Marie-Louise Ricketts,
Mark V Boekschoten,
Arja J Kreeft,
Guido J E J Hooiveld,
Corina J A Moen,
Michael Müller,
Rune R Frants,
Soemini Kasanmoentalib,
Sabine M Post,
Hans Mg Princen,
J Gordon Porter,
Martijn B Katan,
Marten H Hofker,
David D Moore
Baylor College of Medicine, Houston, TX, USA (M.L.R., D.D.M.), Wageningen University, Division of Human Nutrition, The Netherlands (M.V.B., G.J.E.J.H., M.M.), Wageningen Centre for Food Sciences, The Netherlands (M.V.B.), Center for Human and Clinical Genetics, LUMC, Leiden, The Netherlands (A.J.K., C.J.A.M., R.R.F.), Dept. of Medical Statistics, LUMC, Leiden, The Netherlands (S.K.), TNO Pharma, Leiden, The Netherlands (S.M.P., H.M.G.P.), Incyte Corp. Palo Alto CA,(Present address: CV Therapeutics, Palo Alto, CA)(J.G.P.), Vriye Univeriteit Amsterdam, Institute for Health Sciences, The Netherlands (M.B.K.); Department of Pathology and Laboratory Medicine, University Medical Center, Groningen, The Netherlands (M.H.H.).
Cafestol, a diterpene present in unfiltered coffee brews such as Scandinavian boiled, Turkish and Cafetière coffee, is the most potent cholesterol-elevating compound known in the human diet. Several genes involved in cholesterol homeostasis have previously been shown to be targets of cafestol, including CYP7A1, the rate-limiting enzyme in bile acid biosynthesis. We have examined the mechanism by which cafestol elevates serum lipid levels. Changes in several lipid parameters were observed in cafestol-treated APOE3Leiden mice, including a significant increase in serum triglyceride levels. Microarray analysis of these mice identified alterations in hepatic expression of genes involved in lipid metabolism and detoxification, many of which are regulated by the nuclear hormone receptors FXR and PXR. Further studies demonstrate that cafestol is an agonist ligand for FXR and PXR, and that cafestol down-regulates expression of the bile acid homeostatic genes CYP7A1, CYP8B1 and NTCP in the liver of wild type but not FXR null mice. Cafestol did not affect genes known to be up-regulated by FXR in the liver of wild type mice, but did increase expression of the positive FXR-target genes IBABP and FGF15 in the intestine. Since FGF15 has recently been shown to function in an enterohepatic regulatory pathway to repress liver expression of bile acid homeostatic genes, its direct induction in the gut may account for indirect effects of cafestol on liver gene expression. PXR-dependent gene regulation of CYP3A11, and other targets by cafestol was also only seen in the intestine. Using a double FXR/PXR knockout mouse model, we found that both receptors contribute to the cafestol-dependent induction of intestinal FGF15 gene expression. In conclusion, cafestol acts as an agonist ligand for both FXR and PXR and this may contribute to its impact on cholesterol homeostasis.
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Department of Nutrition, Harvard School of Public Health, Boston, MA.
An inverse association between coffee consumption and the risk of colorectal cancer has been found in several case-control studies, but such an association was not consistent in prospective cohort studies. We conducted a systematic meta-analysis of prospective cohort studies on coffee consumption and colorectal cancer published up to June 2008. We combined relative risks (RR) for colorectal cancer comparing high vs. low categories of coffee consumption using random-effects models. We identified 12 eligible cohort studies, which included 646,848 participants and 5,403 cases for colorectal cancer. The summarized result of the meta-analysis comparing high- vs. low-consumption categories showed no significant effect of coffee consumption on colorectal cancer risk (RR = .91; 95% confidence intervals [CI]: .81-1.02). The RR was .93 (95% CI: .71-1.22) when considering 4 studies conducted in the United States of America, .91 (95% CI: .76-1.10) for 5 studies from Europe, and .83 (95% CI: .62-1.10) for 3 Japanese studies. No significant differences by sex and cancer-site were found, but there was a slight suggestion of an inverse association between coffee consumption and colon cancer in women (RR = .79; 95% CI: .60-1.04), especially Japanese women (RR = .62; 95% CI: .37-1.05). The suggestive inverse associations were slightly stronger in studies that controlled for smoking and alcohol, and in studies with shorter follow-up times. Information on coffee type, its serving size, or brewing method may provide a better understanding of this reassuring result and the real role of coffee on colorectal cancer risk.(c) 2008 Wiley-Liss, Inc.
Background:Nutrition science aims to create new knowledge, but scientists rarely sit back to reflect on what nutrition research has achieved in recent decades.Methods:We report the outcome of a 1-day symposium at which the audience was asked to vote on the greatest discoveries in nutrition since 1976 and on the greatest challenges for the coming 30 years. Most of the 128 participants were Dutch scientists working in nutrition or related biomedical and public health fields. Candidate discoveries and challenges were nominated by five invited speakers and by members of the audience. Ballot forms were then prepared on which participants selected one discovery and one challenge.Results:A total of 15 discoveries and 14 challenges were nominated. The audience elected Folic acid prevents birth defects as the greatest discovery in nutrition science since 1976. Controlling obesity and insulin resistance through activity and diet was elected as the greatest challenge for the coming 30 years. This selection was probably biased by the interests and knowledge of the speakers and the audience. For the present review, we therefore added 12 discoveries from the period 1976 to 2006 that we judged worthy of consideration, but that had not been nominated at the meeting.Conclusions:The meeting did not represent an objective selection process, but it did demonstrate that the past 30 years have yielded major new discoveries in nutrition and health.European Journal of Clinical Nutrition advance online publication, 10 October 2007; doi:10.1038/sj.ejcn.1602923.
Other papers by authors:
Arja J Kreeft,
Corina J A Moen,
Gordon Porter,
Soemini Kasanmoentalib,
Ronit Sverdlov,
Patrick J van Gorp,
Louis M Havekes,
Rune R Frants,
Marten H Hofker
Department of Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands.
The mechanisms of diet induced hyperlipidemia and atherosclerosis have been widely studied by delineating the role of candidate genes in transgenic and gene targeted mouse models. However, diet induced hyperlipidemia represents a complex process determined by many lipid genes that is only partly understood. This study is aimed at delineating the events induced by dietary intervention in different mouse models at the level of gene expression using microarray analysis. The focus is on the liver as the organ primarily responding to diet, and crucial in determining plasma lipid levels. Firstly, the effect of the genotype was studied. Expression profiles of liver genes were compared between APOE3Leiden (E3L), APOE knockout (E-/-) and C57BL/6JIco (B6) mice using the Incyte GEM 2.03 array carrying 9552 genes. Several hundred differentially expressed genes were identified indicating that the genotype alone effects gene expression. Secondly, the response of E3L mice to high-fat feeding was investigated using a mild and severe high-fat diet (diet W and N, respectively). Diet W caused differential regulation of 200 genes, while diet N affected the expression of 788 genes in B6 and 1010 genes in E3L mice. Annotation of these genes using the Gene Ontology (GO) database showed that two major processes were strongly affected by genotype and diet, namely lipid metabolism and inflammation, the latter as determined by "immune/defense response and detoxification" processes. Many nuclear receptor target genes were differentially regulated, with the largest effects modulated by the severe high-fat diet N, leading to the suppression of genes involved in bile acid, sterol, steroid, fatty acid, and detoxification metabolism. Strikingly, a substantial part of these nuclear receptor target genes were commonly regulated during the different experimental conditions. The common regulation of many nuclear receptor target genes underlying lipid and detoxification processes as found in this study, suggest a defense mechanism involving many nuclear receptors to protect against the accumulation of toxic endogenous lipids and bile acids. These results further strengthen the close link between hyperlipidemia and inflammatory processes.
Corina J A Moen,
Aart P Tholens,
Peter J Voshol,
Willeke de Haan,
Louis M Havekes,
Peter Gargalovic,
Aldons J Lusis,
Ko Willems van Dijk,
Rune R Frants,
Marten H Hofker,
Patrick C N Rensen
Objective - The Hyplip2 congenic mouse strain contains part of chromosome 15 from MRL/MpJ on BALB/cJ (B/c) background. Hyplip2 mice show elevated plasma levels of cholesterol and predominantly triglycerides (TG), and are susceptible to diet-induced atherosclerosis. This study aimed at elucidation of the mechanism(s) explaining the hypertriglyceridemia. Methods and Results - Hypertriglyceridemia can result from an increased intestinal or hepatic TG production and/or by a decreased LPL-mediated TG clearance. The intestinal TG absorption and chylomicron formation was studied after i.v. injection of Triton WR1339 and intragastric load of olive oil containing glycerol tri[3H]oleate. No difference was found in intestinal TG absorption. Moreover, the hepatic VLDL-TG production rate and VLDL particle production, after injection of Triton WR1339, were also not affected. To investigate the LPL-mediated TG clearance, mice were injected i.v. with glycerol tri[3H]oleate-labeled VLDL-like emulsion particles. In Hyplip2 mice, the particles were cleared at a decreased rate (t(1/2) 25+/-6 vs 11+/-2 min, p< .05) concomitant with decreased uptake of emulsion-TG derived 3H-labeled fatty acids by the liver and white adipose tissue. Conclusion - The increased plasma TG levels in Hyplip2 mice do not result from an enhanced intestinal absorption or increased hepatic VLDL-production, but are caused by decreased LPL-mediated TG clearance.
Saskia T J van Cruchten,
Laura H J de Haan,
Patrick P J Mulder,
Cindy Kunne,
Mark V Boekschoten,
Martijn B Katan,
Jac M M J G Aarts,
Renger F Witkamp
Department of Human Nutrition, Wageningen University, 6700 EV, Wageningen, The Netherlands; Top Institute of Food and Nutrition, 6709 PA, Wageningen, The Netherlands.
Cafestol and kahweol are diterpene compounds present in unfiltered coffees. Cafestol is known as the most potent cholesterol-raising agent that may be present in the human diet. Remarkably, the mechanisms behind this effect have only been partly resolved so far. Even less is known about the metabolic fate of cafestol and kahweol. From the structure of cafestol, carrying a furan moiety, we hypothesized that epoxidation may not only be an important biotransformation route but that this also plays a role in its effects found. In bile duct-cannulated mice, dosed with cafestol, we were able to demonstrate the presence of epoxy-glutathione (GSH) conjugates, GSH conjugates and glucuronide conjugates. In addition, it was shown that cafestol was able to induce an electrophile-responsive element (EpRE). Using a murine hepatoma cell line with a luciferase reporter gene under control of an EpRE from the human NQO1 regulatory region, we also found that metabolic activation by CYP450 enzymes is needed for EpRE induction. Furthermore, raising intracellular GSH resulted in a decrease in EpRE-mediated gene induction, whereas lowering intracellular GSH levels increased EpRE-mediated gene induction. In conclusion, evidence suggests that cafestol induces EpRE, apparently via a bioactivation process that possibly involves epoxidation of the furan ring. The epoxides themselves appear subject to conjugation with GSH. The effects on EpRE can also explain the induction of GSH which seems to be involved in the reported beneficial effects of cafestol, for example, when administered with aflatoxin B1 or other toxic or carcinogenic compounds.
Josep Maria Del Bas,
Marie-Louise Ricketts,
Montserrat Vaqué,
Esther Sala,
Helena Quesada,
Anna Ardevol,
M Josepa Salvadó,
Mayte Blay,
Lluís Arola,
David D Moore,
Gerard Pujadas,
Juan Fernandez-Larrea,
Cinta Bladé
Departament de Bioquímica i Biotecnología, Universitat Rovira i Virgili, Campus Sescelades, Tarragona, Spain. Fax:+34-977558232.
Consumption of dietary flavonoids has been associated with reduced mortality and risk of cardiovascular disease, partially by reducing triglyceridemia. We have previously reported that a grape seed procyanidin extract (GSPE) reduces postprandial triglyceridemia in normolipidemic animals signaling through the orphan nuclear receptor small heterodimer partner (SHP) a target of the bile acid receptor farnesoid X receptor (FXR). Our aim was to elucidate whether FXR mediates the hypotriglyceridemic effect of procyanidins. In FXR-driven luciferase expression assays GSPE dose-dependently enhanced FXR activity in the presence of chenodeoxycholic acid. GSPE gavage reduced triglyceridemia in wild type mice but not in FXR-null mice, revealing FXR as an essential mediator of the hypotriglyceridemic actions of procyanidins in vivo. In the liver, GSPE downregulated, in an FXR-dependent manner, the expression of the transcription factor steroid response element binding protein 1 (SREBP1) and several SREBP1 target genes involved in lipogenesis, and upregulated ApoA5 expression. Altogether, our results indicate that procyanidins lower triglyceridemia following the same pathway as bile acids: activation of FXR, transient upregulation of SHP expression and subsequent downregulation of SREBP1 expression. This study adds dietary procyanidins to the arsenal of FXR ligands with potential therapeutic use to combat hypertriglyceridemia, type 2 diabetes and metabolic syndrome.
Department of Biological Sciences, University of Notre Dame, South Bend, IN 46556.
Pregnane X receptor (PXR) is an important component of the body's adaptive defense system responsible for the elimination of various toxic xenobiotics. PXR activation by endogenous and exogenous chemicals, including steroids, antibiotics, bile acids, and herbal compounds, results in induction of drug metabolism. We investigated the ability of the isoflavones genistein, daidzein, and the daidzein metabolite equol to activate human and mouse PXR in vitro using cell-based transient transfection studies and primary hepatocytes and in vivo in a mouse model. In transient transfection assays, the isoflavones genistein and daidzein activate full-length, wild-type mouse PXR, but not a mutant form, with genistein being the most potent. In contrast, equol was a more potent activator of human PXR than genistein or daidzein. In a mammalian 2-hybrid assay, isoflavones induced recruitment of the coactivator steroid receptor coactivator 1 to PXR. When tested against the native human Cytochrome P450 3A4 (CYP3A4) promoter, equol was the more potent activator and treatment of human hepatocytes with equol increased CYP3A4 mRNA and immunoreactive protein expression. Treatment of wild-type, but not PXR(-/-), mouse hepatocytes showed that genistein and daidzein induced the expression of Cytochrome P450 3A11 (Cyp3A11) mRNA, whereas equol had no effect. Cyp3A11 mRNA was also induced in vivo in mice fed a soy protein-containing diet. The results presented herein demonstrate that there is a species-specific difference in the activation of PXR by isoflavones and equol.
Arne Giebeler,
Mark V Boekschoten,
Christian Klein,
Malgorzata Borowiak,
Carmen Birchmeier,
Nikolaus Gassler,
Hermann E Wasmuth,
Michael Müller,
Christian Trautwein,
Konrad L Streetz
Department of Medicine III, University Hospital Aachen, Pauwelsstr. 30, 52074 Aachen, Germany.
BACKGROUND & AIMS:: The HGF/c-Met system is an essential inducer of hepatocyte growth and proliferation. Although a fundamental role for the HGF receptor c-Met has been demonstrated in acute liver regeneration its cell specific role in hepatocytes during chronic liver injury and fibrosis progression has not been determined yet. METHODS:: Hepatocyte-specific c-Met knockout mice (c-Met?(hepa)) using the Cre-loxP system were studied in a bile-duct ligation model. Microarray analysis were performed to define HGF/c-Met dependent gene expression. RESULTS:: Two strategies for c-Met deletion in hepatocytes to generate hepatocyte-specific c-Met knockout mice were tested. Early deletion during embryonic development was lethal, while post-natal Cre-expression was successful leading to the generation of viable c-Met?(hepa) mice. Bile-duct ligation in these mice resulted in extensive necrosis and lower proliferation rates of hepatocytes. Gene array analysis of c-Met?(hepa) mice revealed a significant reduction of anti-apoptotic genes in c-Met deleted hepatocytes. These findings could be functionally tested as c-Met?(hepa) mice showed a stronger apoptotic response after bile-duct ligation and Jo-2 stimulation. The phenotype was associated with increased expression of pro-inflammatory cytokines (TNF-?? and IL-6) and an enhanced recruitment of neutrophils. Activation of these mechanisms triggered a stronger pro-fibrogenic response as evidenced by increased TGF-?(1), ?? SMA, collagen-1? mRNA expression and enhanced collagen-fibre staining in c-Met?(hepa) mice. CONCLUSIONS:: Our results demonstrate that deletion of c-Met in hepatocytes leads to more liver cell damage and fibrosis in a chronic cholestatic liver injury model as c-Met triggers survival signals important for hepatocyte recovery.
Linda M Sanderson,
Philip J de Groot,
Guido J E J Hooiveld,
Arjen Koppen,
Eric Kalkhoven,
Michael Müller,
Sander Kersten
Nutrigenomics Consortium, Top Institute (TI) Food and Nutrition, Wageningen, The Netherlands.
BACKGROUND: The effect of dietary fats on human health and disease are likely mediated by changes in gene expression. Several transcription factors have been shown to respond to fatty acids, including SREBP-1c, NF-kappaB, RXRs, LXRs, FXR, HNF4alpha, and PPARs. However, it is unclear to what extent these transcription factors play a role in gene regulation by dietary fatty acids in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Here, we take advantage of a unique experimental design using synthetic triglycerides composed of one single fatty acid in combination with gene expression profiling to examine the effects of various individual dietary fatty acids on hepatic gene expression in mice. We observed that the number of significantly changed genes and the fold-induction of genes increased with increasing fatty acid chain length and degree of unsaturation. Importantly, almost every single gene regulated by dietary unsaturated fatty acids remained unaltered in mice lacking PPARalpha. In addition, the majority of genes regulated by unsaturated fatty acids, especially docosahexaenoic acid, were also regulated by the specific PPARalpha agonist WY14643. Excellent agreement was found between the effects of unsaturated fatty acids on mouse liver versus cultured rat hepatoma cells. Interestingly, using Nuclear Receptor PamChip(R) Arrays, fatty acid- and WY14643-induced interactions between PPARalpha and coregulators were found to be highly similar, although several PPARalpha-coactivator interactions specific for WY14643 were identified. CONCLUSIONS/SIGNIFICANCE: We conclude that the effects of dietary unsaturated fatty acids on hepatic gene expression are almost entirely mediated by PPARalpha and mimic those of synthetic PPARalpha agonists in terms of regulation of target genes and molecular mechanism. Use of synthetic dietary triglycerides may provide a novel paradigm for nutrigenomics research.
Traditionally, nutritional science was mainly concentrated on nutrient deficiencies and their effects on health and disease. However, over the past few decades, research emphasis has gradually shifted to the link between (over)-nutrition and chronic diseases. Driven by the continuing and accelerating discoveries in omics technology, unique possibilities have emerged to investigate the genome-wide effects of nutrients at the molecular level. Nutrigenomics uses these techniques in combination with a range of models and molecular tools as a strategy to understand the mechanistic basis of nutrition. As a paradigm for this strategy microarray analysis of genes regulated by peroxisome proliferator-activated receptors (PPARs) can serve. PPARs are ligand-activated transcription factors mediating the effect of unsaturated fatty acids and certain drugs on gene expression. Physiologically they act as fatty acid sensors in metabolic active organs, regulating a wide range of metabolic and signaling pathways. This allows cells to modulate their function and metabolic capacity, for example according to diet/nutrient-related changes in ligand concentration. Although much is already known about PPARs, gaps in our knowledge remain. In so far as the biological role of a particular PPAR is directly coupled to the function of its target genes, probing PPAR-regulated genes via the application of genomics tools can greatly improve our understanding of PPAR function. In this review we summarize and discuss the application of transcriptomics to study PPAR function, and discuss some of the challenges inherent to the application of transcriptomics to nutrigenomics research.
Consumption of soy has been demonstrated to reduce circulating cholesterol levels, most notably reducing low-density lipoprotein (LDL) cholesterol levels in hypercholesterolemic individuals. The component or components that might be responsible for this effect is still a matter of debate or controversy among many researchers. Candidate agents include an activity of soy protein itself, bioactive peptides produced during the digestive process, or the soy isoflavones. Although soy intake may provide other health benefits including preventative or remediative effects on cancer, osteoporosis and symptoms of menopause, this review will focus on isoflavones as agents affecting lipid metabolism. Isoflavones were first discovered as a bioactive agent disrupting estrogen action in female sheep, thereby earning the often-used term 'phytoestrogens'. Subsequent work confirmed the ability of isoflavones to bind to estrogen receptors. Along with the cholesterol-lowering effect of soy intake, research that is more recent has pointed to a beneficial antidiabetic effect of soy intake, perhaps mediated by soy isoflavones. The two common categories of antidiabetic drugs acting on nuclear receptors known as peroxisome proliferator activated receptors (PPARs) are the fibrates and glitazones. We and others have recently asked the research question 'do the soy isoflavones have activities as either "phytofibrates" or "phytoglitazones"?' Such an activity should be able to be confirmed both in vivo and in vitro. In both the in vivo and in vitro cases, this action has indeed been confirmed. Further work suggests a possible action of isoflavones similar to the nonestrogenic ligands that bind the estrogen-related receptors (ERRs). Recently, these receptors have been demonstrated to contribute to lipolytic processes. Finally, evaluation of receptor activation studies suggests that thyroid receptor activation may provide additional clues explaining the metabolic action of isoflavones. The recent advances in the discovery and evaluation of the promiscuous nuclear receptors that bind many different chemical ligands should prove to help explain some of the biological effects of soy isoflavones and other phytochemicals.
Mark V Boekschoten,
Maaike K Hofman,
Rien Buytenhek,
Evert G Schouten,
Hans M G Princen,
Martijn B Katan
Division of Human Nutrition, Wageningen University, Wageningen, The Netherlands. Mark.Boekschoten@wur.nl
Unfiltered coffee brews such as French press and espresso contain a lipid from coffee beans named cafestol that raises serum cholesterol in humans. Cafestol decreases the expression and activity of cholesterol 7alpha-hydroxylase, the rate-limiting enzyme in the classical pathway of bile acid synthesis, in cultured rat hepatocytes and livers of APOE3Leiden mice. Inhibition of bile acid synthesis has been suggested to be responsible for the cholesterol-raising effect of cafestol. Therefore, we assessed whether cafestol decreases the activity of cholesterol 7alpha-hydroxylase in humans. Because liver biopsies were not feasible, we measured plasma levels of 7alpha-hydroxy-4-cholesten-3-one, a marker for the activity of cholesterol 7alpha-hydroxylase in the liver. Plasma 7alpha-hydroxy-4-cholesten-3-one was measured in 2 separate periods in which healthy volunteers consumed coffee oil containing cafestol (69 mg/d) for 5 wk. Plasma levels of 7alpha-hydroxy-4-cholesten-3-one increased by 47 +/- 13%(mean +/- SEM, n = 38, P = .001) in the first period and by 23 +/- 10%(n = 31, P = .03) in the second treatment period. Serum cholesterol was raised by 23 +/- 2%(P < .001) in the first period and by 18 +/- 2%(P < .001) in the second period. We corrected individual 7alpha-hydroxy-4-cholesten-3-one levels for serum cholesterol levels, because coffee oil increases serum cholesterol and 7alpha-hydroxy-4-cholesten-3-one is probably present in the lipoprotein fraction of serum. After correction, the increase in 7alpha-hydroxy-4-cholesten-3-one was 24 +/- 11%(P = .04) in the first period and there was no effect in period 2. Our study showed that coffee oil did not decrease, and actually increased, plasma levels of 7alpha-hydroxy-4-cholesten-3-one in humans in 2 separate treatment periods. Therefore, this study does not support the hypothesis that cafestol decreases bile acid synthesis in humans.
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Laboratory of Organic and Bio-molecular Chemistry, Faculty of Pharmaceutical Science, Hiroshima International University, 5-1-1, Hirokoshingai, Kure, Hiroshima 737-0112, Japan; Division of Clinical Pharmaceutical Science, Programs for Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan.
FXR (farnesoid X receptor) is a bile acid-activated nuclear receptor that regulates not only the biosynthesis and enterohepatic circulation of bile acids, but also triglyceride, cholesterol and glucose metabolism. FXR-mediated signaling pathways have become promising novel drug targets for the treatment of common metabolic and hepatic diseases. With the aim of uncovering novel modulators of FXR and further elucidating the molecular basis of FXR activation, we investigated the structure-activity relationships of a variety of naturally occurring sterols structurally related to bile acids in terms of their FXR agonist activity. Here, we report that the ability of bile alcohols to activate FXR varied with the position and number of hydroxyl groups existing in the steroid side chain of bile alcohols. In addition, we showed that the shortening of the steroid side chain of bile acids as well as bile alcohols resulted in a decline of the ability of these agents to activate FXR. Thus, we provide new insights into the structure-activity relationships of bile acids and bile alcohols as FXR agonists.
Life Sciences Research Division, Korea Institute of Science and Technology, P.O. Box 131, Cheongryang, Seoul 130-650, Republic of Korea; Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Republic of Korea.
Farnesoid X receptor (FXR) serves as a receptor for chenodeoxycholic acid (CDCA) and other bile acids, and coordinates cholesterol and lipid metabolism. Since targeting the FXR-CDCA interaction thus might provide a way to regulate lipid homeostasis, we developed an FXR binding assay based on fluorescence polarization. Employing a fluorescently-labeled CDCA-F, we showed that CDCA-F selectively bound to the ligand binding domain of FXR (FXR-LBD) among nuclear receptors. The assay was then utilized for screening inhibitors against the FXR-CDCA interaction, thereby discovering four relatively potent inhibitors. The selected inhibitors were further studied for changes in intrinsic tryptophan fluorescence of FXR-LBD to gain structural insights of the interaction. Furthermore, transactivation effects of the inhibitors on the human bile salt excretory pump (BSEP) promoter were examined to reveal their cellular activities in the FXR-mediated pathway. Therefore, we demonstrated that the developed assay would offer an efficient primary screening tool for identifying FXR modulators.
Peter Fickert,
Andrea Fuchsbichler,
Tarek Moustafa,
Martin Wagner,
Gernot Zollner,
Emina Halilbasic,
Ulrike Stöger,
Marco Arrese,
Margarita Pizarro,
Nancy Solís,
Gonzalo Carrasco,
Allessandra Caligiuri,
Martina Sombetzki,
Emil Reisinger,
Oleksiy Tsybrovskyy,
Kurt Zatloukal,
Helmut Denk,
Hartmut Jaeschke,
Massimo Pinzani,
Michael Trauner
From the Laboratory of Experimental and Molecular Hepatology,* Division of Gastroenterology and Hepatology, Departments of Medicine and Pathology, Medical University Graz, Austria; the Departments of Medicine and Pathology Escuela de Medicina, Pontificia Universidad Catolica de Chile, Santiago, Chile; the Center of Research, High Education and Transfer, Department of Internal Medicine, Florence, Italy; the Department of Internal Medicine, Rostock, Germany; and the Department of Pharmacology,** Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas.
The nuclear bile acid receptor, farnesoid X receptor (FXR), may play a pivotal role in liver fibrosis. We tested the impact of genetic FXR ablation in four different mouse models. Hepatic fibrosis was induced in wild-type and FXR knock-out mice (FXR(-/-)) by CCl4 intoxication, 3,5-diethoxycarbonyl-1,4-dihydrocollidine feeding, common bile duct ligation, or Schistosoma mansoni (S.m.)-infection. In addition, we determined nuclear receptor expression levels (FXR, pregnane X receptor (PXR), vitamin D receptor, constitutive androstane receptor (CAR), small heterodimer partner (SHP)) in mouse hepatic stellate cells (HSCs), portal myofibroblasts (MFBs), and human HSCs. Cell type-specific FXR protein expression was determined by immunohistochemistry in five mouse models and prototypic human fibrotic liver diseases. Expression of nuclear receptors was much lower in mouse and human HSCs/MFBs compared with total liver expression with the exception of vitamin D receptor. FXR protein was undetectable in mouse and human HSCs and MFBs. FXR loss had no effect in CCl4-intoxicated and S.m.-infected mice, but significantly decreased liver fibrosis of the biliary type (common bile duct ligation, 3,5-diethoxycarbonyl-1,4-dihydrocollidine). These data suggest that FXR loss significantly reduces fibrosis of the biliary type, but has no impact on non-cholestatic liver fibrosis. Since there is no FXR expression in HSCs and MFBs in liver fibrosis, our data indicate that these cells may not represent direct therapeutic targets for FXR ligands.
Pharmacogenetics Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, North Carolina, USA.
OBJECTIVE: We previously found that the lack of nuclear xenobiotic receptor, PXR, decreases femoral bone mineral density (BMD) in Pxr mice. Our present study aims to elucidate the inherited phenotype that correlates with the decreased BMD and to identify the PXR-regulated gene that may link with this phenotype. METHODS: Pxr and Pxr mice were used to measure the serum levels of inorganic phosphate (Pi), calcium and vitamin D3. Real time PCR and western blots were used to determine the intestinal and renal expressions of Pi and calcium transporters and various other genes involved in bone homeostasis. Cell-based reporter and gel shift assays were performed to characterize the promoter of the identified PXR-regulated gene. RESULTS: In both Pxr male and female mice, lumbar, sternum, and skull were all also found to have decreased their BMD values. Serum Pi levels, but not calcium levels, are attenuated in Pxr mice, exhibiting a phenotype of hypophosphatemia. Among the members of the Na/Pi contransporter family, only the SLC34A2 mRNA and protein are repressed in Pxr mice. PXR can directly activate the transcription of the SLC34A2 gene through an ER6 motif on its promoter. CONCLUSION: Pxr mice show the inherited phenotype of hypophosphatemia. The lack of PXR results in a severe repression of the Na/Pi cotransporter NaPi-IIb/Npt2b (SLC34A2), thus leading Pxr males and females to develop a type of hypophosphatemia.
Maria De Lucia,
Lucia Panzella,
Dominique Melck,
Italo Giudicianni,
Andrea Motta,
Alessandra Napolitano,
Marco d'Ischia
Department of Organic Chemistry and Biochemistry and Centro Interdipartimentale di Metodologie Chimico-Fisiche (CIMCF), University of Naples "Federico II", Via Cinthia 4, I-80126 Naples, Italy, and Istituto di Chimica Biomolecolare del CNR, Comprensorio Olivetti, Edificio A, Via Campi Flegrei 34, I-80078 Pozzuoli Naples, Italy.
Cafestol and kahweol, coffee-specific furan diterpenes, are believed to cause various physiological effects in human subjects, including an increase in cholesterol and plasma triacylglycerol levels as well as cancer chemopreventive effects. Despite the increasing interest in these compounds raised by the diverse range of biological activities, their reaction behavior and degradation pathways under physiologically relevant conditions remain uncharted. Herein, we report a detailed investigation of the structural modifications suffered by cafestol and kahweol in the presence of acidic nitrite under conditions mimicking those occurring in the stomach during digestion as well as by action of other oxidants. Prior to the chemical study, an isolation procedure for kahweol from green coffee beans was developed based on Soxhlet extraction followed by preparative HPLC. Preliminary experiments showed that kahweol is much more reactive than cafestol toward nitrite at pH 3, as evidenced by inhibition experiments with the 2,3-diaminonaphthalene assay as well as by product analysis in coffee extracts. When exposed to equimolar nitrite in phosphate buffer, pH 3, kahweol gave as a main product the ring-opened dicarbonyl derivative 1. Under more forcing conditions, cafestol reacted as well to give a main nitrogenous product identified as the 1-hydroxy-2-pyrrolinone 2. It is concluded that the conjugated double bond in kahweol is a critical structural element, increasing the susceptibility of the furan ring to protonation rather than nitrosation and favoring ring-opening routes driven by the irreversible oxidation steps. These results offer a useful background to assess the effects of coffee-specific lipids in association with abnormally high nitrite levels from the diet.
Andrea Mencarelli,
Barbara Renga,
Marco Migliorati,
Sabrina Cipriani,
Eleonora Distrutti,
Luca Santucci,
Stefano Fiorucci
*Dipartimento di Medicina Clinica e Sperimentale, University of Perugia, Perugia, Italy.
Immune-mediated liver diseases including autoimmune and viral hepatitis are a major health problem worldwide. In this study, we report that activation of the farnesoid X receptor (FXR), a member of the ligand-activated nuclear receptor superfamily and bile sensor highly expressed in the liver, attenuates liver injury in a model of autoimmune hepatitis induced by Con A. We found that FXR gene ablation results in a time-dependent increase of liver expression (up to 20-fold in a 9-mo-old mouse) of osteopontin, a NKT cell-derived extracellular matrix protein and immunoregulatory cytokine. In comparison to wild-type, FXR(-/-) mice are more susceptible to Con A-induced hepatitis and react to Con A administration by an unregulated production of osteopontin. Administering wild-type mice with a synthetic FXR agonist attenuated Con A-induced liver damage and liver expression of the osteopontin gene. By in vitro studies, we found that FXR is expressed by primarily isolated NKT cells and its ablation favors ostepontin production in response to Con A. Chromatin immunoprecipitation assay and coimmunoprecipitation experiments demonstrate that the short heterodimer partner (SHP), a nuclear receptor and FXR target, was expressed by NKT cell hybridomas and increased in response to FXR activation. FXR activates SHP that interacts with and inhibits c-Jun binding to the osteopontin promoter. These data indicate that in NKT cells, FXR activation causes a SHP-mediated inhibition of osteopontin production. These data support the notion that the bile acid sensor FXR regulates the activation of liver NKT cells.
*Dipartimento di Medicina Clinica e Sperimentale, Università di Perugia, Perugia, Italy and.
The farnesoid X receptor (FXR) is a bile acid-regulated nuclear receptor expressed in enterohepatic tissues. In this study we investigated whether FXR is expressed by cells of innate immunity and regulates inflammation in animal models of colitis. Acute (7 days) and chronic (8 wk) colitis were induced in wild-type and FXR(-/-) mice by intrarectal administration of trinitrobenzensulfonic acid or by 7-day administration of 5% dextran sulfate in drinking water. The results of this experiment demonstrate that FXR is expressed by and exerts counterregulatory effects on cells of innate immunity. Exposure of LPS-activated macrophages to 6-ethyl chenodeoxycholic acid (6E-CDCA; INT-747) a synthetic FXR ligand, results in a reciprocal regulation of NF-kappaB dependent-genes (TNF-alpha, IL-1beta, IL-6, COX-1, COX-2, and iNOS) and induction of SHP, a FXR-regulated gene. FXR activation stabilizes the nuclear corepressor NCoR on the NF-kappaB responsive element on the IL-1beta promoter. Colon inflammation in Crohn's disease patients and in rodent models of colitis is associated with a reduced expression of FXR mRNA. Using two rodent models of colon inflammation, we show that progression of these immune-mediated disorders is exacerbated in FXR(-/-) mice (p < .01). In vivo treatment with INT-747 attenuates organ injury and immune cell activation. FXR activation increased the colon expression of I-BABP, FXR, and SHP while reducing IL-1beta, IL-2, IL-6, TNF-alpha, and IFN-gamma mRNA expression and attenuating disease severity. In aggregate, these findings provide evidence that FXR is an essential component of a network of nuclear receptors that regulate intestinal innate immunity and homeostasis.
Department of Molecular Biology, Graduate School of Pharmaceutical Sciences,Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan.
The farnesoid X receptor (FXR; NR1H4) is a member of the nuclear receptor superfamily and regulates the expression of genes involved in enterohepatic circulation and the metabolism of bile acids. Based on functional analyses, nuclear receptors are divided into regions A-F. To explore the cofactors interacting with FXR, we performed a pull-down assay using GST fused to the N-terminal A/B region and the C region, which are required for the ligand-independent transactivation and DNA-binding, respectively, of FXR, and nuclear extracts from HeLa cells. We identified DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku80, and Ku70 as FXR-associated factors. These proteins are known to have an important role in DNA repair, recombination and transcription. DNA-PKcs mainly interacted with the A/B region of FXR, whereas the Ku proteins interacted with the C region and with the D region (hinge region). Chromatin immunoprecipitation assays revealed that the Ku proteins associated with FXR on the bile salt export pump (BSEP) promoter. Furthermore, we demonstrated that ectopic expression of the Ku proteins decreased the promoter activity and expression of BSEP gene mediated by FXR. These results suggest that the Ku proteins function as corepressors for FXR.
Mélanie Verreault,
Jenny Kaeding,
Patrick Caron,
Jocelyn Trottier,
Laurent Grosse,
Elise Houssin,
Sophie Pâquet,
Martin Perreault,
Olivier Barbier
Laboratory of Molecular Pharmacology, CHUQ Research Center and Faculty of Pharmacy, Laval University, Québec, Canada.
Recent progresses in molecular pharmacology approaches have allowed the identification and characterization of a series of nuclear receptors (NR) which efficiently control the level UDP-glucuronosyltransferase (UGT) genes expression. These regulatory processes ensure optimized UGT expression in response to specific endogenous and/or exogenous stimuli. Interestingly, numerous endogenous activators of these NRs are conjugated by the UGT enzymes they regulate. In such a case, the NR-dependent regulation of UGT genes corresponds to a feedforward/feedback mechanism by which a bioactive molecule controls its own concentrations. In the present review, we will discuss i) how bilirubin reduces its circulating levels by activating AhR in the liver; ii) how bile acids modulate their hepatic glucuronidation via PXR- and FXR-dependent processes in enterohepatic tissues; and iii) how androgens inhibit their cellular metabolism in prostate cancer cells through an AR-dependent mechanism. Subsequently, with further discussion of the same examples (bilirubin and bile acids), we will illustrate how NR-dependent regulation of UGT enzymes may contribute to the beneficial effects of pharmacological activators of nuclear receptors, such as CAR and PPARa.
J Méndez-González,
S Süren-Castillo,
L Calpe-Berdiel,
N Rotllan,
M Vázquez-Carrera,
J C Escolà-Gil,
F Blanco-Vaca
Servei de Bioquímica and Institut de Recerca, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain.
Disodium ascorbyl phytostanol phosphate (FM-VP4) is a synthetic compound derived from sitostanol and campestanol that has proved to be efficient as a cholesterol-lowering therapy in mice and human subjects. However, the mechanism of action of FM-VP4 remains unknown. The present study tests the ability of FM-VP4 to alter intestinal and liver cholesterol homeostasis in mice. Female C57BL/6J mice were fed either a control chow or a 2 % FM-VP4-enriched diet for 4 weeks. FM-VP4 reduced the in vivo net intestinal cholesterol absorption and plasma and liver cholesterol concentrations by 2.2-, 1.5- and 1.6-fold, respectively, compared with control mice. Furthermore, FM-VP4 also showed an impact on bile acid homeostasis. In FM-VP4 mice, liver and intestinal bile acid content was increased by 1.3- and 2.3-fold, respectively, whereas faecal bile acid output was 3.3-fold lower. FM-VP4 also increased the intestinal absorption of orally administered [3H]taurocholic acid to small intestine in vivo. Inhibition of intestinal cholesterol absorption by FM-VP4 was not mediated via transcriptional increases in intestine liver X receptor (LXR)-alpha, adenosine triphosphate-binding cassette transporter (ABC)-A1, ABCG5/G8 nor to decreases in intestinal Niemann-Pick C1-like 1 (NPC1L1) expression. In contrast, FM-VP4 up-regulated liver LXRalpha, ABCA1, ABCG5, scavenger receptor class BI (SR-BI) and hydroxymethylglutaryl coenzyme A reductase (HMGCoA-R) gene expression, whereas it down-regulated several farnesoid X receptor (FXR)-target genes such as cytochrome P450 family 7 subfamily A polypeptide 1 (CYP7A1) and Na+/taurocholate co-transporter polypeptide (NTCP). In conclusion, FM-VP4 reduced intestinal cholesterol absorption, plasma and liver cholesterol and affected bile acid homeostasis by inducing bile acid intestinal reabsorption and changed the liver expression of genes that play an essential role in cholesterol homeostasis. This is the first phytosterol or stanol that affects bile acid metabolism and lowers plasma cholesterol levels in normocholesterolaemic mice.
