Fig 2: SIRT5-depletion aggravates OND-induced mitochondrial mass reduction. hPTECs were transfected using siRNA against SIRT5 or non-targeting siRNA control. Seventy-two hours post-transfection cells were exposed to 6 h of OND (1%O2 + HBSS) or normoxia and complete medium (21%O2 + CM). (A) Maximum projection laser-scanning confocal micrographs showing lysosomes (cyan; red arrow) and lysosome-engulfed mitochondria (magenta; yellow arrow). hPTECs were incubated with a fluorescent, pH-dependent probe (Mtphagy dye; magenta), exposed to 21%O2 + CM or OND and subsequently, incubated with a lysosomal dye (Lyso dye; cyan). Scale bar: 25 µm. (B) Representative 3D image of engulfed mitochondria (magenta) in lysosomes (cyan). Co-localized areas appear white. Z-stacks were analyzed using Image-Pro Premier 10 (MEDIA CYBERNETICS). (C) Bar graph showing citrate synthase (CS) activity as a measure of mitochondrial mass. CS activity/sample was normalized to total protein content. Data are from three independent experiments with n = 3 technical replicates/group. To determine statistical significance a two-way ANOVA was carried out followed by Tukey’s post hoc test to normalize for multiple comparisons. Data are mean ± SD. *p < 0.05. (D) WB showing protein levels of SDHA, TIM23, TOM20 and VDAC1. Scatter plots showing the densitometric analysis for SDHA, VDAC1, Tim23 and TOM20. VDAC1 was normalized to tubulin and SDHA, TIM23 and TOM20 were normalized to actin. Whole WBs shown in supplementary file (Supplementary Fig. S8g) ImageJ was used for densitometric analysis. Data are from three to four independent experiments with n = 3 technical replicates/group. To determine statistical significance a two-way ANOVA was carried out followed by Tukey’s post hoc test to normalize for multiple comparisons. Data are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Fig 3: Elevated SIRT5 levels were detected in ADMSCs during in vitro expansion. A, Relative mRNA expression levels of Sirt1–7 in passages 3 and 7 of wild-type ADMSCs (n = 3). B,C, Western blot analysis and expression level quantification of SIRT5 in passages 3, 5, and 7 of wild-type ADMSCs (n = 3). Data are expressed as the mean ± SD. * P < .05; ** P < .01

Fig 4: SIRT5 ablation impairs lysine oxidation and glutaryl-CoA oxidation in cells and mice.A, schematic of lysine oxidation enzymes with steps that release carbon dioxide (CO2). B, oxidation of [U-14C]-lysine to [14C]-CO2 in 293T SIRT5 crWT and crKO cells. Data representative of three independent experiments (mean ± SD, n = 6; *p-value = 0.05). C, oxidation of [1, 5-14C]-glutaryl-CoA to [14C]-CO2 in mitochondria isolated from 3 h starved 293T SIRT5 crWT and crKO cells. Data representative of single experiment (mean ± SD, n = 4; *p-value = 0.05). D, oxidation of [1, 5-14C]-glutaryl-CoA to [14C]-CO2 in mitochondria isolated from SIRT5WT and SIRT5KO mouse liver. Data representative of single experiment (mean ± SD, n = 6, 8 for SIRT5WT and SIRT5KO respectively; *p-value = 0.05). SIRT5, sirtuin 5.

Fig 5: (a–f) There were no significant differences in the scotopic a-wave, scotopic b-wave, or photopic b-wave implicit times when comparing hyperglycemic SIRT5 germline knockout (Sirt5-/-) and wild-type control (Sirt5+/+) mice to normoglycemic mice of the same genotype except for one pairwise comparison at a single flash intensity after post-hoc testing (N = 8–13/group; 2-way mixed ANOVA with Bonferroni post-hoc test). Graphs depict mean + S.E.M. (a–f) (^P < 0.0001).