Figure 3

Metformin ameliorates the SFA-induced impairment of autophagic flux and reduces the accumulation of lipid droplets. (A) Human endothelial cells were serum starved for 2 h, and then pretreated with metformin (Met, 100 μM) for 30 min followed by treatment with PA (200 μM, 4 h). The cells were treated without or with NH₄Cl (20 mM)/Leu (200 μM) 1 h prior to cell harvest. The cell lysates were subjected to a western blot with the indicated antibodies. Pre-treatment with metformin enhanced autophagic flux. (B) Human endothelial cells were transfected with siRNA for scrambled (sc) or AMPKα for 48 h. The cells were serum starved for 2 h and then pretreated with metformin (100 μM) for 30 min followed by treatment with palmitate (200 μM, 4 h). The cells were treated without or with NH₄Cl (20 mM)/Leu (200 μM) 1 h prior to cell harvest, and then cell lysates were harvested. The cell lysates were subjected to a western blot with the indicated antibodies. (C) Autophagic flux was calculated by setting the BSA-treated cells as 100%. (D) Endothelial cells were pre-treated with etomoxir (100 μM) 30 min prior to the treatment with PA (200 μM, 4 h) and metformin (100 μM). (E) Inhibition of β-oxidation by etomoxir blunted the effects of metformin. LD numbers were obtained by using Image J as described in “Materials and methods”. The quantification of three independent experiments is shown in bar graphs (mean ± SEM). ***p < 0.001, **p < 0.01, and *p < 0.05 indicate that the samples are statistically different between the indicated samples. The original images are presented in Supplemental Figure S3A,B. The dotted lines in the Supplemental figures indicate the cropped area.