Figure 4. Hyperacetylation of mitochondrial proteins but normal ROS markers in Sirt3^hep−/− and Sirt3^skm−/− mice subjected to HFD.

(A) Protein expression levels of complex I, II, IV and V in isolated mitochondria of Sirt3^skm+/+ and Sirt3^skm−/− muscle (left panels) and Sirt3^hep+/+ and Sirt3^hep−/− livers (right panels). (B, C) Acetylation status of mitochondrial proteins extracted from muscle and liver of Sirt3^skm+/+ and Sirt3^skm−/− mice (B) and Sirt3^hep+/+ and Sirt3^hep−/− mice (C). Mitochondrial protein lysates were subjected to western blotting using an anti-acetylated lysine antibody (Ac-K) and porin as a loading control. (D) Acetylation level of complex I members (top two rows of panels) and Sod2 (bottom two rows of panels) in Sirt3^skm+/+ and Sirt3^skm−/− muscle (left panels) or Sirt3^hep+/+ and Sirt3^hep−/− livers (right panels). Endogenous Ndufa9 or Sod2 were immunopurified from muscle lysates and immunoblotted using Ndufa9/Sod2 and Ac-K antibodies. (E) Complex I (CI), complex IV (CIV) and Citrate synthase (CS) activities in Sirt3^skm+/+ and Sirt3^skm−/− muscle (left panel) or Sirt3^hep+/+ and Sirt3^hep−/− (right panel). (F) Oxygen consumption rate (OCR) from primary hepatocytes obtained from Sirt3^L2/L2 mice infected with the indicated viruses. (G) ATP levels were measured from the indicated tissue lysates. (H–K) Activities of mitochondrial Sod2 (H) and catalase (I) and levels of 4-hydroxy-2-nonenal (HNE; J) and GSH/GSSG ratio (K) in muscle and liver lysates of Sirt3^skm+/+ and Sirt3^skm−/− mice (left panels) or Sirt3^hep+/+ and Sirt3^hep−/− mice (right panels). Measurements in panels E–K were determined using samples from at least four mice per genotype. All the samples used for these studies come from mice subjected to the HFD treatment, sacrificed in fed state. Data are represented as mean ± SEM.