Regulation of Akt mRNA and protein levels by glycogen synthase kinase-3β in adrenal chromaffin cells: Effects of LiCl and SB216763

In cultured bovine adrenal chromaffin cells, where Akt1 is the predominant isoform over Akt2 and Akt3, chronic (≥ 12 h) treatment with 1–20 mM LiCl, an inhibitor of glycogen synthase kinase-3, decreased Akt1 level by ~ 52% (EC50 = 3.7 mM; t1/2 = l2 h); it was associated with LiCl-induced increased levels of Ser9-phosphorylated glycogen synthase kinase-3β (~ 37%) and β-catenin (~ 59%), two hallmarks of glycogen synthase kinase-3β inhibition. The same LiCl treatment did not change phosphoinositide 3-kinase, phosphoinositide-dependent kinase 1, and extracellular signal-regulated kinase-1/2 levels. Treatment with SB216763 [3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione], a selective inhibitor of glycogen synthase kinase-3, lowered Akt1 level by ~ 67% (EC50 = 2 μM; t1/2 = l2 h), when SB216763 caused concentration- and time-dependent increase of β-catenin level by ~ 76%. LiCl- or SB216763-induced Akt1 decrease, as well as increases of Ser9-phosphorylated glycogen synthase kinase-3β and β-catenin were restored to the control levels of nontreated cells after the washout of LiCl (20 mM for 24 h)- or SB216763 (30 μM for 24 h)-treated cells. LiCl-induced Akt1 reduction was not prevented by β-lactone, lactacystin (two inhibitors of proteasome), calpastatin (an inhibitor of calpain), or leupeptin (an inhibitor of lysosome). LiCl decreased Akt1 mRNA level by 20% at 6 h, with no effect on Akt1 mRNA stability. These results suggest that glycogen synthase kinase-3β inhibition caused down-regulation of Akt1 mRNA and Akt1 protein levels; conversely, constitutive activity of glycogen synthase kinase-3β maintains steady-state level of Akt1 in quiescent adrenal chromaffin cells.