Tr0100

TG and cholesterol levels were measured using kits (TR0100 and MAK043, respectively, Sigma) and total BA levels in the liver, intestine, plasma, and gallbladder were measured with a kit (MAK309, Sigma). Plasma metabolites were identified by gas chromatography-mass spectrometry (GC-MS) in the Metabolomics Center at UIUC.

To measure total intracellular triglyceride (TG) content, lipids were extracted from the cultured cells and total TG content was determined by enzymatic assays (TR0100, Sigma-Aldrich) and normalized to protein concentration. The protein concentration was measured using the Bradford assay (Bio-Rad, Munich, Germany). HepG2 cells were seeded in 6-well plates and stained using the Oil red O method. Briefly, after treatment, cells were washed three times with phosphate-buffered saline (PBS) and fixed for 30 minutes with 10% formalin. After fixation, cells were washed again with PBS, stained with Oil-Red O (Sigma-Aldrich) for 1 hour, and then rinsed with distilled water. The cells were observed under a light microscope (Olympus, Tokyo, Japan) with a 400× magnification and images were photographed. For quantitative analysis of cellular lipid, 1 mL isopropanol (Sigma-Aldrich) was added to the stained culture plate and absorbance of the extracted dye was recorded at 540 nm.

Plasma glucose, [³H]glucose, glucagon, canine and human insulin, and nonesterified fatty acid (NEFA) levels and blood lactate and glycerol concentrations were measured using standard methods as previously described (19 (link),21 (link)). Plasma triglycerides were assayed spectrophotometrically (TR0100; Sigma-Aldrich, St. Louis, MO).
Serum concentrations of LY were assayed using a dual-antibody ELISA specific for LY (Charles River Laboratories, Senneville, QC, Canada). The quantitation range was 20–500 pmol/L, with the standard curve range 15–1,000 pmol/L. Samples with concentrations higher than 500 pmol/L were diluted so that the resulting concentration was within the quantitation range. Standard curve and quality control samples were analyzed with each set of study samples and passed acceptance criteria (within ±20% except at lower limit of quantitation: within ±25%).
Western blotting was carried out on hepatic and hypothalamic tissue from control and LY_0.5 dogs; all analytic procedures have previously been described (22 (link)). Antibodies against total and phosphorylated Akt (Ser⁴⁷³), signal transducer and activator of transcription 3 (STAT3) (Tyr⁷⁰⁵), and glycogen synthase kinase-3β (GSK3β) (Ser⁹) were purchased from Cell Signaling (Danvers, MA). Protein bands were quantified using ImageJ software (http://rsb.info.nih.gov/ij/).