Atherosclerosis. 2016 Jan;244:29-37
L-carnitine intake and high trimethylamine N-oxide plasma levels correlate with low aortic lesions in ApoE-/- transgenic mice expressing CETP
Heidi L.Collins Ph.D.1, Denise Drazul-Schrader M.S.1, Anthony C. Sulpizio M.S.1, Paul D. Koster B.A.1, Yuping Williamson M.S.2, Steven J. Adelman Ph.D.1, Kevin Owen Ph.D.2, Toran Sanli Ph.D2 and Aouatef Bellamine Ph.D.3
1VascularStrategies LLC, 2KGK Synergize Inc., 3Lonza Inc.
Objective: Dietary L-carnitine can be metabolized by intestinal microbiota to trimethylamine, which is absorbed by the gut and further oxidized to trimethylamine N-oxide (TMAO) in the liver. TMAO plasma levels have been associated with atherosclerosis development in ApoE-/- mice. To better understand the mechanisms behind this association, we conducted in vitro and in vivo studies looking at the effect of TMAO on different steps of atherosclerotic disease progression. Methods: J774 mouse macrophage cells were used to evaluate the effect of TMAO on foam cell formation. Male ApoE-/- mice transfected with human cholesteryl ester transfer protein (hCETP) were fed L-carnitine and/or methimazole, a flavin monooxygenase 3 (FMO3) inhibitor that prevents the formation of TMAO. Following 12 week treatment, L-carnitine and TMAO plasma levels, aortic lesion development, and lipid profiles were determined. Results: TMAO at concentrations up to 10-fold the Cmax reported in humans did not affect in vitro foam cell formation. In ApoE-/-mice expressing hCETP, high doses of L-carnitine resulted in a significant increase in plasma TMAO levels. Surprisingly, and independently from treatment group, TMAO levels inversely correlated with aortic lesion size in both aortic root and thoracic aorta. High TMAO levels were found to significantly correlate with smaller aortic lesion area. Plasma lipid and lipoprotein levels did not change with treatment nor with TMAO levels, suggesting that the observed effects on lesion area were independent from lipid changes. Conclusion: These findings suggest that TMAO slows aortic lesion formation in this mouse model and may have a protective effect against atherosclerosis development in humans.
KEYWORDS: Atherosclerosis; Cardiovascular disease; Gut microbiota; TMAO; l-carnitine
About two years ago, Robert Koeth and his colleagues (1) published a paper linking atherosclerosis and increased cardiac disease risks to Trimethylamine N-oxide (TMAO), a degradation product of dietary quaternary ammonium compounds such as L-carnitine, Betaine and Choline. When these compounds are not completely absorbed into the intestine, bacterial gut metabolizes them to TMA (Trimethylamine) which is absorbed to the blood and further metabolized by the liver flavin-containing mono-oxygenases (FMOs) to TMAO.
Figure 1. Effects of TMAO on macrophage cholesterol loading and efflux. A) Total, free, and esterified cholesterol content was evaluated in J774 mouse macrophage cells treated with the indicated concentrations of TMAO. Cells were incubated with acetylated LDL (12 μg/ml), and increasing concentrations of TMAO for 24 h. Following treatments, cholesterol mass (free cholesterol, FC or cholesteryl ester, CE) concentrations (μg/mg cell protein) were determined. B) Cholesterol efflux was evaluated using ApoAI and HDL3 as acceptors. J774 cells were treated with the indicated concentrations of TMAO and efflux to media containing either 20 μg/ml human apoA-I or20 μg/ml human HDL3, for 4 h was measured. C) The mean ± SD protein content (mg/well) for each group is also depicted in the table to assess the toxicity of each treatment. TMAO; trimethylamine N-oxide.
Koeth’s observation was based on 1- clinical association between L-carnitine levels increased incidence of major cardiac events, 2- on increased lesion formation in ApoE-/- mouse, a disease model used to study atherosclerosis. The conclusion was that TMAO promotes atherosclerosis. Although the association is established, the cause to effect cannot be clarified given the lack of dose response in this mouse model (a single dose has been used) and the small number of animals in the treatment group making the difference between treatments (3 out of 11 animals). In addition, TMAO has been described to play the role of a molecular chaperone, preventing the protein unfolding. TMAO is also found in fish where it plays an important role in maintaining a normal osmolality. Fish is reported otherwise to be a healthy food source and its consumption is not linked to atherosclerosis occurrence.
Lonza decided to investigate the mechanism (s) behind these observations. First, we showed that increasing TMAO levels up to 10-fold the Cmax as reported in humans, did not affect the foam cell formation in vitro, an obligatory step in the atherosclerosis disease progression (2) (Figure 1). Second, we used an improved version of the ApoE-/- mouse model, expressing the cholesteryl ester transport protein (CETP) lacking normally in rodents. The CETP plays a major role in the cholesterol re-cycling in humans and its inhibition has been studied as a target for atherosclerosis treatment. Further, we used different doses of dietary L-carnitine, leading to different levels of TMAO in an attempt to establish a dose-response curve. We found that TMAO levels inversely correlated with the lesion size (Figure 2.). Significantly reduced aortic lesion size was observed at high levels of TMAO. These effects were independent from lipid changes. These observations suggest that TMAO may play a protective role in atherosclerosis disease development by reducing the lesion formation. When the lesions start developing, the TMAO levels would be up-regulated by a compensatory mechanism (possibly by increasing FMOs expression levels).
Figure 2: Effect of TMAO production on aortic lesions in vivo. Following 12 weeks of treatment, animals were sacrificed for aortic lesion assessments. A) Sections (∼5 mm) of the aortic root were cut, followed by hematoxylin-eosin staining and subsequent Nikon imaging. The lesion area (μm2) for each treatment group was then plotted as the mean ± SD from 15 animals. B) A correlation analysis for all treatments between TMAO and aortic root lesion area was conducted (n = 74 animals). C) A correlation analysis between l-carnitine (352 mg/kg)-induced TMAO levels and aortic root lesion area versus vehicle-treated mice was conducted (n = 28 mice). D) Thoracic aortas were isolated, formalin fixed, and stained with Sudan IV for en face analysis using a Nikon computerized imaging system. The % lesion area in thoracic aortas for each treatment group was then expressed as the mean ± SEM from 14 animals. E) A correlation analysis between % lesion area in thoracic aorta and TMAO concentration was conducted for all treatment points (n = 73). F) A correlation analysis between the thoracic aorta % lesion area and very low (<0.05 ppm), low (<0.1 ppm), moderate (0.1–0.2 ppm), and high (>0.2 ppm) TMAO levels was conducted. The results are expressed as the mean ± SEM from 73 animals. TMAO; trimethylamine N-oxide.
1- Koeth, R. A., Wang, Z., Levison, B. S., Buffa, J. A., Org, E., Sheehy, B. T. et al. (2013). Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis. Nat.Med., 19, 576-585
2- Bellamine A. TMAO Treatment Does Not Affect Foam Cell Formation (Cholesterol Mass Loading) and Cholesterol Efflux in J774 Mouse Macrophage. Experimental Biology meeting, San Diego, 28 April 2014