Ruiyin Chu

Hepatocellular and hepatic peroxisomal alterations in mice with a disrupted peroxisomal fatty acyl-coenzyme A oxidase gene.

Peroxisomal genetic disorders, such as Zellweger syndrome, are characterized by defects in one or more enzymes involved in the peroxisomal β-oxidation of very long chain fatty acids and are associated with defective peroxisomal biogenesis. The biologic role of peroxisomal β-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-month-old 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 β-oxidation system, suggesting that ACOX gene disruption leads to increased endogenous ligand-mediated transcription levels. These observations demonstrate links among peroxisomal β-oxidation, development of severe microvesicular fatty liver, peroxisome assembly, cell death, and cell proliferation in liver.

Protein Profiling of Mouse Livers with Peroxisome Proliferator-Activated Receptor α Activation

ABSTRACT Peroxisome proliferator-activated receptor α (PPARα) 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 PPARα 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 PPARα 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 ∼18-fold decrease in expression and a modest ∼6-fold increase in expression, respectively. In addition, glycine N-methyltransferase, pyrophosphate phosphohydrolase, and protein phosphatase 1D were down-regulated with PPARα activation. These observations establish proteomic profiles reflecting a common and predictable pattern of differential protein expression in livers with PPARα activation. We conclude that livers with PPARα activation are transcriptionally geared towards fatty acid combustion.

Cloning and Identification of Rat Deoxyuridine Triphosphatase as an Inhibitor of Peroxisome Proliferator-activated Receptor α

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily that transcriptionally regulate responsive genes by binding to the peroxisome proliferator response elements. Protein(s) interacting with PPAR isoforms (α, δ, and γ) may modulate the PPAR-mediated transcriptional activation. Using a yeast two-hybrid system to screen a rat liver cDNA library, we have identified rat deoxyuridine-triphosphatase (dUTPase, EC 3.6.1.23) as a PPARα-interacting protein. This cDNA encodes a polypeptide of 203 amino acids; the C-terminal 141-amino acid segment of this protein corresponds to the full-length human enzyme, which exhibits 92% identity with human dUTPase; the N-terminal extra 62-amino acid residue region is arginine-rich. In vitro binding assays indicate that rat dUTPase interacts with all three isoforms of mouse PPAR, but not with retinoid X receptor and thyroid hormone receptor. Interaction of PPARα with dUTPase is with the N-terminal 62-amino acid segment of rat dUTPase. Full-length rat dUTPase prevents PPAR-retinoid X receptor heterodimerization resulting in an inhibition of PPAR activity in a ligand-independent manner. Immunostaining of human kidney tsA201 cells, transiently expressing dUTPase showed that this protein is present predominantly in the cytoplasm but translocates into the nucleus with PPARα when PPARα is coexpressed with dUTPase. Northern blot hybridization shows that rat dUTPase is encoded by an abundant 1kilobase mRNA species present in all rat tissues. The identification of dUTPase as a PPAR-interacting protein suggests a possible link between tumorigenic peroxisome proliferators and the enzyme system involved in the maintenance of DNA fidelity.

Profiling of acyl-CoA oxidase-deficient and peroxisome proliferator Wy14,643-treated mouse liver protein by surface-enhanced laser desorption/ionization ProteinChip Biology System.

Peroxisome proliferators induce hepatic peroxisome proliferation and hepatocellular carcinomas in rodents. These chemicals increase the expression of the peroxisomal beta-oxidation pathway and the cytochrome P-450 4A family, which metabolizes lipids, including fatty acids. Mice lacking fatty acyl-CoA oxidase (AOX-/-), the first enzyme of the peroxisomal beta-oxidation system, exhibit extensive microvesicular steatohepatitis, leading to hepatocellular regeneration and massive peroxisome proliferation. To investigate proteins involved in peroxisome proliferation, we adopted a novel surface-enhanced laser desorption/ionization (SELDI) ProteinChip technology to compare the protein profiles of control (wild-type), AOX-/-, and wild-type mice treated with peroxisome proliferator, Wy-14,643. The results indicated that the protein profiles of AOX-/- mice were similar to the wild-type mice treated with Wy14,643, but significantly different from the nontreated wild-type mice. Using four different ProteinChip Arrays, a total of 40 protein peaks showed more than twofold changes. Among these differentially expressed peaks, a downregulated peak was identified as the major urinary protein in both AOX-/- and Wyl4,643-treated mice by SELDI. The identification of MUP was further confirmed by two-dimensional electrophoresis and liquid chromatography coupled tandem mass spectrometry (LC-MS-MS). This SELDI method offers several technical advantages for detection of differentially expressed proteins, including ease and speed of screening, no need for chromatographic processing, and small sample size.