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 O(2)(*-)-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 Ser(473) and Thr(308) as well as its activity. The inhibitory effect of nitration on Akt activity was confirmed by the ability of SIN-1 to completely block GSK3alpha 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 Tyr(156/139), close to their active site Asp(166/149), 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.