Valproate inhibits mitochondrial bioenergetics and increases glycolysis in Saccharomyces cerevisiae

The widely used mood stabilizer valproate (VPA) causes perturbation of energy metabolism, which is implicated in both the therapeutic mechanism of action of the drug as well as drug toxicity. To gain insight into these mechanisms, we determined the effects of VPA on energy metabolism in yeast. VPA treatment increased levels of glycolytic intermediates, increased expression of glycolysis genes, and increased ethanol production. Increased glycolysis was likely a response to perturbation of mitochondrial function, as reflected in decreased membrane potential and oxygen consumption. Interestingly, yeast, mouse liver, and isolated bovine cytochrome c oxidase were directly inhibited by the drug, while activities of other oxidative phosphorylation complexes (III and V) were not affected. These findings have implications for mechanisms of therapeutic action and toxicity.

www.nature.com/scientificreports/ potential (ΔΨ m ), and ATP levels in hepatocytes (HepG2) after 48 h of treatment 48 . While these studies indicate that VPA inhibits mitochondrial metabolism, the resulting consequences for other metabolic pathways have yet to be established.
The aim of the current study was to investigate the ramifications of VPA-induced inhibition of mitochondrial bioenergetics using the yeast model, in which genetic and biochemical analyses of the metabolic effects of VPA have been uniquely informative 49,50 . In a previous screen of the yeast deletion mutant collection to identify mutants exhibiting sensitivity to VPA, several mutants with defects in mitochondrial functions were identified as hypersensitive to the drug 49 . In addition, a microarray analysis of yeast gene expression revealed that VPA altered the expression of many metabolic genes and increased the expression of several glycolytic genes 50 . In the current study, we report that VPA decreases mitochondrial OCR and ΔΨ m and increases levels of glycolytic metabolites. Interestingly, VPA inhibited cytochrome c oxidase (COX) activity in yeast and mammalian cells, while activities of complex III or V were not affected. These findings suggest that the metabolic effect of VPA is an increase in glycolysis most likely to compensate for the inhibition of mitochondrial bioenergetics.

Results
VpA increases glycolytic activity. We previously conducted a genome-wide microarray analysis to identify pathways affected by VPA 50 . Cells grown in the presence of VPA (0.6 mM) for 5 h exhibit increased expression of glycolytic genes (Table 1). Consistent with an increase in glycolysis, we observed a significant increase in ethanol production after 5 h and to a greater extent after 10 h (Fig. 1).
An increase in carbon flux through glycolysis and/or a decrease in carbon flux to acetyl-CoA due to inhibition of mitochondrial activity can lead to an increase in ethanol production. To test the possibility that VPA Table 1. VPA up-regulates expression of glycolysis genes. Mid-log phase (A 550 = 0.5) cells were cultured in the presence or absence of VPA (0.6 mM) for 5 h, total RNA was extracted, and mRNA levels were determined by microarray analysis and analyzed using Gene Spring software. Statistical significance was calculated using student's t-test with a p-value less than 0.05 for all genes. Fold change reflects expression in cells grown in the presence of VPA relative to vehicle-treated control cells 50 . www.nature.com/scientificreports/ increases glycolysis, cells were treated with VPA and levels of glycolytic intermediates were determined by mass spectrometry. VPA-treated cells exhibited increased levels of glycolytic intermediates fructose-1,6-bisphosphate, 2-phosphoglycerate/3-phosphoglycerate, and phosphoenolpyruvate compared to untreated cells (Fig. 2a). These findings indicate that VPA increases carbon accumulation through the glycolytic pathway, which explains the observed increase in ethanol production. An increase in 6-phosphogluconic acid, which is part of the oxidative phase of the pentose phosphate pathway, was also observed. However, metabolite levels in the non-oxidative phase of the pathway (erythrose-4-phosphate and sedoheptulose-7-phosphate) were not affected (Fig. 2b). This may demonstrate an increased need for NADPH, which is generated only during the oxidative phase. VPA increases reactive oxygen species levels and induces the oxidative stress response in yeast 51,52 . Therefore, cells may require increased NADPH, which is utilized to reduce glutathione that is essential to neutralize reactive oxygen species.
In further support, a metabolic labelling experiment utilizing [U-13 C]-glucose suggests that VPA increased carbon flux to several glycolytic intermediates (Fig. 3a) and to 6-phosphogluconic acid (Fig. 3b), which can explain the accumulation of these metabolites described above.
VpA decreases mitochondrial bioenergetics. One possible explanation for the observed increase in glycolysis is that it is a compensatory mechanism in response to a decrease in mitochondrial bioenergetics. To address this possibility, ΔΨ m was measured using the probe tetramethylrhodamine (TMRM), a cationic fluorescent dye that accumulates in the mitochondria as a function of ΔΨ m . Dye fluorescence was quantified by flow cytometry. Following incubation with VPA, a significant decrease in the ΔΨ m was observed, as indicated by decreased TMRM fluorescence (Fig. 4). A decrease in ΔΨ m in VPA-treated cells suggested that the OCR was reduced. In agreement with this, a rapid decrease was observed in the OCR of cells treated with VPA (Fig. 5).
Decreased ΔΨ m and OCR in response to VPA suggested that the drug may directly target COX, which catalyzes the reduction of oxygen to water and pumps protons to the mitochondrial intermembrane space, generating the ΔΨ m 53, 54 . It has been shown that cytochrome aa 3 depletion caused by chronic VPA administration decreases COX activity 55 . However, the decrease in OCR in this study was observed immediately after addition of the  www.nature.com/scientificreports/ drug. To test the possibility that acute VPA inhibits COX, COX specific activity was assayed in the presence of yeast cytochrome c. VPA in concentrations ranging from 0.25 to 2.5 mM significantly inhibited COX activity by 10-20% (Fig. 6a).
A small inhibition of COX by VPA may be a contributing factor underlying perturbation of mitochondrial energy metabolism. To determine if mammalian COX, similar to the yeast enzyme, is inhibited by VPA, mouse liver homogenates were incubated with VPA at 37 °C for 25 min. Similar to the effect on the yeast enzyme, VPA significantly inhibited COX activity in mouse liver homogenates (Fig. 6b). To ascertain whether VPA inhibits COX directly, VPA was incubated with isolated bovine COX and activity was monitored. Therapeutically relevant concentrations of VPA inhibited activity of the isolated enzyme by ~ 15% (Fig. 6c). The specific activities of complex III and V were not decreased by 1 mM VPA (Fig. 7). These findings suggest that VPA binds to COX and directly inhibits its activity.

Discussion
In this study, we show that clinically relevant concentrations of VPA increase carbon accumulation through glycolysis and decrease mitochondrial bioenergetics. Furthermore, we demonstrate for the first time that VPA directly inhibits mammalian COX activity, which contributes to inhibition of mitochondrial function by the drug. This is the first description of a VPA-mediated increase in glycolysis (Figs. 2a, 3a). Because of its central position in metabolism, glycolysis is very active in rapidly proliferating cells such as yeast and cancer cells 56 . In addition to generating ATP, many glycolytic intermediates are used in other anabolic pathways that support active growth. For example, the pentose phosphate pathway generates ribose (for nucleic acid synthesis) and NADPH (for lipid and nucleic acid synthesis and reduction of glutathione). As Fig. 2b indicates, VPA did not affect levels of metabolites of the non-oxidative phase of the pentose phosphate pathway. Yeast cells ferment the end-product of glycolysis (pyruvate) to ethanol. Because the increase in levels of glycolytic intermediates was accompanied by an increase in ethanol production (Fig. 1), it is likely that the majority of the carbon utilized through glycolysis was converted to ethanol and not incorporated into anabolic pathways associated with cell growth and division. This is further supported by findings showing that VPA inhibited proliferation and cell cycle progression in yeast cells 51,57 .
Activation of glycolysis by VPA is likely the response to inhibition of mitochondrial energy metabolism. VPA-treated cells exhibited a decrease in ΔΨ m as indicated by a 50% reduction of TMRM fluorescence (Fig. 4). This was accompanied by a 30% decrease in OCR (Fig. 5), which occurred immediately after addition of the drug. Our findings agree with those of Komulainen et al. 48 , who reported that VPA decreased ΔΨ m and OCR in HepG2 cells. Here, we show that VPA directly inhibits yeast and mammalian COX activity (Fig. 6). Maximal inhibition of mammalian COX was observed in response to 0.5 to 1 mM VPA (Fig. 6c), a concentration range present in patients' blood during treatment 58 . VPA (1 mM) did not decrease the activities of complexes III and V (Fig. 7). COX is the terminal and proposed rate-limiting enzyme of the electron transport chain [59][60][61][62][63][64] . COX exerts tight control over cellular respiration and membrane potential, as demonstrated in intact HepG2 cells [59][60][61] . Pacelli and co-workers reported that even a small degree of inhibition of COX leads to a significant decrease in membrane potential 61 , which affects not only energy production but also mitochondrial protein import and calcium homeostasis. It is not surprising, therefore, that further inhibition of electron transport enzymes may have detrimental consequences, as suggested by the report of liver failure resulting from VPA treatment of a patient with a COX deficiency 32 . These findings suggest that inhibition of COX by VPA contributes to toxicity, and that VPA may be contraindicated for patients with defects in pathways related to energy metabolism. The most www.nature.com/scientificreports/ likely mechanism of inhibition of energy metabolism by VPA is direct inhibition of COX, although we cannot rule out additional inhibitory effects of the drug on other pathways, such as β-oxidation 35 and the TCA cycle 41 .
In conclusion, VPA inhibits mitochondrial bioenergetics and increases glycolytic activity in yeast. Intriguingly, studies using magnetic resonance spectroscopy of brains of BD patients indicate that BD pathophysiology involves mitochondrial dysfunction and a resultant increase in glycolysis for energy production 65 . However, it is not always possible to separate the effects of the medications from the underlying pathophysiology. Therefore, VPA treatment may contribute to an increase in glycolysis in the brain. This possibility is supported by an increase in lactate production in rat brains after acute VPA 66 . Additionally, the inhibitory effect of VPA on mitochondrial metabolism could explain VPA toxicity, especially hepatotoxicity 29,30 . Alternatively, inhibition of mitochondrial bioenergetics may be a therapeutic response to VPA, as several markers of mitochondrial activity are higher in bipolar mania than in the euthymic and depressive phases of the disorder 13 . This could explain why VPA is mainly prescribed for its acute antimanic effects [67][68][69] . Future studies should further investigate whether the increase in glycolysis caused by VPA is therapeutically relevant, or is a contributing factor to the pathophysiology of BD. Figure 6. VPA inhibits COX activity. COX activity was assayed in yeast mitochondrial extracts (a), mouse liver homogenates (b), and isolated bovine liver (c) as described in Methods with ascorbate as a reductant using a micro Clark-type electrode in a closed chamber. COX specific activity was defined as consumed O 2 (nmol/min/ mg total protein) and reported as % inhibition relative to control. Data shown are mean ± SD (n = 4) (*p < 0.05; **p < 0.01; ***p < 0.001). ethanol. Cells were treated with VPA for the specified times and pelleted. Ethanol concentrations were measured in supernatants using a fluorometric assay kit from BioVision (K620). Ethanol concentration is in mM ethanol normalized to cell density as measured at A 550 .

Scientific RepoRtS
Mass spectrometry of glycolytic and pentose phosphate pathway metabolite levels. Cell cultures (10 mL) were grown to the mid-log phase and treated with VPA (1 mM) or dH 2 O for 5 h. Cells were then quenched, and metabolites were extracted as described 70 . Quantification of metabolites was determined by mass Yeast coX activity. Cells were grown in 1.5 L cultures of YPGal (2% bactopeptone, 1% yeast extract, 2% galactose) until the late-log phase. Cells were pelleted and mitochondria were isolated following zymolase treatment and differential centrifugation as described 71 . Isolated mitochondria were stored at -80 °C. COX activity was assayed in the presence of 20 μM S. cerevisiae cytochrome c (Sigma) and 20 mM ascorbic acid as a reductant. Briefly, mitochondria were incubated with the specified concentrations of VPA and COX activity was assayed using a micro-Clark electrode (Oxygraph system, Hansatech) at 25 °C. Oxygen consumption was recorded on a computer and analyzed with Oxygraph software. Protein concentration was determined with the DC protein assay kit (Bio-Rad, Hercules, CA, USA). COX specific activity is defined as consumed O 2 (nmol)/min/mg total protein and reported as percentage of control.
Mammalian coX activity. Mouse liver tissue or isolated bovine COX was incubated with VPA and COX activity was assayed as described 72,73 . Procedures for acquiring animal tissues were approved by the Wayne State University Institutional Animal Care and Use Committee (IACUC), and all experiments were performed in accordance with relevant guidelines and regulations. Briefly, mouse livers from 8-week old animals were harvested from euthanized animals, minced in incubation buffer (250 mM sucrose, 20 mM Tris buffer pH 7.4, 1 mM PMSF), and incubated at 37 °C for 25 min with different concentrations of VPA. Tissue was collected by centrifugation at 4 °C, the supernatant discarded, and samples stored frozen at − 80 °C. COX activity was measured with a micro Clark-type oxygen electrode in a closed chamber (Oxygraph system, Hansatech) at 25 °C. Frozen pellets were solubilized in 10 mM K-HEPES, 40 mM KCL, 1% Tween-20, 2 mM EGTA, 1 mM Na-vanadate, 1 mM PMSF, 1 μM oligomycin by sonication for 5 s, 2 times. The supernatant was collected, and COX activity measured in the presence of 20 μM cow heart cytochrome c (Sigma) and 20 mM ascorbic acid as a reductant. Oxygen consumption was recorded on a computer and analyzed with the Oxygraph software. Protein concentration was determined with the DC protein assay kit (Bio-Rad, Hercules, CA, USA). COX specific activity is defined as consumed O 2 (nmol)/min/mg total protein and reported as percentage of control. Measurements with COX isolated from bovine liver were performed in a similar manner; see 73 for details.
Complex III and V specific activities. Yeast mitochondria prepared as described above were used to assay complex III and complex V specific activities as described 74 . Briefly, isolated mitochondria were resuspended in potassium phosphate buffer (10 mM), pH 7.4. Samples were exposed to three freeze-thaw cycles. Protein concentration (determined by the Bio-Rad Bradford assay) was normalized to 0.5 µg/µL. For complex III activity, aliquots corresponding to 4-8 µg protein were added to a cuvette containing 200 µL potassium phosphate buffer (250 mM), pH 7.4, 40 µL sodium azide (50 mM), and 50 µL freshly prepared cytochrome c (1 mM). The volume was adjusted to 990 µL with dH 2 O and the baseline was recorded at 550 nm for 2 min. The reaction was initiated by adding 10 µL chemically reduced decylubiquinol (10 mM). Absorbance of the sample was measured for 2 min. Complex III specific activity was determined by subtracting the absorbance of a parallel cuvette containing 10 µL antimycin A (1 mg/mL). VPA (1 mM) effect was assayed in parallel cuvettes incubated for 10 min at 30 °C.