Abstract
This study was conducted to elucidate the molecular mechanisms of thromboxane A2 receptor (TP)-induced insulin resistance in endothelial cells. Exposure of human umbilical vein endothelial cells (HUVECs) or mouse aortic endothelial cells to either IBOP or U46619, two structurally related thromboxane A(2) mimetics, significantly reduced insulin-stimulated phosphorylation of endothelial nitric-oxide synthase (eNOS) at Ser(1177) and Akt at Ser(473). These effects were abolished by pharmacological or genetic inhibitors of TP. TP-induced suppression of both eNOS and Akt phosphorylation was accompanied by up-regulation of PTEN (phosphatase and tension homolog deleted on chromosome 10), Ser(380)/Thr(382/383) PTEN phosphorylation, and PTEN lipid phosphatase activity. PTEN-specific small interference RNA restored insulin signaling in the face of TP activation. The small GTPase, Rho, was also activated by TP stimulation, and pretreatment of HUVECs with Y27632, a Rho-associated kinase inhibitor, rescued TP-impaired insulin signaling. Consistent with this result, pertussis toxin abrogated IBOP-induced dephosphorylation of both Akt and eNOS, implicating the G(i) family of G proteins in the suppressive effects of TP. In mice, high fat diet-induced diabetes was associated with aortic PTEN up-regulation, PTEN-Ser(380)/Thr(382/383) phosphorylation, and dephosphorylation of both Akt (at Ser(473)) and eNOS (at Ser(1177)). Importantly, administration of TP antagonist blocked these changes. We conclude that TP stimulation impairs insulin signaling in vascular endothelial cells by selectively activating the Rho/Rho-associated kinase/LKB1/PTEN pathway.
Highlights
Our results reveal that activation of thromboxane A2 receptor (TP) using synthesis was inhibited using the following oligoribonucleotia potent and stable ligand (IBOP) abrogates insulin signaling in des (TP siRNA): 5Ј-GGAGCUGCUCAUCUACUUG UU-3Ј
HUVECs were in 0.5% serum medium and exposed to the indicated agents maintained in endothelial cell basal medium (EBM) supple- overnight
TP Activation Abrogates Basal and Insulin-stimulated Phosphorylation—Because insulin activates eNOS through Akt eNOS-Ser1177 Phosphorylation—Treatment of HUVECs with [27], we investigated whether TP activation affects both basal the TP agonist IBOP (400 nmol/liter) [13, 22] overnight signif- and insulin-enhanced Akt activation, as assessed by Ser473 icantly inhibited the phosphorylation of eNOS at Ser1177
Summary
Target-specific siRNA or nonspecific control siRNA for 48 h using LipofectamineTM 2000 (Invitrogen) according to the Materials—Human umbilical vein endothelial cells manufacturer’s instructions. Transfected cells were cultured (HUVECs) were purchased from Invitrogen. HUVECs were in 0.5% serum medium and exposed to the indicated agents maintained in endothelial cell basal medium (EBM) supple- overnight. Mouse aortic endo- fusion protein with recombinant Akt in the presence or thelial cells (MAECs) were isolated and maintained in cultured, absence of recombinant ROCK1 or ROCK2 for 20 min at 37 °C. as described previously [21]. Recombinant human ROCK1 and The reaction mixture consisted of kinase buffer (25 mM Tris, ROCK2 proteins and antibodies against PTEN, phospho- pH 7.5, 5 mM -glycerophosphate, 2 mM dithiothreitol, 0.1 mM PTEN-Ser380/Thr382/383, Akt, phospho-Akt-Ser473, eNOS, Na3VO4, 10 mM MgCl2) supplemented with 0.2 mM ATP.
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