Increased levels of ROS, increased apoptosis, and decreased proliferation precede reduction of β cell mass. (a) Readouts from ESR-based detection of superoxide in control islets (upper panel) and knockout islets (lower panel) from 6-week-old animals. Gray lines depict the raw signal with magnetic fields, and black lines depict the filtered signal. Circles indicate peaks and troughs used to determine the type of ROS, in this case specifically indicating the presence of superoxide. a.u., arbitrary units. (b) Fluorescence at 488 nm after loading of islets with H₂DCF, showing higher levels of DCF, an oxidation product of H₂DCF, in knockout islets in the presence of 11.1 mM glucose. Insets depict native islets. (c) Quantification of islet cells immunohistochemically positive for activated caspase-3, a marker of apoptosis, as well as Ki67 and BrdU, both markers for proliferation. Black bars, knockout animals; gray bars, control animals; error bars, SEM. **P < 0.005.