Action of insulin on the subcellular metabolism of polyphosphoinositides in isolated rat hepatocytes

The effect of insulin on 32Pi incorporation into phospholipids in various subcellular sites of isolated rat hepatocytes was investigated. After labeling the phospholipids of hepatocytes from rats previously starved for 24 h with 32Pi (10 mu Ci/10(6) cells) for 90 min, either saline or insulin (32 nM) was added. Following incubations of 1, 5, and 30 min, chilled cells were rapidly washed, homogenized in the presence of inhibitors of phospholipid degradation, and fractionated into the major subcellular organelles. Phospholipids were extracted from plasma membranes, microsomes, lysosomes, mitochondria, and nuclei with acidic chloroform:methanol. The aqueous deacylation products were separated by anion exchange high performance liquid chromatography, and the 32 incorporated into all the major diacylglycerophospholipids was determined. In parallel experiments, the specific radioactivity of 2Pi and [gamma-32P]ATP was determined. The results revealed that insulin had no effect on the turnover of the major phospholipids, including the polyphosphoinositides, of all subcellular compartments analyzed relative to the control. In addition, there were no significant differences in the amount and 32P labeling of cellular orthophosphate between saline- and insulin-treated cells. The specific radioactivity of [gamma-32P]ATP was increased by 20% after 30-min treatment with insulin, requiring appropriate correction of 32P-labeled phosphatidic acid, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate for estimation of mass changes at near steady-state labeling of cellular ATP