Annie M. Bérard

Naringin, the major grapefruit flavonoid, specifically affects atherosclerosis development in diet-induced hypercholesterolemia in mice

Naringin (NAR) from grapefruit has exhibited potential protective effects against atherosclerosis development. However, specific mechanisms responsible for such effects are poorly understood. Thus, we aimed to investigate the antiatherogenic effects of NAR in different mouse models of hypercholesterolemia and decipher its molecular targets in the aorta using transcriptomic approach. Two mouse models of hypercholesterolemia, wild-type mice fed a high-fat/high-cholesterol diet and apolipoprotein E-deficient mice fed a semisynthetic diet, were studied. Mice were fed a respective control diets supplemented or not for 18 weeks with 0.02% of NAR, that is, nutritional supplementation. NAR supplementation reduced plaque progression only in wild-type mice fed the high-fat/high-cholesterol diet (-41%). Consistent with this protective effect, NAR reduced plasma non-high-density lipoprotein cholesterol concentrations as well as biomarkers of endothelial dysfunction. Microarray studies performed on aortas demonstrated differentially expressed genes encoding proteins involved in cell adhesion, actin cytoskeleton organization and cell division. Thus, the changes in gene expression induced by NAR could suggest a limited atherosclerosis progression by preventing immune cell adhesion and infiltration in the intima of vascular wall, as well as smooth muscle cell proliferation. Furthermore, this hypothesis was strengthened by in vitro experiments, which showed the ability of naringenin to reduce monocyte adhesion to endothelial cells and smooth muscle cell proliferation. In conclusion, this study revealed the antiatherogenic effect of NAR supplemented at a nutritionally achievable dose, specifically toward diet-induced atherosclerosis, and depicted its multitarget mode of action at the vascular level.

Low amounts of trans 18:1 fatty acids elevate plasma triacylglycerols but not cholesterol and alter the cellular defence to oxidative stress in mice.

Trans fatty acids are found mainly in processed foods. It has been shown that when their intake is high, total cholesterol, LDL-cholesterol and triacylglycerols are elevated, while HDL-cholesterol decreases. To evaluate a possible effect of these compounds, even in low amounts, C57Bl/6J mice were fed for 7 weeks a diet containing 13.6 % energy as partially hydrogenated rapeseed oil-enriched diet (Trans diet). The Trans diet contained 3 % energy as trans 18:1 fatty acid (elaidic acid). Control mice were on an isologous diet containing native rapeseed oil (Rapeseed diet) in which trans fatty acids were undetectable. Total, free and HDL-cholesterol as well as reverse cholesterol transport did not change. However, plasma triacylglycerol and VLDL levels increased. Hepatic gene expression in the Trans v. Rapeseed diet were compared using quantitative RT-PCR. The Trans diet produced a 2-3-fold elevation in mRNA of fatty acid synthase and microsomal transfer protein mRNA, explaining (at least in part) the observed increase in triacylglycerols and VLDL. In addition, mice on the Trans diet developed a deficiency in plasma vitamin E accompanied by a higher concentration of F2-isoprostanes, indicative of increased oxidative stress. The 78 kDa glucose-related protein (GRP78) mRNA expression increased 3-4-fold in liver, suggesting that a response against apoptosis was provoked by lipid peroxidation.

Dietary fish oil up-regulates cholesterol 7α-hydroxylase mRNA in mouse liver leading to an increase in bile acid and cholesterol excretion

To investigate the molecular events controlling reverse cholesterol transport, we compared gene expression of normal mouse liver to that of mice fed a long chain (LC) ω‐3 fatty acid‐enriched diet. Using cDNA microarrays, we assessed expression levels of 1176 genes, and we found that D‐site binding protein (DBP) was three‐fold increased in mice on a LC ω‐3 fatty acid‐rich diet compared to controls. DBP is known to increase transcriptional level of cholesterol 7α‐hydroxylase (C7α), the rate‐limiting enzyme for bile acid production and cholesterol excretion, and we found that C7α mRNA was also up‐regulated by LC ω‐3 fatty acids. Moreover, liver X receptor‐α, another transcription factor up‐regulating C7α, was three‐ to four‐fold increased in liver of treated mice. On the other hand, we demonstrated that bile acid and cholesterol excretion were two‐fold increased. These results show that LC ω‐3 fatty acids control cholesterol metabolism in mice at a new endpoint.

Novel risk factors for premature peripheral arterial occlusive disease in non-diabetic patients: a case-control study.

Background This study aimed to determine the prevalence of genetic and environmental vascular risk factors in non diabetic patients with premature peripheral arterial disease, either peripheral arterial occlusive disease or thromboangiitis obliterans, the two main entities of peripheral arterial disease, and to established whether some of them are specifically associated with one or another of the premature peripheral arterial disease subgroups. Methods and Results This study included 113 non diabetic patients with premature peripheral arterial disease (diagnosis <45-year old) presenting either a peripheral arterial occlusive disease (N = 64) or a thromboangiitis obliterans (N = 49), and 241 controls matched for age and gender. Both patient groups demonstrated common traits including cigarette smoking, low physical activity, decreased levels of HDL-cholesterol, apolipoprotein A–I, pyridoxal 5′-phosphate (active form of B6 vitamin) and zinc. Premature peripheral arterial occlusive disease was characterized by the presence of a family history of peripheral arterial and carotid artery diseases (OR 2.3 and 5.8 respectively, 95% CI), high lipoprotein (a) levels above 300 mg/L (OR 2.3, 95% CI), the presence of the factor V Leiden (OR 5.1, 95% CI) and the glycoprotein Ia807T,837T,873A allele (OR 2.3, 95% CI). In thromboangiitis obliterans group, more patients were regular consumers of cannabis (OR 3.5, 95% CI) and higher levels in plasma copper has been shown (OR 6.5, 95% CI). Conclusions According to our results from a non exhaustive list of study parameters, we might hypothesize for 1) a genetic basis for premature peripheral arterial occlusive disease development and 2) the prevalence of environmental factors in the development of thromboangiitis obliterans (tobacco and cannabis). Moreover, for the first time, we demonstrated that the 807T/837T/873A allele of platelet glycoprotein Ia may confer an additional risk for development of peripheral atherosclerosis in premature peripheral arterial occlusive disease.

A Protective Role for CD154 in Hepatic Steatosis in Mice

Inflammation and lipid metabolism pathways are linked, and deregulation of this interface may be critical in hepatic steatosis. The importance of the dialog between inflammatory signaling pathways and the unfolded protein response (UPR) in metabolism has been underlined. Herein, we studied the role of CD154, a key mediator of inflammation, in hepatic steatosis. To this end, Balb/c mice, wild‐type or deficient in CD154 (CD154KO), were fed a diet rich in olive oil. In vitro, the effect of CD154 was studied on primary hepatocyte cultures and hepatocyte‐derived cell lines. Results showed that CD154KO mice fed a diet rich in olive oil developed hepatic steatosis associated with reduced apolipoprotein B100 (apoB100) expression and decreased secretion of very low‐density lipoproteins. This phenotype correlated with an altered UPR as assessed by reduced X‐Box binding protein‐1 (XBP1) messenger RNA (mRNA) splicing and reduced phosphorylation of eukaryotic initiation factor 2α. Altered UPR signaling in livers of CD154KO mice was confirmed in tunicamycin (TM) challenge experiments. Treatment of primary hepatocyte cultures and hepatocyte‐derived cell lines with soluble CD154 increased XBP1 mRNA splicing in cells subjected to either oleic acid (OA) or TM treatment. Moreover, CD154 reduced the inhibition of apoB100 secretion by HepG2 cells grown in the presence of high concentrations of OA, an effect suppressed by XBP1 mRNA silencing and in HepG2 cells expressing a dominant negative form of inositol requiring ER‐to‐nucleus signaling protein‐1. The control of the UPR by CD154 may represent one of the mechanisms involved in the pathophysiology of hepatic steatosis. Conclusion: Our study identifies CD154 as a new mediator of hepatic steatosis. (HEPATOLOGY 2010)

CD40 signaling and hepatic steatosis: Unanticipated links

Obesity predisposes to an increased risk of nonalcoholic fatty liver disease (NAFLD). Hepatic steatosis is the key pathological feature of NAFLD and has emerged as a metabolic disorder in which innate and adaptive arms of the immune response play a central role in disease pathogenesis. Recent studies have revealed unexpected relationships between CD40 signaling and hepatic steatosis in high fat diet rodent models. CD154, the ligand of CD40, is a mediator of inflammation and controls several critical events of innate and adaptive immune responses. In the light of these reports, we discuss potential links between CD40 signaling and hepatic steatosis in NAFLD.

Naringin at a nutritional dose modulates expression of genes related to lipid metabolism and inflammation in liver of mice fed a high-fat diet

Epidemiological and clinical studies a role for flavanones (predominately found in citrus fruits) in the prevention of cardiovascular disease. Previously, we have shown that a nutritional dose of naringin exerts anti-atherogenic properties together with non HDL-cholesterol lowering effect in a murin model of dietary-induced hypercholesterolemia (1). The goal of the present study was to explore possible molecular mechanisms of naringin at the hepatic level. To this end, we analyzed the hepatic transcriptome using a microarray approach in response to naringin supplementation (0.02%) in mice fed a high-fat high- cholesterol diet. Naringin was observed to increase hepatic lipid content (triglyceride and cholesterol) without significant liver dysfunction (ALAT and ASAT activities) or histopathological alterations. Naringin supplementation also significantly improved insulin sensitivity as evaluated by the HOMA index and nutrigenomics revealed that naringin modulated the expression of 1,766 genes. These genes encode proteins involved in different cellular processes, such as lipid metabolism, inflammation and insulin signaling. In conclusion, this study revealed that the hypolipemic and anti-atherogenic effects induced by a nutritional- level naringin supplementation in high-fat high-cholesterol diet could be related to changes in hepatic lipid metabolism and inflammatory response, revealing new in vivo targets of this flavanone.

CO.109 - Mécanismes du contrôle de la progression de la stéatose hépatique par le CD154

Introduction Contexte - L’histoire naturelle de la steatopathie metabolique est classiquement divisee en deux etapes, steatose simple et steato-hepatite. Les mecanismes de cette transition restent obscurs. En tentant d’eclaircir les liens entre inflammation et steatose, nous avons observe que les souris dont le gene codant pour le CD154, un acteur cle de la reaction inflammatoire, est invalide (CD154Y/-), developpent une steatose hepatique majeure lorsqu’elles sont soumises a un regime riche en lipides. Cette steatose est liee a une reduction de la secretion des VLDL par le foie et est correlee a une reduction de l’expression de l’apoB100. Hypothese - Des etudes anterieures ont rapporte que le reticulum endoplasmique (RE) est central dans la secretion des VLDL et etabli un lien entre l’expression de l’apoB100 et le stress du RE dans un contexte de surcharge lipidique. Nous avons donc etudie les relations entre CD154 et stress du RE. La reponse au stress du RE correspond a un ensemble de mecanismes visant a adapter les capacites fonctionnelles du RE. Elle se manifeste par l’« Unfolded Protein Response » (reponse UPR), qui se traduit par l’activation de voies de signalisation dont les effecteurs contribuent a adapter les fonctions du RE a une demande accrue, comme par exemple dans un contexte de regime lipidique. Notre hypothese a ete que le CD154 facilite l’adaptation du RE a un apport lipidique excessif en intervenant sur la reponse UPR. Resultats In vivo , nous avons observe l’augmentation de l’expression des marqueurs de la reponse UPR chez les animaux sauvages mais pas chez les animaux CD154Y/- en reponse a un regime riche en lipides ou a un inducteur du stress du RE, la tunicamycine. La sensibilite hepatique a ce toxique est par ailleurs accrue chez les animaux CD154Y/-, soulignant le defaut d’adaptation du RE. Ces resultats demontraient le role du CD154 dans le controle de l’adaptation du RE. In vitro , nous avons etudie le mode d’action du CD154 sur des lignees cellulaires derivees de tumeurs hepatiques humaines. Ces lignees expriment le CD40, le recepteur du CD154. En presence de tunicamycine, le CD154 augmente la reponse UPR et la viabilite cellulaire. Conclusion Nous avons montre que les voies de signalisation activees par le CD154 interagissent avec la reponse UPR pour augmenter la capacite d’adaptation des hepatocytes dans un contexte de stress metabolique. Le controle de l’homeostasie du RE par le CD154 est un mecanisme permettant d’etablir un lien entre CD154 et steatose hepatique. Nos resultats suggerent que le CD154 est un acteur important dans la progression de la steatopathie metabolique. Remerciements, financements, autres Travail soutenu par l’Association francaise pour l’etude du foie et la Societe Echosens SA.