γ-Butyrobetaine is a proatherogenic intermediate in gut microbial metabolism of L-carnitine to TMAO

Abstract

L-carnitine, a nutrient in red meat, was recently reported to accelerate atherosclerosis via a metaorganismal pathway involving gut microbial trimethylamine (TMA) formation and host hepatic conversion into trimethylamine-N-oxide (TMAO). Herein, we show that following L-carnitine ingestion, γ-butyrobetaine (γBB) is produced as an intermediary metabolite by gut microbes at a site anatomically proximal to and at a rate ∼1,000-fold higher than the formation of TMA. Moreover, we show that γBB is the major gut microbial metabolite formed from dietary L-carnitine in mice, is converted into TMA and TMAO in a gut microbiota-dependent manner (like dietary L-carnitine), and accelerates atherosclerosis. Gut microbial composition and functional metabolic studies reveal that distinct taxa are associated with the production of γBB or TMA/TMAO from dietary L-carnitine. Moreover, despite their close structural similarity, chronic dietary exposure to L-carnitine or γBB promotes development of functionally distinct microbial communities optimized for the metabolism of L-carnitine or γBB, respectively.

Figure 1. γBB is a quantitatively significant gut microbe generated metabolite of dietary L-carnitine

(A) Hypothesized scheme of carnitine metabolism to TMA/TMAO through the intermediate production of γBB by gut microbes. (B) Stable isotope dilution LC/MS/MS analyses of plasma γBB, L-carnitine, TMA, and TMAO in plasma of C57BL/6J, Apoe−/− female mice on the indicated respective diets between the ages of weaning and 19 weeks of age. Error bars represent ± SE and P values represent Wilcoxon rank sums test. (C) C57BL/6J, Apoe−/− female mice challenged with d3-carnitine. Post challenge measurement of d3-metabolites was performed in serial venous blood draws by stable isotope dilution LC/MS/MS. (D) Female Swiss Webster Germ Free mice (n=4) challenged with d3-L-carnitine before and after conventionalization. Post challenge measurement of d3-L-carnitine and d3-γBB was performed in serial venous blood draws by stable isotope dilution LC/MS/MS. Data is expressed as means ± SE. (see also Figure S1).

Figure 2. Orally ingested γBB generates TMA and TMAO via a gut microbe dependent pathway

(A) C57BL/6J female mice (n=5) challenged with d9-γBB gastric gavage (left panels; upper (d9TMA/TMAO) and lower (d9-L-carnitine and d9-γBB)) followed with serial blood venous blood draws and quantification of deuterated plasma analytes by stable isotope dilution LC/MS/MS. Repeat gastric gavage with d9-γBB after 1 month gut suppression with a cocktail of broad-spectrum antibiotics as described in Experimental Procedures (middle panels). A final d9-γBB gastric challenge and sequential measurement of deuterated plasma compounds (right panels) was performed after a month long reconventionalization period. (B) Female Swiss Webster Germ Free mice (n=5) were challenged with d9-γBB before and after conventionalization. Post challenge measurement of d9-TMA, d9-TMAO, (upper panels), d9-L-carnitine, and d9-γBB (lower panels) was performed in serial venous blood draws by stable isotope dilution LC/MS/MS. Data is expressed as means ± SE. (see also Figure S2).

Figure 3. Gut microbes convert L-carnitine into γBB anatomically proximal to TMA, and the microbial enzyme yeaW/X shows TMA lyase activity with multiple trimethylamine nutrients

(A) C57BL/6J Female mouse intestines (n=7) were sectioned into two complementary pieces for incubation at 37 °C for 18 hours with equimolar amounts of d3-L-carnitine (middle and right panels) or d9-γBB (left panel) under either aerobic (open bars) or anaerobic(closed bars)conditions, as indicated. Deuterated trimethylamine analytes were quantified by stable isotope dilution LC/MS/MS as described in Experimental Procedures. d3-γBB production from d3-L-carnitine is approximately 1,000-fold higher (lower panel) than d3-TMA production (middle panel). Duo =Duodenum, Jejen=Jejenum, Cec=Cecum (B) Cloning of YeaW/YeaX from E. coli DH10b into pET at NdeI and HindIII sites and transforming E. coli BL21. (C) SDS-PAGE confirmation of the purified yeaW and yeaX from E. coli BL21 lysate transformed with pET-yeaW and pET-yeaX, respectively. Both yeaW and yeaX contain 8xHis Tag. (D) YeaW/X catalyzes production of TMA from multiple TMA containing compounds. Data presented are mean ± SE for triplicate determinations from 2 independent replicates of purified proteins. (see also Figure S3–S7).

Figure 4. Gut microbes promote atherosclerosis in a gut microbe-dependent manner

(A) Oil-red-O stained and hematoxylin counterstained representative aortic root slides of 19 week-old C57BL/6J, Apoe−/− female mice on the indicated diets in the presence versus absence of gut microbe suppression (± antibiotics (ABS)), as described under Experimental Procedures. (B) Quantification of mouse aortic root plaque lesion area of 19 week-old C57BL/6J, Apoe−/− female mice. Mice were started on the indicated diets at the time of weaning (4 weeks of age). Lesion area was quantified as described under Experimental Procedures. (C) Terminal plasma concentrations of γBB, L-carnitine, TMA, and TMAO were determined using stable isotope dilution LC/MS/MS analysis. Data is expressed as means ± SE. (see also Table S1).

Figure 5. Mice chronically exposed to dietary L-carnitine demonstrate selective enhancement in gut microbial functional capacity to produce γBB and TMA/TMAO from L-carnitine, but not from γBB

(A) d3-L-carnitine challenge of mice on an L-carnitine supplemented diet (1.3%) from weanining until 10 weeks of age or age-matched normal chow controls. Plasma concentrations of d3-γBB, d3-L-carnitine, d3-TMA, and d3-TMAO were measured in sequential venous blood draws at the indicated times post d3-L-carnitine challenge using stable isotope dilution LC/MS/MS. Data points represents mean ± SE of 4 replicates per group. (B) The same C57Bl/6J, Apoe−/− mice on L-carnitine and normal chow were challenged with d9-γBB oral gavage followed by venous blood draws at the indicated times over 12 hours. d9-γBB, d9-L-carnitine, d9-TMA, and d9-TMAO levels were analyzed using stable isotope dilution LC/MS/MS. Error bars represent ± SE and P values represent Wilcoxon rank sum test of the mean of the area under the curve for each replicate mouse.

Figure 6. Chronic dietary exposure to L-carnitine or γBB influences global function of the gut microbiome

Plasma (A) γBB or (B) TMAO concentrations were determined by stable isotope dilution LC/MS/MS (plotted on x axes) and the proportion of taxonomic operational units (OTUs, plotted on Y axes) were determined as described in Experimental Procedures. The FDR (for multiple comparisons) P value shown is for comparisons between L-carnitine and normal chow dietary groups. (C) Mice on a 1.3% γBB supplemented diet from weaning until 10 weeks of age (n=4) and age-matched mice on a normal chow diet (n=5) were challenged with d9-γBB oral gavage. Plasma concentrations of d9-γBB, d9-L-carnitine, d9-TMA, and d9-TMAO recovered by sequential venous blood draws were measured using stable isotope dilution LC/MS/MS. P values represent Wilcoxon rank sum test of the mean of the area under the curve for each replicate mouse. (D) Mice on a 1.3% γBB-supplemented diet at 10 weeks of age (n=4) and age-matched mice on a normal chow diet (n=5) were challenged with d3-carnitine oral gavage. Plasma concentrations of d3-γBB, d3-L-carnitine, d3-TMA, and d3-TMAO recovered by sequential venous blood draws were measured using stable isotope dilution LC/MS/MS. P values represent Wilcoxon rank sum test of the mean of the area under the curve for each replicate mouse. (see also Table S2). All Error bars represent ± SE.

Figure 7. Overall scheme showing gut microbe-dependent pathways for conversion of dietary L-carnitine into the pro-atherosclerotic metabolite TMAO

γBB is endogenously produced as part of the L-carnitine biosynthetic pathway from lysine, but can also be produced by the metabolism of L-carnitine by commensal gut microbes. L-carnitine and γBB both serve as sources of TMA production via gut microbes.