Figure 4. Interplay between PI3K/AKT and ERK1/2-MAPK signaling pathways under caloric restriction, NPY, and ghrelin-induced autophagy in 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–F) Cells were incubated with PI3K/AKT inhibitor (LY294002 (LY), 1 μM) or ERK1/2-MAPK inhibitor (U0126 (U0), 1 μM), 30 min before caloric restriction, NPY, or ghrelin treatment. Whole-cell extracts were assayed, phospho-ERK (A, C, E) and phospho-AKT (B, D, F) and ERK or AKT (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<0.05, **p<0.01, ***p<0.001 and ****p<0.0001, significantly different compared to control; $p<0.05 and $$p<0.01, significantly different from stimulus-treated cells as determined by ANOVA, followed by Newman-Keuls multiple comparison test.