13-week-old male DIO mice were purchased and housed as described in “Animals” above. Mice were weight-matched into two groups (p=0.88). After an overnight fast (17:00 to 07:00), mice received either CA7S or PBS via direct duodenal and rectal administration. The optimal, physiologic dose of CA7S was extrapolated from the average pmol concentration of CA7S in cecal samples from SG animals (average of 3000pmol/mg of stool with 500mg of stool per animal corresponds to 0.75mg of CA7S per cecum).
Under isoflurane general anesthesia, 0.25 mg and 0.75 mg of CA7S in PBS (pH 7.2) was delivered by slow infusion (5 min) antegrade into the duodenum and retrograde into the rectum, respectively. The total volume of instillation was 2 mL (0.5 mg CA7S/mL). Control animals received similar volumes of PBS alone. 15 min post infusion, serum glucose was measured via tail vein followed by whole blood collection via cardiac puncture into K+EDTA tubes containing DPPIV inhibitor (Merck Millipore, Billerica, MA), Perfabloc (Sigma), and apoprotinin (Sigma). One glucose reading from the PBS group was occluded by a clot, therefore was excluded from glucose analysis. Whole blood and tissues were harvested for analysis. In order to account for changes in fasting times and hormonal diurnal rhythms, this experiment was carried out on four consecutive days such that only four mice were tested per day.
Under isoflurane general anesthesia, 0.25 mg and 0.75 mg of CA7S in PBS (pH 7.2) was delivered by slow infusion (5 min) antegrade into the duodenum and retrograde into the rectum, respectively. The total volume of instillation was 2 mL (0.5 mg CA7S/mL). Control animals received similar volumes of PBS alone. 15 min post infusion, serum glucose was measured via tail vein followed by whole blood collection via cardiac puncture into K+EDTA tubes containing DPPIV inhibitor (Merck Millipore, Billerica, MA), Perfabloc (Sigma), and apoprotinin (Sigma). One glucose reading from the PBS group was occluded by a clot, therefore was excluded from glucose analysis. Whole blood and tissues were harvested for analysis. In order to account for changes in fasting times and hormonal diurnal rhythms, this experiment was carried out on four consecutive days such that only four mice were tested per day.