[Show abstract][Hide abstract] ABSTRACT: Circulating levels of the gut microbe-derived metabolite trimethylamine-N-oxide (TMAO) have recently been linked to cardiovascular disease (CVD) risk. Here, we performed transcriptional profiling in mouse models of altered reverse cholesterol transport (RCT) and serendipitously identified the TMAO-generating enzyme flavin monooxygenase 3 (FMO3) as a powerful modifier of cholesterol metabolism and RCT. Knockdown of FMO3 in cholesterol-fed mice alters biliary lipid secretion, blunts intestinal cholesterol absorption, and limits the production of hepatic oxysterols and cholesteryl esters. Furthermore, FMO3 knockdown stimulates basal and liver X receptor (LXR)-stimulated macrophage RCT, thereby improving cholesterol balance. Conversely, FMO3 knockdown exacerbates hepatic endoplasmic reticulum (ER) stress and inflammation in part by decreasing hepatic oxysterol levels and subsequent LXR activation. FMO3 is thus identified as a central integrator of hepatic cholesterol and triacylglycerol metabolism, inflammation, and ER stress. These studies suggest that the gut microbiota-driven TMA/FMO3/TMAO pathway is a key regulator of lipid metabolism and inflammation.
[Show abstract][Hide abstract] ABSTRACT: We performed silencing and overexpression studies of flavin containing monooxygenase 3 (FMO3) in hyperlipidemic mouse models to examine its effects on trimethylamine N-oxide (TMAO) levels and atherosclerosis. Knockdown of hepatic FMO3 in LDL receptor null (LDLRKO) mice using an antisense oligonucleotide resulted in decreased circulating TMAO levels and atherosclerosis. Surprisingly, we also observed significant decreases in hepatic lipids and in levels of plasma lipids, ketone bodies, glucose and insulin. FMO3 over-expression in transgenic mice, on the other hand, increased hepatic and plasma lipids. Global gene expression analyses suggested that these effects of FMO3 on lipogenesis and gluconeogenesis may be mediated through the PPARα and KLF15 pathways. In vivo and in vitro results were consistent with the concept that the effects were mediated directly by FMO3 rather than TMA/TMAO; in particular, over-expression of FMO3 in the human hepatoma cell line, Hep3B, resulted in significantly increased glucose secretion and lipogenesis. Our results indicate a major role for FMO3 in modulating glucose and lipid homeostasis in vivo, and they suggest that pharmacologic inhibition of FMO3 to reduce TMAO levels would be confounded by metabolic interactions.
The Journal of Lipid Research 11/2014; 56(1). DOI:10.1194/jlr.M051680 · 4.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We profiled and analyzed 283 metabolites representing eight major classes of molecules including Lipids, Carbohydrates, Amino Acids, Peptides, Xenobiotics, Vitamins and Cofactors, Energy Metabolism, and Nucleotides in mouse liver of 104 inbred and recombinant inbred strains. We find that metabolites exhibit a wide range of variation, as has been previously observed with metabolites in blood serum. Using genome-wide association analysis, we mapped 40% of the quantified metabolites to at least one locus in the genome and for 75% of the loci mapped we identified at least one candidate gene by local expression QTL analysis of the transcripts. Moreover, we validated 2 of 3 of the significant loci examined by adenoviral overexpression of the genes in mice. In our GWAS results, we find that at significant loci the peak markers explained on average between 20 and 40% of variation in the metabolites. Moreover, 39% of loci found to be regulating liver metabolites in mice were also found in human GWAS results for serum metabolites, providing support for similarity in genetic regulation of metabolites between mice and human. We also integrated the metabolomic data with transcriptomic and clinical phenotypic data to evaluate the extent of co-variation across various biological scales.
Molecular Systems Biology 05/2014; 10(5). DOI:10.15252/msb.20135004 · 14.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Identifying environmentally-specific genetic effects is a key challenge in understanding the structure of complex traits. Model organisms play a crucial role in the identification of such gene-by-environment interactions, as a result of the unique ability to observe genetically similar individuals across multiple distinct environments. Many model organism studies examine the same traits but under varying environmental conditions. For example, knock-out or diet-controlled studies are often used to examine cholesterol in mice. These studies, when examined in aggregate, provide an opportunity to identify genomic loci exhibiting environmentally-dependent effects. However, the straightforward application of traditional methodologies to aggregate separate studies suffers from several problems. First, environmental conditions are often variable and do not fit the standard univariate model for interactions. Additionally, applying a multivariate model results in increased degrees of freedom and low statistical power. In this paper, we jointly analyze multiple studies with varying environmental conditions using a meta-analytic approach based on a random effects model to identify loci involved in gene-by-environment interactions. Our approach is motivated by the observation that methods for discovering gene-by-environment interactions are closely related to random effects models for meta-analysis. We show that interactions can be interpreted as heterogeneity and can be detected without utilizing the traditional uni- or multi-variate approaches for discovery of gene-by-environment interactions. We apply our new method to combine 17 mouse studies containing in aggregate 4,965 distinct animals. We identify 26 significant loci involved in High-density lipoprotein (HDL) cholesterol, many of which are consistent with previous findings. Several of these loci show significant evidence of involvement in gene-by-environment interactions. An additional advantage of our meta-analysis approach is that our combined study has significantly higher power and improved resolution compared to any single study thus explaining the large number of loci discovered in the combined study.
[Show abstract][Hide abstract] ABSTRACT: The paraoxonase (PON) gene family consists of three members, PON1, PON2 and PON3. All PON proteins possess antioxidant properties and lipo-lactonase activities, and are implicated in the pathogenesis of several inflammatory diseases including atherosclerosis, Alzheimer's, Parkinson's, diabetes and cancer. Despite the role of PON proteins in critical cellular functions and associated pathologies, the physiological substrates and molecular mechanisms by which PON proteins function as anti-inflammatory proteins remain largely unknown. PON1 is found exclusively extracellular and associated solely with high-density lipoprotein (HDL) particles in the circulation, and, in part, confers the anti-oxidant and anti-inflammatory properties associated with HDL. Recent studies demonstrated that the intracellular PON proteins; PON2 and PON3 (i) are associated with mitochondria and mitochondria-associated membranes, (ii) modulate mitochondria-dependent superoxide production, and (iii) prevent apoptosis. Overexpression of PON2 and PON3 genes protected (i) mitochondria from antimycin or oligomycin mediated mitochondrial dysfunction and (ii) ER stress and ER stress mediated mitochondrial dysfunction. These studies illustrate that the anti-inflammatory effects of PON2 and PON3 may, in part, be mediated by their role in mitochondrial and associated organelle function. Since oxidative stress as a result of mitochondrial dysfunction is implicated in the development of inflammatory diseases including atherosclerosis and cancer, these recent studies on PON2 and PON3 proteins may provide a mechanism for the scores of epidemiological studies that show a link between PON genes and numerous inflammatory diseases. Understanding such mechanisms will provide novel routes of intervention in the treatment of diseases associated with pro-inflammatory oxidative stress.
Advances in Experimental Medicine and Biology 01/2014; 824:33-41. DOI:10.1007/978-3-319-07320-0_5 · 2.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Myeloperoxidase (MPO) and paraoxonase 1 (PON1) are high-density lipoprotein-associated (HDL-associated) proteins mechanistically linked to inflammation, oxidant stress, and atherosclerosis. MPO is a source of ROS during inflammation and can oxidize apolipoprotein A1 (APOA1) of HDL, impairing its atheroprotective functions. In contrast, PON1 fosters systemic antioxidant effects and promotes some of the atheroprotective properties attributed to HDL. Here, we demonstrate that MPO, PON1, and HDL bind to one another, forming a ternary complex, wherein PON1 partially inhibits MPO activity, while MPO inactivates PON1. MPO oxidizes PON1 on tyrosine 71 (Tyr71), a modified residue found in human atheroma that is critical for HDL binding and PON1 function. Acute inflammation model studies with transgenic and knockout mice for either PON1 or MPO confirmed that MPO and PON1 reciprocally modulate each other's function in vivo. Further structure and function studies identified critical contact sites between APOA1 within HDL, PON1, and MPO, and proteomics studies of HDL recovered from acute coronary syndrome (ACS) subjects revealed enhanced chlorotyrosine content, site-specific PON1 methionine oxidation, and reduced PON1 activity. HDL thus serves as a scaffold upon which MPO and PON1 interact during inflammation, whereupon PON1 binding partially inhibits MPO activity, and MPO promotes site-specific oxidative modification and impairment of PON1 and APOA1 function.
The Journal of clinical investigation 08/2013; 123(9). DOI:10.1172/JCI67478 · 13.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We examined the effects of a natural secondary bile acid, hyodeoxycholic acid (HDCA), on lipid metabolism and atherosclerosis in LDL receptor-null (LDLRKO) mice. Female LDLRKO mice were maintained on a Western diet for 8 wk and then divided into 2 groups that received chow, or chow + 1.25% HDCA, diets for 15 wk. We observed that mice fed the HDCA diet were leaner and exhibited a 37% (P<0.05) decrease in fasting plasma glucose level. HDCA supplementation significantly decreased atherosclerotic lesion size at the aortic root region, the entire aorta, and the innominate artery by 44% (P<0.0001), 48% (P<0.01), and 94% (P<0.01), respectively, as compared with the chow group. Plasma VLDL/IDL/LDL cholesterol levels were significantly decreased, by 61% (P<0.05), in the HDCA group as compared with the chow diet group. HDCA supplementation decreased intestinal cholesterol absorption by 76% (P<0.0001) as compared with the chow group. Furthermore, HDL isolated from the HDCA group exhibited significantly increased ability to mediate cholesterol efflux ex vivo as compared with HDL of the chow diet group. In addition, HDCA significantly increased the expression of genes involved in cholesterol efflux, such as Abca1, Abcg1, and Apoe, in a macrophage cell line. Thus, HDCA is a candidate for antiatherosclerotic drug therapy.-Shih, D. M., Shaposhnik, Z., Meng, Y., Rosales, M., Wang, X., Wu, J., Ratiner, B., Zadini, F., Zadini, G., Lusis, A. J. Hyodeoxycholic acid improves HDL function and inhibits atherosclerotic lesion formation in LDLR-knockout mice.
The FASEB Journal 06/2013; 27(9). DOI:10.1096/fj.12-223008 · 5.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We studied the influence of PON1 on metabolic alterations induced by oxidized LDL when incubated with endothelial cells. HUVEC cells were incubated with native LDL, oxidized LDL, oxidized LDL plus HDL from wild type mice, and oxidized LDL plus HDL from PON1-deficient mice. Results showed alterations in carbohydrate and phospholipid metabolism and increased apoptosis in cells incubated with oxidized LDL. These changes were partially prevented by wild type mouse HDL, but the effects were less effective with HDL from PON1-deficient mice. Our results suggest that PON1 may play a significant role in endothelial cell survival by protecting cells from alterations in the respiratory chain induced by oxidized LDL. These results extend current knowledge on the protective role of HDL and PON1 against oxidation and apoptosis in endothelial cells.
Mediators of Inflammation 05/2013; 2013(3):156053. DOI:10.1155/2013/156053 · 3.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the course of studying the effects of ABCC6 deficiency in mice, we observed an enrichment of mitochondrial gene expression signatures. In subsequent studies we found that ABCC6 null mice exhibited abnormal mitochondrial morphology and functional mitochondrial deficiencies.(1) We then carried out subcellular fractionation studies indicating that ABCC6 co-localized with markers of the mitochondria-associated membranes (MAM) in mouse liver and kidney.(1) Our results differed from Le Saux et al (2011), from the Varadi group who had concluded in a recent publication that ABCC6 resided in the plasma membrane.(2) To test whether ABCC6 was localized in plasma membrane, we performed cell surface protein biotin labeling experiments, which were negative for ABCC6.(1)Pomozi et al(3) of the Varadi group have now challenged our conclusions based, as in their previous report,(2) on immunofluorescence imaging of frozen liver sections and cells in culture showing peripheral cellular localization of antibody binding. They argue that cell disruption and subcellular fractionation in our study1 may have resulted in artifactual associations of membrane proteins. However, this seems improbable given that the plasma membrane markers that we examined did not fractionate with ABCC6, and MAM constitutes a very small fraction of the total membranes.(1) [Extract].
Circulation Research 04/2013; 112(11). DOI:10.1161/CIRCRESAHA.113.301666 · 11.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Oxidative stress is a determinant of liver steatosis and the progression to more severe forms of disease. The present study investigated the effect of paraoxonase-1 (PON1) deficiency on histological alterations and hepatic metabolism in mice fed a high-fat high-cholesterol diet. We performed non-targeted metabolomics on liver tissues from 8 male PON1-deficient mice and 8 wild-type animals fed a high-fat, high-cholesterol diet for 22 weeks. We also measured 8-oxo-20-deoxyguanosine, reduced and oxidized glutathione, malondialdehyde, 8-isoprostanes and protein carbonyl concentrations. Results indicated lipid droplets in 14.5% of the hepatocytes of wild-type mice and in 83.3% of the PON1-deficient animals (P < 0.001). The metabolomic assay included 322 biochemical compounds, 169 of which were significantly decreased and 16 increased in PON1-deficient mice. There were significant increases in lipid peroxide concentrations and oxidative stress markers. We also found decreased glycolysis and the Krebs cycle. The urea cycle was decreased, and the pyrimidine cycle had a significant increase in orotate. The pathways of triglyceride and phospholipid synthesis were significantly increased. We conclude that PON1 deficiency is associated with oxidative stress and metabolic alterations leading to steatosis in the livers of mice receiving a high-fat high-cholesterol diet.
Journal of Proteome Research 02/2013; 12(4). DOI:10.1021/pr400050u · 5.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Circulating trimethylamine-N-oxide (TMAO) levels are strongly associated with atherosclerosis. We now examine genetic, dietary, and hormonal factors regulating TMAO levels. We demonstrate that two flavin mono-oxygenase family members, FMO1 and FMO3, oxidize trimethylamine (TMA), derived from gut flora metabolism of choline, to TMAO. Further, we show that FMO3 exhibits 10-fold higher specific activity than FMO1. FMO3 overexpression in mice significantly increases plasma TMAO levels while silencing FMO3 decreases TMAO levels. In both humans and mice, hepatic FMO3 expression is reduced in males compared to females. In mice, this reduction in FMO3 expression is due primarily to downregulation by androgens. FMO3 expression is induced by dietary bile acids by a mechanism that involves the farnesoid X receptor (FXR), a bile acid-activated nuclear receptor. Analysis of natural genetic variation among inbred strains of mice indicates that FMO3 and TMAO are significantly correlated, and TMAO levels explain 11% of the variation in atherosclerosis.
[Show abstract][Hide abstract] ABSTRACT: Hyperlipidemia blunts anabolic effects of intermittent parathyroid hormone (PTH) on cortical bone, and the responsiveness to PTH are restored in part by oral administration of the antioxidant ApoA-I mimetic peptide, D-4F. To evaluate the mechanism of this rescue, mice overexpressing the high-density lipoprotein-associated antioxidant enzyme, paraoxonase 1 (Ldlr(-/-)PON1(tg)) were generated, and daily PTH injections were administered to Ldlr(-/-)PON1(tg) and to littermate Ldlr(-/-) mice. Expression of bone regulatory genes was determined by realtime RT-qPCR, and cortical bone parameters of the femoral bones by micro-computed tomographic analyses. PTH-treated Ldlr(-/-)PON1(tg) mice had significantly greater expression of PTH receptor (PTH1R), activating transcription factor (ATF4), and osteoprotegerin (OPG) in femoral cortical bone, as well as significantly greater bone mineral content, thickness, and area in femoral diaphyses compared with untreated Ldlr(-/-)PON1(tg) mice. In contrast, in control mice (Ldlr(-/-)) without PON1 overexpression, PTH treatment did not induce these markers. Calvarial bone of PTH-treated Ldlr(-/-)PON1(tg) mice also had significantly greater expression of osteoblastic differentiation marker genes as well as BMP-2-target and Wnt-target genes. Untreated Ldlr(-/-)PON1(tg) mice had significantly greater expression of PTHR1 than untreated Ldlr(-/-) mice, whereas sclerostin expression was reduced. In femoral cortical bones expression levels of transcription factors, FoxO1 and ATF4, were also elevated in Ldlr(-/-)PON1(tg) mice, suggesting enhancement of cellular protection against oxidants. These findings suggest that PON1 restores responsiveness to PTH through effects on oxidant stress, PTH receptor expression, and/or Wnt signaling.
Biochemical and Biophysical Research Communications 01/2013; 431(1). DOI:10.1016/j.bbrc.2012.12.114 · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mutations of the orphan transporter ABCC6 (ATP-binding cassette, subfamily C, member 6) cause the connective tissue disorder pseudoxanthoma elasticum. ABCC6 was thought to be located on the plasma membrane of liver and kidney cells.
Mouse systems genetics and bioinformatics suggested that ABCC6 deficiency affects mitochondrial gene expression. We therefore tested whether ABCC6 associates with mitochondria.
We found ABCC6 in crude mitochondrial fractions and subsequently pinpointed its localization to the purified mitochondria-associated membrane fraction. Cell-surface biotinylation in hepatocytes confirmed that ABCC6 is intracellular. Abcc6-knockout mice demonstrated mitochondrial abnormalities and decreased respiration reserve capacity.
Our finding that ABCC6 localizes to the mitochondria-associated membrane has implications for its mechanism of action in normal and diseased states.
Circulation Research 07/2012; 111(5):516-20. DOI:10.1161/CIRCRESAHA.112.276667 · 11.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To achieve malignancy, cancer cells convert numerous signaling pathways, with evasion from cell death being a characteristic hallmark. The cell death machinery represents an anti-cancer target demanding constant identification of tumor-specific signaling molecules. Control of mitochondrial radical formation, particularly superoxide interconnects cell death signals with appropriate mechanistic execution. Superoxide is potentially damaging, but also triggers mitochondrial cytochrome c release. While paraoxonase (PON) enzymes are known to protect against cardiovascular diseases, recent data revealed that PON2 attenuated mitochondrial radical formation and execution of cell death. Another family member, PON3, is poorly investigated. Using various cell culture systems and knockout mice, here we addressed its potential role in cancer. PON3 is found overexpressed in various human tumors and diminishes mitochondrial superoxide formation. It directly interacts with coenzyme Q10 and presumably acts by sequestering ubisemiquinone, leading to enhanced cell death resistance. Localized to the endoplasmic reticulum (ER) and mitochondria, PON3 abrogates apoptosis in response to DNA damage or intrinsic but not extrinsic stimulation. Moreover, PON3 impaired ER stress-induced apoptotic MAPK signaling and CHOP induction. Therefore, our study reveals the mechanism underlying PON3's anti-oxidative effect and demonstrates a previously unanticipated function in tumor cell development. We suggest PONs represent a novel class of enzymes crucially controlling mitochondrial radical generation and cell death.
Cell death and differentiation 03/2012; 19(9):1549-60. DOI:10.1038/cdd.2012.35 · 8.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Homocysteine (Hcy)-thiolactone is toxic, induces epileptic seizures in rodents, and has been implicated in Alzheimer's disease. Paraoxonase 1 (Pon1), a component of high-density lipoprotein, hydrolyzes Hcy-thiolactone in vitro. Whether this reflects a physiological function and whether Pon1 can protect against Hcy-thiolactone toxicity was unknown. Here we show that Hcy-thiolactone was elevated in brains of Pon1-/- mice (1.5-fold, p = 0.047) and that Pon1-/- mice excrete more Hcy-thiolactone than wild type animals (2.4-fold, p = 0.047). The frequency of seizures induced by intraperitoneal injections of L-Hcy-thiolactone was significantly higher in Pon1-/- mice compared with wild type animals (52.8% versus 29.5%, p = 0.042); the latency of seizures was lower in Pon1-/- mice than in wild type animals (31.8 min versus 41.2 min, p = 0.019). Using the Pon1 null mice, we provide the first direct evidence that a specific Hcy metabolite, Hcy-thiolactone, rather than Hcy itself is neurotoxic in vivo. Our findings indicate that Pon1 protects mice against Hcy-thiolactone neurotoxicity by hydrolyzing it in the brain, and suggest a mechanism by which Pon1 can protect against neurodegeneration associated with hyperhomocysteinemia and Alzheimer's disease.
[Show abstract][Hide abstract] ABSTRACT: Chronic infection has long been postulated as a stimulus for atherogenesis. Pseudomonas aeruginosa infection has been associated with increased atherosclerosis in rats, and these bacteria produce a quorum-sensing molecule 3-oxo-dodecynoyl-homoserine lactone (3OC12-HSL) that is critical for colonization and virulence. Paraoxonase 2 (PON2) hydrolyzes 3OC12-HSL and also protects against the effects of oxidized phospholipids thought to contribute to atherosclerosis. We now report the response of human aortic endothelial cells (HAECs) to 3OC12-HSL and oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) in relation to PON2 expression.
Using expression profiling and network modeling, we identified the unfolded protein response (UPR), cell cycle genes, and the mitogen-activated protein kinase signaling pathway to be heavily involved in the HAEC response to 3OC12-HSL. The network also showed striking similarities to a network created based on HAEC response to Ox-PAPC, a major component of minimally modified low-density lipoprotein. HAECs in which PON2 was silenced by small interfering RNA showed increased proinflammatory response and UPR when treated with 3OC12-HSL or Ox-PAPC.
3OC12-HSL and Ox-PAPC influence similar inflammatory and UPR pathways. Quorum sensing molecules, such as 3OC12-HSL, contribute to the proatherogenic effects of chronic infection. The antiatherogenic effects of PON2 include destruction of quorum sensing molecules.
[Show abstract][Hide abstract] ABSTRACT: Therapies that raise levels of HDL, which is thought to exert atheroprotective effects via effects on endothelium, are being examined for the treatment or prevention of coronary artery disease (CAD). However, the endothelial effects of HDL are highly heterogeneous, and the impact of HDL of patients with CAD on the activation of endothelial eNOS and eNOS-dependent pathways is unknown. Here we have demonstrated that, in contrast to HDL from healthy subjects, HDL from patients with stable CAD or an acute coronary syndrome (HDLCAD) does not have endothelial antiinflammatory effects and does not stimulate endothelial repair because it fails to induce endothelial NO production. Mechanistically, this was because HDLCAD activated endothelial lectin-like oxidized LDL receptor 1 (LOX-1), triggering endothelial PKCβII activation, which in turn inhibited eNOS-activating pathways and eNOS-dependent NO production. We then identified reduced HDL-associated paraoxonase 1 (PON1) activity as one molecular mechanism leading to the generation of HDL with endothelial PKCβII-activating properties, at least in part due to increased formation of malondialdehyde in HDL. Taken together, our data indicate that in patients with CAD, HDL gains endothelial LOX-1- and thereby PKCβII-activating properties due to reduced HDL-associated PON1 activity, and that this leads to inhibition of eNOS-activation and the subsequent loss of the endothelial antiinflammatory and endothelial repair-stimulating effects of HDL.
The Journal of clinical investigation 06/2011; 121(7):2693-708. DOI:10.1172/JCI42946 · 13.77 Impact Factor