Pctp−/− and wildtype control mice seven generations backcrossed into FVB/NJ genetic background11 were housed in a standard 12-hour alternate light/dark cycle facility and fed a standard rodent diet 5001 (LabDiets, St. Louis, MO) with free access to drinking Dinaciclib in vitro water. Protocols for animal use and euthanasia were approved by the institutional committees of the Harvard Medical School and the Albert Einstein College of Medicine. Experiments were conducted using male mice. Starting at 4-5 weeks of age, mice were fed a high-fat diet (60% kcal; D12492; Research Diets, New Brunswick, NJ) for periods ranging from 8 to 18 weeks prior to commencing experiments. Mice were weighed

weekly and rates of food consumption were calculated per mouse from the weight of food withdrawn by all of the mice in the cage each week. Prior to selected experiments, mice were anesthetized by intraperitoneal (i.p.) injection of ketamine (87 mg/kg body weight [b.w.]) plus xylazine (13 mg/kg b.w.) (Webster Veterinary, Sterling, MA). In experiments click here designed to test the influence of PC-TP inhibition on glucose homeostasis, administration of compound A1 (see Supporting Information: Materials and Methods, for synthesis and assays of microsomal

stability and pharmacokinetics) or vehicle was initiated concurrently with the high-fat diet. Compound A1 was prepared to a final concentration of 0.6 mg/mL in 4% dimethyl sulfoxide (DMSO) and 96% of 6% hydroxypropyl-β-cyclodextrin (Sigma Aldrich, St. Louis, MO) solution in sterile water. Mice were injected i.p. 5 days per week with 3 mg/kg compound A1 or the equivalent volume of vehicle (5 μL/g). For all experiments, mice were sacrificed after an overnight fast. Plasma nonesterified fatty acid (NEFA), triglyceride, cholesterol, and phospholipid concentrations were determined using reagent kits from Wako (Richmond, VA), Sigma Aldrich, and Roche Diagnostics (Indianapolis, PD184352 (CI-1040) IN), respectively.

Blood glucose was determined using a OneTouch Ultra glucose monitor (LifeScan, Milpitas, CA). Hepatic concentrations of triglycerides and cholesterol were measured enzymatically following hepatic lipid extraction.12 Plasma insulin, leptin, and adiponectin were determined by enzyme-linked immunosorbent assay (ELISA) as a service of the Joslin Diabetes and Endocrinology Research Center Specialized Assay Core (NIH 5P30 DK36836, Joslin Diabetes Center, Boston, MA). Plasma activities of alanine aminotransferase (ALT) and concentrations of bilirubin were determined using standard assays by Charles River Research Animal Diagnostic Services (Wilmington, MA). To assess protein expression, liver tissue or cell lysates were homogenized in RIPA buffer supplemented with protease and phosphatase inhibitors (Roche Diagnostics). Lysates were rotated slowly at 4°C for 30 minutes and then centrifuged at 12,000g for 10 minutes to remove cellular debris.