myotubes. Ang II induced hyperacetylation of mitochondrial proteins with molecular weights of about 55, 48, 44, 38, and 26 kDa, the latter corresponding to MnSOD (Fig 3A). This effect was concomitant with 23840699 the reduction of MnSOD antioxidant activity (Fig 3B). The addition of ALCAR reversed the inhibitory effect of Ang II on Sirt3 activity (Fig 3A, left) and restored the cell antioxidant defense, normalizing MnSOD activity (Fig 3B) and gene expression (S2 Fig) in L6 cells. Similarly, MnTBAP, by fostering MnSOD activity 
(Fig 3B), limited mitochondrial protein acetylation induced by Ang II in L6 myotubes (Fig 3A, right) confirming an inhibitory role for oxygen radicals in Sirt3-dependent mitochondrial protein deacetylation. To assess no matter if impaired Sirt3 deacetylase activity induced by Ang II was because of the deleterious effect of mitochondrial O2opening the mitochondrial permeability transition pore (mPTP) leading for the collapse of C, we investigated the impact of Ang II on C -sensing dye JC-1 in both the absence and presence of ALCAR or MnTBAP. Ang II-treated L6 myotubes showed mitochondrial depolarization, as evidenced by diffuse cytoplasmic JC-1 green staining, that was prevented by both ALCAR and MnTBAP (Fig 4AD) suggesting mitochondrial O2as a mPTP trigger. To confirm the functional hyperlink amongst the mPTP opening and modulation of Sirt3 deacetylase activity, experiments have been repeated inside the presence of CsA, known to inhibit mPTP opening. Prevention of mitochondrial depolarization by CsA inhibited Ang II-induced mitochondrial protein acetylation, reflecting enhanced Sirt3 activity (S3 Fig).
Ang II promotes insulin resistance by means of mitochondrial ROS. Impact of Ang II on (A) mitochondrial O2generation, (B) insulin (Ins)-stimulated GLUT4 transport (densitometric evaluation, best, and representative western blot, bottom) and (C) Ins-stimulated 2-deoxyFatostatin A glucose (2-DG) uptake in L6 myotubes in the absence and presence of ALCAR (0.six mM). MFI, imply fluorescence intensity; CPM, crude plasma membrane. (D) Impact of Ang II on surface GLUT4-myc density in Ins-stimulated cells inside the absence and presence of ALCAR or MnTBAP (0.1 mM). To elucidate the pathways linking Sirt3 dysfunction-induced by Ang II- to impaired GLUT4 translocation, we focused on AMPK, a master regulator of cellular power metabolism involved in glucose transport in skeletal muscles [28, 29]. Ang II lowered the pAMPK/AMPK ratio by about 70% in L6 myotubes (Fig 5A, left). ALCAR normalized AMPK phosphorylation in Ang II-treated L6 myotubes (Fig 5A, left), even though it didn’t have an effect on the pAMPK/AMPK ratio in handle cells (fold alterations vs. control, ALCAR 0.99 0.16 vs. 1.0). Scavenging mitochondrial O2by MnTBAP yielded comparable final results on AMPK phosphorylation in Ang II-treated and control L6 myotubes (Fig 5A, left). Activation of AMPK by AICAR [30] normalized GLUT4�myc levels on the surface of Ang II-treated cells (Fig 5B), although inhibition of AMPK by compound C [31] mimicked the inhibitory impact of Ang II on GLUT4 translocation (Fig 5B) suggesting a functional hyperlink amongst AMPK inactivation and impaired glucose transport. To establish the function of Sirt3 in AMPK regulation we studied the effect of Ang II on the pAMPK/AMPK ratio in Sirt3 overexpressing cells (pSirt3). A 4-fold improve in Sirt3 protein expression (S4 Fig) abrogated Ang II-induced inactivation of AMPK (Fig 5A, middle). Around the other hand, silencing Sirt3 with modest interfering RNA (siSirt3) resulted inside a 69% decrease of Sirt3