To detect IRS pY and IRS/PI3K JNK inhibitor association, liver extracts were subjected to immunoprecipitation with IRS-1 or IRS-2 antibody prior to immunoblotting. Serum hCRP was determined using an enzyme-linked immunosorbent assay (ELISA) kit (Helica, Fullerton, CA), with no crossreactivity with rat CRP. Blood glucose was determined with an Abbott FreeStyle glucometer. [3H]-glucose-specific activity was measured in the supernatants of Ba(OH)2 and Zn2SO4 precipitates of plasma samples

after they were evaporated to dryness to eliminate tritiated water. Plasma insulin was determined using a radioimmunoassay kit (Millipore, Bedford, MA), and FFA measured using an enzymatic assay kit (Wako, Osaka, Japan). Plasma TNF-α and adiponectin were quantified using rat ELISA kits from Invitrogen and Millipore, respectively. IL-6 and leptin were determined using the Luminex technique and a rat MAP multiplex kit (Millipore). Primary rat hepatocytes were isolated by liver perfusion as described,22 with some modifications. Briefly, under 4% isoflurane-induced general anesthesia, livers were isolated from the circulatory system;

the thoracic aorta, the caudal vena cava, the abdominal aorta, and the abdominal vena cava were tied off. The portal vein was severed, and livers were perfused by way of the inferior vena cava with perfusion medium followed by digest medium at 42°C. The liver was excised and minced in wash medium. Digested tissue was filtered through a cell strainer (100 μm), and hepatocytes Apoptosis Compound Library supplier were pelleted by centrifugation, washed, and resuspended in Williams E medium containing 5% fetal bovine serum (FBS), 0.0015 μg/mL insulin, and 0.1% penicillin-streptomycin. Cells were seeded on cell culture plates and incubated for 3 hours (37°C, 5% CO2). Following an overnight serum-free incubation, MycoClean Mycoplasma Removal Kit cells were incubated with U0126 (100 μmol/L) or SB203580 (50 μmol/L). Thirty minutes later, hCRP (30 mg/L) or vehicle was added for 150 minutes. The

hCRP concentration and incubation period were designed to match those in the in vivo study as described above. To determine the time- and concentration-dependency of the effect of hCRP on insulin signaling in vitro, we performed additional studies in which hCRP at 15 mg/L and 30 mg/L, respectively, was incubated with cells for 75 minutes and 150 minutes, respectively. For detection of insulin-stimulated IRS-1/PI3K association and pY, 100 nM human insulin or saline was added for 10 minutes. No insulin was added for measurements of MAPKs and IRS-1 serine phosphorylation. Cell lysates were prepared and subjected to immunoprecipitation and/or immunoblotting analyses. Data were presented as mean ± SE. Comparisons among groups were made using Student’s t tests or repeated measures analysis of variance (ANOVA) followed by pairwise post-hoc analysis.