Peroxisome proliferator-activated receptor alpha regulates a microRNA-mediated signaling cascade responsible for hepatocellular proliferation

Activation of peroxisome proliferator-activated receptor alpha (PPARalpha) leads to hepatocellular proliferation and liver carcinomas. The early events mediating these effects are unknown. A novel mechanism by which PPARalpha regulates gene expression and hepatocellular proliferation was uncovered. MicroRNA (miRNA) expression profiling demonstrated that activated PPARalpha was a major regulator of hepatic miRNA expression. Of particular interest, let-7C, an miRNA important in cell growth, was inhibited following 4-h treatment and 2-week and 11-month sustained treatment with the potent PPARalpha agonist Wy-14,643 in wild-type mice. let-7C was shown to target c-myc via direct interaction with the 3' untranslated region of c-myc. The PPARalpha-mediated induction of c-myc via let-7C subsequently increased expression of the oncogenic mir-17-92 cluster; these events did not occur in Pparalpha-null mice. Overexpression of let-7C decreased c-myc and mir-17 and suppressed the growth of Hepa-1 cells. Furthermore, using the human PPARalpha-expressing mouse model, which is responsive to Wy-14,643 effects on beta-oxidation and serum triglycerides but resistant to hepatocellular proliferation and tumorigenesis, we demonstrated a critical role for let-7C in liver oncogenesis. Wy-14,643 treatment did not inhibit let-7C or induce c-myc and mir-17 expression. These observations reveal a let-7C signaling cascade critical for PPARalpha agonist-induced liver proliferation and tumorigenesis.     

PPARalpha is a key regulator of hepatic FGF21

The metabolic regulator fibroblast growth factor 21 (FGF21) has antidiabetic properties in animal models of diabetes and obesity. Using quantitative RT-PCR, we here show that the hepatic gene expression of FGF21 is regulated by the peroxisome proliferator-activated receptor alpha (PPARalpha). Fasting or treatment of mice with the PPARalpha agonist Wy-14,643 induced FGF21 mRNA by 10-fold and 8-fold, respectively. In contrast, FGF21 mRNA was low in PPARalpha deficient mice, and fasting or treatment with Wy-14,643 did not induce FGF21. Obese ob/ob mice, known to have increased PPARalpha levels, displayed 12-fold increased hepatic FGF21 mRNA levels. The potential importance of PPARalpha for FGF21 expression also in human liver was shown by Wy-14,643 induction of FGF21 mRNA in human primary hepatocytes, and PPARalpha response elements were identified in both the human and mouse FGF21 promoters. Further studies on the mechanisms of regulation of FGF21 by PPARalpha in humans will be of great interest.     

Metabolomic and genetic analysis of biomarkers for peroxisome proliferator-activated receptor alpha expression and activation

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear receptor with manifold effects on intermediary metabolism. To define a set of urinary biomarkers that could be used to determine the efficacy of PPARalpha agonists, a metabolomic investigation was undertaken in wild-type and Pparalpha-null mice fed for 2 wk either a regular diet or a diet containing the PPARalpha ligand Wy-14,643 ([4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid), and their urine was analyzed by ultra-performance liquid chromatography coupled with time-of-flight mass spectrometry. Principal components analysis of 6393 accurate mass positive ions revealed clustering as a single phenotype of the treated and untreated Pparalpha (-/-) mice plus two additional discrete phenotypes for the treated and untreated Pparalpha (+/+) mice. Biomarkers of PPARalpha activation were identified from their accurate masses and confirmed by tandem mass spectrometry of authentic compounds. Biomarkers were quantitated from raw chromatographic data using appropriate calibration curves. PPARalpha urinary biomarkers highly statistically significantly elevated by Wy-14,643 treatment included 11beta-hydroxy-3,20-dioxopregn-4-en-21-oic acid (>3700-fold), 11beta,20-dihydroxy-3-oxopregn-4-en-21-oic acid (50-fold), nicotinamide (>2-fold), nicotinamide 1-oxide (5-fold), 1-methylnicotinamide (1.5-fold), hippuric acid (2-fold), and 2,8-dihydroxyquinoline-beta-d-glucuronide (3-fold). PPARalpha urinary biomarkers highly statistically significantly attenuated by Wy-14,643 treatment included xanthurenic acid (1.3-fold), hexanoylglycine (20-fold), phenylpropionylglycine (4-fold), and cinnamoylglycine (9-fold). These biomarkers arise from PPARalpha effects on tryptophan, corticosterone, and fatty acid metabolism and on glucuronidation. This study underscores the power of mass spectrometry-based metabolomics combined with genetically modified mice in the definition of monogenic metabolic phenotypes.     

The acyl-CoA thioesterase I is regulated by PPARalpha and HNF4alpha via a distal response element in the promoter

The cytosolic acyl-coenzyme A thioesterase I (Acot1) is an enzyme that hydrolyzes long-chain acyl-CoAs of C(12)-C(20)-CoA in chain length to the free fatty acid and CoA. Acot1 was shown previously to be strongly upregulated at the mRNA and protein level in rodents by fibrates. In this study, we show that Acot1 mRNA levels were increased by 90-fold in liver by treatment with Wy-14,643 and that Acot1 mRNA was also increased by 15-fold in the liver of hepatocyte nuclear factor 4alpha (HNF4alpha) knockout animals. Our study identified a direct repeat 1 (DR1) located in the Acot1 gene promoter in mouse, which binds the peroxisome proliferator-activated receptor alpha (PPARalpha) and HNF4alpha. Chromatin immunoprecipitation (ChIP) assay showed that the identified DR1 bound PPARalpha/retinoid X receptor alpha (RXRalpha) and HNF4alpha, whereas the binding in ChIP was abrogated in the PPARalpha and HNF4alpha knockout mouse models. Reporter gene assays showed activation of the Acot1 promoter in cells by the PPARalpha agonist Wy-14,643 after cotransfection with PPARalpha/RXRalpha. However, transfection with a plasmid containing HNF4alpha also resulted in an increase in promoter activity. Together, these data show that Acot1 is under regulation by an interplay between HNF4alpha and PPARalpha.     

Microarray analysis of gene expression changes in mouse liver induced by peroxisome proliferator- activated receptor alpha agonists.

We used a microarray technique to investigate changes of gene expression in liver induced by two peroxisome proliferator-activated receptor alpha (PPARalpha) agonists, a strong PPARalpha agonist, Wy-14,643, and a marketed fibrate drug, fenofibrate. The purposes of this work are: 1) to examine whether or not gene expression is altered in different ways by these two PPARalpha agonists and 2) to find genes whose expression has not been previously reported to be affected by PPARalpha agonists. Mice were treated orally with 100 mg/kg fenofibrate, or 30 mg/kg or 100 mg/kg Wy-14,643, and the liver was collected on Day 2 or 3. mRNA was extraction from liver, and subjected to microarray analysis. Previously reported induction or reduction of gene expression, e.g. genes involved in beta-oxidation and lipid metabolism, was confirmed in our study. Scatter plot analysis indicated that the changes of gene expression pattern induced by fenofibrate and Wy-14,643 were almost identical. However, expression levels of metallothionein 1 and 2 mRNAs were different: no change of hepatic metallothionein 1 and 2 mRNA expression was induced by 100 mg/kg fenofibrate on Day 2 or 3, while 30 mg/kg Wy-14,643 administration increased expression of both genes by 1.8-fold on Day 3. In addition to previously reported gene expression changes by PPARalpha agonists, we found expression changes of other genes, including cis-retinol/3alpha-hydroxysterol short chain dehydrogenase, vanin-1, RecA-like protein, and serum amyloid A (SAA) 2. Among them, the change of SAA2 mRNA level was noteworthy; it showed a decrease to as little as one-seventh. Seven-day fenofibrate pre-treatment of mice completely inhibited the acute-phase elevation of plasma SAA concentration triggered by acetaminophen challenge. This finding suggests that fenofibrate treatment may reduce plasma SAA concentration in patients with secondary amyloidosis.     

Peroxisome proliferator-activated receptor alpha-mediated pathways are altered in hepatocyte-specific retinoid X receptor alpha-deficient mice

Retinoid x receptor alpha (RXRalpha) serves as an active partner of peroxisome proliferator-activated receptor (PPARalpha). In order to dissect the functional role of RXRalpha and PPARalpha in PPARalpha-mediated pathways, the hepatocyte RXRalpha-deficient mice have been challenged with physiological and pharmacological stresses, fasting and Wy14,643, respectively. The data demonstrate that RXRalpha and PPARalpha deficiency are different in several aspects. At the basal untreated level, RXRalpha deficiency resulted in marked induction of apolipoprotein A-I and C-III (apoA-I and apoC-III) mRNA levels and serum cholesterol and triglyceride levels, which was not found in PPARalpha-null mice. Fasting-induced PPARalpha activation was drastically prevented in the absence of hepatocyte RXRalpha. Wy14,643-mediated pleiotropic effects were also altered due to the absence of hepatocyte RXRalpha. Hepatocyte RXRalpha deficiency did not change the basal acyl-CoA oxidase, medium chain acyl-CoA dehydrogenase, and malic enzyme mRNA levels. However, the inducibility of those genes by Wy14,643 was markedly reduced in the mutant mouse livers. In contrast, the basal cytochrome P450 4A1, liver fatty acid-binding protein, and apoA-I and apoC-III mRNA levels were significantly altered in the mutant mouse livers, but the regulatory effect of Wy14,643 on expression of those genes remained the same. Wy14,643-induced hepatomegaly was partially inhibited in hepatocyte RXRalpha-deficient mice. Wy14,643-induced hepatocyte peroxisome proliferation was preserved in the absence of hepatocyte RXRalpha. These data suggested that in comparison to PPARalpha, hepatocyte RXRalpha has its unique role in lipid homeostasis and that the effect of RXRalpha, -beta, and -gamma is redundant in certain aspects.     

Effect of peroxisome proliferators on the methylation and protein level of the c-myc protooncogene in B6C3F1 mice liver

Peroxisome proliferators in general are nongenotoxic mouse liver carcinogens for which DNA hypomethylation and altered gene expression are proposed mechanisms. Therefore, the peroxisome proliferators 2,4-dichlorophenoxyacetic acid (2,4-D), dibutyl phthalate (DBP), gemfibrozil, and Wy-14,643 were evaluated for the ability to alter the methylation and expression of the c-myc protooncogene. Male B6C3F1 mice were administered for 6 days in their diet Wy-14,643 (5-500 ppm), 2,4-D (1,680 ppm), DBP (20,000 ppm), or gemfibrozil (8,000 ppm). All four peroxisome proliferators caused hypomethylation of the c-myc gene in the liver. Wy-14,643 appeared to be the most efficacious with a threshold between 10 and 50 ppm. The level of the c-myc protein was increased by Wy-14,643, but not the other peroxisome proliferators. When female B6C3F1 mice received a two-thirds partially hepatectomy and 16 h later were administered 50 mg/kg Wy-14,643 by gavage, hypomethylation of the gene occurred 24 h later. Hypomethylation was not found in mice that received Wy-14,643 following a sham operation. Hypomethylation of the c-myc gene within 24 h of administering Wy-14,643 after a partial hepatectomy but not after a sham operation supports the hypothesis that the peroxisome proliferators prevent methylation of hemimethylated sites formed by DNA replication.     

Induction of peroxisomal enzymes in livers of neonatal rats exposed to lactating mothers treated with hypolipidaemic drugs. Role of drug metabolite transfer in milk.

Lactating rats were administered by gavage 100 mg/kg body wt. twice a day of either nafenopin or Wy-14,643, two hypolipidaemic drugs with hepatic peroxisome proliferative property. Neonatal rats, after feeding from the drug-treated mothers for 8-14 days, showed sustained increases in both the proliferation of hepatic peroxisomes, as well as in levels of the peroxisome-associated enzymes catalase (3-fold), carnitine acetyltransferase (15-35-fold), peroxisomal enoyl-CoA hydratase (29-46-fold), and palmitoyl-CoA oxidation (12-14-fold). These increases in enzyme activities in suckling rats were similar to those seen in the livers of the drug-treated, lactating mothers after 14 days of treatment. After administering [3H]nafenopin or [3H]Wy-14,643 to lactating rats, significant levels of drug-derived radioactivity were observed in suckling rat gastric milk curds by 2-4 h with significant radioactivity seen in suckling rat livers by 4-6 h. T.l.c. analysis of organic extracts of milk samples from [3H]Wy-14,643 treated animals indicated no detectable levels of the parent drug, only more-polar metabolites. Wy-14,643 metabolites preparatively purified from a rat liver microsomal fraction incubation induced peroxisome proliferation when injected into a neonatal rat. Preparative high pressure liquid chromatography purification and mass spectral analysis has allowed preliminary assessment of the structures of the Wy-14,643 microsomal metabolites. It is concluded that one or more of the metabolite fractions of Wy-14,643 transferred in milk exert the biological ability to induce peroxisome proliferation and peroxisomal enzymes in neonatal livers.     

Wy-14,643 and fenofibrate inhibit mitochondrial respiration in isolated rat cardiac mitochondria

We investigated the direct effects of two selective PPARalpha ligands, fenofibrate and Wy-14,643, on mitochondrial respiratory function using isolated rat cardiac mitochondria. Isolated left ventricular mitochondria were incubated with increasing concentrations of fenofibrate or Wy-14,643 (10, 100, and 500 microM) and mitochondrial respiration determined using: malate/glutamate (complex I), succinate (complex II) and palmitoyl-l-carnitine as oxidative substrates. Our data show that acute exposure to Wy-14,643 and fenofibrate differentially perturb cardiac mitochondrial respiration i.e., fenofibrate more potently inhibited mitochondrial respiration and bioenergetic capacity compared to Wy-14,643. Moreover, we found that both agents increased uncoupling of mitochondrial oxidative phosphorylation.     

Differential effects of phthalates on the testis and the liver

Phthalates have been shown to elicit contrasting effects on the testis and the liver, causing testicular degeneration and promoting abnormal hepatocyte proliferation and carcinogenesis. In the present study, we compared the effects of phthalates on testicular and liver cells to better understand the mechanisms by which phthalates cause testicular degeneration. In vivo treatment of rats with di-(2-ethylhexyl) phthalate (DEHP) caused a threefold increase of germ cell apoptosis in the testis, whereas apoptosis was not changed significantly in livers from the same animals. Western blot analyses revealed that peroxisome proliferator-activated receptor (PPAR) alpha is equally abundant in the liver and the testis, whereas PPAR gamma and retinoic acid receptor (RAR) alpha are expressed more in the testis. To determine whether the principal metabolite of DEHP, mono-(2-ethylhexyl) phthalate (MEHP), or a strong peroxisome proliferator, 4-chloro-6(2,3-xylindino)-2-pyrimidinylthioacetic acid (Wy-14,643), have a differential effect in Sertoli and liver cells by altering the function of RAR alpha and PPARs, their nuclear trafficking patterns were compared in Sertoli and liver cells after treatment. Both MEHP and Wy-14,643 increased the nuclear localization of PPAR alpha and PPAR gamma in Sertoli cells, but they decreased the nuclear localization of RAR alpha, as previously shown. Both PPAR alpha and PPAR gamma were in the nucleus and cytoplasm of liver cells, but RAR alpha was predominant in the cytoplasm, regardless of the treatment. At the molecular level, MEHP and Wy-14,643 reduced the amount of phosphorylated mitogen-activated protein kinase (activated MAPK) in Sertoli cells. In comparison, both MEHP and Wy-14,643 increased phosphorylated MAPK in liver cells. These results suggest that phthalates may cause contrasting effects on the testis and the liver by differential activation of the MAPK pathway, RAR alpha, PPAR alpha, and PPAR gamma in these organs.     

Regulation of peroxisome proliferator-activated receptor alpha by protein kinase C

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear receptor activated by fatty acids, hypolipidemic drugs, and peroxisome proliferators (PPs). Like other nuclear receptors, PPARalpha is a phosphoprotein whose activity is affected by a variety of growth factor signaling cascades. In this study, the effects of protein kinase C (PKC) on PPARalpha activity were explored. In vivo phosphorylation studies in COS-1 cells transfected with murine PPARalpha showed that the level of phosphorylated PPARalpha is increased by treatment with the PP Wy-14,643 as well as the PKC activator phorbol myristol acetate (PMA). In addition, inhibitors of PKC decreased Wy-14,643-induced PPARalpha activity in a variety of reporter assays. Overexpressing PKCalpha, -beta, -delta, and -zeta affected both basal and Wy-14,643-induced PPARalpha activity. Four consensus PKC phosphorylation sites are contained within the DNA binding (C-domain) and hinge (D-domain) regions of rat PPARalpha (S110, T129, S142, and S179), and their contribution to receptor function was examined. Mutation of T129 or S179 to alanine prevented heterodimerization of PPARalpha with RXRalpha, lowered the level of phosphorylation by PKCalpha and PKCdelta in vitro, and lowered the level of phosphorylation of transfected PPARalpha in transfected cells. In addition, the T129A mutation prevented PPARalpha from binding DNA in an electromobility shift assay. Together, these studies demonstrate a direct role for PKC in the regulation of PPARalpha, and suggest several PKCs can regulate PPARalpha activity through multiple phosphorylation sites.     

Metabolism of phytol to phytanic acid in the mouse, and the role of PPARalpha in its regulation

Phytol, a branched-chain fatty alcohol, is the naturally occurring precursor of phytanic and pristanic acid, branched-chain fatty acids that are both ligands for the nuclear hormone receptor peroxisome proliferator-activated receptor alpha (PPARalpha). To investigate the metabolism of phytol and the role of PPARalpha in its regulation, wild-type and PPARalpha knockout (PPARalpha-/-) mice were fed a phytol-enriched diet or, for comparison, a diet enriched with Wy-14,643, a synthetic PPARalpha agonist. After the phytol-enriched diet, phytol could only be detected in small intestine, the site of uptake, and liver. Upon longer duration of the diet, the level of the (E)-isomer of phytol increased significantly in the liver of PPARalpha-/- mice compared with wild-type mice. Activity measurements of the enzymes involved in phytol metabolism showed that treatment with a PPARalpha agonist resulted in a PPARalpha-dependent induction of at least two steps of the phytol degradation pathway in liver. Furthermore, the enzymes involved showed a higher activity toward the (E)-isomer than the (Z)-isomer of their respective substrates, indicating a stereospecificity toward the metabolism of (E)-phytol. In conclusion, the results described here show that the conversion of phytol to phytanic acid is regulated via PPARalpha and is specific for the breakdown of (E)-phytol.     

Changes to the integral membrane protein composition of mouse liver peroxisomes in response to the peroxisome proliferators clofibrate,Wy-14,643 and Di(2-ethyl-hexyl)phthalate

Peroxisomes were purified from livers of control mice and from mice treated with three agents which induce proliferation of hepatic peroxisomes — namely two structurally unrelated hypolipidemic drugs, clofibrate (ethyl--p-chlorophenoxyisobutyrate) and Wy-14,643 (4-chloro-6[2,3-xylidino)-2-pyrimidinylthio] acetic acid), and a plasticizer, DEHP (di-(2-ethylhexyl)phthalate).Membranes were isolated from these purified peroxisomes and analysed by SDS-polyacrylamide gel electrophoresis. All membranes which were tested, displayed two predominant integral membrane proteins of apparent molecular weights of 68 kDa and 70 kDa respectively, as well as a number of minor components. Treatment of animals with clofibrate, Wy-14,643 and DEHP was observed to result in each case in an increased proportion of the 70 kDa protein in the peroxisomal membranes. These treatments also resulted in increased peroxisomal fatty acid oxidation in livers and an increase in the proportion of catalase activity in the cytosolic fraction of liver cells.These results have been discussed in relation to alterations in the molecular composition of the membranes, the mechanisms of peroxisome proliferation and the inducibility of peroxisomal membrane proteins.     

Specific changes in the protein composition of rat liver in response to the peroxisome proliferators ciprofibrate, Wy-14,643 and di-(2-ethylhexyl)phthalate.

The hypolipidaemic agents ciprofibrate and Wy-14,643 ([4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid) and the phthalate-ester plasticizer di-(2-ethylhexyl)-phthalate (DEHP), like other peroxisome proliferators, produce a significant hepatomegaly and induce the peroxisomal fatty acid beta-oxidation enzyme system together with profound proliferation of peroxisomes in hepatic parenchymal cells. Changes in the profile of liver proteins in rats following induction of peroxisome proliferation by ciprofibrate, Wy-14,643 and DEHP have been analysed by high-resolution two-dimensional gel electrophoresis. The proteins of whole liver homogenates from normal and peroxisome-proliferator-treated rats were separated by two-dimensional gel electrophoresis using isoelectric focusing for acidic proteins and nonequilibrium pH gradient electrophoresis for basic proteins. In the whole liver homogenates, the quantities of six proteins in acidic gels and six proteins in the basic gels increased following induction of peroxisome proliferation. Peroxisome proliferator administration caused a repression of three acidic proteins in the liver homogenates. By the immunoblot method using polyspecific antiserum against soluble peroxisomal proteins and monospecific antiserum against peroxisome proliferation associated Mr 80000 polypeptide (polypeptide PPA-80), the majority of basic proteins induced by these peroxisome proliferators appeared to be peroxisomal proteins. Polypeptide PPA-80 becomes the most abundant protein in the total liver homogenates of peroxisome-proliferator-treated rats. These results indicate that ciprofibrate, DEHP and Wy-14,643 induce marked changes in the profile of specific hepatic proteins and that some of these changes should serve as a baseline to identify a set of gene products that may assist in defining the specific 'peroxisome proliferator domain'.