4.5. Gastric Emptying (GE)

We assessed GE in 24-h-fasted mice according to the phenol red method, which has been described previously [44,45,46,47]. The animals (wild-type and human MLNR Tg mice) were administered 0.5 mL of test meal by oral gavage, and then were euthanized by cervical dislocation either immediately (t = 0) or 20 min after gavaging. Following laparotomy, the pylorus and the cardia were ligated, and the stomach was excised. Intracerebroventricular (icv) and intraperitoneal (ip) administration of drugs were performed 30 min before the gavage.

The stomach was cut into pieces and homogenized, with its contents, in 25 mL of 0.1 N NaOH. The homogenate was centrifuged 2500× g for 3 min at 4 °C); an 8-mL aliquot of the resulting supernatant was combined with 1 mL of 33% trichloroacetic acid to precipitate proteins. After another round of centrifugation (2500× g for 30 min at 4 °C), 2 mL of 2 N NaOH were added to the supernatant, and the amount of phenol red was determined by measuring absorbance at 560 nm. This absorbance correlates with the concentration of phenol red in the stomach, which in turn depends on GE. The GE rate (in percent) was calculated as GE = (1−X/Y) × 100, where X and Y are the absorbances of phenol red recovered from the stomach of animals killed 20 min and 0 min (respectively) after gavaging.