All of the described experiments

were performed using male mice aged between 8 and 12 weeks. For quantitative real-time polymerase chain reaction (PCR) messenger RNA (mRNA) was isolated using the RNeasy Mini Kit (Qiagen, Valencia, CA) after complementary DNA synthesis expression was determined using the ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA) (see Supporting Information for details). selleck compound For immunohistochemical analysis, paraffin-embedded tissue slides were stained using a primary anti-Glut2 antibody (1:150) (Abcam, Cambridge, MA) and fluorescence or horseradish peroxidase (HRP)-labeled secondary antibodies (Vectorstain ABC-Kit, Vector Laboratories, Burlingame, CA). Staining was detected using a Nikon light, or fluorescence microscope, respectively (see Supporting Information for details). Proteins were separated by way of sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrotransfered onto nitrocellulose membranes (Invitrogen, Carlsbad, CA), and protein expression was determined using the indicated primary antibodies (Supporting Table 1). Binding of the antibody was detected using HRP-labeled secondary antibodies (BioRad, Hercules, CA) and the Amersham ECL Plus Western Blotting Detection Reagents Rapamycin chemical structure (GE Healthcare, Baie d’Urfe, Quebec, Canada). Chemiluminescence was determined using a KODAK ImageStation

4000MM (Mandel, Guelph, Ontario, Canada). Animals were fed ad libitum using a western diet (TestDiet, Richmond, IN) containing 16.8% protein, 6.5% fiber, 48% carbohydrates, and 20% fat. After 6 weeks of feeding, wild-type and Slco1b2−/− mice were sacrificed and blood samples were collected. The measurement of cholesterol and Akt inhibitor TSH was performed at Charles River Laboratories (Wilmington, MA). Total and free thyroxine (T4) and triiodothyronine (T3) in plasma were determined using enzyme-linked immunosorbent assay (ELISA) kits from Alpha-Diagnostics (San Antonio, TX). Insulin levels were determined using the UltraSensitive Mouse Insulin ELISA kit (Crystal

Chem Inc., Downers Grove, IL). Total bile acids or 7-α-hydroxy-4-cholesten-3-one were determined using a commercially available colorimetric assay (BioQuant, San Diego, CA) or mass spectrometry, respectively (see Supporting Information for details). Glucose tolerance testing and pyruvate challenge were performed using 2 g/kg glucose or pyruvate. Glucose levels were determined using a glucometer (OneTouch, LifeScan Inc., Milpitas, CA). For thyroid hormone (TH) extraction, tissue was homogenized in methanol. After addition of chloroform (2:1) and centrifugation (15 minutes, 1,900g, 4°C), pellets were re-extracted with a chloroform/methanol (2:1) mixture. Both supernatants were combined and further extracted with chloroform/methanol/water (8:4:3) and 0.05% CaCl2. The mixed solution was centrifuged (10 minutes, 800g, 4°C). Lower apolar phase was re-extracted with chloroform/methanol/water (3:49:48).