Fig. 9.
Impact of Vps34 inactivation on insulin-mediated Akt/mTORC1 and AMPK signaling. a Cultured myotubes were starved for 5 h and stimulated for the indicated time points with 100 nM insulin. Cell lysates were immunoblotted with the indicated antibodies. Representative data from three independent experiments are shown. b Primary hepatocytes were cultured overnight in insulin-free HM in the presence of DMSO or Vps34-IN1 at the indicated doses. Cell lysates were immunoblotted with the indicated antibodies. Representative data of three independent experiments are shown. c 2-Deoxy-d-glucose uptake in differentiated myotubes. Myoblasts were differentiated for 6–7 days, starved for 5 h before and stimulated with 100 nM insulin for 20 min. Myotube cultures derived from three independent mice/genotype. Data represent mean ± SEM (Student t-test). d 2-Deoxy-d-glucose uptake in differentiated myotubes. Myoblasts were differentiated for 6–7 days, cultured overnight in DMSO or in presence of 1 µM Vps34-IN1, then starved for 5 h before the assay. Myotube cultures were derived from three independent mice/genotype. Data represent mean ± SEM (Student t-test). e, f Expression levels of genes related to fatty acid β-oxidation in Hepa1.6 cells. e treated overnight in absence or in presence of 1 µM Vps34-IN1 in serum-free media and in liver tissues f from WT and Vps34D761A/+ starved mice. Expression levels were quantified by RT-qPCR from independent cell cultures and from n = 5 mice/genotype. Data represent mean ± SEM (Student t-test). g AICAR tolerance test was performed by intraperitoneal injection of 0.15 g AICAR per kg body weight into overnight-fasted 9–11-week-old mice. ≥ 5–11 mice/genotype were used. Data represent mean ± SEM (non-parametric Mann–Whitney t-test). *p < 0.05, **p < 0.01, ***p < 0.001