Gut Luminal Exosomes in Young and Old Mice: Multi‐Omic Characteristics and Regulation of Gut Permeability

Aging is a multifaceted process impacting physiological, genomic, metabolic, and immune functions. This study investigates the role of luminal fecal exosomes (LFEs) in age-associated metabolic dysfunction. We analyzed LFEs from young (3-month) and old (24-month) male and female C57BL/6 mice to characterize age-related differences in exosomal proteomic and miRNA cargos. To explore interactions between LFEs and the gut microbiome, naïve young mice were gavage fed with LFEs from old donors, followed by 16S rRNA sequencing. Gut permeability in vitro and in vivo and systemic metabolic effects were assessed using ECIS, 3D microfluidic models, and insulin sensitivity assays. Bioinformatic analyses identified specific proteins and miRNAs linked to insulin resistance and barrier dysfunction. Heatmaps and principal component analysis revealed distinct differences in LFE profiles between young and old mice. Notably, LFEs from old mice impaired gut barrier integrity and metabolic function in young recipients, with reciprocal effects noted in older mice when receiving LFEs from young mice. Multi-omics profiling, including proteomics and miRNA sequencing, identified age-dependent and gender-related changes in LFE cargo, encompassing host- and GM-derived proteins and miRNAs. These age-specific profiles were associated with pathways implicated in cancer, neurobehavioral changes, and metabolic dysfunction. Our findings highlight that LFEs from old mice are enriched with proteins and miRNAs involved in insulin resistance and gut barrier disruption. Together, these findings identify gut luminal exosomes as age-dependent mediators of microbiome-host communication that contribute to intestinal barrier dysfunction and metabolic decline.