Fig 2: Model establishment. LCR, low calorie refeeding; SCR, standard calorie refeeding; AAV9-SIRT3 group, AAV9-SIRT3 overexpression group; AAV9-Nc group, adeno-associated virus 9 group; PBS, phosphate buffer saline; EN, enteral nutrition; LPS, lipopolysaccharide.

Fig 3: SIRT3 regulates PDHE1a acetylation and PDH enzyme activity in tubular epithelial cells.TECs incubated with or without HKL then stimulated with TGF-ß1 for 1 h (A–F), TECs stimulated with 3-TYP for 15 min (G–L), and TECs transfected with Sirt3 siRNA (M–R) for 12 h. Mitochondrial lysates immunoprecipitated with anti-acetyl-lysine antibody and analyzed with anti-PDHE1a. (A, C, G, I, M, O). PDHE1a served as the standard (*P < 0.05, †P < 0.05; n = 3). Western blots of phosphorylated and total PDHE1a expression in TEC lysates (B, C, H, I, N, O). PDHE1a served as the standard (*P < 0.05, †P < 0.05; n = 3). Enzyme activity of PDH in TECs incubated with or without HKL, then stimulated with TGF-ß1 (*P < 0.05 vs. control, n = 7; †P < 0.05 vs. TGF-ß1, n = 7) (D), or 3-TYP (*P < 0.05 vs. vehicle, n = 5) (J), or transfected with Sirt3 siRNA (*P < 0.05 vs. NC siRNA transfection, n = 5) (P); levels of ATP in TECs incubated with or without HKL, then stimulated with TGF-ß1 (*P < 0.05 vs. control, n = 3; †P < 0 .05 vs. TGF-ß1 stimulation, n = 3) (E), or 3-TYP (*P < 0.05 vs. vehicle, n = 4) (K), or transfected with Sirt3 siRNA (*P < 0.05 vs. NC siRNA transfection, n = 3) (Q); lactate levels in TECs incubated with or without HKL, then stimulated with TGF-ß1 (*P < 0.05 compared with control, n = 3; †P < 0.05 compared with TGF-ß1 stimulation, n = 3) (F), or by 3-TYP stimulation (*P < 0.05 compared with vehicle treatment, n = 3) (L), or by Sirt3 siRNA transfection (*P < 0.05 compared with NC siRNA transfection, n = 3) (R).

Fig 4: Puerarin restored mitochondrial function in the muscle of diabetic rats a Representative western blots and quantification of mitochondrial proteins used as markers for OXPHOS complexes (I–IV). b Real-time RT-PCR analysis of mRNA levels of mitochondrial proteins used as markers for electron transport chain (MTCO1, COIV) and TAC (CS) and normalized to ß-actin. c Representative western blots and quantification of p-p66Shc(Ser36)/Shc, UCP2, SIRT3 and SOD2. d The mRNA levels of UCP2, UCP3, and SIRT3 were analyzed by real-time RT-PCR and normalized to ß-actin. e Representative TEM images of mitochondrial ultrastructure from muscle. Scale bar: 0.2 µm. Data were normalized to NC and presented as mean ± s.d., n = 5. *P < 0.05, **P < 0.01, vs. NC group; #P < 0.05, ##P < 0.01 vs. DM group

Fig 5: Astrocytic SIRT3 overexpression mitigates the neurotoxicity caused by LPS/IFN-?-stimulated astrocytes. (A) Primary astrocytes were transfected with Flag- or human SIRT3-Flag expression constructs for 3 days, and then immunoblotting was performed. Successful transfection of Flag or SIRT3-Flag in cells was confirmed by immunoblotting using an anti-SIRT3 antibody. SIRT3 protein levels were highly increased in SIRT3-Flag-expressing cells compared with Flag-expressing cells. Tubulin was used as loading control. Data are presented as the mean±SD of 3 independent experiments. ***p<0.001 (unpaired Student’s t-test). (B, C) Flag- or SIRT3-Flag-expressing astrocytes were treated with LPS+IFN-? (100 ng/ml+50 unit/ml) for 24 h and then cocultured with primary cortical neurons in transwell culture inserts. Cell viability was measured using a CMFDA staining (green; B) or CCK-8 assay (C) after a coculture period of 5 days. Data are presented as the mean±SD. **p<0.005 (unpaired Student’s t-test). Scale bars, 20 µm. (D) Flag- or SIRT3-Flag-expressing astrocytes were treated with LPS+IFN-? (100 ng/ml+50 unit/ml) for 24 h, and then, Real Time-PCR was performed. Transcription levels of target genes are presented as the mean±SD. 18S rRNA was used to normalize changes in specific gene expression. *p<0.05; **p<0.005; ***p<0.001 (one-way ANOVA with Tukey’s post-hoc comparison test).