Measurement of Hormones and Gastric Emptying

Hormones were measured using the following assays as described previously [20 (link), 22 (link)]. Total GLP-1, human total GLP-1 (ver. 2) assay kit (K150JVC-2; Mesoscale Discovery, Gaithersburg, MD, USA); total GIP, human GIP (total) ELISA (EZHGIP-54K; Merck Millipore, Darmstadt, Germany); glucagon, glucagon RIA (GL-3K; Merck Millipore); insulin, lumipulse presto insulin (Fujirebio, Tokyo, Japan); and C-peptide, lumipulse presto C-peptide (Fujirebio). Gastric emptying rate was determined by mathematical modelling, based on changes of the ¹³CO₂/¹²CO₂ ratio in breath samples measured by an infrared spectral analyser (POCone, Otsuka Electronics Company) [23 (link)]. Stable ¹³C-labelled sodium acetate is emptied from the stomach following the trituration and liquefaction of the steamed rice, and is transported to the liver via the portal vein, where it is oxidised to ¹³CO₂ and exhaled into breath. The Wagner–Nelson method was applied to adjust the time for ¹³CO₂ distribution [8 (link), 24 (link)]. Other laboratory measurements including HbA_1c and plasma glucose were done by standard assays.