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Intestinal microbiota metabolism of l-carnitine, a nutrient in red meat, promotes atherosclerosis / CROI: Higher Carnitine Levels Almost quadrupled MI Risk in HIV+
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CROI: Higher Carnitine Tied to Almost Quintupled MI Risk in People With HIV Conference on Retroviruses and Opportunistic Infections (CROI), February 13-16, 2017, Seattle - (05/01/17)
"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......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"
"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."
"The association between plasma carnitine concentrations and cardiovascular risks further supports the potential pathophysiological importance of a carnitine gut microbiota TMA/TMAO atherosclerosis pathway.....The association between high plasma carnitine concentration and CVD risk disappeared after TMAO levels were added to the statistical model."
"......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."......."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 diet"
"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."......"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."
"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."
"l-carnitine is essential for the import of activated long-chain fatty acids from the cytoplasm into mitochondria for β-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)."
Intestinal microbiota metabolism of l-carnitine, a nutrient in red meat, promotes atherosclerosis
Nature Medicine 07 April 2013
ABSTRACT
Intestinal microbiota metabolism of choline and phosphatidylcholine produces trimethylamine (TMA), which is further metabolized to a proatherogenic species, trimethylamine-N-oxide (TMAO). We demonstrate here that metabolism by intestinal microbiota of dietary l-carnitine, a trimethylamine abundant in red meat, also produces TMAO and accelerates atherosclerosis in mice. Omnivorous human subjects produced more TMAO than did vegans or vegetarians following ingestion of l-carnitine through a microbiota-dependent mechanism. The presence of specific bacterial taxa in human feces was associated with both plasma TMAO concentration and dietary status. Plasma l-carnitine levels in subjects undergoing cardiac evaluation (n = 2,595) predicted increased risks for both prevalent cardiovascular disease (CVD) and incident major adverse cardiac events (myocardial infarction, stroke or death), but only among subjects with concurrently high TMAO levels. Chronic dietary l-carnitine supplementation in mice altered cecal microbial composition, markedly enhanced synthesis of TMA and TMAO, and increased atherosclerosis, but this did not occur if intestinal microbiota was concurrently suppressed. In mice with an intact intestinal microbiota, dietary supplementation with TMAO or either carnitine or choline reduced in vivo reverse cholesterol transport. Intestinal microbiota may thus contribute to the well-established link between high levels of red meat consumption and CVD risk.
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 β-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.
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