Fig. 7.

ADMA suppressed the expression and transcription of PGC-1α by inhibiting its promoter activity but boosting its protein acetylation and phosphorylation. Western blotting was applied to measure expressions of peroxisome proliferator activated receptor γ coactivator-1α (PGC-1α), Sirt1, total and phosphorylated Akt and AMPK proteins. Reversed transcription PCR was used to detect the transcription of PGC-1α, luciferase assay was performed to analyze the promoter activity of PGC-1α, and immunoprecipitation was employed to determine the post-translational modification including acetylation and phosphorylation of PGC-1α in cardiomyocytes incubated with ADMA (30 μM) and glucose (30 mM), or oleic acid (OA, 100 μM) for 48 h. Panel a & b demonstrate the dose-response (10 ~ 100 μM) and time course (24 ~ 72 h) of asymmetrical dimethylarginine (ADMA) on PGC-1α expression, respectively; Panel c shows the PGC-1α transcription, and panel d displays the promoter activity of PGC-1α. Panel e & f diagram the acetylation (resveratrol as the negative control) and phosphorylation (AICAR as the positive control) of PGC-1α protein, respectively. Panel g & f are the dose-response and time course of ADMA on expressions of sirt1, phosphorylated Akt and AMPK proteins. Graphs in panel a ~ b, e ~ f and g ~ h represent the quantification of PGC-1α vs GAPDH, acetylated and phosphorylated PGC-1α vs its total protein, phosphorylated Akt and AMPK vs their total proteins as well as sirt1 vs β-actin from 3 independent experiments, respectively. Date are expressed as mean ± S.E.M., n = 3, ^*P < 0.05, ^**P < 0.01 vs control