Existence of acetyl-CoA-dependent chain elongation system in hepatic peroxisomes of rat: effects of clofibrate and di-(2-ethylhexyl)phthalate on the activity

The acetyl-CoA-dependent elongation of medium-chain acyl-CoA in the presence of pyridine nucleotide was studied in rat liver. The activity was increased by the administration of peroxisome proliferators, clofibrate and di-(2-ethylhexyl)phthalate, and the change was more remarkable in peroxisomes than in mitochondria. Addition of 0.01% Triton X-100 to the incubation mixture caused an increase in the mitochondrial activity, whereas the peroxisomal activity did not increase significantly. The pH optimum for the peroxisomal activity was in the range of pH 6.5-7.0 and that for the mitochondrial activity was pH 7.5-8.0. The specificities of primer chain length in both organelles were almost the same, and octanoyl-CoA was the preferred substrate. Peroxisomal activity was completely inhibited by the addition of 1 mM N-ethylmaleimide or 1 mM p-hydroxymercuribenzoic acid, while the activity did not change on the addition of 1 mM KCN or an antibody to acyl-CoA oxidase, the first enzyme of the peroxisomal beta-oxidation system. The activity of enoyl-CoA reductase, which catalyzes the last step of the elongation system, was also detected in peroxisomes, although the main activity was localized in microsomes. When the liver peroxisomal fraction of clofibrate-treated rats was incubated with a mixture of octanoyl-CoA, acetyl-CoA, NADH, NADPH, and Triton X-100 in a buffer system, dodecanoyl-CoA was detected as the main product by radio-gas chromatography. On the other hand, the elongation activity was decreased greatly by the addition of NAD+ into the mixture. These results indicate that (i) peroxisomes have activity to elongate medium chain acyl-CoA; (ii) the peroxisomal elongation system may consist of the reverse reaction of the beta-oxidation system except for the last step, which is catalyzed by enoyl-CoA reductase; and (iii) the peroxisomal elongation system is less active than the beta-oxidation system under physiological conditions.     

A novel acyl-CoA oxidase that can oxidize short-chain acyl-CoA in plant peroxisomes

Short-chain acyl-CoA oxidases are beta-oxidation enzymes that are active on short-chain acyl-CoAs and that appear to be present in higher plant peroxisomes and absent in mammalian peroxisomes. Therefore, plant peroxisomes are capable of performing complete beta-oxidation of acyl-CoA chains, whereas mammalian peroxisomes can perform beta-oxidation of only those acyl-CoA chains that are larger than octanoyl-CoA (C8). In this report, we have shown that a novel acyl-CoA oxidase can oxidize short-chain acyl-CoA in plant peroxisomes. A peroxisomal short-chain acyl-CoA oxidase from Arabidopsis was purified following the expression of the Arabidopsis cDNA in a baculovirus expression system. The purified enzyme was active on butyryl-CoA (C4), hexanoyl-CoA (C6), and octanoyl-CoA (C8). Cell fractionation and immunocytochemical analysis revealed that the short-chain acyl-CoA oxidase is localized in peroxisomes. The expression pattern of the short-chain acyl-CoA oxidase was similar to that of peroxisomal 3-ketoacyl-CoA thiolase, a marker enzyme of fatty acid beta-oxidation, during post-germinative growth. Although the molecular structure and amino acid sequence of the enzyme are similar to those of mammalian mitochondrial acyl-CoA dehydrogenase, the purified enzyme has no activity as acyl-CoA dehydrogenase. These results indicate that the short-chain acyl-CoA oxidases function in fatty acid beta-oxidation in plant peroxisomes, and that by the cooperative action of long- and short-chain acyl-CoA oxidases, plant peroxisomes are capable of performing the complete beta-oxidation of acyl-CoA.     

Differential effects of freezing on total hepatic peroxisomal fatty acid oxidation and on the rate-limiting enzyme, acyl-CoA oxidase

Fatty acid oxidation defects can be acutely fatal, leading to the collection of tissues which are frozen for future analysis. Since peroxisomes can also oxidize long-chain fatty acids, differentiation of the contributions from the peroxisome as opposed to the mitochondria is important. We studied the effects of freezing and storage of rat livers on peroxisomal and mitochondrial beta-oxidation as measured by cyanide sensitivity of the oxidation of [1-14C]oleoyl-CoA to 14CO2 and acid-soluble labeled products. In addition, we examined the effects of freezing and storage on the rate-limiting enzyme for peroxisomal beta-oxidation, acyl-CoA oxidase, by the H2O2 generation method. Marked reduction in the oxidation of [1-14C]oleoyl-CoA was found for both peroxisomal and mitochondrial systems upon freezing at -18 or -70 degrees C for 2 days which declined further on storage at these temperatures for 12 weeks. Loss of activity after freezing was greater for the mitochondrial than the peroxisomal beta-oxidation system. By contrast, acyl-CoA oxidase activity was resistant to these changes, maintaining prefrozen activities despite storage for 12 weeks. The contribution of the peroxisomal system to beta-oxidation was 32% of the total rate of oxidation of [1-14C]oleoyl-CoA in the rat liver. These findings indicate that the contributions of the peroxisomal system to total fatty acid oxidation may be considerable, that freezing of the liver results in drastic reduction in enzyme activities of both peroxisomal as well as mitochondrial beta-oxidation, but that the rate-limiting enzyme of the peroxisomal system, acyl-CoA oxidase, retains full activity despite freezing and storage.     

Fatty acid metabolism in liver of rats treated with hypolipidemic sulphur-substituted fatty acid analogues

The purpose of this study was to investigate early biochemical changes and possible mechanisms via which alkyl(C12)thioacetic acid (CMTTD, blocked for beta-oxidation), alkyl(C12)thiopropionic acid (CETTD, undergo one cycle of beta-oxidation) and a 3-thiadicarboxylic acid (BCMTD, blocked for both omega- (and beta-oxidation) influence the peroxisomal beta-oxidation in liver of rats. Treatment of rats with CMTTD caused a stimulation of the palmitoyl-CoA synthetase activity accompanied with increased concentration of hepatic acid-insoluble CoA. This effect was already established during 12-24 h of feeding. From 2 days of feeding, the cellular level of acid-insoluble CoA began to decrease, whereas free CoASH content increased. Stimulation of [1-14C]palmitoyl-CoA oxidation in the presence of KCN, palmitoyl-CoA-dependent dehydrogenase (termed peroxisomal beta-oxidation) and palmitoyl-CoA hydrolase activities were revealed after 36-48 h of CMTTD-feeding. Administration of BCMTD affected the enzymatic activities and altered the distribution of CoA between acid-insoluble and free forms comparable to what was observed in CMTTD-treated rats. It is evident that treatment of peroxisome proliferators (BCMTD and CMTTD), the level of acyl-CoA esters and the enzyme activity involved in their formation precede the increase in peroxisomal and palmitoyl-CoA hydrolase activities. In CMTTD-fed animals the activity of cyanide-insensitive fatty acid oxidation remained unchanged when the mitochondrial beta-oxidation and carnitine palmitoyltransferase operated at maximum rates. The sequence and redistribution of CoA and enzyme changes were interpreted as support for the hypothesis that substrate supply is an important factor in the regulation of peroxisomal fatty acid metabolism, i.e., the fatty acyl-CoA species appear to be catabolized by peroxisomes at high rates only when uptake into mitochondria is saturated. Administration of CETTD led to an inhibition of mitochondrial fatty acid oxidation accompanied with a rise in the concentration of acyl-CoA esters in the liver. Consequently, fatty liver developed. The peroxisomal beta-oxidation was marginally affected. Whether inhibition of mitochondrial beta-oxidation may be involved in regulation of peroxisomal fatty acid metabolism and in development of fatty liver should be considered.     

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.     

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

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

Participation of peroxisomes in the metabolism of xenobiotic acyl compounds: comparison between peroxisomal and mitochondrial beta-oxidation of omega-phenyl fatty acids in rat liver

The peroxisomal beta-oxidation of omega-phenyl fatty acids (PFAs) as model compounds for xenobiotic acyl compounds was investigated. In isolated hepatocytes, omega-phenyllauric acid (PFA12) was chain-shortened to PFAs having an even number of carbon atoms in the acyl side chain. Associated with this reaction, H2O2 generation was observed, the rate of which was markedly enhanced by clofibrate treatment of rats. Also when using isolated peroxisomes, such a chain-shortening of PFA12 occurred, associated with stoichiometrical production of NADH and acetyl-CoA. The CoA-ester form of PFA12 as a substrate and NAD as a cofactor were required in this reaction, indicating the participation of peroxisomal beta-oxidation in the chain-shortening of PFA12. When using PFAs with various chain lengths, the rates of H2O2 generation measured as the peroxisomal beta-oxidation in isolated hepatocytes were similar to those with the corresponding fatty acids, whereas the rates of ketone body production measured as the mitochondrial beta-oxidation were much lower than that with any fatty acid examined. From the study with isolated mitochondria and purified enzymes, it was found that the mitochondrial beta-oxidation of PFAs was carnitine-dependent, and that the activities of carnitine palmitoyltransferase for PFA-CoAs are low. Moreover, the activities of acyl-CoA dehydrogenase for PFA-CoAs were lower than those for fatty acyl-CoAs, while the activities of acyl-CoA oxidase for PFA-CoAs were comparable to those for fatty acyl-CoAs. As a result, relatively long chain PFAs were hardly subjected to mitochondrial beta-oxidation. Based on the maximum enzyme activities of the beta-oxidation, which were measured by following acyl-CoA-dependent NAD reduction in isolated peroxisomes and O2 consumption in isolated mitochondria, about 60% of the beta-oxidation of PFA12 in the rat liver was peroxisomal. In clofibrate-treated rats, the value reached about 85%. From these results it is concluded that the peroxisome is one of the important sites of degradation of xenobiotic acyl compounds.     

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

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

Mitochondrial and Peroxisomal β-Oxidation of Stearic and Lignoceric Acids by Rat Brain

Crude subcellular fractions were prepared from adult rat brains by differential centrifugation of brain homogenates. Greater than 98% of the cellular mitochondrial marker enzyme activity sedimented in the heavy and light mitochondrial pellets, and less than 1% of the activity sedimented in microsomal pellets. Lysosomal marker enzyme activities mainly (71-78% of cellular activity) sedimented in the heavy and light mitochondrial pellets. Significant amounts of the lysosomal marker enzyme activity also sedimented in the crude microsomal pellets (9-13% of total) and high-speed supernatants (14-16% of total). The specific activities of microsomal and peroxisomal marker enzyme activities were highest in the crude microsomal pellets. Fractionation of the crude microsomal pellets on Nycodenz gradients resulted in the separation of the bulk of the remaining mitochondrial, lysosomal, and microsomal enzyme activities from peroxisomes. Fatty acyl-CoA synthetase activities separated on Nycodenz gradients as two distinct peaks, and the minor peak of the activities was in the peroxisomal enriched fraction. Fatty acid beta-oxidation activities also separated as two distinct peaks, and the activities were highest in the peroxisomal enriched fractions. Mitochondria were purified from the heavy mitochondrial pellets by Percoll density gradients. Fatty acyl-CoA synthetase and fatty acid beta-oxidation activities were present in both the purified mitochondrial and peroxisomal enriched fractions. Stearoyl-CoA synthetase activities were severalfold greater compared to lignoceroyl-CoA synthetase, and stearic acid beta-oxidation was severalfold greater compared to lignoceric acid beta-oxidation in purified mitochondrial and peroxisomal enriched fractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucagon and fasting do not activate peroxisomal fatty acid beta-oxidation in rat liver

In a study of the endocrine control of peroxisomes, the effects of acute glucagon treatment and fasting on hepatic peroxisomal beta-oxidation in rats have been investigated. The activity of the rate-limiting peroxisomal beta-oxidation enzyme, fatty acyl-CoA oxidase, was measured to determine whether activation of peroxisomal beta-oxidation could account for the increase in total hepatic fatty acid oxidation following acute glucagon exposure. Catalase, a peroxisomal enzyme not directly involved in beta-oxidation, was also measured as a control for total peroxisomal activity. No changes with acute glucagon treatment of intact animals were observed with either activity as measured in liver homogenates or partially purified peroxisomal fractions. These observations indicate the lack of acute control by glucagon of peroxisomal function at the level of total enzyme activity. Previous work on the effects of fasting on hepatic fatty acid beta-oxidation [H. Ishii, S. Horie, and T. Suga (1980) J. Biochem. 87, 1855-1858] suggested an enhanced role for the peroxisomal beta-oxidation pathway during starvation. It was found that the peroxisomal beta-oxidation system, as measured by fatty acyl-CoA oxidase activity, does increase with duration of fast when expressed on a per gram wet weight liver basis. However, when this activity is expressed as total liver capacity, a decline in activity with increasing duration of fast is observed. Furthermore, this decline in peroxisomal capacity parallels the decline in total liver capacity for citrate synthase, a mitochondrial matrix enzyme, and total liver protein. These data indicate that peroxisomal beta-oxidation activity is neither stimulated nor even preferentially spared from proteolysis during fasting.     

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.     

The involvement of fatty acid binding protein in peroxisomal fatty acid oxidation

Rat liver fatty acid-binding protein (FABP) can function as a fatty acid donor protein for both peroxisomal and mitochondrial fatty acid oxidation, since 14C-labeled palmitic acid bound to FABP is oxidized by both organelles. FABP is, however, not detected in peroxisomes and mitochondria of rat liver by ELISA. Acyl-CoA oxidase activity of isolated peroxisomes was not changed by addition of FABP or flavaspidic acid, an inhibitor of fatty acid binding to FABP, nor by disruption of the peroxisomal membranes. These data indicate that FABP may transfer fatty acids to peroxisomes, but is not involved in the transport of acyl-CoA through the peroxisomal membrane.     

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.     

Chlorpromazine and carnitine-dependency of rat liver peroxisomal beta-oxidation of long-chain fatty acids.

The enzyme targets for chlorpromazine inhibition of rat liver peroxisomal and mitochondrial oxidations of fatty acids were studied. Effects of chlorpromazine on total fatty acyl-CoA synthetase activity, on both the first and the third steps of peroxisomal beta-oxidation, on the entry of fatty acyl-CoA esters into the peroxisome and on catalase activity, which allows breakdown of the H2O2 generated during the acyl-CoA oxidase step, were analysed. On all these metabolic processes, chlorpromazine was found to have no inhibitory action. Conversely, peroxisomal carnitine octanoyltransferase activity was depressed by 0.2-1 mM-chlorpromazine, which also inhibits mitochondrial carnitine palmitoyltransferase activity in all conditions in which these enzyme reactions are assayed. Different patterns of inhibition by the drug were, however, demonstrated for both these enzyme activities. Inhibitory effects of chlorpromazine on mitochondrial cytochrome c oxidase activity were also described. Inhibitions of both cytochrome c oxidase and carnitine palmitoyltransferase are proposed to explain the decreased mitochondrial fatty acid oxidation with 0.4-1.0 mM-chlorpromazine reported by Leighton, Persico & Necochea [(1984) Biochem. Biophys. Res. Commun. 120, 505-511], whereas depression by the drug of carnitine octanoyltransferase activity is presented as the factor responsible for the decreased peroxisomal beta-oxidizing activity described by the above workers.     

Integral membrane polypeptides of rat liver peroxisomes: topology and response to different metabolic states

Rats were treated with clofibrate, a hypolipidemic drug, and with thyroxine. Both drugs which are known to cause peroxisome proliferation, and a concomitant increase in peroxisomal fatty acid beta-oxidation activity in liver increased one of the major integral peroxisomal membrane polypeptides (PMPs), with apparent molecular mass of 69-kDa, six- and twofold, respectively. On the other hand hypothyroidism caused a decrease in peroxisomal fatty acid beta-oxidation activity and considerably lowered the concentration of PMP 69 in the peroxisomal membrane. Two other PMPs with apparent molecular masses of 36 and 22 kDa were not influenced by these treatments. The PMPs with apparent molecular masses of 42, 28, and 26 kDa were shown to be derived from the 69-kDa polypeptide by the activity of a yet uncharacterized endogenous protease during isolation of peroxisomes. Limited proteolysis of intact peroxisomes using proteinase K and subtilisin further substantiated that some portion of the 69-kDa polypeptide extends into the cytoplasm. The 36- and the 22-kDa polypeptides were accessible to proteolytic attack to a much lower extent and, therefore, are supposed to be rather deeply embedded within the peroxisomal membrane. It is demonstrated that peroxisomal acyl-CoA synthetase, an integral PMP extending partially into the cytoplasm, and PMP 69 are not identical polypeptides. Comparison of the peroxisomal membrane with that of mitochondria and microsomes revealed that the 69- and 22-kDa polypeptides as well as the bifunctional protein of the peroxisomal fatty acid beta-oxidation pathway were specifically located only in peroxisomes. Considerable amounts of a polypeptide cross-reacting with the antiserum against the 36-kDa polypeptide were found in mitochondria.     

Metabolic aspects of peroxisomal beta-oxidation

In the course of the last decade peroxisomal beta-oxidation has emerged as a metabolic process indispensable to normal physiology. Peroxisomes beta-oxidize fatty acids, dicarboxylic acids, prostaglandins and various fatty acid analogues. Other compounds possessing an alkyl-group of six to eight carbon atoms (many substituted fatty acids) are initially omega-oxidized in endoplasmic reticulum. The resulting carboxyalkyl-groups are subsequently chain-shortened by beta-oxidation in peroxisomes. Peroxisomal beta-oxidation is therefore, in contrast to mitochondrial beta-oxidation, characterized by a very broad substrate-specificity. Acyl-CoA oxidases initiate the cycle of beta-oxidation of acyl-CoA esters. The next steps involve the bi(tri)functional enzyme, which possesses active sites for enoyl-CoA hydratase-, beta-hydroxyacyl-CoA dehydrogenase- and for delta 2, delta 5 enoyl-CoA isomerase activity. The beta-oxidation sequence is completed by a beta-ketoacyl-CoA thiolase. The peroxisomes also contain a 2,4-dienoyl-CoA reductase, which is required for beta-oxidation of unsaturated fatty acids. The peroxisomal beta-hydroxyacyl-CoA epimerase activity is due to the combined action of two enoyl-CoA hydratases. (For a recent review of the enzymology of beta-oxidation enzymes see Ref. 225.) The broad specificity of peroxisomal beta-oxidation is in part due to the presence of at least two acyl-CoA oxidases, one of which, the trihydroxy-5 beta-cholestanoyl-CoA (THCA-CoA) oxidase, is responsible for the initial dehydrogenation of the omega-oxidized cholesterol side-chain, initially hydroxylated in mitochondria. Shortening of this side-chain results in formation of bile acids and of propionyl-CoA. In relation to its mitochondrial counterpart, peroxisomal beta-oxidation in rat liver is characterized by a high extent of induction following exposure of rats to a variety of amphipathic compounds possessing a carboxylic-, or sulphonic acid group. In rats some high fat diets cause induction of peroxisomal fatty acid beta-oxidation and of trihydroxy-5 beta-cholestanoyl-CoA oxidase. Induction involves increased rates of synthesis of the appropriate mRNA molecules. Increased half-lives of mRNA- and enzyme molecules may also be involved. Recent findings of the involvement of a member of the steroid hormone receptor superfamily during induction, suggest that induction of peroxisomal beta-oxidation represents another regulatory phenomenon controlled by nuclear receptor proteins. This will likely be an area of intense future research. Chain-shortening of fatty acids, rather than their complete beta-oxidation, is the prominent feature of peroxisomal beta-oxidation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Postnatal development of peroxisomal and mitochondrial enzymes in rat liver.

Subcellular organellles from livers of rats three days prenatal to 50 weeks postnatal were separated on sucrose gradients. The peroxisomes had a constant density of 1.243 g/ml throughout the life of the animal. The density of the mitochondria changed from about 1.236 g/ml at birth to a constant value of 1.200 g/ml after two weeks. The peroxisomal and mitochondrial fatty acid beta-oxidation and the peroxisomal and supernatant activities of catalase and glycerol-3-phosphate dehydrogenase were measured at each age, as well as the peroxisomal core enzyme, urate oxidase, and the mitochondrial matrix enzyme, glutamate dehydrogenase. All of these activities were very low or undetectable before birth. Mitochondrial glutamate dehydrogenase and peroxisomal urate oxidase reached maximal activities per g of liver at two and five weeks of age, respectively. Fatty acid beta-oxidation in both peroxisomes and mitochondria and peroxisomal glycerol-3-phosphate dehydrogenase exhibited maximum activities per g of liver between one and two weeks of age before weaning and then decreased to steady state levels in the adult. Peroxisomal beta-oxidation accounted for at least 10% of the total beta-oxidation activity in the young rat liver, but became 30% of the total in the liver of the adult female and 20% in the adult male due to a decrease in mitochondrial beta-oxidation after two weeks of age. The greatest change in beta-oxidation was in the mitochondrial fraction rather than in the peroxisomes. At two weeks of age, four times as much beta-oxidation activity was in the mitochondria as in the peroxisomal fraction. Peroxisomal glycerol-3-phosphate dehydrogenase activity accounted for 5% to 7% of the total activity in animals younger than one week, but only 1% to 2% in animals older than one week. Up to three weeks of age, 85% to 90% of the liver catalase was recovered in the peroxisomes. The activity of peroxisomal catalase per g of rat liver remained constant after three weeks of age, but the total activity of catalase further increased 2.5- to 3-fold, and all of the increased activity was in the supernatant fraction.     

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

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