d into 3 groups, each constituted by four 3-monthand 4 24-month-old rats. Animals from the initially group had been fasted (nutrient withdrawal) 16 h just before euthanizing, these with the second group have been fasted (nutrient withdrawal) 36 h just before euthanizing, and these of your third group were fasted for 36 h then refed for 30 min before euthanizing. The third group was introduced for the purpose of evaluating the adaptation for the fed state following prolonged fasting. Rats were anesthetized by CO2 inhalation and sacrificed by decapitation at 09:30 AM. 2.two. Analytical Procedures Blood was obtained promptly following fasting (16 or 36 h) in the very first and second group and just after 30 min of refeeding within the third group. Serum glucose was measured immediately making use of an Accutrend Glucose Analyzer (Roche Diagnostics Corp., Indianapolis, IN, USA). Serum triacylglycerides (TAG) and nonesterified fatty acid (NEFA) contents were quantified by specific enzymatic kits from Wako Chemical substances (Neuss, Germany). Total-cholesterol and cholesterol-HDL (high-density lipoprotein) levels had been measured, respectively, making use of an enzymatic kit from Stanbio Laboratory (Boerne, TX, USA). Insulin and leptin levels were assayed applying precise rat ELISA kits from Spi-Bio (Montigny le Bretonneaux, France) plus the levels of total ketone bodies and glucagon were 5-HT5 Receptor Agonist Species determined working with an Autokit Total Ketone Bodies and an ELISA glucagon kit, respectively, each from WAKO, Chemical Neus. Ghrelin (acetylated and unacetylated) levels have been assayed in plasma making use of precise rat ELISA kits from Spi-Bio (Montigny le Bretonneaux, France) in accordance with the manufacturer’s guidelines. Liver and visceral fat depots have been carefully dissected and weighed. Then, tissues have been flash frozen in liquid nitrogen and stored at -70 C until made use of. Frozen liver samples had been utilized for glycogen and TAG measurement. Neutral lipids were extracted in the liver as previously described [37] and also the hepatic TAG content was analyzed by the enzymatic kits from Stanbio Laboratory (Boerne, TX, USA). Glycogen levels have been assessed in the liver making use of a glycogen assay kit II (ab 169558, Abcam, Boerne, TX, USA) following the manufacturer’s instruction. Both TAG and glycogen were measured in PAR1 manufacturer triplicate and each contents had been expressed as mg/g wet tissue. 2.3. Total Extract from Liver and Immunoblot Evaluation A piece of fresh liver was thawed, reduce into modest pieces on ice, and suspended (four mL buffer/g tissue) in cold Krebs-Henseleit buffer pH 7.four (116 mM NaCl, four.7 mM KCl, 1.2 mM CaCl2 , 1.2 mM KH2 PO4 , 1.two mM MgSO4 .7H2 O, five.5 mM glucose, 25 mM NaHCO3 , 1 mM PMSF, 10 /mL leupeptin, 1 /mL pestatin, two mM NaF, 1 mM Na3 VO4 ) before homogeneization with ten passes of a loose-fitting B pestle inside a Dounce homogenizer. Then, theAntioxidants 2021, ten,five ofhomogenates have 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. Protein content material of your mitochondrial oxidative phosphorylation OXPHOS complicated was determined with Total OXPHOS rodent WB antibody cocktail (6 /mL, ab110413, Abcam, Cambridge, UK), which include 5 mouse monoclonal antibodies, 1 every single against CI subunit NDUFB8, CII-30kDa, CIII-Core protein, CIV subunit I, and CV alpha subunit of OXPHOS. The antibody cocktail was applied in accordance with the manufacturer’s instructions. In total, 20 of protein were separated under decreasing circumstances on 12.5 SDS-PAGE, transferred to nitrocellulos
