The peroxisome proliferator-activated receptor α-selective activator ciprofibrate upregulates expression of genes encoding fatty acid oxidation and ketogenesis enzymes in rat brain

Abstract

Activated peroxisome proliferator activated receptor alpha (PPARα) protects against the cellular inflammatory response, and is central to fatty acid-mediated upregulation of the gene encoding the key ketogenic enzyme mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHS). We have previously demonstrated both PPARα and mHS expression in brain, implying that brain-targeted PPARα activators may likewise up-regulate mHS expression in brain. Thus, to attempt pharmacological activation of brain PPARα in vivo, we have administered to rats two drugs with previously defined actions in rat brain, namely the PPARα-selective activator ciprofibrate and the pan-PPAR activator valproate. Using the sensitive and discriminatory RNase protection co-assay, we demonstrate that both ciprofibrate and valproate induce mHS expression in liver, the archetypal PPARα-expressing organ. Furthermore, ciprofibrate potently increases mHS mRNA abundance in rat brain, together with lesser increases in two other PPARα-regulated mRNAs. Thus we demonstrate, for the first time, up-regulation of expression of PPARα-dependent genes including mHS in brain, with implications in the increased elimination of neuro-inflammatory lipids and concomitant increased production of neuro-protective ketone bodies.

Introduction

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHS) is the key hepatic ketogenic enzyme responsible for initiating conversion of fatty acid-derived acetyl-CoA into the ketone bodies acetoacetate and β-hydroxybutyrate (Quant, 1994, Hegardt, 1999). Overexpression of mHS is associated with hepatic hyperketogenesis, with consequent lowered blood free fatty acids in transgenic mice expressing a P-enolpyruvate carboxykinase/mHS chimeric gene (Valera et al., 1994). Conversely, genetic mutations resulting in the absence of functional mHS result in profound hypoketonemia in humans (Bouchard et al., 2001). A critical component in the regulation of hepatic fatty acid oxidation and ketogenesis is the peroxisome proliferator-activated receptor alpha (PPARα) transcription factor. PPARα mediates fatty acid induction of mHS expression in rat (Rodriguez et al., 1994) and human (Hsu et al., 2001) hepatocyte-derived cell lines. In vivo, PPARα null mice exhibit profound alterations of hepatic lipid metabolism, especially during fasting (Leone et al., 1999) and, whereas fasting induces major increases in hepatic expression of mHS in PPARα +/+ mice with accompanying hyperketonemia, this effect is abolished in PPARα –/– mice (Le May et al., 2000). Furthermore, in addition to its role as a ketogenic enzyme, human mHS protein acts as a transcription factor co-activator of PPARα that synergistically activates expression of the mHS gene itself (Meertens et al., 1998). Together, such studies indicate that PPARα/mHS ‘systems’, at the genetic and enzymic levels, play a fundamental role in fatty acid homeostasis.

We have recently demonstrated PPARα expression in rat brain, and in primary cultures of rat cortical astrocytes (Cullingford et al., 1998a). Furthermore, we have demonstrated the, hitherto unsuspected, expression of mHS in rat brain (Cullingford et al., 1998b), shown that it is subject to developmental/dietary regulation (Cullingford et al., 1998b), and have further demonstrated its up-regulation by glucocorticoids in primary cultures of neonatal cortical astrocytes (Cullingford et al., 1998c). Based on our novel observations, we now wish to examine the pharmacological activation of brain PPARα/mHS ‘systems’ and hence the potential for in vivo manipulation of brain fatty acid oxidation and ketone body production. This has clinical implications in neuro-inflammatory disease since increased oxidative elimination of pro-inflammatory fatty acids is involved in PPARα-mediated protective action against the inflammatory response, atherosclerosis and aging (Pineda Torra et al., 1999). Furthermore, ketone bodies, the products of such fatty acid oxidation/ketogenesis, possess potential protective actions against epilepsy (Stafstrom, 1999), Alzheimer’s disease (Kashiwaya et al., 2000), Parkinsonism (Kashiwaya et al., 2000) and stroke (Dardzinski et al., 2000).

A range of fatty acids, hypolipidemic drugs and peroxisome proliferators can act as ligands for PPARα in vitro (Forman et al., 1997), some of which also have potent actions within the brain. Included in this category are the anti-hyperlipidemic drug ciprofibrate and the anti-epileptic drug valproate that are peroxisome proliferators in rodents (Keller et al., 1992). Ciprofibrate is a selective and potent PPARα ligand-activator in vitro (Forman et al., 1997, Takada et al., 2000). In vivo, ciprofibrate accesses brain of both neonatal and adult rats (Singh and Lazo, 1992), such that ex vivo brain homogenates derived from ciprofibrate-treated rats exhibit potently increased capacities to oxidize radiolabelled fatty acids (Singh and Lazo, 1992). The branch chain fatty acid valproate is apparently a pan-PPAR activator in vitro (Lampen et al., 1999, Gottlicher et al., 1998) that activates both PPARα and the two remaining receptor isoforms, PPARδ and PPARγ (Lampen et al., 1999, Gottlicher et al., 1998). The potent anti-epileptic action of valproate has likewise established it as a compound with a pharmacological action in brain (Löscher and Nau, 1985).

Thus, we have performed the sensitive and discriminatory RNase protection co-assay (Cullingford et al., 1998b) on brain total RNA derived from control- or drug-treated rats and monitored changes in mHS expression, via changes in mHS mRNA abundance, together with changes in two other hepatic PPARα-regulated genes, namely acyl-CoA oxidase (ACOX) (Marcus et al., 1993) and medium chain acyl-CoA dehydrogenase (MCAD) (Gulick et al., 1994). As a co-assay control, we have analyzed expression of acyl-CoA synthase 2 (ACS2), since it is regulated by another brain PPAR isoform, PPARδ (Basu-Modak et al., 1999). We have also examined liver as a control tissue, since it expresses PPARα at high levels (Cullingford et al., 1998a).