FIG. 5.

p38 activation disrupts insulin-induced Akt and Foxo1 phosphorylation and insulin-regulated gene expression in cardiomyocytes. A: Overexpression of p38 blocked insulin-induced Akt and Foxo1 phosphorylation in NRVMs. Cells were transfected with adenovirus (Ad) expressing GFP, p38-WT, or p38-DN for 8 h and cultured in serum-free medium for another 8 h. Cells were then treated with 100 nmol/L insulin for 0.5 h, and cellular protein lysates were prepared for immunoblotting. *P < 0.05 vs. Ad-GFP treatment. B: Overexpression of p38-WT, rather than p38-DN, blocked insulin-stimulated or insulin-suppressed gene expression, as determined by real-time PCR in NRVMs. Cells were transfected with adenovirus expressing GFP, p38-WT, or p38-DN, followed by another 8 h of serum starvation. Cells were then treated with 100 nmol/L insulin for 18 h, and cellular RNA was prepared for real-time PCR analysis. Graphs indicate relative expression of genes encoding α-MHC, Glut4, Fasn, and ACC1 for insulin stimulation, or β-MHC, BNP, IRS1, and IRS2 for insulin inhibition. Data are expressed as the mean ± SEM from at least three independent experiments. *P < 0.05 vs. Ad-GFP; ⁺P < 0.05 vs. Ad-p38-DN. C: Overexpression of IRS1 or IRS2 restored p38-WT–regulated transcriptional levels of heart failure and metabolic genes in cells. NRVMs were transfected with 50 MOI p38WT and 50 MOI adenovirus expressing GFP, IRS1, or IRS2 for 8 h, followed by another 8 h in serum-rich DMEM medium. The protein or RNA was prepared for Western blot or real-time PCR, respectively. Representative data from Western-blot and quantitative PCR are shown. *P < 0.05 vs. Ad-GFP; ⁺P < 0.05 vs. Ad-p38WT. N.S., no significant difference.