A Cleveland Clinic-led study has revealed new insights into how a diet rich in red meat increases risk for cardiovascular disease. The findings were published in Nature Microbiology and build on more than a decade of research by lead author Stanley Hazen, MD, Ph.D..
In a previous series of landmark studies, Dr. Hazen found that a byproduct that forms when gut bacteria digest certain nutrients abundant in red meat and other animal products – called TMAO (trimethylamine N-oxide) – increases the risk of heart disease and stroke.
The latest findings offer a more comprehensive understanding of the two-step process by which gut microbes convert the nutrient carnitine into TMAO, an atherosclerosis- and blood clot-promoting molecule, following the ingestion of a red meat-rich diet.
“These new studies identify the gut microbial gene cluster responsible for the second step of the process that links a red meat-rich diet to elevated cardiac disease risks,” said Dr. Hazen, who directs the Cleveland Clinic Center for Microbiome & Human Health. “This discovery helps point us towards new therapeutic targets to prevent or reduce diet-associated cardiovascular disease risk.”
In 2018, Dr. Hazen published results in the Journal of Clinical Investigation showing that dietary carnitine is converted into TMAO in the gut through a two-step, two microbe process. An intermediary metabolite in this process is a molecule called γBB (gamma-butyrobetaine).
According to Dr. Hazen, multiple gut microbes can convert dietary carnitine to γBB, but very few can transform the molecule to TMA, the precursor to TMAO.
“In omnivores, Emergencia timonensis is the primary human gut microbe involved in the transformation of γBB to TMA/TMAO. Conversely, long-term vegetarians and vegans have very low levels of this microbe in their gut and therefore have minimal to no capacity to convert carnitine into TMAO.”
The researchers studied the relationship between fasting plasma γBB levels and disease outcomes using samples and clinical data collected from nearly 3,000 patients. Higher γBB levels were associated with cardiovascular disease and major adverse events including death, non-fatal heart attack or stroke.
To understand the mechanistic link between γBB and the observed outcomes in patients, the researchers studied fecal samples collected from mice and patients, as well as preclinical models of arterial injury. They found that introducing E. timonensis completes the transformation of carnitine to TMAO, elevates TMAO levels and enhances blot clot potential.
The researchers used sequencing technology to identify the relevant gut microbial gene cluster. The cluster was named the gbu (gamma-butyrobetaine utilization) gene cluster, based on its newly discovered function, and includes six genes. They found that in the presence of γBB, the expression of all six genes in the gbu gene cluster increases and that four genes (gbuA, gbuB, gbuC and gbuE) are critical in the conversion of γBB to TMA/TMAO.
“By studying patient samples, we saw that the abundance of gbuA is significantly associated with a diet rich in red meat and plasma TMAO levels,” said Dr. Hazen, who is also chair of the Department of Cardiovascular Disease & Metabolic Sciences and a practicing physician.
“Patients who transitioned to a non-meat diet went on to exhibit reduced gut microbial levels of gbuA. Taken together, this suggests that dietary modifications may help reduce diet- and TMAO- associated cardiovascular disease risk. Likewise, the role of the gbu gene cluster may be worth exploring as a potential therapeutic target.”
The studies were performed in part during a collaboration between Dr. Hazen’s team and Procter & Gamble (P&G). Jennifer Buffa, MS; Kymberleigh Romano, Ph.D.; and Matthew Copeland, Ph.D., are co-first authors on the study, which was supported in part by the National Heart, Lung & Blood Institute (part of the National Institutes of Health), the Leducq Foundation and P&G.
Dr. Hazen is named as co-inventor on pending and issued patents held by Cleveland Clinic relating to cardiovascular diagnostics and therapeutics and has the right to receive royalty payment for inventions or discoveries related to cardiovascular diagnostics or therapeutics. Dr. Hazen also reports having been paid as a consultant for P&G, and receiving research funds from P&G. Dr. Hazen holds the Jan Bleeksma Endowed Chair in Vascular Cell Biology & Atherosclerosis.
The human gut microbiota collectively synthesizes an array of small molecule metabolites. The metabolic output of this microbial community varies substantially between individual human subjects, and specific metabolites are strongly associated with health and disease (1⇓–3). In many cases, however, we lack both a molecular understanding of how gut microbial metabolites influence human physiology and how the metabolites themselves are produced.
These gaps in knowledge limit our ability to establish causative effects of microbial metabolites in human disease and to develop microbiota-based strategies to improve human health. Identification of the specific organisms, genes, and enzymes responsible for metabolite production is needed to accurately profile specific metabolic functions in microbial communities, to experimentally investigate links to disease, and to modulate the metabolic output of the gut microbiota.
Trimethylamine (TMA) is a gut microbial metabolite that has been strongly associated with human disease. It is derived from gut microbial transformations of dietary nutrients including phosphatidylcholine, choline, L-carnitine, betaine, and TMA N-oxide (TMAO) (4⇓⇓⇓⇓–9).
Microbially produced TMA is absorbed by the host in the gastrointestinal tract, enters hepatic circulation, and is oxidized to TMAO in the liver by the flavin-dependent monooxygenase FMO3 (10). Genetic mutations in the human FMO3 gene lead to accumulation of TMA in the body, causing the metabolic disorder trimethylaminuria or fish malodor syndrome (11).
In addition, elevated plasma levels of TMA and TMAO have been associated with multiple human diseases, including cardiovascular, chronic kidney, and nonalcoholic fatty liver diseases, obesity, type II diabetes, and colorectal cancer (12). Especially strong correlations between TMAO and its precursors have been demonstrated for cardiovascular disease (CVD). Elevated plasma levels of dietary TMA precursors were also associated with disease risk, but only when they co-occurred with elevated TMAO levels (6, 7, 9).
Furthermore, oral administration of the TMA precursors phosphatidylcholine, choline, and carnitine to atherosclerosis-prone mice resulted in development of atherosclerotic plaques in a gut microbiota-dependent fashion (6, 7). Direct oral administration of TMAO in these mice similarly resulted in phenotypes of atherosclerosis (6).
These observations suggest a causal role for TMAO in animal models of CVD and that gut microbial metabolism is a crucial factor contributing to pathogenesis. However, a causative role of TMA or TMAO in the development or exacerbation of complex diseases has not yet been definitively established in humans. Deciphering the contribution of TMA production to human disease clearly necessitates a better understanding of the gut microbial metabolic pathways that generate this small molecule.
L-Carnitine is an important dietary precursor to TMA, and its metabolism by gut microbes is associated with CVD (13). An essential nutrient for the host, L-carnitine plays a key role in fatty acid β-oxidation by transporting fatty acids across the mitochondrial membrane for metabolism (5, 14). Although it is produced endogenously, humans must uptake additional L-carnitine in the diet to support cellular function (5, 14).
The major sources of L-carnitine are animal-based products, especially red meat (15), but it is also ingested as a supplement for enhanced physical performance (16, 17). In contrast to the host which cannot breakdown L-carnitine, gut bacteria metabolize this molecule in multiple ways.
The most well-known conversion of L-carnitine involves the production of TMA; however, the human gut bacterium Eubacterium limosum was also recently reported to demethylate L-carnitine (18). Studies in rats and human subjects demonstrated that a large proportion of dietary L-carnitine is converted to TMA and that this metabolism is dependent on the gut microbiota (7, 19, 20).
In addition, these studies noted accumulation of an intermediate metabolite identified as γ-butyrobetaine (γbb) that was produced by the gut microbiota (19⇓–21). Furthermore, γbb was shown to be a proatherogenic metabolite in mouse models like its precursor L-carnitine (21).
The well-characterized metabolic pathway that converts L-carnitine to γbb is encoded by the cai gene operon (Fig. 1) and is used during anaerobic respiration by facultative anaerobic Proteobacteria such as Escherichia coli, Salmonella typhimurium, and Proteus mirabilis (5). The microbial genes and enzymes that are responsible for generating TMA from L-carnitine–derived γbb, however, are not fully elucidated.
Specifically, there is a significant gap in our understanding of the molecular basis for TMA production from L-carnitine and γbb precursors under anaerobic conditions. Select facultative anaerobic Proteobacteria and Actinobacteria possess an iron-dependent Rieske-type monooxygenase (CntA) that can directly convert L-carnitine to TMA (21⇓–23).
This enzyme uses dioxygen to hydroxylate L-carnitine at the C4 position, followed by nonenzymatic formation of an aldehyde through elimination of TMA (Fig. 1).
Whereas L-carnitine was the only substrate tested for activity with CntA from Acinetobacter baumannii (22), the E. coli homolog, also known as YeaW (71% amino acid identity), was shown to produce low levels of TMA from both L-carnitine and γbb (21). Although CntA activity was originally proposed to represent the major mechanism for conversion of L-carnitine to TMA by the human gut microbiota, this conclusion has been called into question.
This enzyme strictly requires dioxygen for catalysis; however, dioxygen levels in the colon lumen are <1 mm Hg (24). In addition, a study in humans showed that plasma TMAO levels after a carnitine challenge were not correlated with cntA gene abundance in gut microbiomes (25).
Notably, TMA production from L-carnitine and γbb was demonstrated in anaerobic ex vivo incubations of mice cecal and colon tissues (21). Finally, the human gut isolate Emergencia timonensis, an obligate anaerobe that does not encode a CntA homolog, was recently found to metabolize γbb to TMA under strictly anaerobic growth conditions (13). Collectively, this information indicates the existence of an as-yet-uncharacterized anaerobic pathway for γbb metabolism in the human gut microbiota.
We present here the identification and characterization of the metabolic pathway, genes, and enzymes responsible for anaerobic TMA production from γbb in E. timonensis. The enzyme catalyzing the key C–N bond cleavage reaction that generates TMA is a flavin-dependent, acyl-coenzyme A (CoA) dehydrogenase-like enzyme that uses the activated CoA thioester of γbb as its substrate.
This chemically challenging reaction generates the intermediate crotonyl-CoA, which is further metabolized by E. timonensis for anaerobic respiration and as a source of carbon and energy. Homologous gene clusters for γbb metabolism are present in other cultured and uncultured host-associated bacteria from the Clostridiales order.
We find that anaerobic γbb metabolism is prevalent in human gut microbiomes and is likely a major underappreciated contributor of L-carnitine–derived TMA. Together, this work expands our knowledge of TMA-producing enzymes, pathways, and organisms, providing a more complete understanding of microbial TMA production in the anoxic human gut. These findings identify potential targets for manipulation of this microbial function and will help resolve the major dietary and microbial contributors to TMA production.
REFERENCE LINK :https://www.pnas.org/content/118/32/e2101498118
More information: Stanley Hazen, The microbial gbu gene cluster links cardiovascular disease risk associated with red meat consumption to microbiota l-carnitine catabolism, Nature Microbiology (2021). DOI: 10.1038/s41564-021-01010-x. www.nature.com/articles/s41564-021-01010-x