Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease

Journal name:
Nature
Volume:
472,
Pages:
57–63
Date published:
DOI:
doi:10.1038/nature09922
Received
Accepted
Published online

Abstract

Metabolomics studies hold promise for the discovery of pathways linked to disease processes. Cardiovascular disease (CVD) represents the leading cause of death and morbidity worldwide. Here we used a metabolomics approach to generate unbiased small-molecule metabolic profiles in plasma that predict risk for CVD. Three metabolites of the dietary lipid phosphatidylcholine—choline, trimethylamine N-oxide (TMAO) and betaine—were identified and then shown to predict risk for CVD in an independent large clinical cohort. Dietary supplementation of mice with choline, TMAO or betaine promoted upregulation of multiple macrophage scavenger receptors linked to atherosclerosis, and supplementation with choline or TMAO promoted atherosclerosis. Studies using germ-free mice confirmed a critical role for dietary choline and gut flora in TMAO production, augmented macrophage cholesterol accumulation and foam cell formation. Suppression of intestinal microflora in atherosclerosis-prone mice inhibited dietary-choline-enhanced atherosclerosis. Genetic variations controlling expression of flavin monooxygenases, an enzymatic source of TMAO, segregated with atherosclerosis in hyperlipidaemic mice. Discovery of a relationship between gut-flora-dependent metabolism of dietary phosphatidylcholine and CVD pathogenesis provides opportunities for the development of new diagnostic tests and therapeutic approaches for atherosclerotic heart disease.

At a glance

Figures

  1. Strategy for metabolomics studies to identify plasma analytes associated with cardiovascular risk.
    Figure 1: Strategy for metabolomics studies to identify plasma analytes associated with cardiovascular risk.

    a, Overall schematic to identify plasma analytes associated with cardiac risk over the ensuing 3-year period. CVA, cerebrovascular accident; HPLC, high-performance liquid chromatography; MI, myocardial infarction. b, Odds ratio (OR) and 95% confidence intervals (CI) of incident (3-year) risk for MI, CVA or death of the 18 plasma analytes that met all selection criteria in both Learning and Validation Cohorts; odds ratio and 95% confidence intervals shown are for the highest versus lowest quartile for each analyte. Filled circles represent the analytes (m/z = 76, 104, 118) focused on in this study. m/z, mass to charge ratio.

  2. Identification of metabolites of dietary PC and an obligatory role for gut flora in generation of plasma analytes associated with CVD risks.
    Figure 2: Identification of metabolites of dietary PC and an obligatory role for gut flora in generation of plasma analytes associated with CVD risks.

    a, Summary schematic indicating structure of metabolites and routes (oral or i.p.) of formation observed in choline challenge studies in mice using the indicated isotope-labelled choline. The m/z in plasma observed for the isotopomers of the choline metabolites are shown. b, Plasma levels of d9 metabolites after i.p. challenge with d9(trimethyl)-dipalmitoylphosphatidylcholine (d9-DPPC). c, d9-TMAO production after oral d9-DPPC administration in mice, following suppression of gut flora with antibiotics (3 weeks), and then following placement (4 weeks) into conventional cages with non-sterile mice (‘conventionalized’). Data are presented as mean±standard error (s.e.) from four independent replicates.

  3. Plasma levels of choline, TMAO and betaine are associated with atherosclerosis risks in humans and promote atherosclerosis in mice.
    Figure 3: Plasma levels of choline, TMAO and betaine are associated with atherosclerosis risks in humans and promote atherosclerosis in mice.

    ac, Spline models of the logistic regression analyses reflecting risk of CVD (with 95% CI) according to plasma levels of choline, TMAO and betaine in the entire cohort (n = 1,876 subjects). d, Comparison in aortic lesion area among 20-week-old female C57BL/6J Apoe−/− mice fed with chow diet supplemented with the indicated amounts (wt/wt) of choline or TMAO from time of weaning (4 weeks). e, Relationship between plasma TMAO levels and aortic lesion area. f, Relationship between fasting plasma levels of TMAO versus CAD burden among subjects (N = 1,020). Boxes represent 25th, 50th and 75th percentile, and whiskers 5th and 95th percentile plasma levels. Single, double and triple coronary vessel disease refers to number of major coronary vessels demonstrating≥50% stenosis on diagnostic coronary angiography.

  4. Hepatic Fmo genes are linked to atherosclerosis and dietary PC metabolites enhance macrophage scavenger receptor expression.
    Figure 4: Hepatic Fmo genes are linked to atherosclerosis and dietary PC metabolites enhance macrophage scavenger receptor expression.

    ac, Correlation between hepatic Fmo3 expression and aortic lesion, plasma HDL cholesterol and TMAO in female mice from the F2 intercross between atherosclerosis-prone C57BL/6J Apoe−/− and atherosclerosis-resistant C3H/HeJ Apoe−/− mice. d, Correlation between human hepatic FMO3 expression and plasma TMAO. e, Effect of Fmo3 genotype (SNP rs3689151) on aortic sinus atherosclerosis in male mice from the C57BL6/J Apoe−/− and C3H/HeJ Apoe−/− F2 intercross. f, g, Quantification of scavenger receptor CD36 and SR-A1 surface protein levels in macrophages harvested from C57BL/6J mice (13 week) after three weeks of standard chow versus chow supplemented with the indicated amounts (wt/wt) of choline, TMAO or betaine. Data are presented as mean±s.e. from the indicated numbers of mice in each group.

  5. Obligatory role of gut flora in dietary choline enhanced atherosclerosis.
    Figure 5: Obligatory role of gut flora in dietary choline enhanced atherosclerosis.

    a, Choline supplementation promotes macrophage foam cell formation in a gut-flora-dependent fashion. C57BL/6J Apoe−/− mice at time of weaning (4 weeks) were provided drinking water with or without broad-spectrum antibiotics (Abx), and placed on chemically defined diets similar in composition to normal chow (control diet, 0.08±0.01% total choline, wt/wt) or normal chow with high choline (choline diet, 1.00%±0.01% total choline, wt/wt). Resident peritoneal macrophages were recovered at 20 weeks of age. Typical images of Oil-red-O/haematoxylin-stained macrophages in each diet group are shown. b, Foam cell quantification from peritoneal macrophages recovered from mice in studies described in panel a. c, Macrophage cellular cholesterol content. d, Representative Oil-red-O/haematoxylin-stained aortic root sections from female C57BL/6J Apoe−/− mice fed control and high-choline diets in the presence or absence of antibiotics. e, f, Aortic lesion area in 20 week old C57BL/6J Apoe−/− mice off or on antibiotics and fed with control or choline diet. g, Aortic macrophage quantification with anti-F4/80 antibody staining. h, Quantification of the scavenger receptor CD36 in aorta within the indicated groups. Error bars represent s.e.m. from the indicated numbers of mice.

  6. Gut-flora-dependent metabolism of dietary PC and atherosclerosis.
    Figure 6: Gut-flora-dependent metabolism of dietary PC and atherosclerosis.

    Schematic summary illustrating newly discovered pathway for gut-flora-mediated generation of pro-atherosclerotic metabolite from dietary PC.

References

  1. Epstein, S. E. et al. The role of infection in restenosis and atherosclerosis: focus on cytomegalovirus. Lancet 348 (suppl. 1). S13S17 (1996)
  2. Patel, P. et al. Association of Helicobacter pylori and Chlamydia pneumoniae infections with coronary heart disease and cardiovascular risk factors. Br. Med. J. 311, 711714 (1995)
  3. Danesh, J., Collins, R. & Peto, R. Chronic infections and coronary heart disease: is there a link? Lancet 350, 430436 (1997)
  4. Saikku, P. et al. Serological evidence of an association of a novel Chlamydia, TWAR, with chronic coronary heart disease and acute myocardial infarction. Lancet 332, 983986 (1988)
  5. O’Connor, C. M. et al. Azithromycin for the secondary prevention of coronary heart disease events—the WIZARD study: a randomized controlled trial. J. Am. Med. Assoc. 290, 14591466 (2003)
  6. Cannon, C. P. et al. Antibiotic treatment of Chlamydia pneumoniae after acute coronary syndrome. N. Engl. J. Med. 352, 16461654 (2005)
  7. Andraws, R., Berger, J. S. & Brown, D. L. Effects of antibiotic therapy on outcomes of patients with coronary artery disease: a meta-analysis of randomized controlled trials. J. Am. Med. Assoc. 293, 26412647 (2005)
  8. Wright, S. D. et al. Infectious agents are not necessary for murine atherogenesis. J. Exp. Med. 191, 14371442 (2000)
  9. Backhed, F., Ley, R. E., Sonnenburg, J. L., Peterson, D. A. & Gordon, J. I. Host-bacterial mutualism in the human intestine. Science 307, 19151920 (2005)
  10. Turnbaugh, P. J. et al. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444, 10271031 (2006)
  11. Dumas, M. E. et al. Metabolic profiling reveals a contribution of gut microbiota to fatty liver phenotype in insulin-resistant mice. Proc. Natl Acad. Sci. USA 103, 1251112516 (2006)
  12. Cashman, J. R. et al. Biochemical and clinical aspects of the human flavin-containing monooxygenase form 3 (FMO3) related to trimethylaminuria. Curr. Drug Metab. 4, 151170 (2003)
  13. Al-Waiz, M., Mikov, M., Mitchell, S. C. & Smith, R. L. The exogenous origin of trimethylamine in the mouse. Metabolism 41, 135136 (1992)
  14. Zeisel, S. H., Mar, M. H., Howe, J. C. & Holden, J. M. Concentrations of choline-containing compounds and betaine in common foods. J. Nutr. 133, 13021307 (2003)
  15. Lang, D. H. et al. Isoform specificity of trimethylamine N-oxygenation by human flavin-containing monooxygenase (FMO) and P450 enzymes: selective catalysis by fmo3. Biochem. Pharmacol. 56, 10051012 (1998)
  16. Zhang, A. Q., Mitchell, S. C. & Smith, R. L. Dietary precursors of trimethylamine in man: a pilot study. Food Chem. Toxicol. 37, 515520 (1999)
  17. Mitchell, S. C. & Smith, R. L. Trimethylaminuria: the fish malodor syndrome. Drug Metab. Dispos. 29, 517521 (2001)
  18. Schadt, E. E. et al. An integrative genomics approach to infer causal associations between gene expression and disease. Nature Genet. 37, 710717 (2005)
  19. Dolphin, C. T., Janmohamed, A., Smith, R. L., Shephard, E. A. & Phillips, I. R. Missense mutation in flavin-containing mono-oxygenase 3 gene, FMO3, underlies fish-odour syndrome. Nature Genet. 17, 491494 (1997)
  20. Wang, S. S. et al. Identification of pathways for atherosclerosis in mice: integration of quantitative trait locus analysis and global gene expression data. Circ. Res. 101, e11e30 (2007)
  21. Treberg, J. R., Wilson, C. E., Richards, R. C., Ewart, K. V. & Driedzic, W. R. The freeze-avoidance response of smelt Osmerus mordax: initiation and subsequent suppression of glycerol, trimethylamine oxide and urea accumulation. J. Exp. Biol. 205, 14191427 (2002)
  22. Devlin, G. L., Parfrey, H., Tew, D. J., Lomas, D. A. & Bottomley, S. P. Prevention of polymerization of M and Z α1-Antitrypsin (α1-AT) with trimethylamine N-oxide. Implications for the treatment of α1-AT deficiency. Am. J. Respir. Cell Mol. Biol. 24, 727732 (2001)
  23. Song, J. L. & Chuang, D. T. Natural osmolyte trimethylamine N-oxide corrects assembly defects of mutant branched-chain α-ketoacid decarboxylase in maple syrup urine disease. J. Biol. Chem. 276, 4024140246 (2001)
  24. Bain, M. A., Faull, R., Fornasini, G., Milne, R. W. & Evans, A. M. Accumulation of trimethylamine and trimethylamine-N-oxide in end-stage renal disease patients undergoing haemodialysis. Nephrol. Dial. Transplant. 21, 13001304 (2006)
  25. Dong, C., Yoon, W. & Goldschmidt-Clermont, P. J. DNA methylation and atherosclerosis. J. Nutr. 132, 2406S2409S (2002)
  26. Zaina, S., Lindholm, M. W. & Lund, G. Nutrition and aberrant DNA methylation patterns in atherosclerosis: more than just hyperhomocysteinemia? J. Nutr. 135, 58 (2005)
  27. Salmon, W. D. & Newberne, P. M. Cardiovascular disease in choline-deficient rats. Effects of choline deficiency, nature and level of dietary lipids and proteins, and duration of feeding on plasma and liver lipid values and cardiovascular lesions. Arch. Pathol. 73, 190209 (1962)
  28. Danne, O., Lueders, C., Storm, C., Frei, U. & Mockel, M. Whole blood choline and plasma choline in acute coronary syndromes: prognostic and pathophysiological implications. Clin. Chim. Acta 383, 103109 (2007)
  29. LeLeiko, R. M. et al. Usefulness of elevations in serum choline and free F2-isoprostane to predict 30-day cardiovascular outcomes in patients with acute coronary syndrome. Am. J. Cardiol. 104, 638643 (2009)
  30. Bidulescu, A., Chambless, L. E., Siega-Riz, A. M., Zeisel, S. H. & Heiss, G. Usual choline and betaine dietary intake and incident coronary heart disease: the Atherosclerosis Risk in Communities (ARIC) study. BMC Cardiovasc. Disord. 7, 20 (2007)
  31. Eckburg, P. B. et al. Diversity of the human intestinal microbial flora. Science 308, 16351638 (2005)
  32. Ley, R. E., Turnbaugh, P. J., Klein, S. & Gordon, J. I. Microbial ecology: human gut microbes associated with obesity. Nature 444, 10221023 (2006)
  33. Li, M. et al. Symbiotic gut microbes modulate human metabolic phenotypes. Proc. Natl Acad. Sci. USA 105, 21172122 (2008)
  34. Reigstad, C. S., Lunden, G. O., Felin, J. & Backhed, F. Regulation of serum amyloid A3 (SAA3) in mouse colonic epithelium and adipose tissue by the intestinal microbiota. PLoS ONE 4, e5842 (2009)
  35. Martin, F. P. et al. Probiotic modulation of symbiotic gut microbial–host metabolic interactions in a humanized microbiome mouse model. Mol. Syst. Biol. 4, 157 (2008)
  36. Rizzo, M. L. Statistical Computing with R (Chapman & Hall/CRC, 2008)
  37. Rakoff-Nahoum, S., Paglino, J., Eslami-Varzaneh, F., Edberg, S. & Medzhitov, R. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell 118, 229241 (2004)
  38. Wang, S. et al. Genetic and genomic analysis of a fat mass trait with complex inheritance reveals marked sex specificity. PLoS Genet. 2, e15 (2006)
  39. Baglione, J. & Smith, J. D. Quantitative assay for mouse atherosclerosis in the aortic root. Methods Mol. Med. 129, 8395 (2006)
  40. Folch, J., Lees, M. & Sloane Stanley, G. H. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226, 497509 (1957)
  41. Robinet, P., Wang, Z., Hazen, S. L. & Smith, J. D. A simple and sensitive enzymatic method for cholesterol quantification in macrophages and foam cells. J. Lipid Res. 51, 33643369 (2010)
  42. Millward, C. A. et al. Genetic factors for resistance to diet-induced obesity and associated metabolic traits on mouse chromosome 17. Mamm. Genome 20, 7182 (2009)
  43. Ahn, S. J., Costa, J. & Emanuel, J. R. PicoGreen quantitation of DNA: effective evaluation of samples pre- or post-PCR. Nucleic Acids Res. 24, 26232625 (1996)
  44. Wang, Z. et al. Protein carbamylation links inflammation, smoking, uremia and atherogenesis. Nature Med. 13, 11761184 (2007)
  45. Nicholls, S. J. et al. Lipoprotein (a) levels and long-term cardiovascular risk in the contemporary era of statin therapy. J. Lipid Res. 51, 30553061 (2010)
  46. Stoves, J., Lindley, E. J., Barnfield, M. C., Burniston, M. T. & Newstead, C. G. MDRD equation estimates of glomerular filtration rate in potential living kidney donors and renal transplant recipients with impaired graft function. Nephrol. Dial. Transplant. 17, 20362037 (2002)
  47. Barham, A. H. et al. Appropriateness of cholesterol management in primary care by sex and level of cardiovascular risk. Prev. Cardiol. 12, 95101 (2009)
  48. daCosta, K. A., Vrbanac, J. J. & Zeisel, S. H. The measurement of dimethylamine, trimethylamine, and trimethylamine N-oxide using capillary gas chromatography-mass spectrometry. Anal. Biochem. 187, 234239 (1990)
  49. Schledzewski, K. et al. Lymphatic endothelium-specific hyaluronan receptor LYVE-1 is expressed by stabilin-1+, F4/80+, CD11b+ macrophages in malignant tumours and wound healing tissue in vivo and in bone marrow cultures in vitro: implications for the assessment of lymphangiogenesis. J. Pathol. 209, 6777 (2006)
  50. Cailhier, J. F. et al. Conditional macrophage ablation demonstrates that resident macrophages initiate acute peritoneal inflammation. J. Immunol. 174, 23362342 (2005)
  51. Kunjathoor, V. V. et al. Scavenger receptors class A-I/II and CD36 are the principal receptors responsible for the uptake of modified low density lipoprotein leading to lipid loading in macrophages. J. Biol. Chem. 277, 4998249988 (2002)
  52. Yang, X. et al. Tissue-specific expression and regulation of sexually dimorphic genes in mice. Genome Res. 16, 9951004 (2006)
  53. Zhou, J., Lhotak, S., Hilditch, B. A. & Austin, R. C. Activation of the unfolded protein response occurs at all stages of atherosclerotic lesion development in apolipoprotein E-deficient mice. Circulation 111, 18141821 (2005)
  54. Miles, E. A., Wallace, F. A. & Calder, P. C. Dietary fish oil reduces intercellular adhesion molecule 1 and scavenger receptor expression on murine macrophages. Atherosclerosis 152, 4350 (2000)
  55. Westendorf, T., Graessler, J. & Kopprasch, S. Hypochlorite-oxidized low-density lipoprotein upregulates CD36 and PPARγ mRNA expression and modulates SR-BI gene expression in murine macrophages. Mol. Cell. Biochem. 277, 143152 (2005)
  56. Rasooly, R., Kelley, D. S., Greg, J. & Mackey, B. E. Dietary trans 10, cis 12-conjugated linoleic acid reduces the expression of fatty acid oxidation and drug detoxification enzymes in mouse liver. Br. J. Nutr. 97, 5866 (2007)
  57. Zhang, J. & Cashman, J. R. Quantitative analysis of FMO gene mRNA levels in human tissues. Drug Metab. Dispos. 34, 1926 (2006)
  58. de Vries, T. J., Schoenmaker, T., Hooibrink, B., Leenen, P. J. & Everts, V. Myeloid blasts are the mouse bone marrow cells prone to differentiate into osteoclasts. J. Leukoc. Biol. 85, 919927 (2009)
  59. Chen, F. C. M. & Benoiton, L. N. A new method of quatenizing amines and its use in amino acid and peptide chemistry. Can. J. Chem. 54, 33103311 (1976)
  60. Morano, C., Zhang, X. & Fricker, L. D. Multiple isotopic labels for quantitative mass spectrometry. Anal. Chem. 80, 92989309 (2008)
  61. Greenberg, M. E. et al. The lipid whisker model of the structure of oxidized cell membranes. J. Biol. Chem. 283, 23852396 (2008)
  62. Gauvreau, K. & Pagano, M. Student’s t-test. Nutrition 9, 386 (1993)
  63. Wijnand, H. P. & van de Velde, R. Mann–Whitney/Wilcoxon’s nonparametric cumulative probability distribution. Comput. Methods Programs Biomed. 63, 2128 (2000)
  64. Gaddis, M. L. & Gaddis, G. M. Introduction to biostatistics: part 6, correlation and regression. Ann. Emerg. Med. 19, 14621468 (1990)
  65. Deichmann, M. et al. S100-β, melanoma-inhibiting activity, and lactate dehydrogenase discriminate progressive from nonprogressive American Joint Committee on Cancer stage IV melanoma. J. Clin. Oncol. 17, 18911896 (1999)
  66. Goodall, C. M., Stephens, O. B. & Moore, C. M. Comparative sensitivity of survival-adjusted chi-square and normal statistics for the mutagenesis fluctuation assay. J. Appl. Toxicol. 6, 95100 (1986)
  67. Traissac, P., Martin-Prevel, Y., Delpeuch, F. & Maire, B. Logistic regression vs other generalized linear models to estimate prevalence rate ratios. Rev. Epidemiol. Sante Publique 47, 593604 (1999)
  68. Gautam, S. Test for linear trend in 2 × K ordered tables with open-ended categories. Biometrics 53, 11631169 (1997)

Download references

Author information

Affiliations

  1. Department of Cell Biology, Cleveland Clinic, Cleveland, Ohio 44195, USA

    • Zeneng Wang,
    • Elizabeth Klipfell,
    • Robert Koeth,
    • Bruce S. Levison,
    • Brandon DuGar,
    • Ariel E. Feldstein,
    • Earl B. Britt,
    • Xiaoming Fu,
    • Yoon-Mi Chung,
    • Jonathan D. Smith,
    • W. H. Wilson Tang,
    • Joseph A. DiDonato &
    • Stanley L. Hazen
  2. Center for Cardiovascular Diagnostics and Prevention, Cleveland Clinic, Cleveland, Ohio 44195, USA

    • Zeneng Wang,
    • Elizabeth Klipfell,
    • Bruce S. Levison,
    • Ariel E. Feldstein,
    • Earl B. Britt,
    • Xiaoming Fu,
    • Yoon-Mi Chung,
    • W. H. Wilson Tang,
    • Joseph A. DiDonato &
    • Stanley L. Hazen
  3. Department of Medicine/Division of Cardiology, BH-307 Center for the Health Sciences, University of California, Los Angeles, California 90095, USA

    • Brian J. Bennett &
    • Aldons J. Lusis
  4. Department of Mathematics, Cleveland State University, Cleveland, Ohio 44115, USA

    • Yuping Wu
  5. Bariatric and Metabolic Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA

    • Phil Schauer
  6. Department of Cardiovascular Medicine, Cleveland Clinic, Cleveland, Ohio 44195, USA

    • Jonathan D. Smith,
    • W. H. Wilson Tang &
    • Stanley L. Hazen
  7. Department of Preventive Medicine and Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California 90089, USA

    • Hooman Allayee

Contributions

Z.W. performed metabolomics analyses, and biochemical, cellular, animal model and mass spectrometry studies. He assisted with statistical analyses, and assisted in both drafting and critical review of the manuscript. E.K., B.D. and J.D.S. assisted with performance of animal models and their analyses. B.S.L. synthesized d9-DPPC and assisted in metabolomics/mass spectrometry analyses. B.J.B., H.A. and A.J.L. performed the mouse eQTL experiments and analyses, and assisted in both drafting and critical review of the manuscript. A.J.L. provided some funding for the study. R.K., E.B.B., X.F. and Y.-M.C. performed mass spectrometry analyses of clinical samples. Y.W. performed statistical analysis. A.E.F. and P.S. helped with collection of human liver biopsy material and interpretation of biochemical and pathological examination of animal liver for steatosis. W.H.W.T. assisted in GeneBank study design and enrolment, as well as analyses of clinical studies and critical review of the manuscript. J.A.D. assisted in clinical laboratory testing for human clinical studies, animal model experimental design, and critical review of the manuscript. S.L.H. conceived of the idea, designed experiments, assisted in data analyses, the drafting and critical review of the manuscript, and provided funding for the study.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Information (2M)

    This file contains Supplementary Tables 1-8 and Supplementary Figures 1-24 with legends.

Additional data