Inhibition of hepatic carnitine palmitoyl-transferase I (CPT IA) by valproyl-CoA as a possible mechanism of valproate-induced steatosis
Graphical abstract
Introduction
Carnitine palmitoyl-transferase I (CPT I, EC 2.3.1.21) is a mitochondrial outer membrane protein that is part of the carnitine shuttle, and catalyses the conversion of cytosolic long-chain (LC) acyl-CoA esters to the respective LC-acylcarnitine (AC) esters, allowing the transfer of LC-acyl-CoAs into the mitochondrial matrix for β-oxidation [1], [2]. The activity of this enzyme is inhibited by malonyl-CoA [3], [4], which is the carboxylation product of acetyl-CoA mediated by acetyl-CoA carboxylase (ACC) [5], and is also an intermediate in the de novo synthesis of LC-fatty acids (LCFA) [5], [6], [7]. It has been shown that malonyl-CoA regulates mitochondrial fatty acid oxidation (FAO) in a variety of tissues, including the liver, muscle, the pancreatic β-cell, endothelium, adipocytes and the central nervous system [6]. Consequently, a rise in malonyl-CoA levels (during a fed state) will decrease the mitochondrial fatty acid uptake and oxidation, whereas a decrease in malonyl-CoA (during starvation) promotes LCFA oxidation, since CPT I becomes uninhibited [5], [8].
Valproic acid (VPA) is currently the most widely used anticonvulsive drug worldwide, prescribed for the control of epileptic episodes. This C8-branched chain fatty acid is mainly metabolized in mitochondria by FAO, and via a minor pathway in the endoplasmic reticulum, yielding Δ4-valproic acid. Both free acids can be activated to valproyl-CoA (VP-CoA) and Δ4-valproyl-CoA (Δ4-VP-CoA), respectively, not only in the mitochondrial matrix but also in the extra-mitochondrial compartment [9]. In mitochondria, VP-CoA can be fully metabolized by β-oxidation [10], [11] to acetyl- and propionyl-CoA, but its metabolic fate in the extra-mitochondrial space is still unknown. Consequently, these CoA esters may potentially affect numerous cellular functions besides mitochondrial FAO, triggering important consequences for the imbalance of the energetic state of the cell and for the metabolic fate of the drug.
Three different CPT I isoforms have been described in mammalian tissues: the liver (L-CPT I or CPT IA), muscle (M-CPT I or CPT IB) and brain (CPT IC) isoforms, which are encoded by different genes [12], [13], [14]. The kinetic characteristics of CPT IA and CPT IB differ in several important aspects. CPT IB is more sensitive to malonyl-CoA [2], [15], [16], [17] whereas CPT IA has higher affinity for l-carnitine, one of the substrates [16], [18], [19]. The requirement for carnitine and the sensitivity to malonyl-CoA appear to be inversely related [20].
CPT IA is unique in responding to different physiological states (e.g. starvation and insulin deficiency) by changing its sensitivity to malonyl-CoA several fold. This occurs as a response to changes in the lipid composition of the membrane where it is localized, and with which it interacts through its two transmembrane (TM) domains. The polytopic membrane topology of the protein results in both a short regulatory N-terminal segment and a large catalytic C-terminal segment, both exposed to the cytosolic side of the outer mitochondrial membrane [1], [2], [15], [16], [18]. The N-terminal domain, which contains both TM1 and TM2 domains, was shown to be responsible for mitochondrial import and for maintenance of a folded enzymatically active and malonyl-CoA-sensitive conformation [19]. Moreover, the nature of the cytosolic N–C (N- and C-terminal domain) interactions determines the degree of malonyl-CoA sensitivity of the liver isoform [2], [21]. Faye et al. demonstrated that mutations in the regulatory regions of the N-terminal domain affect the ability of this segment to interact physically with the C-terminal domain, either by increasing [S24A/Q30A] or lowering [E3A] the sensitivity of CPT I for malonyl-CoA [2]. CPT IA adopts different conformational states that differ in their degree of proximity between the cytosolic N-terminal and the C-terminal domains (intramolecular N/C interactions), and this determines its degree of malonyl-CoA sensitivity depending on the physiological state [1], [2], [15], [18], [22], [23], [24]. Recently it was shown that the sequence spanning the intermembrane loop-TM2 boundary determines the disposition of this TM in the membrane so as to alter the conformation of the C-terminal catalytic domain, and thus malonyl-CoA sensitivity [1], [23], [24].
Over the last decades it has become clear that the role of the malonyl-CoA-CPT I interaction is crucial both in hepatic and nonlipogenic tissues, such as heart, skeletal muscle, pancreatic β-cell. It regulates glucose and fatty acid metabolism in response to different physiological and hormonal states. In several clinical studies involving patients subjected to VPA treatment, a significant weight gain is often described [25], [26], [27] as an unwanted side effect. Valproic acid-related weight gain was originally reported to be associated with hyperinsulinemia, probably explained by the fact that VPA might inhibit the metabolism of insulin in the liver. However, it seems that VPA-induced hyperinsulinemia is independent of the drug-related weight gain, and may actually precede weight gain [26]. The mechanisms responsible for this side effect associated with VPA therapy are still not clear and require clarification.
The present studies were designed to test the hypothesis that VP-CoA and Δ4-VP-CoA, formed in the cytosol, may interact with the carnitine shuttle at the level of CPT I activity, since the membrane topography of this enzyme dictates that both the active and regulatory (malonyl-CoA-binding) sites of CPT I are exposed to the cytosolic face of the outer membrane [1], [15], [16], [18], [23], [24]. Our results unequivocally demonstrate that both CoA esters derived from VPA interfere with CPT IA activity, affecting its sensitivity to malonyl-CoA and its intrinsic regulation. As a consequence, the rate of LCFA oxidation and the regulation of mitochondrial FAO is affected, as previously reported by our group [11], [28]. This may partially explain the reported weight gain and fatty liver associated with VPA treatment.
Section snippets
Chemicals
VPA, HSA, l-carnitine, malonyl-CoA, bicinchoninic acid (BCA), digitonin and other standard biochemicals were obtained from Sigma–Aldrich. Complete mini protease inhibitor cocktail tablets were from Roche. The cell culture medium F-10 (Ham) Nutrient Mixture (25 mM HEPES + l-glutamine) and the trypsin-EDTA solution were acquired from Gibco. Potassium cyanide, butanol and acetylchloride were from Merck. Acetonitrile (ACN) gradient grade was obtained from Biosolve. The [2H]3-C3, [2H]3-C8 and [2H]3
CPT I activity in human fibroblasts
The first aim of the present study was to elucidate whether valproyl-CoA had any effect on CPT I activity using control human skin fibroblasts permeabilized with digitonin, using a recently developed method [35]. The hepatic isoform of CPT I (CPT IA) is the only one expressed in human fibroblasts [37], [38]. The results of Fig. 1 show that valproyl-CoA is a potent inhibitor of CPT I comparable to malonyl-CoA, a well-known inhibitor of this enzyme. A similar result was also observed using Δ4
Discussion
A significant weight gain and hepatotoxicity with steatosis have been frequently reported in patients treated with VPA [10], [11], [25], [26]. However, the mechanisms underlying these adverse effects of VPA are not completely understood. It has been well demonstrated that VPA interferes with several metabolic pathways, notably with mitochondrial energy metabolism. Previous studies from our group have shown a clear impairment of mitochondrial LC-FAO [11], [28] induced by this drug, supported by
Acknowledgement
This work was financially supported by Fundação para a Ciência e a Tecnologia (FCT), Lisboa, Portugal, by a grant awarded to Cátia C.P. Aires (SFRH/BD/22420/2005).
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