Accu check compact plus

Prior to the OGTT, male 9–11-week-old mice were fasted five hours before being given oral gavage with 2 g/kg glucose. Blood glucose was measured after tail tip punctures using glucometer (Accu-check compact plus, Roche, Switzerland), at times 0, 15, 30, 45, 60, 75 and 90 min after the glucose load. Simultaneously, blood samples were taken from the retrobulbar plexus at times 0, 15, 30, 60 and 90 min, and plasma samples were transferred to new tubes after 20 min and centrifuged at 2800 g at 4 °C. Insulin levels were subsequently measured by mouse insulin ELISA (Mercodia AB, Sweden) according to the manufacturer's instructions, with a detection limit of 2 ng/ml. The plasma samples from the OGTT and the samples from islet perifusion (see below) were measured without dilution.

Two weeks prior to OGTT, pigs were introduced to a standard meal consisting of 50 g ground pregrower fodder, and acclimated to handling and standing in a sling. OGTT was conducted after overnight fasting. An hour before OGTT, ears were cleaned and Lidocaine/prilocaine cream applied (EMLA Cream; AstraZeneca, Mississauga, Ontario) to reduce pain. After measuring fasting blood glucose (FBG) animals were given the standard meal (50 g) mixed with 2 g/kg glucose solution. Upon finishing the meal (time 0), glucose was measured with a glucometer (Accu-Check Compact Plus; Roche Diagnostics) in whole blood from ear vein at 15, 30, 45, 60, 90, 120, and 180 min. Additional blood samples were collected - during the first 45 min by 70 μl microhematocrit capillary tubes coated with ammonium heparin (Fisher Scientific). Blood samples were centrifuged at 1500 rpm for 10 min at 4 °C and plasma collected and stored at −80 °C until assayed for insulin by ELISA, according to the manufacturer’s instructions (Alpco Diagnostics, Salem, N.H., USA). Blood glucose and plasma insulin concentrations were plotted as a function of time, and area under the curve was calculated using established methods72 (link).

Mice were fasted overnight (16–18 h) with free access to water. Mice were anesthetized (IP injection or gas form) or given a saline injection (IP injection) at time −15 min before oral gavage of glucose, 50% w/v solution, in a dose corresponding to 2 g glucose per kg bodyweight. Control mice were returned to their cages following oral gavage and were allowed to move around between the blood sampling. For pentobarbital injected animals, a lidocaine IP injection was given at time −20 min in the area where pentobarbital was to be injected at time −15 min to minimize the pain induced by pentobarbital. BG was measured after tail tip punctures, at times −15, 0, 20, 40, 60, 90, 120, and 150 min after oral glucose load using a handheld plasma calibrated glucometer (Accu‐check compact plus, F.Hoffmann‐La Roche AG, Bazel, Switzerland). Blood samples (75 μL) were drawn from the retrobulbar plexus at time 0 and 20 min using EDTA coated capillary tubes (Vitrex Medical, Herlev, Denmark, ref 164213), and transferred to chilled Eppendorf tubes. Blood samples were centrifuged at 2800 g at 4°C for 20 min and plasma was transferred to new tubes and immediately frozen on dry ice. Insulin was measured using an insulin ELISA (Mercodia AB, Uppsala, Sweden, catalog no. 10‐1247‐10) according to manufacturers’ protocol; lower detection limit was 200 ng/L.