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. 2014 Feb 10;289(13):9449–9459. doi: 10.1074/jbc.M113.543447

FIGURE 4.

FIGURE 4.

AMPK α2 deficiency exacerbated hepatocyte death and elevated ROS. A, fresh liver cryosections were prepared on 12 day after SL4 injection and incubated with 2 μmol/liter DHE for 30 min at 37 °C. Positive staining of the oxidized dye was identified by fluorescence microscopy, indicating the level of ROS in the tumor-bearing liver. Dashed lines show the tumor region. Scale bar, 100 μm. B, the primary hepatocytes were cultured in Williams's medium E with 5% FBS for 24 h and then transferred to 5% FBS DMEM medium with or without glucose and incubated for 36 h. Cells were trypsinized and oxidized to the FITC-CM H2DCFDA for 20 min. The oxidized dye was identified by fluorescence microscopy, indicating the ROS level in cultured hepatocytes after glucose starvation. DCF indicates FITC-CM H2DCFDA; DIC, differential interference contrast. Scale bar, 50 μm. C, the primary hepatocytes were treated as B and then analyzed using a FACSCalibur flow cytometer. AMPK α2 deficiency aggravated the production of ROS after glucose starvation. D, the WT or AMPK α2−/− primary hepatocytes were infected with Ad-GFP or Ad-AMPK-CA overnight. The cells were then glucose-starved for an additional 36 h and then analyzed by flow cytometer. E, the primary hepatocytes were double stained by PI and Hoechst 33342 after glucose starvation for 36 h, indicating hepatocyte death after glucose starvation. Dead cells included ghost hepatocytes and PI-positive cells. The blue arrow indicates a viable cell, the yellow arrow indicates a PI-positive dead cell, and a white arrow indicates the ghost cell. Scale bar, 50 μm. F, the primary hepatocytes were harvested for analysis by electron microscopy after glucose starvation for 36 h with or without the apoptosis inhibitor, z-VAD. Necrotic death of the primary hepatocytes can be observed. G, the primary hepatocytes were trypsinized, washed with PBS, incubated with PI and Hoechst 33342 for 20 min, and analyzed on MoFlo XDP flow cytometer. AMPK α2 deficiency aggravated hepatocyte death after glucose starvation and N-acetyl-l-cysteine (NAC) partially rescued the cells.