Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 114, Issue 7, Pages E1273-E1281Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1621400114
Keywords
mTOR; N-ethyl-N-nitrosourea; inflammatory bowel disease; glycine amidinotransferase
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Funding
- Crohn's & Colitis Foundation of America Fellowship [2014CCFA00RFA0000090037]
- NIH K08 Grant [DK107886]
- Immunology T32 Training Grant [AI005184]
- NIH [R37 GM066759]
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Creatine, a nitrogenous organic acid, replenishes cytoplasmic ATP at the expense of mitochondrial ATP via the phosphocreatine shuttle. Creatine levels are maintained by diet and endogenous synthesis from arginine and glycine. Glycine amidinotransferase (GATM) catalyzes the rate-limiting step of creatine biosynthesis: the transfer of an amidino group from arginine to glycine to form ornithine and guanidinoacetate. We screened 36,530 third-generation germline mutant mice derived from N-ethyl-N-nitrosourea-mutagenized grandsires for intestinal homeostasis abnormalities after oral administration of dextran sodium sulfate (DSS). Among 27 colitis susceptibility phenotypes identified and mapped, one was strongly correlated with a missense mutation in Gatm in a recessive model of inheritance, and causation was confirmed by CRISPR/Cas9 gene targeting. Supplementation of homozygous Gatm mutants with exogenous creatine ameliorated the colitis phenotype. CRISPR/Cas9-targeted (Gatm(c/c)) mice displayed a normal peripheral immune response and immune cell homeostasis. However, the intestinal epithelium of the Gatm(c/c) mice displayed increased cell death and decreased proliferation during DSS treatment. In addition, Gatm(c/c) colonocytes showed increased metabolic stress in response to DSS with higher levels of phospho-AMPK and lower levels of phosphorylation of mammalian target of rapamycin (phospho-mTOR). These findings establish an in vivo requirement for rapid replenishment of cytoplasmic ATP within colonic epithelial cells in the maintenance of the mucosal barrier after injury.
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