Thromb Haemost 2003; 90(05): 863-871
DOI: 10.1160/TH03-04-0228
Platelets and Blood Cells
Schattauer GmbH

AMP-activated protein kinase (AMPK) regulates the insulin-induced activation of the nitric oxide synthase in human platelets

Ingrid Fleming
1   Institut für Kardiovaskuläre Physiologie, Klinikum der J.W. Goethe-Universität, Frankfurt am Main, Germany
,
Christian Schulz
1   Institut für Kardiovaskuläre Physiologie, Klinikum der J.W. Goethe-Universität, Frankfurt am Main, Germany
,
Birgit Fichtlscherer
1   Institut für Kardiovaskuläre Physiologie, Klinikum der J.W. Goethe-Universität, Frankfurt am Main, Germany
,
Bruce E. Kemp
2   St.Vincent’s Institute of Medical Research, Fitzroy, Victoria, Australia
,
Beate Fisslthaler
1   Institut für Kardiovaskuläre Physiologie, Klinikum der J.W. Goethe-Universität, Frankfurt am Main, Germany
,
Rudi Busse
1   Institut für Kardiovaskuläre Physiologie, Klinikum der J.W. Goethe-Universität, Frankfurt am Main, Germany
› Author Affiliations
Financial support: This study was supported by the Deutsche Forschungsgemeinschaft (SFB 553, B5), the Heinrich and Fritz Riese-Stiftung, NHMRC and National Heart Foundation of Australia, BEK is an NHMRC Fellow and sponsored by the Max Planck Research Award Program.
Further Information

Publication History

Received 15 April 2003

Accepted after revision 11 June 2003

Publication Date:
05 December 2017 (online)

Summary

Little is known about the signaling cascades that eventually regulate the activity of the endothelial nitric oxide synthase (eNOS) in platelets. Here, we investigated the effects of insulin on the phosphorylation and activation of eNOS in washed human platelets and in endothelial cells.

Insulin activated the protein kinase Akt in cultured endothelial cells and increased the phosphorylation of eNOS on Ser1177but failed to increase endothelial cyclic GMP levels or to elicit the relaxation of endothelium-intact porcine coronary arteries. In platelets, insulin also elicited the activation of Akt as well as the phosphorylation of eNOS and initiated NO production which was associated with increased cyclic GMP levels and the inhibition of thrombin-induced aggregation. The insulin-induced inhibition of aggregation was accompanied by a decreased Ca2+response to thrombin and was also prevented by Nωnitro-L- arginine. In platelets, but not in endothelial cells, insulin induced the activation of the AMP-activated protein kinase (AMPK), a metabolic stress-sensing kinase which was sensitive to the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin and the AMPK inhibitor iodotubercidin. Moreover, the insulin-mediated inhibition of thrombin-induced aggregation was prevented by iodotubercidin. Insulin-independent activation of the AMPK using 5-aminoimidazole-4-carboxamide ribonucleoside, increased platelet eNOS phosphorylation, increased cyclic GMP levels and attenuated platelet aggregation.

These results highlight the differences in the signal transduction cascade activated by insulin in endothelial cells and platelets, and demonstrate that insulin stimulates the formation of NO in human platelets, in the absence of an increase in Ca2+, by activating PI3-K and AMPK which phosphorylates eNOS on Ser1177.

 
  • References

  • 1 Steinberg HO, Brechtel G, Johnson A. et al Insulin-mediated skeletal muscle vasodilation is nitric oxide dependent. J Clin Invest 1994; 94: 1172-9.
  • 2 Taddei S, Virdis A, Mattei P. et al Effect of insulin on acetylcholine-induced vasodilation in normotensive subjects and patients with essential hypertension. Circulation 1995; 92: 2911-8.
  • 3 Lembo G, Iaccarino G, Vecchione C. et al Insulin modulation of an endothelial nitric oxide component present in the α2-and β–adrenergic responses in human forearm. J Clin Invest 1997; 100: 2007-14.
  • 4 Zeng G, Quon MJ. Insulin-stimulated production of nitric oxide is inhibited by wortmannin-direct measurement in vascular endothelial cells. J Clin Invest 1996; 98: 894-8.
  • 5 Zeng G, Nystrom FH, Ravichandran LV. et al Roles for insulin receptor, PI3-kinase, and Akt in insulin-signaling pathways related to production of nitric oxide in human vascular endothelial cells. Circulation 2000; 101: 1539-45.
  • 6 Wallerath T, Gath I, Aulitzky WE. et al Identification of the NO synthase isoforms expressed in human neutrophil granulocytes, megakaryocytes and platelets. Thromb Haemost 1997; 77: 163-7.
  • 7 Berkels R, Bertsch A, Zuther T. et al Evidence for a NO synthase in porcine platelets which is stimulated during activation/aggregation. Eur J Haematol 1997; 58: 307-13.
  • 8 Trovati M, Massucco P, Mattiello L. et al The insulin-induced increase of guanosine-3’,5’-cyclic monophosphate in human platelets is mediated by nitric oxide. Diabetes 1996; 45: 768-70.
  • 9 Lantoine F, Brunnet A, Bedioui F. et al Direct measurement of nitric oxide production in platelets: relationship with cytosolic Ca2+ con centration. Biochem Biophys Res Commun 1995; 215: 842-8.
  • 10 Busse R, Mülsch A. Calcium-dependent nitric oxide synthesis in endothelial cytosol is mediated by calmodulin. FEBS Lett 1990; 265: 133-6.
  • 11 Fleming I, Busse R. Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase. Am J Physiol Regul Integr Comp Physiol 2003; 284: R1-R12.
  • 12 Chen Z-P, Mitchelhill KI, Michell BJ. et al AMP-activated protein kinase phosphorylation of endothelial NO synthase. FEBS Lett 1999; 443: 285-9.
  • 13 Fleming I, Hecker M, Busse R. Intracellular alkalinization induced by bradykinin sustains activation of the constitutive nitric oxide syn-thase in endothelial cells. Circ Res 1994; 74: 1220-6.
  • 14 Timmons S, Hawiger J. Separation of human platelets from plasma proteins including factor VIII VWF by a combined albumin gradient-gel filtration method using HEPES buffer. Thromb Res 1978; 12: 297-306.
  • 15 Mülsch A, Mordvincev P, Vanin A. Quantification of nitric oxide in biological samples by electron spin resonance spectroscopy. Neuroprotocols 1992; 1: 165-73.
  • 16 Fleming I, Fisslthaler B, Dimmeler S. et al Phosphorylation of Thr495 regulates Ca2+/cal-modulin-dependent endothelial nitric oxide synthase activity. Circ Res 2001; 88: e68-e75.
  • 17 Malinski T, Radomski M, Taha Z. et al Direct electrochemical measurement of nitric oxide released from human platelets. Biochem Biophys Res Commun 1993; 194: 960-5.
  • 18 Viollet B, Andreelli F, Jorgensen SB. et al The AMP-activated protein kinase alpha2 catalytic subunit controls whole-body insulin sensitivity. J Clin Invest 2003; 111: 91-8.
  • 19 Corton JM, Gillespie JG, Hawley SA. et al 5-aminoimidazole-4-carboxamide ribonucleoside. A specific method for activating AMP-activated protein kinase in intact cells?. Eur J Biochem 1995; 229: 558-65.
  • 20 Russell III RR, Bergeron R, Shulman I G. et al Translocation of myocardial GLUT-4 and increased glucose uptake through activation of AMPK by AICAR. Am J Physiol 1999; 277: H643-H649.
  • 21 Sase K, Michel T. Expression of constitutive endothelial nitric oxide synthase in human blood platelets. Life Sci 1995; 57: 2049-55.
  • 22 Rao GH, Krishnamurthi S, Raij L. et al Influence of nitric oxide on agonist-mediated calcium mobilization in platelets. Biochem Med Metab Biol 1990; 43: 271-5.
  • 23 Trovati M, Anfossi G, Massucco P. et al Insulin stimulates nitric oxide synthesis in human platelets and, through nitric oxide, increases platelet concentrations of both guanosine-3’,5’-cyclic monophosphate and adenosine-3’,5’-cyclic monophosphate. Diabetes 1997; 46: 742-9.
  • 24 Fontana J, Fulton D, Chen Y. et al Domain mapping studies reveal that the M domain of hsp90 serves as a molecular scaffold to regulate Akt-dependent phosphorylation of endothelial nitric oxide synthase and NO release. Circ Res 2002; 90: 866-73.
  • 25 Hardie DG, Salt IP, Hawley SA. et al AMP-activated protein kinase: an ultrasensitive system for monitoring cellular energy charge. Biochem J 1999; 338: 717-22.
  • 26 Chen ZP, McConell GK, Michell BJ. et al AMPK signaling in contracting human skeletal muscle: acetyl-CoA carboxylase and NO synthase phosphorylation. Am J Physiol Endocrinol Metab 2000; 279: E1202-E1206.
  • 27 Zou MH, Hou XY, Shi CM. et al Modulation by peroxynitrite of Akt-and AMP-activated kinase-dependent Ser1179 phosphorylation of endothelial nitric oxide synthase. J Biol Chem 2002; 277: 32552-7.
  • 28 Ruderman NB, Saha AK, Vawas D. et al Malonyl-CoA, fuel sensing, and insulin resistance. Am J Physiol 1999; 276: E1-E18.
  • 29 Lochhead PA, Salt IP, Walker KS. et al 5-aminoimidazole-4-carboxamide riboside mimics the effects of insulin on the expression of the 2 key gluconeogenic genes PEPCK and glucose-6-phosphatase. Diabetes 2000; 49: 896-903.
  • 30 Kishi K, Yuasa T, Minami A. et al AMP-Activated protein kinase is activated by the stimulations of Gq-coupled receptors. Biochem Biophys Res Commun 2000; 276: 16-22.
  • 31 Moule SK, Denton RM. The activation of p38 MAPK by the beta-adrenergic agonist isoproterenol in rat epididymal fat cells. FEBS Lett 1998; 439: 287-90.
  • 32 Yamauchi T, Kamon J, Minokoshi Y. et al Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase. Nat Med 2002; 8: 1288-95.
  • 33 Minokoshi Y, Kim YB, Peroni OD. et al Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature 2002; 415: 339-43.
  • 34 Witters LA, Kemp BE. Insulin activation of acetyl-CoA carboxylase accompanied by inhibition of the 5’-AMP-activated protein kinase. J Biol Chem 1992; 267: 2864-7.
  • 35 Freedman JE, Michelson AM, Barnard MR. et al Nitric oxide release from activated platelets inhibits platelet recruitment. J Clin Invest 1997; 100: 350-6.
  • 36 Freedman JE, Sauter R, Battinelli EM. et al Deficient platelet-derived nitric oxide and enhanced hemostasis in mice lacking the NOSIII gene. Circ Res 1999; 84: 1416-21.
  • 37 Winder WW, Hardie DG. AMP-activated protein kinase, a metabolic master switch: possible roles in type 2 diabetes. Am J Physiol 1999; 277: E1-E10.