FIGURE 2.

a and c, impact of diamide on the membrane potential of mitochondria isolated from skeletal muscle and BAT. Skeletal muscle and BAT mitochondria from WT and UCP3 null (KO) mice were treated acutely with different concentrations of diamide for 10 min and tested for mitochondrial inner membrane polarization using safranin O. Reactions were performed under oligomycin-induced state 4 conditions. *, p < 0.05; **, p < 0.02, n = 5–7, Student's t test. The single and double asterisks correspond to significance when compared with the WT control (0 μm diamide). Error bars in a are S.E. b and d, oligomycin-induced state 4 oxygen consumption in skeletal muscle mitochondria isolated from WT and UCP3 null (KO) mice. Mitochondria were treated with or without 2 μm diamide (skeletal muscle) and 100 μm diamide (BAT) with oligomycin and then tested for state 4 respiration. *, p < 0.05, n = 4–6, Student's t test. e, diamide-induced glutathionylation inhibits state 4 respiration in myotubes from WT but not UCP3 null (KO) mice. Shown is an in situ assessment of the impact of diamide on oligomycin-induced state 4 respiration using the Seahorse extracellular flux analyzer. Following treatment of WT and UCP3 null (KO) myotubes for 15 min with or without 100 μm diamide, the state 4 OCR (nmol/min) was tested. State 4 respiration was initiated following diamide treatment. p < 0.05, n = 4, Student's t test. The asterisk corresponds to significance when compared with no diamide treatment at each interval. f, In situ analysis of the diamide-mediated control of UCP3 carrying mutations at Cys²⁵, Cys²³⁰, and Cys²⁵⁹. Analyses were conducted using the Seahorse extracellular flux analyzer. Confluent HEK293 cells transiently transfected with WT or mutant (C25A, C230A, or C259A) UCP3 genes were acutely treated for 15 min with or without 100 μm diamide, and then oligomycin-induced state 4 OCR was assessed. Basal OCR was measured prior to diamide treatment, and data were expressed as a percentage of basal OCR. p < 0.05, n = 4, Student's t test. The asterisk corresponds to significance when compared with no diamide treatment. Error bars, S.D.