Biodistribution and racemization of gut-absorbed L/D-alanine in germ-free mice

Microbiome-derived metabolites are important for the microbiome-gut-brain axis and the discovery of new disease treatments. D-Alanine (D-Ala) is found in many animals as a potential co-agonist of the N-methyl-D-aspartate receptors (NMDAR), receptors widely used in the nervous and endocrine systems. The gut microbiome, diet and putative endogenous synthesis are the potential sources of D-Ala in animals, although there is no direct evidence to show the distribution and racemization of gut-absorbed L-/D-Ala with regards to host-microbe interactions in mammals. In this work, we utilized germ-free mice to control the interference from microbiota and isotopically labeled L-/D-Ala to track their biodistribution and racemization in vivo. Results showed time-dependent biodistribution of gut-absorbed D-Ala, particularly accumulation of gut-absorbed D-Ala in pancreatic tissues, brain, and pituitary. No endogenous synthesis of D-Ala via racemization was observed in germ-free mice. The sources of D-Ala in mice were revealed as microbiota and diet, but not endogenous racemization. This work indicates the importance of further investigating the in vivo biological functions of gut-microbiome derived D-Ala, particularly on NMDAR-related activities, for D-Ala as a potential signaling molecules in the microbiome-gut-brain axis.