Role And Mechanism Of AMPK In Platelet Activation In Type 2 Diabetic Patients

Part ? AMPK and Related Pathways in Platelet from Patients with Type 2 Diabetes MellitusObjective:To investigate the changes of AMPK/m TOR signaling pathway molecules AMPK,S6K1 and PI3 K signaling Akt in type 2 diabetes mellitus(T2DM).Methods: 20 cases of T2 DM patients admitted to Department of Endocrinology,the First Affiliated Hospital of Soochow University from September 2016 to January 2017,and 20 healthy volunteers enrolled in the physical examination center were included in this study.After washing the platelets by centrifugation,every sample was divided equally into two portions,one of which was incubated with 20 m M AMPK activator metformin for 3 min.Platelet aggregation test was performed.Platelets were prepared in the same way and the phosphorylation of platelets AMPK,Akt and S6K1 were detected by Western-blotting before and after incubation,respectively.Results: Compared with normal control platelets,phosphorylation of AMPK in platelets of T2 DM decreased and phosphorylation of Akt and S6K1 increased.Platelet aggregation was inhibited after addition of 20 m M metformin.AMPK phosphorylation in platelets of T2 DM recovered and phosphorylation of Akt and S6K1 increased after addition of 20 m M metformin.Conclusions: Decreased activity of AMPK/mTOR pathway and increased activity of PI3 K pathway were presented in T2 DM platelets.Part ? Study on Mechanism of AMPK Regulating Platelet ActivationObjective:To observe the changes of platelet AMPK and related molecules in the presence of different stimulants,and to explore the mechanism of AMPK and related molecules involved in platelet activation in the collagen-induced platelet activation model.Methods: In vitro,5 m M and 20 m M AMPK agonist metformin was used to treat platelet aggregation and secretion after stimulated with adenosine diphosphate(ADP),collagen and thrombin respectively.Western blotting was used to detect platelet phosphorylation and quantification of AMPK,PI3 K and S6K1.Collagen induced platelet aggregation and secretion models were used in combination with AMPK inhibitor Compound C(10?M),AMPK activators metformin(20m M),m TOR inhibitor rapamycin(10n M),PI3 K inhibitor LY294002(25?M)and c GMP analog 8-p CPT-c GMP(50?M)for further assessment of the role of AMPK/m TOR signaling pathway and its possible cross-talk with PI3K/NO/c GMP signaling pathway.Results: Both 5 m M and 20 m M AMPK agonist metformin inhibited platelet aggregation and secretion induced by ADP,collagen and thrombin,while AMPK phosphorylation was a possible mechanism.Inhibition of AMPK and m TOR by Compound C and rapamycin,respectively,in the collagen induced platelet aggregation and secretion reversed the inhibitory effect of metformin on platelet aggregation and secretion.Western-blotting assay suggested that Akt phosphorylation increased,and adding LY294002 inhibit PI3 K can inhibit platelet aggregation and secretion.Further detection of PI3 K pathway downstream showed that c GMP analog 8-p CPT-c GMP can cancel the inhibitory effect of platelet AMPK activation on aggregation and secretion.Western-blotting indicated increased phosphorylation of VASP,a substrate for c GMP-dependent protein kinase in platelets,confirmed that NO/c GMP pathway is involved in AMPK-mediated platelet activation.Conclusions:Cross-talk between AMPK/m TOR pathway and PI3 K pathway was involved in collagen induced platelet activation.