Fatty Acid Β-oxidation System Research Articles

Pharmacological and Toxicological Advances in PPAR-Related Medicines

Pharmacological and Toxicological Advances in PPAR-Related Medicines

Fatty Acid Accumulation and Resulting PPARαActivation in Fibroblasts due to Trifunctional Protein Deficiency

To examine fatty acid accumulation and its toxic effects in cells, we analyzed skin fibroblasts from six patients with mitochondrial trifunctional protein deficiency, who had abnormalities in the second through fourth reactions in fatty acid β-oxidation system. We found free fatty acid accumulation, enhanced three acyl-CoA dehydrogenases, catalyzing the first reaction in the β-oxidation system and being assumed to have normal activities in these patients, and PPARα activation that was confirmed in the experiments using MK886, a PPARα specific antagonist and fenofibrate, a PPARα specific agonist. These novel findings suggest that the fatty acid accumulation and the resulting PPARα activation are major causes of the increase in the β-oxidation ability as probable compensation for fatty acid metabolism in the patients' fibroblasts, and that enhanced cell proliferation and increased oxidative stress due to the PPARα activation relate to the development of specific clinical features such as hypertrophic cardiomyopathy, slight hepatomegaly, and skeletal myopathy. Additionally, significant suppression of the PPARα activation by means of MK886 treatment is assumed to provide a new method of treating this deficiency.

Sustained activation of PPARα by endogenous ligands increases hepatic fatty acid oxidation and prevents obesity in ob/ob mice

Obesity, a major health concern, results from an imbalance between energy intake and expenditure. Leptin-deficient ob/ob mice are paradigmatic of obesity, resulting from excess energy intake and storage. Mice lacking acyl-CoA oxidase 1 (Acox1), the first enzyme of the peroxisomal fatty acid β-oxidation system, are characterized by increased energy expenditure and a lean body phenotype caused by sustained activation of peroxisome proliferator-activated receptor α (PPARα) by endogenous ligands in liver that remain unmetabolized in the absence of Acox1. We generated ob/ob mice deficient in Acox1 (Acox1(-/-)) to determine how the activation of PPARα by endogenous ligands might affect the obesity of ob/ob mice. In contrast to Acox1(-/-) (14.3±1.2 g at 6 mo) and the Acox1-deficient (ob/ob) double-mutant mice (23.8±4.6 g at 6 mo), the ob/ob mice are severely obese (54.3±3.2 g at 6 mo) and had significantly more (P<0.01) epididymal fat content. The resistance of Acox1(-/-)/ob/ob mice to obesity is due to increased PPARα-mediated up-regulation of genes involved in fatty acid oxidation in liver. Activation of PPARα in Acox1-deficient ob/ob mice also reduces serum glucose and insulin (P<0.05) and improves glucose tolerance and insulin sensitivity. Further, PPARα activation reduces hepatic steatosis and increases hepatocellular regenerative response in Acox1(-/-)/ob/ob mice at a more accelerated pace than in mice lacking only Acox1. However, Acox1(-/-)/ob/ob mice manifest hepatic endoplasmic reticulum (ER) stress and also develop hepatocellular carcinomas (8 of 8 mice) similar to those observed in Acox1(-/-) mice (10 of 10 mice), but unlike in ob/ob (0 of 14 mice) and OB/OB (0 of 6 mice) mice, suggesting that superimposed ER stress and PPARα activation contribute to carcinogenesis in a fatty liver. Finally, absence of Acox1 in ob/ob mice can impart resistance to high-fat diet (60% fat)-induced obesity, and their liver had significantly (P<0.01) more cell proliferation. These studies with Acox1(-/-)/ob/ob mice indicate that sustained activation of lipid-sensing nuclear receptor PPARα attenuates obesity and restores glucose homeostasis by ameliorating insulin resistance but increases the risk for liver cancer development, in part related to excess energy combustion.

Reduction of liver fructokinase expression and improved hepatic inflammation and metabolism in liquid fructose-fed rats after atorvastatin treatment

Consumption of beverages that contain fructose favors the increasing prevalence of metabolic syndrome alterations in humans, including non-alcoholic fatty liver disease (NAFLD). Although the only effective treatment for NAFLD is caloric restriction and weight loss, existing data show that atorvastatin, a hydroxymethyl-glutaryl-CoA reductase inhibitor, can be used safely in patients with NAFLD and improves hepatic histology. To gain further insight into the molecular mechanisms of atorvastatin's therapeutic effect on NAFLD, we used an experimental model that mimics human consumption of fructose-sweetened beverages. Control, fructose (10% w/v solution) and fructose + atorvastatin (30 mg/kg/day) Sprague–Dawley rats were sacrificed after 14 days. Plasma and liver tissue samples were obtained to determine plasma analytes, liver histology, and the expression of liver proteins that are related to fatty acid synthesis and catabolism, and inflammatory processes. Fructose supplementation induced hypertriglyceridemia and hyperleptinemia, hepatic steatosis and necroinflammation, increased the expression of genes related to fatty acid synthesis and decreased fatty acid β-oxidation activity. Atorvastatin treatment completely abolished histological signs of necroinflammation, reducing the hepatic expression of metallothionein-1 and nuclear factor kappa B binding. Furthermore, atorvastatin reduced plasma (× 0.74) and liver triglyceride (× 0.62) concentrations, decreased the liver expression of carbohydrate response element binding protein transcription factor (×0.45) and its target genes, and increased the hepatic activity of the fatty acid β-oxidation system (× 1.15). These effects may be related to the fact that atorvastatin decreased the expression of fructokinase (× 0.6) in livers of fructose-supplemented rats, reducing the metabolic burden on the liver that is imposed by continuous fructose ingestion.

Immunohistochemical localization of mitochondrial fatty acid β-oxidation enzymes in Müller cells of the retina

The presence of a mitochondrial fatty acid β-oxidation system in the retina was shown by immunohistochemistry. Fatty acids are considered to serve as a major energy source metabolized by fatty acid β-oxidation together with glucose metabolized by glycolysis in the organs of the entire body, but almost nothing is known about this metabolic system in the retina. Adult rat retinae were subjected to immunofluorescence and immuno-electron microscopy for the localization of fatty acid β-oxidation enzymes, together with western blot analysis for quantitation of the amount of enzyme proteins and DNA microarray analysis for gene expression. All the enzymes examined were shown to be present in the retina, but in small amounts, with the amount of protein and gene expression in the retina being about 1/10 of those in the liver. Immunohistochemistry at light and electron microscopic levels revealed the enzymes to be more preferentially localized to the mitochondria of Müller cells than the retinal neurons. The Müller cells were isolated from the retina and confirmed for the presence of mitochondrial fatty acid β-oxidation enzymes. A mitochondrial fatty acid β-oxidation system was thus shown to be present in the retina heterogeneously.

A Novel Fluorescent Sensor Protein for Visualization of Redox States in the Cytoplasm and in Peroxisomes

Reactive oxygen species are generated within peroxisomes during peroxisomal metabolism. However, due to technological difficulties, the intraperoxisomal redox state remain elusive, and the effect of peroxisome deficiency on the intracellular redox state is controversial. A newly developed, genetically encoded fluorescence resonance energy transfer (FRET) probe, Redoxfluor, senses the physiological redox state via its internal disulfide bonds, resulting in a change in the conformation of the protein leading to a FRET response. We made use of Redoxfluor to measure the redox states at the subcellular level in yeast and Chinese hamster ovary (CHO) cells. In wild-type peroxisomes harboring an intact fatty acid beta-oxidation system, the redox state within the peroxisomes was more reductive than that in the cytosol, despite the fact that reactive oxygen species were generated within the peroxisomes. Interestingly, we observed that the redox state of the cytosol of cell mutants for peroxisome assembly, regarded as models for a neurological metabolic disorder, was more reductive than that of the wild-type cells in yeast and CHO cells. Furthermore, Redoxfluor was utilized to develop an efficient system for the screening of drugs that moderate the abnormal cytosolic redox state in the mutant CHO cell lines for peroxisome assembly without affecting the redox state of normal cells.

Effect of a Large Dose of Di (2-ethylhexyl) phthalate (DEHP) on Hepatic Peroxisome in Cynomolgus Monkeys (Macaca Fascicularis)

To elucidate the effect of a large dose of di (2-ethylhexyl) phthalate (DEHP), a plasticizer and peroxisome proliferator-activated receptor-α (PPARα) agonist, on hepatic peroxisomes, we orally administered 1,000 mg/kg/day, once daily, to 3 male and 4 female cynomolgus monkeys for 28 days consecutively. Light-microscopic and electron microscopic examinations of the liver were carried out in conjunction with measurement of the hepatic fatty acid β-oxidation system (FAOS), carnitine acetyltransferase (CAT) and carnitine palmitoyltransferase (CPT) activities, which are peroxisomal and/or mitochondrial enzyme activities. Electron microscopically, enlargement of the mitochondria was observed with lamellar orientation of the cristae along the major axis. Although the number of peroxisomes showed a tendency to increase when compared with those in a biopsied specimen before treatment, no abnormality in morphology was observed. A slight increase in CPT activity was noted at termination. No changes were noted in hepatic FAOS or CAT activity. In conclusion, although repeated oral treatment of cynomolgus monkeys with a large dose of DEHP induced a subtle increase in the numbers of peroxisomes with slight enlargements of the mitochondria, this low-sensitivity response to peroxisome proliferators in cynomolgus monkeys was considered to be closer to the response in humans than that in rodents.

Immunohistochemical Localization of Mitochondrial Fatty Acid β-Oxidation Enzymes in Rat Testis

The testis consists of two types of tissues, the interstitial tissue and the seminiferous tubule, which have different functions and are assumed to have different nutritional metabolism. The localization of enzymes of the mitochondrial fatty acid β-oxidation system in the testis was investigated to obtain a better understanding of nutrient metabolism in the testis. Adult rat testis tissues were subjected to immunoblot analysis for quantitation of the amounts of enzyme proteins, to DNA microarray analysis for gene expression, and to immunofluorescence and immunoelectron microscopy for localization. Quantitative analysis by immunoblot and DNA microarray revealed that enzymes occur abundantly in Leydig cells in the interstitial tissue but much less so in the seminiferous tubules. Immunohistochemistry revealed that Leydig cells in the interstitial tissue and Sertoli cells in the seminiferous tubules contain a full set of mitochondrial fatty acid β-oxidation enzymes in relatively plentiful amounts among the cells in the testis, but that this is not so in spermatogenic cells. This characteristic localization of the mitochondrial fatty acid β-oxidation system in the testis needs further elucidation in terms of a possible role for it in the nutritional metabolism of spermatogenesis.

Activation of lipid catabolism by the water-soluble fraction of petroleum in the crustacean Macrobrachium borellii

Little is known about the effect of the water-soluble fraction of crude oil (WSF) on lipid metabolism in invertebrates. The effect of the WSF on the triacylglycerol (TAG) mobilization, fatty acid activation and degradation was evaluated in the decapod Macrobrachium borellii, exposing adult and eggs at different stages of development for 7 days to a sublethal concentration of WSF. Using radioactive tracers, mitochondrial palmitoyl-CoA synthetase (ACS), triacylglycerol lipase (TAG-lipase) and fatty acid β-oxidation system activities were assayed. Before studying the effect of WSF, the kinetic parameters of ACS were determined in purified mitochondria. Its optimal temperature and pH were 32 °C and 8.0, respectively, the apparent K m 2.48 μmol l −1, and its V max of 1.93 nmol min −1 mg protein −1. These kinetic parameters differed significantly from this shrimp's microsomal isoform. After 7 days exposure to a sublethal concentration of WSF (0.6 mg/l), changes were observed in the enzymatic activity of all enzymes or enzymatic system assayed in adult midgut gland as well as in stage 5 eggs, a period of active organogenesis. An increase in the mobilization of energy stores was detected as early as stage 4, where TAG-lipase activity increased by 27% in exposed eggs. The increase was even more marked in exposed eggs at stage 5 where a three-fold rise (154%) was determined. Exposed adult shrimp also showed an augmented lipase activity by 38%. Fatty acid β-oxidation increased by 51.0 and 35.5% in midgut gland and eggs at stage 5, respectively, but no changes were observed at less-developed stages. Mitochondrial fatty acid activation by ACS also increased in adults and stage 5 eggs by 7.4 and 52.0%, respectively. A similar response of the lipid catabolic pathways to WSF contamination in both adult and eggs, suggests that the exposure to this pollutant causes an increase in the energy needs of this shrimp. When validated by field studies, these catabolic enzymes could be employed as early pollution biomarkers.

Conjugated docosahexaenoic acid inhibits lipid accumulation in rats

Conjugated linoleic acid (CLA), which contains a conjugated double-bond system, and n-3 highly unsaturated fatty acids such as docosahexaenoic acid (DHA) are widely known to improve lipid metabolism. To examine the possibility that a fatty acid with a combination of these structural features might have stronger physiological effects, we prepared conjugated DHA (CDHA) by alkaline isomerization of DHA and examined its effects on lipid and sugar metabolism in rats. Rats were force fed with 200 mg of test oils [linoleic acid (LA), DHA, CLA or CDHA] everyday for 4 weeks. Compared with the animals from the other groups, those in the CDHA group showed a significant weight loss in white adipose tissue (57% of adipose tissue weight in the LA group) and significant decreases in the levels of liver triacylglycerol (TG; 65% of TG level in the LA group) as well as total cholesterol (TC; 88% of TC level in the LA group), indicating suppression of lipid accumulation in the liver and adipose tissue. In addition, plasma TG and TC levels significantly decreased (69% of TG level and 82% of TC level in the LA group), indicating improved lipid metabolism. In the liver, the fatty acid synthesis system was inhibited and the fatty acid β-oxidation system was activated, whereas the free fatty acid, glucose and tumor necrosis factor α levels in the plasma were lowered following CDHA administration. Hence, intake of CDHA appears to suppress the accumulation of fat in the liver and epididymal adipose tissue and improves lipid and sugar metabolism in rats.

Peroxisomal and Mitochondrial Oxidation of Fatty Acids in the Heart, Assessed from the 13C Labeling of Malonyl-CoA and the Acetyl Moiety of Citrate

We previously showed that a fraction of the acetyls used to synthesize malonyl-CoA in rat heart derives from partial peroxisomal oxidation of very long and long-chain fatty acids. The 13C labeling ratio (malonyl-CoA)/(acetyl moiety of citrate) was >1.0 with 13C-fatty acids, which yields [13C]acetyl-CoA in both mitochondria and peroxisomes and < 1.0 with substrates, which yields [13C]acetyl-CoA only in mitochondria. In this study, we tested the influence of 13C-fatty acid concentration and chain length on the labeling of acetyl-CoA formed in mitochondria and/or peroxisomes. Hearts were perfused with increasing concentrations of labeled docosanoate, oleate, octanoate, hexanoate, butyrate, acetate, or dodecanedioate. In contrast to the liver, peroxisomal oxidation of 1-13C-fatty acids in heart does not form [1-13C]acetate. With [1-13C]docosanoate and [1,12-13C2]dodecanedioate, malonyl-CoA enrichment plateaued at 11 and 9%, respectively, with no detectable labeling of the acetyl moiety of citrate. Thus, in the intact rat heart, docosanoate and dodecanedioate appear to be oxidized only in peroxisomes. With [1-13C]oleate or [1-13C]octanoate, the labeling ratio >1 indicates the partial peroxisomal oxidation of oleate and octanoate. In contrast, with [3-13C]octanoate, [1-13C]hexanoate, [1-13C]butyrate, or [1,2-13C2]acetate, the labeling ratio was <0.7 at all concentrations. Therefore, in rat heart, (i) n-fatty acids shorter than 8 carbons do not undergo peroxisomal oxidation, (ii) octanoate undergoes only one cycle of peroxisomal beta-oxidation, (iii) there is no detectable transfer to the mitochondria of acetyl-CoA from the cytosol or the peroxisomes, and (iv) the capacity of C2-C18 fatty acids to generate mitochondrial acetyl-CoA decreases with chain length.

Up-regulation of the peroxisomal β-oxidation system occurs in butyrate-grown Candida tropicalis following disruption of the gene encoding peroxisomal 3-ketoacyl-CoA thiolase

In the yeast Candida tropicalis, two thiolase isozymes, peroxisomal acetoacetyl-CoA thiolase and peroxisomal 3-ketoacyl-CoA thiolase, participate in the peroxisomal fatty acid β-oxidation system. Their individual contributions have been demonstrated in cells grown on butyrate, with C. tropicalis able to grow in the absence of either one. In the present study, a lack of peroxisomal 3-ketoacyl-CoA thiolase protein resulted in increased expression (up-regulation) of acetoacetyl-CoA thiolase and other peroxisomal proteins, whereas a lack of peroxisomal acetoacetyl-CoA thiolase produced no corresponding effect. Overexpression of the acetoacetyl-CoA thiolase gene did not suppress the up-regulation or the growth retardation on butyrate in cells without peroxisomal 3-ketoacyl-CoA thiolase, even though large amounts of the overexpressed acetoacetyl-CoA thiolase were detected in most of the peroxisomes of butyrate-grown cells. These results provide important evidence of the greater contribution of 3-ketoacyl-CoA thiolase to the peroxisomal β-oxidation system than acetoacetyl-CoA thiolase in C. tropicalis and a novel insight into the regulation of the peroxisomal β-oxidation system.

Hydrogen peroxide generation in peroxisome proliferator-induced oncogenesis

Peroxisome proliferators are a structurally diverse group of non-genotoxic chemicals that induce predictable pleiotropic responses including the development of liver tumors in rats and mice. These chemicals interact variably with peroxisome proliferator-activated receptors (PPARs), which are members of the nuclear receptor superfamily. Evidence derived from mice with PPARα gene disruption indicates that of the three PPAR isoforms (α, β/δ and γ), the isoform PPARα is essential for the pleiotropic responses induced by peroxisome proliferators. Peroxisome proliferator-induced activation of PPARα leads to profound transcriptional activation of genes encoding for the classical peroxisomal β-oxidation system and cytochrome P450 CYP 4A isoforms, CYP4A1 and CYP4A3, among others. Livers with peroxisome proliferation manifest substantial increases in the expression of H 2O 2-generating peroxisomal fatty acyl-CoA oxidase, the first enzyme of the classical peroxisomal fatty acid β-oxidation system, and of microsomal cytochrome P450 4A1 and 4A3 genes. Disproportionate increases in H 2O 2-generating enzymes and H 2O 2-degrading enzyme catalase and reductions in glutathione peroxidase activity by peroxisome proliferators, lead to increased oxidative stress in liver cells. Sustained oxidative stress resulting from chronic increases in H 2O 2-generating enzymes manifests as massive accumulation of lipofuscin in hepatocytes, and increased levels of 8-hydroxydeoxyguanosine adducts in liver DNA; this supports the hypothesis that oxidative stress plays a critical role in the development of liver tumors induced by these non-genotoxic chemical carcinogens. Evidence also indicates that cells stably overexpressing H 2O 2-generating fatty acyl-CoA oxidase or urate oxidase, when exposed to appropriate substrate(s), reveal features of neoplastic conversion including growth in soft agar and formation of tumors in nude mice. Mice with disrupted fatty acyl-CoA oxidase gene (AOX −/− mice), which encodes the first enzyme of the PPARα regulated peroxisomal β-oxidation system, exhibit profound spontaneous peroxisome proliferation, including development of liver tumors, indicative of sustained activation of PPARα by the unmetabolized substrates of acyl-CoA oxidase. With the exception of fatty acyl-CoA oxidase, all PPARα responsive genes including CYP4A1 and CYP4A3 are up-regulated in the livers of these AOX −/− mice. Thus, the substrates of acyl-CoA oxidase serve as endogenous ligands for this receptor leading to a receptor–enzyme cross-talk, because acyl-CoA oxidase gene is transcriptionally regulated by PPARα. Peroxisome proliferators induce only a transient increase in liver cell proliferation and this may serve as an additional contributory factor, rather than play a primary role in liver tumor development. Thus, sustained activation of PPARα by either synthetic or natural ligands leads to reproducible pleiotropic responses culminating in the development of liver tumors. This phenomenon of peroxisome proliferation provides fascinating challenges in exploring the molecular mechanisms of cell specific transcription, and in identifying the PPARα responsive target genes, as well as events involved in their regulation. Genetically altered animals and cell lines should enable investigations on the role of H 2O 2-producing enzymes in neoplastic conversion.

Peroxisomal Acyl-Coenzyme A Oxidase Is a Rate-Limiting Enzyme in a Very Long-Chain Fatty Acid β-Oxidation System

The contents of peroxisomal fatty acid β-oxidation enzymes in three rat hepatoma cell lines, i.e., H4IIEC3 (H4), N1S1, and McA-RH7777 (H7), were measured by immunoblot analysis, and a significant difference in acyl-coenzyme A oxidase (AOX) content became evident. These cell lines were respectively infected with a recombinant virus to express significant amounts of AOX protein. The expressed AOX mainly localized in organelle, supposing peroxisomes, and was catalytically active. The cDNA-expression in H4, N1S1, and H7 cells enhanced 2.6-, 2.2-, and 1.0-fold β-oxidation activity of lignoceric acid, respectively. The enhancement in H4 and NISI cells suggests that AOX is a rate-limiting enzyme in the very-long-chain fatty acid β-oxidation system, in these cell lines.

Comparison of molecular structures and regulation of biosynthesis of unique thiolase isozymes localized only in peroxisomes of n-alkane-utilizable yeast, Candida tropicalis

Two transcribed genes encoding 3-ketoacyl-CoA thiolase [(EC 2.3.1.16) Thiolase III], one of the peroxisomal thiolase isozymes, of n-alkane-utilizable yeast, Candida tropicalis, were isolated. Restriction maps of these two genes were very similar. Each of the genes had only one open reading frame consisting of 1224 bp corresponding to 408 amino acid residues. The nucleotide sequences of another thiolase isozyme in peroxisomes of C. tropicalis, acetoacetyl-CoA thiolase (Thiolase IA and IB), have been already determined (Kurihara et al., Eur. J. Biochem., 210, 999–1005, 1992). The amino acid sequences of these peroxisomal thiolase isozymes (Thiolases I and III) showed 35% homology. The regulation of biosynthesis of these thiolases in C. tropicalis was compared by RNA and Western blotting analyses. The results revealed that the biosynthesis of Thiolase III in C. tropicalis was strongly induced in a medium containing butyrate or alkanes as a carbon source and a peroxisome proliferator, while Thiolase I was constitutively expressed even in a medium with glucose or acetate, although a slight induction was observed at the levels of transcription and translation in the medium containing butyrate or alkanes. Thus, the regulations of biosyntheses of Thiolases I and III were found to be quite different, in spite of the enzymes in the final step of the peroxisomal fatty acid β-oxidation system.

Microbial physiology of sidechain degradation of sterols

A large number of valuable starting materials for steroids synthesis (e.g. 4-androstene-3,17-dione, 1,4-androstadiene-3,17-dione, 9α-hydroxy-4-androsten-17-one) have been produced by microbial transformation methods. This review helps to evaluate the microbial physiological interest of the widely used sterol sidechain degradation processes. Four inducible groups of the catabolic enzymes are involved in the sterol sidechain degradation pathway; the fatty acid β-oxidation system, the ω-oxidase reaction, a methyl-crotonyl-CoA carboxylation system and the propionyl-CoA carboylase system. Altogether nine catabolic enzymes are involved in the β-sitosterol sidechain degradation pathway. They work in 14 consecutive enzymatic steps. Summing up: three molecules of FADH2, three molecules of propionyl-SCoA, three of NADH and one molecule of acetic acid are formed, while the sidechain of one mole of sitosterol is removed selectively. The metabolism of the propionates and the acetate yield 18 molecules of NADH and 7 molecules of FADH2. Taking into consideration the whole process more than 80 molecules of ATP could be formed during the sitosterol sidechain degradation process.

Proliferation of peroxisomes without simultaneous induction of the peroxisomal fatty acid βoxidation

Marked proliferation of rat hepatic peroxisomes is observed after treatment with a new potent hypolipidemic drug BM 15766, as well as after bezafibrate. Whereas the relative specific activity of the peroxisomal fatty acid βoxidation system is not affected by BM 15766 it is significantly increased after bezafibrate. This is also confirmed by immunoblot analysis of individual β-oxidation enzymes in highly purified peroxisome fractions. These observations suggest that peroxisome proliferation and the induction of the fatty acid β-oxidation are regulated separately.

Characterization of peroxisomes in an n-alkane-utilizable yeast, Candida tropicalis, grown on glucose and propionate

Two types of peroxisomes (microbodies) in different states of development, which are attractive for investigating the biogenesis of the organelles, were first isolated from the glucose- and propionate-grown cells of an n-alkane-utilizable yeast, Candida tropicalis pK 233, and their properties were disclosed. Peroxisomes isolated from the glucose-grown cells contained high levels of isocitrate lyase and malate synthase, whereas the levels of catalase and enzymes constituting the fatty acid β-oxidation system were demonstrated to be very low by means of subcellular fractionation and Western blot analysis. Citrate synthase common to the tricarboxylic acid and glyoxylate cycles was localized only in mitochondria. We have proposed that the peroxisomes in the glucose-grown cells are called ‘pro-peroxisomes’ or ‘prooxisomes’ to distinguish them from the ‘mature’ organelles in the alkane-grown cells which have high levels of catalase and the β-oxidation enzymes. The peroxisomes isolated from the propionate-grown cells exhibited higher levels of isocitrate lyase and malate synthase together with moderate levels of catalase and acyl-CoA oxidase inducibly synthesized in the cells. That is, the peroxisomes in the propionate-grown cells showed characteristics of both ‘prooxisomes’ and ‘mature’ peroxisomes, and were supposed to be an intermediate form deficient in some of the developmental processes.

Co-induction of microsomal cytochrome P-452 and the peroxisomal fatty acid β-oxidation pathway in the rat by clofibrate and di-(2-ethylhexyl)phthalate: Dose-response studies

Male Wistar rats have been pretreated with either clofibrate or diethylhexylphthalate and the dose-dependency of induction of the microsomal, cytochrome P-452-driven fatty acid hydroxylase and peroxisomal fatty acid β-oxidation system investigated. Both clofibrate and DEHP specifically induced (approximately 10-fold) the 12-hydroxylation of lauric acid in a dose-dependent manner and only marginally increased the associated 11-hydroxylase activity. This dose-dependent increase in fatty acid hydroxylase activity was accompanied by a similar ten-fold increase in the specific content of the cytochrome P-452 isoenzyme responsible for this activity, as assessed by an immunochemical-based ELISA method. Similarly, both clofibrate and DEHP induced the peroxisomal fatty acid β-oxidation pathway in a dose-dependent manner. Furthermore, our results provide evidence that, after oral administration, clofibrate has a higher in vivo potency in inducing the above enzymes of fatty acid metabolism than is exhibited by DEHP. A correlation matrix analysis of the above data indicated a close association between the induction of microsomal cytochrome P-452 (and its associated fatty acid hydroxylase activity) and peroxisomal β-oxidation enzymes, implicating a mechanistic inter-relationship between changes in fatty acid metabolising enzymes in these two hepatic subcellular organelles.

Occurrence and possible roles of acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase in peroxisomes of an n-alkane-grown yeast, Candida tropicalis

Two kinds of 3-ketoacyl-CoA thiolases were found in the peroxisomes of Candida tropicalis cells grown on n-alkanes (C 10C 13). One was a typical acetoacetyl-CoA thiolase specific only to acetoacetyl-CoA, while another was 3-ketoacyl-CoA thiolase showing high activities on the longer chain substrates. A high level of the latter thiolase activity in alkane-grown cells was similar to that of other enzymes constituting the fatty acid β-oxidation system in yeast peroxisomes. These facts suggest that the complete degradation of fatty acids to acetyl-CoA is carried out in yeast peroxisomes by the cooperative contribution of acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase.