Research Paper Volume 13, Issue 6 pp 7872—7882

PI3K/AKT/MTOR and ERK1/2-MAPK signaling pathways are involved in autophagy stimulation induced by caloric restriction or caloric restriction mimetics in cortical neurons

PI3K inhibition decreases MTOR activity in primary rat cortical neurons. Primary rat cortical neurons were exposed to caloric restriction mimic medium (CR) - DMEM low glucose, NPY (100 nM), or ghrelin (GHRL, 10 nM) for 6 h. Untreated cells were used as control (Ctrl). (A–C) Cells were incubated with PI3K/AKT inhibitor (LY294002 (LY), 1 μM), 30 min before caloric restriction, NPY, or ghrelin treatment. Whole-cell extracts were assayed, phospho-MTOR (A–C), and MTOR or α-Actin (loading control) immunoreactivity through Western blotting analysis, as described in Materials and Methods. Representative Western blots for each protein are presented above each respective graph. The results represent the mean±SEM of, at least, four independents experiments, and are expressed as a percentage of control. **p$$p$$$p$$$$p

Figure 2. PI3K inhibition decreases MTOR activity in primary rat cortical neurons. Primary rat cortical neurons were exposed to caloric restriction mimic medium (CR) - DMEM low glucose, NPY (100 nM), or ghrelin (GHRL, 10 nM) for 6 h. Untreated cells were used as control (Ctrl). (AC) Cells were incubated with PI3K/AKT inhibitor (LY294002 (LY), 1 μM), 30 min before caloric restriction, NPY, or ghrelin treatment. Whole-cell extracts were assayed, phospho-MTOR (AC), and MTOR or α-Actin (loading control) immunoreactivity through Western blotting analysis, as described in Materials and Methods. Representative Western blots for each protein are presented above each respective graph. The results represent the mean±SEM of, at least, four independents experiments, and are expressed as a percentage of control. **p<0.01, ***p<0.001 and ****p<0.0001, significantly different compared to control; $$p<0.01, $$$p<0.001 and $$$$p<0.0001, significantly different from stimulus-treated cells, as determined by ANOVA, followed by Newman-Keuls multiple comparison test.