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

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 αketoglutarate (α-KG) by mutant isocitrate dehydrogenase (ICDH), while L (S) 2-HG is generated by lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) in response to hypoxia. Since 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 α-KG levels further stimulated this elevation. Studies with isolated LDH-1 and MDH-2 revealed that generation of 2-HG by both enzymes was stimulated several-fold 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 ICDH (carboxylation of αKG to isocitrate). Furthermore, since 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 over-expressing L-2HGDH 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 (2)) is an elevated level of the small metabolic acid 2hydroxyglutarate (2-HG), derived from the TCA cycle intermediate α-ketoglutarate (α-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 α-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)(9)(10)(11). However, the importance of these dehydrogenases in regulating 2-HG levels in other settings is not clear.
The downstream signaling roles of D-2-HG in cancer biology and of L-2-HG in hypoxia or stem cell biology, are thought to be mediated by epigenetic effects (12)(13)(14), owing to competitive inhibition of the α-KG-dependent dioxygenase superfamily of enzymes. This includes the JmjC domain-containing histone demethylases, the TET 5-methylcytosine hydroxylases, the EGLN prolylhydroxylases that regulate hypoxia inducible factor (HIF), and the AlkB homolog family of DNA/RNA demethylases (15)(16)(17)(18). As such, 2-HG is a potentially important link between metabolism and epigenetic signaling.
A common feature of hypoxia, as well as the tumor and stem cell microenvironments, is metabolic acidosis. However, the role of pH in regulating 2-HG formation and disposal has not been considered. We tested the hypothesis that acidic pH is a regulator of 2-HG metabolism at the cellular, mitochondrial, and isolated enzyme levels. Our results indicate that acidic pH can independently drive elevated 2-HG levels, and we propose that pH regulation of 2-HG may have important implications for 2-HG signaling in hypoxia and other settings.

RESULTS & DISCUSSION
We investigated the role of pH as an independent variable in the regulation of 2-HG levels in HEK293 cells, using an ammonium plus 5-(N-Ethyl-N-isopropyl) amiloride (EIPA) pH clamp system (19). This system was capable of depressing and maintaining intracellular pH to 6.8 within 2 hrs. (Figures 1A-C). This magnitude of pH depression (approximately 0.5 units) is similar to that previously reported upon exposure of cells to hypoxia/anoxia (20)(21)(22). As such, the pHdependent signaling effects measured herein can be considered to represent those that may be expected to occur in hypoxic tissues.
Although this degree of acidification drove a 4-fold elevation in 2-HG 1 levels ( Figure  1D, left two bars), this was somewhat lower than the >8-fold elevation reported in the same cell type in response to hypoxia (5,6). Since 2-HG generation also depends on the availability of substrate α-KG, which is elevated in hypoxia (6), we hypothesized that α-KG may be limiting in acidic pH alone. As such, we found that inhibiting the TCA cycle enzyme α-KGDH with ketomethyvalerate (KMV) boosted cellular α-KG levels by ~5-fold regardless of pH ( Figure 1E). Furthermore, while KMV alone drove a small increase in 2-HG levels at pH 7.4 ( Figure 1D), the combination of KMV plus acidic pH yielded an overall 13-fold increase in 2-HG levels. Together these data indicate that reproducing the acidic pH and elevated α-KG levels seen in hypoxia is sufficient to increase 2-HG in cells. Acidosis may therefore be an important variable that drives 2-HG elevation in hypoxia.
Operating on the premise that the 2-HG observed in cells was the L-isomer (see footnote 1 ), we next investigated the mechanism of acidinduced 2-HG elevation, by examining the effects of pH on the known enzymes that generate and remove this metabolite -namely LDH, MDH, ICDH and 2-HGDH. The purity of commercial isolated LDH-1, MDH-2 and NADP + -dependent ICDH were verified by gel electrophoresis, with ICDH found to be somewhat impure (Figure 2A). A spectrophotometric assay for the consumption of NADH by LDH in the presence of α-KG ( Figure 3A inset) was performed across a range of pH values spanning 6.6-7.8 (i.e. encompassing the pH values studied in intact cells), and revealed that NADH consumption was approximately 2.5-fold faster at pH 6.8 than at pH 7.4 ( Figure 3A). In agreement with the cell data ( Figure 1D), the rate was also [α-KG] dependent, being faster at 10 mM than at 1 mM α-KG. The spectrophotometric data were confirmed by LC-MS/MS measurement of 2-HG generation ( Figure 3B), with approximate molar equivalents of 2-HG produced across the pH range 6.6-7.8 ( Figure 2B, r 2 = 0.97).
The native activity of LDH (i.e., conversion of pyruvate to lactate) was unaffected by pH ( Figure 3C); unsurprising for an enzyme that typically drives metabolic acidosis. Fractionally, the 2-HG generating capacity of LDH was at least 4 orders of magnitude lower than its native activity ( Figure 3A vs. 3C), indicating that acidic pH unmasks a latent 2-HG generating activity in LDH without impacting its native activity. This observation also suggests that the generation of 2-HG is not a quantitatively important metabolic sink for NADH.
As a control, the effect of hypoxia on the ability of isolated LDH to generate 2-HG at pH 7.4 was also studied. Hypoxia was verified by measuring the spectrum of oxy-vs. deoxyhemoglobin in the cuvet ( Figure 4A). In contrast to the situation seen in cells (5, 6), no effect of pO 2 on 2-HG generation by isolated LDH was seen ( Figures 4B and 4C). These data suggest that hypoxic stimulation of 2-HG generation by LDH is not a property of the isolated enzyme, but rather it requires the intact hypoxic cell environment, likely including acidosis.
Similar to LDH, the generation of 2-HG by MDH was also found to be higher at pH 6.8 than at pH 7.4 ( Figures 3D and 3E). As seen with LDH, spectrophotometric and LC-MS/MS data for MDH correlated well ( Figure 2C, r 2 = 0.98), with approximate molar equivalents of 2-HG produced across the pH range 6.6-7.8. Notably, unlike LDH ( Figure 3C) the native activity of MDH was itself pH sensitive, being inhibited ~30% at pH 6.8 versus pH 7.4 ( Figure 3F). At first glance, this result might suggest an acid-induced activity switch, away from native and toward 2-HGgeneration. However, it should again be noted that the 2-HG generating activity of MDH was at least 3 orders of magnitude lower than its native activity. Although 2-HG generation has been termed a "metabolic error" and the 2-HG dehydrogenases referred to as metabolite repair enzymes (11), our data suggest that the 2-HG synthetic capacity of MDH unmasked by acidic pH is fractionally small, and does not represent a major diversion of carbon away from the TCA cycle.
Comparing LDH and MDH, at the pH range seen in cells (6.8 vs. 7.4), and at the same α-KG level (10 mM), the relative acid-induced stimulation of 2-HG generation was slightly larger for LDH (2-fold) vs. MDH (1.3-fold). In addition, the pH response of LDH appeared to initiate at slightly more neutral pH, whereas the MDH response was flatter until more acidic pH values were attained. These data, plus the >3-fold greater specific activity for 2-HG generation by LDH vs. MDH, suggest that LDH may be a quantitatively more important source of 2-HG in response to acid.
The ability of native (non-mutant) ICDH to convert α-KG to 2-HG in a pH-sensitive manner was also tested. While ICDH in the presence of α-KG indeed consumed NADPH in a manner that was accelerated at acidic pH ( Figure 5A), no 2-HG generation was seen ( Figure 5B). Instead, ICDH was observed to carboxylate α-KG to isocitrate, and this reaction was stimulated at acidic pH ( Figure 5C). This is somewhat expected because the reaction uses protons as a substrate ( Figure 5C inset). Such reversal of ICDH activity was previously shown to be triggered under hypoxic conditions (23), and is an important pathway for diversion of carbon from anaplerotic glutamine toward lipid synthesis (via ATP citrate lyase) in cancer cells. Thus, while native ICDH does not appear to be a source for acid-triggered 2-HG generation, these data suggest that acidic pH may regulate numerous oncometabolic pathways, including reductive α-KG carboxylation.
Since cellular 2-HG levels are also regulated by specific dehydrogenases, we next tested the effects of pH on mitochondrial L-2-HGDH activity, using both a formazan-linked colorimetric assay (10) and LC-MS/MS to quantify α-KG generation from the substrate L-2-HG. Both assays correlated well ( Figure 2D, r 2 = 0.98), and showed that L-2-HGDH activity was inhibited by acidic pH ( Figure 6). Together with the data in Figure 3 this result indicates that acidic pH both promotes the formation of L-2-HG and inhibits its removal, suggesting a coordinated metabolic response to elevate L-2-HG under acidic conditions. Based on the data in Figures 1, 3 and 6, we conclude that pH is an important determinant of cellular 2-HG levels, and that acidosis may play a key role in the L-2-HG elevation observed in hypoxia (5,6).
Biochemically, the reaction catalyzed by LDH and MDH during generation of 2-HG is the same as the native reactions of these enzymes, namely the NADH dependent conversion of an αketo acid to its corresponding α-hydroxy acid ( Figure 7). The only difference between substrates is the corresponding R group, and our current findings suggest that the mechanism of acidinduced 2-HG generation by LDH and MDH may involve changes in access to the active site, permitting entry of a larger R group. Such a model leads to a simple prediction: if LDH under acidic conditions can accommodate the R group of α-KG ((CH 2 ) 2 -COOH)), it should also be able to accommodate oxaloacetate (OAA). Figures 7B  and 7C show that this prediction is correct, with LDH able to convert OAA to malate (i.e. to perform the MDH reaction) in an acid-sensitive manner. A crystal structure for LDH in the presence of the inhibitor oxamate (R group = NH 2 , PDB 1I0Z), shows the region of the protein that may accommodate the R group occupied by 2 molecules of water, with no acid-sensitive residues such as histidine nearby (24). As such, the mechanism by which acidic pH may allow larger substrates into the active site of LDH remains to be elucidated. In addition, the ability of LDH (or MDH) to accept even larger substrates such as αketobutyrate is unknown, but could be worthy of investigation because elevated α-hydroxybutyrate has been shown to be a component of the metabolic signature of mitochondrial dysfunction across multiple organisms (25,26). In general, the potential for acidic pH to remodel metabolite profiles by promoting α-keto to α-hydroxy acid conversion, remains largely unexplored.
Downstream signaling by 2-HG is thought to be mediated by the inhibition of α-KGdependent dioxygenases, such as the EGLN prolyl-hydroxylases that regulate HIF (15). It is known that acidic pH alone can activate HIF-1α (27), and therefore we hypothesized that 2-HG may play a role in acid-induced HIF activation. To test this, a cell line with stable transgenic overexpression of L-2-HGDH was generated ( Figure  8). The L-2-HGDH-Tg cells (clone #2) exhibited 12-fold greater L-2-HGDH activity compared to mock transfected controls ( Figure 8B). Furthermore, exposure of cells to acidosis plus KMV as in Figure 1 resulted in elevated 2-HG in control cells with a blunted response in L-2-HGDH-Tg cells ( Figure 8C). Measurement of HIF levels by western blot (Figure 9) revealed that HIF stabilization in response to acid plus KMV was significantly blunted in L-2-HGDH-Tg cells (lane 3 vs. 4). In contrast, HIF induction by hypoxia was similar between both control and L-2-HGDH-Tg cells, indicating that canonical HIF signaling was intact. These data indicate that 2-HG generation is required for acid-induced HIF stabilization.
Although the current data suggest that pH is an important regulator of 2-HG signaling, the relative importance of the pH  2-HG axis in cancer or stem cell biology remains to be determined. While acidic pH is often associated with the high glycolytic metabolism of cancer cells, the regulation of pH in cancer is complex, with extracellular pH (pH ex ) tending more toward acidity than intracellular pH (pH in ) (28,29). Furthermore, although it has been suggested that exposure to acidic pH ex may play a role in promoting "stem-ness" (30), there has been much recent controversy regarding the effects of acid exposure on stem cell phenotype (31). Thus, while it could be speculated that a simple elevation in 2-HG levels can transmit a pH signal to the epigenetic machinery and bring about a cancer or stem cell phenotype, the situation is likely to be far more complex in-vivo. As the data in Figure 1 suggest, the availability of substrate α-KG is also a major determinant of 2-HG generation, and the fact that α-KG is also a substrate for the dioxygenase signaling enzymes that 2-HG inhibits, suggests that the α-KG/2-HG ratio is likely to be an important determinant of 2-HG signaling outcomes.
Finally, the current findings may also hold therapeutic implications for the hydroxyglutaric acidurias. Lactic acidosis has been reported in some 2-HG aciduric patients (32), and it could be speculated that acidotic episodes may trigger 2-HG accumulation. As such, the careful management of pH to avoid acidosis, for example by treatment with dichloroacetate, (33) may be an overlooked therapeutic approach in these patients.

EXPERIMENTAL PROCEDURES
All reagents including isolated enzymes were purchased from Sigma (St. Louis, MO) unless otherwise stated. Male C57BL/6J mice were bred in-house from stocks obtained from Jax Labs (Bar Harbor, ME) and were maintained in a thermoneutral environment on a 12 hr. light/dark cycle with food and water available ad libitum. All experiments were in compliance with the NIH "Guide for the Care and Use of Laboratory Animals" and were approved by a local ethics committee (UCAR protocol #2007-087). Enzyme activities were assayed in 25 mM potassium phosphate buffer at 37°C, adjusted to specific pH values as indicated in the figures. Conversion of α-KG to L-2-HG (mediated by LDH, MDH, or ICDH) and was monitored as the oxidation of NADH (0.1 mM) spectrophotometrically at 340 nm. For hypoxia experiments on isolated enzymes, an "open-flow" apparatus was used, in which the spectrophotometer cuvet was fitted with a lid and stirring apparatus, allowing purging of the headspace with gas mixtures defined by mass-flow controllers (34). Gas mixtures were humidified to prevent sample evaporation, and successful achievement of anoxia after 1 hr. of purging with argon was verified by the spectrum of oxy/deoxyhemoglobin (Hb, 100 μM) in a matching openflow neighboring cuvet. Oxy-Hb was freshly prepared by dithionite reduction followed by gel filtration and incubation under 100% O 2 and white light.
The disappearance and formation of L-2-HG, α-KG, citrate/isocitrate and other metabolites was determined by LC-MS/MS analysis of the cuvet reactions. Metabolites were extracted in 80% methanol and then analyzed using reverse phase chromatography with an ion-paring reagent in a Shimadzu HPLC coupled to a Thermo Quantum triple-quadrupole mass spectrometer (7). Data were analyzed using MzRock machine learning tool kit (http://code.google.com/p/mzrock/), which automates analysis of targeted metabolites data based on chromatographic retention time, whole molecule mass, collision energy, and resulting fragment mass. Metabolite concentrations were determined from standard curves constructed using known concentrations.
To create a stable cell line expressing L-2-HGDH, the expression vector pTS4 was created by PCR cloning of the rat L-2-HGDH open reading frame into pcDNA3.1-V5HisC (Invitrogen, Carlsbad, CA) as a BstEII fragment. Rat cDNA was amplified using primers F-5'-ACA CGG TCA CCA TGT GGC CGA CCC TGC GCT ACG-3' and R-5'-ACA CGG TCA CCT AAC TTA AAC CTT TGC TGT GCT TCC TC-3'. HEK293 cells were transfected with pTFS4 using Lipofectamine 3000 (Invitrogen). Stable clonal isolates were expanded in DMEM with 10 % heat inactivated FBS under 1 mg/ml G418 selective pressure for 10 days and thereafter maintained in media containing 200μg/ml G418. The presence of transgenic L-2-HGDH was monitored by enzymatic assay, and by western blotting for V5 and mCherry tags. pcDNA3.1mCherry was included with pTFS4 at 1:10 to visualize transfected cells, and was transfected on its own to create a negative control (mock transfected) cell line.
All experiments were performed a minimum of 3 times. In purified enzyme studies (Figures 3-5 and Figure 6), "N" represents a single measurement, with multiple measurements made on each of multiple batches of enzyme (i.e. technical replicates, with the number of replicates overall exceeding the number of independent batches of enzyme / biological replicates). In all other Figures, "N" refers to independent biological preparations. Statistical significance between groups was calculated using one-way or two-way ANOVA with post-hoc Student's t-testing.

NOTE ADDED IN PROOF
During revision of this manuscript, a report emerged on LDH as a source of high levels of 2-HG found in testis (37), including data showing this phenomenon to be pH sensitive. The relative role of acid as a 2-HG stimulus in testis under physiologically relevant conditions is unclear. We consider this study complimentary to our current findings which span multiple enzymes and cell systems, and overall these discoveries highlight the importance of pH as a driver of metabolic signaling.   Native MDH activity (oxaloacetate to malate) at various pH values, assayed spectrophotometrically as NADH consumption. (Note -MDH activity was measured in the reverse direction, i.e., oxaloacetate to malate, due to thermodynamic constraints as detailed in the supplemental methods). All data are means ± SEM, N>3. *p<0.05 (two-way ANOVA followed by Student's t-test) compared to corresponding value at pH 7.4. Where error bars appear absent, they are sufficiently small as to be wholly contained within the data symbols.    The ability of LDH to catalyze the native back reaction of MDH (i.e. OAA to malate) was measured at various pH values, assayed spectrophotometrically as NADH consumption. (C): Generation of malate in this system was also assayed by LC-MS/MS. Data are means ± SEM, N=4. *p<0.05 (two-way ANOVA followed by Student's t-test) compared to corresponding value at pH 7.4.  For normoxia and acid plus KMV conditions, high exposure blots were used. However, due to saturation of the HIF signal, low exposure blots were used for the hypoxia condition. Data are means ± SEM, N=4. *p<0.05 (paired Student's t-test) between mock and L-2-HGDH-Tg cells under the same condition.