Murine Methyl Donor Deficiency Impairs Early Growth in Association with Dysmorphic Small Intestinal Crypts and Reduced Gut Microbial Community Diversity

Background: Folate and choline are essential methyl donor nutrients throughout the life span; however, the adverse effects of combined deficiency on early growth, intestinal epithelial morphology, and the gut microbiome remain only partially understood. Objectives: We investigated the effects of dietary folate and choline deficiency on early growth, small intestinal (SI) epithelial architecture, and the gut microbiota of mice. To explore potential mechanisms for adverse effects on gut epithelial morphology, we also evaluated gene expression and DNA methylation in mouse intestinal epithelial organoids (enteroids) maintained in methyl donor-deficient (MDD) conditions. Methods: Pregnant dams were administered 1 of 4 diets: 1) control diet (CD-), 2) an isocaloric MDD- diet, or 3) CD+ and 4) MDD+ formulations containing 1% succinylsulfathiazole to inhibit folate-producing gut bacteria. We weaned pups to their dams' diet at 3 wk of age and monitored body weight and tail length pre- and postweaning. We measured serum folate, SI crypt morphology, and microbiota composition at 7 wk of age. Results: Both MDD+ and MDD- diets impaired early ponderal and linear growth, lowered serum folate concentrations, and produced patchy areas of increased crypt depth throughout the SI. Succinylsulfathiazole increased crypt depth independently of diet. MDD or succinylsulfathiazole, alone or in combination, altered the gut microbiome, with decreased Bacteroidales and Clostridiales, increased Lactobacillales and Erysipelotrichaceae taxa, and decreased alpha-diversity indexes. Enteroids maintained in MDD media displayed dysmorphic crypt domains, altered expression of stem cell and secretory differentiation genes, and decreased DNA methylation of the glycosylation genes Beta-1,4-N-Acetyl-Galactosaminyltransferase-1 (B4galnt1) and Phosphoethanolamine/Phosphocholine-Phosphatase (Phospho1). Conclusions: MDD impairs ponderal and linear growth in mice in association with dysmorphic SI crypts and reduced gut microbial diversity. In vitro methyl donor deficiency similarly induced dysmorphic crypts in mouse enteroids in conjunction with altered gene expression and DNA methylation.