Constitutive activity of glycogen synthase kinase-3β: Positive regulation of steady-state levels of insulin receptor substrates-1 and -2 in adrenal chromaffin cells

In cultured bovine adrenal chromaffin cells, 12-h treatment with 1–20 mM LiCl, an inhibitor of glycogen synthase kinase-3 (GSK-3), increased Ser9 phosphorylation of GSK-3β by ∼ 44%, while decreasing insulin receptor substrate-1 (IRS-1) and IRS-2 protein levels by ∼ 38 and ∼ 62% in a concentration-dependent manner. Treatment with SB216763 (0.1–30 μM for 12 h), a selective inhibitor of GSK-3, lowered IRS-1 and IRS-2 levels by ∼ 38 and ∼ 48%, while increasing β-catenin protein level by ∼ 47%, due to the prevention of GSK-3-induced degradation of β-catenin by SB216763. Insulin (100 nM for 24 h) increased Ser9 phosphorylation of GSK-3β by ∼ 104%, while decreasing IRS-1 and IRS-2 levels by ∼ 41 and ∼ 72%; the insulin-induced Ser9 phosphorylation of GSK-3β, as well as down-regulations of IRS-1 and IRS-2 levels were restored to the control levels of nontreated cells at 24 h after the washout of the insulin (100 nM for 12 h)-treated cells. Either clasto-lactacystin β-lactone or lactacystin (an inhibitor of proteasome) prevented LiCl- or SB216763-induced decreases of IRS-1 and IRS-2 levels by ∼ 100 and ∼ 69%, respectively. In contrast, calpastatin (an inhibitor of calpain) and leupeptin (an inhibitor of lysosome) failed to prevent the decreases of IRS-1 and IRS-2 levels caused by LiCl or SB216763. LiCl or SB216763 lowered IRS-2 mRNA level, with no effect on IRS-1 mRNA level. These results suggest that constitutive activity of GSK-3β in quiescent cells positively maintains steady-state levels of IRS-1 and IRS-2 via regulating proteasomal degradation and/or synthesis of IRS-1 and IRS-2 proteins.