Fig 2: Effects of Sirt5 on FA uptake in diabetic mice and PA-treated NMCMs. A, Representative X-ray and fluorescent images of cardiac BODIPY™ FL C16 uptake in WT and Sirt5−/− mice after HFD/STZ induction (n = 6/group). FL: Fluorescence. B, Statistical analysis of epi-fluorescence of X-ray and fluorescent images of the above mice. C, Representative fluorescent images of Bodipy™ FL C16 uptake in WT and Sirt5−/− NMCMs treated with BSA or 200 μM PA for 48 h (n = 4/group). Green: Bodipy™ FL C16. Blue: DAPI. Scale bars: 20 μm. D, Statistical analysis of fluorescence intensity of the fluorescence images of the above NMCMs. E, Representative Western blot of myocardial FA transporter CD36 protein expression in WT and Sirt5−/− mice after HFD/STZ induction (n = 6/group), TUBA (α-tubulin) was used as the loading control. F, Statistical analysis of the Western blot. G, Representative Western blot of myocardial CD36 protein expression in db/db mice transfected with AAV9-control or AAV9-Sirt5 (n = 6/group), TUBA (α-tubulin) was used as the loading control. H, Statistical analysis of the Western blot. Data are expressed as mean ± SEM. ns, not significant, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data in B was analyzed using Student's t-test. Data in D was analyzed using one-way ANOVA followed by Bonferroni post hoc analysis. Data in F and H was analyzed using multiple unpaired t tests. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig 3: Inhibition of succinylation by glycine reversed metabolic alterations and reduced proliferation in Sirt5‐knockout ADMSCs. A, Lysine succinylation levels in wild‐type (WT) ADMSCs, Sirt5‐knockout (Sirt5−/−) ADMSCs, and Sirt5−/− ADMSCs treated with different concentrations of glycine. B, CCK8 cell viability assay in ADMSCs 24 h after seeding (n = 8). C, Cell counting assay of WT ADMSCs, Sirt5−/− ADMSCs, and glycine‐treated (25 mM) Sirt5−/− ADMSCs at days 2 and 4 after seeding (n = 3). D, Dihydroethidium (DHE) staining (red) of WT ADMSCs, Sirt5−/− ADMSCs, and glycine‐treated (25 mM) Sirt5−/− ADMSCs under basal conditions and after 12 h of H2O2 (200 μM) treatment. Scale bars: upper panel, 100 μm; lower panel, 50 μm. E, Relative fluorescence intensities of DHE staining (n = 3, different fields). F, Representative flow cytometry assay image (DCFH‐DA staining) of WT ADMSCs, Sirt5−/− ADMSCs, and glycine‐treated (25 mM) Sirt5−/− ADMSCs under basal conditions and after 12 h of H2O2 (200 μM) treatment. G, Mean fluorescent intensity of DCFH‐DA staining (n = 3). H, Oxygen consumption rates (OCRs) of WT ADMSCs, Sirt5−/− ADMSCs, and glycine‐treated (25 mM) Sirt5−/− ADMSCs. I, Extracellular acidification rates (ECARs) of WT ADMSCs, Sirt5−/− ADMSCs, and glycine‐treated (25 mM) Sirt5−/− ADMSCs. J, ECAR/OCR ratios. # P < .05 versus glycine‐treated Sirt5−/− ADMSCs, ## P < .01 versus glycine‐treated Sirt5−/− ADMSCs, * P < .05 versus WT, ** P < .01 versus WT (n = 4). K, Relative mRNA expression levels of glycolysis‐related genes (n = 6). L, Relative mRNA expression levels of oxidative (G6pd, Pgls, Pgd) and nonoxidative (Rpia, Tkt, Rpe) pentose phosphate pathway‐related genes (n = 6). Data are expressed as the mean ± SD. * P < .05; ** P < .01; *** P < .001; **** P < .0001

Fig 4: Sirt5 re‐expression reversed metabolic pattern and proliferative phenotype of Sirt5‐knockout ADMSCs: A, Protein expression levels of SIRT5 in control virus transfected Sirt5−/− ADMSCs (NC) and Sirt5 overexpression virus transfected Sirt5−/− ADMSCs (Sirt5 RE). B, Cell counting assay of NC and Sirt5 RE ADMSCs at days 2, 4, and 6 after seeding (n = 4). C, Representative flow cytometry assay image (DCFH‐DA staining) of NC and Sirt5 RE ADMSCs. D, Mean fluorescent intensity of DCFH‐DA staining (n = 3). E, Senescence‐associated β‐galactosidase (SA‐β‐gal) staining (blue) of NC and Sirt5 RE ADMSCs at passages 3 and 7 (upper panel: passage 3; lower panel: passage 7). F, Oxygen consumption rates (OCRs) of NC and Sirt5 RE ADMSCs (n = 6). G, Extracellular acidification rates (ECARs) of NC and Sirt5 RE ADMSCs (n = 6). Data are expressed as the mean ± SD. * P < .05; ** P < .01; *** P < .001; **** P < .0001

Fig 5: The regulation of Sirt5 expression parallels ATF4.A Scatter plots showing Sirt5 mRNA levels correlates with ATF4 mRNA levels. Data were collected from TCGA breast cancer dataset. B Western blot analysis of WCL of MDA-MB-231 cells ± CB-839 (1 μM) for 24 h. Blots are representative of two independent experiments. C, D RT-qPCR quantification of Sirt5 mRNA in MDA-MB-231 and BT549 cells treated ± CB-839 (1 μM) or ± glutamine (2 mM) at the indicated times. Data shown are representative of two independent experiments and expressed as means ± SD for triplicate measurements. E RT-qPCR quantification of Sirt5 mRNA in MDA-MB-231 cells treated with CB-839 (1 μM) ± DM-αKG (2 mM) for 24 h. Data shown are representative of two independent experiments and expressed as means ± SD for triplicate measurements. F Western blot analysis of WCL from BT549 cells treated with CB-839 (1 μM) ± Torin1 (500 nM) for 4 h. Blots are representative of two independent experiments. G RT-qPCR quantification of Sirt5 mRNA and Western blot analysis of WCL in control and sin1 KD MDA-MB-231 cells treated with menadione (10 μM) for 24 h. Data shown are representative of two independent experiments and expressed as means ± SD for triplicate measurements. H Western blot analysis of WCL from control or sin1 KD MDA-MB-231 cells treated with CB-839 (1 μM) for 24 h. Blots are representative of two independent experiments. I, J Western blot analysis of WCL and RT-qPCR quantification of Sirt5 mRNA in control and PKCα KD MDA-MB-231 cells treated with CB-839 (1 μM) for 24 h. Blots are representative of two independent experiments. qPCR data shown are representative of two independent experiments and expressed as means ± SD for triplicate measurements. K RT-qPCR quantification of Sirt5 mRNA in control and Nrf2 KD MDA-MB-231 cells treated with menadione (10 μM) for 24 h. Data shown are representative of two independent experiments and expressed as means ± SD for triplicate measurements. L Scatter plots show Sirt5 mRNA levels correlates with sin1, PKCA and NRF2 mRNA levels in breast cancer. Data are from TCGA breast cancer dataset.