Over supplementation with vitamin B12 alters microbe-host interactions in the gut leading to accelerated Citrobacter rodentium colonization and pathogenesis in mice.

BACKGROUND: Vitamin B12 supplements typically contain doses that far exceed the recommended daily amount, and high exposures are generally considered safe. Competitive and syntrophic interactions for B12 exist between microbes in the gut. Yet, to what extent excessive levels contribute to the activities of the gut microbiota remains unclear. The objective of this study was to evaluate the effect of B12 on microbial ecology using a B12 supplemented mouse model with Citrobacter rodentium, a mouse-specific pathogen. Mice were fed a standard chow diet and received either water or water supplemented with B12 (cyanocobalamin: ~120 µg/day), which equates to approximately 25 mg in humans. Infection severity was determined by body weight, pathogen load, and histopathologic scoring. Host biomarkers of inflammation were assessed in the colon before and after the pathogen challenge. RESULTS: Cyanocobalamin supplementation enhanced pathogen colonization at day 1 (P < 0.05) and day 3 (P < 0.01) postinfection. The impact of B12 on gut microbial communities, although minor, was distinct and attributed to the changes in the Lachnospiraceae populations and reduced alpha diversity. Cyanocobalamin treatment disrupted the activity of the low-abundance community members of the gut microbiota. It enhanced the amount of interleukin-12 p40 subunit protein (IL12/23p40; P < 0.001) and interleukin-17a (IL-17A; P < 0.05) in the colon of naïve mice. This immune phenotype was microbe dependent, and the response varied based on the baseline microbiota. The cecal metatranscriptome revealed that excessive cyanocobalamin decreased the expression of glucose utilizing genes by C. rodentium, a metabolic attribute previously associated with pathogen virulence. CONCLUSIONS: Oral vitamin B12 supplementation promoted C. rodentium colonization in mice by altering the activities of the Lachnospiraceae populations in the gut. A lower abundance of select Lachnospiraceae species correlated to higher p40 subunit levels, while the detection of Parasutterella exacerbated inflammatory markers in the colon of naïve mice. The B12-induced change in gut ecology enhanced the ability of C. rodentium colonization by impacting key microbe-host interactions that help with pathogen exclusion. This research provides insight into how B12 impacts the gut microbiota and highlights potential consequences of disrupting microbial B12 competition/sharing through over-supplementation. Video Abstract.

Intestinal Phospholipid Disequilibrium Initiates an ER Stress Response That Drives Goblet Cell Necroptosis and Spontaneous Colitis in Mice.

BACKGROUND & AIMS: Patients with ulcerative colitis have low concentrations of the major membrane lipid phosphatidylcholine (PC) in gastrointestinal mucus, suggesting that defects in colonic PC metabolism might be involved in the development of colitis. To determine the precise role that PC plays in colonic barrier function, we examined mice with intestinal epithelial cell (IEC)-specific deletion of the rate-limiting enzyme in the major pathway for PC synthesis: cytidine triphosphate:phosphocholine cytidylyltransferase-α (CTαIKO mice). METHODS: Colonic tissue of CTαIKO mice and control mice was analyzed by histology, immunofluorescence, electron microscopy, quantitative polymerase chain reaction, Western blot, and thin-layer chromatography. Histopathologic colitis scores were assigned by a pathologist blinded to the experimental groupings. Intestinal permeability was assessed by fluorescein isothiocyanate-dextran gavage and fecal microbial composition was analyzed by sequencing 16s ribosomal RNA amplicons. Subsets of CTαIKO mice and control mice were treated with dietary PC supplementation, antibiotics, or 4-phenylbutyrate. RESULTS: Inducible loss of CTα in the intestinal epithelium reduced colonic PC concentrations and resulted in rapid and spontaneous colitis with 100% penetrance in adult mice. Colitis development in CTαIKO mice was traced to a severe and unresolving endoplasmic reticulum stress response in IECs with altered membrane phospholipid composition. This endoplasmic reticulum stress response was linked to the necroptotic death of IECs, leading to excessive loss of goblet cells, formation of a thin mucus barrier, increased intestinal permeability, and infiltration of the epithelium by microbes. CONCLUSIONS: Maintaining the PC content of IEC membranes protects against colitis development in mice, showing a crucial role for IEC phospholipid equilibrium in colonic homeostasis. SRA accession number: PRJNA562603.

Insufficient dietary choline aggravates disease severity in a mouse model of Citrobacter rodentium-induced colitis.

Dietary choline, which is converted to phosphatidylcholine (PC) in intestinal enterocytes, may benefit inflammatory bowel disease patients who typically have reduced intestinal choline and PC. The present study investigated the effect of dietary choline supplementation on colitis severity and intestinal mucosal homoeostasis using a Citrobacter rodentium-induced colitis model. C57BL/6J mice were fed three isoenergetic diets differing in choline level: choline-deficient (CD), choline-sufficient (CS) and choline-excess (CE) for 3 weeks prior to infection with C. rodentium. The effect of dietary choline levels on the gut microbiota was also characterised in the absence of infection using 16S rRNA gene amplicon sequencing. At 7 d following infection, the levels of C. rodentium in CD mice were significantly greater than that in CS or CE groups (P < 0·05). CD mice exhibited greater damage to the surface epithelium and goblet cell loss than the CS or CE mice, which was consistent with elevated pro-inflammatory cytokine and chemokine levels in the colon. In addition, CD group exhibited decreased concentrations of PC in the colon after C. rodentium infection, although the decrease was not observed in the absence of challenge. Select genera, including Allobaculum and Turicibacter, were enriched in response to dietary choline deficiency; however, there was minimal impact on the total bacterial abundance or the overall structure of the gut microbiota. Our results suggest that insufficient dietary choline intake aggravates the severity of colitis and demonstrates an essential role of choline in maintaining intestinal homoeostasis.

Pea polyphenolics and hydrolysis processing alter microbial community structure and early pathogen colonization in mice.

Health benefits associated with pea consumption have been attributed to the fiber and polyphenolic content concentrated within the pea seed coat. However, the amount of pea polyphenols can vary between cultivars, and it has yet to be studied whether pea polyphenols impact the intestinal microbiota. We hypothesized that pea polyphenols promote a healthy microbiome that supports intestinal integrity and pathogen colonization resistance. To investigate the effects of pea polyphenols, pea cultivars rich and poor in proanthocyanidins were supplemented in raw or acid hydrolyzed form to an isocaloric diet in mice. Acid hydrolysis increases the absorption of pea polyphenols by cleaving polymeric proanthocyanidins to their readily absorbable anthocyanidin monomers. After 3 weeks of diet, mice were challenged with Citrobacter rodentium and pathogen colonization and inflammation were assessed. Counter to our hypothesis, pea seed coat fraction supplementation, especially the non-hydrolyzed proanthocyanidin-rich fraction diet adversely increased C. rodentium pathogen load and inflammation. Ileal, cecal and colon microbial communities were notably distinct between pea seed cultivar and hydrolysis processing. The consumption of intact proanthocyanidins decreased microbial diversity indicating that proanthocyanidins have antimicrobial properties. Together our results indicate supplementation of raw pea seed coat rich in proanthocyanidins adversely affect intestinal integrity. However, acid hydrolysis processing restored community structure and colonization resistance, and the anthocyanidin-rich fractions reduced weight gain on a high fat diet. Establishing a clear understanding of the effects of pea fiber and polyphenolic form on health will help to develop research-based pea products and dietary recommendations.

Hepatic Expression of PEMT, but Not Dietary Choline Supplementation, Reverses the Protection against Atherosclerosis in Pemt-/-/Ldlr-/- Mice.

Background: Phosphatidylethanolamine N-methyltransferase (PEMT) converts phosphatidylethanolamine to phosphatidylcholine. Pemt-/-/low density lipoprotein receptor (Ldlr)-/- mice have significantly reduced plasma lipids and are protected against atherosclerosis. Recent studies have shown that choline can be metabolized by the gut flora into trimethylamine-N-oxide (TMAO), which is an emerging risk factor for atherosclerosis. Objective: The objective of this study was to determine whether ectopic hepatic PEMT expression or choline supplementation would promote atherosclerosis in Pemt-/-/Ldlr-/- mice. Methods: Male 8- to 10-wk-old Pemt+/+/Ldlr-/- (SKO) and Pemt-/-/Ldlr-/- (DKO) mice were injected with an adeno-associated virus (AAV) expressing green fluorescent protein (GFP) or human PEMT and fed a Western diet (40% of calories from fat, 0.5% cholesterol) for 8 wk. In a separate experiment, 8- to 10-wk-old SKO and half of the DKO male mice were fed a Western diet with normal (3 g/kg) choline for 12 wk. The remaining DKO mice [choline-supplemented (CS) DKO] were fed a CS Western diet (10 g choline/kg). Plasma lipid concentrations, choline metabolites, and aortic atherosclerosis were measured. Results: Plasma cholesterol, plasma TMAO, and aortic atherosclerosis were reduced by 60%, 40%, and 80%, respectively, in DKO mice compared with SKO mice. AAV-PEMT administration increased plasma cholesterol and TMAO by 30% and 40%, respectively, in DKO mice compared with AAV-GFP-treated DKO mice. Furthermore, AAV-PEMT-injected DKO mice developed atherosclerotic lesions similar to SKO mice. In the second study, there was no difference in atherosclerosis or plasma cholesterol between DKO and CS-DKO mice. However, plasma TMAO concentrations were increased 2.5-fold in CS-DKO mice compared with DKO mice. Conclusions: Reintroducing hepatic PEMT reversed the atheroprotective phenotype of DKO mice. Choline supplementation did not increase atherosclerosis or plasma cholesterol in DKO mice. Our data suggest that plasma TMAO does not induce atherosclerosis when plasma cholesterol is low. Furthermore, this is the first report to our knowledge that suggests that de novo choline synthesis alters TMAO status.

Effects of different methionine sources on production and reproduction performance, egg quality and serum biochemical indices of broiler breeders.

OBJECTIVE: The study was conducted to evaluate the effects of different methionine (Met) sources on production performance, reproduction performance, egg quality and serum biochemical indices in broiler breeders. METHODS: After receiving a basal diet (containing 0.25% Met) for a 2-wk pretreatment period, a total of 360 39-wk-old Lingnan yellow broiler breeders were randomly allocated to four treatments with six replicates each (15 birds per replicate). Breeders were fed with basal diets (control) or diets supplemented with DL-methionine (DLM), DL-2-hydroxy-4-methylthio butytric calcium (MHA-Ca) and coated DL-Met (CME) respectively. RESULTS: The results showed that CME supplementation promoted laying rate and decreased feed-to-egg ratio (F/E) (p<0.05), DLM and MHA-Ca supplementation decreased F/E (p<0.05) when compared with control group. The rate of fertility, hatchability and birthrate were higher (p<0.05) in DLM, MHA-Ca, and CME groups than control group. Compared with control group, CME increased the eggshell thickness (p<0.05); MHA-Ca improved the eggshell thickness, shell ratio and eggshell strength (p<0.05). Results also showed that CME elevated the concentration of total protein in serum (p<0.05); MHA-Ca improved the concentration of calcium in serum (p<0.05). The concentration of serum uric acid in DLM, MHA-Ca, and CME groups was lower than that in control group (p<0.05). Besides, CME had higher concentrations of serum taurine, cysteine and cystanthionine (p<0.05) while MHA-Ca and DLM promoted the serum taurine concentration (p<0.05) compared with control group. CONCLUSION: Based on the results, it was concluded that Met supplementation could enhance the production and reproduction performance as well as the antioxidant status and egg quality of broiler breeders. In terms of improving the production performance, reproduction performance and antioxidant performance, CME was superior to DLM and MHA-Ca; but in regard to the enhancement of eggshell quality and serum Ca concentration, MHA-Ca was superior to DLM and CME.

S-adenosylmethionine-induced adipogenesis is accompanied by suppression of Wnt/ß-catenin and Hedgehog signaling pathways.

S-Adenosylmethionine (SAM) plays a crucial role as a methyl donor in various biological processes and has been previously shown to be involved in adipogenesis in skeletal muscle. This study was conducted to explore the mechanism of SAM inducing adipogenesis in skeletal muscle. Adipose precursor cells, 3T3-L1, and C2C12 cells, were induced into adipogenic differentiation by addition of SAM in MDI-differentiation media (0.5 mmol/L isobutylmethylxanthine, 1 µm/L dexamethasone, and 10 µg/mL insulin) to explore the role of SAM in promoting adipogenesis. Subsequently, cells were cultured with a medium containing SAM alone at the beginning of differentiation to test the relationship between SAM-induced adipogenesis and Wnt/ß-catenin, and Hedgehog signaling pathways that control the cell commitment to adipogenic- or myogenic-differentiation. We found SAM possessed an additive effect with MDI in promoting adipogenesis of 3T3-L1 and C2C12 cells at the beginning of adipogenic differentiation. SAM could also individually induce cell adipogenesis in a dose-dependent manner. Moreover, the expression of Wnt/ß-catenin and Hedgehog signals and their targets were suppressed by SAM (P < 0.05). These results demonstrate that SAM, as an increasingly accepted nutritional supplement, can initiate adipogenesis of adipose precursor cells derived from adipose and muscle tissues, a function at least partly correlated with the suppression of Wnt/ß-catenin and Hedgehog pathways.