Role of bile acid receptor FXR in development and function of brown adipose tissue.

Bile acids act as signalling molecules that contribute to maintenance of energy homeostasis in mice and humans. Activation of G-protein-coupled bile acid receptor TGR5 induces energy expenditure in brown adipose tissue (BAT). However, a role for the nuclear bile acid receptor Farnesoid X receptor (FXR) in BAT has remained ambiguous. We aimed to study the potential role of FXR in BAT development and functioning. Here we demonstrate low yet detectable expression of the α1/2 isoforms of FXR in murine BAT that markedly decreases upon cold exposure. Moderate adipose tissue-specific FXR overexpression in mice induces pronounced BAT whitening, presenting with large intracellular lipid droplets and extracellular collagen deposition. Expression of thermogenic marker genes including the target of Tgr5, Dio2, was significantly lower in BAT of chow-fed aP2-hFXR mice compared to wild-type controls. Transcriptomic analysis revealed marked up-regulation of extracellular matrix formation and down-regulation of mitochondrial functions in BAT from aP2-hFXR mice. In addition, markers of cell type lineages deriving from the dermomyotome, such as myocytes, as well as markers of cellular senescence were strongly induced. The response to cold and ß3-adrenergic receptor agonism was blunted in these mice, yet resolved BAT whitening. Newborn cholestatic Cyp2c70-/- mice with a human-like bile acid profile also showed distinct BAT whitening and upregulation of myocyte-specific genes, while thermogenic markers were down-regulated. Ucp1 expression inversely correlated with plasma bile acid levels. Therefore, bile acid signalling via FXR has a role in BAT function already early in tissue development. Functionally, FXR activation appears to oppose TGR5-mediated thermogenesis.

Skeletal muscle functions around the clock.

In mammals, the central clock localized in the central nervous system imposes a circadian rhythmicity to all organs. This is achieved thanks to a well-conserved molecular clockwork, involving interactions between several transcription factors, whose pace is conveyed to peripheral tissues through neuronal and humoral signals. The molecular clock plays a key role in the control of numerous physiological processes and takes part in the regulation of metabolism and energy balance. Skeletal muscle is one of the peripheral organs whose function is under the control of the molecular clock. However, although skeletal muscle metabolism and performances display circadian rhythmicity, the role of the molecular clock in the skeletal muscle has remained unappreciated for years. Peripheral organs such as skeletal muscle, and the liver, among others, can be desynchronized from the central clock by external stimuli, such as feeding or exercise, which impose a new rhythm at the organism level. In this review, we discuss our current understanding of the clock in skeletal muscle circadian physiology, focusing on the control of myogenesis and skeletal muscle metabolism.

Fibrates suppress bile acid synthesis via peroxisome proliferator-activated receptor-alpha-mediated downregulation of cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase expression.

Fibrates are hypolipidemic drugs that affect the expression of genes involved in lipid metabolism by activating peroxisome proliferator-activated receptors (PPARs). Fibrate treatment causes adverse changes in biliary lipid composition and decreases bile acid excretion, leading to an increased incidence of cholesterol gallstones. In this study, we investigated the effect of fibrates on bile acid synthesis. Ciprofibrate and the PPARalpha agonist Wy14,643 decreased bile acid synthesis in cultured rat hepatocytes and suppressed cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase activities, paralleled by a similar reduction of the respective mRNAs. Treatment of rats with 0.05% (wt/wt) ciprofibrate decreased cholesterol 7alpha-hydroxylase enzyme activity and mRNA. The functional involvement of PPARalpha in the suppression of both enzymes was proven with the use of PPARalpha-null mice. In wild-type mice, ciprofibrate reduced cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase enzyme activities and mRNA. The decrease in mRNA of both enzymes is regulated transcriptionally and posttranscriptionally, respectively, resulting in a decline in the output of fecal bile acids (-45%) and a 3-fold increase in fecal cholesterol secretion. These effects were completely abolished in PPARalpha-null mice. A decreased bile acid production by PPARalpha-mediated downregulation of cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase may contribute to the increased risk of gallstone formation after fibrate treatment.

PPARS in inflammation, atherosclerosis and thrombosis.

PPARs are transcription factors which regulate lipid and lipoprotein metabolism, glucose homeostasis and cellular differentiation. PPARalpha enhances fatty acid oxidation whereas PPARgamma promotes adipogenesis and fatty acid storage in adipose tissue. Both PPARalpha and PPARgamma improve glucose homeostasis. PPARalpha and PPARgamma are activated by the pharmacological agents fibrates and glitazones respectively, and by natural fatty acid derivatives, including inflammation mediators. PPARs are expressed in the different cell types of human atherosclerotic lesions where they regulate the expression of genes involved in the inflammatory response and lipid homeostasis. PPARs modulate the recruitment and adhesion of leukocytes and monocytes to the atherosclerotic lesion. PPARs furthermore control macrophage lipid homeostasis through their action on scavenger receptors and by regulating genes involved in the first steps of the reverse cholesterol transport pathway. Finally, PPARs regulate genes controlling thrombogenicity associated with plaque rupture. These observations suggest that PPARs modulate atherosclerosis development by acting at both metabolic and vascular levels. This review will essentially focus on the functions of PPARalpha and PPARgamma in immunoregulation, vascular inflammation and thrombosis associated to atherosclerosis.

Statin-induced inhibition of the Rho-signaling pathway activates PPARalpha and induces HDL apoA-I.

Statins are inhibitors of the rate-limiting enzyme in cholesterol synthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. In addition to reducing LDL cholesterol, statin treatment increases the levels of the antiatherogenic HDL and its major apolipoprotein apoA-I. Here, we investigated the molecular mechanisms of apoA-I regulation by statins. Treatment with statins increased apoA-I mRNA levels in human HepG2 hepatoma cells, and this effect was reversed by the addition of mevalonate, implicating HMG-CoA reductase as the relevant target of these drugs. Pretreatment with Actinomycin D abolished the increase of apoA-I mRNA, indicating that statins act at the transcriptional level. Indeed, statins increased the human apoA-I promoter activity in transfected cells, and we have identified a statin response element that coincides with a PPARalpha response element known to confer fibrate responsiveness to this gene. The statin effect could be abolished not only by mevalonate, but also by geranylgeranyl pyrophosphate, whereas inhibition of geranylgeranyl transferase activity or treatment with an inhibitor of the Rho GTP-binding protein family increased PPARalpha activity. Using dominant negative forms of these proteins, we found that Rho A itself mediates this response. Because cotreatment with statins and fibrates activated PPARalpha in a synergistic manner, these observations provide a molecular basis for combination treatment with statins and fibrates in coronary heart disease.

Transcriptional regulation of apolipoprotein C-III gene expression by the orphan nuclear receptor RORalpha.

Triglyceride-rich remnant lipoproteins are considered as major risk factors contributing to the pathogenesis of atherosclerosis. Because apolipoprotein (apo) C-III is a major determinant of plasma triglyceride and remnant lipoprotein metabolism, it is important to understand how the expression of this gene is regulated. In the present study, we identified the orphan nuclear receptor RORalpha1 as a regulator of human and mouse apo C-III gene expression. Plasma triglyceride and apo C-III protein concentrations in staggerer (sg/sg) mice, homozygous for a deletion in the RORalpha gene, were significantly lower than in wild type littermates. The lowered plasma apo C-III levels were associated with reduced apo C-III mRNA levels in liver and intestine of sg/sg mice. Transient transfection experiments in human hepatoma HepG2, human colonic CaCO2, and rabbit kidney RK13 cells demonstrated that overexpression of the human RORalpha1 isoform specifically increases human apo C-III promoter activity, indicating that RORalpha1 enhances human apo C-III gene transcription. RORalpha1 response elements were mapped by promoter deletion analysis and gel shift experiments to two AGGTCA half-sites located at positions -83/-78 (within the C3P site) and -23/-18 (downstream of the TATA box) in the human apo C-III promoter, with the -23/-18 site exhibiting the highest binding affinity. Transfection of site-directed mutated constructs in HepG2 cells indicated that the RORalpha1 effect is predominantly mediated by the -23/-18 site. This site is conserved in the mouse apo C-III gene promoter. Moreover, RORalpha binds to the equivalent mouse site and activates constructs containing three copies of the mouse site cloned in front of an heterologous promoter. Taken together, our data identify RORalpha as a transcriptional regulator of apo C-III gene expression, providing a novel, physiological role for RORalpha1 in the regulation of genes controlling triglyceride metabolism.

A colony immunoblotting method for quantitative detection of a Bifidobacterium animalis probiotic strain in human faeces.

A colony immunoblotting method has been developed to allow detection of the probiotic Bifidobacterium animalis strain DN-173 010 in human faecal samples. Rabbits were immunized with heat-killed DN-173 010 bacteria resulting in the production of an antiserum highly specific for bacteria belonging to Bif. animalis species. Of the 89 strains representative of 29 different bifidobacterial species tested, only the 15 strains of the Bif. animalis species could be detected with the antiserum. In Western immunoblotting the serum reacts with a protein of 45-kDa apparent molecular weight. None of the bacteria classically encountered in human faecal samples and able to grow on non-selective Columbia blood agar (enterobacteria, Bacteroides or Lactobacillus for instance) reacted with the antiserum. Taking advantage of the high specificity of the antiserum and of the absence of Bif. animalis bacteria in faeces samples of five human volunteers, we demonstrated that strain DN-173 010 survives the intestinal transit. Being based on a combination of semiselective cultivation and colony immunoblotting techniques, the method allowed detection of the Bif. animalis strain even when it represented only one thousandth of the total bifidobacterial population.

Activation of PPARdelta alters lipid metabolism in db/db mice.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors, which heterodimerize with the retinoid X receptor and bind to peroxisome proliferator response elements in the promoters of regulated genes. Despite the wealth of information available on the function of PPARalpha and PPARgamma, relatively little is known about the most widely expressed PPAR subtype, PPARdelta. Here we show that treatment of insulin resistant db/db mice with the PPARdelta agonist L-165041, at doses that had no effect on either glucose or triglycerides, raised total plasma cholesterol concentrations. The increased cholesterol was primarily associated with high density lipoprotein (HDL) particles, as shown by fast protein liquid chromatography analysis. These data were corroborated by the chemical analysis of the lipoproteins isolated by ultracentrifugation, demonstrating that treatment with L-165041 produced an increase in circulating HDL without major changes in very low or low density lipoproteins. White adipose tissue lipoprotein lipase activity was reduced following treatment with the PPARdelta ligand, but was increased by a PPARgamma agonist. These data suggest both that PPARdelta is involved in the regulation of cholesterol metabolism in db/db mice and that PPARdelta ligands could potentially have therapeutic value.

Protective role of interleukin-10 in atherosclerosis.

The potential role of anti-inflammatory cytokines in the modulation of the atherosclerotic process remains unknown. Interleukin (IL)-10 has potent deactivating properties in macrophages and T cells and modulates many cellular processes that may interfere with the development and stability of the atherosclerotic plaque. IL-10 is expressed in human atherosclerosis and is associated with decreased signs of inflammation. In the present study, we show that IL-10-deficient C57BL/6J mice fed an atherogenic diet and raised under specific pathogen-free conditions exhibit a significant 3-fold increase in lipid accumulation compared with wild-type mice. Interestingly, the susceptibility of IL-10-deficient mice to atherosclerosis was exceedingly high (30-fold increase) when the mice were housed under conventional conditions. Atherosclerotic lesions of IL-10-deficient mice showed increased T-cell infiltration, abundant interferon-gamma expression, and decreased collagen content. In vivo, transfer of murine IL-10 achieved 60% reduction in lesion size. These results underscore the critical roles of IL-10 in both atherosclerotic lesion formation and stability. Moreover, IL-10 appears to be crucial as a protective factor against the effect of environmental pathogens on atherosclerosis.