Kharchenko V. Role of sphingolipids in the modulation of phospholipase D-dependent insulin signaling in old age.

In the present work the functions and regulation of insulin-induced phospholipase D (PLD) were inverstigated in old age and under experimental changes of hormonal sensitivity of the target cells and tissues. The experimental and age-dependent changes of PLD activation, glucose uptake and storage were modulated by inhibitors of sphingolipid metabolism. The activation of PC-specific PLD by insulin in classical target tissues (liver and muscles) and novel target (neocortex) was established. Halopemide, an inhibitor of PLD, suppressed the activity of PLD and significantly reduced the glucose uptake and the glycogen synthesis activated by insulin in hepatocytes. The insulin-induced activation of PLD was blocked by specific inhibitors of phosphatidylinositol-3-kinase (PI3-kinase) wortmannin and LY294002 and by the inhibitors of Akt/protein kinase B (Akt/PKB) apigenin-7-glucoside and luteolin-7-glucoside. These results indicated that PLD is activated downstream P3-kinase and Akt/PKB. Regulation of PLD activity and glucose uptake and storage by insulin in primary hepatocytes was sensitive to the ceramide, the antagonist of P3-kinase/Akt/PKB-signaling pathways. The sensitivity of the PLD to the insulin was decreased in hepatocytes, diaphragm tissue and neocortex of 24-month-old rats by elevated ceramide level. A high fat saturated diet induced the synthesis of free fatty acids and their derivatives such as DAG, TAG and ceramide in the diaphragm, liver and neocortex of 3-month-old rats. There were found disturbances in PLD activation by insulin in young rats caused by high-fat diet induced ceramide accumulation. The endogenous ceramide accumulation and the suppression of activation PLD and glucose metabolism by insulin in young rat muscle tissue, liver cells and neocortex were found by modeling of the impaired sensitivity of the target tissues to the action of insulin with palmitic acid, C2-ceramide, cytotoxic drugs – doxorubicin and paclitaxel. Using specific inhibitors of the sphingolipid synthesis (myriocin, fumonisin B1) and sphingomyelinase (SPMase) activity (imipramine, GW4869) it was determined that palmitic acid and C2-ceramide induce the endogenous ceramide de novo synthesis, while doxorubicin and paclitaxel increase the sphingolipid content by de novo synthesis and SPMase activation. The combination of inhibitors for both SPMase and sphingolipid synthesis promotes recovery of the insulin dependent PLD activation, glucose uptake and glycogen formation through the downregulation of endogenous ceramide levels accompanied with normalization of the target cells and tissues sensitivity for the hormonal action.