Peroxisomal Fatty acyl-CoA Oxidase Research Articles

Pex20p functions as the receptor for non-PTS1/non-PTS2 acyl-CoA oxidase import into peroxisomes of the yeast Yarrowia lipolytica.

Most soluble proteins targeted to the peroxisomal matrix contain a C-terminal peroxisome targeting signal type 1 (PTS1) or an N-terminal PTS2 that is recognized by the receptors Pex5p and Pex7p, respectively. These receptors cycle between the cytosol and peroxisome and back again for multiple rounds of cargo delivery to the peroxisome. A small number of peroxisomal matrix proteins, including all six isozymes of peroxisomal fatty acyl-CoA oxidase (Aox) of the yeast Yarrowia lipolytica, contain neither a PTS1 nor a PTS2. Pex20p has been shown to function as a co-receptor for Pex7p in the import of PTS2 cargo into peroxisomes. Here we show that cells of Y. lipolytica deleted for the PEX20 gene fail to import not only the PTS2-containing protein 3-ketoacyl-CoA thiolase (Pot1p) but also the non-PTS1/non-PTS2 Aox isozymes. Pex20p binds directly to Aox isozymes Aox3p and Aox5p, which requires the C-terminal Wxxx(F/Y) motif of Pex20p. A W411G mutation in the C-terminal Wxxx(F/Y) motif causes Aox isozymes to be mislocalized to the cytosol. Pex20p interacts physically with members of the peroxisomal import docking complex, Pex13p and Pex14p. Our results are consistent with a role for Pex20p as the receptor for import of the non-PTS1/non-PTS2 Aox isozymes into peroxisomes.

Physiological effects of γ-linolenic acid and sesamin on hepatic fatty acid synthesis and oxidation

Interrelated effects of γ-linolenic acid (GLA) and sesamin, a sesame lignan, on hepatic fatty acid synthesis and oxidation were examined. Rats were fed experimental diets supplemented with 0 or 2 g/kg sesamin (1:1 mixture of sesamin and episesamin) and containing 100 g/kg of palm oil (saturated fat), safflower oil rich in linoleic acid, or oil of evening primrose origin containing 43% GLA (GLA oil) for 18 days. In rats fed sesamin-free diets, GLA oil, compared with other oils, increased the activity and mRNA levels of various enzymes involved in fatty acid oxidation, except for some instances. Sesamin greatly increased these parameters, and the enhancing effects of sesamin on peroxisomal fatty acid oxidation rate and acyl-CoA oxidase, enoyl-CoA hydratase and acyl-CoA thioesterase activities were more exaggerated in rats fed GLA oil than in the animals fed other oils. The combination of sesamin and GLA oil also synergistically increased the mRNA levels of some peroxisomal fatty acid oxidation enzymes and of several enzymes involved in fatty acid metabolism located in other cell organelles. In the groups fed sesamin-free diets, GLA oil, compared with other oils, markedly reduced the activity and mRNA levels of various lipogenic enzymes. Sesamin reduced all these parameters, except for malic enzyme, in rats fed palm and safflower oils, but the effects were attenuated in the animals fed GLA oil. These changes by sesamin and fat type accompanied profound alterations in serum lipid levels. This may be ascribable to the changes in apolipoprotein-B-containing lipoproteins.

Altered hepatic lipid metabolism in C57BL/6 mice fed alcohol: a targeted lipidomic and gene expression study

Chronic alcohol consumption is associated with fatty liver disease in mammals. The object of this study was to gain an understanding of dysregulated lipid metabolism in alcohol-fed C57BL/6 mice using a targeted lipidomic approach. Liquid chromatography tandem mass spectrometry was used to analyze several lipid classes, including free fatty acids, fatty acyl-CoAs, fatty acid ethyl esters, sphingolipids, ceramides, and endocannabinoids, in plasma and liver samples from control and alcohol-fed mice. The interpretation of lipidomic data was augmented by gene expression analyses for important metabolic enzymes in the lipid pathways studied. Alcohol feeding was associated with i) increased hepatic free fatty acid levels and decreased fatty acyl-CoA levels associated with decreased mitochondrial fatty acid oxidation and decreased fatty acyl-CoA synthesis, respectively; ii) increased hepatic ceramide levels associated with higher levels of the precursor molecules sphingosine and sphinganine; and iii) increased hepatic levels of the endocannabinoid anandamide associated with decreased expression of its catabolic enzyme fatty acid amide hydrolase. The unique combination of lipidomic and gene expression analyses allows for a better mechanistic understanding of dysregulated lipid metabolism in the development of alcoholic fatty liver disease.

AoxA is a major peroxisomal long chain fatty acyl-CoA oxidase required for β-oxidation in A. nidulans

Filamentous fungi can use a variety of fatty acids (FA) as sole carbon and energy sources. Aspergillus nidulans has been shown to possess both peroxisomal and mitochondrial beta-oxidation pathways. In these studies, the major peroxisomal long chain fatty acyl coenzyme A oxidase AoxA was identified. AoxA was shown to be localised to peroxisomes and deletion of the aoxA gene leads to reduced growth on long chain FA, but not on short chain FA. AoxA is predicted to be part of the same peroxisomal beta-oxidation pathway as the bifunctional protein FoxA. In addition, an aoxA(p)lacZ reporter gene construct is induced by short and long chain FA and the induction is dependent on the transcriptional regulators FarA, FarB and ScfA with FarA being required for the induction by short chain as well as long chain FA and FarB and ScfA being required for induction of aoxA by short chain FA. It is proposed that there are additional peroxisomal beta-oxidation pathways in A. nidulans, which include fatty acyl-CoA dehydrogenases with a partially overlapping substrate range and include a pathway for short chain FA.

Induction of time-dependent oxidative stress and related transcriptional effects of perfluorododecanoic acid in zebrafish liver

The effects of acute perfluorododecanoic acid (PFDoA) exposure on the induction of oxidative stress and alteration of mitochondrial gene expression were studied in the livers of female zebrafish ( Danio rerio). Female zebrafish were exposed to PFDoA via a single intraperitoneal injection (0, 20, 40, or 80 μg PFDoA/g body weight) and were then sacrificed 48 h, 96 h, or seven days post-PFDoA administration. PFDoA-treated fish exhibited histopathological liver damage, including swollen hepatocytes, vacuolar degeneration, and nuclei pycnosis. Glutathione (GSH) content and catalase (CAT) activity decreased significantly at 48 h post-injection while superoxide dismutase (SOD) activity was initially decreased at 48 h post-injection but was then elevated by seven days post-injection. The activity of glutathione peroxidase (GPx) increased at 48 h and seven days compared to control fish, although the increased level at seven days post-injection was decreased compared to the level at 48 h post-injection. Lipid peroxidation levels were increased at seven days post-injection, while no apparent induction was observed at 48 h or 96 h post-injection. The mRNA expression of medium-chain fatty acid dehydrogenase (MCAD) was induced, while the transcriptional expression of liver fatty acid binding protein (L-FABP), peroxisome proliferating activating receptor α (PPARα), carnitine palmitoyl-transferase I (CPT-I), uncoupling protein 2 (UCP-2), and Bcl-2 were significantly inhibited. Furthermore, the transcriptional expression of peroxisomal fatty acyl-CoA oxidase (ACOX), very long-chain acyl-CoA dehydrogenase (VLCAD), long-chain acyl-CoA dehydrogenase (LCAD) did not exhibit significant changes following PFDoA treatment. No significant changes were noted in the transcriptional expression of genes involved in mitochondrial respiratory chain and ATP synthesis, including cytochrome c oxidase subunit I (COXI), NADH dehydrogenase subunit I (NDI), and ATP synthase F0 subunit 6 (ATPo6). These results demonstrate that turbulence of fatty acid β-oxidation and oxidative stress responses were involved in the PFDoA-induced hepatotoxicity.

Suppression of peroxisomal enzyme activities and cytochrome P450 4A isozyme expression by congeneric polybrominated and polychlorinated biphenyls.

The purpose of this study was to determine the effects of PCBs and PBBs on peroxisome proliferator-activated receptor--(PPAR-) associated enzyme activities or protein levels. Male Sprague-Dawley rats were administered a single IP injection (150 μ mol/kg) of either 3,3′,4,4′-tetrabromobiphenyl, 3,3′,4,4′-tetrachlorobiphenyl, 3,3′,5,5′-tetrabromobiphenyl, 2′,3,3′,4,5-pentachlorobiphenyl, 3,3′,4,4′,5-pentachlorobiphenyl, 2,2′,3,3′,5,5′-hexachlorobiphenyl, or 3,3′,4,4′,5,5′-hexabromobiphenyl in corn oil (10 ml/kg). One week later, the activities of catalase, peroxisomal fatty acyl-CoA oxidase, and peroxisomal beta-oxidation as well as cytochrome P450 4A (CYP4A) protein content were determined in subcellular liver fractions. None of the peroxisomal enzyme activities were significantly increased by any of the halogenated biphenyl congeners tested. Except for minor (approx. 25%) increases in the total CYP4A content following treatment with 2,2′,3,3′,5,5′-hexachlorobiphenyl and 3,3′,5,5′-tetrabromobiphenyl, CYP4A protein contents were not increased by any treatment. The two Ah receptor agonists, 3,3′,4,4′-tetrabromobiphenyl and 3,3′,4,4′,5-pentachlorobiphenyl, significantly diminished the liver content of CYP4A proteins and activities of the peroxisomal enzymes studied. Since a range of congeners with different biologic and toxicologic activities were selected for this study, it may be concluded that the polyhalogenated biphenyls do not induce peroxisome proliferation in the male rat, but rather certain members of this class of compounds down regulate peroxisome-associated enzymes. Since PCBs and PBBs do not increase enzyme activities and expression of proteins associated with PPAR, these agents are therefore exerting their carcinogenic and promoting activities by some other mechanism.

Alcoholic liver injury in the rat is associated with reduced expression of peroxisome proliferator-alpha (PPARalpha)-regulated genes and is ameliorated by PPARalpha activation.

Alcoholic liver disease is associated with a state of hepatic fatty acid overload. We examined the effect of ethanol and different types of dietary fat on the expression of mRNA for liver fatty acid binding protein (L-FABP), peroxisome proliferator-activated receptor-alpha (PPARalpha), and peroxisomal fatty acyl CoA oxidase (FACO). Four groups of rats (n = 5) were fed intragastrically, a liquid diet with or without ethanol (10-16 g/kg/day) for 4 weeks. Pair-fed controls received isocaloric amounts of dextrose. The source of fat was either corn oil or fish oil. Ethanolfed rats developed fatty liver, necrosis, and inflammation; the changes were more severe in the fish oil-ethanol (FE) rats. PPARalpha mRNA levels were not different between groups, although there was a trend toward increased levels in ethanol-fed rats. We calculated L-FABP/PPARalpha and FACO/PPARalpha ratios as a measure of FACO and L-FABP up-regulation relative to PPARalpha expression. Both FACO/PPARalpha and L-FABP/PPARalpha ratios were significantly decreased in FE rats. However, only L-FABP/PPARalpha was decreased in corn oil plus ethanol rats. Also, the level of L-FABP/mRNA correlated inversely with the degree of fatty liver in ethanol-fed rats. Since expression of PPARalpha response genes was impaired in ethanol-fed rats, we determined whether activation of PPARalpha would normalize the PPARalpha response and prevent the pathological changes in ethanol-fed rats. Treatment with clofibrate, a PPARalpha-activating ligand, led to a marked decrease in fatty liver and complete abrogation of necroinflammatory changes in FE rats. Also, nuclear factor kappaB activation and up-regulation of tumor necrosis factor-alpha and cyclooxygenase-2 was also abolished in clofibrate-treated rats. We conclude that adaptive gene regulation of FACO and L-FABP by PPARalpha is impaired in ethanol-fed rats and that treatment with clofibrate, a PPARalpha ligand, prevents alcohol-induced pathological liver injury, possibly by reversing the above changes.

The peroxisome-proliferator-activated receptor alpha agonist ciprofibrate severely aggravates hypercholesterolaemia and accelerates the development of atherosclerosis in mice lacking apolipoprotein E.

Mice lacking apolipoprotein E (apoE) are characterized by severe hypercholesterolaemia, caused by an abnormal accumulation of apolipoprotein B-48 (apoB-48)-carrying remnants of chylomicrons and very-low-density lipoproteins (VLDL) in the plasma, and by the spontaneous development of atherosclerotic lesions. Ciprofibrate is a hypolipidaemic compound that acts primarily by enhancing the oxidation of fatty acids in the liver and, consequently, decreasing the production of hepatic VLDL. In the present study, homozygous apoE-deficient mice were fed with a normal chow diet, supplemented with ciprofibrate. We report that, as anticipated, ciprofibrate treatment (a) stimulated hepatic fatty acid oxidation, as indicated by an increase in the mRNA levels of peroxisomal fatty acyl-CoA oxidase (AOX) and peroxisomal bifunctional enzyme, and (b) decreased the hepatic secretion of VLDL into the plasma, as determined by treating the animals with Triton WR-1339. Paradoxically, the apoE-deficient mice developed a 3-4-fold increase in their plasma cholesterol levels. A similar effect was observed in apoE-deficient mice treated with other peroxisome-proliferator-activated receptor alpha agonists (fenofibrate, bezafibrate and WY14,643). By FPLC of the plasma and Western-blot analysis, we determined that the enhanced hypercholesterolaemia was due to an increased accumulation of apoB-48-carrying lipoprotein remnants in the plasma. Consistent with this finding, atherosclerotic lesions in animals treated with ciprofibrate for 90 days were considerably more advanced than in untreated animals. These results indicate that the ciprofibrate-induced accumulation of apoB-48-carrying remnants in apoE-deficient mice is caused by the inhibition of an as yet uncharacterized apoE-independent mechanism of removal of remnant from the circulation by the liver.

Pitavastatin ameliorates severe hepatic steatosis in aromatase-deficient (Ar-/-) mice.

Tamoxifen is a potent antagonist of estrogen, and hepatic steatosis is a frequent complication in adjuvant tamoxifen for breast cancer. Impaired hepatic FA beta-oxidation in peroxisomes, microsomes, and mitochondria results in progression of massive hepatic steatosis in estrogen deficiency. This impairment, although latent, is potentially serious: About 3% of the general population in the United States is now suffering from nonalcoholic steatohepatitis associated with obesity and hyperlipidemia. Therefore, in the present study we tried to restore impaired hepatic FA beta-oxidation by administering a novel statin, pitavastatin, to aromatase-deficient (Ar-/-) mice defective in intrinsic estrogen synthesis. Northern blot analysis of Ar-/- mice liver revealed a significant restoration of mRNA expression of essential enzymes involved in FA beta-oxidation such as very long fatty acyl-CoA synthetase in peroxisome, peroxisomal fatty acyl-CoA oxidase, and medium-chain acyl-CoA dehydrogenase. Severe hepatic steatosis observed in Ar-/- mice substantially regressed. Consistent findings were obtained in the in vitro assays of FA beta-oxidation activity. These findings demonstrate that pitavastatin is capable of restoring impaired FA beta-oxidation in vivo via the peroxisome proliferator-activated receptor-alpha-mediated signaling pathway and is potent enough to ameliorate severe hepatic steatosis in mice deficient in intrinsic estrogen.

Identification and cloning of two forms of liver peroxisomal fatty Acyl CoA Oxidase from the koala ( Phascolarctos cinereus)

In the present study, the cloning, expression and characterization of the rate-limiting enzyme of the peroxisomal β-oxidation spiral, acyl CoA oxidase (AOX), from koala ( Phascolarctos cinereus) liver is described. It has been previously reported that peroxisomal cyanide-insensitive palmitoyl-CoA oxidation activity was absent in koala liver [Comp. Biochem. Physiol. (C) 127 (2000) 327]. This activity is a measure of the overall peroxisomal β-oxidation minus the final step catalysed by thiolase. Two 2039 bp koala liver AOX cDNAs, designated AOX1 and AOX2, were cloned by reverse transcription–polymerase chain reaction and rapid amplification of cDNA ends. The koala AOX cDNAs encode proteins of 662 amino acids. Transfection of the koala AOX cDNAs into Cos-7 cells resulted in the expression of proteins with palmitoyl-CoA oxidase activity. The apparent K m values for AOX1 and AOX2 cDNA-expressed enzymes were 28 and 38 μM, respectively, which are within the range of order of magnitude reported for rat and human purified AOX enzymes (approximately 10 μM). Northern analysis, utilizing the koala AOX1 cDNA as probe, detected a more intense AOX mRNA band in the koala liver as compared to rats and humans. Southern blot analysis of liver genomic DNA samples revealed a single AOX gene fragment of less than 14 kb in koalas, rat and humans, suggesting a single AOX gene. Collectively, the results of this study suggest that the absence of peroxisomal cyanide-insensitive palmitoyl-CoA oxidation activity in the koala liver is possibly due to deficiencies of one or more enzymes downstream of acyl-CoA oxidase and/or deficiencies of mitochondrial β-oxidation enzymes.

Pseudolaric acid analogs as a new class of peroxisome proliferator-activated receptor agonists.

Extracts of the root and trunk barks of the Chinese tree Pseudolarix kaempferi, which contain pseudolaric acids, are used in Chinese medicine for treatment of fungal infections. Pseudolaric acid B (PLAB) is the major constituent that exhibits anti-fungal activity. The nuclear peroxisome proliferator-activator receptors (PPAR) were proposed as a cellular target for the action of PLAB and its analogs. PLAB and two derivatives were tested for the activation of PPAR isoforms in two mammalian cell lines. CV-1 and H4IIEC3 cells were transfected with phorbol ester response element or PPAR response element reporter constructs, and CV-1 cells were co-transfected with the individual PPAR isoform expression plasmids. PLAB showed similar concentration-dependent effects for the activation of PPAR alpha, gamma and delta isoforms in CV-1 and H4IIEC3 cells. O-Deacetylation of PLAB (PLAC) or esterification of the free carboxy group of PLAB with beta-D-O-glucopyranoside (PLAG) markedly reduced or abolished the activation of these PPAR isoforms. In H4IIEC3 cells, PLAB increased the activation of endogenous PPARalpha and the phospholipase C signaling pathway; and stimulated peroxisomal fatty acyl-CoA oxidase activity. These effects of PLAB on the activation of endogenous PPARalpha and phospholipase C-dependent pathway were blocked by staurosporine. These results suggest that the action of PLAB on PPARalpha in H4IIEC3 cells is mediated by a protein kinase C dependent phosphorylation. Based upon these findings, the chemical class of biologically active diterpene acids related to PLAB may have promise for the treatment of metabolic and pathophysiological disorders that are regulated by these nuclear receptor isoforms.

Aromatase-deficient (ArKO) mice are retrieved from severe hepatic steatosis by peroxisome proliferator administration

Tamoxifen is a potent antagonist of estrogen, and hepatic steatosis is a frequent complication in adjuvant tamoxifen for breast cancer. Recently, aromatase-deficient (ArKO, Ar−/−) mice lacking intrinsic estrogen was developed and the molecular mechanism involved in progression of massive hepatic steatosis in estrogen-deficiency was elucidated; impairment in hepatic fatty acid β-oxidation of peroxisomes, microsomes and mitochondria. This impairment is latent, but is potentially serious, because hepatic energy supply depends greatly on fatty acid β-oxidation. Therefore in the present study, we tried to conquer impaired hepatic fatty acid β-oxidation by administrating bezafibrate, a potent peroxisome proliferator, to Ar−/− mice through activating fatty acid β-oxidation via the peroxisome proliferator activated receptor-α mediated signaling pathway. Northern blot analysis of Ar−/− mice liver revealed a significant restoration of mRNA expression of very long fatty acyl-CoA synthetase in peroxisome, peroxisomal fatty acyl-CoA oxidase, and medium-chain acyl-CoA dehydrogenase in mitochondria, essential enzymes in fatty acid β-oxidation by administration of bezafibrate. Severe hepatic steatosis observed in Ar−/− mice regressed dramatically. Consistent findings were obtained in the in vitro assays of fatty acid β-oxidation activity. These findings demonstrate that bezafibrate is capable of restoring impaired fatty acid β-oxidation in vivo via the peroxisome proliferator-activated receptor-α mediated signaling pathway and is potent enough to regress severe hepatic steatosis in mice deficient in intrinsic estrogen.

Peroxisome Proliferator-Activated Receptor α-Responsive Genes Induced in the Newborn but Not Prenatal Liver of Peroxisomal Fatty Acyl-CoA Oxidase Null Mice

Mice deficient in fatty acyl-CoA oxidase (AOX−/−), the first enzyme of the peroxisomal β-oxidation system, develop specific morphological and molecular changes in the liver characterized by microvesicular fatty change, increased mitosis, spontaneous peroxisome proliferation, increased mRNA and protein levels of genes regulated by peroxisome proliferator-activated receptor α (PPARα), and hepatocellular carcinoma. Based on these findings it is proposed that substrates for AOX function as ligands for PPARα. In this study we examined the sequential changes in morphology and gene expression in the liver of wild-type and AOX−/− mice at Embryonic Day 17.5, and during postnatal development up to 2 months of age. In AOX−/− mice high levels of expression of PPARα-responsive genes in the liver commenced on the day of birth and persisted throughout the postnatal period. We found no indication of PPARα activation in the livers of AOX−/− mice at embryonic age E17.5. In AOX−/− mice microvesicular fatty change in liver cells was evident at 7 days. At 2 months of age livers showed extensive steatosis and the presence in the periportal areas of clusters of hepatocytes with abundant granular eosinophilic cytoplasm rich in peroxisomes. These results suggest that the biological ligands for PPARα vis a vis substrates for AOX either are not functional in fetal liver or do not cross the placental barrier during the fetal development and that postnatally they are likely derived from milk and diet.

Peroxisomal beta-oxidation and peroxisome proliferator-activated receptor alpha: an adaptive metabolic system.

beta-Oxidation occurs in both mitochondria and peroxisomes. Mitochondria catalyze the beta-oxidation of the bulk of short-, medium-, and long-chain fatty acids derived from diet, and this pathway constitutes the major process by which fatty acids are oxidized to generate energy. Peroxisomes are involved in the beta-oxidation chain shortening of long-chain and very-long-chain fatty acyl-coenzyme (CoAs), long-chain dicarboxylyl-CoAs, the CoA esters of eicosanoids, 2-methyl-branched fatty acyl-CoAs, and the CoA esters of the bile acid intermediates di- and trihydroxycoprostanoic acids, and in the process they generate H2O2. Long-chain and very-long-chain fatty acids (VLCFAs) are also metabolized by the cytochrome P450 CYP4A omega-oxidation system to dicarboxylic acids that serve as substrates for peroxisomal beta-oxidation. The peroxisomal beta-oxidation system consists of (a) a classical peroxisome proliferator-inducible pathway capable of catalyzing straight-chain acyl-CoAs by fatty acyl-CoA oxidase, L-bifunctional protein, and thiolase, and (b) a second noninducible pathway catalyzing the oxidation of 2-methyl-branched fatty acyl-CoAs by branched-chain acyl-CoA oxidase (pristanoyl-CoA oxidase/trihydroxycoprostanoyl-CoA oxidase), D-bifunctional protein, and sterol carrier protein (SCP)x. The genes encoding the classical beta-oxidation pathway in liver are transcriptionally regulated by peroxisome proliferator-activated receptor alpha (PPAR alpha). Evidence derived from mice deficient in PPAR alpha, peroxisomal fatty acyl-CoA oxidase, and some of the other enzymes of the two peroxisomal beta-oxidation pathways points to the critical importance of PPAR alpha and of the classical peroxisomal fatty acyl-CoA oxidase in energy metabolism, and in the development of hepatic steatosis, steatohepatitis, and liver cancer.

Identification of Peroxisome Proliferator-responsive Human Genes by Elevated Expression of the Peroxisome Proliferator-activated Receptor α in HepG2 Cells

In mice and other sensitive species, PPARalpha mediates the induction of mitochondrial, microsomal, and peroxisomal fatty acid oxidation, peroxisome proliferation, liver enlargement, and tumors by peroxisome proliferators. In order to identify PPARalpha-responsive human genes, HepG2 cells were engineered to express PPARalpha at concentrations similar to mouse liver. This resulted in the dramatic induction of mRNAs encoding the mitochondrial HMG-CoA synthase and increases in fatty acyl-CoA synthetase (3-8-fold) and carnitine palmitoyl-CoA transferase IA (2-4-fold) mRNAs that were dependent on PPARalpha expression and enhanced by exposure to the PPARalpha agonist Wy14643. A PPAR response element was identified in the proximal promoter of the human HMG-CoA synthase gene that is functional in its native context. These data suggest that humans retain a capacity for PPARalpha regulation of mitochondrial fatty acid oxidation and ketogenesis. Human liver is refractory to peroxisome proliferation, and increased expression of mRNAs for the peroxisomal fatty acyl-CoA oxidase, bifunctional enzyme, or thiolase, which accompanies peroxisome proliferation in responsive species, was not evident following Wy14643 treatment of cells expressing elevated levels of PPARalpha. Additionally, no significant differences were seen for the expression of apolipoprotein AI, AII, or CIII; medium chain acyl-CoA dehydrogenase; or stearoyl-CoA desaturase mRNAs.

Identification of novel peroxisome proliferator-activated receptor alpha (PPARalpha) target genes in mouse liver using cDNA microarray analysis.

Peroxisome proliferators, which function as peroxisome proliferator-activated receptor-alpha (PPARalpha) agonists, are a group of structurally diverse nongenotoxic hepatocarcinogens including the fibrate class of hypolipidemic drugs that induce peroxisome proliferation in liver parenchymal cells. Sustained activation of PPARalpha by these agents leads to the development of liver tumors in rats and mice. To understand the molecular mechanisms responsible for the pleiotropic effects of these agents, we have utilized the cDNA microarray to generate a molecular portrait of gene expression in the liver of mice treated for 2 weeks with Wy-14,643, a potent peroxisome proliferator. PPARalpha activation resulted in the stimulation of expression (fourfold or greater) of 36 genes and decreased the expression (fourfold or more decrease) of 671 genes. Enhanced expression of several genes involved in lipid and glucose metabolism and many other genes associated with peroxisome biogenesis, cell surface function, transcription, cell cycle, and apoptosis has been observed. These include: CYP2B9, CYP2B10, monoglyceride lipase, pyruvate dehydrogenase-kinase-4, cell death-inducing DNA-fragmentation factor-alpha, peroxisomal biogenesis factor 11beta, as well as several cell recognition surface proteins including annexin A2, CD24, CD39, lymphocyte antigen 6, and retinoic acid early transcript-gamma, among others. Northern blotting of total RNA extracted from the livers of PPARalpha-/- mice and from mice lacking both PPARalpha and peroxisomal fatty acyl-CoA oxidase (AOX), that were fed control and Wy-14,643-containing diets for 2 weeks, as well as time course of induction following a single dose of Wy-14,643, revealed that upregulation of genes identified by microarray procedure is dependent upon peroxisome proliferation vis-à-vis PPARalpha. However, cell death-inducing DNA-fragmentation factor-alpha mRNA, which is increased in the livers of wild-type mice treated with peroxisome proliferators, was not enhanced in AOX-/- mice with spontaneous peroxisome proliferation. These observations indicate that the activation of PPARalpha leads to increased and decreased expression of many genes not associated with peroxisomes, and that delayed onset of enhanced expression of some genes may be the result of metabolic events occurring secondary to PPARalpha activation and alterations in lipid metabolism.

Peroxisome proliferator-activated receptor alpha-dependent induction of cell surface antigen Ly-6D gene in the mouse liver.

Spontaneous peroxisome proliferation-related pleiotropic responses occurring in the liver of mice lacking peroxisomal fatty acyl-CoA oxidase (AOX-/-) are attributed to sustained activation of peroxisome proliferator-activated receptor alpha (PPARalpha) by its putative natural ligands that require AOX for their metabolism. In this study, using a gene expression screen, we show that Ly-6 (lymphocyte antigen 6 complex, locus D; mouse ThB), which belongs to a distinctive family of low molecular weight phosphatidyl inositol anchored cell surface glycoproteins, is upregulated in mouse liver with peroxisome proliferation. Increases in Ly-6D mRNA levels are observed in AOX-/- mouse liver with spontaneous peroxisome proliferation and also in the liver of wild-type mice treated with synthetic peroxisome proliferators. Peroxisome proliferators failed to increase hepatic Ly-6D mRNA levels in mice lacking PPARalpha (PPARalpha-/-), suggesting a regulatory role for PPARalpha in the induction of Ly-6D. These observations suggest that changes in certain cell surface proteins also form part of the pleiotropic responses associated with peroxisome proliferation.

Growth reduction in glioma cells after treatment with tetradecylthioacetic acid: changes in fatty acid metabolism and oxidative status

During aerobic metabolism, a small amount of partially reduced oxygen is produced, yielding reactive oxygen species (ROS). Peroxisomes and mitochondria are major contributors to cellular ROS production, which is normally balanced by consumption by antioxidants. The fatty acid analogue tetradecylthioacetic acid (TTA) promotes mitochondrial and peroxisomal proliferation, and may induce oxidative stress and change the growth potential of cancer cells. In the present study, we found that TTA reduced [ 3H]thymidine incorporation in the glioma cell lines BT4Cn (rat), D54Mg (human), and GaMg (human) in a dose- and time-dependent manner. The 50% inhibitory TTA doses were approximately 125 μM for BT4Cn and D54Mg cells and 40 μM for GaMg cells after 4 days. α-Tochopherol counteracted this inhibition in GaMg cells. TTA enhanced the oxidation of [1- 14C]palmitic acid, which could be explained by stimulation of enzymes involved in peroxisomal (fatty acyl-CoA oxidase) and/or mitochondrial (carnitine palmitoyltransferase) fatty acid oxidation. The glutathione content and the activities of glutathione peroxidase, glutathione reductase, and glutathione S-transferase were differentially affected. Increased malondialdehyde (MDA) production was seen in TTA-treated GaMg and D54Mg cells, but not in BT4Cn cells, in vitro. In BT4Cn tumor tissue from TTA-treated rats, MDA was increased while the α-tocopherol content tended to decrease. TTA increased the level of cytosolic cytochrome c in BT4Cn cells, which suggests induction of apoptotic cascades. Although several mechanisms are likely to be involved in the TTA-mediated effects on growth, we propose that modulation of cellular redox conditions caused by changes in fatty acid metabolism may be of vital importance.

Peroxisomal beta-oxidation and steatohepatitis.

Fatty acid beta-oxidation occurs in both mitochondria and peroxisomes. Mitochondria catalyze the beta-oxidation of the bulk of short-, medium-, and long-chain fatty acids derived from diet, and this pathway constitutes the major process by which fatty acids are oxidized to generate energy. Peroxisomes are involved, preferentially, in the beta-oxidation chain shortening of very long chain fatty acids (VLCFAs) and in the process produce H2O2. Long-chain fatty acids and VLCFAs are also metabolized by the cytochrome P450 CYP4A omega-oxidation system to toxic dicarboxylic acids (DCAs) that serve as substrates for peroxisomal beta-oxidation, and this process also leads to the production of superoxide and H2O2. The genes encoding peroxisomal, microsomal, and certain mitochondrial fatty acid metabolizing enzymes in liver are transcriptionally regulated by peroxisome proliferator-activated receptor alpha (PPAR alpha). Deficiencies of the enzymes of peroxisomal beta-oxidation have been recognized as important causes of disease. Evidence from mice deficient in PPAR alpha (PPAR alpha-/-), deficient in peroxisomal fatty acyl-CoA oxidase (AOX-/-), the first enzyme of the classical beta-oxidation system, and deficient in both PPAR alpha and AOX (PPAR alpha-/-AOX-/-) points to the critical importance of PPAR alpha-inducible peroxisomal and microsomal oxidation systems that metabolize LCFAs and VLCFAs in the pathogenesis of nonalcoholic microvesicular hepatic steatosis and steatohepatitis. These and other mouse models should provide greater understanding of the molecular mechanism responsible for hepatic steatosis and steatohepatitis. Deficiency of AOX disrupts the oxidation of VLCFAs, DCAs, and other substrates leading to extensive microvesicular steatosis and steatohepatitis. Loss of this enzyme also causes sustained hyperactivation of PPAR alpha, leading to transcriptional up-regulation of PPAR alpha-regulated genes, indicating that unmetabolized substrates of AOX function as ligands of PPAR alpha. beta-Oxidation is the major process by which fatty acids are oxidized to generate energy, especially when glucose availability is low during periods of starvation. Mice deficient in PPAR alpha and those nullizygous for both PPAR alpha and AOX show a minimal steatotic phenotype under fed conditions but manifest an exaggerated steatotic response to fasting, indicating that defects in PPAR alpha-inducible fatty acid oxidation determine the severity of fatty liver phenotype to conditions reflecting energy-related stress.

Defect in Peroxisome Proliferator-activated Receptor α-inducible Fatty Acid Oxidation Determines the Severity of Hepatic Steatosis in Response to Fasting

Fasting causes lipolysis in adipose tissue leading to the release of large quantities of free fatty acids into circulation that reach the liver where they are metabolized to generate ketone bodies to serve as fuels for other tissues. Since fatty acid-metabolizing enzymes in the liver are transcriptionally regulated by peroxisome proliferator-activated receptor alpha (PPARalpha), we investigated the role of PPARalpha in the induction of these enzymes in response to fasting and their relationship to the development of hepatic steatosis in mice deficient in PPARalpha (PPARalpha(-/-)), peroxisomal fatty acyl-CoA oxidase (AOX(-/-)), and in both PPARalpha and AOX (double knock-out (DKO)). Fasting for 48-72 h caused profound impairment of fatty acid oxidation in both PPARalpha(-/-) and DKO mice, and DKO mice revealed a greater degree of hepatic steatosis when compared with PPARalpha(-/-) mice. The absence of PPARalpha in both PPARalpha(-/-) and DKO mice impairs the induction of mitochondrial beta-oxidation in liver following fasting which contributes to hypoketonemia and hepatic steatosis. Pronounced steatosis in DKO mouse livers is due to the added deficiency of peroxisomal beta-oxidation system in these animals due to the absence of AOX. In mice deficient in AOX alone, the sustained hyperactivation of PPARalpha and up-regulation of mitochondrial beta-oxidation and microsomal omega-oxidation systems as well as the regenerative nature of a majority of hepatocytes containing numerous spontaneously proliferated peroxisomes, which appear refractory to store triglycerides, blunt the steatotic response to fasting. Starvation for 72 h caused a decrease in PPARalpha hepatic mRNA levels in wild type mice, with no perceptible compensatory increases in PPARgamma and PPARdelta mRNA levels. PPARgamma and PPARdelta hepatic mRNA levels were lower in fed PPARalpha(-/-) and DKO mice when compared with wild type mice, and fasting caused a slight increase only in PPARgamma levels and a decrease in PPARdelta levels. Fasting did not change the PPAR isoform levels in AOX(-/-) mouse liver. These observations point to the critical importance of PPARalpha in the transcriptional regulatory responses to fasting and in determining the severity of hepatic steatosis.