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Gemfibrozil increases the specific binding of rat-cortex nuclear extracts to a PPRE probe
Life Sciences 73 (2003) 2927 – 2937 www.elsevier.com/locate/lifescie
Gemfibrozil increases the specific binding of rat-cortex nuclear extracts to a PPRE probe Elena Sanguino, Miguel Ramo´n, Nu´ria Roglans, Marta Alegret, Rosa M. Sa´nchez, Manuel Va´zquez-Carrera, Juan C. Laguna * Unidad de Farmacologı´a y Farmacognosia, Facultad de Farmacia, Universidad de Barcelona, Nucleo Univ. de Pedralbes, Avda Diagonal 643, 08028 Barcelona, Spain Received 16 January 2003; accepted 9 April 2003
Abstract PPAR agonists have been shown to elicit beneficial responses in several cell- and tissue-models of neurotoxicity. To determine if brain PPARs are responsive to the in vivo administration of PPAR agonists in a similar way to those receptors present in other anatomical localizations, such as liver, we fed rats with gemfibrozil incorporated in the diet at a dose that activates hepatic PPARa and produces its typical hypolipidemic effect. Rat cortex nuclear extracts presented a pattern of two specific shifted bands when incubated with a PPRE oligonucleotide. Samples from gemfibrozil-treated rats showed a significant increase in the intensity of the two shifted bands regarding control values (2.4- and 1.8-fold for the specific bands 1 and 2, respectively), indicating that orally administered gemfibrozil reaches brain tissues at concentrations sufficient to increase the specific binding of cortex nuclear extracts to an oligonucleotide mimicking a bona fide PPRE, although no changes in cortex ACO mRNA levels were produced. D 2003 Elsevier Inc. All rights reserved. Keywords: PPAR; Gemfibrozil; Rat cortex; Fibrates; ACO
Introduction Gemfibrozil is a member of the fibrate class of hypolipidaemic drugs mainly used in the treatment of hypertriglyceridaemia and mixed hyperlipidaemia (Spencer and Barradell, 1996). Fibrates elicit their
* Corresponding author. Tel.: +34-934-024-531; fax: +34-934-035-982. E-mail address: [email protected] (J.C. Laguna). 0024-3205/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2003.04.001
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biological effects by activating a type of nuclear receptors, the peroxisome proliferator-activated receptors (PPARs), acting mainly on the PPARa isoform (Kersten et al., 2000). As a consequence of fibrate administration, marked changes are produced in the expression of genes bearing a peroxisome proliferator response element (PPRE) in their promoter region (Keller et al., 2000). Among those genes, the expression of the aco gene, whose product, the enzyme acyl-CoA oxidase (ACO), controls the activity of the peroxisomal fatty acid h-oxidation system, is typically induced in liver of rats and mice after fibrate administration (Alegret et al., 1994; Berthou et al., 1995). Three isoforms of PPARs have been described, PPARa (NR1C1), PPARh (NR1C2), and PPARg (NR1C3), with differentiated tissue and cell-type distribution, and biological properties. Agonists of PPARa and PPARg, fibrates and thiazolidinediones respectively, are being increasingly used in the therapeutic management of dyslipidemia and diabetes. As knowledge of PPARs functions in the organism builds up, other therapeutic possibilities of PPAR agonists, especially as anti-inflammatory drugs, are being explored (Kersten et al., 2000; Keller et al., 2000). In this regard, the presence of the three isoforms of PPAR has been demonstrated in several areas of the Central Nervous System (CNS) (Cullingford et al., 1998) and, at least for the PPARg isoform, an increased expression has been detected in brain tissue necropsies obtained from patients suffering Alzheimer’s dementia (Kitamura et al., 1999). Further, PPARh, the most ubiquitous and abundant isoform of PPARs in the CNS, regulates the expression of neuronal acyl-CoA synthetase-2, and probably participates in the process of neural maturation and myelination (Basu-Modak et al., 1999). Finally, RXR, the heterodimerization partner of PPAR is also expressed in brain tissues, together with the mRNA encoding ACO (Cullingford et al., 1998; Knoll et al., 2000). PPAR agonists have been shown to elicit beneficial responses in several cell- and tissue-models of neurotoxicity (Combs et al., 2000; Saluja et al., 2001; Combs et al., 2001). Nevertheless, cardinal to the issue of a possible pharmacological use of PPAR agonists for the treatment of CNS diseases is to determine if brain PPARs are accessible and respond to the in vivo administration of PPAR agonists in a similar way to those receptors present in other anatomical localizations, such as liver or adipose tissue. The present report provides evidence that the oral administration of gemfibrozil to rats is able to increase the specific binding of cortex nuclear extracts to an oligonucleotide mimicking a bona fide PPRE, although no changes in cortex ACO mRNA levels are produced.
Methods Animals Male Sprague-Dawley rats (Criffa, Barcelona, Spain) weighing 207 F 11 g were divided at random into two groups of treatment and fed, respectively, 28 g/day of a standard diet (Panlab) or the same diet supplemented with gemfibrozil (0.3% weight/weight). After three days, animals were killed by decapitation under ketamine (100 mg/kg, i.p.) anesthesia between 9–10 a.m., at the beginning of the light period. Both treatment diets were prepared as described by Alegret et al. (1994). Before treatment, the animals were maintained with water and food ad libitum at constant humidity and temperature with a light/dark cycle of 12 hours (8:00 AM–8:00 PM) for a minimum of five days. All procedures were conducted in accordance with the principles and guidelines established by the University of Barcelona Bioethics Committee, as stated in Law 5/1995, 21st July, from the Generalitat de Catalunya.
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Sample preparation Blood samples were collected at the time of death in 5% EDTA-tubes; plasma was obtained by centrifugation and stored at 80 jC until needed. Livers were excised and perfused in a 150 mM NaCl, 1 mM dithiotreitol, 30 mM EDTA, 50 mM KH2PO4, pH 7.4 buffer; 0.5 g of liver tissue of each rat was homogenised in the same buffer and used for obtaining the postmitochondrial fraction by centrifugation (Montgomery and Cinti, 1977) and frozen at 80 jC until needed. 10–100 mg of liver (L), and cortex tissue of each rat was immediately frozen in liquid N2 and stored at 80 jC until used for the extraction of total RNA. Further, a fresh sample of cortex tissue of each rat was immediately used for obtaining nuclear extracts. Plasma lipids and enzyme assays Plasma total cholesterol, triglyceride, and phospholipid concentrations were measured with the Boehringer Mannheim GmbH colorimetric tests (Monotest Cholesterol CHODPAP Nj 290319, Peridochrom Triglyceride GPOPAP Nj 701882, and MPR2 Nj 691844 phospholipid, respectively). By using reagent Nj 543004, also from Boehringer Mannheim, VLDL and LDL from plasma samples were precipitated, and HDL-cholesterol concentration was determined in the supernatant. The hepatic peroxisomal fatty acid h-oxidation system was determined as described previously (Alegret et al., 1994). Protein concentration was determined by the method of Bradford (Bradford, 1976). RNA preparation and analysis Total RNA was isolated by using the Ultraspec reagent (Biotecx, Houston, USA). Relative levels of specific mRNAs were assessed by the reverse transcription-polymerase chain reaction (RT-PCR). Complementary DNA was synthesized from RNA samples by mixing 1 Ag of total RNA, 125 ng of random hexamers as primers in the presence of 50 mM Tris-HCl buffer (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 200 U Moloney murine leukemia virus reverse transcriptase (Life Technologies, Gaithersburg, USA), 20 U RNAsin (Life Technologies) and 0.5 mM of each dNTP (Sigma) in a total volume of 20 Al. Samples were incubated at 37 jC for 60 min. A 5 Al aliquot of the RT reaction was then used for subsequent PCR amplification with specific primers. Each 25-Al PCR reaction contained 5 Al of the RT reaction, 1.2 mM MgCl2, 200 AM dNTPs, 1.25 ACi [32P]-dATP (3000 Ci/mmol, Amersham pharmacia biotech), 1 unit of Taq polymerase (Life Technologies), 0.5 Ag of each primer and 20 mM Tris-HCl, pH 8.5. To avoid unspecific annealing, cDNA and Taq polymerase were separated from primers and dNTPs by using a layer of paraffin (reaction components contact only when paraffin fuses, at 60 jC). The sequences of the sense and antisense primers used for amplification were: PPARa, 5V-GGCTCGGAGGGCTCTGTCATC-3V and 5V-ACATGCACTGGCAGCAGTGGA-3V; ACO 5V-ACTATATTTGGCCAATTTTGTG3V and 5V-TGTGGCAGTGGTTTCCAAGCC-3V; RXRa, 5V-GCTCTCCAACGGGTCGAGGCT-3V and 5V-TGGGTGTGGTGGGTACCGACA-3V; PPARh, 5V-GAGGAAGTGGCCACGGGTGAC-3V and 5VCCACCTGAGGCCCCATCACAG-3V; PPARg, 5V-TGGGGATGTCTCACAATGCCA-3V and 5VTTCCTGTCAAGATCGCCCTCG-3V; and APRT (adenosyl phosphoribosyl transferase), 5VAGCTTCCCGGACTTCCCCATC-3V and 5V-GACCACTTTCTGCCCCGGTTC-3V. The aprt gene
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was used as internal control in the PCR reaction to normalize the results. PCR was performed in an MJ Research Thermocycler (Ecogen, Barcelona, Spain) equipped with a peltier system and temperature probe. After an initial denaturation for 1 min at 94 jC, PCR was performed for 23 cycles. Each cycle consisted of denaturation at 92 jC for 1 min, primer annealing at 60 jC (except 58 jC for ACO), and primer extension at 72 jC for 1 min and 50 s. A final 5-min extension step at 72 jC was performed. Five microliters of each PCR sample was electrophoresed on a 1-mmthick 5% polyacrylamide gel. The gels were dried and subjected to autoradiography using Agfa Xray films (Danny Commercial, Barcelona, Spain) to show the amplified DNA products. Amplification of each gene yielded a single band of the expected size (PPARa: 645 bp, PPARh: 151 bp, PPARg: 200 bp, ACO: 195 bp, RXRa: 202 bp, and APRT: 329 bp). Preliminary experiments were carried out with various amounts of cDNA to determine non-saturating conditions of PCR amplification for all the genes studied. Thus cDNA amplification was performed in comparative and semiquantitative conditions (Freeman et al., 1999). Radioactive bands were quantified by videodensitometric scanning (Vilbert Lourmat Imaging). The results for the expression of specific mRNAs are always presented relative to the expression of the control gene (aprt). Isolation of nuclear extracts Nuclear extracts were isolated using the Dignam method (Dignam et al., 1983) with the modifications described by Sonnenberg et al. (1989). Briefly, fresh cortex tissues were weighed and homogenized by a Potter Elvehjem homogenizer in 4 volumes (w/v) of buffer A containing 0.25M sucrose, 15 mM Tris HCl pH 7.9, 15 mM NaCl, 60 mM KCl, 1 mM EGTA, 5mM EDTA, 0.15 mM spermine, 0.5 mM spermidine and a mixture of protease inhibitors (0.1 mM phenylmethylsolfonyl fluoride, 1.0 mM dithiothreitol, 5 Ag/ml aprotinin, 2 Ag/ml leupeptin). Homogenates were incubated for 10 min. on ice and centrifuged (2,000 g, 10 min, 4 jC). Pellets were resuspended in 4 vol of buffer B (10 mM Hepes pH 7.9, 1.5 mM MgCl2, 10 mM KCl and protease inhibitors as above) and then centrifuged at 4,000 g, 4 jC, for 10 min. Supernatants were discarded and pellets were resuspended in 1 vol. of buffer C (0.5 M HEPES, pH 7.9, 0.75 mM MgCl2, 0.5 M KCl, 12.5% glycerol and the protease inhibitors). Homogenates were kept for 30 min at 4 jC under continuous rotary shaking, and then centrifuged at 14,000 g for 30 min at 4 jC. Finally, the resulting supernatants were dialyzed overnight at 4 jC with buffer D (10 mM TrisHCl, pH 7.9, 5 mM MgCl2, 10 mM KCl, 1.0 mM EDTA, 10% glycerol and the protease inhibitors). Nuclear extracts were collected in microfuge tubes and stored in aliquots at 80 jC The protein concentration of the nuclear extracts was determined by the method of Bradford (1976). Electrophoretic mobility shift assays (EMSA) The DNA sequence of the double-stranded oligonucleotide used was a consensus binding site of a PPAR response element, 5V-CAAAACTAGGTCAAAGGTCA-3V (Santa Cruz Biotechnology, Santa Cruz, CA). Oligonucleotides were labeled in the following reaction: 1 Al of oligonucleotide (20 ng/Al), 2 Al of 5x kinase buffer, 5 U. of T4 polynucleotide kinase, and 3 Al of [g-32P] ATP (3000 Ci/mmol at 10 mCi/ml, Amersham) incubated at 37 jC for 1 h. The reaction was stopped by adding 90 Al of TE buffer (10 mM Tris-HCl pH 7.4 and 1 mM EDTA). To separate the labeled probe from the unbound ATP, the reaction mixture was eluted in a Nick column (Pharmacia, Sant Cugat, Spain) according to the
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manufacturer’s instructions. 10 micrograms of crude nuclear proteins were incubated for 10 min on ice in binding buffer (10 mM Tris-HCl pH 8.0, 25 mM KCl, 0.5 mM DTT, 0.1 mM EDTA pH 8.0, 5% glycerol, 5 mg/ml BSA, 100 Ag/ml tRNA and 50 Ag/ml poly(dI-dC)), in a final volume of 15 Al. Labeled probe (approximately 50,000 cpm) was added and the reaction was incubated for 20 min. at room temperature. Where indicated, non-radioactive specific competitor or mutated PPRE oligonucleotide was added before the addition of labeled probe and incubated for 15 min on ice. Supershift assays were carried out by adding 8 Ag of a specific antibody against PPARh (Santa Cruz Biotechnology) 90 min before the radiolabelled probe. Statistics The results are the mean F standard deviation of the mean of n (3–5) experiments. Plasma samples were assayed in duplicate. Significant differences were established by an ANOVA test, using the computer program GraphPad- InStat-tm (GraphPad Software V2.03). When the number of animals was too small or the variance was not homogeneous, a non-parametric test was performed (Kruskal-Wallis test). When significant variations were found, the Student-Newman-Keuls multiple comparisons test was performed. The level of statistical significance was set at P < 0.05.
Results Gemfibrozil administration to rats reduced plasma cholesterol concentrations by 19%, affecting equally to both free and esterified cholesterol. This reduction was at the expenses of a marked decrease (40%) in the apo B-cholesterol fraction, while HDL cholesterol was not modified. Plasma triglyceride and phospholipid concentrations were also decreased by 43 and 25%, respectively, in gemfibrozil-treated animals (Table 1). It has been shown that gemfibrozil, as well as other fibrate drugs, exerts its hypolipidemic effect by activating hepatic PPARa. Consequently, hepatic levels of the specific mRNA for ACO and the activity of the peroxisomal fatty acid h-oxidation system were increased by 117 and 64%, respectively (Fig. 1). Rat cortex expressed detectable amounts of specific mRNA for ACO, RXRa and the three isoforms of PPAR (Fig. 2). As previously described (Cullingford et al., 1998), PPARh was the most abundant PPAR isoform in cortex, followed by PPARa and PPARg. Fig. 3 shows the pattern of retardation bands
Table 1 Concentrations (mg/dL) of plasma lipids in control - (CTRL) and gemfibrozil - (GFB) treated animals CTRL GFB
TC
FC
CE
HDLc
apoBc
TG
PL
108 F 12 87 F 9b
34 F 5 27 F 5a
74 F 9 60 F 7b
71 F 8 65 F 14
37 F 11 22 F 14c
117 F 18 67 F 18b
198 F 26 148 F 11b
Values are means F S.D. of 6 animals per group. TC: Total cholesterol; FC: free cholesterol; CE: cholesteryl esters; HDLc: HDL cholesterol; apoBc: apo B cholesterol; TG: triglycerides; PL: phospholipids. a p < 0.05. b p < 0.01. c Marginal statistical significance, p < 0.07.
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Fig. 1. A. Peroxisomal h-oxidation activity. Bars represent the mean F sd of the values obtained from 6 animals in each treatment group. B. Relative levels of mRNA for ACO. Bars represent the mean F sd of the values obtained from 6 animals in each treatment group. A representative autoradiography is shown in the upper part of the figure, with signals corresponding to ACO and the reference gene APRT, obtained from one control (CTR) and one gemfibrozil (GFB) treated-rat. * p < 0.05.
obtained in an EMSA performed by incubating a PPRE oligonucleotide probe and cortex nuclear extracts obtained from a control rat and a gemfibrozil-treated rat. In both samples, two retardation bands, 1 and 2, were obtained. Both bands were fully specific for the PPRE probe, as shown by the fact that they were effectively competed by incubating with increasing amounts of unlabeled probe, while no change in band intensity was detected in the presence of identical amounts of an unlabeled mutated PPRE probe. Incubation in the presence of a specific PPARh antibody produced a supershifted band
Fig. 2. Representative autoradiography showing the bands corresponding to the specific mRNAs for RXRa, PPARa, PPARh, PPARg, and the reference gene APRT in cortex samples obtained from two different rats.
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Fig. 3. EMSA assay showing the specificity of the binding of cortex nuclear extracts (NE) obtained from control (CT) rats and gemfibrozil-treated (GFB) rats to a PPRE oligonucleotide; the shifted bands are effectively competed in the presence of unlabeled probe, while no changes in band intensity are produced in the presence of mutated PPRE oligonucleotide.
(Fig. 4), indicating the participation of this transcription factor in the formation of the specific bands. When the intensities of the shifted bands were compared between samples from control and gemfibroziltreated animals, drug treatment produced a marked significant increase in the intensities of band 1 (2.4fold) and 2 (1.8-fold) (Fig. 5). However, the levels of the specific mRNA for ACO in rat cortex were not
Fig. 4. EMSA assay showing the appearance of a super-shifted band when cortex nuclear extract (NE) obtained from a gemfibrozil treated rat is incubated with a PPRE oligonucleotide in the presence of a specific PPARh antibody (see Material and Methods for details).
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Fig. 5. A. EMSA assay showing changes in the intensity of the specific retarded bands 1 and 2 obtained by incubating cortex nuclear extracts (NE) from control (CT) and gemfibrozil-treated (GFB) rats with a PPRE oligonucleotide. B. Quantification of the intensity of shifted bands 1 and 2 corresponding to the EMSA presented in Fig. 5A. * p < 0.05, ** p < 0.01.
increased in gemfibrozil-treated animals (0.54 F 0.04 vs. 0.55 F 0.09 ACO mRNA arbitrary units for control and gemfibrozil-treated rats, respectively).
Discussion Gemfibrozil is a selective, low affinity ligand for PPARa (Krey et al., 1997) and its pharmacological effects in experimental animals and humans are mainly due to the activation of the transcriptional activity of PPARa (Kersten et al., 2000; Keller et al., 2000; Alegret et al., 1994; Verthou et al., 1995). Although its therapeutic niche is in the treatment of dyslipidemia, there is an increasing interest in other possible therapeutic applications of PPARa agonists, such as putative anti-inflammatory agents for the treatment of chronic inflammatory diseases, acting by a mechanism not related to COX inhibition (Cabrero et al., 2002). Among those diseases, a growing interest in the study of anti-inflammatory therapy for the prevention of neurodegenerative diseases has been aroused, after the publication of data
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relating chronic use of antiinflammatory agents as a protective factor for Alzheimer’s disease (McGreer et al., 1996; McGreer and McGreer, 1999); classical non-steroidal anti-inflammatory drugs, such as indomethacin and ibuprofen, have been shown to activate PPARa and PPARg (Lehmann et al., 1997). Anxiety, dizziness and sleep disorders have been described as adverse effects of gemfibrozil administration to humans (Todd and Ward, 1988); nevertheless, there is no data about the penetration of gemfibrozil into the central nervous system when used at pharmacological doses. As shown in this work and previous ones from our laboratory and others (Alegret et al., 1994; Berthou et al., 1995; Todd and Ward, 1988), administration of gemfibrozil to rats, by activating hepatic PPARa, typically reduces plasma triglycerides and non-HDL colesterol, and produces hepatic peroxisome proliferation, with increases in the expression of marker enzymes such as acyl-CoA oxidase (Table 1, Fig. 1). When we assayed the binding of cortex nuclear extracts obtained from the same gemfibroziltreated animals to a PPRE oligonucleotide probe, we detected a significant increase in the intensity of the specific bands obtained in the gel-shift assay. Thus, orally administered gemfibrozil reaches brain tissues at concentrations sufficient to elicit a typical PPAR-mediated pharmacological response. However, although gemfibrozil administration increased the relative levels of ACO mRNA in liver samples, no changes in ACO mRNA expression in cortex of treated animals was observed, despite the increase intensity of the PPRE specific bands detected in the gel-shift assay. Two possible mechanisms can be put forward to explain this situation: a) Increases in the intensity of specific bands obtained after incubation of liver nuclear extracts from fibrate-treated rats to a PPRE oligonucleotide are transient in nature, preceding the increase in liver ACO mRNA observed after fibrate administration (Rodrı´guez et al., 2000). In rats and other laboratory animals, gemfibrozil presents a peak of plasma concentration after 1–2 hours of administration, with two elimination phases, with half-lives of 0.7 and 33 hours (Cayen, 1985). Thus, assuming a slow distribution of gemfibrozil in organs such as brain, there is a possibility that we failed to detect an increase in cortex ACO mRNA from gemfibrozil-treated rats because we actually obtained the samples before such phenomenon was produced. b) We and others have shown that PPARh is the most abundant PPAR isoform in brain tissues (Fig. 2, Cullingford et al., 1998). Further, PPARh, by binding to a PPRE and associating with corepressors proteins, is able to inhibit the expression of endogenous PPARa target genes, such as aco (Jow and Mukherjee, 1995; Mochizuki et al., 2001; Shi et al., 2002). As PPARh is contributing to the formation of the specific bands obtained by incubating cortex nuclear extracts with a PPRE probe (Fig. 4), it could be argued that its presence is sufficient to inhibit the gemfibrozil-induced transcriptional activity of the low levels of PPARa present
Fig. 6. Comparison of PPARa/PPARh mRNA ratios in rat cortex and liver. Bars represent the mean F sd of the values obtained from 3 animals in each treatment group. * p < 0.02.
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in cortex. Further supporting this hypothesis is the fact that the relative proportion of the mRNAs of PPARa and PPARh is 2.4-fold higher in rat liver than in cortex (Fig. 6). In conclusion, orally administered gemfibrozil, at a dose that activates hepatic PPARa and induces its typical hypolipidemic effect, is able to penetrate into the brain tissue, increasing the specific binding of cortex nuclear proteins to a PPRE oligonucleotide.
Acknowledgements This work was supported by grants from FPCNL, CICYT (SAF00/0201), FISss (01/0075-01), MCyT (BFI2002-05167), Generalitat de Catalunya (2001SGR00141) and SEA/Almirall Prodesfarma 2001. E. Sanguino holds a fellowship for Training in Teaching and Research from the University of Barcelona.
References Alegret, M., Ferrando, R., Va´zquez, M., Adzet, T., Merlos, M., Laguna, J.C., 1994. Relationship between plasma lipids and palmitoyl-CoA hydrolase and synthetase activities with peroxisomal proliferation in rats treated with fibrates. British Journal of Pharmacology 112, 551 – 556. Basu-Modak, S., Braissant, O., Escher, P., Desvergne, B., Honegger, P., Wahli, W., 1999. Peroxisome proliferator-activated receptor h regulates acyl-CoA synthetase 2 in reaggregated rat brain cell cultures. Journal of Biological Chemistry 274, 35881 – 35888. Berthou, L., Saladin, R., Yaqoob, P., Branellec, D., Calder, P., Fruchart, J.-C., Dene`fle, P., Auwerx, J., Staels, B., 1995. Regulation of rat liver apolipoprotein A-I, apolipoprotein A-II and acyl-coenzyme A oxidase gene by fibrates and dietary fatty acids. European Journal of Biochemistry 232, 178 – 187. Bradford, M., 1976. A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding. Analytical Biochemistry 72, 248 – 254. Cabrero, A., Laguna, J.C., Va´zquez, M., 2002. Peroxisome proliferator-activated receptors and the control of inflammation. Current Drug Targets in Inflammation and Alergy 1, 243 – 248. Cayen, M.N., 1985. Disposition, metabolism and pharmacokinetics of antihyperlipidemic agents in laboratory animals and man. Pharmacology and Therapeutics 29, 157 – 204. Combs, C.K., Bates, P., Colleen Karlo, J., Landreth, G.E., 2001. Regulation of h-amyloid stimulated proinflammatory responses by peroxisome proliferator-activated receptor a. Neurochemistry International 39, 449 – 457. Combs, C.K., Johnson, D.E., Colleen Karlo, J., Cannady, S.B., Landreth, G.E., 2000. Inflammatory mechanisms in Alzheimer’s disease: Inhibition of h-amyloid-stimulated proinflammatory responses and neurotoxicity by PPARg agonists. Journal of Neuroscience 20, 558 – 567. Cullingford, T.E., Bhakoo, K., Peuchen, S., Dolphin, C.T., Patel, R., Clark, J.B., 1998. Distribution of mRNAs encoding the peroxisome proliferator-activated receptor a, h and g and the retinoid X receptor a, h and g in rat central nervous system. Journal of Neurochemistry 70, 1366 – 1375. Dignam, J.D., Lebovitz, R.M., Roeder, R.G., 1983. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acid Research 11, 1475 – 1489. Freeman, W.M., Walker, S.J., Vrana, E.V., 1999. Quantitative RT-PCR: Pitfalls and potential. Biological Tecniques 26, 112 – 125. Jow, L., Mukherjee, R., 1995. The human peroxisome proliferator-activated receptor (PPAR) subtype NUC1 represses the activation of hPPARa and thyroid hormone receptors. Journal of Biological Chemistry 270, 3836 – 3840. Keller, J.M., Collet, P., Bianchi, A., Huin, C., Bouillaud-Kremarik, P., Becuwe, P., Schohn, H., Domenjoud, L., Daucßa, M., 2000. Implications of peroxisome proliferator-activated receptors (PPARs) in development, cell life status and disease. International Journal of Developmental Biology 44, 429 – 442. Kersten, S., Desvergne, B., Wahli, W., 2000. Roles of PPARs in health and disease. Nature 405, 421 – 424.
E. Sanguino et al. / Life Sciences 73 (2003) 2927–2937
2937
Kitamura, Y., Shimohama, S., Koike, H., Kakimura, J.-i., Matsuoka, Y., Nomura, Y., Gebicke-Haerter, P.J., Taniguchi, T., 1999. Increased expression of cyclooxygenases and peroxisome proliferator-activated receptor-g in Alzheimer’s disease brain. Biochemical Biophysical Research Communications 254, 582 – 586. Knoll, A., Salles, J., Sargueil, F., Cassagne, C., Garbay, B., 2000. Peroxisomal beta-oxidation enzyme gene expression in the developing mouse brain. Neuroscience Letters 285, 201 – 204. Krey, G., Braissant, O., L’Horset, F., Kalkhoben, E., Perroud, M., Parker, M.G., Wahli, W., 1997. Fatty acids, eicosanoids and hypolipidemic agents identified as ligands of peroxisome proliferator-activated receptors by CARLA. Molecular Endocrinology 11, 779 – 791. Lehmann, J.M., Lenhard, J.M., Oliver, B.B., Ringold, G.M., Kliewer, S.A., 1997. Peroxisome proliferator-activated receptors a and g are activated by indomethacin and other non-steroidal anti-inflammatory drugs. Journal of Biological Chemistry 272, 3406 – 3410. McGreer, E.G., McGreer, P.L., 1999. Brain inflammation in Alzheimer disease and the therapeutic implications. Current Pharmaceutical Design 5, 821 – 836. McGreer, P.L., Schulzer, M., McGeer, E.G., 1996. Arthritis and anti-inflammatory agents as possible protective factors for Alzheimer’s disease: A review of 17 epidemiologic studies. Neurology 47, 425 – 432. Mochizuki, K., Suruga, K., Yagi, E., Takase, S., Goda, T., 2001. The expression of PPAR-associated genes is modulated through postnatal development of PPAR subtypes in the small intestine. Biochimica Biophysica Acta 1531, 68 – 76. Montgomery, M.R., Cinti, D.L., 1977. Pyridine nucleotide-dependent electron transport in kidney cortex microsomes: interaction between desaturase and other mixed function oxidases. Molecular Pharmacology 13, 60 – 69. Rodrı´guez, C., Noe´, V., Cabrero, A., Ciudad, C.J., Laguna, J.C., 2000. Differences in the formation of PPARa-RXR/ acoPPRE complexes between responsive and nonresponsive species upon fibrate administration. Molecular Pharmacology 58, 185 – 193. Saluja, I., Granneman, J.G., Skoff, R.P., 2001. PPARy agonists stimulate oligodendrocyte differentiation in tissue culture. Glia 33, 191 – 204. Shi, Y., Hon, M., Evans, R.M., 2002. The peroxisome proliferator-activated receptor y, an integrator of transcriptional repression and nuclear receptor signaling. Proceedings of the National Academy of Sciences USA 99, 2613 – 2618. Sonnenberg, J.L., MacGregor-Leon, P.F., Curran, T., Morgan, J.I., 1989. Dynamic alterations occur in the levels and composition of transcription factor AP-1 complexes after seizures. Neuron 3, 359 – 365. Spencer, C.M., Barradell, L.B., 1996. Gemfibrozil. A reappraisal of its pharmacological properties and place in the management of dyslipidemia. Drugs 51, 982 – 1018. Todd, P.A., Ward, A., 1988. Gemfibrozil. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use un dyslipidemia. Drugs 36, 314 – 339.
Gemfibrozil increases the specific binding of rat-cortex nuclear extracts to a PPRE probe Elena Sanguino, Miguel Ramo´n, Nu´ria Roglans, Marta Alegret, Rosa M. Sa´nchez, Manuel Va´zquez-Carrera, Juan C. Laguna * Unidad de Farmacologı´a y Farmacognosia, Facultad de Farmacia, Universidad de Barcelona, Nucleo Univ. de Pedralbes, Avda Diagonal 643, 08028 Barcelona, Spain Received 16 January 2003; accepted 9 April 2003
Abstract PPAR agonists have been shown to elicit beneficial responses in several cell- and tissue-models of neurotoxicity. To determine if brain PPARs are responsive to the in vivo administration of PPAR agonists in a similar way to those receptors present in other anatomical localizations, such as liver, we fed rats with gemfibrozil incorporated in the diet at a dose that activates hepatic PPARa and produces its typical hypolipidemic effect. Rat cortex nuclear extracts presented a pattern of two specific shifted bands when incubated with a PPRE oligonucleotide. Samples from gemfibrozil-treated rats showed a significant increase in the intensity of the two shifted bands regarding control values (2.4- and 1.8-fold for the specific bands 1 and 2, respectively), indicating that orally administered gemfibrozil reaches brain tissues at concentrations sufficient to increase the specific binding of cortex nuclear extracts to an oligonucleotide mimicking a bona fide PPRE, although no changes in cortex ACO mRNA levels were produced. D 2003 Elsevier Inc. All rights reserved. Keywords: PPAR; Gemfibrozil; Rat cortex; Fibrates; ACO
Introduction Gemfibrozil is a member of the fibrate class of hypolipidaemic drugs mainly used in the treatment of hypertriglyceridaemia and mixed hyperlipidaemia (Spencer and Barradell, 1996). Fibrates elicit their
* Corresponding author. Tel.: +34-934-024-531; fax: +34-934-035-982. E-mail address: [email protected] (J.C. Laguna). 0024-3205/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2003.04.001
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biological effects by activating a type of nuclear receptors, the peroxisome proliferator-activated receptors (PPARs), acting mainly on the PPARa isoform (Kersten et al., 2000). As a consequence of fibrate administration, marked changes are produced in the expression of genes bearing a peroxisome proliferator response element (PPRE) in their promoter region (Keller et al., 2000). Among those genes, the expression of the aco gene, whose product, the enzyme acyl-CoA oxidase (ACO), controls the activity of the peroxisomal fatty acid h-oxidation system, is typically induced in liver of rats and mice after fibrate administration (Alegret et al., 1994; Berthou et al., 1995). Three isoforms of PPARs have been described, PPARa (NR1C1), PPARh (NR1C2), and PPARg (NR1C3), with differentiated tissue and cell-type distribution, and biological properties. Agonists of PPARa and PPARg, fibrates and thiazolidinediones respectively, are being increasingly used in the therapeutic management of dyslipidemia and diabetes. As knowledge of PPARs functions in the organism builds up, other therapeutic possibilities of PPAR agonists, especially as anti-inflammatory drugs, are being explored (Kersten et al., 2000; Keller et al., 2000). In this regard, the presence of the three isoforms of PPAR has been demonstrated in several areas of the Central Nervous System (CNS) (Cullingford et al., 1998) and, at least for the PPARg isoform, an increased expression has been detected in brain tissue necropsies obtained from patients suffering Alzheimer’s dementia (Kitamura et al., 1999). Further, PPARh, the most ubiquitous and abundant isoform of PPARs in the CNS, regulates the expression of neuronal acyl-CoA synthetase-2, and probably participates in the process of neural maturation and myelination (Basu-Modak et al., 1999). Finally, RXR, the heterodimerization partner of PPAR is also expressed in brain tissues, together with the mRNA encoding ACO (Cullingford et al., 1998; Knoll et al., 2000). PPAR agonists have been shown to elicit beneficial responses in several cell- and tissue-models of neurotoxicity (Combs et al., 2000; Saluja et al., 2001; Combs et al., 2001). Nevertheless, cardinal to the issue of a possible pharmacological use of PPAR agonists for the treatment of CNS diseases is to determine if brain PPARs are accessible and respond to the in vivo administration of PPAR agonists in a similar way to those receptors present in other anatomical localizations, such as liver or adipose tissue. The present report provides evidence that the oral administration of gemfibrozil to rats is able to increase the specific binding of cortex nuclear extracts to an oligonucleotide mimicking a bona fide PPRE, although no changes in cortex ACO mRNA levels are produced.
Methods Animals Male Sprague-Dawley rats (Criffa, Barcelona, Spain) weighing 207 F 11 g were divided at random into two groups of treatment and fed, respectively, 28 g/day of a standard diet (Panlab) or the same diet supplemented with gemfibrozil (0.3% weight/weight). After three days, animals were killed by decapitation under ketamine (100 mg/kg, i.p.) anesthesia between 9–10 a.m., at the beginning of the light period. Both treatment diets were prepared as described by Alegret et al. (1994). Before treatment, the animals were maintained with water and food ad libitum at constant humidity and temperature with a light/dark cycle of 12 hours (8:00 AM–8:00 PM) for a minimum of five days. All procedures were conducted in accordance with the principles and guidelines established by the University of Barcelona Bioethics Committee, as stated in Law 5/1995, 21st July, from the Generalitat de Catalunya.
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Sample preparation Blood samples were collected at the time of death in 5% EDTA-tubes; plasma was obtained by centrifugation and stored at 80 jC until needed. Livers were excised and perfused in a 150 mM NaCl, 1 mM dithiotreitol, 30 mM EDTA, 50 mM KH2PO4, pH 7.4 buffer; 0.5 g of liver tissue of each rat was homogenised in the same buffer and used for obtaining the postmitochondrial fraction by centrifugation (Montgomery and Cinti, 1977) and frozen at 80 jC until needed. 10–100 mg of liver (L), and cortex tissue of each rat was immediately frozen in liquid N2 and stored at 80 jC until used for the extraction of total RNA. Further, a fresh sample of cortex tissue of each rat was immediately used for obtaining nuclear extracts. Plasma lipids and enzyme assays Plasma total cholesterol, triglyceride, and phospholipid concentrations were measured with the Boehringer Mannheim GmbH colorimetric tests (Monotest Cholesterol CHODPAP Nj 290319, Peridochrom Triglyceride GPOPAP Nj 701882, and MPR2 Nj 691844 phospholipid, respectively). By using reagent Nj 543004, also from Boehringer Mannheim, VLDL and LDL from plasma samples were precipitated, and HDL-cholesterol concentration was determined in the supernatant. The hepatic peroxisomal fatty acid h-oxidation system was determined as described previously (Alegret et al., 1994). Protein concentration was determined by the method of Bradford (Bradford, 1976). RNA preparation and analysis Total RNA was isolated by using the Ultraspec reagent (Biotecx, Houston, USA). Relative levels of specific mRNAs were assessed by the reverse transcription-polymerase chain reaction (RT-PCR). Complementary DNA was synthesized from RNA samples by mixing 1 Ag of total RNA, 125 ng of random hexamers as primers in the presence of 50 mM Tris-HCl buffer (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 200 U Moloney murine leukemia virus reverse transcriptase (Life Technologies, Gaithersburg, USA), 20 U RNAsin (Life Technologies) and 0.5 mM of each dNTP (Sigma) in a total volume of 20 Al. Samples were incubated at 37 jC for 60 min. A 5 Al aliquot of the RT reaction was then used for subsequent PCR amplification with specific primers. Each 25-Al PCR reaction contained 5 Al of the RT reaction, 1.2 mM MgCl2, 200 AM dNTPs, 1.25 ACi [32P]-dATP (3000 Ci/mmol, Amersham pharmacia biotech), 1 unit of Taq polymerase (Life Technologies), 0.5 Ag of each primer and 20 mM Tris-HCl, pH 8.5. To avoid unspecific annealing, cDNA and Taq polymerase were separated from primers and dNTPs by using a layer of paraffin (reaction components contact only when paraffin fuses, at 60 jC). The sequences of the sense and antisense primers used for amplification were: PPARa, 5V-GGCTCGGAGGGCTCTGTCATC-3V and 5V-ACATGCACTGGCAGCAGTGGA-3V; ACO 5V-ACTATATTTGGCCAATTTTGTG3V and 5V-TGTGGCAGTGGTTTCCAAGCC-3V; RXRa, 5V-GCTCTCCAACGGGTCGAGGCT-3V and 5V-TGGGTGTGGTGGGTACCGACA-3V; PPARh, 5V-GAGGAAGTGGCCACGGGTGAC-3V and 5VCCACCTGAGGCCCCATCACAG-3V; PPARg, 5V-TGGGGATGTCTCACAATGCCA-3V and 5VTTCCTGTCAAGATCGCCCTCG-3V; and APRT (adenosyl phosphoribosyl transferase), 5VAGCTTCCCGGACTTCCCCATC-3V and 5V-GACCACTTTCTGCCCCGGTTC-3V. The aprt gene
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was used as internal control in the PCR reaction to normalize the results. PCR was performed in an MJ Research Thermocycler (Ecogen, Barcelona, Spain) equipped with a peltier system and temperature probe. After an initial denaturation for 1 min at 94 jC, PCR was performed for 23 cycles. Each cycle consisted of denaturation at 92 jC for 1 min, primer annealing at 60 jC (except 58 jC for ACO), and primer extension at 72 jC for 1 min and 50 s. A final 5-min extension step at 72 jC was performed. Five microliters of each PCR sample was electrophoresed on a 1-mmthick 5% polyacrylamide gel. The gels were dried and subjected to autoradiography using Agfa Xray films (Danny Commercial, Barcelona, Spain) to show the amplified DNA products. Amplification of each gene yielded a single band of the expected size (PPARa: 645 bp, PPARh: 151 bp, PPARg: 200 bp, ACO: 195 bp, RXRa: 202 bp, and APRT: 329 bp). Preliminary experiments were carried out with various amounts of cDNA to determine non-saturating conditions of PCR amplification for all the genes studied. Thus cDNA amplification was performed in comparative and semiquantitative conditions (Freeman et al., 1999). Radioactive bands were quantified by videodensitometric scanning (Vilbert Lourmat Imaging). The results for the expression of specific mRNAs are always presented relative to the expression of the control gene (aprt). Isolation of nuclear extracts Nuclear extracts were isolated using the Dignam method (Dignam et al., 1983) with the modifications described by Sonnenberg et al. (1989). Briefly, fresh cortex tissues were weighed and homogenized by a Potter Elvehjem homogenizer in 4 volumes (w/v) of buffer A containing 0.25M sucrose, 15 mM Tris HCl pH 7.9, 15 mM NaCl, 60 mM KCl, 1 mM EGTA, 5mM EDTA, 0.15 mM spermine, 0.5 mM spermidine and a mixture of protease inhibitors (0.1 mM phenylmethylsolfonyl fluoride, 1.0 mM dithiothreitol, 5 Ag/ml aprotinin, 2 Ag/ml leupeptin). Homogenates were incubated for 10 min. on ice and centrifuged (2,000 g, 10 min, 4 jC). Pellets were resuspended in 4 vol of buffer B (10 mM Hepes pH 7.9, 1.5 mM MgCl2, 10 mM KCl and protease inhibitors as above) and then centrifuged at 4,000 g, 4 jC, for 10 min. Supernatants were discarded and pellets were resuspended in 1 vol. of buffer C (0.5 M HEPES, pH 7.9, 0.75 mM MgCl2, 0.5 M KCl, 12.5% glycerol and the protease inhibitors). Homogenates were kept for 30 min at 4 jC under continuous rotary shaking, and then centrifuged at 14,000 g for 30 min at 4 jC. Finally, the resulting supernatants were dialyzed overnight at 4 jC with buffer D (10 mM TrisHCl, pH 7.9, 5 mM MgCl2, 10 mM KCl, 1.0 mM EDTA, 10% glycerol and the protease inhibitors). Nuclear extracts were collected in microfuge tubes and stored in aliquots at 80 jC The protein concentration of the nuclear extracts was determined by the method of Bradford (1976). Electrophoretic mobility shift assays (EMSA) The DNA sequence of the double-stranded oligonucleotide used was a consensus binding site of a PPAR response element, 5V-CAAAACTAGGTCAAAGGTCA-3V (Santa Cruz Biotechnology, Santa Cruz, CA). Oligonucleotides were labeled in the following reaction: 1 Al of oligonucleotide (20 ng/Al), 2 Al of 5x kinase buffer, 5 U. of T4 polynucleotide kinase, and 3 Al of [g-32P] ATP (3000 Ci/mmol at 10 mCi/ml, Amersham) incubated at 37 jC for 1 h. The reaction was stopped by adding 90 Al of TE buffer (10 mM Tris-HCl pH 7.4 and 1 mM EDTA). To separate the labeled probe from the unbound ATP, the reaction mixture was eluted in a Nick column (Pharmacia, Sant Cugat, Spain) according to the
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manufacturer’s instructions. 10 micrograms of crude nuclear proteins were incubated for 10 min on ice in binding buffer (10 mM Tris-HCl pH 8.0, 25 mM KCl, 0.5 mM DTT, 0.1 mM EDTA pH 8.0, 5% glycerol, 5 mg/ml BSA, 100 Ag/ml tRNA and 50 Ag/ml poly(dI-dC)), in a final volume of 15 Al. Labeled probe (approximately 50,000 cpm) was added and the reaction was incubated for 20 min. at room temperature. Where indicated, non-radioactive specific competitor or mutated PPRE oligonucleotide was added before the addition of labeled probe and incubated for 15 min on ice. Supershift assays were carried out by adding 8 Ag of a specific antibody against PPARh (Santa Cruz Biotechnology) 90 min before the radiolabelled probe. Statistics The results are the mean F standard deviation of the mean of n (3–5) experiments. Plasma samples were assayed in duplicate. Significant differences were established by an ANOVA test, using the computer program GraphPad- InStat-tm (GraphPad Software V2.03). When the number of animals was too small or the variance was not homogeneous, a non-parametric test was performed (Kruskal-Wallis test). When significant variations were found, the Student-Newman-Keuls multiple comparisons test was performed. The level of statistical significance was set at P < 0.05.
Results Gemfibrozil administration to rats reduced plasma cholesterol concentrations by 19%, affecting equally to both free and esterified cholesterol. This reduction was at the expenses of a marked decrease (40%) in the apo B-cholesterol fraction, while HDL cholesterol was not modified. Plasma triglyceride and phospholipid concentrations were also decreased by 43 and 25%, respectively, in gemfibrozil-treated animals (Table 1). It has been shown that gemfibrozil, as well as other fibrate drugs, exerts its hypolipidemic effect by activating hepatic PPARa. Consequently, hepatic levels of the specific mRNA for ACO and the activity of the peroxisomal fatty acid h-oxidation system were increased by 117 and 64%, respectively (Fig. 1). Rat cortex expressed detectable amounts of specific mRNA for ACO, RXRa and the three isoforms of PPAR (Fig. 2). As previously described (Cullingford et al., 1998), PPARh was the most abundant PPAR isoform in cortex, followed by PPARa and PPARg. Fig. 3 shows the pattern of retardation bands
Table 1 Concentrations (mg/dL) of plasma lipids in control - (CTRL) and gemfibrozil - (GFB) treated animals CTRL GFB
TC
FC
CE
HDLc
apoBc
TG
PL
108 F 12 87 F 9b
34 F 5 27 F 5a
74 F 9 60 F 7b
71 F 8 65 F 14
37 F 11 22 F 14c
117 F 18 67 F 18b
198 F 26 148 F 11b
Values are means F S.D. of 6 animals per group. TC: Total cholesterol; FC: free cholesterol; CE: cholesteryl esters; HDLc: HDL cholesterol; apoBc: apo B cholesterol; TG: triglycerides; PL: phospholipids. a p < 0.05. b p < 0.01. c Marginal statistical significance, p < 0.07.
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Fig. 1. A. Peroxisomal h-oxidation activity. Bars represent the mean F sd of the values obtained from 6 animals in each treatment group. B. Relative levels of mRNA for ACO. Bars represent the mean F sd of the values obtained from 6 animals in each treatment group. A representative autoradiography is shown in the upper part of the figure, with signals corresponding to ACO and the reference gene APRT, obtained from one control (CTR) and one gemfibrozil (GFB) treated-rat. * p < 0.05.
obtained in an EMSA performed by incubating a PPRE oligonucleotide probe and cortex nuclear extracts obtained from a control rat and a gemfibrozil-treated rat. In both samples, two retardation bands, 1 and 2, were obtained. Both bands were fully specific for the PPRE probe, as shown by the fact that they were effectively competed by incubating with increasing amounts of unlabeled probe, while no change in band intensity was detected in the presence of identical amounts of an unlabeled mutated PPRE probe. Incubation in the presence of a specific PPARh antibody produced a supershifted band
Fig. 2. Representative autoradiography showing the bands corresponding to the specific mRNAs for RXRa, PPARa, PPARh, PPARg, and the reference gene APRT in cortex samples obtained from two different rats.
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Fig. 3. EMSA assay showing the specificity of the binding of cortex nuclear extracts (NE) obtained from control (CT) rats and gemfibrozil-treated (GFB) rats to a PPRE oligonucleotide; the shifted bands are effectively competed in the presence of unlabeled probe, while no changes in band intensity are produced in the presence of mutated PPRE oligonucleotide.
(Fig. 4), indicating the participation of this transcription factor in the formation of the specific bands. When the intensities of the shifted bands were compared between samples from control and gemfibroziltreated animals, drug treatment produced a marked significant increase in the intensities of band 1 (2.4fold) and 2 (1.8-fold) (Fig. 5). However, the levels of the specific mRNA for ACO in rat cortex were not
Fig. 4. EMSA assay showing the appearance of a super-shifted band when cortex nuclear extract (NE) obtained from a gemfibrozil treated rat is incubated with a PPRE oligonucleotide in the presence of a specific PPARh antibody (see Material and Methods for details).
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Fig. 5. A. EMSA assay showing changes in the intensity of the specific retarded bands 1 and 2 obtained by incubating cortex nuclear extracts (NE) from control (CT) and gemfibrozil-treated (GFB) rats with a PPRE oligonucleotide. B. Quantification of the intensity of shifted bands 1 and 2 corresponding to the EMSA presented in Fig. 5A. * p < 0.05, ** p < 0.01.
increased in gemfibrozil-treated animals (0.54 F 0.04 vs. 0.55 F 0.09 ACO mRNA arbitrary units for control and gemfibrozil-treated rats, respectively).
Discussion Gemfibrozil is a selective, low affinity ligand for PPARa (Krey et al., 1997) and its pharmacological effects in experimental animals and humans are mainly due to the activation of the transcriptional activity of PPARa (Kersten et al., 2000; Keller et al., 2000; Alegret et al., 1994; Verthou et al., 1995). Although its therapeutic niche is in the treatment of dyslipidemia, there is an increasing interest in other possible therapeutic applications of PPARa agonists, such as putative anti-inflammatory agents for the treatment of chronic inflammatory diseases, acting by a mechanism not related to COX inhibition (Cabrero et al., 2002). Among those diseases, a growing interest in the study of anti-inflammatory therapy for the prevention of neurodegenerative diseases has been aroused, after the publication of data
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relating chronic use of antiinflammatory agents as a protective factor for Alzheimer’s disease (McGreer et al., 1996; McGreer and McGreer, 1999); classical non-steroidal anti-inflammatory drugs, such as indomethacin and ibuprofen, have been shown to activate PPARa and PPARg (Lehmann et al., 1997). Anxiety, dizziness and sleep disorders have been described as adverse effects of gemfibrozil administration to humans (Todd and Ward, 1988); nevertheless, there is no data about the penetration of gemfibrozil into the central nervous system when used at pharmacological doses. As shown in this work and previous ones from our laboratory and others (Alegret et al., 1994; Berthou et al., 1995; Todd and Ward, 1988), administration of gemfibrozil to rats, by activating hepatic PPARa, typically reduces plasma triglycerides and non-HDL colesterol, and produces hepatic peroxisome proliferation, with increases in the expression of marker enzymes such as acyl-CoA oxidase (Table 1, Fig. 1). When we assayed the binding of cortex nuclear extracts obtained from the same gemfibroziltreated animals to a PPRE oligonucleotide probe, we detected a significant increase in the intensity of the specific bands obtained in the gel-shift assay. Thus, orally administered gemfibrozil reaches brain tissues at concentrations sufficient to elicit a typical PPAR-mediated pharmacological response. However, although gemfibrozil administration increased the relative levels of ACO mRNA in liver samples, no changes in ACO mRNA expression in cortex of treated animals was observed, despite the increase intensity of the PPRE specific bands detected in the gel-shift assay. Two possible mechanisms can be put forward to explain this situation: a) Increases in the intensity of specific bands obtained after incubation of liver nuclear extracts from fibrate-treated rats to a PPRE oligonucleotide are transient in nature, preceding the increase in liver ACO mRNA observed after fibrate administration (Rodrı´guez et al., 2000). In rats and other laboratory animals, gemfibrozil presents a peak of plasma concentration after 1–2 hours of administration, with two elimination phases, with half-lives of 0.7 and 33 hours (Cayen, 1985). Thus, assuming a slow distribution of gemfibrozil in organs such as brain, there is a possibility that we failed to detect an increase in cortex ACO mRNA from gemfibrozil-treated rats because we actually obtained the samples before such phenomenon was produced. b) We and others have shown that PPARh is the most abundant PPAR isoform in brain tissues (Fig. 2, Cullingford et al., 1998). Further, PPARh, by binding to a PPRE and associating with corepressors proteins, is able to inhibit the expression of endogenous PPARa target genes, such as aco (Jow and Mukherjee, 1995; Mochizuki et al., 2001; Shi et al., 2002). As PPARh is contributing to the formation of the specific bands obtained by incubating cortex nuclear extracts with a PPRE probe (Fig. 4), it could be argued that its presence is sufficient to inhibit the gemfibrozil-induced transcriptional activity of the low levels of PPARa present
Fig. 6. Comparison of PPARa/PPARh mRNA ratios in rat cortex and liver. Bars represent the mean F sd of the values obtained from 3 animals in each treatment group. * p < 0.02.
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in cortex. Further supporting this hypothesis is the fact that the relative proportion of the mRNAs of PPARa and PPARh is 2.4-fold higher in rat liver than in cortex (Fig. 6). In conclusion, orally administered gemfibrozil, at a dose that activates hepatic PPARa and induces its typical hypolipidemic effect, is able to penetrate into the brain tissue, increasing the specific binding of cortex nuclear proteins to a PPRE oligonucleotide.
Acknowledgements This work was supported by grants from FPCNL, CICYT (SAF00/0201), FISss (01/0075-01), MCyT (BFI2002-05167), Generalitat de Catalunya (2001SGR00141) and SEA/Almirall Prodesfarma 2001. E. Sanguino holds a fellowship for Training in Teaching and Research from the University of Barcelona.
References Alegret, M., Ferrando, R., Va´zquez, M., Adzet, T., Merlos, M., Laguna, J.C., 1994. Relationship between plasma lipids and palmitoyl-CoA hydrolase and synthetase activities with peroxisomal proliferation in rats treated with fibrates. British Journal of Pharmacology 112, 551 – 556. Basu-Modak, S., Braissant, O., Escher, P., Desvergne, B., Honegger, P., Wahli, W., 1999. Peroxisome proliferator-activated receptor h regulates acyl-CoA synthetase 2 in reaggregated rat brain cell cultures. Journal of Biological Chemistry 274, 35881 – 35888. Berthou, L., Saladin, R., Yaqoob, P., Branellec, D., Calder, P., Fruchart, J.-C., Dene`fle, P., Auwerx, J., Staels, B., 1995. Regulation of rat liver apolipoprotein A-I, apolipoprotein A-II and acyl-coenzyme A oxidase gene by fibrates and dietary fatty acids. European Journal of Biochemistry 232, 178 – 187. Bradford, M., 1976. A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding. Analytical Biochemistry 72, 248 – 254. Cabrero, A., Laguna, J.C., Va´zquez, M., 2002. Peroxisome proliferator-activated receptors and the control of inflammation. Current Drug Targets in Inflammation and Alergy 1, 243 – 248. Cayen, M.N., 1985. Disposition, metabolism and pharmacokinetics of antihyperlipidemic agents in laboratory animals and man. Pharmacology and Therapeutics 29, 157 – 204. Combs, C.K., Bates, P., Colleen Karlo, J., Landreth, G.E., 2001. Regulation of h-amyloid stimulated proinflammatory responses by peroxisome proliferator-activated receptor a. Neurochemistry International 39, 449 – 457. Combs, C.K., Johnson, D.E., Colleen Karlo, J., Cannady, S.B., Landreth, G.E., 2000. Inflammatory mechanisms in Alzheimer’s disease: Inhibition of h-amyloid-stimulated proinflammatory responses and neurotoxicity by PPARg agonists. Journal of Neuroscience 20, 558 – 567. Cullingford, T.E., Bhakoo, K., Peuchen, S., Dolphin, C.T., Patel, R., Clark, J.B., 1998. Distribution of mRNAs encoding the peroxisome proliferator-activated receptor a, h and g and the retinoid X receptor a, h and g in rat central nervous system. Journal of Neurochemistry 70, 1366 – 1375. Dignam, J.D., Lebovitz, R.M., Roeder, R.G., 1983. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acid Research 11, 1475 – 1489. Freeman, W.M., Walker, S.J., Vrana, E.V., 1999. Quantitative RT-PCR: Pitfalls and potential. Biological Tecniques 26, 112 – 125. Jow, L., Mukherjee, R., 1995. The human peroxisome proliferator-activated receptor (PPAR) subtype NUC1 represses the activation of hPPARa and thyroid hormone receptors. Journal of Biological Chemistry 270, 3836 – 3840. Keller, J.M., Collet, P., Bianchi, A., Huin, C., Bouillaud-Kremarik, P., Becuwe, P., Schohn, H., Domenjoud, L., Daucßa, M., 2000. Implications of peroxisome proliferator-activated receptors (PPARs) in development, cell life status and disease. International Journal of Developmental Biology 44, 429 – 442. Kersten, S., Desvergne, B., Wahli, W., 2000. Roles of PPARs in health and disease. Nature 405, 421 – 424.
E. Sanguino et al. / Life Sciences 73 (2003) 2927–2937
2937
Kitamura, Y., Shimohama, S., Koike, H., Kakimura, J.-i., Matsuoka, Y., Nomura, Y., Gebicke-Haerter, P.J., Taniguchi, T., 1999. Increased expression of cyclooxygenases and peroxisome proliferator-activated receptor-g in Alzheimer’s disease brain. Biochemical Biophysical Research Communications 254, 582 – 586. Knoll, A., Salles, J., Sargueil, F., Cassagne, C., Garbay, B., 2000. Peroxisomal beta-oxidation enzyme gene expression in the developing mouse brain. Neuroscience Letters 285, 201 – 204. Krey, G., Braissant, O., L’Horset, F., Kalkhoben, E., Perroud, M., Parker, M.G., Wahli, W., 1997. Fatty acids, eicosanoids and hypolipidemic agents identified as ligands of peroxisome proliferator-activated receptors by CARLA. Molecular Endocrinology 11, 779 – 791. Lehmann, J.M., Lenhard, J.M., Oliver, B.B., Ringold, G.M., Kliewer, S.A., 1997. Peroxisome proliferator-activated receptors a and g are activated by indomethacin and other non-steroidal anti-inflammatory drugs. Journal of Biological Chemistry 272, 3406 – 3410. McGreer, E.G., McGreer, P.L., 1999. Brain inflammation in Alzheimer disease and the therapeutic implications. Current Pharmaceutical Design 5, 821 – 836. McGreer, P.L., Schulzer, M., McGeer, E.G., 1996. Arthritis and anti-inflammatory agents as possible protective factors for Alzheimer’s disease: A review of 17 epidemiologic studies. Neurology 47, 425 – 432. Mochizuki, K., Suruga, K., Yagi, E., Takase, S., Goda, T., 2001. The expression of PPAR-associated genes is modulated through postnatal development of PPAR subtypes in the small intestine. Biochimica Biophysica Acta 1531, 68 – 76. Montgomery, M.R., Cinti, D.L., 1977. Pyridine nucleotide-dependent electron transport in kidney cortex microsomes: interaction between desaturase and other mixed function oxidases. Molecular Pharmacology 13, 60 – 69. Rodrı´guez, C., Noe´, V., Cabrero, A., Ciudad, C.J., Laguna, J.C., 2000. Differences in the formation of PPARa-RXR/ acoPPRE complexes between responsive and nonresponsive species upon fibrate administration. Molecular Pharmacology 58, 185 – 193. Saluja, I., Granneman, J.G., Skoff, R.P., 2001. PPARy agonists stimulate oligodendrocyte differentiation in tissue culture. Glia 33, 191 – 204. Shi, Y., Hon, M., Evans, R.M., 2002. The peroxisome proliferator-activated receptor y, an integrator of transcriptional repression and nuclear receptor signaling. Proceedings of the National Academy of Sciences USA 99, 2613 – 2618. Sonnenberg, J.L., MacGregor-Leon, P.F., Curran, T., Morgan, J.I., 1989. Dynamic alterations occur in the levels and composition of transcription factor AP-1 complexes after seizures. Neuron 3, 359 – 365. Spencer, C.M., Barradell, L.B., 1996. Gemfibrozil. A reappraisal of its pharmacological properties and place in the management of dyslipidemia. Drugs 51, 982 – 1018. Todd, P.A., Ward, A., 1988. Gemfibrozil. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use un dyslipidemia. Drugs 36, 314 – 339.