Figure 1.
Effect of ROS and RNS modulators on Ang II-regulated insulin-mediated GLUT 4 translocation and Akt phosphorylation.
L6 myotubes were incubated with AEBSF (0.5 mM) for 30 min, 1400 W (10 µM) for 60 min and myricetin (100 µM) for 30 min, prior to incubation with Ang II (10 nM) for 30 min and subsequent stimulation with insulin 100 nM for 60 min. A: Blots of the plasma membrane or total cell lysate proteins were probed with anti-GLUT4 antibody. Results are expressed as fold change of untreated control (means ± s.d.) and the blot is representative of three independent experiments (*P<0,05 vs control, °P<0,05 vs Ang II + insulin). B: Western blots were probed with anti-phosphoSer473-Akt and anti-Akt antibodies. The blot displayed is representative of three independent experiments. Results are expressed as the fold increase of the ratio of phospho-Akt/Akt over controls (means ± s.d.) (*P<0,05 vs control, °P<0,05 vs Ang II, #P<0,05 vs Ang II + Insulin).
Figure 2.
Kinetics of insulin-induced Akt phosphorylation and inhibitory effect of Ang II on insulin-induced Akt activation.
L6 myotubes were exposed to insulin (100 nM) for 0–120 min. Blots were probed with anti-Akt and anti-phosphoSer473-Akt (A) or anti-phosphoThr308-Akt (B). L6 myotubes were pretreated with Ang II 10 nM for 30 min and stimulated with insulin (100 nM) for 60 min. Blots were probed with anti-Akt and anti-phosphoSer473-Akt (C) or anti-phosphoThr308-Akt (D) antibodies. Results are expressed as fold change of the ratio of phospho-Akt/Akt over controls (means ± s.d.) and the blots are representative of three independent experiments (*P<0,05 vs control, °P<0,05 vs Ang II, #P<0,05 vs Ang II + insulin).
Figure 3.
Kinetics of Ang II-mediated Akt nitration.
L6 myotubes were exposed to Ang II (10 nM) for 0–120 min. Western blot of nitrated proteins from the soluble fractions were immunoprecipitated with anti-nitrotyrosine antibody and probed with anti-Akt antibody. Results are expressed as the fold increase over controls (means ± s.d.) and the blot is representative of three independent experiments (*P<0,05 vs. control).
Figure 4.
Effect of ROS and RNS modulators and of nitration on Akt ativity.
A: L6 myotubes were pretreated with either AEBSF (0.5 mM) for 30 min, 1400 W (10 µM) for 60 min or myricetin (100 µM) for 30 min prior to addition of Ang II (10 nM) for 30 min and subsequent stimulation with insulin 100 nM for 60 min. The activity of Akt immunoprecipitated from the cell lysates was determined by its ability to phosphorylate GSK3α. Western blots were probed with anti-phosphoSer21-GSK3α. The blot displayed is representative of three independent experiments. Results are expressed as the fold increase over controls (means ± s.d.) (*P<0,05 vs control, °P<0,05 vs Insulin, #P<0,05 vs Ang II, §P<0,05 vs Ang II + Insulin). B: Recombinant human PKB/Akt1 (1.5 µg) was nitrated with SIN-1 (1, 10 and 100 µM) for 1 hour at 30°C, immunoprecipitated and incubated with GST-GSK-3α peptide in the presence of Mg++/ATP for 2 hours. Blots were probed with a polyclonal rabbit anti-phosphoSer21-GSK-3α and a monoclonal mouse anti-nitrotyrosine antibody. The blots are representative of three independent experiments.
Figure 5.
Effect of the MEK inhibitor U0126 on insulin-stimulated Akt phosphorylation.
L6 myotubes were treated with U0126 (10 µM) for 30 min prior to addition of Ang II (10 nM) for 30 min with subsequent stimulation by insulin 100 nM for 60 min. Western blots were probed with anti-phosphoSer473-Akt and anti-Akt antibodies. The blot is representative of three independent experiments. Results are expressed as the fold increase of the ratio of phospho-Akt/Akt over controls (means ± s.d.). (*P<0,05 vs control, #P<0,05 vs Ang II + Insulin).
Figure 6.
Kinetics of Ang II-dependent phosphorylation and nitration of ERK 1/2.
L6 myotubes were exposed to Ang II (10 nM) for 0–120 min. (A) Blots were probed with anti-phosphoERK1/2 and anti-ERK1/2 antibodies. (B) Samples were immunoprecipitated with anti-nitrotyrosine antibody and probed with anti-Akt antibody. (C) Effect of ROS and RNS modulators on ERK 1/2 phosphorylation. L6 myotubes were pretreated with either AEBSF (0.5 mM) for 30 min, 1400 W (10 µM) for 60 min or myricetin (100 µM) for 30 min, prior to addition of Ang II (10 nM) for 30 min and subsequent stimulation with insulin 100 nM for 60 min. Western blots were probed with anti-phosphoERK and anti-ERK antibodies. The blots are representative of three independent experiments. Results are expressed as the fold increase of the ratio of phospho-ERK/ERK over controls (means ± s.d.) (*P<0,05 vs control, °P<0,05 vs Ang + Insulin, #P<0,05 vs 1400 W + Ang, +P<0,05 vs AEBSF + Ang, ∧P<0,05 vs Myricetine + Ang).
Figure 7.
Mass spectra of tryptic peptides of nitrated ERK1.
Tryptic peptides of nitrated recombinant ERK1 were immunoprecipitated with anti-nitrotyrosine antibodies. A: Mass spectrum (500–3000 Da) shows a major peak with a monoisotopic mass of 1552.91 corresponding to GLKY156-(NO2)IHSANVHR. B: Detailed spectrum (2742–2755.5 Da) shows a major peak with a monoisotopic mass of 2749.28 corresponding to the peptide ASTLEAMRDVYIVQDLMETDLYK containing one nitrated Tyr, Tyr119 or Tyr130. Other peaks correspond to peptides which do not contain Tyr and were adsorbed non-specifically to the anti-nitrotyrosine agarose beads.
Figure 8.
Schematic representation of the putative mechanisms involved in the regulation of Akt activity by insulin and Ang II.
Activating pathways are shown in green and inhibitory mechanisms in red.