Abraham Demoz

Structural and metabolic requirements for activators of the peroxisome proliferator-activated receptor

Fatty acids have recently been demonstrated to activate peroxisome proliferator-activated receptors (PPARs) but specific structural requirements of fatty acids to produce this response have not yet been determined. Importantly, it has hitherto not been possible to show specific binding of these compounds to PPAR. To test whether a common PPAR binding metabolite might be formed, we tested the effects of long-chain omega-3 polyunsaturated fatty acids, differentially beta-oxidizable fatty acids and inhibitors of fatty acid metabolism. We determined the activation of a reporter gene by a chimaeric receptor encompassing the DNA binding domain of the glucocorticoid receptor and the ligand binding domain of PPAR. The omega-3 unsaturated fatty acids were slightly more potent PPAR activators in vitro than saturated fatty acids. The peroxisomal proliferation-inducing, non-beta-oxidizable, tetradecylthioacetic acid activated PPAR to the same extent as the strong peroxisomal proliferator WY 14,643, whereas the homologous beta-oxidizable tetradecylthiopropionic acid was only as potent as a non-substituted fatty acid. Cyclooxygenase inhibitors, radical scavengers or cytochrome P450 inhibitors did not affect activation of PPAR. In conclusion, beta-oxidation is apparently not required for the formation of the PPAR-activating molecule and this moiety might be a fatty acid, its ester with CoA, or a further derivative of the activated fatty acid prior to beta-oxidation of the acyl-CoA ester. These data should aid understanding of signal transduction via PPAR and the identification of a receptor ligand.

Eicosapentaenoic acid at hypotriglyceridemic dose enhances the hepatic antioxidant defense in mice

The effect of oral administration of purified (95%) eicosapentaenoic acid on serum lipids, hepatic peroxisomal enzymes, antioxidant enzymes and lipid peroxidation was compared with that of palmitic acid fed mice and corresponding controls. After 10 d, a dose of 1000 mg eicosapentaenoic acid per day/kg body weight lowered serum triglycerides by 45%, while no significant change in serum cholesterol level was noted in comparison to palmitic acid fed mice and controls. Hepatic acyl-CoA oxidase and catalase activities increased by 50% and 30%, respectively, in the eicosapentaenoic acid fed group. In addition, the hepatic reduced glutathione content and the activities of glutathione transferase, glutathione peroxidase and glutathione reductase, increased significantly during eicosapentaenoic acid treatment. The levels of hepatic lipid peroxides were lower after eicosapentaenoic acid feeding, while no significant change was noted in the palmitic acid fed mice when compared to the controls. Taken together, the present data demonstrate for the first time that at hypolipidemic doses eicosapentaenoic acid feeding i) enhances the hepatic antioxidant defense, and ii) does not cause a significant differential induction of the two peroxisomal enzymes, acyl-CoA oxidase and catalase, as was noted after administration of hypolipidemic peroxisome proliferating compounds, such as clofibrate in rodents.

Rapid method for the separation and detection of tissue short-chain coenzyme A esters by reversed-phase high-performance liquid chromatography

A simple and rapid method for the separation and identification of tissue levels of short chain coenzyme A (CoA) esters by a reversed-phase high-performance liquid chromatography with ultraviolet-visible adsorbance detection is described. Samples of liver, heart and kidney tissues were homogenised in 5% sulfosalicylic acid containing 50 microM of dithioerythritol in 1:9 w/v proportion. Following centrifugation, 20 microliters of the supernatant were directly injected onto a 3-micron ODS C18 column (100 x 4.6 mm I.D.). The separation of acetyl-CoA, malonyl-CoA, methylmalonyl-CoA, succinyl-CoA, propionyl-CoA and free CoASH was achieved in less than 20 min using gradient elution with sodium phosphate, sodium acetate and methanol at a constant flow-rate of 1.5 ml/min. The lowest detection limit was 3 pmol.

LONG-TERM EFFECT OF TETRADECYLTHIOACETIC ACID : A STUDY ON PLASMA LIPID PROFILE AND FATTY ACID COMPOSITION AND OXIDATION IN DIFFERENT RAT ORGANS

Administration of tetradecylthioacetic acid (a 3-thia fatty acid) increases mitochondrial and peroxisomal beta-oxidative capacity and carnitine palmitoyltransferase activity, but reduces free fatty acid and triacylglycerol levels in plasma compared to palmitic acid-treated rats and controls. The decrease in plasma triacylglycerol was accompanied by a reduction (56%) in VLDL-triacylglycerol. Prolonged supplementation of tetradecylthioacetic acid caused a significant increase in lipogenic enzyme activities (ATP-citrate lyase and acetyl-CoA carboxylase) and diacylglycerol acyltansferase, but did not affect phosphatidate phosphohydrolase. Plasma cholesterol, LDL- and HDL-cholesterol levels were reduced. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase activity was, however, stimulated in 3-thia fatty acid-treated rats compared to controls. In addition. the mRNAs of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and LDL-receptor were increased. Tetradecylthioacetic acid administration affected the fatty acid composition in plasma and liver by increasing the amount of monoenes, especially 18:1(n-9), mostly at the expense of omega-3 fatty acids. Compared to liver a large amount of tetradecylthioacetic acid accumulated in the heart, and this accumulation was accompanied by an increase in omega-3 fatty acids, particularly 22:6(n-3) and a decrease in omega-6 fatty acids, mainly 20:4(n-6). The results show that the hypolipidemic effect of tetradecylthioacetic acid is sustained after prolonged administration and may, at least in part, be due to increased fatty acid oxidation and upregulated LDL-receptor gene expression. The increase in lipogenic enzyme activities as well as increased 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity, may be compensatory mechanisms to maintain cellular integrity. Decreased level of 20:4(n-6) combined with increased omega-3/omega-6 ratio in cardiac tissue after tetradecylthioacetic acid treatment may have influence on membrane dynamics and function.

Early effects on mitochondrial and peroxisomal β-oxidation by the hypolipidemic 3-thia fatty acids in rat livers

A single administration of 3-thiadicarboxylic and tetradecylthioacetic acids stimulates both mitochondrial and peroxisomal beta-oxidation and lowers plasma triacylglycerol levels. An increased rate of mitochondrial beta-oxidation and carnitine palmitoyl-transferase activity was established after 3 h and this was accompanied by a lowering of plasma triacylglycerol. Peroxisomal beta-oxidation, however, remained unchanged up to 8 h and was significantly increased after 12 h. These results suggest that after a single administration of 3-thia fatty acids mitochondrial beta-oxidation precedes peroxisomal beta-oxidation. Furthermore, they show that the observed tricylglycerol-lowering effect, which is established early (3-4 h) after the administration of 3-thia fatty acids, is initially due to an increased mitochondrial beta-oxidation.

Modulation of plasma and hepatic oxidative status and changes in plasma lipid profile by n-3 (EPA and DHA), n-6 (corn oil) and a 3-thia fatty acids in rats

This manuscript describes changes in plasma lipid profiles and parameters of oxidative status in the plasma and liver of rats fed 5 different fatty acids: 95% eicosapentaenoic acid, 92% docosahexaenoic acid (DHA), corn oil (n-6), 1-mono-(carboxymethylthio)-tetradecane (CMTTD) and palmitic acid (controls) for 3 months. At the given doses both EPA and the 3-thia fatty acid, CMTTD, caused a significant decrease in plasma triglycerides, phospholipids, free fatty acids and cholesterol. DHA decreased plasma free fatty acids and cholesterol, while corn oil feeding reduced only plasma free fatty acids. Plasma and hepatic vitamin E levels were significantly decreased in EPA, DHA and CMTTD fed rats, but remained unchanged in corn oil fed rats. Plasma glutathione was noted to decrease after EPA and DHA feeding but remained unchanged in other groups. However, hepatic glutathione content was increased in EPA, DHA and CMTTD fed rats, whereas cysteine levels were noted to decrease. As hepatic levels of cysteinylglycine remained unchanged, increased rate of cellular glutathione synthesis rather than its decreased degradation is likely to contribute to the increased hepatic glutathione content in EPA, DHA and CMTTD fed rats. Except for reduction in the levels of plasma lipid peroxidation caused by CMTTD, no significant changes were noted between the different treatment groups. Hepatic lipid peroxidation was elevated only in rats given DHA. Furthermore, our results show that EPA and DHA cause minimal imbalance of the peroxisomal H2O2 metabolising enzymes as compared to CMTTD. In addition, contrary to the potent peroxisome proliferator compound CMTTD which decreased the activities of glutathione transferase and glutathione peroxidase, EPA and DHA increased the activities of these detoxification enzymes.

Separation and detection of tissue CoASH and longchain acyl-CoA by reversed-phase high-performance liquid chromatography after precolumn derivatization

Abstract A method for the determination of tissue levels of free coenzyme A (CoASH) and long-chain acyl-CoA was developed using reversed-phase high-performance

Tetradecylthioacetic acid reduces the amount of lipid droplets, induces megamitochondria formation and increases the fatty acid oxidation in rat heart

The effects of prolonged administration (3 months) of a 3-thia fatty acid analogue and omega-3-fatty acids on cardiac fatty acid oxidation and the volume fraction of lipid droplets and mitochondria in cardiomyocytes were investigated. Doses were 1 g/day/kg body weight, except 150 mg/day/kg body weight for tetradecylthioacetic acid (a 3-thia fatty acid). One group served as control and did not receive any treatment. The volume fraction of lipid droplets in cardiomyocytes was significantly lower in the tetradecylthioacetic acid group compared to the other groups. Mitochondrial beta-oxidation was 60% greater and fatty acyl-CoA oxidase activity was increased by 430% in the tetradecylthioacetic acid group compared to control. This was accompanied by a greater volume fraction of mitochondria in cardiomyocytes (0.514 +/- 0.032% in tetradecylthioacetic acid v 0.318 +/- 0.007% in control) which was due to an increased size of mitochondria. The volume fraction of mitochondria was also greater in eicosapentaenoic acid (EPA) treated rats compared to control, but the enzymic activities were unaffected. Docosahexaenoic acid (DHA) treatment resulted in a greater volume fraction of lipid droplets in the cardiomyocytes, but the volume fraction of mitochondria and enzyme activities were unaltered. These results indicate that EPA and DHA have different effects on the modulation of mitochondrial biogenesis. Tetradecylthioacetic acid treatment results in megamitochondria formation and increased peroxisomal and mitochondrial beta-oxidation with a concomitant reduction of lipid droplets in the cardiomyocytes.

Coordinate induction of hepatic fatty acyl-CoA oxidase and P4504A1 in rat after activation of the peroxisome proliferator-activated receptor (PPAR) by sulphur-substituted fatty acid analogues

1. In the liver of rat fed a single dose of 3-thia fatty acids, 3-dithiahexadecanedioic acid (3-thiadicarboxylic acid) and tetradecylthioacetic acid, steady-state levels of P4504A1 and fatty acyl-CoA oxidase mRNAs increased in parallel. The increases were significant 8 h after administration, reaching a maximum after 12 h and decreased from 12 to 24 h after administration. 2. The corresponding enzyme activities of P4504A1 and fatty acyl-CoA oxidase were also induced in a parallel manner by the 3-thia fatty acids. The enzyme activities were significantly increased 12 h after administration and increased further after 24 h. This may reflect a possible effect of the 3-thia fatty acids not only on mRNA levels, but also on the translation and degradation rate of the two enzymes. 3. Repeated administration of 3-thia fatty acids resulted in an increase of the specific P4504A1 protein accompanied with an increased lauric acid hydroxylase activity. The correlation between induction of P4504A1 and fatty acyl-CoA oxidase mRNAs and their enzyme activities may reflect a coordinated rather than a causative induction mechanism, and that these genes respond to a common signal. This suggests that the increased P450 activity may not be responsible or be a prerequisite for fatty acyl-CoA oxidase induction. 4. Since the peroxisome proliferator-activated receptor (PPAR) plays a role in mediating the induction of fatty acyl-CoA oxidase, we analysed the activation of PPAR by fatty acids and sulphur-substituted analogues utilizing a chimera between the N-terminal and DNA-binding domain of the glucocorticoid receptor and the putative ligand-binding domain of PPAR. Arachidonic acid activated this chimeric receptor in Chinese hamster ovary cells. Inhibitors of P450 did not affect the activation of PPAR by arachidonic acid. Furthermore, dicarboxylic acids including 1,12-dodecanedioic acid or 1,16-hexadecanedioic acid only weakly activated the chimera. 3-Thidicarboxylic acid, however, was a much more effective activator than the non-sulphur-substituted analogues. In conclusion, the data suggest that the most likely mechanism of the induction process is fatty acid-induced activation of PPAR, which then leads to a coordinated induction of P4504A1 and fatty acyl-CoA oxidase.

Relationship between peroxisome-proliferating sulfur-substituted fatty acid analogs, hepatic lipid peroxidation and hydrogen peroxide metabolism

The effect of the administration of three peroxisome-proliferating sulfur-substituted fatty acid analogs on hepatic antioxidant status and lipid peroxidation was studied in rats. After 14 days of treatment, the ratio of induction of peroxisomal fatty acyl-CoA oxidase to catalase was 4.2 and 3.5 in rats treated with 1,10 bis-(carboxymethylthio)decane (BCMTD) and 1-mono (carboxymethylthio)tetradecane (CMTTD), respectively, while the corresponding ratio was 1.3 in 1-mono (carboxyethylthio)tetradecane (CETTD)-treated rats. As compared to the controls an increase in hepatic hydrogen peroxide content was noted in BCMTD- and CMTTD-treated rats, but not CETTD-treated rats. Hepatic lipid peroxidation was increased in all the three treatment groups in a manner not related to the potency of the compounds to induce the peroxisomal hydrogen peroxide metabolizing enzymes. Hepatic glutathione content increased while the activities of its associated enzymes such as glutathione transferase, glutathione peroxidase and glutathione reductase decreased in all the treated rats. Taken together, our data show a relationship between the levels of hydrogen peroxide and lipid peroxidation in rat livers treated with BCMTD and CMTTD. However, increased hepatic lipid peroxidation in CETTD-treated rats cannot be accounted for by the changes in the peroxisomal enzymes.