Acidic pH Is a Metabolic Switch for 2-Hydroxyglutarate Generation and Signaling

Abstract

2-Hydroxyglutarate (2-HG) is an important epigenetic regulator, with potential roles in cancer and stem cell biology. The D-(R)-enantiomer (D-2-HG) is an oncometabolite generated from ␣-ketoglutarate (␣-KG) by mutant isocitrate dehydrogenase, whereas L-(S)-2-HG is generated by lactate dehydrogenase and malate dehydrogenase in response to hypoxia. Because acidic pH is a common feature of hypoxia, as well as tumor and stem cell microenvironments, we hypothesized that pH may regulate cellular 2-HG levels. Herein we report that cytosolic acidification under normoxia moderately elevated 2-HG in cells, and boosting endogenous substrate ␣-KG levels further stimulated this elevation. Studies with isolated lactate dehydrogenase-1 and malate dehydrogenase-2 revealed that generation of 2-HG by both enzymes was stimulated severalfold at acidic pH, relative to normal physiologic pH. In addition, acidic pH was found to inhibit the activity of the mitochondrial L-2-HG removal enzyme L-2-HG dehydrogenase and to stimulate the reverse reaction of isocitrate dehydrogenase (carboxylation of ␣-KG to isocitrate). Furthermore, because acidic pH is known to stabilize hypoxia-inducible factor (HIF) and 2-HG is a known inhibitor of HIF prolyl hydroxylases, we hypothesized that 2-HG may be required for acid-induced HIF stabilization. Accordingly, cells stably overexpressing L-2-HG dehydrogenase exhibited a blunted HIF response to acid. Together, these results suggest that acidosis is an important and previously overlooked regulator of 2-HG accumulation and other oncometabolic events, with implications for HIF signaling.The field of cancer biology has long been rapt by the notion of a cancer-specific metabolic phenotype, perhaps most famously embodied in the "Warburg effect," wherein glycolytic metabolism predominates in cancer cells despite O 2 availability and largely intact mitochondrial respiratory function (1). A prominent feature of the cancer metabolic phenotype (reviewed in Ref. 2) is an elevated level of the small metabolic acid 2-hydroxyglutarate (2-HG), 2 derived from the TCA cycle intermediate ␣-ketoglutarate (␣-KG) (3). The D-(R)-enantiomer of 2-HG (D-2-HG) was shown to be generated by mutant forms of isocitrate dehydrogenase (ICDH) that are associated with a variety of cancers including aggressive gliomas (4). In addition, more recently the L-(S)-enantiomer (L-2-HG) was shown to be generated under hypoxic conditions by lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) (5, 6). We have also reported elevated D/L-2-HG levels in the heart following ischemic preconditioning (7).In addition to synthesis, 2-HG levels are regulated by a pair of dehydrogenases that convert 2-HG back to ␣-KG (i.e. L-2-HGDH and D-2-HGDH). Mutations in these enzymes manifest as the hydroxyglutaric acidurias, devastating inherited metabolic diseases with symptoms including epilepsy and cerebellar ataxia (8 -11). However, the importance of these dehydrogenases in regulating 2-HG levels in other settings is not clear.The downstream signaling roles ...

Potential mechanisms linking SIRT activity and hypoxic 2-hydroxyglutarate generation: no role for direct enzyme (de)acetylation

“…Activities of LDH, IDH or MDH were measured spectrophotometrically at 340 nm, representing either the consumption of NADH (for LDH and MDH) or the generation of NADPH (for IDH) [6]. In addition, aliquots from spectrophotometric cuvets were withdrawn for quantitation of selected reaction substrates or products by LC-MS/MS as previously reported [6].…”

“…In addition, aliquots from spectrophotometric cuvets were withdrawn for quantitation of selected reaction substrates or products by LC-MS/MS as previously reported [6]. Note : The LC-MS/MS method employed was not capable of discerning L vs. D enantiomers of 2-HG.…”

“…as previously described [6]. Primary adult mouse cardiomyocytes were isolated as previously described [20].…”

“…Primary adult mouse cardiomyocytes were isolated as previously described [20]. Cytosolic pH was measured by fluorescent microscopy using the pH-sensitive probe BCECF as previously described [6]. To model ischemia, cardiomyocytes were plated on laminin-coated 25 mm glass cover slips (25,000 cells per slide) in 35 mm dishes, followed by over placement of a second cover slip to exclude oxygen and limit extracellular volume.…”

“…In addition, hypoxia [3–5] and acidic pH [6–8] trigger 2-HG production by MDH2 and LDH. Acidic pH is thought to protonate α-KG, enhancing its access to the substrate binding pocket of these enzymes [7].…”