Activation of promoter activity of the catalytic subunit of γ-glutamylcysteine ligase (GCL) in brain endothelial cells by insulin requires antioxidant response element 4 and altered glycemic status: implication for GCL expression and GSH synthesis.

Our recent finding that insulin increased the expression of the glutamate-cysteine ligase catalytic subunit (GCLc) with coincident increases in GCL activity and cellular glutathione (GSH) in human brain microvascular endothelial cells (IHECs) suggests a role for insulin in vascular GSH maintenance. Here, using IHECs stably transfected with promoter-luciferase reporter vectors, we found that insulin increased GCLc promoter activity, which required a prerequisite increase or decrease in medium glucose. An intact antioxidant response element-4 was essential for promoter activation, which was attenuated by inhibitors of PI3-kinase/Akt/mTOR signaling. Interestingly, only under low-glucose conditions did promoter activation correlate with increased GCLc expression and GSH synthesis. Low tert-butylhydroperoxide (tBH) concentrations similarly mediated promoter activation, but the maximal activation dose was decreased 10-fold by insulin. Insulin-tBH coadministration abrogated the low or high glucose requirement for promoter activation, suggesting possible ROS involvement. ROS production was elevated at low glucose without or with insulin; however, GSH increases were not inhibited by tempol, suggesting that ROS did not achieve the threshold for driving GCLc promoter activation and de novo GSH synthesis. The minor effect of pyruvate also ruled out a major role for hypoglycemia (±insulin)-induced metabolic stress on GSH induction under these conditions.