Alanyl-glutamine promotes intestinal epithelial cell homeostasis in vitro and in a murine model of weanling undernutrition

Abstract

Alanyl-glutamine (Ala-Gln) has recently been shown to enhance catch-up growth and gut integrity in undernourished children from Northeast Brazil. We hypothesized that the intestinal epithelial effects of Ala-Gln in malnourished weanling mice and mouse small intestinal epithelial (MSIE) cells would include modulation of barrier function, proliferation, and apoptosis. Dams of 10-day-old suckling C57BL/6 pups were randomized to a standard diet or an isocaloric Northeast Brazil "regional basic diet," moderately deficient in protein, fat, and minerals. Upon weaning to their dam's diet on day of life 21, pups were randomized to Ala-Gln solution or water. At 6 wk of age, mice were killed, and jejunal tissue was collected for morphology, immunohistochemistry, and Ussing chamber analysis of transmucosal resistance and permeability. Proliferation of MSIE cells in the presence or absence of Ala-Gln was measured by MTS and bromodeoxyuridine assays. MSIE apoptosis was assessed by annexin and 7-amino-actinomycin D staining. Pups of regional basic diet-fed dams exhibited failure to thrive. Jejunal specimens from undernourished weanlings showed decreased villous height and crypt depth, decreased transmucosal resistance, increased permeability to FITC-dextran, increased claudin-3 expression, and decreased epithelial proliferation and increased epithelial apoptosis (as measured by bromodeoxyuridine and cleaved caspase-3 staining, respectively). Undernourished weanlings supplemented with Ala-Gln showed improvements in weight velocity, villous height, crypt depth, transmucosal resistance, and epithelial proliferation/apoptosis compared with unsupplemented controls. Similarly, Ala-Gln increased proliferation and reduced apoptosis in MSIE cells. In summary, Ala-Gln promotes intestinal epithelial homeostasis in a mouse model of malnutrition-associated enteropathy, mimicking key features of the human disease.

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

“…In this study, using ex vivo mouse jejunal enteroids to complement an in vivo finding of crypt preservation in undernourished mice treated with oral Ala-Gln, a stable dipeptide previously shown to promote growth and gut integrity in undernourished children and mice (7,23), we demonstrate important effects of Gln and Ala-Gln on intestinal epithelial homeostasis. In enteroids, we found that Gln or Ala-Gln promoted mTOR signal activation, epithelial proliferation, and crypt expansion.…”

“…We randomized wild-type C57/B6 dams with 10-day-old suckling pups to a multideficient regional basic diet (RBD) or an isocaloric, balanced control diet (CD), as previously described (22,23). Pups were weaned to their dams' diet (RBD vs. control) at 21 days of age and then subrandomized to ad libitum oral supplementation with a 111 mM solution of Ala-Gln (AminoStable, Ajinomoto) or plain drinking water.…”

“…Pups were weaned at 21 days of age to their dams' diet and randomized to plain drinking water or 111 mM Ala-Gln solution. We previously reported the composition of the RBD and protective effects of Ala-Gln on jejunal villus height, crypt depth, barrier function, and catch-up growth in RBD-challenged mice (23). In the current study we detected no differences in weight-based food consumption between groups (2.48 Ϯ 0.11, 2.61 Ϯ 0.37, and 2.64 Ϯ 0.48 g feed·g body wt Ϫ1 ·day Ϫ1 in CD, RBD, and RBD ϩ Ala-Gln, respectively; P ϭ 0.66, by ANOVA).…”

“…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 marker for actively cycling ISCs (Lgr5) (2), along with the development of ex vivo intestinal organoid systems to study ISC dynamics (17), provides new approaches to address this question.To elucidate the role of ISCs in Gln-mediated intestinal homeostasis, we examined jejunal crypt dynamics in undernourished wild-type mice treated with L-alanyl-L-glutamine (Ala-Gln), a stable Gln dipeptide shown to improve gut integrity in undernourished children and animal models (7,23). Enteroids are a nontransformed tissue culture system containing ISCs and differentiated intestinal epithelial cells within an organotypic three-dimensional structure containing crypt-, villus-, and lumenlike domains (12,17).…”

Morphology of the Intestinal Barrier in Different Physiological and Pathological Conditions

“…MTS and bromodeoxyuridine assays and increased epithelial cell apoptosis as measured by annexin and 7-aminoactinomycin D staining. All these changes were associated with decreased transmucosal resistance and increased permeability to FITC-dextran indicating intestinal barrier dysfunction [12].…”