conditional model of POR deletion to investigate the mechanism underlying these changes. We demonstrate that P450 induction, liver growth and hepatic triglyceride homeostasis are intimately linked and provide evidence that the observed phenotypes result from hepatic accumulation of unsaturated fatty acids, which mediate these phenotypes by activation of the nuclear receptor CAR and, to a lesser degree, PXR. To our knowledge this is the first direct evidence that P450s play a major role in controlling unsaturated fatty acid homeostasis via CAR. The regulation of P450s involved in xenobiotic metabolism by this mechanism has potentially significant implications for individual responses to drugs and environmental chemicals.

INTRODUCTION

Cytochrome P450 enzymes (P450s) play a major role in the metabolism and disposition of chemicals and their activities are critical in determining the efficacy or side effects of drugs. These enzymes also have additional functions in adrenal steroid hormone biosynthesis and the hepatic synthesis of cholesterol and its degradation to bile acids [START_REF] Ortiz De Montellano | Cytochrome P450: structure, mechanism and biochemistry[END_REF]. The P450s involved in these different pathways are distinct and their regulation has conventionally been considered to be by different mechanisms. This is manifested in that many of the hepatic enzymes involved in foreign compound metabolism are expressed constitutively at low levels but become highly induced on exogenous chemical exposure, resulting in an increased rate of excretion of the compound(s). Significant advances have been made in understanding this adaptive response through the identification of transcription factors, such as the Aryl-hydrocarbon receptor (AhR), the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR) which have the capacity to bind drugs and exogenous chemicals resulting in transcriptional activation of genes that mediate their detoxification [START_REF] Okey | Regulatory gene product of the Ah locus. Characterization of the cytosolic inducer-receptor complex and evidence for its nuclear translocation[END_REF][START_REF] Kliewer | An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway[END_REF][START_REF] Lehmann | The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions[END_REF][START_REF] Choi | Differential transactivation by two isoforms of the orphan nuclear hormone receptor CAR[END_REF]. Although distinct classes of compounds bind to each receptor, due to the diversity of these compounds' chemical structures, promiscuity exists in their specificity, particularly in the case of PXR and CAR [START_REF] Jones | The pregnane X receptor: a promiscuous xenobiotic receptor that has diverged during evolution[END_REF][START_REF] Maglich | Nuclear Pregnane X receptor and Constitutive Androstane receptor regulate overlapping but distinct sets of genes involved in Xenobiotic detoxification[END_REF].

We, and the group of Ding, et.al. [START_REF] Gu | Liver-specific deletion of the NADPH-cytochrome P450 reductase gene: impact on plasma cholesterol homeostasis and the function and regulation of microsomal cytochrome P450 and heme oxygenase[END_REF], have recently reported a mouse model where hepatic P450 activity has been reduced by > 95% by the conditional deletion of the P450 oxidoreductase (POR) gene. This was achieved by crossing mice carrying the Por gene flanked by loxP sites with mice expressing Cre recombinase under the control of the rat albumin promoter. This resulted in the specific hepatic deletion of POR neonatally with a concomitant reduction in P450 activity [START_REF] Gu | Liver-specific deletion of the NADPH-cytochrome P450 reductase gene: impact on plasma cholesterol homeostasis and the function and regulation of microsomal cytochrome P450 and heme oxygenase[END_REF][START_REF] Henderson | Inactivation of the hepatic cytochrome P450 system by conditional deletion of hepatic cytochrome P450 reductase[END_REF]. Although hepatic reductase null (HRN) mice developed normally and were fertile, there were a number of intriguing phenotypic changes including an enlarged liver and hepatic steatosis. Also, there was a profound increase in the expression of a range of P450s, with the most marked changes being in the expression of Cyp2b10 and Cyp3a11 [START_REF] Wang | Relationship between hepatic phenotype and changes in gene expression in cytochrome P450 reductase (POR) null mice[END_REF]. These enzymes, which are expressed at low levels in untreated animals, are highly inducible by exogenous chemicals such as barbiturates and synthetic glucocorticoids [START_REF] Simmons | Induction of the hepatic mixed-function oxidase system by synthetic glucocorticoids. Transcriptional and posttranscriptional regulation[END_REF][START_REF] Meehan | Regulation of phenobarbital-inducible cytochrome P-450s in rat and mouse liver following dexamethasone administration and hypophysectomy[END_REF]. These data show that the P450 system is a key regulator of hepatic lipid homeostasis and liver growth and that this metabolic homeostasis can have a profound effect on the control of P450 gene expression and as a consequence on the metabolism/deposition of drugs. The HRN mouse therefore provides a model for investigating the endogenous factors involved in regulating P450 genes and the role of the P450 system in controlling hepatic steatosis and liver growth.

Studies of the temporal events that lead to the hepatic phenotype are, however, not possible in the HRN mice because the deletion of the Por gene occurs neonatally. To overcome this we have created a model where the POR null genotype is regulated conditionally and in an inducible manner. This was achieved by crossing Por floxed mice with mice where Cre recombinase is conditionally regulated by the rat CYP1A1 promoter. This promoter is activated through the Ah receptor and administration of a single dose of 3methlycholanthrene at 40 mg/kg results in a specific, time-dependent, hepatic deletion of POR and a phenotype identical to that observed in HRN mice [START_REF] Finn | Conditional deletion of cytochrome P450 oxidoreductase in the liver and gastrointestinal tract: a new model for studying the functions of the P450 system[END_REF].

In this study, we have used the new model to study the temporal biochemical changes that control lipid accumulation and P450 over-expression. We provide evidence that dietary unsaturated fatty acids play a key role in both lipid accumulation and cytochrome P450 induction. Also, using nuclear receptor/HRN double and triple knockout mice we show that P450 over-expression occurs predominantly through the activation of CAR.

EXPERIMENTAL Reagents

All chemicals were purchased from Sigma-Aldrich (Dorset, UK), except where indicated.

Production of Transgenic Mice

Por lox/lox /Cre Alb (HRN) and Por lox/lox /Cre CYP1A1 mice on a C57/BL6 genetic background were generated as previously described [START_REF] Henderson | Inactivation of the hepatic cytochrome P450 system by conditional deletion of hepatic cytochrome P450 reductase[END_REF][START_REF] Finn | Conditional deletion of cytochrome P450 oxidoreductase in the liver and gastrointestinal tract: a new model for studying the functions of the P450 system[END_REF]. Littermates with the Por lox/lox genotype were used as controls. PXR null (Pxr -/-) and CAR null (Car -/-) mice on a C57/BL6 genetic background were crossed onto the HRN line to generate Pxr -/-/Por lox/lox Cre Alb (Pxr -/-/HRN) and Car -/- /Por lox/lox Cre Alb (Car -/-/HRN) double knockout mice respectively. Pxr -/-/HRN and Car -/-/HRN were crossed to generate Car -/-/Pxr -/-/Por lox/lox Cre Alb (Car -/-/Pxr -/-/HRN) triple knockout mice. Pxr -/-/Por lox/lox , Car -/-/Por lox/lox and Car -/-/Pxr -/-/Por lox/lox littermates were used as controls. Pxr - /-and Car -/-mice were identified by PCR as previously described [14,[START_REF] Wei | The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolism[END_REF].

All mice were maintained under standard animal house conditions with a 12hr light/12hr dark cycle and free access to water and RM1 (control) diet (Special Diets Services, Essex, UK). All studies were carried out on 12 week old male mice, except where indicated, in accordance with the Animal Scientific Procedures Act (1986) and after local ethical review.

Drug Treatments

3-Methylcholanthrene (3MC) in corn oil was administered to Por lox/lox /Cre CYP1A1 and Por lox/lox mice by intraperitoneal (i.p.) injection at 40 mg/kg body weight, and animals harvested at the indicated times.

Dietary Restrictions

Por lox/lox /Cre CYP1A1 and Por lox/lox mice were weaned and maintained on a control diet until 8 weeks old before being transferred to either a fat-deficient diet (SDS, 821459) or a fatty acid supplemented fat-deficient diet (Table S1), for an additional 6 weeks. As controls, both Por lox/lox /Cre CYP1A1 and Por lox/lox mice were weaned and maintained on the control diet for the duration of the experiment. All diets are isocaloric and free of cholesterol. At 12 weeks of age mice were administered either a single i.p. dose of 3MC (40 mg/kg body weight) or corn oil, and harvested 14 days later.

For linoleic acid treatment, Por lox/lox /Cre CYP1A1 were maintained on the fat-deficient diet as described above. At 12 weeks of age mice were administered a single i.p. dose of 3MC (40 mg/kg body weight), followed by an oral dose of linoleic acid of 65 mg/day from day 5 after 3MC administration until being harvested 9 days later.

Immunoblotting and Biochemical Analysis

Microsomal fractions were prepared by differential centrifugation [START_REF] Meehan | Regulation of phenobarbital-inducible cytochrome P-450s in rat and mouse liver following dexamethasone administration and hypophysectomy[END_REF] and protein concentrations were determined using the Bio-Rad Protein Assay Reagent (Bio-Rad, Herts., UK). Western blot analysis was carried out as described previously [16] using 5µg microsomal protein per lane and polyclonal antisera raised against human POR [START_REF] Smith | Dissection of NADPH-cytochrome P450 oxidoreductase into distinct functional domains[END_REF], murine Cyp7a1 [START_REF] Finn | Conditional deletion of cytochrome P450 oxidoreductase in the liver and gastrointestinal tract: a new model for studying the functions of the P450 system[END_REF] or rat P450s [16]. Purified human POR, rat CYP4A1 and the following murine P450s -Cyp2b10, His 6 -Cyp3a11, His 6 -Cyp7a1 -were used as standards. Immunoreactive proteins were detected using donkey anti-rabbit horseradish peroxidase IgG as a secondary antibody (DAKO, Ely, UK) and visualised by chemiluminescence (ECL+, GE Healthcare, Bucks, UK). The relative protein content of individual P450s in microsomal fractions was determined by direct scanning of ECL membranes with Bio-Rad Quantity One densitometry software. POR activity was determined by NADPH-dependent reduction of cytochrome c as previously described [START_REF] Smith | Dissection of NADPH-cytochrome P450 oxidoreductase into distinct functional domains[END_REF].

RNA isolation and Real-Time Quantitative PCR analysis

Total RNA was isolated from snap-frozen liver samples using Trizol (Invitrogen, Paisley, UK). Genomic DNA was removed using RQ1 DNase (Promega, Southampton, UK), and RNA (600 ng) reverse transcribed using random hexamers and Superscript II RNase H reverse transcriptase polymerase (Invitrogen, Paisley UK). Real-Time Quantitative PCR analysis was carried out with gene-specific primers for Cyp2b10, Cyp3a11 and CAR as previously described [START_REF] Wang | Relationship between hepatic phenotype and changes in gene expression in cytochrome P450 reductase (POR) null mice[END_REF] and CD36 (Mm01135198_m1), fatty acid synthase (Mm01253292_m1) and carnitine-palmitoyltransferase 1a (Mm00550438_m1) supplied by Applied Biosystems (Cheshire, UK). Reactions were carried out in triplicate and monitored by measuring fluorescence at 518 nm with excitation at 494 nm. mRNA levels were quantified using the Prism 7700 associated software and GAPDH as an internal standard.

Blood Chemistry

Blood was collected by cardiac puncture into heparinised tubes and plasma analysed for nonfasting total cholesterol, and non-fasting triglycerides using assay kits (Thermo Trace, Eastleigh, UK) on a Cobas Fara II centrifugal analyser (Roche, Hertfordshire, UK).

Histopathology

Tissue samples were snap-frozen in Cryo-M-Bed (Bright Instrument Co, Huntington, UK), cryo-sectioned (10μM) and stained with Oil Red O (lipid content) or haematoxylin and eosin (morphological analysis).

Lipid Isolation and Analysis

A small piece of liver was homogenised in phosphate-buffered saline containing 0.1% Triton X-100 and hepatic triglyceride and cholesterol levels determined using assay kits (Thermo Trace, Eastleigh, UK) on a Cobas Fara II centrifugal analyser (Roche, Herts., UK) [18].

Detailed fatty acid analysis of the various diets was carried out by Special Diet Services, Essex, UK (Tables S2 andS3).

Plasmid Construction

The plasmids pCMX Gal4-hPXR-LBD and p4*4xGal4-UAS-luciferase were a generous gift from Luisella Vignati (Department of Pharmacokinetics, Dynamics and Metabolism, Gruppo Pfizer Inc., Italy) [19] and the former was used as a source of the pCMX Gal4 vector to facilitate the generation of further reporter constructs. The mouse CAR ligand binding domain from amino acids 118-358 (mCAR-LBD) was amplified using the following: 5′-GCGGAATTCGGTACTAAAGCATCTTTGCAACTGAA-3′ and 5′-GCGGATCCTCAACTGCAAATCTCCCCGAGC-3′ as forward and reverse primers respectively, incorporating 5′ EcoRI and 3′ BamHI sites to facilitate cloning. The resulting 738bp PCR product was cloned as an EcoRI/BamHI fragment into pCMX Gal4-hPXR-LBD replacing the hPXR-LDB and generating the vector, pCMX Gal4-mCAR-LBD.

Cell culture, transient transfection and cell treatments

All cell culture media and supplements were supplied by Invitrogen, Paisley, UK. HepG2 cells were cultured in DMEM containing 10% heat-inactivated foetal bovine serum, 1X non essential amino acids, 100U/ml penicillin, 100μg/ml streptomycin and 2mM L-glutamine at 37 o C and 5% CO 2 .

HepG2 cells were transiently transfected with pCMX Gal4-mCAR-LBD/p4*4xGal4-UAS-luciferase according to the method of Vignati et.al. [19], with the following modifications -Fugene6 (Roche, Herts., UK) was used instead of Lipofectamine Plus (Invitrogen, Paisley, UK), assays were carried out in serum-containing media and 0.2μg of the

RESULTS

Temporal analysis of the phenotypic changes observed in the Hepatic POR null mice

Por lox/lox /Cre CYP1A1 mice, in the absence of a chemical inducer, were phenotypically normal and indistinguishable from wild type animals. Treatment with a single dose of 3MC (40 mg/kg) led to a time-dependent complete deletion of the hepatic POR and the development of a phenotype that was identical to the original HRN model [START_REF] Finn | Conditional deletion of cytochrome P450 oxidoreductase in the liver and gastrointestinal tract: a new model for studying the functions of the P450 system[END_REF]. We therefore used this model to study the sequential development of the HRN phenotypic traits over a 28 day period following 3MC treatment.

Six days after 3MC administration a > 85% reduction in the expression of POR protein, determined both by immunoblotting and cytochrome c activity, had occurred (Figures 1A andB(i)), increasing to approximately 96% by day 14. These data were confirmed by immunohistochemistry where staining was reduced in all hepatocytes by day 3 and undetectable on day 14 and remained absent for at least 28 days (data not shown). Treatment with 3MC resulted in the anticipated induction of Cyp1a1 as a consequence of the activation of the Ah receptor. By day 7 -9 Cyp1a1 levels, presumably due to the metabolism and elimination of the 3MC, were undetectable, indicating that the level of Cyp1a1 was not induced by the POR deletion (Figure 1A). The transient changes observed in Cyp1a1 expression imply that Cre recombinase expression would follow the same pattern and suggest that the deletion of POR is reversible. Cre recombinase is indeed expressed transiently, similar to Cyp1a1, however return of POR is dependent on cell turnover, which in the case of hepatocytes is several weeks. In Por lox/lox /Cre CYP1A1 mice, no return of POR is observed 60 days post 3MC treatment (data not shown). In contrast, very marked increases in the expression of other P450s were detected (Figure 1A). Cyp2b, Cyp2c, Cyp3a and Cyp7a1 levels increased profoundly as a function of time and in association with the decrease in POR expression. Particularly marked changes were obtained in P450s identified as Cyp2b10 (corresponding to the upper band and Cyp2b9 to the lower band according to the findings of Li-Masters and Morgan [20]) and Cyp3a11. Interestingly, a stepwise increase in the level of Cyp2b proteins occurred between day 9 and 11, whereas the increase in Cyp2c and Cyp3a proteins appeared to occur more gradually. An induction of Cyp7a1 occurred on day 7 and continued to increase until day 21 (Figure 1A). Cyp4a levels decreased from day 11 in both control (Por lox/lox ) as well as POR null mice, after which time they remained slightly higher in POR nulls (data not shown). No changes were observed in the expression levels of Cyp2d proteins in any samples. In control, Por lox/lox animals, 3MC treatment had no effect on any of the P450s measured other than the induction of Cyp1a1 (data not shown).

Associated with the time-dependent reduction in POR were changes in liver size and non-fasting plasma total cholesterol and triglycerides levels (Figures 1B(ii), (iii) and (iv)). Significant consistent changes in liver/body weight ratio were observed from day 11, suggesting that other phenotypic changes must precede this effect (Figure 1B(ii)). A significant decrease in non-fasting plasma triglyceride levels also occurred at this time (Figure 1B(iv)). A reduction in non-fasting total plasma cholesterol levels was also observed which reduced steadily from day 3, in parallel with POR reduction (Figure 1B(iii)), indicating that the reduction in triglycerides occurs independently of the changes in cholesterol but in parallel with liver enlargement.

Loss of POR also led to a profound increase in hepatic lipids (Figure 1C). This accumulation occurred rapidly after 3MC administration and paralleled the loss of POR. At early time points, increases in lipid appeared to be localised to the perivenous region, the major site of POR and P450 expression, and not to the periportal region [START_REF] Wolf | Characterization, localization and regulation of a novel phenobarbital-inducible form of cytochrome P450, compared with three further P450isoenzymes, NADPH P450-reductase, glutathione transferases and microsomal epoxide hydrolase[END_REF]22]. At later time points this zonal localisation was lost, leading to lipid accumulation throughout the liver. Quantification of hepatic lipid content demonstrated that the hepatic steatosis was due to increases in hepatic triglycerides by day 11 (Figure 2A). No significant changes in hepatic cholesterol levels were observed over the entire time course of the experiment (Figure 2B). The changes in hepatic triglyceride content were closely correlated with changes in both liver/body weight ratio and the reduction in plasma triglycerides (Figures 2C andD). This suggests that the level of hepatic triglycerides determines the circulating triglyceride levels and that they are related to liver enlargement.

Changes in hepatic lipid homeostasis mediate P450 induction

Hepatic deletion of POR led to a profound time-dependent induction of several P450s, including members of the Cyp2b and Cyp3a families. This appeared to occur both at the protein and mRNA levels [START_REF] Henderson | Inactivation of the hepatic cytochrome P450 system by conditional deletion of hepatic cytochrome P450 reductase[END_REF][START_REF] Wang | Relationship between hepatic phenotype and changes in gene expression in cytochrome P450 reductase (POR) null mice[END_REF]. Changes in Cyp2b10 expression were first seen on day 3, with a very marked increase between days 9 and 11 (Figure 1A). The increases in Cyp2b10 protein and mRNA were not correlated with the decrease in POR activity (Figure S1A(i) and (ii)) and therefore did not appear to be due to changes in the metabolism of an endogenous inducing agent. However, Cyp2b10 protein and mRNA levels were correlated with each other indicating that the induction of the Cyp2b10 gene occurs mainly at a transcriptional level (Figure S1A(iii)). In addition, Cyp2b10 mRNA levels were correlated with hepatic triglyceride concentration (Figure S1A(iv)). Similar analysis for Cyp3a11 indicated that the increased protein expression occurred more rapidly than Cyp2b10, steadily increasing from day two, reaching a maximum by day 21 but also did not directly correlate with changes in POR activity (Figure S1B). Although Cyp3a11 mRNA levels were increased in POR nulls this did not correlate with the level of Cyp3a11 protein, indicating a more complex mechanism of regulation of this gene (Figure S1B(iii)) possibly involving both transcriptional and post-translational mechanisms. Unlike Cyp2b10, only a weak correlation between the increases in Cyp3a11 mRNA levels and hepatic triglyceride levels was observed (Figure S1B(iv)). Thus differences observed in the induction kinetics of Cyp2b10 and Cyp3a11, both at the protein and mRNA levels, suggests that the mechanisms regulating their overexpression are at least in part distinguishable.

To determine the contributions of lipid uptake, oxidation and synthesis to the development of the fatty liver phenotype and the induction of P450s, changes in the expression levels of the lipid transporter CD36, fatty-acid synthase (FAS) and carnitinepalmitoyltransferase 1a (Cpt1a) were determined in 3MC-treated Por lox/lox /Cre CYP1A1 mice compared to Por lox/lox mice (Figure S2). No significant changes in the expression levels of these genes were observed, except for Cpt1a on day 28 which showed a minor but significant increase and may possibly be an attempt to reverse lipid accumulation by increasing βoxidation. Increased expression of CD36 was observed on days 21 and 28 in Por lox/lox /Cre CYP1A1 mice which may contribute to the increase in hepatic lipid at these time points, however no correlation could be made earlier than day 21. There was no correlation between the expression levels of these genes and the increases in P450 expression. They therefore do not appear to contribute to the lipid accumulation.

Dietary fatty acids mediate P450 induction in hepatic POR null mice

In view of the finding that changes in hepatic fatty acid homeostasis correlated with the increases in P450 expression, detailed fatty acid analysis was carried out on livers from HRN and Por lox/lox (control) mice (Tables S4 andS5). Changes in all fatty acid classes occurred; however, the most significant changes were in monounsaturated and polyunsaturated fatty acids (Table S5), with C16:1, C18:1 and C20:3 increasing by 6.78, 14.06 and 6.19-fold respectively (Table S4). These data are in close agreement with the report of Weng, et.al., [START_REF] Weng | Hepatic Gene Expression Changes in Mouse Models with Liver-specific Deletion or Global Suppression of the NADPH-Cytochrome P450 Reductase Gene: MECHANISTIC IMPLICATIONS FOR THE REGULATION OF MICROSOMAL CYTOCHROME P450 AND THE FATTY LIVER PHENOTYPE[END_REF] who, for their hepatic POR null model, reported additional fold changes of 7.3 and 14.9 for C18:3 and C20:1 unsaturated fatty acids respectively. Together these findings suggest that the accumulated hepatic triglycerides preferentially contain unsaturated fatty acid chains and that these fatty acid classes may mediate the P450 induction.

In the light of this possibility, Por lox/lox /Cre CYP1A1 mice were placed on a fat-deficient diet for 4 weeks prior to 3MC administration. This treatment completely prevented the increases in P450 protein and mRNA expression as a consequence of POR deletion (Figure 3A). Maintenance of control Por lox/lox mice on a fat-deficient diet caused slight reductions in the constitutive expression of Cyp7a and Cyp2c protein, and did not affect the levels of the other P450s (data not shown). In addition to the changes in hepatic P450 expression, maintenance of Por lox/lox /Cre CYP1A1 mice on a fat-deficient diet prevented the accumulation of lipid in the livers of these animals (Figure 3C), resulting in an Oil red O staining pattern that was indistinguishable from that of Por lox/lox mice on a control diet. Also no significant changes in total plasma cholesterol or triglyceride levels were measured on deletion of POR in Por lox/lox /Cre CYP1A1 mice fed a fat-deficient diet (Figure S3). These data indicate that hepatic triglyceride accumulation in POR null mice originates from the diet and that as a consequence of the inactivation of the P450 system an inability to maintain hepatic lipid homeostasis occurs which triggers increased P450 expression (possibly in an attempt to restore normal hepatic lipid levels). It is worth noting that the fat-deficient diet also prevented the liver enlargement seen on POR deletion, thus providing a metabolic link to all three phenomena.

In order to identify the fatty acids that mediated P450 induction, various fatty acids were added back to the fat-deficient diet as outlined in Table S1. Por lox/lox /Cre CYP1A1 and Por lox/lox were maintained on the supplemented diets and analysed 14 days following 3MC administration. In diets containing unsaturated fatty acids the loss of P450 gene expression observed with the fat-deficient diet was reversed. This was particularly the case for diets containing sunflower oil (which is composed of approximately 88% unsaturated fatty acids) (Figure 3B, Table S2). The inclusion of saturated fatty acids in the diet was essentially without effect. None of the treatments had effects on POR or P450 gene expression in the control Por lox/lox animals, with the exception of Cyp4a which was induced by all three unsaturated fatty acid diets (data not shown). Analysis of Cyp2b10 and Cyp3a11 mRNA levels confirmed that the changes in protein expression were at least in part a result of transcriptional activation, the highest increases in mRNA being found in mice on the sunflower oil supplemented diet (Figures 3B(ii) and (iii)). Analysis of plasma lipid levels revealed some minor decreases in both total cholesterol and triglyceride levels between Por lox/lox /Cre CYP1A1 and Por lox/lox for all dietary treatments, however, these were only significant for the sunflower oil supplemented diet where a 1.5-(p = 0.036) and 1.8-fold (p = 0.013) decrease was observed, respectively (Figures S3A andB). Hepatic lipid accumulation was observed for all diets, except the fat-deficient diet, indicating that lipid accumulation alone is not sufficient to cause P450 induction (Figure 3C). In addition, no significant increases in liver/body weight ratio were observed for the diets used, except that containing sunflower oils (Figure S3C). These data indicate that lipid accumulation alone was not the cause of liver enlargement but that unsaturated fatty acids had the capacity to induce the effect. Comparison of the fatty acid composition of the diets (Tables S2 andS3), identified C18:2, linoleic acid, an n-6 PUFA, as a candidate fatty acid mediator of both P450 induction and liver growth as it was the only fatty acid present in the diets that correlated with the effects observed. This hypothesis suggests that the addition of linoleic acid as the sole dietary fatty acid should be sufficient to induce P450 expression in Por lox/lox /Cre CYP1A1 mice maintained on a fat-deficient diet. This was indeed found to be the case, with oral administration of linoleic acid (65 mg/day for 9 days) to Por lox/lox /Cre CYP1A1 mice on a fatdeficient diet resulting in induction of Cyp2b10, Cyp3a11 and Cyp2c proteins (Figure 4A). Analysis of mRNA levels for Cyp2b10 and Cyp3a11 showed this induction to occur at the transcription level (Figure 4B). In addition, administration of linoleic acid was found to promote hepatic lipid accumulation (Figure 4C). This lipid accumulation was reduced compared to Por lox/lox /Cre CYP1A1 mice maintained on a control diet and appeared concentrated around the blood vessels, a pattern similar to that observed at early time points following 3MC administration (Figure 1C), however in this case is probably due to differences in the amount of fat intake. These results therefore indicate that linoleic acid, an n-6 dietary essential fatty acid, is a key regulator of P450 expression in hepatic POR null models and that members of the Cyp2b, Cyp3a and Cyp2c, in a normal physiological situation, are involved in its metabolism.

Role of Nuclear receptors in P450 induction in HRN mice

Nuclear receptors that have been shown to mediate the regulation of many P450 genes in response to exogenous chemicals [START_REF] Honkakoski | Regulation of cytochrome P450 (CYP) genes by nuclear receptors[END_REF][START_REF] Xie | Reciprocal activation of xenobiotic response genes by nuclear receptors SXR/PXR and CAR[END_REF], have also been shown to play an important role in controlling liver growth [START_REF] Huang | Xenobiotic stress induces hepatomegaly and liver tumors via the nuclear receptor constitutive androstane receptor[END_REF][START_REF] Zhou | A novel pregnane X receptor-mediated and sterol regulatory elementbinding protein-independent lipogenic pathway[END_REF]. Of particular interest with regards to this study were CAR and PXR, which have been intimately linked to the regulation of CYP2B and CYP3A proteins in response to both exogenous and endogenous inducing agents [28-30] and PXR has been linked to triglyceride homeostasis [START_REF] Zhou | A novel pregnane X receptor-mediated and sterol regulatory elementbinding protein-independent lipogenic pathway[END_REF]. In order to investigate whether these nuclear receptors mediate the P450 over-expression in the HRN mice, we crossed the HRN animals with mice nulled at either the Car or Pxr locus alone or in combination, to generate Car -/-/HRN, Pxr -/- /HRN and Pxr -/-/Car -/-/HRN genotypes.

Loss of PXR did not affect POR deletion or the induction of P450 proteins observed in HRN mice (Figure 5A). In agreement with previous findings Pxr -/-null mice exhibited slight increases in Cyp3a11 protein [14], and also Cyp2b and Cyp2c proteins (Figure 5A(i)); Cyp3a11 mRNA was also elevated relative to control Por lox/lox mice (Figure 5A(ii)). Basal expression of Cyp2b10 mRNA was also very slightly increased (Figure 5A(ii)), although this was not reflected in detectable protein expression (Figure 5A(i)). The deletion of PXR did not affect the increases in both Cyp2b10 and Cyp3a11 protein in hepatic POR null animals, however a reduction in both Cyp2b10 and Cyp3a11 mRNA levels was observed, but this was not significant compared to HRN mice (Figure 5A(ii)). No changes in plasma and hepatic lipid levels were observed in Pxr -/-/HRN mice compared to HRN mice (Table S6 and Figure 5D), however, in Pxr -/-mice non-fasting plasma total cholesterol and triglyceride levels were reduced, the former significantly, in comparison to wild-type mice (Table S6).

In relation to HRN mice nulled for CAR, profound changes in the HRN phenotype were observed. The elevated expression of P450s Cyp2b, Cyp7a and Cyp2c was markedly attenuated, however, Cyp3a11 expression appeared unaffected (Figure 5B). Analysis of mRNA levels also showed that no induction of Cyp2b10 mRNA occurred in Car -/-/HRN mice, indicating that the increase in the expression of this P450 observed in HRN mice was a consequence of CAR activation (Figure 5B(ii)). Interestingly, deletion of CAR in both control and HRN mice further increased Cyp3a11 mRNA levels (Figure 5B(ii)). The reduction in non-fasting plasma total cholesterol and triglyceride levels observed in the HRN mice were however not reversed on the CAR null background (Table S6), however a significant reduction in hepatic lipid content for Car -/-/HRN mice compared to HRN mice was observed (Figure 5D). These data suggest that, in addition to mediating P450 induction in HRN mice, CAR plays a role in hepatic lipid accumulation.

Finally, due to the cross-talk between PXR and CAR [START_REF] Pascussi | The Tangle of Nuclear Receptors that Controls Xenobiotic Metabolism and Transport: Crosstalk and Consequences[END_REF], the effect of deleting both receptors on the development of the HRN phenotype was analysed. In Pxr -/-/ Car -/-/HRN mice, consistent with the CAR nulls, the expression of Cyp2b and Cyp2c proteins was profoundly reduced, whereas in contrast Cyp7a expression levels were only slightly affected (Figure 5C). In mice nulled at both the Car and Pxr gene loci a marked decrease in Cyp3a11 protein was also observed (Figure 5C(i)). The change in Cyp2b protein expression was also reflected in a reduction in Cyp2b10 mRNA level (Figure 5C(ii)). A slight decrease in Cyp3a11 mRNA was observed in Pxr -/-/ Car -/-/HRN mice, although this was not significantly different from HRN or Pxr -/-/Car -/-mice (Figure 5C(ii)). Deletion of both CAR and PXR on an HRN background resulted in an increase in non-fasting plasma total cholesterol level that was not significantly different from control (Por lox/lox ) mice. Although an increase in plasma triglycerides was also observed, this was not significantly different to the HRN value (Table S6). Hepatic lipid content was reduced in Pxr -/-/Car -/-/HRN mice compared to HRN mice, similar to levels observed in Car -/-/HRN mice but still elevated compared to controls (Figure 5D); however this reduction did result in a change in liver size (Table S6).

Direct activation of the nuclear receptor CAR by linoleic acid in Hepatic POR null mice

In order to establish whether PUFAs could activate CAR directly we carried out transactivation assays using a Gal4-CAR-LBD reporter assay in HepG2 cells [32]. In these experiments, both TCPOBOP and linoleic acid could activate CAR directly (Figure 6). Phenobarbital, which is known to activate CAR by an indirect mechanism, was not active in this assay. In view of reports that the fatty acid sensitive transcription factor, peroxisome proliferator-activated receptor α (PPARα) can effect CAR expression [START_REF] Wieneke | PPARalpha-dependent induction of the energy homeostasis-regulating nuclear receptor NR1i3 (CAR) in rat hepatocytes: potential role in starvation adaptation[END_REF][START_REF] Guo | Induction of nuclear translocation of constitutive androstane receptor by peroxisome proliferator-activated receptor alpha synthetic ligands in mouse liver[END_REF] and that the effects in the POR null mice may be indirect, we measured CAR mRNA expression in Por lox/lox /Cre CYP1A1 mice. Expression levels of CAR were found to be unchanged (Figure S2D).

DISCUSSION

The liver is the major organ controlling metabolic homeostasis due to dietary change. It is also the major tissue responsible for drug and dietary chemical detoxification through the actions of phase I and phase II drug metabolism in which the P450 system plays a pivotal role. Although it has been known for some time that alterations in diet and disease states such as diabetes affect the P450 system, there has been little evidence that P450s can play a direct role in metabolic control [START_REF] Kato | The effect of dietary fatty acids on the expression levels and activities of hepatic drug metabolizing enzymes[END_REF][36][START_REF] Khemawoot | Obesityinduced increase of CYP2E1 activity and its effect on disposition kinetics of chlorzoxazone in Zucker rats[END_REF]. However, there is a growing body of evidence, as demonstrated in this paper, that these pathways are intimately linked. This has a wide range of implications not only for how alterations in P450 function may influence the pathogenesis of disease, but also how diet and disease states may influence the metabolism and disposition of drugs and environmental agents; for example, obesity in rats has been shown to reduce expression levels of CAR and impaired CYP2B induction by phenobarbital [START_REF] Xiong | Role of constitutive androstane receptor in the in vivo induction of Mrp3 and CYP2B1/2 by phenobarbital[END_REF].

The metabolic dialogue between the pathways of detoxification and metabolic homeostasis are manifested in the findings that when the P450 system is inactivated profound changes in lipid homeostasis and liver size occur which are paralleled by the marked induction of P450s associated with drug metabolism in the absence of an exogenous inducing agent. We have demonstrated that P450s from a range of gene families are induced and that this induction parallels the accumulation of hepatic triglycerides. We have also shown that the regulation of different P450s is in part distinguishable, as exemplified by Cyp2b10 and Cyp3a11. Cyp2b10 induction is almost entirely controlled at the transcriptional level and occurs at a time point when there is a stepwise increase in triglyceride accumulation, where as in the case of Cyp3a11 there is only a poor correlation between mRNA induction and protein levels indicating that both transcriptional and post-translational mechanisms are involved. The latter mechanism could possibly occur via phosphorylation or ubiquitination [START_REF] Posti | Modulation of murine phenobarbital-inducible CYP2A5, CYP2B10 and CYP1A enzymes by inhibitors of protein kinases and phosphatases[END_REF][40][START_REF] Zangar | Cytochrome P450 3A conjugation to ubiquitin in a process distinct from classical ubiquitination pathway[END_REF]. This may also be the case for Cyp2c proteins. Evidence for distinct pathways of regulation are also reflected in the finding that the over-expression of these proteins is at least in part mediated by different transcription factors (see below). We have also demonstrated that hepatic steatosis per se is not sufficient to induce the hepatic P450 expression but this effect is mediated by dietary unsaturated fatty acids such as linoleic acid, which we have found is sufficient on its own to reverse the fat-deficient phenotype.

Cytochromes P450 of the CYP2B family are the major enzymes induced in the liver by barbiturate drugs such as phenobarbital [42] and have the capacity to metabolise fatty acids such as arachidonic acid [43]. Interestingly, Fulco and co-workers demonstrated over 20 years ago that a fatty acid monooxygenase involved in fatty acid oxidation in Bacillus megaterium is highly inducible by phenobarbital [START_REF] Ruettinger | Epoxidation of unsaturated fatty acids by a soluble cytochrome P-450-dependent system from Bacillus megaterium[END_REF][START_REF] Narhi | Phenobarbital induction of a soluble cytochrome P-450-dependent fatty acid monooxygenase in Bacillus megaterium[END_REF]. In fact studies on this pathway demonstrated that its induction provided an adaptive response against unsaturated fatty acid toxicity [START_REF] Palmer | The repressor protein, Bm3R1, mediates an adaptive response to toxic fatty acids in Bacillus megaterium[END_REF]. Whether the induction of enzymes such as Cyp2b10 and Cyp3a11 in mammals are part of a similar detoxification pathway against dietary unsaturated fatty acids which are not produced endogenously, such as linoleic acid, remains to be determined. However the data presented here does raise the possibility that, under certain circumstances, diets containing unsaturated fatty acids may influence hepatic detoxification systems.

A wide range of transcription factors have now been discovered which control fatty acid homeostasis and liver growth, including PPARα, CAR, PXR, liver X receptor (LXR) and hepatocyte nuclear factor 4 α (HNF4α) [START_REF] Huang | Xenobiotic stress induces hepatomegaly and liver tumors via the nuclear receptor constitutive androstane receptor[END_REF][START_REF] Zhou | A novel pregnane X receptor-mediated and sterol regulatory elementbinding protein-independent lipogenic pathway[END_REF][START_REF] Li | Peroxisome proliferator-activated receptor alpha is required for feedback regulation of highly unsaturated fatty acid synthesis[END_REF][START_REF] Hayhurst | Hepatocyte nuclear factor 4alpha (nuclear receptor 2A1) is essential for maintenance of hepatic gene expression and lipid homeostasis[END_REF][49]. For example, it has been demonstrated that PPARα mediates liver growth induced by perturbations in fatty acid homeostasis as a consequence of peroxisome proliferation and that PXR plays a role in controlling hepatic triglyceride homeostasis [START_REF] Zhou | A novel pregnane X receptor-mediated and sterol regulatory elementbinding protein-independent lipogenic pathway[END_REF][START_REF] Mccarthy | Disruption of hepatic lipid homeostasis in mice after amiodarone treatment is associated with peroxisome proliferator-activated receptor-alpha target gene activation[END_REF][START_REF] Yang | Hepatocyte-restricted constitutive activation of PPAR alpha induces hepatoproliferation but not hepatocarcinogenesis[END_REF]. It was therefore intriguing that the majority of the phenotypes resulting from inactivation of the hepatic P450 system and the downstream effects on fatty acid homeostasis were mediated by the transcription factor CAR. It has been postulated for some time that CAR function is regulated by both endogenous and exogenous inducing agents including metabolic control through AMP kinase [START_REF] Rencurel | AMP-activated protein kinase mediates phenobarbital induction of CYP2B gene expression in hepatocytes and a newly derived human hepatoma cell line[END_REF], interaction with the forkhead transcription factor FoxO1 [START_REF] Kodama | Nuclear receptors CAR and PXR cross talk with FOXO1 to regulate genes that encode drug-metabolizing and gluconeogenic enzymes[END_REF] and activation during fasting [START_REF] Maglich | The nuclear receptor CAR is a regulator of thyroid hormone metabolism during caloric restriction[END_REF]. It has also been postulated that CAR function can be modulated by the fatty acid sensitive transcription factor PPARα [START_REF] Wieneke | PPARalpha-dependent induction of the energy homeostasis-regulating nuclear receptor NR1i3 (CAR) in rat hepatocytes: potential role in starvation adaptation[END_REF][START_REF] Guo | Induction of nuclear translocation of constitutive androstane receptor by peroxisome proliferator-activated receptor alpha synthetic ligands in mouse liver[END_REF]. However, this mechanism of CAR activation would not appear to explain the hepatic phenotype as no changes in CAR expression were found and also linoleic acid activated CAR directly. However, to our knowledge the finding that CAR may play a role in linking the control of hepatic lipid homeostasis and endogenous P450 expression has not been previously reported. We provide additional evidence that for one group of enzymes in the Cyp3a gene family, PXR may also play a role in defining the response to unsaturated fatty acids. The finding that feeding conditional POR nulls a fatdeficient diet prevents the induction of Cyp3a11 suggests that fatty acids, as well as bile acids [14], can influence PXR function.

A possible pathway linking P450 function to the findings observed here is shown in Figure 7. As described in this model dietary unsaturated fatty acids enter the liver via fatty acids transporters, where in wild type mice they are oxidised for energy purposes or detoxified by P450s. Inactivation of the P450 system as a result of POR deletion leads to hepatic accumulation of unsaturated fatty acids initially via increase import due to the induction of lipid transporters such as CD36 [START_REF] Weng | Hepatic Gene Expression Changes in Mouse Models with Liver-specific Deletion or Global Suppression of the NADPH-Cytochrome P450 Reductase Gene: MECHANISTIC IMPLICATIONS FOR THE REGULATION OF MICROSOMAL CYTOCHROME P450 AND THE FATTY LIVER PHENOTYPE[END_REF] and a reduction of plasma lipid levels. Hepatic lipid accumulation triggers the activation of the nuclear receptor, CAR (and possibly PXR) leading to P450 induction and liver growth through the activation of other downstream growth/transcription factors [START_REF] Huang | Xenobiotic stress induces hepatomegaly and liver tumors via the nuclear receptor constitutive androstane receptor[END_REF][START_REF] Zhou | A novel pregnane X receptor-mediated and sterol regulatory elementbinding protein-independent lipogenic pathway[END_REF]. The over-expression of P450s is aimed at re-establishing hepatic lipid homeostasis and the prevention of toxicity via increased metabolism of unsaturated fatty acids. Inactivation of POR prevents this increased metabolism and ultimately leads to hepatic steatosis, which establishes a cyclical process that promotes further hepatic lipid accumulation and growth. The ability to control the hepatic deletion of POR conditionally using the CYP1A1-Cre system has allowed us to provide evidence in support of this pathway. Activation of Cre through the administration of 3MC resulted in the rapid deletion of POR, in that over 85% of hepatocytes did not express the protein after 6 days. Although accumulation of lipid was already occurring at this time point, there appears to be a threshold at which point the CAR pathway becomes activated. This hypothesis is supported by the finding that it was only after 9 -11 days that a marked change in triglyceride accumulation occurred which was directly linked to changes in plasma lipids, liver growth and P450 induction. Loss of P450 activity per se was not responsible for its auto-induction (although the corresponding accumulation of a metabolite produced e.g. a bile acid precursor, which binds to Cyp3a11 could explain the stabilisation of this protein). Whether the inactivation of the P450 system causes the triglyceride accumulation and liver growth and whether their induction is to attenuate the phenotypic changes produced remains uncertain; however, it is quite clear that the accumulation of hepatic triglycerides activates CAR. The demonstration of cross talk between drugs and lipids on CAR implies there will be drug/lipid interactions associated with effects on this receptor and evidence for this is provided by the work of Li, et.al. who showed that polyunsaturated fatty acids could influence the induction of P450 genes by phenobarbital [START_REF] Li | n-6 and n-3 polyunsaturated fatty acids down-regulate cytochrome P-450 2B1 gene expression induced by phenobarbital in primary rat hepatocytes[END_REF]. Why the HRN phenotype results in the reduction in circulating triglycerides and cholesterol remains to be established. It is interesting to note, however, that the cholesterol 7α-hydroxylase enzyme (Cyp7a1) involved in bile acid homeostasis also appears to be affected by unsaturated fatty acid levels in a CAR-dependent manner and possibly a PXR-dependent manner as suggested by the opposing effects on this protein's expression observed in Car -/-and Pxr -/-/Car -/-HRN mice. The effect observed here on this enzyme's expression is intriguing as a previous report suggested that CAR activation represses Cyp7a1 expression [START_REF] Miao | Functional inhibitory cross-talk between constitutive androstane receptor and hepatic nuclear factor-4 in hepatic lipid/glucose metabolism is mediated by competition for binding to the DR1 motif and to the common coactivators, GRIP-1 and PGC-1alpha[END_REF]. The contrasting results may be a consequence of the fact that fasted, as opposed to non-fasted, animals were used in Miao's study. Despite this, these 13 In summary, we have used a novel conditional hepatic POR null model to investigate the sequential development of the phenotypic changes observed in mice on deletion of POR. POR deletion and the associated inactivation of hepatic P450 function leads to accumulation of dietary fatty acids in the liver, in the form of triglycerides of unsaturated fatty acids, leading to organ enlargement and increased P450 expression, in the case of Cyp2b, Cyp2c and Cyp7a1 via a CAR-dependent signalling pathway. The molecular mechanism underlying Cyp3a11 expression appears to involve CAR, PXR and protein stabilisation. Induction of the P450 system appears to be mediated by the accumulation of PUFAs, such as linoleic acid (C18:2n-6) and suggests that this may be part of an adaptive response to prevent the deleterious effects of PUFA accumulation. The finding that the major P450s inducible by exogenous chemicals are also regulated by changes in fatty acid homeostasis has potentially significant implications for the variability observed in drug responses in man. Dietary unsaturated fatty acids enter the liver via fatty acids transporters where they are oxidised for energy purposes or detoxified by P450s. Inactivation of the P450 system as a result of POR deletion leads to hepatic accumulation of unsaturated fatty acids and a reduction of plasma lipid levels. Hepatic lipid accumulation triggers the activation of the nuclear receptor, CAR (and possibly PXR) leading to P450 induction and liver growth. The over-expression of P450s is aimed at re-establishing hepatic lipid homeostasis and the prevention of toxicity via increased metabolism of unsaturated fatty acids. Inactivation of POR prevents this increased metabolism and ultimately leads to hepatic steatosis. The development of steatosis establishes a cyclical process that potentiates hepatic lipid accumulation and growth. The adverse effect of P450 induction is altered drug metabolism and environmental chemical detoxification but in addition the increased production of oxidised lipids could result in development of oxidative stress.

Biochemical

  Journal Immediate Publication. Published on 09 Sep 2008 as manuscript BJ20080740 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080740 A c c e p t e d M a n u s c r i p t data suggest the existence of a new pathway for the dietary regulation of Cyp7a1 by unsaturated fatty acids.

Biochemical

  Journal Immediate Publication. Published on 09 Sep 2008 as manuscript BJ20080740 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080740A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

FIGURE LEGENDS Figure 1 .

 1 FIGURE LEGENDS Figure 1. Time-course of the phenotypic changes in conditional hepatic P450 oxidoreductase null mice. Por lox/lox /Cre CYP1A1 and Por lox/lox mice were treated with a single dose of 3MC (40 mg/kg) following which plasma and liver samples were collected at the time points shown. (A) POR and P450 protein expression. Samples represent a pool of three animals per time point and 5 μg of hepatic microsomal protein per lane. Purified human POR and murine P450s were used as standards. (B) (i) Hepatic microsomal POR activity, (ii) Liver to body weight ratios, = Por lox/lox /Cre CYP1A1 and = Por lox/lox , (iii) Non-fasting plasma total cholesterol and (iv) Nonfasting plasma triglyceride levels. Values represent mean ± S.E.M. of three animals per time point and in the case of POR activity and plasma lipid levels are expressed as a % compared to Por lox/lox mice at the same time point. p ≤ 0.05 = *, p ≤ 0.01 = **, p ≤ 0.001 = ***, compared to Por lox/lox mice. (C) Hepatic lipid content was visualised by Oil red O staining of snap-frozen sections. Photomicrographs were taken at 10x magnification, bright field, and are representative of the results from at least three mice per group.

Figure 2 .

 2 Figure 2. Hepatic lipid accumulation and its relationship with organ weight and plasma lipid levels in conditional hepatic P450 oxidoreductase null mice. Por lox/lox /Cre CYP1A1 and Por lox/lox mice were treated with a single dose of 3MC (40 mg/kg), and harvested on the indicated days. (A) Hepatic triglyceride and (B) cholesterol concentrations, and the correlation of hepatic triglyceride concentration with (C) liver/body weight ratio and (D) non-fasting plasma triglyceride levels. Values represent the mean ± S.E.M. of three animals per time point. p ≤ 0.05 = *, p ≤ 0.01 = **, p ≤ 0.001 = ***, compared to lipid level on day one.

Figure 3 .

 3 Figure 3. Development of phenotypic changes in conditional hepatic P450 oxidoreductase null mice on restricted fatty acid diets.Por lox/lox /Cre CYP1A1 mice were maintained on a control diet until 8 weeks old, and then either placed on a fat-deficient or fatty acid supplemented diet, or continued on the control diet, for the following 6 weeks. At 12 weeks old, they were treated with a single dose of 3MC (40 mg/kg) and harvested 14 days later. (A) POR and P450 expression levels in Por lox/lox /Cre CYP1A1 and Por lox/lox mice maintained on a control or fat-deficient diet treated with 3MC (+) or corn oil (CO) (-); (i) Protein expression determined by Western blotting and hepatic mRNA levels of (ii) Cyp2b10 and (iii) Cyp3a11 expressed as a fold change compared to Por lox/lox mice. (B) POR and P450 expression levels in Por lox/lox /Cre CYP1A1 mice maintained on fatty acid supplemented diets and treated with 3MC; (i) Protein expression determined by Western blotting and hepatic mRNA levels of (ii) Cyp2b10 and (iii) Cyp3a11 expressed as a fold change compared to Por lox/lox mice. (C) Hepatic lipid content determined by Oil red O staining of snap-frozen liver sections of Por lox/lox /Cre CYP1A1 mice maintained on fatty acid supplemented diets and treated with 3MC. Photomicrographs were taken at 10x magnification, bright field, and are representative of the results from at least three mice per group. For mRNA analysis, all values represent the mean ± S.E.M., n = 3. p ≤ 0.05 = *, p ≤ 0.01 = **, p ≤ 0.001 = ***, compared to Por lox/lox mice.= Por lox/lox /Cre CYP1A1 and = Por lox/lox . SFA = plus saturated fatty acids, MUFA = plus monounsaturated fatty acids, PUFA = plus polyunsaturated fatty acids and SunFl = plus 1% sunflower oil.

Figure 4 .

 4 Figure 4. Induction of hepatic P450 expression in conditional hepatic P450 oxidoreductase null mice by linoleic acid.Por lox/lox /Cre CYP1A1 mice were maintained on a control diet until 8 weeks old, and then placed on a fat-deficient diet for the following 6 weeks. At 12 weeks old, they were treated with a

Figure 5 .

 5 Figure 5. Induction of hepatic P450 expression and other phenotypes in HRN mice on nuclear receptor null backgrounds. (A) POR and P450 expression levels in Pxr -/-/HRN mice; (i) protein expression determined by Western blotting and (ii) hepatic mRNA levels of Cyp2b10 and Cyp3a11 expressed as a fold change compared to Por lox/lox mice. (B) POR and P450 expression levels in Car -/-/HRN mice; (i) protein expression determined by Western blotting and (ii) hepatic mRNA levels of Cyp2b10 and Cyp3a11 expressed as a fold change compared to Por lox/lox mice. (C) POR and P450 expression levels in Car -/-/Pxr -/-/HRN mice; (i) protein expression determined by Western blotting and (ii) hepatic mRNA levels of Cyp2b10 and Cyp3a11 expressed as a fold change compared to Por lox/lox mice. (D) Hepatic lipid content determined by Oil red O staining of snap-frozen liver sections. Photomicrographs were taken at 10x magnification, bright field, and are representative of the results from at least three mice per group. For mRNA analysis, all values represent the mean ± S.E.M. of n = 3. p ≤ 0.05 = *, p ≤ 0.01 = **, p ≤ 0.001 = ***, compared to Por lox/lox mice. = Cyp2b10 and = Cyp3a11.

Figure 6 .

 6 Figure 6. Activation of the nuclear receptor CAR by linoleic acid. HepG2 cells were transfected with pCMX Gal4-mCAR-LBD and p4*4xGal4-UAS4luciferase as outlined in the Experimental procedures. Transfected cells were subsequently treated with linoleic acid at a concentration ranging from 0 -12.5μM and luciferase activity analysed 24hr later. Transfected cells were treated with 0.25 -0.5μM TCPOBOP or 1mM PB as positive and negative controls. All values represent the mean ± S.E.M. of n = 6 determinations and are expressed as a fold change compared cells treated with either DMSO in the case of TCPOBOP and PB or the 0 μM sample in the case of linoleic acid. p ≤ 0.05 = *, p ≤ 0.01 = **, p ≤ 0.001 = ***.

Figure 7 .

 7 Figure 7. A schematic representation of the phenotypic changes that occur in hepatic P450 oxidoreductase null mouse models.Dietary unsaturated fatty acids enter the liver via fatty acids transporters where they are oxidised for energy purposes or detoxified by P450s. Inactivation of the P450 system as a result of POR deletion leads to hepatic accumulation of unsaturated fatty acids and a reduction of plasma lipid levels. Hepatic lipid accumulation triggers the activation of the nuclear receptor, CAR (and possibly PXR) leading to P450 induction and liver growth. The over-expression of P450s is aimed at re-establishing hepatic lipid homeostasis and the prevention of toxicity via increased metabolism of unsaturated fatty acids. Inactivation of POR prevents this increased metabolism and ultimately leads to hepatic steatosis. The development of steatosis establishes a cyclical process that potentiates hepatic lipid accumulation and growth. The adverse effect of P450 induction is altered drug metabolism and environmental chemical detoxification but in addition the increased production of oxidised lipids could result in development of oxidative stress.

Biochemical Journal Immediate Publication. Published on 09 Sep 2008 as manuscript BJ20080740 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080740 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society

A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgements:

The authors would like to thank Dr. Steven Kliewer for his gift of Pxr and Car null mice and Dr. Alexander Hill for carrying out the hepatic fatty acid analysis. Dianne Carrie and Catherine Hughes are thanked for excellent technical assistance with the animal work. This work was funded by Cancer Research UK programme grant to C.R.W.