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J. Biol. Chem., Vol. 282, Issue 36, 25986-25992, September 7, 2007

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

ROLE OF THE ALDO-KETO REDUCTASE AKR7A2^*

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

-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.

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

^* 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.

¹ Supported by a Bell College Research Fund studentship.

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.

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