Abstract
Phosphatidylinositol-3-phosphate (PI3P) is a lipid that accumulates in the early endosomal membrane, and acts as a scaffold to recruit proteins that contain a PI3P-binding domain, such as the FYVE domain. In this study, we examined the effect of PI3P depletion on the insulin response in rat hepatoma-derived H4IIEC3 cells. We found that insulin treatment induced the transient formation of an actin domain structure, a mesh-like tangled network of actin filaments where phosphorylated Akt, endosomal proteins, and PI3P accumulated. Actin domain formation was repressed by the depletion of PI3P by SAR405, an inhibitor of the class III PI3 kinase, Vps34, by the inhibition of PI3P function by the competitive binding of an excess amount of GST-fused 2xFYVE protein to intracellular PI3P, and by the use of diabetic model cells, in which PI3P was depleted. SAR405 did not affect the phosphorylation level of Akt, and the transcriptional regulation of gluconeogenic and cholesterol synthetic genes after insulin treatment. Interestingly, insulin-induced DNA synthesis was specifically inhibited by SAR405, cytochalasin B, and also in diabetic model cells. These results suggest that PI3P is required for the formation of actin domains, which affected a signaling pathway downstream of Akt associated with DNA synthesis in H4IIEC3 cells.