The Peroxisomal 3-keto-acyl-CoA thiolase B Gene Expression Is under the Dual Control of PPARα and HNF4α in the Liver.

PPARα and HNF4α are nuclear receptors that control gene transcription by direct binding to specific nucleotide sequences. Using transgenic mice deficient for either PPARα or HNF4α, we show that the expression of the peroxisomal 3-keto-acyl-CoA thiolase B (Thb) is under the dependence of these two transcription factors. Transactivation and gel shift experiments identified a novel PPAR response element within intron 3 of the Thb gene, by which PPARα but not HNF4α transactivates. Intriguingly, we found that HNF4α enhanced PPARα/RXRα transactivation from TB PPRE3 in a DNA-binding independent manner. Coimmunoprecipitation assays supported the hypothesis that HNF4α was physically interacting with RXRα. RT-PCR performed with RNA from liver-specific HNF4α-null mice confirmed the involvement of HNF4α in the PPARα-regulated induction of Thb by Wy14,643. Overall, we conclude that HNF4α enhances the PPARα-mediated activation of Thb gene expression in part through interaction with the obligate PPARα partner, RXRα.

Increased entropy production in diaphragm muscle of PPAR alpha knockout mice.

Peroxisome proliferator activated receptor alpha (PPAR alpha) regulates fatty acid beta-oxidation (FAO) and plays a central role in the metabolic and energetic homeostasis of striated muscles. The thermodynamic consequences of the absence of PPAR alpha were investigated in diaphragm muscle of PPAR alpha knockout mice (KO). Statistical mechanics provides a powerful tool for determining entropy production, which quantifies irreversible chemical processes generated by myosin molecular motors and which is the product of thermodynamic force A/T (chemical affinity A and temperature T) and thermodynamic flow (myosin crossbridge (CB) cycle velocity upsilon). The behavior of both wild type (WT) and KO diaphragm was shown to be near-equilibrium and in a stationary state, but KO was farther from equilibrium than WT. In KO diaphragm, a substantial decrease in contractile function was associated with an increase in both A/T and upsilon and with profound histological injuries such as contraction band necrosis. There were no changes in PPAR delta and gamma expression levels or myosin heavy chain (MHC) patterns. In KO diaphragm, a marked increase in entropy production (A/T x upsilon) accounted for major thermodynamic dysfunction and a dramatic increase in irreversible chemical processes during the myosin CB cycle.

PPARalpha transcriptionally induces AhR expression in Caco-2, but represses AhR pro-inflammatory effects.

In this work we demonstrate that Caco-2 cell treatment with WY-14643 (a potent PPARalpha agonist) causes an increase in AhR expression. Luciferase assays and directed mutagenesis experiments showed that induction mainly occurred at transcriptional level and involved a PPRE site located within the AhR promoter. These results were further confirmed by the use of PPARalpha knockout mice in which AhR induction by WY14643 was abrogated. In addition to CYP1 regulation, AhR has been described as being involved in inflammation, so we also studied the effect of AhR regulation by PPARalpha on the expression of some inflammation target genes. 3-Methylcholanthrene (a potent AhR agonist) increased the expression (mRNA) of the major inflammatory targets IL-1beta and MMP9. WY-14643 co-treatment abrogated the 3-methylcholanthrene pro-inflammatory effect. Hence the anti-inflammatory effect of PPARalpha overrides the pro-inflammatory effect of AhR.

Dehydroepiandrosterone up-regulates the Adrenoleukodystrophy-related gene (ABCD2) independently of PPARalpha in rodents.

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disease caused by mutations in the ABCD1 gene, which encodes a peroxisomal ABC transporter, ALDP, supposed to participate in the transport of very long chain fatty acids (VLCFA). The adrenoleukodystrophy-related protein (ALDRP), which is encoded by the ABCD2 gene, is the closest homolog of ALDP and is considered as a potential therapeutic target since functional redundancy has been demonstrated between the two proteins. Pharmacological induction of Abcd2 by fibrates through the activation of PPARalpha has been demonstrated in rodent liver. DHEA, the most abundant steroid in human, is described as a PPARalpha activator and also as a prohormone able to mediate induction of several genes. Here, we explored the in vitro and in vivo effects of DHEA on the expression of peroxisomal ABC transporters. We show that Abcd2 and Abcd3 but not Abcd4 are induced in primary culture of rat hepatocytes by DHEA-S. We also demonstrate that Abcd2 and Abcd3 but not Abcd4 are inducible by an 11-day treatment with DHEA in the liver of male rodents but not in brain, testes and adrenals. Finally and contrary to Abcd3, we show that the mechanism of induction of Abcd2 is independent of PPARalpha.

Evidence for a new human CYP1A1 regulation pathway involving PPAR-alpha and 2 PPRE sites.

BACKGROUND AND AIMS: Cytochrome P450 1A1 catalyzes the degradation of endobiotics (estradiol, fatty acids, and so on) and the bioactivation of numerous environmental procarcinogens, such as arylamines and polycyclic aromatic hydrocarbons, that are found in food. Several peroxisome proliferators and arachidonic acid derivatives enhance cytochrome P450 1A1 activity, but the mechanisms involved remain unknown. The aim of this work was to study the role of peroxisome proliferator-activated receptors in cytochrome P450 1A1 gene induction. METHODS: The role of peroxisome proliferator-activated receptor transcription factors in cytochrome P450 1A1 induction was assessed by means of enzymatic activities, quantitative real-time polymerase chain reaction, gene reporter assays, mutagenesis, and electrophoretic mobility shift assay. RESULTS: We show that peroxisome proliferator-activated receptor-alpha agonists (WY-14643, bezafibrate, clofibrate, and phthalate) induce human cytochrome P450 1A1 gene expression, whereas 2,4-thiazolidinedione, a specific peroxisome proliferator-activated receptor-gamma agonist, represses it. The induction of cytochrome P450 1A1 transcripts by WY-14643 was associated with a marked increase of ethoxyresorufin O -deethylase activity (10-fold at 200 mumol/L). Transfection of peroxisome proliferator-activated receptor-alpha complementary DNA enhanced cytochrome P450 1A1 messenger RNA induction by WY-14643, although WY-14643 failed to activate xenobiotic responsive element sequences. Two peroxisome proliferator response element sites were located at positions -931/-919 and -531/-519 of the cytochrome P450 1A1 promoter. Their inactivation by directed mutagenesis suppressed the inductive effect of WY-14643 on cytochrome P450 1A1 promoter activation. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay experiments showed that the 2 cytochrome P450 1A1 peroxisome proliferator response element sites bind the peroxisome proliferator-activated receptor-alpha/retinoid X receptor-alpha heterodimer. CONCLUSIONS: We describe here a new cytochrome P450 1A1 induction pathway involving peroxisome proliferator-activated receptor-alpha and 2 peroxisome proliferator response element sites, indicating that peroxisome proliferator-activated receptor-alpha ligands, which are common environmental compounds, may be involved in carcinogenesis.

Purification and characterization of a glutathione S-transferase Omega in pig: evidence for two distinct organ-specific transcripts.

A cytosolic glutathione S-transferase (GST, EC 2.5.1.18) from the recently characterized Omega class [GSTO; Board et al. 2000, J. Biol. Chem. 275, 24798-24806] has been identified in pig organs. It was found widely distributed in the different tissues investigated and especially abundant in liver and muscle. The hepatic enzyme has been purified to homogeneity by using its selective affinity for S-hexylglutathione over GSH, thus providing a simple method to isolate mammalian GSTO. The dimeric protein has a subunit molecular mass of 27328 Da as measured by electrospray ionization MS. Internal peptide sequencing and complete cDNA sequencing revealed strong similarities with its human recombinant orthologue and two rodent GST-like proteins with the ability to catalyse the GSH-dependent reduction of dehydroascorbate. Additional similarities, including the presence of a specific N-terminal extension and of immunological cross-reactivity, support the results. Moreover, this gene encoding GSTO generates two organ-specific transcripts, suggesting transcriptional mechanisms with a significance that is as yet uncharacterized.

Fibrate induction of the adrenoleukodystrophy-related gene (ABCD2): promoter analysis and role of the peroxisome proliferator-activated receptor PPARalpha.

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disease due to a defect in the ABCD1 (ALD) gene. ABCD1, and the two close homologues ABCD2 (ALDR) and ABCD3 (PMP70), are genes encoding ATP-binding cassette half-transporters of the peroxisomal membrane. As overexpression of the ABCD2 or ABCD3 gene can reverse the biochemical phenotype of X-ALD (reduced beta-oxidation of very-long-chain fatty acids), pharmacological induction of these partially redundant genes may represent a therapeutic approach to X-ALD. We previously reported that the ABCD2 and ABCD3 genes could be strongly induced by fibrates, which are hypolipidaemic drugs and peroxisome-proliferators in rodents. We provide evidence that the induction is dependent on peroxisome proliferator-activated receptor (PPARalpha) as both genes were not induced in fenofibrate-treated PPARalpha -/- knock-out mice. To further characterize the PPARalpha pathway, we cloned and analysed the promoter of the ABCD2 gene, the closest homologue of the ABCD1 gene. The proximal region (2 kb) of the rat promoter displayed a high conservation with the human and mouse cognate sequences suggesting an important role of the region in regulation of the ABCD2 gene. Classically, fibrate-induction involves interaction of PPARalpha with a response element (PPRE) characterized by a direct repeat of the AGGTCA-like motif. Putative PPRE motifs of the rat ABCD2 promoter were studied in the isolated form or in their promoter context by gel-shift assay and transfection of COS-7 cells. We failed to characterize a functional PPRE, suggesting a different mechanism for the PPARalpha-dependent regulation of the ABCD2 gene.

Differential involvement of peroxisome-proliferator-activated receptors alpha and delta in fibrate and fatty-acid-mediated inductions of the gene encoding liver fatty-acid-binding protein in the liver and the small intestine.

Liver fatty-acid-binding protein (L-FABP) is a cytoplasmic polypeptide that binds with strong affinity especially to long-chain fatty acids (LCFAs). It is highly expressed in both the liver and small intestine, where it is thought to have an essential role in the control of the cellular fatty acid (FA) flux. Because expression of the gene encoding L-FABP is increased by both fibrate hypolipidaemic drugs and LCFAs, it seems to be under the control of transcription factors, termed peroxisome-proliferator-activated receptors (PPARs), activated by fibrate or FAs. However, the precise molecular mechanism by which these regulations take place remain to be fully substantiated. Using transfection assays, we found that the different PPAR subtypes (alpha, gamma and delta) are able to mediate the up-regulation by FAs of the gene encoding L-FABP in vitro. Through analysis of LCFA- and fibrate-mediated effects on L-FABP mRNA levels in wild-type and PPARalpha-null mice, we have found that PPARalpha in the intestine does not constitute a dominant regulator of L-FABP gene expression, in contrast with what is known in the liver. Only the PPARdelta/alpha agonist GW2433 is able to up-regulate the gene encoding L-FABP in the intestine of PPARalpha-null mice. These findings demonstrate that PPARdelta can act as a fibrate/FA-activated receptor in tissues in which it is highly expressed and that L-FABP is a PPARdelta target gene in the small intestine. We propose that PPARdelta contributes to metabolic adaptation of the small intestine to changes in the lipid content of the diet.

Long-chain fatty acids regulate liver carnitine palmitoyltransferase I gene (L-CPT I) expression through a peroxisome-proliferator-activated receptor alpha (PPARalpha)-independent pathway.

Liver carnitine palmitoyltransferase I (L-CPT I) catalyses the transfer of long-chain fatty acid (LCFA) for translocation across the mitochondrial membrane. Expression of the L-CPT I gene is induced by LCFAs as well as by lipid-lowering compounds such as clofibrate. Previous studies have suggested that the peroxisome-proliferator-activated receptor alpha (PPARalpha) is a common mediator of the transcriptional effects of LCFA and clofibrate. We found that free LCFAs rather than acyl-CoA esters are the signal metabolites responsible for the stimulation of L-CPT I gene expression. Using primary culture of hepatocytes we found that LCFAs failed to stimulate L-CPT I gene expression both in wild-type and PPARalpha-null mice. These results suggest that the PPARalpha-knockout mouse does not represent a suitable model for the regulation of L-CPT I gene expression by LCFAs in the liver. Finally, we determined that clofibrate stimulates L-CPT I through a classical direct repeat 1 (DR1) motif in the promoter of the L-CPT I gene while LCFAs induce L-CPT I via elements in the first intron of the gene. Our results demonstrate that LCFAs can regulate gene expression through PPARalpha-independent pathways and suggest that the regulation of gene expression by dietary lipids is more complex than previously proposed.

New molecular aspects of regulation of mitochondrial activity by fenofibrate and fasting.

Fenofibrate and fasting are known to regulate several genes involved in lipid metabolism in a similar way. In this study measuring several mitochondrial enzyme activities, we demonstrate that, in contrast to citrate synthase and complex II, cytochrome c oxidase (COX) is a specific target of these two treatments. In mouse liver organelles, Western blot experiments indicated that mitochondrial levels of p43, a mitochondrial T3 receptor, and mitochondrial peroxisome proliferator activated receptor (mt-PPAR), previously described as a dimeric partner of p43 in the organelle, are increased by both fenofibrate and fasting. In addition, in PPAR alpha-deficient mice, this influence was abolished for mt-PPAR but not for p43, whereas the increase in COX activity was not altered. These data indicate that: (1) PPAR alpha is involved in specific regulation of mt-PPAR expression by both treatments; (2) fenofibrate and fasting regulate the mitochondrial levels of p43 and thus affect the efficiency of the direct T3 mitochondrial pathway.

Reduced hepatic fatty acid oxidation in fasting PPARalpha null mice is due to impaired mitochondrial hydroxymethylglutaryl-CoA synthase gene expression.

Glucose and fatty acid metabolism (oxidation versus esterification) has been measured in hepatocytes isolated from 24 h starved peroxisome proliferator-activated receptor-alpha (PPARalpha) null and wild-type mice. In PPARalpha null mice, the development of hypoglycemia during starvation was due to a reduced capacity for hepatic gluconeogenesis secondary to a 70% lower rate of fatty acid oxidation. This was not due to inappropriate expression of the hepatic CPT I gene, which was similar in both genotypes, but to impaired mitochondrial hydroxymethylglutaryl-CoA synthase gene expression in the PPARalpha null mouse liver. We also demonstrate that hepatic steatosis of fasting PPARalpha null mice was not due to enhanced triglyceride synthesis.

Effects of AFB1 on CYP 1A1, 1A2 and 3A6 mRNA, and P450 expression in primary culture of rabbit hepatocytes.

Although numerous studies report strong hepatic cytochrome P450 decrease during aflatoxicosis, the mechanisms involved in this decrease remain to be established. The purpose of this work is to investigate whether decreased CYP mRNA expression could explain decreased P450 expression and activity. Studies were conducted in primary cultures of rabbit hepatocytes exposed to 0.1 and 1 microM aflatoxin B1 (AFB1) incubated in the culture medium for 72 h. In order to confirm the effects of the mycotoxin, 30 microM beta-naphthoflavone or rifampicin were used as respective inductors of P450 1A1 and 1A2 or 3A6. Dose-dependent decreases of CYP mRNA expression were observed in all AFB1-treated cells; however, these decreases were not specific. Moreover, P450 expression and activity are far less decreased by the AFB1 treatment than their corresponding mRNA. Taken together, these results suggest that the specific P450 decrease observed during aflatoxicosis was not the consequence of a specific decrease of their mRNA expression.

Thymocyte development in Ah-receptor-deficient mice is refractory to TCDD-inducible changes.

The arylhydrocarbon receptor (AhR), a ligand-activated transcription factor, is differentially distributed in tissues and abundant in the thymus epithelium. The activated AhR can induce the transcription of an array of genes, including genes of cell growth and differentiation. Neither the physiological function of the AhR nor its putative natural ligand is known. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a xenobiotic high-affinity activator of the AhR, and appears to be essential for most of the multifold toxic effects of TCDD. Activation of the AhR by even low doses of TCDD results in general immunosuppression and thymus hypoplasia. TCDD exposure interferes with thymocyte development; for instance, it reduces the proliferation rate of the very immature (CD4- CD8- and CD4- CD8+ HSA+) thymocytes, leads to preferential emigration of very immature cells, and drastically skews the differentiation of thymocyte subpopulations towards mature CD4- CD8+ alphabeta TCRhigh thymocytes. As shown here, in fetal thymi of AhR-deficient mice, thymocyte differentiation kinetics as defined by CD4 and CD8 surface markers, was comparable to AhR+/+ C57BL/6 mice. Also, the cell emigration characteristics were similar to AhR+/+ mice. These parameters were refractory to TCDD exposure in the AhR-/- mice, but not in the C57BL/6 mice. However, in AhR deficient mice at gestation day 15 more CD4- CD8- immature cells bore high amounts of the (alphabeta-T-cell receptor. Also, fetal thymocyte numbers were significantly lower, as compared to strain C57BL/6. Thus, the AhR is the mediator of thymotoxic effects of TCDD.

Cytochrome P450 CYP1B1 determines susceptibility to 7, 12-dimethylbenz[a]anthracene-induced lymphomas.

CYP1B1-null mice, created by targeted gene disruption in embryonic stem cells, were born at the expected frequency from heterozygous matings with no observable phenotype, thus establishing that CYP1B1 is not required for mouse development. CYP1B1 was not detectable in cultured embryonic fibroblast (EF) or in different tissues, such as lung, of the CYP1B1-null mouse treated with the aryl hydrocarbon receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin whereas the equivalent wild-type EF cells express basal and substantial inducible CYP1B1 and lung expresses inducible CYP1B1. CYP1A1 is induced to far higher levels than CYP1B1 in liver, kidney, and lung in wild-type mice and is induced to a similar extent in CYP1B1-null mice. 7,12-dimethylbenz[a]anthracene (DMBA) was toxic in wild-type EFs that express CYP1B1 but not CYP1A1. These cells effectively metabolized DMBA, consistent with CYP1B1 involvement in producing the procarcinogenic 3,4-dihydrodiol as a major metabolite, whereas CYP1B1-null EF showed no significant metabolism and were resistant to DMBA-mediated toxicity. When wild-type mice were administered high levels of DMBA intragastrically, 70% developed highly malignant lymphomas whereas only 7.5% of CYP1B1-null mice had lymphomas. Skin hyperplasia and tumors were also more frequent in wild-type mice. These results establish that CYP1B1, located exclusively at extrahepatic sites, mediates the carcinogenicity of DMBA. Surprisingly, CYP1A1, which has a high rate of DMBA metabolism in vitro, is not sufficient for this carcinogenesis, which demonstrates the importance of extrahepatic P450s in determining susceptibility to chemical carcinogens and validates the search for associations between P450 expression and cancer risk in humans.

In vivo involvement of cytochrome P450 4A family in the oxidative metabolism of the lipid peroxidation product trans-4-hydroxy-2-nonenal, using PPARalpha-deficient mice.

Trans-4-hydroxy-2-nonenal (HNE) is a potent cytotoxic and genotoxic compound originating from the peroxidation of n-6 polyunsaturated fatty acids. Its metabolism has been previously studied in the rat (Alary et al. 1995. Chem. Res. Toxicol., 8: 35-39). In addition to major urinary mercapturic derivatives, some polar urinary metabolites were isolated and could correspond to hydroxylated compounds. 4-Hydroxynonenoic acid (HNA), resulting from the oxidation of the HNE carbonyl group, is a medium chain fatty acid and its omega-hydroxylation might be hypothesized. Therefore, the involvement of the CYP 4A family isoenzymes in the metabolism of [3H]HNE has been investigated in vivo using inducer treatments (fibrates) in wild-type or in peroxisome proliferator-activated receptor alpha (PPARalpha)-deficient mice. In wild-type mice, but not in PPARalpha (-/-) mice, fibrate treatments resulted in an increase of two urinary metabolites characterized, after HPLC purifications and mass spectrometry analyses, as the omega-hydroxylated metabolite of HNA, i.e., 4,9-dihydroxy-2-nonenoic acid, and its oxidized form, 4-hydroxy-2-nonene-1,9-dicarboxylic acid. The formation of the latter is correlated accurately to laurate hydroxylase activity studied concurrently in microsomes prepared from the liver of these animals. Basal levels of these two metabolites were measured in urine of normal and PPARalpha-deficient mice. These results are in accord with an implication of the P450 4A family in the extended oxidative metabolism of 4-HNE.

Comparative effects of cytokines on constitutive and inducible expression of the gene encoding for the cytochrome P450 3A6 isoenzyme in cultured rabbit hepatocytes: consequences on progesterone 6beta-hydroxylation.

Cultured rabbit hepatocytes were used to compare the relative activities of cytokines to inhibit the constitutive or rifampicin (RIF)-induced expression of the cytochrome P450 3A6 gene (CYP3A6). Human recombinant cytokines tested were interleukin-1beta (IL-1beta) (2 U/mL), interleukin-2 (IL-2) (5,000 U/mL) and interferon-gamma (IFN-gamma) (50 U/mL). Hepatocytes were cultured in the presence or absence of 25 microM RIF for 24 hr, with or without cytokines alone or in combination. All these cytokines inhibited RIF-induced P4503A6 expression without apparent cellular toxicity. By contrast, only IFN-gamma treatment provided a significant decrease (41%) in the constitutive P4503A6 protein level. Moreover, cytokines differed in their ability to repress RIF-dependent transcriptional induction of CYP3A6: IL-1beta and IL-2 were approximately equipotent, causing an almost 40-50% suppression of CYP3A6 mRNA and protein levels, whereas IFN-gamma exerted repressive effects only on P4503A6-related erythromycin N-demethylase activity and inducible protein expression. In fact, although strongly reducing P4503A6 protein content (an approximate 70% decrease), IFN-gamma did not exhibit any influence on CYP3A6 mRNAs with the exception of its association with interleukins. All these results suggest that IL-1beta and IL-2 mainly promote a transcriptional repression mechanism, given the absence of effect of these cytokines on the basal P4503A6 level, whereas IFN-gamma exerts a post-transcriptional suppressive action on both induced and constitutive P4503A6 expression. Consequently, P4503A6-dependent progesterone 6beta-hydroxylase activity also presented a cytokine-specific pattern of inhibition, with a much greater sensitivity than P4503A6 immunoreactive protein to IL-1beta and IL-2 + IFN-gamma treatments. Thus, this study underlines the significant impact of inflammation on steroid metabolism.

Peroxisome proliferator-activated receptor alpha controls the hepatic CYP4A induction adaptive response to starvation and diabetes.

The hepatic CYP4A enzymes are important fatty acid and prostaglandin omega-hydroxylases that are highly inducible by fibric acid hypolipidemic agents and other peroxisome proliferators. Induction of the CYP4A enzymes by peroxisome proliferators is mediated through the nuclear peroxisome proliferator-activated receptor alpha (PPARalpha). Fatty acids have recently been identified as endogenous ligands of PPARalpha, and this receptor has been implicated in the regulation of lipid homeostasis. In the present report we characterized the induction of the hepatic CYP4A genes in rats during the altered lipid metabolism associated with starvation and diabetes. The mRNA levels of CYP4A1, CYP4A2, and CYP4A3 were induced 7-17-fold in the livers of fasted animals and 3-8-fold in the livers of diabetic animals. This was accompanied by corresponding changes in CYP4A protein levels and arachidonic and lauric acid omega-hydroxylase activity. Interestingly, feeding animals after the fasting period caused as much as an 80% suppression of CYP4A mRNA levels, whereas CYP4A protein levels and functional activity returned to control values. A second PPARalpha-responsive gene, acyl-CoA oxidase, was also induced in rat liver by diabetes and fasting. By using PPARalpha-deficient mice, we unambiguously demonstrated that PPARalpha is strictly required for hepatic CYP4A induction by starvation and diabetes. Similarly, induction of hepatic thiolase and bifunctional enzyme also required expression of PPARalpha. This represents the first evidence for the pathophysiologically induced activation of a nuclear receptor.

Peroxisome proliferator-activated receptor alpha-isoform deficiency leads to progressive dyslipidemia with sexually dimorphic obesity and steatosis.

The alpha-isoform of the peroxisome proliferator-activated receptor (PPARalpha) is a nuclear transcription factor activated by structurally diverse chemicals referred to as peroxisome proliferators. Activators can be endogenous molecules (fatty acids/steroids) or xenobiotics (fibrate lipid-lowering drugs). Upon pharmacological activation, PPARalpha modulates target genes encoding lipid metabolism enzymes, lipid transporters, or apolipoproteins, suggesting a role in lipid homeostasis. Transgenic mice deficient in PPARalpha were shown to lack hepatic peroxisomal proliferation and have an impaired expression and induction of several hepatic target genes. Young adult males show hypercholesterolemia but normal triglycerides. Using a long term experimental set up, we identified these mice as a model of monogenic, spontaneous, late onset obesity with stable caloric intake and a marked sexual dimorphism. Serum triglycerides, elevated in aged animals, are higher in females that develop a more pronounced obesity than males. The latter show a marked and original centrilobular-restricted steatosis and a delayed occurrence of obesity. Fat cells from their liver express substantial levels of PPARgamma2 transcripts when compared with lean cells. These studies demonstrate, in rodents, the involvement of PPARalpha nuclear receptor in lipid homeostasis, with a sexually dimorphic control of circulating lipids, fat storage, and obesity. Characterization of this pathological link may help to delineate new molecular targets for therapeutic intervention and could lead to new insights into the etiology and heritability of mammalian obesity.

Fenofibrate modifies transaminase gene expression via a peroxisome proliferator activated receptor alpha-dependent pathway.

Fibrates modify the expression of genes implicated in lipoprotein and fatty acid metabolism via the peroxisome proliferator-activated receptor alpha(PPARalpha), leading to reductions in serum triglycerides and cholesterol. The expression of certain genes regulated by PPARalpha have been shown to be modified in a species dependent manner. Aspartate aminotransferase (AspAT or GOT) and alanine aminotransferase (AlaAT or GPT) are enzymes involved in intermediate metabolism in all cells and in hepatic gluconeogenesis. These enzymes are also widely used as serum markers of possible tissue damage. This study investigated whether fenofibrate could modify the expression of liver AspAT and/or AlaAT and thus possibly alter transaminase levels independently of a cytotoxic effect. In human Hep G2 cells, fenofibrate increased cytosolic AspAT (cAspAT) activity by 40% and AlaAT activity by 100%, as well as both mRNAs. Nuclear run on assays showed that this effect was, at least in part, transcriptional. Increases in mRNA were also observed in human hepatocyte cultures at concentrations of the drug attained in patients. In C57BL/6 mice, fenofibrate decreased cAspAT and cAlaAT mRNA, while these effects were abolished in PPARalpha knock-out mice. In conclusion, fenofibrate has been shown to modify cAspAT and AlaAT gene expression in a species and PPARalpha dependent manner. This is the first demonstration that cAspAT and AlaAT activities may be pharmacologically altered, independently of a toxic phenomenon.

Modulation of prostaglandin H synthase-2 mRNA expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin in mice.

Prostaglandin endoperoxide H synthases (PGHS-1 and PGHS-2) catalyze an intermediate step in the biosynthesis of prostaglandins and thromboxanes. Recently, it was observed that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) modulates the expression of PGHS-2 mRNA in different cell lines. The main aim of this study was to examine whether PGHS-2 mRNA expression can be changed by acute TCDD in vivo and, second, we were also interested in whether modulation of PGHS-2 is mediated by the aryl hydrocarbon receptor (AhR) which is known to be involved in the transcriptional control of TCDD-induced phase 1 and phase 2 enzymes. Initially C57BL/6J mice were treated with a single dose of 10,000 ng TCDD/kg and the PGHS-1 and PGHS-2 mRNAs were analyzed in liver, lung, thymus, kidney, and spleen. In all tissues examined the expression of PGHS-1 mRNA was not affected by TCDD. However, TCDD treatment enhanced the PGHS-2 mRNA levels in lung and spleen. No effect of TCDD on PGHS-2 expression was found in liver and kidney. For dose-response studies C57BL/6J and DBA/2J mice were treated for 24 h with various doses of TCDD (1-50,000 ng/kg) and the PGHS-2 mRNA increases were analyzed in lungs and spleens. A significant increase of PGHS-2 mRNA in lungs of C57BL/6J mice was found at a dose of 100 ng TCDD/kg, whereas a nearly 100-fold higher TCDD dose was needed to increase PGHS-2 in DBA/2J mice. A similar dose-dependent induction of PGHS-2 was found in spleens of C57BL/6J mice; however, no significant increase of PGHS-2 was found in spleens of DBA/2 mice. These results indicate an involvement of AhR in TCDD-mediated changes of PGHS-2 expression. This suggestion is supported by studies in AhR-deficient animals which showed that TCDD had no effect on PGHS-2 mRNA. When changes of PGHS-2 mRNA expression are compared with those of CYP1A1 between 4 and 72 h after TCDD, it is noteworthy that TCDD led to a delayed and more transient increase of PGHS-2. These data suggest that the mechanism of modulation of both genes by TCDD may be different.