Joseph Gresti

CB1 Antagonism Exerts Specific Molecular Effects on Visceral and Subcutaneous Fat and Reverses Liver Steatosis in Diet-Induced Obese Mice

OBJECTIVE The beneficial effects of the inactivation of endocannabinoid system (ECS) by administration of antagonists of the cannabinoid receptor (CB) 1 on several pathological features associated with obesity is well demonstrated, but the relative contribution of central versus peripheral mechanisms is unclear. We examined the impact of CB1 antagonism on liver and adipose tissue lipid metabolism in a mouse model of diet-induced obesity. RESEARCH DESIGN AND METHODS Mice were fed either with a standard diet or a high-sucrose high-fat (HSHF) diet for 19 weeks and then treated with the CB1-specific antagonist SR141716 (10 mg · kg−1 · day−1) for 6 weeks. RESULTS Treatment with SR141716 reduced fat mass, insulin levels, and liver triglycerides primarily increased by HSHF feeding. Serum adiponectin levels were restored after being reduced in HSHF mice. Gene expression of scavenger receptor class B type I and hepatic lipase was induced by CB1 blockade and associated with an increase in HDL-cholesteryl ether uptake. Concomitantly, the expression of CB1, which was strongly increased in the liver and adipose tissue of HSHF mice, was totally normalized by the treatment. Interestingly, in visceral but not subcutaneous fat, genes involved in transport, synthesis, oxidation, and release of fatty acids were upregulated by HSHF feeding, while this effect was counteracted by CB1 antagonism. CONCLUSIONS A reduction in the CB1-mediated ECS activity in visceral fat is associated with a normalization of adipocyte metabolism, which may be a determining factor in the reversion of liver steatosis induced by treatment with SR141716.

Method for evaluating the bioconversion of radioactive polyunsaturated fatty acids by use of reversed-phase liquid chromatography

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.

Depletion of phytosterols from the plant plasma membrane provides evidence for disruption of lipid rafts

Involvement of sterols in membrane structural properties has been extensively studied in model systems but rarely assessed in natural membranes and never investigated for the plant plasma membrane (PM). Here, we address the question of the role of phytosterols in the organization of the plant PM. The sterol composition of tobacco BY‐2 cell PM was determined by gas chromatography. The cyclic oligosaccharide methyl‐β‐cyclodextrin, commonly used in animal cells to decrease cholesterol levels, caused a drastic reduction (50%) in the PM total free sterol content of the plant material, without modification in amounts of steryl‐conjugates. Fluorescence spectroscopy experiments using DPH, TMA‐DPH, Laurdan, and di‐4‐ANEPPDHQ indicated that such a depletion in sterol content increased lipid acyl chain disorder and reduced the overall liquid‐phase heterogeneity in correlation with the disruption of phytosterol‐rich domains. Methyl‐β‐cyclodextrin also prevented isolation of a PM fraction resistant to solubilization by nonionic detergents, previously characterized in tobacco, and induced redistribution of the proteic marker of this fraction, NtrbohD, within the membrane. Altogether, our results support the role of phytosterols in the lateral structuring of the PM of higher plant cells and suggest that they are key compounds for the formation of plant PM microdomains.— Roche, Y., Gerbeau‐Pissot, P., Buhot, B., Thomas, D., Bonneau, L., Gresti, J., Mongrand, S., Perrier‐Cornet, J.‐M., Simon‐Plas, F. Depletion of phytosterols from the plant plasma membrane provides evidence for disruption of lipid rafts. FASEB J. 22, 3980–3991 (2008)

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.

Altered adipose tissue metabolism in offspring of dietary obese rat dams

To investigate further the mechanisms of developmental programming, we analysed the effects of maternal overnutrition and of postnatal high-fat feeding on adipose tissue metabolism in the offspring. Postnatal changes in serum adiponectin, leptin and TAG [triacylglycerol (triglyceride)] levels, adipose tissue TAGs, fatty acids and enzyme activities were determined in offspring of cafeteria-diet-fed dams during gestation and lactation, weaned on to standard chow or on to cafeteria diet. Obese rats showed higher adiposity (+35% to 85%) as well as a significant increase in serum glucose, insulin, leptin, adiponectin and TAG levels (P<0.01) and adipose tissue LPL (lipoprotein lipase) and GPDH (glycerol-3-phosphate dehydrogenase) activities (P<0.01), compared with control pups at weaning (day 21) and at adulthood (day 90). Adipose HSL (hormone-sensitive lipase) activity was increased only at day 90 (P<0.05), and FAS (fatty acid synthase) activity remained unchanged. The proportions of SFAs (saturated fatty acids) and MUFAs (mono-unsaturated fatty acids) and the Δ(9)-desaturation index were significantly increased (P<0.05), whereas PUFAs (polyunsaturated fatty acids) were decreased (P<0.01) in serum and adipose TAGs of obese pups compared with controls. The cafeteria diet at weaning induced more severe abnormalities in obese rats. In conclusion, maternal overnutrition induced permanent changes in adipose tissue metabolism of the offspring. These pre-existing alterations in offspring were worsened under a high-fat diet from weaning to adulthood. Consequently, adipose adipokines and enzymes could provide a potential therapeutic target, and new investigations in this field could constitute strategies to improve the impact of early-life overnutrition.

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.

Antagonism of peripheral hepatic cannabinoid receptor-1 improves liver lipid metabolism in mice: Evidence from cultured explants†‡

It is well established that inactivation of the central endocannabinoid system (ECS) through antagonism of cannabinoid receptor 1 (CB1R) reduces food intake and improves several pathological features associated with obesity, such as dyslipidemia and liver steatosis. Nevertheless, recent data indicate that inactivation of peripheral CB1R could also be directly involved in the control of lipid metabolism independently of central CB1R. To further investigate this notion, we tested the direct effect of the specific CB1R antagonist, SR141716, on hepatic carbohydrate and lipid metabolism using cultured liver slices. CB1R messenger RNA expression was strongly decreased by SR141716, whereas it was increased by the CB1R agonist, arachidonic acid N‐hydroxyethylamide (AEA), indicating the effectiveness of treatments in modulating ECS activity in liver explants both from lean or ob/ob mice. The measurement of O2 consumption revealed that SR141716 increased carbohydrate or fatty acid utilization, according to the cellular hormonal environment. In line with this, SR141716 stimulated ß‐oxidation activity, and the role of CB1R in regulating this pathway was particularly emphasized when ECS was hyperactivated by AEA and in ob/ob tissue. SR141716 also improved carbohydrate and lipid metabolism, blunting the AEA‐induced increase in gene expression of proteins related to lipogenesis. In addition, we showed that SR141716 induced cholesterol de novo synthesis and high‐density lipoprotein uptake, revealing a relationship between CB1R and cholesterol metabolism. Conclusion: These data suggest that blocking hepatic CB1R improves both carbohydrate and lipid metabolism and confirm that peripheral CB1R should be considered as a promising target to reduce cardiometabolic risk in obesity. (HEPATOLOGY 2011)

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.

Time course of changes in serum oxidant/antioxidant status in overfed obese rats and their offspring.

The aim of the present study was to determine the time course of changes in oxidant/antioxidant status, as well as serum glucose, insulin, leptin and lipid levels, liver adipose tissue and muscle lipid and protein contents, in cafeteria-diet-fed dams during gestation and lactation, and in their offspring throughout adulthood. Food intake was also evaluated. The cafeteria diet induced a significant increase in maternal body and relative adipose tissue weights, daily energy intake, and plasma glucose, insulin, leptin and lipid levels at parturition (day 0) and at the end of lactation (day 21). Plasma total antioxidant status [ORAC (oxygen radical absorbance capacity)], erythrocyte catalase and SOD (superoxide dismutase) activities were lower, whereas plasma hydroperoxide and carbonyl protein levels were higher in cafeteria-diet-fed mothers compared with control mothers at days 0 and 21. Pups from cafeteria-diet-fed dams, both males and females, also had consistently higher body and relative adipose tissue weights, and plasma glucose, insulin, leptin, triacylglycerol (triglyceride) and cholesterol levels at birth (day 0), weaning (day 21) and 3 months of age (day 90). These offspring had significantly lower ORAC and catalase activity, and higher plasma hydroperoxide and carbonyl protein levels and SOD activity at birth, at days 21 and 90 compared with control offspring. In conclusion, excessive maternal fat and energy intake can play an important role in the development of metabolic disorders in the offspring. Maternal oxidative stress may be among the responsible factors. Fetal oxidative stress may present an additional confounding influence and probably contributes to additional disorders, aggravating features of the metabolic syndrome. An improvement in maternal oxidant/antioxidant status during pregnancy and lactation, with adequate nutrition, could have beneficial effects on the progeny.