d into 3 groups, every constituted by 4 3-monthand 4 24-month-old rats. Animals on the first group have been fasted (nutrient withdrawal) 16 h just before euthanizing, these of the second group have been fasted (nutrient withdrawal) 36 h ahead of euthanizing, and these from the third group have been fasted for 36 h and after that refed for 30 min just before euthanizing. The third group was introduced for the goal 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 quickly after fasting (16 or 36 h) inside the 1st and second group and soon after 30 min of refeeding in the third group. Serum glucose was MNK1 Synonyms measured straight away making use of an Accutrend Glucose Analyzer (Roche Diagnostics Corp., Indianapolis, IN, USA). Serum triacylglycerides (TAG) and nonesterified fatty acid (NEFA) contents have been quantified by particular enzymatic kits from Wako Chemical substances (Neuss, Germany). Total-cholesterol and cholesterol-HDL (high-density lipoprotein) levels were measured, respectively, employing an enzymatic kit from Stanbio Laboratory (Boerne, TX, USA). Insulin and leptin levels had been assayed applying particular rat ELISA kits from Spi-Bio (Montigny le Bretonneaux, France) and also the levels of total ketone bodies and glucagon had been determined working with an Autokit Total Ketone Bodies and an ELISA glucagon kit, respectively, each from WAKO, Chemical Neus. Ghrelin (acetylated and unacetylated) levels were assayed in plasma making use of precise rat ELISA kits from Spi-Bio (Montigny le Bretonneaux, France) based on the manufacturer’s directions. Liver and visceral fat depots had been cautiously dissected and weighed. Then, tissues have been flash frozen in liquid nitrogen and stored at -70 C until used. Frozen liver samples were used 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 have been assessed inside the liver applying a glycogen assay kit II (ab 169558, Abcam, Boerne, TX, USA) following the manufacturer’s instruction. Both TAG and glycogen were measured in triplicate and each contents had been expressed as mg/g wet tissue. two.three. Total Extract from Liver and Immunoblot Analysis A piece of fresh liver was thawed, reduce into compact pieces on ice, and suspended (4 mL buffer/g tissue) in cold Krebs-Henseleit buffer pH 7.four (116 mM NaCl, 4.7 mM KCl, 1.2 mM CaCl2 , 1.2 mM KH2 PO4 , 1.two mM MgSO4 .7H2 O, 5.five mM glucose, 25 mM NaHCO3 , 1 mM PMSF, ten /mL leupeptin, 1 /mL pestatin, 2 mM NaF, 1 mM Na3 VO4 ) just before homogeneization with ten passes of a loose-fitting B pestle in a Dounce homogenizer. Then, theAntioxidants 2021, ten,5 ofhomogenates have been incubated for 1 h at four C and centrifuged at 800g for 15 min at 4 C. The supernatant (total extract) was collected and frozen at -70 C till use. Protein content of your mitochondrial oxidative phosphorylation OXPHOS complex was determined with Total OXPHOS rodent WB PKC Storage & Stability antibody cocktail (6 /mL, ab110413, Abcam, Cambridge, UK), which include 5 mouse monoclonal antibodies, a single every against CI subunit NDUFB8, CII-30kDa, CIII-Core protein, CIV subunit I, and CV alpha subunit of OXPHOS. The antibody cocktail was employed in line with the manufacturer’s guidelines. In total, 20 of protein were separated below decreasing circumstances on 12.5 SDS-PAGE, transferred to nitrocellulos
