Uncoupling protein-3 lowers reactive oxygen species production in isolated mitochondria

Abstract

Mitochondria are the major cellular producers of reactive oxygen species (ROS), and mitochondrial ROS production increases steeply with increased proton-motive force. The uncoupling proteins (UCP1, UCP2, and UCP3) and adenine nucleotide translocase induce proton leak in response to exogenously added fatty acids, superoxide, or lipid peroxidation products. "Mild uncoupling" by these proteins may provide a negative feedback loop to decrease proton-motive force and attenuate ROS production. Using wild-type and Ucp3(-/-) mice, we found that native UCP3 actively lowers the rate of ROS production in isolated energized skeletal muscle mitochondria, in the absence of exogenous activators. The estimated specific activity of UCP3 in lowering ROS production was 90 to 500 times higher than that of the adenine nucleotide translocase. The mild uncoupling hypothesis was tested by measuring whether the effect of UCP3 on ROS production could be mimicked by chemical uncoupling. A chemical uncoupler mimicked the effect of UCP3 at early time points after mitochondrial energization, in support of the mild uncoupling hypothesis. However, at later time points the uncoupler did not mimic UCP3, suggesting that UCP3 can also affect ROS production through a membrane potential-independent mechanism.

Figure 1. Rate of ROS production in WT and Ucp3KO skeletal muscle mitochondria

The rate of H₂O₂ production was measured in WT and Ucp3KO skeletal muscle mitochondria as described in Materials and Methods. (A) The cumulative amount of ROS produced is shown over 10 min after energization with succinate. (B) The rate of ROS production is shown over 10 min after energization with succinate. 2.5 μM carboxyatractylate (CAT) was present as indicated. (C) The average rate of ROS production calculated over the first 2 min after energization with succinate, with or without 500 μM GDP. Values are means ± SEM of triplicate experiments performed on 9 separate preparations. One-way ANOVA was performed for (C), with Tukey's multiple comparison test used comparing the bars: *P<0.05, ***P<0.001

Figure 2. Effect of UCP3 and ANT in lowering ROS production

The effect of UCP3 on ROS production was calculated for each time point after energization with succinate by subtracting the rate of ROS production in Ucp3KO mitochondria from the rate in WT mitochondria. The effect of ANT was calculated in Ucp3KO mitochondria by subtracting the rate of ROS production in the presence of 2.5 μM CAT from the rate without CAT.

Figure 3. Effect of UCP3 on membrane potential 0–2 min after energization

The membrane potential was measured in WT and Ucp3KO skeletal muscle mitochondria over the first two min after energization, as described in Materials and Methods. Values are means ± SEM of triplicate experiments performed on 9 separate preparations.

Figure 4. FCCP can mimic the effect of UCP3 on ROS production and membrane potential 0–2 min after energization

Mitochondria from WT and Ucp3KO mice were independently energized with different amounts of FCCP from 0–60 nM. ROS production rates over the first 2 min are plotted against average membrane potential over the same period. Nonlinear regression analysis (Prism) using exponential and polynomial models found that a single curve best describes the data in all cases. Dashed lines highlight the ROS production rate and membrane potential for WT mitochondria without FCCP, and for Ucp3KO mitochondria in the presence of 10 nM FCCP. Values are means ± SEM of triplicate experiments performed on 11 separate preparations.

Figure 5. Effect of UCP3 in skeletal muscle mitochondria 8–10 min after energization

(A) The average rate of ROS production in WT and Ucp3KO mitochondria was measured 8–10 min after energization, with or without 500 μM GDP. (B) The average membrane potential in WT and Ucp3KO mitochondria was measured 8–10 min after energization, with or without 500 μM GDP. (C) Mitochondria from WT and Ucp3KO mice were independently energized with different amounts of FCCP from 0–60 nM. ROS production rates over 8–10 min after energization are plotted against average membrane potential over the same period. Linear and nonlinear regression analysis (Prism) show that the data are best modelled by two different curves (P<0.0001). Dashed lines highlight the ROS production rate and membrane potential for WT mitochondria without FCCP, and for Ucp3KO mitochondria in the presence of 10 nM FCCP. Values are means ± SEM of triplicate experiments performed on 9 (A, B) or 11 (C) separate preparations.