Mitochrondrial fragmentation is necessary for HG-induced ROS overproduction and respiration increase. (A and B) ROS increase is not required for mitochondrial fragmentation in HG incubation. Cells pretreated with 100 nM carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) blocked ROS increase (A) but mitochondria were still fragmented upon HG exposure (B). (C and D) Inhibition of mitochondrial fission prevented HG-induced ROS production. (C) Cells transfected with GFP-DLP1-K38A showed bright aggregates of GFP signals (Left, arrows and arrowheads). Under normal glucose conditions, ethidium fluorescence of transfected cells (Upper Right, arrows) was similar to that in untransfected cells. At 30-min exposure to 25 mM glucose, ethidium fluorescence remained low in transfected cells (Lower Right, arrowheads) whereas more intense ethidium fluorescence was observed in untransfected cells. (D) Quantification of fluorescence intensity is shown. (E and F) Inhibition of mitochondrial fission prevents respiration increase in HG conditions. (E) Oxygen consumption profiles of cells in normal and HG conditions. DLP1-K38A overexpression was induced by tetracycline (Tet-induced). (F) Respiration rates show that DLP1-K38A overexpression (Tet-induced) blocked HG-induced respiration increase (arrowheads).