A. Rehman et al.
The Journal of Nutrition xxx (xxxx) xxx
the Frui fl ow group and Bacteroides acidifaciens and related spe- cies Parabacteroides goldsteinii whenFrui fl ow was compared with placebo. Cruden and Galask et al . [42] identi fi ed that Bacteroides sp. could reduce choline to TMA [43]. Moreover, Bacteroides sp. has been suggested to reduce TMAO to TMA [44]. Thus, it is reasonable to hypothesize that a reduction in Bacteroidetes via Fruit fl ow is one mechanism explaining the observed effects on TMAO. We also observed a reduction in Hungatella hathewayi and Rumonococcus faecis within the Frui fl ow group. Hungatella hathewayi is known to be a TMA producer [45], whereas Rumo- nococcus was positively correlated with plasma TMAO in a study in rats [46]. Moreover, we found an increase in Alistipes with Fruit fl ow when comparing between groups , another member of the Bacteroidetes phylum. This is in line with a recent clinical trial investigating the effect of short period lifestyle changes on car- diovascular disease markers and gut microbiome in which TMAO levels were inversely correlated with the relative abun- dances of Alistipes [47]. Finally, we observed an increase in Clostridium carnis when Frui fl ow was compared with placebo. Although the Clostridium group is generally known to produce TMA, which would contradict our observations, it is thought to be related to C. asparagiforme , C. hathewayi , and C. sporogenes [48, 49]. On the other hand, the Clostridium group is also known to produce large amounts of SCFA from several nutrients and therefore often cited for its health bene fi ts [50], although we did not fi nd an increase in fecal SCFA in our study. Other studies have also reported a broad range of antimicrobial mechanisms by which polyphenols modulate gut microbial communities as well as prebiotic-like properties favoring the growth of key bene fi cial species such as A. muciniphila, B. thetaiotaomicrom, F. prausnitzii, Bi fi dobacteria, and Lactobacilli [51]. However, under our experimental conditions, we did not observe similar effects. As mentioned above, TMAO may promote atherosclerosis by several mechanisms, including inhibiting hepatic BA synthesis [13, 15, 16, 38]. In the study in mice by Chen et al. [10], resveratrol attenuated TMAO-induced atherosclerosis not only by inhibiting microbial TMA production but also by increasing hepatic BA syn- thesis. This was thought to be due to resveratrol that increased the relative abundance of Lactobacillus and Bi fi dobacterium , which resulted in an increased bile salt hydrolase activity, BA deconju- gation, and subsequently increased fecal excretion of bile. The decrease in ileal BA content and repression of the enterohepatic farnesoid X receptor (FXR)- fi broblast growth factor 15 (FGF15) axis increased hepatic BA synthesis. We found that plasma CDCA, GCA, GCDCA, and GDCA rose signi fi cantly in the Fruit fl owgroup but not in the placebo group which may suggest an increase in hepatic BA synthesis. However, we did not observe changes in Lactobacillus or Bi fi dobacterium or reduced fecal BA contents. Thus, Fruit fl ow or its metabolites may alter FXR receptor activity and plasma BA pro fi les through other mechanisms as has been reported for metformin or acarbose, and this may further depend on gut microbiota compositions before treatments [52, 53]. Clearly further data are required for a better understanding of these effects. There was no signi fi cant difference in plasma LPS when comparing changes in both groups; however, we observed a signi fi cant within-group decrease in plasma LPS with Fruit fl ow but not placebo. LPS is a gut microbiota-derived factor and has been suggested to drive onset and progression of chronic in fl ammation-related diseases such as obesity, T2DM, or non- alcoholic fatty liver disease (NAFLD) [14, 54 – 56] Normally,
Our fi ndings are in line with the studies showing an overall reduction in plasma TMAO [10, 11, 19]. The mean decrease of 1.51 μ M in plasma TMAO within the Fruit fl ow group is similar to what was observed in the study by Annunziata et al. [9] (mean reduction of 1.2 μ M) and suggests reproducible effects with polyphenol-rich extracts. Although the reduction in our study was not signi fi cantly different between groups, most likely because of a lack of power in the per-protocol analysis, the observed effects size may be clinically relevant. A recent cross-sectional study including 377 patients with acute ischemic stroke and 50 healthy controls found that plasma TMAO levels were higher in patients with ischemic stroke than in healthy controls (median 5.1 vs. 3.0 μ mol/L; P < 0.001), and every 1- μ mol/L increase in TMAO was associated with a 1.13-point in- crease in the National Institutes of Health Stroke Scale and 1.69-mL increase in infarct volume after adjustment for vascular risk factors. We also investigated postprandial changes in plasma TMAO in a subgroup of participants that completed an acute egg chal- lenge. Fasting plasma TMAO levels were recently suggested to underestimate the TMAO-producing function of gut microbiota because of host ’ sef fi cient renal clearance of TMAO [40, 41]. As a result, oral challenge tests using egg or carnitine have been used to investigate postprandial increases in plasma TMAO [40, 41]. However, we did not observe an effect of Fruit fl ow on post- prandial plasma TMAO concentrations which may be related to the large variation in postprandial responses between in- dividuals, limited power ( n ¼ 9 subjects) and sampling time of only 8 hours which in turn requires independent research. Our observations support the notion that changes in plasma and urine TMAO are dependent on changes in the gut micro- biota. We found signi fi cant changes in several microbial taxa, although these were not re fl ected in microbial alpha and global beta diversity indices but a signi fi cant shift in Jaccard PC1 indicating that Fruit fl ow consumption can affect gut microbial composition. One strategy to alter plasma TMAO is to selectively decrease the proportion of bacteria that produce TMA and/or increase the proportion of microbes metabolizing TMA ef fi - ciently. We observed a signi fi cant decrease in the relative abundance of Bacteroides uniformis and Bacteroides ovatus within FIGURE5. Effects of 4 wk of supplementation of Fruit fl ow or placebo (maltodextrin) on plasma lipopolysaccharides (LPS) in overweight and obese adults. Boxes represent median and interquartile range (IQR), and the diamond shape, symbol mean values of absolute changes in plasma LPS from baseline to end of the intervention in each group. There were within-group changes, i.e. different from baseline at P 0.05. N ¼ 22.
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