Very long chain fatty acid beta-oxidation by rat liver mitochondria and peroxisomes

Crude mitochondrial fractions were isolated by differential centrifugation of rat liver homogenates. Subfractionation of these fractions on self-generating continuous Percoll gradients resulted in clearcut separation of peroxisomes from mitochondria. Hexacosanoic acid beta-oxidation was present mainly in peroxisomal fractions whereas hexacosanoyl CoA oxidation was present in the mitochondrial as well as in the peroxisomal fractions. The presence of much greater hexacosanoyl CoA synthetase activity in the purified preparations of microsomes and peroxisomes compared to mitochondria, suggests that the synthesis of coenzyme A derivatives of very long chain fatty acids (VLCFA) is limited in mitochondria. We postulate that a specific VLCFA CoA synthetase may be required to effectively convert VLCFA to VLCFA CoA in the cell. This specific synthetase activity is absent from the mitochondrial membrane, but present in the peroxisomal and the microsomal membranes. We postulate that substrate specificity and the subcellular localization of the specific VLCFA CoA synthetase directs and regulates VLCFA oxidation in the cell.     

3-Hydroxyacyl-CoA epimerases of rat liver peroxisomes and Escherichia coli function as auxiliary enzymes in the beta-oxidation of polyunsaturated fatty acids

The beta-oxidation of 2-trans,4-cis-decadienoyl-CoA, an assumed metabolite of linoleic acid, by purified enzymes from mitochondria, peroxisomes, and Escherichia coli was studied. 2-trans,4-cis-Decadienoyl-CoA is an extremely poor substrate of the beta-oxidation system reconstituted from mitochondrial enzymes. The results of a kinetic evaluation lead to the conclusion that in mitochondria 2-trans,4-cis-decadienoyl-CoA is not directly beta-oxidized, but instead is reduced by NADPH-dependent 2,4-dienoyl-CoA reductase prior to its beta-oxidation. Hence, the mitochondrial beta-oxidation of 2-trans,4-cis-decadienoyl-CoA does not require 3-hydroxyacyl-CoA epimerase, a conclusion which agrees with the finding that 3-hydroxyacyl-CoA epimerase is absent from mitochondria (Chu, C.-H., and Schulz, H. (1985) FEBS Lett. 185, 129-134). However, 2-trans,4-cis-decadienoyl-CoA can be slowly oxidized by the bifunctional beta-oxidation enzyme from rat liver peroxisomes, as well as by the fatty acid oxidation complex from E. coli. The observed rates of 2-trans,4-cis-decadienoyl-CoA degradation by these two multi-functional proteins were significantly higher than the values calculated according to steady-state velocity equations derived for coupled enzyme reactions. This is attributed to the direct transfer of L-3-hydroxy-4-cis-decenoyl-CoA from the active site of enoyl-CoA hydratase to that of 3-hydroxyacyl-CoA dehydrogenase on the same protein molecule. All observations together lead to the suggestion that the chain shortening of 2-trans,4-cis-decadienoyl-CoA in peroxisomes and in E. coli occurs simultaneously by two different pathways. The major pathway involves the NADPH-dependent 2,4-dienoyl-CoA reductase, whereas 3-hydroxyacyl-CoA epimerase functions in the metabolism of D-3-hydroxyoctanoyl-CoA which is formed via the minor pathway.     

beta-Oxidation of polyunsaturated fatty acids by rat liver peroxisomes. A role for 2,4-dienoyl-coenzyme A reductase in peroxisomal beta-oxidation

beta-Oxidation of unsaturated fatty acids was studied with isolated solubilized or nonsolubilized peroxisomes or with perfused liver isolated from rats treated with clofibrate. gamma-Linolenic acid gave the higher rate of beta-oxidation, while arachidonic acid gave the slower rate of beta-oxidation. Other polyunsaturated fatty acids (including docosahexaenoic acid) were oxidized at rates which were similar to, or higher than, that observed with oleic acid. Experiments with 1-14C-labeled polyunsaturated fatty acids demonstrated that these are chain-shortened when incubated with nonsolubilized peroxisomes. Spectrophotometric investigation of solubilized peroxisomal incubations showed that 2,4-dienoyl-CoA esters accumulated during peroxisomal beta-oxidation of fatty acids possessing double bond(s) at even-numbered carbon atoms. beta-Oxidation of [1-14C]docosahexaenoic acid by isolated peroxisomes was markedly stimulated by added NADPH or isocitrate. This fatty acid also failed to cause acyl-CoA-dependent NADH generation with conditions of assay which facilitate this using other acyl-CoA esters. These findings suggest that 2,4-dienoyl-CoA reductase participation is essential during peroxisomal beta-oxidation if chain shortening is to proceed beyond a delta 4 double bond. Evidence obtained using arachidionoyl-CoA, [1-14C]arachidonic acid, and [5,6,8,9,11,12,14,15-3H]arachidonic acid suggests that peroxisomal beta-oxidation also can proceed beyond a double bond positioned at an odd-numbered carbon atom. Experiments with isolated perfused livers showed that polyunsaturated fatty acids also in the intact liver are substrates for peroxisomal beta-oxidation, as judged by increased levels of the catalase-H2O2 complex on infusion of polyunsaturated fatty acids.     

Peroxisomal beta-oxidation of long-chain fatty acids possessing different extents of unsaturation.

Rates of peroxisomal beta-oxidation were measured as fatty acyl-CoA-dependent NAD+ reduction, by using solubilized peroxisomal fractions isolated from livers of rats treated with clofibrate. Medium- to long-chain saturated fatty acyl-CoA esters as well as long-chain polyunsaturated fatty acyl-CoA esters were used. Peroxisomal beta-oxidation shows optimal specificity towards long-chain polyunsaturated acyl-CoA esters. Eicosa-8,11,14-trienoyl-CoA, eicosa-11,14,17-trienoyl-CoA and docosa-7,10,13,16-tetraenoyl-CoA all gave Vmax. values of about 150% of that obtained with palmitoyl-CoA. The Km values obtained with these fatty acyl-CoA esters were 17 +/- 6, 13 +/- 4 and 22 +/- 3 microM respectively, which are in the same range as the value for palmitoyl-CoA (13.8 +/- 1 microM). Myristoyl-CoA gave the higher Vmax. (110% of the palmitoyl-CoA value) of the saturated fatty acyl-CoAs tested. Substrate inhibition was mostly observed with acyl-CoA esters giving Vmax. values higher than 50% of that given by palmitoyl-CoA.     

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.     

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.     

Effects of norepinephrine on the metabolism of fatty acids with different chain lengths in the perfused rat liver

The effects of norepinephrine on ketogenesis in isolated hepatocytes have been reported as ranging from stimulation to inhibition. The present work was planned with the aim of clarifying these discrepancies. The experimental system was the once-through perfused liver from fasted and fed rats. Fatty acids with chain lengths varying from 8-18 were infused. The effects of norepinephrine depended on the metabolic state of the rat and on the nature of the fatty acid. Norepinephrine clearly inhibited ketogenesis from long-chain fatty acids (stearate > palmitate > oleate), but had little effect on ketogenesis from medium-chain fatty acids (octanoate and laureate). With palmitate the decrease in oxygen uptake was restricted to the substrate stimulated portion; with stearate, the decrease exceeded the substrate stimulated portion; with oleate, oxygen uptake was transiently inhibited. Withdrawal of Ca²⁺ attenuated the inhibitory effects. ¹⁴CO₂ production from [1-¹⁴C]oleate was inhibited. Net uptake of the fatty acids was not affected by norepinephrine. In livers from fed rats, oxygen uptake and ketogenesis from stearate were only transiently inhibited. The conclusions are: (a) in the fasted state norepinephrine reduces ketogenesis and respiration by means of a Ca²⁺-dependent mechanism; (b) the degree of inhibition varies with the chain length and the degree of saturation of the fatty acids; (c) norepinephrine favours esterification of the activated long-chain fatty acids in detriment to oxidation; (d) in the fed state the stimulatory action of norepinephrine on glycogen catabolism induces conditions which are able to reverse inhibition of ketogenesis and oxygen uptake.     

Influence of chain length and unsaturation on the effects of fatty acids on phosphoglyceride biosynthesis in isolated rat and pig hepatocytes.

Hepatocytes isolated from rat or pig by collagenase perfusion were incubated with [3H]glcyerol and different albumin-bount fatty acids. Among C22 fatty acids docosahexaenoic acid stimulated phosphatidylethanolamine synthesis in rat hepatocytes most effectively. Addition of docosahexaenoic acid plus either palmitic or stearic acid resulted almost in the same stimulation whereas combinations of this acid with lauric or myristic acid had no effect. Lauric acid and myristic acid alone inhibited phosphatidylethanolamine synthesis. The chain length specificity for monoenoic fatty acids was similar, the hexadecenoic and octadecenoic acids (both cis and trans) being most stimulatory. The addition of 0.2 mM ethanolamine markedly stimulated phosphatidylethanolamine synthesis, but most effects of fatty acids were similar in its presence or absence.     

Distinct long chain and very long chain fatty acyl CoA synthetases in rat liver peroxisomes and microsomes

Mitochondria, peroxisomes, and microsomes were isolated from rat liver homogenates, and stearic acid and lignoceric acid beta-oxidation, as well as stearoyl CoA synthetase and lignoceroyl CoA synthetase activities in the three organelles, were compared. Stearic acid beta-oxidation in peroxisomes was sixfold greater compared to the oxidation in mitochondria. Lignoceric acid beta-oxidation, observed only in peroxisomes, was fivefold lower compared to stearic acid beta-oxidation. Stearoyl CoA synthetase was present whereas lignoceroyl CoA synthetase was absent in mitochondria. Stearoyl CoA synthetase and lignoceroyl CoA synthetase activities were present in microsomes and peroxisomes, but the activity of stearoyl CoA synthetase was several-fold greater compared to lignoceroyl CoA synthetase in both organelles. The differing responses to detergents and phospholipids of stearoyl CoA and lignoceroyl CoA synthetase activities in microsomes as well as peroxisomes indicated that each activity was catalyzed by a separate enzyme. Differences in detergent and phospholipid response were also noted when either stearoyl CoA or lignoceroyl CoA synthetase activity in one organelle was compared with the corresponding activity in the other organelle, suggesting that the same activity in different organelles may be catalyzed by separate enzyme proteins.     

Comparison of fatty acid oxidation in mitochondria and peroxisomes from rat liver

Oxygen uptake with succinate or palmitoyl-CoA as substrates can be measured in rat liver mitochondria that have been isolated by sucrose density gradient centrifugation providing the fractions are diluted with a 30 mM phosphate buffer rather than with an isotonic medium. Separate assay procedures were used to measure peroxisomal and mitochondrial β-oxidation of palmitoyl-CoA in the fractions of a sucrose gradient used to separate these organelles. A preliminary estimate of the ratio of palmitoyl-CoA oxidation by the mitochondrial fraction relative to the surviving peroxisomes from livers of male rats was 3.2.     

Activation and peroxisomal beta-oxidation of fatty acids and bile acid intermediates in liver from Bombina orientalis and from the rat

1. Bombina orientalis excretes mainly C27 bile acids: trihydroxycoprostanic and varanic acids. More than 90% of the trihydroxycoprostanic acid (THCA) present in the bile, was conjugated with taurine; varanic acid was present in the unconjugated form. 2. Trihydroxycoprostanoyl-CoA (THC-CoA) synthetase activity, required for the formation of the taurine conjugate, was present in the liver of Bombina orientalis. 3. Peroxisomal beta-oxidation, which catalyzes the oxidation of fatty acids as well as the conversion of C27 bile acids into C24 bile acids in rat and human liver, could be detected in liver of Bombina orientalis when palmitoyl-CoA was used as substrate, but not when trihydroxycoprostanoyl-CoA (THC-CoA) was used.     

Peroxisomal beta-oxidation of branched chain fatty acids in human skin fibroblasts.

Human skin fibroblasts in suspension are able to degrade [1-14C]-labeled alpha- and gamma-methyl branched chain fatty acids such as pristanic and homophytanic acid. Pristanic acid was converted to propionyl-CoA, whereas homophytanic acid was beta-oxidized to acetyl-CoA. Incubation of skin fibroblasts with [1-14C]-labeled fatty acids for longer periods produced radiolabeled carbon dioxide, presumably by further degradation of acetyl-CoA or propionyl-CoA generated by beta-oxidation. Under the same conditions similar products were produced from very long chain fatty acids, such as lignoceric acid. Inclusion of digitonin (> 10 micrograms/ml) in the incubations strongly inhibited carbon dioxide production but stimulated acetyl-CoA or propionyl-CoA production from fatty acids. ATP, Mg2+, coenzyme A, NAD+ and L-carnitine stimulated acetyl-CoA or propionyl-CoA production from [1-14C]-labeled fatty acids in skin fibroblast suspensions. Branched chain fatty acid beta-oxidation was reduced in peroxisome-deficient cells (Zellweger syndrome and infantile Refsum's disease) but they were beta-oxidized normally in cells from patients with X-linked adrenoleukodystrophy (ALD). Under the same conditions, lignoceric acid beta-oxidation was impaired in the above three peroxisomal disease states. These results provide evidence that branched chain fatty acid, as well as very long chain fatty acid, beta-oxidation occurs only in peroxisomes. As the defect in X-linked ALD is in a peroxisomal fatty acyl-CoA synthetase, which is believed to be specific for very long chain fatty acids, we postulate that different synthetases are involved in the activation of branched chain and very long chain fatty acids in peroxisomes.     

Phosphoinositide synthesis and degradation in isolated rat liver peroxisomes

Analyzing peroxisomal phosphoinositide (PId(#)) synthesis in highly purified rat liver peroxisomes we found synthesis of phosphatidylinositol 4-phosphate (PtdIns4P), PtdIns(4,5)P(2) and PtdIns(3,5)P(2). PtdIns3P was hardly detected in vitro, however, was observed in vivo after [(32)P]-phosphate labeling of primary rat hepatocytes. In comparison with other subcellular organelles peroxisomes revealed a unique PId pattern suggesting peroxisomal specificity of the observed synthesis. Use of phosphatase inhibitors enhanced the amount of PtdIns4P. The results obtained provide evidence that isolated rat liver peroxisomes synthesize PIds and suggest the association of PId 4-kinase and PId 5-kinase and PId 4-phosphatase activities with the peroxisomal membrane.     

Arsenite inhibits beta-oxidation in isolated rat liver mitochondria.

A partial inhibition of acylcarnitine oxidation by arsenite in rat liver mitochondria has been studied. This inhibition is confined to the thiolase(s). The inhibition was observed also in the presence of malate, indicating no selective effect on ketogenesis. Ketogenesis from acetyl-CoA was inhibited by arsenite. Mitochondrial CoA was acylated by acylcarnitine nearly as rapidly in the presence of arsenite as in its absence. Thus, arsenite did not interfere with the availibility of CoA in the mitochondria. No effect of arsenite on enzymes of beta-oxidation other than the thiolase(s) was observed. When arsenite and acylcarnitine were added simultaneously to mitochondria, there was a delay before maximal inhibition of oxygen uptake occurred. When the mitochondria were preincubated with arsenite before addition of acylcarnitine, the inhibitory effect on oxygen utilization was initially large, but then partially repealed. Similar time delays were observed in the activity of acetoacetyl-CoA thiolase of disrupted mitochondria depending on the sequence of arsenite and acetoacetyl-CoA addition. It is suggested that substrate and arsenite complete for the reactive sulfhydryl group at the active site of the thiolase(s).     

Proteomic analysis of highly purified peroxisomes from etiolated soybean cotyledons

To identify previously unknown peroxisomal proteins, we establishedan optimized method for isolating highly purified peroxisomesfrom etiolated soybean cotyledons using Percoll density gradientcentrifugation followed by iodixanol density gradient centrifugation.Proteins in highly purified peroxisomes were separated by two-dimensionalPAGE. We performed peptide mass fingerprinting of proteins separatedin the gel with matrix-assisted laser desorption ionizationtime-of-flight mass spectrometry and used the peptide mass fingerprintsto search a non-redundant soybean expressed sequence tag database.We succeeded in assigning 92 proteins to 70 sequences in thedatabase. Among them, proteins encoded by 30 sequences werejudged to be located in peroxisomes. These included enzymesfor fatty acid β-oxidation, the glyoxylate cycle, photorespiratoryglycolate metabolism, stress response and metabolite transport.We also show experimental evidence that plant peroxisomes containa short-chain dehydrogenase/reductase family protein, enoyl-CoAhydratase/isomerase family protein, 3-hydroxyacyl-CoA dehydrogenase-likeprotein and a voltage-dependent anion-selective channel protein.     

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.     

Induction by perfluorinated fatty acids with different carbon chain length of peroxisomal beta-oxidation in the liver of rats

The potency of the induction of peroxisomal beta-oxidation was compared between perfluorinated fatty acids (PFCAs) with different carbon chain lengths in the liver of male and female rats. In male rats, perfluoroheptanoic acid (PFHA) has little effect, although perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA) and perfluorodecanoic acid (PFDA) potentially induced the activity. By contrast, PFHA and PFOA did not induce the activity of peroxisomal beta-oxidation in the liver of female rats while PFNA and PFDA effectively induced the activity. The induction of the activity by these PFCAs was in a dose-dependent manner, and there is a highly significant correlation between the induction and hepatic concentrations of PFCAs in the liver regardless of their carbon chain lengths. These results strongly suggest that the difference in their chemical structure is not the cause of the difference in the potency of the induction. Hepatic concentrations of PFOA and PFNA was markedly higher in male compared with female rats. Castration of male rats reduced the concentration of PFNA in the liver and treatment with testosterone entirely restored the reduction. In contrast to the results obtained from the in vivo experiments, the activity of peroxisomal beta-oxidation was induced by PFDA and PFOA to the same extent in cultured hepatocytes prepared from both male and female rats. These results, taken together, indicate that difference in accumulation between PFCAs in the liver was responsible for the different potency of the induction of peroxisomal beta-oxidation between PFCAs with different carbon chain lengths and between sexes.     

Effect of fatty acids on physical properties of microsomes from isolated perfused rat liver

A computer-centered spectrofluorimeter was used to examine the physicochemical properties of hepatic microsomes and microsomal lipids obtained from isolated rat livers perfused with medium containing palmitate or oleate. The fatty acid composition and degree of unsaturation of the liver microsomal lipids reflected that the fatty acid present in the perfusate. The absorption corrected fluorescence, relative fluorescence efficiency, polarization, and fluorescence anisotropy of several fluorescent probe molecules were measured to determine if their different microenvironments may be altered by the type of fatty acid infused. The probe molecules β-parinaric acid and 1.6-diphenyl-1,3,5-hexatriene had higher values for each of these parameters when incorporated into microsomes obtained from livers perfused with a medium containing palmitate than with oleate. The same parameters measured for cholesta-5,7,9(11)-trien-3β-ol and N-phenyl-1-naphthylamine were not altered. These differences appeared to be primarily due to alterations in microviscosity of the probe microenvironments since the rotational correlation time of 1,6-diphenyl-1,3,5-hexatriene was 25% lower in the microsomes from livers perfused with oleate as compared to livers perfused with palmitate. Thermal discontinuities in Arrhenius plots were noted in the intact microsomes but not in the isolated microsomal lipids with the fluorescence probe molecule β-parinaric acid. Break points occurred at 10°C and 26°C for microsomes from livers perfused with palmitate and at 12°C and 17°C for microsomes from livers perfused with oleate containing medium. These results suggest that the physicochemical properties of liver microsomes were determined in part by the fatty acid in the perfusate.