Measurements of Cellular Phosphoinositide Levels in 3T3-L1 Adipocytes

Phosphoinositides are a family of minor membrane phospholipids that have important roles in cell signaling pathways. Seven phosphoinositides have been identified in eukaryotic cells (all are found in 3T3-L1 cells), including phosphatidylinositol 3-phosphate [PtdIns(3)P], phosphatidylinositol 4-phosphate [PtdIns(4)P], phosphatidylinositol 5-phosphate [PtdIns(5)P], phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P₂ ], phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P₂ ], phosphatidylinositol4,5-bisphosphate [PtdIns(4,5)P₂ ], and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P₃ ]. PtdIns(3)P and PtdIns(4)P are the products of phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase action. In eukaryotic cells, PtdIns(3)P and PtdIns(4)P have critical roles in membrane transport. PtdIns(5)P has been identified at low concentrations in some eukaryotic cells, but its role is unknown. PtdIns(4,5)P₂ can be produced by the action of PtdIns 4- or 5-kinases on PtdIns(5)P or PtdIns(4)P, respectively, and is involved in signaling via G-protein-coupled receptors, regulating vesicle-mediated protein traffic, and actin filament polymerization. PtdIns(4,5)P₂ also serves as a precursor for inositol 1,4,5-trisphosphate that is known to cause cytosolic calcium mobilization in some cell types. PtdIns(3,5)P₂ is produced via phosphatidylinositol 5-kinase action on PtdIns(3)P and has roles in membrane transport and stress signaling. PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃ are found at nominal levels in some unstimulated mammalian cells and their levels can increase rapidly following growth factor receptor and phosphatidylinositol 3-kinase activation.