Abstract 16708: Inhibition of Alpha-Ketoglutarate Dehydrogenase Activates Autophagy

Introduction: Metabolic reprogramming is a hallmark of cancer and heart failure. In isocitrate dehydrogenase 1 and 2 mutant tumors, increased plasma levels of the oncometabolite D-2-hydroxyglutarate (D2-HG) inhibit α-ketoglutarate dehydrogenase (α-KGDH) in the heart. This inhibition is associated with muscle atrophy and reduced ATP and NADH provision. The connection between metabolic changes and proteome remodeling are still poorly understood. Hypothesis: Inhibition of α-KGDH promotes autophagy activation through the sirtuin family of NAD + -dependent deacetylases. Methods: We treated both male and female wild-type C57BL/J6 mice (n=12) with D2-HG in vivo, and cultured L6 myocytes (L6Ms) and adult mouse ventricular cardiomyocytes (AMVMs) with D2-HG. We constructed LC3 mutants with a lysine-to-arginine (GFP-LC3K49R-K51R) and lysine-to-glutamine (GFP-LC3K49Q-K51Q) replacement at Lys49 and Lys51 mimicking deacetylated and acetylated LC3, respectively. Results: Inhibition of α-KGDH by D2-HG is accompanied by heart and skeletal muscle atrophy, as well as increased LC3 and p62 expression in mice treated over 30-days. In vivo and in vitro treatment with D2-HG for 24h, increases phosphorylation and activation of AMPK, while phosphorylation of mTOR and p70S6K decreases. Thus, acute α-KGDH inhibition impacts protein turnover. Autophagy activation is highly dependent on D2-HG concentration and duration. ATP levels significantly decrease in differentiated L6Ms and AMVMs when treated with D2-HG levels above 0.13 mM over 24 to 96 h. GFP-tagged LC3 co-localizes with lysosomes labeled with LysoTracker Blue dye and mitochondria labeled with MitoTracker Red CMXRos dye upon α-KGDH inhibition, which indicates the presence of autophagosomes and mitophagosomes. We show that upon α-KGDH inhibition and subsequent NAD + increase, Sirt1 forms a molecular complex with LC3 and activates autophagy through deacetylation of LC3. In these conditions, LC3K49R-K51R mutants show puncta formation in the cytoplasm indicating that in addition to NAD + increase other factors are driving autophagy activation. Mass spectrometry-based proteomics and metabolomics revealed that α-KGDH inhibition induces upregulation of tropomyosin, NADH dehydrogenase and proteins involved in glycolysis and pentose phosphate pathway. Conclusions: Inhibition of α-KGDH promotes autophagy activation through deacetylation of LC3 by Sirt1 and culminates in proteome remodeling.