Insulin secretion and glucose measurements

Insulin was determined in serum from blood drawn after feeding or intraperitoneal (ip) injections of Exendin-4 or sulfonylurea, and also during oral (o) and ip glucose tolerance tests (oGTT and ipGTT, respectively), or L-arginine tolerance tests (ArgGTT). Blood was extracted from the mandibular vein at the indicated time points and glucose was quantified immediately using an automatic analyzer (One Touch Ultra, LifeScan). In Fig. 5c, d, glucose was quantified using blood from the tail vein.

For the analysis of insulin levels, blood was allowed to clot after collection by leaving it undisturbed at room temperature for 30 min. The clot was removed by centrifugation at 1000g for 15 min in a refrigerated centrifuge, the resulting supernatant constituting the serum. Insulin content was measured in 10 μl of serum using an ELISA assay (Mouse Ultrasensitive Insulin ELISA, Mercodia).

To quantify insulin secretion after feeding, mice were fasted for 24 h and then allowed to eat standard diet pellet (Diet 150, Safe Diets) during the indicated time periods. For oGTT and ipGTTs, mice were fasted overnight for 14 h and 2 g/kg glucose (Merck, 0.2 g/ml dissolved in 0.9% NaCl) was administered by gavage or ip injection, respectively. To assess responses to GLP-1R agonists, Exendin-4 (MedChem Express, 5 μg/kg body weight dissolved in 0.2 g/ml glucose saline solution) was administered ip [20].

To study the dynamics of insulin release, L-arginine (Merck, 1 g/kg body weight dissolved in 0.9% NaCl) was injected ip in animals fasted for 14 h to depolarize the β-cell [40, 90] and thus elicit the secretion of insulin from the readily releasable pool (RRP) of insulin granules. A second ip administration of L-arginine (1 g/kg body weight) was performed 10 min after 1st ip injection to assess the replenishment of the RRP from the RP (Releasable Pool) [40].

To explore EPAC2-mediated insulin secretion mice were fasted for 1 h and injected ip with EPAC2-activating (glibenclamide, 5 mg/kg) or non-activating (glicazide, 10 mg/kg) sulfonylureas (MedChem Express) in 5% DMSO in sunflower oil [43].