This study investigated the effect of clofibrate treatment on expression of target genes of peroxisome proliferator-activated receptor (PPAR)-α and various genes of the lipid metabolism in liver and adipose tissue of pigs. An experiment with 18 pigs was performed in which pigs were fed either a control diet or the same diet supplemented with 5 g clofibrate/kg for 28 days. Pigs treated with clofibrate had heavier livers, moderately increased mRNA concentrations of various PPAR-α target genes in liver and adipose tissue, a higher concentration of 3-hydroxybutyrate, and markedly lower concentrations of triglycerides and cholesterol in plasma and lipoproteins than control pigs (P < 0.05). mRNA concentrations of sterol regulatory element-binding proteins (SREBP)-1 and -2, insulin-induced genes (Insig)-1 and Insig-2, and the SREBP target genes acetyl-CoA carboxylase, 3-methyl-3-hydroxyglutaryl-CoA reductase, and low-density lipoprotein receptor in liver and adipose tissue and mRNA concentrations of apolipoproteins A-I, A-II, and C-III in the liver were not different between both groups of pigs. In conclusion, this study shows that clofibrate treatment activates PPAR-α in liver and adipose tissue and has a strong hypotriglyceridemic and hypocholesterolemic effect in pigs. The finding that mRNA concentrations of some proteins responsible for the hypolipidemic action of fibrates in humans were not altered suggests that there were certain differences in the mode of action compared with humans. It is also shown that PPAR-α activation by clofibrate does not affect hepatic expression of SREBP target genes involved in synthesis of triglycerides and cholesterol homeostasis in liver and adipose tissue of pigs.
- peroxisome proliferator-activated receptor-α
fibrates are a group of hypolipidemic agents that have been in clinical use for several decades in humans (46). It is well established that these agents act as synthetic agonists of peroxisome proliferator-activating receptor-α (PPAR-α), a nuclear receptor also activated by natural ligands such as free fatty acids or some eicosanoids. PPAR-α is an important regulator of cellular fatty acid uptake and intracellular fatty acid transport, mitochondrial and peroxisomal fatty acid oxidation, ketogenesis, and gluconeogenesis (48). In humans, the most pronounced effect of fibrates is a decrease in plasma triglyceride-rich lipoproteins. Concentrations of low-density lipoprotein (LDL) cholesterol generally decrease in individuals with elevated baseline plasma concentrations, and plasma high-density lipoprotein (HDL) cholesterol concentrations are usually increased when baseline concentrations are low (46). Effects of PPAR-α activation have been mostly studied in rodents, which exhibit a strong expression of PPAR-α in liver and show peroxisome proliferation in the liver in response to PPAR-α activation (36). Expression of PPAR-α and sensitivity to peroxisomal induction by PPAR-α agonists, however, vary greatly among species (19, 21). In contrast to rats and mice, which are highly sensitive to induction by peroxisome proliferators, guinea pigs, monkeys, pigs, and humans are relatively insensitive (21, 32, 39, 48). In these nonproliferating species, expression of PPAR-α in the liver is much lower, and the response of many genes to PPAR-α activation is weaker than in proliferating species (8).
In contrast to rodents, there is little information to date about the effects of PPAR-α agonists on lipid metabolism in pigs, which are not only of agricultural importance but are also a valuable model for studying the lipid metabolism because of their close relationship to humans (6). It has been shown that treatment of pigs with clofibrate stimulates mitochondrial and peroxisomal β-oxidation in liver, muscle, and kidney (34, 56). Moreover, it was found that pigs express functional PPAR-α in the liver, and several target genes induced in the liver by PPAR-α activation have been identified (8). However, effects of PPAR-α activation on lipid concentrations in plasma and liver of pigs have not yet been investigated. In contrast to rats, mice, or humans in which PPAR-α is predominant in liver (11), pigs exhibit also a high expression of PPAR-α in adipose tissue, and it has been suggested that pigs have a considerable capacity for β-oxidation in adipose tissue (13). The effect of PPAR-α agonists on gene expression of PPAR-α target genes in adipose tissue of pigs, however, has not yet been investigated.
Recent studies (17, 23, 25, 33) in rodents suggested that activation of PPAR-α influences hepatic triglyceride synthesis and cholesterol homeostasis by interacting with gene expression or proteolytic activation of sterol regulatory element-binding proteins (SREBPs), key regulators of lipid synthesis and homeostasis. SREBP-1 preferentially activates genes required for fatty acid synthesis, whereas SREBP-2 preferentially activates the LDL receptor gene and various genes required for cholesterol synthesis (22). SREBPs are synthesized as inactive precursors bound to the endoplasmatic reticulum membranes. For activation to occur, they have to be cleaved by two resident proteases within the Golgi, which sequentially cleave the SREBPs and release the amino-terminal bHLH-Zip-containing domain from the membrane, allowing it to translocate to the nucleus and activate transcription of target genes. Insulin-induced genes (Insig)-1 and Insig-2 are modulators of SREBP activity (53, 55). They block the proteolytic cleavage and transcriptional activation of SREBP. In pigs, in contrast to rodents in which lipogenesis takes place primarily in the liver (15, 44), adipose tissue is the major site of lipogenesis. It has been shown that SREBP-1 and its target gene fatty acid synthase, one of the key enzymes of de novo fatty acid synthesis, are expressed at a higher level in pig adipose tissue than in pig liver (12, 13). Whether a link exists also in pigs between PPAR-α activation and gene expression or proteolytic activation of SREBPs in liver or adipose tissue, which in turn could influence lipid synthesis, is presently unknown.
The objective of the present study was to investigate the effects of clofibrate treatment on expression of genes involved in lipid metabolism in liver and adipose tissue of pigs. We were particularly interested in what extent clofibrate upregulates PPAR-α target genes in liver and adipose tissue of pigs and whether there is an interaction between PPAR-α activation and gene expression or proteolytic activation of SREBPs in these tissues. Therefore, we performed an experiment in which pigs were treated with clofibrate. We used relatively young pigs with a body weight slightly in excess of 10 kg because it has been recently shown that such young pigs express a functional PPAR-α in the liver (8). To characterize hepatic PPAR-α expression in the pig model, we compared mRNA concentration of PPAR-α in pig liver with those of rat and human livers. To assess PPAR-α expression in pig adipose tissue, we compared PPAR-α mRNA concentration in pig liver and pig adipose tissue. To examine PPAR-α activation in this animal model by clofibrate, we determined mRNA concentrations of several PPAR-α target genes in liver and adipose tissue and also plasma concentration of 3-hydroxybutyrate because it is known that PPAR-α activation leads to stimulation of ketogenesis (28). To find out whether activation of PPAR-α by clofibrate treatment also affects expression or proteolytic processing of SREBPs in pig liver or adipose tissue, we determined gene expression of SREBP-1, SREBP-2, Insigs, and target genes of SREBP-1 [acetyl-CoA carboxylase (ACC)] and SREBP-2 [3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and LDL receptor] in these tissues. To explore the molecular basis of alterations of plasma lipoprotein concentrations, we also determined genes involved in lipoprotein metabolism such as apolipoproteins (apo)A-I, apoA-II, and apoC-III and microsomal triglyceride transfer protein (MTP).
MATERIALS AND METHODS
Animals and treatments.
Eighteen male 8-wk-old cross-bred pigs [(German Landrace × Large White) × Pietrain] were kept in a room under controlled temperature at 23 ± 2°C and 55 ± 5% relative humidity with lights on from 0600 to 1800. One day before the beginning of the experimental feeding period, the pigs were weighed and randomly allocated to two groups, with body weights of 12.0 ± 0.4 kg in the control group and 11.9 ± 0.2 kg in the treatment group (means ± SE). Both groups of pigs received a nutritionally adequate diet (31) for growing pigs, which contained (in g/kg) 400 wheat, 230 soybean meal, 150 wheat bran, 100 barley, and 90 sunflower oil, as well as a mineral premix that included l-lysine, dl-methionine, and l-threonine (30). This diet contained 14.4 MJ metabolizable energy and 185 g crude protein/kg. The diet of the treatment group was supplemented with 5 g clofibrate/kg diet. Diet intake was controlled, and each animal in the experiment was offered an identical amount of diet per day. The amount of diet administered was ∼15% below that consumed ad libitum by pigs of a similar weight (as assessed in a previous unpublished study). Therefore, the diet offered was completely taken in by all pigs in the experiment. During the feeding period, the amount of diet offered each day was increased continuously from 400 to 1,200 g. The pigs had free access to water via nipple drinking systems. The experimental diets were administered for 28 days. All experimental procedures in animals described followed established guidelines for the care and use of laboratory animals and were approved by the Council of Saxony-Anhalt.
After completion of the feeding period, the animals were killed under light anesthesia. Four hours before euthanasia, each pig was fed its respective diet. After death, blood was collected into heparinized polyethylene tubes. Plasma was obtained by centrifugation of the blood (1,100 g at 4°C for 10 min). Plasma lipoproteins were separated by step-wise ultracentrifugation (Mikro-Ultrazentrifuge; Sorvall Products, Bad Homburg, Germany) at 900,000 g at 4°C for 1.5 h. Plasma densities were adjusted by sodium chloride and potassium bromide, and the lipoprotein fractions ρ < 1.006 kg/l [very low-density lipoproteins (VLDL) plus chylomicrons], 1.006 kg/l < ρ < 1.063 kg/l (LDL), and ρ > 1.063 kg/l (HDL) were removed by suction. The liver was dissected and weighed, and samples of liver, skeletal muscle (longissimus dorsal muscle), and subcutaneous adipose tissue (backfat, at the level of the 13th/14th rib) were stored at −80°C until analysis. For comparison of PPAR-α expression in rats, humans, and pigs, liver samples of three male adult rats (362 ± 25 g) and three male adult humans (collected during a resection of a test sample for a histopathological evaluation) and liver and adipose tissue of three randomly selected piglets of the control group were used. The human probes were obtained following ethical principles for medical research according to the World Medical Association Declaration of Helsinki.
Lipids from liver were extracted with a mixture of n-hexane and isopropanol (3:2, vol/vol) (18). After lipid extracts were dried, aliquots were dissolved with Triton X-100 (10). The concentrations of cholesterol and triglycerides in the lipoprotein fractions, plasma, and liver were determined with enzymatic kits (no. 113009990314 for cholesterol and no. 157609990314 for triglycerides, Ecoline S+; DiaSys, Holzheim, Germany).
Determination of 3-hydroxybutyrate.
Concentration of 3-hydroxybutyrate in plasma was determined with an enzymatic assay (no. 10907979035; R-BIOPHARM, Darmstadt, Germany).
Total RNAs from liver tissue, skeletal muscle, and adipose tissue were isolated by a tissue lyser (Qiagen, Hilden, Germany) using Trizol reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer's protocol. RNA concentration and purity were estimated from optical densities at 260 and 280 nm (SpectraFluor Plus; Tecan, Crailsheim, Germany). The quality of all RNA samples was furthermore assessed by agarose gel electrophoresis. Total RNA (1.2 μg) was used for cDNA synthesis as described previously (24). The mRNA concentration of genes was measured by real-time PCR, using SYBR green I and an MJ Research Opticon system (Biozym Diagnostik, Oldendorf, Germany). Real-time PCR was performed with 1.25 U of Taq DNA polymerase, 500 μM dNTPs, and 26.7 pmol of the specific primers. Amplification efficiencies for all primer pairs were determined by template dilution series. Calculation of the relative mRNA concentration was made with the amplification efficiencies and the threshold cycle values (37). The housekeeping gene GAPDH was used for normalization. The PCR primers used for real-time RT-PCR were obtained from Operon (Köln, Germany) and Roth (Karlsruhe, Germany), respectively, and are listed in Table 1.
Quantification of PPAR-α mRNA.
For the quantification of copy numbers of PPAR-α mRNA, isolation of total RNA and cDNA synthesis from rat, pig, and human liver tissues and pig white adipose tissue were performed as described above. Real-time PCR was carried out with specific primers (Table 1) as described above; afterward, an aliquot of 10 μl per PCR product was submitted to agarose gel electrophoresis to create standard templates. After dissection from ethidium bromide-stained gel, probes from each species and tissue were pooled and eluted with the peqGOLD gel extraction kit (PeqLab Biotechnologie, Erlangen, Germany). For the generation of standard curves, the concentration of double-stranded cDNA was measured by a Pico green double-stranded DNA quantitation kit (Molecular Probes, Leiden, The Netherlands). The calculated molecular weight of each PCR product was converted into copy numbers by using Avogadro's number (1 mol = 6.022 × 1023 molecules). The threshold cycles measured by real-time PCR were plotted vs. the copy numbers of diluted standard templates to create a standard curve. On the basis of the respective standard curve, the threshold cycle of each probe was used to calculate the copy number of PPAR-α mRNA and was normalized to 1 ng of total RNA.
The results were analyzed with Minitab (State College, PA) statistical software (release 13). Statistical significances of differences between control group and treatment group were evaluated with Student's t-test. Mean values were considered significantly different at P < 0.05. Data in the text are presented as means ± SE.
Comparison of mRNA concentrations of PPAR-α in human, rat, and pig liver and in pig adipose tissue.
To characterize PPAR-α gene expression in the pig model used, we determined mRNA concentrations of PPAR-α in liver and adipose tissue of three control pigs and compared them with those in livers of three adult rats and three male human subjects. PPAR-α mRNA concentrations, corrected for total RNA concentration, were similar in human and in pig liver; PPAR-α mRNA concentrations in human or pig liver were, however, ∼10-fold lower than concentrations in rat liver (Fig. 1). In pigs, mRNA concentrations of PPAR-α were similar in adipose tissue and in liver (Fig. 1).
Food intake and body and liver weights in control pigs and pigs treated with clofibrate.
Because we used a controlled feeding system, food intake throughout the feeding period was the same for each pig in the experiment, averaging 696 ± 2 g/day. Body weight after the 28-day experiment period did not differ between control pigs and pigs treated with clofibrate (Table 2). Relative liver weights, expressed per kilogram of body weight, were higher in pigs treated with clofibrate than in control pigs (P < 0.05; Table 2).
Gene expression in the liver of control pigs and pigs treated with clofibrate.
mRNA concentration of PPAR-α in liver did not differ between both groups of pigs (control: 1.00 ± 0.13; clofibrate: 0.92 ± 0.07; n = 9), whereas relative mRNA concentrations of acyl-CoA oxidase (ACO), carnitine palmitoyltransferase (CPT) 1, liver fatty acid binding protein (l-FABP), mitochondrial HMG-CoA synthase, and stearoyl-CoA desaturase (SCD) in the liver were moderately increased (1.7- to 2.5-fold) in pigs treated with clofibrate compared with control pigs (P < 0.05; Fig. 2). Relative mRNA concentrations of MTP and apoA-I, apoA-II, and apoC-III were not different between the two groups of pigs (relative mRNA concentrations in control pigs and pigs treated with clofibrate were 1.00 ± 0.10 vs. 0.90 ± 0.14 for MPT, 1.00 ± 0.15 vs. 1.10 ± 0.10 for apoA-I, 1.00 ± 0.17 vs. 0.86 ± 0.11 for apoA-II, and 1.00 ± 0.19 vs. 0.85 ± 0.18 for apoC-III, respectively; n = 9 for each group). Relative mRNA concentrations of SREBP-1, SREBP-2, Insig-1, and Insig-2 in the liver did also not differ between both groups of pigs (relative mRNA concentrations in control pigs and pigs treated with clofibrate were 1.00 ± 0.10 vs. 0.93 ± 0.13 for SREBP-1, 1.00 ± 0.20 vs. 1.16 ± 0.27 for SREBP-2, 1.00 ± 0.16 vs. 0.97 ± 0.15 for Insig-1, and 1.00 ± 0.20 vs. 1.18 ± 0.21 for Insig-2, respectively; n = 9 for each group). Hepatic mRNA concentrations of ACC, a target gene of SREBP-1, and HMG-CoA reductase, as well as LDL receptor, target genes of SREBP-2, were also not different between both groups (relative mRNA concentrations in control pigs and pigs treated with clofibrate were 1.00 ± 0.09 vs. 0.82 ± 0.14 for ACC, 1.00 ± 0.12 vs. 0.84 ± 0.10 for HMG-CoA reductase, and 1.00 ± 0.11 vs. 0.88 ± 0.12 for LDL receptor, respectively; n = 9 for each group). mRNA concentrations of cholesterol-7α-hydroxylase (CYP7) in liver (control: 1.00 ± 0.09; clofibrate: 1.03 ± 0.13; n = 9) and of lipoprotein lipase in muscle (control: 1.00 ± 0.13; clofibrate: 1.13 ± 0.15; n = 9) were also not different between both groups of pigs. mRNA concentrations of lipoprotein lipase in the liver were not detectable by mRNA analysis in pigs of both groups.
Gene expression in adipose tissue of control pigs and pigs treated with clofibrate.
mRNA concentration of PPAR-α in adipose tissue did not differ between both groups of pigs (control: 1.00 ± 0.17; clofibrate: 1.12 ± 0.16; n = 9), whereas pigs treated with clofibrate had moderately increased mRNA concentrations of the PPAR-α target genes ACO, CPT-1, and SCD (P < 0.05; Fig. 3). mRNA concentration of lipoprotein lipase, another PPAR-α target gene, in adipose tissue was not different between both groups (control: 1.00 ± 0.11; clofibrate: 0.90 ± 0.17; n = 9). mRNA concentrations of SREBP-1, SREBP-2, Insig-1, and Insig-2, and SREBP downstream genes ACC, HMG-CoA reductase, and LDL receptor in adipose tissue did not differ between both groups of pigs (relative mRNA concentrations in control pigs and pigs treated with clofibrate were 1.00 ± 0.06 vs. 1.10 ± 0.08 for SREBP-1, 1.00 ± 0.14 vs. 0.87 ± 0.16 for SREBP-2, 1.00 ± 0.18 vs. 0.94 ± 0.13 for Insig-1, 1.00 ± 0.20 vs. 0.92 ± 0.16 for Insig-2, 1.00 ± 0.19 vs. 1.19 ± 0.18 for ACC, 1.00 ± 0.10 vs. 1.10 ± 0.14 for HMG-CoA reductase, and 1.00 ± 0.08 vs. 1.07 ± 0.07 for LDL receptor, respectively; n = 9 for each group).
Concentration of 7β-hydroxybutyrate in plasma of control pigs and pigs treated with clofibrate.
Pigs treated with clofibrate had a higher concentration of 7β-hydroxybutyrate in plasma than control pigs (control: 0.52 ± 0.09 mmol/l; clofibrate: 2.17 ± 0.18 mmol/l; n = 9, P < 0.05).
Concentrations of triglycerides and cholesterol in liver plasma and lipoproteins of control pigs and pigs treated with clofibrate.
Pigs treated with clofibrate had lower concentrations of triglycerides in plasma and triglyceride rich-lipoproteins (VLDL + chylomicrons) than control pigs (P < 0.05); triglyceride concentrations in the liver did not differ between both groups of pigs (Table 2). Pigs treated with clofibrate also had lower concentrations of total cholesterol in plasma and lower LDL and HDL levels than control pigs (P < 0.05); cholesterol concentrations in liver, however, did not differ between the two groups of pigs (Table 2).
To study the effect of clofibrate treatment on lipid metabolism and gene expression in pigs, we performed an experiment with young pigs. As in other studies dealing with the effects of clofibrate on metabolism in experimental animals, we added clofibrate to the diet. The concentration of clofibrate in the diet of 5 g/kg diet was adopted from other studies with pigs (8, 34, 56), resulting in a daily dose of 220 mg/kg body wt. This dose is relatively high compared with doses used in humans for treatment of hyperlipidemia, which are usually in the range between 25 and 30 mg/kg body wt.
The present study confirms that expression of PPAR-α in pig liver is much lower than that in rat liver. In the young control pigs, PPAR-α abundance in the liver was ∼10-fold lower than that in the rat liver. The finding that hepatic PPAR-α abundance was similar in the liver of the pigs used in this study as in liver of adult humans indicates that young pigs may be a useful model for studying the response of PPAR-α activation. The finding that PPAR-α mRNA concentrations in human livers are much lower than concentrations in rat liver also agrees with literature data. In the study of Tugwood et al. (50), the abundance of PPAR-α transcript in human liver vs. that in rat liver was 1:5, which is close to the ratio of 1:10 found in the present study.
The finding that mRNA concentrations of the PPAR-α target genes ACO, CPT-1, l-FABP, mitochondrial HMG-CoA synthase, and SCD in the liver were increased by 50–150% compared with results shown in control animals clearly indicates that clofibrate treatment caused PPAR-α activation in the liver of the pigs. This is confirmed by an increased concentration of 3-hydroxybutyric acid, indicative of a stimulation of hepatic ketogenesis, which is a typical response of PPAR-α activation (28). The finding that clofibrate causes a moderate upregulation of PPAR-α target genes agrees well with recent studies conducted in piglets that were treated with clofibrate. In these studies, mRNA concentrations and activities of ACO and CPT-1 were two to four times higher in livers of piglets treated with clofibrate in doses similar to those used in the present study than in untreated piglets (34, 56). Interestingly, in our study, clofibrate treatment caused a significant increase in liver weights of pigs by ∼15%, indicative of moderate peroxisome proliferation. Therefore, the present study suggests that clofibrate not only upregulated PPAR-α target genes in the liver but also caused a moderate peroxisome proliferation in the pigs. It should be noted that upregulation of PPAR-α target genes was much lower than that shown in rodents, where treatment with PPAR-α agonists typically increases mRNA concentrations of ACO 10- to 20-fold compared with untreated controls (14, 20, 23, 25). The reason for the comparatively low upregulation of these enzymes in pigs by clofibrate might be the lower hepatic PPAR-α expression in pigs compared with rodents. Furthermore, the presence of an alternative spliced PPAR-α isoform, which lacks the ligand-binding domain, could contribute to the lower responsiveness of the pig to clofibrate (49).
It has been found that pigs, in contrast to humans or rodents, have a high concentration of PPAR-α in adipose tissue. Ding et al. (12, 13) found that PPAR-α mRNA concentration, corrected for 18S ribosomal RNA, was three to four times higher in subcutaneous adipose tissue than in liver of young pigs with a body weight of 30 kg. In the young pigs used in the present study, expression of PPAR-α in adipose tissue, corrected for total mRNA content, was at a level in subcutaneous adipose tissue similar to that shown in liver. This also confirms that pig adipose tissue has a comparatively high expression of PPAR-α. To our knowledge, this is the first study that investigated the effect of treatment with a PPAR-α agonist on expression of PPAR-α target genes in adipose tissue of pigs. Our study shows that the PPAR-α target genes ACO, CPT-1, and SCD are indeed significantly upregulated by clofibrate, although only to a moderate extent. Although we did not perform a direct activation assay, we conclude that PPAR-α in adipose tissue is functional and is activated by PPAR-α agonists. In this study, we did not directly determine fatty acid oxidation in adipose tissue. The finding that genes involved in mitochondrial and peroxisomal were upregulated by clofibrate suggests that PPAR-α agonists indeed could stimulate β-oxidation in pig adipose tissue, which should be investigated in future studies.
To elucidate a possible link between PPAR-α activation and SREBP-mediated lipid homeostasis in pigs, we determined relative mRNA concentrations of SREBP-1 and -2, Insig-1 and -2, and some target genes of SREBP-1 (ACC) and SREBP-2 (HMG-CoA reductase, LDL receptor) in the liver and in the adipose tissue, which is the major site of lipogenesis in the pig. We found that mRNA concentrations of SREBP-1 and -2, Insig-1 and -2, and target genes of SREBP-1 and -2 in liver and adipose tissue were not different in clofibrate-treated and control pigs. This indicates that activation of PPAR-α by clofibrate did not influence expression and activity of SREBP-1 and SREBP-2 in both tissues. SCD is another target gene of SREBP-1 involved in lipogenesis, which also has a PPAR response element in its promoter and is upregulated by PPAR-α activation (30). We assume that an upregulation of SCD in liver and adipose tissue of pigs treated with clofibrate was probably due to PPAR-α activation. An upregulation of SCD in the liver of pigs by treatment with clofibrate has also been observed by Cheon et al. (8). SREBP-1 controls fatty acid and triglyceride synthesis, and SREBP-2 controls cholesterol synthesis and cholesterol uptake in cells via the LDL receptor (22). It is assumed that these SREBP-controlled processes were not altered by PPAR-α activation in liver and adipose tissue. This assumption is in contrast to recent findings in mice in which WY-14,643, a synthetic PPAR-α agonist, stimulated SREBP-1-mediated fatty acid synthesis in the liver (23) and findings in rats and hamsters, in which fibrates decreased SREBP-2-mediated expression of HMG-CoA reductase and LDL receptor (17, 25).
The present study shows for the first time that clofibrate treatment strongly reduces triglyceride concentrations in plasma and triglyceride-rich lipoproteins in pigs. These results agree with observations in humans and rodents (2, 16). Studies in humans and rodents have shown that this effect is in part due to increased oxidation of fatty acids in the liver, leading to reduced triglyceride synthesis and secretion, and in part due to increased lipolysis caused by induction of lipoprotein lipase activity and repressed transcription of apoC-III in the liver (14, 17, 41, 54). The observation that the PPAR-α target genes l-FABP, ACO, and CPT-1 were increased indicates that clofibrate increased the uptake of fatty acids into hepatocytes and stimulated both peroxisomal and mitochondrial β-oxidation in pigs. To find out whether clofibrate stimulates lipolysis, we determined mRNA concentrations of lipoprotein lipase in liver, adipose tissue, and muscle, extrahepatic tissues that also express comparatively high levels of PPAR-α (4), and mRNA concentration of apoC-III, an inhibitor of lipoprotein lipase. The observation that lipoprotein lipase mRNA could not be detected in liver of pigs of both groups suggests that the pig has generally a low expression of lipoprotein lipase in the liver. This suggestion is confirmed by recent studies that also showed a very low gene expression of lipoprotein lipase in liver of pigs (27, 44). The finding that clofibrate does not reduce apoC-III agrees with observations in monkeys (42) but disagrees with observations in human and rat hepatocytes and livers of hamsters in which apoC-III expression was downregulated by fibrates (9, 17, 26). Because lipoprotein lipase in adipose tissue and muscle was also not upregulated, the data of this study do not give any indication of increased lipolysis in pigs treated with clofibrate. It has recently been shown in monkeys that administration of a PPAR-α agonist increases serum levels of apoA-V, which has recently been recognized as a key regulator of serum triglyceride concentrations (42). Overexpression of apoA-V in mice caused a strong reduction of serum triglyceride concentrations (35, 51), and upregulation of apoA-V could be involved in the hypotriglyceridemic effect of PPAR-α agonists. For technical reasons, we were unable to determine gene expression of apoA-V, but it is possible that clofibrate reduced triglyceride concentrations in pigs by upregulation of apoA-V.
In this study, we also determined hepatic mRNA concentrations of MTP, the rate-limiting protein for assembly and secretion of VLDL in the liver, which has recently been demonstrated in mice to be a PPAR-α target gene (1). Our study shows that activation of PPAR-α by clofibrate does not stimulate gene expression of MTP in pigs, in contrast to rats, and probably does not stimulate secretion of lipids from the liver into the blood via VLDL. This could help to explain the observation that concentrations of triglycerides and cholesterol in the liver remained unchanged in pigs after clofibrate treatment.
In humans, treatment with fibrates usually reduces plasma and LDL cholesterol concentrations and increases HDL cholesterol (46). The present study shows that clofibrate reduces plasma and LDL cholesterol concentrations in pigs, as also shown in humans. The effect of clofibrate on HDL cholesterol, however, is opposite to that observed in humans. The elevation of HDL cholesterol by fibrate treatment in humans is caused primarily by increased gene expression of apoA-I and apoA-II in the liver (3, 52). In contrast to humans, fibrates lower plasma HDL concentrations in rats and hamsters because of a decrease of liver apoA-I and apoA-II gene expression (17, 45, 47). The finding that mRNA concentrations of apoA-I and apoA-II in the liver did not differ between both groups of pigs suggests that clofibrate reduced HDL cholesterol by a mechanism other than HDL reduction.
It has been shown in mice that activation of PPAR-α leads to downregulation of CYP7, the key enzyme of hepatic bile acid formation, which is probably because of reduced availability of hepatic nuclear factor-4, a transcription factor involved in the basal expression of CYP7 (29, 38). The present study shows that activation of PPAR-α by clofibrate does not alter hepatic mRNA concentrations of CYP7, which, along with LDL receptor and HMG-CoA reductase, is a key factor of hepatic cholesterol homeostasis (40). The finding that mRNA concentrations of these three genes were not altered by clofibrate treatment agrees with a recent study in which pigs were treated with clofibrate (34) and is in accordance with the observation that hepatic cholesterol concentrations were also unchanged in pigs treated with clofibrate compared with control pigs. These data also suggest that the greatly reduced concentration of LDL cholesterol is not because of a lowered cholesterol synthesis, enhanced elimination of cholesterol from the liver via bile acid formation, or upregulation of LDL receptor. It has been shown in humans that fibrate treatment enhances LDL uptake via the LDL receptor, not as a result of increased LDL receptor expression but as a result of the formation of LDL particles with a higher affinity to the LDL receptor (5, 7). It is possible that a similar effect is responsible for the strongly reduced LDL levels in pigs treated with clofibrate. This should be investigated further in future studies.
In conclusion, this study shows that clofibrate treatment causes an increase in liver weights, indicative of peroxisome proliferation, and moderate upregulation of PPAR-α target genes involved in liver and adipose tissue. Clofibrate treatment causes a strong reduction of triglyceride and cholesterol concentrations in plasma and lipoproteins, which agrees with findings in rodents. Gene expression analysis of lipoprotein lipase, apoA-I, apoA-II, and apoC-III in the liver suggests that biochemical mechanisms underlying these effects might be in part different from those in humans or rodents. In pigs, unlike rodents, hepatic concentrations of triglycerides and cholesterol are not altered by clofibrate. We also showed that PPAR-α activation by clofibrate does not affect expression of SREBP target genes, which are involved in synthesis of triglycerides and cholesterol, in liver and adipose tissue of pigs.
The authors thank Bettina König for critical discussion of the manuscript.
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