Figure 6.

Constitutively active GSK3 or dominant negative AKT are sufficient to increase ROS and enhance the mitochondrial membrane potential in REDD1 KO cells exposed to hyperglycemic conditions. (A–C) REDD1 KO (sgREDD1) R28 cells were maintained in DMEM containing 5 mmol/L glucose supplemented with 10% FBS. Cells were transfected with either an empty vector (EV) control plasmid or constructs that express Hemagglutinin (HA)-tagged REDD1, HA-tagged constitutively active GSK3^S9A (caGSK3), or HA-tagged dominant negative Akt^K147M (dnAkt). Cells were then exposed to culture medium containing 30 mmol/L glucose for 24 hours. DCFDA was used to visualize ROS by confocal microscopy (A) and quantified (B). (B) HA-tagged protein expression was assessed via Western blotting. Gel loading was evaluated by protein stain. (C) Mitochondrial membrane potential was evaluated with JC-1 dye. The ratio of JC-1 aggregates to monomers were quantified. Results are representative for three experiments. Within each experiment, seven independent samples were analyzed. Values are means + SEM. *, P < 0.05 versus EV. (D) Working model for the mechanism whereby REDD1 expression promotes a ROS-generating feedback loop in response to hyperglycemia. ns, nonspecific.