Infinity triglyceride liquid stable reagent

Triglyceride measurements were carried out as described (Tsedensodnom et al., 2013 (link)). Briefly, 20 livers were dissected from 5-dpf larvae and pooled in 0.5% Triton X-100 (VWR International, West Chester, PA, USA) and the Infinity™ Triglyceride Liquid Stable Reagent (Thermo Fisher Scientific, Waltham, MA, USA) was used following the manufacturer’s instructions. Triglyceride levels were normalized to the total protein concentration as determined by BCA Assay (Thermo Fisher Scientific, Waltham, MA).

In order to determine tissue triglyceride content, lipids were extracted from 50-200 mg of tissue by overnight incubation at 55°C in ethanolic KOH (66.6% ethanol with 33.3% 30% KOH). Samples were brought to a volume of 1200 μl with 50% ethanol then spun for 5 minutes at maximum speed in a microcentrifuge. After mixing 100 μl of supernatant with 100 μl of 0.5 M MgCl₂, samples were kept on ice for 10 minutes. They were then centrifuged for 5 minutes at maximum speed in a microcentrifuge and supernatant was moved to a new tube. Glycerol standards were prepared ranging from 1,000 mg/dl down to 1 mg/dl. In a standard 96-well plate, 3 μl of sample or standard was mixed with 300 μl of Infinity Triglyceride Liquid Stable Reagent (#TR22421, Thermo Fisher Scientific) and incubated at 37°C for 10 minutes before reading. The unknown triolein equivalents (TE) were interpolated from the standard curve using a least squares (ordinary) fit line calculated with Prism Graphpad software. The total liver triglycerides per sample was calculated in mg/g tissue by TE^∗2^∗0.012/(tissue weight in grams).

Mouse plasma samples were analyzed using ELISA assays to measure insulin (Ultra-Sensitive Mouse Insulin ELISA Kit, #90080, Crystal Chem, USA), leptin (Mouse/Rat Leptin Immunoassay, #MOB00B, R&D Systems, USA), total ghrelin (Rat/Mouse Total Ghrelin, #EZRGRT-91K, EMD Millipore, USA), GDF15 (Rat/mouse GDF15 Quantikine ELISA kit, #MGD150, R&D Systems, USA) and FGF21 (Fibroblast Growth Factor 21 Mouse/Rat ELISA, #RD291108200R, BioVendor R&D, Czech Republic) using manufacturer instructions. Plasma was diluted 1:5 for total ghrelin measurement, 1:20 (1:60 for ExpOF d14 samples) for leptin measurements, 1:10 for FGF21 (1:3 for controls) and 1:5 for GDF15 measurements. Blood glucose was measured from tail blood in awake mice using a glucometer. Plasma total cholesterol (Infinity Cholesterol Liquid Stable Reagent, #TR13421, Thermo Fisher Scientific, USA), total glycerol (triglycerides) (Infinity Triglyceride Liquid Stable Reagent, #TR22421, Thermo Fisher Scientific, USA), and free fatty acids (HR Series NEFA-HR(2), #434-91795, #436-91995, #270-77000, Fujifilm Wako Chemicals Europe) concentrations were measured by using commercially available kits.