Transcriptomic modifications of the thyroid gland upon exposure to phytosanitary-grade fipronil: Evidence for the activation of compensatory pathways.

Fipronil is a phenylpyrazole insecticide used for the control of a variety of pest for domestic, veterinary and agricultural uses. Fipronil exposure is associated to thyroid disruption in the rat. It increases thyroid hormone (TH) hepatic clearance. The effect on thyroxine (T4) clearance is about four fold higher than the effect on T4 plasma concentrations suggesting that the thyroid gland might develop compensatory mechanisms. The aim of this study was to document the potential effects of fipronil treatment on the thyroid transcriptome together with its effects on TSH and TH blood levels under well characterized internal exposure to fipronil and its main metabolite fipronil sulfone. Fipronil (3 mg/kg/d by gavage for 14 days) clearance increased while its half-life decreased (about 10 fold) throughout treatment. Fipronil treatment in adult female rats significantly decreased total T4 and free triiodothyronine (T3) concentrations. Key genes related to thyroid hormone synthesis and/or cellular dynamic were modulated by fipronil exposure. RT-PCR confirmed that thyroglobulin gene expression was upregulated. A trend toward higher Na/I symporter expression was also noted, while sulfotransferase 1a1 gene expression was down-regulated. The expression of genes potentially involved in thyroid cell dynamic were upregulated (e.g. prostaglandin synthase 1, amphiregulin and Rhoa). Our results indicate that both pathways of TH synthesis and thyroid cell dynamics are transcriptional targets of fipronil and/or its main sulfone metabolite. The underlying mechanisms remain to be elucidated.

Liver PPARα is crucial for whole-body fatty acid homeostasis and is protective against NAFLD.

OBJECTIVE: Peroxisome proliferator-activated receptor α (PPARα) is a nuclear receptor expressed in tissues with high oxidative activity that plays a central role in metabolism. In this work, we investigated the effect of hepatocyte PPARα on non-alcoholic fatty liver disease (NAFLD). DESIGN: We constructed a novel hepatocyte-specific PPARα knockout (Pparα(hep-/-)) mouse model. Using this novel model, we performed transcriptomic analysis following fenofibrate treatment. Next, we investigated which physiological challenges impact on PPARα. Moreover, we measured the contribution of hepatocytic PPARα activity to whole-body metabolism and fibroblast growth factor 21 production during fasting. Finally, we determined the influence of hepatocyte-specific PPARα deficiency in different models of steatosis and during ageing. RESULTS: Hepatocyte PPARα deletion impaired fatty acid catabolism, resulting in hepatic lipid accumulation during fasting and in two preclinical models of steatosis. Fasting mice showed acute PPARα-dependent hepatocyte activity during early night, with correspondingly increased circulating free fatty acids, which could be further stimulated by adipocyte lipolysis. Fasting led to mild hypoglycaemia and hypothermia in Pparα(hep-/-) mice when compared with Pparα(-/-) mice implying a role of PPARα activity in non-hepatic tissues. In agreement with this observation, Pparα(-/-) mice became overweight during ageing while Pparα(hep-/-) remained lean. However, like Pparα(-/-) mice, Pparα(hep-/-) fed a standard diet developed hepatic steatosis in ageing. CONCLUSIONS: Altogether, these findings underscore the potential of hepatocyte PPARα as a drug target for NAFLD.

Activation of the Constitutive Androstane Receptor induces hepatic lipogenesis and regulates Pnpla3 gene expression in a LXR-independent way.

The Constitutive Androstane Receptor (CAR, NR1I3) has been newly described as a regulator of energy metabolism. A relevant number of studies using animal models of obesity suggest that CAR activation could be beneficial on the metabolic balance. However, this remains controversial and the underlying mechanisms are still unknown. This work aimed to investigate the effect of CAR activation on hepatic energy metabolism during physiological conditions, i.e. in mouse models not subjected to metabolic/nutritional stress. Gene expression profiling in the liver of CAR knockout and control mice on chow diet and treated with a CAR agonist highlighted CAR-mediated up-regulations of lipogenic genes, concomitant with neutral lipid accumulation. A strong CAR-mediated up-regulation of the patatin-like phospholipase domain-containing protein 3 (Pnpla3) was demonstrated. Pnpla3 is a gene whose polymorphism is associated with the pathogenesis of nonalcoholic fatty liver disease (NAFLD) development. This observation was confirmed in human hepatocytes treated with the antiepileptic drug and CAR activator, phenobarbital and in immortalized human hepatocytes treated with CITCO. Studying the molecular mechanisms controlling Pnpla3 gene expression, we demonstrated that CAR does not act by a direct regulation of Pnpla3 transcription or via the Liver X Receptor but may rather involve the transcription factor Carbohydrate Responsive Element-binding protein. These data provide new insights into the regulation by CAR of glycolytic and lipogenic genes and on pathogenesis of steatosis. This also raises the question concerning the impact of drugs and environmental contaminants in lipid-associated metabolic diseases.

Scientific literature on infectious diseases affecting livestock animals, longitudinal worldwide bibliometric analysis.

The objectives of this bibliometric analysis of the scientific literature were to describe the research subjects and the international collaborations in the field of research on infectious diseases in livestock animals including fishes and honeybees. It was based on articles published worldwide from 2006 through 2013. The source of data was the Web of Science, Core collection(®) and only papers fully written in English were considered. Queries were built that combined 130 descriptors related to animal species and 1213 descriptors related to diseases and pathogens. To refine and assess the accuracy of the extracted database, supplementary filters were applied to discard non-specific terms and neighbouring topics, and numerous tests were carried out on samples. For pathogens, annotation was done using a thematic terminology established to link each disease with its corresponding pathogen, which was in turn classified according to its family. A total of 62,754 articles were published in this field during this 8-year period. The average annual growth rate of the number of papers was 5%. This represents the reference data to which we compared the average annual growth rate of articles produced in each of the sub-categories that we defined. Thirty-seven percent of the papers were dedicated to ruminant diseases. Poultry, pigs and fishes were covered by respectively 21, 13 and 14% of the total. Thirty-seven percent of papers concerned bacteria, 33% viruses, 19% parasites, 2% prions, the remaining being multi-pathogens. Research on virology, especially on pigs and poultry, is increasing faster than the average. There also is increasing interest in monogastric species, fish and bees. The average annual growth rate for Asia was 10%, which is high compared to 3% for Europe and 2% for the Americas, indicating that Asia is currently playing a leading role in this field. There is a well established network of international collaborations. For 75% of the papers, the co-authors were from the same country, for 10%, they were from different countries on the same continent, and for 15%, they were from different continents. The annual growth rate of papers representing international collaborations generally is increasing more quickly than the overall average.

Essential fatty acids deficiency promotes lipogenic gene expression and hepatic steatosis through the liver X receptor.

BACKGROUND & AIMS: Nutrients influence non-alcoholic fatty liver disease. Essential fatty acids deficiency promotes various syndromes, including hepatic steatosis, through increased de novo lipogenesis. The mechanisms underlying such increased lipogenic response remain unidentified. METHODS: We used wild type mice and mice lacking Liver X Receptors to perform a nutrigenomic study that aimed at examining the role of these transcription factors. RESULTS: We showed that, in the absence of Liver X Receptors, essential fatty acids deficiency does not promote steatosis. Consistent with this, Liver X Receptors are required for the elevated expression of genes involved in lipogenesis in response to essential fatty acids deficiency. CONCLUSIONS: This work identifies, for the first time, the central role of Liver X Receptors in steatosis induced by essential fatty acids deficiency.

Tempol prevents cardiac oxidative damage and left ventricular dysfunction in the PPAR-α KO mouse.

Peroxisome proliferator-activated receptor (PPAR)-α deletion induces a profound decrease in MnSOD activity, leading to oxidative stress and left ventricular (LV) dysfunction. We tested the hypothesis that treatment of PPAR-α knockout (KO) mice with the SOD mimetic tempol prevents the heart from pathological remodelling and preserves LV function. Twenty PPAR-α KO mice and 20 age-matched wild-type mice were randomly treated for 8 wk with vehicle or tempol in the drinking water. LV contractile parameters were determined both in vivo using echocardiography and ex vivo using papillary muscle mechanics. Translational and posttranslational modifications of myosin heavy chain protein as well as the expression and activity of major antioxidant enzymes were measured. Tempol treatment did not affect LV function in wild-type mice; however, in PPAR-α KO mice, tempol prevented the decrease in LV ejection fraction and restored the contractile parameters of papillary muscle, including maximum shortening velocity, maximum extent of shortening, and total tension. Moreover, compared with untreated PPAR-α KO mice, myosin heavy chain tyrosine nitration and anion superoxide production were markedly reduced in PPAR-α KO mice after treatment. Tempol also significantly increased glutathione peroxidase and glutathione reductase activities (~ 50%) in PPAR-α KO mice. In conclusion, these findings demonstrate that treatment with the SOD mimetic tempol can prevent cardiac dysfunction in PPAR-α KO mice by reducing the oxidation of contractile proteins. In addition, we show that the beneficial effects of tempol in PPAR-α KO mice involve activation of the glutathione peroxidase/glutathione reductase system.

Low doses of bisphenol A induce gene expression related to lipid synthesis and trigger triglyceride accumulation in adult mouse liver.

UNLABELLED: Changes in lifestyle are suspected to have strongly influenced the current obesity epidemic. Based on recent experimental, clinical, and epidemiological work, it has been proposed that some food contaminants may exert damaging effects on endocrine and metabolic functions, thereby promoting obesity and associated metabolic diseases such as nonalcoholic fatty liver disease (NAFLD). In this work, we investigated the effect of one suspicious food contaminant, bisphenol A (BPA), in vivo. We used a transcriptomic approach in male CD1 mice exposed for 28 days to different doses of BPA (0, 5, 50, 500, and 5,000 µg/kg/day) through food contamination. Data analysis revealed a specific impact of low doses of BPA on the hepatic transcriptome, more particularly on genes involved in lipid synthesis. Strikingly, the effect of BPA on the expression of de novo lipogenesis followed a nonmonotonic dose-response curve, with more important effects at lower doses than at the higher dose. In addition to lipogenic enzymes (Acc, Fasn, Scd1), the expression of transcription factors such as liver X Receptor, the sterol regulatory element binding protein-1c, and the carbohydrate responsive element binding protein that govern the expression of lipogenic genes also followed a nonmonotonic dose-response curve in response to BPA. Consistent with an increased fatty acid biosynthesis, determination of fat in the liver showed an accumulation of cholesteryl esters and of triglycerides. CONCLUSION: Our work suggests that exposure to low BPA doses may influence de novo fatty acid synthesis through increased expression of lipogenic genes, thereby contributing to hepatic steatosis. Exposure to such contaminants should be carefully examined in the etiology of metabolic diseases such as NAFLD and nonalcoholic steatohepatitis.

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 (15)N(2)-guanido-arginine and measuring urinary (15)NO(3) and [(15)N]-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.

Issues and special features of animal health research.

In the rapidly changing context of research on animal health, INRA launched a collective discussion on the challenges facing the field, its distinguishing features, and synergies with biomedical research. As has been declared forcibly by the heads of WHO, FAO and OIE, the challenges facing animal health, beyond diseases transmissible to humans, are critically important and involve food security, agriculture economics, and the ensemble of economic activities associated with agriculture. There are in addition issues related to public health (zoonoses, xenobiotics, antimicrobial resistance), the environment, and animal welfare.Animal health research is distinguished by particular methodologies and scientific questions that stem from the specific biological features of domestic species and from animal husbandry practices. It generally does not explore the same scientific questions as research on human biology, even when the same pathogens are being studied, and the discipline is rooted in a very specific agricultural and economic context.Generic and methodological synergies nevertheless exist with biomedical research, particularly with regard to tools and biological models. Certain domestic species furthermore present more functional similarities with humans than laboratory rodents.The singularity of animal health research in relation to biomedical research should be taken into account in the organization, evaluation, and funding of the field through a policy that clearly recognizes the specific issues at stake. At the same time, the One Health approach should facilitate closer collaboration between biomedical and animal health research at the level of research teams and programmes.

Phenylbutyrate up-regulates the adrenoleukodystrophy-related gene as a nonclassical peroxisome proliferator.

X-linked adrenoleukodystrophy (X-ALD) is a demyelinating disease due to mutations in the ABCD1 (ALD) gene, encoding a peroxisomal ATP-binding cassette transporter (ALDP). Overexpression of adrenoleukodystrophy-related protein, an ALDP homologue encoded by the ABCD2 (adrenoleukodystrophy-related) gene, can compensate for ALDP deficiency. 4-Phenylbutyrate (PBA) has been shown to induce both ABCD2 expression and peroxisome proliferation in human fibroblasts. We show that peroxisome proliferation with unusual shapes and clusters occurred in liver of PBA-treated rodents in a PPARalpha-independent way. PBA activated Abcd2 in cultured glial cells, making PBA a candidate drug for therapy of X-ALD. The Abcd2 induction observed was partially PPARalpha independent in hepatocytes and totally independent in fibroblasts. We demonstrate that a GC box and a CCAAT box of the Abcd2 promoter are the key elements of the PBA-dependent Abcd2 induction, histone deacetylase (HDAC)1 being recruited by the GC box. Thus, PBA is a nonclassical peroxisome proliferator inducing pleiotropic effects, including effects at the peroxisomal level mainly through HDAC inhibition.

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.

Transcriptional regulation of hepatic fatty acid metabolism.

The liver is a major site of fatty acid synthesis and degradation. Transcriptional regulation is one of several mechanisms controlling hepatic metabolism of fatty acids. Two transcription factors, namely SREBP1-c and PPARalpha, appear to be the main players controlling synthesis and degradation of fatty acids respectively. This chapter briefly presents fatty acid metabolism. The first part focuses on SREBP1-c contribution to the control of gene expression relevant to fatty acid synthesis and the main mechanisms of activation for this transcriptional program. The second part reviews the evidence for the involvement of PPARalpha in the control of fatty acid degradation and the key features of this nuclear receptor. Finally, the third part aims at summarizing recent advances in our current understanding of how these two transcription factors fit in the regulatory networks that sense hormones or nutrients, including cellular fatty acids, and govern the transcription of genes implicated in hepatic fatty acid metabolism.

Possible involvement of pregnane X receptor-enhanced CYP24 expression in drug-induced osteomalacia.

Vitamin D controls calcium homeostasis and the development and maintenance of bones through vitamin D receptor activation. Prolonged therapy with rifampicin or phenobarbital has been shown to cause vitamin D deficiency or osteomalacia, particularly in patients with marginal vitamin D stores. However, the molecular mechanism of this process is unknown. Here we show that these drugs lead to the upregulation of 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24) gene expression through the activation of the nuclear receptor pregnane X receptor (PXR; NR1I2). CYP24 is a mitochondrial enzyme responsible for inactivating vitamin D metabolites. CYP24 mRNA is upregulated in vivo in mice by pregnenolone 16alpha-carbonitrile and dexamethasone, 2 murine PXR agonists, and in vitro in human hepatocytes by rifampicin and hyperforin, 2 human PXR agonists. Moreover, rifampicin increased 24-hydroxylase activity in these cells, while, in vivo in mice, pregnenolone 16alpha-carbonitrile increased the plasma concentration of 24,25-dihydroxyvitamin D(3). Transfection of PXR in human embryonic kidney cells resulted in rifampicin-mediated induction of CYP24 mRNA. Analysis of the human CYP24 promoter showed that PXR transactivates the sequence between -326 and -142. We demonstrated that PXR binds to and transactivates the 2 proximal vitamin D-responsive elements of the human CYP24 promoter. These data suggest that xenobiotics and drugs can modulate CYP24 gene expression and alter vitamin D(3) hormonal activity and calcium homeostasis through the activation of PXR.

Effect of breed upon cytochromes P450 and phase II enzyme expression in cattle liver.

Cattle represent an important source of animal-derived food-products; nonetheless, our knowledge about the expression of drug-metabolizing enzymes (DMEs) in present and other food-producing animals still remains superficial, despite the obvious toxicological consequences. Breed represents an internal factor that modulates DME expression and catalytic activity. In the present work, the effect of breed upon relevant phase I and phase II DMEs was investigated at the pretranscriptional and post-translational levels in male Charolais (CH), Piedmontese (PM) and Blonde d'Aquitaine (BA) cattle. Because specific substrates for cattle have not yet been identified, the breed effect upon specific cytochrome P450 (P450), UDP-glucuronosyltransferase (UGT), or glutathione S-transferase (GST) DMEs, in terms of catalytic activity, was determined by using human marker substrates. Among P450s, benzphetamine N-demethylase, 16beta-, 6beta-, and 2beta-testosterone hydroxylase, aniline and p-nitrophenol hydroxylase, and alpha-naphthol and p-nitrophenol UGT activities were significantly higher in CH; in contrast, lower levels of CYP1A1-, CYP1A2-, CYP2B6-, CYP2C9-, CYP2C18-, CYP3A4-, and UGT1A1-like mRNAs were noticed, with CH < PM < or = BA as a trend. CYP2B and CYP3A mRNA results were confirmed with immunoblotting, too. As regards conjugative DMEs, UGT1A6-like mRNA levels were consistent with respective catalytic activities. Both 1-chloro-2,4-dinitrobenzene and 3,4-dichloronitrobenzene GST activities were higher in BA, and these results agreed with GSTA1-, GSTM1-, and GSTP1-like mRNA amounts. Correlation analysis between catalytic activities and mRNAs showed either significant or uneven results, depending on the substrate. These findings confirm previous data obtained in laboratory species; however, further studies are required to ascribe this behavior to pretranscriptional or post-translational phenomena.

Cytochrome P450 inhibition profile in liver of veal calves administered a combination of 17beta-estradiol, clenbuterol, and dexamethasone for growth-promoting purposes.

The effects of the administration of a combination of 17beta-estradiol (10mg i.m. for three times at 17 days intervals), dexamethasone (4 mg/day for 6 days and 5mg/day for further 6 days, dissolved in milk), and clenbuterol (20 microg/kg b.w./day, dissolved in milk, for the last 40 days before slaughtering) for growth-promoting (GP) purposes on liver drug metabolising capacity were studied in crossbred Friesian male calves. Compared to controls, liver preparations from GP-treated calves showed an overall reduction in the extent of the in vitro ability to metabolize testosterone and a number of substrates, most notably those associated with CYP 2C or CYP 3A, which also displayed a reduced expression on western blotting. By contrast, the tested hydrolytic and conjugative pathways were not significantly affected. As measured by northern blot, the lack of significant differences in CYP mRNA abundance point to a post-transcriptional effect of the GP combination. The remarkable involvement of the affected hepatic CYPs in the biotransformation of both steroid hormones and a large array of commonly used drugs may result in the further accumulation of undesirable residues in meat and offals of illegally treated calves.

Fumonisin B1 exposure and its selective effect on porcine jejunal segment: sphingolipids, glycolipids and trans-epithelial passage disturbance.

Fumonisin B(1) (FB(1)) is a mycotoxin produced by Fusarium verticillioides, the cause of Fusarium kernel rot in maize. FB(1) is toxic in domestic and laboratory animals, including pigs. This study investigated the effects of a seven-days-exposure of 1.5mg/kg b.w. FB(1) on the porcine intestinal epithelium. Statistically significant increases in the ratio of sphinganine to sphingosine, as well as alterations of the glycolipid distribution were observed in the jejunum. Using a porcine intestinal epithelial cell line (IPEC-1) derived from jejunum and ileum, we tested the effect of FB(1)in vitro in a time- and dose-dependent fashion. A significant increase in sphinganine concentration was observed after 2 days of FB(1) exposure at concentrations >100 microM, or from 6 days of FB(1) exposure at concentration >20 microM. We were also able to show that FB(1) exposure at 200 microM during 16 days increased the intestinal trans-epithelial flux of FB(1). These data indicate that, in pigs, this mycotoxin acts selectively on jejunum cells as follows: (i) FB(1) affects sphingolipid metabolism, as demonstrated by an increase of the amount of free sphingoid bases in a time- and dose-dependent manner, (ii) a depletion of the glycolipids in plasma membranes is observed, and (iii) an increase occurs in the trans-epithelial flux.

Interaction of macrocyclic lactones with P-glycoprotein: structure-affinity relationship.

P-glycoprotein (P-gp) is involved in the ATP-dependant cellular efflux of a large number of drugs including ivermectin, a macrocyclic lactone (ML) endectocide, widely used in livestock and human antiparasitic therapy. The interactions of P-gp with ivermectin and other MLs were studied. In a first approach, the ability of ivermectin (IVM), eprinomectin (EPR), abamectin (ABA), doramectin (DOR), selamectin (SEL), or moxidectin (MOX) to inhibit the rhodamine123 efflux was measured in recombinant cells overexpressing P-gp. Then, the influence of these compounds on the P-gp ATPase activity was tested on membrane vesicles prepared from fibroblasts overexpressing P-gp. All the MLs tested increased the intracellular rhodamine123. However, the potency of MOX to inhibit P-gp function was 10 times lower than the other MLs. They all inhibited the basal and decreased the verapamil-stimulated P-gp ATPase activity. But SEL and MOX were less potent than the other MLs when competing with verapamil. According to the structural specificity of SEL and MOX, we conclude that the integrity of the sugar moiety is determinant to achieve the optimal interaction of macrocyclic lactones with P-gp. The structure-affinity relationship for interaction with P-gp is important information for improving ML bioavailability and reversal of multidrug resistance (MDR).

Fasting induces hyperlipidemia in mice overexpressing proprotein convertase subtilisin kexin type 9: lack of modulation of very-low-density lipoprotein hepatic output by the low-density lipoprotein receptor.

Several proprotein convertase subtilisin kexin type 9 (PCSK9) mutations lead to familial hypercholesterolemia by virtue of its role as a negative modulator of the low-density lipoprotein receptor (LDLr). Here, we uncover that upon dietary challenge, the down-regulation of the LDLr is also a key mechanism by which PCSK9 modulates the hepatic production of apolipoprotein-B-containing lipoproteins. Thus, adenoviral-mediated overexpression of PCSK9 in 24-h fasted mice results in massive hyperlipidemia, due to a striking increase in very-low-density lipoprotein (VLDL) triglycerides and apolipoprotein B100 hepatic output. Similar studies in LDLr (-/-) mice demonstrate that PCSK9-mediated alteration of VLDL output in the fasted state requires the LDLr. This increased production of VLDL was associated with a concomitant reduction of intrahepatic lipid stores as well as a lack of down-regulation of peroxisome proliferator-activated receptor-alpha activity and target genes expression. Finally, we show that PCSK9 hepatic expression is inhibited by the hypotriglyceridemic peroxisome proliferator-activated receptor-alpha agonist fenofibrate. In summary, the negative modulation of LDLr expression by PCSK9, which decreases plasma LDL clearance, also promotes an overproduction of nascent VLDL in vivo upon fasting.

Effects of peroxisome proliferator-activated receptor alpha activation on pathways contributing to cholesterol homeostasis in rat hepatocytes.

Peroxisome proliferator-activated receptor alpha (PPARalpha) activation by fibrates controls expression of several genes involved in hepatic cholesterol metabolism. Other genes could be indirectly controlled in response to changes in cellular cholesterol availability. To further understand how fibrates may affect cholesterol synthesis, we investigated in parallel the changes in the metabolic pathways contributing to cholesterol homeostasis in liver. Ciprofibrate increased HMG-CoA reductase and FPP synthase mRNA levels in rat hepatocytes, together with cholesterogenesis from [(14)C] acetate and [(3)H] mevalonate. The up-regulation observed in fenofibrate- and WY-14,643-treated mice was abolished in PPARalpha-null mice, showing an essential role of PPARalpha. Among the three sterol regulatory element-binding protein (SREBP) mRNA species, only SREBP-1c level was significantly increased. In ciprofibrate-treated hepatocytes, cholesterol efflux was decreased, in parallel with cholesteryl ester storage and bile acids synthesis. As expected, AOX expression was strongly induced, supporting evidence of the peroxisome proliferation. Taken together, these results show that fibrates can cause cholesterol depletion in hepatocytes, possibly in part as a consequence of an important requirement of cholesterol for peroxisome proliferation, and increase cholesterogenesis by a compensatory phenomenon afterwards. Such cholesterogenesis regulation could occur in vivo, in species responsive to the peroxisome proliferative effect of PPARalpha ligands.

Transcriptional modulations by RXR agonists are only partially subordinated to PPARalpha signaling and attest additional, organ-specific, molecular cross-talks.

Nuclear hormone receptors (NR) are important transcriptional regulators of numerous genes involved in diverse pathophysiological and therapeutic functions. Following ligand activation, class II NR share the ability to heterodimerize with the retinoid X receptor (RXR). It is established that RXR activators, rexinoids, transactivate several peroxisome proliferator-activated receptor alpha (PPARalpha) target genes in a PPARalpha-dependent manner. We hypothesized that, once activated, RXR might signal through quiescent NR other than PPARalpha, in an organ-specific manner. To study this putative phenomenon in vivo, we developed an array of 120 genes relevant to the class II NR field. The genes were selected using both published data and high-density screenings performed on RXR or PPARalpha agonist-treated mice. Wild-type C57BL/6J and PPARalpha-deficient mice were treated with fenofibrate (PPARalpha activator) or LGD1069 (RXR activator). Using our customized array, we studied the hepatic, cardiac, and renal expression of this panel of 120 genes and compared them in both murine genotypes. The results obtained from this study confirmed the ability of an RXR agonist to modulate PPARalpha-restricted target genes in the liver and the kidney. Furthermore, we show that various organ-specific regulations occurring in both genotypes (PPARalpha +/+ or -/-) are highly indicative of the ability of RXR to recruit other class II NR pathways. Further development of this molecular tool may lead to a better understanding of the permissiveness of class II nuclear receptor dimers in vivo.