Transcriptomic analysis of Sirt3 knock-out mouse embryonic fibroblasts reveals major differences in metabolism and stress-response between sexes

In this study, we investigated sex-specific differences in gene expression caused by Sirt3 knockout. SIRT3, a mitochondrial deacetylase, plays a crucial role in maintaining cellular health. Through activation or suppression of a large number of target proteins, it integrates various metabolic processes, including energy production and antioxidant defense. Using NGS mRNA-seq on wild-type and Sirt3-knock-out male and female mouse embryonic fibroblasts (MEFs) we detected significant changes in both global gene signature and affected cellular pathways. More importantly, we describe major differences in metabolic status of male and female KO MEFs which include fundamental processes like glycolysis, TCA cycle and beta-oxidation. We describe a sex-specific response to Sirt3 loss including maintainance of cellular oxidative status, Hif1a stabilization and induction of Integrated Stress Response (ISR). While female MEFs are significantly more able to tolerate Sirt3 loss, male cells adapt to knock-out by entering a persistent state of cellular stress. Considering many known and potential roles of Sirt3 in metabolism of both normal and cancer cells, aging and longevity, this study emphasizes the crucial role of including sex as a variable in biomedical research. Sirt3 knock-out mouse embryonic fibroblasts were isolated from 13 days old embryos of 129S1/SvImJ WT (Stock No: 002448 Jackson Laboratory, Bar Harbor, ME, USA) Sirt3+/+ and Sirt3-/- (Stock No: 012755, Jackson Laboratory, Bar Harbor, ME, USA) female mice and stable cells were generated using SV40 promoter-containing plasmid. To investigate sex-specific effects of Sirt3 knock-out, we performed mRNA-seq, in triplicates, on wild-type male (WT_M), wild-type female (WT_F), Sirt3 knock-out male (KO_M) and female (KO_F) cells.