Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease

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.

©2011 Macmillan Publishers Limited. All rights reserved

Figures

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. b, Odds ratio (OR) and 95% confidential intervals (CI) of prospective (3 year) risk for myocardial infarction (MI), stroke (CVA) or death of the 18 plasma analytes that met all selection criteria in the Learning and Validation Cohorts, OR and CI shown are for the highest vs. 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 of an analyte monitored in positive MS1 mode.

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 intraperitoneal, i.p.) of formation observed in choline challenge studies in mice using the indicated isotope labeled choline. The m/z in plasma observed for the isotopmers of the choline metabolites are shown. b, Plasma levels of d9-metabolites following i.p. challenge with d9(trimethyl)-dipalmitoylphosphatidylcholine (d9-DPPC). c, d9-TMAO production following oral d9-DPPC in mice, following suppression of gut flora with antibiotics (3 weeks), and then following placement (4 weeks) into conventional cages with non-sterile mice (i.e. – “conventionalized”). Data are presented as mean ± SE from 4 independent replicates.

Figure 3. Choline, TMAO and betaine are associated with atherosclerosis risks in humans and promote atherosclerosis in mice

a–c, Spline models of the logistic regression analyses reflecting risk of cardiovascular disease (CVD) (with 95% CI) according to plasma levels of choline, TMAO and betaine in the entire cohort (n = 1876 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=1020). 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.

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

a–c, 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 in macrophages harvested from C57BL/6J mice (13 week) following three weeks of standard chow verses chow supplemented with the indicated amounts (wt/wt) of choline, TMAO or betaine. Data are presented as mean ± SE from the indicated numbers of mice in each group.

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

a, Choline supplementation promotes macrophage foam cell formation in gut flora dependent fashion. C57BL/6J.Apoe−/− mice at time of weaning (4 weeks) were provided drinking water with versus 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/hematoxylin 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/hematoxylin stained aortic root sections from female C57BL/6J.Apoe−/− mice fed control and high choline diets in the presence versus absence of Abx. e,f, Aortic lesion area in 20 week old C57BL/6J.Apoe−/− mice off versus on Abx and fed with control versus choline diet. g, Aortic macrophage quantification with anti-F4/80 antibody staining. h, Quantitation of scavenger receptor CD36 in aorta within the indicated groups. Error bars represent s.e.m. from the indicated numbers of mice.

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.