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Title: Ex vivo enteroids recapitulate in vivo citrulline production in mice

item WANG, XIAOYING - Children'S Nutrition Research Center (CNRC)
item YUAN, YANG - Children'S Nutrition Research Center (CNRC)
item DIDELIJA, INKA - Children'S Nutrition Research Center (CNRC)
item MOHAMMAD, MAHMOUD - Children'S Nutrition Research Center (CNRC)
item MARINI, JUAN - Children'S Nutrition Research Center (CNRC)

Submitted to: Journal of Nutrition
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/24/2018
Publication Date: 8/31/2018
Citation: Wang, X., Yuan, Y., Didelija, I.C., Mohammad, M.A., Marini, J.C. 2018. Ex vivo enteroids recapitulate in vivo citrulline production in mice. Journal of Nutrition. 148(9):1415-1420.

Interpretive Summary: There is a need for in vitro systems to study intestinal citrulline production, which is the endogenous precursor for the amino acid arginine. Here we report a novel system (enteroids) derived from intestinal stem cells. We used wild type control mice and mutant mice that have a reduced capacity to produce citrulline. Enteroids derived from control and mutant mice produced citrulline, but as expected the mutant enteroids produced less. This in vitro system allows for genetic and biochemical manipulation which is needed to gain a better understanding of citrulline production and arginine synthesis.

Technical Abstract: The endogenous production of arginine relies on the synthesis of citrulline by enteral ornithine transcarbamylase (OTC). Mutations in the gene coding for this enzyme are the most frequent cause of urea cycle disorders. There is a lack of correlation between in vivo metabolic function and DNA sequence, transcript abundance or in vitro enzyme activity. The goal of the present work was to test the hypothesis that enteroids, a novel ex vivo model, is able to recapitulate the in vivo citrulline production of wild type and mutant mice. Six week old male wild type (WT) and OTC deficient mice (spf-ash mutation) were studied. Urea and citrulline fluxes were determined in vivo and OTC abundance measured in liver and gut tissue. Intestinal crypts were isolated and cultured to develop enteroids. Ex vivo citrulline production and OTC abundance were determined in these enteroids. Liver OTC abundance was lower (mean +/- SE; 0.16 +/- 0.01 vs. 1.85 +/- 0.18 a.u.; P < 0.001) in spf-ash than in WT mice, but there was no difference in urea production. In gut tissue, OTC was barely detectable in mutant mice; despite of this, a lower but substantial citrulline production (6 +/- 3 vs 167+/-8 µmol/kg-1/h-1, P < 0.001) was determined in the mutant animals. Enteroids recapitulated the in vivo findings of a very low OTC content accompanied by a reduced citrulline production (1.07 +/- 0.20 vs 4.64 +/- 0.44 nmole/µg DNA-1/d-1, P < 0.001). Enteroids recapitulate in vivo citrulline production and offer the opportunity to study the regulation of citrulline production in a highly manipulable system.