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The sarcoplasmic reticulum (SR) plays an important role in calcium homeostasis. SR calcium mishandling is described in pathological conditions such as myopathies. Here, we investigated whether the nuclear receptor Rev-erb-α regulates skeletal muscle SR calcium homeostasis. Our data demonstrate that Rev-erbα invalidation in mice impairs SERCA-dependent SR calcium uptake. Rev-erb-α acts on calcium homeostasis by repressing the SERCA inhibitor Myoregulin, through direct binding to its promoter. Restoration of Myoregulin counteracts the effects of REV-ERB-α overexpression on SR calcium content. Interestingly, myoblasts from Duchenne myopathy patients display downregulated REV-ERBα expression, whereas pharmacological Rev-erb activation ameliorates SR calcium homeostasis, and improves muscle structure and function in dystrophic mdx/Utr+/- mice. Our findings demonstrate that Rev-erb-α regulates muscle SR calcium homeostasis, pointing to its therapeutic interest for mitigating myopathy.

International audience; AIMS:Not all people with obesity become glucose intolerant, suggesting differential activation of cellular pathways. The unfolded protein response (UPR) may contribute to the development of insulin resistance in several organs, but its role in skeletal muscle remains debated. Therefore, we explored the UPR activation in muscle from non-diabetic glucose tolerant or intolerant patients with obesity and the impact of bariatric procedures.METHODS:Muscle biopsies from 22 normoglycemic (NG, blood glucose measured 120 min after an oral glucose tolerance test, G120

The genomic CDKN2A/B locus, encoding p16(INK4a) among others, is linked to an increased risk for cardiovascular disease and type 2 diabetes. Obesity is a risk factor for both cardiovascular disease and type 2 diabetes. p16(INK4a) is a cell cycle regulator and tumour suppressor. Whether it plays a role in adipose tissue formation is unknown. p16(INK4a) knock-down in 3T3/L1 preadipocytes or p16(INK4a) deficiency in mouse embryonic fibroblasts enhanced adipogenesis, suggesting a role for p16(INK4a) in adipose tissue formation. p16(INK4a)-deficient mice developed more epicardial adipose tissue in response to the adipogenic peroxisome proliferator activated receptor gamma agonist rosiglitazone. Additionally, adipose tissue around the aorta from p16(INK4a)-deficient mice displayed enhanced rosiglitazone-induced gene expression of adipogenic markers and stem cell antigen, a marker of bone marrow-derived precursor cells. Mice transplanted with p16(INK4a)-deficient bone marrow had more epicardial adipose tissue compared to controls when fed a high-fat diet. In humans, p16(INK4a) gene expression was enriched in epicardial adipose tissue compared to other adipose tissue depots. Moreover, epicardial adipose tissue from obese humans displayed increased expression of stem cell antigen compared to lean controls, supporting a bone marrow origin of epicardial adipose tissue. These results show that p16(INK4a) modulates epicardial adipose tissue development, providing a potential mechanistic link between the genetic association of the CDKN2A/B locus and cardiovascular disease risk.

The genomic CDKN2A/B locus, encoding p16INK4a among others, is linked to an increased risk for cardiovascular disease and type 2 diabetes. Obesity is a risk factor for both cardiovascular disease and type 2 diabetes. p16INK4a is a cell cycle regulator and tumour suppressor. Whether it plays a role in adipose tissue formation is unknown. p16INK4a knock-down in 3T3/L1 preadipocytes or p16INK4a deficiency in mouse embryonic fibroblasts enhanced adipogenesis, suggesting a role for p16INK4a in adipose tissue formation. p16INK4a-deficient mice developed more epicardial adipose tissue in response to the adipogenic peroxisome proliferator activated receptor gamma agonist rosiglitazone. Additionally, adipose tissue around the aorta from p16INK4a-deficient mice displayed enhanced rosiglitazone-induced gene expression of adipogenic markers and stem cell antigen, a marker of bone marrow-derived precursor cells. Mice transplanted with p16INK4a-deficient bone marrow had more epicardial adipose tissue compared to controls when fed a high-fat diet. In humans, p16INK4a gene expression was enriched in epicardial adipose tissue compared to other adipose tissue depots. Moreover, epicardial adipose tissue from obese humans displayed increased expression of stem cell antigen compared to lean controls, supporting a bone marrow origin of epicardial adipose tissue. These results show that p16INK4a modulates epicardial adipose tissue development, providing a potential mechanistic link between the genetic association of the CDKN2A/B locus and cardiovascular disease risk.

The nuclear receptor Rev-erb-α modulates hepatic lipid and glucose metabolism, adipogenesis and thermogenesis. We have previously demonstrated that Rev-erb-α is also an important regulator of skeletal muscle mitochondrial biogenesis and function, and autophagy. As such, Rev-erb-α over-expression in skeletal muscle or its pharmacological activation improved mitochondrial respiration and enhanced exercise capacity. Here, in gain- and loss-of function studies, we show that Rev-erb-α also controls muscle mass. Rev-erb-α-deficiency in skeletal muscle leads to increased expression of the atrophy-related genes (atrogenes), associated with reduced muscle mass and decreased fiber size. By contrast, in vivo and in vitro Rev-erb-α over-expression results in reduced atrogenes expression and increased fiber size. Finally, Rev-erb-α pharmacological activation blocks dexamethasone-induced upregulation of atrogenes and muscle atrophy. This study identifies Rev-erb-α as a promising pharmacological target to preserve muscle mass.

The nuclear receptor Rev-erb-α modulates hepatic lipid and glucose metabolism, adipogenesis and the inflammatory response in macrophages. We show here that Rev-erb-α is highly expressed in oxidative skeletal muscle and that its deficiency in muscle leads to reduced mitochondrial content and oxidative function, as well as upregulation of autophagy. These cellular effects resulted in both impaired mitochondrial biogenesis and increased clearance of this organelle, leading to compromised exercise capacity. On a molecular level, Rev-erb-α deficiency resulted in deactivation of the Lkb1-Ampk-Sirt1-Ppargc-1α signaling pathway. These effects were recapitulated in isolated fibers and in muscle cells after knockdown of the gene encoding Rev-erb-α, Nr1d1. In complementary experiments, Rev-erb-α overexpression in vitro increased the number of mitochondria and improved respiratory capacity, whereas muscle overexpression or pharmacological activation of Rev-erb-α in vivo increased exercise capacity. This study identifies Rev-erb-α as a pharmacological target that improves muscle oxidative function by modulating gene networks controlling mitochondrial number and function.

Obesity is a low-grade chronic inflammatory disease associated with an increased number of macrophages (adipose tissue macrophages) in adipose tissue. Within the adipose tissue, adipose tissue macrophages are the major source of visfatin/pre-B-cell colony-enhancing factor/nicotinamide phosphoribosyl transferase. The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) exerts anti-inflammatory effects in macrophages by inhibiting cytokine production and enhancing alternative differentiation. In this study, we investigated whether PPARgamma modulates visfatin expression in murine (bone marrow-derived macrophage) and human (primary human resting macrophage, classical macrophage, alternative macrophage or adipose tissue macrophage) macrophage models and pre-adipocyte-derived adipocytes. We show that synthetic PPARgamma ligands increase visfatin gene expression in a PPARgamma-dependent manner in primary human resting macrophages and in adipose tissue macrophages, but not in adipocytes. The threefold increase of visfatin mRNA was paralleled by an increase of protein expression (30%) and secretion (30%). Electrophoretic mobility shift assay experiments and transient transfection assays indicated that PPARgamma induces visfatin promoter activity in human macrophages by binding to a DR1-PPARgamma response element. Finally, we show that PPARgamma ligands increase NAD(+) production in primary human macrophages and that this regulation is dampened in the presence of visfatin small interfering RNA or by the visfatin-specific inhibitor FK866. Taken together, our results suggest that PPARgamma regulates the expression of visfatin in macrophages, leading to increased levels of NAD(+).

Here we show that gene expression of the nuclear receptor RORalpha is induced during adipogenesis, with RORalpha4 being the most abundantly expressed isoform in human and murine adipose tissue. Over-expression of RORalpha4 in 3T3-L1 cells impairs adipogenesis as shown by the decreased expression of adipogenic markers and lipid accumulation, accompanied by decreased free fatty acid and glucose uptake. By contrast, mouse embryonic fibroblasts from staggerer mice, which carry a mutation in the RORalpha gene, differentiate more efficiently into mature adipocytes compared to wild-type cells, a phenotype which is reversed by ectopic RORalpha4 restoration.

Objective—Macrophages play a central role in the immune response against infectious organisms. Once activated, macrophages secrete proinflammatory cytokines and chemokines. Interleukin (IL)-8 and related CXC chemokines play a role in the recruitment and activation of phagocytes acting through CXCR1 and CXCR2 receptors. The nuclear receptor peroxisome proliferator-activated receptor (PPAR) γ exerts antiinflammatory properties in macrophages, by inhibiting cytokine and CC chemokine production. In this study, we investigated whether PPAR-γ also plays a role in the regulation of the CXC chemokine pathway.Methods and Results—Synthetic PPAR-γ ligands increase CXCR2 but not CXCR1 gene expression in a PPAR-γ-dependent manner in primary human macrophages in vitro and in atherosclerotic plaques in vivo. The increase of CXCR2 mRNA was paralleled by an increase in membrane protein expression. EMSA, ChIP, and transient transfection assays indicate that PPAR-γ activates the CXCR2 promoter by binding to a PPAR response element (PPRE). Finally, human macrophages acquire responsiveness to the CXCR2 ligands (IL-8 and Groβ), as measured by superoxide anion production, after induction of CXCR2 expression by PPAR-γ ligands.Conclusions—Our results provide a novel mechanism via which PPAR-γ can enhance the immune response in human macrophages.

Objective— The newly identified apolipoprotein A5 ( APOA5 ), selectively expressed in the liver, is a crucial determinant of plasma triglyceride levels. Because elevated plasma triglyceride concentrations constitute an independent risk factor for cardiovascular diseases, it is important to understand how the expression of this gene is regulated. In the present study, we identified the retinoic acid receptor-related orphan receptor-α (RORα) as a regulator of human APOA5 gene expression. Methods and Results— Using electromobility shift assays, we first demonstrated that RORα1 and RORα4 proteins can bind specifically to a direct repeat 1 site present at the position −272/−260 in the APOA5 gene promoter. In addition, using transient cotransfection experiments in HepG2 and HuH7 cells, we demonstrated that both RORα1 and RORα4 strongly increase APOA5 promoter transcriptional activity in a dose-dependent manner. Finally, adenoviral overexpression of hRORα in HepG2 cells led to enhanced hAPOA5 mRNA accumulation. We show that the homologous region in mouse apoa5 promoter is not functional. Moreover, we show that in staggerer mice, apoa5 gene is not affected by RORα. Conclusions— These findings identify RORα1 and RORα4 as transcriptional activators of human APOA5 gene expression. These data suggest an additional important physiological role for RORα in the regulation of genes involved in lipid homeostasis and probably in the development of atherosclerosis.

BACKGROUND Resveratrol (a naturally occurring phytoalexin found in grapes and wine) has cardiovascular protective effects that suggest the antiatherogenic (ie, antiinflammatory) activities of the compound on endothelial cells. OBJECTIVE The antiinflammatory activity of resveratrol could be mediated by its interference with nuclear factor-kappaB (NF-kappaB)-dependent transcription. Thus, we studied the in vitro influence of physiologic concentrations of resveratrol (

Background Atherosclerosis is an inflammatory disease in which macrophages play a crucial role. Macrophages are present in different phenotypes, with at the extremes of the spectrum the classical M1 pro-inflammatory and the alternative M2 anti-inflammatory macrophages. The neuron-derived orphan receptor 1 (NOR1), together with Nur77 and Nurr1, are members of the NR4A orphan nuclear receptor family, expressed in human atherosclerotic lesion macrophages. However, the role of NOR1 in human macrophages has not been studied yet. Objectives To determine the expression and the functions of NOR1 in human alternative macrophages. Methods and results In vitro IL-4 polarization of primary monocytes into alternative M2 macrophages enhances NOR1 expression in human but not in mouse macrophages. Moreover, NOR1 expression is most abundant in CD68+MR+ alternative macrophage-enriched areas of human atherosclerotic plaques in vivo . Silencing NOR1 in human alternative macrophages decreases the expression of several M2 markers such as the Mannose Receptor (MR), Interleukin-1 Receptor antagonist (IL-1Ra), CD200 Receptor (CD200R), coagulation factor XIII A1 polypeptide (F13A1), Interleukin 10 (IL-10) and the Peroxisome Proliferator-Activated Receptor (PPAR)γ. Bioinformatical analysis identified F13A1, IL-1Ra, IL-10 and the Matrix Metalloproteinase-9 (MMP9) as potential target genes of NOR1 in human alternative macrophages. Moreover, expression and enzymatic activity of MMP9 are induced by silencing and repressed by NOR1 overexpression in M2 macrophages. Conclusions These data identify NOR1 as a transcription factor induced during alternative differentiation of human macrophages and demonstrate that NOR1 modifies the alternative macrophage phenotype.

Purpose. The objective of this work was to assess, in vitro, the passage of P-glycoprotein dependent drugs across brain capillary endothelial cells, when these drugs are associated with a reversing agent.

Objective— 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) catalyzes the intracellular reduction of inactive cortisone to active cortisol, the natural ligand activating the glucocorticoid receptor (GR). Peroxisome proliferator– activated receptor-γ (PPARγ) is a nuclear receptor controlling inflammation, lipid metabolism, and the macrophage polarization state. In this study, we investigated the impact of macrophage polarization on the expression and activity of 11β-HSD1 and the role of PPARγ therein. Methods and Results— 11β-HSD1 gene expression is higher in proinflammatory M1 and anti-inflammatory M2 macrophages than in resting macrophages, whereas its activity is highest in M2 macrophages. Interestingly, PPARγ activation induces 11β-HSD1 enzyme activity in M2 macrophages but not in resting macrophages or M1 macrophages. Consequently, human M2 macrophages displayed enhanced responsiveness to the 11β-HSD1 substrate cortisone, an effect amplified by PPARγ induction of 11β-HSD1 activity, as illustrated by an increased expression of GR target genes. Conclusion— Our data identify a positive cross-talk between PPARγ and GR in human M2 macrophages via the induction of 11β-HSD1 expression and activity.

Retinoic acid-related Orphan Receptor alpha (RORα; NR1F1) is a widely distributed nuclear receptor involved in several (patho)physiological functions including lipid metabolism, inflammation, angiogenesis, and circadian rhythm. To better understand the role of this nuclear receptor in liver, we aimed at displaying genes controlled by RORα in liver cells by generating HepG2 human hepatoma cells stably over-expressing RORα. Genes whose expression was altered in these cells versus control cells were displayed using micro-arrays followed by qRT-PCR analysis. Expression of these genes was also altered in cells in which RORα was transiently over-expressed after adenoviral infection. A number of the genes found were involved in known pathways controlled by RORα, for instance LPA, NR1D2 and ADIPOQ in lipid metabolism, ADIPOQ and PLG in inflammation, PLG in fibrinolysis and NR1D2 and NR1D1 in circadian rhythm. This study also revealed that genes such as G6PC, involved in glucose homeostasis, and AGRP, involved in the control of body weight, are also controlled by RORα. Lastly, SPARC, involved in cell growth and adhesion, and associated with liver carcinogenesis, was up-regulated by RORα. SPARC was found to be a new putative RORα target gene since it possesses, in its promoter, a functional RORE as evidenced by EMSAs and transfection experiments. Most of the other genes that we found regulated by RORα also contained putative ROREs in their regulatory regions. Chromatin immunoprecipitation (ChIP) confirmed that the ROREs present in the SPARC, PLG, G6PC, NR1D2 and AGRP genes were occupied by RORα in HepG2 cells. Therefore these genes must now be considered as direct RORα targets. Our results open new routes on the roles of RORα in glucose metabolism and carcinogenesis within cells of hepatic origin.

BACKGROUND & AIMS: Glucagon-like peptide (GLP)-1, an intestinal incretin produced by L cells through proglucagon processing, is secreted after nutrient ingestion and acts on endocrine pancreas beta cells to enhance insulin secretion. Peroxisome proliferator-activated receptor (PPAR) β/δ is a nuclear receptor that improves glucose homeostasis and pancreas islet function in diabetic animal models. Here, we investigated whether PPARβ/δ activation regulates L cell GLP-1 production. METHODS: Proglucagon regulation and GLP-1 release were evaluated in murine GLUTag and human NCI-H716 L cells and in vivo using wild-type, PPARβ/δ-null, and ob/ob C57Bl/6 mice treated with the PPARβ/δ synthetic agonists GW501516 or GW0742. RESULTS: PPARβ/δ activation increased proglucagon expression and enhanced glucose- and bile acid-induced GLP-1 release by intestinal L cells in vitro and ex vivo in human jejunum. In vivo treatment with GW0742 increased proglucagon messenger RNA levels in the small intestine in wild-type but not in PPARβ/δ-deficient mice. Treatment of wild-type and ob/ob mice with GW501516 enhanced the increase in plasma GLP-1 level after an oral glucose load and improved glucose tolerance. Concomitantly, proglucagon and GLP-1 receptor messenger RNA levels increased in the small intestine and pancreas, respectively. Finally, PPARβ/δ agonists activate the proglucagon gene transcription by interfering with the β-catenin/TCF-4 pathway. CONCLUSIONS: Our data show that PPARβ/δ activation potentiates GLP-1 production by the small intestine. Pharmacologic targeting of PPARβ/δ is a promising approach in the treatment of patients with type 2 diabetes mellitus, especially in combination with dipeptidyl peptidase IV inhibitors.

The bile acid receptor farnesoid X receptor (FXR) is expressed in adipose tissue, but its function remains poorly defined. Peroxisome proliferator-activated receptor-gamma (PPAR gamma) is a master regulator of adipocyte differentiation and function. The aim of this study was to analyze the role of FXR in adipocyte function and to assess whether it modulates PPAR gamma action. Therefore, we tested the responsiveness of FXR-deficient mice (FXR-/-) and cells to the PPAR gamma activator rosiglitazone. Our results show that genetically obese FXR-/-/ob/ob mice displayed a resistance to rosiglitazone treatment. In vitro, rosiglitazone treatment did not induce normal adipocyte differentiation and lipid droplet formation in FXR-/- mouse embryonic fibroblasts (MEFs) and preadipocytes. Moreover, FXR-/- MEFs displayed both an increased lipolysis and a decreased de novo lipogenesis, resulting in reduced intracellular triglyceride content, even upon PPAR gamma activation. Retroviral-mediated FXR re-expression in FXR-/- MEFs restored the induction of adipogenic marker genes during rosiglitazone-forced adipocyte differentiation. The expression of Wnt/beta-catenin pathway and target genes was increased in FXR-/- adipose tissue and MEFs. Moreover, the expression of several endogenous inhibitors of this pathway was decreased early during the adipocyte differentiation of FXR-/- MEFs. These findings demonstrate that FXR regulates adipocyte differentiation and function by regulating two counteracting pathways of adipocyte differentiation, the PPAR gamma and Wnt/beta-catenin pathways.

A role of the nuclear receptor Rev-erbalpha in the regulation of transcription pathways involving other nuclear receptors is emerging. Indeed, Rev-erbalpha is a negative regulator of transcription by binding to overlapping response elements shared with various nuclear receptors, including the peroxisome proliferator-activated receptors and the retinoid-related orphan receptor alpha (RORalpha). Here, we show that Rev-erbalpha is expressed in primary human macrophages and that its expression is induced by synthetic ligands for the liver X receptors (LXRs), which control cholesterol homeostasis, inflammation, and the immune response in macrophages. LXRalpha binds to a specific response element in the human Rev-erbalpha promoter, thus inducing Rev-erbalpha transcriptional expression. Interestingly, Rev-erbalpha does not influence basal or LXR-regulated cholesterol homeostasis. However, Rev-erbalpha overexpression represses the induction of toll-like receptor (TLR)-4 by LXR agonists, whereas Rev-erbalpha silencing by short interfering RNA results in enhanced TLR-4 expression upon LXR activation. Electrophoretic mobility shift, chromatin immunoprecipitation, and transient transfection experiments demonstrate that Rev-erbalpha represses human TLR-4 promoter activity by binding as a monomer to a RevRE site overlapping with the LXR response element site in the TLR-4 promoter. These data identify Rev-erbalpha as a new LXR target gene, inhibiting LXR-induction of TLR-4 in a negative transcriptional feedback loop, but not cholesterol homeostasis gene expression.

Background & Aims: Conversion into bile acids represents an important route to remove excess cholesterol from the body. Rev-erb alpha is a nuclear receptor that participates as one of the clock genes in the control of circadian rhythmicity and plays a regulatory role in lipid metabolism and adipogenesis. Here, we investigate a potential role for Rev-erb alpha in the control of bile acid metabolism via the regulation of the neutral bile acid synthesis pathway. Methods: Bile acid synthesis and CYP7A1 gene expression were studied in vitro and in vivo in mice deficient for or over expressing Rev-erb alpha. Results: Rev-erb alpha-deficient mice display a lower synthesis rate and an impaired excretion of bile acids into the bile and feces. Expression of CYP7A1, the rate-limiting enzyme of the neutral pathway, is decreased in livers of Rev-erb alpha-deficient mice, whereas adenovirus-mediated hepatic Rev-erb alpha overexpression induces its expression. Moreover, bile acid feeding resulted in a more pronounced suppression of hepatic CYP7A1 expression in Rev-erb alpha-deficient mice. Hepatic expression of E4BP4 and the orphan nuclear receptor small heterodimer partner (SHP), both negative regulators of CYP7A1 expression, is increased in Rev-erb alpha-deficient mice. Promoter analysis and chromatin immunoprecipitation experiments demonstrated that SHP and E4BP4 are direct Rev-erb alpha target genes. Finally, the circadian rhythms of liver CYP7A1, SHP, and E4BP4 messenger RNA levels were perturbed in Rev-erb alpha-deficient mice. Conclusions: These data identify a role for Rev-erb alpha in the regulatory loop of bile acid synthesis, likely acting by regulating both hepatic SHP and E4BP4 expression.

FXR (farnesoid X receptor), a nuclear receptor activated by BAs (bile acids), is a key factor in the regulation of BA, lipid and carbohydrate metabolism. The recent development of synthetic FXR agonists and knockout mouse models has accelerated the discovery of FXR target genes. In the present study, we identify human fetuin-B as a novel FXR target gene. Treatment with FXR agonists increased fetuin-B expression in human primary hepatocytes and in the human hepatoma HepG2 cell line. In contrast, fetuin-B expression was not responsive to FXR agonist treatment in murine primary hepatocytes. Fetuin-B induction by FXR agonist was abolished upon FXR knockdown by siRNA (small interfering RNA). In addition to the previously described P1 promoter, we show that the human fetuin-B gene is also transcribed from an alternative promoter, termed P2. Transcription via the P2 promoter was induced by FXR agonist treatment, whereas P1 promoter activity was not sensitive to FXR agonist treatment. Two putative FXR-response elements [IR-1 (inverted repeat-1)] were identified in the region –1.6 kb upstream of the predicted P2 transcriptional start site. Both motifs bound FXR–RXR (retinoid X receptor) complexes in vitro and were activated by FXR in transient transfection reporter assays. Mutations in the IR-1 sites abolished FXR–RXR binding and activation. Taken together, these results identify human fetuin-B as a new FXR target gene in human hepatocytes.

Statins are inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase used in the prevention of cardiovascular disease (CVD). In addition to their cholesterol-lowering activities, statins exert pleiotropic antiinflammatory effects, which might contribute to their beneficial effects not only on CVD but also on lipid-unrelated immune and inflammatory diseases, such as rheumatoid arthritis, asthma, stroke, and transplant rejection. However, the molecular mechanisms involved in these antiinflammatory properties of statins are unresolved. Here we show that the peroxisome proliferator-activated receptor (PPAR) alpha mediates antiinflammatory effects of simvastatin in vivo in models of acute inflammation. The inhibitory effects of statins on lipopolysaccharide-induced inflammatory response genes were abolished in PPARalpha-deficient macrophages and neutrophils. Moreover, simvastatin inhibited PPARalpha phosphorylation by lipopolysaccharide-activated protein kinase C (PKC) alpha. A constitutive active form of PKCalpha inhibited nuclear factor kappaB transrepression by PPARalpha whereas simvastatin enhanced transrepression activity of wild-type PPARalpha, but not of PPARalpha mutated in its PKC phosphorylation sites. These data indicate that the acute antiinflammatory effect of simvastatin occurs via PPARalpha by a mechanism involving inhibition of PKCalpha inactivation of PPARalpha transrepression activity.

Peroxisome proliferator-activated receptor (PPAR) alpha and gamma are ligand-activated transcription factors belonging to the nuclear receptor family. PPAR alpha mediates the hypolipidemic action of the fibrates, whereas PPAR gamma is a receptor for the antidiabetic glitazones. In the present study, the UDP-glucuronosyltransferase (UGT) 1A9 enzyme is identified as a PPAR alpha and PPAR gamma target gene. UGTs catalyze the glucuronidation reaction, which is a major pathway in the catabolism and elimination of numerous endo- and xenobiotics. Among the UGT1A family enzymes, UGT1A9 metabolizes endogenous compounds, including catecholestrogens, and xenobiotics, such as fibrates and to a lesser extent troglitazone. Treatment of human hepatocytes and macrophages and murine adipocytes with activators of PPAR alpha or PPAR gamma resulted in an enhanced UGT1A9 expression and activity. In addition, disruption of the PPAR alpha gene in mice completely abolished the PPAR alpha agonist-induced UGT1A9 mRNA and activity levels. A PPAR response element was identified in the promoter of UGT1A9 at positions -719 to -706 bp by transient transfection and electromobility shift assays. Considering the role of UGT1A9 in catecholestrogen metabolism, PPAR alpha and PPAR gamma activation may contribute to the protection against genotoxic catecholestrogens by stimulating their inactivation in glucuronide derivatives. Furthermore, since UGT1A9 is involved in the catabolism of fibrates, these results suggest that PPAR alpha and PPAR gamma may control the intracellular level of active fibrates.

The passage of substances across the blood-brain barrier (BBB) is regulated in the cerebral capillaries, which possess certain distinct different morphological and enzymatic properties compared with the capillaries of other organs. Investigations of the functional characteristics of brain capillaries have been facilitated by the use of cultured brain endothelial cells, but in most studies some characteristics of the in vivo BBB are lost. To provide an in vitro system for studying brain capillary functions, we have developed a process of coculture that closely mimics the in vivo situation by culturing brain capillary endothelial cells on one side of a filter and astrocytes on the other. In order to assess the drug transport across the blood-brain barrier, we compared the extraction ratios in vivo to the permeability of the in vitro model. The in vivo and the in vitro values showed a strong correlation. The relative ease with which such cocultures can be produced in large quantities facilitates the screening of new centrally active drugs. This model provides an easier, reproducible and mass-production method to study the blood-brain barrier in vitro.

Introduction L'incidence de l'obesite et des maladies metaboliques en general ne cesse d'augmenter a travers le monde. Ces desordres metaboliques sont associes a des perturbations de l'expression des genes impliques dans le metabolisme lipidique et glucidique et l'inflammation. L'expression d'un certain nombre de ces genes est regulee par les membres de la superfamille des recepteurs nucleaires. Le recepteur nucleaire Farnesoid X Receptor (FXR) est exprime dans le tissu adipeux, cependant son role reste encore inconnu. L'objectif de ce projet est d'elucider le role de FXR dans la fonction adipocytaire et de determiner les mecanismes moleculaires impliques dans ce processus. Resultats Nous montrons ainsi que les cellules MEFs (mouse embryonic fibroblasts) deficientes en FXR (FXR-/-) presentent une augmentation de la lipolyse et une diminution de la neolipogenese par rapport aux cellules MEFs controles (FXR +/+ ) ce qui a pour consequence une diminution des triglycerides stockees. Ce phenotype est associe a une diminution de l'expression des genes marqueurs de l'adipogenese et les genes codant pour les proteines des gouttelettes lipidiques a la fois dans les MEFs FXR -/- et les pre-adipocytes isolees a partir du tissu adipeux des souris FXR -/- . L'introduction de FXR, par la voie d'un retrovirus sur-exprimant FXR, restaure une differentiation adipocytaire normale. L'absence de FXR induit une augmentation de l'expression des genes de la voie Wnt/β-catenine et une augmentation de l'expression de la proteine β-catenine. D'ailleurs, l'expression des genes codant pour les inhibiteurs de cette voie de signalisation sont inhibes dans les MEFs FXR -/- . Un traitement par l'agoniste synthetique de PPARγ, la rosiglitazone, ne restaure pas une differenciation adipocytaire normale et des gouttelettes lipidiques comparables aux MEFs FXR +/+ . De meme, les souris FXR -/- sont resistantes au traitement rosiglitazone en comparaisons aux controles. Conclusion Cette etude montre clairement que FXR controle la differenciation adipocytaire en interferant avec la voie Wnt/β-catenine et la voie d'activation de PPARγ

Introduction La visfatin est une cytokine pro-inflammatoire mais aussi une enzyme limitante de la synthese du NAD + qui est un cofacteur de nombreuses enzymes cellulaires. L’obesite est associee a une infiltration de macrophages au sein du tissu adipeux. Ces macrophages sont la principale source de visfatin dont l’implication dans le developpement du diabete de type2 est largement reconnue. Dans cette etude, la capacite de PPARγ et/ou de LXR (des modulateurs de la reponse inflammatoire des macrophages) a reguler l’expression de la visfatin dans differents types de macrophages primaires (humains et murins) a ete analysee, de meme que l’influence de cette regulation sur la production intracellulaire de NAD + . Patients et Methodes Les macrophages primaires humains (« resting macrophages ») provenant d’individus minces ou obeses rendus pro-inflammatoires M1 ou anti-inflammatoires M2 in vitro ont ete utilises, de meme que des macrophages du tissu adipeux visceral d’individus obeses. Ces differents types macrophages ont ete traites avec des ligands synthetiques de PPARγ et/ou de LXR. L’expression genique et proteique de la visfatin ainsi que sa secretion ont ete analysees par Q-PCR, Western blot, Elisa, de meme que la production de NAD + Resultats L’activation de LXR ou de PPARγ par leurs ligands entraine respectivement une diminution ou une augmentation de l’expression genique de la visfatin. L’augmentation de l’expression de l’ARNm de la visfatin suite a l’activation de PPARγ s’accompagne d’une augmentation de l’expression et de la secretion proteique. La diminution de l’expression de l’ARNm suite a l’activation de LXR s’accompagne quant a elle, d’une diminution de la secretion mais pas de l’expression proteique. L’activation de LXR diminue l’expression et la secretion de la visfatin induite par l’activation de PPARγ. L’activation de LXR ou de PPARγ regule de facon opposee la production de NAD + dans les resting macrophages. Conclusion L’identification de la visfatin comme etant un gene cible de LXR et de PPARγ differentiellement regule au sein des macrophages permettra une meilleure evaluation des therapeutiques impliquant leurs agonistes lors du traitement des pathologies associees a l’obesite telles que le diabete type 2.

We have developed two methods for measuring the alpha-tocopherol content in plasma and lipoproteins (LDL and HDL). In procedure 1, plasma or lipoproteins are deproteinized with ethanol containing delta-tocopherol as internal standard and then extracted with hexane or ethyl acetate. The organic layer is removed and evaporated, and the residue is redissolved in methanol and injected into a reversed-phase HPLC. In procedure 2, plasma or lipoproteins are diluted in a methanol and ethanol mixture containing the same internal standard. The solution is vortex-mixed, centrifuged, and directly injected into the column. The tocopherols are eluted with an isocratic methanol mobile phase at a flow rate of 1 mL/min and detected by fluorescence (lambda(exc)= 295 nm, lambda(em)= 330nm). Recoveries are approximately 100% in both cases. Between-run CVs were 8.39% for procedure 1 and 6.55% for procedure 2. Small sample requirement, simplicity of sample preparation, short assay time, and good reproducibility make procedure 2 ideal for clinical or research use. This method was applied to determination of alpha-tocopherol in plasma of patients whose diet was supplemented with alpha-tocopherol and in LDL and HDL.