Effect of metformin on hepatic mitochondrial biogenesis.

<p><b>A)</b> Mouse primary hepatocytes were cultured on permanox Lab-Tek 8-well chamber slides with 1 mM metformin for 48 hours. Cells were fixed with 4% PFA, immunostained as described and mounted with Fluoroshield with DAPI. MTCO1 stained green, cell nuclei blue. Representative images are shown. <b>B)</b> Western blot of whole cell lysates from mouse primary hepatocytes incubated with or without metformin for 48 hours. Upper panel: immunostaining with anti-MTCO1; lower panel: β-actin was detected as a loading control. <b>C)</b> HepG2 cells were cultured on permanox Lab-Tek 8-well chamber slides with 3 mM metformin for 48 hours, incubated with Mitotracker Green FM (100 nM) for 1 hour and mounted with Fluoroshield with DAPI. Mitochondria stained green, cell nuclei blue. Representative images are shown. <b>D)</b> Mitochondrial mass of HepG2 cells, treated with 3 mM metformin for 48 hours, was measured by FACS and compared with untreated cells by fluorescence levels upon staining with MitoTracker Green FM. A representative experiment is shown, which was repeated two additional times with similar results. MFI (Mean Fluorescence Intensity), ANOVA, Neuman–Keuls post-hoc test, *** P<0.001. Relative mitochondrial DNA level <b>E)</b> in mouse primary hepatocytes (n = 6) treated with metformin for 48 hours, <b>F)</b> in livers of mice treated with metformin for 7 days (300 mg/kg) (control n = 6, metformin n = 7), <b>G)</b> in HepG2 cells (n = 3) treated with 3 mM metformin for either 24 or 48 hours. In panels E-G, * P<0.05, ** P<0.01 (Student one tailed t-test).