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 CaCl
2 ⋅ 2H
2O, 1.2 KH
2PO
4, 1.2 MgSO
4 ⋅ 7H
2O, and 24.6 NaHCO
3, 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 10,000
g 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.