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Insulin levels were measured by a radioimmunoassay kit obtained from the Northern Bioengineering Institute (Beijing, China). Insulin sensitivity was evaluated by the oral glucose tolerance test (OGTT) and the homeostasis model assessment of insulin resistance (HOMA-IR) index. Mice were fasted for 4?h before OGTT. Glucose (2?g/kg body weight) was given by oral gavage, and blood droplets were collected from the tail at 0, 30, 60, and 120?min after glucose loading. Blood glucose concentration was immediately determined, with the use of a glucose analyzer (EKF Diagnostic Company, Germany). The areas under the glucose concentration�Ctime curve (AUC) were calculated. The HOMA-IR indices were calculated according to the Oxacillin following formula, as reported previously11: HOMA�\IR=fastinginsulin��fastingbloodglucose/22.5. The expression of insulin signaling proteins was evaluated using standard Western blot procedures, in total tissue homogenate of the liver. Tissues were homogenized in 100?mmol/L HEPES buffer, pH 7.9, containing 100?mmol/L KCl, 1?mmol/L DTT, 5% (v/v) glycerol, 5?mmol/L EDTA, 5?mmol/L EGTA, 50?��mol/L phenylmethylsulfonylfluoride (PMSF) and 25?ng/mL leupeptin (Roche). Samples were centrifuged at 12,000��g, at 4?��C, for 60?min, to remove tissue debris, and the supernatant EPZ5676 cost fraction was kept as a tissue homogenate for subsequent analysis. Protein concentration was assayed by the Bradford method, using the Bio-Rad protein assay kit (USA). Aliquots of the tissue homogenate (containing 30?��g protein) were subjected to electrophoresis on 6% or 10% SDS-polyacrylamide gels. Immunoblot analysis was performed using an enhanced chemiluminescence (ECL) detection system, and images were acquired using the Fluorchem model-5500 gel analysis system (Alpha Innotech, USA). The antibodies for AMP-activated protein kinase (AMPK) (#2532), phosphorylated AMP-activated protein kinase (p-AMPK) (#2531), and insulin receptor (��-subunit) (#3025) were purchased from Cell Signaling Technology Inc., USA. Antibodies to insulin receptor substrate 1 (IRS1) (sc-559) and ��-actin (sc-1616R) were LY2109761 molecular weight obtained from Santa Cruz Biotechnology Inc., USA. All data are expressed as means��SE. Statistical significance of differences among various groups was analyzed using one-way ANOVA (SPSS version 10.0, USA). P