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Molecule in meat may increase heart disease risk
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Gut bacteria transform compound into artery hardener
"Discovery of a link between l-carnitine ingestion, gut microbiota metabolism and CVD risk has broad health-related implications. Our studies reveal a new pathway potentially linking dietary red meat ingestion with atherosclerosis pathogenesis. The role of gut microbiota in this pathway suggests new potential therapeutic targets for preventing CVD. Furthermore, our studies have public health relevance, as l-carnitine is a common over-the-counter dietary supplement. Our results suggest that the safety of chronic l-carnitine supplementation should be examined, as high amounts of orally ingested l-carnitine may under some conditions foster growth of gut microbiota with an enhanced capacity to produce TMAO and potentially advance atherosclerosis."
By Meghan Rosen
Web edition: April 8, 2013
http://www.sciencenews.org
Drop that hamburger, put down the can of Monster Energy and back away from the body building pills.
A nutrient found in red meat and added to energy drinks and supplements may crank up people's risk of heart disease, a new study suggests. Bacteria in the gut digest the nutrient, L-carnitine, and help turn it into an artery-hardening chemical - particularly in meat eaters, researchers report April 7 in Nature Medicine.
The intestinal microbes of vegetarians and vegans didn't make much of the chemical, even when researchers fed them an 8-ounce sirloin steak.
"I always thought that what I ate mattered, but I never realized that my gut bacteria might matter more," says biochemist Harry Ischiropoulos of the University of Pennsylvania in Philadelphia, who was not involved with the study.
What's more, high blood levels of the bacterial by-product of L-carnitine, called trimethylamine N-oxide or TMAO, were an "astoundingly good" warning sign of impending heart attack, stroke and death, says study coauthor Stanley Hazen of Cleveland Clinic. A test for TMAO, which will become commercially available this year, could give physicians a new tool for gauging heart disease risk.
Scientists have long known that eating red meat jacks up a person's chances of developing heart disease, but reliable biomarkers - blood-borne indicators of disease or health - have been hard to find. One way physicians gauge risk is with blood tests for cholesterol, a greasy molecule in meat and other foods, which gums up arteries. But tests for cholesterol and other molecules don't wholly explain meat's link to heart disease, Hazen says. "Cholesterol, saturated fat and salt only account for a tiny little piece of the risk."
Gut bacteria might account for a bit more. Hazen's team first linked intestinal microbes to heart disease in 2011, when they spotted TMAO in blood collected from people who later suffered heart attacks, had strokes or died (SN Online: 4/7/11).
For the new study, Hazen zeroed in on L-carnitine because the nutrient is structurally similar to a compound that gut microbes can convert to TMAO.
Volunteers - a mix of omnivores, vegetarians and vegans - ate steak and L-carnitine capsules, and then researchers measured TMAO levels in the blood. Only meat eaters could make TMAO from L-carnitine, Hazen's team found, and they needed their gut bacteria to do it. TMAO production shut down when researchers wiped out volunteers' intestinal microbes with antibiotics.
L-carnitine passed right through the guts of long-term vegans and vegetarians, leaving their blood practically TMAO-free. When researchers examined volunteers' stool, they found different groups of bacteria in people who did and didn't eat meat.
Hazen's group also found that blood levels of TMAO and L-carnitine could predict heart disease risk, which they learned by collecting blood samples from 2,595 patients and tracking their health for three years.
The findings are new and exciting but need to be confirmed, says cardiovascular researcher Ishwarlal Jialal of the University of California, Davis Medical Center. Molecules proposed as biomarkers for heart disease often look promising in initial studies but fizzle out clinically. "We've been down this road so many times before."
But one message is clear, Jialel says: "L-carnitine is not good for you. It's not good as a supplement and it's not good in red meat. That's one thing you can take to the bank."
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Intestinal microbiota metabolism of l-carnitine, a nutrient in red meat, promotes atherosclerosis
Nature Medicine
07 April 2013
Introduction
The high level of meat consumption in the developed world is linked to CVD risk, presumably owing to the large content of saturated fats and cholesterol in meat1, 2. However, a recent meta-analysis of prospective cohort studies showed no association between dietary saturated fat intake and CVD, prompting the suggestion that other environmental exposures linked to increased meat consumption are responsible3. In fact, the suspicion that the cholesterol and saturated fat content of red meat may not be sufficiently high enough to account for the observed association between CVD and meat consumption has stimulated investigation of alternative disease-promoting exposures that accompany dietary meat ingestion, such as high salt content or heterocyclic compounds generated during cooking4, 5. To our knowledge, no studies have yet explored the participation of commensal intestinal microbiota in modifying the diet-host interaction with reference to red meat consumption.
The microbiota of humans has been linked to intestinal health, immune function, bioactivation of nutrients and vitamins, and, more recently, complex disease phenotypes such as obesity and insulin resistance6, 7, 8. We recently reported a pathway in both humans and mice linking microbiota metabolism of dietary choline and phosphatidylcholine to CVD pathogenesis9. Choline, a trimethylamine-containing compound and part of the head group of phosphatidylcholine, is metabolized by gut microbiota to produce an intermediate compound known as TMA (Fig. 1a). TMA is rapidly further oxidized by hepatic flavin monooxygenases to form TMAO, which is proatherogenic and associated with cardiovascular risks. These findings raise the possibility that other dietary nutrients possessing a trimethylamine structure may also generate TMAO from gut microbiota and promote accelerated atherosclerosis. TMAO has been proposed to induce upregulation of macrophage scavenger receptors and thereby potentially contribute to enhanced "forward cholesterol transport."10. Whether TMAO is linked to the development of accelerated atherosclerosis through additional mechanisms, and which specific microbial species contribute to TMAO formation, have not been fully clarified.
l-carnitine is an abundant nutrient in red meat and contains a trimethylamine structure similar to that of choline (Fig. 1a). Although dietary ingestion is a major source of l-carnitine in omnivores, it is also endogenously produced in mammals from lysine and serves an essential function in transporting fatty acids into the mitochondrial compartment10, 11. l-Carnitine ingestion and supplementation in industrialized societies have markedly increased12. Whether there are potential health risks associated with the rapidly growing practice of consuming l-carnitine supplements has not been evaluated.
Herein we examine the gut microbiota-dependent metabolism of l-carnitine to produce TMAO in both rodents and humans (omnivores and vegans or vegetarians). Using isotope tracer studies in humans, clinical studies to examine the effects on cardiovascular disease risk, and animal models including germ-free mice, we demonstrate a role for gut microbiota metabolism of l-carnitine in atherosclerosis pathogenesis. We show that TMAO, and its dietary precursors choline and carnitine, suppress reverse cholesterol transport (RCT) through gut microbiota-dependent mechanisms in vivo. Finally, we define microbial taxa in feces of humans whose proportions are associated with both dietary carnitine ingestion and plasma TMAO concentrations. We also show microbial compositional changes in mice associated with chronic carnitine ingestion and a consequent marked enhancement in TMAO synthetic capacity in vivo.
DISCUSSION
The dietary nutrient l-carnitine has been studied for over a century30. Although eukaryotes can endogenously produce l-carnitine, only prokaryotic organisms can catabolize it11. A role for intestinal microbiota in TMAO production from dietary l-carnitine was first suggested by studies in rats31. Although TMAO production from alternative dietary trimethylamines has been suggested in humans, a role for the microbiota in the production of TMAO from dietary l-carnitine in humans has not previously been demonstrated31, 32, 33. The present studies reveal an obligatory role of gut microbiota in the production of TMAO from ingested l-carnitine in humans. They also suggest a new nutritional pathway in CVD pathogenesis that involves dietary l-carnitine, the intestinal microbial community and production of the proatherosclerotic metabolite TMAO. Finally, these studies show that TMAO modulates cholesterol and sterol metabolism at multiple anatomic sites and processes in vivo, with a net effect of increasing atherosclerosis.
Our results also suggest a previously unknown mechanism for the observed relationship between dietary red meat ingestion and accelerated atherosclerosis. Consuming foods rich in l-carnitine (predominantly red meat) can increase fasting human l-carnitine concentrations in the plasma34. Meats and full-fat dairy products are abundant components of the Western diet and are commonly implicated in CVD. Together, l-carnitine and choline-containing lipids can constitute up to 2% of a Western diet14, 15, 35. Numerous studies have suggested a decrease in atherosclerotic disease risk in vegan and vegetarian individuals compared to omnivores; reduced levels of dietary cholesterol and saturated fat have been suggested as the mechanism explaining this decreased risk36, 37. Notably, a recent 4.8-year randomized dietary study showed a 30% reduction in cardiovascular events in subjects consuming a Mediterranean diet (with specific avoidance of red meat) compared to subjects consuming a control diet38. The present studies suggest that the reduced ingestion of l-carnitine and total choline by vegans and vegetarians, with attendant reductions in TMAO levels, may contribute to their observed cardiovascular health benefits. Conversely, an increased capacity for microbiota-dependent production of TMAO from l-carnitine may contribute to atherosclerosis, particularly in omnivores who consume high amounts of l-carnitine.
One proatherosclerotic mechanism observed for TMAO in the current studies is suppression of RCT (Fig. 6c). Dietary l-carnitine and choline each suppressed RCT (P < 0.05), but only in mice with intact intestinal microbiota and increased TMA and TMAO concentrations. Suppression of the intestinal microbiota completely eliminated choline- and l-carnitine-dependent suppression of RCT. Moreover, direct dietary supplementation with TMAO promoted a similar suppression of RCT.
These results are consistent with a gut microbiota-dependent mechanism whereby generation of TMAO impairs RCT, potentially contributing to the observed proatherosclerotic phenotype of these interventions. Another mechanism by which TMAO may promote atherosclerosis is through increasing macrophage SRA and CD36 surface expression and foam cell formation9 (Fig. 6c). Within macrophages, TMAO does not seem to alter known cholesterol biosynthetic and uptake pathways24, 39 or the more recently described regulatory role of desmosterol in integrating macrophage lipid metabolism and inflammatory gene responses25. In the liver, TMAO decreased the bile acid pool size and lowered the expression of key bile acid synthesis and transport proteins (Fig. 6c). However, it is unclear whether these changes contribute to the impairment of RCT. Of note, TMAO lowered expression of Cyp7a1, the major bile acid synthetic enzyme and rate-limiting step in the catabolism of cholesterol. The effect of TMAO is thus consistent with reports of human Cyp7a1 gene variants that are associated with reduced expression of Cyp7a1, leading to decreased bile acid synthesis, decreased bile acid secretion and enhanced atherosclerosis40, 41, 42. Furthermore, upregulation (as opposed to downregulation) of Cyp7a1 has been reported to lead to expansion of the bile acid pool, increased RCT and reduced atherosclerotic plaque area in susceptible mice43, 44, 45. Within the intestine, we found that TMAO concentration was also associated with changes in cholesterol metabolism.
However, the reduction in cholesterol absorption observed, although consistent with the reduction in intestinal Npc1L1 expression46 (as well as hepatic Cyp7a1 and Cyp27a1 expression28, 29), cannot explain the suppression of RCT observed after dietary supplementation with TMAO.
Thus, the molecular mechanisms through which gut microbiota formation of TMAO leads to inhibition of RCT are not entirely clear. It is also not known whether TMAO interacts directly with a specific receptor or whether it acts to alter signaling pathways indirectly by altering protein conformation (that is, via allosteric effects). Whereas TMA has been reported to influence signal transduction by direct interaction with a family of G protein-coupled receptors47, 48, TMAO, a small quaternary amine with aliphatic character, is reportedly capable of directly inducing conformational changes in proteins, stabilizing protein folding and acting as a small-molecule protein chaperone49, 50. It is thus conceivable that TMAO may alter many signaling pathways without directly acting at a 'TMAO receptor'.
A noteworthy finding is the magnitude with which long-term dietary habits affect TMAO synthetic capacity in both humans (vegans and vegetarians versus omnivores) and mice (normal chow versus chronic l-carnitine supplementation). Analyses of microbial composition in human feces and mice cecal contents revealed specific taxa that segregate with both dietary status and plasma TMAO concentrations.
Recent studies have shown that changes in enterotype are associated with long-term dietary patterns19. We observed that plasma TMAO concentration varied significantly (P < 0.05) according to previously reported enterotypes. We also showed an obligatory role for gut microbiota in TMAO formation from dietary l-carnitine in mice and humans. The differences observed in TMAO production after an l-carnitine challenge in omnivore versus vegan subjects is striking, and is consistent with the observed differences in microbial community composition. Recent reports have shown differences in microbial communities among vegetarians and vegans versus omnivores51. Of note, we observed an increase in baseline plasma TMAO concentrations in what has historically been called enterotype 2 (Prevotella), a relatively rare enterotype that in one study was associated with low animal-fat and protein consumption19. In our study, three of the four individuals classified into enterotype 2 are self-identified omnivores, suggesting more complexity in the human gut microbiome than anticipated. Indeed, other studies have demonstrated variable results in associating human bacterial genera, including Bacteroides and Prevotella, to omnivorous and vegetarian eating habits18, 52. This complexity is no doubt in part attributable to the fact that there are many species within any genus, and distinct species within the same genus may have different capacities to use l-carnitine as a fuel and form TMA. Indeed, prior studies have suggested that multiple bacterial strains can metabolize l-carnitine in culture53, and species within the genus Clostridium differ in their ability to use choline as the sole source of carbon and nitrogen in culture54. Our results suggest that multiple 'proatherogenic' (that is, TMA- and TMAO-producing) species probably exist. Consistent with this supposition, others have reported that several bacterial phylotypes are associated with a history of atherosclerosis and that human microbiota biodiversity may in part be influenced by carnivorous eating habits16, 19, 55.
The association between plasma carnitine concentrations and cardiovascular risks further supports the potential pathophysiological importance of a carnitine gut microbiota TMA/TMAO atherosclerosis pathway (Fig. 6c). The association between high plasma carnitine concentration and CVD risk disappeared after TMAO levels were added to the statistical model. These observations are consistent with a proposed mechanism whereby oral l-carnitine ingestion contributes to atherosclerotic CVD risk via the microbiota metabolite TMAO. There are only a few reports of specific intestinal anaerobic and aerobic bacterial species that can use l-carnitine as a carbon nitrogen source10, 11, 56.
l-carnitine is essential for the import of activated long-chain fatty acids from the cytoplasm into mitochondria for ß-oxidation, and dietary supplementation with l-carnitine has been widely studied. Some case reports of l-carnitine supplementation have reported beneficial effects in individuals with inherited primary and acquired secondary carnitine deficiency syndromes13. l-Carnitine supplementation studies in chronic disease states have reported both positive and negative results57, 58. Oral l-carnitine supplementation in subjects on hemodialysis raises plasma l-carnitine concentrations to normal levels but also substantially increases TMAO levels57. A broader potential therapeutic scope for l-carnitine and two related metabolites, acetyl-l-carnitine and propionyl-l-carnitine, has also been explored for the treatment of acute ischemic events and cardiometabolic disorders (reviewed in ref. 58). Here too, both positive and negative results have been reported. Potential explanations for the discrepant findings of various l-carnitine intervention studies are differences in the duration of dosing or in the route of administration. In many studies, l-carnitine or a related molecule is administered over a short interval or via the parenteral route, thereby bypassing gut microbiota (and hence TMAO formation).
Discovery of a link between l-carnitine ingestion, gut microbiota metabolism and CVD risk has broad health-related implications. Our studies reveal a new pathway potentially linking dietary red meat ingestion with atherosclerosis pathogenesis. The role of gut microbiota in this pathway suggests new potential therapeutic targets for preventing CVD. Furthermore, our studies have public health relevance, as l-carnitine is a common over-the-counter dietary supplement. Our results suggest that the safety of chronic l-carnitine supplementation should be examined, as high amounts of orally ingested l-carnitine may under some conditions foster growth of gut microbiota with an enhanced capacity to produce TMAO and potentially advance atherosclerosis.
RESULTS
Metabolomic studies link l-carnitine with CVD
Given the similarity in structure between l-carnitine and choline (Fig. 1a), we hypothesized that dietary l-carnitine in humans, like choline and phosphatidylcholine, might be metabolized to produce TMA and TMAO in a gut microbiota-dependent fashion and be associated with atherosclerosis risk. To test this hypothesis, we initially examined data from our recently published unbiased small-molecule metabolomics analyses of plasma analytes and CVD risks9.
An analyte with identical molecular weight and retention time to l-carnitine was not in the top tier of analytes that met the stringent P value cutoff for association with CVD. However, a hypothesis-driven examination of the data using less stringent criteria (no adjustment for multiple testing) revealed an analyte with the appropriate molecular weight and retention time for l-carnitine that was associated with cardiovascular event risk (P = 0.04) (Supplementary Table 1). In further studies we were able to confirm the identity of the plasma analyte as l-carnitine and develop a quantitative stable-isotope-dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) method for measuring endogenous l-carnitine concentrations in all subsequent investigations (Supplementary Figs. 1-3).
Human gut microbiota is required to form TMAO from l-carnitine
The participation of gut microbiota in TMAO production from dietary l-carnitine in humans has not previously been shown. In initial subjects (omnivores), we developed an l-carnitine challenge test in which the subjects were fed a large amount of l-carnitine (an 8-ounce sirloin steak, corresponding to an estimated 180 mg of l-carnitine)13, 14, 15, together with a capsule containing 250 mg of a heavy isotope-labeled l-carnitine (synthetic d3-(methyl)-l-carnitine). At visit 1 post-prandial increases in plasma d3-TMAO and d3- l-carnitine concentrations were readily detected, and 24-h urine collections also revealed the presence of d3-TMAO (Fig. 1b-e and Supplementary Figs. 4 and 5). Figure 1 and Supplementary Figure 4 show tracings from a representative omnivorous subject, of five studied with sequential serial blood draws after carnitine challenge. In most subjects examined, despite clear increases in plasma d3-carnitine and d3-TMAO concentrations over time (Fig. 1e), post-prandial changes in endogenous (unlabeled) carnitine and TMAO concentrations were modest (Supplementary Fig. 5), consistent with total body pools of carnitine and TMAO that are relatively very large in relation to the amounts of carnitine ingested and TMAO produced from the carnitine challenge.
To examine the potential contribution of gut microbiota to TMAO formation from dietary l-carnitine, we placed the five volunteers studied above on oral broad-spectrum antibiotics to suppress intestinal microbiota for a week and then performed a second l-carnitine challenge (visit 2). We noted near complete suppression of detectable endogenous TMAO in both plasma and urine after a week-long treatment with the antibiotics (visit 2) (Fig. 1b-e and Supplementary Fig. 5). Moreover, we observed virtually no detectable formation of either native or d3-labeled TMAO in all post-prandial plasma samples or 24-h urine samples examined after carnitine challenge, consistent with an obligatory role for gut microbiota in TMAO formation from l-carnitine (Fig. 1b-e and Supplementary Fig. 4). In contrast, we detected both d3- l-carnitine and unlabeled l-carnitine after the l-carnitine challenge, and there was little change in the overall time course before (visit 1) versus after (visit 2) antibiotic treatment (Fig. 1e and Supplementary Fig. 5). We rechallenged the same subjects several weeks after discontinuation of antibiotics (visit 3). Baseline and post-l-carnitine challenge plasma and urine samples again showed TMAO and d3-TMAO formation, consistent with intestinal recolonization (Fig. 1b-e and Supplementary Figs. 4 and 5). Collectively, these data show that TMAO production from dietary l-carnitine in humans is dependent on intestinal microbiota.
Vegans and vegetarians produce less TMAO from l-carnitine
The capacity to produce TMAO (native and d3-labeled) after l-carnitine ingestion was variable among individuals. A post hoc nutritional survey that the volunteers completed suggested that antecedent dietary habits (red meat consumption) may influence the capacity to generate TMAO from l-carnitine (data not shown). To test this prospectively, we examined TMAO and d3-TMAO production after the same l-carnitine challenge, first in a long-term (>5 years) vegan who consented to the carnitine challenge (including both steak and d3-(methyl)-carnitine consumption) (Fig. 2a). Also shown for comparison are data from a single representative omnivore with self-reported frequent (near daily) dietary consumption of red meat (beef, venison, lamb, mutton, duck or pork).
Post-prandially, the omnivore showed increases in TMAO and d3-TMAO concentrations in both sequential plasma measurements (Fig. 2a) and in a 24-h urine collection sample (Fig. 2b). In contrast, the vegan showed nominal plasma and urine TMAO levels at baseline, and virtually no capacity to generate TMAO or d3-TMAO in plasma after the carnitine challenge (Fig. 2a,b). The vegan subject also had lower fasting plasma levels of l-carnitine compared to the omnivorous subject (Supplementary Fig. 6).
To confirm and extend these findings, we examined additional vegans and vegetarians (n = 23) and omnivorous subjects (n = 51). Fasting baseline TMAO levels were significantly lower among vegan and vegetarian subjects compared to omnivores (Fig. 2c). In a subset of these individuals, we performed an oral d3-(methyl)-carnitine challenge (but with no steak) and confirmed that long-term (all >1 year) vegans and vegetarians have a markedly reduced capacity to synthesize TMAO from oral carnitine (Fig. 2c,d). Vegans and vegetarians challenged with d3-(methyl)-carnitine also had significantly higher post-challenge plasma concentrations of d3-(methyl)-carnitine compared to omnivorous subjects (Supplementary Fig. 7), perhaps due to decreased intestinal microbial metabolism of carnitine before absorption.
TMAO levels are associated with human gut microbial taxa
Dietary habits (for example, vegan or vegetarian versus omnivore or carnivore) are associated with significant alterations in intestinal microbiota composition16, 17, 18. To determine microbiota composition, we sequenced the gene encoding bacterial 16S rRNA in fecal samples from a subset of the vegans and vegetarians (n = 23) and omnivores (n = 30) studied above. In parallel, we quantified plasma TMAO, carnitine and choline concentrations by stable-isotope-dilution LC-MS/MS. Global analysis of taxa proportions (Supplementary Methods) revealed significant associations with plasma TMAO concentrations (P = 0.03), but not with plasma carnitine (P = 0.77) or choline (P = 0.74) concentrations.
After false discovery rate (FDR) adjustment for multiple comparisons, several bacterial taxa remained significantly (FDR-adjusted P < 0.10) associated with plasma TMAO concentration (Supplementary Fig. 8). When we classified subjects into previously reported enterotypes19 on the basis of fecal microbial composition, individuals with an enterotype characterized by enriched proportions of the genus Prevotella (n = 4) had higher (P < 0.05) plasma TMAO concentrations than did subjects with an enterotype notable for enrichment in the Bacteroides (n = 49) genus (Fig. 2e). Examination of the proportion of specific bacterial genera and subject plasma TMAO concentrations revealed several taxa (at the genus level) that simultaneously were significantly associated with both vegan or vegetarian versus omnivore status and plasma TMAO concentration (Fig. 2f). These results indicate that preceding dietary habits may modulate both intestinal microbiota composition and ability to synthesize TMA and TMAO from dietary l-carnitine.
TMAO production from dietary l-carnitine is inducible
We next investigated the ability of chronic dietary l-carnitine intake to induce gut microbiota-dependent production of TMA and TMAO in mice. Initial LC-MS/MS studies in germ-free mice showed no detectable plasma d3-(methyl)-TMA or d3-(methyl)-TMAO after oral (gastric gavage) d3-(methyl)-carnitine challenge. However, after a several-week period in conventional cages to allow for microbial colonization ('conventionalization'), previously germ-free mice acquired the capacity to produce both d3-(methyl)-TMA and d3-(methyl)-TMAO following oral d3-(methyl)-carnitine challenge (Supplementary Fig. 9). Parallel studies with non-germ-free ('conventional') Apoe-/- mice (lacking apolipoprotein E; on a C57BL/6J background) that had been placed on a cocktail of oral, relatively nonabsorbable broad-spectrum antibiotics previously shown to suppress intestinal microbiota9, 20 showed similar results (complete suppression of both TMA and TMAO formation; Supplementary Fig. 10). Collectively, these studies confirm in mice an obligatory role for gut microbiota in TMA and TMAO production from dietary l-carnitine.
To examine whether dietary l-carnitine can induce TMA and TMAO production from intestinal microbiota, we compared the pre- and post-prandial plasma profiles of Apoe-/- mice on normal chow diet versus a normal chow diet supplemented with l-carnitine for 15 weeks. The production of both d3-(methyl)TMA and d3-(methyl)TMAO after gastric gavage of d3-(methyl)-carnitine was induced by approximately tenfold in mice on the l-carnitine-supplemented diet (Fig. 3a). Furthermore, plasma post-prandial d3-(methyl)-carnitine levels in mice in the l-carnitine-supplemented diet arm were substantially lower than those observed in mice on the l-carnitine-free diet (normal chow), consistent with enhanced microbiota-dependent catabolism before absorption in the l-carnitine-supplemented mice.
Plasma TMA and TMAO associate with mouse gut microbial taxa
The marked effects of an acute l-carnitine challenge (d3-(methyl)-carnitine by gavage) on TMA and TMAO production suggested that chronic l-carnitine supplementation may significantly alter intestinal microbial composition, with an enrichment for taxa better suited for TMA production from l-carnitine. To test this hypothesis, we first identified the cecum as the segment of the entire intestinal tract of mice showing the highest synthetic capacity to form TMA from carnitine (data not shown). We then sequenced 16S rRNA gene amplicons from ceca of mice on either normal chow (n = 10) or l-carnitine-supplemented (n = 11) diets and in parallel quantified plasma concentrations of TMA and TMAO (Fig. 3b). Global analyses of the presence of the microbiota taxa revealed that, in general, taxa that were at a relatively high proportion coincident with high TMA plasma concentrations also tended to be a relatively high proportion coincident with high TMAO plasma concentrations. Several bacterial taxa remained significantly associated with plasma TMA and/or TMAO levels after FDR adjustment for multiple comparisons (Fig. 3b).
Further analyses revealed several bacterial taxa whose proportion was significantly associated (some positively, others inversely) with dietary l-carnitine and with plasma TMA or TMAO concentrations (P < 0.05) (Fig. 3c and Supplementary Fig. 11). Notably, a direct comparison of taxa associated with plasma TMAO concentrations in humans versus in mice failed to identify common taxa. These results are consistent with prior reports that microbes identified from the distal gut of the mouse represent genera that are typically not detected in humans16, 21.
High plasma l-carnitine concentration is associated with CVD
We next investigated the relationship of fasting plasma concentrations of l-carnitine with CVD risk in an large, independent cohort of stable subjects (n = 2,595) undergoing elective cardiac evaluation. Patient demographics, laboratory values and clinical characteristics are provided in Supplementary Table 2. We observed significant dose-dependent associations between carnitine concentration and risks of prevalent coronary artery disease (CAD) (P < 0.05), peripheral artery disease (PAD) (P < 0.05) and overall CVD (P < 0.05) (Fig. 4a-c). Moreover, these associations remained significant following adjustments for traditional CVD risk factors (P < 0.05) (Fig. a-c). Plasma concentrations of carnitine were high in subjects with angiographic evidence of CAD (≥50% stenosis), regardless of the extent (for example, single- versus multivessel) of CAD, as revealed by diagnostic cardiac catheterization (Kruskal-Wallis P < 0.001) (Fig. 4d).
We also examined the relationship between fasting plasma concentrations of carnitine and incident (3-year) risk for major adverse cardiac events (MACE: composite of death, myocardial infarction, stroke and revascularization).
Elevated carnitine (4th quartile) concentration was an independent predictor of MACE, even after adjustments for traditional CVD risk factors (Fig. 4e). After further adjustment for both plasma TMAO concentration and a larger number of comorbidities that might be known at time of presentation (for example, extent of CAD, ejection fraction, medications and estimated renal function), the significant relationship between carnitine and MACE risk was completely abolished (Fig. 4e). Notably, we observed a significant association between carnitine concentration and incident cardiovascular event risks in Cox regression models after multivariate adjustment, but only among those subjects with concurrent high plasma TMAO concentrations (P < 0.001) (Fig. 4f). Thus, although plasma concentrations of carnitine seem to be associated with both prevalent and incident cardiovascular risks, these results suggest that TMAO, rather than carnitine, is the primary driver of the association of carnitine with cardiovascular risks.
Dietary l-carnitine promotes microbiota-dependent atherosclerosis
We next investigated whether dietary l-carnitine has an impact on the extent of atherosclerosis in the presence or absence of TMAO formation. We fed Apoe-/- mice from the time of weaning a normal chow diet versus the same diet supplemented with l-carnitine. Aortic root atherosclerotic plaque quantification revealed approximately a doubling of disease burden in l-carnitine supplemented mice compared to normal chow-fed mice (Fig. 5a,b). Parallel studies in mice placed on an oral antibiotic cocktail to suppress intestinal microbiota showed marked reductions in plasma TMA and TMAO concentrations (Fig. 5c) and complete inhibition of the dietary l-carnitine-dependent increase in atherosclerosis (Fig. 5b). Of note, the increase in atherosclerotic plaque burden with dietary l-carnitine occurred in the absence of proatherogenic changes in plasma lipid, lipoprotein, glucose or insulin levels; moreover, both biochemical and histological analyses of livers from any group of the mice failed to show evidence of steatosis (Supplementary Tables 3 and 4 and Supplementary Fig. 12).
Plasma concentrations of carnitine were significantly higher in l-carnitine-fed mice compared to normal chow-fed controls (P < 0.05) (Fig. 5c). Plasma carnitine concentrations were even higher in mice supplemented with l-carnitine in the antibiotic arm of the study (Fig. 5c), presumably as a result of the reduced capacity of microbiota to catabolize l-carnitine. However, as the l-carnitine-supplemented mice that received antibiotics did not show enhanced atherosclerosis, these results are consistent with the notion that it is a downstream microbiota-dependent metabolite, not l-carnitine itself, that promotes atherosclerosis.
TMAO inhibits RCT
To identify additional mechanisms by which TMAO might promote atherosclerosis, we first noted that TMAO and its trimethylamine nutrient precursors are all cationic quaternary amines that could potentially compete with arginine, thereby limiting its bioavailability and reducing nitric oxide synthesis. However, a direct test of this hypothesis with competition studies using [14C]arginine and TMAO in bovine aortic endothelial cells demonstrated no decrease in [14C]arginine transport (Supplementary Fig. 13).
In recent studies we showed that TMAO can promote macrophage cholesterol accumulation in a microbiota-dependent manner by increasing cell surface expression of two proatherogenic scavenger receptors, CD36 and scavenger receptor A (SRA)9, 22, 23. We envisioned three non-exclusive mechanisms through which cholesterol can accumulate within cells of the artery wall: enhancing the rate of influx (as noted above), enhancing synthesis or diminishing the rate of efflux. To test whether TMAO might alter the canonical regulation of cholesterol biosynthesis genes24, we loaded macrophages with cholesterol in the presence or absence of physiologically relevant TMAO concentrations. However, TMAO failed to alter mRNA levels of the low-density lipoprotein (LDL) receptor or cholesterol synthesis genes (Supplementary Fig. 14). Parallel studies examining macrophage inflammatory gene expression25 and desmosterol levels in the culture medium also failed to show any effect of TMAO (Supplementary Figs. 14 and 15).
We next examined whether TMAO might inhibit cholesterol removal from peripheral macrophages by testing whether dietary sources of TMAO (choline or l-carnitine) inhibit RCT in vivo using an adaptation of an established model system26. Mice on either choline (1.3% choline chloride by mass)- or l-carnitine-supplemented diets showed significantly less (~30%, P < 0.05) RCT compared to normal chow-fed controls (Fig. 5d). Furthermore, suppression of intestinal microbiota (and plasma TMAO concentrations) with oral broad-spectrum antibiotics completely blocked the diet-dependent (for both choline and l-carnitine) suppression of RCT (Fig. 5d), suggesting that a microbiota-generated product (for example, TMAO) inhibits RCT (Supplementary Fig. 16). To further test this hypothesis, we placed mice on a TMAO-containing diet. They showed a 35% decrease in RCT relative to mice on a normal chow diet (Fig. 5d, P < 0.05). Further examination of plasma, liver and bile showed significantly less [14C]cholesterol recovered from plasma of TMAO-fed compared to chow-fed mice (16% lower, P < 0.05) but no changes in counts recovered from liver or bile (Supplementary Fig. 17).
TMAO alters sterol metabolism in vivo
To better understand the molecular mechanisms through which TMAO suppresses RCT, we examined candidate genes and biological processes in compartments (macrophages, plasma, liver and intestine) known to participate in cholesterol and sterol metabolism and RCT. We exposed peritoneal macrophages recovered from wild-type C57BL/6J mice to TMAO in vitro and quantified mRNA levels of the cholesterol transporters Abca1, Srb1 and Abcg1. TMAO treatment led to modest but statistically significant increases in expression of Abca1 and Abcg1 (P < 0.05; Supplementary Fig. 18). Parallel studies showed corresponding modest TMAO-dependent increases in Abca1-dependent cholesterol efflux to apoA1 as cholesterol acceptor in RAW 264.7 macrophages (P < 0.01; Supplementary Fig. 19). Collectively, these results suggest that the observed global reduction in RCT in vivo induced by TMAO is unlikely to be accounted for by changes in the expression of these transporters. Parallel examination of plasma recovered from mice in the RCT experiments showed no differences in total cholesterol and high-density lipoprotein cholesterol concentrations (Supplementary Table 5).
In parallel studies, we examined the mRNA levels of known cholesterol transporters (Srb1, Abca1, Abcg1, Abcg5, Abcg8 and Shp) in mouse liver but found only a modest difference for Srb1 expression (Supplementary Fig. 20). Western blot analysis of liver from TMAO-supplemented mice, however, showed no change in the abundance of Srb1 protein compared to chow (control) mouse livers (Supplementary Fig. 21). In contrast, mRNA levels in the liver of the key bile acid synthetic enzymes Cyp7a1 and Cyp27a1 were significantly lower in mice supplemented with dietary TMAO, with no change in expression of the upstream regulator Shp (P < 0.05 for each; Fig. 5e and Supplementary Fig. 20). Multiple bile acid transporters in the liver (Oatp1, Oatp4, Mrp2, and Ntcp) also showed significant dietary TMAO-induced decreases in expression (P < 0.05 each); however, Bsep and Ephx1 did not (Fig. 5f). In contrast to the liver, TMAO-induced changes in bile acid transporter gene expression were not observed in the gut (Supplementary Fig. 22). Taken together, these data show that the gut microbiota-dependent metabolite TMAO affects a major pathway for cholesterol elimination from the body, the bile acid synthetic pathway, at multiple levels.
Consistent with the effects of TMAO on bile acid transporter gene expression, mice supplemented with TMAO had a significantly smaller total bile acid pool size compared to normal chow-fed mice (P < 0.01) (Fig. 6a). Dietary supplementation with TMAO also markedly lowered mRNA expression of both types of intestinal cholesterol transporters: Npc1L1, which transports cholesterol into enterocyte from the gut lumen27, and Abcg5-Abcg8, which transport cholesterol out of enterocytes into the gut lumen27 (Supplementary Fig. 23). Previous studies using either Cyp7a1- or Cyp27a1-null mice demonstrated a reduction in cholesterol absorption28, 29. In separate studies, dietary TMAO supplementation compared to normal chow similarly promoted a decrease (26% reduced compared to normal chow-fed mice, P < 0.01) in total cholesterol absorption (Fig. 6b).
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