FIGURE 8.

G6PD inhibition blocks the effect of both high glucose and palmitate on ROS generation and chemotactic factor gene expression. 3T3-L1 adipocytes were exposed to low or high glucose and/or palmitate (250 μm) with or without the G6PD inhibitors, 6-AN (5 μm) or DHEA (100 nm), for 7 days with daily medium changes. For silencing experiments, some 3T3-L1 adipocytes were transfected with an siRNA specific for G6PD or a scrambled siRNA (negative control) as indicated. 24 h later, the cells were exposed to low or high glucose with/without palmitate (250 μm) for 7 days with daily medium changes. A, total RNA and lysates from transfected cells were analyzed by multiplex real time RT-PCR using G6pd-specific primers and normalized to Gapdh, or immunoblot using a G6PD antibody and normalized to β-actin. B, FACS analysis was used to sort CM-H₂DCFDA fluorescence in cells. Results are plotted as counts (number of cells) on the vertical axis versus DCF fluorescence intensity on the horizontal axis. Cells exposed to 5 mm glucose are shown in blue, and cells exposed to the indicated treatments are shown in red. Total RNA from cells treated with siRNA for G6PD (C and E), 6-AN and DHEA (D and F), as described above, was isolated and analyzed by multiplex real time RT-PCR using Saa3-specific (C and D) or Mcp-1-specific (E and F) primers and normalized to Gapdh. *, p < 0.001 versus negative control in 25 mm glucose control; **, p < 0.001 versus negative control plus palmitate in 25 mm glucose control; #, p < 0.001 versus 25 mm glucose control.