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.     

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.     

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.     

Effect of clofibrate on peroxisomal and mitochondrial beta-oxidation in chicken liver

The effect of a 0.25% clofibrate diet for 2 weeks on peroxisomal and mitochondrial beta-oxidation in chicken liver was studied. The activities of antimycin antimycin A-insensitive palmitoyl-CoA oxidation (peroxisomal beta-oxidation) and carnitine acetyltransferase increased about two-fold. The activities of palmitoyl-CoA-dependent O2 consumption (mitochondrial beta-oxidation) and carnitine palmitoyltransferase were also slightly activated by the administration of clofibrate, but not significant. Thus, clofibrate may be a typical drug which activates the peroxisomal beta-oxidation more than the mitochondrial one in various species. The effect of clofibrate on peroxisomal carnitine acetyltransferase was the same as that on the mitochondrial one in chicken liver. Serum lipids were not lowered, but hepatomegaly was observed in the present experiment with chicken.     

Purification and properties of peroxisomal carnitine palmitoyltransferase in chick embryo liver

Peroxisomal carnitine palmitoyltransferase was purified by solubilization using Tween 20 and KCl from the large granule fraction of the liver of clofibrate-treated chick embryo, DEAE-Sephacel and blue Sepharose CL-6B column chromatography. The peroxisomal carnitine palmitoyltransferase was an Mr 64,000 polypeptide; the mitochondrial carnitine palmitoyltransferase had a subunit molecular weight of 69,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The carnitine acetyltransferase was an Mr 64,000 polypeptide. Antibody against purified peroxisomal carnitine palmitoyltransferase reacted only with peroxisomal carnitine palmitoyltransferase, but not with mitochondrial carnitine palmitoyltransferase or carnitine acetyltransferase. In addition, anti-peroxisomal carnitine palmitoyltransferase reacted only with the protein in peroxisomes purified from chick embryo liver by sucrose density gradient centrifugation. Thus, it was confirmed that purified peroxisomal carnitine palmitoyltransferase was a peroxisomal protein. Compared with mitochondrial carnitine palmitoyltransferase, peroxisomal carnitine palmitoyltransferase was extremely resistant to inactivation by trypsin. The pH optimum of peroxisomal carnitine palmitoyltransferase was 8.5, differing from that of mitochondrial carnitine palmitoyltransferase. The Km value of peroxisomal carnitine palmitoyltransferase for palmitoyl-CoA (32 microM) was similar to that of the mitochondrial one, whereas those values for L-carnitine (140 microM), palmitoyl-L-carnitine (43 microM) and CoA (9 microM) were lower than those of mitochondrial carnitine palmitoyltransferase. Peroxisomal carnitine palmitoyltransferase exhibited similar substrate specificities in both the forward and reverse reactions, with the highest activity toward lauroyl derivatives. Furthermore, this enzyme showed relatively high affinities for long-chain acyl derivatives (C10-C16) and similar Km values (30-50 microM) for acyl-CoAs, acylcarnitine and CoA, and a constant Km value (approximately 150 microM) for carnitine. These results indicate that peroxisomal carnitine palmitoyltransferase played a role in the modulation of the intracellular CoA/long-chain acyl-CoA ratio at the hatching stage of chicken when long-chain fatty acids are actively oxidized in peroxisomes.     

Correlation between the cellular level of long-chain acyl-CoA, peroxisomal beta-oxidation, and palmitoyl-CoA hydrolase activity in rat liver. Are the two enzyme systems regulated by a substrate-induced mechanism?

Data obtained in earlier studies with rats fed diets containing high doses of peroxisome proliferators (niadenate, tiadenol, clofibrate, or nitotinic acid) are used to look for a quantitative relationship between peroxisomal beta-oxidation, palmitoyl-CoA hydrolase, palmitoyl-CoA synthetase and carnitine palmitoyltransferase activities, and the cellular concentration of their substrate and reaction products. The order of the hyperlipidemic drugs with regard to their effect on CoA derivatives and enzyme activities was niadenate greater than tiadenol greater than clofibrate greater than nicotinic acid. Linear regression analysis of long-chain acyl-CoA content versus palmitoyl-CoA hydrolase and peroxisomal beta-oxidation activity showed highly significant linear correlations both in the total liver homogenate and in the peroxisome-enriched fractions. A dose-response curve of tiadenol showed that carnitine palmitoyltransferase and palmitoyl-CoA synthetase activities and the ratio of long-chain acyl-CoA to free CoASH in total homogenate rose at low doses before detectable changes occurred in the peroxisomal beta-oxidation and palmitoyl-CoA hydrolase activity. A plot of this ratio parallelled the palmitoyl-CoA synthetase activity. The specific activity of microsomally localized carnitine palmitoyl-transferase was low and unchanged up to a dose where no enhanced peroxisomal beta-oxidation was observed, but over this dose the activity increased considerably so that the specific of the enzyme in the mitochondrial and microsomal fractions became comparable. The mitochondrial palmitoyl-CoA synthetase activity decreased gradually. The correlations may be interpreted as reflecting a common regulation mechanism for palmitoyl-CoA hydrolase and peroxisomal beta-oxidation enzymes, i.e., the cellular level of long-chain acyl-CoA acting as the metabolic message for peroxisomal proliferation resulting in induction of peroxisomal beta-oxidation and palmitoyl-CoA hydrolase activity. The findings are discussed with regard to their possible consequences for mitochondrial fatty acid oxidation and the conversion of long-chain acyl-L-carnitine to acyl-CoA derivatives.     

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.     

Developmental changes in the activities of peroxisomal and mitochondrial beta-oxidation in chicken liver

The activities of peroxisomal and mitochondrial beta-oxidation and carnitine acyltransferases changed during the process of development from embryo to adult chicken, and the highest activities of peroxisomal beta-oxidation, palmitoyl-CoA oxidase, and carnitine acetyltransferase were found at the hatching stage of the embryo. The profiles of these alterations were in agreement with those of the contents of triglycerides and free fatty acids in the liver. The highest activities of mitochondrial beta-oxidation and palmitoyl-CoA dehydrogenase were observed at the earlier stages of the embryo; then the activities decreased gradually from embryo to adult chicken. The ratio of activities of carnitine acetyltransferase in peroxisomes and mitochondria (peroxisomes/mitochondria) increased from 0.54 to 0.82 during the development from embryo to adult chicken. The ratio of activities of carnitine palmitoyltransferase decreased from 0.82 to 0.25 during the development. The affinity of fatty acyl-CoA dehydrogenase toward the medium-chain acyl-CoAs (C6 and C8) was high in the embryo and decreased with development, whereas the substrate specificity of fatty acyl-CoA oxidase did not change. The substrate specificity of mitochondrial carnitine acyltransferases did not change with development. The affinity of peroxisomal carnitine acyltransferases toward the long-chain acyl-CoAs (C10 to C16) was high in the embryo, but low in adult chicken.     

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

The subcellular distribution of carnitine acetyl-, octanoyl-, and palmitoyl- transferase in the livers of normal and clofibrate-treated male rats was studied with isopycnic sucrose density gradient fractionation. 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 most 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 palmitoyltransferase activities were located in mitochondria but a considerable amount of both activities was found also in the soluble fraction of liver.     

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.     

The effect of di(ethylhexyl)phthalate on fatty acid oxidation and carnitine palmitoyltransferase in various rat tissues

Male rats were fed a diet with or without 2% di(2-ethylhexyl)phthalate (DEHP) for 12 days. Total and peroxisomal oxidation rates of palmitic and arachidonic acid were increased in homogenates of liver and kidney after DEHP administration. The relative peroxisomal contribution to the total oxidation was only higher in liver. The activities of acyl-CoA oxidase and carnitine palmitoyltransferase were also higher in both tissues. Immunoblots showed that the increase of fatty acid oxidation was associated with a higher concentration of enzymes of peroxisomal and mitochondrial beta-oxidation. DEHP did not change total and peroxisomal fatty acid oxidation and activity of carnitine palmitoyltransferase of homogenates of heart and skeletal muscle. The cause for the tissue-specific response is discussed.     

Comparison of the carnitine acyltransferase activities from rat liver peroxisomes and microsomes

Carnitine acyltransferase activities for acetyl- and octanoyl-CoA (coenzyme A) occur in isolated peroxisomal, mitochondrial, and microsomal fractions from rat and pig liver. Solubility studies indicated that both peroxisomal carnitine acyltransferases were in the soluble matrix. In contrast, the microsomal carnitine acyltransferases were tightly associated with their membrane. The microsomal short-chain transferase, carnitine acetyltransferase, was solubilized and stabilized by extensive treatment of the membrane with 0.4 m KCl or 0.3 m sucrose in 0.1 m pyrophosphate at pH 7.5. The same treatment only partially solubilized the microsomal medium-chain transferase, carnitine octanoyltransferase.Although half of the total carnitine acetyltransferase activity in rat liver resides in peroxisomes and microsomes, previous reports have only investigated the mitochondrial activity. Transferase activity for acetyl- and octanoyl-CoA were about equal in peroxisomal and in microsomal fractions. A 200-fold purification of peroxisomal and microsomal carnitine acetyltransferases was achieved using O-(diethylaminoethyl)-cellulose and cellulose phosphate chromatography. This short-chain transferase preparation contained less than 5% as much carnitine octanoyltransferase and acyl-CoA deacylase activities. This fact, plus differences in solubility and stability of the microsomal transferase system for acetyl- and octanoyl-CoA indicate the existence of two separate enzymes: a carnitine acetyltransferase and a carnitine octanoyltransferase in peroxisomes and in microsomes.Peroxisomal and microsomal carnitine acetyltransferases had similar properties and could be the same protein. They showed identical chromatographic behavior and had the same pH activity profiles and major isoelectric points. They also had the same apparent molecular weight by gel filtration (59,000) and the same relative velocities and K_m values for several short-chain acyl-CoA substrates. Both were active with propionyl-, acetyl-, malonyl-, and acetyacetyl-CoA, but not with succinyl- and β-hydroxy-β-methylglutaryl-CoA as substrates.     

The membrane of peroxisomes in Saccharomyces cerevisiae is impermeable to NAD(H) and acetyl-CoA under in vivo conditions.

We investigated how NADH generated during peroxisomal beta-oxidation is reoxidized to NAD+ and how the end product of beta-oxidation, acetyl-CoA, is transported from peroxisomes to mitochondria in Saccharomyces cerevisiae. Disruption of the peroxisomal malate dehydrogenase 3 gene (MDH3) resulted in impaired beta-oxidation capacity as measured in intact cells, whereas beta-oxidation was perfectly normal in cell lysates. In addition, mdh3-disrupted cells were unable to grow on oleate whereas growth on other non-fermentable carbon sources was normal, suggesting that MDH3 is involved in the reoxidation of NADH generated during fatty acid beta-oxidation rather than functioning as part of the glyoxylate cycle. To study the transport of acetyl units from peroxisomes, we disrupted the peroxisomal citrate synthase gene (CIT2). The lack of phenotype of the cit2 mutant indicated the presence of an alternative pathway for transport of acetyl units, formed by the carnitine acetyltransferase protein (YCAT). Disruption of both the CIT2 and YCAT gene blocked the beta-oxidation in intact cells, but not in lysates. Our data strongly suggest that the peroxisomal membrane is impermeable to NAD(H) and acetyl-CoA in vivo, and predict the existence of metabolite carriers in the peroxisomal membrane to shuttle metabolites from peroxisomes to cytoplasm and vice versa.     

Properties of purified carnitine acyltransferases of mouse liver peroxisomes

The purpose of this study was to characterize the physical, kinetic, and immunological properties of carnitine acyltransferases purified from mouse liver peroxisomes. Peroxisomal carnitine octanoyltransferase and carnitine acetyltransferase were purified to apparent homogeneity from livers of mice fed a diet containing the hypolipidemic drug Wy-14,643 [( 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]-acetic acid). Both enzymes have a molecular weight of 60,000 and a similar pH optimum. Carnitine octanoyltransferase had a maximum activity for C6 moieties while the maximum for carnitine acetyltransferase was with C3 and C4 moieties. The apparent Km values were between 2 and 20 microM for the preferred acyl-CoA substrates, and the Km values for L-carnitine varied depending on the acyl-CoA cosubstrates used. The Hill coefficient, n, was approximately 1 for all acyl-CoAs tested, indicating Michaelis-Menten kinetics. Carnitine octanoyltransferase retained its maximum activity when preincubated with 5,5'-dithiobis-(2-nitrobenzoate) at pH 7.0 or 8.5. Neither carnitine octanoyltransferase nor carnitine acetyltransferase were inhibited by malonyl-CoA. The immunology of carnitine octanoyltransferase is discussed. These data indicate that peroxisomal carnitine octanoyltransferase and carnitine acetyltransferase function in vivo in the direction of acylcarnitine formation, and suggest that the concentration of L-carnitine could influence the specificity for different acyl-CoA substrates.     

Molecular cloning and expression of human carnitine octanoyltransferase: evidence for its role in the peroxisomal beta-oxidation of branched-chain fatty acids.

To study the putative role of human carnitine octanoyltransferase (COT) in the beta-oxidation of branched-chain fatty acids, we identified and cloned the cDNA encoding human COT and expressed it in the yeast Saccharomyces cerevisiae. Enzyme activity measurements showed that COT efficiently converts one of the end products of the peroxisomal beta-oxidation of pristanic acid, 4, 8-dimethylnonanoyl-CoA, to its corresponding carnitine ester. Production of the carnitine ester of this branched/medium-chain acyl-CoA within the peroxisome is required for its transport to the mitochondrion where further beta-oxidation occurs. In contrast, 4, 8-dimethylnonanoyl-CoA is not a substrate for carnitine acetyltransferase, another acyltransferase localized in peroxisomes, which catalyzes the formation of carnitine esters of the other products of pristanic acid beta-oxidation, namely acetyl-CoA and propionyl-CoA. Our results shed new light on the function of COT in fatty acid metabolism and point to a crucial role of COT in the beta-oxidation of branched-chain fatty acids.     

Carnitine-dependent transport of acetyl coenzyme A in Candida albicans is essential for growth on nonfermentable carbon sources and contributes to biofilm formation

In eukaryotes, acetyl coenzyme A (acetyl-CoA) produced during peroxisomal fatty acid beta-oxidation needs to be transported to mitochondria for further metabolism. Two parallel pathways for acetyl-CoA transport have been identified in Saccharomyces cerevisiae; one is dependent on peroxisomal citrate synthase (Cit), while the other requires peroxisomal and mitochondrial carnitine acetyltransferase (Cat) activities. Here we show that the human fungal pathogen Candida albicans lacks peroxisomal Cit, relying exclusively on Cat activity for transport of acetyl units. Deletion of the CAT2 gene encoding the major Cat enzyme in C. albicans resulted in a strain that had lost both peroxisomal and mitochondrion-associated Cat activities, could not grow on fatty acids or C(2) carbon sources (acetate or ethanol), accumulated intracellular acetyl-CoA, and showed greatly reduced fatty acid beta-oxidation activity. The cat2 null mutant was, however, not attenuated in virulence in a mouse model of systemic candidiasis. These observations support our previous results showing that peroxisomal fatty acid beta-oxidation activity is not essential for C. albicans virulence. Biofilm formation by the cat2 mutant on glucose was slightly reduced compared to that by the wild type, although both strains grew at the same rate on this carbon source. Our data show that C. albicans has diverged considerably from S. cerevisiae with respect to the mechanism of intracellular acetyl-CoA transport and imply that carnitine dependence may be an important trait of this human fungal pathogen.     

Stimulation of carnitine acetyltransferase in PC12 cells by nerve growth factor: Relationship to choline acetyltransferase stimulation

The activity of carnitine acetyltransferase (acetyl-CoA:L-carnitine O-acetyltransferase) was found to be at least 50-fold higher than that of choline acetyltransferase in PC12 cells. Nerve growth factor stimulated both enzymes in a parallel manner with respect to concentration of NGF and culture time. The stimulation of both enzymes was completely inhibited by 10 M 6-thioguanine, an inhibitor of protein kinase N. Results are discussed with reference to the hypothesis that the two enzymes may be functionally related in neuronal cells.     

Purification of heart and liver mitochondrial carnitine acetvltransferase

Heart and liver mitochondrial, as well as liver peroxisomal, carnitine acetyltransferase was purified to apparent homogeneity and some properties, primarily of heart mitochondrial carnitine acetyltransferase, were determined. Hill coefficients for propionyl-CoA are 1.0 for each of the enzymes. The molecular weight of heart mitochondrial carnitine acetyltransferase, determined by SDS-PAGE, is 62,000. It is monomeric in the presence of catalytic amounts of substrate. Polyclonal antibodies against purified rat liver peroxisomal carnitine acetyltransferase precipitate liver and heart mitochondrial and liver peroxisomal carnitine acetyltransferase, but not liver peroxisomal carnitine octanoyltransferase. Liver peroxisomes, mitochondria, and microsomes and heart mitochondria all give multiple bands on Western blotting with the antibody against carnitine acetyltransferase. Major protein bands occur at the molecular weight of carnitine acetyltransferase and at 33 to 35 kDa.     

Induction of fatty acid binding protein by peroxisome proliferators in primary hepatocyte cultures and its relationship to the induction of peroxisomal beta-oxidation

The induction of liver fatty acid binding protein (L-FABP) by the peroxisome proliferators bezafibrate and clofibrate was compared with the induction of peroxisomal (cyanide-insensitive) palmitoyl-CoA oxidation in cultured rat hepatocytes maintained on a substratum of laminin-rich (EHS) gel. This substratum was chosen because marked induction of both L-FABP and peroxisomal palmitoyl-CoA oxidation was effected by bezafibrate in hepatocytes supported on EHS gel, whereas only peroxisomal palmitoyl-CoA oxidation was induced in hepatocytes maintained on collagen-coated plates. In control cells on EHS, activity of peroxisomal palmitoyl-CoA oxidation remained stable, while L-FABP abundance declined with time, and L-FABP mRNA was undetectable after 5 days. In cultures exposed to bezafibrate or clofibrate, peroxisomal palmitoyl-CoA oxidation activity was induced earlier and more rapidly than L-FABP. When fibrates were withdrawn, peroxisomal palmitoyl-CoA oxidation declined rapidly, whereas L-FABP continued to increase. L-FABP induction was accompanied by a striking increase in mRNA specifying this protein. Tetradecylglycidic acid, an inhibitor of carnitine palmitoyltransferase I, effectively doubled peroxisomal palmitoyl-CoA oxidation activity. However, tetradecylglycidic acid markedly inhibited fibrate induction of L-FABP and peroxisomal palmitoyl-CoA oxidation but, unexpectedly, did not prevent the fibrate-induced proliferation of peroxisomes. Maximal induction of both L-FABP and peroxisomal palmitoyl-CoA oxidation was produced at a bezafibrate concentration in the culture medium (0.05 mM) much lower than that of clofibrate (0.3 mM). Also, bezafibrate, but not clofibrate, inhibited [1-14C]oleic acid binding to L-FABP with a Ki = 9.5 microM. We conclude that hepatocytes maintained on EHS gel provide an important tool for investigating the regulation of L-FABP. These studies show that the induction of peroxisomal beta-oxidation and L-FABP by peroxisome proliferators are temporally consecutive but closely related processes which may be dependent on a mechanism distinct from that which leads to peroxisome proliferation. Furthermore, the mechanism of action of the more potent peroxisome proliferator, bezafibrate, may be mediated, in part, by interaction of this agent with L-FABP.