Creatine maintains intestinal homeostasis and protects against colitis

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 (Gatmc/c) mice displayed a normal peripheral immune response and immune cell homeostasis. However, the intestinal epithelium of the Gatmc/c mice displayed increased cell death and decreased proliferation during DSS treatment. In addition, Gatmc/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.

ATP dependence of K-Cl cotransport in dog red blood cells

Swelling-induced K-Cl cotransport in resealed dog red blood cell ghosts requires the presence of an ATP-generating system (G. C. Colclasure and J. C. Parker. J. Gen. Physiol. 100: 1-10, 1992). The present study shows that the endogenous adenine nucleotide present in the dog ghosts is sufficient to activate K-Cl cotransport, provided that creatine phosphate is incorporated in them. Creatine kinase is not required, because dog red blood cells, unlike those of humans, possess this enzyme. Although some ATP appears to be required for K-Cl cotransport by dog ghosts, an excess of this nucleotide is inhibitory. Creatine phosphate appears to play a special role in generating the ATP required for activation of K-Cl cotransport. If ghost ATP content is manipulated in the absence of creatine phosphate, by simply adding ATP to the hemolysate, no stimulation of K-Cl cotransport occurs. On the other hand, when creatine phosphate is present, K-Cl cotransport is activated. The results are discussed in relation to current views regarding the role of ATP in activation of K-Cl cotransport and the concept of the "phosphocreatine shuttle."