Quantification of Hepatic Triglycerides

Triglyceride in the liver was measured as previously described (Le Marchand et al., 1973 (link); Fujii et al., 2008 (link); Komazaki et al., 2017 (link); Sasaki et al., 2018 (link)). Briefly, the aliquots of liver lysates were added to a microcentrifuge tubes containing 37.5% KOH and heated at 70°C for 30 min. The tubes were placed in 55°C water bath overnight (n = 4). Subsequently, 50% ethanol was added, and the tubes were centrifuged. The supernatants were separated, treated with MgCl₂, left on ice for 10 min, and then centrifuged again. The supernatants and a triglyceride standard (Sigma Aldrich [St. Louis, MO, USA]) were placed in a 96 well black plate with a clear flat bottom, and triglyceride levels were measured using a commercially available kit (Triglyceride quantification kit, Sigma Aldrich [St. Louis, MO, USA]). Preparation was performed by following the manufacturer’s instruction. Fluorescence intensity (λex = 540 nm/λem = 590 nm) was measured with a plate reader (BMG Labtech FLUOstar OPTIMA-6).