F. Lasserre

Di-(2-ethylhexyl)-phthalate (DEHP) activates the constitutive androstane receptor (CAR): A novel signalling pathway sensitive to phthalates

Di-(2-ethylhexyl)-phthalate (DEHP), a widely used plasticizer, is detected in consumers body fluids. Contamination occurs through environmental and food chain sources. In mouse liver, DEHP activates the peroxisome proliferator-activated receptor alpha (PPARalpha) and regulates the expression of its target genes. Several in vitro investigations support the simultaneous recruitment of additional nuclear receptor pathways. We investigated, in vivo, the hepatic impact of low doses of DEHP on PPARalpha activation, and the putative activation of additional signalling pathways. Wild-type and PPARalpha-deficient mice were exposed to different doses of DEHP. Gene expression profiling delineated the role of PPARalpha and revealed a PPARalpha-independent regulation of several prototypic constitutive androstane receptor (CAR) target genes. Thus, we developed an original hepatic cell line expressing CAR to investigate its activation by DEHP. By means of a pharmacological inhibitor or CAR-targeting shRNAs, we established that CAR is required for the effect of DEHP on Cyp2b10, a recognized CAR target gene. Moreover, DEHP dose-dependently induced CYP2B6 in human primary hepatocyte cultures. This finding demonstrates that CAR also represents a transcriptional regulator sensitive to phthalates. CAR-mediated effects of DEHP provide a new rationale for most endpoints of phthalates toxicity described previously, including endocrine disruption, hepatocarcinogenesis and the metabolic syndrome.

Novel aspects of PPARα-mediated regulation of lipid and xenobiotic metabolism revealed through a nutrigenomic study

Peroxisome proliferator‐activated receptor‐α (PPARα) is a major transcriptional regulator of lipid metabolism. It is activated by diverse chemicals such as fatty acids (FAs) and regulates the expression of numerous genes in organs displaying high FA catabolic rates, including the liver. The role of this nuclear receptor as a sensor of whole dietary fat intake has been inferred, mostly from high‐fat diet studies. To delineate its function under low fat intake conditions (4.8% w/w), we studied the effects of five regimens with contrasted FA compositions on liver lipids and hepatic gene expression in wild‐type and PPARα‐deficient mice. Diets containing polyunsaturated FAs reduced hepatic fat stores in wild‐type mice. Only sunflower, linseed, and fish oil diets lowered hepatic lipid stores in PPARα−/− mice, a model of progressive hepatic triglyceride accumulation. These beneficial effects were associated, in particular, with dietary regulation of Δ9‐desaturase in both genotypes, and with a newly identified PPARα‐dependent regulation of lipin. Furthermore, hepatic levels of 18‐carbon essential FAs (C18:2ω6 and C18:3ω3) were elevated in PPARα−/− mice, possibly due to the observed reduction in expression of the Δ6‐desaturase and of enoyl‐coenzyme A isomerases. Effects of diet and genotype were also observed on the xenobiotic metabolism‐related genes Cyp3a11 and CAR. Conclusion: Together, our results suggest that dietary FAs represent—even under low fat intake conditions—a beneficial strategy to reduce hepatic steatosis. Under such conditions, we established the role of PPARα as a dietary FA sensor and highlighted its importance in regulating hepatic FA content and composition. (HEPATOLOGY 2007;45:767–7777.)

Germline transmission of exogenous genes in chickens using helper-free ecotropic avian leukosis virus-based vectors

We have used vectors derived from avian leukosis viruses to transduce exogenous genes into early somatic stem cells of chicken embryos. The ecotropic helper cell line, Isolde, was used to generate stocks of NL-B vector carrying theNeor selectable marker and theEscherichia coli lacZ gene. Microinjection of the NL-B vector directly beneath unincubated chicken embryo blastoderms resulted in infection of germline stem cells. One of the 16 male birds hatched (6.25%) from the injected embryos contained vector DNA sequences in its semen. Vector sequences were transmitted to G1 progeny at a frequency of 2.7%.Neor andlacZ genes were transcribedin vitro in chicken embryo fibroblast cultures from transgenic embryos of the G2 progeny.

Identification of potential mechanisms of toxicity after di-(2-ethylhexyl)-phthalate (DEHP) adult exposure in the liver using a systems biology approach

Phthalates are industrial additives widely used as plasticizers. In addition to deleterious effects on male genital development, population studies have documented correlations between phthalates exposure and impacts on reproductive tract development and on the metabolic syndrome in male adults. In this work we investigated potential mechanisms underlying the impact of DEHP on adult mouse liver in vivo. A parallel analysis of hepatic transcript and metabolic profiles from adult mice exposed to varying DEHP doses was performed. Hepatic genes modulated by DEHP are predominantly PPARalpha targets. However, the induction of prototypic cytochrome P450 genes strongly supports the activation of additional NR pathways, including Constitutive Androstane Receptor (CAR). Integration of transcriptomic and metabonomic profiles revealed a correlation between the impacts of DEHP on genes and metabolites related to heme synthesis and to the Rev-erbalpha pathway that senses endogenous heme level. We further confirmed the combined impact of DEHP on the hepatic expression of Alas1, a critical enzyme in heme synthesis and on the expression of Rev-erbalpha target genes involved in the cellular clock and in energy metabolism. This work shows that DEHP interferes with hepatic CAR and Rev-erbalpha pathways which are both involved in the control of metabolism. The identification of these new hepatic pathways targeted by DEHP could contribute to metabolic and endocrine disruption associated with phthalate exposure. Gene expression profiles performed on microdissected testis territories displayed a differential responsiveness to DEHP. Altogether, this suggests that impacts of DEHP on adult organs, including testis, could be documented and deserve further investigations.

Effects of dexamethasone, administered for growth promoting purposes, upon the hepatic cytochrome P450 3A expression in the veal calf.

Dexamethasone (DEX) exerts its known anti-inflammatory and immunosuppressant activities through the interaction with the glucocorticoid receptor (GR). In human liver, DEX is metabolized by cytochrome P450 3A (CYP3A); moreover, it is among those xenobiotics which induce CYP3A itself. The transcriptional regulation of CYP3A involves GR and nuclear receptors (NRs). In cattle, DEX is used at low dosages as a growth promoter; besides, CYP3A is expressed in the liver. In the present study, the effects of two illicit DEX protocols upon liver CYP3A were investigated in the veal calf. Dexamethasone, administered per os (DOS) or injected intramuscularly (DIM) at growth promoting purposes, increased GR mRNA (+25.62% and +73.02% of CTRL for DOS and DIM, respectively), while tyrosine aminotransferase (TAT) and NRs gene expression profiles were unaffected; decreased CYP3A mRNA (-20.64% and -16.07% with Q RT-PCR; -30.55% and -34.31% with Northern blotting); at the post-translational level, decreased TAT activity (-19.84% and 44.34%), CYP3A apoprotein (-27.65% and -42.85%) and CYP3A-dependent enzyme activities (erythromycin N-demethylase, -78.89% and -23.87%; ethylmorphine N-demethylase, -44.26% and -28.37%; testosterone 6beta-hydroxylase, -44.60% and -18.07%; testosterone 2beta-hydroxylase, -43.95% and -11.69%); by contrast, an increase (about 2-fold) of the urinary 6beta-hydroxycortisol:cortisol ratio was observed in vivo. In summary, DEX modulates cattle liver CYP3A at pre- and post-translational level. Species-differences in GR-NRs-CYP3A regulation and in their response to differing DEX dosages might justify present results. Furthermore, the urinary 6beta-hydroxycortisol:cortisol ratio is not useful to monitor in vivo CYP3A activity in DEX-treated individuals.

A role for PPARα in the regulation of arginine metabolism and nitric oxide synthesis

The pleiotropic effects of PPARα may include the regulation of amino acid metabolism. Nitric oxide (NO) is a key player in vascular homeostasis. NO synthesis may be jeopardized by a differential channeling of arginine toward urea (via arginase) versus NO (via NO synthase, NOS). This was studied in wild-type (WT) and PPARα-null (KO) mice fed diets containing either saturated fatty acids (COCO diet) or 18:3 n-3 (LIN diet). Metabolic markers of arginine metabolism were assayed in urine and plasma. mRNA levels of arginases and NOS were determined in liver. Whole-body NO synthesis and the conversion of systemic arginine into urea were assessed by using 15N2-guanido-arginine and measuring urinary 15NO3 and [15N]-urea. PPARα deficiency resulted in a markedly lower whole-body NO synthesis, whereas the conversion of systemic arginine into urea remained unaffected. PPARα deficiency also increased plasma arginine and decreased citrulline concentration in plasma. These changes could not be ascribed to a direct effect on hepatic target genes, since NOS mRNA levels were unaffected, and arginase mRNA levels decreased in KO mice. Despite the low level in the diet, the nature of the fatty acids modulated some effects of PPARα deficiency, including plasma arginine and urea, which increased more in KO mice fed the LIN diet than in those fed the COCO diet. In conclusion, PPARα is largely involved in normal whole-body NO synthesis. This warrants further study on the potential of PPARα activation to maintain NO synthesis in the initiation of the metabolic syndrome.

Intratesticular Inoculation of Avian Leukosis Virus (ALV) in Chickens-Production of Neutralizing Antibodies and Lack of Virus Shedding into Semen

In order to investigate the possibility of producing transgenic chickens by injection of avian leukosis virus-based vectors into testis, we have analyzed the infection rate of testicular cells following inoculation of Rous-associated virus type 1 (RAV-1) into the gonads of adult and 1-wk-old brown leghorn males. Viroproduction, neutralizing antibody production, and vital DNA presence in testis, blood, muscle, and semen were analyzed at various times after infection. Inoculation of RAV-1 into the gonads of adult males resulted in a low level of viroproduction in testis and blood, followed by the appearance of neutralizing antibody 2 or 3 wk later. Neither viroproduction in semen nor viral DNA presence in sperm were detected even though the infected chickens were found to produce RAV-1 in testis. One week after intratesticular inoculation of 1-wk-old males with RAV-1, a high level of viroproduction was found in blood and testis, and viral DNA was detected in gonadal cells. Further, by 6 wk after inoculation, the production of virus decreased in all tissues, viral DNA could not longer be detected in the testis, and neutralizing antibodies appeared in blood. All together these data show that it is possible to infect testicular cells by direct inoculation of RAV-1 in the testis, and that the immune response of both adult and young chickens seems to reduce this infection. Moreover, no evidence of spermatozoa infection was found; this result suggests that RAV-1 inoculation into testis may not induce genetic transmission of virus, and consequently would not be useful in the production of transgenic chickens.

Insights into the role of hepatocyte PPARα activity in response to fasting

The liver plays a central role in the regulation of fatty acid metabolism. Hepatocytes are highly sensitive to nutrients and hormones that drive extensive transcriptional responses. Nuclear hormone receptors are key transcription factors involved in this process. Among these factors, PPARα is a critical regulator of hepatic lipid catabolism during fasting. This study aimed to analyse the wide array of hepatic PPARα-dependent transcriptional responses during fasting. We compared gene expression in male mice with a hepatocyte specific deletion of PPARα and their wild-type littermates in the fed (ad libitum) and 24-h fasted states. Liver samples were acquired, and transcriptome and lipidome analyses were performed. Our data extended and confirmed the critical role of hepatocyte PPARα as a central for regulator of gene expression during starvation. Interestingly, we identified novel PPARα-sensitive genes, including Cxcl-10, Rab30, and Krt23. We also found that liver phospholipid remodelling was a novel fasting-sensitive pathway regulated by PPARα. These results may contribute to investigations on transcriptional control in hepatic physiology and underscore the clinical relevance of drugs that target PPARα in liver pathologies, such as non-alcoholic fatty liver disease.

Hepatic Fasting-Induced PPARα Activity Does Not Depend on Essential Fatty Acids

The liver plays a central role in the regulation of fatty acid metabolism, which is highly sensitive to transcriptional responses to nutrients and hormones. Transcription factors involved in this process include nuclear hormone receptors. One such receptor, PPARα, which is highly expressed in the liver and activated by a variety of fatty acids, is a critical regulator of hepatic fatty acid catabolism during fasting. The present study compared the influence of dietary fatty acids and fasting on hepatic PPARα-dependent responses. Pparα−/− male mice and their wild-type controls were fed diets containing different fatty acids for 10 weeks prior to being subjected to fasting or normal feeding. In line with the role of PPARα in sensing dietary fatty acids, changes in chronic dietary fat consumption influenced liver damage during fasting. The changes were particularly marked in mice fed diets lacking essential fatty acids. However, fasting, rather than specific dietary fatty acids, induced acute PPARα activity in the liver. Taken together, the data imply that the potent signalling involved in triggering PPARα activity during fasting does not rely on essential fatty acid-derived ligand.

O29: Régulation circadienne et nutritionnelle de FGF21 par PPARalpha

Introduction et but de l’etude Le « Fibroblast Growth Factor 21 » (FGF21) est une hepatokine qui exerce de nombreux effets endocriniens. Elle exerce des effets insulino-sensibilisateurs et des effets sur le brunissement des adipocytes qui en font une cible phar-macologique privilegiee pour le traitement des maladies metaboliques associees a l’obesite. Pendant longtemps le foie a ete considere comme la seule source de FGF21. Cependant, il est desormais clair que d’autres tissus comme le coeur, le pancreas et les tissus adipeux expriment FGF21. Dans ce projet nous avons etudie la regulation nutritionnelle de l’expression de FGF21 au niveau du foie et mesure la contribution de PPARalpha a ces regulations. Materiel et methodes Nous avons teste in vivo l’effet des acides gras polyinsatures alimentaires, de l’apport en lipides et du jeune sur l’expression de FGF21 hepatique et plasmatique. De plus, nous avons manipule la prise alimentaire pour reprogrammer l’horloge hepatique et etudier la regulation circadienne de l’expression de FGF21. Nous avons realise la deletion hepato-specifique de PPARalpha afin de mesurer son importance dans cette regulation de FGF21. Resultats et Analyse statistique Dans les conditions testees, seul le jeune exerce un effet marque sur l’expression de FGF21. Nous montrons que l’expression hepatique de PPARalpha regule l’expression de FGF21 hepatique et plasmatique en reponse au jeune. Nous montrons egalement que cette regulation est specifique de certaines periodes du cycle circadien et que le controle de cette rythmicite est central. Conclusion Ce travail valide pour la premiere fois l’hypothese que l’activite hepatique de PPARalpha exerce des effets endocriniens en controlant l’expression plasmatique de FGF21.