Glucose uptake assay

Female mice were orally administered a radiolabeled glucose solution through oral gavage. Unlabeled 40% glucose was combined with 370 Bq/µl [¹⁴C(U)]-D-glucose (50 µCi/1.85 MBq; PerkinElmer) and with 370 Bq/µl [1-³H(N)]-D-Mannitol (Perkin Elmer) (2 µCi), the latter necessary for correct glucose uptake into tissue for the adherent extracellular fluid phase⁷⁸. Mice were challenged with glucose at 4 g/kg. After 15 min, blood was collected and mice were euthanized in a CO₂ chamber. Blood was centrifuged at 1200 × g for 20 min before ¹⁴C tracer contents in plasma were measured in a liquid scintillation counter. For intestinal glucose uptake analysis, the whole small intestine was longitudinally opened and washed in ice-cold Krebs buffer for 10 min (119 mM NaCl, 4,7 mM KCl, 2,5 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 25 mM NaHCO₃, pH 7.4). Tissues were then solubilized in Soluene (Perkin Elmer) and radioactivity was measured with a liquid scintillation counter. Background counts from nonradioactive control samples were subtracted from each sample.