Fig. 4

The role of SIRT1 inhibition and palmitic acid (PA)-induced insulin resistance on the levels of mitochondrial membrane potential (MMP) and ATP production. MMP and ATP level are measured in control (C) cells incubated with and without SIRT1 inhibitor (EX527;10µM), PA-treated insulin-resistant cells incubated with and without SIRT1 activator (SRT1720;2µM) for 24 h. The representative ratio-metric original confocal traces (A) to determine the level of the mitochondrial membrane potential (MMP) in cells loaded with cell permeable JC-1 dye (5µM, 30 min) and (B) their mean values as bar graph. The dye was excited at 488 nm, and the emission was collected at both 535 nm (green signal; cytosolic accumulation of the dye) and 585 nm (red signal; mitochondrial accumulation of the dye). The mitochondria were depolarized, to calibrate the changes in MMP, with Carbonyl cyanide 4-trifluoromethoxy phenylhydrazone (FCCP; 5 µM). (C) Total ATP production was measured using a Luminescent based cell viability assay kit (Promega, G9241). EX527 was used as an inhibitor, and SRT1720 as an activator of SIRT1. The mean values represented as bar graph. Bars represent as mean (± SEM). The total number of cells used per group; n_cell=90–70. Significance level accepted at *p < 0.05 vs. C, and ^†p < 0.05 vs. PA