Cetaben-induced changes on the morphology and peroxisomal enzymes in MH1C1 rat hepatoma cells and HepG2 human hepatoblastoma cells

Human HepG2 and rat MH1C1 hepatoma cell lines were examined for their response to cetaben, an exceptional type of peroxisome proliferator. Shape change and proliferation of peroxisomes as well as induction of selected peroxisomal enzymes catalase, acyl-CoA oxidase, and peroxisomal bifunctional enzyme, were assessed in response to cetaben. In MH1C1 cells, peroxisomes were seen in clusters displaying typical features of microperoxisomes. Cetaben caused little but reversible proliferation and morphological heterogeneity with the occurrence of dumbbell-shaped and cup-shaped peroxisomal profiles. Peroxisomes in HepG2 cells showed marked variation in size and shape. Cetaben treatment of HepG2 cells caused disintegration of Golgi regions and augmented mitochondrial matrix. Interestingly, MH1C1 cells showed different subunit composition of acyl-CoA oxidase in immunoblot analysis: only subunit A at 72 kDa was detected but not the cleavage products. In situ hybridization underlined the marked morphological heterogeneity observed, and both cell lines revealed different stages of gene expression. Our results indicate that cetaben represents an extraordinary type of peroxisomal proliferator with pleiotropic effects on human and rat hepatoma cells, and, at least in the human hepatoma cell line HepG2, these effects are not restricted to peroxisome proliferation.     

Import of human bifunctional enzyme into peroxisomes of human hepatoma cells in vitro.

A polypeptide containing the carboxyl-terminal fragment of human peroxisomal enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme was synthesized in vitro from its cDNA clone. This expression polypeptide was transported into purified rat liver peroxisomes. When the expression polypeptide was incubated with postnuclear supernatant fractions of human hepatoma cells and analyzed by Nycodenz gradient SDS-PAGE and fluorography, it was imported specifically into peroxisomes as indicated by its resistance to proteinase K degradation. A deletion of the last nine amino acid residues at the carboxyl-terminus of this polypeptide prevents its peroxisomal import. A tripeptide sequence, SKL, located at the carboxyl-terminus of human bifunctional enzyme appears to be the targeting signal for the peroxisomal importation of bifunctional enzyme in human cells.     

Acyl-CoA synthetase and the peroxisomal enzymes of beta-oxidation in human liver. Quantitative analysis of their subcellular localization.

The presence of acyl-CoA synthetase (EC 6.2.1.3) in peroxisomes and the subcellular distribution of beta-oxidation enzymes in human liver were investigated by using a single-step fractionation method of whole liver homogenates in metrizamide continuous density gradients and a novel procedure of computer analysis of results. Peroxisomes were found to contain 16% of the liver palmitoyl-CoA synthetase activity, and 21% and 60% of the enzyme activity was localized in mitochondria and microsomal fractions respectively. Fatty acyl-CoA oxidase was localized exclusively in peroxisomes, confirming previous results. Human liver peroxisomes were found to contribute 13%, 17% and 11% of the liver activities of crotonase, beta-hydroxyacyl-CoA dehydrogenase and thiolase respectively. The absolute activities found in peroxisomes for the enzymes investigated suggest that in human liver fatty acyl-CoA oxidase is the rate-limiting enzyme of the peroxisomal beta-oxidation pathway, when palmitic acid is the substrate.     

Separate peroxisomal oxidases for fatty acyl-CoAs and trihydroxycoprostanoyl-CoA in human liver

Fatty acyl-CoAs as well as the CoA esters of the bile acid intermediates di- and trihydroxycoprostanic acids are beta-oxidized in peroxisomes. The first reaction of peroxisomal beta-oxidation is catalyzed by acyl-CoA oxidase. We recently described the presence of two fatty acyl-CoA oxidases plus a trihydroxycoprostanoyl-CoA oxidase in rat liver peroxisomes (Schepers, L., P. P. Van Veldhoven, M. Casteels, H. J. Eyssen, and G. P. Mannaerts. 1990. J. Biol. Chem. 265: 5242-5246). We have now developed methods for the measurement of palmitoyl-CoA oxidase and trihydroxycoprostanoyl-CoA oxidase in human liver. The activities were measured in livers from controls and from three patients with peroxisomopathies. In addition, the oxidase activities were partially purified from control livers by ammonium sulfate fractionation and heat treatment, and the partially purified enzyme preparation was subjected to chromatofocusing, hydroxylapatite chromatography, and gel filtration. In earlier experiments this allowed for the separation of the three rat liver oxidases. The results show that human liver, as rat liver, contains a separate trihydroxycoprostanoyl-CoA oxidase. In contrast to the situation in rat liver, no conclusive evidence was obtained for the presence of two fatty acyl-CoA oxidases in human liver. Our results explain why bile acid metabolism is normal in acyl-CoA oxidase deficiency, despite a severely disturbed peroxisomal fatty acid oxidation and perhaps also why, in a number of other cases of peroxisomopathy, di- and trihydroxycoprostanic acids are excreted despite a normal peroxisomal fatty acid metabolism.     

Immunocytochemical localization of serine: pyruvate aminotransferase in peroxisomes of the human liver parenchymal cells

The localization of serine:pyruvate aminotransferase (SPT) in human liver was investigated by indirect immunoenzyme and protein A-gold techniques. By light microscopy, diaminobenzidine reaction product was present in cytoplasmic granules of the parenchymal cells. By electron microscopy, gold particles indicating the antigenic sites for SPT were exclusively confined to peroxisomes but not to mitochondria. By double labeling technique, both peroxisomal marker enzyme, catalase and SPT were detected in the same peroxisomes. Quantitative analysis of the labeling density showed that SPT is contained only in peroxisomes. The results indicate that in human liver most of SPT is contained in the peroxisomes.     

Immunocytochemical localization of serine: pyruvate aminotransferase in peroxisomes of the human liver parenchymal cells

Summary The localization of serine:pyruvate aminotransferase (SPT) in human liver was investigated by indirect immunoenzyme and protein A-gold techniques. By light microscopy, diaminobenzidine reaction product was present in cytoplasmic granules of the parenchymal cells. By electron microscopy, gold particles indicating the antigenic sites for SPT were exclusively confined to peroxisomes but not to mitochondria. By double labeling technique, both peroxisomal marker enzyme, catalase and SPT were detected in the same peroxisomes. Quantitative analysis of the labeling density showed that SPT is contained only in peroxisomes. The results indicate that in human liver most of SPT is contained in the peroxisomes.     

Induction of peroxisomal enzymes in cultured porcine endothelial cells by the hypolipidemic drug ciprofibrate

The purpose of this study was to determine if the hypolipidemic peroxisome proliferator ciprofibrate, which induces peroxisomes in the liver, can induce peroxisomes in cultured porcine pulmonary endothelial cells. Ciprofibrate was added at three concentrations to cell cultures for a 6-day period. The induction of peroxisomes in the cells was detected by determining total peroxisomal beta-oxidation and peroxisomal catalase activity. The addition of ciprofibrate was found to increase peroxisomal enzyme activities in a dose-dependent manner, with the highest activity being reached at 1000 microM ciprofibrate. Ciprofibrate also caused an increased transfer of albumin across endothelial cells cultured on micropore filters. This study shows that peroxisomal enzyme activities can be induced by ciprofibrate in endothelial cells, which may have implications in diseases mediated by vascular injury.     

Cytochemical and biochemical demonstration of an ATPase in membranes of human peroxisomes.

We demonstrated a neutral Mg-ATPase activity in human peroxisomal membranes. To establish the precise experimental conditions for detection of this ATPase, both cytochemical and biochemical characterizations were first carried out in liver peroxisomes from control and cipofibrate-treated rats. The results demonstrated an Mg-ATPase reaction in both normal and proliferated peroxisomes. The nucleotidase activity, with marked preference for ATP, was sensitive to the inhibitors N-ethylmaleimide and 7-chloro-4-nitro-benzo-2-oxadiazole (NBDCl). An ultrastructural cytochemical analysis was developed to evaluate the peroxisomal localization, which localized the reaction product to the peroxisomal membrane. These characteristics can help to differentiate the peroxisomal ATPase from the activity found in mitochondria and endoplasmic reticulum. The conditions established for detecting the rat peroxisomal ATPase were then applied to human peroxisomes isolated from liver and skin fibroblasts in culture. A similar Mg-ATPase activity was readily shown, both cytochemically and biochemically, in the membranes of human peroxisomes. These results, together with previous evidence, strongly support the presence of a specific ATPase in the human peroxisomal membrane. This ATPase may play a crucial role in peroxisome biogenesis.     

Peroxisome through cell differentiation and neoplasia

Summary— Peroxisomes are essential in cellular metabolism as their dysgenesis or defects in single enzymes or impairment of multiple peroxisomal enzymatic functions have been found in several inherited metabolic diseases with serious clinical sequelae. The assembly and formation of these cytoplasmic organelles constitute a major and intringuing research topic. In the present study the biogenesis of peroxisomes and the developmental patterns of their enzymes have been reviewed during embryonic and/or post-embryonic ontogenesis of lower (amphibians) and higher (avians, mammals) vertebrates. In developing vertebrates, epithelial cell differentiation is accompanied by increases in frequency and size of peroxisomes. The tissue-specific expression of peroxisomal enzymes contributes substantially to the biochemical maturation of epithelial cells. The relationship between biogenesis of peroxisomes, expression of peroxisomal enzymes and structural and functional cellular phenotype has also been investigated in differentiating epithelial cells along the crypt-villus axis of the adult rat intestine. Cytochemical studies at the ultrastructural level have provided evidence that peroxisomes are already present in proliferating cells of the intestinal crypt region before they begin to differentiate. Migration and differentiation of intestinal epithelial cells from crypt to villus compartments are marked by significant increases in number and size of catalase-positive structures. Increasing activity gradients from crypt to surface areas are found for the peroxisomal oxidases examined (enzymes of the peroxisomal β-oxidation system, d -amino acid oxidase and polyamine oxidase). Thus, peroxisomes are more and more involved in oxidative metabolic pathways as intestinal epithelial cells differentiate. Finally, we have analyzed the peroxisomal behaviour in human neoplastic epithelial cells. The presence of peroxisomes has been cytochemically revealed in human breast and colon carcinomas. Peroxisomal enzyme specific activities are significantly lower in human breast and colon carcinomas than in the adjacent healthy mucosa. Furthermore, a relationship is found between the specific activities of some peroxisomal enzymes and the histological tumour grades.     

Dihydroxyacetone phosphate acyltransferase and alkyldihydroxyacetone phosphate synthase activities in rat liver subcellular fractions and human skin fibroblasts

Dihydroxyacetone phosphate acyltransferase (DHAP-AT) and alkyldihydroxyacetone phosphate synthase (DHAP-synthase) activities were examined in subcellular fractions of rat liver. The results indicate that at least 80% of DHAP-AT (assays carried out at pH 5.4) activity in rat liver is in peroxisomes, and the remaining activity is mitochondrial. In contrast to DHAP-AT, DHAP-synthase was detected in all subcellular fractions analyzed but the activity in peroxisomes was 208-fold and 42-fold greater compared to mitochondria and microsomes, respectively. We estimate that at least 70% of the DHAP-synthase activity in rat liver is in peroxisomes. DHAP-AT and DHAP-synthase activities were also examined in homogenates of skin fibroblasts from patients with inherited defects in peroxisomal structure and/or function. Both the enzyme activities were deficient in Zellweger syndrome whereas the activities were only partially deficient in infantile Refsum's disease. Greater reduction in DHAP-synthase activity, but only a partial reduction in DHAP-AT activity was observed in rhizomelic chondrodysplasia punctata. However, both DHAP-AT and DHAP-synthase activities were either normal or near normal in Refsum's disease or X-linked adrenoleukodystrophy. The results reported suggest that various peroxisomal disease states can be identified based on DHAP-AT and DHAP-synthase activities in skin fibroblasts of patients.     

In vitro processing of the human alkyl-dihydroxyacetonephosphate synthase precursor.

Alkyl-dihydroxyacetonephosphate synthase, a peroxisomal enzyme involved in the biosynthesis of ether phospholipids, is synthesized with a cleavable N-terminal presequence containing the peroxisomal targeting signal type 2. The human alkyl-dihydroxyacetonephosphate synthase precursor produced in vitro or expressed in Escherichia coli could be processed to a lower molecular weight protein by incubation at 37 degrees C with a guinea pig liver fraction, enriched in mitochondria, lysosomes, and peroxisomes. This lower molecular weight protein was identified as the mature human alkyl-dihydroxyacetonephosphate synthase by radiosequencing, indicating that the processing protease is present in this organellar fraction. Characterization of the processing protease indicated that it is a cysteine protease with a pH optimum of 6.5. Furthermore, it was demonstrated that exogenously added pre-alkyl-dihydroxyacetonephosphate synthase was imported and processed in purified peroxisomes in vitro. Processing of alkyl-dihydroxyacetonephosphate synthase did not increase the activity of the enzyme. This indicates that the presence of the presequence does not affect the activity of the enzyme.     

Microsomal and cytosolic epoxide hydrolases, the peroxisomal fatty acid beta-oxidation system and catalase. Activities, distribution and induction in rat liver parenchymal and non-parenchymal cells

A number of structurally unrelated hypolipidaemic agents and certain phthalate-ester plasticizers induce hepatomegaly and proliferation of peroxisomes in rodent liver, but there is relatively limited data regarding the specific effects of these drugs on liver non-parenchymal cells. In the present study, liver parenchymal, Kupffer and endothelial cells from untreated and fenofibrate-fed rats were isolated and the activities of two enzymes associated with peroxisomes (catalase and the peroxisomal fatty acid beta-oxidation system) as well as cytosolic and microsomal epoxide hydrolase were measured. Microsomal epoxide hydrolase, cytosolic epoxide hydrolase and catalase activities were 7-12-fold higher in parenchymal cells than in Kupffer or endothelial cells from untreated rats; the peroxisomal fatty acid beta-oxidation activity was only detected in parenchymal cells. Fenofibrate increased catalase, cytosolic epoxide hydrolase and peroxisomal fatty acid beta-oxidation activities in parenchymal cells by about 1.5-, 3.5- and 20-fold, respectively. The induction of catalase (2-3-fold) and cytosolic epoxide hydrolase (3-5-fold) was also observed in Kupffer and endothelial cells; furthermore, a low peroxisomal fatty acid beta-oxidation activity was detected in endothelial cells. Morphological examination by electron microscopy showed that peroxisomes were confined to liver parenchymal cells in untreated animals, but could also be observed in endothelial cells after administration of fenofibrate.     

Insulin-degrading enzyme exists inside of rat liver peroxisomes and degrades oxidized proteins

Insulin-degrading enzyme (IDE) was detected by immunoblot analysis in highly purified rat liver peroxisomes. IDE in the peroxisomal fraction was resistant to proteolysis by trypsin and chymotrypsin under conditions where the peroxisomal membranes remained intact. After sonication of the peroxisomal fraction, IDE was recovered in the supernatant fraction. Further, the localization of IDE in the peroxisomes was shown by immunoelectron microscopy. In addition, IDE isolated from peroxisomes degraded insulin as well as oxidized lysozyme as a model substrate for oxidized proteins. These results suggest that IDE exists in an active form in the matrix of rat liver peroxisomes and is involved in elimination of oxidized proteins in peroxisomes.     

Effect of clofibrate on peroxisomal lignoceroyl-CoA ligase activity

The effect of a 2-week clofibrate (0.5%)-fortified diet on peroxisomal palmitoyl-CoA and lignoceroyl-CoA ligases was studied. The activities of palmitoyl-CoA and lignoceroyl-CoA ligases in peroxisomes isolated from clofibrate-treated animals were 4.4- and 4.0-fold higher than those of the controls. The different degrees of increases in these two enzyme activities support the previous conclusions that in peroxisomes palmitoyl-CoA ligase and lignoceroyl-CoA ligase are different enzymes. Since clofibrate treatment increases both of these peroxisomal acyl-CoA ligase activities and normal palmitoyl-CoA ligase is the source of the partial activity for the oxidation of lignoceric acid in X-ALD, treatment with a hypolipidemic drug, which can increase human peroxisomal enzyme activities, may be helpful in lowering the amount of the pathogen, VLC fatty acids, in X-ALD.     

Immunological analyses of alkyl-dihydroxyacetone-phosphate synthase in human peroxisomal disorders.

Alkyl-dihydroxyacetonephosphate synthase (alkyl-DHAP synthase) is a peroxisomal enzyme involved in the biosynthesis of ether phospholipids. To localize the enzyme in human peroxisomal disorders, indirect immunofluorescence and immunoblot analysis was performed. In Zellweger syndrome and rhizomelic chondrodysplasia punctata fibroblast cell lines, alkyl-DHAP synthase protein levels on immunoblots were strongly decreased and residual immunofluorescence was diffusely localized throughout the cytoplasm. In a particular neonatal adrenoleukodystrophy cell line, characterized by the absence of a functional peroxisomal targeting signal 1 receptor, the precursor form of the enzyme was detected in Western blots at levels comparable to that of the mature enzyme in control fibroblasts. Similarly, fibroblasts from patients with a single deficiency in the activity of either alkyl-DHAP synthase or DHAP-acyltransferase showed normal levels of the mature alkyl-DHAP synthase protein on immunoblots. Immunofluorescence experiments revealed a peroxisomal localization of both the precursor and the mature form of the enzyme. Collectively, these results visualize the peroxisomal localization of alkyl-DHAP synthase, indicate that the enzyme is unstable outside its target organelle and explain that normal enzyme protein levels found in some peroxisomal disorders result from protection against cytoplasmic degradation through import into peroxisomes. Additionally, alkyl-DHAP synthase could be detected in rat mesangial cells and murine NIH-3R3 fibroblasts by immunofluorescence as well as immunoblot analysis. Immunoelectron microscopy showed that the enzyme is predominantly located on the lumenal side of the peroxisomal membrane in rat and guinea pig liver.     

Thermogenic flux induced by lignoceric acid in peroxisomes isolated from HepG2 cells and from X-adrenoleukodystrophy and control fibroblasts

This work analyzes the thermogenic flux induced by the very long-chain fatty acid (VLCFA) lignoceric acid (C24:0) in isolated peroxisomes. Specific metabolic alterations of peroxisomes are related to a variety of disorders, the most frequent one being the neurodegenerative inherited disease X-linked adrenoleukodystrophy (X-ALD). A peroxisomal transport protein is mutated in this disorder. Due to reduced catabolism and enhanced fatty acid (FA) elongation, VLCFA accumulates in plasma and in all tissues, contributing to the clinical manifestations of this disorder. During peroxisomal metabolism, heat is produced but it is considered lost. Instead, it is a form of energy that could play a role in molecular mechanisms of this pathology and other neurodegenerative disorders. The thermogenic flux induced by lignoceric acid (C24:0) was estimated by isothermal titration calorimetry in peroxisomes isolated from HepG2 cells and from fibroblasts obtained from patients with X-ALD and healthy subjects. Heat flux induced by lignoceric acid in HepG2 peroxisomes was exothermic, indicating normal peroxisomal metabolism. In X-ALD peroxisomes the heat flux was endothermic, indicating the requirement of heat/energy, possibly for cellular metabolism. In fibroblasts from healthy subjects, the effect was less pronounced than in HepG2, a kind of cell known to have greater FA metabolism than fibroblasts. Our hypothesis is that heat is not lost but it could act as an activator, for example on the heat-sensitive pathway related to TRVP2 receptors. To investigate this hypothesis we focused on peroxisomal metabolism, considering that impaired heat generation could contribute to the development of peroxisomal neurodegenerative disorders.     

Identification and characterization of HAOX1, HAOX2, and HAOX3, three human peroxisomal 2-hydroxy acid oxidases

Computer-based approaches identified three distinct human 2-hydroxy acid oxidase genes, HAOX1, HAOX2, and HAOX3, that encode proteins with significant sequence similarity to plant glycolate oxidase, a prototypical 2-hydroxy acid oxidase. The products of these genes are targeted to peroxisomes and have 2-hydroxy acid oxidase activities. Each gene displays a distinct tissue-specific pattern of expression, and each enzyme exhibits distinct substrate preferences. HAOX1 is expressed primarily in liver and pancreas and is most active on the two-carbon substrate, glycolate, but is also active on 2-hydroxy fatty acids. HAOX2 is expressed predominantly in liver and kidney and displays highest activity toward 2-hydroxypalmitate. HAOX3 expression was detected only in pancreas, and this enzyme displayed a preference for the medium chain substrate 2-hydroxyoctanoate. These results indicate that all three human 2-hydroxy acid oxidases are involved in the oxidation of 2-hydroxy fatty acids and may also contribute to the general pathway of fatty acid alpha-oxidation. Primary hyperoxaluria type 1 (PH1) is caused by defects in peroxisomal alanine-glyoxylate aminotransferase, the enzyme that normally eliminates intraperoxisomal glyoxylate. The presence of HAOX1 in liver and kidney peroxisomes and the ability of HAOX1 to oxidize glyoxylate to oxalate implicate HAOX1 as a mediator of PH1 pathophysiology.     

Ubiquinone biosynthesis in rat liver peroxisomes

The possibility that ubiquinone biosynthesis is present in rat liver peroxisomes was investigated. The specific activity of trans-prenyltransferase was 30% that of microsomes, with a pH optimum of around 8. trans-Geranyl pyrophosphate was required as a substrate and maximum activity was achieved with Mn(2+). Several detergents specifically inactivated the peroxisomal enzyme. The peroxisomal transferase is present in the luminal soluble contents, in contrast to the microsomal enzyme which is a membrane component. The treatment of rats with a number of drugs has demonstrated that the activities in the two organelles are subjected to separate regulation. Nonaprenyl-4-hydroxybenzoate transferase has about the same specific activity in peroxisomes as in microsomes and like the transferase activity, its regulation differs from the microsomal enzyme. The results demonstrate that peroxisomes are involved in ubiquinone biosynthesis, and at least two enzymes of the biosynthetic sequence are present in this organelle.     

Peroxisomes and peroxisomal functions in muscle. Studies with muscle cells from controls and a patient with the cerebro-hepato-renal (Zellweger) syndrome

In the present study we investigated peroxisomal functions in cultured human muscle cells from control subjects and from a patient with the Zellweger syndrome, a genetic disease characterized by the absence of morphologically distinguishable peroxisomes in liver and kidney. In homogenates of cultured muscle cells from control subjects, catalase is contained within subcellular particles, acyl-CoA:dihydroxyacetonephosphate acyltransferase activity is present and palmitoyl-CoA can be oxidized by a peroxisomal beta-oxidative pathway; these findings are indicative of the presence of peroxisomes in the cells. In homogenates of cultured muscle cells from the patient with the Zellweger syndrome, acyl-CoA:dihydroxyacetonephosphate acyltransferase activity was deficient, peroxisomal beta-oxidation of palmitoyl-CoA was impaired and catalase was not particle-bound. These findings indicate that functional peroxisomes are absent in muscle from patients with the Zellweger syndrome. We conclude that cultured human muscle cells can be used as a model system to study peroxisomal functions in muscle and the consequences for this tissue of a generalized dysfunction of peroxisomes.