Abstract
Gut microbes play a pivotal role in host physiology by producing beneficial or detrimental metabolites. Gut bacteria metabolize dietary choline and L-carnitine to trimethylamine (TMA) which is then converted to trimethylamine-N-oxide (TMAO). An elevated circulating TMAO is associated with diabetes, obesity, cardiovascular disease, and cancer in humans. In the present study, we investigated the effect of dietary blueberries and strawberries at a nutritional dosage on TMA/TMAO production and the possible role of gut microbes. Blueberry cohort mice received a control (C) or freeze-dried blueberry supplemented (CB) diet for 12 weeks and subgroups received an antibiotics cocktail (CA and CBA). Strawberry cohort mice received a control (N) or strawberry-supplemented (NS) diet and subgroups received antibiotics (NA and NSA). Metabolic parameters, choline, TMA, and TMAO were assessed in addition to microbial profiling and characterization of berry powders. Blueberry supplementation (equivalent to 1.5 human servings) reduced circulating TMAO in CB vs C mice (~48%) without changing choline or TMA. This effect was not mediated through alterations in metabolic parameters. Dietary strawberries did not reduce choline, TMA or TMAO. Depleting gut microbes with antibiotics in these cohorts drastically reduced TMA and TMAO to not-quantified levels. Further, dietary blueberries increased the abundance of bacterial taxa that are negatively associated with circulating TMA/TMAO suggesting the role of gut microbes. Our phenolic profiling indicates that this effect could be due to chlorogenic acid and increased phenolic contents in blueberries. Our study provides evidence for considering dietary blueberries to reduce TMAO and prevent TMAO-induced complications.
Keywords: chlorogenic acid, blueberry, strawberry, gut microbes, trimethylamine, trimethylamine N-oxide
Graphical Abstract
1. INTRODUCTION
Gut microbes play a fundamental role in host physiology and pathophysiology. Diet is a key factor in shaping gut microbes that interact with the host by producing beneficial or detrimental microbial metabolites such as short-chain fatty acids and trimethylamine N-oxide (TMAO) (1). TMAO is one of the major microbial metabolites produced through a microbiome-host metabolic axis and is implicated in several metabolic disorders (2–4, 1). Red meat, egg yolk, fish, and dairy products are the main sources of TMAO precursors through nutrients such as phosphatidylcholine, choline, and L-carnitine (1). Gut microbes metabolize choline and phosphatidylcholine to trimethylamine (TMA) using the microbial enzyme TMA lyase (CutC) that is encoded by the bacterial choline utilization (Cut) gene cluster (2, 5). TMA is then transported to the liver and oxidized into TMAO by the liver enzyme flavin-containing monooxygenases (FMOs) (5). TMAO can exert its effects when it enters the circulation and is finally eliminated by the kidneys (5). The role of gut microbiota in the production of TMAO is well-documented in humans (6). Antibiotic treatment suppresses the production of TMAO while the TMAO production recovers after the removal of antibiotics indicating the effects are gut-microbiota dependent (7).
Human studies indicate that TMAO is associated with a higher risk of developing insulin resistance, diabetes, obesity, cardiovascular disease, kidney disease, and cancer (2–4, 1). TMAO regulates various physiological processes involved in the development and progression of atherosclerosis (8, 9). In metabolic disorders, dysbiosis associated with an increased intake of TMA precursors leads to an increase in plasma TMAO, which is involved in the pathogenesis of metabolic disease comorbidities including cardiovascular diseases (1). Many TMA precursors are cardioprotective in contexts and found in heart-healthy diets such as seafood (10). Hence, limiting the intake of TMA precursor may not be a potential strategy to reduce circulating TMAO. Evidence suggests that targeting gut microbes and suppressing TMA formation reduce circulating TMAO levels and its pathological effects (10). However, to date, there does not exist an FDA-approved drug to manage TAMO levels (11).
Emerging evidence indicates nutritional intervention such as supplementation of phenolic compounds may be an efficient strategy to manage TMAO (11). Indeed, several classes of phenolic compounds (i.e., anthocyanins, flavonols, and hydroxycinnamic acids) have shown the potential to reduce the formation of TMA or TMAO in different in vitro and in vivo models. A human study showed that administering a black raspberry extract rich in anthocyanins for 4 weeks reduces plasma TMAO levels in smokers (12). Similarly, Lonicera caerulea berry extract rich in anthocyanins reduced plasma TMAO levels in a chronic administration study (12 weeks) in the Sprague-Dawley rats (13). Chlorogenic acid, a hydroxycinnamic acid, was shown to inhibit the formation of TMAO in L-carnitine-fed mice (14). We previously showed that chlorogenic acid and other polyphenols inhibit choline microbial transformation into TMA in vitro (15, 16).
Human studies support the cardioprotective effects of dietary blueberries and strawberries (17, 18). Recently we showed that blueberry or strawberry supplementation at a nutritional dose suppresses vascular inflammation and improves vascular dysfunction in diabetic mice (19, 20). Blueberries and strawberries are rich in bioactive phenolic compounds (21). Hence, blueberry and strawberry supplementation could be a potential nutritional strategy to manage TMAO levels and prevent complications associated with TMAO. In the present study, we investigated whether dietary supplementation of blueberries or strawberries reduces circulating TMAO and the possible role of gut microbes in mediating the effect of berries on TMAO.
2. MATERIALS AND METHODS
2.1. Chemicals and reagents
Ampicillin, vancomycin, neomycin sulfate, metronidazole, ammonium formate, ammonia, ethyl bromoacetate, choline, choline-d9, TMA, TMA-d9, TMAO, TMAO-d9, gallic acid, malvidin-3-O-glucoside, chlorogenic acid, delphinidin-3-O-glucoside and procyanidin dimer B2 were purchased from Sigma-Aldrich/Millipore (St. Louis, MO, USA). Petunidin chloride, cyanidin-3-O-xyloside, pelargonidin-3-O-glucoside chloride, malvidin-3-O-glucoside, and bilberry anthocyanidins algycans kit were purchased from Chromadex (Los Angeles, CA, USA). Delphinidin-3-O-galactoside, kuromanin chloride (cyanidin glucoside), ideain chloride (cyanidin galactoside), cyanidin-3-O-arabinoside, petunidin-3-O-glucoside, peonidin-3-O-glucoside, peonidin-3-O-galactoside, and peonidin-3-O-arabinoside were purchased from Extrasynthese (Genay, France). Acetonitrile and water (HPLC grade) were purchased from VWR International (Suwanee, GA, USA). Freeze-dried wild blueberry powder and freeze-dried strawberry powder were provided by the Wild Blueberry Association of North America (Maine, USA) and FutureCeuticals (Momence, IL, USA), respectively.
2.2. Experimental design
Six-week-old male C57BL/6J mice (Stock number: 000664) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Mice were maintained in a 12-hour light/dark cycle, 23 ± 1 °C and 45 ± 5 % humidity under artificial light. The Institutional Animal Care and Use Committee at the University of Utah approved all protocols (Protocol Numbers: 18–10005 and 18–09003). These protocols conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Housed at the University of Utah Comparative Medicine Center Vivarium under humane conditions, the mice were acclimated for a week and were 7 weeks old before experiments began. Animals were fed a diet supplemented with blueberry or strawberry powder as described below. The blueberry cohort includes mice that received a control diet (C) or a diet supplemented with 3.8 % freeze-dried wild blueberries (CB) for 12 weeks. Subgroups of mice were treated with or without an antibiotics cocktail in drinking water (CA and CBA). The antibiotic dosage was gradually increased with the final dosage of 1 g/L ampicillin, 500 mg/L vancomycin, 1 g/L neomycin sulfate, and 1 g/L metronidazole. 3.8 % freeze-dried blueberry powder in the diet (w/w) used in the present study is equivalent to human consumption of ~240 g of blueberries (1.5 servings) per day (20). The strawberry cohort includes mice that received a control diet (N) or a diet supplemented with 2.35 % freeze-dried strawberries (NS) for 12 weeks. Subgroups of mice were treated with or without an antibiotics cocktail in drinking water (NA and NSA) with a gradual increase in the dosage of antibiotics. 2.35 % freeze-dried strawberry powder in the diet (w/w) used in the present study is equivalent to human consumption of ~160 g of strawberries (1 serving) per day (19). The blueberry or strawberry-supplemented diets were matched for the sugar and fiber contents of control diets (Supplementary Tables S1 and S2). The diets were irradiated and supplied by Research Diets Inc. (New Brunswick, NJ, USA). The broad-spectrum antibiotic cocktail (1 g/L ampicillin, 500 mg/L vancomycin, 1 g/L neomycin sulfate, and 1 g/L metronidazole) in drinking water was shown to deplete gut bacteria in experimental mice (22, 23). The gut microbial depletion with the antibiotics cocktail in the present study was confirmed by 16s rRNA amplification of cecum bacterial DNA as described below.
2.3. Characterization and phenolic compound profiling of freeze-dried blueberry and strawberry powders
Freeze-dried blueberry and strawberry powders were extracted (n=4; each) following the methods described by Dorenkott et al. (24). A general profiling of the phenolic content of berries was conducted by quantifying the total polyphenol content (TPC), the total anthocyanin content (TAC) and total procyanidin content (PCA) of extracts as described by Racine et al. (25), Iglesias-Carres et al. (26), and Neilson et al. (27), respectively. A more in-depth analysis of the phenolic profile of berries was performed by LC-MS following the methods reported by Mohamedshah et al. (28).
2.4. Measurement of metabolic parameters and collection of tissue samples
Body weight, blood glucose and body composition were measured after the treatment as we described previously (19, 20). Fasting blood glucose concentrations were measured in tail vein blood samples (Bayer Contour Next One blood glucose monitoring system, Parsippany, NJ). Body composition (fat and lean body mass) was measured by TD-NMR using the LF50 body composition mice analyzer (Minispec; Bruker, Germany). After 12 weeks of treatment, mice were anesthetized using 2–5% isoflurane and blood was collected via cardiac puncture. Cecum contents were collected, flash-frozen in liquid nitrogen, and stored at −80 °C for microbial profiling.
2.5. Extraction and quantification of choline, TMA, and TMAO from plasma
Extraction and quantification of choline, TMA and TMAO was carried out as described by Griffin et al. (29), with some modifications as described below. The volumes of plasma used for choline, TMA and TMAO extraction were 10 – 12 μL of sample; 20 μL of a mixture of choline-d9, TMA-d9 and TMAO-d9 (IS; 2.5 μM) were added to samples in 96-well plates; and plating of plasma was performed manually, while other reagents were plated using an OT-2 liquid handler (Opentrons, NY, USA). After extraction, choline, TMA and TMAO were analyzed through UPLC-ES-MS/MS as described by Iglesias-Carres et al. (16). Multi-reaction monitoring (MRM) fragmentation conditions of analytes and internal standard (IS) compounds are reported in Supplementary Table S3. For sample quantification, HPLC water was spiked with 13 different concentrations (0 – 50 μM) of choline, TMA and TMAO standards to obtain external calibration. Samples were quantified by interpolating the analyte/IS peak abundance ratio in the standard curves. Data acquisition was carried out using Masslynx software (V4.1 version, Waters). Method sensitivity was determined by limit of detection (LOD) and limit of quantification (LOQ), respectively defined as the concentration of analyte corresponding to 3 and 10 times the signal/noise ratio. Method quality parameters are reported in Supplementary Table S4.
2.6. Microbial community profiling
Bacterial genomic DNA was extracted from cecal contents using the DNeasy PowerSoil Kit (Qiagen, USA). Fifty nanograms of genomic DNA were utilized to amplify the V4 variable region of the 16S rRNA gene using 515F/806R primers. Forward and reverse primers were barcoded to accommodate multiplexing up to 384 samples per run as described (30). Paired-end sequencing (2 X 250 bp) of pooled amplicons was carried out using Illumina Miseq platform with ~30% PhiX DNA.
2.7. Bioinformatics analysis
Demultiplexing, adapter trimming, and generating the fastq files were performed automatically using the Miseq Reporter on the instrument computer. Bioinformatics analysis was then conducted using the QIIME 2 platform (31). Denoising was done with an initial quality filtering followed by the Deblur algorithm (32, 33). Representative amplicon sequence variants (ASVs) were used to generate a phylogenetic tree with FastTree (34) and taxonomy was assigned using a Naives Bayes classifier trained on the Greengenes 13_8 reference (35). α-Diversity metrics for richness, diversity, and evenness were determined using the observed genus, Shannon diversity index, and Pielou’s evenness index, respectively. β-Diversity was assessed using the weighted Unifrac distances (36) and visualized using the principal coordinate analysis (PCoA) plot.
2.8. Statistics
Prism 8.0 (GraphPad, La Jolla, CA, USA) or SPSS (Version 25; IBM) was used for statistical analyses and graph creation. Microbial data was analyzed using R programming language (version R-4.2.2). One-way ANOVA was used to compare groups for metabolic parameters. When the main effects were significant, Tukey post hoc tests were performed. Two-way ANOVA was used to estimate differences in the plasma levels of choline, TMA, and TMAO. Groups with non-quantifiable levels of analytes were excluded from the analyses and were plotted in graphs as 0 μM. When only two groups reported quantifiable levels of analytes, changes in their levels were evaluated through a student’s t-test. In all cases, statistical significance was established a priori as p < 0.05. Spearman’s correlations were performed using the Shiny App between bacterial abundance data and plasma TMA/TMAO. All data are considered statistically significant where p<0.05, expressed as mean ± SEM, where appropriate.
3. RESULTS
3.1. Characterization of freeze-dried blueberry and strawberry powders indicates an extensive compositional difference in their polyphenol and anthocyanins profiles
The general phenolic profile of blueberries and strawberries is reported in Table 1. The detailed profile of blueberries and strawberries is reported in Table 2. Blueberries were rich in anthocyanins, especially glycosides of delphinidins and petunidin. Blueberries also presented relevant quantities of other flavonoids, especially flavonols (i.e., quercetin-3-O-rutinoside). However, the single most abundant measured phenolic compound in blueberries was chlorogenic acid (~15 mg/g dry weight of the powder). On the other hand, the levels of chlorogenic acid in strawberries were minimal. Moreover, the phenolic profile of strawberries was much less diverse as compared to blueberries. Strawberries were rich in anthocyanins like blueberries, but the most abundant ones were glycosides of pelargonidin. Overall, the quantification of individual polyphenols was in line with the quantification of general phenolic families (Table 1).
Table 1.
General phenolic content of blueberries and strawberries
| Phenolic families | Blueberry | Strawberry |
|---|---|---|
| Total phenolic content (mg GAE/g dw) | 30.08 ± 0.57 | 20.68 ± 2.02 |
| Total anthocyanin content (mg MvG/g dw) | 12.33 ± 0.59 | 8.13 ± 0.36 |
| Total procyanidin content (mg PCB2/g dw) | 12.94 ± 0.44 | 5.36 ± 0.41 |
Results are expressed as mean ± SEM (n=4). GAE, gallic acid equivalents; dw, dry weight; MvG, Malvidin-3-O-glucoside; and PCB2, procyanidin dimer B2.
Table 2.
Detailed polyphenol profile of berries (mg/g dry weight)
| Compound | Freeze-dried blueberry powder | Freeze-dried strawberry powder |
|---|---|---|
| p-Coumaric acid | N.D. | 0.04 ± 0.00 |
| Ferulic acid | 0.04 ± 0.01 | N.Q. |
| Caffeic acid | 0.02 ± 0.00 | N.D. |
| Gallic acid | N.D. | 0.02 ± 0.01 |
| Chlorogenic acid | 14.94 ± 0.86 | 0.02 ± 0.01 |
| Epicatechin | 0.22 ± 0.03 | 0.83 ± 0.08 |
| Catechin | 0.12 ± 0.02 | 0.05 ± 0.01 |
| Quercetin | 0.02 ± 0.00 | 0.01 ± 0.00 |
| Kaempferol | N.D. | N.D. |
| Quercetin-3-O-Glucoside | 2.29 ± 0.18 | 0.08 ± 0.02 |
| Kaempferol-3-O-Glucoside | 0.21 ± 0.03 | 0.33 ± 0.05 |
| Quercetin-3-O-Rutinoside | 0.88 ± 0.13 | 0.01 ± 0.00 |
| Cyanidin | 0.02 ± 0.00 | 0.02 ± 0.00 |
| Cyanidin-3-O-Arabinoside | 0.38 ± 0.03 | N.D. |
| Cyanidin-3-O-Glucoside | 0.48 ± 0.03 | 0.35 ± 0.1 |
| Cyanidin-3-O-Galactoside | 0.83 ± 0.06 | N.D. |
| Peonidin-3-O-Arabinoside | 0.11 ± 0.01 | N.D. |
| Peonidin-3-O-Glucoside | 0.43 ± 0.02 | N.D. |
| Peonidin-3-O-Galactoside | 0.12 ± 0.01 | N.D. |
| Delphinidin-3-O-Arabinosidea | 0.59 ± 0.06 | N.D. |
| Delphinidin-3-O-Glucoside | 3.83 ± 0.19 | N.D. |
| Delphinidin-3-O-Galactosideb | 2.78 ± 0.20 | N.D. |
| Petunidin-3-O-Arabinosidec | 1.33 ± 0.11 | 0.01 ± 0.00 |
| Petunidin-3-O-Glucoside | 1.25 ± 0.08 | N.D. |
| Petunidin-3-O-Galactosidec | 0.92 ± 0.06 | N.D. |
| Malvidin-3-O-Arabinosidea | 0.70 ± 0.04 | N.D. |
| Malvidin-3-O-Glucosided | 0.02 ± 0.00 | N.D. |
| Malvidin-3-O-Galactosidee | 0.03 ± 0.00 | N.D. |
| Pelargonidin | N.D. | 0.36 ± 0.09 |
| Pelargonidin-3-O-Glucosided | N.D. | 11.08 ± 2.12 |
| Pelargonidin-3-O-Rutinosided | N.D. | 0.26 ± 0.08 |
Results are expressed as mean ± SEM (n=4). Quantified using the calibration curve of cyanidin-3-O-arabinoside
delphinidin-3-O-glucoside
petunidin-3-O-glucoside
cyanidin-3-O-glucoside
and cyanidin-3-O-galactoside
N.D., Not detected; N.Q., Not quantified.
3.2. Antibiotics cocktail was effective in depleting gut microbes in experimental mice
In the present study, the blueberry cohort includes mice that received a control diet (C) or a diet supplemented with 3.8 % freeze-dried wild blueberries (CB) for 12 weeks. Subgroups of mice were treated with or without an antibiotics cocktail in drinking water (CA and CBA). 3.8 % freeze-dried blueberry powder in the diet (w/w) is equivalent to human consumption of ~240 g of blueberries (1.5 servings) per day (20). The strawberry cohort includes mice that received a control diet (N) or a diet supplemented with 2.35 % freeze-dried strawberries (NS) for 12 weeks. Subgroups of mice were treated with or without an antibiotics cocktail in drinking water (NA and NSA). 2.35 % freeze-dried strawberry powder in the diet (w/w) is equivalent to human consumption of ~160 g of strawberries (1 serving) per day (19). The microbial depletion in the present study was validated by 16s rRNA amplification. The diversity plots (α-Diversity indices and β-Diversity) and rarefaction graph indicate that the antibiotic cocktail is effective in depleting gut microbes both in the blueberry cohort (CA and CBA mice) (Fig. 1A–C) and strawberry cohort (NA and NSA mice) (Fig. 2A–C).
Fig 1.
Blueberry cohort: Microbial Profiling. α-Diversity indices (A), β-diversity plot (B), and rarefaction plot (C) of gut microbial communities. C, Standard diet-fed mice; CB, blueberry-fed mice; CA, standard diet-fed mice treated with antibiotics in drinking water; CBA, blueberry-fed mice treated with antibiotics in drinking water. Values are mean ± SEM (n=5–10). * vs. C; # vs. C; ✢ vs. CB, p < 0.05.
Fig 2.
Strawberry cohort: Microbial Profiling. α-Diversity indices (A), β-diversity plot (B), and rarefaction plot (C) of gut microbial communities. C, Standard diet-fed mice; NS, strawberry-fed mice; NA, standard diet-fed mice treated with antibiotics in drinking water; NSA, strawberry-fed mice treated with antibiotics in drinking water. Values are mean ± SEM (n=5–10). * vs. N; # vs. N; ✢ vs. NS, p < 0.05.
3.3. Blueberry supplementation greatly reduced plasma concentrations of TMAO without altering the concentrations of choline or TMA
The plasma concentrations of choline in the blueberry cohort ranged from 58.26 – 67.58 μM. Two-way ANOVA indicated a statistical reduction in the plasma levels of choline promoted by antibiotic administration (Fig. 3A). TMA was quantified only in non-antibiotics treated groups [C (2.13 μM) and CB (1.2 μM)]. Student’s t-test indicated that there were no differences in the plasma levels of TMA in C vs. CB (Fig. 3B). Plasma TMAO was also quantified only in non-antibiotic treated groups [C (3.15 μM) and CB (1.63 μM)]. Student’s t-test indicated a significant decrease of TMAO levels with blueberry supplementation in CB vs C mice (p<0.001) (Fig. 3C). Importantly, blueberry supplementation reduced plasma TMAO levels by ~1.52 μM (reduction of ~48 %).
Fig 3.
Blueberry cohort: Plasma choline (A), TMA (B), and TMAO (C). TMA, Trimethylamine; TMAO, trimethylamine N-oxide; C, standard diet-fed mice; CB, blueberry-fed mice; CA, standard diet-fed mice treated with antibiotics in drinking water; CBA, blueberry-fed mice treated with antibiotics in drinking water. Values are mean ± SEM (n=8). ** vs. C, p < 0.001 by Student’s t-test.
3.4. Strawberry supplementation did not alter the plasma concentrations of choline, TMA or TMAO
The plasma concentrations of choline in the strawberry cohort ranged from 33.86–54.59 μM. Two-way ANOVA indicated that plasma choline levels were significantly reduced (p<0.01) due to antibiotic administration. This can be seen by the statistical significance of the student’s t-test (p<0.05) between NS (47.17 μM) and NSA (33.86 μM) (Fig. 4A). TMA levels were quantified only in non-antibiotic treated groups [N (3.03 μM) and NS mice (3.06 μM)] (Fig. 4B). Plasma TMAO levels were also quantified only in non-antibiotic treated groups [N (1.18 μM) and NS (1.53 μM)]. Student’s t-test indicated that plasma TMA or TMAO levels were not statistically modulated by strawberry administration in NS vs. N mice (Fig. 4C).
Fig 4.
Strawberry cohort: Plasma choline (A), TMA (B), and TMAO (C). TMA, Trimethylamine; TMAO, trimethylamine N-oxide; C, standard diet-fed mice; NS, strawberry-fed mice; NA, standard diet-fed mice treated with antibiotics in drinking water; NSA, strawberry-fed mice treated with antibiotics in drinking water. Values are mean ± SEM (n=8). ✢ vs. NS, p < 0.05 by Student’s t-test.
3.5. Effect of blueberry supplementation on TMAO was not mediated through metabolic alterations
Blueberry or strawberry supplementation did not alter body weight, lean body mass, fat body mass, fasting blood glucose, and non-fasting glucose in experimental mice (C vs CB mice and N vs NS mice) (Table 3 & 4). Further, antibiotics treatment did not alter the body weight, lean body mass, fat body mass, and blood glucose in CA vs C, CBA vs CB, NA vs N and NSA vs NS mice.
Table 3.
Blueberry Cohort: Body weight, blood glucose and body composition in mice treated with or without blueberries and/or antibiotics for 12 weeks.1
| Characteristics | C | CB | CA | CBA |
|---|---|---|---|---|
| Body weight (g) | 28.9 ± 0.7 | 27.7 ± 0.7 | 27.4 ± 0.7 | 26.3 ± 0.8 |
| Fasting blood glucose (mg/dL) | 77 ± 3 | 83 ± 3 | 73 ± 2 | 72 ± 3✢ |
| Body composition | ||||
| Lean (%) | 70.4 ± 0.5 | 69.6 ± 0.9 | 71.9 ± 0.5 | 69.9 ± 0.8 |
| Fat (%) | 13.5 ± 0.8 | 15 ± 1.6 | 10.2 ± 1.2 | 12.5 ± 1.3 |
| Fluid (%) | 12.8 ± 0.2 | 13 ± 0.2* | 14.3 ± 0.2# | 14.2 ± 0.3 |
Values are mean ± SEM (n=10). Comparison among groups was made using one-way ANOVA and Tukey post hoc tests were performed when significant main effects were obtained.
vs. C;
vs. C;
vs. CB, p < 0.05.
C, Standard diet fed mice; CB, blueberry fed mice; CA, standard diet fed mice treated with antibiotics in drinking water; CBA, blueberry fed mice treated with antibiotics in drinking water.
Table 4.
Strawberry Cohort: Body weight, food intake, blood glucose and body composition in mice treated with or without strawberries and/or antibiotics for 12 weeks.1
| Characteristics | N | NS | NA | NSA |
|---|---|---|---|---|
| Body weight (g) | 28.9 ± 0.6 | 27.5 ± 0.7 | 27.4 ± 0.5 | 27.8 ± 0.3 |
| Fasting blood glucose (mg/dL) | 58 ± 1 | 55 ± 1 | 55 ± 1 | 56 ± 1 |
| Body composition | ||||
| Lean (%) | 70.5 ± 1 | 70.5 ± 1 | 72.1 ± 0.6 | 73.6 ± 0.6 |
| Fat (%) | 13.2 ± 1.7 | 12.2 ± 1.4 | 9.61 ± 0.6 | 7.9 ± 0.8 |
| Fluid (%) | 13.3 ± 0.3 | 13.4 ± 0.3 | 14.6 ± 0.1# | 15.2 ± 0.1✢ |
Values are mean ± SEM (n=10). Comparison among groups was made using one-way ANOVA and Tukey post hoc tests were performed when significant main effects were obtained.
vs. N;
vs. NS, p < 0.05.
N, Standard diet fed mice; NS, strawberry fed mice; NA, standard diet fed mice treated with antibiotics in drinking water; NSA, strawberry fed mice treated with antibiotics in drinking water.
3.6. Blueberry supplementation increased the abundance of bacterial taxa that are negatively associated with circulating TMA/TMAO
Spearman’s correlation indicates that specific gut microbes are positively or negatively associated with circulating TMA and TMAO (Fig. 5A). Genera such as Allobaculum and Dorea is positively associated with TMAO. Several taxa (unclassified genus of Order RF39, Turicibacter genus, unclassified genus of order Clostradiales, and Ruminococcaceae family) are negatively associated with circulating TMA. Taxa such as unclassified genera of the families Coriobacteriaceae and S24–7, Oscillospira genus, unclassified genus of the order Clostridiales, Ruminococcus genus, and unclassified genus of the family Ruminococcaceae are negatively associated with circulating TMAO. Further, the relative abundance of the microbial population indicates that blueberry supplementation increases the abundance of bacterial taxa that are negatively associated with circulating TMA or TMAO (Fig. 5B). Blueberry supplementation greatly increases (p=0.006) the abundance of an unclassified genus of the family Coribacteriaceae. In addition, an increased trend is observed in the relative abundance of S24–7 (p=0.06), Turicibacter (p=0.06), and Oscillospira (p=0.07) with blueberry supplementation (CB vs C).
Fig 5.
Spearman’s correlations between bacterial abundance data and TMA/TMAO using Shiny App (n=5–8) (A). The relative abundance of the microbial population at the genus level (n=5–10) (B). TMA, Trimethylamine; TMAO, trimethylamine N-oxide; C, standard diet-fed mice; CB, blueberry-fed mice; CA, standard diet-fed mice treated with antibiotics in drinking water; CBA, blueberry-fed mice treated with antibiotics in drinking water. * vs. C; # vs. C; ✢ vs. CB, p < 0.05.
4. DISCUSSION
Human studies indicate elevated circulating levels of microbiota-dependent metabolite TMAO are associated with a higher risk of developing CVD (1). Emerging evidence supports the cardiovascular benefits of blueberry and strawberry consumption (17, 18). We tested the hypothesis that supplementation of blueberry or strawberry at a nutritional dose reduces TMAO and this effect is associated with specific microbes. First, in our study, blueberry supplementation reduced circulating TMAO in experimental mice but this effect was not observed in the strawberry cohort. Second, the effect of blueberry on TMAO was not mediated through metabolic alterations. Third, the characterization of freeze-dried blueberry and strawberry powders indicates extensive compositional differences in their polyphenol and anthocyanins profiles suggesting the bioactive components of blueberry may be more efficient in reducing TMAO. Fourth, dietary blueberry increased the abundance of bacterial taxa that are negatively associated with circulating TMA/TMAO suggesting the possible role of gut microbes in mediating the effect of blueberry on TMAO. Collectively, blueberry supplementation reduces TMAO possibly by modulating the gut microbes that are negatively associated with the circulating TMAO.
Evidence indicates a strong link between diet, gut microbes, and cardiovascular health (9, 37). Indeed, the effect of diet-derived microbial metabolites such as short-chain fatty acids and TMAO on vasculature is well-documented (1, 9). Human studies showed that circulating TMAO and its precursors are associated with an increased risk of cardiovascular disease, atherosclerotic burden, and death due to arterial diseases (8, 9). In addition, preclinical studies provide the possible mechanisms involved in TMAO accelerated atherosclerosis (9). As TMAO is implicated in several metabolic disorders, reducing circulating TMAO can have a significant effect on the healthcare system. Several therapeutic strategies are proposed to reduce TMAO that includes dietary intervention, modulating the gut microbes, inhibiting TMA production, and inhibiting the conversion of TMA to TMAO (38).
Dietary consumption of phenolic compounds may be one of the potential nutritional strategies to reduce the circulating TMAO (12, 39, 13, 15, 11, 16). In the present study, we investigated whether dietary supplementation of blueberry or strawberry reduces the circulating levels of TMA and TMAO in healthy mice. Blueberry supplementation greatly reduced the circulating levels of TMAO (p<0.001) in CB vs C mice though plasma choline and TMA levels were not significantly altered between these groups. However, strawberry supplementation did not significantly alter the circulating levels of choline, TMA, or TMAO (NS vs. N mice). Depleting gut microbes with antibiotics drastically reduced TMA and TMAO to non-detected or not-quantified levels in the blueberry cohort (CA and CBA mice) and the strawberry cohort (NA and NSA mice). This is consistent with previous studies that demonstrated the reduction in TMA and TMAO with antibiotic treatment (40, 7, 11).
Blueberry supplementation did not alter body weight, blood glucose, and body composition (lean body mass and fat mass) in CB vs C mice indicating the effect of blueberry on TMAO is not mediated through alterations in the metabolic parameters. The characterization of freeze-dried blueberry and strawberry powder indicates an extensive compositional difference, especially polyphenol and anthocyanin profiles. Both blueberries and strawberries are rich in anthocyanins as evidenced by total anthocyanin content values and individual polyphenol profiling in the present study. However, blueberries were also rich in hydroxycinnamic acids which is consistent with previous studies (41, 42). Importantly, chlorogenic acid (hydroxycinnamic acid derivative) was reported to reduce TMAO in both in vitro and in vivo studies (39, 15, 11, 16). Chlorogenic acid is abundant in blueberries but not in strawberries suggesting chlorogenic acid may be one of the bioactive components responsible for the effect of blueberries on TMAO observed in the present study. In addition, another possible contributing factor could be the total amount of phenolic compounds administered through each treatment, as blueberries presented a higher total polyphenolic content and total anthocyanin content than strawberries consistent with previous studies (43, 41). Also, other food matrix components, such as fiber type and content, could have a relevant effect, especially if these have prebiotic-like effects on the gut microbiota like fiber (44, 45).
Spearman’s correlation indicated that several gut microbes are positively or negatively associated with circulating TMA or TMAO. Further, blueberry supplementation increased the abundance of selected gut microbes that are negatively associated with TMA or TMAO. Importantly, blueberry supplementation increased the abundance of an unclassified genus of the family Coriobacteriaceae that was negatively associated with circulating TMAO. A recent human study showed a negative association between the Coriobacteriaceae and TMAO (46). In addition, a decrease in the abundance of Coriobacteriaceae was reported in heart failure patients suggesting the beneficial role of this bacterial family (47). In the present study, blueberry supplementation increased the abundance of Coriobacteriaceae which is consistent with a previous human study that showed the modulation of this bacterial family with raspberry consumption (46). Oscillospira and the unclassified genus of S24–7 were negatively associated with circulating TMAO whereas Turicibacter was negatively associated with TMA. The protective role of Oscillospira against atherosclerosis and a significant negative correlation with atherosclerotic plaque was reported previously (48). Further, S24–7 showed an inverse association with both TMA levels and atherosclerotic plaque area and a trend toward decreased plasma TMAO (49). A study investigated the gut microbial composition on TMAO response in healthy humans and this study identified S24–7 as a low TMAO-producer (50). Blueberry supplementation showed a trend towards an increased abundance of Oscillospira, Turicibacter, and the unclassified genus of S24–7 in CB vs. C mice. Berberine, an alkaloid commonly found in many plants including barberry, increased the abundance of S24–7 and inhibited TMA/TMAO by modulating gut microbiota in Apo E knockout mice (51). The present study indicates that the effect of dietary blueberries on TMAO could possibly be mediated by increasing the abundance of gut microbes that are negatively associated with TMA/TMAO.
5. CONCLUSION
Blueberry but not strawberry supplementation at a physiologically relevant dosage suppresses TMAO formation possibly by increasing the abundance of gut microbes that are negatively associated with circulating TMAO. This could be due to the extensive compositional difference between blueberry and strawberry, especially chlorogenic acid content. Our study suggests that blueberry supplementation could be a potential nutritional intervention to manage TMAO. However, further investigation is required to identify the efficiency of dietary blueberries in managing circulating levels of TMAO by using rodent models supplemented with high doses of choline and to determine the possible molecular mechanisms involved. Nevertheless, our study provides substantial proof of concept for further considering dietary blueberries to reduce circulating TMAO and to improve TMAO-mediated complications.
Supplementary Material
ACKNOWLEDGMENTS
Supported by research funds from the NIH/NCCIH: R01AT010247, USDA/NIFA: 2018-67018-27510, and USDA/NIFA: 2019-67017-29253 (to P.V.A.B.); and USDA/NIFA Predoctoral Fellowship Award: 2021-67034-35128 (to C.P.). Freeze-dried wild blueberry powder and freeze-dried strawberry powder were provided by the Wild Blueberry Association of North America (Maine, USA) and FutureCeuticals (Momence, IL, USA), respectively.
Footnotes
CONFLICT OF INTEREST
The authors declare that they have no competing interests. All authors have read and approved the submission of the manuscript and have provided consent for publication.
DATA AVAILABILITY STATEMENT
Raw sequencing reads for all samples described in this project have been deposited in the NCBI Sequence Read Archive under the accession number: PRJNA927009. All other relevant data are provided in the article, Supplementary information, or available from the corresponding author upon reasonable request. Bioinformatic tools, software versions, and parameters used in the present study are described in the “Methods” section. Additional details regarding the code to reproduce the analyses are available upon request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
Raw sequencing reads for all samples described in this project have been deposited in the NCBI Sequence Read Archive under the accession number: PRJNA927009. All other relevant data are provided in the article, Supplementary information, or available from the corresponding author upon reasonable request. Bioinformatic tools, software versions, and parameters used in the present study are described in the “Methods” section. Additional details regarding the code to reproduce the analyses are available upon request.