ToxSci Advance Access originally published online on June 30, 2008
Toxicological Sciences 2008 105(2):384-394; doi:10.1093/toxsci/kfn130
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PPAR alpha, more than PPAR delta, Mediates the Hepatic and Skeletal Muscle Alterations Induced by the PPAR Agonist GW0742
* Safety Assessment Department, GlaxoSmithKline, Research Triangle Park, North Carolina 27709
Department of Toxicology, Alcon Research Labs, Fort Worth, Texas 76134
Safety Assessment Department, GlaxoSmithKline, The Frythe, UK
1 To whom correspondence should be addressed at GSK, PO Box 13398, 5 Moore Dr., Research Triangle Park, NC 27709. Fax: (919) 483-6858. E-mail: Brenda.x.Faiola{at}gsk.com.
Received April 1, 2008; accepted June 23, 2008
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Therapeutic use of certain peroxisome proliferator–activated receptor (PPAR) alpha agonists (fibrates) for the treatment of dyslipidemia has infrequently been associated with the untoward side effect of myopathy. With interest in PPAR-
as a therapeutic target, this study assessed whether a PPAR- agonist induced similar hepatic and skeletal muscle alterations as noted with some fibrates. PPAR-
null (KO) and corresponding wild-type (WT) mice were administered toxicological dosages of a potent PPAR- agonist tool ligand (GW0742; which also has weak PPAR- agonist activity) or a potent PPAR- agonist (WY-14,643) for 10 days. Increases in liver weights and clinical chemistry indicators of skeletal muscle damage and/or liver injury were more pronounced in WT mice compared with KO mice administered the PPAR- agonist. Likewise, the incidence and severity of skeletal myopathy were greater in WT mice given GW0742 compared with KO mice. Ultrastructural and immunohistochemical analyses revealed significant peroxisome proliferation in muscle and liver of WT mice treated with each agonist; however, KO animals showed little or no evidence of hepatic and muscle peroxisome proliferation. PMP-70 protein expression in liver was consistent with these results. The hepatomegaly, hepatic and skeletal muscle peroxisome proliferation, and skeletal myopathy induced by this PPAR- ligand was predominantly mediated by its cross-activation of PPAR-, though PPAR- agonism contributed slightly to these effects. Key Words: peroxisome proliferator–activated receptor; myopathy; mice.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Peroxisome proliferator–activated receptors (PPAR)-
, - (also known as β), and -
are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily (reviewed in Peraza et al., 2006
and Shearer and Hoekstra, 2003). PPAR- is expressed in tissues having high rates of β-oxidation such as brown adipose tissue, liver, kidney, heart, and skeletal muscle (Braissant et al., 1996; reviewed in Shearer and Hoekstra, 2003 and Willson et al., 2000). PPAR- is ubiquitously expressed, with the highest levels in the intestine, colon, brain, skeletal muscle, kidney, and skin (Braissant et al., 1996; reviewed in Willson et al., 2000). PPAR-, present in humans as 2 protein isoforms, is highly expressed in brown and white adipose tissue, and to a lesser degree in the colon, immune system (macrophages, spleen), heart, and skeletal muscle (Braissant et al., 1996; reviewed in Willson et al., 2000). PPARs bind peroxisome proliferator response elements in the promoter region of target genes as a heterodimer with retinoid X receptor and activate transcription in response to ligand binding of one or both receptors in the complex. PPARs are important for the regulation of lipid and glucose metabolism, cell proliferation and differentiation, adipogenesis, and inflammatory signaling in numerous tissues (reviewed in Kersten et al., 2000). Thus, PPARs are attractive targets for the development of therapeutic agents for a wide variety of disorders, and several PPAR agonists are currently in clinical use.
PPAR- agonists such as glitazones (thiazolidinediones) are commonly used as glucose lowering agents in diabetic patients whereas PPAR- agonists such as the fibrates are efficacious as lipid lowering agents (reviewed in Peraza et al., 2006 and Shearer and Hoekstra 2003). However, the ability to modulate PPAR- activity with the therapeutic use of fibrates has been associated with the untoward, albeit infrequent, side effect of skeletal myopathy (Hodel, 2002). It is well known that activation of PPAR- in rodents induces peroxisome proliferation and hepatomegaly and can also induce skeletal myopathy (Hodel, 2002; NTP, 2007). The mechanism for the PPAR-–induced myopathy is unclear but may, at least in part, result from increased mitochondrial and peroxisomal β-oxidation of fatty acids leading to oxidative stress and tissue damage. Our findings in rats treated with fenofibrate (a lipid lowering PPAR- agonist) indicate that slow-twitch (Type 1) oxidative fibers are more sensitive to myopathic damage than fast-twitch (Type 2) glycolytic fibers and that genes associated with oxidative stress are more highly upregulated in muscles composed primarily of oxidative fibers (Bordelon et al., 2005). In addition, long-term administration of certain PPAR- agonists is hepatocarcinogenic in rodents but to date, there is no evidence of increased hepatocarcinogenesis in man with therapeutic use of fibrates (Peraza et al., 2006), presumably due to the lower expression of PPAR- in human liver compared with rodent liver as well as other differences in receptor transactivation and transcriptional events.
Much less is known about PPAR- compared with what is known about the ligands and functions of the other two PPAR subtypes. PPAR- regulates expression of acyl-CoA synthetase 2 in the brain (implicating PPAR- in lipid metabolism) and may be involved in the potentiation and/or attenuation of certain cancers in rodents (Marin et al., 2006; reviewed in Kersten et al., 2000; Peraza et al., 2006). Recently, selective PPAR- agonists have been developed which have enabled investigations into the role of this receptor. In vitro studies with human and rat myotubes showed that treatment with GW0742, a potent PPAR- agonist with weak PPAR- agonist activity, increased fatty acid oxidation levels and induced mRNA expression of genes involved in fatty acid uptake and β-oxidation similar to that of the PPAR- agonist GW647 (Muoio et al., 2002). Experiments which characterized the gene expression in the C2C12 myotube culture system following treatment with GW501516, a potent PPAR- agonist related to GW0742 that has weak PPAR- and PPAR- agonist activity, suggested a role for PPAR- in energy uncoupling, lipid uptake and storage, triglyceride hydrolysis, cholesterol efflux, and fatty acid oxidation (Dressel et al., 2003). The increased fatty acid oxidation induced in primary cultured human muscle cells by GW501516 was dependent on both PPAR- and AMP-activated protein kinase (Kramer et al., 2007). In addition, studies using cardiomyocytes and H9c2 myoblasts and myotubes implicated PPAR-, along with PPAR-, in the regulation of cardiac fatty acid metabolism (Gilde et al., 2003). In a transgenic mouse model, PPAR- overexpression led to prolonged exercise endurance by increasing the number of slow-twitch (Type 1) oxidative muscle fibers and to decreased weight gain (Wang et al., 2004). From these in vitro and in vivo studies, a clinical interest in PPAR- agonists for the treatment of metabolic syndrome and obesity has been stimulated. The potential of PPAR- agonists to induce myopathy similar to that noted with some PPAR- agonists must therefore be a consideration in the development of such therapeutic agents.
Using the recently described potent PPAR- tool ligand, GW0742 (Sznaidman et al., 2003), studies were conducted in wild-type (WT) and PPAR- null (KO) mice to elucidate the role of PPAR- in the morphologic and cellular effects in skeletal muscle and liver induced by this PPAR- agonist. This PPAR- ligand is also able to bind and activate murine and human PPAR- (as well as human PPAR-) at high dosages, such as those used here. The EC50 of GW0742 in a cell-based transactivation assay is approximately 30nM for murine PPAR- and 8.8µM for murine PPAR- (Sznaidman et al., 2003). It is well established that PPAR- null mice do not exhibit the pleiotropic response (hepatocellular proliferation, hepatic peroxisome proliferation, hepatomegaly, and transcriptional activation of peroxisomal β-oxidation and microsomal 
-oxidation enzymes) to prototypical PPAR- agonists such as clofibrate and WY-14,643 (Lee et al., 1995). In addition, PPAR- null mice are resistant to hepatocarcinogenesis after long-term exposure to WY-14,643 (Peters et al., 1997) and the PPAR pan agonist bezafibrate (Hays et al., 2005). Thus, this model system is well suited for assessing the biologic responses to PPAR- activation in the absence of PPAR- activation, thereby clarifying the role of each receptor. WY-14,643 was used as the prototypical PPAR- agonist in this study based on its hepatic effects that had been well characterized in WT and PPAR- null mice at the time our experiments began. After completion of our investigations, a recent report has also shown high doses (> 100 mg/kg/day) of WY-14,643 given for 3 months induces skeletal myopathy in rats and mice (NTP, 2007). The EC50 of WY-14,643 in the same cell-based transactivation assay mentioned above is approximately 0.63µM for murine PPAR- (unpublished data).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mice.
Groups of 10-week-old, male PPAR-
null (KO) mice (B6.129S2-PparatmlN12) and the corresponding C57BL/6 WT control mice (Taconic, Germantown, NY) (10 per group) were administered 0 (vehicle), 100, or 250 mg/kg/day GW0742 (potent PPAR-, weak PPAR- agonist in rodents), or 50 mg/kg/day WY-14,643 (potent PPAR- agonist, very weak PPAR- agonist; Sigma, St Louis, MO) in 0.5% aqueous hydroxypropyl methylcellulose (Dow Chemical Co., Midland, MI) once daily for 10 days by oral gavage (10 ml/kg/day). The doses of GW0742 selected were known to induce hepatic changes associated with peroxisome proliferation in WT mice, and the high dose was known to induce skeletal myopathy in WT mice (unpublished data). The dose of WY-14,643 was shown to increase liver weights in male B6C3F1 mice following oral gavage for 3 days (Anderson et al., 2001), and was anticipated to induce liver weight increases of similar magnitude in WT mice as the low dose of GW0742 used here. Mice were single-housed in polycarbonate solid-bottom cages with Bed-O'Cobs (The Andersons, Maumee, OH) in a temperature- and humidity-controlled environment with a 12-hour light/dark cycle. Mice were fed LabDiet brand Certified Rodent Diet 5002 pellets (PMI Nutrition International, Richmond, IN) and provided reverse osmosis-treated water ad libitum. Clinical observations were performed daily (approximately 1-2 h after dosing). Body weights and food consumption measurements were taken on Days 1, 5, and 10. On Day 11 (approximately 24 h after administration of the final dose), terminal body weights were taken and animals were euthanized by exsanguination after administration of isoflurane anesthesia. Blood was collected from the abdominal vena cava into serum separator tubes without additives for measurement of serum clinical chemistry parameters using the Olympus AU640 chemistry analyzer. Liver weights were recorded and tissues were collected for histological and/or immunohistochemical examination, ultrastructural analysis (liver only), and protein expression (liver only). Statistically significant differences (p value 
0.05) in clinical chemistry parameters and liver weights from treated groups compared with the appropriate vehicle control group were determined by the Dunnett's test. Statistically significant differences (p value 0.05) between strains were determined by a Student's t-test. Samples of liver and muscle were also collected for gene expression analysis (manuscripts in preparation). The Institutional Animal Care and Use Committee of GlaxoSmithKline approved all animal use.
Histological and immunohistochemical examination.
All tissues collected were fixed in 10% neutral buffered formalin, embedded in paraffin wax, sectioned, and stained with hematoxylin and eosin (H&E) for light microscopy. Severity grades of skeletal myopathy were assigned numeric values (minimal = 1; mild = 2; moderate = 3; marked = 4), and scores from all five muscles (diaphragm, gastrocnemius, intercostals, quadriceps, and soleus) from each animal evaluated were summed. These five muscles were selected as they represent various regions of the body and various fiber-type compositions. Additional sections of quadriceps muscle from all animals, intercostal muscle from selected animals, and liver from five animals per group were labeled with rabbit polyclonal antibody specific for the 70-kDa peroxisomal membrane protein (PMP-70; also known as ABCD3 and PXMP1) (Affinity Bioreagents, Golden, CO). Serial sections of quadriceps and intercostal muscles were labeled with mouse monoclonal antibody specific for myosin heavy chain (MHC) slow or MHC fast (Novocastra, Newcastle Upon Tyne, UK) using the Animal Research Kit (Dako, Carpinteria, CA) to identify Type 1 or Type 2 fibers, respectively. PMP-70 labeled slides were developed using either 3,3'-diaminobenzidine (DAB) chromagen (Dako, Carpinteria, CA) or streptavidin-conjugated Alexa 488 (Invitrogen, Carlsbad CA). MHC-labeled slides were developed using Permanent Red or DAB chromagen (Dako) for MHC fast or slow, respectively. Sections were counterstained with DAPI (Invitrogen) to label the nuclei. A qualitative assessment of PMP-70 immunoreactivity in muscle was performed and a quantitative assessment of PMP-70 immunoreactivity in liver was performed using the iCyte Laser Scanning Cytometer (CompuCyte Corporation, Cambridge, MA). Statistically significant differences (p value 0.05) in liver from treated groups compared with the appropriate vehicle control group were determined by the Dunnett's test. Statistically significant differences (p value 0.05) between strains were determined by a Student's t-test (two tailed, homoscedastic).
Ultrastructural analysis.
Liver samples (right median lobe; adjacent to slices taken for histology) were fixed in Trump's fixative and stored at 2–8°C. Samples from five animals per group were processed and embedded in Spurr's resin. An area from Zone 3 (centrilobular) of the liver was sectioned (
90 nm), stained with 5% methanolic uranyl acetate and Reynold's lead citrate, and examined on a JEOL 1010 transmission electron microscope (TEM). The total number of mitochondria and peroxisomes in each photomicrograph (x1000) were manually counted. Histomorphometric analysis was also performed on the same images using Weibel's modified principles (Weibel, 1979) and Image-Pro-Plus software (MediaCybernetics, Silver Spring, MD). The central (or largest) hepatocyte in each image was selected for histomorphometric evaluation. For each grid point overlying this central hepatocyte, a tag was placed based upon the organelle over which the grid point was superimposed (nucleus, peroxisomes, or mitochondria). If the grid point was not superimposed on a nucleus, peroxisome, or mitochondria then the point was tagged as "cytoplasm."
Hepatic protein expression.
After removal of the gallbladder, remaining liver tissue was frozen in liquid nitrogen and stored at or below –70°C. Protein fractions from individual liver samples were prepared according to the T-PER Tissue Protein Extraction Reagent protocol (Pierce, Rockford, IL). Approximately 200 mg of tissue was homogenized in the extraction reagent containing a protease inhibitor cocktail (Sigma). Homogenized samples were centrifuged at 10,000 x g for 5 min at 4°C. The resulting supernatant was filtered through glass wool and analyzed for protein concentration via the bicinchoninic acid protein assay (Pierce). Liver protein samples from five animals in each group (five or six replicates per animal) were analyzed by immunoblot for PMP-70 expression. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was performed using gradient 4–12% Tris-Bis precast gels under conditions supplied by the manufacturer (Bio-Rad Laboratories, Inc., Hercules, CA), and the separated proteins were electrophoretically transferred to a PVDF membrane (Bio-Rad Laboratories, Inc.) for 30 min at 100 V. The Odyssey protocol for 2-color immunoblotting was used to allow for simultaneous detection of PMP-70 and β-actin (control for total protein loaded). Briefly, the membrane was blocked for 1 h at room temperature in Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE), then incubated for 3 h in Odyssey blocking solution containing both the rabbit anti-PMP-70 antibody (Affinity Bioreagents) diluted 1:1000 and the mouse anti-β-actin antibody (Abcam, Inc., Cambridge, MA) diluted 1:4000. The membrane was washed in PBS with Tween 80 several times and incubated for 1 h at room temperature in Odyssey blocking solution containing a 1:1000 dilution of IRDye 800 conjugated goat anti-rabbit IgG (Rockland, Gilbertsville, PA) and a 1:5000 dilution of Alexa Fluor 680 conjugated goat anti-mouse IgG (Molecular Probes, Eugene, OR). The membrane was then washed with PBST several times, rinsed in PBS, and allowed to air dry. Scanning of the membrane was performed using the 700 and 800 nm channels of an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE) for the detection of β-actin and PMP-70 protein levels, respectively. Quantitation of PMP-70 levels per protein sample was determined by normalizing the signal for PMP-70 to the signal obtained with β-actin. Statistically significant differences (p value 0.05) in the mean normalized signal intensity among the groups were determined by the Dunnett's test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Clinical Chemistry Results, In-Life Data, and Liver Weights
In WT mice, administration of 100 and 250 mg/kg/day GW0742 was associated with dose-dependent elevations in the skeletal myopathy and liver injury markers creatine kinase (CK), aldolase (ALD), and lactate dehydrogenase (LDH); the prototypical liver injury markers alanine aminotransferase (ALT) and aspartate aminotransferase (AST); as well as albumin/globulin (A/G) ratio and alkaline phosphatase (ALP) (Table 1). The increases in ALT and AST likely reflect damage to both the liver and the skeletal muscle in these animals. In addition, albumin was increased, whereas globulin was decreased, in WT mice given 250 mg/kg/day GW0742. Administration of WY-14,643 was associated with mildly decreased LDH and globulin as well as mildly increased ALP and A/G ratio.
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In PPAR-
KO mice, administration of 100 and 250 mg/kg/day GW0742 was also associated with dose-dependent increases in CK, ALD, and LDH (Table 1). A/G ratio, ALT, and AST were slightly increased in KO mice given 250 mg/kg/day GW0742. The clinical chemistry changes were more pronounced in the WT mice compared with the KO mice, though there was considerable individual animal variation. Similar to WT mice, administration of WY-14,643 was associated with mildly decreased LDH activity in the KO mice, the significance of which is unknown.
Administration of each PPAR agonist to KO and WT mice resulted in no treatment-related clinical signs, changes in body weight and food consumption, or macroscopic findings. Dose-dependent increases in mean absolute and relative liver weights were induced by treatment with GW0742 in both strains, but the response was approximately 3-fold greater in WT mice compared with the PPAR- KO mice. WY-14,643 increased liver weights in WT mice to a similar extent as observed for the low dose of GW0742 but had no effect on the liver weight of PPAR- KO mice.
Histological Examination of Liver and Muscle
In general, microscopic changes in the liver were dose related and were more severe in WT than PPAR- KO mice (Fig. 1). Marked diffuse hepatocellular hypertrophy, accompanied by marked granular eosinophilia was observed in all ten WT mice treated with 250 mg/kg/day GW0742. The same dose in PPAR- KO mice induced only minimal to mild centrilobular hepatocellular hypertrophy in all ten mice, which was accompanied by minimal hepatocellular granular eosinophilia in six mice. At 100 mg/kg/day GW0742, moderate to marked centrilobular hepatocellular hypertrophy, accompanied by mild hepatocellular granular eosinophilia was noted in all ten WT mice, whereas eight PPAR- KO mice showed just minimal centrilobular hepatocellular hypertrophy. An additional microscopic liver finding present in 50% of WT mice given 250 mg/kg/day GW0742 was minimal to mild hepatocellular apoptosis. Administration of GW0742 to both strains at both doses also resulted in a minimal to mild increase in mitotic figures in a few mice. WY-14,643 at 50 mg/kg/day induced minimal to mild centrilobular hepatocellular hypertrophy and granular eosinophilia in WT mice and produced no histologic alterations in the liver of PPAR- KO mice. The hepatocellular hypertrophy correlated with the increased liver weights noted for both strains and both agonists.
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Skeletal myopathy was observed in multiple muscles in both WT and KO mice given 250 mg/kg/day GW0742; however, both the severity and incidence of the lesions were greater in WT mice resulting in an overall 6.2-fold higher total myopathy score in WT mice compared with KO mice (Table 2). At 100 mg/kg/day, myopathy was observed in the quadriceps of just one WT mouse. The skeletal myopathy was predominately degenerative in nature. The myopathy was characterized by a mixture of very acute myofibril degeneration (cytoplasmic disruption with vacuolation, pallor or eosinophilia of myofibril cytoplasm) accompanied by more advanced degeneration in which a primarily mononuclear inflammatory component had become established (intrafibrillar and interstitial inflammatory cell infiltrates, with low-grade interstitial edema). Regeneration of the more long-standing damaged myofibrils was a minor component of the myopathy, characterized by occasional myofibrils with basophilic cytoplasm and centrally arranged linear nuclei (Fig. 2). The most severely affected muscle was the quadriceps, followed by the intercostal, gastrocnemius, soleus, and diaphragm. As expected with this dose and duration, WY-14,643 did not induce myopathy in either strain; however, minimal myofiber regeneration was noted in 7 of 10 WT and 7 of 10 KO mice given WY-14,643 compared with 0 and 3 of 10 WT and KO controls, respectively. Minimal myofiber regeneration was also noted in 10 of 20 WT mice and 6 of 20 KO mice treated with GW0742. There were no microscopic lesions in the hearts of WT or PPAR-
null mice attributable to administration of GW0742 or WY-14,643.
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Hepatic Peroxisome Proliferation
The mean number and percent area of peroxisomes in the liver observed by TEM increased in a statistically significant, dose-dependent manner in WT mice given GW0742 compared with vehicle control mice (Table 3). WT mice treated with 50 mg/kg/day WY-14,643 also had a statistically significant increase in the number and area of peroxisomes, similar to the level seen in WT mice treated with 100 mg/kg/day GW0742, as well as a slight decrease in the mitochondrial area. Although a few individual PPAR-
KO mice given 250 mg/kg/day GW0742 showed an increase in peroxisomes, due to the high interanimal variability, a significant difference from controls was not detected for this group.
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Immunohistochemical analysis of hepatic PMP-70, a peroxisomal membrane protein often used as a marker of peroxisome proliferation (Colton et al., 2004
), confirmed the results of the TEM evaluation (Fig. 3). In general, DAB labeling yielded a more robust signal intensity than the Alexa 488 staining. The analysis showed that with one or both detection methods, there was a subtle increase in mean PMP-70 signal intensity in GW0742-treated WT and KO mice compared with their respective controls. Quenching of the signal may account for the lack of dose responsiveness observed in WT mice. PMP-70 mean signal intensity was significantly higher in the liver of WT controls than KO controls. Both WT and PPAR- KO mice administered 50 mg/kg/day WY-14,643 had similar or slightly higher signal intensities for PMP-70 in the liver compared with their respective strain control; however, the standard deviations were large and statistical significance was not demonstrated.
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) indicates a statistically significant difference (p Western blot analysis of PMP-70 supports the results of the TEM and immunohistochemical evaluations. PMP-70 was detected in liver protein samples from all WT and PPAR-
KO mice tested. The constitutive level of PMP-70 protein expression was greater in the WT control mice compared with the PPAR- KO vehicle controls, confirming the results of the immunohistochemical analysis. PMP-70 expression showed a statistically significant, dose-dependent increase in WT and PPAR- KO mice after treatment with GW0742 for 10 days; the induction was greater in WT mice (Fig. 4). In mice administered 250 mg/kg/day GW0742, the PMP-70 protein levels in WT and KO were increased 5.8- and 4.1-fold, respectively, compared with their respective vehicle controls. PMP-70 expression was also significantly induced in WY-14,643–treated WT mice to a similar level as observed for 100 mg/kg/day GW0742-treated WT mice; however, PPAR- KO mice treated with WY-14,643 did not show induction of PMP-70.
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Skeletal Muscle Peroxisome Proliferation
PMP-70 protein was also detected by immunohistochemical methods in the skeletal muscle of WT and KO mice administered each PPAR agonist. The incidence and/or intensity of PMP-70 labeling showed a dose dependence in both WT and KO mice treated with GW0742 (Table 4), with the WT mice exhibiting a higher incidence and intensity of staining than the KO mice. Treatment of WT and KO mice with WY-14,643 resulted in similar levels of skeletal muscle PMP-70 immunoreactivity as that seen with the low, essentially non-myopathic dose of GW0742 treatment in the same strain. Fiber typing in the quadriceps and intercostal muscles revealed that the PMP-70 immunoreactivity was generally observed in Type 1 (MHC slow) or mixed fibers rather than in Type 2 (MHC fast) fibers; in general, the necrotic/myopathic fibers were also found to be Type 1 or mixed (Fig. 5). Treatment with GW0742 did not impact the fiber-type composition of the quadricep or intercostal muscles in either WT or KO mice; on average, the quadriceps contained
95% Type 2 fibers, whereas the intercostal muscle was composed of approximately 75% Type 2 fibers and 25% Type 1 and mixed fibers (Table 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Treatment of WT mice with the potent PPAR-
agonist GW0742 using dosages of 100 and 250 mg/kg/day resulted in dose-related effects in the liver (increased liver weight, hepatocellular hypertrophy and granular eosinophilia, and in a few mice apoptosis and/or a mitotic increase) and skeletal muscles (myopathy). These changes were associated with dose-related serum chemistry alterations (increased CK, ALD, LDH, AST, ALT, and ALP levels as well as some mild serum protein changes) and peroxisome proliferation in the liver and muscle. As anticipated, administration of the potent PPAR- agonist WY-14,643 to WT mice at 50 mg/kg/day resulted in mild hepatic (increased liver weight, hepatocellular hypertrophy and granular eosinophilia) and clinical chemistry (decreased LDH and globulin, increased ALP and A/G ratio) changes, but not skeletal myopathy.
The increased granular eosinophilia observed microscopically in hepatocytes of GW0742-treated WT and KO mice and WY-14,643–treated WT mice represented peroxisome proliferation as indicated by increased PMP-70 protein expression and confirmed by TEM. The number of hepatic peroxisomes determined by TEM was higher in the WT control mice compared with the KO control mice, and correlated with the higher level of PMP-70 protein expression detected in WT control mice by immunohistochemistry and western blot. The low-grade hepatocellular granular eosinophilia noted in the PPAR- KO mice given the high dose of GW0742 indicates susceptibility to peroxisome proliferation with sufficient stimulation of PPAR-.
As anticipated, the hepatic effects of the PPAR- agonist WY-14,643 were abolished in PPAR- KO mice. The hepatic and myopathic effects of the PPAR- agonist GW0742 were greatly reduced but not completely eliminated in PPAR- KO mice, suggesting that the liver and skeletal muscle alterations were primarily due to PPAR- activation. It should be noted that although GW0742 is quite potent against human PPAR- (EC50 of approximately 1nM with 1000-fold selectivity over human PPAR- in a cell-based transactivation assay) (Sznaidman et al., 2003), this molecule is potent against murine PPAR- (EC50 of approximately 30nM) but also has weak activity on PPAR- (EC50 of approximately 8.8µM for PPAR-, thus a selectivity of 300-fold for PPAR- vs. PPAR-) (Sznaidman et al., 2003). In a preliminary toxicokinetic study, C57BL/6 mice given 250 mg/kg/day GW0742 for 10 days had a maximal GW0742 plasma concentration of 142 µg/ml, which translates to 34-fold over the 4.15 µg/ml (8.8µM) EC50 for PPAR- (unpublished data); consequently, agonism of both PPAR- and PPAR- was expected in the WT mice in this study. Thus, the results of this study suggest that most, but not all, of the myopathic and hepatic injury properties of this PPAR- agonist were mediated through the PPAR- agonist activity inherent in this molecule, but PPAR- activation contributed somewhat to the liver and skeletal muscle effects. Recent studies using a rat myotube culture system further support the hypothesis that PPAR- agonism, more so than PPAR- or PPAR- agonism, mediates the PPAR-induced muscle toxicity (Johnson et al., 2005). Thus, development of a PPAR- agonist with the desired pharmacological effects (such as glucose lowering and insulin sensitivity), while guarding against a risk for potential skeletal myopathy and hepatic effects requires identification of a selective PPAR- agonist with little to no PPAR- agonist activity at efficacious dosages.
The mechanism of PPAR agonist-induced skeletal myopathy is unclear, but may in part be due to oxidative stress. Evidence from in vivo studies in our laboratory revealed that PPAR agonist-induced hepatic peroxisome proliferation and production of H2O2 as a result of increased hepatic peroxisomal β-oxidation of fatty acids results in rapid, systemic decreases in glutathione levels in rats (Faiola et al., 2006). Under such pro-oxidant conditions, continued mitochondrial and/or peroxisomal β-oxidation in liver and/or skeletal muscle due to PPAR agonism may then lead to further oxidative stress such that cells/tissues may be more susceptible to oxidative damage and skeletal myopathy may result. Our laboratory has also observed decreased blood glutathione levels suggestive of oxidative stress and skeletal myopathy in C57BL/6 mice given 250 mg/kg/day GW0742 for 10 days (unpublished data). If the hepatic effects of PPAR agonism are blunted such that the levels of glutathione, which is primarily synthesized in the liver, do not decrease, less skeletal myopathy may be expected; studies to test this hypothesis are ongoing. Here we showed that treatment of WT, but not PPAR- KO mice, with WY-14,643 resulted in increased hepatic peroxisome proliferation yet skeletal myopathy was not observed, suggesting that increased hepatic peroxisomal β-oxidation was not solely responsible for induction of skeletal myopathy, and that other biological and/or pharmacological factors may be involved. Although WY-14,643 is approximately 5- and 14-fold more potent against human and murine PPAR-, respectively, compared with GW0742, it is weakly active on murine PPAR- (EC50 of approximately 32µM) and is inactive on murine PPAR- at 100µM (Willson et al., 2000). The weak PPAR- activity in WY-14,643 may be one of the possible pharmacologic factors as to why this potent PPAR- molecule did not induce skeletal myopathy. In addition, after completion of our study, recently published literature suggests that the duration and dose of WY-14,643 used in our study may not have been sufficient to elicit a myopathic response (NTP, 2007; De Souza et al., 2006).
Evidence of increased fatty acid oxidation and/or oxidative stress as a mechanism for PPAR-induced muscle effects has also been noted in gene expression analyses. In vitro studies have shown that PPAR- is required for increased fatty acid transport and oxidation and increased expression of genes involved in lipid metabolism (FABP3, CPT1, and PDK4) in primary human muscle cells treated with GW501516 (a potent PPAR- agonist closely related to the GW0742 used here) (Kramer et al., 2007). Our laboratory has demonstrated induction of various genes in the quadriceps muscle of mice treated with GW0742 indicative of oxidative stress, cytokine response, and decreased glucose utilization which correlated well with levels of a serum marker of skeletal myopathy, CK, suggesting oxidative stress and decreased glycolytic activity are involved in PPAR agonist-induced skeletal myopathy (Casey et al., 2008).
Interestingly, rats and mice show differences in susceptibility to PPAR-induced myopathy. Studies in our laboratory have shown that in the rat the soleus and intercostal muscles, both predominantly Type 1 muscles, are most affected (Bordelon et al., 2005; Faiola et al., 2006) whereas the quadriceps and intercostal muscles are the most, and the soleus the least, affected in mice. Fiber-type composition may partially explain this species difference as the soleus in rat contains approximately 90% Type 1 fibers whereas in the mouse, the soleus contains approximately 35–60% Type 1 and 40–50% Type 2a fibers (Burkholder et al., 1994; Hitomi et al., 2005; our laboratory's unpublished results). As noted in this study, although the quadriceps and intercostals of mice are mainly comprised of Type 2 fibers, the peroxisome proliferation and myopathy/necrosis tend to manifest mostly in the Type 1 or mixed fibers present in these muscles. In rats and mice, PPAR- mRNA levels in the slow/oxidative soleus muscle have been shown to be higher than in the fast/glycolytic extensor digitorum longus (EDL) muscle (Lunde et al., 2007; Wang et al., 2004). Likewise, preliminary gene expression analysis results from our laboratory indicate that PPAR- mRNA levels are higher in the rat soleus compared with the EDL (unpublished data). Thus, the higher expression of PPAR- and PPAR- in slow/oxidative muscles may partially explain the observed propensity of Type 1 fibers to be more susceptible to the skeletal myopathy induced by GW0742 and various PPAR- ligands. In addition, gene expression analysis of different skeletal muscles from C57BL/10SnJ mice revealed that the soleus and diaphragm, which are the two least affected muscles in our studies, have unique expression profiles that separate these two muscles from each other as well as from the quadriceps, gastrocnemius, tibialis anterior, and EDL (the intercostal muscle was not analyzed in the study) (Haslett et al., 2005). Despite the significant difference in fiber-type proportions, soleus having approximately 50–60% and diaphragm with approximately 10–12% Type 1 fibers, these investigators observed only 15 genes differentially expressed between these two muscles in WT mice (Haslett et al., 2005). Similar gene expression differences in various muscles are likely to occur in the C57BL/6NTac mice used in our studies. Thus, molecular as well as biomechanical differences and fiber-type proportion may all contribute to the differential response to PPAR agonism in the various muscles.
Recent efforts to identify gene expression profiles following PPAR- agonist treatment have begun to elucidate the fiber-type selectivity of the response but have not illuminated the off-target responses that may be responsible for the untoward side effects of these compounds (De Souza et al., 2006). We have begun to evaluate the transcriptomic responses of the WT and PPAR- null mice discussed here as gene expression analysis may reveal key differences in the response of the liver and quadriceps muscle to the selective PPAR- tool compound (GW0742) and may provide insight into why PPAR- activity appears to mediate the hepatic and skeletal muscle effects more so than PPAR- activity (manuscripts in preparation).
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ACKNOWLEDGMENTS |
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We would like to thank the GSK SA resource staff for carrying out the in-life portions of the study, the GSK SA necropsy and histopathology staff for obtaining and processing tissue samples for analysis, the GSK SA clinical pathology staff for serum chemistry analyses, David Krull for performing the automated image analysis, Carie Kimbrough for statistical analysis, Hong Ni for glutathione analysis, Jack Chism for toxicokinetic analysis, Warren Casey for scientific input, and Chris Merrill for providing the histopathology images.
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