Disruption of the gut microbiota in regulator of G protein signaling 14 knockout (RGS14 KO) mice alters the metabolome and reduces enhanced exercise capacity

Regulator of G-protein Signaling 14 Knockout (RGS14 KO) mice exhibit enhanced exercise capacity and health span, however the contribution of the gut microbiota to this phenotype remains unclear. This study integrated long-read rRNA operon amplicon sequencing and metabolomics to first determine how microbial composition and tissue metabolite profiles differ between RGS14 KO and their wild-type littermates. Next, we investigated how administration of antibiotics to perturb the gut microbiota may affect the RGS14 KO phenotype. Prior to antibiotic treatment (ABX), RGS14 KO mice outperformed WT littermates in maximal running distance and work performed, accompanied by elevated skeletal muscle citrate synthase, complex IV activity, and nitric oxide production. One week of ABX significantly reduced exercise capacity in both genotypes and markedly suppressed mitochondrial activity in RGS14 KO skeletal muscle. Gut microbiota profiling revealed similar phylum-level structure between genotypes but distinct species- and strain-level signatures. Metabolomics of brown adipose tissue (BAT) and quadriceps identified genotype-specific metabolic programs that were disrupted by ABX, including pathways related to amino acid metabolism, nucleotide turnover, and mitochondrial energetics. Collectively, these findings demonstrate that RGS14 KO mice harbor unique microbial and metabolic networks that support enhanced thermogenesis and exercise performance, and that microbiota depletion eliminates these advantages. This work establishes a mechanistic foundation connecting the gut microbiota to BAT and skeletal muscle metabolism, highlighting potential microbiome-targeted strategies to improve metabolic health and physical performance.