Control of Muscle Mitochondria by Insulin Entails Activation of Akt2-mtNOS Pathway: Implications for the Metabolic Syndrome
Figure 3
Phosphorylation allows translocation of Akt1 and Akt2 to mitochondria, but only p-Akt2 increases matrix NO.
(A) Translocation of Akt1 and 2 was tested ex vivo in purified mitochondria isolated from rat muscle Mitochondria were suspended in MSHE buffer with NADH and ATP (pH7.4), incubated with recombinant His-tagged p-Akt1 and p-Akt2 proteins for 30 min at 30°C and centrifuged at 10000 g by 10 min to separate supernatant from mitochondrial pellet further resuspended in phosphate buffer saline (PBS) and incubated with proteinase K and compared with non-phosphorylated Akt. Western blots were performed with anti-His-tag (upper), anti-total Akt (middle) and anti-p-Akt antibodies (bottom). (B) To detect mitochondrial NO, flow cytometry histograms from isolated mitochondria (1 mg protein/ml) were obtained with DAF-FM in the same conditions of (A) and after incubating the organelles for 30 min a 37°C with recombinant phosphorylated and non-phosphorylated Akt1 and Akt2 alone or plus 3 mM L-NMMA. The histograms were obtained in a mitochondrial population previously delimited as in Figures 1 and 2.