To test for a role of AMPK signaling in the inhibition of G6PD expression, accumulation of G6PD mRNA was measured after treatment with AICAR, a pharmacological activator of AMPK [20]. Insulin-induced G6PD mRNA accumulation 5-fold compared to no addition, and the addition of AICAR to the hepatocytes inhibited this increase by 50% (Fig. 2A). This mimics the extent of inhibition of G6PD mRNA by arachidonic
VX950 [12]. The abundance of G6PD mRNA is expressed relative to RPL32 mRNA abundance. In all experiments, RPL32 expression was not regulated by AICAR (data not shown). Hepatocytes were also treated with metformin, another pharmacological activator of AMPK, to test if AMPK activation by a different compound can also inhibit G6PD expression [21]. Like AICAR, metformin also inhibited the insulin-induction of G6PD mRNA by 50% or more (data not shown). As expected, AICAR enhanced AMPK phosphorylation in these cells (Fig. 2B). In addition, this stimulation of AMPK was accompanied by a 5-fold increase in p38 MAPK activation as compared to insulin alone (Fig. 2B and C). Thus, activation of AMPK by AICAR mimics the effect of arachidonic
acid on G6PD expression and p38 MAPK activation.
