Fatty acid oxidation drives mitochondrial hydrogen peroxide production by α-ketoglutarate dehydrogenase.

In the present study, we examined the mitochondrial hydrogen peroxide (mH2O2) generating capacity of α-ketoglutarate dehydrogenase (KGDH) and compared it to components of the electron transport chain using liver mitochondria isolated from male and female C57BL6N mice. We show for the first time there are some sex dimorphisms in the production of mH2O2 by electron transport chain complexes I and III when mitochondria are fueled with different substrates. However, in our investigations into these sex effects, we made the unexpected and compelling discovery that 1) KGDH serves as a major mH2O2 supplier in male and female liver mitochondria and 2) KGDH can form mH2O2 when liver mitochondria are energized with fatty acids but only when malate is used to prime the Krebs cycle. Surprisingly, 2-keto-3-methylvaleric acid (KMV), a site-specific inhibitor for KGDH, nearly abolished mH2O2 generation in both male and female liver mitochondria oxidizing palmitoyl-carnitine. KMV inhibited mH2O2 production in liver mitochondria from male and female mice oxidizing myristoyl-, octanoyl-, or butyryl-carnitine as well. S1QEL 1.1 (S1) and S3QEL 2 (S3), compounds that inhibit reactive oxygen species generation by complexes I and III, respectively, without interfering with OxPhos and respiration, had a negligible effect on the rate of mH2O2 production when pyruvate or acyl-carnitines were used as fuels. However, inclusion of KMV in reaction mixtures containing S1 and/or S3 almost abolished mH2O2 generation. Together, our findings suggest KGDH is the main mH2O2 generator in liver mitochondria, even when fatty acids are used as fuel.

Mitochondrial hydrogen peroxide production by pyruvate dehydrogenase and α-ketoglutarate dehydrogenase in oxidative eustress and oxidative distress.

Pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (KGDH) are vital entry points for monosaccharides and amino acids into the Krebs cycle and thus integral for mitochondrial bioenergetics. Both complexes produce mitochondrial hydrogen peroxide (mH2O2) and are deactivated by electrophiles. Here, we provide an update on the role of PDH and KGDH in mitochondrial redox balance and their function in facilitating metabolic reprogramming for the propagation of oxidative eustress signals in hepatocytes and how defects in these pathways can cause liver diseases. PDH and KGDH are known to account for ∼45% of the total mH2O2 formed by mitochondria and display rates of production several-fold higher than the canonical source complex I. This mH2O2 can also be formed by reverse electron transfer (RET) in vivo, which has been linked to metabolic dysfunctions that occur in pathogenesis. However, the controlled emission of mH2O2 from PDH and KGDH has been proposed to be fundamental for oxidative eustress signal propagation in several cellular contexts. Modification of PDH and KGDH with protein S-glutathionylation (PSSG) and S-nitrosylation (PSNO) adducts serves as a feedback inhibitor for mH2O2 production in response to glutathione (GSH) pool oxidation. PSSG and PSNO adduct formation also reprogram the Krebs cycle to generate metabolites vital for interorganelle and intercellular signaling. Defects in the redox modification of PDH and KGDH cause the over generation of mH2O2, resulting in oxidative distress and metabolic dysfunction-associated fatty liver disease (MAFLD). In aggregate, PDH and KGDH are essential platforms for emitting and receiving oxidative eustress signals.

Ablating the glutaredoxin-2 (Glrx2) gene protects male mice against non-alcoholic fatty liver disease (NAFLD) by limiting oxidative distress.

In the present study, we investigated the consequences of deleting the glutaredoxin-2 gene (Glrx2-/-) on the development of non-alcoholic fatty liver disease (NAFLD) in male and female C57BL6N mice fed a control (CD) or high-fat diet (HFD). We report that the HFD induced a significant increase in body mass in the wild-type (Wt) and Glrx2-/- male, but not female, mice, which was associated with the hypertrophying of the abdominal fat. Interestingly, while the Wt male mice fed the HFD developed NAFLD, the deletion of the Glrx2 gene mitigated vesicle formation, intrahepatic lipid accumulation, and fibrosis in the males. The protective effect associated with ablating the Glrx2 gene in male mice was due to enhancement of mitochondrial redox buffering capacity. Specifically, liver mitochondria from male Glrx2-/- fed a CD or HFD produced significantly less hydrogen peroxide (mtH2O2), had lower malondialdehyde levels, greater activities for glutathione peroxidase and thioredoxin reductase, and less protein glutathione mixed disulfides (PSSG) when compared to the Wt male mice fed the HFD. These effects correlated with the S-glutathionylation of α-ketoglutarate dehydrogenase (KGDH), a potent mtH2O2 source and key redox sensor in hepatic mitochondria. In comparison to the male mice, both Wt and Glrx2-/- female mice displayed almost complete resistance to HFD-induced body mass increases and the development of NAFLD, which was attributed to the superior redox buffering capacity of the liver mitochondria. Together, our findings show that modulation of mitochondrial S-glutathionylation signaling through Glrx2 augments resistance of male mice towards the development of NAFLD through preservation of mitochondrial redox buffering capacity. Additionally, our findings demonstrate the sex dimorphisms associated with the manifestation of NAFLD is related to the superior redox buffering capacity and modulation of the S-glutathionylome in hepatic mitochondria from female mice.