Figure 2: Deletion of FoxOs prevents muscle loss and weakness during fasting.
(a) Frequency histograms of cross-sectional areas (μm2) of FoxO1,3,4f/f (black bars) and FoxO1,3,4−/− (magenta bars) fibres in fed (upper panel) and fasted (lower panel) conditions, n=4, each group. (b) Force measurements preformed in vivo on gastrocnemius showed that FoxO1,3,4−/− muscles preserve maximal tetanic force after fasting; n=6 muscles in each group. (c) Force/frequency curve of starved gastrocnemius muscle underlines the important protection achieved by the absence of FoxOs; n=6 muscles in each group. (d) Immunoblot of protein extracts from gastrocnemius muscles. Phosphorylation of AKT and S6 is reduced in fed and starved FoxO1,3,4−/− muscles when compared with controls. Data are representative of three independent experiments. (e) Immunoblot analysis of p62 and LC3 in homogenates of gastrocnemius muscles from fed and starved FoxO1,3,4−/− or controls. Fasting did not induce LC3 lipidation and p62 upregulation in FoxO-deficient muscles. Data are representative of three independent experiments. (f) Quantification of GFP–LC3-positive vesicles in FoxO1,3,4f/f and FoxO1,3,4−/− TA muscles; n=4 muscles in each group (g) Autophagy flux is not increased in FoxO-deficient TA muscles. Inhibition of autophagy–lysosome fusion by colchicine treatment induces accumulation of LC3II band in starved control but not in starved FoxO1,3,4−/− muscles. Upper panel: immunoblot analysis of gastrocnemius homogenates. Lower panel: quantification of LC3 lipidation. n=4 muscles in each group (h) The scheme shows the overlap between FoxO-dependent genes, identified by gene expression profiling of fed (n=4) and fasted (n=4) muscles from FoxO1,3,4f/f and FoxO1,3,4−/− and atrophy-related genes or atrogenes. The data in the graphs are shown as mean±s.e.m. Error bars indicate s.e.m. *P<0.05, **P<0.01 (Student's t-test).
