2-Deoxyglucose Uptake Assay

Glucose uptake was initiated as previously described (15), with minor modifications. The differentiated 3T3-L1 adipocytes and L6 myocytes were co-cultured for 24 h. L6 cells were serum-starved for 6 h in KRP-HEPES buffer [10 mM HEPES pH 7.4, 131.2 mM sodium chloride (NaCl), 4.7 mM potassium chloride (KCl), 1.2 mM magnesium sulfate (MgSO₄), 2.5 mM calcium chloride (CaCl₂), and 2.5 mM monosodium phosphate (NaH₂PO₄)] for 30 min at 37°C. In case of insulin stimulation, we used 100 ng/mL concentration of insulin. Glucose uptake was determined in triplicates at each point of addition of 2-DOG ([³H] 0.1 μCi at a final concentration of 0.1 mM) in the KRP-HEPES buffer for 5 min at 37°C. Cells were washed thrice with ice-cold PBS and suspended in 1N sodium hydroxide (NaOH). All samples were subjected to quantification of radioactivity using LSC-6100 liquid scintillation counter (Aloka, Tokyo, Japan).