Synthesis and Catabolism of γ-Hydroxybutyrate in SH-SY5Y Human Neuroblastoma Cells

Abstract

␥-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.

ADHFE1 is a breast cancer oncogene and induces metabolic reprogramming

“…in Figure 1D, MYC induced not only ADHFE1 in HMEC-MYC cells but also the aldo-keto reductase AKR7A2, a key enzyme in the synthesis of 4-hydroxybutyrate in the brain (20). Additionally, ENCODE ChIP-seq data show that MYC has several binding sites in the AKR7A2 promoter region (Supplemental Figure 5).…”

Chronic Cerebral Hypoperfusion Induced Synaptic Proteome Changes in the rat Cerebral Cortex

“…In the present study, we identified (with at least 3 peptides) 23 proteins with significantly increased (Table 1) and 23 proteins with decreased ( Table 2) (1) - (Chihara et al, 2007), (2) - (Folci et al, 2014), (3) - (Chen et al, 2002), (4) - (Tanaka et al, 2000), (5) - (Ding and Shen, 2008), (6) - (Na et al, 2012), (7) - (Lyon et al, 2007), (8) - (Sweatt et al, 2004), (9) - (Bahn et al, 2002), (10) - (Manya et al, 1998), (11) - (Kamada et al, 2003), (12) - (McKenna et al, 2000), (13) - (Christel et al, 2012), (GO) -According to Gene Ontology database. …”

The Aldo-Keto Reductase Superfamily and its Role in Drug Metabolism and Detoxification

“…The AKR7A2 and 1A1 enzymes are expressed in the brain and neuronal cells and thus could participate in SSA metabolism (Hoffman et al, 1980;Ireland et al, 1998). Recently, using siRNA, Lyon et al have demonstrated that in 2 human neuronal cell lines: neuroblastoma derived SH-SY5Y and astrocytoma 1321N1, AKR7A2 is responsible for over 80% of the SSA reductase activity (Lyon et al, 2007). Golgi localization of this enzyme has been speculated to facilitate the secretion of GHB (Kelly et al, 2002).…”

“…The ubiquitously expressed AKR7A2 (human) and 7A4 (rat) enzymes have high affinity and catalytic efficiency for succinic semialdehyde (SSA), which is a metabolite of the neurotransmitter GABA. SSA is considered to be a physiological substrate of AKR7A2, which converts it to γ-hydroxybutyrate (GHB) and has been demonstrated to represent the major SSA reductase activity in cell lines of neuronal origin (Lyon et al, 2007). The second enzyme, AKR7A3, and its rat counterpart (AKR7A1) are significantly more efficient in reducing aflatoxin B 1 -dialdehyde (Guengerich et al, 2001).…”

“…The reverse reaction involving the oxidation of GHB to succinic semialdehyde is not inhibited by AKR7A2 siRNA and was attributed to alcohol dehydrogenase and aldehyde reductase (AKR1A1). The GHB dehydrogenase activity is mainly localized to the mitochondria, where succinic semialdehyde is deemed to enter tricarboxylic acid cycle after oxidation to succinate (Lyon et al, 2007).…”