C57BL/6J mice upregulate catalase to maintain the hydrogen peroxide buffering capacity of liver mitochondria

Here, we demonstrate that the upregulation of catalase is required to compensate for the loss of nicotinamide nucleotide transhydrogenase (NNT) to maintain hydrogen peroxide (H₂O₂) steady-state levels in C57BL/6J liver mitochondria. Our investigations using the closely related mouse strains C57BL/6NJ (6NJ; +NNT) and C57BL/6J (6J; -NNT) revealed that NNT is required for the provision of NADPH and that the upregulation of isocitrate dehydrogenase-2 (IDH2) activity is not enough to compensate for the absence of NNT, which is consistent with previous observations. Intriguingly, despite the absence of NNT, 6J mitochondria had rates of H₂O₂ production (58.56 ± 3.79 pmol mg^-1 min^-1) that were similar to samples collected from 6NJ mice (72.75 ± 14.26 pmol mg^-1 min^-1) when pyruvate served as the substrate. However, 6NJ mitochondria energized with succinate produced significantly less H₂O₂ (59.95 ± 2.13 pmol mg^-1 min^-1) when compared to samples from 6J mice (116.39 ± 20.74 pmol mg^-1 min^-1), an effect that was attributed to the presence of NNT. Further investigations into the H₂O₂ eliminating capacities of these mitochondria led to the novel observation that 6J mitochondria compensate for the loss of NNT by upregulating catalase. Indeed, 6NJ and 6J mitochondria energized with pyruvate or succinate displayed similar rates for H₂O₂ elimination, quenching ~84% and ~86% of the H₂O₂, respectively, in the surrounding medium within 30 s. However, inclusion of palmitoyl-CoA, an NNT inhibitor, significantly limited H₂O₂ degradation by 6NJ mitochondria only (~55% of H₂O₂ eliminated in 30 s). Liver mitochondria from 6J mice treated with palmitoyl-CoA still cleared ~80% of the H₂O₂ from the surrounding environment. Inhibition of catalase with triazole compromised the capacity of 6J mitochondria to maintain H₂O₂ steady-state levels. By contrast, disabling NADPH-dependent antioxidant systems had a limited effect on the H₂O₂ clearing capacity of 6J mitochondria. Liver mitochondria collected from 6NJ mice, on the other hand, were more reliant on the GSH and TRX systems to clear exogenously added H₂O₂. However, catalase still played an integral in eliminating H₂O₂ in 6NJ liver mitochondria. Immunoblot analyses demonstrated that catalase protein levels were ~7.7-fold higher in 6J mitochondria. Collectively, our findings demonstrate for the first time that 6J liver mitochondria compensate for the loss of NNT by increasing catalase levels for the maintenance of H₂O₂ steady-state levels. In general, our observations reveal that catalase is an integral arm of the antioxidant response in liver mitochondria.