Figure 3.

Effect of CaMKK inhibitors on contraction-stimulated AMPK phosphorylation and glucose uptake in mouse skeletal muscles. (A) Schematic depicting the signaling pathways and experimental strategy to study the potential role for CaMKK2 in stimulating AMPK and glucose uptake in response to contractions in skeletal muscle. (B) Isolated extensor digitorum longus (EDL) muscles from wild-type C57BL/6 mice (male, an age range of 10–20 weeks) were incubated in Krebs–Ringer bicarbonate buffer ex vivo with vehicle (0.1% DMSO) or the indicated compounds (10 μM) for 1 h. During the last 10 min of incubation, muscle contractions were evoked by electrical stimulation. Muscle tissue extracts were subjected to immunoblot analysis with the indicated antibodies. Representative immunoblots are shown. (C and D) Quantification of AMPKα and ACC2 phosphorylation from (B). Data are n = 6 from two separate membranes, ∗P < 0.05 (Basal vs. contracted treatments) as indicated. (E) At the end of the 50 min incubation period with or without contractions, EDL muscles were further incubated for 10 min and [³H]-2-deoxy-glucose uptake was assessed. Shown data from n = 9–11 from two separate experiments. ∗P < 0.05 (Basal vs. indicated treatment) and ^#P < 0.05 (Vehicle contracted vs. indicated treatment) (F and G) Quantification of peak force production (n = 6–7) and force kinetics during the 10-min contractions in the presence of the indicated treatments. (H–J) Isolated EDL muscles were subjected to the indicated treatments and [³H]-2-deoxy-glucose uptake following the 50 min incubation period. Data are n = 5–6 (H), n = 11–12 from two separate experiments (I) and n = 6–7 (J). Data shown are ∗P < 0.05 (Basal vs. indicated treatment) and ^#P < 0.05 (Vehicle contracted vs. indicated treatment) (H) and ^#P < 0.05 (treatment vs. treatment + SGC-CAMKK2-1 (I and J). One-way ANOVA (F, I, J) and two-way ANOVA (C-E, H) tests were performed with Tukey's correction for multiple comparisons respectively. All data points shown are mean ± S.E.M. NS; not significant. PI3K; phosphoinositide-3-kinase, PDK1; phosphoinositide-dependent kinase 1, mTORC2; mTOR complex 2, GLUT4: glucose transporter 4.