Insulin enhances the expression of the endothelial nitric oxide synthase in native endothelial cells: a dual role for Akt and AP-1.
Insulin-induced vasodilatation in vivo has been attributed to the activation of the endothelial nitric oxide (NO) synthase (eNOS). The present study addressed the effects of insulin on the activity and expression of eNOS in native and cultured endothelial cells. Insulin applied to native porcine aortic endothelial cells elicited the tyrosine phosphorylation of the insulin receptor and receptor substrate, the subsequent activation of phosphatidylinositol 3-kinase (PI 3-K), Akt (protein kinase B), and ERK1/2. Insulin did not activate eNOS in cultured endothelial cells nor relax endothelium-intact arterial segments. However, 4h after application of insulin to native endothelial cells eNOS mRNA was increased 2-fold. A comparable increase in eNOS protein was detected after 18-24h and associated with an increase in intracellular cyclic GMP. In native endothelial cells, insulin enhanced the DNA-binding activity of Sp1 and AP-1, but not that of NF-kappaB. The insulin-induced increase in eNOS expression was prevented by wortmannin as well as by AP-1 decoy oligonucleotides. The MEK1 inhibitor, PD 98059, also enhanced eNOS expression in native and cultured endothelial cells, an effect which was independent of ERK1/2 and associated with an increase in the DNA-binding activity of AP-1 and Sp1. These results demonstrate that insulin activates multiple signalling pathways in endothelial cells but does not acutely activate eNOS. Insulin however enhances eNOS mRNA and protein by a mechanism involving the combined activation of a PI 3-K- and AP-1-dependent pathway.
Fisslthaler
Beate
B
Institut für Kardiovaskuläre Physiologie, Klinikum der J. W. Goethe-Universität, Theodor-Stern-Kai 7, Frankfurt am Main D-60590, Germany.
Benzing
Thomas
T
Busse
Rudi
R
Fleming
Ingrid
I
D000818:Animals; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D007328:Insulin / Q000502:physiology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism; D016328:NF-kappa B / Q000187:drug effects / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000235:genetics; D052250:Nitric Oxide Synthase Type III; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt; D016760:Proto-Oncogene Proteins c-fos / Q000187:drug effects / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000187:drug effects / Q000378:metabolism; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor / Q000187:drug effects / Q000378:metabolism; D013552:Swine; D018808:Transcription Factor AP-1 / Q000502:physiology*
D004791:Enzyme Inhibitors; D007328:Insulin; D016328:NF-kappa B; D011518:Proto-Oncogene Proteins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D018808:Transcription Factor AP-1; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases