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Mussels may concentrate pollutants, with possibly significant side effects on human health. Therefore, mussels (Mytilus galloprovincialis) from two sites of the Moroccan Atlantic coast (Jorf Lasfar [JL], an industrial site, and Oualidia [OL], a vegetable-growing area), were subjected to biochemical analyses to quantify the presence of heavy metals (Cd, Cr, and Pb) and to establish the lipid profile: fatty acid, cholesterol, oxysterol, phytosterol and phospholipid content. In addition, mussel lipid extracts known to accumulate numerous toxic components were tested on murine pancreatic ß-cells (MIN6), and their biological activities were measured with various flow cytometric and biochemical methods to determine their impacts on cell death induction, organelle dysfunctions (mitochondria, lysosomes, and peroxisomes), oxidative stress and insulin secretion. The characteristics of JL and OL lipid extracts were compared with those of commercially available mussels from Spain (SP) used for human consumption. OL and JL contained heavy metals, high amounts of phospholipids, and high levels of oxysterols; the [(unsaturated fatty acids)/(saturated fatty acids)] ratio, which can be considered a sign of environmental stress leading to lipid peroxidation, was low. On MIN6 cells, JL and OL lipid extracts were able to trigger cell death. This event was associated with overproduction of H2 O2 , increased catalase activity, a decreased GSH level, lipid peroxidation and stimulation of insulin secretion. These effects were not observed with SP lipid extracts. These data suggest that some components from OL and JL lipid extracts might predispose to pancreatic dysfunctions. Epidemiological studies would be needed to assess the global risk on human health and the metabolic disease incidence in a context of regular seafood consumption from the OL and JL areas.

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Consumption of trans fatty acids is positively correlated with cardiovascular diseases and with atherogenic risk factors. Trans fatty acids might play their atherogenic effects through lipid metabolism alteration of vascular cells. Accumulation of lipids in vascular smooth muscle cells is a feature of atherosclerosis and a consequence of lipid metabolism alteration. Stearoyl-CoA desaturase 1 (scd1) catalyses the production of monounsaturated fatty acids (e.g. oleic acid) and its expression is associated with lipogenesis induction and with atherosclerosis development. We were interested in analysing the regulation of delta-9 desaturation rate and scd1 expression in human aortic smooth muscle cells (HASMC) exposed to cis and trans C18:1 fatty acid isomers (cis-9 oleic acid, trans-11 vaccenic acid or trans-9 elaidic acid) for 48 h at 100 µM. Treatment of HASMC with these C18:1 fatty acid isomers led to differential effects on delta-9 desaturation; oleic acid repressed the desaturation rate more potently than trans-11 vaccenic acid, whereas trans-9 elaidic acid increased the delta-9 desaturation rate. We then correlated the delta-9 desaturation rate with the expression of scd1 protein and mRNA. We showed that C18:1 fatty acids controlled the expression of scd1 at the transcriptional level in HASMC, leading to an increase in scd1 mRNA content by trans-9 elaidic acid treatment, whereas a decrease in scd1 mRNA content was observed with cis-9 oleic acid and trans-11 vaccenic acid treatments. Altogether, this work highlights a differential capability of C18:1 fatty acid isomers to control scd1 gene expression, which presumes of different consequent effects on cell functions.

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Decreased body fat mass and liver steatosis have been reported in mice fed diets containing the conjugated linoleic acid trans-10,cis-12-C18:2 (CLA2), but not in those fed diets containing cis-9,trans-11-C18:2 (CLA1). Because the decrease in fatty acid (FA) oxidation may cause fat accumulation, we questioned whether the effects of both CLAs on enzyme activities and mRNA expression were related to liver FA oxidation. To address this question, 7-wk-old male C57BL/6J mice were fed for 4 wk a diet supplemented with 1% CLA1, CLA2, or cis-9-C18:1 (control) esterified as triacylglycerols. In CLA2-fed mice, the proportions of CLA2 in the total FA of liver lipids were substantially lower than those of CLA1 in mice fed CLA1. The mitochondrial protein content per total liver was about 56% greater in CLA2-fed mice than in CLA1-fed mice and controls. Mitochondrial carnitine palmitoyltransferase I (CPT I) and carnitine-dependent palmitate oxidation activities were also significantly greater in CLA2-fed mice than in the two other groups. The amounts of malonyl-CoA per gram of liver and the sensitivity of CPT I to malonyl-CoA inhibition were greater in both groups of CLA-fed mice than in the controls. L-CPT I mRNA expression doubled in CLA2-fed mice and was 3 and 2 times greater for M-CPT I in the CLA1 and CLA2 groups, respectively, compared with controls. Peroxisomal FA oxidation-related activities and acyl-CoA oxidase mRNA expression were increased in CLA1-fed mice, and to a larger extent in CLA2-fed mice, relative to controls. These data indicate that FA oxidation capacities were increased in mice fed CLA2, but were likely depressed in vivo through malonyl-CoA inhibition.

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Rumenic acid (cis-9, trans-11-C(18:2)) represents approx. 80% of conjugated linoleic acid (CLA) in dairy products. CLA has been shown to exert beneficial effects on health, but little work has been devoted to the ability to oxidize CLA isomers and the role of these isomers in the modulation of beta-oxidation flux. In the present study, respiration on rumenic acid was compared with that on linoleic acid (cis-9, cis-12-C(18:2)) with the use of rat liver mitochondria. In state-3, respiration was decreased by half with rumenic acid in comparison with linoleic acid. In the uncoupled state, respiration on CLA remained 30% lower. The lower ability to oxidize CLA was investigated through characterization of the enzymic steps. Rumenic acid was 33% less activated by acyl-CoA synthase than was linoleic acid. However, after such activation, the transfer of both acyl moieties to carnitine by carnitine acyltransferase I (CAT I) was of the same order. Moreover, CAT II activity was comparable with either isomer. After prior incubation with rumenic acid, oxidation of octanoic acid by re-isolated mitochondria was unimpaired, but that of palmitoleic acid was impaired unless linoleic acid was used in the prior incubation. The slower respiration on cis-9, trans-11-C(18:2) is suggested to arise from lower carnitine-acylcarnitine translocase activity towards the acylcarnitine form, causing an upstream increase in the corresponding acyl-CoA.

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(i) Highly purified mitochondrial fractions were practically devoid of microsomal contamination and of acyl-CoA ligase activity. (ii) In mitochondria, glycerol-3-phosphate acyltransferase (GPAT) activity was supported by two enzymes, the first being very active at low palmitoyl-CoA/albumin ratios and sensitive to external agents (external form), the second being detected only at higher palmitoyl-CoA/albumin ratios and insensitive to external agents (internal form). (iii) Carnitine palmitoyltransferase I (CPT I) activity was shown to inhibit external GPAT activity only. (iv) Glycerol-3-phosphate exerted an inhibitory effect on CPT I, even when GPAT was inactive. Reciprocal interaction of CPT I and GPAT was discussed with regard to the balance existing between fatty acid oxidation and esterification metabolic pathways.

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(1) Effects of dietary treatment of male albino rats with eicosapentaenoic acid (EPA) or docosahexaenoic acid on hepatic mitochondrial lipid metabolism have been investigated. (2) Mitochondria isolated from rats given these treatments were shown to have increased ability to respire on acyl-CoA esters in the presence of malonate. This effect was expressed with most of the long-chain acyl-CoA esters used as substrates. When malonate in the incubations was replaced with malate, mitochondria from treated animals were found to exhibit diminished rates of respiration on polyunsaturated acyl-CoA esters, in particular linolenoyl-, eicosapentaenoyl- and docosahexaenoyl-CoA. This phenomenon could not be attributed to changes in activity of carnitine palmitoyltransferase I or in peroxisomal beta-oxidation. (3) Uncoupled respiration on glutamate, malate or succinate was also affected by treatment with EPA. With liver mitochondria isolated from rats that had been treated with a omega-3 fatty acid in the fasted state, the respiratory rates were lower than those observed with mitochondria isolated from control rats. Respiratory rates with mitochondria isolated from rats given the omega-3 fatty acid in the fed state was not significantly different from control rates. (4) In rats treated with EPA in the fed state, the amount of EPA incorporated into mitochondrial lipids was markedly more increased as compared to rats given omega-3 fatty acid in the fasted state. Incorporation of dietary EPA into tissue lipids was investigated, also following mildronate treatment of rats (an inhibitor of carnitine biosynthesis). (5) A hypolipidaemic effect of dietary EPA was only observed when the fatty acid was given to fed rats. Rats treated with EPA in the fasted state, in contrast, exhibited hypoglycaemia, the hypolipidaemic effects now being absent. (6) These results suggest that hypolipidaemia is most pronounced when the metabolic state favours incorporation of dietary EPA into body lipids rather than its beta-oxidation, as mediated by the fed/fasted transition or by treatment with mildronate.

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The five-fold higher carnitine content in the liver of fenofibrate-treated rats addresses the question about the possible role of this enhancement in the hypolipidaemic effect of the drug and the underlying mechanisms. When fenofibrate was administered with mildronate (a gamma-butyrobetaine hydroxylase inhibitor) in suitable amount, the content in carnitine was found to be normalized in liver. However, triglyceride contents of liver and serum were then at least as low as in rats treated by fenofibrate only. When carnitine concentration was lowered by mildronate to the third of the normal value, a marked increase in triglycerides occurred both in liver and serum, while the five-fold increase in carnitine due to fenofibrate enhanced blood ketone body concentration with no effect on liver and serum triglycerides. Data suggest that the normal carnitine concentration is largely sufficient to meet the usual requirement for carnitine palmitoyltransferase I activity (CPT I). In rat liver, increase in mitochondrial CPT I activity and in peroxisomal fatty acid oxidation may constitute part of the hypolipidaemic effect of fenofibrate.

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This study was designed to determine which enzyme activities were first impaired in mitochondria exposed to 2,2'-azobis-(2-amidinopropane) dihydrochloride (AAPH), a known radical initiator. EPR spin-trapping revealed generation of reactive oxygen species although malondialdehyde formation remained very low. With increasing AAPH concentrations, State-3 respiration was progressively depressed with unaltered ADP/O ratios. A top-down approach demonstrated that alterations were located at the phosphorylation level. As shown by inhibitor titrations, ATP/ADP translocase activity was unaffected in the range of AAPH concentrations used. In contrast, AAPH appeared to exert a deleterious effect at the level of F1F0-ATPase, comparable with dicyclohexylcarbodi-imide, which alters Fo proton channel. A comparison of ATP hydrolase activity in uncoupled and broken mitochondria reinforced this finding. In spite of its pro-oxidant properties, AAPH was shown to act as a dose-dependent inhibitor of cyclosporin-sensitive permeability transition initiated by Ca2+, probably as a consequence of its effect on F1F0-ATPase. Resveratrol, a potent antiperoxidant, completely failed to prevent the decrease in State-3 respiration caused by AAPH. The data suggest that AAPH, when used under mild conditions, acted as a radical initiator and was capable of damaging F1F0-ATPase, thereby slowing respiratory chain activity and reducing mitochondrial antioxidant defences.

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This study was designed to examine whether short- and long-term treatments by a low level of dietary L-carnitine are capable of altering enzyme activities related to fatty acid oxidation in normal Wistar rats. Under controlled feeding, ten days of treatment changed neither body weights nor liver and gastrocnemius weights, but succeeded in reducing the weight of peri-epididymal adipose tissues. Triacylglycerol contents were lowered in liver and ketone body concentrations were found slightly more elevated in blood. In the liver, mitochondrial carnitine palmitoyltransferase I (CPT I) exhibited a slightly higher specific activity and a lower sensitivity to malonyl-CoA inhibition, while peroxisomal fatty acid oxidizing system (PFAOS) was found to be less active. Carnitine supplied for one month reduced the mass of the periepididymal fat tissue, but not those of the other studied organs, and produced a slight but non-significant gain in body weight after ten days of treatment. In the liver, CPTI characteristics were comparable in control and treated groups, while PFAOS activity was less in rats receiving carnitine. Data show that L-carnitine at a low level in the diet exerted two paradoxical effects before and after ten days of treatment. Results are discussed in regard to fatty acid oxidation in mitochondria and peroxisomes, and to the possible altered acyl-CoA/acylcarnitine ratio with increased concentrations of L-carnitine in the liver.

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The first rate limiting step in the conversion of alpha-linolenic acid is catalyzed by the delta-6-desaturase enzyme. The activity of such an enzyme was studied in order to investigate the n-3 Polyunsaturated Fatty Acid biogenesis during hypertension. Rat isolated hepatocyte n-3 delta-6-desaturase activity was higher in 1 month old Spontaneously Hypertensive Rats -- prehypertensive period-as compared to normotensive Wistar Kyoto rats, whereas there was no significant differences at 12 months -- hypertensive period-. Our data indicate no correlation between the directly measured enzyme activity and the changes in hepatocyte n-3 fatty acid compositions. The loss of hepatocyte n-3 delta-6-desaturase activity in the Spontaneously Hypertensive Rat may be a key factor in the evolution of hypertension related to aging through altering the eicosanoid balance.

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The synthesis of rac-1-palmitoyl-2-oleoylglycero-3-phospho-[3H]choline of high specific activity was carried out on a microscale by making 7 mumol of rac-1-palmitoyl-2-oleoylglycerol react first with an equimolar amount of POCl3 and then of [3H]choline. After purification by thin-layer chromatography and normal-phase high-performance liquid chromatography and normal-phase high-performance liquid chromatography (HPLC), the yield of the synthesis of [3H]phosphatidylcholine (120 microCi/mumol) was 22%. rac-1-Palmitoyl-2-oleoylglycerol was purified before use by reversed-phase HPLC under conditions which were nonisomerizing and allowed the separation of 1,2- and 1,3-isomers of diacylglycerol. Ethanol, but not benzene, was shown to cause isomerization of long-chain diacylglycerol and, therefore, was not used for drying the substrate before reaction. A rapid and complete separation of 1,2- and 1,3-isomers of long-chain phosphatidylcholine was obtained by reversed-phase HPLC using 20 mM choline chloride in methanol/acetonitrile/water (50:50:1, by vol) isocratically as the mobile phase. Under these conditions, analysis of the synthesized rac-1-palmitoyl-2-oleoylglycero-3-phospho-[3H]choline showed a total absence of 1,3-isomer.

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This study was designed to examine whether the depletion of L-carnitine may induce compensatory mechanisms allowing higher fatty acid oxidative activities in liver, particularly with regard to mitochondrial carnitine palmitoyltransferase I activity and peroxisomal fatty acid oxidation. Wistar rats received D-carnitine for 2 days and 3-(2,2,2,-trimethylhydrazinium)propionate (mildronate), a noncompetitive inhibitor of gamma-butyrobetaine hydroxylase, for 10 days. They were starved for 20 hr before being sacrificed. A dramatic reduction in carnitine concentration was observed in heart, skeletal muscles and kidneys, and to a lesser extent, in liver. Triacylglycerol content was found to be significantly more elevated on a gram liver and whole liver basis as well as per mL of blood (but to a lesser extent), while similar concentrations of ketone bodies were found in the blood of D-carnitine/mildronate-treated and control rats. In liver mitochondria, the specific activities of acyl-CoA synthetase and carnitine palmitoyltransferase I were enhanced by the treatment, while peroxisomal fatty acid oxidation was higher per gram of tissue. It is suggested that there may be an enhancement of cellular acyl-CoA concentration, a signal leading to increased liver fatty acid oxidation in acute carnitine deficiency.

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Liver mitochondrial fractions as normally isolated contain only 10-20% of total mitochondria and may not be representative of the whole mitochondrial population. This study was designed to evaluate the dependence of the sensitivity of carnitine palmitoyl-transferase I (CPT I) to malonyl-CoA inhibition in mitochondrial fractions that are not normally studied. Four fractions prepared from rat liver were found to be contaminated to different extents by microsome vesicles, on the basis of marker-enzyme activities and micrographic data. Purification of mitochondrial fractions on a Percoll gradient decreased to some extent the microsomal contamination, which was due in part to the existence of close bonds between microsomes and the outer membranes of mitochondria. A greater degree of contamination of mitochondrial fractions by microsomes was correlated with a greater sensitivity of CPT I to malonyl-CoA inhibition. Attempts were made to enhance the sensitivity of CPT I to malonyl-CoA with the use of microsomes. Measurements performed by adding mitochondria and microsomes in the same CPT I assay failed to demonstrate any significant enhancement of malonyl-CoA inhibition. However, addition of ATP to a mixture of mitochondria and microsomes was shown to trigger the binding of both particles, as assessed by enzymic and micrographic data, and to increase the sensitivity of CPT I to malonyl-CoA inhibition. These results demonstrated that the binding of microsomes to mitochondria, unlike the simple mixing of both particles, was capable of altering the sensitivity of CPT I to malonyl-CoA. The data also suggest that this process could be of physiological importance, owing to the frequency of contiguous zones between mitochondria and endoplasmic reticulum observed in sections of intact liver cells.

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This study was designed to examine whether n-3 and n-6 polyunsaturated fatty acids at a very low dietary level (about 0.2%) would alter liver activities in respect to fatty acid oxidation. Obese Zucker rats were used because of their low level of fatty acid oxidation, which would make increases easier to detect. Zucker rats were fed diets containing different oil mixtures (5%, w/w) with the same ratio of n-6/n-3 fatty acids supplied either as fish oil or arachidonic acid concentrate. Decreased hepatic triacylglycerol levels were observed only with the diet containing fish oil. In mitochondrial outer membranes, which support carnitine palmitoyltransferase I activity, cholesterol content was similar for all diets, while the percentage of 22:6n-3 and 20:4n-6 in phospholipids was enhanced about by 6 and 3% with the diets containing fish oil and arachidonic acid, respectively. With the fish oil diet, the only difference found in activities related to fatty acid oxidation was the lower sensitivity of carnitine palmitoyltransferase I to malonyl-CoA inhibition. With the diet containing arachidonic acid, peroxisomal fatty acid oxidation and carnitine palmitoyltransferase I activity were markedly depressed. Compared with the control diet, the diets enriched in fish oil and in arachidonic acid gave rise to a higher specific activity of aryl-ester hydrolase in microsomal fractions. We suggest that slight changes in composition of n-3 or n-6 polyunsaturated fatty acids in mitochondrial outer membranes may alter carnitine palmitoyltransferase I activity.

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Triacylglycerols from bovine milk fat were fractionated by reversed-phase liquid chromatography. The fatty acid and triacylglycerol compositions of each fraction were determined by capillary gas chromatography. These data were used to determine the accurate proportions of 223 individual molecular species of even-numbered triacylglycerols, accounting for 80% of total triacylglycerols (all percentages are expressed as moles per 100 mol). The three major triacylglycerols were butyroylpalmitoylacylglycerols, namely butyroylpalmitoyloleoylglycerol (4.2%), butyroyldipalmitoylglycerol (3.2%), and butyroylmyristoylpalmitoylglycerol (3.1%). Twenty-two triacylglycerols (> 1%) contained at least two of the four major long-chain fatty acids (C14:0, C16:0, C18:0, and C18:1). Among them were eight butyroyldiacylglycerols, the proportions of which reached 19% in total but only 12% when calculated on the basis of a random distribution of the fatty acids in the triacylglycerol molecules. More generally, most of the triacylglycerols that are composed of a short-chain fatty acid (C4:0 or C6:0) and two fatty acids in the range of C12 to C18 are preferentially synthesized by the mammary gland; their proportions (36% in total) were higher than the corresponding random values (24% in total). Conversely, the total amounts of simple (.4%) and mixed (2.9%) saturated long-chain (C14:0 to C18:0) triacylglycerols were much lower than those expected from random calculation (1.9 and 6.1%, respectively).

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Triacylglycerols (TGs) from a sample of summer butterfat (bovine milk) were analysed and fractionated by reversed-phase liquid chromatography (RPLC). Fatty acid and TG composition of eac of the 47 RPLC fractions ranging from 0.1 to 6.9% were determined by capillary gas chromatography. The data were used together to determine the quantitative composition of the molecular species of TGs. A large number of TG species, accounting for 80% of the total, could be unequivocally identified and individually determined. The combination of the chromatographic methods used proved to be a powerful and accurate approach for the determination of molecular species of TGs in a complex fat, but also a difficult and time-consuming task.

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Reversed-phase high-performance liquid chromatography on a thermostatted octadecylsilyl column was used to separate mixtures of labelled polyunsaturated fatty acids (as their methyl esters) formed by successive desaturations and elongations of labelled linoleic (18:2 n - 6) or linolenic (18:3 n - 3) acid by rat liver microsomes. Acetonitrile-water mixtures were used for elution of the esters. Unsaturated and saturated esters were detected by their refractive indices. The order of elution of fatty acid methyl esters in complex mixtures varies as a function of the chain length and unsaturation, analysis temperature, water concentration and solvent flow-rate. The peak areas vary as a function of the unsaturation. Specific radioactivities of 14C-labelled fatty acids and the percentage distribution of radioactivity among fatty acids from complex mixtures can be efficiently determined by collection and direct measurement of the radioactivity in the solvent by liquid scintillation counting. The method can be applied to complete compositional analysis, but is especially useful for determination of specific radioactivities during studies on the metabolic conversion of labelled polyunsaturated fatty acids.

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