7-Hydroxystaurosporine (UCN-01) Inhibition of Akt Thr308 but not Ser473 Phosphorylation

Abstract

7-Hydroxystaurosporine (UCN-01) infused for 72 hours by continuous i.v. infusion induced insulin resistance during phase I clinical trials. To understand the mechanism for this observation, we examined the effect of UCN-01 on insulin-stimulated glucose transport activity with 3-O-methylglucose in isolated rat adipose cells. UCN-01 inhibits glucose transport activity in a dose-dependent manner at all insulin concentrations. At the clinically relevant concentration of 0.25 μmol/L UCN-01, glucose transport is inhibited 66, 29, and 26% at insulin concentrations of 10, 50, and 100,000 (100K) microunits/mL respectively, thus shifting the dose-response curve to the right. Increasing concentrations of UCN-01 up to 2.5 μmol/L progressively shift the insulin dose-response curve even further. As Akt is known to mediate in part action initiated at the insulin receptor, we also studied the effect of UCN-01 on Akt activation in whole-cell homogenates of these cells. Decreased glucose transport activity directly parallels decreased Akt Thr³⁰⁸ phosphorylation in both an insulin and UCN-01 dose-dependent manner, whereas Akt Ser⁴⁷³ phosphorylation is inhibited only at the lowest insulin concentration, and then, only modestly. UCN-01 also inhibits insulin-induced Thr³⁰⁸ but not Ser⁴⁷³ phosphorylation of Akt associated with the plasma membranes and low-density microsomes and inhibits translocation of GLUT4 from low-density microsomes to plasma membranes as expected from the glucose transport activity measurements. These data suggest that UCN-01 induces clinical insulin resistance by blocking Akt activation and subsequent GLUT4 translocation in response to insulin, and this effect appears to occur by inhibiting Thr³⁰⁸ phosphorylation even in the face of almost completely unaffected Ser⁴⁷³ phosphorylation.