Biodistribution and racemization of gut-absorbed l/d-alanine in germ-free mice

The microbial metabolite, d-alanine, cannot be endogenously synthesized by mice, and is instead derived from the food and gut microbiota, with gut-absorbed d-alanine accumulating in the mouse pancreas, brain, and pituitary. 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.