Abstract--The three peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors of the nuclear hormone receptor superfamily. They share a high degree of structural homology with all members of the superfamily, particularly in the DNA-binding domain and ligand-and cofactor-binding domain. Many cellular and systemic roles have been attributed to these receptors, reaching far beyond the stimulation of peroxisome proliferation in rodents after which they were initially named. PPARs exhibit broad, isotype-specific tissue expression patterns. PPAR␣ is expressed at high levels in organs with significant catabolism of fatty acids. PPAR␤/␦ has the broadest expression pattern, and the levels of expression in certain tissues depend on the extent of cell proliferation and differentiation. PPAR␥ is expressed as two isoforms, of which PPAR␥2 is found at high levels in the adipose tissues, whereas PPAR␥1 has a broader expression pattern. Transcriptional regulation by PPARs requires heterodimerization with the retinoid X receptor (RXR). When activated by a ligand, the dimer modulates transcription via binding to a specific DNA sequence element called a peroxisome proliferator response element (PPRE) in the promoter region of target genes. A wide variety of natural or synthetic compounds was identified as PPAR ligands. Among the synthetic ligands, the lipidlowering drugs, fibrates, and the insulin sensitizers, thiazolidinediones, are PPAR␣ and PPAR␥ agonists, respectively, which underscores the important role of PPARs as therapeutic targets. Transcriptional control by PPAR/RXR heterodimers also requires interaction with coregulator complexes. Thus, selective action of PPARs in vivo results from the interplay at a given time point between expression levels of each of the three PPAR and RXR isotypes, affinity for a specific promoter PPRE, and ligand and cofactor availabilities.

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.

Reddy, Janardan K., and M. Sambasiva Rao. Lipid Metabolism and Liver Inflammation. II. Fatty liver disease and fatty acid oxidation.

The Ah receptor (AHR) is a ligand-activated transcription factor that mediates a pleiotropic response to environmental contaminants such as benzo [a]pyrene and 2, 3,7,8-tetrachlorodibenzo-p-dioxin. In an effort to gain insight into the physiological role of the AHR and to develop models useful in risk assessment, gene targeting was used to inactivate the murineAhr gene by homologous recombination. Ahr-l mice are viable and fertile but show a spectrum of hepatic defects that indicate a role for the AHR in normal liver growth and development. TheAhr-1-phenotype is most severe between 0-3 weeks ofage and involves slowed early growth and hepatic defects, including reduced liver weight, transient microvesicular fatty metamorphosis, prolonged extramedullary hematopoiesis, and portal hypercellularity with thickening and fibrosis.The Ah receptor (AHR) is a ligand-activated transcription factor that regulates a biphasic pleiotropic response to a variety of structurally related environmental contaminants (1, 2). Upon binding polycyclic aromatic hydrocarbons (PAHs), such as benzo [a]pyrene, the AHR increases the expression of xenobiotic metabolizing enzymes, including the cytochrome P450IA1, P450IA2 and P450IB1-dependent monooxygenases, the glutathione S-transferase Ya subunit, quinone oxidoreductase, and UDP-glucuronosyltransferase (3-8). In response to more potent halogenated aromatic agonists like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the AHR induces xenobiotic metabolism and also mediates a spectrum of toxic responses, including thymic involution, teratogenesis, tumor promotion, wasting, and epithelial hyperplasia and metaplasia (9-11).The AHR is a member of a family of transcription factors containing basic/helix-loop-helix and PAS homology domains (bHLH-PAS) (1). In response to agonist binding within the PAS domain, the cytosolic AHR undergoes a conformational change, translocates to the nucleus, dissociates from the 90-kDa heat shock protein, and dimerizes with a second bHLH-PAS protein known as the Ah receptor nuclear translocator (ARNT) (12-16). This heterodimer interacts with dioxin-responsive enhancer elements upstream of target genes and activates their transcription. Despite our mechanistic understanding of the role of the AHR in regulating xenobiotic metabolism, we still have little understanding of how the AHR mediates the toxic responses of halogenated agonists and why such responses are produced only by high affinity, poorly metabolized ligands such as TCDD.To examine the importance of the AHR in normal vertebrate biology, we have used gene targeting to create mice with a mutation at theAhr locus. We anticipated that such a mutant could provide insights into additional physiological roles of the AHR and would represent a powerful model to understand the toxicology of halogenated aromatic pollutants like TCDD.

Peroxisome proliferator activated-receptor (PPAR) isoforms, ␣ and ␥, function as important coregulators of energy (lipid) homeostasis. PPAR␣ regulates fatty acid oxidation primarily in liver and to a lesser extent in adipose tissue, whereas PPAR␥ serves as a key regulator of adipocyte differentiation and lipid storage. Of the two PPAR␥ isoforms, PPAR␥1 and PPAR␥2 generated by alternative splicing, PPAR␥1 isoform is expressed in liver and other tissues, whereas PPAR␥2 isoform is expressed exclusively in adipose tissue where it regulates adipogenesis and lipogenesis. Since the function of PPAR␥1 in liver is not clear, we have, in this study, investigated the biological impact of overexpression of PPAR␥1 in mouse liver. Adenovirus-PPAR␥1 injected into the tail vein induced hepatic steatosis in PPAR␣ ؊/؊ mice. Northern blotting and gene expression profiling results showed that adipocyte-specific genes and lipogenesis-related genes are highly induced in PPAR␣ ؊/؊ livers with PPAR␥1 overexpression. These include adipsin, adiponectin, aP2, caveolin-1, fasting-induced adipose factor, fat-specific gene 27 (FSP27), CD36, ⌬ 9 desaturase, and malic enzyme among others, implying adipogenic transformation of hepatocytes. Of interest is that hepatic steatosis per se, induced either by feeding a diet deficient in choline or developing in fasted PPAR␣ ؊/؊ mice, failed to induce the expression of these PPAR␥-regulated adipogenesis-related genes in steatotic liver. These results suggest that a high level of PPAR␥ in mouse liver is sufficient for the induction of adipogenic transformation of hepatocytes with adipose tissue-specific gene expression and lipid accumulation. We conclude that excess PPAR␥ activity can lead to the development of a novel type of adipogenic hepatic steatosis.

To gain insight into the regulation of expression of peroxisome proliferator-activated receptor (PPAR) isoforms, we have determined the structural organization of the mouse PPAR 'y (mPPARy) gene. This gene extends > 105 kb and gives rise to two mRNAs (mPPARy1 and mPPARy2) that differ at their 5' ends. The mPPARy2 cDNA encodes an additional 30 amino acids N-terminal to the first ATG codon of mPPARyl and reveals a different 5' untranslated sequence.We show that mPPARy1 mRNA is encoded by eight exons, whereas the mPPARy2 mRNA is encoded by seven exons.Most of the 5' untranslated sequence of mPPARy1 mRNA is encoded by two exons, whereas the 5' untranslated sequence and the extra 30 N-terminal amino acids of mPPARy2 are encoded by one exon, which is located between the second and third exons coding for mPPARyl. The last six exons of mPPARy gene code for identical sequences in mPPARyl and mPPARy2 isoforms. The mPPARyl and mPPARy2 isoforms are transcribed from different promoters. The mPPARy gene has been mapped to chromosome 6 E3-F1 by in situ hybridization using a biotin-labeled probe. These results establish that at least one of the PPAR genes yields more than one protein product, similar to that encountered with retinoid X receptor and retinoic acid receptor genes. The existence of multiple PPAR isoforms transcribed from different promoters could increase the diversity of ligand and tissue-specific transcriptional responses.

Genetically modified mice have been extensively used for analyzing the molecular events that occur during tumor development. In many, if not all, cases, however, it is uncertain to what extent the mouse models reproduce features observed in the corresponding human conditions. This is due largely to lack of precise methods for direct and comprehensive comparison at the molecular level of the mouse and human tumors. Here we use global gene expression patterns of 68 hepatocellular carcinomas (HCCs) from seven different mouse models and 91 human HCCs from predefined subclasses to obtain direct comparison of the molecular features of mouse and human HCCs. Gene expression patterns in HCCs from Myc, E2f1 and Myc E2f1 transgenic mice were most similar to those of the better survival group of human HCCs, whereas the expression patterns in HCCs from Myc Tgfa transgenic mice and in diethylnitrosamine-induced mouse HCCs were most similar to those of the poorer survival group of human HCCs. Gene expression patterns in HCCs from Acox1(-/-) mice and in ciprofibrate-induced HCCs were least similar to those observed in human HCCs. We conclude that our approach can effectively identify appropriate mouse models to study human cancers.

Peroxisomal ␤-oxidation system consists of four consecutive reactions to preferentially metabolize very long chain fatty acids. The first step of this system, catalyzed by acyl-CoA oxidase (AOX), converts fatty acylCoA to 2-trans-enoyl-CoA. Herein, we show that mice deficient in AOX exhibit steatohepatitis, increased hepatic H 2 O 2 levels, and hepatocellular regeneration, leading to a complete reversal of fatty change by 6 to 8 months of age. The liver of AOX؊/؊ mice with regenerated hepatocytes displays profound generalized spontaneous peroxisome proliferation and increased mRNA levels of genes that are regulated by peroxisome proliferator-activated receptor ␣ (PPAR␣). Hepatic adenomas and carcinomas develop in AOX؊/؊ mice by 15 months of age due to sustained activation of PPAR␣. These observations implicate acyl-CoA and other putative substrates for AOX, as biological ligands for PPAR␣; thus, a normal AOX gene is indispensable for the physiological regulation of PPAR␣.