Cholesterol e test

The liver glycogen content was measured as previously reported [12 (link),19 (link)]. Liver lipids were extracted using the Bligh and Dyer method [21 (link)], and measured using triglyceride (Wako Pure Chemical Industries) and cholesterol E-tests (Wako Pure Chemical Industries). Liver fructose contents were measured by enzymatic methods [22 ]. Briefly, freeze-clamped tissues (100 mg) were homogenized in 2 mL of cold 6% perchloric acid, neutralized, and centrifuged. The assay is based on the oxidation of glucose as glucose-6-phosphate (G6P) using G6PDH. Fructose-6-phosphate is converted to G6P by the phosphoglucose isomerase enzyme, and subsequently oxidized by the G6PDH in the assay mixture. The fructose concentration is determined as the difference in G6P concentration before and after phosphoglucose isomerase treatment. All enzymes were purchased from Roche Custom Biotech Inc. Blood plasma was collected from the retro-orbital venous plexus following ad libitum feeding or after a 6-h fast. Blood glucose levels were measured using a FreeStyle Freedom monitoring system (Nipro, Osaka, Japan). Plasma triglycerides and total cholesterol levels were determined using the commercial kits, triglyceride E-test (Wako Pure Chemical Industries), and cholesterol E-test (Wako Pure Chemical Industries), respectively.

Liver lipids were extracted using the Bligh and Dyer method [31 (link)]; they were measured using triglyceride (Wako Pure Chemicals, Osaka, Japan) and cholesterol E-tests (Wako). Blood plasma was collected from the retro–orbital venous plexus, following ad libitum feeding or after a 6-h fast. Blood glucose and beta-hydroxybutyrate (β-OHB) levels were measured using a FreeStyle Freedom monitoring system (Nipro, Osaka, Japan). Plasma insulin, FFA, fibroblast growth factor 21 (FGF21), triglyceride, and total cholesterol levels were determined using commercial assay kits, as follows: mouse insulin enzyme-linked immunosorbent assay (ELISA) (H type) (Shibayagi, Gunma, Japan), NEFA C-test (Wako Pure Chemicals, Tokyo, Japan), mouse/rat Fgf21 ELISA (R&D Systems, Minneapolis, MN, USA), triglyceride E-test (Wako), and the cholesterol E-test (Wako), respectively.

Plasma TC, HDL-C and TG were enzymatically measured by using commercial kits (Cholesterol E-test, HDL-Cholesterol E-test and Triglyceride E-test, Wako Pure Chemical Industries, Osaka, Japan). The non-HDL-C concentration was calculated as (non-HDL-C) = (TC) − (HDL-C). The hepatic and fecal lipids were extracted by the ordinary method of Folch et al. [36 (link)]. The hepatic cholesterol, TG, and fecal cholesterol concentrations were enzymatically determined by using commercial kits (Cholesterol E-test and Triglyceride E-test, Wako Pure Chemical Industries, Osaka, Japan). Fecal bile acids were extracted with hot ethanol (70 °C, 60 min) from freeze-dried feces. The extracts were enzymatically determined by using commercial kits (Total bile acid test, Wako Pure Chemical Industries, Osaka, Japan).