Background: Trimethylamine-N-oxide (TMAO), a choline-derived gut microbiota-dependent metabolite, is a newly recognized risk marker for cardiovascular disease. We sought to determine: (1) TMAO response to meals containing free versus lipid-soluble choline and (2) effects of gut microbiome on TMAO response. Methods: In a randomized, controlled, double-blinded, crossover study, healthy men (n = 37) were provided meals containing 600 mg choline either as choline bitartrate or phosphatidylcholine, or no choline control. Results: Choline bitartrate yielded three-times greater plasma TMAO AUC (p = 0.01) and 2.5-times greater urinary TMAO change from baseline (p = 0.01) compared to no choline and phosphatidylcholine. Gut microbiota composition differed (permutational multivariate analysis of variance, PERMANOVA; p = 0.01) between high-TMAO producers (with ≥40% increase in urinary TMAO response to choline bitartrate) and low-TMAO producers (with <40% increase in TMAO response). High-TMAO producers had more abundant lineages of Clostridium from Ruminococcaceae and Lachnospiraceae compared to low-TMAO producers (analysis of composition of microbiomes, ANCOM; p < 0.05). Conclusion: Given that phosphatidylcholine is the major form of choline in food, the absence of TMAO elevation with phosphatidylcholine counters arguments that phosphatidylcholine should be avoided due to TMAO-producing characteristics. Further, development of individualized dietary recommendations based on the gut microbiome may be effective in reducing disease risk

Excess vitamin intake during pregnancy leads to obesogenic phenotypes, and folic acid accounts for many of these effects in male, but not in female, offspring. These outcomes may be modulated by another methyl nutrient choline and attributed to the gut microbiota. Pregnant Wistar rats were fed an AIN-93G diet with recommended vitamin (RV), high 10-fold multivitamin (HV), high 10-fold folic acid with recommended choline (HFol) or high 10-fold folic acid without choline (HFol-C) content. Male and female offspring were weaned to a high-fat RV diet for 12 weeks post-weaning. Removing choline from the HFol gestational diet resulted in obesogenic phenotypes that resembled more closely to HV in male and female offspring with higher body weight, food intake, glucose response to a glucose load and body fat percentage with altered activity, concentrations of short-chain fatty acids and gut microbiota composition. Gestational diet and sex of the offspring predicted the gut microbiota differences. Differentially abundant microbes may be important contributors to obesogenic outcomes across diet and sex. In conclusion, a gestational diet high in vitamins or imbalanced folic acid and choline content contributes to the gut microbiota alterations consistent with the obesogenic phenotypes of in male and female offspring.

Objectives Trimethylamine-N-oxide (TMAO), a choline-derived gut microbiota-dependent metabolite, is a newly recognized risk marker for cardiovascular disease. However, the contributions of different forms of choline and gut microbiota composition on TMAO production are largely unknown. The objectives of this study were to: 1) compare acute TMAO response to meals containing free choline (choline bitartrate) versus fat-soluble choline (phosphatidylcholine) and 2) to determine the effects of gut microbiota composition on TMAO response. Methods In a controlled, double-blinded, cross-over study, healthy men (n = 37) were provided meals containing (i) 600 mg choline as choline bitartrate (free choline); (ii) 600 mg choline as phosphatidylcholine; or (iii) no choline control in a random order. Blood and urine samples were collected at baseline and throughout the 6-h study period; a one-time stool sample was collected at baseline. Results Compared to no choline and phosphatidylcholine, free choline yielded 295% higher plasma TMAO (P = 0.002) and 250% higher urinary TMAO (P = 0.01), with no difference in TMAO response between phosphatidylcholine and no choline. High-TMAO producers (those with ≥40% increase in urinary TMAO response to free choline) had significantly different beta-diversity measures (unweighted UniFrac; PERMANOVA P = 0.01) compared to low-TMAO producers (those with &lt;40% increase in TMAO response) but showed no difference in alpha-diversity. Analysis of Composition of Microbiomes (ANCOM) revealed that high-TMAO producers had more abundant lineages of Clostridium from Ruminococcaceae and Lachnospiraceae (in phylum Firmicutes) compared to low-TMAO producers (P &lt; 0.05 with the strength of the ANCOM test W = 11 and W = 8, respectively). Conclusions Given that the majority of choline in food is in the form of phosphatidylcholine, the absence of TMAO elevation with phosphatidylcholine counters arguments that dietary choline should be avoided for TMAO-producing characteristics. Further, development of individualized dietary recommendations based on the gut microbiota composition may be a more appropriate strategy to reduce risk of cardiovascular disease. Funding Sources This research was supported by the Utah Agricultural Experiment Station Seed Grants Program.

Micronutrients consumed in excess or imbalanced amounts during pregnancy may increase the risk of metabolic diseases in offspring but the mechanisms underlying these effects are unknown. Serotonin (5-hydroxytryptamine, 5-HT), a multifunctional indoleamine in the brain and the gut, may have key roles regulating metabolism. We investigated the effects of gestational micronutrient intakes on the central and peripheral serotonergic systems as modulators of the offspring metabolic phenotypes. Pregnant Wistar rats were fed an AIN-93G diet with 1-fold recommended vitamins (RV), high 10-fold multivitamins (HV), high 10-fold folic acid with recommended choline (HFolRC) or high 10-fold folic acid with no choline (HFolNC). Male and female offspring were weaned to a high-fat RV diet for 12 weeks. We assessed the central function using the 5-HT2C receptor agonist, 1-(3-chlorophenyl)piperazine (mCPP), and found that male offspring from the HV- or HFolRC-fed dams were less responsive (P<0.05) whereas female HFolRC offspring were more responsive to mCPP (P<0.01) at 6 weeks post-weaning. Male and female offspring from the HV and HFolNC groups, and male HFolRC offspring had greater food intake (males P<0.001; females P<0.001) and weight gain (males P<0.0001; females P<0.0001), elevated colon 5-HT (males P<0.01; females P<0.001) and fasting glucose concentrations (males P<0.01; females P<0.01) as well as body composition toward obesity (males P<0.01; females P<0.01) at 12 weeks post-weaning. Colon 5-HT was correlated with fasting glucose concentrations (males R2=0.78, P<0.0001; females R2=0.71, P<0.0001). Overall, the serotonergic systems are sensitive to the composition of gestational micronutrients, with alterations consistent with metabolic disturbances in offspring.

Objectives Excess gestational folic acid and insufficient choline intakes as observed in the North American populations may increase the risk of obesity in offspring. It is well-established that adverse health outcomes may arise due to shifts in the gut microbial communities, but whether high vitamin intakes or an imbalance between methyl nutrients contributes to gut microbiota alterations is unclear. The objective of this research was to determine the gut microbiota composition of male and female offspring in relation to the vitamin composition of the gestational diet. Methods Pregnant Wistar rats (n = 10/group) were fed the AIN-93G diet with either the recommended vitamin (RV), high multivitamin (HV), high folic acid (HFol) or high folic acid without choline (HFol-C) content. Male and female offspring were weaned to a high-fat control diet for 12 weeks. Fecal samples were collected from the colon upon termination for gut microbiota profiling by 16S rRNA sequencing and data analyses in QIIME2. Results The overall gut microbial communities as assessed by unweighted UniFrac distances differed among the gestational diet groups for male (PERMANOVA P = 0.04) and female (PERMANOVA P = 0.05) offspring. The covariates gestational diet and sex predicted the gut microbiota differences in the offspring (Q2 = 0.07 in Songbird) whereas diet alone resulted in overfitting of the multinomial regression model (Q2 &lt; 0). High ranked features from the natural log-ratios of microbial abundance were Shigella, Clostridiales, Clostridiaceae for HV, and Odoribacter, Akkermansia muciniphila, Blautia for both HFol and HFol-C compared to RV. Low ranked features were Odoribacter for HV, Clostridiaceae and Clostridiales for HFol, and Bifidobacterium, Allobaculum, Lactobacillus vaginalis for HFol-C compared to RV. In male offspring, Lactobacillus vaginalis, Sutterella and Clostridiales were high ranked and Odoribacter was low ranked compared to female offspring. These differentially abundant microbes may be important contributors to obesity across diet and sex. Conclusions Increased vitamin content or an imbalance between folic acid and choline in the gestational diet leads to a shift in the gut microbiota composition in the offspring toward obesity. These effects differed by sex. Funding Sources Utah Agricultural Experiment Station and USU Research Catalyst. UNM supported by USU URCO.