Mitochondrial l-2-hydroxyglutarate is a physiological signalling metabolite

Key Points:

• The study utilized 143B human osteosarcoma cells and mouse embryonic stem (ES) cells cultured under specific media conditions, with various drug treatments applied for 16-24 hours, including mitochondrial inhibitors and metabolic modulators.
• Generation of genetically modified 143B ΔCYTB cell lines with MDH2 knockout and lentiviral overexpression of metabolic enzymes was performed, with validation by Simple Western analysis and fluorescence-activated cell sorting.
• Multiple high-throughput techniques were employed, including bulk RNA-seq, m6A sequencing, PRO-seq, ChIP-seq, ATAC-seq, mRRBS DNA methylation analysis, and single-cell RNA-seq, to investigate transcriptomic, epigenomic, and metabolic changes.
• Metabolite profiling and enzyme activity assays were conducted using ultrahigh-performance liquid chromatography and mass spectrometry (UHPLC–MS/MS), alongside oxygen consumption rate measurements to assess cellular metabolic states.
• Mouse models with targeted genetic modifications were developed and analyzed for phenotypic effects, including teratoma formation assays, histological assessments of fibrosis and glomerular density, and CUT&RUN assays for chromatin profiling, ensuring comprehensive in vivo validation.