Liver peroxisomes in newborns from clofibrate-treated rats. I. A morphometric study of the recovery period.

Morphological and morphometric parameters (volume density (Vv), numerical density (NA) and mean diameter (D)) of newborn liver peroxisomes were measured throughout the first week of life in rats born to mothers treated with clofibrate (ethyl 2 p-chlorophenoxy isobutyrate) during the last five days of pregnancy. In control studies the same analyses were carried out in newborns from untreated rats. At birth (day 0), treated animals exhibited a proliferated, pleiomorphic peroxisomal population (higher Vv, NA and D, and a spread distribution of profile diameter with respect to the controls). In the subsequent two days, many peroxisomes disappeared (decrease of Vv and NA to values even lower than controls), with a persisting high pleiomorphism (no change of D and diameter distribution) in residual ones. Starting from day 3, and up to day 6, larger peroxisomes were no longer detectable in test animals, and a significant, not pleiomorphic proliferation took place (D and diameter distributions strictly comparable to the controls and progressively increasing Vv and NA). The correlation analysis validated these morphological results, from which it can be surmised that the postnatal peroxisome recovery period consists of a destructive phase followed by a proliferative one. The possible mechanism(s) of disposal of the excess of drug-induced peroxisomes are discussed.     

Changes to the integral membrane protein composition of mouse liver peroxisomes in response to the peroxisome proliferators clofibrate,Wy-14,643 and Di(2-ethyl-hexyl)phthalate

Peroxisomes were purified from livers of control mice and from mice treated with three agents which induce proliferation of hepatic peroxisomes — namely two structurally unrelated hypolipidemic drugs, clofibrate (ethyl--p-chlorophenoxyisobutyrate) and Wy-14,643 (4-chloro-6[2,3-xylidino)-2-pyrimidinylthio] acetic acid), and a plasticizer, DEHP (di-(2-ethylhexyl)phthalate).Membranes were isolated from these purified peroxisomes and analysed by SDS-polyacrylamide gel electrophoresis. All membranes which were tested, displayed two predominant integral membrane proteins of apparent molecular weights of 68 kDa and 70 kDa respectively, as well as a number of minor components. Treatment of animals with clofibrate, Wy-14,643 and DEHP was observed to result in each case in an increased proportion of the 70 kDa protein in the peroxisomal membranes. These treatments also resulted in increased peroxisomal fatty acid oxidation in livers and an increase in the proportion of catalase activity in the cytosolic fraction of liver cells.These results have been discussed in relation to alterations in the molecular composition of the membranes, the mechanisms of peroxisome proliferation and the inducibility of peroxisomal membrane proteins.     

The effect of clofibrate on amphibian hepatic peroxisomes.

Liver peroxisomes of two anuran amphibian species, Rana esculenta and Xenopus laevis, were studied in untreated and in clofibrate-treated adults by means of complementary technical approaches, ie, ultrastructural cytochemistry, cell fractionation and marker enzyme activity assays. In untreated adults, hepatic peroxisomes were found to be very scarce in Xenopus when compared to Rana. Activities of catalase, D-amino acid oxidase and of the three first enzymes of the peroxisomal beta-oxidation system were detected in the light mitochondrial fractions enriched in peroxisomes and prepared from livers of both species. Administration of clofibrate at a daily dose level of 60 mg (Rana) and 90 mg (Xenopus) during ten days induced a drastic peroxisome proliferation in Rana hepatocytes but had no visible effect on the hepatic peroxisomal population of Xenopus. The catalase activity and the peroxisomal beta-oxidation system of liver cells were enhanced in Rana as well as in Xenopus. The hepatic D-amino acid oxidase specific activity was increased in Rana whereas it remained rather constant in Xenopus. Taking advantage of the behaviors of Rana and Xenopus hepatic peroxisomes, the molecular mechanisms of clofibrate induction are now investigated in the target liver cells of the two amphibian species.     

The effect of clofibrate on the phospholipid composition of the peroxisomal membranes in mouse liver

Membranes were prepared from peroxisomes which had been isolated from the livers of normal mice and from mice treated with clofibrate (a hypolipidemic drug and peroxisome proliferator). Phospholipid analysis of these membranes revealed that clofibrate treatment resulted in a decrease in the membrane content of phosphatidylcholine, the most abundant phospholipid, and a concomitant increase in the amount of lysophosphatidylcholine, this latter component reaching a level of almost 6% of the total membrane phospholipid. The concentrations of other phospholipids in these membranes were not significantly altered. The parallel analysis of microsomal membranes demonstrated an analogous increase in the level of lysophosphatidylcholine following clofibrate treatment. In control experiments with microsomal membranes employing quinacrine, an inhibitor of phospholipase A2, the increased lysophosphatidylcholine concentration was still observed in clofibrate-treated animals. As well, a decrease in the proportion of microsomal phosphatidylcholine with clofibrate treatment was seen when quinacrine was used. Fatty acid analysis of the phosphatidylcholines from peroxisomal membranes showed some minor changes, including an increase in one component tentatively identified as docosahexaenoic acid, in clofibrate-treated animals. Overall, these data demonstrate that clofibrate causes a marked perturbation of the phospholipid composition of peroxisomal membranes, and are interpreted as indicating that the main site of action of the drug is the deacylation-reacylation cycle between phosphatidylcholine and lysophosphatidylcholine.     

The involvement of selenium in peroxisome proliferation caused by dietary administration of clofibrate to rats.

The effects of dietary treatment with clofibrate (0.5% w/w for 10 days) on the livers of selenium-deficient male rats were examined. The peroxisome proliferation (as determined by electron microscopy) in the livers of selenium-deficient animals was much less pronounced than in the case of selenium-adequate rats and no increase in peroxisomal fatty acid beta-oxidation (assayed both as antimycin-insensitive palmitoyl-CoA oxidation and lauroyl-CoA oxidase activity) was observed in the deficient animals. On the other hand, in selenium-deficient rats clofibrate caused increases in the specific activity of microsomal lauric acid omega- and omega-1-hydroxylation and an apparent change in mitochondrial size, seen as a redistribution of mitochondria from the 600 x g(av) pellet to the 10,000 x g(av) pellet, which were approximately 50% as great as the corresponding effects on control animals. Obviously, then, these three different effects of clofibrate are not strictly coupled and may involve at least partially distinct underlying mechanisms. Initial experiments demonstrated that peroxisome proliferation could be obtained by exposing primary hepatocyte cultures derived from selenium-deficient rats to clofibric acid (an in vivo hydrolysis product of clofibrate which is the proximate peroxisome proliferator), nafenopin or mono(2-ethylhexyl)phthalate. This finding suggests that selenium deficiency does not have a direct influence on the basic process(es) underlying peroxisome proliferation, but rather has indirect effects, influencing, for example, the pharmacokinetics of clofibrate and/or hormonal factors.     

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.     

Peroxisome proliferation and oxidative stress mediated by activated oxygen species in plant peroxisomes

The existence of a relationship between clofibrate-induced peroxisome proliferation and oxidative stress mediated by activated oxygen species was studied in intact peroxisomes purified from Pisum sativum L. plants. Incubation of leaves with 1 mM clofibrate produced a remarkable increase in the peroxisomal activity of acyl-CoA oxidase and, to a lesser extent, of xanthine oxidase, whereas there was a nearly complete loss of catalase activity and a decrease in Mn-superoxide dismutase. Ultrastructural studies of intact leaves showed that clofibrate induced a five- and twofold proliferation of the peroxisomal and mitochondrial populations, respectively, in comparison with those in control leaves. Prolonged incubation with clofibrate produced considerable alterations in the ultrastructure of cells. In peroxisomal membranes, the NADH-induced generation of O2- radicals, as well as the lipid peroxidation of membranes, increased as a result of treatment of plants with clofibrate. In intact peroxisomes treated with this hypolipidemic drug, the H2O2 concentration was higher than in peroxisomes from control plants. These results demonstrate that clofibrate stimulates the production of activated oxygen species (O2- and H2O2) inside peroxisomes, as well as the lipid peroxidation of peroxisomal membranes. This effect is concomitant with a decrease of catalase and Mn-SOD activities, the main peroxisomal enzymatic defenses against H2O2 and O2-, and indicates that in the toxicity of clofibrate, at the level of peroxisomes, an oxidative stress mechanism mediated by activated oxygen species is involved.     

Partial purification and immunoreactivity of an 80000-molecular-weight polypeptide associated with peroxisome proliferation in rat liver

Hypolipidaemic drugs and industrial plasticizers such as di-(2-ethylhexyl) phthalate, which cause proliferation of hepatic peroxisomes, also cause an increase in an 80000-mol.wt. polypeptide in the liver of rats and mice. This polypeptide has been designated as PPA-80 (PPA, for peroxisome-proliferation-associated; 80 for 80000mol.wt.). The polypeptide PPA-80 was purified to over 90% purity from livers of rats treated with the peroxisome proliferators Wy-14,643, nafenopin, tibric acid and clofibrate by a single-step preparative sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic procedure. The antibodies raised against the PPA-80 polypeptide isolated from livers of rats treated with Wy-14,643 cross-reacted with polypeptide PPA-80 purified from the livers of rats treated with Wy-14,643, as well as from the livers of rats treated with nafenopin, tibric acid and clofibrate. The anti-(polypeptide PPA-80) antibodies did not cross-react with catalase, a marker enzyme for peroxisomes, or with NADPH–cytochrome P-450 reductase, which has the same approximate mol.wt., 80000. The intensity of immunoprecipitin bands formed with microsomal, large-particle and postnuclear fractions from livers of animals pretreated with peroxisome proliferators was significantly greater compared with equal amounts of protein from corresponding fractions obtained from control animals, suggesting that these agents all enhance the synthesis of the same 80000-mol.wt. polypeptide. Although the polypeptide PPA-80 was increased in the postnuclear, large-particle and microsomal fractions of livers of rats pretreated with peroxisome proliferators, the relative abundance of this peptide in the peroxisome-rich light-mitochondrial fraction and its lack in highly purified mitochondrial fractions suggest the localization of this polypeptide in peroxisomes and/or microsomal fraction. Additional studies are needed to establish unequivocally the subcellular localization of the polypeptide PPA-80 and to ascertain if this polypeptide is identical with the multi-functional protein displaying enoyl-CoA hydratase and β-hydroxyacyl-CoA dehydrogenase activities that was purified by Osumi & Hashimoto [(1979) Biochem. Biophys. Res. Commun. 89, 580–584].     

Association of the liver peroxisomal fatty acyl-CoA beta-oxidation system with the synthesis of bile acids

The association of liver peroxisomal fatty acyl-CoA beta-oxidizing system (FAOS) with the synthesis of bile acids was investigated. When rats were given clofibrate, a peroxisome proliferator and stimulator of peroxisomal FAOS, the biosynthesis of bile acids was significantly increased. Di(2-ethylhexyl)phthalate, another peroxisome proliferator, also increased the biosynthesis of bile acids. On the other hand, administration of orotate, an inhibitor of mitochondrial FAOS activity, did not affect the biosynthesis. It is known that fatty acyl-CoA oxidase [EC 1.3.99.3] in peroxisomal FAOS conjugates with catalase [EC 1.11.1.6]. When the catalase activity of liver peroxisomes was irreversibly inhibited by administration of 3-amino-1,2,4-triazole (amino-triazole), the biosynthesis of bile acids was suppressed to about one-third, and the serum cholesterol level was increased. However, the bile acid components of the bile obtained from aminotriazole-treated rats were not essentially different from those of control rats, and no accumulation of intermediates of bile acid synthesis was found in this experiment. Peroxisomal FAOS activity of the liver from amino-triazole-treated rats was considerably lower than that of control liver. The above results indicate that liver peroxisomes play a role in the biosynthesis of bile acids in vivo.     

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.     

Effect of clofibrate application on morphology and enzyme content of liver peroxisomes.

Male albino rats (Sprague Dawley) were fed for 2-6 weeks on a diet containing 0.75% clofibrate. Liver cell fractions obtained from these animals were assayed for peroxisomal enzymes. In the cell homogenate the catalase activity was doubled, whereas the activity of urate oxidase was found to be only slightly depressed. The activity of carnitine acetyltransferase increased several times. In liver peroxisomes purified by isopycnic gradient centrifugation the specific activity of urate oxidase decreased appreciably showing that peroxisomes formed under the proliferative influence of clofibrate are not only modified with respect to their morphological characteristics but also to their enzymic equipment. This is also obvious from the changes in peroxisomal carnitine acetyltransferase activity which was enhanced by clofibrate to more than the fivefold amount. In purified mitochondria this enzyme was even more active: clofibrate advances both, the peroxisomal and the mitochondrial moiety of carnitine acetyltransferase. Morphological and cytochemical studies showed an increase in the number of microbodies and as compared to the controls microbodies were lying in groups more frequently. Small particles located closely adjacent to "normal" sized peroxisomes were found particularly after short feeding periods. While the number of coreless microbodies increased studies gave no clear evidence for an increase in marked shape irregularities of the peroxisomes.     

Isolation and Characterization of Rat and Human cDNAs Encoding a Novel Putative Peroxisomal Enoyl-CoA Hydratase

We have used a PCR-based subtractive hybridization method to identify upregulated cDNAs in the livers of rats treated with a peroxisome proliferator [clofibrate or di(2-ethylhexyl) phthalate]. After four rounds of subtractive hybridization 62 differentially hybridizing clones were partially sequenced and analyzed by sequence homology searching. Of 62, 49 were identical to 14 different upregulated rat sequences in the databank (mostly genes encoding microsomal or peroxisomal enzymes), 4 of 62 were fragments of three previously unknown genes, and 9 of 62 were false positives. Two of the unknown fragments hybridized to a single novel cDNA that was found to be more than 20-fold induced by both peroxisome proliferators. The 36-kDa predicted protein product of this cDNA shows a high degree of sequence homology to enoyl-CoA hydratases of several different species and has a C-terminal peroxisomal targeting sequence. An epitope-tagged protein product of a full-length cDNA was targeted to peroxisomes in a human cell line. We named this gene, which encodes an apparent peroxisomal enoyl-CoA hydratase, ECH1. We have also identified human ECH1 cDNA and mapped its structural gene to 19q13, 3′ to the ryanodine receptor, by hybridization to somatic cell hybrid DNA and chromosome 19-specific cosmid arrays. Possible roles for the ECH1 protein product in peroxisomal β-oxidation are discussed.     

Morphometric analysis of the ultrastructural changes in rat liver induced by the peroxisome proliferator SaH 42-348

The changes occurring in hepatocytes of F-344 male rats during a 3-wk treatment with a hypolipidemic agent, 1-methyl-4-piperidyl-bis [p- chlorophenoxy]acetate (SaH 42-348), have been evaluated by morphometric and biochemical methods. The twofold increase in liver weight resulted from a significant increase in hepatocyte cytoplasm as well as a moderate increase in the number of liver cells. The peroxisome population and SER played an overwhelming part in the hypertrophy of hepatocytic cytoplasm. The relative volume and the surface density of peroxisomes volume resulted from an increased ninefold and sevenfold, respectively. The increase in the collective peroxisome volume resulted from an increase in both the number and the average volume of peroxisomes. The SER also demonstrated a substantial increase in these values. The relative volume and surface density of mitochondria were not significantly altered in comparison to controls, while these values for RER decreased onefold. Studies on the lobular distribution of cytoplasmic organelles before and during treatment revealed that the relative volume and surface density of peroxisomes and SER increased from periportal to centrilobular cells of the hepatic lobule, whereas mitochondrial values decreased from periportal to centrilobular cells. The RER values were fairly constant in different parts of the hepatic lobule. The increase in peroxisome and SER volume and surface area was first evident within the first 3 days of SaH 42-348 treatment and these values continued to increase, reaching a steady state within 2 wk. The time course of increase in catalase and carnitine acetyltransferase activities correlated with the morphometric data on the peroxisomes. After cessation of SaH 42-348 treatment, the peroxisome values decreased rapidly within the first 3 days and reached control levels within 1 wk. Moderate reduction in SER values occurred after withdrawal of the drug, but these values remained higher than controls even after 2 wk, suggesting that the reduction in the amount of circulating peroxisome proteins may result in empty SER channels. On the 4th day of drug withdrawal a significant increase in the relative volume and surface density of lysosomes was observed, suggesting that these organelles may play some part in the removal of cellular membranes. However, the rapid reduction in peroxisome values after SaH 42-348 withdrawal appears to be due to cessation of enhanced peroxisome protein synthesis.     

Liver and kidney peroxisomes in lactating rats and their pups after treatment with ciprofibrate. Biochemical and morphometric analysis.

We administered the hypolipidemic drug ciprofibrate to lactating rats and examined the enzymatic content and ultrastructural features of liver and kidney peroxisomes, both in treated animals and in their pups. The peroxisomal morphometric parameters, in particular, were measured in specimens submitted to the cytochemical reaction for the marker enzyme catalase. In liver of treated rats, the activities of peroxisomal enzymes involved in the fatty acid catabolism were significantly increased, while D-amino acid oxidase activity was lower than in controls; increments were also found in relative volume and pleiomorphism degree of the peroxisomal compartment, where a catalase dilution was supposed to occur. In the kidney, the treatment induced generalized increases of all examined enzymes; values significantly higher than controls were found in peroxisomal relative volume and numerical density, while the peroxisomal mean diameter practically did not change. The two organs, moreover, were affected by the drug in an age-dependent way, the pups being more responsive than the adults. The organ- and age-specific responses to the drug are interpreted as possibly related to the tissue-specific distribution of the peroxisomal proliferator activated receptor isotypes.     

Lysosomal involvement in the removal of clofibrate-induced rat liver peroxisomes. A biochemical and morphological analysis

Peroxisomal proliferators induce in rodents hepatic hyperplasia and hypertrophy; the significant increase in the peroxisomal population is accompanied by specific and reversible induction of some peroxisomal enzymes. In suckling rats born from clofibrate-treated mothers, a massive removal of proliferated organelles occurs within 3 days of recovery. In the present paper we examined the early stages of the recovery period in liver of male rats treated with clofibrate for 5 days. The lysosomal involvement in the removal of drug-induced peroxisomes was investigated under physiological conditions, ie in the absence of inhibitors of the autophagic process. Biochemical results indicate that peroxisomal β-oxidation, but not catalase activity, returns to the control values within the examined period. Total acid phosphatase activity is not affected by clofibrate treatment, but following fractionation on a linear density gradient the lysosomal marker enzyme activity is shifted towards lower density values, particularly at day 1 and 2 of recovery. This class of organelles possibly represents lysosomes involved in active autophagic processes. Acid phosphatase cytochemistry shows an increase of lysosome number at day 1 of recovery. Combination of acid phosphatase cytochemistry either with catalase cytochemistry or with catalase immunogold labelling allows to reveal organelles containing both marker enzymes. These results strongly support the involvement of autophagic processes in the removal of proliferated peroxisomes.     

Rat liver peroxisomes catalyze the beta oxidation of fatty acids

Peroxisomes were purified by differential and equilibrium density centrifugation from the livers of rats treated with clofibrate to enhance their peroxisomal system of fatty acid oxidation. These purified peroxisomes were tested for the presence of crotonase, beta-hydroxybutyryl-CoA dehydrogenase and thiolase using spectroscopic techniques that utilize the characteristic absorption bands of the appropriate 4-carbon acyl-CoA substrates. All three enzymes were found. Analysis of the fractions from equilibrium density centrifugation revealed major peaks of these enzyme activities in peroxisomes and excluded contamination by mitochondria as an explanation of the results. In the presence of excess CoA the purified peroxisomes oxidized palmitoyl-CoA to acetyl-CoA, and reduced NAD, with a 1:5:5 stoichiometry. The peroxisomes were inactive with butyryl-CoA and less active with octanoyl-CoA than with lauroyl-CoA or palmitoyl-CoA; they appear specialized for the beta oxidation of long chain fatty acids.     

Peroxisomal proliferation induced by treatment with clofibrate in a patient with a peroxisomal disease

The induction of peroxisomal proliferation in liver parenchymal cells of rats fed a diet containing clofibrate, a hypolipidemic drug, is a well-established event. However, the available data on human hepatocytes in vivo and in vitro indicate that agents that induce peroxisomal proliferation in rats and mice have no effect on human liver cells. The authors are reporting the case of a patient with clinical and laboratory diagnosis of X-linked-adrenoleukodistrophy. In an initial liver biopsy, a reduced volume fraction of peroxisomes was found (Vv.=.012) after a morphometric analysis, initiating treatment with clofibrate at a dose of 1.5 g/d. The administration of clofibrate was maintained for 7 yr. Liver biopsies were taken after 2, 4, and 7 yr, to follow the peroxisomal response. Results demonstrated a 500% increase in peroxisomal Vv. (.060) after 2 yr of treatment, compared with the pretreatment Vv. In subsequent biopsies, the peroxisomal Vv. value was maintained at 225 and 183% increases above the pretreatment biopsy (.027 and .022, respectively).     

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.     

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.     

Effect of clofibrate treatment on carnitine acyltransferases in different subcellular fractions of rat liver

The subcellular distribution of carnitine acetyl-, octanoyl-, and palmitoyltransferase in the livers of normal and clofibrate-treated male rats was studied with isopycnic sucrose density gradient fraction.In normal liver 48% of total carnitine acetyltransferase activity was peroxisomal, 36% of the activity located in mitochondria and 16% in a membranous fraction containing microsomes. Carnitine octanoyltransferase and carnitine palmitoyltransferase were confined almost totally (77–81%) to mitochondria in normal liver.Clofibrate treatment increased the total activity of carnitine acetyltransferase over 30 times, whereas the total activities of the other two transferases were increased only 5-fold.From the three different subcellular carnitine acetyltransferases the mitochondrial one was not responsive to clofibrate treatment, i.e. the rise in mitochondrial activity was over 70-fold as contrasted to the 6- and 14-fold rises in peroxisomal and microsomal activities, respectively. After treatment mitochondria contained 79% of total activity.It is concluded that the clofibrate-induced increase of carnitine acetyltransferase activity is not due to the peroxisomal proliferation that occurs during clofibrate treatment. The rise in peroxisomal activity contributed only 8% to the total increase.After clofibrate treatment the greatest part of carnitine octanoyl- and palmitoyltrnasferase activities were located in mitochondria but a considerable amount of both activities was found also in the soluble fraction of liver.