Hyperinsulinemia exists before the onset of overt type 2 diabetes mellitus. This response is at least partly due to enhanced insulin release from pancreatic beta-cells. Increased insulin secretion can be mimicked in vitro by acute culture of 832/13 rat insulinoma cells with phosphatidylinositol 3-kinase (PI-3K) inhibitors, a treatment that would theoretically simulate insulin resistance. We demonstrate in this study that while the PI-3K inhibitors Wortmannin and LY294002 both block Akt phosphorylation, only LY29002 significantly augments insulin secretion. LY294002 treatment potentiates insulin secretion over both basal and stimulatory glucose concentrations. This effect correlates with a significant increase in action potential duration. There was no change in resting or peak membrane potential under any of the treatment conditions, demonstrating that the cells remain healthy under the acute treatments used in this study. By contrast, Wortmannin has no effect on action potential duration. A partial explanation for these findings is that LY294002 potently inhibits voltage-dependent potassium channels, but does not affect voltage-gated calcium currents. We conclude that while PI-3K may play a role in regulating insulin secretion, there are diverse effects of the established inhibitors of this enzyme on beta-cell insulin secretory responses. (C) 2004 Elsevier Inc. All rights reserved.