Ex vivo Muscle Glucose Uptake Assay

Ex vivo muscle [³H]-2-deoxy-d-glucose uptake was assessed using methods adapted from Hinkley et al. (22 (link)). Muscles were preincubated in continuously oxygenated 37°C Krebs-Ringer bicarbonate buffer (KRBB) composed of the following (in mmol/L): 117 NaCl, 4.7 KCl, 2.5 CaCl₂ ⋅ 2H₂O, 1.2 KH₂PO₄, 1.2 MgSO₄ ⋅ 7H₂O, and 24.6 NaHCO₃, pH 7.5, supplemented with 2 mmol/L pyruvate. For glucose uptake, muscles were incubated in KRBB supplemented with 1.5 μCi/mL [³H]-2-deoxy-d-glucose, 1 mmol/L 2-deoxy-d-glucose, 0.45 μCi/mL [¹⁴C]-mannitol, and 7 mmol/L mannitol, unless otherwise indicated. All radioactive incubations were conducted at 30°C for 10 min. Cytochalasin B and phloridzin were added into buffers as described in the figure legends.
Hexose competition experiments were performed using methods adapted from Ryder et al. (23 (link)). Muscles were preincubated in KRBB plus pyruvate and then incubated in 90% KRBB supplemented with 1.5 μCi/mL [³H]-2-deoxy-d-glucose, 1 mmol/L 2-deoxy-d-glucose, 0.45 μCi/mL [¹⁴C]-mannitol, and 35 mmol/L d-fructose, d-galactose, d-glucose, d-xylose, or l-glucose. The osmolarity of the radioactive solution was kept at ∼310 mOsm by the 10% dilution of the KRBB and the removal of nonradiolabeled mannitol.
After radioactive incubations, muscles were frozen in liquid nitrogen, weighed, and solubilized in 1 mol/L NaOH at 80°C for 15 min. Solubilized muscles were neutralized with 1 mol/L HCl. Nonsoluble particulates were precipitated by centrifugation at 10,000g for 1 min. Aliquots were removed for scintillation counting of the [³H] and [¹⁴C] labels, and the extracellular and intracellular spaces calculated to determine [³H]-2-deoxy-d-glucose uptake.