Modulation of rat liver apolipoprotein gene expression and serum lipid levels by tetradecylthioacetic acid (TTA) via PPARα activation
1999; Elsevier BV; Volume: 40; Issue: 11 Linguagem: Inglês
10.1016/s0022-2275(20)32433-0
ISSN1539-7262
AutoresEric Raspé, Lise Madsen, Anne‐Marie Lefebvre, Iris Leitersdorf, Laurent Gelman, Julia Peinado‐Onsurbe, Jean Dallongeville, JC Fruchart, Rolf K. Berge, Bart Staels,
Tópico(s)Drug Transport and Resistance Mechanisms
Resumo3-Thia fatty acids are modified fatty acids that promote hepatic peroxisome proliferation and decrease serum triacylglycerol, cholesterol and free fatty acid levels in rats. In vivo administration of tetradecylthioacetic acid (TTA) to rats led to a significant decrease in liver apolipoproteins apoA-I, A-II, A-IV, and C-III mRNA levels, and to an increase of liver acyl-CoA oxidase (ACO), carnitine palmitoyltransferase-II, and 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMG-CoA synthase) mRNA levels and activities. By contrast, no significant changes of lipoprotein lipase (LPL) mRNA levels were detected in rat epididymal adipose tissue. Liver carnitine palmitoyltransferase-I, apoB, apoE, and LDL receptor mRNA levels were not significantly affected. When tested in vitro, TTA increased rat ACO and carnitine palmitoyltransferase-I mRNA levels in primary rat hepatocytes and also LPL mRNA levels in 3T3-L1 preadipocytes. TTA also enhanced the transcriptional activity of chimeras containing the DNA binding domain of the yeast transcription factor Gal4 fused to the ligand binding domain of either human PPARα or human PPARγ. The effect depended on the concentration tested and the cell type. In conclusion, our data suggest that in vitro, TTA activates both PPARα and PPARγ, but the latter with much lower affinity. TTA affects serum lipid levels in vivo in rats by acting mainly on the liver via PPARα where it decreases the liver expression of genes involved in vascular lipid transport and increases the expression of genes involved in intracellular fatty acid metabolism.—Raspé, E., L. Madsen, A-M. Lefebvre, I. Leitersdorf, L. Gelman, J. Peinado-Onsurbe, J. Dallongeville, J-C. Fruchart, R. Berge, and B. Staels. Modulation of rat liver apolipoprotein gene expression and serum lipid levels by tetradecylthioacetic acid (TTA) via PPARα activation. J. Lipid Res. 1999. 40: 2099–2110. 3-Thia fatty acids are modified fatty acids that promote hepatic peroxisome proliferation and decrease serum triacylglycerol, cholesterol and free fatty acid levels in rats. In vivo administration of tetradecylthioacetic acid (TTA) to rats led to a significant decrease in liver apolipoproteins apoA-I, A-II, A-IV, and C-III mRNA levels, and to an increase of liver acyl-CoA oxidase (ACO), carnitine palmitoyltransferase-II, and 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMG-CoA synthase) mRNA levels and activities. By contrast, no significant changes of lipoprotein lipase (LPL) mRNA levels were detected in rat epididymal adipose tissue. Liver carnitine palmitoyltransferase-I, apoB, apoE, and LDL receptor mRNA levels were not significantly affected. When tested in vitro, TTA increased rat ACO and carnitine palmitoyltransferase-I mRNA levels in primary rat hepatocytes and also LPL mRNA levels in 3T3-L1 preadipocytes. TTA also enhanced the transcriptional activity of chimeras containing the DNA binding domain of the yeast transcription factor Gal4 fused to the ligand binding domain of either human PPARα or human PPARγ. The effect depended on the concentration tested and the cell type. In conclusion, our data suggest that in vitro, TTA activates both PPARα and PPARγ, but the latter with much lower affinity. TTA affects serum lipid levels in vivo in rats by acting mainly on the liver via PPARα where it decreases the liver expression of genes involved in vascular lipid transport and increases the expression of genes involved in intracellular fatty acid metabolism.—Raspé, E., L. Madsen, A-M. Lefebvre, I. Leitersdorf, L. Gelman, J. Peinado-Onsurbe, J. Dallongeville, J-C. Fruchart, R. Berge, and B. Staels. Modulation of rat liver apolipoprotein gene expression and serum lipid levels by tetradecylthioacetic acid (TTA) via PPARα activation. J. Lipid Res. 1999. 40: 2099–2110. A group of modified fatty acids, in which a sulfur atom replaces a methylene group in a defined position of the carbon chain of an ordinary fatty acid (n-thia fatty acid), has been developed and extensively tested as hypolipidemic drugs (1Berge R.K. Hvattum E. Impact of cytochrome P450 system on lipoprotein metabolism. Effect of abnormal fatty acids (3-thia fatty acids).Pharmacol. Ther. 1994; 61: 345-383Google Scholar). 3-Thia fatty acids such as tetradecylthioacetic acid or TTA, a saturated fatty acid where the 3 methylene group is substituted (CH3-(CH2)13-S-CH2-COOH), are non-β oxidizable but can undergo ω-oxidation (1Berge R.K. Hvattum E. Impact of cytochrome P450 system on lipoprotein metabolism. Effect of abnormal fatty acids (3-thia fatty acids).Pharmacol. Ther. 1994; 61: 345-383Google Scholar). The other n-thia fatty acids can be metabolized both through β- or ω-oxidation and have different biological activities (1Berge R.K. Hvattum E. 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The recently reported absence of peroxisome proliferation in PPARα-deficient mice supports previous evidence suggesting the direct implication of this receptor in the action of peroxisome proliferators including fibrates (40Lee S.S. Pineau T. Drago J. Lee E.J. Owens J.W. Kroetz D.L. Fernandez-Salguero P.M. Westphal H. Gonzalez F.J. Targeted disruption of the α isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators.Mol. Cell. Biol. 1995; 15: 3012-3022Google Scholar). By contrast, thiazolidinedione PPARγ activators such as BRL49653 enhance epididymal adipose tissue LPL mRNA accumulation but do not affect liver apolipoprotein or ACO gene expression (41Lefebvre A-M. Peinado-Onsurbe J. Leitersdorf I. Briggs M.R. Paterniti J.R. Fruchart J-C. Fiévet C. Auwerx J. Staels B. Regulation of lipoprotein metabolism by thiazolidinediones occurs through a distinct, but complementary mechanism relative to fibrates.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1756-1764Google Scholar). Interestingly, simultaneous administration of BRL49653 and fenofibrate resulted in a more pronounced lowering of serum triacylglycerols than each drug alone, suggesting that drugs with combined PPARα and PPARγ agonist activity will display an increased hypotriglyceridemic activity (41Lefebvre A-M. Peinado-Onsurbe J. Leitersdorf I. Briggs M.R. Paterniti J.R. Fruchart J-C. Fiévet C. Auwerx J. Staels B. Regulation of lipoprotein metabolism by thiazolidinediones occurs through a distinct, but complementary mechanism relative to fibrates.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1756-1764Google Scholar). As fatty acids are PPARα and PPARγ activators (27Krey G. Braissant O. L'Horset F. Kalkhoven E. Perroud M. Parker M.G. Wahli W. Fatty acids, eicosanoids, and hypolipidemic agents identified as ligands of peroxisome proliferator-activated receptors by coactivator-dependent receptor ligand assay.Mol. Endocrinol. 1997; 11: 779-791Google Scholar, 28Forman B.M. Chen J. Evans R.M. Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors α and ∂.Proc. Natl. Acad. Sci. USA. 1997; 94: 4312-4317Google Scholar), 3-thia fatty acids are good pharmacological candidates for such combined action. The first goal of this study was therefore to determine whether 3-thia fatty acids act by regulating the expression of genes encoding proteins involved in lipid transport and lipoprotein metabolism in hepatic and adipose tissue in vivo. Second, we aimed to determine whether the hypolipidemic effects of 3-thia fatty acids could be associated with activation of PPARα and/or PPARγ in vitro. Tetradecylthioacetic acid (TTA) was prepared as described earlier (42Spydevold O. Bremer J. Induction of peroxisomal β-oxidation in 7800 C1 Morris hepatoma cells in steady state by fatty acids and fatty acid analogues.Biochim. Biophys. Acta. 1989; 1003: 72-79Google Scholar). BRL49653 and fenofibric acid were gifts respectively from Dr. J-J. Berthelon (Merck-Lipha, Lyon, France) and Dr. A. Edgar (Laboratoires Fournier, Daix, France). Pirixinic acid (Wy 14643) was from Chemsyn Science Laboratories (Lenexa, KS). The cationic lipid RPR 120535B (WO patent 97/18185) was a generous gift from Dr. G. Byk (Rhône-Poulenc-Rorer, Paris, France). Male Wistar rats weighing approximately 150 g were housed in metal cages and maintained under 12-h light/dark cycles at a constant temperature of 20 ± 3°C. The animals were acclimatized for at least 1 week before the beginning of the experiments and randomized with respect to body weight. TTA was suspended in 0.5% sodium carboxymethylcellulose and administered by gavage once a day for 7 days at a dose of 300 mg/day per kg body weight. The animals had free access to food and water. At the end of the experiment, the rats were weighed and killed by cardiac puncture under neuroleptic anaestesia. Blood was collected in EDTA-containing tubes. Plasma was prepared by centrifugation at 3000 rpm for 20 min. Liver and epididymal fat pads were removed immediately, weighed, and frozen in liquid nitrogen. All animals received appropriate care as outlined in the "Guide for the Care and Use of Laboratory Animals." Serum lipid concentrations (total and free cholesterol, triacylglycerols, phospholipids) were measured colorimetrically using enzymatic test kits from Boehringer Mannheim (Mannheim, Germany). For fast protein liquid chromatography (FPLC) size fractionation of lipoproteins, 300 μl of serum isolated from individual rats were injected on a Sepharose 6HR 10/30 prepacked column (Pharmacia, Uppsala, Sweden) and eluted at a constant flow rate of 0.2 ml/min with PBS pH 7.2. The effluent was monitored at 280 nm, collected in 0.3-ml fractions and cholesterol and triacylglycerol concentrations were determined in 0.1 ml of each fraction. Rat hepatocytes were isolated by collagenase perfusion (25Berthou L. Saladin R. Yaqoob P. Branellec D. Calder P. Fruchart J.C. Denefle P. Auwerx J. Staels B. Regulation of rat liver apolipoprotein A-I, apolipoprotein A-II, and acyl-coenzyme A oxidase gene expression by fibrates and dietary fatty acids.Eur. J. Biochem. 1995; 232: 179-187Google Scholar) of livers from male rats of mass ranging between 150 and 250 g. When cell viability judged by the Trypan Blue exclusion test was higher than 90%, the cells were seeded in 10-cm2 Petri dishes (Falcon, Lincoln Park, NJ) and cultured as a monolayer (1.5 × 105 cells/cm2) at 37°C in a humidified atmosphere of 5% CO2/95% air. The culture medium used was Leibowitz-15 medium (Gibco, Paisley, UK) supplemented with fetal calf serum (10% by vol), fatty acid-free BSA (0.2% mass/vol), NaHCO3 (26 mm), l-glutamine (2 mm), glucose (3 g/l), dexamethasone (1 μm, from a 104-fold concentrated stock in ethanol), penicillin (100 U/ml), and streptomycin (100 μg/ml). Agents were dissolved in ethanol or DMSO (1000-fold concentrated stock) and added to the culture medium immediately after seeding. Control cells received vehicle only (0.1% by vol, final concentration). After 24 h incubation, the medium was removed, cells were quickly washed with ice-cold 0.15 m NaCl, 0.01 m sodium phosphate buffer, pH 7.2, lysed and scraped in 2 ml of ice-cold 4 m guanidinium isothiocyanate. 3T3-L1 cells obtained from E.C.A.C.C. (Porton Down, Salisbury, U.K.) were cultured as previously described (26Schoonjans K. Peinado-Onsurbe J. Lefebvre A-M. Heyman R.A. Briggs M. Deeb S. Staels B. Auwerx J. PPARα and PPARγ activators direct a distinct tissue-specific transcriptional response via a PPRE in the lipoprotein lipase gene.EMBO J. 1996; 15: 5336-5348Google Scholar) and incubated for the time indicated in the presence of tetradecylthioacetic acid. At the end of the experiment, cells were quickly washed with ice-cold 0.15 m NaCl, 0.01 m sodium phosphate buffer, pH 7.2, lysed and scraped in 2 ml of ice-cold 4 m guanidinium isothiocyanate. Human hepatoma HepG2 cells were obtained from E.C.A.C.C. (Porton Down, Salisbury, U.K.). Cells were maintained in standard culture conditions (Dulbecco's modified Eagle's minimal essential medium (DMEM), supplemented with 10% fetal calf serum at 37°C in a humidified atmosphere of 5% CO2/95% air). Medium was changed every 2 days. The livers from individual rats were homogenized in ice-cold sucrose medium (0.25 m sucrose, 10 mm HEPES (pH 7.4) and 2 mm EDTA). Postnuclear, mitochondrial, and peroxisomal fractions were prepared using preparative differential centrifugation as earlier described (43Berge R.K. Flatmark T. Osmudsen H. Enhancement of long-chain acyl-CoA hydrolase activity in peroxisomes and mitochondria of rat liver by peroxisomal proliferators.Eur. J. Biochem. 1984; 141: 637-644Google Scholar). The fractionation process was carried out at 0–4°C. Protein content of the homogenates was evaluated by the Bradford assay using the kit from Bio-Rad (Bio-Rad, München, Germany). BSA in distillated water was used as standard. Fatty acyl-CoA oxidase activity in the peroxisomal fraction was measured as previously described (5Asiedu D.K. Al-Shurbaji A. Rustan A.C. Björkhem I. Berge R.K. Hepatic fatty acid metabolism as a determinant of plasma and liver triacylglycerol levels. Studies on tetradecylthioacetic and tetradecylthiopropionic acids.Eur. J. Biochem. 1995; 227: 715-722Google Scholar). Acid-soluble products were measured using palmitoyl-CoA as substrate (44Willumsen N. Hexeberg S. Skorve J. Lundquist M. Berge R.K. Docosahexaenoic acid shows no triglyceride-lowering effects but increases the peroxisomal fatty acid oxidation in liver of rats.J. Lipid Res. 1993; 34: 13-22Google Scholar). Briefly, the assay mix (0.3 ml) contained 12 mm HEPES buffer (pH 7.3), 11 mm MgCl2, 12 mm dithiothreitol, 5.6 mm ADP, 0.2 mm NAD+, 0.6 mm EDTA, 125 mm KCl, and 1.0 mg mitochondrial protein. Palmitoyl-CoA oxidation was measured with 80 μm [1-14C]palmitoyl-CoA supplemented with 1 mm l-carnitine. After 2 min incubation at 30°C, the reaction was terminated with 150 μl 1.5 m KOH. Then, 2.5 mg BSA and 500 μl 4 m HClO4 were added.
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