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Choline and butyrate beneficially modulate the gut microbiome without affecting atherosclerosis in APOE*3-Leiden.CETP mice

  • Cong Liu
    Affiliations
    Department of Medicine, Division of Endocrinology, Leiden University Medical Center, Leiden, the Netherlands

    Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, the Netherlands
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  • Zhuang Li
    Affiliations
    Department of Medicine, Division of Endocrinology, Leiden University Medical Center, Leiden, the Netherlands

    Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, the Netherlands
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  • Zikuan Song
    Affiliations
    Department of Medicine, Division of Endocrinology, Leiden University Medical Center, Leiden, the Netherlands

    Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, the Netherlands
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  • Xiayue Fan
    Affiliations
    Med-X Institute, Center for Immunological and Metabolic Diseases, Department of Endocrinology, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an Jiaotong University, Xi'an, China
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  • Hua Shao
    Affiliations
    Med-X Institute, Center for Immunological and Metabolic Diseases, Department of Endocrinology, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an Jiaotong University, Xi'an, China
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  • Milena Schönke
    Affiliations
    Department of Medicine, Division of Endocrinology, Leiden University Medical Center, Leiden, the Netherlands

    Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, the Netherlands
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  • Mariëtte R. Boon
    Affiliations
    Department of Medicine, Division of Endocrinology, Leiden University Medical Center, Leiden, the Netherlands

    Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, the Netherlands
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  • Patrick C.N. Rensen
    Affiliations
    Department of Medicine, Division of Endocrinology, Leiden University Medical Center, Leiden, the Netherlands

    Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, the Netherlands

    Med-X Institute, Center for Immunological and Metabolic Diseases, Department of Endocrinology, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an Jiaotong University, Xi'an, China
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  • Yanan Wang
    Correspondence
    Corresponding author. Department of Endocrinology, The First Affiliated Hospital of Xi'an Jiaotong University, 277 West Yanta Road, Xi'an, Shaanxi, China.
    Affiliations
    Department of Medicine, Division of Endocrinology, Leiden University Medical Center, Leiden, the Netherlands

    Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, the Netherlands

    Med-X Institute, Center for Immunological and Metabolic Diseases, Department of Endocrinology, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an Jiaotong University, Xi'an, China
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Open AccessPublished:October 18, 2022DOI:https://doi.org/10.1016/j.atherosclerosis.2022.10.009

      Highlights

      • Choline does not aggravate atherosclerosis development in APOE*3-Leiden.CETP mice.
      • Co-administration of choline and butyrate increases plasma trimethylamine-N-oxide (TMAO) levels.
      • Plasma TMAO levels do not associate with atherosclerotic lesion size.

      Abstract

      Background and aims

      Choline has been shown to exert atherogenic effects in Apoe−/− and Ldlr−/− mice, related to its conversion by gut bacteria into trimethylamine (TMA) that is converted by the liver into the proinflammatory metabolite trimethylamine-N-oxide (TMAO). Since butyrate beneficially modulates the gut microbiota and has anti-inflammatory and antiatherogenic properties, the aim of the present study was to investigate whether butyrate can alleviate choline-induced atherosclerosis. To this end, we used APOE*3-Leiden.CETP mice, a well-established atherosclerosis-prone model with human-like lipoprotein metabolism.

      Methods

      Female APOE*3-Leiden.CETP mice were fed an atherogenic diet alone or supplemented with choline, butyrate or their combination for 16 weeks.

      Results

      Interestingly, choline protected against fat mass gain, increased the abundance of anti-inflammatory gut microbes, and increased the expression of gut microbial genes involved in TMA and TMAO degradation. Butyrate similarly attenuated fat mass gain and beneficially modulated the gut microbiome, as shown by increased abundance of anti-inflammatory and short chain fatty acid-producing microbes, and inhibited expression of gut microbial genes involved in lipopolysaccharide synthesis. Both choline and butyrate upregulated hepatic expression of flavin-containing monooxygenases, and their combination resulted in highest circulating TMAO levels. Nonetheless, choline, butyrate and their combination did not influence atherosclerosis development, and TMAO levels were not associated with atherosclerotic lesion size.

      Conclusions

      While choline and butyrate have been reported to oppositely modulate atherosclerosis development in Apoe−/− and Ldlr−/− mice as related to changes in the gut microbiota, both dietary constituents did not affect atherosclerosis development while beneficially modulating the gut microbiome in APOE*3-Leiden.CETP mice.

      Graphical abstract

      Keywords

      1. Introduction

      Atherosclerosis, the main underlying cause of cardiovascular diseases (CVD), is a chronic disease arising from an imbalanced cholesterol metabolism and a maladaptive immune response [
      • Back M.
      • Yurdagul Jr., A.
      • Tabas I.
      • Oorni K.
      • Kovanen P.T.
      Inflammation and its resolution in atherosclerosis: mediators and therapeutic opportunities.
      ]. Hypercholesterolemia induces the retention of apolipoprotein (Apo) B-containing cholesterol-rich lipoproteins in the arterial intima, which triggers infiltration of circulating monocytes into the intima, induces cholesterol-laden foam cell formation and accumulation, and subsequently initiates and aggravates atherosclerosis [
      • Back M.
      • Yurdagul Jr., A.
      • Tabas I.
      • Oorni K.
      • Kovanen P.T.
      Inflammation and its resolution in atherosclerosis: mediators and therapeutic opportunities.
      ]. Therefore, cholesterol-lowering therapy potently reduces morbidity and mortality of atherosclerotic CVD (asCVD) [
      • Hegele R.A.
      • Gidding S.S.
      • Ginsberg H.N.
      • McPherson R.
      • Raal F.J.
      • Rader D.J.
      • Robinson J.G.
      • Welty F.K.
      Nonstatin low-density lipoprotein-lowering therapy and cardiovascular risk reduction-statement from ATVB Council.
      ]. Yet, a significant burden of asCVD remains, at least partly due to the residual inflammatory risk [
      • Aday A.W.
      • Ridker P.M.
      Targeting residual inflammatory risk: a shifting paradigm for atherosclerotic disease.
      ]. Therefore, there is an urgent need to search for additional therapeutic targets which govern atherogenesis, particularly those regulating both cholesterol metabolism and inflammation.
      Several lines of evidence have linked the gut microbiota to atherogenesis [
      • Jonsson A.L.
      • Backhed F.
      Role of gut microbiota in atherosclerosis.
      ,
      • Tang W.H.W.
      • Li D.Y.
      • Hazen S.L.
      Dietary metabolism, the gut microbiome, and heart failure.
      ]. The gut microbiota is mainly shaped by dietary factors. Bacteria in the gut can metabolize complex dietary components to generate various functional small-molecule metabolites [
      • Jonsson A.L.
      • Backhed F.
      Role of gut microbiota in atherosclerosis.
      ,
      • Tang W.H.W.
      • Li D.Y.
      • Hazen S.L.
      Dietary metabolism, the gut microbiome, and heart failure.
      ]. Trimethylamine N-oxide (TMAO) is a gut-derived metabolite that has been described to aggravate atherosclerosis in Apoe−/− and Ldlr−/− mice [
      • Wang Z.
      • Klipfell E.
      • Bennett B.J.
      • Koeth R.
      • Levison B.S.
      • Dugar B.
      • Feldstein A.E.
      • Britt E.B.
      • Fu X.
      • Chung Y.M.
      • et al.
      Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease.
      ,
      • Seldin M.M.
      • Meng Y.
      • Qi H.
      • Zhu W.
      • Wang Z.
      • Hazen S.L.
      • Lusis A.J.
      • Shih D.M.
      Trimethylamine N-oxide promotes vascular inflammation through signaling of mitogen-activated protein kinase and nuclear factor-kappaB.
      ]. Gut microbiota generate TMAO from dietary choline via a two-step meta-organismal pathway to first produce trimethylamine (TMA) that is delivered via the portal vein to the liver where it can be rapidly oxidized into TMAO by flavin monooxygenases (FMOs). TMAO aggravates atherosclerosis via various mechanisms, such as promoting foam cell formation and activation of the inflammatory response [
      • Wang Z.
      • Klipfell E.
      • Bennett B.J.
      • Koeth R.
      • Levison B.S.
      • Dugar B.
      • Feldstein A.E.
      • Britt E.B.
      • Fu X.
      • Chung Y.M.
      • et al.
      Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease.
      ,
      • Collins H.L.
      • Drazul-Schrader D.
      • Sulpizio A.C.
      • Koster P.D.
      • Williamson Y.
      • Adelman S.J.
      • Owen K.
      • Sanli T.
      • Bellamine A.
      L-Carnitine intake and high trimethylamine N-oxide plasma levels correlate with low aortic lesions in ApoE(-/-) transgenic mice expressing CETP.
      ]. Hence, TMAO-lowering interventions may have therapeutic potential to reduce asCVD risk. Interestingly, butyrate, a short chain fatty acid (SCFA), has been shown to protect against atherosclerosis in Apoe−/− and Ldlr−/− mice [
      • Kasahara K.
      • Krautkramer K.A.
      • Org E.
      • Romano K.A.
      • Kerby R.L.
      • Vivas E.I.
      • Mehrabian M.
      • Denu J.M.
      • Backheds F.
      • Lusis A.
      • et al.
      Interactions between Roseburia intestinalis and diet modulate atherogenesis in a murine model.
      ,
      • Chen Y.
      • Xu C.
      • Huang R.
      • Song J.
      • Li D.
      • Xia M.
      Butyrate from pectin fermentation inhibits intestinal cholesterol absorption and attenuates atherosclerosis in apolipoprotein E-deficient mice.
      ,
      • Du Y.
      • Li X.
      • Su C.
      • Xi M.
      • Zhang X.
      • Jiang Z.
      • Wang L.
      • Hong B.
      Butyrate protects against high-fat diet-induced atherosclerosis via up-regulating ABCA1 expression in apolipoprotein E-deficiency mice.
      ,
      • Brandsma E.
      • Kloosterhuis N.J.
      • Koster M.
      • Dekker D.C.
      • Gijbels M.J.J.
      • van der Velden S.
      • Rios-Morales M.
      • van Faassen M.J.R.
      • Loreti M.G.
      • de Bruin A.
      • et al.
      A proinflammatory gut microbiota increases systemic inflammation and accelerates atherosclerosis.
      ]. This is in part mediated through its action on the gut microbiota, since butyrate suppresses the overgrowth of pathogenic gut microbes, inhibits the synthesis of endotoxins, and prevents bacterial translocation. As a result, butyrate alleviates systemic inflammation, thereby halting atherosclerosis progression [
      • Kasahara K.
      • Krautkramer K.A.
      • Org E.
      • Romano K.A.
      • Kerby R.L.
      • Vivas E.I.
      • Mehrabian M.
      • Denu J.M.
      • Backheds F.
      • Lusis A.
      • et al.
      Interactions between Roseburia intestinalis and diet modulate atherogenesis in a murine model.
      ].
      Based on the hypothesis that butyrate may be able to protect against dietary choline-induced atherosclerosis development, the aim of the present study was to examine the effects of choline, butyrate and their combination on atherosclerosis in APOE*3-Leiden.CETP mice, a well-established translational model for human-like lipoprotein metabolism. In contrast to our expectations, we demonstrate that both choline and butyrate beneficially modulate the gut microbiome without affecting atherosclerosis development, and TMAO levels were not associated with atherosclerotic lesion size.

      2. Materials and methods

      For details of animals and antibodies used, please see the Major Resources Table in the Supplementary Materials.

      2.1 Mice

      Female APOE*3-Leiden.CETP mice were generated as previously described [
      • Westerterp M.
      • van der Hoogt C.C.
      • de Haan W.
      • Offerman E.H.
      • Dallinga-Thie G.M.
      • Jukema J.W.
      • Havekes L.M.
      • Rensen P.C.
      Cholesteryl ester transfer protein decreases high-density lipoprotein and severely aggravates atherosclerosis in APOE*3-Leiden mice.
      ]. Mice at the age of 8–12 weeks were housed under standard conditions (22 °C; 12/12-h light/dark cycle) with ad libitum access to water and a cholesterol-containing Western-type diet (WTD; 0.15% cholesterol and 16% fat; ssniff, Soest, Germany). All mice were acclimatized to housing and WTD for 3 weeks prior to the dietary intervention. Then, based on 4-h fasted plasma lipid levels, body weight as well as body composition, these mice were randomized to 4 treatment groups using RandoMice [
      • van Eenige R.
      • Verhave P.S.
      • Koemans P.J.
      • Tiebosch I.
      • Rensen P.C.N.
      • Kooijman S.
      RandoMice, a novel, user-friendly randomization tool in animal research.
      ] (n = 17 per group) receiving either WTD (ctrl group), WTD + choline (1.2% w/w, according to previous studies [
      • Wang Z.
      • Klipfell E.
      • Bennett B.J.
      • Koeth R.
      • Levison B.S.
      • Dugar B.
      • Feldstein A.E.
      • Britt E.B.
      • Fu X.
      • Chung Y.M.
      • et al.
      Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease.
      ,
      • Tang W.H.
      • Wang Z.
      • Levison B.S.
      • Koeth R.A.
      • Britt E.B.
      • Fu X.
      • Wu Y.
      • Hazen S.L.
      Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk.
      ]; choline group), WTD + butyrate (5% w/w, according to previous studies [
      • Li Z.
      • Yi C.X.
      • Katiraei S.
      • Kooijman S.
      • Zhou E.
      • Chung C.K.
      • Gao Y.
      • van den Heuvel J.K.
      • Meijer O.C.
      • Berbee J.F.P.
      • et al.
      Butyrate reduces appetite and activates brown adipose tissue via the gut-brain neural circuit.
      ,
      • Gao Z.
      • Yin J.
      • Zhang J.
      • Ward R.E.
      • Martin R.J.
      • Lefevre M.
      • Cefalu W.T.
      • Ye J.
      Butyrate improves insulin sensitivity and increases energy expenditure in mice.
      ]; butyrate group) or WTD + butyrate + choline (1.2% w/w choline and 5% w/w butyrate; butyrate + choline group) for 16 weeks according to a well-established protocol in our group [
      • Liu C.
      • Schonke M.
      • Zhou E.
      • Li Z.
      • Kooijman S.
      • Boon M.R.
      • Larsson M.
      • Wallenius K.
      • Dekker N.
      • Barlind L.
      • et al.
      Pharmacological treatment with FGF21 strongly improves plasma cholesterol metabolism to reduce atherosclerosis.
      ,
      • Berbee J.F.
      • Boon M.R.
      • Khedoe P.P.
      • Bartelt A.
      • Schlein C.
      • Worthmann A.
      • Kooijman S.
      • Hoeke G.
      • Mol I.M.
      • John C.
      • et al.
      Brown fat activation reduces hypercholesterolaemia and protects from atherosclerosis development.
      ,
      ]. The sample size was calculated based on the average atherosclerotic lesion area of 1 × 105 μm [
      • Hegele R.A.
      • Gidding S.S.
      • Ginsberg H.N.
      • McPherson R.
      • Raal F.J.
      • Rader D.J.
      • Robinson J.G.
      • Welty F.K.
      Nonstatin low-density lipoprotein-lowering therapy and cardiovascular risk reduction-statement from ATVB Council.
      ] in the ctrl group with a standard deviation lesion size of 0.3 × 105 μm [
      • Hegele R.A.
      • Gidding S.S.
      • Ginsberg H.N.
      • McPherson R.
      • Raal F.J.
      • Rader D.J.
      • Robinson J.G.
      • Welty F.K.
      Nonstatin low-density lipoprotein-lowering therapy and cardiovascular risk reduction-statement from ATVB Council.
      ]. We considered a difference in plaque size of 30% to be biologically relevant. To achieve the differences with α = 5% and a power of 80%, 17 animals per group were therefore needed. Mice were group housed (4–5 per cage) during the experimental period to avoid stress caused by single housing. All animal experiments were performed in accordance with the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals, and were approved by the National Committee for Animal Experiments and by the Ethics Committee on Animal Care (Protocol No. AVD1160020172927) and Experimentation of the Leiden University Medical Center (Protocol No. PE.18.063.006). All animal procedures were conform the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animal used for scientific purposes.

      2.2 Body weight and body composition

      Body weight was measured weekly with a scale, and body composition of conscious mice was measured biweekly using an EchoMRI-100 analyzer (EchoMRI, Houston, TX, USA).

      2.3 Plasma lipid profiles and choline metabolites

      Every 4 weeks, after 4 h of fasting (9:00–13:00), tail vein blood was collected into paraoxon-coated glass capillaries. These capillaries were placed on ice and centrifuged, and plasma was collected and stored at −20 °C. Plasma total cholesterol (TC) and triglyceride (TG) levels were determined (n = 17 per group) using commercial enzymatic kits from Roche Diagnostics (Mannheim, Germany). Plasma high density lipoprotein cholesterol (HDL-C) and non-HDL-C levels (n = 17 per group) were determined as previously described [
      • Berbee J.F.
      • Boon M.R.
      • Khedoe P.P.
      • Bartelt A.
      • Schlein C.
      • Worthmann A.
      • Kooijman S.
      • Hoeke G.
      • Mol I.M.
      • John C.
      • et al.
      Brown fat activation reduces hypercholesterolaemia and protects from atherosclerosis development.
      ]. At week 16, plasma choline, betaine, TMA and TMAO levels were quantified (n = 10 per group) using liquid chromatography-tandem mass spectrometry as described under Expanded Methods in the Supplementary Materials.

      2.4 Gene expression

      Total RNA was extracted from snap-frozen tissues using the Tripure RNA isolation reagent (Roche, Mijdrecht, The Netherlands), according to the manufacturer's instructions. Complementary DNA for quantitative reverse transcriptase-PCR was generated as previously described [
      • Liu C.
      • Schonke M.
      • Zhou E.
      • Li Z.
      • Kooijman S.
      • Boon M.R.
      • Larsson M.
      • Wallenius K.
      • Dekker N.
      • Barlind L.
      • et al.
      Pharmacological treatment with FGF21 strongly improves plasma cholesterol metabolism to reduce atherosclerosis.
      ]. The expression of mRNA was normalized to Actb and Rplp0 mRNA levels and expressed as fold change compared with the ctrl group. The primer sequences are listed in the Supplementary Materials.

      2.5 Genomic DNA extraction and metagenomic sequencing

      At week 16, cecal contents were collected, and genomic bacterial DNA was isolated with the fast DNA stool mini kit (QIAamp, Germany) following the manufacturer's instructions. Then, these DNA samples were used for determination of gut microbial gene expression via qPCR as well as metagenomics sequencing. Sequencing data was processed as described under Expanded Methods in the Supplementary Materials.

      2.6 Atherosclerotic plaque characterization and quantification

      Hearts were collected and fixated in phosphate-buffered 4% formaldehyde, embedded in paraffin after dehydration in 70–100% ethanol and cross-sectioned (5 μm) perpendicular to the axis of the aorta throughout the aortic root area, starting from the appearance of open aortic valve leaflets. Per mouse, 4 sections with 50 μm intervals were used for atherosclerosis measurements. Sections were stained with haematoxylin-phloxine-saffron for histological analysis. Lesions were categorized by severity according to the guidelines of the American Heart Association adapted for mice [
      • Wong M.C.
      • van Diepen J.A.
      • Hu L.
      • Guigas B.
      • de Boer H.C.
      • van Puijvelde G.H.
      • Kuiper J.
      • van Zonneveld A.J.
      • Shoelson S.E.
      • Voshol P.J.
      • et al.
      Hepatocyte-specific IKKbeta expression aggravates atherosclerosis development in APOE*3-Leiden mice.
      ]. Sirius Red staining was used to quantify the collagen area. Monoclonal mouse antibody M0851 (1:800; Dako, Heverlee, The Netherlands) against smooth muscle cell actin was used to quantify the smooth muscle cell area. Rat monoclonal anti-mouse MAC-3 antibody (1:1000; BD Pharmingen, San Diego, CA, USA) was used to quantify the macrophage area. Immunostainings were amplified using Vector Laboratories Elite ABC kit (Vector Laboratories Inc., Burlingame, CA, USA) and the immune-peroxidase complex was visualized with Nova Red (Vector Laboratories Inc., Burlingame, CA, USA). Lesion area and composition were analyzed using Image J software. The stability index was calculated by dividing the relative collagen and smooth muscle cell area by the relative area of macrophages within the same lesion.

      2.7 Statistical analyses

      Statistical analyses among these 4 groups were assessed using One-way ANOVA followed by a Fisher's LSD post hoc test, unless indicated otherwise. The square root of the lesion area was taken to linearize the relationship with the plasma TC, non-HDL-C, HDL-C and TG exposures and plasma TMAO levels (at 16 week). To assess significant correlations between atherosclerotic lesion size and plasma lipids and TMAO, univariate regression analyses were performed. Then, to predict the contribution of these plasma parameters to the atherosclerotic lesion size, multiple regression analysis was performed. Data are presented as mean ± SEM, and a p value less than 0.05 is considered statistically significant. All statistical analyses were performed with GraphPad Prism 9 (GraphPad Software Inc., California, CA, USA) except for univariate and multiple regression analyses which were performed with SPSS 20.0 (SPSS, Chicago, IL USA) for Windows and metagenomic data analysis using R packages.

      3. Results

      3.1 Choline and butyrate attenuate WTD-induced fat mass gain in APOE*3-Leiden.CETP mice

      To address how choline and butyrate affect atherosclerosis in a mouse model for human-like lipoprotein metabolism, we fed female APOE*3-Leiden.CETP mice a cholesterol-containing WTD alone or supplemented with choline, butyrate or a combination of both supplements for 16 weeks (Fig. 1A). Neither choline nor butyrate affected food intake (Fig. 1B). Despite this, choline and butyrate attenuated WTD-induced body weight gain (Fig. 1C), which was explained by reduced fat mass gain (Fig. 1D) without affecting body lean mass (Supplementary Fig. 1).
      Fig. 1
      Fig. 1Choline and butyrate attenuate WTD-induced fat mass gain in APOE*3-Leiden.CETP mice.
      (A) Experimental set up. (B) Cumulative food intake (n = 4–5 per group), (C) body weight (n = 16–17 per group), and (D) body fat mass (n = 16–17 per group) were monitored throughout the experimental period. Data are shown as mean ± SEM. Differences were assessed using one-way ANOVA followed by a Fisher's LSD post-test. *p < 0.05; **p < 0.01, ***p < 0.001, compared with the control (ctrl) group. WTD, Western-type diet.

      3.2 Choline and butyrate beneficially modulate the gut microbiome in APOE*3-Leiden-CETP mice

      Previous studies have shown that the gut microbiota participates in choline- and butyrate-induced modulation of body weight and atherogenesis [
      • Li Z.
      • Yi C.X.
      • Katiraei S.
      • Kooijman S.
      • Zhou E.
      • Chung C.K.
      • Gao Y.
      • van den Heuvel J.K.
      • Meijer O.C.
      • Berbee J.F.P.
      • et al.
      Butyrate reduces appetite and activates brown adipose tissue via the gut-brain neural circuit.
      ,
      • Agus A.
      • Clement K.
      • Sokol H.
      Gut microbiota-derived metabolites as central regulators in metabolic disorders.
      ,
      • Schugar R.C.
      • Gliniak C.M.
      • Osborn L.J.
      • Massey W.
      • Sangwan N.
      • Horak A.
      • Banerjee R.
      • Orabi D.
      • Helsley R.N.
      • Brown A.L.
      • et al.
      Gut microbe-targeted choline trimethylamine lyase inhibition improves obesity via rewiring of host circadian rhythms.
      ]. We thus performed whole metagenome shotgun sequencing to assess the impact of choline, butyrate and their combination on the gut microbiota composition and function. While principal component analysis revealed great similarities of the gut microbiome structure among the groups (Fig. 2A), choline reduced the gut microbial α diversity, regardless of butyrate supplementation (Fig. 2B). At the phylum level, most gut commensal microbes belonged to Bacteroidetes, Firmicutes, Proteobacteria, which, along with Actinobacteria and Verrucomicrobia, represented approximately 95% of the total microbial community (Supplementary Fig. 2A). At the species level, Faecalibaculum rodentium (F. rodentium), Parabacteroides distasonis (P. distasonis) and Bacteroides uniforms (B. uniforms) were abundant among the groups (Fig. 2C). As compared to control treatment, butyrate enriched species with proposed anti-inflammatory properties, such as Duncaniella spB8 (D. spB8) [
      • Forster S.C.
      • Clare S.
      • Beresford-Jones B.S.
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      Identification of gut microbial species linked with disease variability in a widely used mouse model of colitis.
      ,
      • Feng P.
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      • Tao Y.
      • Huang P.
      • Wang P.
      Crocetin prolongs recovery period of DSS-induced colitis via altering intestinal microbiome and increasing intestinal permeability.
      ] and Blautia producta (B. producta) [
      • Aoki R.
      • Onuki M.
      • Hattori K.
      • Ito M.
      • Yamada T.
      • Kamikado K.
      • Kim Y.G.
      • Nakamoto N.
      • Kimura I.
      • Clarke J.M.
      • et al.
      Commensal microbe-derived acetate suppresses NAFLD/NASH development via hepatic FFAR2 signalling in mice.
      ], Faecalibaculum prausnitzii (F. prausnitzii) [
      • Machiels K.
      • Joossens M.
      • Sabino J.
      • De Preter V.
      • Arijs I.
      • Eeckhaut V.
      • Ballet V.
      • Claes K.
      • Van Immerseel F.
      • Verbeke K.
      • et al.
      A decrease of the butyrate-producing species Roseburia hominis and Faecalibacterium prausnitzii defines dysbiosis in patients with ulcerative colitis.
      ,
      • Olsson L.M.
      • Boulund F.
      • Nilsson S.
      • Khan M.T.
      • Gummesson A.
      • Fagerberg L.
      • Engstrand L.
      • Perkins R.
      • Uhlen M.
      • Bergstrom G.
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      ] and Ruthenibacterium lactatiformans (R. lactatiformans) [
      • Shkoporov A.N.
      • Chaplin A.V.
      • Shcherbakova V.A.
      • Suzina N.E.
      • Kafarskaia L.I.
      • Bozhenko V.K.
      • Efimov B.A.
      Ruthenibacterium lactatiformans gen. nov., sp. nov., an anaerobic, lactate-producing member of the family Ruminococcaceae isolated from human faeces.
      ] (Fig. 2D). Simultaneously, butyrate downregulated gut microbial genes involved in lipopolysaccharide (LPS) biosynthesis when compared to control treatment (Fig. 2E, and Supplementary Fig. 2B).
      Fig. 2
      Fig. 2Choline and butyrate beneficially modulate the gut microbiome in APOE*3-Leiden-CETP mice.
      At the end of the study, the cecal content was collected and sequenced using metagenomics sequencing (n = 10 per group). (A) Principal component analysis (PCA) at the species level. (B) The Shannon index at the species level. (C) The abundance of the top 15 microbial species. (D and F) Linear discrimination analysis (LDA) effect size analysis was performed, and LDA scores calculated for differences in species-level abundance between groups. (E and I) Relative changes of the gut microbial genes involved in lipopolysaccharide (LPS) signaling and the metabolic pathway of TMA. (G) Top 30 significantly regulated KEGG pathways between the ctrl and choline groups. (H) Relative gut microbial choline TMA-lyase (CutC) gene expression. (B, H) Data are represented as means ± SEM. Differences were assessed using one-way ANOVA followed by a Fisher's LSD post-test. (E, G, I) Comparisons between groups were performed using Wilcoxon test. *p < 0.05; **p < 0.01, ***p < 0.001, compared with the ctrl group. ##p < 0.01, compared to the butyrate group.
      In addition, choline increased several bacterial species compared to control treatment, including a probiotic microbe Lactobacillus reuteria (L. reuteria) [
      • Zhou Q.
      • Wu F.
      • Chen S.
      • Cen P.
      • Yang Q.
      • Guan J.
      • Cen L.
      • Zhang T.
      • Zhu H.
      • Chen Z.
      Lactobacillus reuteri improves function of the intestinal barrier in rats with acute liver failure through Nrf-2/HO-1 pathway.
      ] and three anti-inflammatory species of the Olsenella genus [
      • Mager L.F.
      • Burkhard R.
      • Pett N.
      • Cooke N.C.A.
      • Brown K.
      • Ramay H.
      • Paik S.
      • Stagg J.
      • Groves R.A.
      • Gallo M.
      • et al.
      Microbiome-derived inosine modulates response to checkpoint inhibitor immunotherapy.
      ] (Fig. 2F). Interestingly, choline treatment did not affect TMA-producing bacteria, had no impact on gut microbial genes associated with TMA synthesis (Fig. 2G), and did not affect the expression of choline trimethylamine-lyase (CutC), an essential bacterial choline TMA-lyase gene (Fig. 2H). Rather, choline treatment upregulated gene expression of enzymes involved in TMA and TMAO degradation, including TMA corrinoid protein and TMAO reductase (Fig. 2I), effects that were blunted upon concomitant butyrate administration (Supplementary Figs. 2C–E). Moreover, choline treatment upregulated gut microbial genes involved in starch and sugar metabolism (Fig. 2G), which are associated with SCFA production [
      • Arpaia N.
      • Campbell C.
      • Fan X.
      • Dikiy S.
      • van der Veeken J.
      • deRoos P.
      • Liu H.
      • Cross J.R.
      • Pfeffer K.
      • Coffer P.J.
      • et al.
      Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation.
      ]. Furthermore, the combination group shared greater similarities in the gut composition and function compared to the choline group than the butyrate group (Supplementary Figs. 3A–E). Taken together, both choline and butyrate beneficially affected the gut microbial composition and function in WTD-fed APOE*3-Leiden-CETP mice, with a greater impact on the gut microbiome induced by choline versus butyrate.

      3.3 Co-administration of choline and butyrate, instead of choline or butyrate alone, increases plasma TMAO levels in APOE*3-Leiden-CETP mice

      Then, we investigated the role of dietary choline and butyrate in choline metabolism. To this end, we performed targeted metabolomics analyses to measure choline-related metabolites in plasma. Dietary choline increased plasma levels of choline and its oxidation product betaine (Fig. 3A and B) without affecting plasma TMA levels (Fig. 3C), while butyrate did not alter the levels of choline and its metabolites (Fig. 3A–D). Of note, only combination treatment increased TMAO levels (Fig. 3D). We observed that both choline and butyrate upregulated the hepatic expression of Fmos (i.e., Fmo2 and Fmo3; Fig. 3E). Co-administration of choline and butyrate caused the highest expression of Fmo3 in the liver (Fig. 3E), which may explain the highest plasma TMAO levels in the combination group.
      Fig. 3
      Fig. 3Co-administration of choline and butyrate, instead of choline or butyrate alone, increases plasma TMAO levels in APOE*3-Leiden-CETP mice.
      At week16, plasma levels of (A) choline, (B) betaine, (C) TMA and (D) TMAO were determined. (E) At the end of the study, the relative mRNA expression of flavin-containing monooxygenases (Fmos) was determined in the liver. Data are shown as mean ± SEM (n = 10 per group). Differences were assessed using one-way ANOVA followed by a Fisher's LSD post hoc test. *p < 0.05; **p < 0.01, ***p < 0.001, compared with the ctrl group; #p < 0.05, ##p < 0.01, ###p < 0.001, compared to the butyrate group; $$p < 0.01, $$$p < 0.001, compared to the choline group. Fmos, flavin monooxygenases; TMA, trimethylamine; TMAO, trimethylamine N-oxide.

      3.4 Choline and butyrate do not affect plasma lipid levels in APOE*3-Leiden.CETP mice

      Choline and butyrate have been suggested to modulate reverse cholesterol transport (RCT) [
      • Du Y.
      • Li X.
      • Su C.
      • Xi M.
      • Zhang X.
      • Jiang Z.
      • Wang L.
      • Hong B.
      Butyrate protects against high-fat diet-induced atherosclerosis via up-regulating ABCA1 expression in apolipoprotein E-deficiency mice.
      ,
      • Koeth R.A.
      • Wang Z.
      • Levison B.S.
      • Buffa J.A.
      • Org E.
      • Sheehy B.T.
      • Britt E.B.
      • Fu X.
      • Wu Y.
      • Li L.
      • et al.
      Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis.
      ]. However, choline upregulated to only some extent the expression of genes involved in HDL assembly (e.g. ATP-binding cassette subfamily A member 1, Abca1; Fig. 4A), HDL uptake (e.g. scavenger receptor class B type 1, Srb-1; Fig. 4B), bile acid secretion (e.g. bile salt export pump, Bsep; Fig. 4C), sterol secretion (e.g. ATP-binding cassette transporter G member 5, Abcg5; Fig. 4D), Ldlr and CETP (Fig. 4E). And, butyrate had minor impact on the expression of these genes (Fig. 4A–E). Similarly, neither choline nor butyrate had any evident effects on levels of plasma TG, TC, HDL-C and non-HDL-C (Supplementary Figs. 4A–B, and 4F-G).
      Fig. 4
      Fig. 4Choline and butyrate do not affect plasma lipid levels in APOE*3-Leiden.CETP mice.
      The relative expression of genes involved in high-density lipoprotein (HDL) assembly and clearance (A and B), bile acid synthesis and secretion (C), and sterol secretion (D) was determined. (E) The mRNA levels of low-density lipoprotein receptor and cholesteryl ester transfer protein (CETP) were measured. Fasting plasma levels of total cholesterol (TC; F) and non-high-density lipoprotein cholesterol (non-HDL-C; G) were determined throughout the experimental period. Data are represented as mean ± SEM (A-E, n = 9–10 per group; F-G, n = 16–17 per group). Differences were assessed using one-way ANOVA followed by a Fisher's LSD post hoc test. *p < 0.05; **p < 0.01, ***p < 0.001, compared with the ctrl group; #p < 0.05, compared to the butyrate group. Abca1, ATP-binding cassette subfamily A member 1; Abcg5, ATP-binding cassette transporter G member 5; Bsep, bile salt export pump; Cyp27a1, sterol 27-hydroxylase; Cyp7a1, cholesterol 7α-hydroxylase; Ldlr, low density lipoprotein receptor; Srb-1, scavenger receptor class B type 1.

      3.5 Choline and butyrate have no impact on atherosclerosis development in APOE*3-Leiden-CETP mice

      We next assessed the size, severity and composition of atherosclerotic lesions throughout the aortic root in the heart isolated after 16 weeks of treatment. Neither choline, butyrate nor their combination affected atherosclerotic plaque area, severity and composition (Fig. 5A–D, and Supplementary Fig. 5A-C). In accordance, choline and butyrate had no impact on atherosclerotic plaque stability (Fig. 5E), as calculated from dividing the relative collagen and smooth muscle cell area by the relative area of macrophages within the same lesion. While univariate regression analysis revealed that the atherosclerotic lesion area was to some extent predicted by plasma cholesterol levels (Fig. 5F, and Supplementary Fig. 5D-F), plasma TMAO levels were not associated with atherosclerotic lesion size in APOE*3.Leiden-CETP mice (Fig. 5G).
      Fig. 5
      Fig. 5Choline and butyrate have no impact on atherosclerosis development in APOE*3-Leiden-CETP mice.
      At 16 weeks, hearts were collected, and the valve area in the aortic root was stained with haematoxylin–phloxine–saffron (HPS). To quantify the contents of collagen, smooth muscle cells and macrophages within the lesion, the valve area in the aortic root was stained with Picrosirius red (PSR), anti-α-smooth muscle cell actin (α-SMC actin) antibody and anti-MAC3 antibody, respectively. (A) Representative pictures of every staining. (B) The relationship between atherosclerotic lesion area and the distance from aortic valve was determined by calculating the lesion area of 4 consecutive sections (with 50 μm intervals) beginning with the appearance of open aortic valve leaflets (n = 13–16 per group). Lesions were categorized into undiseased (C), mild, and severe (D) lesions (n = 13–16 per group). (E) The stability index was calculated by dividing the smooth muscle cell and collagen area by macrophage area within the same lesion. The square root (SQRT) of the atherosclerotic lesion area plotted against plasma (F) non-HDL-C exposure and TMAO (G) levels, and linear regression analyses were performed. Data are represented as means ± SEM (B–F, n = 13–16 per group; G, n = 10 per group). Comparisons among four groups were performed using one-way ANOVA followed by a Fisher's LSD post hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

      4. Discussion

      Choline has been reported to aggravate atherosclerosis development, as caused by generation of TMAO through the gut-liver axis [
      • Wang Z.
      • Klipfell E.
      • Bennett B.J.
      • Koeth R.
      • Levison B.S.
      • Dugar B.
      • Feldstein A.E.
      • Britt E.B.
      • Fu X.
      • Chung Y.M.
      • et al.
      Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease.
      ,
      • Koeth R.A.
      • Wang Z.
      • Levison B.S.
      • Buffa J.A.
      • Org E.
      • Sheehy B.T.
      • Britt E.B.
      • Fu X.
      • Wu Y.
      • Li L.
      • et al.
      Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis.
      ], while butyrate beneficially modulates the gut microbiota and shows antiatherogenic effects, in Apoe−/− and Ldlr−/− mice [
      • Kasahara K.
      • Krautkramer K.A.
      • Org E.
      • Romano K.A.
      • Kerby R.L.
      • Vivas E.I.
      • Mehrabian M.
      • Denu J.M.
      • Backheds F.
      • Lusis A.
      • et al.
      Interactions between Roseburia intestinalis and diet modulate atherogenesis in a murine model.
      ,
      • Chen Y.
      • Xu C.
      • Huang R.
      • Song J.
      • Li D.
      • Xia M.
      Butyrate from pectin fermentation inhibits intestinal cholesterol absorption and attenuates atherosclerosis in apolipoprotein E-deficient mice.
      ,
      • Du Y.
      • Li X.
      • Su C.
      • Xi M.
      • Zhang X.
      • Jiang Z.
      • Wang L.
      • Hong B.
      Butyrate protects against high-fat diet-induced atherosclerosis via up-regulating ABCA1 expression in apolipoprotein E-deficiency mice.
      ]. Based on our hypothesis that butyrate is able to alleviate choline-induced atherosclerosis, we set out to evaluate the effects of choline, butyrate and their combination in APOE*3.Leiden-CETP mice, a well-established model for human-like lipid metabolism and atherosclerosis development. In contrast to expectations, we here report that both choline and butyrate beneficially modulate the gut microbiome, without affecting atherosclerosis development. Likewise, the combination of choline and butyrate did not influence atherosclerosis development, and TMAO levels were not associated with atherosclerotic lesion size.
      First, we demonstrated that choline exerts beneficial effects on the gut microbial composition and function without influencing atherosclerosis. Choline increased cecal abundance of L. reuteria and three species in the Olsenella genus. L. reuteria has been reported to improve gut barrier function via reducing inflammation [
      • Zhou Q.
      • Wu F.
      • Chen S.
      • Cen P.
      • Yang Q.
      • Guan J.
      • Cen L.
      • Zhang T.
      • Zhu H.
      • Chen Z.
      Lactobacillus reuteri improves function of the intestinal barrier in rats with acute liver failure through Nrf-2/HO-1 pathway.
      ,
      • Wu H.
      • Xie S.
      • Miao J.
      • Li Y.
      • Wang Z.
      • Wang M.
      • Yu Q.
      Lactobacillus reuteri maintains intestinal epithelial regeneration and repairs damaged intestinal mucosa.
      ], and bacteria of the Olsenella genus enhance the efficacy of immune checkpoint inhibitors in cancer by enhancing anti-inflammatory capacities of T cells [
      • Mager L.F.
      • Burkhard R.
      • Pett N.
      • Cooke N.C.A.
      • Brown K.
      • Ramay H.
      • Paik S.
      • Stagg J.
      • Groves R.A.
      • Gallo M.
      • et al.
      Microbiome-derived inosine modulates response to checkpoint inhibitor immunotherapy.
      ]. In this study, choline did not affect the cecum content expression of bacterial CutC, a key gene responsible for converting choline into TMA by the gut microbiota. In line with this finding, choline did not affect plasma TMA levels. Choline did upregulate hepatic expression of Fmos (especially Fmo3), encoding the rate-limiting enzymes responsible for the oxidation of TMA to TMAO [
      • Tang W.H.
      • Wang Z.
      • Levison B.S.
      • Koeth R.A.
      • Britt E.B.
      • Fu X.
      • Wu Y.
      • Hazen S.L.
      Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk.
      ,
      • Koeth R.A.
      • Wang Z.
      • Levison B.S.
      • Buffa J.A.
      • Org E.
      • Sheehy B.T.
      • Britt E.B.
      • Fu X.
      • Wu Y.
      • Li L.
      • et al.
      Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis.
      ]. However, choline did not influence plasma TMAO levels, which may be explained by upregulated gut microbial gene expression of TMAO reductase that reduces TMAO to TMA, and TMA corrinoid that catabolizes TMA to methane [
      • Ferguson Jr., D.J.
      • Krzycki J.A.
      Reconstitution of trimethylamine-dependent coenzyme M methylation with the trimethylamine corrinoid protein and the isozymes of methyltransferase II from Methanosarcina barkeri.
      ,
      • Ellenbogen J.B.
      • Jiang R.
      • Kountz D.J.
      • Zhang L.
      • Krzycki J.A.
      The MttB superfamily member MtyB from the human gut symbiont Eubacterium limosum is a cobalamin-dependent gamma-butyrobetaine methyltransferase.
      ]. It has been shown that excess choline can also be metabolized to other metabolites, such as phosphocholine and acetylcholine [
      • Corbin K.D.
      • Zeisel S.H.
      Choline metabolism provides novel insights into nonalcoholic fatty liver disease and its progression.
      ,
      • Zeisel S.H.
      Metabolic crosstalk between choline/1-carbon metabolism and energy homeostasis.
      ,
      • Garcia-Molina P.
      • Sola-Leyva A.
      • Luque-Navarro P.M.
      • Laso A.
      • Rios-Marco P.
      • Rios A.
      • Lanari D.
      • Torretta A.
      • Parisini E.
      • Lopez-Cara L.C.
      • et al.
      Anticancer activity of the choline kinase inhibitor PL48 is due to selective disruption of choline metabolism and transport systems in cancer cell lines.
      ], and the mouse model used here may have different choline metabolism compared to other mouse models such as Apoe−/− and Ldlr−/− mice. Thus, future studies are needed to explore the differences in choline metabolism between the various mouse models. In addition, we observed that choline slightly upregulated hepatic expression of RCT-related genes. This is in contrast to previous studies in Apoe−/− mice, which proposed that choline suppresses expression of RCT-related genes to aggravate atherosclerosis [
      • Koeth R.A.
      • Wang Z.
      • Levison B.S.
      • Buffa J.A.
      • Org E.
      • Sheehy B.T.
      • Britt E.B.
      • Fu X.
      • Wu Y.
      • Li L.
      • et al.
      Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis.
      ], although it is in fact in line with the absence of effects of choline on atherosclerosis in APOE*3-Leiden.CETP mice. We did observe that choline profoundly upregulates hepatic expression of CETP and Ldlr. Therefore, the seeming discrepancies between the different mouse models might be explained by a choline-mediated increase of CETP-mediated transfer of neutral lipids between lipoproteins and a APOE-LDLR mediated clearance pathway of triglyceride-rich lipoprotein remnants, which are both operational in APOE*3-Leiden.CETP mice but not in ApoE−/− mice. In fact, and in line with our findings, a very recent study showed that choline does not affect atherosclerosis in CETP-expressing Apoe−/− mice [
      • Collins H.L.
      • Adelman S.J.
      • Butteiger D.N.
      • Bortz J.D.
      Choline supplementation does not promote atherosclerosis in CETP-expressing male apolipoprotein E knockout mice.
      ].
      Similarly to choline, butyrate also beneficially modulated the gut microbiome although in different aspects. Butyrate increased cecal abundance of the anti-inflammatory species D. spB8 [
      • Forster S.C.
      • Clare S.
      • Beresford-Jones B.S.
      • Harcourt K.
      • Notley G.
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      • Kumar N.
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      • Adoum A.
      • Wong H.
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      Identification of gut microbial species linked with disease variability in a widely used mouse model of colitis.
      ,
      • Feng P.
      • Li Q.
      • Liu L.
      • Wang S.
      • Wu Z.
      • Tao Y.
      • Huang P.
      • Wang P.
      Crocetin prolongs recovery period of DSS-induced colitis via altering intestinal microbiome and increasing intestinal permeability.
      ] as well as three strains known for producing SCFAs, including acetate-producing B. producta [
      • Aoki R.
      • Onuki M.
      • Hattori K.
      • Ito M.
      • Yamada T.
      • Kamikado K.
      • Kim Y.G.
      • Nakamoto N.
      • Kimura I.
      • Clarke J.M.
      • et al.
      Commensal microbe-derived acetate suppresses NAFLD/NASH development via hepatic FFAR2 signalling in mice.
      ], butyrate-producing F. Prausnitzii [
      • Machiels K.
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      • De Preter V.
      • Arijs I.
      • Eeckhaut V.
      • Ballet V.
      • Claes K.
      • Van Immerseel F.
      • Verbeke K.
      • et al.
      A decrease of the butyrate-producing species Roseburia hominis and Faecalibacterium prausnitzii defines dysbiosis in patients with ulcerative colitis.
      ,
      • Olsson L.M.
      • Boulund F.
      • Nilsson S.
      • Khan M.T.
      • Gummesson A.
      • Fagerberg L.
      • Engstrand L.
      • Perkins R.
      • Uhlen M.
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      ] and lactate-producing R. lactatiformans [
      • Shkoporov A.N.
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      • Shcherbakova V.A.
      • Suzina N.E.
      • Kafarskaia L.I.
      • Bozhenko V.K.
      • Efimov B.A.
      Ruthenibacterium lactatiformans gen. nov., sp. nov., an anaerobic, lactate-producing member of the family Ruminococcaceae isolated from human faeces.
      ]. Furthermore, butyrate downregulated cecal microbial genes involved in LPS synthesis, which may imply that butyrate alleviates LPS-induced damage of gut barrier integrity [
      • Guo S.H.
      • Nighot M.
      • Al-Sadi R.
      • Alhmoud T.
      • Nighot P.
      • Ma T.Y.
      Lipopolysaccharide regulation of intestinal tight junction permeability is mediated by TLR4 signal transduction pathway activation of FAK and MyD88.
      ]. Previous studies showed that butyrate reduces atherosclerotic lesion size in Apoe−/− and Ldlr−/− mice partially via improving gut barrier function [
      • Kasahara K.
      • Krautkramer K.A.
      • Org E.
      • Romano K.A.
      • Kerby R.L.
      • Vivas E.I.
      • Mehrabian M.
      • Denu J.M.
      • Backheds F.
      • Lusis A.
      • et al.
      Interactions between Roseburia intestinalis and diet modulate atherogenesis in a murine model.
      ,
      • Chen Y.
      • Xu C.
      • Huang R.
      • Song J.
      • Li D.
      • Xia M.
      Butyrate from pectin fermentation inhibits intestinal cholesterol absorption and attenuates atherosclerosis in apolipoprotein E-deficient mice.
      ,
      • Du Y.
      • Li X.
      • Su C.
      • Xi M.
      • Zhang X.
      • Jiang Z.
      • Wang L.
      • Hong B.
      Butyrate protects against high-fat diet-induced atherosclerosis via up-regulating ABCA1 expression in apolipoprotein E-deficiency mice.
      ,
      • Brandsma E.
      • Kloosterhuis N.J.
      • Koster M.
      • Dekker D.C.
      • Gijbels M.J.J.
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      • Loreti M.G.
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      A proinflammatory gut microbiota increases systemic inflammation and accelerates atherosclerosis.
      ,
      • Fang W.
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      • Ling W.
      Supplementation with sodium butyrate modulates the composition of the gut microbiota and ameliorates high-fat diet-induced obesity in mice.
      ]. This was attributed to maintained gut microbiota homeostasis and inhibited LPS synthesis, which reduces gut barrier permeability and thus reduces systemic inflammation [
      • Fang W.
      • Xue H.
      • Chen X.
      • Chen K.
      • Ling W.
      Supplementation with sodium butyrate modulates the composition of the gut microbiota and ameliorates high-fat diet-induced obesity in mice.
      ,
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      • Jia J.
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      • Shi Y.
      • Rong S.
      • Yuan W.
      Butyrate ameliorates skeletal muscle atrophy in diabetic nephropathy by enhancing gut barrier function and FFA2-mediated PI3K/Akt/mTOR signals.
      ,
      • Bach Knudsen K.E.
      • Laerke H.N.
      • Hedemann M.S.
      • Nielsen T.S.
      • Ingerslev A.K.
      • Gundelund Nielsen D.S.
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      • Hald S.
      • Schioldan A.G.
      • et al.
      Impact of diet-modulated butyrate production on intestinal barrier function and inflammation.
      ]. Probably as a combined result, butyrate inhibited macrophage infiltration into atherosclerotic plaques and halts plaque progression [
      • Kasahara K.
      • Krautkramer K.A.
      • Org E.
      • Romano K.A.
      • Kerby R.L.
      • Vivas E.I.
      • Mehrabian M.
      • Denu J.M.
      • Backheds F.
      • Lusis A.
      • et al.
      Interactions between Roseburia intestinalis and diet modulate atherogenesis in a murine model.
      ,
      • Aguilar E.C.
      • Leonel A.J.
      • Teixeira L.G.
      • Silva A.R.
      • Silva J.F.
      • Pelaez J.M.
      • Capettini L.S.
      • Lemos V.S.
      • Santos R.A.
      • Alvarez-Leite J.I.
      Butyrate impairs atherogenesis by reducing plaque inflammation and vulnerability and decreasing NFkappaB activation.
      ]. In contrast, we found no influence of butyrate on atherosclerotic size, severity and composition including macrophage content within atherosclerotic lesions in APOE*3-Leiden.CETP mice. In addition to beneficially modulating gut microbiome, butyrate has been demonstrated to promote RCT and thus to improve cholesterol metabolism, by primarily stimulating ABCA1-mediated cholesterol efflux in macrophages and increasing hepatic bile acid synthesis and secretion, thereby alleviating atherosclerosis in Apoe−/− mice [
      • Du Y.
      • Li X.
      • Su C.
      • Xi M.
      • Zhang X.
      • Jiang Z.
      • Wang L.
      • Hong B.
      Butyrate protects against high-fat diet-induced atherosclerosis via up-regulating ABCA1 expression in apolipoprotein E-deficiency mice.
      ]. In our mouse model, butyrate only slightly affected hepatic expression of RCT-related genes including Abca1 and did not affect cholesterol levels. Moreover, plasma non-HDL-C levels were positively correlated to atherosclerotic size. Therefore, it is likely that atherosclerosis is more inflammation-driven in Apoe−/− and Ldlr−/− mice, and more cholesterol-driven in APOE*3-Leiden.CETP mice, explaining why butyrate is not atheroprotective in our model.
      TMAO has been identified as the main mediator of the previously described atherogenic effects of choline in Apoe−/− and Ldlr−/− mice [
      • Wang Z.
      • Klipfell E.
      • Bennett B.J.
      • Koeth R.
      • Levison B.S.
      • Dugar B.
      • Feldstein A.E.
      • Britt E.B.
      • Fu X.
      • Chung Y.M.
      • et al.
      Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease.
      ,
      • Koeth R.A.
      • Wang Z.
      • Levison B.S.
      • Buffa J.A.
      • Org E.
      • Sheehy B.T.
      • Britt E.B.
      • Fu X.
      • Wu Y.
      • Li L.
      • et al.
      Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis.
      ]. In the present study, we observed that treatment with the combination of choline and butyrate increased plasma TMAO levels as compared to single treatments. This is likely due to the observation that butyrate impaired the choline-induced TMA and TMAO degradation signaling. Indeed, the gut microbial gene expression of TMA corrinoid protein and TMAO reductase in the combination group was comparable to that of the control and butyrate groups. However, in spite of increased plasma TMAO levels, combined choline and butyrate administration did not affect atherosclerosis. Of note, plasma TMAO levels were not associated with atherosclerotic lesion size. The fact that TMAO induces atherosclerosis in Apoe−/− and Ldlr−/− mice but not in APOE*3-Leiden.CETP mice, may suggest that TMAO lacks atherogenic properties in humans. Indeed, many human dietary trials did not find any association between plasma TMAO and CVD risk [
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      • Jacobs R.L.
      • Field C.J.
      Impact of egg consumption on cardiovascular risk factors in individuals with type 2 diabetes and at risk for developing diabetes: a systematic review of randomized nutritional intervention studies.
      ,
      • Shin J.Y.
      • Xun P.
      • Nakamura Y.
      • He K.
      Egg consumption in relation to risk of cardiovascular disease and diabetes: a systematic review and meta-analysis.
      ]. Multiple meta-analyses and systematic reviews have shown that the intake of eggs, rich in TMAO precursors, is not correlated to heart disease risk and mortality [
      • Meyer K.A.
      • Shea J.W.
      Dietary choline and betaine and risk of CVD: a systematic review and meta-analysis of prospective studies.
      ]. Similarly, another systematic review and a cohort study have concluded that TMAO does not associate with CVD risk [
      • Nagata C.
      • Wada K.
      • Tamura T.
      • Konishi K.
      • Kawachi T.
      • Tsuji M.
      • Nakamura K.
      Choline and betaine intakes are not associated with cardiovascular disease mortality risk in Japanese men and women.
      ].
      In conclusion, we demonstrate that in APOE*3-Leiden.CETP mice, a well-established model for human-like lipoprotein metabolism, both choline and butyrate beneficially modulate the gut microbiome and increase TMAO, however without affecting atherosclerosis.

      Financial support

      This work was supported by the Leiden University Fund/Mulder-Hamelers Fonds ( W18307-2-53 to YW), Leiden University Fund/Elise Mathilde Fund ( W213045-2 to ZL), Department of Science and Technology foundation of Shaanxi province ( 2021SF-021 to YW), the Dutch Diabetes Research Foundation ( 2015.81.1808 to MRB), an NWO-VENI grant ( 09150161910073 to M.R.B.) and the Netherlands Cardiovascular Research Initiative: an initiative with support of the Dutch Heart Foundation (CVON-GENIUS-2 to PCNR). ZL is supported by the China Scholarship Council (CSC 201506170051 ); MS is supported by Novo Nordisk Foundation ( NNF18OC0032394 to MS); YW is supported by the China “Thousand Talents Plan” (Young Talents), Shaanxi province “Thousand Talents Plan” (Young Talents) and Foundation of Xi'an Jiaotong University (Plan A).

      CRediT authorship contribution statement

      Cong Liu: Methodology, carried out the research, Formal analysis, Writing – original draft. Zhuang Li: carried out the research, interpreted the results, Writing – review & editing, obtained funding. Zikuan Song: carried out the research, interpreted the results, Writing – review & editing. Xiayue Fan: Formal analysis, Writing – review & editing. Hua Shao: Formal analysis, Writing – review & editing. Milena Schönke: interpreted the results, reviewed, Writing – review & editing. Mariëtte R. Boon: advised the study, Writing – review & editing. Patrick C.N. Rensen: Methodology, interpreted the results, Writing – review & editing, obtained funding. Yanan Wang: Methodology, interpreted the results, Writing – review & editing, obtained the funding.

      Declaration of competing interest

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

      Acknowledgments

      We thank T.C.M. Streefland, A.C.M. Pronk, R.A. Lalai and S. Afkir from the Department of Medicine, Division of Endocrinology, Leiden University Medical Center for technical assistance.

      Appendix A. Supplementary data

      The following is the Supplementary data to this article:

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