Acylated ghrelin

To mimic a caloric restriction condition, primary rat cortical neuron cultures were exposed to a Dulbecco’s Modified Eagle Medium (DMEM; Sigma- Aldrich) low glucose medium (1 g.L^-1 glucose, 100 U.mL^-1 penicillin and, 100 μg.mL^-1 streptomycin, without B27 supplementation) or, NPY (100 nM; Phoenix Europe GmbH) or acylated ghrelin (10 nM; Bachem) as previous described [18 (link)]. Protein kinase inhibitors were used as follow: PI3K/AKT inhibitor (LY294002; 10 μM) and ERK1/2-MAPK inhibitor (U0126; 10 μM), both from Merck Millipore. Inhibitors were added to cell culture medium 30 min before the addition of caloric restriction mimetics NPY or ghrelin for 6 h.

Cells were exposed to nutrient deprivation to mimic a caloric restriction condition, Dulbecco's Modified Eagle Medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) low glucose medium (1 g.L⁻¹ glucose, 100 U.mL⁻¹ penicillin and 100 μg.mL⁻¹ streptomycin, without B27 supplementation), NPY (100 nM; Phoenix Europe GmbH, Karlsruhe, Germany) or acylated ghrelin (10 nM; Bachem, Bubendorf, Switzerland). Cells were also exposed to the lysosomal protein degradation inhibitor, chloroquine (100 μM; Sigma-Aldrich), NPY receptors selective antagonists (all at 1 μM; all from Bachem, Bubendorf, Switzerland; NPY Y₁ antagonist (BIBP3226), NPY Y₂ antagonist (BIIE0246) and NPY Y₅ antagonist (L-152,804)), and/or ghrelin receptor antagonist ([D-Lys³]-GHRP-6) (100 μM; Tocris Bioscience, Bristol, UK), added to the cell culture medium 30 min prior to caloric restriction, NPY or ghrelin treatment for 6 h.