Insulin Signaling in Vascular Endothelial Cells: A Key Role for Heterotrimeric G Proteins Revealed by siRNA-Mediated G1 Knockdown † Hongjie Chen ‡ and Thomas Michel* ,‡,§ CardioVascular DiVision, Brigham and Women’s Hospital, HarVard Medical School, Boston, Massachusetts 02115, and Veterans Affairs Boston Healthcare System, West Roxbury, Massachusetts 02132 ReceiVed January 16, 2006; ReVised Manuscript ReceiVed May 8, 2006 ABSTRACT: Activation of insulin receptors stimulates the phosphoinositide 3-kinase (PI3-K)/Akt signaling pathway in vascular endothelial cells. Heterotrimeric G proteins appear to modulate some of the cellular responses that are initiated by receptor tyrosine kinases, but the roles of specific G protein subunits in signaling are less clearly defined. We found that insulin treatment of cultured bovine aortic endothelial cells (BAEC) activates the R isoform of PI3-K (PI3-KR) and discovered that purified G protein G1γ2 inhibits PI3-KR enzyme activity. Transfection of BAEC with a duplex siRNA targeting bovine G1 leads to a 90% knockdown in G1 protein levels, with no effect on expression of other G protein subunits. siRNA-mediated G1 knockdown markedly and specifically potentiates insulin-dependent activation of kinase Akt, likely reflecting the removal of the inhibitory effect of Gγ on PI3-KR activity. Insulin- induced tyrosine phosphorylation of insulin receptors is unaffected by G1 siRNA. By contrast, G1 knockdown leads to a significant decrease in the level of serine phosphorylation of the insulin receptor substrate IRS-1. We explored the effects of siRNA on several serine/threonine protein kinases that have been implicated in insulin signaling. G1 siRNA significantly attenuates phosphorylation of the 70 kDa ribosomal protein S6 kinase (p70S6K) in the basal state and following insulin treatment. We also found that IGF-1-initiated activation of Akt is significantly enhanced after siRNA-mediated G1 knockdown, while IGF-1-induced p70S6K activation is markedly suppressed following transfection of G1 siRNA. We propose that G1 participates in the activation of p70S6K, which in turn promotes the serine phosphorylation and inhibition of IRS-1. Taken together, these studies suggest that G1 plays an important role in insulin and IGF-1 signaling in endothelial cells, both by inhibiting the activity of PI3-KR and by stimulating pathways that lead to activation of protein kinase p70S6K and to the serine phosphorylation of IRS-1. Cellular responses to insulin have been extensively characterized in diverse tissues, including liver, muscle, and fat, but the role of insulin in vascular endothelial cell signaling is less completely understood. Activation of the insulin receptor tyrosine kinase stimulates autophosphory- lation of the receptor on specific tyrosine residues, which promotes the binding and tyrosine phosphorylation of a family of insulin receptor substrates (IRS) 1 and leads to the activation of diverse downstream protein kinases and other modulators (1). A key downstream modulator of in- sulin responses is the phosphoinositide 3-kinase (PI3-K) (2, 3). The PI3-Ks belong to a family of receptor-activated lipid kinases that phosphorylate the D-3 position on the inositol ring in phosphoinositides, which in turn activate specific phospholipid-dependent kinases (PDKs) and lead to the phosphorylation and activation of protein kinase Akt. Activated kinase Akt phosphorylates several sub- strate proteins, such as the endothelial isoform of nitric oxide synthase (eNOS), glycogen synthase 3 (GSK3), and mammalian target of rapamycin (mTOR), and appears to be a key determinant of insulin-mediated responses, including cell growth, cell survival, and glucose uptake (1-3). Cellular responses to insulin receptor (IR) activation are dependent upon the receptor-mediated tyrosine phosphory- lation of IRS proteins (1-3). However, in contrast to the stimulation of IRS by tyrosine phosphorylation, the phos- phorylation of IRS proteins on serine and threonine residues attenuates insulin signaling by decreasing the level of insulin- stimulated tyrosine phosphorylation of IRS (4). Activation of intracellular serine/threonine kinases that phosphorylate IRS may provide a mechanism for receptor-mediated inhibi- tion of insulin signaling and may be altered in the clinical † This work was supported by NIH Grants HL48743 and GM36259 to T.M. * To whom correspondence should be addressed: Cardiovascular Division, Brigham and Women’s Hospital, Thorn Building 1210A, 75 Francis St., Boston, MA 02115. Telephone: (617) 732-7376. Fax: (617) 732-5132. E-mail: tmichel@research.bwh.harvard.edu. ‡ Harvard Medical School. § Veterans Affairs Boston Healthcare System. 1 Abbreviations: siRNA, small interfering RNA; G1 siRNA, small interfering RNA targeted to the bovine G1 mRNA; BAEC, bovine aortic endothelial cells; PI3-K, phosphatidylinositol 3-kinase; IR, insulin receptor; IRS, insulin receptor substrate(s); PIP3, phosphatidylinositol 3-monophosphate; PDK, phospholipid-dependent kinase; mTOR, mam- malian target of rapamycin; eNOS, endothelial isoform of nitric oxide synthase; GSK3, glycogen synthase kinase 3; p70S6K, 70 kDa ribosomal protein S6 kinase; rpS6, ribosomal protein S6; S1P, sphin- gosine 1-phosphate; VEGF, vascular endothelial growth factor; IGF- 1, insulin-like growth factor-1; SE, standard error. 8023 Biochemistry 2006, 45, 8023-8033 10.1021/bi0600906 CCC: $33.50 © 2006 American Chemical Society Published on Web 06/10/2006