d into 3 groups, each constituted by four 3-monthand four 24-month-old rats. Animals of the initial group have been fasted (nutrient withdrawal) 16 h before euthanizing, those on the second group were fasted (nutrient withdrawal) 36 h before euthanizing, and those of the third group were fasted for 36 h and then refed for 30 min just before euthanizing. The third group was introduced for the purpose of evaluating the adaptation for the fed state following prolonged fasting. Rats had been anesthetized by CO2 inhalation and sacrificed by decapitation at 09:30 AM. two.2. Analytical Procedures Blood was obtained immediately right after fasting (16 or 36 h) in the first and second group and right after 30 min of refeeding within the third group. Serum glucose was measured instantly utilizing an Accutrend Glucose Analyzer (Roche Diagnostics Corp., Indianapolis, IN, USA). Serum triacylglycerides (TAG) and nonesterified fatty acid (NEFA) contents have been quantified by precise enzymatic kits from Wako Chemical substances (Neuss, Germany). Total-cholesterol and cholesterol-HDL (high-density lipoprotein) levels had been measured, respectively, applying an enzymatic kit from Stanbio Laboratory (Boerne, TX, USA). Insulin and leptin levels had been assayed working with certain rat ELISA kits from Spi-Bio (Montigny le Bretonneaux, France) plus the levels of total ketone bodies and glucagon were determined making use of an Autokit Total Ketone Bodies and an ELISA glucagon kit, respectively, both from WAKO, Chemical Neus. Ghrelin (acetylated and unacetylated) levels were assayed in plasma using certain rat ELISA kits from Spi-Bio (Montigny le Bretonneaux, France) based on the manufacturer’s guidelines. Liver and visceral fat depots had been very carefully dissected and weighed. Then, tissues have been flash frozen in liquid nitrogen and stored at -70 C till made use of. Frozen liver samples were utilized for glycogen and TAG measurement. Neutral lipids have been extracted in the liver as previously described [37] plus the hepatic TAG content was analyzed by the enzymatic kits from Stanbio Laboratory (Boerne, TX, USA). Glycogen levels were assessed within the liver applying a glycogen assay kit II (ab 169558, Abcam, Boerne, TX, USA) following the manufacturer’s instruction. Each TAG and glycogen had been measured in triplicate and each contents were expressed as mg/g wet tissue. two.three. Total Extract from Liver and Immunoblot Analysis A piece of fresh liver was thawed, cut into tiny pieces on ice, and suspended (four mL buffer/g tissue) in cold Krebs-Henseleit buffer pH 7.4 (116 mM NaCl, 4.7 mM KCl, 1.2 mM CaCl2 , 1.two mM KH2 PO4 , 1.2 mM MgSO4 .7H2 O, 5.5 mM glucose, 25 mM NaHCO3 , 1 mM PMSF, 10 /mL leupeptin, 1 /mL pestatin, two mM NaF, 1 mM Na3 VO4 ) prior to homogeneization with ten passes of a loose-fitting B pestle inside a Dounce homogenizer. Then, theAntioxidants 2021, ten,five ofhomogenates had been incubated for 1 h at 4 C and centrifuged at 800g for 15 min at four C. The supernatant (total extract) was collected and frozen at -70 C till use. NOX4 Gene ID protein content with the mitochondrial oxidative phosphorylation OXPHOS complicated was determined with Total OXPHOS rodent WB antibody cocktail (six /mL, ab110413, Abcam, Cambridge, UK), which include 5 mouse monoclonal antibodies, one each and every against CI subunit NDUFB8, CII-30kDa, CIII-Core protein, CIV subunit I, and CV alpha subunit of OXPHOS. The antibody cocktail was made use of based on the manufacturer’s directions. In total, 20 of protein were separated below reducing conditions on 12.five SDS-PAGE, TIP60 Compound transferred to nitrocellulos