Triglycerides fs

At PND60, blood samples (300 µL) were collected from 6 h fasted offspring. An oral glucose tolerance test (OGTT) was performed in offspring at PND61 and PND62. Following 6 hours of fasting, all rats received a 2 g/kg BW dose of glucose by gavage. Blood samples (100 µL) were collected to determine plasma insulin concentration before (T0) and after 15 (T15) and 30 min (T30) gavage with glucose. Blood glucose was measured at T0, T15, T30, T45, T60, T90, and T120 after glucose intake, using a Performa Accu-Chek® glucometer (Roche Diabetes Care France, Meylan, France).
Insulin (Rat Insulin ELISA kit®, ALPCO, Salem, USA), glucose (Glucose GOD FS®, DiaSys, Holzheim, Germany), triglycerides (Triglycerides FS®, DiaSys, Holzheim, Germany), and cholesterol (Cholesterol FS®, DiaSys, Holzheim, Germany) were measured in plasma, following manufacturers’ instructions. Optical density was read with a microplate reader Varioskan Lux® (ThermoFisher Scientific, Waltham, USA).

Frozen small pieces of liver were placed in 2 ml tubes with Ceramic Beads (for Precellys homogenizer) and were homogenized in sodium acetate (0.2 M, pH 4.5) using the Precellys homogenizer. After centrifugation, the supernatant was stored at −80°C. The TGs in homogenates were measured according to the reagent kit instruction (Triglycerides FS, DiaSys Diagnostic Systems GmbH, Holzheim, Germany).

Portions of frozen liver from mice were homogenized in chloroform-methanol (2:1) in order to extract total lipids and TGs were separated by thin layer chromatography. TGs were extracted from the silica plate with acetone, measured with a colorimetric diagnostic kit (Triglycerides FS; Diasys, Holzheim, Germany) and expressed in mg of TGs per mg of liver. To quantify liver glycogen, we used a glycogen assay kit (MAK016, Sigma-Aldrich, Missouri, USA). Briefly, 10 mg of liver is homogenized in 100 μL of water at 4°C and boiled for 5 minutes to inactivate enzymes. Samples were centrifugated at 13,000g for 5 min. 50 μL of samples were incubated in the Master mix as described by the purchaser. The absorbance was measured at 570 nm. Glycogen measurement was performed on mice after 12 weeks of diet, since livers of mice after 16 weeks of dier were used for other experiments.

Circulating levels of insulin, triglycerides and total cholesterol were evaluated by using specific ELISA or colorimetric kits (Mouse Insulin ELISA (10-1247-01), Mercodia, Paris, France; Triglycerides FS (1 5760 99 10 021) and Cholesterol FS (1 1350 99 10 021), DiaSys, Nivelles, Belgium). Adiponectin and leptin concentrations were evaluated in both plasma and visceral adipose tissue by using Mouse Adiponectin (ELM-Adiponectin-1) and Mouse Leptin (ELM-Leptin-1) ELISA kits, respectively (RayBiotech^®, Peachtree Corners, GA, USA). The absorbance was determined at 450 nm (FLUOstar Optima, Bmg Labtech, Ortenberg, Germany), except for triglycerides and cholesterol assays at 500 nm. For the analysis of markers from the visceral adipose tissue, absolute absorbance values were normalized to the total protein contents evaluated by BCA assay.

Body composition was analyzed by NMR (Minispec LF50; Bruker). Glucose tolerance was determined after 16 h of fasting and the intraperitoneal injection of glucose (2 g/kg) and repeated blood glucose measurements from the tail vein using glucose test strips (Contour Next; Bayer). Serum insulin was measured by ELISA (Rat Insulin ELISA; Crystal Chem). Serum NEFA and triglycerides were analyzed biochemically using commercial NEFA-HR (2 (link)) (FUJIFILM Wako Chemicals Europe) and triglycerides FS (DiaSys Diagnostic Systems) kits, respectively. Serum β-hydroxybutyrate was analyzed by test strips (FreeStyle precision pro; Abbott). Serum FGF21 was determined by ELISA (Quantikine ELISA mouse/rat FGF-21; R&D Systems). Food intake was determined in single-housed mice for 24 h.

Mice were fasted for 4 h and ip injected with 400 μl of 7.5% tyloxapol solution (Sigma) in PBS. Blood was collected over a time course and plasma triglyceride content was quantified using a triglyceride assay kit (Triglycerides FS; Diasys).