Glucose uptake assay

The glucose uptake assay (Abcam, Inc.,Cambridge, MA) was performed according to the manufacturer’s instructions. Briefly, pre-adipocytes were differentiated to adipocytes as described and maintained for an additional 4 days. Cultured cells were then starved overnight as described. Cultured cells were washed and starved in KRPH + 2% BSA buffer. Then 1 µM insulin and 2-deoxyglucose (2-DG) were added to activate glucose transporters to take up 2-DG and metabolize it to 2-DG-6-phosphate (2-DG6P). The accumulated 2-DG6P was oxidized to NADPH which generated an oxidized substrate that was measured at OD_412nm using a microplate reader. A 2-DG6P standard curve was also measured at the same time. Absorbance values were normalized to the mean “blank” absorbance value. Cells not treated with insulin or 2-DG were used as sample background controls. 2-DG uptake [µM] was calculated as: (Calculated concentration of 2-DG6P/Sample volume) × Sample dilution. The experiment was performed three times, and data were presented as the mean ± s.e.m. Results were analyzed with Microsoft Excel software by applying the equal variance t Test. P ≤ 0.05 was considered statistically significant.