Research Paper Volume 8, Issue 7 pp 1470—1484

Figure 7. Ghrelin increases NPY content and NPY receptor antagonists block the stimulatory role of ghrelin on autophagy in rat cortical neurons. Primary rat cortical neuronal cultures were exposed to ghrelin (GHRL, 10 nM) for 6 h. Untreated cells were used as control (Ctrl). (A) Total RNA was isolated and the transcript levels of NPY were analyzed by qPCR, as described in Materials and Methods. The results represent the mean ± SEM of five independents experiments and are expressed as the relative amount compared to control. *p<0.05, significantly different compared to control, as determined by Student’s t test. (B) Ghrelin leads to increased NPY protein content, using an Enzyme-Linked Immunosorbent Assay, as described in Material and Methods. The results represent the mean ± SEM of three independents experiments and are expressed as the relative amount compared to control. (C and D) Cells were incubated with NPY Y₁ receptor antagonist BIBP3226 (Y₁ant, 1 μM), NPY Y₂ receptor antagonist BIIE0246 (Y₂ant, 1 μM) or NPY Y₅ receptor antagonist L152,800 (Y₅ant, 1 μM), 30 min before ghrelin (GHRL, 10 nM) treatment for 6 h. Whole cell extracts were assayed for LC3B-II (C), SQSTM1 (D) and β-tubulin (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, five independents experiments, and are expressed as percentage of control. *p<0.05 significantly different compared to control; ^#p<0.05 and ^##p<0.01, significantly different from ghrelin treatment, as determined by ANOVA, followed by Bonferroni’s post test.