The role of peroxisomal fatty acyl-CoA beta-oxidation in bile acid biosynthesis

The physiological role of the peroxisomal fatty acyl-CoA beta-oxidizing system (FAOS) is not yet established. We speculated that there might be a relationship between peroxisomal degradation of long-chain fatty acids in the liver and the biosynthesis of bile acids. This was investigated using [1-14C]butyric acid and [1-14C]lignoceric acid as substrates of FAOS in mitochondria and peroxisomes, respectively. The incorporation of [14C]lignoceric acid into primary bile acids was approximately four times higher than that of [14C]butyric acid (in terms of C-2 units). The pools of these two fatty acids in the liver were exceedingly small. The incorporations of radioactivity into the primary bile acids were strongly inhibited by administration of aminotriazole, which is a specific inhibitor of peroxisomal FAOS in vivo [F. Hashimoto and H. Hayashi (1987) Biochim. Biophys. Acta 921, 142-150]. Aminotriazole inhibited preferentially the formation of cholate, the major primary bile acid, from both [14C]lignoceric acid and [14C]butyric acid, rather than the formation of chenodeoxycholate. The former inhibition was about 70% and the latter was approximately 40-50%. In view of reports that cholate is biosynthesized from endogenous cholesterol, the above results indicate that peroxisomal FAOS may have an anabolic function, supplying acetyl CoA for bile acid biosynthesis.     

Role of peroxisomal fatty acyl-CoA beta-oxidation in phospholipid biosynthesis

We have already reported that peroxisomal beta-oxidation has an anabolic function, supplying acetyl-CoA for bile acid biosynthesis [H. Hayashi and A. Miwa, 1989, Arch. Biochem. Biophys. 274, 582-589]. The anabolic significance of peroxisomal beta-oxidation was further investigated in the present study by using clofibrate, a peroxisome proliferator, as an experimental tool. Clofibrate suppressed 3-hydroxymethylglutaryl-CoA reductase activity (the key enzyme of cholesterol synthesis) and enhanced fatty acyl-CoA oxidase activity (the rate-limiting enzyme of beta-oxidation). Rats were fed a chow containing 0.25% clofibrate for 2 weeks, and then a bile duct fistula was implanted. [1-14C]lignoceric acid, which is degraded exclusively by peroxisomal FAOS, was injected into the rats 24 h after the operation. By this time, the secondary bile acids and pooled cholesterol which would normally be secreted into the bile are considered to have been exhausted from the liver. Clofibrate significantly decreased the incorporations of radioactivity into biliary bile acid (40% of the control) and cholesterol (50%), but did not affect biliary lipid contents. [14C]Acetyl-CoA formed by peroxisomal beta-oxidation of [1-14C]lignoceric acid was preferentially utilized for syntheses of long-chain fatty acids and phospholipids rather than synthesis of cholesterol or triglyceride. The radioactivities incorporated into the former two lipids were increased 2-fold over the control by administration of clofibrate, while the incorporation into triglyceride was decreased to approximately half. In particular, the incorporation into phosphatidylethanolamine was increased as much as 3.5-fold over the control. The contents of these lipids in the liver were not affected by clofibrate. The results suggest that peroxisomal beta-oxidation plays an important role in the biosynthesis of functional lipids such as phospholipids (this work), in addition to bile acids and cholesterol (previous report) by supplying acetyl-CoA.     

Characterization of an acyl-coA thioesterase that functions as a major regulator of peroxisomal lipid metabolism.

Peroxisomes function in beta-oxidation of very long and long-chain fatty acids, dicarboxylic fatty acids, bile acid intermediates, prostaglandins, leukotrienes, thromboxanes, pristanic acid, and xenobiotic carboxylic acids. These lipids are mainly chain-shortened for excretion as the carboxylic acids or transported to mitochondria for further metabolism. Several of these carboxylic acids are slowly oxidized and may therefore sequester coenzyme A (CoASH). To prevent CoASH sequestration and to facilitate excretion of chain-shortened carboxylic acids, acyl-CoA thioesterases, which catalyze the hydrolysis of acyl-CoAs to the free acid and CoASH, may play important roles. Here we have cloned and characterized a peroxisomal acyl-CoA thioesterase from mouse, named PTE-2 (peroxisomal acyl-CoA thioesterase 2). PTE-2 is ubiquitously expressed and induced at mRNA level by treatment with the peroxisome proliferator WY-14,643 and fasting. Induction seen by these treatments was dependent on the peroxisome proliferator-activated receptor alpha. Recombinant PTE-2 showed a broad chain length specificity with acyl-CoAs from short- and medium-, to long-chain acyl-CoAs, and other substrates including trihydroxycoprostanoyl-CoA, hydroxymethylglutaryl-CoA, and branched chain acyl-CoAs, all of which are present in peroxisomes. Highest activities were found with the CoA esters of primary bile acids choloyl-CoA and chenodeoxycholoyl-CoA as substrates. PTE-2 activity is inhibited by free CoASH, suggesting that intraperoxisomal free CoASH levels regulate the activity of this enzyme. The acyl-CoA specificity of recombinant PTE-2 closely resembles that of purified mouse liver peroxisomes, suggesting that PTE-2 is the major acyl-CoA thioesterase in peroxisomes. Addition of recombinant PTE-2 to incubations containing isolated mouse liver peroxisomes strongly inhibited bile acid-CoA:amino acid N-acyltransferase activity, suggesting that this thioesterase can interfere with CoASH-dependent pathways. We propose that PTE-2 functions as a key regulator of peroxisomal lipid metabolism.     

Presence and features of fatty acyl-CoA binding activity in rat hepatic peroxisomes

We studied the fatty acyl-CoA binding activity of rat liver peroxisomes. After subcellular fractionation of rat liver treated with or without clofibrate, a peroxisome proliferator, the binding activity with [1-(14)C]palmitoyl-CoA was detected in the light mitochondrial fraction in addition to the mitochondrial and cytosol fractions. After Nycodenz centrifugation of the light mitochondrial fraction, the binding activity was detected in peroxisomes. The peroxisomal activity depended on the incubation temperature and peroxisome concentration. The activity also depended on the concentration of 2-mercaptoethanol, and a plateau of activity was unexpectedly found at 2-mercaptoethanol concentrations from 20 to 40 mM. Clofibrate increased the total and specific activity of the fatty acyl-CoA binding of peroxisomes by 7.9 and 2.5 times compared with the control, respectively. In the presence of 20% glycerol at 0 degree C, approximately 90% of the binding activity was maintained for up to at least 3 wk. After successive treatment with an ultramembrane Amicon YM series, about 70% of the binding activity was detected in the M.W. 30,000-100,000 fraction. When the M.W. 30,000-100,000 fraction was added to the incubation mixture of the peroxisomal fatty acyl-CoA beta-oxidation system, a slight increase in the beta-oxidation activity was found. 2-Mercaptoethanol (20 mM) significantly activated the fatty acyl-CoA beta-oxidation system to 1.4 times control. After gel filtration of the M.W. 30,000-100,000 fraction, the peaks of fatty acyl-CoA binding protein showed broad elution profiles from 45,000 to 75,000. These results suggest that fatty acyl-CoA binding activity can be detected directly in peroxisomes and is increased by peroxisome proliferators. The high binding activity in the presence of higher concentrations of 2-mercaptoethanol indicates the importance of the SH group for binding. The apparent molecular weight of the binding protein may be from 45,000 to 75,000.     

Mitochondrial and peroxisomal beta oxidation of the branched chain fatty acid 2-methylpalmitate in rat liver

A number of isoprenoids (e.g. pristanic acid and the side chains of fat soluble-vitamins) is degraded or shortened via beta oxidation. We synthesized 2-methyl-palmitate and 2-methyl[1-14C] palmitate as a model substrate for the study of the beta oxidation of branched (isoprenoid) fatty acids in rat liver. 2-Methylpalmitate was well oxidized by isolated hepatocytes and its oxidation was stimulated after treatment of the animals with a peroxisome proliferator. Subcellular fractionation of rat liver demonstrated that 2-methylpalmitate is activated to its CoA ester in endoplasmic reticulum, mitochondria, and peroxisomes and that mitochondria and peroxisomes are capable of beta-oxidizing 2-methylpalmitate. At low unbound 2-methylpalmitate concentrations and in the presence of competing straight chain fatty acids, a condition encountered in vivo, peroxisomal 2-methyl-palmitate oxidation was 2- to 4-fold more active than mitochondrial oxidation. Treatment of rats with a peroxisome proliferator markedly stimulated mitochondrial but only slightly peroxisomal 2-methylpalmitate oxidation. The same treatment dramatically induced palmitoyl-CoA oxidase but did not change 2-methyl-palmitoyl-CoA oxidase activity. Our results indicate 1) that in untreated rats peroxisomes contribute for an important part to the oxidation of 2-methylpalmitate; 2) that treatment with a peroxisome proliferator stimulates mainly the mitochondrial component of 2-methylpalmitate oxidation; and 3) that palmitoyl-CoA and 2-methylpalmitoyl-CoA are oxidized by different peroxisomal oxidases.     

Protein organization in mouse liver peroxisomes.

Peroxisomes from mouse liver were fractionated with Triton X-114, a procedure which yields a detergent phase consisting of proteins containing hydrophobic binding sites, and a nondetergent, or aqueous, phase containing hydrophilic proteins. When this method was applied to peroxisomes from control mice, catalase and fatty acyl-CoA oxidase distributed to the aqueous phase, whereas the integral membrane protein, PMP68, and the bifunctional protein were recovered exclusively in the detergent phase. Urate oxidase distributed intermediate between these two phases. With peroxisomes from mice treated with the peroxisome proliferator clofibrate, the bifunctional protein was recovered in both the detergent and the aqueous phases, and urate oxidase was shifted toward the aqueous phase. Other analyses of the subperoxisomal distribution of the bifunctional protein were consistent with a proportion of this protein being tightly associated with the peroxisomal membrane, or with some other uncharacterized, poorly soluble, component. Sucrose gradient centrifugation of the aqueous phase resulting from Triton X-114 fractionation of peroxisomes revealed that a major proportion of catalase, fatty acyl-CoA oxidase, the bifunctional protein, and other unidentified proteins behaved as if associated under these conditions. In this respect, use of a higher concentration of Triton X-114 for peroxisome fractionation led to the partitioning of some catalase and fatty acyl-CoA oxidase to the detergent phase, indicating the presence of some detergent-accessible hydrophobic binding sites even on these proteins. These data have been interpreted as indicating matrix protein associations in vivo, associations which may be responsive to proliferator treatment.     

Significance of catalase in peroxisomal fatty acyl-CoA beta-oxidation

Catalase activity was inhibited by aminotriazole administration to rats in order to evaluate the influence of catalase on the peroxisomal fatty acyl-CoA beta-oxidation system. 2 h after the administration of aminotriazole, peroxisomes were prepared from rat liver, and the activities of catalase, the beta-oxidation system and individual enzymes of beta-oxidation (fatty acyl-CoA oxidase, crotonase, beta-hydroxybutyryl-CoA dehydrogenase and thiolase) were determined. Catalase activity was decreased to about 2% of the control. Among the individual enzymes of the beta-oxidation system, thiolase activity was decreased to 67%, but the activities of fatty acyl-CoA oxidase, crotonase and beta-hydroxybutyryl-CoA dehydrogenase were almost unchanged. The activity of the peroxisomal beta-oxidation system was assayed by measuring palmitoyl-CoA-dependent NADH formation, and the activity of the purified peroxisome preparation was found to be almost unaffected by the administration of aminotriazole. The activity of the system in the aminotriazole-treated preparation was, however, significantly decreased to 55% by addition of 0.1 mM H2O2 to the incubation mixture. Hydrogen peroxide (0.1 mM) reduced the thiolase activity of the aminotriazole-treated peroxisomes to approx. 40%, but did not affect the other activities of the system. Thiolase activity of the control preparation was decreased to 70% by addition of hydrogen peroxide (0.1 mM). The half-life of 0.1 mM H2O2 added to the thiolase assay mixture was 2.8 min in the case of aminotriazole-treated peroxisomes, and 4 s in control peroxisomes. The ultraviolet spectrum of acetoacetyl-CoA (substrate of thiolase) was clearly changed by addition of 0.1 mM H2O2 to the thiolase assay mixture without the enzyme preparation; the absorption bands at around 233 nm (possibly due to the thioester bond of acetoacetyl-CoA) and at around 303 nm (due to formation of the enolate ion) were both significantly decreased. These results suggest that H2O2 accumulated in peroxisomes after aminotriazole treatment may modify both thiolase and its substrate, and consequently suppress the fatty acyl-CoA beta-oxidation. Therefore, catalase may protect thiolase and its substrate, 3-ketoacyl-CoA, by removing H2O2, which is abundantly produced during peroxisomal enzyme reactions.     

Acyl-Coenzyme A synthetase and fatty acid oxidation in rat liver peroxisomes.

Rat liver peroxisomes oxidized palmitate in the presence of ATP, CoA and NAD+, and the rate of palmitate oxidation exceeded that of palmitoyl-CoA oxidation. Acyl-CoA synthetase [acid: CoA ligase (AMP-forming); EC 6.2.1.3] was found in peroxisomes. The substrate specificity of the peroxisomal synthetase towards fatty acids with various carbon chain lengths was similar to that of the microsomal enzyme. The peroxisomal synthetase activity toward palmitate (40--100 nmol/min per mg protein) was higher than the rate of palmitate oxidation by the peroxisomal system (0.7--1.7 nmol/min per mg protein). The data show that peroxisomes activate long chain fatty acids and oxidize their acyl-CoA derivatives.     

Developmental changes of bile acid composition and conjugation in L- and D-bifunctional protein single and double knockout mice

Peroxisomal beta-oxidation is an essential step in bile acid synthesis, since it is required for shortening of C27-bile acid intermediates to produce mature C24-bile acids. D-Bifunctional protein (DBP) is responsible for the second and third step of this beta-oxidation process. However, both patients and mice with a DBP deficiency still produce C24-bile acids, although C27-intermediates accumulate. An alternative pathway for bile acid biosynthesis involving the peroxisomal L-bifunctional protein (LBP) has been proposed. We investigated the role of LBP and DBP in bile acid synthesis by analyzing bile acids in bile, liver, and plasma from LBP, DBP, and LBP:DBP double knock-out mice. Bile acid biosynthesis, estimated by the ratio of C27/C24-bile acids, was more severely affected in double knock-out mice as compared with DBP-/- mice but was normal in LBP-/- mice. Unexpectedly, trihydroxycholestanoyl-CoA oxidase was inactive in double knock-out mice due to a peroxisomal import defect, preventing us from drawing any firm conclusion about the potential role of LBP in an alternative bile acid biosynthesis pathway. Interestingly, the immature C27-bile acids in DBP and double knock-out mice remained unconjugated in juvenile mice, whereas they occurred as taurine conjugates after weaning, probably contributing to the minimal weight gain of the mice during the lactation period. This correlated with a marked induction of bile acyl-CoA:amino acid N-acyltransferase expression and enzyme activity between postnatal days 10 and 21, whereas the bile acyl-CoA synthetases increased gradually with age. The nuclear receptors hepatocyte nuclear factor-4alpha, farnesoid X receptor, and peroxisome proliferator receptor alpha did not appear to be involved in the up-regulation of the transferase.     

Immunocytochemical localization of urate oxidase, fatty acyl-CoA oxidase, and catalase in bovine kidney peroxisomes

We investigated the localization of urate oxidase, peroxisomal fatty acyl-CoA oxidase, and catalase in bovine kidney by immunoblot analysis and protein A-gold immunocytochemistry, using the respective polyclonal monospecific antibodies raised against the enzymes purified from rat liver. By immunoblot analysis, these three proteins were detected in bovine kidney and bovine liver homogenates. Subcellular localization of these three enzymes in kidney was ascertained by protein A-gold immunocytochemical staining of Lowicryl K4M-embedded tissue. Peroxisomes in bovine kidney cortical epithelium possessed crystalloid cores or nucleoids, which were found to be the exclusive sites of urate oxidase localization. The limiting membrane, the marginal plate, and the matrix of renal peroxisomes were negative for urate oxidase staining. In contrast, catalase and fatty acyl-CoA oxidase were found in the peroxisome matrix. These results demonstrate that, unlike rat kidney peroxisomes which lack urate oxidase, peroxisomes of bovine kidney contain this enzyme as well as peroxisomal fatty acyl-CoA oxidase.     

Participation of peroxisomes in lipid biosynthesis in the harderian gland of guinea pig.

Peroxisomal enzyme activities in the guinea-pig harderian gland, which has a unique lipid composition, were studied. Activities of catalase, acyl-CoA oxidase and the cyanide-insensitive acyl-CoA beta-oxidation system in this tissue were comparable with those in rat liver. The activities of dihydroxyacetone phosphate acyltransferase (DHAPAT, EC 2.3.1.42) and alkyl-DHAP synthase (EC 2.5.1.26) were appreciable, and the distributions of both activities were consistent with that of sedimentable catalase activity. Glycerol-3-phosphate acyltransferase (GPAT, EC 2.3.1.15), which is localized in both microsomes (microsomal fractions) and mitochondria in the rat liver, was a peroxisomal enzyme in the harderian gland, though the activity was only about one-tenth of the DHAPAT activity. These enzymes had different pH profiles and substrate specificity. The existence of high activities of enzymes of the acyl-DHAP pathway in peroxisomes suggests the physiological significance of peroxisomes in the biosynthesis of glycerol ether phospholipid and 1-alkyl-2,3-diacylglycerol in the guinea-pig harderian gland.     

Characteristics of the suppressive effect of nicardipine on peroxisome induction in rat liver

In vivo administration of nicardipine, a known calcium antagonist, suppressed the clofibrate-evoked induction of activities of peroxisomal enzymes, such as catalase, the peroxisomal fatty acyl-CoA oxidizing system, carnitine acetyltransferase and mitochondrial carnitine palmitoyltransferase in rat liver. On a time-course study, the suppression of induction in the activities of the peroxisomal fatty acyl-CoA oxidizing system and carnitine acetyltransferase was found at 5 days after the treatment, whereas the induction by clofibrate was already observed at 1 day after the treatment, suggesting that in the process of peroxisome induction by clofibrate there might be two steps, i.e., a triggering step and an enhancing step, and nicardipine might act as suppressor for the later step. The precursor-incorporation studies with [3H]leucine showed that the rate of the synthesis of the peroxisomal bifunctional enzyme was increased by 4.2-fold after clofibrate-treatment, whereas nicardipine suppressed this enhancement to only 2.2-fold of the control. The rate of degradation of this enzyme was not affected by any treatment. These results show that nicardipine affects the regulation mechanism of the biosynthesis of this enzyme. Nicardipine showed hardly any suppressive-effect on the hepatic peroxisomal enzyme induction observed in high-fat diet fed rat. Furthermore, the suppression of clofibrate-evoked induction of peroxisomal enzymes was observed also in mice. These interesting findings suggest that there is a difference in the mechanism of peroxisome proliferation and/or the induction of peroxisomal enzymes between clofibrate and physiological conditions, such as high-fat diet feeding. The suppression of drug-induced peroxisome proliferation by calcium antagonists may help in dissecting the causal relationship between the multiple effects mediated by peroxisomal proliferators.     

Response of chemically induced hepatocytelike cells in hamster pancreas to methyl clofenapate, a peroxisome proliferator

Administration of N-nitrosobis (2-oxopropyl)amine during peak DNA synthesis of regenerating pancreas in hamsters has been shown to induce hepatocytelike cells in pancreas. We now present evidence to demonstrate that such cells respond to methyl clofenapate, a peroxisome proliferator. The response includes a marked proliferation of peroxisomes and enhanced activity of peroxisomal enzymes enoyl-CoA hydratase (8.5- to 13-fold), [1-14C]-palmitoyl-CoA oxidation (2.8- to 3.9-fold), catalase (1.6 to 3.4-fold), and carnitine acetyltransferase (greater than 2,000-fold). Cytochemical localization of catalase by the alkaline 3,3'-diaminobenzidine procedure and immunofluorescence localization of heat-labile enoyl-CoA hydratase showed that these peroxisome-associated enzymes are localized strictly in pancreatic hepatocytelike cells, while adjacent acinar, duct, and islet cells appeared consistently negative. Morphometric analyses of hepatocytelike cells showed a significant increase in the numerical density and an eightfold increase in the volume density of peroxisomes in methyl clofenapate treated animals. These results demonstrate that the hepatocytelike cells are responsible for the observed peroxisomal enzyme activity in pancreas of hamsters and suggest that the derepressed peroxisome specific genes in these cells respond to a peroxisome proliferator as do parenchymal cells in hamster liver.     

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.     

Significance of catalase in peroxisomal fatty acyl-CoA beta-oxidation: NADH oxidation by acetoacetyl-CoA and H2O2

To clarify the significance of catalase in peroxisomes, we have examined the effect of aminotriazole treatment of rats on the activity of beta-hydroxybutyryl-CoA dehydrogenase in liver peroxisomes. When the effect of H2O2 on the dehydrogenase activity was examined using an extract of liver peroxisomes from aminotriazole-treated rats, the acetoacetyl-CoA-dependent oxidation of NADH was found to increase considerably on the addition of dilute H2O2. Such an effect of H2O2 was not seen on the beta-hydroxybutyryl-CoA-dependent reduction of NAD nor with extracts from untreated animals. We then noticed that similar NADH oxidation was caused non-enzymatically by a mixture of acetoacetyl-CoA and H2O2. The oxidation was dependent on both acetoacetyl-CoA and H2O2, and was blocked by scavengers of oxyradicals such as ascorbate and ethanol. Degradation products formed during the reaction of acetoacetyl-CoA with H2O2 had no NADH oxidizing activity, indicating that effective oxidant(s) were generated during the reaction of H2O2 with acetoacetyl-CoA. No other fatty acyl-CoA so far examined nor acetoacetate could replace acetoacetyl-CoA in this reaction. Therefore, if H2O2 were to be accumulated in peroxisomes, it would decrease both NADH and acetoacetyl-CoA, thus affecting the fatty acyl-CoA beta-oxidation system. These results, together with our previous finding that peroxisomal thiolase was significantly inactivated by H2O2 [Hashimoto, F. & Hayashi, H. (1987) Biochim. Biophys. Acta 921, 142-150] suggest that the role of catalase in peroxisomes is at least in part to protect the fatty acyl-CoA beta-oxidation system from the deleterious action of H2O2.     

Proliferation of peroxisomes and induction of peroxisomal beta-oxidation enzymes in rat hepatoma H4IIEC3 by ciprofibrate

For the analysis of the molecular mechanism of the action of peroxisome proliferators, we attempted to establish the optimal conditions for obtaining the effects of the chemicals in vitro, employing an established cell line, Reuber rat hepatoma H4IIEC3. Histochemical analyses revealed a marked increase in the number, size, and catalase content of peroxisomes in the cells cultured on a medium containing 0.5 mM ciprofibrate, a peroxisome proliferator. The activity of acyl-CoA oxidase, the initial enzyme of the peroxisomal beta-oxidation system, was increased by more than 10-fold by the same treatment. Catalase was also induced significantly, whereas the activities of glutamate dehydrogenase and lactate dehydrogenase, mitochondrial and cytosolic marker enzymes, did not change upon the treatment. Immunoblotting and RNA-blotting analyses confirmed the increases in the amount of protein and mRNA for all the three enzymes of the peroxisomal beta-oxidation system. Cell fractionation experiments gave a partial separation of peroxisomes from other organelles for the induced culture. Thus, H4IIEC3 cells offer a good in vitro model system of the induction of peroxisomes and peroxisomal beta-oxidation enzymes by peroxisome proliferators.     

Characteristics of dehydroepiandrosterone as a peroxisome proliferator.

Treatment of rats with dehydroepiandrosterone (300 mg/kg body weight, per os, 14 days) caused a remarkable increase in the number of peroxisomes and peroxisomal beta-oxidation activity in the liver. The activities of carnitine acetyltransferase, microsomal laurate 12-hydroxylation, cytosolic palmitoyl-CoA hydrolase, malic enzyme and some other enzymes were also increased. The increases in these enzyme activities were all greater in male rats than in female rats. Immunoblot analysis revealed remarkable induction of acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme in the liver and to a smaller extent in the kidney, whereas no significant induction of these enzymes was found in the heart. The increase in the hepatic peroxisomal beta-oxidation activity reached a maximal level at day 5 of the treatment of dehydroepiandrosterone and the increased activity rapidly returned to the normal level on discontinuation of the treatment. The increase in the activity was also dose-dependent, which was saturable at a dose of more than 200 mg/kg body weight. All these features in enzyme induction caused by dehydroepiandrosterone correlate well with those observed in the treatment of clofibric acid, a peroxisome proliferator. Co-treatment of dehydroepiandrosterone and clofibric acid showed no synergism in the enhancement of peroxisomal beta-oxidation activity, suggesting the involvement of a common process in the mechanism by which these compounds induce the enzymes. These results indicate that dehydroepiandrosterone is a typical peroxisome proliferator. Since dehydroepiandrosterone is a naturally occurring C19 steroid in mammals, the structure of which is novel compared with those of peroxisome proliferators known so far, this compound could provide particular information in the understanding of the mechanisms underlying the induction of peroxisome proliferation.     

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.     

Identification of a catalase-negative sub-population of peroxisomes induced in mouse liver by clofibrate.

The peroxisomal compartment in mouse liver was investigated using rate sedimentation of liver subfractions on sucrose density gradients. Treatment of mice with clofibrate, a hypolipidemic agent and peroxisome proliferator, resulted in the formation of small particles which were devoid of catalase and urate oxidase, but which were identified as peroxisomal on the basis of content of the clofibrate-induced peroxisomal beta-oxidation enzymes (fatty acyl-CoA oxidase, hydratase/dehydrogenase bifunctional protein, and thiolase) and the 68 kDa peroxisomal integral membrane protein. Immunoelectron microscopy confirmed the membrane-bound organellar nature and enzyme composition of these particles. These particles were absent in normal mice, and were increased to a maximal level within 2 days of clofibrate treatment. These data have been taken as indicative of a role of these particles in the mechanism of drug-induced peroxisome proliferation.