Fig. 1Metformin (Met) inhibits cell viability and induces apoptosis in H4IIE cells. (A) H4IIE cells were pre-incubated in serum-free Dulbecco's minimal essential medium (DMEM, 1 g/L glucose) for 24 hours and then pretreated with compound C (CC, 10 µM) in glucose-free DMEM (GFM) for 30 minutes. Cells were further treated with Met and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) for 4 hours (A) or 24 hours (B-E). The levels of phospho-AMP-activated protein kinase (p-AMPK) and actin were detected by Western blotting analyses, and the density of p-AMPK was normalized against actin. Each bar represents the average value of the duplicated experiments (n=2) (A). Cell viability was analyzed using the 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and each bar represents the mean±standard error (n=3) (B, C). The onset of apoptosis was detected using H33342 staining to observe nuclear chromatin condensation (D) and Western blotting analyses for poly ADP ribose polymerase (PARP) and cleaved caspase-3 (E). CTL, control. aP<0.001 vs. the non-treated control, bP<0.05 vs. cells treated with 1 mM AICAR, cP<0.001 vs. cells treated with 2 mM Met.
Fig. 2Inhibition of AMP-activated protein kinase stimulates the formation of acidic intracellular vesicles, a marker of the onset of autophagy, and protected metformin-induced apoptosis in glucose-free DMEM (GFM), but not in amino acid (AA)-free medium. H4IIE cells were pre-incubated in serum-free Dulbecco's minimal essential medium (DMEM, 1 g/L glucose) for 24 hours and then pretreated with compound C (CC, 10 µM) in fresh GFM (A, B), AA-free Hank's-balanced salt solution (1 g/L glucose) (C, D), or AA-containing DMEM (1 g/L glucose) (D) for 30 minutes. Cells were further treated with 2 mM metformin (Met) and 1 mM 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR, AIC) for 24 hours. After treatment, cells were stained with AO and H33342 (A-C) and subjected to the 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay (D). Arrows (B) indicates condensed chromatin (apoptotic bodies). Each bar represents the mean±standard error (n=3) (D). CTL, control. aP<0.05 vs. the non-treated control (dark bar), bP<0.05 vs. cells treated with 2 mM Met (dark bar), cP<0.01 vs. the non-treated control (white bar).
Fig. 3Metformin decreases the expression levels of autophagy-related proteins. H4IIE cells were pre-incubated in serum-free Dulbecco's minimal essential medium (DMEM, 1 g/L glucose) for 24 hours and then pretreated with compound C (CC, 10 µM) in glucose-free DMEM (GFM) for 30 minutes. Cells were further treated with metformin (Met, 0 to 2 mM) for 24 hours and subjected to Western blotting analyses. The density of light chain 3B (LC3B) or beclin-1 was normalized against actin. Each bar represents the average value of the duplicated experiments (n=2).
Fig. 4Inhibition of p38 mitogen-activated protein kinase (p38MAPK) as well as AMP-activated protein kinase protected cells from apoptosis induced by metformin (Met). H4IIE cells were pre-incubated in serum-free Dulbecco's minimal essential medium (DMEM, 1 g/L glucose) for 24 hours and then pretreated with various inhibitors against signaling proteins (20 µM compound C [CC], 50 µM SB202190, 50 µM SP600125, 10 µM U0126, 100 nM rapamycin, and 100 nM wortmannin) for 30 minutes. Cells were further treated 1 mM Met for 24 hours. After the treatments, the protein levels of cleaved caspase-3, beclin-1, light chain 3B (LC3B), and actin were detected using Western blotting analyses (A, C, D), and cell viability was analyzed using the 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay (B, E). Each bar represents the mean±standard error (n=3) (B, E). Rapa, rapamycin; SB, SB202190; SP, SP600125; U, U0126; Wt, wortmannin. aP<0.01 vs. cells treated with 2 mM Met (n=3) (B).