A noninvasive ¹³ C-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 non-invasive 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 ¹³ C-labeled mannitol is metabolized by murine cecal microbiota, producing measurable ¹³ CO₂ in exhaled breath. Using indirect calorimetry combined with real-time measurement of the ¹³ C/ ¹² C ratio, this approach provides a highly granular, non-invasive 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.