Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

There are three related members of the peroxisome proliferator-activated receptor (PPAR) familyPPAR, PPAR, and PPARthat are subjected to regulation by fatty acids and lipid metabolites (Schoonjans et al., 1996). Individual PPARs dimerize with the retinoid X receptor and the PPAR-retinoid X receptor complex binds to specific DNA response elements [peroxisome proliferator response elements (PPREs) composed of hexanucleotide direct repeats] in gene promoters and functions as a ligand-activated transcription factor (Gearing et al., 1993). After ligand binding, the PPAR ligand binding domain undergoes specific conformational changes allowing for the recruitment of one or more coactivator proteins (such as steroid receptor coactivator 1 or cAMP-response element-binding protein). The receptor-coactivator complex then interacts with components of the basal transcriptional apparatus, resulting in the induction of RNA transcription (McInerney et al., 1998).

PPAR is expressed in liver and other tissues including kidney, heart, and to a lesser degree, muscle and brown fat. Activation of PPAR in liver of mice or rats stimulates fatty acid oxidation and peroxisome proliferation (PP). Known PPAR target genes include the enzymes of peroxisomal and mitochondrial fatty acid -oxidation, liver fatty acid-binding protein lfabp, and carnitine palmitoyl transferase (Kaikaus et al., 1993). PPAR is expressed at highest levels in adipose tissue where it mediates transcriptional activation of the promoters for aP2 (adipocyte fatty acid-binding protein) and other adipocyte genes involved in regulation of lipid uptake and lipogenesis (Schoonjans et al., 1996). PPAR is widely expressed; however, its target genes and physiologic effects are not known.

Compounds that cause PP in rodents are generally believed to act exclusively through the PPAR receptor because the potent and selective mPPAR agonist WY14643 is ineffective in inducing PP in PPAR-null mice (Lee et al., 1995; Gonzalez et al., 1998). We have observed, however, that selected compounds that are specific PPAR agonists, such as some of the insulin-sensitizing thiazolidinediones (TZDs) (Lehmann et al., 1995; Berger et al., 1996; Elbrecht et al., 1996), can cause PP in mice when administered at high dose levels. There are at least four potential mechanisms that might account for this observation: 1) compounds that lack significant murine PPAR activity based on in vitro assays may possess weak PPAR activity in vivo; 2) such compounds may undergo in vivo metabolism, resulting in de novo generation of PPAR agonists; 3) high occupancy of PPAR in liver may be sufficient to mediate some effects normally attributed to PPAR; 4) compounds exhibiting these effects might act through another related receptor, such as PPAR, which is expressed in the liver and could function as a surrogate for PPAR. Because it would be desirable to develop therapeutic agents lacking the liability of rodent PP and the associated risk of tumorigenesis, the question arises of whether it would be possible to eliminate PP potential by increasing the PPAR specificity of compound candidates. We report here the results of studies with PPAR-null mice investigating whether PPAR- or -/-specific agonists are capable of inducing PP in the absence of PPAR.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

Compounds. Compounds used in this study include a potent known PPAR-specific TZD agonist {5-(4-[2-[methyl-(2-pyridyl)amino]ethoxy]benzyl)thiazolidine-2,4-dione}, previously described in (Berger et al., 1996). A non-TZD compound with potent murine PPAR and PPAR (but not PPAR) agonist activity, L-783,483 (Berger et al., 1999), provided by Dominick F. Gratale (Merck Research Laboratories, Rahway, NJ), was also used. A known PPAR selective agonist (Kliewer et al., 1994), WY14643 {[4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid}, was purchased from Chemsyn Science Laboratory (Lenexa, KS).

Animals and Assessment of In Vivo Parameters. CD-1 (ICR) BR mice (approximately 5 weeks of age at study initiation; Charles River, Raleigh, NC) or homozygous PPAR-null or sex- and age-matched wild-type (WT) Sv/129 mice (approximately 9 weeks of age at study initiation) (Lee et al., 1995) were allowed ad libitum access to rodent chow (Purina #5001; Purina Mills Inc., Brentwood, MO) and water. Animals were dosed daily by gavage with vehicle (0.5% carboxymethylcellulose) with or without PPAR agonists at the indicated doses. After treatment for 4 or 6 days, the animals were euthanized by isofluorane asphyxiation (CD-1) or pneumothorax under Nembutal anesthesia. Livers were rapidly removed and weighed, and samples were placed in ice-cold phosphate buffer for immediate processing or in liquid nitrogen and stored at 80°C for subsequent preparation of total RNA or measurement of enzyme levels or activity. Additional samples were taken for light and electron microscopy. Plasma glucose, triglyceride, and free fatty acid (FFA) concentrations were determined from blood obtained at necropsy. Glucose and triglyceride determinations were performed using standard glucose oxidase (Sigma, St. Louis, MO) and glycerol kinase (Boehringer Mannheim Biochemica, Indianapolis, IN) assays, respectively. FFA levels were determined by the acyl-coenzyme A (CoA) synthetase/acyl-CoA oxidase/peroxidase method (Boehringer Mannheim Biochemica). All in vivo experiments were approved by the Institutional Animal Care and Use Committee.

Measurement of Hepatic Acyl-CoA-Oxidase (ACO) and CYP4A Levels. Hepatic ACO activity was measured spectrophotometrically using leuco-dichlorofluorescein as the chromophore (Walusimbi-Kisitu and Harrison, 1983). Briefly, 20 µl of whole liver homogenate, diluted 1:40 with 12 mM sodium/potassium phosphate buffer, pH 7.4 (dilutions were increased as necessary to keep the rate of increase in A < 0.08 absorbance units/min) was incubated with 0.02% Triton X-100, 40 mM aminotriazole, 2.3 U of horseradish peroxidase, 0.6 µM leuco-dichlorofluorescein, and 33 µM palmitoyl-CoA in a 30°C cuvette. The increase in A at 502 nm was monitored for ~1.5 min after a lag period of 2 min against a blank lacking palmitoyl-CoA in a final volume of 0.92 ml.

Measurement of PPAR Target Gene Expression. Frozen tissues were pulverized and then homogenized in 10 volumes of ULTRA SPEC buffer (Biotecx Laboratories, Inc. Houston, TX) according to the manufacturer's specifications, followed by extraction of total RNA as described previously (Chomczynski and Sacchi, 1987). Aliquots of total RNA (15 µg) were denatured in a solution of 2.2 M formaldehyde and 50% formamide, 1× MOPS/EDTA buffer (Digene Diagnostics, Inc., Beltsville, MD), and 0.01 mg/ml ethidium bromide by heating at 70°C for 10 min. The samples were cooled, and gel loading buffer (0.5% xylene cyanol, 0.5% bromophenol blue, 40% sucrose, 2.2 M formaldehyde, and 50% formamide) was added. The samples were then loaded onto 1.2% SeaKem Gold agarose (FMC BioProducts, Rockland, ME) gels in 1× MOPS. After electrophoresis, RNA was transferred to Duralon-UV membranes (Stratagene, La Jolla, CA) overnight in 20× saline/sodium phosphate/EDTA (SSPE) (Digene Diagnostics, Inc.) (Ausubel et al., 1987). After UV cross-linking with UV Crosslinker 1800 (Stratagene), Northern blots were prehybridized in Express-Hyb (CLONTECH Laboratories, Inc., Palo Alto, CA) at 60°C for 1 h then hybridized to specific ³²P-labeled cDNA probes at a concentration of 1 to 4 × 10⁶ cpm/ml. The hybridization was carried out for at least 16 h at 60°C. The blots were washed twice in 2× SSPE, 0.1% SDS at 60°C for 30 min each and then twice in 0.1× SSPE, 0.1% SDS at 50°C for 10 min. The membranes were sealed in plastic and exposed to a PhosphorImager screen. The screens were analyzed on a Molecular Dynamics PhosphorImager with ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

Microscopic Examination. At necropsy, tissue samples were placed in 10% neutral buffered Formalin. Tissues were processed by routine methods and embedded in paraffin. Sections of liver, approximately 5 µm thick, were immunohistochemically stained with a rabbit polyclonal antibody to rat and mouse PMP70 (Affinity Bioreagents, Golden, CO), a peroxisomal membrane protein known to be induced by peroxisome proliferators (Baumgart et al., 1989). For transmission electron microscopy, liver sections were fixed in 4% formaldehyde + 1% glutaraldehyde solution, and 1 mm³ blocks were cut, washed in 0.15 M phosphate buffer, postfixed in a 1% osmium tetroxide solution, and embedded in Epon 812 (Polysciences Inc., Warrington, PA) after alcoholic dehydration. Blocks of liver from all control and treated animals were cut using a Reichert Ultracut ultramicrotome (Leica, Deerfield, IL). Toluidine blue-stained semi-thin sections were examined by light microscopy to assess the adequacy of tissue preservation for ultrastructural examination and to select similar centrilobular and periportal areas in the liver of control and treated animals. Uranyl acetate- and lead citrate-contrasted ultrathin sections were then examined using a CM12 (Philips/FEI Co., Hillsboro, OR) transmission electron microscope at 80 keV. From each animal, micrographs of hepatocytes were randomly obtained during microscopic observation of the liver for examination of the peroxisome content. The number of hepatic peroxisomes (differentiated from mitochondria based on ultrastructure) was semiquantitatively assessed (from + = few to ++ = numerous) for each group, based on the observation of approximately 20 micrographs per group.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

PP Can Be Caused by Compounds Lacking In Vitro PPAR Activity. Using cloned isoforms of PPAR, PPAR, and PPAR, we previously established (Berger et al., 1996) that the TZD compound used in this study was a potent (K_i < 50 nM) PPAR agonist without detectable PPAR or PPAR activity (up to 30 µM in in vitro assays). L-783,483 was also previously shown (Berger et al., 1999) to lack murine PPAR activity, although it has potent activity on both murine PPAR (29 nM) and PPAR (30 nM). Finally, the potent rodent PPAR agonist WY14643 was shown not to have detectable in vitro PPAR or PPAR agonist activity (data not shown). In studies to determine whether compounds with PPAR (and PPAR) activity might be able to induce hepatic PP, liver weight, ACO activity, and CYP4A levels were measured after four daily doses (500 mg/kg/day) of TZD or L-783,483 were administered to CD-1 mice (n = 4, each sex). The results of these studies are shown in Table 1. At these dose levels, both compounds were effective in inducing increases in all three parameters. Treatment with the TZD compound resulted in increases in mean liver weight of 17 to 27% versus 36 to 59% increases with L-783,483. Mean hepatic ACO activity was increased by 245 to 260% with TZD and by 460 to 720% with L-783,483 treatment; in addition, mean CYP4A levels were increased by 125 to 221% with TZD and by 206 to 466% with L-783,483. Thus, in vivo treatment of normal mice with a PPAR-selective agonist or a dual PPAR/PPAR agonist resulted in substantial hepatic effects that were consistent with induction of the PP pathway. With the possible exception of the observations of Kolattukudy et al. of PP in the uropygial glands of mallard ducks in response to estradiol (Bohnet et al., 1991; Ma et al., 1998), this represents the first demonstration of induction of hepatic ACO or CYP4A enzymes by compounds with PPAR and/or PPAR activity.

Effects of PPAR Agonists in PPAR-Null Mice. In vivo experiments were conducted using male PPAR-null mice and matched controls (WT) to assess whether the effects seen in CD-1 mice were mediated by spillover onto PPAR or by independent mechanisms (e.g., occupancy and activation of hepatic PPAR and/or PPAR mimicking the effect of PPAR activation). In an initial study, both PPAR WT and PPAR-null mice (n = 4 males in each group) were treated with four daily doses of vehicle, TZD, or L-783,483 just as CD-1 mice had been. There was clear evidence of PP in the PPAR WT mice; in the null mice, the results were suggestive of a modest degree of inductionin particular, both TZD and L-783,483 appeared to retain the capacity to induce ACO activity (data not shown). To firmly establish that induction of parameters related to PP could occur in the absence of PPAR, a second study was performed in which groups of four male mice were treated with six daily doses of TZD or L-783,483 (500 mg/kg/day, each). A separate group of mice also received a PPAR-specific agonist, WY14643 (50 mg/kg/day); we have also verified that this compound is a potent (75 nM) and specific (no PPAR or PPAR activity) PPAR agonist using cloned murine isoforms (Berger et al., 1999).

Effects on Organ Weights and Clinical Parameters. Table 2 shows the effects of these compounds on liver weight and blood levels of glucose, triglycerides, and FFAs. In PPAR WT animals, all three compounds caused significant decreases in serum triglyceride levels. In the null mice, however, no decrease in triglycerides was seen with any of the compounds. The effect of fibrates and compounds such as WY14643 to lower triglyceride levels has been proven to be mediated by PPAR (Peters et al., 1997). Our results, therefore, suggested that triglyceride lowering was mediated almost exclusively by PPAR with little contribution from PPAR (or PPAR) and, further, that some degree of PPAR activation was being effected by the PPAR/-specific compounds in the intact animal, in contrast with the results seen in vitro with cloned receptors. In accord with the known physiologic effects of PPAR activation on lipid metabolism (Schoonjans et al., 1997), FFA levels were significantly lowered by both compounds with PPAR activity (in both WT and null mice) but not by WY14643. PPAR agonists are effective in lowering glucose in insulin-resistant hyperglycemic states but do not elicit hypoglycemia; thus, as expected, glucose levels were not lowered in any treatment group below the normal levels present in both WT or null mice.

View this table: [in this window] [in a new window]   TABLE 2 Effect of PPAR agonist treatment on in vivo parameters and liver weight in WT versus PPAR-null mice Mice were treated as indicated for 6 days. All parameters are expressed as mean values (±S.E.).

Induction of PPAR Target Genes. Table 3 shows the effect of treatment on ACO activity and CYP4A content, two widely used markers of PP. PPAR WT animals displayed highly significant increases in both parameters in response to treatment with all three compounds. Although the extent of CYP4A induction was similar among compounds, WY14643 and L-783,483 caused larger increases in ACO activity (~18× and ~15×, respectively) versus only a modest increase of ~4.5× with TZD. In PPAR-null animals, WY14643, as expected, had no effect on either ACO activity or CYP4A content. In contrast, treatment with either TZD or L-783,483 increased both parameters in PPAR-null mice. The increases in ACO activity were modest relative to WT animals, but the induced levels of CYP4A were equivalent to WT for L-783,483 and approximately half that seen in WT for TZD (percentage increases were actually greater than those in WT mice due to low basal levels CYP4A).

View this table: [in this window] [in a new window]   TABLE 3 Effect of PPAR agonist treatment on ACO activity and CYP4A content in WT versus PPAR-null mice Mice were treated as indicated for 6 days. All parameters are expressed as mean values (±S.E.). CYP4A values have had an average "no sample" background of 0.10 AU subtracted.

View larger version (42K): [in this window] [in a new window]   Fig. 1.   mRNA expression levels. mRNA was isolated from the livers of WT or PPAR-null mice treated as indicated. A and B, duplicate lanes of Northern blots probed with cDNAs corresponding to two PPAR target genes, liver FABP (A) and ACO (B). C, mRNA expression levels of a gene not containing a PPRE, 23-kDa highly basic protein (HBP), used to normalize for RNA loading and transfer differences. D and E, quantitation of mean mRNA expression levels for liver FABP (D) or ACO (E) after normalizing against minor variation in 23-kDa highly basic protein expression. Shaded columns, WT; open columns, knockout (KO). F, expression of PPAR in the livers of WT and PPAR-null mice compared with levels in brown adipose tissue (BAT).

Effect of PPAR Agonists on Liver Histomorphology. Ultimately, hepatic PP is defined as an increase in the peroxisomal content of hepatocytes. Consequently, liver sections from each of the treatment groups were processed for both light and electron microscopy. The sections processed for light microscopy were stained with antisera to a peroxisomal membrane protein, PMP70. Examples of control, L-783,483-, and WY14643-treated liver sections are shown in Fig. 2. Significant induction was seen in both L-783,483- and WY14643-treated WT mice, but only L-783438 (and, to a lesser extent, TZD; data not shown) caused induction in null mice. The sections processed for transmission electron microscopy were used both to confirm that the changes seen in PMP70 were reflected in an actual increase in ultrastructurally identifiable peroxisomes as well as being subjected to semiquantitative analysis, as shown in Table 4. It can be seen that, on the basis of ultrastructural identification, the increase in PMP70 caused by L-783,483 in null mice does correlate with an increase in the number of peroxisomes. In addition, the lesser degree of PP caused by TZD, as determined from the electron micrographs (Table 4) also correlated with the level of PMP70 expression (data not shown). Although the zonal gradation of staining does not appear as clear-cut in the null mice as it is in the WT mice, this is likely due to the obscuring effect of the numerous vacuoles. Specific staining indicated that these vacuoles were lipid-filled and probably are indicative of an inability of the null mice to maintain lipid homeostasis in the face of metabolic stress produced by the administration of the PPAR/ agonist.

View larger version (174K): [in this window] [in a new window]   Fig. 2.   Light micrographs of liver sections. Sections of liver from WT and PPAR-null mice were immunohistochemically stained for the peroxisomal membrane protein PMP70 and examined by light microscopy. A (WT) and B (PPAR-null), vehicle-treated mice; C (WT) and D (PPAR-null), L-783,483-treated mice; E (WT) and F (PPAR-null), WY14643-treated mice.