Glucose production and uptake assays of HepG2 cells

For the glucose production assay, the HepG2 cells were rinsed with PBS to remove glucose and then incubated in glucose production assay medium (glucose- and phenol red-free DMEM) for 3 h. Subsequently, the supernatant was collected for glucose concentration measurement by using a commercial colorimetric glucose assay kit (Sigma-Aldrich; Merck KGaA). The readings were then normalized to the total protein content determined from the whole-cell lysates.

The glucose uptake rate was measured using the methods established by Yoshioka et al (19) with slight modifications. Briefly, HepG2 cells were washed twice with PBS and then incubated with 200 µM 2-NBDG in glucose-free culture medium for 30 min. Cells incubated with glucose-free medium without 2-NBDG served as a negative control. Finally, the cells were rinsed with PBS and fluorescence was determined using a microplate reader (Infinite M1000, Tecan Austria GmbH) with excitation at 488 nm and emission at 520 nm.