Figure 4.

Exendin-4 exhibits protective effects through PI3K-Akt axis in H9c2 cells. (A–G) H9c2 cells were pretreated without or with either 10 μM PKI (PKA inhibitor), 10 μM ESI-09 (Epac inhibitor), 10 μM PI3K inhibitor (LY294002) or 1 μM Akti-1/2 (Akt inhibitor; Akt inh) for 1 h. After 1 h, cells were treated with either vehicle (control), or 20 nM exendin-4 (Ex-4) for 3 h, and further induced with methylglyoxal (MG) for the indicated time. (A) The intracellular ROS production was quantified and expressed as the percentage relative to vehicle group (control). (B) The mitochondrial ROS level was detected by staining with MitoSOX (red), and DAPI (blue) to show nuclei. The fluorescence values were quantified using the corrected total cell fluorescence (CTCF) and expressed as the percentage of control. Scale bar, 10 μm. (C) The relative mitochondrial membrane potential (MMP) levels were evaluated and represented as the percentage of control. (D) Apoptotic cells were assayed by TUNEL staining (green) and counterstained with DAPI (blue) to show nuclei. The number of apoptotic cells was determined by the percentage of control, Scale bar, 10 μm. (E, F) The relative mRNA levels were quantified and expressed as fold increase over control. (G) Representative immunoblots are presented for protein expression of pro-apoptotic markers. The relative protein levels were quantified and expressed as fold increase over basal (vehicle) using GAPDH as a loading control. Data are presented as means ± SEM (N = 4). *P < 0.05 vs. vehicle; ^#P < 0.05 vs. MG; ^‡P < 0.05 vs. MG+Ex-4.