De novo phosphatidylcholine synthesis in the small intestinal epithelium is required for normal dietary lipid handling and maintenance of the mucosal barrier.

Cytidine triphosphate:phosphocholine cytidylyltransferase-α (CTα) is the rate limiting enzyme in the major pathway for de novo phosphatidylcholine (PC) synthesis. When CTα is deleted specifically in intestinal epithelial cells of adult mice (CTαIKO mice) fed a high-fat diet they present with weight loss, lipid malabsorption, and high postprandial GLP-1 levels. The current study aimed to characterize the changes that occur in the small intestines of CTαIKO mice using transcriptomics and to determine whether intestinal function could be rescued in CTαIKO mice. We found that impaired de novo PC synthesis in the gut is linked to lower abundance of transcripts related to lipid metabolism and higher abundance of transcripts related to ER stress and cell death, together with loss of goblet cells from the small intestinal epithelium. Furthermore, impaired movement of fatty acids from the intestinal lumen into enterocytes was observed in isolated intestinal sacs derived from CTαIKO mice, a model that excludes factors such as bile, gastric emptying, the nervous system, and circulating hormones. Antibiotic treatment prevented acute weight loss and normalized jejunum TG concentrations after refeeding but did not prevent ER stress or loss of goblet cells in CTαIKO mice. Dietary PC supplementation partially prevented loss of goblet cells but was unable to normalize jejunal TG concentrations after refeeding in CTαIKO mice. High postprandial plasma GLP-1 levels were present in CTαIKO mice regardless of antibiotic treatment, dietary PC content, or dietary fat content. Together, these data show that there is a specific requirement from de novo PC synthesis in maintaining small intestinal homeostasis, including dietary lipid uptake, normal hormone secretion, and barrier function.

Timing of Tributyrin Supplementation Differentially Modulates Gastrointestinal Inflammation and Gut Microbial Recolonization Following Murine Ileocecal Resection.

BACKGROUND: Gastrointestinal surgery imparts dramatic and lasting imbalances, or dysbiosis, to the composition of finely tuned microbial ecosystems. The aim of the present study was to use a mouse ileocecal resection (ICR) model to determine if tributyrin (TBT) supplementation could prevent the onset of microbial dysbiosis or alternatively enhance the recovery of the gut microbiota and reduce gastrointestinal inflammation. METHODS: Male wild-type (129 s1/SvlmJ) mice aged 8-15 weeks were separated into single cages and randomized 1:1:1:1 to each of the four experimental groups: control (CTR), preoperative TBT supplementation (PRE), postoperative TBT supplementation (POS), and combined pre- and postoperative supplementation (TOT). ICR was performed one week from baseline assessment with mice assessed at 1, 2, 3, and 4 weeks postoperatively. Primary outcomes included evaluating changes to gut microbial communities occurring from ICR to 4 weeks. RESULTS: A total of 34 mice that underwent ICR (CTR n = 9; PRE n = 10; POS n = 9; TOT n = 6) and reached the primary endpoint were included in the analysis. Postoperative TBT supplementation was associated with an increased recolonization and abundance of anaerobic taxa including Bacteroides thetaiotomicorn, Bacteroides caecimuris, Parabacteroides distasonis, and Clostridia. The microbial recolonization of PRE mice was characterized by a bloom of aerotolerant organisms including Staphylococcus, Lactobacillus, Enteroccaceae, and Peptostreptococcacea. PRE mice had a trend towards decreased ileal inflammation as evidenced by decreased levels of IL-1ß (p = 0.09), IL-6 (p = 0.03), and TNF-α (p < 0.05) compared with mice receiving TBT postoperatively. In contrast, POS mice had trends towards reduced colonic inflammation demonstrated by decreased levels of IL-6 (p = 0.07) and TNF-α (p = 0.07). These changes occurred in the absence of changes to fecal short-chain fatty acid concentrations or histologic injury scoring. CONCLUSIONS: Taken together, the results of our work demonstrate that the timing of tributyrin supplementation differentially modulates gastrointestinal inflammation and gut microbial recolonization following murine ICR.

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.

Sex-Specific Differences in the Gut Microbiome in Response to Dietary Fiber Supplementation in IL-10-Deficient Mice.

There is growing interest in studying dietary fiber to stimulate microbiome changes that might prevent or alleviate inflammatory bowel disease (IBD). However, dietary fiber effects have shown varying degrees of efficacy, for reasons that are unclear. This study examined whether the effects of isomaltodextrin on gut microbiota and IBD were dependent on dose or host sex, using an Interleukin (IL)-10 deficient murine colitis model. After 12 weeks, colonic IL-12p70 was depressed in male mice receiving high-dose isomaltodextrin supplementation compared to the control group (p = 0.04). Male mice receiving high-dose isomaltodextrin exhibited changes in microbial alpha-diversity, including enhanced richness and evenness (p = 0.01) and limited reduction in the relative abundance of Coprococcus (q = 0.08), compared to the control group. These microbial compositional changes were negatively associated with IL-12p70 levels in the male group (rs ≤ -0.51, q ≤ 0.08). In contrast, female mice receiving isomaltodextrin displayed a reduction in alpha-diversity and Coprococcus abundance and a high level of IL-12p70, as did the control group. Together, these results indicate that isomaltodextrin altered the gut microbial composition linking specific immune-regulatory cytokine responses, while the interactions among fiber, microbiota and immune response were dose dependent and largely sex specific. The results further indicate that interactions between environmental and host factors can affect microbiome manipulation in the host.

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.

Prebiotic Supplementation Following Ileocecal Resection in a Murine Model is Associated With a Loss of Microbial Diversity and Increased Inflammation.

Background: Individuals with Crohn's disease frequently require ileocecal resection (ICR), and inflammation often recurs in the neoterminal ileum following surgery. Fructooligosaccharide (FOS) is a fermentable prebiotic that stimulates the growth of bifidobacteria and may promote anti-inflammatory activity. The aim of this study was to determine if supplementation of a postICR diet with FOS in a mouse model would be effective in stimulating the growth of bifidobacteria and reducing systemic and local inflammation. Methods: ICR was performed in IL10-/- mice (129S1/SvlmJ) with colitis. Following surgery, nonICR control and ICR mice were fed a chow diet ± 10% FOS for 28 days. Serum, colon, and terminal ileum (TI) were analyzed for cytokine expression by MesoScale discovery platform. DNA extracted from stool was analyzed using 16s rRNA sequencing and qPCR. Expression of occludin and ZO1 was assessed using qPCR. Short-chain fatty acid (SCFA) concentrations were assessed using gas chromatography. Results: ICR led to increased systemic inflammation (P < 0.05) and a significant decline in fecal microbial diversity (P < 0.05). Mice on the FOS diet had a greater reduction in microbial diversity and also had worsened inflammation as evidenced by increased serum IL-6 (P < 0.05) and colonic IFNγ and TNFα (P < 0.05). Expression of occludin and ZO1 were significantly reduced in FOS-supplemented mice. There was a correlation between loss of diversity and the bifidogenic effectiveness of FOS (r = -0.61, P < 0.05). Conclusions: FOS-supplementation of a postICR diet resulted in a decrease in fecal bacterial diversity, reduction in barrier function, and increased gut inflammation.

Choline supplementation protects against liver damage by normalizing cholesterol metabolism in Pemt/Ldlr knockout mice fed a high-fat diet.

Dietary choline is required for proper structure and dynamics of cell membranes, lipoprotein synthesis, and methyl-group metabolism. In mammals, choline is synthesized via phosphatidylethanolamine N-methyltransferase (Pemt), which converts phosphatidylethanolamine to phosphatidylcholine. Pemt(-/-) mice have impaired VLDL secretion and developed fatty liver when fed a high-fat (HF) diet. Because of the reduction in plasma lipids, Pemt(-/-)/low-density lipoprotein receptor knockout (Ldlr(-/-)) mice are protected from atherosclerosis. The goal of this study was to investigate the importance of dietary choline in the metabolic phenotype of Pemt(-/-)/Ldlr(-/-) male mice. At 10-12 wk of age, Pemt(+/+)/Ldlr(-/-) (HF(+/+)) and half of the Pemt(-/-)/Ldlr(-/-) (HF(-/-)) mice were fed an HF diet with normal (1.3 g/kg) choline. The remaining Pemt(-/-)/Ldlr(-/-) mice were fed an HF diet supplemented (5 g/kg) with choline (HFCS(-/-) mice). The HF diet contained 60% of calories from fat and 1% cholesterol, and the mice were fed for 16 d. HF(-/-) mice lost weight and developed hepatomegaly, steatohepatitis, and liver damage. Hepatic concentrations of free cholesterol, cholesterol-esters, and triglyceride (TG) were elevated by 30%, 1.1-fold and 3.1-fold, respectively, in HF(-/-) compared with HF(+/+) mice. Choline supplementation normalized hepatic cholesterol, but not TG, and dramatically improved liver function. The expression of genes involved in cholesterol transport and esterification increased by 50% to 5.6-fold in HF(-/-) mice when compared with HF(+/+) mice. Markers of macrophages, oxidative stress, and fibrosis were elevated in the HF(-/-) mice. Choline supplementation normalized the expression of these genes. In conclusion, HF(-/-) mice develop liver failure associated with altered cholesterol metabolism when fed an HF/normal choline diet. Choline supplementation normalized cholesterol metabolism, which was sufficient to prevent nonalcoholic steatohepatitis development and improve liver function. Our data suggest that choline can promote liver health by maintaining cholesterol homeostasis.

Effects of Lactobacillus helveticus on murine behavior are dependent on diet and genotype and correlate with alterations in the gut microbiome.

Modulation of the gut microbiota with diet and probiotic bacteria can restore intestinal homeostasis in inflammatory conditions and alter behavior via the gut-brain axis. The purpose of this study was to determine whether the modulatory effects of probiotics differ depending on diet and mouse genotype. At weaning, wild type (WT) and IL-10 deficient (IL-10(-/-)) 129/SvEv mice were placed on a standard mouse chow or a Western-style diet (fat 33%, refined carbohydrate 49%)±Lactobacillus helveticus ROO52 (10(9)cfu/d) for 21 days. Animal weight and food eaten were monitored weekly. Intestinal immune function was analysed for cytokine expression using the Meso Scale Discovery platform. Spatial memory and anxiety-like behavior was assessed in a Barnes maze. Terminal restriction fragment length polymorphism (TRFLP) was used to analyze the fecal microbiota. Both WT and IL-10(-/-) mice on a Western diet had increased weight gain along with changes in gut microbiota and cytokine expression and altered anxiety-like behavior. The ability of L. helveticus to modulate these factors was genotype- and diet-dependent. Anxiety-like behavior and memory were negatively affected by Western-style diet depending on inflammatory state, but this change was prevented with L. helveticus administration. However, probiotics alone decreased anxiety-like behavior in WT mice on a chow diet. Mice on the Western diet had decreased inflammation and fecal corticosterone, but these markers did not correlate with changes in behavior. Analysis of bacterial phyla from WT and IL-10(-/-)mice showed discrete clustering of the groups to be associated with both diet and probiotic supplementation, with the diet-induced shift normalized to some degree by L. helveticus. These findings suggest that the type of diet consumed by the host and the presence or absence of active inflammation may significantly alter the ability of probiotics to modulate host physiological function.

Dietary lipids alter the effect of steroids on transport of glucose after intestinal resection: Part II. Signalling of the response.

BACKGROUND/PURPOSE: Glucocorticosteroids alter the function of the intestine. Budesonide (Bud) increases the jejunal D-glucose uptake, and this effect is prevented through a polyunsaturated fatty acid (PUFA) diet. This study was undertaken to assess the possible signalling effect of budesonide, prednisone (Pred), or dexamethasone (Dex) in animals with a 50% intestinal resection and fed chow or a diet enriched with saturated (SFA) or polyunsaturated fatty acids. METHODS: Northern blots were performed. RESULTS: Steroids reduced the jejunal but not the ileal expression of proglucagon. Ornithine decarboxylase (ODC) expression was reduced in the jejunum. CONCLUSIONS: c-jun, ODC, and proglucagon may be involved in the adaptive response that occurs with steroids and variations in dietary lipids after intestinal resection.

Dietary lipids alter the effect of steroids on the transport of glucose after intestinal resection: Part I. Phenotypic changes and expression of transporters.

BACKGROUND/PURPOSE: Glucocorticosteroids alter the function of the intestine. This study was undertaken to assess the effect on D-glucose uptake of budesonide (Bud), prednisone (Pred), or dexamethasone (Dex) in animals with a 50% intestinal resection and fed chow or a diet enriched with saturated (SFA) or polyunsaturated fatty acids (PUFA). METHODS: In vitro ring uptake technique, Western blots, and Northern blots were performed. RESULTS: Bud increased the jejunal D-glucose uptake, and this effect was prevented by feeding PUFA. SGLT1 and Na+/K+ ATPase protein and mRNA abundance did not correlate with the change in the rate of uptake of glucose. CONCLUSIONS: (1) Bud increased the jejunal glucose uptake, (2) the activity of the sugar transporter does not correlate with the abundance of protein or their respective mRNAs, (3) th Bud effect on glucose uptake is prevented by feeding PUFA. Thus, the desired intestinal adaptive response after intestinal resection may be enhanced further by the administration of the locally acting steroid budesonide and by feeding a saturated compared with a polyunsaturated fatty acid diet.