Abstract 12314: β-2-Hydroxyglutarate Inhibits Glycolysis in Pulmonary Vascular Cells
β-2-hydroxyglutarate (L2HG) is a metabolite of unknown function that we have found to increase in pulmonary arterial endothelial cells, smooth muscle cells, and lung fibroblasts (PAEC, PASMC, LF) in response to hypoxia. L2HG is produced from the reduction of mitochondrial 2-oxoglutarate (2OG) and is oxidized back to 2OG by L2HG dehydrogenase (L2HGDH) in a redox cycle coupled to the oxidation of NADH and reduction of FAD, respectively. As these are the only known reactions in which L2HG participates, we hypothesized that L2HG metabolism plays an important role in cellular redox homeostasis. To test this hypothesis, we measured the effect of L2HGDH levels on cellular NADH/NAD+ in LF using an enzymatic cycling assay. L2HGDH knockdown potentiated hypoxia-mediated increases in cellular NADH/NAD+ (138 ± 10% v. 169 ± 14%, siCTL v. siL2HGDH, p = 0.08) while L2HGDH overexpression had the opposite effect (139 ± 10% v. 127 ± 7%, Vector v. L2HGDH, p = 0.08). Given these results, we next examined the effect of L2HGDH knockdown on mitochondrial electron transport and glycolysis using the Seahorse Extracellular Flux Analyzer. We found that siL2HGDH decreased maximal, FCCP-stimulated oxygen consumption rate in LF (10.8 ± 1.0 v. 9.8 ± 0.9 nmol O2/min/106 cells, p < 0.001) and inhibited maximal, oligomycin-stimulated glycolytic flux (790 ± 42 v. 519 ± 61 mpH/min/106 cells, p = 0.02). The effects of siL2HGDH on glycolysis were confirmed in PAEC and PASMC, where the knockdown decreased oligomycin-stimulated extracellular acidification rate (ECAR) by 24% and 16%, respectively. Cell-permeable L2HG inhibited ECAR to a similar degree. Metabolomic profiling of LF treated with siL2HGDH identified increases in the pentose phosphate pathway intermediates 6-phosphogluconate and pentose monophosphate, suggesting phosphofructokinase as a potential target of L2HG. Together, these data indicate that L2HG accumulation in hypoxia reduces mitochondrial reductant stress.
Author Disclosures: W. Oldham: None. J. Loscalzo: None.
- © 2015 by American Heart Association, Inc.