Glutamine and alanyl-glutamine promote crypt expansion and mTOR signaling in murine enteroids

Abstract

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...

Distinct Effects of Growth Hormone and Glutamine on Activation of Intestinal Stem Cells

“…Furthermore, combined GH and GLN treatments have been found to have synergistic effects on intestinal adaptation in animal studies and in patients with SBS . Although these viewpoints have been widely accepted for many years, the impact on ISCs has been reported only in very few studies . In our research, in vitro and in vivo studies revealed that GH treatment enhances the formation of crypt organoids, increases ISC stemness markers in the intestinal organoids, and increases proliferation.…”

“…[1][2][3][4]10,12 Although these viewpoints have been widely accepted for many years, the impact on ISCs has been reported only in very few studies. 36 In our research, in vitro and in vivo studies revealed that GH treatment enhances the formation of crypt organoids, increases ISC stemness markers in the intestinal organoids, and increases proliferation. In addition, GH upregulates the expression of lysozyme and villin, indicating an increased number of Paneth cells and enterocytes.…”

“…The interactions between GH and GLN were speculated to be dependent on multiple factors such as the dose or concentration, route of administration, treatment duration, and treatment sequence of GH and GLN . The dose/concentration of GH and GLN may affect the results we report; however, the doses/concentrations of GH and GLN we used for in vitro and in vivo studies were chosen according to previous reports . For the in vivo experiments, GLN treatment in mice might only elevate the plasma GLN concentration for a short time and GH alone can increase the plasma GLN level, which suggest the dose of GLN present in the GH + GLN group may not necessarily increase the plasma GLN concentration enough to differentiate GH + GLN effects from those of GH alone .…”

“…[1][2][3][4][5][6]8,12,14 The dose/concentration of GH and GLN may affect the results we report; however, the doses/concentrations of GH and GLN we used for in vitro and in vivo studies were chosen according to previous reports. 2,4,13,14,36,38,45,46 For the in vivo experiments, GLN treatment in mice might only elevate the plasma GLN concentration for a short time 44 and GH alone can increase the plasma GLN level, which suggest the dose of GLN present in the GH + GLN group may not necessarily increase the plasma GLN concentration enough to differentiate GH + GLN effects from those of GH alone. 10,47 Different route of administration may also affect the absorption efficiency and plasma levels of GH and GLN.…”

New insights into environmental enteric dysfunction

“…Trials using specific amino acids such as arginine and glutamine,34 proteins such as lactoferrin and lysozyme,35 and legumes as supplementary foods for children during their period of weaning36 all have potential for demonstrating improvements in EED and stunting. Foods, either genetically modified or natural, that are high in growth factors that reduce apoptosis, enhance enterocyte proliferation and stimulate villous growth are prime targets for investigation.…”