palmitoyl-CoA Synthetase Activity Research Articles

Activation of lipid catabolism by the water-soluble fraction of petroleum in the crustacean Macrobrachium borellii

Little is known about the effect of the water-soluble fraction of crude oil (WSF) on lipid metabolism in invertebrates. The effect of the WSF on the triacylglycerol (TAG) mobilization, fatty acid activation and degradation was evaluated in the decapod Macrobrachium borellii, exposing adult and eggs at different stages of development for 7 days to a sublethal concentration of WSF. Using radioactive tracers, mitochondrial palmitoyl-CoA synthetase (ACS), triacylglycerol lipase (TAG-lipase) and fatty acid β-oxidation system activities were assayed. Before studying the effect of WSF, the kinetic parameters of ACS were determined in purified mitochondria. Its optimal temperature and pH were 32 °C and 8.0, respectively, the apparent K m 2.48 μmol l −1, and its V max of 1.93 nmol min −1 mg protein −1. These kinetic parameters differed significantly from this shrimp's microsomal isoform. After 7 days exposure to a sublethal concentration of WSF (0.6 mg/l), changes were observed in the enzymatic activity of all enzymes or enzymatic system assayed in adult midgut gland as well as in stage 5 eggs, a period of active organogenesis. An increase in the mobilization of energy stores was detected as early as stage 4, where TAG-lipase activity increased by 27% in exposed eggs. The increase was even more marked in exposed eggs at stage 5 where a three-fold rise (154%) was determined. Exposed adult shrimp also showed an augmented lipase activity by 38%. Fatty acid β-oxidation increased by 51.0 and 35.5% in midgut gland and eggs at stage 5, respectively, but no changes were observed at less-developed stages. Mitochondrial fatty acid activation by ACS also increased in adults and stage 5 eggs by 7.4 and 52.0%, respectively. A similar response of the lipid catabolic pathways to WSF contamination in both adult and eggs, suggests that the exposure to this pollutant causes an increase in the energy needs of this shrimp. When validated by field studies, these catabolic enzymes could be employed as early pollution biomarkers.

Peroxisomal Proliferation in Rat Liver and Essential Fatty Acid Deficiency

The effects of essential fatty acid (EFA) deficiency were studied on male Wistar rats membrane lipid composition, enzyme activities and proliferation of liver peroxisome by feeding the animals, during 4 weeks, with fat free diets supplemented or not with a peroxisome proliferator, clofibrate (CLO).In deficient (E) goup, peroxisomal membrane was characterized by higher level of total de novo synthesized unsaturated (n-9 and n-7 series) fatty acids and lower content of EFA (n-6 and n-3 series) and exhibited cholesterol (CHOL) to phospholipids (PLs) ratio diminution compared with that of control (C) group. Co-administration of CLO with the fat-free diet (EC group) enhanced the membrane uptake of n-9 fatty acids. Slight increase of membrane dihydroxyacetone-phosphate acyl-transferase (DHAP-AT) and decrease of palmitoyl-CoA synthetase activities were found in E group which featured normal-shaped peroxisomes.In EC group, enzyme inductions of peroxisomal pamitoyl-CoA oxidase and palmitoyl-CoA synthetase were slightly higher and significantly lower respectively compared with those of C group treated with CLO (CC group) whereas activity of DHAP-AT was stable. Identical marked proliferation of peroxisomes were observed in both groups.It is inferred that, despite the parallel alterations of membrane lipid composition and of enzyme activities of peroxisomes upon 4 weeks of EFA deficientdiet, the biogenesis and proliferation of the organelles were independently processed at a normal level. The possibility of homeostatic regulation and its physiological importance were discussed.

Evaluation of Adenosine 5'-Hexadecylphosphate as an Inhibitor of Acyl-CoA Synthetase Isozyme Functional for Phospholipid Reconstitution in the Yeast Saccharomyces cerevisiae.

Adenosine 5'-hexadecylphosphate (AMPC16) is a mimic of palmitoyl-AMP, a transient intermediate of the acyl-CoA synthetic reaction, and thus could effectively inhibit the activity of palmitoyl-CoA synthetase of Saccharomyces cerevisiae. AMPC16 inhibited the cellular incorporation of [3H]palmitic acid into phospholipids, in which such an inhibition was most significantly detected in the fraction containing both phosphatidylinositol (PI) and phosphatidylserine (PS). A pulse-chase experiment revealed the loss of PI and PS accompanied with accumulation of free fatty acids in AMPC16-treated cells. AMPC16-induced events suggested that Faa1p, the major acyl-CoA synthetase isozyme, is functional for phospholipid reconstitution in the exponential growth phase.

Evaluation of adenosine 5′-hexadecylphosphate as an inhibitor of acyl-CoA synthetase isozyme functional for phospholipid reconstitution in the yeast Saccharomyces cerevisiae

Adenosine 5′-hexadecylphosphate (AMPC16) is a mimic of palmitoyl-AMP, a transient intermediate of the acyl-CoA synthetic reaction, and thus could effectively inhibit the activity of palmitoyl-CoA synthetase of Saccharomyces cerevisiae. AMPC16 inhibited the cellular incorporation of [ 3H]palmitic acid into phospholipids, in which such an inhibition was most significantly detected in the fraction containing both phosphatidylinositol (PI) and phosphatidylserine (PS). A pulse-chase experiment revealed the loss of PI and PS accompanied with accumulation of free fatty acids in AMPC16-treated cells. AMPC16-induced events suggested that Faa1p, the major acyl-CoA synthetase isozyme, is functional for phospholipid reconstitution in the exponential growth phase.

The Fatty Acid Transport Protein (FATP1) Is a Very Long Chain Acyl-CoA Synthetase

The primary sequence of the murine fatty acid transport protein (FATP1) is very similar to the multigene family of very long chain (C20-C26) acyl-CoA synthetases. To determine if FATP1 is a long chain acyl coenzyme A synthetase, FATP1-Myc/His fusion protein was expressed in COS1 cells, and its enzymatic activity was analyzed. In addition, mutations were generated in two domains conserved in acyl-CoA synthetases: a 6- amino acid substitution into the putative active site (amino acids 249-254) generating mutant M1 and a 59-amino acid deletion into a conserved C-terminal domain (amino acids 464-523) generating mutant M2. Immunolocalization revealed that the FATP1-Myc/His forms were distributed between the COS1 cell plasma membrane and intracellular membranes. COS1 cells expressing wild type FATP1-Myc/His exhibited a 3-fold increase in the ratio of lignoceroyl-CoA synthetase activity (C24:0) to palmitoyl-CoA synthetase activity (C16:0), characteristic of very long chain acyl-CoA synthetases, whereas both mutant M1 and M2 were catalytically inactive. Detergent-solubilized FATP1-Myc/His was partially purified using nickel-based affinity chromatography and demonstrated a 10-fold increase in very long chain acyl-CoA specific activity (C24:0/C16:0). These results indicate that FATP1 is a very long chain acyl-CoA synthetase and suggest that a potential mechanism for facilitating mammalian fatty acid uptake is via esterification coupled influx.

The inhibitory guanine nucleotide-binding protein Gi2alpha induces and potentiates adipocyte differentiation.

The present study further elucidates the involvement of the alpha-subunit of the GTP-binding protein Gi2 in the differentiation of murine 3T3-L1 cells. Control and vector-transfected cells attained a fully differentiated adipocyte phenotype showing ample lipid droplets. Cells expressing wild type (WT)-Gi2alpha or the constitutively active R179E-Gi2alpha, however, became enlarged, less confluent, and produced large amounts of lipids. Differentiation consistently increased the triglyceride (TAG) content in control cells. In both WT-Gi2alpha and R179E-Gi2alpha clones, a marked increase in TAG could be detected even prior to insulin/dexamethasone/isobutyl methylxanthine exposure. The activity of palmitoyl-CoA synthetase (PCS) and glycerophosphate acyltransferase (GPAT) also increased upon differentiation. WT-Gi2alpha and R179E-Gi2alpha overexpression also enhanced PCS and GPAT activities even before differentiation medium was added. The total amount of phospholipids (PL) generally increased upon differentiation; however, pre- and postdifferentiation values were insignificantly different in cells expressing WT-Gi2alpha and R179E-Gi2alpha. Differentiation altered the PL profile with a relative shift from phosphatidylcholine and phosphatidylethanolamine to phosphatidylinositol (PI) in differentiated cells. Finally, differentiation yielded a general increase in the activity of basal PI-phospholipase-C activity. Again, cells expressing WT-Gi2alpha and R179E-Gi2alpha demonstrated elevated enzyme activity and enhanced second messenger accumulation subsequent to differentiation. In summary, cells with the R179E-mutants of Gi2alpha exhibited stimulated lipid turnover and accumulation in both undifferentiated and differentiated cells.

Phytanic acid activation in rat liver peroxisomes is catalyzed by long-chain acyl-CoA synthetase

In Refsum disease, disorders of peroxisome biogenesis, and rhizomelic chondrodysplasia punctata, pathological accumulation of phytanic acid results from impaired alpha-oxidation of this branched-chain fatty acid. Previous studies from this laboratory indicated that activation of phytanic acid to its CoA derivative precedes its alpha-oxidation in peroxisomes. It was reported that this reaction is catalyzed by a unique phytanoyl-CoA synthetase in human peroxisomes. We wanted to determine whether phytanic acid activation in rats required long-chain acyl-CoA synthetase (LCS), very long-chain acyl-CoA synthetase (VLCS), or a different enzyme. To test directly whether LCS could activate phytanic acid, rat liver cDNA encoding this enzyme was transcribed and translated in vitro. The expressed enzyme had both LCS activity (assayed with palmitic acid, C16: 0) and phytanoyl-CoA synthetase activity; VLCS activity (assayed with lignoceric acid, C24: 0) was not detectable. The ratio of phytanoyl-CoA synthetized activity to palmitoyl-CoA synthetase activity for LCS synthetized in vitro (approximately 205) was higher than that observed in peroxisomes isolated from rat liver (5-10%), suggesting that the expressed enzyme contained sufficient phytanoyl-Coa synthetase activity to account for all activity observed in intact peroxisomes. Further experiments were carried out to verify that phytanic acid was activated by LCS in rat liver peroxisomes. Attempts to separate LCS from phytanoyl-CoA synthetase by chromatography on several matrices were unsuccessful. Preparative isoelectric focusing revealed that phytanoyl-CoA synthetase and LCS had indistinguishable isoelectric points. Phytanoyl-CoA synthetase activity was inhibited by unlabeled palmitic acid but not by lignoceric acid. Heat treatment inactivated both phytanoyl-CoA and palmitoyl-CoA synthetase activities at similar rates. 5,8,11,14-Eicosatetraynoic acid inhibited activation of phytanic acid and palmitic acid in a parallel dose-dependent manner, whereas activation of lignoceric acid was not affected. These data support our conclusion that rat liver LCS, an enzyme known to be present in peroxisomal membranes, has phytanoyl-CoA synthetase activity.

Complementation of Saccharomycescerevisiae Strains Containing Fatty Acid Activation Gene (FAA) Deletions with a Mammalian Acyl-CoA Synthetase

Four unlinked fatty acid activation (FAA) genes encoding acyl-CoA synthetases have been identified in Saccharomyces cerevisiae and characterized by noting the phenotypes of isogenic strains containing all possible combinations of faa null alleles. None of these genes is required for vegetative growth when acyl-CoA production by the fatty acid synthetase (Fas) complex is active. When Fas is inhibited by cerulenin, exponentially growing cells are not viable on media containing a fermentable carbon source unless supplemented with fatty acids such as myristate, palmitate, or oleate. The functionally interchangeable FAA1 and FAA4 genes are responsible for activation of these imported fatty acids. Analysis of lysates prepared from isogenic FAA1FAA4 and faa1 delta faa4 delta strains indicated that Faa1p and Faa4p together account for 99% of total cellular myristoyl-CoA and palmitoyl-CoA synthetase activities. Genetic complementation studies revealed that rat liver acyl-CoA synthetase (RLACS) rescues the viability of faa1 delta faa4 delta cells in media containing a fermentable carbon source, myristate or palmitate, plus cerulenin. Rescue is greater at 37 degrees C compared with 24 degrees C, paralleling the temperature-dependent changes in RLACS activity in vitro as well as the enzyme's ability to direct incorporation of tritiated myristate and palmitate into cellular phospholipids in vivo. Complementation by RLACS is blocked by treatment of cells with triacsin C (1-hydroxy-3-(E,E,E,2',4',7'- undecatrienylidine)triazene). Even though Faa1p, Faa4p, and RLACS are all able to activate imported myristate and palmitate in S. cerevisiae, the sensitivity of Faa4p and RLACS, but not Faa1p, to inhibition by triacsin C suggests that the rat liver enzyme is functionally more analogous to Faa4p than to Faa1p. Finally, an assessment of myristate and palmitate import into FAA1FAA4 and faa1 delta faa4 delta strains, with or without episomes that direct overexpression of Faa1p, Faa4p or RLACS, indicated that fatty acid uptake is not coupled to activation in S. cerevisiae.

Selective inhibition of lignoceroyl-CoA synthetase by adenosine 5′-alkylphosphates

Structural analogs of adenosine 5′-acylphosphates, which are intermediates of the reaction catalysed by acyl-CoA synthetases, were synthesized by condensing primary alcohols with AMP to examine the inhibitory effects on the lignoceroyl-CoA and palmitoyl-CoA synthetase activities. Hexadecyl, octadecyl, eicosyl, docosyl and tetracosyl esters of AMP were remarkably potent inhibitors of the lignoceroyl-CoA formation. On the other hand, the eicosyl, docosyl or tetracosyl esters of AMP did not behave as significant inhibitors of the palmitoyl-CoA formation at the concentration at which the two other shorter chain analogs were effective. Namely, these longer alkyl esters of AMP have selective inhibitory effects on the lignoceroyl-CoA synthetase activity. The K i value of adenosine 5′-tetracosylphosphate, the most potent inhibitor, was about one tenth lower than the K m value for the substrate lignoceric acid. Furthermore, the results support the notion that lignoceroyl-CoA synthetase is distinct from palmitoyl-CoA synthetase.

Relationship between plasma lipids and palmitoyl‐CoA hydrolase and synthetase activities with peroxisomal proliferation in rats treated with fibrates

1. The time-course of the effect of clofibrate (CFB), bezafibrate (BFB) and gemfibrozil (GFB) on lipid plasma levels and palmitoyl-CoA hydrolase and synthetase activities, as well as the correlations with the peroxisomal proliferation phenomenon have been studied in male Sprague-Dawley rats. 2. The administration of the three drugs caused a significant reduction in body weight gain, accompanied with a paradoxical increase in food intake in groups treated with BFB and GFB. 3. Drug treatment produced gross hepatomegaly and increase in peroxisomal beta-oxidation, and these parameters were strongly correlated. The order of potency was BFB > CFB > or = GFB. 4. Both plasma cholesterol (BFB approximately CFB > GFB) and triglyceride (BFB approximately GFB > CFB) levels were reduced in treated animals. There was an inverse correlation between these parameters and peroxisomal beta-oxidation, although the peroxisomal proliferation seemed to explain only a small part of the hypolipidemic effect observed. 5. Cytosolic and microsomal (but not mitochondrial) palmitoyl-CoA hydrolase activities were increased by the three drugs (BFB > CFB > GFB), probably by inducing the hydrolase I isoform, which is insensitive to inhibition by fibrates in vitro. The increased hydrolase activities were directly and strongly correlated with peroxisomal beta-oxidation. 6. Palmitoyl-CoA synthetase activity was also increased by the treatment with fibrates (BFB > CFB > GFB), probably as a consequence of the enhancement of hydrolase activities. 7. Some of the effects of fibrate treatment can be explained, at least in part, in terms of peroxisomal induction and caution should be exercised in the extrapolation of these results to species, such as man,that are insensitive to peroxisomal proliferation.

Substrate and hormone regulation of palmitoyl-CoA synthetase in 7800 C1 Morris hepatoma cells and cultured rat hepatocytes

The effects of tetradecylthioacetic acid (TTA), insulin and dexamethasone on palmitoyl-CoA synthetase activity and its mRNA both in 7800 Cl hepatoma cells and cultured rat hepatocytes were studied. (1) When the hepatoma cells were cultivated in the presence of fatty acids or alkyl thioacetic acids (3-thia fatty acids) palmitoyl-CoA synthetase activity was increased several fold. The stronger effect was obtained with TTA, which also increased long-chain acyl-CoA synthetase mRNA significantly. TTA has no inducing effect on butyryl-CoA synthetase and little effect on octanoyl-CoA synthetase in the same cells. Dexamethasone also had inducing effect on palmitoyl-CoA synthetase in the hepatoma cells. Insulin counteracted the induction given by TTA. All of these regulation actions take place at the pretranslational level. (2) In isolated hepatocytes the activity of palmitoyl-CoA synthetase was much higher than in hepatoma cells, but it was lost rapidly in culture. The loss of the enzyme activity was slowed down in the presence of TTA and insulin, either alone or combined. Dexamethasone combined with TTA reversed the loss of enzyme activity, while dexamethasone alone even increased the loss. Analysis of palmitoyl-CoA synthetase mRNA shows that TTA prevents the loss of the enzyme activity by inducing mRNA of the enzyme, dexamethasone enhances the effect of TTA, while insulin stabilizes the enzyme activity in the cultured cells without increasing the mRNA level.

Studies on the effect of fenoprofen on the activation and oxidation of long chain and very long chain fatty acids in hepatocytes and subcellular fractions from rat liver

We studied the effect of fenoprofen on the activation of palmitic acid (C 16:0), lignoceric acid (C 24:0) and cerotic acid (C 26:0) in microsomal and peroxisomal fractions from rat liver. Fenoprofen was found to inhibit the formation of palmitoyl-CoA in both microsomal and peroxisomal fractions whereas the formation of lignoceroyl-CoA and cerotoyl-CoA was not inhibited at all. In freshly isolated rat hepatocytes palmitic acid β-oxidation was progressively inhibited at increasing concentrations of fenoprofen, most probably due to its inhibitory effect on palmitoyl-CoA synthetase activity. On the other hand, fenoprofen was also found to inhibit the β-oxidation of lignoceric acid and cerotic acid in rat hepatocytes. It is shown that the acyl-CoA oxidase activity with lignoceroyl-CoA as substrate was inhibited by fenoprofen whereas the palmitoyl-CoA and pristanoyl-CoA oxidase activities were not inhibited by fenoprofen. This finding provides an explanation for the inhibitory effect of fenoprofen on lignocerate and cerotate β-oxidation in hepatocytes.

Docosahexaenoic acid shows no triglyceride-lowering effects but increases the peroxisomal fatty acid oxidation in liver of rats.

The effect of docosahexaenoic acid (DHA) on mitochondrial and peroxisomal fatty acid oxidation and on key enzymes in triglyceride metabolism was investigated in the liver of rats fed a standard diet, a cholesterol diet, and a pelleted chow diet. Unexpectedly, in all three rat models repeated administration of highly purified DHA (92% pure) at different doses and times, at a dose of 1000 mg/day per kg body weight, resulted in no significant decrease of hepatic and plasma concentration of triglycerides. The serum concentrations of cholesterol and phospholipids showed an increase in a time-dependent manner in rats fed the pelleted chow diet. The hepatic concentration of cholesterol was increased in rats fed the cholesterol diet and pelleted chow diet after administration of DHA compared to palmitic acid. In all rat models, treatment with DHA tended to increase the peroxisomal beta-oxidation. This was accompanied with a significant increase (1.5-fold) of fatty acyl-CoA oxidase activity. The mitochondrial fatty acid oxidation system and carnitine palmitoyl-transferase activity, however, were almost unchanged. Moreover, palmitoyl-CoA synthetase activity was increased, whereas the palmitoyl-CoA hydrolase activity was decreased. Neither microsomal phosphatidate phosphohydrolase activity nor cytosolic phosphatidate phosphohydrolase activity was affected by DHA feeding in the three rat models. Acyl-CoA:1,2-diacylglycerol acyltransferase activity was also unaffected. In contrast to docosahexanoic acid feeding, eicosapentaenoic acid (EPA) administration possessed a hypotriglyceridemic effect and resulted in an increase of mitochondrial and peroxisomal oxidation of fatty acids. Carnitine palmitoyltransferase activity was also stimulated. Phosphatidate phosphohydrolase activity was unaffected whereas diacylglycerol acyltransferase activity was increased by EPA treatment compared with palmitic acid feeding. The results indicate that docosahexaenoic acid, in contrast to eicosapentaenoic acid, does not inhibit the synthesis and secretion of triglycerides in the liver. In addition, the results emphasize the importance that stimulation of peroxisomal beta-oxidation by these n-3 fatty acids is not sufficient to decrease the serum levels of triglycerides. In addition, increased mitochondrial beta-oxidation of fatty acids and thereby decreased availability of nonesterified fatty acids may be a mechanism by which EPA inhibits triglyceride, and subsequently very low density lipoprotein-triglyceride, production. Whether DHA and EPA possess different metabolic properties should be considered.

Fasting induced alterations in mitochondrial palmitoyl-coa metabolism may inhibit adipocyte pyruvate dehydrogenase activity

1. 1. Adipocytes from fed and fasted (24 hr) groups of rats were fractionated into mitochondria, microsomes and plasma membranes. 2. 2. Fasting significantly decreased the mitochondrial activity of palmitoyl-CoA synthetase, palmitoyl-CoA hydrolase, β -oxidation and pyruvate dehydrogenase. 3. 3. Fasting elevated intramitochondrial long-chain acyl-CoA. 4. 4. Pyruvate dehydrogenase was inhibited 50% by addition of 30 μM palmitoyl-CoA. 5. 5. Fasting-induced changes in palmitoyl-CoA metabolism may modulate pyruvate dehydrogenase activity in adipocyte mitochondria.

Presence and properties of acyl coenzyme A synthetase for medium-chain fatty acids in rat intestinal mucosa.

A system was developed for assay of acyl coenzyme A (acyl CoA) activity on medium-chain fatty acids in rat intestinal mucosa. Using this system, we compared the characteristics of octanoyl (C8:0) CoA synthetase activity and palmitoyl (C16:0) CoA synthetase activity. Palmitoyl CoA synthetase activity as a function of palmitate concentration followed Michaelis-Menten kinetics, but octanoyl CoA synthetase activity as a function of octanoate concentration showed a biphasic reaction curve. The distributions of octanoyl CoA synthetase activity and palmitoyl CoA synthetase activity along the gastrointestinal tract were similar, both activities being present mainly in the middle portion of the small intestine. Incubation of octanoate with a homogenate of intestinal mucosa revealed that octnoate, like palmitate, is incorporated into phospholipids and triglycerides after its CoA activation. Oral administration of medium chain triglycerides induced a nearly 2-fold increase in octanoyl CoA synthetase activity in rat intestinal mucosa, whereas oral administration of long chain triglycerides did not affect the palmitoyl CoA synthetase activity. These results indicate that acyl CoA synthetase for medium-chain fatty acids in rat intestinal mucosa plays a key role in the utilization of medium-chain fatty acids for lipid synthesis, and that it is regulated in a different manner from acyl CoA synthetase for long-chain fatty acids.

Identity between palmitoyl-CoA synthetase and arachidonoyl-CoA synthetase in human platelet?

Apparent Km values have been determined for the substrates ATP, CoA and fatty acids for the long-chain acyl-CoA synthetase (EC 6.2.1.3) reaction in lysates of human blood platelets. The apparent Km for ATP was higher for saturated fatty acids (C12:0 to C18:0) than for unsaturated acids (C18:1 to C22:6). Other apparent Km values were very similar for all long-chain fatty acids tested. Palmitic acid inhibited the formation of [14C]arachidonoyl-CoA, and arachidonic acid inhibited the formation of [14C]palmitoyl-CoA, with [14C]arachidonate or [14C]palmitate respectively as substrate. After chromatography of Triton X-100-extracted platelet protein in several systems (hydroxyapatite, DEAE-Sepharose, Sephacryl S-200 HR, CoA-Sepharose, Sephadex G-100 and AcA 34), both arachidonoyl-CoA synthetase and palmitoyl-CoA synthetase activities were eluted together in the various protein peaks, and with approximately the same ratio of activities in all peaks. After some purification steps (DEAE-Sepharose and Sephacryl S-200 HR), the acyl-CoA synthetase activity was up to 37 nmol/min per mg of protein with [14C]palmitate as substrate, and up to 116 nmol/min per mg of protein with [14C]arachidonate as substrate. The purification was respectively about 8- and 10-fold. The results indicate that palmitoyl-CoA (or unspecific) synthetase and arachidonoyl-CoA (or specific) synthetase are in fact the same enzyme, in agreement with previously reported results from this laboratory.

Effects of sodium 2-[5-(4-chlorophenyl)pentyl]-oxirane-2-carboxylate (POCA) on fatty acid oxidation in fibroblasts from patients with peroxisomal diseases

The effects of sodium 2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA), a potent inhibitor of carnitine palmitoyltransferase I, on fatty acid oxidation were investigated using fibroblasts from control subjects and from patients with peroxisomal disorders. [1- 14C]Palmitate oxidation was inhibited by 8% of the control value when 15 μM POCA was added to the medium. The inhibition by POCA was significantly (P < 0.05) stronger in fibroblasts from patients with Zellweger syndrome or with neonatal adrenoleukodystrophy, in which peroxisomes and peroxisomal β-oxidation enzymes were absent. However, the inhibition in fibroblasts from patients with X-linked adrenoleukodystrophy, in which a specific defect of peroxisomal lignoceroyl-CoA synthetase was speculated, was similar to that in the controls. [1- 14C]Lignocerate oxidation was not influenced by the addition of POCA, in samples from the controls and from the patients. These results indicate that peroxisomes account for a small but demonstrable proportion of palmitate oxidation, and add new evidence to the concept that lignocerate is oxidized exclusively in the peroxisomes. Our findings also support the hypotheses that the activity of palmitoyl-CoA synthetase and the enzymes of β-oxidation cycle in peroxisomes are normal in patients with X-linked adrenoleukodystrophy and that a specific defect of lignoceroyl-CoA synthetase is responsible for the accumulation of very long chain fatty acids in these patients.

Peroxisome proliferating sulphur- and oxy-substituted fatty acid analogues are activated to acyl coenzyme a thioesters

In liver homogenates from untreated rats the sulphur-substituted fatty acid analogues tetradecylthioacetic acid (CMTTD) was activated to its acyl-coenzyme A thioester. The activation was found to take place in the mitochondrial, microsomal and peroxisomal fractions. The activity of CMTTD-CoA synthetase was 50% compared to palmitoyl-CoA synthetase in all cellular fractions. When rats were treated with the peroxisome proliferating sulphur-substituted fatty acid analogues CMTTD and 3-dithiahexadecanedioic acid (BCMTD), the CMTTD-CoA synthetase activity was induced in mitochondrial, peroxisomal and microsomal fractions. Palmitoyl-CoA synthetase was induced proportionally. In rats treated with tetradecylthiopropionic acid (CETTD) of low peroxisome proliferating potency, the activities of CMTTD-CoA synthetase and palmitoyl-CoA synthetase were inhibited in mitochondrial and microsomal fractions. In contrast, all three sulphur-substituted acids induced the activity of palmitoyl-CoA synthetase and CMTTD-CoA synthetase in peroxisomes. Both the CMTTD-CoA and palmitoyl-CoA synthetase activities were induced by CMTTD and BCMTD, in close correlation to the induction of peroxisomal β-oxidation. During the three treatment regimes, CMTTD-CoA synthetase activity ran parallel to the palmitoyl-CoA synthetase activity at a rate of 50% in all cellular fractions. Thus, CMTTD is assumed to be activated by the long-chain acyl-CoA synthetase enzyme. Rats were treated for 5 days with sulphur- and oxy-substituted fatty acid analogues, clofibric acid and fenofibric acid. All compounds which induced peroxisomal β-oxidation activity in vivo could be activated to their respective CoA thioesters in liver homogenate. CETTD which induced peroxisomal β-oxidation only two-fold, was activated at a rate of 50% compared to palmitate. Fenofibric acid induced peroxisomal β-oxidation 9.6-fold, while it was activated at a rate of only 10% compared to palmitate. Thus, no correlation was found between rate of activation in vitro and induction of peroxisomal activity in vivo. On the other hand, tetradecylsulfoxyacetic acid (TSOA) and tetradecylsulfonacetic acid (TSA) (sulphuroxygenated metabolites of CMTTD) with no inductive effects, were not activated to their respective CoA derivatives. Altogether the data suggest that the enzymatic activation of the peroxisome proliferating compounds is essential for their proliferating activity, but the rate of activation does not determine the potency of the proliferators. The role of the xenobiotic-CoA pool in relation to the whole coenzyme A profile during peroxisome proliferation is discussed.

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 β-oxidation), alkyl(C12)thiopropionic acid (CETTD, undergo one cycle of β-oxididation) and a 3-thiadicarboxylic acid (BCMTD, blocked for both ω- (and β-oxidation) influence the peroxisomal β-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 β-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 β-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 β-oxidation was marginally affected. Whether inhibition of mitochondrial β-oxidation may be involved in regulation of peroxisomal fatty acid metabolism and in development of fatty liver should be considered.

Effects of the tumor promoter 12- O-tetradecanoylphorbol 13-acetate on peroxisomal activities and enzyme activities involved in lipid metabolism in rat liver

The effects of 12- O-tetradecanoylphorbol 13-acetate (TPA) on hepatic lipids and key enzymes involved in esterification, hydrolysis and oxidation of long-chain fatty acids at increasing doses were investigated in rats. TPA administration tended to decrease the mitochondrial activities of palmitoyl-CoA synthetase and carnitine palmitoyltransferase. The microsomal palmitoyl-CoA synthetase activity was increased. TPA administration was also associated with a dose-dependent increase of glycerophosphate acyltransferase activity both in the mitochondrial and microsomal fractions in particular. The data are consistent with a decreased catabolism of long-chain fatty acids at the mitochondrial level, and an increased capacity for esterification of fatty acids in the microsomal fraction. Peroxisomal β-oxidation was increased about 2-fold in the peroxisome-enriched fraction of TPA-treated rats while the catalase and urate oxidase activities were only marginally affected. TPA administration revealed elevated capacity for hydrolysis of palmitoyl-CoA and palmitoyl- l-carnitine in the microsomal fraction. Neither increased cytosolic palmitoyl-CoA hydrolase activity nor increased hydroxylation of lauric acid nor changes of the hepatic content of cytochrome P-450 isoenzymic forms were observed in the TPA-treated animals. There was no induction of the protein content of the bifunctional enoyl-CoA hydratase. Thus, TPA behaves more like choline-deficient diet and ethionine treatment than well-known peroxisome proliferators. It seems possible that TPA selectively stimulated the peroxisomal activities, i.e., peroxisomal β-oxidation rather than evoking a peroxisome proliferation capacity.