Moore SR, Guedes MM, Costa TB, Vallance J, Maier EA, Betz KJ, Aihara E, Mahe MM, Lima AA, Oriá RB, Shroyer NF. Glutamine and alanyl-glutamine promote crypt expansion and mTOR signaling in murine enteroids. Am J Physiol Gastrointest Liver Physiol 308: G831-G839, 2015. First published March 19, 2015; doi:10.1152/ajpgi.00422.2014.-L-Glutamine (Gln) is a key metabolic fuel for intestinal epithelial cell proliferation and survival and may be conditionally essential for gut homeostasis during catabolic states. We show that L-alanyl-L-glutamine (Ala-Gln), a stable Gln dipeptide, protects mice against jejunal crypt depletion in the setting of dietary protein and fat deficiency. Separately, we show that murine crypt cultures (enteroids) derived from the jejunum require Gln or Ala-Gln for maximal expansion. Once expanded, enteroids deprived of Gln display a gradual atrophy of cryptlike domains, with decreased epithelial proliferation, but stable proportions of Paneth and goblet cell differentiation, at 24 h. Replenishment of enteroid medium with Gln selectively activates mammalian target of rapamycin (mTOR) signaling pathways, rescues proliferation, and promotes crypt regeneration. Gln deprivation beyond 48 h leads to destabilization of enteroids but persistence of EGFP-Lgr5-positive intestinal stem cells with the capacity to regenerate enteroids upon Gln rescue. Collectively, these findings indicate that Gln deprivation induces a reversible quiescence of intestinal stem cells and provides new insights into nutritional regulation of intestinal epithelial homeostasis. intestinal organoids; L-glutamine; L-alanyl-L-glutamine; ERK; mammalian target of rapamycin IMPORTANT QUESTIONS regarding the maintenance of gastrointestinal (GI) epithelial homeostasis by the intestinal stem cells (ISCs) residing within the crypts of Lieberkühn are 1) how decisions about cellular growth, differentiation, and death are made and 2) how nutritional status and specific nutrients influence ISC dynamics (8,11, 28). Food provides a key stimulus for preserving the growth and normal crypt-villus architecture of the GI epithelium (19). Hence, atrophy of the GI mucosa is a common gut manifestation of both undernutrition and exclusive parenteral nutrition in humans and laboratory animals (24).Several gut trophic nutrients, including select amino acids, have been shown to promote GI epithelial homeostasis and barrier function in human and animal models of disease (3, 29). Among the amino acids with purported gut trophic effects, L-glutamine (Gln) continues to be a topic of keen interest, both as an important fuel for enterocytes and, more controversially, as a "conditionally essential" nutrient for GI epithelial homeostasis during severe illness (26). Despite several advances in understanding mechanisms of Gln in the gut (9), specific effects of Gln on ISC activation and differentiation have yet to be elucidated. This gap in knowledge is due, in part, to the long and elusive search for bona fide markers of ISCs. Thus the identification of a definitive m...
BackgroundFolate 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.ObjectivesWe 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.MethodsPregnant 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.ResultsBoth 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 α-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).ConclusionMDD 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.
BackgroundPolymeric immunoglobulin receptor (pIgR) transport of secretory immunoglobulin A (SIgA) to mucosal surfaces is thought to promote gut integrity and immunity to Salmonella enterica serovar Typhimurium (S. Typhimurium), an invasive pathogen in mice. To elucidate potential mechanisms, we assessed intestinal barrier function and both oral and systemic S. Typhimurium virulence in pIgR knockout (KO) and wildtype (WT) mice.MethodsIn uninfected animals, we harvested jejunal segments for Ussing chamber analyses of transepithelial resistance (TER); mesenteric lymph nodes (mLN) for bacterial culture; and serum and stool for IgA. Separately, we infected mice either orally or intravenously (IV) with S. Typhimurium to compare colonization, tissue dynamics, and inflammation between KOs and WTs.ResultsUninfected KOs displayed decreased TER and dramatically increased serum IgA and decreased fecal IgA vs. WT; however, KO mLNs yielded fewer bacterial counts. Remarkably, WTs challenged orally with S. Typhimurium exhibited increased splenomegaly, tissue colonization, and pro-inflammatory cytokines vs. pIgR KOs, which showed increased survival following either oral or IV infection.ConclusionsAbsence of pIgR compromises gut integrity but does not exacerbate bacterial translocation nor S. Typhimurium infection. These findings raise the possibility that immune adaptation to increased gut permeability and elevated serum IgA in the setting of SIgA deficiency provides compensatory protection against invasive gut pathogens.
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