Fig 2: Palmitoleic acid reduced the expression of SIRT3 and MDH2 but did not change the protein levels of PC, PEPCK, and SIRT1 in the liver of mice under HFD. C57BL/6N male mice of 5–6 weeks of age were fed with HFD for 12 weeks. At the 13th week, BSA, oleic acid, or palmitoleic acid were administered intragastrically. At the 18th week, mice were euthanized, and liver tissues were collected. (A) Expression of SIRT3, SIRT1, and gluconeogenic enzymes in the liver of mice under HFD. Protein expression was detected using Western blot analysis. (B) Quantification of SIRT3 protein level. (C) Quantification of PC protein level. (D) Quantification of PEPCK protein level. (E) Quantification of MDH2 protein level. (F) Quantification of SIRT1 protein level. AU, arbitrary units. The data are mean ± s.e. (error bars). n = 4–6 mice per group. *, p < 0.05, **, p < 0.01, HFD BSA vs. HFD palmitoleic acid; †, p < 0.05, oleic acid vs. palmitoleic acid.

Fig 3: SIRT3 overexpression increases the activities of gluconeogenic enzymes. C57BL/6N male mice of 5–6 weeks of age were injected with AAV-GFP or AAV-SIRT3 via a tail vein and fed with LFD or HFD for 12 weeks. At the 14th week of feeding, mice were euthanized, and the liver tissues were collected. (A) Enzymatic activity of MDH2. (B) Enzymatic activity of PC. (C) Enzymatic activity of PEPCK. The data are mean ± s.e. (error bars). n = 4–7 mice per group *, p < 0.05, LFD AAV-GFP vs. HFD AAV-GFP; ‡, p < 0.05, ‡‡, p < 0.01, LFD AAV-GFP vs. LFD AAV-SIRT3; †, p < 0.05, ††, p < 0.01, HFD AAV-GFP vs. HFD AAV-SIRT3.

Fig 4: MDH2 Mutations Cause Loss of MDH2 Levels and Enzymatic Activity(A) Western blot analysis with anti-MDH2 antibodies in fibroblasts from subject 1 (S1, top) and subject 3 (S3, bottom). Additional abbreviations are as follows: C1–C3, control individuals; M, mother of S1; and F, father of S1. Commercial MDH2-specific antisera at 1:250 (HPA019716, Sigma-Aldrich) or 1:1,000 (8610S, Cell Signaling Technology) were used for the upper and lower panels, respectively. GAPDH and a-tubulin were used as loading controls.(B) Representation of MDH2 activity, measured with the Mitochondrial Malate Dehydrogenase (MDH2) Activity Assay Kit (Ab119693, Abcam), in the fibroblasts from S1, his parents, and control individuals (top) and in those from S3 and control individuals (bottom). Data represent three independent experiments performed in duplicate.(C) Schematic of the Krebs cycle.(D) Metabolite ratios assessed by liquid chromatographic tandem-mass spectrometry in fibroblasts from S1 are compared with those from his parents and control individuals. Experiments were performed as previously described.4 Differences between fibroblast cells were analyzed by Student’s t test: ***p < 0.001 (extremely significant).

Fig 5: SIRT3 overexpression increases glucose production and the protein level of MDH2 in primary hepatocytes. Primary hepatocytes from HFD or LFD mice were isolated and infected with Ad-GFP or Ad-SIRT3 adenovirus. (A) Infection status after 48 h as fluorescent detection of GFP. (B) Glucose production from primary hepatocytes. In the presence of substrates (20 mM lactic acid and 2 mM pyruvate), the primary hepatocytes were stimulated with 100 nM glucagon for 3 h with or without 50 uM 3-TYP treatment for 24 h. a, p < 0.05, LFD Ad-GFP vs. LFD Ad-GFP+3-TYP; b, p < 0.05, LFD Ad-GFP vs. LFD Ad-SIRT3; c, p < 0.05, LFD Ad-GFP+3-TYP vs. LFD Ad-SIRT3; d, p <0.05, LFD Ad-GFP vs. HFD Ad-GFP; e, p < 0.05, HFD Ad-GFP vs. HFD Ad-GFP+3-TYP; f, p < 0.05, HFD Ad-GFP vs. HFD Ad-SIRT3; g, p < 0.01, HFD Ad-GFP+3-TYP vs. HFD Ad-SIRT3. (C) SIRT3, PC and MDH2 protein levels. Primary hepatocytes infected with adenovirus were treated with DMSO or 3-TYP for 24 h and 100 nM glucagon for 1 h. Cells were collected for protein extraction. Western blot analysis was used to detect the expression of SIRT3, PC, and MDH2. *, p < 0.05, LFD Ad-GFP vs. LFD Ad-SIRT3; ††, p < 0.01 HFD Ad-GFP vs. HFD Ad-SIRT3. The data are mean ± s.e. (error bars). n = 5 from three independent experiments.