The Arabidopsis acyl-CoA oxidase gene family

A family of acyl-CoA oxidase isozymes catalyse the first step in the peroxisomal fatty acid beta-oxidation spiral. Our group and others have recently characterized four genes from this family in the model oilseed Arabidopsis. These genes encode isozymes with different acyl-CoA substrate specificities, which together encompass the full range of fatty acid chain lengths that exist in vivo. Here we review the biochemical properties and physiological roles of the acyl-CoA oxidase isozymes.     

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.     

Arabidopsis mutants in short- and medium-chain acyl-CoA oxidase activities accumulate acyl-CoAs and reveal that fatty acid beta-oxidation is essential for embryo development

The short-chain acyl-CoA oxidase (ACX4) is one of a family of ACX genes that together catalyze the first step of peroxisomal fatty acid beta-oxidation during early, postgerminative growth in oilseed species. Here we have isolated and characterized an Arabidopsis thaliana mutant containing a T-DNA insert in ACX4. In acx4 seedlings, short-chain acyl-CoA oxidase activity was reduced by greater than 98%, whereas medium-chain activity was unchanged from wild type levels. Despite the almost complete loss of short-chain activity, lipid catabolism and seedling growth and establishment were unaltered in the acx4 mutant. However, the acx4 seedlings accumulated high levels (31 mol %) of short-chain acyl-CoAs and showed resistance to 2,4-dichlorophenoxybutyric acid, which is converted to the herbicide and auxin analogue 2,4-dichlorophenoxyacetic acid by beta-oxidation. A mutant in medium-chain length acyl-CoA activity (acx3) (1) shows a similar phenotype to acx4, and we show here that acx3 seedlings accumulate medium-chain length acyl-CoAs (16.4 mol %). The acx3 and acx4 mutants were crossed together, and remarkably, the acx3acx4 double mutants aborted during the first phase of embryo development. We propose that acx3acx4 double mutants are nonviable because they have a complete block in short-chain acyl-CoA oxidase activity. This is the first demonstration of the effects of eliminating (short-chain) beta-oxidation capacity in plants and shows that a functional beta-oxidation cycle is essential in the early stages of embryo development.     

Intramitochondrial fatty acid metabolism: riboflavin deficiency and energy production.

Inborn errors of fatty acid beta-oxidation have contributed significantly to our understanding of intracellular fatty acid metabolism. The first intramitochondrial step in beta-oxidation of fatty acyl-CoA of different chain lengths is catalyzed by the three chain length specific acyl-CoA dehydrogenases. Inherited deficiency of these enzymes has been reported. Some are riboflavin responsive. The first step of fatty acid oxidation is reviewed with specific emphasis on beta-oxidation in newborn infants, rendered riboflavin deficient by phototherapy. Given that medium chain fatty acids are not stored as triacylglycerols and undergo rapid beta-oxidation, they have been proposed as superior substrates compared with long chain triglycerides in times of metabolic stress. This review also examines medium chain triglycerides as an alternate energy source. When medium chain triglycerides were fed as 50% of total energy, glucose sparing was present with little loss of energy as dicarboxylic acids.     

Peroxisomal oxidases and catalase in liver and kidney homogenates of normal and di(ethylhexyl)phthalate-fed rats.

1. Activities of peroxisomal oxidases and catalase were assayed at neutral and alkaline pH in liver and kidney homogenates from male rats fed a diet with or without 2% di(2-ethylhexyl)phthalate (DEHP) for 12 days. 2. All enzyme activities were higher at alkaline than at neutral pH in both groups. 3. The effect of the DEHP-diet on the peroxisomal enzymes was different in kidney and liver. Acyl-CoA oxidase activity was raised three- and sixfold in kidney and liver homogenates, respectively. The activity of D-amino acid oxidase decrease in liver, but increased in kidney homogenates. In liver homogenates, urate oxidase activity was not affected by the DEHP diet. The catalase activity was twofold induced in liver, but not in kidney. 4. The differences suggest that the changes of peroxisomal enzyme activities by DEHP treatment are not directly related to peroxisome proliferation. 5. DEHP treatment caused a marked increase of total and peroxisomal fatty acid oxidation in rat liver homogenates. 6. In the control group the rate of peroxisomal fatty acid oxidation was higher at alkaline pH than at neutral pH. 7. This rate was equal at both pH values in the DEHP-fed group, in contrast to the acyl-CoA oxidase activity. These results indicate that after DEHP treatment other parameters than acyl-CoA oxidase activity become limiting for peroxisomal beta-oxidation.     

Effects of thia-substituted fatty acids on mitochondrial and peroxisomal beta-oxidation. Studies in vivo and in vitro.

1. The effects of 3-, 4- and 5-thia-substituted fatty acids on mitochondrial and peroxisomal beta-oxidation have been investigated. When the sulphur atom is in the 4-position, the resulting thia-substituted fatty acid becomes a powerful inhibitor of beta-oxidation. 2. This inhibition cannot be explained in terms of simple competitive inhibition, a phenomenon which characterizes the inhibitory effects of 3- and 5-thia-substituted fatty acids. The inhibitory sites for 4-thia-substituted fatty acids are most likely to be the acyl-CoA dehydrogenase in mitochondria and the acyl-CoA oxidase in peroxisomes. 3. The inhibitory effect of 4-thia-substituted fatty acids is expressed both in vitro and in vivo. The effect in vitro is instantaneous, with up to 95% inhibition of palmitoylcarnitine oxidation. The effect in vivo, in contrast, is dose-dependent and increases with duration of treatment. 4. Pretreatment of rats with a 3-thia-substituted fatty acid rendered mitochondrial beta-oxidation less sensitive to inhibition by 4-thia-substituted fatty acids.     

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.     

Presence of three acyl-CoA oxidases in rat liver peroxisomes. An inducible fatty acyl-CoA oxidase, a noninducible fatty acyl-CoA oxidase, and a noninducible trihydroxycoprostanoyl-CoA oxidase

Mammalian liver peroxisomes are capable of beta-oxidizing a variety of substrates including very long chain fatty acids and the side chains of the bile acid intermediates di- and trihydroxycoprostanic acid. The first enzyme of peroxisomal beta-oxidation is acyl-CoA oxidase. It remains unknown whether peroxisomes possess one or several acyl-CoA oxidases. Peroxisomal oxidases from rat liver were partially purified by (NH4)2SO4 precipitation and heat treatment, and the preparation was subjected to chromatofocusing, chromatography on hydroxylapatite and dye affinity matrices, and gel filtration. The column eluates were assayed for palmitoyl-CoA and trihydroxycoprostanoyl-CoA oxidase activities and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results revealed the presence of three acyl-CoA oxidases: 1) a fatty acyl-CoA oxidase with a pI of 8.3 and an apparent molecular mass of 145 kDa. The enzyme consisted mainly of 52- and 22.5-kDa subunits and could be induced by clofibrate treatment; 2) a noninducible fatty acyl-CoA oxidase with a pI of 7.1 and an apparent molecular mass of 427 kDa. It consisted mainly, if not exclusively, of one polypeptide component of 71 kDa; and 3) a noninducile trihydroxycoprostanoyl-CoA oxidase with a pI of 7.1 and an apparent molecular mass of 139 kDa. It consisted mainly, if not exclusively, of one polypeptide component of 69 kDa. Our findings are probably related to the recent discovery of two species of acyl-CoA oxidase mRNA in rat liver (Miyazawa, S., Hayashi, H., Hijikata, M., Ishii, N., Furata, S., Kagamiyama, H., Osumi, T., and Hashimoto, T. (1987) J. Biol. Chem. 262, 8131-8137) and they probably also explain why in human peroxisomal beta-oxidation defects an accumulation of very long chain fatty acids is not always accompanied by an excretion of bile acid intermediates and vice versa.     

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.     

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.     

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.     

Inducible beta-oxidation pathway in Neurospora crassa.

An inducible beta-oxidation system was demonstrated in a particulate fraction from Neurospora crassa. The activities of all individual beta-oxidation enzymes were enhanced in cells after a shift from a sucrose to an acetate medium. The induction was even more pronounced in transfer to a medium containing oleate as sole carbon and energy source. Since an acyl-coenzyme A (CoA) dehydrogenase was detected instead of acyl-CoA oxidase, the former enzyme seems to catalyze the first step of the beta-oxidation sequence in N. crassa. After isopycnic centrifugation in a linear sucrose gradient, the intracellular organelles housing the fatty acid degradation pathway cosedimented (1.21 g/cm3) with the glyoxylate bypass enzymes isocitrate lyase and malate synthase and were clearly resolved from both mitochondrial marker enzymes (1.19 g/cm3) and catalase (1.26 g/cm3). On the basis of biochemical as well as morphological properties, these particles from N. crassa have recently been designated as glyoxysome-like particles (G. Wanner and T. Theimer, Ann. N.Y. Acad. Sci. 386:269-284, 1982). The failure to detect catalase, urate oxidase, and acyl-CoA oxidase indicate that these glyoxysome-like microbodies in N. crassa lack peroxisomal function and thus are clearly different from the various microbodies reported so far to contain a beta-oxidation pathway.     

Acyl-CoA oxidase 1 from Arabidopsis thaliana. Structure of a key enzyme in plant lipid metabolism

The peroxisomal acyl-CoA oxidase family plays an essential role in lipid metabolism by catalyzing the conversion of acyl-CoA into trans-2-enoyl-CoA during fatty acid beta-oxidation. Here, we report the X-ray structure of the FAD-containing Arabidopsis thaliana acyl-CoA oxidase 1 (ACX1), the first three-dimensional structure of a plant acyl-CoA oxidase. Like other acyl-CoA oxidases, the enzyme is a dimer and it has a fold resembling that of mammalian acyl-CoA oxidase. A comparative analysis including mammalian acyl-CoA oxidase and the related tetrameric mitochondrial acyl-CoA dehydrogenases reveals a substrate-binding architecture that explains the observed preference for long-chained, mono-unsaturated substrates in ACX1. Two anions are found at the ACX1 dimer interface and for the first time the presence of a disulfide bridge in a peroxisomal protein has been observed. The functional differences between the peroxisomal acyl-CoA oxidases and the mitochondrial acyl-CoA dehydrogenases are attributed to structural differences in the FAD environments.     

Altered acyl-CoA metabolism in riboflavin deficiency

We have recently described the effects of riboflavin deficiency on the metabolism of dicarboxylic acids (Draye et al. (1988) Eur. J. Biochem. 178, 183-189). As both mitochondria and peroxisomes are thought to be involved, we have examined the activities of various enzymes in these organelles in the livers of riboflavin-deficient rats. Mitochondrial beta-oxidation of fatty acids was severely depressed due to loss of activity of the three fatty acyl-CoA dehydrogenases, whereas there was an enhancement of peroxisomal beta-oxidation due to an increased activity of the FAD-dependent fatty acyl-CoA oxidase, although the activities of other peroxisomal flavoproteins, D-amino acid oxidase and glycolate oxidase, were lowered. Hepatocyte morphometry revealed an increase in the numbers of peroxisomes, indicating a proliferation induced by the deficiency. The mitochondrial acyl-CoA dehydrogenases involved in branched-chain amino acid metabolism were also severely decreased leading to characteristic organic acidurias. There was some loss of activity of the flavin-dependent sections of the electron transport chain (complexes I and II), but these were probably not sufficient to affect normal function in vivo. The specificity of these effects allows the use of the riboflavin-deficient rat as a model for the study of dicarboxylate metabolism.     

Acyl-CoA synthetase and the peroxisomal enzymes of beta-oxidation in human liver. Quantitative analysis of their subcellular localization.

The presence of acyl-CoA synthetase (EC 6.2.1.3) in peroxisomes and the subcellular distribution of beta-oxidation enzymes in human liver were investigated by using a single-step fractionation method of whole liver homogenates in metrizamide continuous density gradients and a novel procedure of computer analysis of results. Peroxisomes were found to contain 16% of the liver palmitoyl-CoA synthetase activity, and 21% and 60% of the enzyme activity was localized in mitochondria and microsomal fractions respectively. Fatty acyl-CoA oxidase was localized exclusively in peroxisomes, confirming previous results. Human liver peroxisomes were found to contribute 13%, 17% and 11% of the liver activities of crotonase, beta-hydroxyacyl-CoA dehydrogenase and thiolase respectively. The absolute activities found in peroxisomes for the enzymes investigated suggest that in human liver fatty acyl-CoA oxidase is the rate-limiting enzyme of the peroxisomal beta-oxidation pathway, when palmitic acid is the substrate.     

Separate peroxisomal oxidases for fatty acyl-CoAs and trihydroxycoprostanoyl-CoA in human liver

Fatty acyl-CoAs as well as the CoA esters of the bile acid intermediates di- and trihydroxycoprostanic acids are beta-oxidized in peroxisomes. The first reaction of peroxisomal beta-oxidation is catalyzed by acyl-CoA oxidase. We recently described the presence of two fatty acyl-CoA oxidases plus a trihydroxycoprostanoyl-CoA oxidase in rat liver peroxisomes (Schepers, L., P. P. Van Veldhoven, M. Casteels, H. J. Eyssen, and G. P. Mannaerts. 1990. J. Biol. Chem. 265: 5242-5246). We have now developed methods for the measurement of palmitoyl-CoA oxidase and trihydroxycoprostanoyl-CoA oxidase in human liver. The activities were measured in livers from controls and from three patients with peroxisomopathies. In addition, the oxidase activities were partially purified from control livers by ammonium sulfate fractionation and heat treatment, and the partially purified enzyme preparation was subjected to chromatofocusing, hydroxylapatite chromatography, and gel filtration. In earlier experiments this allowed for the separation of the three rat liver oxidases. The results show that human liver, as rat liver, contains a separate trihydroxycoprostanoyl-CoA oxidase. In contrast to the situation in rat liver, no conclusive evidence was obtained for the presence of two fatty acyl-CoA oxidases in human liver. Our results explain why bile acid metabolism is normal in acyl-CoA oxidase deficiency, despite a severely disturbed peroxisomal fatty acid oxidation and perhaps also why, in a number of other cases of peroxisomopathy, di- and trihydroxycoprostanic acids are excreted despite a normal peroxisomal fatty acid metabolism.     

The regulation of peroxisomal enzyme systems of Tetrahymena pyriformis by fatty acid composition, glucose and oxygen in the medium

The effect of the chain length of fatty acids on peroxisomal enzyme activities of Tetrahymena pyriformis was investigated. The growth of cells and the activities of peroxisomal enzymes were inhibited markedly by the addition of medium-chain fatty acids (C6-C12) to the culture medium, whereas the addition of longer-chain fatty acids (C14-C18) resulted in a slight increase of growth and in the marked stimulation of enzyme activities concerned with fatty acid beta-oxidation and the glyoxylate cycle in peroxisomes. Peroxisomal beta-oxidation (fatty acyl-CoA oxidase) was more potent towards longer-chain fatty acids than the mitochondrial activity (fatty acyl-CoA dehydrogenase). The induction of the peroxisomal beta-oxidation system by palmitate was repressed both by the addition of glucose and the aeration of the culture medium, whereas that of the peroxisomal glyoxylate cycle was repressed only by the addition of glucose to the medium. These results indicate that peroxisomal enzyme systems related to the beta-oxidation of fatty acids and the glyoxylate cycle are regulated by the compositions of fatty acids, glucose, and oxygen in the medium.     

Peroxisomal straight-chain Acyl-CoA oxidase and D-bifunctional protein are essential for the retroconversion step in docosahexaenoic acid synthesis

Docosahexaenoic acid (DHA, C22:6n-3) is essential for normal brain and retinal development. The nature and subcellular location of the terminal steps in DHA biosynthesis have been controversial. Rather than direct Delta4-desaturation of C22:5n-3, it has been proposed that this intermediate is elongated to C24:5n-3, desaturated to C24:6n-3, and "retroconverted" to DHA via peroxisomal beta-oxidation. However, this hypothesis has recently been challenged. The goal of this study was to determine the mechanism and specific enzymes required for the retroconversion step in human skin fibroblasts. Cells from patients with deficiencies of either acyl-CoA oxidase or D-bifunctional protein, the first two enzymes of the peroxisomal straight-chain fatty acid beta-oxidation pathway, exhibited impaired (5-20% of control) conversion of either [1-14C]18:3n-3 or [1-14C]22:5n-3 to DHA as did cells from peroxisome biogenesis disorder patients comprising eight distinct genotypes. In contrast, normal DHA synthesis was observed in cells from patients with rhizomelic chondrodysplasia punctata, Refsum disease, X-linked adrenoleukodystrophy, and deficiency of mitochondrial medium- or very long-chain acyl-CoA dehydrogenase. Acyl-CoA oxidase-deficient cells accumulated 2-5 times more radiolabeled C24:6n-3 than did controls. Our data are consistent with the retroconversion hypothesis and demonstrate that peroxisomal beta-oxidation enzymes acyl-CoA oxidase and D-bifunctional protein are essential for this process in human skin fibroblasts.     

Peroxisome proliferator-activated receptor alpha (PPARalpha) agonist treatment reverses PPARalpha dysfunction and abnormalities in hepatic lipid metabolism in ethanol-fed mice

Proper function of the peroxisome proliferator-activated receptor alpha (PPARalpha) is essential for the regulation of hepatic fatty acid metabolism. Fatty acid levels are increased in liver during the metabolism of ethanol and should activate PPARalpha. However, recent in vitro data showed that ethanol metabolism inhibited the function of PPARalpha. We now report that ethanol feeding impairs fatty acid catabolism in the liver in part via blocking PPARalpha-mediated responses in C57BL/6J mice. Ethanol feeding decreased PPARalpha/retinoid X receptor alpha binding in electrophoretic mobility shift assay of liver nuclear extracts. mRNAs for PPAR-regulated genes were reduced (long chain and medium chain acyl-CoA dehydrogenases) or failed to be induced (acyl-CoA oxidase, liver carnitine palmitoyl-CoA transferase, very long chain acyl-CoA synthetase, very long chain acyl-CoA dehydrogenase) in livers of the ethanol-fed animals, and ethanol feeding did not increase the rate of fatty acid beta-oxidation. Wy14,643, a PPARalpha agonist, restored the DNA binding activity of PPARalpha/retinoid X receptor alpha, induced mRNA levels of PPARalpha target genes, stimulated the rate of fatty acid beta-oxidation, and prevented fatty liver in ethanol-fed animals. Impairment of PPARalpha function during ethanol consumption contributes to the development of alcoholic fatty liver, which can be overcome by Wy14,643.