A noninvasive ¹³C-mannitol breath test to monitor semaglutide treatment-induced delayed oral-cecal transit in mice

Glucagon-like peptide-1 receptor agonists (GLP-1RAs), such as semaglutide, are highly effective treatments for obesity and type 2 diabetes but are often associated with gastrointestinal side effects including delayed transit times through the gastrointestinal tract. We present a noninvasive breath test to measure oral-cecal transit time (OCTT) in mice that could be readily translated to clinical studies. Current methods for measuring OCTT are limited by invasiveness, terminal procedures, anesthesia, radioactive tracers, or stress-induced influence on the outcomes. Here we demonstrate that ¹³C-labeled mannitol is metabolized by murine cecal microbiota, producing measurable ¹³CO₂ in exhaled breath. Using indirect calorimetry combined with real-time measurement of the ¹³C/¹²C ratio, this approach provides a highly granular, noninvasive method to measure OCTT in mice. This technique is performed in unrestrained freely moving animals while enabling high-throughput measurements performed longitudinally. The method improves experimental rigor and reproducibility and provides a valuable tool for studying gastrointestinal physiology and the effects of pharmacologic therapies.NEW & NOTEWORTHY A novel noninvasive assay uses orally administered ¹³C-mannitol, metabolized by cecal microbiota into exhaled ¹³CO₂, to enable continuous, longitudinal measurement of oral-cecal transit time in unanesthetized, freely moving mice via indirect calorimetry with isotope ratio analysis. Pharmacological validation confirmed sensitivity to delayed transit, including a pronounced semaglutide-induced delay in obese mice that models a clinically relevant glucagon-like peptide-1 (GLP-1) receptor agonist adverse effect.