D-609

Abstract Glucose uptake is stimulated by insulin via stimulation of glucose transporter 4 (GLUT4) translocation to the plasma membrane from intracellular compartments in adipose tissue and muscles. Insulin stimulation for prolonged periods depletes GLUT4 protein, particularly in highly insulin-responsive GLUT4 storage vesicles. This depletion mainly occurs via H2O2-mediated retromer inhibition. However, the post-receptor mechanism of insulin activation of oxidative stress remains unknown. Here, we show that phosphatidylcholine-specific phospholipase C (PC-PLC) plays an important role in insulin-mediated downregulation of GLUT4. In the study, 3T3-L1 adipocytes were exposed to a PC-PLC inhibitor, tricyclodecan-9-yl-xanthogenate (D609), for 30 min prior to the stimulation with 500 nM insulin for 4 h, weakening the depletion of GLUT4. D609 also prevents insulin-driven H2O2 generation in 3T3-L1 adipocytes. Exogenous PC-PLC and its product, phosphocholine (PCho), also caused GLUT4 depletion and promoted H2O2 generation in 3T3-L1 adipocytes. Furthermore, insulin-mediated the increase in the cellular membrane PC-PLC activity was observed in Amplex Red assays. These results suggested that PC-PLC plays an important role in insulin-mediated downregulation of GLUT4 and that PCho may serve as a signaling molecule.

Sphingomyelin (SM) is an essential component of biological lipid rafts, and it plays an indispensable role in maintaining plasma membrane stability and in mediating signal transduction. The ultimate biosynthesis of SM is catalyzed by two sphingomyelin synthases (SMSs) namely SMS1 and SMS2, which are selectively distributed in the trans-Golgi apparatus and the plasma membrane. It has been demonstrated that SMS2 acts as an irreplaceable molecule in the regulation of transmembrane signaling, and loss of SMS2 has been reported to worsen atherosclerosis and liver steatosis. However, the function of SMS2 in platelet activation and its association with the pathological process of thrombosis in acute coronary syndrome (ACS) and portal hypertension (PH) remain unclear.

In this study, we tested the role of SMS2 in platelet activation and thrombosis using SMS2 knockout (SMS2 –/–) mice and SMS2-specific inhibitor, D609. Furthermore, we detected SMS2 expression in patients with ACS and PH.

SMS2 –/– platelets showed significant reduction in platelet aggregation, spreading, clot retraction and in vivo thrombosis. Similar inhibitory effects on platelet activation were detected in D609-treated wild-type platelets. PLCγ/PI3K/Akt signaling pathway was inhibited in SMS2 –/– platelets and D609-treated wild-type platelets. In addition, we discovered that platelet SMS2 expression was remarkably increased in patients with ACS and PH, compared with healthy subjects.

Our study indicates that SMS2 acts as a positive regulator of platelet activation and thrombosis, and provides a theoretical basis for the potential use of D609 in anti-thrombosis treatment.