Valproate uncompetitively inhibits arachidonic acid acylation by rat acyl-CoA synthetase 4: Relevance to valproate's efficacy against bipolar disorder

Abstract

Background: The ability of chronic valproate (VPA) to reduce arachidonic acid (AA) turnover in brain phospholipids of unanesthetized rats has been ascribed to its inhibition of acyl-CoA synthetase (Acsl)-mediated activation of AA to AA-CoA. Our aim was to identify a rat Acsl isoenzyme that could be inhibited by VPA in vitro. Methods: Rat Acsl3-, Acsl6v1- and Acsl6v2-, and Acsl4-flag proteins were expressed in E. coli, and the ability of VPA to inhibit their activation of long-chain fatty acids to acyl-CoA was estimated using Michaelis–Menten kinetics. Results: VPA uncompetitively inhibited Acsl4-mediated conversion of AA and of docosahexaenoic (DHA) but not of palmitic acid to acyl-CoA, but did not affect AA conversion by Acsl3, Acsl6v1 or Acsl6v2. Acsl4-mediated conversion of AA to AA-CoA showed substrate inhibition and had a 10-times higher catalytic efficiency than did conversion of DHA to DHA-CoA. Butyrate, octanoate, or lithium did not inhibit AA activation by Acsl4. Conclusions: VPA's ability to inhibit Acsl4 activation of AA and of DHA to their respective acyl-CoAs, when related to the higher catalytic efficiency of AA than DHA conversion, may account for VPA's selective reduction of AA turnover in rat brain phospholipids, and contribute to VPA's efficacy against bipolar disorder.

Introduction

Valproate (VPA), a branched-chain achiral eight-carbon isooctanoic acid, is approved by the FDA for treating bipolar disorder (BD), but it is teratogenic and its efficacy against BD is incomplete [1]. Understanding the mechanism of action of VPA and its brain target as a basis of its efficacy against BD may help to design more effective, less toxic drugs [2].

One suggested mechanism of action of VPA against BD, as well as of carbamazepine and lithium, is based on evidence that each of these mood stabilizers, when given chronically to rats to produce therapeutically relevant plasma concentrations, downregulated arachidonic acid (AA, 20:4n−6) but not docosahexaenoic acid (DHA, 22:6n−3) or (in the case of lithium) palmitate (16:0) turnover in brain phospholipids. This common effect agrees with the fact that the postmortem BD brain demonstrates upregulated markers of AA metabolism, associated with neuroinflammation, excitotoxicity and apoptosis [2], [3], [4], [5]. Similar associations between these neuropathological processes and upregulated brain AA metabolic markers have been identified in animal models [6], [7], [8].

Reduced brain AA turnover in unanesthetized rats given lithium or carbamazepine correlates with downregulation of mRNA, protein and activity of Ca²⁺-dependent cytosolic phospholipase A₂ (cPLA₂) type IV, which selectively hydrolyzes AA (compared with DHA) from membrane phospholipid in vitro [2], [9], [10], as well with reduced activity of cyclooxygenase (COX)-2; COX-2 is functionally coupled to cPLA₂ and converts AA to prostaglandin E₂ and other proinflammatory eicosanoids [11], [12], [13], [14]. Chronic VPA did not downregulate rat brain cPLA₂, but did reduce net brain COX activity and protein levels of both COX-1 and COX-2 [15]. VPA also reduced the rate of conversion of AA to AA-CoA, but not of DHA to DHA-CoA, in a rat brain microsomal fraction having acyl-CoA synthetase (Acsl, E.C.6.2.1.3) activity [16]. VPA itself is not a substrate for rat brain microsomal Acsl [16], and neither valproyl-CoA nor esterified VPA was found in the brain of rats following chronic administration of the drug [17]. Inhibition of conversion to acyl-CoA may underlie VPA's selective reduction of AA turnover in rat brain phospholipids in vivo [18].

Twenty-six human ACSL genes have been identified within five subfamilies, based on chain length of the preferred acyl groups [19]. Five human long-chain Acsl proteins have been characterized, represented by multiple splice variants, yielding 15 isoenzymes that preferably acylate fatty acids of 12–22 carbon length. In rats, each of four ACSL genes (ACSL1, ACSL3, ACSL4, and ACSL5) is represented by only one variant, whereas ACSL6 is represented by two splice variants (ACSL6v1 and ACSL6v2) [20]. Each isozyme has a distinct tissue distribution, subcellular location and fatty acid preferences. Acsl3, Acsl6v1, and Acsl6v2 are the predominant isoforms in rat brain, whereas Acsl1 and Acsl5 are expressed mainly in liver and adipose tissue [21], [22], [23]. Acsl3 and Acsl6 act on AA preferentially among C₁₄–C₂₂ unsaturated fatty acids, compared to Acsl1 and Acsl5 [23]. Acsl4 has a marked preference for AA [23], and is expressed in neurons but not glial cells in the human cerebellum and hippocampus, where it may be required for dendritic spine formation [24], [25]. No mammalian Acsl enzyme has been crystallized or has a published structure.

In view of the finding that conversion of AA compared with DHA to its respective acyl-CoA by the rat brain microsomal fraction was inhibited by VPA [16], we thought it worthwhile to try to identify a specific rat Acsl whose conversion of AA to AA-CoA could be inhibited by VPA. To do this, we quantified inhibition by VPA of AA, DHA and palmitic acid conversion to their respective acyl-CoAs with recombinant rat Acsl3, Acsl4 and Acsl6 isoenzymes. We did not study Acsl1 or Acsl5 because of their reported low selectivity for AA and their low distribution in the brain compared to the other three Acsl isoenzymes (see above).