Peroxisomal Β-oxidation Enzymes Research Articles

The roles of anabolic and catabolic reactions in the synthesis and recycling of polyunsaturated fatty acids

Generally the biosynthesis and degradation of compounds take place in separate subcellular compartments. The synthesis of 22 carbon acids, with their first double bond at position 4, requires anabolic enzymes in the endoplasmic reticulum as well as peroxisomal β-oxidation enzymes. Partial degradation–resynthesis cycles, using enzymes in these two subcellular compartments, may play an important role in determining what PUFA are available for membrane lipid biosynthesis.

Elevated Basal Expression of Liver Peroxisomal β-Oxidation Enzymes and CYP4A Microsomal Fatty Acid ω-Hydroxylase in STAT5b−/− Mice: Cross-Talk in Vivo between Peroxisome Proliferator-Activated Receptor and Signal Transducer and Activator of Transcription Signaling Pathways

Long-term treatment of rodents with peroxisome proliferator chemicals, a group of structurally diverse nongenotoxic carcinogens, leads to liver cancer in a process dependent on the nuclear receptor peroxisome proliferator-activated receptor-α (PPARα). Previous in vitro studies have shown that growth hormone (GH) can inhibit PPARα-dependent gene expression by down-regulation of PPARα expression and by a novel inhibitory cross-talk involving the GH-activated transcription factor STAT5b. Presently, we evaluate the role of STAT5b in mediating these inhibitory actions of GH on PPAR function using a STATb-deficient mouse model. Protein levels of three PPARα-responsive peroxisomal β-oxidation pathway enzymes (fatty acyl-CoA oxidase, 3-ketoacyl-CoA thiolase, and l-bifunctional enzyme) were increased up to two- to threefold in STAT5b−/− relative to wild-type control mouse liver, as was the basal expression of two PPARα-regulated cytochrome P450 4A proteins. In contrast, protein levels of two PPARα-unresponsive peroxisomal enzymes, catalase and urate oxidase, were not affected by the loss of STAT5b. A corresponding increase in expression of fatty acyl-CoA oxidase and l-bifunctional enzyme mRNA, as well as PPARα mRNA, was observed in the STAT5b-deficient mice, suggesting a transcriptional mechanism for the observed increases. Although basal liver expression of PPARα and its target genes was thus elevated in STAT5b−/− mice, the clofibrate-induced level of enzyme expression was unaffected, suggesting that the inhibitory effects of STAT5b are overcome at high concentrations of PPARα activators. These findings support the hypothesis that GH and potentially other endogenous activators of STAT5b help to maintain liver PPARα function at a low basal level and may thereby moderate PPARα-dependent hepatocarcinogenesis and other responses stimulated by exposure to low levels of environmental chemicals of the peroxisome proliferator class.

Identification of the peroxisomal β-oxidation enzymes involved in the degradation of leukotrienes

Leukotrienes (LTs) are metabolically inactivated via ω-oxidation and subsequent β-oxidation from the ω-end. This β-oxidation process takes place in peroxisomes. In this study we investigated the role of different enzymes involved in peroxisomal β-oxidation in the degradation of LTs. We analyzed LTB 4, LTE 4, and their oxidation products in urine of patients with Infantile Refsum's disease (IRD), d-bifunctional protein (DBP) deficiency, Rhizomelic Chondrodysplasia Punctata (RCDP) type 1, and X-linked adrenoleukodystrophy (XALD). We found that patients with IRD and DBP deficiencies excrete increased amounts of LTB 4, LTE 4, ω-carboxy-LTB 4, and ω-carboxy-LTE 4 in their urine, whereas the β-oxidation products were not detectable. These results show that DBP plays an essential role in the degradation of LTs. In urine of patients with XALD and RCDP type 1 we found normal levels of LTB 4, LTE 4, and their oxidation products, indicating that the adrenoleukodystrophy protein and peroxisomal 3-ketoacyl-CoA thiolase are not involved in the metabolic inactivation of LTs.

Identification of the peroxisomal β-oxidation enzymes involved in the biosynthesis of docosahexaenoic acid

DHA (C22:6n-3) is an important PUFA implicated in a number of (patho)physiological processes. For a long time, the exact mechanism of DHA formation has remained unclear, but now it is known that it involves the production of tetracosahexaenoic acid (C24:6n-3) from dietary linolenic acid (C18:3n-3) via a series of elongation and desaturation reactions, followed by beta-oxidation of C24:6n-3 to C22:6n-3. Although DHA is deficient in patients lacking peroxisomes, the intracellular site of retroconversion of C24:6n-3 has remained controversial. By making use of fibroblasts from patients with defined mitochondrial and peroxisomal fatty acid oxidation defects, we show in this article that peroxisomes, and not mitochondria, are involved in DHA formation by catalyzing the beta-oxidation of C24:6n-3 to C22:6n-3. Additional studies of fibroblasts from patients with X-linked adrenoleukodystrophy, straight-chain acyl-CoA oxidase (SCOX) deficiency, d-bifunctional protein (DBP) deficiency, and rhizomelic chondrodysplasia punctata type 1, and of fibroblasts from l-bifunctional protein and sterol carrier protein X (SCPx) knockout mice, show that the main enzymes involved in beta-oxidation of C24:6n-3 to C22:6n-3 are SCOX, DBP, and both 3-ketoacyl-CoA thiolase and SCPx. These findings are of importance for the treatment of patients with a defect in peroxisomal beta-oxidation.

D-bifunctional protein deficiency with fetal ascites, polyhydramnios, and contractures of hands and toes

Fetal abnormalities including chylous ascites, polyhydramnios, claw hands, and hammer toes were identified in an infant who had a missense mutation R106P and a 52bp deletion in the gene for a peroxisomal β-oxidation enzyme, D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase, D-bifunctional protein. The patient had psychomotor retardation and craniofacial dysmorphism and died at 7 months of age. The patient had atypical fetal manifestations of this enzyme deficiency. (J Pediatr 2001;139:865–7)

Urinary organic acids in peroxisomal disorders: a simple screening method

Using GC–MS, we studied urinary organic acids in 20 Japanese patients with peroxisomal disorders, including Zellweger syndrome (ZS), neonatal adrenoleukodystrophy, and single deficiency of peroxisomal β-oxidation enzymes. Non-ketotic dicarboxylic aciduria with elevated sebacate/adipate molar ratio was observed in 19 of the 20 patients. Elevation of 2-hydroxysebacate and epoxydicarboxylic acids were seen in 13 and 18, respectively. Tyrosyluria was remarkable in all patients. In two ZS patients, we tracked the time course from birth to infancy, and all the above stated findings were detected, except for one sample. Urinary organic acid analysis is indeed useful for screening subjects with peroxisomal disorders.

The short heterodimer partner receptor differentially modulates peroxisome proliferator-activated receptor α-mediated transcription from the peroxisome proliferator-response elements of the genes encoding the peroxisomal β-oxidation enzymes acyl-CoA oxidase and hydratase-dehydrogenase

The promoter regions of the genes encoding the first two enzymes of the peroxisomal β-oxidation pathway, acyl-CoA oxidase (AOx) and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD), contain transcriptional regulatory sequences termed peroxisome proliferator-response elements (PPRE) that are bound by the peroxisome proliferator-activated receptor α (PPARα) and 9- cis-retinoic acid receptor (RXRα) heterodimeric complex. In this study, the role of the short heterodimer partner (SHP) receptor in modulating PPARα-mediated gene transcription from the PPREs of the genes encoding AOx and HD was investigated both in vitro and in vivo. In vitro binding assays using glutathione- S-transferase-tagged chimeric receptors for PPARα and SHP were used to verify the interaction between PPARα and SHP. This interaction was unaffected by the presence of the peroxisome proliferator, Wy-14,643. SHP has been proposed to act as a negative regulator of nuclear hormone receptor activity, and SHP inhibited transcription by PPARα/RXRα heterodimers from the AOx-PPRE. Surprisingly, SHP potentiated transcription by PPARα/RXRα heterodimers from the HD-PPRE. This is the first demonstration of positive transcriptional activity attributable to SHP. Together, these results suggest that SHP can modulate PPARα/RXRα-mediated transcription in a response element-specific manner.

Pristanic acid is activator of peroxisome proliferator activated receptor alpha

lsoprenoid phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is degraded in peroxisomes by α-oxidation to pristanic acid (2,6,10,14-tetramethylpentadecanoic acid) and then via β-oxidation. Branched-chain phytanic acid is an activator of the peroxisome proliferator activated receptor α (PPAR ) which in liver cells regulates expression of genes encoding peroxisomal and mitochondrial β-oxidative enzymes as well as cytosolic/nuclear liver-type fatty acid binding protein (L-FABP). In this report we address the question whether pristanic acid also acts as activator of PPARα and thus mediates the expression of its catabolizing enzymes. In a first in vivo approach we fed pristanic acid for 14 days to wildtype mice and to mice lacking sterol carrier protein 2/sterol carrier protein x which Ieads to a phenotype having high concentrations of branched-chain fatty acids. In either genotype, feeding pristanic acid was associated with a strong induction of peroxisomal β-oxidation enzymes tested (acyl-CoA oxidase, bifunctional enzyme, thiolase) as well as of L-FABP. The link between pristanic acid and protein expression observed was established by carrying out assays for transactivation of PPARα in transfected HepG2 cells. In comparison to hypolipidemic drugs and to straight-chain fatty acids known to be PPARα agonists, branched-chain phytanic and pristanic acids were substantially stronger activators, pristanic acid being even superior to phytanic acid.

Less Extrahepatic Induction of Fatty Acid β-Oxidation Enzymes by PPARα

The peroxisome proliferator-activated receptor α (PPARα) is a nuclear receptor that transcriptionally regulates mitochondrial and peroxisomal fatty acid β-oxidation enzymes in the liver. Ligands include synthetic peroxisome proliferators and some fatty acids. PPARα activation leads to predictable pleiotropic responses in liver including peroxisome proliferation, increased fatty acid oxidation, and hepatocellular carcinoma. In the current study, the response to PPARα-activation was compared in the heart, kidney, and liver since the role of PPARα in extrahepatic fatty acid-oxidizing organs has not been fully explored. Basal expression of mitochondrial β-oxidation enzymes was comparable in the three tissues, but peroxisomal β-oxidation enzymes were most abundant in the liver and less so in the kidney and especially in the heart. After PPARα activation with ciprofibrate, both mitochondrial and peroxisomal β-oxidation enzymes were induced, with the strongest response seen in the liver, a moderate response in the kidney, and no significant response in the heart. PPARα mRNA analysis suggested that the differential response may be related to PPARα expression.

Gene expression of peroxisomal β-oxidation enzymes in rat brain

Despite increasing insight into peroxisomal β-oxidation, it is still not clear which enzymes catalyze very-long-chain fatty acid degradation. Using the northern blot and RT-PCR techniques, a brain-specific expression is demonstrated for acyl-CoA oxidase 3II mRNA, thiolase-A and trans2,3enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase multifunctional enzyme type 2.

Immunocytochemical investigation of catalase and peroxisomal lipid β-oxidation enzymes in human hepatocellular tumors and liver cirrhosis

A significant reduction of catalase activity, a peroxisomal marker enzyme, occurs in human hepatic neoplasias, but no information is available on other peroxisomal proteins. We have studied by means of immunohistochemistry four specific proteins of peroxisomes (catalase and three enzymes of lipid beta-oxidation) in human hepatocellular tumors of various differentiation grades from adenoma to anaplastic carcinoma. In all tumors, except the adenomas, the tumor cells contained fewer peroxisomes than extrafocal hepatocytes and the reduction of antigenic sites in the tumor types generally correlated with the degree of tumor dedifferentiation as assessed by classical histopathological criteria. Two poorly differentiated tumors had no detectable peroxisomes at all. There were no major differences in the intensities of the immunocytochemical staining for all four studied peroxisomal antigens in different tumors, suggesting that the neoplastic transformation affects the biogenesis of the entire organelle and not merely the individual peroxisomal enzyme proteins. Some tumors exhibited a distinct peripheral distribution of peroxisomes. In cases with associated liver cirrhosis, the hepatocytes in the adjacent liver showed marked peroxisome proliferation, forming large perinuclear aggregates, occupying occasionally the entire cytoplasm. Taken together, our observations indicate that peroxisomes are significantly altered in both hepatocellular tumors and liver cirrhosis and, thus, could be responsible for some of the metabolic derangements observed in those disease processes.

Characterization of the 70-kDa Peroxisomal Membrane Protein, an ATP Binding Cassette Transporter

The 70-kDa peroxisomal membrane protein (PMP70) is one of the major components of rat liver peroxisomal membranes and belongs to a superfamily of proteins known as ATP binding cassette transporters. PMP70 is markedly induced by administration of hypolipidemic agents in parallel with peroxisome proliferation and induction of peroxisomal fatty acid beta-oxidation enzymes. To characterize the role of PMP70 in biogenesis and function of peroxisomes, we transfected the cDNA of rat PMP70 into Chinese hamster ovary cells and established cell lines stably expressing PMP70. The content of PMP70 in the transfectants increased about 5-fold when compared with the control cells. A subcellular fractionation study showed that overexpressed PMP70 was enriched in peroxisomes. This peroxisomal localization was confirmed by immunofluorescence and immunoelectron microscopy. The number of immuno-gold particles corresponding to PMP70 on peroxisomes increased markedly in the transfectants, but the size and the number of peroxisomes were essentially the same in both the transfectants and the control cells. beta-Oxidation of palmitic acid increased about 2-3-fold in the transfectants, whereas the oxidation of lignoceric acid decreased about 30-40%. When intact peroxisomes prepared from both the cell lines were incubated with palmitoyl-CoA, oxidation was stimulated with ATP, but the degree of the stimulation was higher in the transfectants than in the control cells. Furthermore, we established three Chinese hamster ovary cell lines stably expressing mutant PMP70. In these cells, beta-oxidation of palmitic acid decreased markedly. These results suggest that PMP70 is involved in metabolic transport of long chain acyl-CoA across peroxisomal membranes and that increase of PMP70 is not associated with proliferation of peroxisomes.

Maturation of the liver-specific peroxisome versus laminin, collagen IV and integrin expression

The interaction of cells with extracellular matrix components contributes to their specific differentiation. We studied hepatic peroxisomes and their changing features during embryonic development, and we immunolocalized in the same tissue the extracellular matrix components laminin and collagen IV as well as the integrin receptor subunits α 1, α 2, β 1 and β 4. Rat and human embryonic liver peroxisome expression were studied at the light- and electron-microscopic level by means of localizing catalase-, D-amino acid oxidase- and polyamine oxidase activities and by means of the immunocytochemistry of six peroxisomal proteins. The successive import of catalase and the peroxisomal β-oxidation enzymes, the late appearance of the other enzymes, and the gradual increase of peroxisomal size and number to adult values occurred as asynchronous events. Although still immature, peroxisomes were recognized at every stage examined and coexisted with laminin and collagen IV in both species. The β 1 integrin subunit was immunodetected as early as at 12.5 days in rat. It was concluded that these extracellular matrix factors may be important for the differentiation of liver parenchyma from the liverbud stage onwards. However, the stepwise maturation of the liver-specific peroxisome suggests the involvement of many other regulating factors.

Early modulation of genes encoding peroxisomal and mitochondrial β-oxidation enzymes by 3-thia fatty acids

The aim of the present study was to elucidate the effects of a single dose of 3-thia fatty acids (tetradecylthioacetic acid and 3-thiadicarboxylic acid) over a 24-hr study period on the expression of genes related to peroxisomal and mitochondrial β-oxidation in liver of rats. The plasma triglyceride level decreased at 2–4 hr, 4–8 hr, and 8–24 hr, respectively, after a single dose of 150, 300, or 500 mg of 3-thia fatty acids/kg body weight. Four to eight hours after administration of 3-thia fatty acids, a several-fold-induced gene expression of peroxisomal multifunctional protein, fatty acyl-CoA oxidase (EC 1.3.3.6), fatty acid binding protein, and 2,4-dienoyl-CoA reductase (EC 1.3.1.43) resulted, concomitant with increased activity of 2,4-dienoyl-CoA reductase and fatty acyl-CoA oxidase. The expression of carnitine palmitoyltransferase-I and carnitine palmitoyltransferase-II increased at 2 and 4 hr, respectively, although at a smaller scale. In cultured hepatocytes, 3-thia fatty acids stimulated fatty acid oxidation after 4 hr, and this was both L-carnitine- and L-aminocarnitine-sensitive. The hepatic content of eicosapentaenoic acid and docosahexaenoic acid decreased throughout the study period. In contrast, the hepatic content of oleic acid tended to increase after 24 hr and was significantly increased after repeated administration of 3-thia fatty acids. Similarly, the expression of Δ 9-desaturase was unchanged during the 24-hr study, but increased after feeding for 5 days. To conclude, carnitine palmitoyltransferase-I expression seemed to be induced earlier than 2,4-dienoyl-CoA reductase and fatty acid binding protein, and not later than the peroxisomal fatty acyl-CoA oxidase. The expression of Δ 9-desaturase showed a more delayed response.

Dietary lipids regulate beta-oxidation enzyme gene expression in the developing rat kidney.

This study examines the ability of dietary lipids to regulate gene expression of mitochondrial and peroxisomal fatty acid beta-oxidation enzymes in the kidney cortex and medulla of 3-wk-old rats and evaluates the role of glucagon or of the alpha-isoform of peroxisome proliferator-activated receptor (PPARalpha) in mediating beta-oxidation enzyme gene regulation in the immature kidney. The long-chain (LCAD) and medium-chain acyl-CoA dehydrogenases (MCAD) and acyl-CoA oxidase (ACO) mRNA levels were found coordinately upregulated in renal cortex, but not in medulla, of pups weaned on a high-fat diet from day 16 to 21. Further results establish that switching pups from a low- to a high-fat diet for only 1 day was sufficient to induce large increases in cortical LCAD, MCAD, and ACO mRNA levels, and gavage experiments show that this upregulation of beta-oxidation gene expression is initiated within 6 h following lipid ingestion. Treatment of pups with clofibrate, a PPARalpha agonist, demonstrated that PPARalpha can mediate regulation of cortical beta-oxidation enzyme gene expression, whereas glucagon was found ineffective. Thus dietary lipids physiologically regulate gene expression of mitochondrial and peroxisomal beta-oxidation enzymes in the renal cortex of suckling pups, and this might involve PPARalpha-mediated mechanisms.

Inhibition of liver cell proliferation by dexamethasone does not prevent tumor induction by ciprofibrate in the rat liver

The mechanism of peroxisome proliferator-induced hepatocarcinogenesis in rats and mice remains controversial. The suggested mechanisms by which peroxisome proliferators cause tumors include oxidative stress resulting from elevated levels of hydrogen peroxide (H 2O 2) generating peroxisomal β-oxidation enzymes and increased cell proliferation. The objective of this study was to delineate the role of cell proliferation in ciprofibrate-induced hepatocarcinogenesis in rats in which liver cell proliferation is inhibited by concomitant administration of dexamethasone. Male Fischer 344 rats were fed a diet containing 0.025% ciprofibrate with or without dexamethasone (0.5 mg kg −1 diet) for 61 weeks. Rats fed dexamethasone alone or no added chemicals in the diet served as controls. The incidence of neoplastic nodules (NN) and hepatocellular carcinomas (HCC) was 100% in both ciprofibrate and ciprofibrate and dexamethasone groups. Interestingly, the multiplicity and the number of large tumors per liver was higher in the ciprofibrate and dexamethasone group. No hepatic tumors developed in rats fed control diets. The carcinogenicity of ciprofibrate in rats was independent of cell proliferation, as measurement of hepatocyte proliferation at 4 and 61 weeks showed a significant decrease in rats treated with dexamethasone and ciprofibrate compared to rats treated with ciprofibrate alone; whereas, the levels of peroxisomal enoyl CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme mRNA was markedly increased in both the groups.

Effect of the peroxisome proliferator ciprofibrate on hepatic cyclooxygenase and phospholipase A 2 in rats

Peroxisome proliferators, which include several hypolipidemic drugs, plasticizers and other chemicals, induce hepatic tumors in rodents. These chemicals alter the expression of enzymes involved in lipid metabolism, such as the cytochrome P450 4A family and peroxisomal β-oxidation enzymes. Previous studies have shown that the peroxisome proliferator ciprofibrate reduces eicosanoid concentrations in rat livers and primary hepatocyte cultures, yet the mechanism is still unclear. In this study we examined cyclooxygenases 1 and 2 (COX-1 and COX-2) and cytosolic phospholipase A 2 (cPLA 2) to determine whether the rate-limiting enzymes in the eicosanoid synthetic pathway are altered by ciprofibrate. Rats were fed 0.01% ciprofibrate for 3, 6, or 10 days. Western analysis revealed that COX-2 protein was induced by ciprofibrate (up to 13-fold at day 10), but that calcium-dependent (Ca-D) cPLA 2 protein was not different from controls. The enzyme activity of calcium-independent (Ca-I) cPLA 2 in ciprofibrate-treated rats was increased 2-fold, whereas Ca-D cPLA 2 and total COX activities were not affected. Using enzyme kinetics, we found that COX-1 ( K i=143 μM) and Ca-I cPLA 2 ( K i=121 μM) were competitively inhibited by ciprofibrate, but the inhibition was not physiologically significant. COX-2 and Ca-D cPLA 2 were not inhibited by ciprofibrate. These results show that ciprofibrate increases Ca-I cPLA 2 enzyme activity and COX-2 protein expression.

Morphological adaptations of human liver peroxisomes in cholestasis.

Part of the bile acid synthesis takes place in peroxisomes. An altered enterohepatic circulation of bile acids might influence peroxisomal beta-oxidation enzymes and peroxisomal morphology. We performed a morphological and morphometric investigation of peroxisomes in liver biopsy samples of eight patients with cholestasis of different origin: graft versus host reaction (n = 1), obstruction of the bile flow (n = 3), and drug-induced cholestatic hepatitis (n = 4). Peroxisomes were identified using catalase cytochemistry. They were regularly shaped and showed individual differences in electron density. A perinuclear distribution was observed in a variable number of hepatocytes in each sample. Morphometric analysis of peroxisomes revealed an increase in numerical density and surface density in all, and a decreased mean diameter in four liver samples. Based on previously obtained data in experimental animals, we hypothesize that the observed alterations in peroxisomal morphology indicate an enhanced metabolic activity of the enzymes in the peroxisomal matrix. Among them are enzymes involved in bile acid synthesis.

Reduction of the concentrations of prostaglandins E 2 and F 2α, and thromboxane B 2 in cultured rat hepatocytes treated with the peroxisome proliferator ciprofibrate

Several hypolipidemic drugs, plasticizers and other chemicals induce peroxisome proliferation and hepatic tumors in rodents, but the mechanism by which they induce tumors is not fully understood. Their carcinogenic activity may be related to alterations in gene expression, such as induction of peroxisomal β-oxidation enzymes or of the cytochrome P450 4A family. These enzymes metabolize lipids, including eicosanoids and their precursor fatty acids. Because eicosanoids likely play a role in the carcinogenic process, alterations in their concentration by xenobiotics may be important in their carcinogenic or promoting activities. In this study we used isolated hepatocytes to study if peroxisome proliferators alter the metabolism of prostaglandins (PG) and thromboxanes (Tx). Isolated rat hepatocytes were cultured for 4 days with 2 concentrations of ciprofibrate (CIP): 100 and 400 μM. Fatty acyl CoA oxidase activities of the 100 and 400 μM CIP treatment groups at the end of the experiment were increased 5.3 and 9.6 times, respectively. TxB 2 and PGF 2α concentrations in cultures treated with CIP were significantly lower than the control at days 3 and 4, whereas a lower concentration of PGE 2 was seen at day 4 only. These studies show that PG and Tx concentrations in cultured hepatocytes are lowered by the peroxisome proliferator CIP.

PPAR: a mediator of peroxisome proliferator action

Peroxisome proliferators are a diverse group of rodent hepatocarcinogens that include hypolipidemic drugs, plasticizers and herbicides. These compounds when administered to rats and mice produce a dramatic increase in the size and number of hepatic peroxisomes and increase the capacity of the hepatocyte to metabolise fatty acids by inducing peroxisomal β-oxidation enzymes such as acyl CoA oxidase. Members of the steroid hormone receptor superfamily of ligand-activated transcription factors have been identified that can be activated by peroxisome proliferators and are therefore called ‘peroxisome proliferator activated receptors’ (PPAR). There appear to be four PPAR isoforms within vertebrates termed α, β, γ and δ and the α isoform appears to be the one that is most strongly activated by synthetic peroxisome proliferators such as Wy-14,643. It has been demonstrated that PPAR α forms a heterodimer with the retinoid X receptor (RXR) and binds to specific DNA sequences located upstream of peroxisome proliferator responsive genes. It is therefore suggested that PPARs mediate the pleiotropic effects of peroxisome proliferators including the regulation of gene expression and rodent hepatocarcinogenesis. Rodents fed a high-fat diet develop peroxisome proliferation and many of the enzymes induced by peroxisome proliferators are involved in fatty acid metabolism. Furthermore, PPARs are activated by a wide range of fatty acids and hypolipidemic drugs, such as clofibrate, that lower triglyceride levels in man. Although it remains to be determined whether fatty acids and peroxisome proliferators bind directly to any PPAR these data suggest that the natural role of PPARs in man is to regulate lipid homeostasis. Interestingly, hypolipidaemic drugs fail to elicit peroxisome proliferation in human hepatocytes although hypolipidaemic effects are observed in patients. A further understanding of the role of PPAR in man will require: (1) the identification of additional human PPARs combined with functional analyses using these and other nuclear receptors that can antagonise PPAR action; (2) a comparison of the expression of these different receptors in human tissues; (3) a clearer understanding of how peroxisome proliferators and fatty acids activate PPAR; and (4) sequence analysis of the regulatory regions in the human counterparts of rodent peroxisome proliferator responsive genes. Together, these data will provide an important mechanism-based framework to assess the hazard of peroxisome proliferators to humans.