till For Western blot analysis, 40 ug of protein was denatured by heating 100 C for 10 min in SDS sample buffer, loaded onto and separated by 10% or 12% SDS polyacrylamide gels, and then transferred electrically to a polyvinylidene fluoride membrane. The mem brane was blocked in 5% nonfat milk with 0. 05% Tween 20 TBS buffer for 1 h and then was incubated overnight with the following different primary antibodies monoclonal anti Akt and anti p Akt, monoclonal anti cleave caspase 3, mono clonal anti PARP, monoclonal anti p ERK1/2, monoclonal anti ERK1/2, and anti B actin antibody was used to show equal loading of the protein in the western blotting and quantita tive analysis. The membranes were incubated with horseradish peroxidase linked anti mouse or anti rabbit secondary antibody at 1 3000 dilutions for 1 h at 37 C and, after washes, visualized for immunoreactivity using an Enhanced Chemiluminescence System.

Statistical analysis Quantitative data are presented as the means SE deter mined from at least three independent of experiments. Statistical analysis was based on Students t test for com parison of two groups or one way ANOVA for multiple comparisons. P value 0. 05 was considered significant. Results Palmitate induced H9c2 cells apoptosis through activation of caspase 3 and PARP In order to determine the toxic effects of palmitate on H9c2 cells, cells were treated with increasing palmitate from 0 to 250 uM for 12 h. An increase in the number of apoptotic cells was observed in H9c2 cells by Hoechst 33342 staining, and decreased cell viability was measured by a MTT assay.

Next, we choose the 150 uM palmitate in subsequent experiments to analyze cleaved caspase 3 and the cleav age of poly polymerase, two well established hallmarks of apoptosis. Immunoblot and quantitative analysis results showed that expression of cleaved caspase 3 was detected at 2 h after treatment with 150 uM palmitate, and increased gradually at 6 h, 12 h and 24 h. The analysis result of PARP cleavage was similar to that of the cleaved caspase 3. These results suggested that caspase 3 and PARP activation were involved in the apoptotic pathway induced by palmitate in H9c2 cells. Adiponectin attenuated palmitate induced H9c2 cells apoptosis through reduced the activation of caspase 3 and PARP Adiponectin exists in the circulation as a full length pro tein and cleaved globular C terminal domain, both of which are pharmacologically active.

In this study, there were three groups, 1% BSA control, palmitate treated group as well as globular adiponectin and palmitate treated group, and the concentra tion of 2. 5 ug/mL globular adiponectin was chosen refer ence from. Cells were treated with 150 uM palmitate for 12 h or pretreated with 2. 5 ug/mL globular adiponectin for 1 h and then treated with 150 uM palmitate for 12 GSK-3 h. BSA treated cells were used as the control.