Trimethylamine-N-oxide, a metabolite associated with atherosclerosis, exhibits complex genetic and dietary regulation

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.

Figure 1. FMO3 is the Major FMO Family Member Responsible for the Conversion of TMA to TMAO

(A–D) Overexpression of untagged or FLAG-tagged human FMO1-5 in HEK292Ad cells. (A) Western blotting analysis of FLAG-tagged FMO1-5 transfected into HEK293Ad cells. GFP was co-transfected to normalize transfection efficiency. (B) FMO3 mRNA levels were determined by RT-qPCR and normalized to GFP mRNA levels. (C and D) TMAO and TMA levels determined in the media of transfected cells treated with d9-TMA and then analyzed for d9-TMA and d9-TMAO levels using mass spectrometry (see Experimental Procedures). TMAO production was determined from triplicate wells for each condition and normalized to the amount of protein per well. (E) Relative specific activity of FMO1-5 determined by dividing normalized TMAO levels by relative FMO protein levels calculated by densitometry analysis and expressed relative to FLAG-FMO3 activity. (F) Relative Fmo3 mRNA levels in various mouse tissues from C57BL/6 mice (n=3 mice/sex). Expression was determined by RT-qPCR and was normalized to 36B4 expression levels. (G) Relative abundance of FMO1-3 determined by RNA-Seq analysis from male and female C57BL/6 mice. (H) Relative FMO3 expression in human liver determined by microarray expression profile from 317 Caucasian human individuals segregated by sex. (I) RT-qPCR analysis of hepatic mRNA levels from human liver biopsies obtained as described in Experimental Procedures. Data are presented as mean ± SEM. Significance was measured with Student’s t-test. * indicates p < 0.05 and *** indicates p < 0.001.

Figure 2. Modulation of Hepatic FMO3 levels in Mice Regulates Plasma TMAO Levels

(A–C) FMO3 overexpression in the livers of male C57BL/6 mice infected with either Ad-control or Ad-FMO3 for 7 days (n=8 mice/group). (D–F) FMO3 knockdown in livers of female C57BL/6 mice treated with either vehicle (saline) or FMO3 antisense oligonucleotide (ASO) once weekly (50mg/kg body weight) for 7 weeks (n=4 mice/group). (A, D) Hepatic FMO3 mRNA levels determined by RT-qPCR and normalized to 36B4 or cyclophilin levels. (B, E) Hepatic FMO3 protein levels determined by Western blotting analysis normalized to β-Actin levels. (C, E) Plasma TMAO levels determined from plasma isolated from adenovirus-treated or ASO- or vehicle-treated mice. TMAO levels were determined by LC/MS/MS. Data are presented as mean ± SEM. Significance was measured with Student’s t-test. * indicates p < 0.05, ** indicates p < 0.01 and *** indicates p < 0.001.

Figure 3. Sex and Dietary Choline Regulate Hepatic FMO3 Expression and Plasma TMAO Levels in Mice

(A–D) Male and female ApoE^−/− mice were fed either a control (chow) or choline-rich diet (1% choline) (n=7–10 mice/group). (A) Hepatic Fmo3 mRNA levels determined by RT-qPCR and normalized to 36B4 expression. (B) Plasma TMAO and (C) TMA levels from male and female mice determined by LC/MS/MS. (D) Conversion of TMA into TMAO by liver homogenates (FMO activity) of male and female mice normalized to total hepatic protein levels. Data are presented as mean ± SEM. Significance was measured with One-way ANOVA and different letters (a–d) indicate statistically significant (p<0.05 or greater) differences between groups.

Figure 4. Gonadal Hormones Regulate Hepatic FMO3 Expression and Plasma TMAO Levels

(A–C) Male and female C57BL/6 mice were either untreated or gonadectomized or ovariectomized and then treated with vehicle or DHT (males) or estrogen (females) (n=4–6 mice/group). (A) Hepatic FMO3 expression was determined by RT-qPCR and normalized to 36B4. (B) FMO3 protein levels were determined by Western blot analysis and GAPDH was used as a control. (C) Plasma TMAO levels from male and female mice were determined by LC/MS/MS. Data are presented as mean ± SEM. Significance was measured with One-way ANOVA and different letters (a–e) indicate statistically significant (p<0.05 or greater) differences between groups.

Figure 5. Dietary Cholic Acid Induces Fmo3 Expression and TMAO Levels in Common Inbred Mouse Strains

Female (A, C) and male (B, D) “humanized” ApoB100 transgenic mice were fed a high fat diet with cholic acid (HF+CA) or without cholic acid (HF) (n=2–9 mice/strain/gender). (A, B) Hepatic FMO3 expression was determined by RT-qPCR normalized to 36B4 expression. (C, D) Plasma TMAO levels from male and female mice of different strains were determined by LC/MS/MS. Data are presented as mean ± SEM. Significance was measured with Student’s t-test. *indicates p < 0.05, ** indicates p < 0.01 and *** indicates p < 0.001.

Figure 6. FMO3 is an FXR Target Gene and FXR Activation Induces FMO3 and Increases Circulating TMAO Levels in vivo

(A) Hepatic Bsep and (B) Fmo3 mRNA levels normalized to 36B4, were determined in male and female wild-type and Fxr^−/− mice treated with GSK2324 at 30 mpk/day for 3 days (n=7–10 mice/group). (C) Plasma TMAO levels from the same mice were determined by LC/MS/MS. (D) Luciferase reporter plasmids under the control of either 2.5kb of the wild type mouse FMO3 promoter or the promoter containing a mutant FXRE, were transfected into Hep3B cells (6 wells per condition) in the presence of increasing amounts of pcDNA FXRα2 and treated with vehicle (water) or GSK2324 (1µM). Promoter activity was determined by luciferase assay and normalized to β-galactosidase (co-transfected to account for transfection efficiency). Data are presented as mean ± SEM. Significance was measured with Student’s t-test. * indicates p < 0.05, ** indicates p < 0.01 and *** indicates p < 0.001.

Figure 7. Natural Genetic Variation of Hepatic FMO3, Plasma TMAO Levels and Atherosclerosis among Common Inbred Strains of Mice

(A) Identification of a cis-acting expression Quantitative Trait Locus (eQTL) for FMO3 among the 100 HMDP strains of mice. Approximately 135,000 SNPs varying across the HMDP panel were examined for association with FMO3 transcript levels following correction for population structure (see Experimental Procedures). The location of each SNP across the 19 autosomes and the X chromosome is indicated on the X-axis and the strength of association is shown on the Y-axis. Signals shown in red indicate genome-wide significant results. (B) Relationship between hepatic FMO3 transcript levels and circulating TMAO levels in 22 strains (males and females) carrying the human ApoB transgene fed a Western diet. (C) TMAO levels in female mice of the 22 hyperlipidemic strains on a Western diet. (D) Relationship between atherosclerotic lesions and TMAO levels in female mice of the 22 hyperlipidemic strains (points represent single animals). Atherosclerotic lesions were quantitated following the sectioning of the aortic sinus and proximal aorta. (E) A high-resolution image of panel A showing that the peak SNPs reside over the location of the FMO3 gene on Chromosome 1. (F) Three copies of the DNA region containing the A or G SNP adjacent to the intergenic FXRE were cloned into a minimal TK promoter luciferase reporter plasmid. SNP reporter plasmids were transfected into Hep3B cells (6 wells/condition) with increasing amounts of pcDNA FXRα2 and treated with vehicle (water) or GSK2324 (1µM). Promoter activity was determined by luciferase assay and normalized to β-galactosidase (co-transfected to account for transfection efficiency). Data are presented as mean ± SEM.