Changes in polyphosphoinositides and phosphatidic acid of erythrocyte membranes in diabetes

We studied metabolic pool size of polyphosphoinositides and phosphatidate of erythrocyte membranes from normal and diabetic subjects using 32P for 20-h incubation, a sufficiently long period to reach isotopic equilibrium between monoesterphosphate bond and gamma-phosphate of ATP. Phosphatidylinositol 4-monophosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and phosphatidate were the phospholipids labelled. Metabolic pools of individual phospholipids were estimated, based on their proportionate and absolute radioactivity. A significant decline in radioactivity of phosphatidate and PtdIns(4,5)P2 was seen in erythrocytes from the diabetic subjects, indicating suppression of the metabolically labile pool of these two phospholipids. There was no significant change in PtdIns4P radioactivity between the groups. The direct effect of insulin on phosphorylation of polyphosphoinositides and phosphatidate was also evaluated by a short incubation period of erythrocyte membranes with [gamma-32P]-ATP. Added insulin increased the incorporation of 32P into phosphatidate in a dose-dependent manner that reached a steady state at 2 nM. We conclude that the metabolically labile pool size of phosphatidate is decreased and that of polyphosphoinositides is altered in erythrocyte membranes from diabetic patients.