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 metabolic and respiratory inhibitors applied for experimental treatments, including hypoxia/anoxia conditions.
• Genetic manipulations included CRISPR-Cas9 knockout of MDH2 in 143B ΔCYTB cells and lentiviral overexpression of metabolic enzymes such as MDH2, L2hgdh, and LbNOX variants, with validation by Simple Western analysis.
• Comprehensive multi-omics approaches were employed, including metabolomics via UHPLC-MS/MS, proteomics with TMT labeling and mass spectrometry, RNA sequencing (bulk and single-cell), m6A RNA methylation sequencing, PRO-seq, ChIP-seq, CUT&RUN, ATAC-seq, and mRRBS for DNA methylation profiling.
• Functional assays measured mitochondrial respiration (OCR), extracellular acidification, NADH consumption, and NADH/NAD+ ratios, alongside histological analyses for fibrosis and glomerular density in mouse kidney tissues, supported by in vivo mouse models with genetic modifications.
• Data processing and statistical analyses were performed using established bioinformatics pipelines and software tools, including DESeq2 for differential expression, MetaboAnalyst for metabolomics, and GraphPad Prism for statistical testing, ensuring reproducibility and rigorous validation of findings.