FIG. 4.
Ex vivo-incubated MRic−/− muscle shows insulin-responsive glycogen synthase activity even with increased basal glycogen synthase activity. EDL muscles were incubated in Krebs-Henseleit buffer without (−) or with (+) insulin (100 mU/ml) for 30 min. The rapamycin-treated muscles were first incubated with rapamycin (200 nM final concentration) alone and then with rapamycin and insulin for 30 min. (A) Glycogen synthase activity was measured in EDL as described in the legend of Fig. 3A (means ± SE, n = 4 per group; *, P < 0.025; **, P < 0.006). (B) Protein extracts prepared from EDL muscles were subjected to SDS-PAGE and immunoblotted with phospho-specific antibodies to Akt, S6K1, GSK-3β, and glycogen synthase and antibodies to actin and total glycogen synthase. (C) Quantification of GSK-3β Ser9 phosphorylation (means ± SE, n = 4; *, P < 0.01) after normalization to actin. arb. units, arbitrary units.