Rat liver dihydroxyacetone-phosphate acyltransferases and their contribution to glycerolipid synthesis

Differential and isopycnic centrifugation of rat liver homogenates showed that, besides its established localization in peroxisomes and endoplasmic reticulum, dihydroxyacetone-phosphate acyltransferase is also present in mitochondria. The three activities differed in a number of properties (pH optimum, palmitoyl-CoA and dihydroxyacetone-phosphate dependence, and sensitivity toward N-ethylmaleimide) and are therefore likely associated with three distinct proteins. Glycerol 3-phosphate (5 mM) did not inhibit peroxisomal dihydroxyacetone-phosphate acyltransferase but inhibited the extraperoxisomal activities virtually completely. Peroxisomal dihydroxyacetone-phosphate acyltransferase was located at the inner aspect of the peroxisomal membrane, but the enzyme was not latent. Purified microsomes, from which intact peroxisomes had been removed, were still contaminated with peroxisomal membranes as deduced from the presence of two dihydroxyacetone-phosphate acyltransferase activities: a glycerol 3-phosphate-resistant activity with properties similar to those of peroxisomal dihydroxyacetone-phosphate acyltransferase and a glycerol 3-phosphate-sensitive "true" microsomal dihydroxyacetone-phosphate acyltransferase. We propose that, assayed in the presence of 5mM glycerol 3-phosphate, dihydroxyacetone-phosphate acyltransferase can be used as a marker enzyme for peroxisomal membranes. Such a marker enzyme has not hitherto been available. The differential effect of 5 mM glycerol 3-phosphate on peroxisomal and extraperoxisomal dihydroxyacetone-phosphate acyltransferases enabled us to determine the relative contribution of these activities to overall dihydroxyacetone-phosphate acylation in whole liver homogenates. At near-physiological pH and at near-physiological concentrations of unbound palmitoyl-CoA and of dihydroxyacetone-phosphate plus glycerol 3-phosphate, peroxisomes contributed 50-75%. The remaining percentage was mostly accounted for by the microsomal enzyme. At near-physiological concentrations of glycerol 3-phosphate plus dihydroxyacetone-phosphate, glycerolphosphate acyltransferase contributed 93% and dihydroxyacetone-phosphate acyltransferase 7% to overall glycerolipid synthesis in homogenates. This suggests that the dihydroxyacetone-phosphate pathway is of minor quantitative importance in overall hepatic glycerolipid synthesis but that its main function lies in the synthesis of ether lipids, which have acyldihydroxyacetone-phosphate as obligatory precursor.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Developmental changes in the characteristics of peroxisomal fatty acid oxidation system in rat liver

Developmental changes in fatty acid oxidation system of rat liver peroxisomes were studied to compare with that of mitochondria. More apparent enhancement of peroxisomal palmitoyl-CoA oxidase was observed than mitochondrial palmitoyl-CoA dehydrogenase during prenatal (20-day fetal) to neonatal (1-day after birth) period. The characteristics of peroxisomal enzymes, fatty acyl-CoA oxidase and carnitime acyltransferase, on the bases of substrate specificities, were rapidly established within the 1 day after birth accompanied by the marked enhancement of these activities. These findings indicate that peroxisomal fatty acid oxidation system plays an important role for early growth of neonatal rats; this system may contribute to supplying short- to medium-chain fatty acyl-CoA and NADH₂ for mitochondrial energy formation system.     

Effects of dietary corn oil and salmon oil on the oxidation of fatty acids and prostaglandin E2 in rat gastric mucosa

The investigations previously carried out by Grataroli and colleagues (1) to elucidate the relationships between dietary fatty acids, lipid composition, prostaglandin E2 production and phospholipase A2 activity in the rat gastric mucosa are, here, extended. In the present investigations, fatty acid and prostaglandin E2 catabolizing enzymes were assayed in gastric mucosa from rats fed either a low fat diet (corn oil: 4.4% w/w) (referred as control group), a corn oil-enriched diet (17%) or a salmon oil-enriched diet (12.5%) supplemented with corn oil (4.5%) (referred as groups of treated animals) for eight weeks. Peroxisomal fatty acyl-CoA beta-oxidation was induced in the treated animals whereas the activities of catalase and mitochondrial tyramine oxidase were increased and normal, respectively. Mitochondrial acyl-CoA dehydrogenations occurred at higher rates and carnitine acyltransferase activities were enhanced. In addition, the induction of peroxisomal but not mitochondrial prostaglandoyl-E2-CoA beta-oxidation could be demonstrated. Induction of peroxisomal oxidation of fatty acids and prostaglandins is suggested to contribute to the decrease of prostaglandin E2 production in the treated animals, especially those receiving the salmon oil diet, that the above mentioned authors originally reported.     

Inhibition of peroxisomal fatty acyl-CoA oxidase by antimycin A.

Peroxisomal fatty acyl-CoA oxidase was inhibited by micromolar concentrations of antimycin A, an inhibitor of mitochondrial respiration. The inhibition was observed with all three substrates tested, i.e. palmitoyl-CoA, trihydroxycoprostanoyl-CoA and hexadecanedioyl-CoA. The peroxisomal D-amino acid oxidase was also inhibited by antimycin, but the peroxisomal L-alpha-hydroxyacid oxidase and uric acid oxidase and the mitochondrial monoamine oxidase were not. The degree of inhibition of acyl-CoA oxidase by antimycin was strongly dependent on the amount of cellular protein present in the assay mixture: at a fixed antimycin concentration, the inhibition was gradually lost with increasing protein concentrations. At a fixed cellular protein concentration in the assay mixtures, the mitochondrial oxidation of glutamate or palmitoylcarnitine was inhibited at antimycin concentrations that were much lower than those required for the inhibition of fatty acyl-CoA oxidase. Our results, nevertheless, demonstrate that antimycin A must be used with caution, when it is added to homogenates or subcellular fractions in order to distinguish between mitochondrial and peroxisomal fatty acid oxidation.     

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.     

The inhibition by valproic acid of the mitochondrial oxidation of monocarboxylic and omega-hydroxymonocarboxylic acids: possible implications for the metabolism of gamma-aminobutyric acid

The interactions of 1-5 mM valproic acid with the hepatic fatty acid oxidation are here described. Valproic acid was not substrate for hepatic peroxisomal fatty acid oxidation. Its activation outside the mitochondrial matrix compartment was poor when compared to that of octanoic acid, a fatty acid containing the same number of carbones. Valproic acid did not inhibit the fatty acyl-CoA oxidase nor the cyanide-insensitive acyl-CoA oxidation. Valproic acid inhibited the mitochondrial oxidations of both long-chain monocarboxylyl-CoAs and omega-hydroxymonocarboxylyl-CoAs. Valproic acid prevented the oxidation by coupled mitochondria of decanoic and 10-hydroxydecanoic acids. Both butyric and 4-hydroxybutyric acids were oxidized by coupled mitochondria. These activities were abolished by preincubating the enzyme source with valproic acid. Administration to rats of 0.5% (w/w)- or 1% (w/w)-valproate containing diets were efficient in producing increased liver peroxisomal population and beta-oxidation. Preliminary investigations on the effects of valproic acid on mitochondrial fatty acid oxidation as a function of the animal used for the experiments pointed out an association of the protection of the mitochondrial process against the toxicity of the drug with enhanced carnitine acyltransferase and acyl-CoA hydrolase activities.     

Effects of dietary corn oil and salmon oil on the oxidation of fatty acids and prostaglandin E2 in rat gastric mucosa

The investigations previously carried out by Grataroli and colleagues (1) to elucidate the relationships between dietary fatty acids, lipid composition, prostaglandin E2 production and phospholipase A2 activity in the rat gastric mucosa are, here, extended. In the present investigations, fatty acid and prostaglandin E2 catabolizing enzymes were assayed in gastric mucosa from rats fed either a low fat diet (corn oil: 4.4% w/w) (referred as control group), a corn oil-enriched diet (17%) or a salmon oil-enriched diet (12.5%) supplemented with corn oil (4.5%) (referred as groups of treated animals) for eight weeks.Peroxisomal fatty acyl-CoA β-oxidation was induced in the treated animals whereas the activities of catalase and mitochondrial tyramine oxidase were increased and normal, respectively. Mitochondrial acyl-CoA dehydrogenations occured at higher rates and carnitine acyltransferase activities were enhanced. In addition, the induction of peroxisomal but not mitochondrial prostaglandoyl-E2-CoA β-oxidation could be demonstrated. Induction of peroxisomal oxidation of fatty acids and prostaglandins is suggested to contribute to the decrease of prostaglandin E2 production in the treated animals, especially those receiving the salmon oil diet, that the above mentioned authors originally reported.     

Glycerolipid synthetic capacity of rat liver peroxisomes

Investigations on rat liver peroxisomal glycerolipid synthetic capability were performed. Highly purified peroxisomal preparations contained dihydroxyacetone-phosphate acyltransferase, acyldihydroxyacetone-phosphate reductase, and fatty acid-CoA ligase activities. Glycerol-3-phosphate acyltransferase, lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, diacylglycerol acyltransferase, diacylglycerol cholinephosphotransferase, diacylglycerol ethanolaminephosphotransferase and ethanol acyltransferase activities were low in activity or not detected. These results suggest that the peroxisomes are specialized to contribute to the synthesis of ether-linked glycerolipids. If peroxisomes contribute towards the synthesis of non-ether-linked glycerolipids (i.e., ester-linked) then translocation of acyl glycerophosphatide (acyl dihydroxyacetone phosphatide) from peroxisomes to endoplasmic reticulum would be expected to occur.     

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 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.     

Acyl-CoA oxidase activity and peroxisomal fatty acid oxidation in rat tissues

Acyl-CoA oxidase, the first enzyme of the peroxisomal β-oxidation, was proved to be rate-limiting for this process in homogenates of rat liver, kidney, adrenal gland, heart and skeletal muscle. Acyl-CoA oxidase activity, based on H₂O₂-dependent leuko-dichlorofluorescein oxidation in tissue extract, was compared with radiochemically assayed peroxisomal β-oxidation rates. Dichlorofluorescein production was a valid measure of peroxisomal fatty acid oxidation only in liver and kidney, but not in adrenal gland, heart or skeletal muscle. Production of ¹⁴C-labeled acid-soluble products from 1-¹⁴C-labeled fatty acids in the presence of antimycin-rotenone appears to be a more accurate and sensitive estimate of peroxisomal β-oxidation than the acyl-CoA oxidase activity on base of H₂O₂ production. Chain-length specificity of acyl-CoA oxidase changed with the acyl-CoA concentrations used. Below 80 μM, palmitoyl-CoA showed the highest activity of the measured substrates in rat liver extract. No indications were obtained for the presence in rat liver of more forms of acyl-CoA oxidase with different chain-length specificity.     

Interactions between the omega- and beta-oxidations of fatty acids

Long-chain monocarboxylic, omega-hydroxymonocarboxylic and dicarboxylic acids were activated approximately at the same rate by rat liver homogenates into their CoA esters (2-3 U/g liver). These acyl-CoA were substrates for rat liver peroxisomal beta-oxidation. The distribution of the peroxisomal oxidation of these substrates was also studied in various tissues. Rat liver mitochondria were capable of oxidizing long-chain monocarboxyl- and omega-hydroxymonocarboxylyl-CoAs but not dicarboxylyl-CoAs. When the mitochondrial preparations were incubated in coupling conditions, the addition of either free decanoic acid or free 10-hydroxydecanoic acid resulted in an increase of the oxygen uptake conversely to the addition of decanedioic acid. The comparative study of the chain-length substrate specificity of peroxisomal fatty acyl-CoA oxidase and mitochondrial fatty acyl-CoA dehydrogenase activities revealed that, actually, both types of organelles, peroxisomes and mitochondria, contain "oxido-reductases" active on long-chain monocarboxylyl-CoAs, omega-hydroxymonocarboxylyl-CoAs and dicarboxylyl-CoAs.     

Effect of dietary alpha-linolenic acid on the activity and gene expression of hepatic fatty acid oxidation enzymes

The activities of hepatic fatty acid oxidation enzymes in rats fed linseed and perilla oils rich in alpha-linolenic acid (alpha-18:3) were compared with those in the animals fed safflower oil rich in linoleic acid (18:2) and saturated fats (coconut or palm oil). Mitochondrial and peroxisomal palmitoyl-CoA (16:0-CoA) oxidation rates in the liver homogenates were significantly higher in rats fed linseed and perilla oils than in those fed saturated fats and safflower oil. The fatty oxidation rates increased as dietary levels of alpha-18:3 increased. Dietary alpha-18:3 also increased the activity of fatty acid oxidation enzymes except for 3-hydroxyacyl-CoA dehydrogenase. Unexpectedly, dietary alpha-18:3 caused great reduction in the activity of 3-hydroxyacyl-CoA dehydrogenase measured with short- and medium-chain substrates but not with long-chain substrate. Dietary alpha-18:3 significantly increased the mRNA levels of hepatic fatty acid oxidation enzymes including carnitine palmitoyltransferase I and II, mitochondrial trifunctional protein, acyl-CoA oxidase, peroxisomal bifunctional protein, mitochondrial and peroxisomal 3-ketoacyl-CoA thiolases, 2, 4-dienoyl-CoA reductase and delta3, delta2-enoyl-CoA isomerase. Fish oil rich in very long-chain n-3 fatty acids caused similar changes in hepatic fatty acid oxidation. Regarding the substrate specificity of beta-oxidation pathway, mitochondrial and peroxisomal beta-oxidation rate of alpha-18:3-CoA, relative to 16:0- and 18:2-CoAs, was higher irrespective of the substrate/albumin ratios in the assay mixture or dietary fat sources. The substrate specificity of carnitine palmitoyltransferase I appeared to be responsible for the differential mitochondrial oxidation rates of these acyl-CoA substrates. Dietary fats rich in alpha-18:3-CoA relative to safflower oil did not affect the hepatic activity of fatty acid synthase and glucose 6-phosphate dehydrogenase. It was suggested that both substrate specificities and alterations in the activities of the enzymes in beta-oxidation pathway play a significant role in the regulation of the serum lipid concentrations in rats fed alpha-18:3.     

Subcellular localization of acyl coenzyme A: dihydroxyacetone phosphate acyltransferase in rat liver peroxisomes (microbodies).

Upon differential centrifugation of rat liver homogenate, the enzyme acyl-CoA:dihydroxyacetone-phosphate acyltransferase (EC 2.3.1.42) was found to be localized in the light mitochondrial (L) fraction which is enriched with lysosomes and peroxisomes. Peroxisomes were separated from lysosomes in a density gradient centrifugation using rats which were injected with Triton WR 1339. By comparing the enzyme distribution with the distribution of different marker enzymes, it was concluded that dihydroxyacetone phosphate acyltransferase is primarily localized in rat liver peroxisomes (microbodies). Similarly, the enzyme acyl dihydroxyacetone-phosphate:NADPH oxidoreductase (EC 1.1.1.101) was shown to be enriched in the peroxisomal fraction, although a portion of this reductase is also present in the microsomal fraction.     

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 lipid biosynthesis in the harderian gland of guinea pig.

Peroxisomal enzyme activities in the guinea-pig harderian gland, which has a unique lipid composition, were studied. Activities of catalase, acyl-CoA oxidase and the cyanide-insensitive acyl-CoA beta-oxidation system in this tissue were comparable with those in rat liver. The activities of dihydroxyacetone phosphate acyltransferase (DHAPAT, EC 2.3.1.42) and alkyl-DHAP synthase (EC 2.5.1.26) were appreciable, and the distributions of both activities were consistent with that of sedimentable catalase activity. Glycerol-3-phosphate acyltransferase (GPAT, EC 2.3.1.15), which is localized in both microsomes (microsomal fractions) and mitochondria in the rat liver, was a peroxisomal enzyme in the harderian gland, though the activity was only about one-tenth of the DHAPAT activity. These enzymes had different pH profiles and substrate specificity. The existence of high activities of enzymes of the acyl-DHAP pathway in peroxisomes suggests the physiological significance of peroxisomes in the biosynthesis of glycerol ether phospholipid and 1-alkyl-2,3-diacylglycerol in the guinea-pig harderian gland.     

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

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