Journal of Cardiovascular Pharmacology:
Fenofibrate Increases Homocystinemia Through a PPARα-Mediated Mechanism
Luc, Gérald MD*; Jacob, Nelly MD†; Bouly, Muriel PhD*; Fruchart, Jean-Charles PhD*; Staels, Bart PhD*; Giral, Philippe PhD‡
From the *Department of Atherosclerosis, Institute Pasteur of Lille, France and Faculté de Pharmacie, University Lille II, France; †Laboratoire de Biochimie C, Groupe Hospitalier Pitié-Salpêtrière, Paris, France; and ‡Service d'Endocrinologie-Métabolisme, Hôpital de la Pitié, Paris, France.
Received for publication September 6, 2003; accepted December 8, 2003.
Reprints: Gérald Luc, Department of Atherosclerosis, Insert U545, Institute Pasteur of Lille, France and Faculté de Pharmacie, University Lille II, France (e-mail: Gerald.Luc@pasteur-lille.fr).
Plasma homocysteine levels increase in humans treated with fibrates but the molecular mechanisms are unknown. The goal of the present study was to determine the mechanism of this increase using animal models. Firstly, an increase in homocysteine was observed in mice treated with fenofibrate irrespective of the genetic background C57BL/6 or SV129. Secondly, as the effect of fenofibrate on gene expression is mediated through activation of the peroxisome proliferator-activated receptor alpha (PPARα), a transcription factor belonging to the nuclear receptor family, it was determined whether the effect of fenofibrate on homocysteine levels were modulated through PPARα activation. Using PPARα-deficient mice, it was shown that the homocysteine increase after fenofibrate treatment was completely abolished in these animals. It can be concluded that fibrates increase homocystinemia through a PPARα-mediated mechanism and that mice constitute an animal model for analyzing the molecular mechanisms behind the homocysteine increase after fibrate therapy in dyslipidemic patients.
Fibrates constitute a class of frequently used normolipidemic drugs that effectively decrease triglyceride-rich lipoprotein and low-density lipoprotein and increase high-density lipoprotein (HDL) plasma levels in humans. They exert their effects, at least in part, through alterations in the transcription of genes encoding for proteins that control lipoprotein metabolism. 1 Indeed, fibrates activate specific transcription factors termed peroxisome proliferator-activated receptors (PPARs), a subfamily of nuclear receptors. 2 Particularly, the PPARα form mediates fibrate action on a number of genes involved in lipoprotein metabolism, fatty acid metabolism, and inflammation in the vascular wall. 1
Homocysteine is an intermediate compound formed during metabolism of methionine. It depends on a number of enzymes involved in the methionine cycle, folate cycle, and transsulfuration pathway. Folic acid, vitamin B6, and vitamin B12 serve as cofactors for a number of these enzymes. 3
Besides the effect on lipids, fenofibrate and bezafibrate were reported to increase plasma homocysteine concentrations in dyslipidemic patients in non-randomized, non-controlled clinical trials. 4–7 Among fibrates, only gemfibrozil has been demonstrated to increase homocysteine levels in a randomized controlled trial. 8 The mechanism of homocysteine increase in patients treated with fibrates remains to be established. Since fibrates induce the expression of several genes through PPARα activation, the aim of the present study was to investigate the role of PPARα in the fenofibrate-induced increase in homocysteine levels. To this end, we studied PPARα wild type (PPARα +/+) and homozygous-deficient mice (PPARα −/−).
MATERIALS AND METHODS
Mice used in this study were non-transgenic C57BL/6 mice, homozygous PPARα deficient mice (PPARα −/−), 9 and their wild type controls (PPARα+/+). PPARα−/− mice were obtained by disrupting the ligand-binding domain of the PPARα by homologous recombination. 9 PPARα−/− and PPARα+/+ mice had the SV129 genetic background; 10- to 16-week-old mice were caged in an animal room with an alternating 12-hour light (7 am to 7 pm) and dark (7 pm to 7 am) cycle. Every group of mice was equally subdivided and fed either the control or fenofibrate (0.2% wt/wt) mixed in chow diet ad libitum for 2 weeks. Body weight and food intake of the mice were regularly checked during the treatment period. No toxic side effect of fenofibrate treatment as checked by body weight changes was observed. Blood was taken from the retroorbital plexus from mice after a 2-hour fast. Plasma was immediately separated by centrifugation at 3000 rpm for 15 minutes, then frozen at −80°C until analysis. Plasma thiols were reduced by tri-n-butylphosphine, then derivated with sulfhydryl 7-fluoro-2,1,3-benzoxadialzole-4-sulfonamide. Total homocysteine was determined by high-performance liquid chromatography and fluorescence detection, as previously described. 10 The coefficient of variation of homocysteine levels was 5.5%.
Effect of Fenofibrate in C57BL/6 Mice
In a first experiment, it was evaluated whether mice increase plasma homocysteine levels when treated with fenofibrate in a manner similar to humans. Therefore animals (n = 5) were treated with fenofibrate mixed in diet (0.2%, wt/wt) while a similar group of animals were fed with chow diet. Fenofibrate treatment increased homocysteine levels from 7.6 ± 0.9 μmol/L in nontreated mice to 15.0 ± 3.1 μmol/L in mice treated with fenofibrate. This difference was significant (P < 0.01). Thus, as in humans, fenofibrate treatment increased homocysteine levels in C57BL/6 mice.
Effect of Fenofibrate in PPARα-Deficient Mice
To determine whether fenofibrate increased homocysteine through PPARα activation, mice deficient in PPARα and their wild type littermates were treated with fenofibrate 0.2%. Results are presented in Table 1. Fenofibrate significantly increased homocysteine in PPARα+/+ mice by 2.5-fold (ie, to the same extent as in C57BL/6 mice) whereas no significant changes were observed in PPARα−/− mice. Thus, fenofibrate exerts its effect on homocysteine levels via PPARα.
Since fibrate therapy in humans increases plasma homocysteine, the objective of this study was to examine if this increase is mediated by PPARα activation. Therefore, different mice were studied. In the first experiment, it was shown that, as in humans, fenofibrate treatment increased plasma homocysteine levels in wild type mice. The increase in homocysteine was observed in mice with 2 different genetic backgrounds. Secondly, this increase did not occur in PPARα-deficient mice. It can be concluded that fenofibrate increases homocysteine through PPARα activation. Several molecular mechanisms may lie at the basis of these effects. In humans, clinical studies suggest that fibrates such as fenofibrate and bezafibrate increase homocysteine, cysteine, and creatinine plasma levels. 4,7,10 Furthermore, it has been demonstrated that the increase in creatinine was not the consequence of a decrease in the glomerular filtration rate. 11 These findings therefore suggest that one or several enzymes involved in the metabolism of these substrates are modulated by fibrate treatment. A key enzyme common to the metabolism of these substrates is S-adenosyl transferase whose increase in activity could explain the metabolic findings. Therefore, a hypothesis is that PPARα increases the expression of methionine-adenosyl transferase, which transforms methionine into S-adenosyl methionine. However, as several enzymes contribute to the synthesis and catabolism of homocysteine, it is plausible that one or several enzymes involved in homocysteine synthesis are regulated by PPARα activation and/or that other enzymes metabolizing homocysteine to cystathionine or back to methionine are repressed. Just as for genes modulated by the PPARα pathway, it appears likely that the promoters of these genes may contain a functional PPRE.
Alternatively, as folic acid, vitamin B6, and vitamin B12 are importantly involved in homocysteine metabolism, it could be possible that fibrates modify the vitamin status in mice, although this hypothesis has been excluded in humans. 4
In conclusion, our results show that the homocysteine level increases in mice treated with fenofibrate; this effect is mediated by PPARα. Thus, mice are an excellent model to analyze the molecular mechanism of the homocysteine increase after fibrate therapy in dyslipidemic patients.
The authors gratefully acknowledge Frank J. Gonzales, Laboratory of Metabolism, National Cancer Institute, National Institute of Health, Bethesda, Maryland, USA for obtaining PPARα-deficient mice.
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