2-Aminobutyric acid modulates glutathione homeostasis in the myocardium

A previous report showed that the consumption of glutathione through oxidative stress activates the glutathione synthetic pathway, which is accompanied by production of ophthalmic acid from 2-aminobutyric acid (2-AB). We conducted a comprehensive quantification of serum metabolites using gas chromatography-mass spectrometry in patients with atrial septal defect to find clues for understanding myocardial metabolic regulation, and demonstrated that circulating 2-AB levels reflect hemodynamic changes. However, the metabolism and pathophysiological role of 2-AB remains unclear. We revealed that 2-AB is generated by an amino group transfer reaction to 2-oxobutyric acid, a byproduct of cysteine biosynthesis from cystathionine. Because cysteine is a rate-limiting substrate for glutathione synthesis, we hypothesized that 2-AB reflects glutathione compensation against oxidative stress. A murine cardiomyopathy model induced by doxorubicin supported our hypothesis, i.e., increased reactive oxygen species are accompanied by 2-AB accumulation and compensatory maintenance of myocardial glutathione levels. Intriguingly, we also found that 2-AB increases intracellular glutathione levels by activating AMPK and exerts protective effects against oxidative stress. Finally, we demonstrated that oral administration of 2-AB efficiently raises both circulating and myocardial glutathione levels and protects against doxorubicin-induced cardiomyopathy in mice. This is the first study to demonstrate that 2-AB modulates glutathione homeostasis in the myocardium.


Figure 1. 2-AB reflects excessive load conditions in hearts.
(a) 2-AB levels in serum in healthy volunteers (n = 11) and ASD patients before (pre) and after (post) transcatheter closure (n = 8). (b) Correlation between 2-AB concentration and TRPG score. c, Illustration of stretch assay. H9c2 cells were stretched by 10% for 1 h. d, e, Mechanical stress induces 2-AB production by cells (c) and can be detected in the culture medium (d).
Scientific RepoRts | 6:36749 | DOI: 10.1038/srep36749 increased compared with that in healthy volunteers. One month after closure of ASD, serum 2-AB levels in patients decreased to almost the same levels as in healthy volunteers (Fig. 1a). Subsequently, we examined the relationship between the levels of 2-AB and clinical data (Supplementary Table 2). Specifically, 2-AB concentrations were significantly correlated with tricuspid regurgitation peak gradient (TRPG) assessed by echocardiography, which is equal to a peak systolic pressure gradient between the right ventricle and the right atrium (Fig. 1b).
Because TRPG elevation reflects right-sided heart overload as a sequela of left-to-right shunting across the ASD, we examined whether mechanical stress to cardiomyocytes induces intracellular 2-AB accumulation. Differentiated H9c2 cardiomyocytes were plated on a collagen-coated chamber, and the cells were stretched by 10% for 1 h (Fig. 1c). This mechanical stress increased the 2-AB levels in H9c2 cells and the culture medium (Fig. 1d,e).
2-AB is an amino transfer enzyme-mediated byproduct in the cysteine biosynthesis pathway. Next, we sought to identify the 2-AB synthesis pathway. Given that 2-AB has a similar structural formula to cysteine (Fig. 2a), we hypothesized that 2-AB is produced using the cysteine biosynthesis pathway. When cysteine is biosynthesized from cystathionine, 2-oxobutyric acid (2-OBA) is generated simultaneously (Fig. 2b). Considering the structure of 2-AB, we hypothesized that 2-AB is generated by an amino group transfer reaction to 2-OBA by amino transfer enzymes, such as aspartate aminotransferase (AST). To test this hypothesis, 2-OBA was incubated with glutamic acid and AST. We found that 2-AB was produced in accordance with increasing 2-OBA concentration (Fig. 2c). We then investigated whether 2-AB was generated from 2-OBA in cells. Because 2-OBA was not incorporated into the cells, we prepared ethyl 2-OBA to promote the incorporation into cells. We confirmed that 2-AB was biosynthesized from the incorporated 2-OBA (Fig. 2d). To examine the involvement of amino transfer enzymes for 2-AB biosynthesis, we used an inhibitor of AST, aminooxyacetic acid (AOA). Although AOA treatment had no effect on the incorporation of 2-OBA, 2-AB production was significantly suppressed (Fig. 2e). These results suggest that 2-AB is an AST-mediated byproduct in the cysteine biosynthesis pathway.

GSH compensation against oxidative stress accompanies elevation of 2-AB. Because cysteine is
a rate-limiting substrate for de novo GSH synthesis 13 , we hypothesized that 2-AB reflects a compensatory maintenance of GSH under oxidative stress conditions. We found that oxidative stress caused by H 2 O 2 administration increased the 2-AB levels in both cardiomyocytes and culture medium (Fig. 3a,b), depending on AST activity (Supplementary Figure 1). Oxidative stress also increased the total GSH levels, which suggests that GSH production is increased to counteract oxidative stress (Fig. 3c). Mechanical stress-induced 2-AB accumulation may also be mediated by oxidative stress, because stretch stress has been reported to increase ROS levels in cardiomyocytes 14 . We also assessed whether 2-AB reflects the myocardial redox state in murine models of cardiomyopathy induced by doxorubicin (DOX), which causes cardiac damage via oxidative stress. Echocardiographic analysis of cardiac function revealed that the administration of DOX decreased the percentage of fractional shortening (%FS) (Fig. 3d). The levels of 2-AB in both plasma and hearts were also increased after the administration of DOX (Fig. 3e,f). In addition, the 2-AB levels in plasma were associated with %FS (Fig. 3g). Reactive oxygen species (ROS) abundance was increased in DOX-treated hearts, consistent with previous reports (Fig. 3h). However, the total GSH levels were retained (Fig. 3i), which suggests that increased 2-AB reflected a compensation in GSH levels for consumption by ROS. Previous reports suggested that the detection of GSH deficiency in blood could be a novel strategy for the early diagnosis of heart failure 15 . However, because the extracellular GSH levels are 100-to 1000-fold lower than the intracellular levels 16 , we could not determine the circulating GSH levels using a popular enzymatic recycling assay. Moreover, as shown in the present study, the GSH levels do not necessarily decrease in parallel with ROS production because of its compensatory mechanism. On the other hand, the present findings suggest that the monitoring of circulating 2-AB levels has a potential to provide a better understanding of GSH homeostasis.

2-AB increases intracellular GSH levels and exerts protective effects against oxidative stress.
Next, we investigated the physiological roles of 2-AB. When 2-AB was treated with H9c2 cells, 2-AB was easily incorporated into cells, the most probable mechanism being via amino acid transporters (Fig. 4a). Surprisingly, intracellular GSH levels were increased after incubation with 2-AB (Fig. 4b). To examine how 2-AB influences GSH levels in cells, we metabolically profiled 2-AB treated cells. Metabolic profiles of cardiomyocytes stimulated by 2-AB imply the activation of the serine biosynthesis pathway (Fig. 4c). A decrease in serine indicates the satisfaction of glycine requirements, which is mainly biosynthesized from serine and components of GSH. Moreover, serine is required for the reaction that produces cystathionine from homocysteine. Glutamine, a precursor of glutamic acid, was also decreased. Although we cannot conclude without metabolic flux analysis, these results might reflect that 2-AB alters metabolism to meet the demands of the intermediates for GSH synthesis.
Recent studies have revealed that AMPK has a key function in nicotinamide adenine dinucleotide phosphate (NADPH) maintenance 17 . The oxidized form of GSH (GSSG) is reduced with NADPH. Thus, we hypothesized that 2-AB regulates AMPK activity. In support of this notion, AMPK was activated in H9c2 cells treated with 2-AB (Fig. 4d). As further confirmation of the involvement AMPK on GSH production induced by 2-AB, we inhibited AMPK using an AMPK inhibitor and assessed GSH productivity of 2-AB. Remarkably, the treatment of AMPK inhibitor decreased GSH production (Fig. 4e). Given that AMPK allosterically activated by AMP 18 , we measured the levels of AMP in 2-AB treated cells. The treatment of 2-AB increased the intracellular levels of AMP (Fig. 4f). Together, these data support the idea that 2-AB regulates AMPK activity to increase the GSH levels.
To test whether 2-AB mediated elevation of GSH possesses cell protective effects, we examined the roles of 2-AB in cell viability. Pretreatment with 2-AB suppressed the cell death observed when H9c2 cardiomyocytes were incubated with H 2 O 2 (Fig. 4g).
Scientific RepoRts | 6:36749 | DOI: 10.1038/srep36749 To confirm 2-AB mediated GSH production in vivo, 2-AB was administered to mice in drinking water for 1 week. Oral administration of 2-AB had no effect on body weight (Fig. 5a). The amounts of 2-AB were increased in both plasma and hearts in a dose-dependent manner (Fig. 5b,c). Coincident with 2-AB increase, GSH levels in plasma and hearts increased (Fig. 5d,e). Finally, we also sought to demonstrate the cardioprotective effect of 2-AB in vivo (Fig. 5f). Pretreatment with 2-AB protected against DOX-induced cardiomyopathy in mice concomitantly with elevation of myocardial GSH levels (Fig. 5g,h).
Recently, the elevation of 2-AB levels in heart tissue has also been observed in global metabolomics analysis of a hamster model for dilated cardiomyopathy 19 , supporting the notion that 2-AB could facilitate early detection    of heart failure. Exogenous GSH preserves mitochondrial energetic/redox balance and exerts cardioprotective effects [20][21][22][23] . However, oral administration of GSH does not efficiently increase circulating levels of GSH [24][25][26] . Whereas 2-OBA requires esterification for cellular uptake, 2-AB is incorporated directly into cells, followed by an increase in intracellular GSH levels. Furthermore, oral intake of 2-AB raised both circulating and myocardial GSH levels and provided a cardioprotective effect. Taken together, the current findings suggest the potential of 2-AB modulation as a novel therapeutic strategy for targeting dysregulation in cellular GSH homeostasis.

Human study. This study was approved by the Institutional Review Board of Kobe University Graduate
School of Medicine and conducted according to the principles expressed in the Declaration of Helsinki. All patients and healthy volunteers provided their written informed consent.

Quantification of 2-AB levels in serum.
Metabolites were extracted from serum as previously described 27 . Analysis of 2-AB by GC-MS was performed on a GCMS-QP2010 Ultra (Shimadzu). The data were acquired with selected ion monitoring and calibrated using the peak height of 2-isopropylmalic acid (internal standard).

Measurement of metabolites in cells.
Cell metabolites were extracted with cold methanol. After centrifugation to remove cell debris, the metabolites were freeze-dried overnight. Lyophilized metabolites were derivatized and analyzed with GC-MS as previously described 28 .
Cell debris was lysed in 20 mM HEPES (pH 7.4), 150 mM NaCl, 1% NP-40, and 1% sodium dodecyl sulfate (SDS). The protein concentration of the lysate was measured with a commercially available BCA Protein Assay kit (Pierce). Metabolite measurements were normalized to protein content. Animal experiments. The care and use of the animals were followed the animal welfare guidelines, and all the experimental protocols were approved the committee of Kobe University. Male C57/BL6J mice (10 weeks old) were purchased from CLEA Japan, Inc. and maintained and treated under specific pathogen-free conditions. 2-AB was orally administrated in drinking water for 1 week. Animals were euthanized, and their hearts and plasma were extracted.
For the doxorubicin (DOX)-induced cardiomyopathy models, 10 week old C57/BL6J mice were administrated a single injection (10 mg/kg intraperitoneally). A 10 mM 2-AB solution was administrated orally 14 days before DOX injection and 8 days after DOX injection.
Reactive oxygen species (ROS) levels were measured with lucigenin-enhanced chemiluminescence method as previously described 29 . Briefly, heart homogenates were incubated with 5 μ M lucigenin, 100 μ M NADPH in the presence or absence of superoxide dismutase (SOD), a ROS scavenger. Light emission was recorded with a 96-well microplate luminometer.

2-AB metabolism in vitro and in vivo.
For in vitro synthesis of 2-AB, 2-oxobutyric acid was mixed with 50 mM Tris-HCl, 0.1 mM pyridoxal-5-phosphate, 0.1 mM glutamic acid, and 5 units of aspartate aminotransferase (WAKO). After incubation at 30 °C for 30 min, the reaction was stopped by adding 0.1 N HCl. GC-MS was used to measure 2-AB yield.
For in vivo synthesis of 2-AB, H9c2 cells were treated with 1 mM or 5 mM ethyl 2-OBA and incubated for 1 h. The production of 2-AB was measured using GC-MS.
Cell viability assay. H9c2 cells were seeded in 96-well plates, and cells were differentiated in DMEM containing 1% FBS. The cells were treated with either water (control) or 2-AB at varying concentrations overnight. The cells were then stimulated with 0.5 mM H 2 O 2 for 24 h, and the number of cells was determined with a Cell Counting Kit-8 (Dojindo) according to the manufacturer's protocol.

Measurement of glutathione.
Glutathione levels in mouse plasma were determined using liquid chromatography-tandem mass spectrometry (AB SCIEX 6500 Qtrap) as described previously 30  Preparation of ethyl 2-oxobutyrate. According to a previously described procedure 31 , ethyl iodide (0.3 mL, 4 mmol) was added to a solution of sodium 2-oxobutyrate (248 mg, 2 mmol) in hexamethylphosphoramide (HMPA) (3 mL) at room temperature. After being stirred for 1 h, the reaction mixture was diluted with 5% Scientific RepoRts | 6:36749 | DOI: 10.1038/srep36749 HCl and extracted with Et 2 O. The combined organic phase was washed with 10% aqueous Na 2 S 2 O 3 followed by saturated aqueous NaCl, and dried over MgSO 4 . The organic phase was concentrated under reduced pressure and the resulting residue was purified by flash column chromatography on silica gel [pentane:Et 2 O (10:3)] to produce ethyl 2-oxobutyrate (60 mg, 23%). The spectral data were identical with those reported in the literature 32,33 . Statistics. An unpaired two-tailed Student's t-test or one-way analysis of variance (ANOVA) with Tukey's post-hoc test was used to make comparisons as indicated. The relationship between the 2-AB levels and clinical data was analyzed using Pearson's correlation. All statistical analyses were performed using GraphPad Prism software.