Figure 1. Insulin selectively increases p-Akt2 signal in skeletal muscle mitochondria.

(A) Representative western blots of Akt1, Akt2, and p-Akt in proteins of rat skeletal muscle during hyperinsulinemic-normoglycemic status as achieved by s.c. administration of human insulin glargine plus oral sucrose (see Methods and Supporting Figure S2). Respective densitometries from 6–7 samples from separate experiments were obtained after digital image analysis and normalized to the actin band for cytosol fraction and to the complex IV subunit I band for mitochondria (Akt2 mitochondria: F = 39.03, DF = 23, P = 0.000; p-Akt cytosol: F = 111.8, DF = 23, P = 0.000; p-Akt mitochondria: F = 77.48, DF = 25, P = 0.000). Figure shows that after 12 hours increase of Akt2 and p-Akt2 is selectively confined to mitochondria. (B) Differential flow cytometry histograms of 100 µg of purified mitochondria isolated from controls (C) and insulin-treated animals were obtained with 1: 1000 fluorescent antibody anti-p-Akt in a mitochondrial population previously delimited with MitoTracker Red 580. Histograms show a kinetics similar to western blotting. (C) An Akt activity assay was performed three times by immunoprecipitation of mitochondria and cytosol proteins with anti-p-Akt antibody. After further immobilization, the proteins were detected with the anti-p-Ser21/9-GSK-3α/β antibody (1∶1000). The assay confirms the insulin-induced kinetics of p-Akt in the fractions as in (A) and (B) (Cytosol: F = 8.46, DF = 15, P = 0.003, Mitochondria: F = 8.15, DF = 15, P = 0.003). (D) Western blot shows submitochondrial localization of p-Akt at the inner mitochondrial membrane, as controlled by duplicate with an antibody specific for subunit IV of cytochrome oxidase. Data are mean±SEM.; * p<.05 respect to controls by ANOVA and Dunnett test.