Figure 4. Metformin treatment impairs mitochondrial function inducing cell death.

(A) Calu-1 cells were treated for 1 hr with different doses of metformin; ATP and AMP concentrations (μMol/mg of proteins) were determined by enzymatic assay. Measured AMP/ATP ratio increased as a function of metformin concentration. * = p < 0.05, ** = p < 0.01. Error bars indicate standard error. (B) 10 mM metformin exposure of Calu-1 cells for 24 hr affect mitochondrial potential (ΔΨm). Upper pannels: Bivariate JC-1 analysis of mitochondrial membrane potential by flow cytometry. Botton pannels: Immunofluorescence analysis of Calu-1 cells treated with JC-1.Metformin treatment induced an increase of depolarized regions as indicated by the disappearance of red and increase of green fluorescence. (C) Apoptosis was evaluated after treating Calu-1 cells with metformin and staining with Annexin-V and propidium iodide (PI) at 24 hr. The values indicate the percentage of death (Annexin-V−, PI+), early apoptotic (Annexin-V+, PI−) and necrotic/advanced apoptotic stage (Annexin-V+, PI+) cells.