Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M702465200 on June 25, 2007

J. Biol. Chem., Vol. 282, Issue 36, 25986-25992, September 7, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
282/36/25986    most recent
M702465200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lyon, R. C.
Right arrow Articles by Ellis, E. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lyon, R. C.
Right arrow Articles by Ellis, E. M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Synthesis and Catabolism of {gamma}-Hydroxybutyrate in SH-SY5Y Human Neuroblastoma Cells

ROLE OF THE ALDO-KETO REDUCTASE AKR7A2*

Robert C. Lyon1, Stuart M. Johnston, David G. Watson, Gail McGarvie, and Elizabeth M. Ellis2

From the Strathclyde Institute of Pharmacy and Biomedical Sciences, Univesity of Strathclyde, Glasgow G1 1XW and the School of Science and Technology, Bell College, Hamilton, ML3 0JB Scotland, United Kingdom

Received for publication, March 22, 2007 , and in revised form, June 21, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
{gamma}-Hydroxybutyrate (GHB) is an endogenous metabolite synthesized in the brain. There is strong evidence to suggest that GHB has an important role as a neurotransmitter or neuromodulator. The human aldo-keto reductase AKR7A2 has been proposed previously to catalyze the NADPH-dependent reduction of succinic semialdehyde (SSA) to GHB in human brain. In this study we have used RNA interference to evaluate the role of AKR7A2 in GHB biosynthesis in human neuroblastoma SH-SY5Y cells. Quantitative reverse transcription-PCR analysis and immunoblotting revealed that short interfering RNA molecules directed against AKR7A2 led to a significant reduction in both AKR7A2 transcript and protein levels 72 h post-transfection. We have shown that reduced expression of AKR7A2 results in a 90% decrease in SSA reductase activity of cell extracts. Furthermore, we have shown using gas chromatography-mass spectrometry that a decrease in the level of AKR7A2 was paralleled with a significant reduction in intracellular GHB concentration. This provides conclusive evidence that AKR7A2 is the major SSA reductase in these cells. In contrast, short interfering RNA-dependent reduction in AKR7A2 levels had no effect on the GHB dehydrogenase activity of the extracts, and inhibitor studies suggest that another enzyme characteristic of an NAD-dependent alcohol dehydrogenase may be responsible for catalyzing this reverse reaction. Together these findings delineate pathways for GHB metabolism in the brain and will enable a better understanding of the relationship between GHB biosynthesis and catabolism in disease states and in drug overdose.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
{gamma}-Hydroxybutyrate (GHB)3 is a naturally occurring short-chain fatty acid, synthesized in regions of the mammalian brain (1–3). It has been used clinically as an anesthetic (4) and in the treatment of sleep disorders (5). It has also gained notoriety as a recreational drug and as a drug used by sexual predators in date rape cases (6).

GHB in the brain is thought to derive predominantly from the metabolism of {gamma}-aminobutyrate (GABA). GABA is transaminated to succinic semialdehyde (SSA) through the action of the mitochondrial enzyme GABA transaminase (7). SSA can be subsequently reduced to form GHB (Fig. 1). Two enzymes have been shown to catalyze the NADPH-dependent reduction of SSA in human brain (8). Both these enzymes have been identified as members of the aldo-keto reductase (AKR) family of enzymes as follows: aldehyde reductase AKR1A1 (9); and a dimeric SSA reductase AKR7A2, previously characterized as aflatoxin aldehyde reductase (10). AKR7A2 has high affinity for SSA with a Km of ~20 µM, whereas AKR1A1 exhibits a lower affinity for SSA with a Km of 231 µM (11). The high affinity of AKR7A2 for SSA coupled with its presence in human brain suggested that it could serve as the major succinic semialdehyde reductase (SSAR) in this tissue, but the presence of other AKR has previously prevented a definitive role from being established.

Not all of the SSA produced by transamination of GABA is reduced to GHB. Studies of rat brain have shown that less than 0.15% of the metabolic flux from GABA takes this reductive pathway (12). SSA can also be oxidized by the mitochondrially located enzyme succinic semialdehyde dehydrogenase (SSADH) producing succinate for entry into the Krebs cycle. Defects in SSADH are found in individuals suffering from {gamma}-hydroxybutyric aciduria, which results in an abnormal accumulation of GHB (13). SSA that cannot be oxidized in these individuals is reduced to GHB by SSAR, and it is the high levels of GHB that are presumed to cause the clinical syndrome associated with SSADH deficiency, characterized by psychomotor retardation, hypotonia, ataxia, and poorly developed to absent speech (13).

GHB catabolism has been studied in rat brain, showing oxidation of GHB to SSA, through the action of an NAD(P)-dependent GHB dehydrogenase (GHB-DH) followed by oxidation to succinate by SSADH (14) or metabolism to GABA by GABA transaminase (15) (Fig. 1). We and others have suggested previously that SSAR (AKR7A2) may be responsible for GHB oxidation in a coupled system (16) where the reaction is driven by removal of the product SSA. More recently, however, it has been shown that GHB can be metabolized to SSA by a hydroxyacid-oxoacid transhydrogenase, which appears to be dependent on the co-substrate {alpha}-ketoglutarate rather than cofactor (17–19).


Figure 1
View larger version (22K):
[in this window]
[in a new window]

  		FIGURE 1.
Pathways of GHB metabolism. Succinic semialdehyde can be either reduced to GHB by an NADPH-dependent succinic semialdehyde reductase (1), oxidized to succinate by an NAD-dependent succinic semialdehyde dehydrogenase (3), or converted to GABA by GABA transaminase (5). In addition, a hydroxyacid-oxoacid transhydrogenase enzyme (4) can convert SSA to GHB through co-metabolism of {alpha}-ketoglutarate to D -2-hydroxyglutarate. GHB can also be synthesized from 1,4-butanediol by the sequential action of a cofactor-dependent alcohol dehydrogenase (7) and aldehyde dehydrogenase (6).

 
GHB can penetrate the brain freely. It has been shown to transport across the blood-brain barrier by a carrier-mediated mechanism (20) and acts through both a specific GHB receptor (21) and the GABA type B receptor (22). It is believed that large increases in brain GHB concentration following external administration hyperstimulates the GHB receptor(s) (23), and this mechanism is likely to be the basis of the major pharmacological effects of GHB. In individuals with {gamma}-hydroxybutyric aciduria, the absence of SSADH leads to a 30-fold increase of GHB in the brain compared with normal brain concentration (13), and its believed that chronic hyperstimulation of the GABA type B receptor plays a role in the pathogenesis of this disease (24).

With increased recreational usage, the therapeutic potential of GHB and the occurrence of disease states resulting from defects in enzymes involved in GHB metabolism, it was important to develop a clearer understanding of the key enzymes involved in the synthesis and degradation of GHB in the brain. In this study, we have investigated the role of AKR7A2 in acting as a SSA reductase in human SH-SY5Y neuroblastoma cells using RNA interference (RNAi) to knockdown expression of AKR7A2 (25, 26). We studied the effect of reduced expression on the ability of cells to reduce SSA, and on intracellular GHB levels. We also investigated whether AKR7A2 is the major GHB-DH.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines—Human SH-SY5Y neuroblastoma cells (27) were obtained from the American Type Culture Collection (ATCC) and maintained in 1:1 mixture consisting of Ham's F-12 and modified Eagle's medium supplemented with nonessential amino acids, sodium pyruvate, L-glutamate, 100 units/ml each of penicillin and streptomycin, and 10% fetal bovine serum. Human 1321N1 astrocytoma cells were a gift from Dr. Eve Lutz maintained in Dulbecco's modified Eagle's medium supplemented with L-glutamate and 100 units/ml each of penicillin and streptomycin.

Chemicals—All chemicals were obtained from Sigma, except for deuterated GHB (GHB-D6), obtained from Cerilliant Corp., Round Rock, TX, and zopolrestat, a gift from Pfizer, Groton, CT.

Antibodies—Antibodies to human AKR7A2 were raised in rabbits (28). Antibodies to AKR1A1 were a gift from John Hayes, University of Dundee (11). Antibodies to glyceraldehyde-3-phosphate dehydrogenase were purchased from Santa Cruz Biotechnology, Santa Cruz, CA.

RNAi—The siRNA sequence targeting AKR7A2, 5'-GUACAAGUAUGAGGACAAGTT-3' and a control, scrambled oligonucleotide sequence were both chemically synthesized by MWG (Ebersbeg, Germany). SH-SY5Y cells were seeded into 6-well plates (1 x 106 cells per well) and transfected with AKR7A2 siRNA at a final concentration of 5 nM using Ribojuice siRNA transfection reagent (Novagen). The expression of AKR7A2 was assayed by quantitative RT-PCR and Western blotting 72 h after transfection.

Quantitative RT-PCR—Total RNA was isolated from SH-SY5Y using the Absolutely RNA RT-PCR miniprep kit (Stratagene) according to the manufacturer's instructions. First strand cDNA was synthesized from 5 µg of total RNA using the SuperScript First Strand Synthesis System for RT-PCR (Invitrogen). Relative quantitation of AKR7A2 expression levels was carried out with the LightCycler instrument (Roche Diagnostics) using a set of oligonucleotide primers (forward, 5'-AAC TGG ACA CGG CCT TCA TG-3'; reverse, 5'-CAG GAT CCA GCC ATT GCT CT-3') to suit amplification under the specific cycling conditions for the LightCycler. PCRs were set up in 20-µl volumes with 0.5 µl of first strand cDNA, 10 pmol of each primer, and LightCycler FastStart DNA Master SYBR Green I (Roche Diagnostics) as detailed in the manual. The relative amount of cDNA synthesized in each RT-PCR was compared with beta-actin mRNA levels detected using specific primers.

Preparation of Cell Extracts—Protein was extracted from SH-SY5Y cells in 75-cm3 flasks or 6-well plates using a "freezethaw lysis" protocol that does not affect enzyme activity. Briefly, the media were aspirated, and the cells were washed twice in phosphate-buffered saline (PBS: 137 mM NaCl, 10 mM sodium phosphate, 2.7 mM KCl, pH 7.4). Cells were detached in 1x SSC (150 mM NaCl, 15 mM sodium citrate) and microcentrifuged for 1 min at 4 °C, and the cell pellet was resuspended in 250 mM Tris-Cl, pH 7.5. The cell suspension was frozen in liquid nitrogen for 5 min and then transferred to 37 °C for 5 min. The freeze-thaw procedure was repeated twice. The cell lysate was microcentrifuged for 5 min at 4 °C, and the supernatant was retained. Protein concentrations were determined using the method of Bradford against bovine serum albumen standards (50).

Subcellular Fractionation of SH-SY5Y Cells—Subcellular fractionation was performed using differential centrifugation. 4 x 175-cm3 flasks of SH-SY5Y cells were washed twice in phosphate-buffered saline before harvesting and pelleting at 1,000 x g for 3 min at 4 °C. Cells were resuspended in 1 ml of homogenization media (0.25 M sucrose, neutralized to pH 7, 10 mM sodium HEPES, pH 7.5) and homogenized using a Teflon pestle. Homogenization media was added to 5 ml, and the homogenate was centrifuged at 2,000 x g for 10 min at 4 °C. The pellet was retained as the nuclei-enriched fraction. The supernatant was removed and centrifuged at 9,000 x g for 10 min at 4 °C, and the pellet was retained as the mitochondrial fraction. The supernatant was removed and centrifuged at 34,000 x g for 30 min at 4 °C, and the pellet was retained as the fraction enriched for Golgi. The supernatant was removed and centrifuged at 80,000 x g for 1 h to pellet the endoplasmic reticulum fraction. The remaining supernatant was retained as the cytosolic fraction.

Protein Gels and Western Blots—Protein samples were separated on 10% polyacrylamide resolving gels with the buffer system described by Laemmli (29). For Western blots, transfer to nitrocellulose was carried out using a Bio-Rad mini gel apparatus for 2 h at 250 mA. Protein-binding sites on the nitrocellulose were blocked overnight at room temperature in TBST (20 mM Tris-HCl, 150 mM NaCl, 0.01% (v/v) Tween) containing 10% (w/v) skimmed milk. The nitrocellulose membranes were probed for 2 h with primary antibodies (at 1:2000 dilutions in TBST-skimmed milk). Membranes were washed with TBST and probed with horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit IgG at 1:3000 dilution in TBST-skimmed milk) for 2 h. Membranes were washed with TBST and antibodies detected using enhanced chemiluminescence (ECL; Amersham Biosciences). Quantification of band intensities was performed using Kodak one-dimensional image analysis software.

Enzyme Assays—Reductase and dehydrogenase activities of SH-SY5Y protein extracts were measured by following the change in absorbance of cofactor (NAD(P)(H) at 340 nm, using a Beckman DU650 UV single-beam recording spectrophotometer. All assays were performed at 25 °C in reaction volumes of 1 ml. Substrates used were SSA, GHB, and 1,4-butanediol; and cofactors used were NADPH, NADP, and NAD. For determination of SSA reductase activity in protein extracts, 10 µM or 1 mM SSA substrate was used, and assays were performed in 100 mM NaPO4 buffer, pH 6.6, 50 µM NADPH. For determination of 1,4-butanediol dehydrogenase activity in protein extracts, 1 mM 1,4-butanediol substrate was used, and assays were performed in 100 mM NaPO4 buffer, pH 8, 50 µM NAD. For determination of GHB dehydrogenase activity in protein extracts, 1 or 10 mM GHB substrate was used, and assays were performed in 100 mM NaPO4 buffer, pH 8, 50 µM NADP. The inhibitor zopolrestat was used at a concentration of 1 mM, sodium valproate was used at a concentration of 0.5 mM, and 4-methylpyrazole was used at a concentration of 2.5 mM (30).

GC-MS Measurement of GHB Levels—GHB levels were measured in cells 72 h following siRNA treatment (adapted from Ref. 31). Cells and media were separated by centrifugation. 500 µl of CH3CN was added along with 10 µlof1 µg/ml deuterated GHB (GHB-d6) as an internal standard. Samples were dried and derivatized with 100 µlof N,O-bis(trimethylsilyl)trifluoroacetamide by heating at 60 °C for 30 min. 200 µlof ethyl acetate was added to each vial and mixed thoroughly, and an aliquot was analyzed by GC-MS. Analysis was performed on a trace GC-2000 series and MD 800 detector with an AS 2000 autosampler. The software used was XcaliburTM homepage version 1.2 and QualTM browser version 1.2.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of RNAi Knockdown of AKR7A2 on GHB Synthesis—A double-stranded siRNA molecule designed to silence by binding to the 3' end of AKR7A2 mRNA was transiently transfected into SH-SY5Y cells. This human neuroblastoma cell line was selected as it is known to synthesize several neurotransmitters and has proved useful as a model for studying function in neuronal cells (27, 32). The effect of the siRNA molecule on AKR7A2 mRNA levels was measured after 72 h using quantitative RT-PCR with primers specific for AKR7A2. This revealed that treatment with AKR7A2 siRNA led to an 86% reduction in AKR7A2 transcript levels (Table 1) at 72 h post-transfection. No mRNA for the highly similar AKR7A3 could be detected in control SH-SY5Y cells using primers that distinguish between the two genes (data not shown).


View this table:
[in this window]
[in a new window]

  		TABLE 1
Levels of AKR7A2 mRNA in siRNA-treated cells

Quantitative RT-PCR analysis of mRNA levels of control and siRNA-treated SH-SY5Y cells determined using the LightCycler real time PCR machine. Values are the means ± S.D. (n = 3) and represent number of copies of AKR7A2 per 1000 copies of beta-actin measured in each sample.

 
To study the effect on AKR7A2 protein levels, cell extracts were prepared from siRNA-transfected cells, and the levels of AKR7A2 were detected using specific antibodies and Western blots (Fig. 2A). Analysis revealed over 90% reduction in AKR7A2 protein levels compared with control cells 72 h post-transfection, indicating that the RNA interference has been effective.

To test the effect of inhibiting AKR7A2 expression on the ability of the cell to reduce SSA, cell extracts were assayed for SSA reductase activity, using SSA at concentrations of 10 µM and 1 mM. The results in Fig. 2B show that 72 h after siRNA treatment, SSA reductase activity is reduced by ~80% using a high (1 mM) concentration of SSA and by 90% using a low (10 µM) concentration of SSA (Fig. 2C) This indicates that even at high SSA concentrations, the "low Km" SSA reductase, AKR7A2, constitutes the major SSA reductase in this cell line. To determine the contribution of aldehyde reductase (AKR1A1) to SSA reduction, enzyme assays were carried out on extracts from siRNA-treated cells in the presence of zopolrestat, an inhibitor of AKR1A1 (33). This revealed that at 1 mM SSA, ~50% of the residual activity in silenced cells was because of a zopolrestat-inhibitable enzyme, most likely the "high Km" aldehyde reductase AKR1A1 (Fig. 2B). However, by using 10 µM SSA, there was no further inhibition of SSAR activity, proving that when SSA levels are low, AKR7A2 is the predominant SSA reductase in these cells (Fig. 2C). siRNA-dependent inhibition of AKR7A2 in human 1321N1 astrocytoma cells also led to an 90% reduction in AKR7A2 expression and in SSA reductase activity (data not shown), indicating that this is not a cell-specific phenomenon.


Figure 2
View larger version (18K):
[in this window]
[in a new window]

  		FIGURE 2.
Silencing of AKR7A2 in SH-SY5Y cells. A, SH-SY5Y neuroblastoma cells were transfected with AKR7A2-targeted siRNA, and at 72 h post-transfection, levels of AKR7A2, AKR1A1, and glyceraldehyde-3-phosphate dehydrogenase (loading control) were analyzed by immunoblotting. s.s., scrambled sequence of siRNA; vehicle, transfection reagent only. Enzyme assays using 1 mM (B) or 10 µM SSA (C) as substrate followed the change in cofactor (NADPH) in extracts from control, vehicle, or silenced cells 72 h after transfection. The AKR inhibitor zopolrestat was used at a concentration of 1 mM. Values represent mean ± S.D. (n = 6). Statistically significant differences relative to control were determined using one way ANOVA (***, p ≤ 0.001; ns, not significant). Differences between silenced and silenced in the presence of inhibitor were compared using one-wayANOVA(**, p < 0.01). D, GHB content of extracts from control or silenced cells 72 h after transfection. GHB levels were determined using GC-MS using deuterated GHB-d6 as internal standard. Values represent mean ± S.D. (n = 3). Statistically significant differences relative to control were determined using an unpaired t test (**, p ≤ 0.01; ns, not significant).

 
Analysis of SH-SY5Y cells from siRNA-treated and control cells by GC-MS showed a 35% reduction in intracellular GHB content after 72 h (Fig. 2D). This is a significant decrease and confirms the important role of AKR7A2 in GHB synthesis. It is apparent, however, that the cells maintain intracellular GHB levels to a limited extent even with the loss of over 90% SSA reductase activity. Interestingly, we observed that knockdown of AKR7A2 led to a significant increase in 1,4-butanediol dehydrogenase activity in SH-SY5Y cells (Fig. 3), suggesting that a compensatory pathway for GHB synthesis from 1,4-butanediol is being evoked to counter the lower levels of GHB. This may explain why GHB levels are not as low as might be predicted from the decrease in SSAR activity.


Figure 3
View larger version (20K):
[in this window]
[in a new window]

  		FIGURE 3.
1,4-Butanediol dehydrogenase activity in AKR7A2-silenced cells. Enzyme assays using 1 mM 1,4-butanediol as substrate followed the change in cofactor (NAD) in extracts from control or silenced cells 72 h after transfection. Values represent mean ± S.D. (n = 6). Statistically significant differences relative to control were determined using an unpaired t test (**, p ≤ 0.01).

 
Effect of AKR7A2 Knockdown on GHB Dehydrogenase Activity—After having established that AKR7A2 is the major SSA reductase in SH-SY5Y cells, we examined whether knockdown of AKR7A2 had an effect on the ability of cells to oxidize GHB. Extracts prepared from siRNA-treated cells were assayed for GHB-DH activity (Fig. 4). It is apparent that knockdown of AKR7A2 had no observable effect on GHB-DH activity, indicating that despite acting as the major SSA reductase, AKR7A2 is not responsible for the oxidation of GHB in these cells.

Characterization of GHB Dehydrogenase Enzymes in SH-SY5Y Cells—Measurement of GHB-DH activity in SH-SY5Y cells showed that both NAD and NADP-dependent activity is present but that the predominant activity is NAD-dependent, showing over 8-fold greater activity than the NADP-dependent activity (Fig. 5). Enzyme assays carried out in the presence of sodium valproate (an AKR inhibitor) and 4-methylpyrazole (an ADH inhibitor) were used to characterize the key enzymes further. The major NAD-dependent activity is inhibitable by 4-methylpyrazole but not sodium valproate, whereas the NADP-dependent GHB-DH activity is not inhibited by 4-methylpyrazole but is inhibited by sodium valproate. These data indicate that the major enzyme responsible for GHB-DH catabolism is an NAD-dependent alcohol dehydrogenase.

Subcellular fractionation of SH-SY5Y cells revealed that the NAD-dependent GHB-DH activity is concentrated within the mitochondrial fraction, where the specific activity is over 20 times that found in the cytosol (Fig. 5C). A mitochondrial location would be appropriate for an enzyme that can produce SSA for entry into the tricarboxylic acid cycle via succinate.


Figure 4
View larger version (24K):
[in this window]
[in a new window]

  		FIGURE 4.
Effect of silencing of AKR7A2 on GHB-DH activity. Enzyme assays using 1 (A) or 10 mM (B) GHB as substrate followed the change in cofactor (NAD) in extracts from control, vehicle, or silenced cells 72 h after transfection. Values represent mean ± S.D. (n = 6). Statistically significant differences relative to control were tested using one-way ANOVA (ns, not significant).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
There has been considerable speculation over the identity of the enzymes responsible for the reduction of SSA to GHB, originally identified as NADPH-dependent aldehyde reductases (3, 8). Later work added to the intrigue as it appeared that an aflatoxin aldehyde reductase, AKR7A2, might be responsible (10). In this study we provide proof that AKR7A2 is responsible for the vast proportion of SSA reductase activity in SH-SY5Y cells when SSA concentrations are between 10 µM and 1 mM.

There is a small proportion (10%) of SSA reductase activity remaining in siRNA-treated cells. By using inhibitors, we have shown that about half of this activity (5%) is attributable to a zopolrestat-inhibitable enzyme. This would be consistent with aldehyde reductase AKR1A1 (8).

The levels of AKR7A2 expressed in SH-SY5Y cells are similar to those found in the hippocampus from human brain, at around 0.1% of total cell protein, indicating that this cell line is a valid model for examining AKR7A2 function in human brain (34). However, the expression of AKR7A2 is not restricted to the brain; it is present in a range of human tissues, including high levels in kidney, pancreas, small intestine, and skeletal muscle with moderate levels in liver, heart, testis, and ovary. Low levels of AKR7A2 mRNA can be detected in brain, colon, lung, prostate, and thymus (35). If the sole purpose of AKR7A2 is the synthesis of GHB, then it appears that GHB has a role in tissues other than brain. GHB has been detected in a range of other tissues, with highest levels in kidney, brown fat, heart, and muscle, as well as being found in brain, liver, and lung (36). The role of GHB in tissues other than brain is not completely understood. GHB can cause alterations in glucose metabolism, promotes the formation of NADPH, and has also been shown to protect against oxidative stress in heart, muscle, and kidney as well as brain (reviewed in Ref. 37).


Figure 5
View larger version (18K):
[in this window]
[in a new window]

  		FIGURE 5.
GHB dehydrogenase activity in SH-SY5Y cells. Enzyme assays using GHB as substrate followed the change in cofactor NAD (A) or NADP (B) in SH-SY5Y cell extracts in the presence or absence of the enzyme inhibitors sodium valproate or 4-methylpyrazole. Values represent mean ± S.D. (n = 6). Statistically significant differences relative to control were determined using one-way ANOVA (***, p ≤ 0.001; ns, not significant). C, subcellular localization of NAD-dependent GHB-DH. Enzyme assays using GHB as substrate followed the change in cofactor, NAD, in SH-SY5Y cytosolic or mitochondrial fractions. Values represent mean ± S.D. (n = 6).

 
We have shown that silencing of AKR7A2 leads to a significant reduction in intracellular GHB levels. However, 65% of the GHB content remained after 72 h, despite blocking its synthesis from SSA by over 80%. This could be due to other compensatory mechanisms for GHB synthesis that occur when the reduction of SSA to GHB is compromised. Other possible routes for GHB biosynthesis have been proposed previously, specifically from 1,4-butanediol and {gamma}-butyrolactone (38, 39). Both of these compounds are present in rat brain at concentrations of ~1/10 of those of GHB (40, 41). These alternate routes may be of relevance to the synthesis of GHB under limiting conditions. In support of this proposition, we observed that 1,4-butanediol dehydrogenase levels rose by ~2-fold in cells in which GHB was lowered, indicating the presence of a homeostatic mechanism that acts to increase GHB levels. This type of regulation would be appropriate for the biosynthesis of a molecule whose function included acting as a pool for either neurotransmitter synthesis or for producing reducing power in the form of NADPH. Elevated GHB levels have been observed in tissues in response to anoxia (42), but the mechanism by which this is achieved is not known. Our results indicate that a 1,4-butanediol dehydrogenase may be involved in this phenomenon and may function as part of an adaptive response to anoxia and/or oxidative stress.

In addition to understanding pathways of GHB synthesis, there has been a considerable history of work aimed at identifying the enzymes responsible for GHB breakdown, following the discovery that GHB is metabolized to CO2 and water via succinate and the tricarboxylic acid cycle (43). Early pioneering work by Kaufman et al. (14) indicated that one of the enzymes responsible in hamster brain was a cytosolic NADP-dependent enzyme, and it was proposed that this enzyme was identical to one of the SSA reductases, most likely the high Km aldehyde reductase (AKR1A1) which showed a Km for GHB of 10 mM (15, 16, 44). A mitochondrial hydroxyacid-oxoacid transhydrogenase was also identified that was capable of converting GHB to SSA using {alpha}-ketoglutarate as co-substrate (45). This enzyme contributed about 20% of the total GHB-DH activity in rat kidney (45).

Our results prove that AKR7A2 is not the enzyme responsible for the NADP- or NAD-dependent oxidation of GHB to SSA in SH-SY5Y cells, indicating that another enzyme is involved. The preference of the total cellular activity we detected was for NAD, in contrast to the results from hamster brain cytosol that was previously identified as being NADP-dependent, although there was some NAD-dependent activity at high concentrations of GHB (14). However, the NAD-dependent GHB-DH that we have detected appears to be mostly mitochondrial, and this location may have precluded its detection in these earlier studies that focused only on the cytosol (14). Given that most alcohol dehydrogenase enzymes are NAD-dependent, and coupled with the fact that the NAD-dependent activity we observe is inhibitable by 4-methylpyrazole, the evidence suggests that the predominant GHB-DH in SH-SY5Y cells is an alcohol dehydrogenase. Several mitochondrial ADH from the ADH4 family have been identified in yeast and other fungi (46), but to date only one mitochondrial ADH enzyme has been identified in mammals (47).

Recent purification of a mitochondrially located hydroxyacid-oxoacid transhydrogenase that catalyzes the oxidation of GHB in rat liver revealed its identity as an iron-dependent alcohol dehydrogenase related to the human ADHFE1 gene (19, 47). This enzyme is related to an NAD-dependent GHB-DH from Clostridium kluyveri (48), and an NAD-dependent Escherichia coli lactaldehyde reductase (49), and it is likely to contain a tightly bound NAD (19, 45). However, the hydroxyacid-oxoacid transhydrogenase enzyme does not appear to require added NAD, and its activity is dependent on the presence of {alpha}-ketoglutarate (19), features that appear to distinguish it from the activity we have detected.

In addition to the NAD-dependent GHB-DH activity we detected, we have also shown that there is an NADP-dependent GHB-DH present that is inhibitable by sodium valproate. This enzyme is likely to be an AKR, although the RNAi experiments show that it is not AKR7A2. A likely contender would be the high Km aldehyde reductase AKR1A1, which is known to exist in brain and which has been proposed previously to catalyze the oxidation of GHB to SSA (14).

In summary, we have provided strong evidence that AKR7A2 is the major SSA reductase in SH-SY5Y cells, and we have also shown that it is not the enzyme responsible for GHB oxidation, contrary to earlier speculation. We have proposed that an alcohol dehydrogenase is responsible for the majority of cellular GHB-DH activity. Identification of the enzymes responsible for GHB oxidation will be the focus of future studies.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported by a Bell College Research Fund studentship. Back

2 To whom correspondence should be addressed: Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 204 George St., Glasgow G1 1XW, Scotland, UK. Tel.: 44-141-548-2122; Fax: 44-141-553-4124; E-mail: elizabeth.ellis{at}strath.ac.uk.

3 The abbreviations used are: GHB, {gamma}-hydroxybutyrate; SSA, succinic semialdehyde; AKR, aldo-keto reductase; SSAR, succinic semialdehyde reductase; GHB-DH, GHB dehydrogenase; SSADH, succinic semialdehyde dehydrogenase; RNAi, RNA interference; RT, reverse transcription; GABA, {gamma}-aminobutyrate; siRNA, short interfering RNA; ANOVA, analysis of variance; GC-MS, gas chromatography-mass spectrometry; ADH, alcohol dehydrogenase. Back


   ACKNOWLEDGMENTS
 
We thank Eve Lutz for expert advice on cell lines.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bessman, S. P., and Fishbein, W. N. (1963) Nature 200, 1207–1208[CrossRef][Medline] [Order article via Infotrieve]
  2. Roth, R., and Giarman, J. (1970) Biochem. Pharmacol. 19, 1087–1093[CrossRef]
  3. Tabakoff, B., and Wartburg, J.-P. (1975) Biochem. Biophys Res. Commun. 63, 957–966[CrossRef][Medline] [Order article via Infotrieve]
  4. Kleinschmidt, S., Schellhase, C., and Mertzlufft, F. (1999) Eur. J. Anaesthesiol. 16, 23–30[Medline] [Order article via Infotrieve]
  5. Tunnicliff, G., and Raess, B. U. (2002) Curr. Opin. Investig. Drugs 3, 278–283[Medline] [Order article via Infotrieve]
  6. Nicholson, K., and Robert, L. (2001) Drug Alcohol Depend. 63, 1–22[Medline] [Order article via Infotrieve]
  7. Park, J., Osei, Y., and Churchich, J. (1993) J. Biol. Chem. 268, 7636–7639[Abstract/Free Full Text]
  8. Cash, C. D., Maitre, M., and Mandel, P. (1979) J. Neurochem. 33, 1169–1175[CrossRef][Medline] [Order article via Infotrieve]
  9. Hoffman, P., Wermuth, B., and von Wartburg, J.-P. (1980) J. Neurochem. 35, 354–366[CrossRef][Medline] [Order article via Infotrieve]
  10. Schaller, M., Schaffhauser, M., Sans, N., and Wermuth, B. (1999) Eur. J. Biochem. 265, 1056–1060[Medline] [Order article via Infotrieve]
  11. O'Connor, T., Ireland, L. S., Harrison, D. J., and Hayes, J. D. (1999) Biochem. J. 343, 487–504[CrossRef][Medline] [Order article via Infotrieve]
  12. Gold, B. I., and Roth, R. H. (1977) J. Neurochem. 28, 1069–1073[CrossRef][Medline] [Order article via Infotrieve]
  13. Gibson, K. M., Hoffmann, G. F., Hodson, A. K., Bottiglieri, T., and Jakobs, C. (1998) Neuropediatrics 29, 14–22[Medline] [Order article via Infotrieve]
  14. Kaufman, E. E., Nelson, T., Goochee, C., and Sokoloff, L. (1979) J. Neurochem. 32, 699–712[CrossRef][Medline] [Order article via Infotrieve]
  15. Vayer, P., Schmitt, M., Bourguignon, J. J., Mandel, P., and Maitre, M. (1985) FEBS Lett. 190, 55–60[CrossRef][Medline] [Order article via Infotrieve]
  16. Kaufman, E. E., Relkin, N., and Nelson, T. (1983) J. Neurochem. 40, 1639–1646[CrossRef][Medline] [Order article via Infotrieve]
  17. Struys, E. A., Verhoeven, N. M., Jansen, E. E., Ten Brink, H. J., Gupta, M., Burlingame, T. G., Quang, L. S., Maher, T., Rinaldo, P., Snead, O. C., Goodwin, A. K., Weerts, E. M., Brown, P. R., Murphy, T. C., Picklo, M. J., Jakobs, C., and Gibson, K. M. (2006) Metabolism 55, 353–358[CrossRef][Medline] [Order article via Infotrieve]
  18. Struys, E. A., Verhoeven, N. M., Ten Brink, H. J., Wickenhagen, W. V., Gibson, K. M., and Jakobs, C. (2005) J. Inherit. Metab. Dis. 28, 921–930[CrossRef][Medline] [Order article via Infotrieve]
  19. Kardon, T., Noel, G., Vertommen, D., and Schaftingen, E. V. (2006) FEBS Lett. 580, 2347–2350[CrossRef][Medline] [Order article via Infotrieve]
  20. Bhattacharya, I., and Boje, K. (2004) J. Pharmacol. Exp. Ther. 311, 92–98[Abstract/Free Full Text]
  21. Snead, O. C. (2000) J. Neurochem. 75, 1986–1996[CrossRef][Medline] [Order article via Infotrieve]
  22. Gervasi, N., Monnier, Z., Vincent, P., Paupardin-Tritsch, D., Hughes, S., Crunelli, V., and Leresche, N. (2003) J. Neurosci. 23, 11469–11478[Abstract/Free Full Text]
  23. Muller, C., Viry, S., Miche, M., Andriamampandry, C., Aunis, D., and Maitre, M. (2002) J. Neurochem. 80, 899–904[CrossRef][Medline] [Order article via Infotrieve]
  24. Buzzi, A., Wu, Y., Frantseva, M. V., Perez Velazquez, J. L., Cortez, M. A., Liu, C. C., Shen, L. Q., Gibson, K. M., and Snead, O. C., III (2006) Brain Res. 1090, 15–22[CrossRef][Medline] [Order article via Infotrieve]
  25. Elbashir, S., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001) Nature 411, 494–498[CrossRef][Medline] [Order article via Infotrieve]
  26. Caplen, N., Parrish, S., Imani, F., Fire, A., and Morgan, R. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 9742–9747[Abstract/Free Full Text]
  27. Biedler, J. L., Roffler-Tarlov, S., Schachner, M., and Freedman, L. S. (1978) Cancer Res. 38, 3751–3757[Abstract/Free Full Text]
  28. Li, D., Hinshelwood, A., Gardner, R., McGarvie, G., and Ellis, E. M. (2006) Toxicology 226, 172–180[CrossRef][Medline] [Order article via Infotrieve]
  29. Laemmli, U. K. (1970) Nature 227, 680–685[CrossRef][Medline] [Order article via Infotrieve]
  30. Short, D. M., Lyon, R., Watson, D. G., Barski, O. A., McGarvie, G., and Ellis, E. M. (2006) Toxicol. Appl. Pharmacol. 210, 163–170[CrossRef][Medline] [Order article via Infotrieve]
  31. Villain, M., Cirimele, V., Ludes, B., and Kintz, P. (2003) J. Chromatogr. B 792, 83–87[CrossRef]
  32. Kuhla, B., Luth, H. J., Haferburg, D., Weick, M., Reichenbach, A., Arendt, T., and Munch, G. (2006) J. Neurosci. Res. 83, 1591–1600[CrossRef][Medline] [Order article via Infotrieve]
  33. Barski, O. A., Gabbay, K. H., Grimshaw, C. E., and Bohren, K. M. (1995) 34, 11264–11275
  34. Picklo, M. J., Olson, S. J., Hayes, J. D., Markesbery, W. R., and Montine, T. J. (2001) Brain Res. 916, 229–238[CrossRef][Medline] [Order article via Infotrieve]
  35. Ireland, L. S., Harrison, D. J., Neal, G. E., and Hayes, J. D. (1998) Biochem. J. 332, 21–34[Medline] [Order article via Infotrieve]
  36. Nelson, T., Kaufman, E., Kline, J., and Sokoloff, L. (1981) J. Neurochem. 37, 1345–1348[CrossRef][Medline] [Order article via Infotrieve]
  37. Mamelak, M. (2007) Neurobiol. Aging 28, 1340–1360[CrossRef][Medline] [Order article via Infotrieve]
  38. Roth, R. H., Delgado, J. M., and Giarman, N. J. (1966) Int. J. Neuropharmacol. 5, 421–428[CrossRef][Medline] [Order article via Infotrieve]
  39. Roth, R. H., and Giarman, N. J. (1968) Biochem. Pharmacol. 17, 735–739[CrossRef][Medline] [Order article via Infotrieve]
  40. Barker, S. A., Snead, O. C., Poldrugo, F., Liu, C. C., Fish, F. P., and Settine, R. L. (1985) Biochem. Pharmacol. 34, 1849–1852[CrossRef][Medline] [Order article via Infotrieve]
  41. Doherty, J. D., Snead, O. C., and Roth, R. H. (1975) Anal. Biochem. 69, 268–277[CrossRef][Medline] [Order article via Infotrieve]
  42. Mamelak, M. (1989) Neurosci. Biobehav. Rev. 13, 187–198[CrossRef][Medline] [Order article via Infotrieve]
  43. Walkenstein, S. S., Wiser, R., Gudmundsen, C., and Kimmel, H. (1964) Biochim. Biophys. Acta 86, 640–642[Medline] [Order article via Infotrieve]
  44. Cromlish, J., and Flynn, T. (1985) J. Neurochem. 44, 1485–1493[CrossRef][Medline] [Order article via Infotrieve]
  45. Kaufman, E., Nelson, T., Fales, H., and Levin, D. (1988) J. Biol. Chem. 263, 16872–16879[Abstract/Free Full Text]
  46. Crichton, P. G., Affourtit, C., and Moore, A. L. (2007) Biochem. J. 401, 459–464[CrossRef][Medline] [Order article via Infotrieve]
  47. Deng, Y., Wang, Z., Gu, S., Ji, C., Ying, K., Xie, Y., and Mao, Y. (2002) DNA Seq. 13, 301–306[Medline] [Order article via Infotrieve]
  48. Sohling, B., and Gottschalk, G. (1996) J. Bacteriol. 178, 871–880[Abstract/Free Full Text]
  49. Montella, C., Bellsolell, L., Perez-Luque, R., Badia, J., Baldoma, L., Coll, M., and Aguilar, J. (2005) J. Bacteriol. 187, 4957–4966[Abstract/Free Full Text]
  50. Bradford, M. M. (1976) Anal Biochem. 72, 248–254[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
282/36/25986    most recent
M702465200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lyon, R. C.
Right arrow Articles by Ellis, E. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lyon, R. C.
Right arrow Articles by Ellis, E. M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement