Total cholesterol kit

An Analox GM9 Glucose Analyzer was used to measure glucose levels via the glucose oxidase method. Plasma insulin levels were determined using a Stellux Chemiluminescent Rodent Insulin ELISA (Alpco). Plasma and liver triglycerides were measured using Infinity Triglyceride Reagent (Thermo Fisher TR22421). Liver triglycerides were extracted using a methanol/chloroform-based method^{15 (link)} and the colorimetric Infinity Triglyceride Reagent. Plasma cholesterol levels were measured using Wako Diagnostics Total Cholesterol Kit (999-02601). Plasma fatty acids were measured using Wako Diagnostics NEFA-HR kit (999-34691, 995-34791, 991-34891, 993-35191). Plasma liver enzymes were assessed using Thermo Fisher Infinity alanine aminotransferase and aspartate aminotranferase activity kits (TR71121, TR70121).

Glucose tolerance tests (GTT) were performed by intraperitoneal administration of 2 g/kg body weight of glucose after a 16 h overnight fast. Blood glucose was monitored using Accu-Check blood glucose strips and glucometer (Roche, Penzberg, Germany). Insulin tolerance tests (ITT) were performed in the random-fed state at 11:00. Animals were injected with 1 U/kg body weight of human regular insulin (Humulin regular, Eli Lilly, Indianapolis, IN) and blood glucose levels were measured at indicated times. ITT data are presented as percentage of initial blood glucose concentration. Insulin ELISA (Millipore, Billerica, MA) was performed with plasma samples obtained from 16 h overnight fasted mice. Total cholesterol kit (Wako, Saitama, Japan) and total triglycerides kit (Thermo Fisher, Waltham, MA) were used with plasma and liver samples obtained from mice fasted for 24 h followed by 1 h of refeeding. Hepatic lipids were isolated as described (Folch et al., 1957 (link)).