Peroxisomal genetic disorders, such as Zellweger syndrome, are characterized by defects in one or more enzymes involved in the peroxisomal ␤-oxidation of very long chain fatty acids and are associated with defective peroxisomal biogenesis. The biologic role of peroxisomal ␤-oxidation system, which consists of three enzymes: fatty acyl-CoA oxidase (ACOX), enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD), and thiolase, has been examined in mice by disrupting ACOX gene, which encodes the first and rate-limiting enzyme of this system. Homozygous (ACOX ؊/؊) mice lacked the expression of ACOX protein and accumulate very long chain fatty acids in blood. However, these homozygous mice are viable, but growth-retarded and infertile. During the first 3-4 months of age, the livers of ACOX ؊/؊ mice reveal severe microvesicular fatty metamorphosis of hepatocytes. In such steatotic cells, peroxisome assembly is markedly defective; as a result, they contain few or no peroxisomes. Few hepatocytes in 1-3-monthold ACOX ؊/؊ mice contain numerous peroxisomes, and these peroxisome-rich hepatocytes show no fatty change. At this stage, the basal mRNA levels of HD, thiolase, and other peroxisome proliferator-induced target genes were elevated in ACOX ؊/؊ mouse liver, but these mice, when treated with a peroxisome proliferator, showed no increases in the number of hepatic peroxisomes and in the mRNAs levels of these target genes. Between 4 and 5 months of age, severe steatosis resulted in scattered cell death, steatohepatitis, formation of lipogranulomas, and focal hepatocellular regeneration. In 6 -7-month-old animals, the newly emerging hepatocytes, which progressively replaced steatotic cells, revealed spontaneous peroxisome proliferation. These livers showed marked increases in the mRNA levels of the remaining two genes of the ␤-oxidation system, suggesting that ACOX gene disruption leads to increased endogenous ligand-mediated transcription levels. These observations demonstrate links among peroxisomal ␤-oxidation, development of severe microvesicular fatty liver, peroxisome assembly, cell death, and cell proliferation in liver.

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Peroxisome proliferators cause a rapid and coordinated transcriptional activation of genes encoding the enzymes of the peroxisomal beta-oxidation pathway in rats and mice. Cis-acting peroxisome proliferator responsive elements (PPREs) have been identified in the 5'-flanking region of H202-producing rat acyl-CoA oxidase (ACOX) gene and in other genes inducible by peroxisome proliferators. To gain more insight into the purported nonresponsiveness of human liver cells to peroxisome volume density and in the activity of the beta-oxidation enzyme system, we have previously cloned the human ACOX gene, the first and rate-limiting enzyme of the peroxisomal beta-oxidation system. We now present information on a regulatory element for the peroxidase proliferator-activated receptor (PPAR)/retinoid X receptor (RXR) heterodimers. The PPRE, consists of AGGTCA C TGGTCA, which is a direct repeat of hexamer half-sites interspaced by a single nucleotide (DR1 motif). It is located at -1918 to -1906 base pairs upstream of the transcription initiation site of this human ACOX gene. This PPRE specifically binds to baculovirus-expressed recombinant rat PPAR alpha/RXR alpha heterodimers. In transient transfection experiments, the maximum induction of luciferase expression by ciprofibrate and/or 9-cis-retinoic acid is dependent upon cotransfection of expression plasmids for PPAR alpha and RXR alpha. The functionally of this human ACOX promoter was further demonstrated by linking it to a beta-galactosidase reporter gene or to a rat urate oxidase cDNA and establishing stably transfected African green monkey kidney (CV1) cell lines expressing reporter protein. The human ACOX promoter has been found to be responsive to peroxisome proliferators in CV1 cells stably expressing PPAR alpha, whereas only a basal level of promoter activity is detected in stably transfected cells lacking PPAR alpha. The presence of a PPRE in the promoter of this human peroxisomal ACOX gene and its responsiveness to peroxisome proliferators suggests that factors other than the PPRE in the 5'-flanking sequence of the human ACOX gene may account for differences, if any, in the pleiotropic responses of humans to peroxisome proliferators.

Peroxisomal acyl-CoA oxidase (ACOX; EC 1.3.3.6) is the first enzyme of the fatty acid 1-oxidation pathway, which catalyzes the desaturation of acyl-CoAs to 2-tnns-enoyl-CoAs, and it donates electrons directly to molecular oxygen, thereby producing H202. The discovery of carcinogenic peroxisome proliferators, which markedly increase the levels ofthis H202-producing ACOX in rat and mouse liver, generated interest in peroxisomal «-oxidation system genes. The present study deals with the structural organization of human ACOX gene. This gene spans :33 kb and consists of 14 exons and 13 introns. Primer-extension analysis revealed three principal cap sites, which were mapped at 50, 52, and 53 nt upstream of the initiator methionine codon. The 5' flanking region of the ACOX gene was sequenced up to 500 bp ups ofthe cap sites. This promoter region is G+C-rich and contains three copies of the "GC box" hexanucleofides. Multiple GC boxes are a characteristic feature of the rat ACOX and functional protein genes ofthe 1oxidation system. A+T-rich TATA-boxilike sequences, TTTATTT and TTATY, have also been identified in this human ACOX gene, but typical CCAAT motifs are absent. This ACOX gene has been mapped to chromosome 17q25 by in situ hybridization, using a biotinlabeled probe.Peroxisomes are cellular organelles that are present in virtually all eukaryotic cells. These organelles contain hydrogen peroxide-producing flavin oxidases together with catalase, which decomposes hydrogen peroxide (1). At present >50 enzymes have been found in mammalian peroxisomes, and more than half of these play a role in lipid metabolism (1

Peroxisome proliferators cause rapid and coordinated transcriptional activation of genes encoding peroxisomal 3-oxidation system enzymes by activating peroxisome proliferator-activated receptor (PPAR) isoform(s). Since the thyroid hormone (T3; 3,3',5-triiodothyronine) receptor (TR), another member of the nuclear hormone receptor superfamily, regulates a subset of fatty acid metabolism genes shared with PPAR, we examined the possibility of interplay between peroxisome proliferator and T3 signaling pathways. T3 inhibited ciprofibrate-induced luciferase activity as well as the endogenous peroxisomal 13-oxidation enzymes in transgenic mice carrying a 3.2-kb 5'-flanking region of the rat peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase gene fused to the coding region of luciferase. Transfection assays in hepatoma H4-II-E-C3 and CV-1 cells indicated that this inhibition is mediated by TR in a liganddependent fashion. Gel shift assays revealed that modulation of PPAR action by TR occurs through titration of limiting amounts of retinoid X receptor (RXR) required for PPAR activation. Increasing amounts of RRXR partially reversed the inhibition in a reciprocal manner; PPAR also inhibited TR activation. Results with heterodimerization-deficient TR and PPAR mutants further confirmed that interaction between PPAR and TR signaling systems is indirect. These results suggest that a convergence of the peroxisome proliferator and T3 signaling pathways occurs through their common interaction with the heterodimeric partner RXR.

Human liver peroxisomes contain two acyl-CoA oxidases, namely, palmitoyl-CoA oxidase and a branched chain acyl-CoA oxidase. The palmitoyl-CoA oxidase (ACOX) oxidizes the CoA esters of straight chain fatty acids and prostaglandins and donates electrons directly to molecular oxygen, thereby producing H2O2. The inducibility of this H2O2-generating ACOX in rat and mouse liver by peroxisome proliferators and the postulated role of the resulting oxidative stress in hepatocarcinogenesis generated interest in characterizing the structure and function of human ACOX. We have constructed a full-length cDNA encoding a 660-amino acid residue human ACOX and produced a catalytically active human ACOX protein at high levels in Spodoptera frugiperda (Sf9) insect cells using the baculovirus vector. Immunoblot analysis demonstrated that the full-length 72-kDa polypeptide (component A) was partially processed into its constituent 51-kDa (component B) and 21-kDa (component C) products, respectively. Recombinant protein (approximately 20 mg/l x 10(9) cells) was purified to homogeneity by a single-step procedure on a nickel-nitrilo-triacetic acid affinity column. Using the purified enzyme, Km and Vmax values for palmitoyl-CoA were found to be 10 microM and 1.4 units/mg of protein, respectively. The maximal activities for saturated fatty acids were observed with C12-18 substrates. The overexpressed human ACOX protein was identified in the cytoplasm of the insect cells by immunocytochemical staining. Individual expression of either the truncated ACOX 51-kDa (component B) or the 21-kDa (component C) revealed lack of enzyme activity, but co-infection of the insect cells with recombinant viruses expressing components B and C resulted in the formation of an enzymatically active heterodimeric B+C complex which could subsequently be inactivated by dissociating with detergent.

Peroxisome proliferator-activated receptor ␣ (PPAR␣) is important in the induction of cell-specific pleiotropic responses, including the development of liver tumors, when it is chronically activated by structurally diverse synthetic ligands such as Wy-14,643 or by unmetabolized endogenous ligands resulting from the disruption of the gene encoding acyl coenzyme A (CoA) oxidase (AOX). Alterations in gene expression patterns in livers with PPAR␣ activation were delineated by using a proteomic approach to analyze liver proteins of Wy-14,643-treated and AOX ؊/؊ mice. We identified 46 differentially expressed proteins in mouse livers with PPAR␣ activation. Up-regulated proteins, including acetyl-CoA acetyltransferase, farnesyl pyrophosphate synthase, and carnitine O-octanoyltransferase, are involved in fatty acid metabolism, whereas down-regulated proteins, including ketohexokinase, formiminotransferase-cyclodeaminase, fructose-bisphosphatase aldolase B, sarcosine dehydrogenase, and cysteine sulfinic acid decarboxylase, are involved in carbohydrate and amino acid metabolism. Among stress response and xenobiotic metabolism proteins, selenium-binding protein 2 and catalase showed a dramatic ϳ18-fold decrease in expression and a modest ϳ6-fold increase in expression, respectively. In addition, glycine N-methyltransferase, pyrophosphate phosphohydrolase, and protein phosphatase 1D were down-regulated with PPAR␣ activation. These observations establish proteomic profiles reflecting a common and predictable pattern of differential protein expression in livers with PPAR␣ activation. We conclude that livers with PPAR␣ activation are transcriptionally geared towards fatty acid combustion.

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