Pierre Clouet

Association of liver steatosis with lipid oversecretion and hypotriglyceridaemia in C57BL/6j mice fed trans-10,cis-12-linoleic acid

Conjugated linoleic acids (CLA) have recently been recognized to reduce body fat and plasma lipids in some animals. This study demonstrated that the steatosis accompanying the fat loss induced by trans‐10,cis‐12‐C18:2 (CLA2) and not cis‐9,trans‐11‐C18:2 (CLA1) isomer in C57BL/6j mice was not due to an alteration of the liver lipoprotein production that was even increased. The 3‐fold decrease in plasma triacylglycerol contents and the induction of mRNA expression of low‐density lipoprotein receptors concomitantly observed in CLA2‐fed mice suggested an increase in the lipoprotein clearance at the level of the liver itself. CLA1 feeding produced similar but attenuated effects on triglyceridaemia only.

Upregulation of liver VLDL receptor and FAT/CD36 expression in LDLR−/− apoB100/100 mice fed trans-10,cis-12 conjugated linoleic acid

This study explores the mechanisms responsible for the fatty liver setup in mice fed trans-10,cis-12 conjugated linoleic acid (t10c12 CLA), hypothesizing that an induction of low density lipoprotein receptor (LDLR) expression is associated with lipid accumulation. To this end, the effects of t10c12 CLA treatment on lipid parameters, serum lipoproteins, and expression of liver lipid receptors were measured in LDLR−/− apoB100/100 mice as a model of human familial hypercholesterolemia itself depleted of LDLR. Mice were fed t10c12 CLA over 2 or 4 weeks. We first observed that the treatment induced liver steatosis, even in the absence of LDLR. Mice treated for 2 weeks exhibited hypertriglyceridemia with high levels of VLDL and HDL, whereas a 4 week treatment inversely induced a reduction of serum triglycerides (TGs), essentially through a decrease in VLDL levels. In the absence of LDLR, the mRNA levels of other proteins, such as VLDL receptor, lipoprotein lipase, and fatty acid translocase, usually not expressed in the liver, were upregulated, suggesting their involvement in the steatosis setup and lipoprotein clearance. The data also suggest that the TG-lowering effect induced by t10c12 CLA treatment was attributable to both the reduction of circulating free fatty acids in response to the severe lipoatrophy and the high capacity of liver to clear off plasma lipids.

Biochemical hepatic alterations and body lipid composition in the herbivorous grass carp (Ctenopharyngodon idella) fed high-fat diets.

High-fat diets may have favourable effects on growth of some carnivorous fish because of the protein-sparing effect of lipids, but high-fat diets also exert some negative impacts on flesh quality. The goal of the study was therefore to determine the effects of fat-enriched diets in juvenile grass carp (Ctenopharyngodon idella) as a typical herbivorous fish on growth and possible lipid metabolism alterations. Three isonitrogenous diets containing 2, 6 or 10 % of a mixture of lard, maize oil and fish oil (1:1:1, by weight) were applied to fish for 8 weeks in a recirculation system. Data show that feeding diets with increasing lipid levels resulted in lowered feed intake, decreased growth and feed efficiency, and increased mesenteric fat tissue weight. Concomitantly, alteration of lipoprotein synthesis and greater level of lipid peroxidation were apparent in blood. In liver, muscle and mesenteric fat tissue, the percentages of alpha-linolenic acid and DHA were significantly increased or tended to increase with higher dietary lipid levels. Biochemical activity measurements performed on liver showed that, with the increase in dietary lipid level, there was a decrease in both mitochondrial and peroxisomal fatty acid oxidation capacities, which might contribute, at least in part, to the specific accumulation of alpha-linolenic acid and DHA into cells more active in membrane building. On the whole, grass carp have difficulty in energetically utilising excess dietary fat, especially when enriched in n-3 PUFA that are susceptible to peroxidation.

Conjugated linoleic acid isomers in mitochondria evidence for an alteration of fatty acid oxidation

The beneficial effects exerted by low amounts of conjugated linoleic acids (CLA) suggest that CLA are maximally conserved and raise the question about their mitochondrial oxidizability. Cis-9,trans-11-C18:2 (CLA1) and trans-10,cis-12-C18:2 (CLA2) were compared to cis-9,cis-12-C18:2 (linoleic acid; LA) and cis-9-C16:1 (palmitoleic acid; PA), as substrates for total fatty acid (FA) oxidation and for the enzymatic steps required for the entry of FA into rat liver mitochondria. Oxygen consumption rate was lowest when CLA1 was used as a substrate with that on CLA2 being intermediate between it and the respiration on LA and PA. The order of the radiolabeled FA oxidation rate was PA >> LA > CLA2 > CLA1. Transesterification to acylcarnitines of the octadecadienoic acids were similar, while uptake across inner membranes of CLA1 and, to a lesser extent, of CLA2 was greater than that of LA or PA. Prior oxidation of CLA1 or CLA2 made re-isolated mitochondria much less capable of oxidising PA or LA under carnitine-dependent conditions, but without altering the carnitine-independent oxidation of octanoic acid. Therefore, the CLA studied appeared to be both poorly oxidizable and capable of interfering with the oxidation of usual FA at a step close to the beginning of the β-oxidative cycle.

Enhancement of activities relative to fatty acid oxidation in the liver of rats depleted of l-carnitine by d-carnitine and a γ-butyrobetaine hydroxylase inhibitor

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.

EFFECT OF DIETARY N-3 AND N-6 POLYUNSATURATED FATTY ACIDS ON LIPID-METABOLIZING ENZYMES IN OBESE RAT LIVER

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.

Fatty acid oxidation and related gene expression in heart depleted of carnitine by mildronate treatment in the rat

The metabolic and genic effects induced by a 20-fold lowering of carnitine content in the heart were studied in mildronate-treated rats. In the perfused heart, the proportion of palmitate taken up then oxidized was 5–10% lower, while the triacylglycerol (TAG) formation was 100% greater than in controls. The treatment was shown to increase the maximal capacity of heart homogenates to oxidize palmitate, the mRNA level of carnitine palmitoyltransferase I (CPT-I) isoforms, the specific activity of CPT-I in subsarcolemmal mitochondria and the total carnitine content of isolated mitochondria. Concomitantly, the increased mRNA expression of lipoprotein lipase, fatty acid translocase and enzymes of TAG synthesis was associated with a 5- and 2-times increase in serum TAG and free fatty acid contents, respectively. The compartmentation of carnitine at its main functional location was expected to allow the increased CPT-I activity to ensure in vivo correct fatty acid oxidation rates. All the inductions related to fatty acid transport, oxidation and esterification most likely stem from the abundance of blood lipids providing cardiomyocytes with more fatty acids.

Hypolipidaemic effects of fenofibrate and fasting in the herbivorous grass carp ( Ctenopharyngodon idella) fed a high-fat diet.

We investigated whether the hypolipidaemic effect of fenofibrate and fasting observed in most omnivorous mammals may also apply to herbivorous fish. Grass carp (Ctenopharyngodon idella) fed a high-fat (8 %) diet exhibited a marked increase in blood lipids and body fat after 6 weeks. They were then treated with fenofibrate (100 mg/kg body weight) in the same high-fat diet for 2 weeks, followed by fasting for 1 week. Plasma lipid concentration, body fat amount, fatty acid composition, plasma thiobarbituric acid-reactive substances and some parameters related to hepatic fatty acid oxidation were measured, and liver samples were stained for histological examination. Fenofibrate treatment decreased TAG and cholesterol concentrations in plasma, total lipids of the whole body and liver, and EPA and DHA contents in tissues. Further, a mobilisation of mesenteric fat concomitant with an increase in hepatic peroxisomal fatty acid oxidation and lipid peroxidation was observed. Compared with fenofibrate treatment, fasting decreased body weight and plasma TAG, but not plasma cholesterol. It also reduced the fat content of the whole body and increased the EPA and DHA contents in the liver and other tissues. Fatty acid oxidation was stimulated by fasting in mitochondria, but not in peroxisomes. These data suggest that fenofibrate and fasting regulate the lipid metabolism in grass carp through different metabolic pathways. The grass carp is moderately responsive to a fibrate derivative in comparison with the well-known excess responsiveness of the rat model, and so it could be used for the study of lipid abnormalities as a herbivorous model.

Molecular cloning and characterization of the cDNA encoding the rat liver gamma-butyrobetaine hydroxylase

Carnitine biosynthesis from lysine and methionine involves five enzymatic reactions. gamma-butyrobetaine hydroxylase (BBH; EC 1.14. 11.1) is the last enzyme of this pathway. It catalyzes the reaction of hydroxylation of gamma-butyrobetaine to carnitine. The cDNA encoding this enzyme has been isolated and characterized. The cDNA contained an open reading frame of 1161 bp encoding a protein of 387 amino acids with a deduced molecular weight of 44.5 kDa. The sequence of the cDNA showed an important homology with the human cDNA recently isolated. Northern analysis showed gamma-butyrobetaine hydroxylase expression in the liver and in some extend in the testis and the epididymis. During this study, it also appeared that BBH mRNA expression was undetectable by Northern analysis during the perinatal period. During the development of the rat, the amount of BBH mRNA appeared after the weaning of the young rat and reached a maximal expression at the adult stage.

Effect of short and long-term treatments by a low level of dietary L-carnitine on parameters related to fatty acid oxidation in Wistar rat

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.