Stg 1 nc

Hepatic cholesterol and fatty acid composition was analyzed using gas chromatography on a Shimadzu GC-17A gas chromatograph with a flame ionization detector using a SAC-5 capillary column (30m × 0.25mm × 0.25mm, Supelco, Bellefonte, CA) according to our previously published procedures (Rideout et al., 2010 (link), Carrier et al., 2014 (link)). Relative hepatic FA content was calculated by using individual FA peak area relative to the total area and expressed as the percentage of total FA. Frozen pulverized liver tissue (100 mg) was homogenized in 1 mL of aqueous 5% NP-40 solution, heated at 90°C for 10 minutes, and spun at top speed in a microcentrifuge for 2 minutes to obtain hepatic extracts for TG analyses (Zenbio, STG-1-NC) (Green, 1974 ).

Hepatic cholesterol was extracted and analyzed according to our previously published procedures [41 (link), 42 (link)]. Approximately 0.5 mL of serum or 0.5 g of pulverized liver was spiked with α-cholestane as internal standard and saponified in freshly prepared KOH–methanol at 100°C for 1 h. The non-saponifiable sterol fraction was extracted with petroleum diethyl ether and dried under N₂ gas. Sterol fractions were analyzed on a Shimadzu GC-17A gas chromatograph fitted with a flame ionization detector using a SAC-5 capillary column (30m × 0·25mm × 0·25 mm, Supelco, Bellefonte, CA). This methodology was recently validated through an international ‘Survey for Sterols and Oxysterols ST1/14’ which included total cholesterol and phytosterol external reference materials from Referenzinstitut für Bioanalytik (Bonn, Germany). For hepatic triglyceride (TG) analysis, liver tissue (100 mg) was homogenized in 1 mL of an aqueous 5% NP-40 solution, heated at 90°C for 10 minutes, and spun at top speed in a microcentrifuge for 2 minutes. TG concentration in extracts were measured with a commercial kit (Zenbio, STG-1-NC) according to manufacturer’s instructions.

Serum TC, high-density lipoprotein cholesterol (HDL-C), and LDL/VLDL cholesterol were determined with enzymatic analysis (BioAssay, EHDL-100). Serum LDL-C was measured by direct automated enzymatic assay (Liposcience). Non-HDL-C was calculated by subtracting HDL-C from TC. Serum TG were determined by automated enzymatic analysis (Zenbio, STG-1-NC). Serum insulin was analysed by ELISA (ThermoScientific, EMINS) and glucose was measured by colorimetric analysis (Invitrogen, EIAGLUC). Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using an Element DC Chemistry Analyzer (Heska) in a pooled subset of animals.