2.10. 2-Deoxy glucose uptake assays

Primary mouse adipocytes isolation was performed as described in the literature [24,25]. Briefly, mouse epididymal adipose tissue was minced and digested in collagenase buffer (1 mg/ml) for 30 min at 37 °C with shaking. The digests were filtered through 100 μm cell strainers and centrifuged at 500 g for 5 min. The floating adipocyte layer was saved and washed with fresh PBS 3 times before being subjected to glucose uptake assays. For glucose uptake assays, isolated primary mouse adipocytes were incubated in Hepes-Salt buffer (20 mM Hepes, 40 mM potassium chloride, 125 mM sodium chloride, 0.85 mM potassium phosphate monobasic, 1.25 mM sodium phosphate dibasic, 1 mM magnesium chloride, 1 mM calcium chloride, 0.1% fatty acid-free BSA) +/− 100 nM insulin for 20 min at 37 °C. The cells were then incubated with [¹⁴C] 2-deoxy-d-glucose at a final concentration of 3 μmol/L for 20 min. After 3 washings in ice-cold PBS, the cells were lysed in lysis buffer with 0.1% sodium dodecyl sulfate and subjected to scintillation counting to determine their ¹⁴C radioactivity. The protein concentrations were determined by using a bicinchoninic acid assay kit (Pierce Chemical Co., Rockford, IL), and the radioactivities were normalized by total protein contents.