Gastric emptying measurement.

The gastric emptying rate for a solid meal was measured in male rats (n = 6) using the ¹³C octanoic acid breath test. After 3 days of acclimation to the testing chamber (1 rat per chamber) and pancake meal, rats were fasted overnight with ad libitum access to water before placement in testing chamber. Testing chambers had a controlled air flow rate with CO₂ levels maintained between 1000 and 2000 ppm. After 60 minutes of baseline recordings, rats were given 1 g of pancake (Pillsbury Homestyle Pancakes, General Mills) treated with 4 μL of [¹³C]-octanoic acid (Cambridge Isotope Laboratories, Inc.). Rats that did not consume the entire pancake within 5 minutes were removed from the study. Air from the 4 testing chambers was automatically sampled one at a time for 30 seconds each at a sample rate of 1 Hz for 8 hours. The air sample was analyzed by off-axis integrated cavity output spectroscopy using a multiple input unit and carbon dioxide carbon isotope analyzer (Los Gatos Research).

For each 30-second time group of data, the first 10 seconds were removed to ensure a complete flush of the previous air sample from the tubing (76). The remaining 20 seconds of data were averaged for a single data point at a given time. The concentration of ¹³CO₂ was then calculated and expressed as change over the baseline. The change of concentration of ¹³CO₂ versus time (t) was fitted by a nonlinear regression curve with the following equation: y = at^be–ct where y is the percentage of the ¹³C excretion in the breath per hour (t) and a, b, and c are regression constants estimated for each breath versus time curve. The gastric half-emptying time (T_1/2) was calculated from a numerical integration procedure using an inverse γ function.

Three baseline measurements were taken with a minimum of 4 days between testing. Rats were exposed to an HFD and gastric emptying was measured at 1 and 4 days of HFD exposure.