Immunocytochemical localization of 2,4-dienoyl-CoA reductase in the liver of normal and di-(2-ethylhexyl)phthalate-fed rats

Summary Localization of 2,4-dienoyl-CoA reductase (DCR) in rat liver was studied using immunoenzyme and immunogold techniques. The animals were fed on a laboratory diet with or without 2% di-(2-ethylhexyl)phthalate (DEHP), a peroxisome proliferator, for two weeks. For light microscopy (LM), semithin Epon sections were stained by immunoenzyme technique after removal of the epoxy resin. For electron microscopy (EM), ultrathin Lowicryl K4M sections were stained by the protein A-gold technique. By LM, in untreated rats reaction deposits showing the antigenic sites for DCR were present in the cytoplasmic granules. Hepatocytes, epithelial cells of interlobular bile duct, and sinus-lining cells contained these granules. After administration of DEHP, the cytoplasmic granules stained similarly. The staining intensity of the heaptocytes increased markedly, but that of the other cells decreased. The sinus-lining cells became mostly negative. By EM, gold particles indicating the antigenic sites for DCR were present in both the mitochondria and peroxisomes of hepatocytes of untreated rats. In the other cells, the gold label was confined to the mitochondria. After administration of DEHP, labelling intensity of the hepatocyte mitochondria increased markedly, but that of the peroxisomes conversely decreased. Quantitative analysis of labelling density showed that the mitochondrial DCR increased to about three times that in the untreated rat, but the peroxisomal DCR decreased to 1/6. The results show that in the rat liver, DCR exists in both, mitochondria and peroxisomes. DEHP can induce mitochondrial DCR, but not peroxisomal DCR.     

Detection of heat-stable delta 3,delta 2-enoyl-CoA isomerase in rat liver mitochondria and peroxisomes by immunochemical study using specific antibody

The subcellular distribution of delta 3,delta 2-enoyl-CoA isomerase [EC 5.3.3.8] and the inducing effect of clofibrate, a peroxisomal proliferator, on the enzyme activity were examined in rat liver. From the results of spectrophotometric investigation of the fractions, which were prepared by sucrose discontinuous gradient centrifugation from the light mitochondrial fraction, the isomerase activity was found in the fractions enriched in mitochondria and those enriched in peroxisomes of the control and the clofibrate treated rat livers. The anti-isomerase antibody reacted with both the mitochondrial isomerase and the peroxisomal isomerase, revealing a single band with an apparent molecular weight of 30,000. However, the isomerase was induced by clofibrate administration mainly in the mitochondrial fraction. These results suggest that delta 3,delta 2-enoyl-CoA isomerase is located in the mitochondria and the peroxisomes of the normal rat liver, and that the isomerase in the mitochondria is induced by clofibrate administration.     

Quantitative immunocytochemical studies on differential induction of serine:pyruvate aminotransferase in mitochondria and peroxisomes of rat liver cells by administration of glucagon or di-(2-ethylhexyl)phthalate

Differential induction of serine: pyruvate amino-transferase (SPT) in rat liver parenchymal cells by administration of glucagon or di-(2-ethylhexyl)phthalate (DEHP) was studied using post-embedding immunocytochemical techniques and morphometric methods. Two groups of rats were fasted for 5 days and daily received peritoneal injection of glucagon (300 micrograms/100 g) or physiological saline. Another two groups of rats were fed on laboratory chow with or without 2% DEHP for 2 weeks. Livers were perfusion-fixed, cut into tissue sections (50-100 micron), and processed to cytochemistry for catalase, immunocytochemistry for SPT, and conventional procedures for electron microscopy. The morphometric analysis showed that glucagon injection has negligible effect on the volume and numerical density and mean diameter of peroxisomes, whereas volume density of mitochondria was decreased by 25%. By DEHP administration peroxisomes were about 3-fold increased in the volume and numerical density. Mitochondria was increased about 40% in the numerical density, but unchanged in the volume density. Light and electron microscopic immunocytochemistry demonstrated that glucagon injection exclusively enhanced mitochondrial SPT, whereas DEHP administration exclusively induced in peroxisomal SPT. Quantitative analysis showed that by the glucagon injection, the labeling density of mitochondria was increased about 4-fold, but that of peroxisomes was 1.6 times as much as control, while by DEHP administration, the labeling density of peroxisomes was enhanced about 3-fold but that of mitochondria was decreased by 13%. The results clearly indicate that glucagon induces mitochondrial SPT, whereas peroxisome proliferator, DEHP induces peroxisomal SPT.     

Occurrence of a long-chain delta 3,delta 2-enoyl-CoA isomerase in rat liver.

Isoproteins of delta 3,delta 2-enoyl-CoA isomerase (EC 5.3.3.8), an auxiliary enzyme in the beta-oxidation of unsaturated fatty acids having double bonds at odd-numbered positions, were studied in livers of control and clofibrate-treated rats. When liver extracts were applied to a hydroxyapatite column at pH 7.0, the previously characterized peroxisomal trifunctional hydratase-dehydrogenase-isomerase enzyme and the mitochondrial isomerase, which shows a preference for short-chain substrates, were eluted almost in parallel. In addition to these activities, a separate isomerase was observed to elute at a lower potassium phosphate concentration in the gradient. Experiments with extracts of purified mitochondria and peroxisomes demonstrated the mitochondrial origin of this third activity. Studies on the kinetic properties of the third isomerase showed that it has a preference for C10-C12 substrates. An Mr of 200,000 was obtained for the native protein by gel-filtration chromatography. Antibodies to mitochondrial short-chain isomerase and peroxisomal trifunctional enzyme did not recognize this novel mitochondrial isoenzyme. The immunological non-cross-reactivity can be interpreted as suggesting that the different isomerases are not closely related at the level of the primary structure of the polypeptide chain. The present data demonstrate that, similar to many other enzymes of beta-oxidation, delta 3,delta 2-enoyl-CoA isomerase has at least three isoenzymes in rat liver: mitochondrial short- and long-chain isomerases and an additional peroxisomal isoenzyme, which in this case is a part of a multifunctional protein.     

Quantitative immunocytochemical studies on differential induction of serine

Summary Differential induction of serine: pyruvate aminotransferase (SPT) in rat liver parenchymal cells by administration of glucagon or di-(2-ethylhexyl)phthalate (DEHP) was studied using post-embedding immunocytochemical techniques and morphometric methods. Two groups of rats were fasted for 5 days and daily received peritoneal injection of glucagon (300 g/100 g) or physiological saline. Another two groups of rats were fed on laboratory chow with or without 2% DEHP for 2 weeks. Livers were perfusionfixed, cut into tissue sections (50–100 ), and processed to cytochemistry for catalase, immunocytochemistry for SPT, and conventional procedures for electron microscopy. The morphometric analysis showed that glucagon injection has negligible effect on the volume and numerical density and mean diameter of peroxisomes, whereas volume density of mitochondria was decreased by 25%. By DEHP administration peroxisomes were about 3-fold increased in the volume and numerical density. Mitochondria was increased about 40% in the numerical density, but unchanged in the volume density. Light and electron microscopic immunocytochemistry demonstrated that glucagon injection exclusively enhanced mitochondrial SPT, whereas DEHP administration exclusively induced in peroxisomal SPT. Quantitative analysis showed that by the glucagon injection, the labeling density of mitochondria was increased about 4-fold, but that of peroxisomes was 1.6 times as much as control, while by DEHP administration, the labeling density of peroxisomes was enhanced about 3-fold but that of mitochondria was decreased by 13%. The results clearly indicate that glucagon induces mitochondrial SPT, whereas peroxisome proliferator, DEHP induces peroxisomal SPT.     

Hepatic subcellular distribution of short-chain beta-ketoacyl coenzyme A reductase and trans-2-enoyl coenzyme A hydratase: 25- to 50-fold stimulation of microsomal activities by the peroxisome proliferator, di-(2-ethylhexyl)phthalate

The present study demonstrates unequivocally the existence of short-chain trans-2-enoyl coenzyme A (CoA) hydratase and beta-ketoacyl CoA reductase activities in the endoplasmic reticulum of rat liver. Subcellular fractionation indicated that all four fractions, namely, mitochondrial, peroxisomal, microsomal, and cytosolic contained significant hydratase activity when crotonyl CoA was employed as the substrate. In the untreated rat, based on marker enzymes and heat treatment, the hydratase activity, expressed as mumol/min/g liver, wet weight, in each fraction was: mitochondria, 684; peroxisomes, 108; microsomes, 36; and cytosol, 60. Following di-(2-ethylhexyl)phthalate (DEHP) treatment (2% (v/w) for 8 days), there was only a 20% increase in mitochondrial activity; in contrast, peroxisomal hydratase activity was stimulated 33-fold, while microsomal and cytosolic activities were enhanced 58- and 14-fold respectively. A portion of the cytosolic hydratase activity can be attributed to the component of the fatty acid synthase complex. Although more than 70% of the total hydratase activity was associated with the mitochondrial fraction in the untreated rat, DEHP treatment markedly altered this pattern; only 11% of the total hydratase activity was present in the mitochondrial fraction, while 49 and 29% resided in the peroxisomal and microsomal fractions, respectively. In addition, all four subcellular fractions contained the short-chain NADH-specific beta-ketoacyl CoA (acetoacetyl CoA) reductase activity. Again, in the untreated animal, reductase activity was predominant in the mitochondrial fraction; following DEHP treatment, there was marked stimulation in the peroxisomal, microsomal, and cytosolic fractions, while the activity in the mitochondrial fraction increased by only 39%. Hence, it can be concluded that both reductase and hydratase activities exist in the endoplasmic reticulum in addition to mitochondria, peroxisomes, and soluble cytoplasm.     

Beta-oxidation of polyunsaturated fatty acids in peroxisomes. Subcellular distribution of delta 3,delta 2-enoyl-CoA isomerase activity in rat liver

The metabolism of the double bonds at the delta 3 position in fatty acids was studied in rat liver. Infusion of delta 3-trans-dodecenoic acid into isolated perfused liver and subcellular fractionation studies showed the presence of both peroxisomal and mitochondrial delta 3,delta 2-enoyl-CoA isomerase activity (EC 5.3.3.8). These findings together with the previous demonstration of peroxisomal 2,4-dienoyl-CoA reductase (EC 1.3.1.34) [(1981) J. Biol. Chem. 256, 8259-8262] and D-3-OH-acyl-CoA epimerase (EC 5.1.2.3) [(1985) FEBS Lett. 185, 129-134] activities show that peroxisomes possess all the auxiliary enzymes required for the beta-oxidation of unsaturated fatty acids.     

Peroxisomal bifunctional protein from rat liver is a trifunctional enzyme possessing 2-enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and delta 3, delta 2-enoyl-CoA isomerase activities

Peroxisomal delta 3, delta 2-enoyl-CoA isomerase (EC 5.3.3.8) was studied in the liver of rats treated with clofibrate. The mitochondrial and peroxisomal isoenzymes were separated chromatographically and the peroxisomal isomerase purified to apparent homogeneity. In addition to the isomerization of 3-enoyl-CoA esters, the purified protein also catalyzed hydration of trans-2-enoyl-CoA and oxidation of L-3-hydroxyacyl-CoA. Incubation of the purified protein with trans-3-decenoyl-CoA, NAD+, and Mg2+ resulted in an increase in absorbance at 303 nm, indicating the formation of 3-ketoacyl-CoA. The protein purified was monomeric, with an estimated molecular weight of 78,000. In immunoblotting it was recognized by the antibody to peroxisomal bifunctional protein from rat liver. Comparison of the amino acid sequences of cyanogen bromide cleaved isomerase with the known sequence of the peroxisomal bifunctional protein from the rat identified them as the same molecule. In control experiments, the peroxisomal bifunctional protein purified according to published methods also catalyzed delta 3, delta 2-enoyl-CoA isomerization. This means that the bifunctional protein of rat liver is in fact a trifunctional enzyme possessing delta 3, delta 2-enoyl-CoA isomerase, 2-enoyl-CoA hydratase (EC 4.2.1.17), and L-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) activities in the same polypeptide.     

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.     

Delta 3,delta 2-enoyl-CoA isomerases. Characterization of the mitochondrial isoenzyme in the rat

Delta 3,delta 2-Enoyl-CoA isomerase (EC 5.3.3.8), an obligatory auxiliary enzyme for the metabolism of double bonds at odd-numbered positions of fatty acids during their beta-oxidation, was studied in hearts and livers of normal and clofibrate-treated rats. Hepatic peroxisomal and mitochondrial isoenzymes were separable by dye-ligand chromatography. The mitochondrial one was further purified to apparent homogeneity. An isomerase was also purified from heart muscle, a peroxisome-poor tissue. These enzymes were dimeric basic proteins (pI 9.5) with a subunit molecular weight of 30,000. Both cis- and trans-enoyl-CoA served as substrates for the hepatic enzyme studied. The velocity ratio for the C6-, C10-, and C12-trans-3-enoyl substrates was 9:2.5:1. By immunoelectron microscopy the enzyme protein selected for purification was found to be mitochondrial both in liver and heart. Chromatographic evidence, immunoelectron microscopy, and immunoblotting indicated that in the liver but not in the heart, the enzyme underwent an induction of 1 order of magnitude during clofibrate treatment. Antibodies towards the rat isomerases detected cross-reactive proteins in bovine and pig liver and heart and human placenta. The estimated subunit sizes varied from species to species, being 31,000 in bovine liver and heart, 29,000 in pig liver and heart, and 30,000 in human placenta. The data are in accord with the notion of a dual location of the delta 3,delta 2-enoyl-CoA isomerase. Mitochondrial origin of one of the isoenzymes and its tissue-specific induction by clofibrate were verified by immunochemistry and the identity of the peroxisomal one revealed by the chromatographic behavior of the proteins.     

Alkylthioacetic acid (3-thia fatty acids)--a new group of non-beta-oxidizable, peroxisome-inducing fatty acid analogues. I. A study on the structural requirements for proliferation of peroxisomes and mitochondria in rat liver

The induction of peroxisome proliferation was examined in rat liver after administration of equal concentrations (1 mmol/kg body weight) of 1,10-bis(carboxymethylthiodecane) (BCMTD), 1-mono(carboxymethylthiotetradecane) (CMTTD), 1-mono(carboxymethylthiooctane) (CMTO), 1-mono(carboxyethylthiotetradecane) (CETTD), palmitic acid and hexadecanedioic acid (HDDA). BCMTD, a non-beta-oxidizable and non-omega-oxidizable sulphur-substituted fatty acid analogue was considerably more potent than CMTTD (only non-beta-oxidizable) in inducing enlargement of the liver and increasing peroxisomal activities (monitored by peroxisomal beta-oxidation, palmitoyl-CoA hydrolase and catalase activities). Morphometric analysis of randomly selected hepatocytes revealed that BCMTD and CMTTD treatment increased the number and size of peroxisomes and the relative volume fraction of the peroxisomes. All these cellular responses were more marked with BCMTD than compared with CMTTD. CMTO, a non-beta-oxidizable fatty acid analogue containing a lower hydrophobic alkyl-end than CMTTD and CETTD (a beta-oxidizable fatty acid analogue), showed a slight increase (1.4-1.8-fold) of peroxisomal beta-oxidation and caused marginally morphological changes of peroxisomes compared with CMTTD and BCMTD. The most striking effect of the alkylthiopropionic acid (CETTD) was an enhancement of the hepatic triacylglycerol level. Palmitic acid and hexadecanedioic acid only marginally affected the peroxisomal activities, but no morphological changes of peroxisomes and fat droplets were observed. The presented data strongly suggest that a minimal structural requirement for a peroxisome proliferator may be (1) a carboxylic acid group linked to (2) a hydrophobic backbone which (3) cannot be beta-oxidized i.e., the fatty acid analogues have a sulphur atom in the beta-position. It is also conceivable that blockage for omega-oxidation may potentiate the peroxisome-proliferating activities in as much as BCMTD was more potent than CMTTD. Two mitochondrial marker enzymes, carnitine palmitoyltransferase and succinate phenazine methosulphate oxidoreductase were differently affected after administration of the investigated compounds. Furthermore, BCMTD and CMTTD as well as HDDA treatments increased the number of mitochondria, but the mitochondria tended to be smaller. The overall results presented here indicate that the structural requirements for proliferation of mitochondria are not identical to those for proliferation of peroxisomes.     

Characterization of the 70-kDa peroxisomal membrane protein, an ATP binding cassette transporter

The 70-kDa peroxisomal membrane protein (PMP70) is one of the major components of rat liver peroxisomal membranes and belongs to a superfamily of proteins known as ATP binding cassette transporters. PMP70 is markedly induced by administration of hypolipidemic agents in parallel with peroxisome proliferation and induction of peroxisomal fatty acid beta-oxidation enzymes. To characterize the role of PMP70 in biogenesis and function of peroxisomes, we transfected the cDNA of rat PMP70 into Chinese hamster ovary cells and established cell lines stably expressing PMP70. The content of PMP70 in the transfectants increased about 5-fold when compared with the control cells. A subcellular fractionation study showed that overexpressed PMP70 was enriched in peroxisomes. This peroxisomal localization was confirmed by immunofluorescence and immunoelectron microscopy. The number of immuno-gold particles corresponding to PMP70 on peroxisomes increased markedly in the transfectants, but the size and the number of peroxisomes were essentially the same in both the transfectants and the control cells. beta-Oxidation of palmitic acid increased about 2-3-fold in the transfectants, whereas the oxidation of lignoceric acid decreased about 30-40%. When intact peroxisomes prepared from both the cell lines were incubated with palmitoyl-CoA, oxidation was stimulated with ATP, but the degree of the stimulation was higher in the transfectants than in the control cells. Furthermore, we established three Chinese hamster ovary cell lines stably expressing mutant PMP70. In these cells, beta-oxidation of palmitic acid decreased markedly. These results suggest that PMP70 is involved in metabolic transport of long chain acyl-CoA across peroxisomal membranes and that increase of PMP70 is not associated with proliferation of peroxisomes.     

Morphologic effects of sulfur-substituted fatty acids on rat hepatocytes with special reference to proliferation of peroxisomes and mitochondria

The morphologic effects of different sulfur-substituted mono- and dicarboxylic fatty acids on rat hepatocytes have been examined. The substance 1,10-biscarboxymethylthiodecane (BCMTD) is blocked for both beta- and omega-oxidation, whereas 1-monocarboxymethylthiodecane (CMTTD) is only non-beta-oxidizable. At equimolar doses BCMTD was considerably more potent than CMTTD in hypertrophic liver enlargement. At the ultrastructural level, BCMTD increased the volume fraction of the peroxisomes by a factor of 8, and their size and number by factors of 2.1 and 6.4, respectively. Furthermore, the frequency of dense cores in the peroxisomes decreased from 60 to 8%. CMTTD resulted in an increased volume fraction of peroxisomes (4.5-fold), in the mean volume (1.9-fold), and in the number of peroxisomes (3.7-fold). At the mitochondrial level, a gradual development toward megamitochondria was observed after CMTTD administration. BCMTD, however, increased the number of mitochondria but they tended to be smaller. Administration of both acids increased peroxisomal beta-oxidation and mitochondrial carnitine palmitoyltransferase activity, whereas the lipid content of hepatocytes was reduced with increasing doses of CMTTD and especially BCMTD. The acid 1-mono(carboxyethylthio)tetradecane (CETTD), which is able to undergo one cycle of beta-oxidation, caused no change in liver weight, and only marginal effects on peroxisomes and mitochondria were observed. In contrast to the BCMTD and CMTTD feeding, the animals developed a tremendous accumulation of fat in the livers: the volume fraction of lipid droplets increased 23-fold after CETTD feeding.     

Beta-oxidation in hepatocyte cultures from mice with peroxisomal gene knockouts

Beta-oxidation of carboxylates takes place both in mitochondria and peroxisomes and in each pathway parallel enzymes exist for each conversion step. In order to better define the substrate specificities of these enzymes and in particular the elusive role of peroxisomal MFP-1, hepatocyte cultures from mice with peroxisomal gene knockouts were used to assess the consequences on substrate degradation. Hepatocytes from mice with liver selective elimination of peroxisomes displayed severely impaired oxidation of 2-methylhexadecanoic acid, the bile acid intermediate trihydroxycholestanoic acid (THCA), and tetradecanedioic acid. In contrast, mitochondrial beta-oxidation rates of palmitate were doubled, despite the severely affected inner mitochondrial membrane. As expected, beta-oxidation of the branched chain compounds 2-methylhexadecanoic acid and THCA was reduced in hepatocytes from mice with inactivation of MFP-2. More surprisingly, dicarboxylic fatty acid oxidation was impaired in MFP-1 but not in MFP-2 knockout hepatocytes, indicating that MFP-1 might play more than an obsolete role in peroxisomal beta-oxidation.     

Mitochondrial and peroxisomal fatty acid oxidation in liver homogenates and isolated hepatocytes from control and clofibrate-treated rats.

Mitochondrial and peroxisomal fatty acid oxidation were compared in whole liver homogenates. Oxidation of 0.2 mM palmitoyl-CoA or oleate by mitochondria increased rapidly with increasing molar substrate:albumin ratios and became saturated at ratios below 3, while peroxisomal oxidation increased more slowly and continued to rise to reach maximal activity in the absence of albumin. Under the latter condition mitochondrial oxidation was severely depressed. In homogenates from normal liver peroxisomal oxidation was lower than mitochondrial oxidation at all ratios tested except when albumin was absent. In contrast with mitochondrial oxidation, peroxisomal oxidation did not produce ketones, was cyanide-insensitive, was not dependent on carnitine, and was not inhibited by (+)-octanoylcarnitine, malonyl-CoA and 4-pentenoate. Mitochondrial oxidation was inhibited by CoASH concentrations that were optimal for peroxisomal oxidation. In the presence of albumin, peroxisomal oxidation was stimulated by Triton X-100 but unaffected by freeze-thawing; both treatments suppressed mitochondrial oxidation. Clofibrate treatment increased mitochondrial and peroxisomal oxidation 2- and 6- to 8-fold, respectively. Peroxisomal oxidation remained unchanged in starvation and diabetes. Fatty acid oxidation was severely depressed by cyanide and (+)-octanoylcarnitine in hepatocytes from normal rats. Hepatocytes from clofibrate-treated rats, which displayed a 3- to 4-fold increase in fatty acid oxidation, were less inhibited by (+)-octanoylcarnitine. Hydrogen peroxide production was severalfold higher in hepatocytes from treated animals oxidizing fatty acids than in control hepatocytes. Assuming that all H2O2 produced during fatty acid oxidation was due to peroxisomal oxidation, it was calculated that the contribution of the peroxisomes to fatty acid oxidation was less than 10% both in cells from control and clofibrate-treated animals.     

Differential induction of peroxisomal populations in subcellular fractions of rat liver

In rat liver, peroxisome proliferators induce profound changes in the number and protein composition of peroxisomes, which upon subcellular fractionation is reflected in heterogeneity in sedimentation properties of peroxisome populations. In this study we have investigated the time course of induction of the peroxisomal proteins catalase, acyl-CoA oxidase (ACO) and the 70 kDa peroxisomal membrane protein (PMP70) in different subcellular fractions. Rats were fed a di(2-ethylhexyl)phthalate (DEHP) containing diet for 8 days and livers were removed at different time-points, fractionated by differential centrifugation into nuclear, heavy and light mitochondrial, microsomal and soluble fractions, and organelle marker enzymes were measured. Catalase was enriched mainly in the light mitochondrial and soluble fractions, while ACO was enriched in the nuclear fraction (about 30%) and in the soluble fraction. PMP70 was found in all fractions except the soluble fraction. DEHP treatment induced ACO, catalase and PMP70 activity and immunoreactive protein, but the time course and extent of induction was markedly different in the various subcellular fractions. All three proteins were induced more rapidly in the nuclear fraction than in the light mitochondrial or microsomal fractions, with catalase and PMP70 being maximally induced in the nuclear fraction already at 2 days of treatment. Refeeding a normal diet quickly normalized most parameters. These results suggest that induction of a heavy peroxisomal compartment is an early event and that induction of 'small peroxisomes', containing PMP70 and ACO, is a late event. These data are compatible with a model where peroxisomes initially proliferate by growth of a heavy, possibly reticular-like, structure rather than formation of peroxisomes by division of pre-existing organelles into small peroxisomes that subsequently grow. The various peroxisome populations that can be separated by subcellular fractionation may represent peroxisomes at different stages of biogenesis.     

Physiological role of peroxisomal beta-oxidation in liver of fasted rats

In the livers of fasted rats, the activity of peroxisomal palmitocyl-CoA oxidation (NADH production) was increased more rapidly and markedly than that of mitochondrial carnitine palmitoyltransferase, which is the rate limiting enzyme of mitochondrial beta-oxidation. The peroxisomal oxidizing activity was about twice that of the control throughout the period of fasting (1-7 days). carnitine acetyltransferase activity was increased to a similar extent in both peroxisomes and mitochondria. A possible physiological role of liver peroxisomes may thus be as an effective supply of NADH2, acetyl residues and short and medium-length fatty acyl-CoA in the cells on the enhancement of peroxisomal beta-oxidation of the animals under starvation; these substances thus produced may be transported into the mitochondria as energy sources.     

The involvement of carnitine intermediates in peroxisomal fatty acid oxidation: a study with 2-bromofatty acids

Metabolism-dependent inactivators of 3-ketothiolase I and carnitine acyltransferase I (CAT I) have been used to study the oxidation of fatty acids in intact hepatocytes. 2-Bromooctanoate inactivates mitochondrial and peroxisomal 3-ketothiolases I in a time-dependent manner. During the first 5 min of incubation, inactivation of 3-ketothiolase in mitochondria is five times faster than its inactivation in peroxisomes. Almost complete inactivation of 3-ketothiolase I in both types of organelle is achieved after incubation with 1 mM 2-bromooctanoate for 40 min. The inactivation is not affected by preincubating hepatocytes with 20 microM tetradecylglycidate (TDGA), an inactivator of CAT I, under conditions which cause greater than 95% inactivation of CAT I. 2-Bromododecanoate (1 mM) causes 60% inactivation of mitochondrial and peroxisomal 3-ketothiolases I in 40 min. These inactivations are greatly reduced by preincubating hepatocytes with 20 microM TDGA, demonstrating that 2-bromododecanoate enters both mitochondria and peroxisomes via its carnitine ester. 2-Bromopalmitate (1 mM) causes less than 5% inactivation of mitochondrial and peroxisomal 3-ketothiolases I in 40 min, but causes 95% inactivation of CAT I during this time. Incubation of hepatocytes with 10-200 microM 2-bromopalmitoyl-L-carnitine causes inactivation of mitochondrial and peroxisomal 3-ketothiolases I at similar rates. This inactivation is decreased by palmitoyl-D-carnitine during the first 5 min of incubation. Pretreating hepatocytes with 20 microM TDGA does not affect the inactivation of mitochondrial or peroxisomal 3-ketothiolase I by 2-bromopalmitoyl-L-carnitine. These results demonstrate that in intact hepatocytes, peroxisomes oxidize fatty acids of medium-chain length by a carnitine-independent mechanism, whereas they oxidize long-chain fatty acids by a carnitine-dependent mechanism.     

Localization of cytosolic NADP-dependent isocitrate dehydrogenase in the peroxisomes of rat liver cells: biochemical and immunocytochemical studies.

Two types of NADP-dependent isocitrate dehydrogenases (ICDs) have been reported: mitochondrial (ICD1) and cytosolic (ICD2). The C-terminal amino acid sequence of ICD2 has a tripeptide peroxisome targeting signal 1 sequence (PTS1). After differential centrifugation of the postnuclear fraction of rat liver homogenate, approximately 75% of ICD activity was found in the cytosolic fraction. To elucidate the true localization of ICD2 in rat hepatocytes, we analyzed the distribution of ICD activity and immunoreactivity in fractions isolated by Nycodenz gradient centrifugation and immunocytochemical localization of ICD2 antigenic sites in the cells. On Nycodenz gradient centrifugation of the light mitochondrial fraction, ICD2 activity was distributed in the fractions in which activity of catalase, a peroxisomal marker, was also detected, but a low level of activity was also detected in the fractions containing activity for succinate cytochrome C reductase (a mitochondrial marker) and acid phosphatase (a lysosomal marker). We have purified ICD2 from rat liver homogenate and raised a specific antibody to the enzyme. On SDS-PAGE, a single band with a molecular mass of 47 kD was observed, and on immunoblotting analysis of rat liver homogenate a single signal was detected. Double staining of catalase and ICD2 in rat liver revealed co-localization of both enzymes in the same cytoplasmic granules. Immunoelectron microscopy revealed gold particles with antigenic sites of ICD2 present mainly in peroxisomes. The results clearly indicated that ICD2 is a peroxisomal enzyme in rat hepatocytes. ICD2 has been regarded as a cytosolic enzyme, probably because the enzyme easily leaks out of peroxisomes during homogenization. (J Histochem Cytochem 49:1123-1131, 2001)     

Transient complex interactions of mammalian peroxisomes without exchange of matrix or membrane marker proteins

Peroxisomes and mitochondria show a much closer interrelationship than previously anticipated. They co-operate in the metabolism of fatty acids and reactive oxygen species, but also share components of their fission machinery. If peroxisomes - like mitochondria - also fuse in mammalian cells is a matter of debate and was not yet systematically investigated. To examine potential peroxisomal fusion and interactions in mammalian cells, we established an in vivo fusion assay based on hybridoma formation by cell fusion. Fluorescence microscopy in time course experiments revealed a merge of different peroxisomal markers in fused cells. However, live cell imaging revealed that peroxisomes were engaged in transient and long-term contacts, without exchanging matrix or membrane markers. Computational analysis showed that transient peroxisomal interactions are complex and can potentially contribute to the homogenization of the peroxisomal compartment. However, peroxisomal interactions do not increase after fatty acid or H(2) O(2) treatment. Additionally, we provide the first evidence that mitochondrial fusion proteins do not localize to peroxisomes. We conclude that mammalian peroxisomes do not fuse with each other in a mechanism similar to mitochondrial fusion. However, they show an extensive degree of interaction, the implication of which is discussed.