erse transcription reagent kit according to manufacturer instructions.Heart Plasma Membrane Preparation Heart plasma membrane fraction was performed as described previously. Ventricle tissue was homogenized in buffer A containing: 10 NaHCO3, 5 NaN3, and then centrifuged at 70006g for 20 min. The pellet was resuspended in buffer B, and centrifuged at 2006g for 20 min. The supernatant was gently layered on top of a 20% Percoll gradient in buffer C, 2 EDTA) and centrifuged at 55,0006g for 1 h. The band at density of 1.030 was aspirated and pelleted by centrifugation at 170,0006g for 1 h and resuspended in buffer C as PM solution. Protein concentration of PM solution was determined with BCA protein assay. Glucose transporter 4 content in PM was determined by Western blot. The table shows general characteristics of the experimental animals at baseline and following MI/R. STZ, streptozotocin; MI/R, 30 min of myocardial ischemia and 1 h of reperfusion. Values presented are means 6 SEM; n = 10/group. P,0.05, P,0.01 vs. Con-baseline. P,0.05, vs. STZ-baseline. doi:10.1371/journal.pone.0069910.t001 Preconditioning markedly reduced infarct size, plasma CK activity and myocardial apoptosis compared with the MI/R group in normal MedChemExpress TAK-438 (free base) Hearts whereas STZ treatment abolished all these 10646850 beneficial effects of IPC. Consistently, IPC significantly 26507655 improved myocardial functions during reperfusion as evidenced by increased LVDP,+LV dP/dtmax and -LV dP/dtmax in normal hearts although there were no significant differences in HR and MABP. However, its improvement of cardiac functions were significantly blunted in STZ-induced insulin-deficient rats. Western Blot Analysis The protein expression and phosphorylation were measured using Western blot as described previously. The immunoblots were probed with anti-GLUT4 , anti-phospho-Akt, anti-Akt, anti-glycogen synthase kinase3b, anti-pGSK3b, anti-AMP-activated protein kinase, anti-pAMPK, anti-acetyl-CoA carboxylase and anti-pACC antibodies overnight at 4uC followed by incubation with the corresponding secondary antibodies at room temperature for 1 h. The blots were visualized with ECL-plus reagent. GAPDH or btubulin were used as the internal loading control. IPC Enhanced Glucose Uptake During Post-ischemic Reperfusion in I/R Hearts As shown in Fig. 3C, there were no significant differences in blood glucose concentrations among all groups after 1 h of reperfusion, indicating IPC has no effect on systemic glucose metabolism. In addition, it seems plasma insulin levels were slightly increased in IPC group, however, there was no significant difference compared with MI/R group. To examine whether IPC increases myocardial 
glucose uptake during reperfusion, we measured myocardial accumulation of the positronemitting glucose analogue FDG. PET imaging and gammacounter biodistribution studies were performed. Representative examples of PET images revealed that IPC plus MI/R group showed more obvious FDG uptake in the hearts compared with MI/R group. Consistently, the results of biodistribution studies also indicated IPC stimulated glucose uptake in I/R hearts. These results illustrated that IPC significantly increased myocardial glucose uptake following MI/R. Importantly, pretreatment of the specific phosphoinositide 3-kinase inhibitor wortmannin significantly blocked the metabolic effects of IPC, suggesting that PI3K plays a role in the metabolic modulation of IPC. Statistical Analysis All values are presented as means 6 SEM. Diffe