Glucose rtu

All parameters were quantified as previously reported, such as in [24 (link),25 (link),26 (link)]. Briefly, glucose concentrations in plasma and liver were evaluated using glucose RTU (bioMerieux, Craponne, France). Triglycerides (TG) were measured by colorimetric methods (RANDOX, Crumlin, Co., Antrim, UK).

Plasma glucose, triglycerides, cholesterol and free fatty acids (FFA) were analyzed with Glucose RTU (BioMerieux, Marcy-l’Étoile, France), PAP 150 (BioMerieux), FUJIFILM WAKO (Sobioda) and NEFA C (Wako Chemicals GmbH, Neuss, Germany) kits, respectively, according to the recommendations of each manufacturer.
Sex steroids were extracted from total plasma samples as described by McMaster et al. (1992) , using 100 μl of serum as starting volume for repeated diethyl ether extraction, and a final assay buffer reconstitution volume buffer of 500 μl. Isolated steroid fractions were used for quantification of 17β-estradiol in female, and testosterone in male serum samples using the Estradiol ELISA kit (Cayman Chemicals, Ann Arbor, MI, United States) and TEST96 ELISA kit (Teco Diagnostics, Anaheim, CA, United States) according to the manufacturers’ instructions. Samples were prediluted to fall into assay sensitivity range based on expected seasonal ranges of circulating sex steroids (Baynes and Scott, 1985 (link); Liley et al., 1986a (link),b (link); Sundling et al., 2014 (link)). Serum steroid concentrations were calculated based on a regression formula obtained from standard curve absorbance readings fitted by 4 parameters logistic regression with R² > 0.99, taking into account additional predilution of samples.

At weeks 20 and 24, chemical composition, including protein, fat and ash of the livers and muscles (five fish/replication, n = 30 per experimental group), was assessed according to Association of Official Analytical Chemists [AOAC] (1990) . The livers (100 mg) and muscles (200 mg) were analyzed for glycogen (two fish/replication, n = 12 per experimental group). The glycogen content was determined using a hydrolysis technique that was previously described by Good et al. (1933) . Each sample was ground in 1 M HCl (VWR, United States). An aliquot was saved at this stage and neutralized by 5 M KOH (VWR) to measure the free glucose content in the samples. After 10 min of centrifugation at 10,000 × g at 4°C, free glucose was measured using a plasma glucose kit (Glucose RTU; bioMérieux, Marcy-l’Étoile, France) according to the manufacturer’s instructions. The remaining ground tissue was boiled at 100°C for 2.5 h and then neutralized by 5 M KOH (VWR). After 10 min of centrifugation at 10,000 × g at 4°C, total glucose (free glucose + glucose obtained from glycogen hydrolysis) was measured using the same kit as before. The glycogen content was evaluated by subtracting free glucose levels.

Glucose tolerance was evaluated by measuring intraperitoneal glucose tolerance (IpGTT) of fasting rats. Capillary glycaemia at baseline and 15, 30, 60, and 120 min after an intraperitoneal (IP)-injection of 2 g/kg glucose (20 % solution) was measured with a glucometer (Accu-Chek Performa®, Roche Diagnostic, France). Blood samples were collected from the tail vein at 0 and 60 min after injection, in order to measure blood glucose (glucose RTU®, Biomérieux, France) and C-peptide levels (Elisa C-peptide kit, Mercodia, Uppsala, Sweden) to evaluate insulin sensitivity. Measuring C-peptide was preferred to measuring insulin for evaluating insulinemia, because it is more stable in blood and is not affected by haemolysis [31 (link)]. Results were expressed in g/L for plasma glucose and in pmol/L for plasma C-peptide. Fasting leptin was measured by ELISA (Elisa Leptin kit, Linco Research Inc., St Louis, MO, USA) as An index of fat mass [32 (link)]. Plasmatic cholesterol was quantified by a colorimetric method Cholesterol RTU™ (BioMérieux, Lyon, France) using a cholesterol calibrator. Insulin resistance was evaluated using the homeostasis model assessment (HOMA2). HOMA2-IR was calculated for fasting plasma glucose and fasting C-peptide using the HOMA2 model calculator (http://www.dtu.ox.ac.uk/homacalculator). All parameters were measured once a month.

Plasma Glycemia and Triglyceridemia were quantified by the colorimetric method using enzymatic kits (Glucose RTU, Biomérieux, France and Triglycérides LDB, Biodirect, France).

Liver and muscle glycogen levels were measured using the amyloglucosidase method [38] on tissues sampled 8 h after treatment (n = 6/line). Plasma glucose (Glucose RTU, bioMérieux, Marcy l’Etoile, France), triglycerides (PAP 150, bioMérieux) and free fatty acids (NEFA C kit, Wako Chemicals, Neuss, Germany) levels were determined in all samples using commercial kits adapted to a microplate format, according to the manufacturer’s recommendations.