Abstract
Angiotensin II (Ang II) plays a major role in the pathogenesis of insulin resistance and diabetes by inhibiting insulin's metabolic and potentiating its trophic effects. Whereas the precise mechanisms involved remain ill-defined, they appear to be associated with and dependent upon increased oxidative stress. We found Ang II to block insulin-dependent GLUT4 translocation in L6 myotubes in an NO- and O2 .−-dependent fashion suggesting the involvement of peroxynitrite. This hypothesis was confirmed by the ability of Ang II to induce tyrosine nitration of the MAP kinases ERK1/2 and of protein kinase B/Akt (Akt). Tyrosine nitration of ERK1/2 was required for their phosphorylation on Thr and Tyr and their subsequent activation, whereas it completely inhibited Akt phosphorylation on Ser473 and Thr308 as well as its activity. The inhibitory effect of nitration on Akt activity was confirmed by the ability of SIN-1 to completely block GSK3α phosphorylation in vitro. Inhibition of nitric oxide synthase and NAD(P)Hoxidase and scavenging of free radicals with myricetin restored insulin-stimulated Akt phosphorylation and GLUT4 translocation in the presence of Ang II. Similar restoration was obtained by inhibiting the ERK activating kinase MEK, indicating that these kinases regulate Akt activation. We found a conserved nitration site of ERK1/2 to be located in their kinase domain on Tyr156/139, close to their active site Asp166/149, in agreement with a permissive function of nitration for their activation. Taken together, our data show that Ang II inhibits insulin-mediated GLUT4 translocation in this skeletal muscle model through at least two pathways: first through the transient activation of ERK1/2 which inhibit IRS-1/2 and second through a direct inhibitory nitration of Akt. These observations indicate that not only oxidative but also nitrative stress play a key role in the pathogenesis of insulin resistance. They underline the role of protein nitration as a major mechanism in the regulation of Ang II and insulin signaling pathways and more particularly as a key regulator of protein kinase activity.
Highlights
Increasing evidence from in vitro studies and animal models using ACE inhibitors (ACEI), Angiotensin II (Ang II) AT1 receptor antagonists (ARB) and more recently renin inhibitors (RI), indicates that Ang II is involved in insulin resistance [1].Most important, several clinical trials with ACEI and ARBs have shown that blockade of the renin-angiotensin system slows down the progression of cardiovascular morbidity and mortality in type 2 diabetic patients [2] and reduces the risk of developing diabetes among hypertensive patients
Superoxide ions (O22.) and hydrogen peroxide (H2O2) have been reported to affect numerous signaling pathways, the precise molecular mechanisms through which they alter specific enzymatic activities have often not been elucidated. With regard to this question, we have shown previously that reactive oxygen species (ROS)-dependent activation of ERK1/2 by Ang II [16] is due to the production of the RNS peroxynitrite (ONOO2), which leads to their nitration on tyrosine residues [17]
As we showed previously that Ang II induces the nitration rather than the S-nitrosation of other kinases [17], implying the activation of pathways generating more ONOO2 than required for S-nitrosation [21], we hypothesized that this might be the case with Akt, especially as it has been recently reported that stimulation of peroxynitrite catalysis restored Akt phosphorylation and insulin-stimulated glucose uptake in insulinresistant mice [25]
Summary
Increasing evidence from in vitro studies and animal models using ACE inhibitors (ACEI), Ang II AT1 receptor antagonists (ARB) and more recently renin inhibitors (RI), indicates that Ang II is involved in insulin resistance [1].Most important, several clinical trials with ACEI and ARBs have shown that blockade of the renin-angiotensin system slows down the progression of cardiovascular morbidity and mortality in type 2 diabetic patients [2] and reduces the risk of developing diabetes among hypertensive patients (reviewed in [3,4]). Increasing evidence from in vitro studies and animal models using ACE inhibitors (ACEI), Ang II AT1 receptor antagonists (ARB) and more recently renin inhibitors (RI), indicates that Ang II is involved in insulin resistance [1]. Despite the clinical implications of these observations, most investigators have focused their efforts on studying the interference of Ang II with insulin signaling pathways in the vascular wall rather than on major metabolic target tissues like the liver, adipose tissue and skeletal muscle [1,6]. Ang II has been shown to stimulate the production of reactive oxygen species (ROS) in endothelial and vascular smooth muscle cells as well as in various tissues including skeletal muscle in various animal models [8,11,14] and this pathway has been suggested to play a major role in its insulin desensitizing effects [1,8,11,14,15]
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