Acquisition of unprecedented phosphatidylinositol 3,5-bisphosphate rise in hyperosmotically stressed 3T3-L1 adipocytes, mediated by ArPIKfyve-PIKfyve pathway

Unlike yeast, where hyperosmotic stress induces a dramatic increase in phosphatidylinositol 3,5-bisphosphate (PtdIns 3,5-P-2) synthesis, in mammalian cells, although activating a complex array of signaling events, hyperosmotic stress fails to up-regulate PtdIns 3,5-P-2, indicating the PtdIns 3,5-P-2 pathway is not involved in mammalian osmo-protective responses. Here we report an unexpected and marked PtdIns 3,5-P-2 increase in response to hyperosmotic stress in differentiated 3T3-L1 adipocytes. Because this effect was not observed in the precursor preadipocytes, a specific role during acquisition of the adipocyte phenotype and transition into insulin-responsive cells could be suggested. However, acute insulin action did not result in a measurable PtdIns 3,5-P-2 rise, indicating the PtdIns 3,5-P-2 pathway is a specific hyperosmotically activated signaling cascade selectively operating in differentiated 3T3-L1 adipocytes. Hyperosmolarity activates different components of several kinase cascades, including p38 mitogen-activated protein and tyrosine kinases, but these appear to be separate from the activated PtdIns 3,5-P-2 pathway. Because PtdIns 3,5-P-2 is primarily produced by PIKfyve-catalyzed synthesis and requires the upstream activator hVac14 ( called herein ArPIKfyve) that physically associates with and activates PIKfyve, we examined the contribution of ArPIKfyve-PIKfyve for the hyperosmotic stress-induced rise in PtdIns 3,5-P-2. Small interfering RNA-directed gene silencing to selectively deplete ArPIKfyve or PIKfyve in 3T3-L1 adipocytes determined the ArPIKfyve-PIKfyve axis fully accountable for the hyperosmotically activated PtdIns 3,5-P-2. Together these results reveal a previously uncharacterized PtdIns 3,5-P-2 pathway activated selectively in hyperosmotically stressed 3T3-L1 adipocytes and suggest a plausible role for PtdIns 3,5-P-2 in the osmo-protective response mechanism in this cell type.