FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2070299412> ?p ?o ?g. }

• W2070299412 endingPage "31527" @default.
• W2070299412 startingPage "31521" @default.
• W2070299412 abstract "We compared the ability of rat and human hepatocytes to respond to fenofibric acid and a novel potent phenylacetic acid peroxisome proliferator-activated receptor (PPAR) α agonist (compound 1). Fatty acyl-CoA oxidase (FACO) activity and mRNA were increased after treatment with either fenofibric acid or compound 1 in rat hepatocytes. In addition, apolipoprotein CIII mRNA was decreased by both fenofibric acid and compound 1 in rat hepatocytes. Both agonists decreased apolipoprotein CIII mRNA in human hepatocytes; however, very little change in FACO activity or mRNA was observed. Furthermore, other peroxisome proliferation (PP)-associated genes including peroxisomal 3-oxoacyl-CoA thiolase (THIO), peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD), peroxisomal membrane protein-70 (PMP-70) were not regulated by PPARα agonists in human hepatocytes. Moreover, other genes that are regulated by PPARα ligands in human hepatocytes such as mitochondrial HMG-CoA synthase and carnitine palmitoyl transferase-1 (CPT-1) were also regulated in HepG2 cells by PPARα agonists. Several stably transfected HepG2 cell lines were established that overexpressed human PPARα to levels between 6- and 26-fold over normal human hepatocytes. These PPARα-overexpressing cells had higher basal mRNA levels of mitochondrial HMG-CoA synthase and CPT-1; however, basal FACO mRNA levels and other PP-associated genes including THIO, HD, or PMP-70 mRNA were not substantially affected. In addition, FACO, THIO, HD, and PMP-70 mRNA levels did not increase in response to PPARα agonist treatment in the PPARα-overexpressing cells, although mitochondrial HMG-CoA synthase and CPT-1 mRNAs were both induced. These results suggest that other factors besides PPARα levels determine the species-specific response of human and rat hepatocytes to the induction of PP. We compared the ability of rat and human hepatocytes to respond to fenofibric acid and a novel potent phenylacetic acid peroxisome proliferator-activated receptor (PPAR) α agonist (compound 1). Fatty acyl-CoA oxidase (FACO) activity and mRNA were increased after treatment with either fenofibric acid or compound 1 in rat hepatocytes. In addition, apolipoprotein CIII mRNA was decreased by both fenofibric acid and compound 1 in rat hepatocytes. Both agonists decreased apolipoprotein CIII mRNA in human hepatocytes; however, very little change in FACO activity or mRNA was observed. Furthermore, other peroxisome proliferation (PP)-associated genes including peroxisomal 3-oxoacyl-CoA thiolase (THIO), peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD), peroxisomal membrane protein-70 (PMP-70) were not regulated by PPARα agonists in human hepatocytes. Moreover, other genes that are regulated by PPARα ligands in human hepatocytes such as mitochondrial HMG-CoA synthase and carnitine palmitoyl transferase-1 (CPT-1) were also regulated in HepG2 cells by PPARα agonists. Several stably transfected HepG2 cell lines were established that overexpressed human PPARα to levels between 6- and 26-fold over normal human hepatocytes. These PPARα-overexpressing cells had higher basal mRNA levels of mitochondrial HMG-CoA synthase and CPT-1; however, basal FACO mRNA levels and other PP-associated genes including THIO, HD, or PMP-70 mRNA were not substantially affected. In addition, FACO, THIO, HD, and PMP-70 mRNA levels did not increase in response to PPARα agonist treatment in the PPARα-overexpressing cells, although mitochondrial HMG-CoA synthase and CPT-1 mRNAs were both induced. These results suggest that other factors besides PPARα levels determine the species-specific response of human and rat hepatocytes to the induction of PP. peroxisome proliferator-activated receptor α apolipoprotein CIII peroxisome proliferator response element human PPARα 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonic acid glutathione S-transferase ligand binding domain CREB-binding protein polymerase chain reaction reverse transcriptase fatty acyl-CoA oxidase carnitine palmitoyl transferase-1 peroxisomal 3-oxoacyl-CoA thiolase peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase cytochrome P450 4A peroxisomal membrane protein-70 mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase Chemicals that elicit the phenomenon of peroxisome proliferation in rodents are associated with hepatocarcinogenesis in long-term studies (reviewed in Ref. 1Lawrence J.W. Eacho P. Plaa G.L. Hewitt W.R. Toxicology of the Liver. Taylor & Francis, Washington, D. C1998: 125-157Google Scholar). The tumorigenic response seems related to both the oxidative stress and increased cell proliferation observed after treatment with these chemicals. It has been demonstrated that non-rodent species are refractory to the induction of peroxisome proliferation (2Foxworthy P.S. White S.L. Hoover D.M. Eacho P.I. Toxicol. Appl. Pharmacol. 1990; 104: 386-394Crossref PubMed Scopus (57) Google Scholar, 3Eacho P.I. Foxworthy P.S. Johnson W.D. Hoover D.M. White S.L. Toxicol. Appl. Pharmacol. 1986; 83: 430-437Crossref PubMed Scopus (81) Google Scholar, 4Lake B.G. Evans J.G. Gray T.J. Korosi S.A. North C.J. Toxicol. Appl. Pharmacol. 1989; 99: 148-160Crossref PubMed Scopus (120) Google Scholar); however, humans are not totally unresponsive to treatment with PPARα1agonists. For example, the expression of apo CIII, a lipoprotein involved in triglyceride transport, is suppressed by PPARα agonsits in rodents and humans and is involved in the hypotriglyceridemic effects of PPARα agonists observed clinically (5Staels B. VuDac N. Kosykh V.A. Saladin R. Fruchart J.C. Dallongeville J. Auwerx J. J. Clin. Invest. 1995; 95: 705-712Crossref PubMed Google Scholar). In addition, mitochondrial HMG-CoA synthase, a gene involved in regulating ketogenesis, is responsive to PPARα agonist treatment independently of the ability of the species to respond by inducing peroxisome proliferation (6Rodriguez J.C. Gilgomez G. Hegardt F.G. Haro D. J. Biol. Chem. 1994; 269: 18767-18772Abstract Full Text PDF PubMed Google Scholar). Thus, species differences in response to peroxisome proliferation induction appear distinct from PPARα activation. There are several potential explanations for the species-specific differences in response to the induction of peroxisome proliferation. One hypothesis suggests that the difference between rodents and non-rodents in their susceptibility to peroxisome proliferation is related to the difference in relative expression of PPARα between the species (7Gonzalez F.J. Peters J.M. Cattley R.C. J. Natl. Cancer Inst. 1998; 90: 1702-1709Crossref PubMed Scopus (269) Google Scholar, 8Tugwood J.D. Aldridge T.C. Lambe K.G. Macdonald N. Woodyatt N.J. Ann. N. Y. Acad. Sci. 1996; 804: 252-265Crossref PubMed Scopus (153) Google Scholar, 9Kliewer S.A. Forman B.M. Blumberg B. Ong E.S. Borgmeyer U. Mangelsdorf D.J. Umesono K. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7355-7359Crossref PubMed Scopus (1281) Google Scholar, 10Tugwood J.D. Holden P.R. James N.H. Prince R.A. Roberts R.A. Arch. Toxicol. 1998; 72: 169-177Crossref PubMed Scopus (95) Google Scholar). For example, human liver seems to express PPARα at levels that are approximately an order of magnitude lower than rat liver (11Palmer C.A. Hsu M.H. Griffin K.J. Raucy J.L. Johnson E.F. Mol. Pharmacol. 1998; 53: 14-22Crossref PubMed Scopus (424) Google Scholar). It is possible, if the amount of the PPARα is limiting, that only a subset of genes may be induced upon exposure to ligand. Another hypothesis is that the response element is defective in non-rodent species (12Woodyatt N.J. Lambe K.G. Myers K.A. Tugwood J.D. Roberts R.A. Carcinogenesis. 1999; 20: 369-372Crossref PubMed Scopus (99) Google Scholar, 13Lambe K.G. Woodyatt N.J. Macdonald N. Chevalier S. Roberts R.A. Toxicol. Lett. 1999; 110: 119-127Crossref PubMed Scopus (58) Google Scholar) and that the receptor cannot bind to or regulate the genes because they do not have functional PPREs within their promoters. Indeed, when several human genomic samples were analyzed, sequence analysis found that their fatty acyl-CoA oxidase promoter contained disrupted PPRE sequences. In these studies, we compared the response of rat and human hepatocytes to the effects of fenofibric acid and a novel potent phenylacetic acid PPARα agonist (compound 1). In addition, we developed several HepG2 cell lines with different levels of human PPARα overexpression and determined the effect of overexpression on basal and ligand-stimulated expression of several genes known to be responsive in both rat and human hepatocytes as well as peroxisome proliferation-associated genes that are only responsive in rat hepatocytes. Rat hepatocytes were isolated by a standard two-stage collagenase perfusion (14Foxworthy P.S. Eacho P.I. Toxicol. Lett. 1986; 30: 189-196Crossref PubMed Scopus (39) Google Scholar), plated in Williams E medium containing 10% fetal bovine serum, 1 µmdexamethasone, 1 µm insulin, 20 µg/ml gentamicin, 292 mg/ml l-glutamine, and 50 mm HEPES on rat-tail collagen, and allowed to attach and recover from isolation overnight at 37 °C. Approximately 24 h after isolation, fresh medium with all additions except fetal bovine serum and containing the appropriate concentration of fenofibric acid or compound 1((3-chloro-4-((3-((3-phenyl-7-propyl-1-benzofuran-6-yl)oxy)propyl)thio)phenyl)acetic acid, Merck Research Laboratories, Rahway, NJ) was added to the cells as a 100× stock in Me2SO. The culture medium was replaced at 24 h and after 48 h of treatment, the cells were rinsed with 100 mm NaPO4, pH 7.4, and harvested by scraping. The cells were disrupted by sonication and the resulting homogenates were assayed for protein concentration by the Lowry procedure (15Lowry O.H. Roseborough N.J. Carr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) and frozen at −70 °C. Human hepatocytes were obtained commercially from Clonetics Corp. (San Diego, CA) as cultures already attached to collagen in proprietary medium. They arrived ∼24 h post-isolation and were allowed to recover from shipping overnight at 37 °C prior to use. Subsequently, the human hepatocytes were treated and processed as indicated above with the exception that the Clonetics proprietary medium was used at medium changes and during treatment. A homogeneous time-resolved fluorescence assay-based nuclear receptor-coactivator assay was used to examine the interaction of hPPARα with compound 1. A complete description of this assay has been published elsewhere (16Zhou G. Cummings R. Li Y. Mitra S. Wilkinson H.A. Elbrecht A. Hermes J.D. Schaeffer J.M. Smith R.G. Moller D.E. Mol. Endocrinol. 1998; 12: 1594-1604Crossref PubMed Scopus (160) Google Scholar). Briefly, 198 µl of reaction mixture (100 mm HEPES, 125 mm KF, 0.125% (w/v) CHAPS, 0.05% dry milk, 5 nm GST-hPPARαLBD, 2 nm anti-GST-(Eu)K, 10 nm biotin-CBP-(1–453), and 20 nmSA/XL665) were added to each well followed by the addition of 2 µl of Me2SO or compound 1 (in Me2SO) in appropriate wells. The plates were mixed by hand and covered with TopSeal. The plates were incubated overnight at 4 °C followed by fluorescence measurement on a Discovery instrument (Packard). The data were reported as a ratio of the fluorescence at 665 nm (XL665, A counts) to the fluorescence at 620 nm (Eu(K), B counts) multiplied by 10,000 (to give a whole number). The plasmid expressing GST-hPPARαLBD was constructed by PCR the cloning of the hPPARα sequence encoding the ligand binding domain (from amino acid 167 to 468) into a GST vector. The GST-hPPARαLBD fusion protein was purified as described (16Zhou G. Cummings R. Li Y. Mitra S. Wilkinson H.A. Elbrecht A. Hermes J.D. Schaeffer J.M. Smith R.G. Moller D.E. Mol. Endocrinol. 1998; 12: 1594-1604Crossref PubMed Scopus (160) Google Scholar). Transactivation by human or murine PPARα was determined in transiently cotransfected in COS-1 cells as described (17Berger J. Leibowitz M.D. Doebber T.W. Elbrecht A. Zhang B. Zhou G.C. Biswas C. Cullinan C.A. Hayes N.S. Li Y. Tanen M. Ventre J. Wu M.S. Berger G.D. Mosley R. Marquis R. Santini C. Sahoo S.P. Tolman R.L. Smith R.G. Moller D.E. J. Biol. Chem. 1999; 274: 6718-6725Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar) using pSG5-hPPARα-GAL4 or pSG5-mPPARα-GAL4, and both pUAS(5X)-tk-luciferase and pCMV-lacz were then incubated with the indicated concentrations of compound 1 for 48 h. HepG2 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. The hPPARα expression construct, pIRES1neo-hPPARα, was constructed by PCR subcloning of the sequence encoding full-length hPPARα into pIRES1neo (CLONTECH). The hPPARα coding sequence was resequenced to confirm the absence of PCR error. HepG2 cells were transfected with pIRES1neo-hPPARα using a standard LipofectAMINE method (Life Technologies, Inc.). Stably transfected cell lines were selected in the presence of 0.5 mg/ml G418 for 3 weeks with the culture medium changed twice a week. Individual G418-resistant colonies were clone-purified followed by immunoblot and RNA analysis by the 5′-nuclease RT-PCR assay to verify hPPARα expression. The cell lysates were obtained by the direct addition of SDS sample buffer without dithiothreitol. The protein concentration was determined using the BCA method (Pierce). Western blots were performed as described using an anti-PPARα antibody (18Shu H. Wong B. Zhou G. Li Y. Berger J. Woods J.W. Wright S.D. Cai T.Q. Biochem. Biophys. Res. Commun. 2000; 267: 345-349Crossref PubMed Scopus (219) Google Scholar). Fatty acyl-CoA oxidase activity was assayed by monitoring the evolution of H2O2 as described by Poosch and Yamazaki (19Poosch M.S. Yamazaki R.K. Biochim. Biophys. Acta. 1986; 884: 585-593Crossref PubMed Scopus (100) Google Scholar) using lauryl-CoA as the substrate and 1 mmhydroxyphenylacetic acid as the indicator. Incubations were carried out at 37 °C for 10 min for the rat homogenates and 15 min for the human homogenates and stopped with 2 mm KCN in carbonate buffer. All samples were assayed in duplicate with a corresponding blank (lacking lauryl-CoA) subtracted. The results were converted to nanomoles of product by comparison to an H2O2 standard curve and normalized to milligrams of protein. Total RNA was isolated from cells with 1 ml of Triazol reagent (Life Technologies, Inc.) following manufacturer instructions. RNA was quantified by spectrophotometry at 260 nm. Rat and human apo CIII DNA probes were generated by RT-PCR from isolated rat or human total RNA using primers derived from published sequences (20Haddad I.A. Ordovas J.M. Fitzpatrick T. Karathanasis S.K. J. Biol. Chem. 1986; 261: 13268-13277Abstract Full Text PDF PubMed Google Scholar, 21Sharpe C.R. Sidoli A. Shelley C.S. Lucero M.A. Shoulders C.C. Baralle F.E. Nucleic Acids Res. 1984; 12: 3917-3932Crossref PubMed Scopus (87) Google Scholar). Purified PCR-generated DNA fragments of rat and human apo CIII and an 18S rRNA DNA template were labeled with a psoralen-biotin kit. Northern analysis was performed using a Tris borate EDTA-urea polyacrylamide gel electrophoresis gel system. RNA was then electroblotted to a positively charged nylon membrane and cross-linked with UV irradiation. Blots were hybridized with a biotinylated apo CIII probe overnight and washed, and the biotinylated probe was detected with a streptavidin-alkaline phosphatase conjugate and a chemiluminescent substrate. The chemiluminescent signal was detected by exposure to x-ray film and quantified by densitometry. Probes were stripped, and the filter was reprobed with biotinylated 18S rRNA probe, detected by chemiluminescence, and quantified as above. The data are expressed as the ratio of apo CIII to 18S rRNA. Fatty acyl-CoA oxidase (FACO), carnitine pamitoyltransferase-1 (CPT-1), peroxisomal 3-oxoacyl-CoA thiolase (THIO), peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD), cytochrome P450 4A (CYP4A), peroxisomal membrane protein-70 (PMP-70), and mitochondrial HMG-CoA synthase (HMG-CoA synthase) mRNAs were quantified using real-time RT-PCR. cDNA synthesis was performed in 25 µl with 0.1 µg total RNA with the TAQMAN® RT kit (PerkinElmer Life Sciences) with the following conditions: 25 °C for 10 min, 48 °C for 30 min, and 95 °C for 5 min. After reverse transcription, a 3-µl aliquot was transferred into a 25-µl TAQMAN® amplification reaction (TAQMAN® PE 2× PCR Mastermix diluted to a 1× final concentration) containing primer/probes for 18S rRNA (JOE-tagged: 6-carboxy-4′,5′-dichloro-2′,7′-dimethylfluorescein) and either rat or human FACO, CPT-1, THIO, HD, CYP4A, PMP-70, or HMG-CoA synthase (6-FAM tagged: 6-carboxyfluorescein) with the following conditions: 50 °C for 2 min, 95 °C for 10 min and amplified at 95 °C for 15 s, and 60 °C for 1 min for 40 cycles. The specific sequences of the primers and probes are listed in Table I.Table IPrimer/Probe sequencesGeneSpeciesF PrimerR PrimerProbePeroxisomal fatty acyl-CoA oxidaseRatGGCCAACTATGGTGGACATCATACCAATCTGGCTGCACGAACTTGTAGGCTTCTGTCAGGCCCTCCAHumanTCCAGACGGCTAGGTTCCTGGTTCAAATAGGACACCATGCCAAAGTTATGATCAGGTGCACTCAGGAAAGTTGGPeroxisomal 3-oxoacyl-CoA thiolaseRatCCTGACATCATGGGCATCGAGTCAGCCCTGCTTTCTGCAACCTGCCTATGCCATCCCTGCGHumanGCAGAAGGCAGCAAGAGCCTGGACCGTGGTGGTCACAGAGAGCAAGGGCTGTTTCCAAGCTGAGATTPeroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenaseRatAAGATAATCACTTTCACCTTGGAGTTTGGTTTTGCCGAAGCTTGAGCATCCAGAGCGCATCAGAACGGHumanGGCCCTGGGCTGTCACTATAGAAGTCCAAGTGTAACTTCTGGTATTGCCCACGCAGACGCTCAAGTTPeroxisomal membrane protein-70RatGTAGTTGGGTACCTGGTTGTCAGTCAGTGGGTGCTGTGAAGGTGTCCCCGTTCCTAGACCTGGCGCATCHumanAAGTTAACGAGTGCAATTGGAGCAGCCCAGAAACAACCAAGTAGGCAGGGCCCAGCGAGCATGATGCytochrome P450 4A1RatCCCGACACAGCCACTCATTCCCTTCAGCTCATTCATGGCAACTTCAGGAGGAGCAAGGAACTGCATTGGCytochrome P450 4A11HumanGTGGCCCAACCCAGAGGTTCCCAATGCAGTTCCTTGATCCTGCTCAACACAGCCACGCTTTCCTCarnitine palmitoyltransferase-1HumanCGTCTTTTGGGATCCACGATTTGTGCTGGATGGTGTCTGTCTCTTAAACATCCGCTCCCACTGAGCGGMitochondrial HMG-CoA synthaseHumanTGAGGGCATAGATACCACCAATGTGGCATAACGACCATCCCAATGCTGCCAACTGGATGGAGTCCAGSequences are 5′ to 3′. Probes contained 5′-6FAM and 3′-TAMRA as the fluorescent tag and quencher, respectively. Open table in a new tab Sequences are 5′ to 3′. Probes contained 5′-6FAM and 3′-TAMRA as the fluorescent tag and quencher, respectively. After treatment with fenofibric acid for 48 h, a dose-dependent increase in FACO activity was observed in rat hepatocytes (Fig.1). At 100 µm, fenofibric acid produced a 6-fold increase in FACO activity. No increase in FACO activity was observed in human hepatocytes. In parallel culture dishes, fenofibric acid produced a dose-dependent suppression of apo CIII mRNA levels in both rat and human at the doses that corresponded to those that induced FACO activity in rat hepatocytes. A novel highly potent phenylacetic acid PPARα agonist (compound1) was used to assess the effects of a more potent PPARα agonist in this system. This compound has high potency on the human PPARα receptor but is a weak agonist on the murine receptor. Based on the ability of PPARα to recruit the CBP coactivator in vitro, the estimated EC50 for compound 1 on human PPARα is 16 nm and is ∼10 µm on the murine receptor (Fig. 2A). Concentrations greater than 10 µm could not be tested in the homogeneous time-resolved fluorescence-based nuclear receptor association assay because of the quenching effect of the compound. These values corresponded well with results using chimeric human or murine PPARα-GAL4 receptor transactivation reporter assays (Fig.2 B). When compound 1 was assessed in rat hepatocytes, FACO activity was induced slightly at doses above 1 µm, corresponding to the weak potency on rodent PPARα (Fig. 3). Doses higher than 10 µm of compound 1 were cytotoxic to the rat and human hepatocytes. Compound 1 also suppressed apo CIII mRNA at doses above 1 µm. Only a very modest effect of compound 1 was observed in human hepatocytes on FACO activity (∼2-fold) at doses up to 10 µm; however, apo CIII mRNA was suppressed at doses as low as 10 nm. The effects on apo CIII corresponded with its potency on the human PPARα.Figure 2Compound 1 is a potent human but weak murine PPARα agonist. A, the ability of compound 1 (theinset shows the structure) to promote human (●) or murine (○) PPARα and CBP interaction was analyzed using an homogeneous time-resolved fluorescence-based nuclear receptor assay in the presence of 10 nm SA/XL665, 10 nmbiotin-CBP-(1–453), 1 nm GST-PPARαLBD, and 2 nm anti-GST-(Eu)K as described under “Experimental Procedures.” The 665 nm/620 nm ratio for each well was normalized by subtracting that obtained from the control well followed by calculation of the percentage maximum activation (using maximal ligand-induced fluorescence resonance energy transfer as 100%). The titration using human GST-PPARαLBD is shown. Each point represents the mean ± S.E. of three determinations. The experiment was repeated with similar results. B, transactivation by human (●) or murine (○) PPARα in COS-1 cells transiently cotransfected with pSG5-hPPARα-GAL4 or pSG5-mPPARα-GAL4 chimeric constructs and both pUAS(5X)-tk-luciferase and pCMV-lacz then incubated with the indicated concentrations of compound 1 for 48 h. The data are expressed as the mean ± S.E. of normalized luciferase activity (n = 3).View Large Image Figure ViewerDownload (PPT)Figure 3Compound 1 induces FACO activity and suppresses apo CIII mRNA in rat hepatocytes but only suppresses apo CIII mRNA in human hepatocytes. Rat or human primary hepatocytes were cultured for 48 h in the presence or absence of various concentrations of compound 1 and then harvested for FACO activity determinations or apo CIII mRNA Northern analysis as described under “Experimental Procedures.” The apo CIII signal was corrected by reprobing with an 18S rRNA probe and dividing the apo CIII signal by the 18S rRNA signal. The data are expressed as nmol of H2O2/min/mg of protein for FACO activity or relative to untreated controls for apo CIII (mean ± S.E., n = 4). ○, rat hepatocytes; ●, human hepatocytes.View Large Image Figure ViewerDownload (PPT) The effects of fenofibric acid and compound 1were assessed on genes potentially regulated by PPARα in rat and human hepatocytes. Messenger RNA for the peroxisome proliferation-associated genes including FACO,THIO, HD, CYP4A1, andPMP-70 were induced by fenofibric acid and compound1 in rat hepatocytes (Fig.4A). In rat hepatocytes, fenofibric acid was more efficacious than compound 1probably because of the ability to test higher doses of fenofibric acid in rat hepatocytes without inducing frank cytotoxicity. Because of the decreased potency and limited concentration of compound 1that could be tested, a more modest increase was observed. In human hepatocytes, no increase in the peroxisome proliferation-associated genes was observed (Fig. 4 B). A small increase in CYP4A11 mRNA was observed in human hepatocytes with compound 1; however, the magnitude was more than 25-fold less than that observed in rat hepatocytes. Two genes were responsive to fenofibric acid and compound 1 in human hepatocytes, CPT-1 andmtHMGS. When the same set of genes was examined in HepG2 cells, no response to either fenofibric acid or compound 1was observed for the mRNA for the peroxisome proliferation-associated genes FACO, THIO, HD, FACO, or PMP-70 (Fig. 4 C). Similar to what was observed in human hepatocytes, CPT-1 and mtHMGS both were induced after treatment. Interestingly, mtHMGS and CPT-1 basal mRNA levels were much lower in the HepG2 cells than in human hepatocytes. Basal mRNA levels of FACO, THIO, and HD were similar to those in human hepatocytes, whereas basal mRNA levels of PMP-70 were 4–5-fold higher than those in human hepatocytes. There was no detectable CYP4A11 mRNA in HepG2 cells. Several stable HepG2 cell lines were created that overexpress human PPARα. The cell lines expressed from 6- to 26-fold higher levels of human PPARα mRNA than in primary human hepatocytes (Fig.5). When three of these cell lines were assessed by Western analysis, they demonstrated markedly increased expression of PPARα protein. Some cell lines had higher protein levels than would be expected from their mRNA analysis (compare cell line J35 to J14). Basal levels of the selected mRNAs mentioned above were measured via the 5′-nuclease RT-PCR assay. Increasing levels of PPARα overexpression in these cells increased the basal levels of mtHMGS and CPT-1 mRNA (Fig. 6). The effect on basal levels of mtHMGS mRNA appeared to be saturable; however, the basal regulation of CPT-1 mRNA was not. In contrast, the peroxisome proliferation-associated genes FACO,THIO, HD, and PMP-70 were not mRNA affected substantially by increasing the amount of PPARα in the cell. PMP-70 did increase modestly, but that may be because of the much higher basal levels in HepG2 cells compared with human hepatocytes. The regulation of the same set of genes was assessed in the J35 cell line (10-fold higher PPARα levels than human hepatocytes) after treatment with 100 nm compound 1 (Fig.7). Similar to the results found in primary human hepatocytes, both mtHMGS and CPT-1 mRNA were strongly induced after treatment; however, mRNA for the peroxisome proliferation-associated genes FACO, THIO,HD, and PMP-70 were not affected substantially by compound 1 treatment (Fig. 7). PMP-70 mRNA levels were increased modestly by compound 1; however, the magnitude of this change was small (less than 2-fold) compared with the induction observed in rat hepatocytes (6–7-fold). Thus, no meaningful induction of peroxisome proliferator-associated genes was observed regardless of the amount of PPARα in the cell.Figure 6Basal mRNA levels of mitochondrial HMG-CoA synthase and CPT-1 are regulated in HepG2 cells expressing increased hPPARα levels, but peroxisome proliferator-associated genes are not. Stable hPPARα-overexpressing cells were harvested, and total RNA was isolated as described under “Experimental Procedures.” The specific mRNAs were quantified by the 5′-nuclease RT-PCR assay and expressed relative to untreated human hepatocytes. Open bars, parent HepG2; solid bars, J38 line with 6-fold higher PPARα levels than human hepatocytes; right-hatched bars, J35 line with 10-fold higher PPARα levels than human hepatocytes;left-hatched bars, J14 line with 16-fold higher PPARα levels than human hepatocytes; cross-hatched bars, B56 line with 26-fold higher PPARα levels than human hepatocytes.View Large Image Figure ViewerDownload (PPT)Figure 7Compound 1 induces mRNA for mitochondrial HMG-CoA synthase and CPT-1 in HepG2 cells expressing hPPARα at levels similar to rat hepatocytes but still does not induce peroxisome proliferator-associated genes. Stable hPPARα-overexpressing cells (J35 clone) were cultured for 48 h in the absence (open bars) or presence (solid bars) of 100 nm compound 1 and harvested, and total RNA was isolated as described under “Experimental Procedures.” The specific mRNAs were quantified by the 5′-nuclease RT-PCR assay and expressed relative to untreated human hepatocytes (mean ± S.D.;n = 2–4).View Large Image Figure ViewerDownload (PPT) We compared rat and human cells to investigate mutualversus species-specific responses to PPARα agonists. Rat hepatocytes responded to PPARα agonist treatment by inducing the mRNA for several peroxisome proliferation-related genes includingFACO, HD, THIO, CYP4A, andPMP-70 and had increased fatty acyl-CoA oxidase activity. In addition, PPARα agonist treatment suppressed apo CIII mRNA at the same doses that induced FACO activity. Fibrates regulate apo CIII through a PPRE in the promoter of the apo CIIIgene (5Staels B. VuDac N. Kosykh V.A. Saladin R. Fruchart J.C. Dallongeville J. Auwerx J. J. Clin. Invest. 1995; 95: 705-712Crossref PubMed Google Scholar), and the regulation of apo CIII is absent in PPARα knockout mice (data not shown). Thus, suppression of apo CIII mRNA can be considered a measure of PPARα activation in vivo. Consistent with their in vitro pharmacologic profile on murine PPARα, the potency of fenofibric acid and compound1 were similar at suppressing apo CIII mRNA levels in rat hepatocytes. In addition, compound 1 produced similar effects as other PPARα agonists in rat hepatocytes on FACO activity. Both compound 1 and fenofibric acid suppressed apo CIII mRNA levels in human hepatocytes; however, compound 1was found to have at least a 1000-fold greater potency than fenofibric acid in these cells. This difference in potency correlated with the ability of compound 1 to function as a very potenthuman PPARα agonist as assessed by its ability to transactivate a reporter construct or recruit CBP in the in vitro interaction assay, which is a more direct measure of PPARα affinity. Mukherjee et al. (22Mukherjee R. Jow L. Noonan D. McDonnell D.P. J. Steroid Biochem. Mol. Biol. 1994; 51: 157-166Crossref PubMed Scopus (263) Google Scholar) have observed that WY14,643 was more potent at activating the rat PPARα than the human PPARα. In addition, they observed that ETYA was more potent on the human PPARα than on the rat PPARα. Our results are consistent with these findings and suggest that human and rodent PPARαs can be distinguished pharmacologically. There are two amino acids that differ in the ligand-binding domain of PPARα between rat and human. These differences in amino acid sequence may explain the differences in the potency of the various ligands for each of the species receptors. In contrast to rat hepatocytes, th" @default.
• W2070299412 created "2016-06-24" @default.
• W2070299412 creator A5004343492 @default.
• W2070299412 creator A5025204088 @default.
• W2070299412 creator A5048401897 @default.
• W2070299412 creator A5055429671 @default.
• W2070299412 creator A5060002817 @default.
• W2070299412 creator A5067269768 @default.
• W2070299412 creator A5071210464 @default.
• W2070299412 creator A5082280074 @default.
• W2070299412 date "2001-08-01" @default.
• W2070299412 modified "2023-09-27" @default.
• W2070299412 title "Differential Gene Regulation in Human Versus Rodent Hepatocytes by Peroxisome Proliferator-activated Receptor (PPAR) α" @default.
• W2070299412 cites W1521754963 @default.
• W2070299412 cites W1526348057 @default.
• W2070299412 cites W1775749144 @default.
• W2070299412 cites W1963874292 @default.
• W2070299412 cites W1965397552 @default.
• W2070299412 cites W1971835418 @default.
• W2070299412 cites W1974367132 @default.
• W2070299412 cites W1978362901 @default.
• W2070299412 cites W1983623331 @default.
• W2070299412 cites W1985400591 @default.
• W2070299412 cites W1985946610 @default.
• W2070299412 cites W1987774915 @default.
• W2070299412 cites W1999982978 @default.
• W2070299412 cites W2003960793 @default.
• W2070299412 cites W2010578427 @default.
• W2070299412 cites W2013127992 @default.
• W2070299412 cites W2017564027 @default.
• W2070299412 cites W2019212738 @default.
• W2070299412 cites W2024411017 @default.
• W2070299412 cites W2044359996 @default.
• W2070299412 cites W2063043675 @default.
• W2070299412 cites W2065624367 @default.
• W2070299412 cites W2078124375 @default.
• W2070299412 cites W2080721799 @default.
• W2070299412 cites W2087936004 @default.
• W2070299412 cites W2107752044 @default.
• W2070299412 cites W2120865762 @default.
• W2070299412 cites W2121914083 @default.
• W2070299412 cites W2123998830 @default.
• W2070299412 cites W2131037449 @default.
• W2070299412 cites W2139717510 @default.
• W2070299412 cites W2147627625 @default.
• W2070299412 cites W2168300082 @default.
• W2070299412 cites W4291113916 @default.
• W2070299412 doi "https://doi.org/10.1074/jbc.m103306200" @default.
• W2070299412 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11418601" @default.
• W2070299412 hasPublicationYear "2001" @default.
• W2070299412 type Work @default.
• W2070299412 sameAs 2070299412 @default.
• W2070299412 citedByCount "171" @default.
• W2070299412 countsByYear W20702994122012 @default.
• W2070299412 countsByYear W20702994122013 @default.
• W2070299412 countsByYear W20702994122014 @default.
• W2070299412 countsByYear W20702994122015 @default.
• W2070299412 countsByYear W20702994122016 @default.
• W2070299412 countsByYear W20702994122017 @default.
• W2070299412 countsByYear W20702994122018 @default.
• W2070299412 countsByYear W20702994122019 @default.
• W2070299412 countsByYear W20702994122020 @default.
• W2070299412 countsByYear W20702994122021 @default.
• W2070299412 countsByYear W20702994122022 @default.
• W2070299412 countsByYear W20702994122023 @default.
• W2070299412 crossrefType "journal-article" @default.
• W2070299412 hasAuthorship W2070299412A5004343492 @default.
• W2070299412 hasAuthorship W2070299412A5025204088 @default.
• W2070299412 hasAuthorship W2070299412A5048401897 @default.
• W2070299412 hasAuthorship W2070299412A5055429671 @default.
• W2070299412 hasAuthorship W2070299412A5060002817 @default.
• W2070299412 hasAuthorship W2070299412A5067269768 @default.
• W2070299412 hasAuthorship W2070299412A5071210464 @default.
• W2070299412 hasAuthorship W2070299412A5082280074 @default.
• W2070299412 hasBestOaLocation W20702994121 @default.
• W2070299412 hasConcept C104317684 @default.
• W2070299412 hasConcept C127078168 @default.
• W2070299412 hasConcept C130241916 @default.
• W2070299412 hasConcept C170493617 @default.
• W2070299412 hasConcept C185592680 @default.
• W2070299412 hasConcept C187345961 @default.
• W2070299412 hasConcept C188413054 @default.
• W2070299412 hasConcept C18903297 @default.
• W2070299412 hasConcept C2778914748 @default.
• W2070299412 hasConcept C2910266098 @default.
• W2070299412 hasConcept C55493867 @default.
• W2070299412 hasConcept C63932345 @default.
• W2070299412 hasConcept C86339819 @default.
• W2070299412 hasConcept C86803240 @default.
• W2070299412 hasConcept C95444343 @default.
• W2070299412 hasConceptScore W2070299412C104317684 @default.
• W2070299412 hasConceptScore W2070299412C127078168 @default.
• W2070299412 hasConceptScore W2070299412C130241916 @default.
• W2070299412 hasConceptScore W2070299412C170493617 @default.
• W2070299412 hasConceptScore W2070299412C185592680 @default.
• W2070299412 hasConceptScore W2070299412C187345961 @default.
• W2070299412 hasConceptScore W2070299412C188413054 @default.
• W2070299412 hasConceptScore W2070299412C18903297 @default.

• next