Cholescolor liquidfc

Mice were anesthetized with isoflurane, and whole blood was collected directly from the heart following 16 h of fasting. Serum insulin levels were measured using the Mouse Insulin ELISA kit (Mercodia AB, Uppsala, Sweden), adiponectin with the Mouse Adiponectin ELISA kit (CEMA2500-1; Assaypro LL, St. Charles, MO, USA), and TNF-α with the Quantikine ELISA Mouse TNF-α kit (R&D Systems, Minneapolis, MN, USA). Serum levels of triglyceride, NEFA (free fatty acid), and cholesterol were measured with enzyme assay kits (LabAssay Triglyceride, LabAssay NEFA, and LabAssay Cholesterol: Wako Pure Chemical Industries, Osaka, Japan). Following 16 h of fasting, blood was obtained from the tail vein, and glucose levels were measured using a portable blood glucose analyzer (Glutest Neo; Sanwa Kagaku Kenkyusho Co., LTD., Aichi, Japan).
The liver was rapidly removed, and lipids were extracted from these tissues according to the method of Folch et al.^{49 (link)}. Briefly, snap frozen livers were homogenized and extracted with a chloroform/methanol (2:1 v/v) solution. Total cholesterol, triglyceride, and free cholesterol were measured with enzyme assay kits (CholestestCHO, CholestestTG: Shimizu Medical, Tokyo, Japan; Cholescolor LiquidFC, Toyobo, Osaka, Japan).

Quantification of intrahepatic lipids and total bile acid (TBA) was conducted at Skylight Biotech, Inc. (Akita, Japan). Lipids and TBA were extracted by the Folch method and ethanol, respectively [29 (link)]. Triglyceride (TG), total cholesterol (T-CHO), free cholesterol (F-CHO), and TBA concentrations were quantified using the Cholestest^® TG, Cholestest^® CHO (Sekisui Medical, Tokyo, Japan), Cholescolor Liquid FC (Toyobo, Osaka, Japan), and Total Bile Acids Assay Kit (Diazyme Laboratories, Inc., Poway, CA, USA), respectively. The TG, T-CHO, F-CHO, and TBA levels are presented as mg/g, and were calculated by dividing the amount of detected analyte by the weight of liver tissue.