Ghrelin

MILLIPLEX Rat Metabolic Hormone Magnetic Bead Panel—Metabolism Multiplex Assay (Millipore Sigma # RMHMAG-84K, EMD Millipore Corporation, Massachusetts, USA) was used to simultaneously analyze Amylin (Active), C-Peptide 2, Ghrelin (Active), GIP (Total), GLP-1 (Active), Glucagon, IL-6, Insulin, Leptin, MCP-1, PP, PYY, TNF-α. Blood samples were collected in tubes containing EDTA (5ul; 0.5M), dipeptidyl peptidase (DPP-4) inhibitor (15 ul; 10mM), and Fisher Halt Protease Inhibitor Cocktail (5ul). Following blood collection, samples were centrifuged at 1000× g for 20 min. Plasma was transferred into a fresh tube on ice and stored at −20 ℃ until the day of further analysis. On the day of analysis, plasma samples were thawed on ice and assayed in triplicates according to the kit manufacturer’s instructions.

At 16 weeks of postnatal age, blood samples were obtained from the mother (3 ml) and the infant (0.5 ml) during fasting (12 hr for mothers and 4 hr for infants). To stabilize appetite‐regulating hormones, blood samples were placed in Vacutainer® EDTA anticoagulant tubes treated with aprotinin protease inhibitor (500 KIU/ml, EMD Millipore, Billerica, MA, USA) and inhibitor of dipeptidyl peptidase‐4 (10 μl/ml, EMD Millipore). After collection, plasma was separated by centrifugation at 3000 g for 15 min at 4°C, aliquoted in 0.6‐ml tubes, and then stored at −80°C. The leptin hormones (R&D Systems, Minneapolis, MN, USA), ghrelin, peptide YY, and GLP‐1 (all three from EMD Millipore) were analyzed with commercial enzyme‐linked immunosorbent assay kits. For the determination of leptin, the samples were diluted 1:20. The sensitivity of the assay for leptin, ghrelin, peptide YY, and GLP‐1 was 7.8 pg/ml, 30 pg/ml, 6.5 pg/ml, and 1.5 pM, respectively. The concentrations of the hormones were determined from curves generated from the standards of each kit, using a logistic regression model of four parameters.

Mice at 12 weeks old were housed in individual cages two days before the beginning of the experiment. Mice were deprived of food for 2 h after the dark period and i.p. injected with ghrelin (0.4 μg/g of body weight; Merck Millipore, Cat# 494127-100G) in physiological saline solution (B. Braun; Cat# 12260029_1019) at 0 min and again at 30 min. We monitored the eating time and food intake for 1 h after the first injection [44 (link)].

Blood was sampled via venepuncture prior to the control period, and then at 08:00 on each morning of bed rest. Since the CONTINUOUS group were constantly in a fed state, all blood samples during bed rest were taken in a “postprandial” state in this group, vs a fasted state in the INTERMITTENT group. In order to ascertain an effect of feeding pattern independent from feeding status, a final blood sample was collected at 08:00 (in the fasted state for both groups) on the day after bed rest (day 8) prior to the hyperinsulinemic-euglycemic clamp. Samples were collected in EDTA-coated tubes and immediately centrifuged at 1000g for 10 min at 4 °C. Plasma was divided into aliquots, snap frozen in liquid nitrogen and stored at − 80 °C until subsequent determination of glucose (GLUC3, reference 05168791 190, Roche; detection limits; 0.11–41.6 mmol L⁻¹), glycated haemoglobin (determined in 4 mL venous blood by high-performance liquid chromatography; Bio-Rad Diamat, Munich, Germany) insulin (Immunologic, reference 12017547 122, Roche; detection limits; 1.39–6945 pmol L⁻¹), GLP-1_Total (Epitope Diagnostics Inc. CA, USA; detection limits; 0.6–54 pmol L⁻¹) ghrelin (EMD Millipore, Germany; detection limits; 50–5000 pg mL⁻¹) glucagon (Mercodia AB, Sweden; detection limits; 0.024–100,000 pmol L⁻¹) leptin (Mercodia AB, Sweden). Intra- and inter-assay co-efficients of variation were all < 8%.

Cord venous blood samples were collected at delivery and centrifuged at 3500 x g for 30 min at 4 °C to separate the serum. The serum samples were stored at − 80 °C. IGF-I (R&D Systems, Inc. Minneapolis, MN, USA), IGFBP-3 (R&D Systems, Inc. Minneapolis, MN, USA), insulin ((R&D Systems, Inc. Minneapolis, MN, USA) and ghrelin (EMD Millipore Corporation, St. Charles, MO, USA) levels were measured using ELISA following the manufacturers’ instructions.

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.