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Peroxisome Proliferator-activated Receptor-α Regulates Lipid Homeostasis, but Is Not Associated with Obesity

2001; Elsevier BV; Volume: 276; Issue: 42 Linguagem: Inglês

10.1074/jbc.m107073200

1083-351X

Taro E. Akiyama, Christopher J.B. Nicol, Catherine Fiévet, Bart Staels, Jerrold M. Ward, Johan Auwerx, Susanna S.T. Lee, Frank J. Gonzalez, Jeffrey M. Peters,

Lipid metabolism and disorders

Considerable controversy exists in determining the role of peroxisome proliferator-activated receptor-α (PPARα) in obesity. Two purebred congenic strains of PPARα-null mice were developed to study the role of this receptor in modulating lipid transport and storage. Weight gain and average body weight in wild-type and PPARα-null mice on either an Sv/129 or a C57BL/6N background were not markedly different between genotypes from 3 to 9 months of age. However, gonadal adipose stores were significantly greater in both strains of male and female PPARα-null mice. Hepatic accumulation of lipids was greater in both strains and sexes of PPARα-null mice compared with wild-type controls. Administration of the peroxisome proliferator WY-14643 caused hepatomegaly, alterations in mRNAs encoding proteins that regulate lipid metabolism, and reduced serum triglycerides in a PPARα-dependent mechanism. Constitutive differences in serum cholesterol and triglycerides in PPARα-null mice were found between genetic backgrounds. Results from this work establish that PPARα is a critical modulator of lipid homeostasis in two congenic mouse lines. This study demonstrates that disruption of the murine gene encoding PPARα results in significant alterations in constitutive serum, hepatic, and adipose tissue lipid metabolism. However, an overt, obese phenotype in either of the two congenic strains was not observed. In contrast to earlier published work, this study establishes that PPARα is not associated with obesity in mice. Considerable controversy exists in determining the role of peroxisome proliferator-activated receptor-α (PPARα) in obesity. Two purebred congenic strains of PPARα-null mice were developed to study the role of this receptor in modulating lipid transport and storage. Weight gain and average body weight in wild-type and PPARα-null mice on either an Sv/129 or a C57BL/6N background were not markedly different between genotypes from 3 to 9 months of age. However, gonadal adipose stores were significantly greater in both strains of male and female PPARα-null mice. Hepatic accumulation of lipids was greater in both strains and sexes of PPARα-null mice compared with wild-type controls. Administration of the peroxisome proliferator WY-14643 caused hepatomegaly, alterations in mRNAs encoding proteins that regulate lipid metabolism, and reduced serum triglycerides in a PPARα-dependent mechanism. Constitutive differences in serum cholesterol and triglycerides in PPARα-null mice were found between genetic backgrounds. Results from this work establish that PPARα is a critical modulator of lipid homeostasis in two congenic mouse lines. This study demonstrates that disruption of the murine gene encoding PPARα results in significant alterations in constitutive serum, hepatic, and adipose tissue lipid metabolism. However, an overt, obese phenotype in either of the two congenic strains was not observed. In contrast to earlier published work, this study establishes that PPARα is not associated with obesity in mice. peroxisome proliferator-activated receptor Peroxisome proliferators are a diverse class of compounds that include commercially used plasticizers (e.g. phthalates), industrial solvents (e.g. trichloroethylene), herbicides (e.g. lactofen), hypolipidemic drugs (e.g.fibrates), naturally occurring chemicals (e.g. phenyl acetate), and hormones (e.g. dehydroepiandrosterone sulfate) (1Desvergne B. Wahli W. Endocr. Rev. 1999; 20: 649-688Crossref PubMed Scopus (2727) Google Scholar, 2Gonzalez F.J. Peters J.M. Cattley R.C. J. Natl. Cancer Inst. 1998; 90: 1702-1709Crossref PubMed Scopus (264) Google Scholar). Administration of peroxisome proliferators to rodents results in numerous hepatic alterations, including an increase in the number and size of peroxisomes; hepatomegaly; increased expression of genes encoding peroxisomal, mitochondrial, and microsomal fatty acid-metabolizing enzymes; and subsequent modulation of lipid homeostasis characterized by increased oxidation of fatty acids, decreased serum lipids, and reduced adipose stores (1Desvergne B. Wahli W. Endocr. Rev. 1999; 20: 649-688Crossref PubMed Scopus (2727) Google Scholar). All of these effects are mediated by PPARα1 since PPARα-null mice are refractory to these changes when administered the prototypical peroxisome proliferator WY-14643 (3Lee S.S. Pineau T. Drago J. Lee E.J. Owens J.W. Kroetz D.L. Fernandez-Salguero P.M. Westphal H. Gonzalez F.J. Mol. Cell. Biol. 1995; 15: 3012-3022Crossref PubMed Scopus (1498) Google Scholar, 4Peters J.M. Hennuyer N. Staels B. Fruchart J.C. Fievet C. Gonzalez F.J. Auwerx J. J. Biol. Chem. 1997; 272: 27307-27312Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 5Aoyama A. Peters J.M. Iritani N. Nasu-Nakajima T. Furihata K. Hashimoto T. Gonzalez F.J. J. Biol. Chem. 1998; 273: 5678-5684Abstract Full Text Full Text PDF PubMed Scopus (750) Google Scholar). In addition to modulation of lipid metabolism induced by peroxisome proliferators, a central role for PPARα in lipid homeostasis during periods of fasting and in response to dietary fatty acids has also been established (6Leone T.C. Weinheimer C.J. Kelly D.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7473-7478Crossref PubMed Scopus (819) Google Scholar, 7Kersten S. Seydoux J. Peters J.M. Gonzalez F.J. Desvergne B. Wahli W. J. Clin. Invest. 1999; 103: 1489-1498Crossref PubMed Scopus (1360) Google Scholar, 8Kroetz D.L. Yook P. Costet P. Bianchi P. Pineau T. J. Biol. Chem. 1998; 273: 31581-31589Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 9Ren B. Thelen A.P. Peters J.M. Gonzalez F.J. Jump D.B. J. Biol. Chem. 1997; 272: 26827-26832Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Thus, it is clear that PPARα regulates lipid homeostasis in response to treatment with peroxisome proliferators, dietary fatty acids, and possibly endogenous fatty acids released during fasting. The PPARα-null mouse was generated to identify PPARα-dependent regulation induced by a variety of stimuli. Most of the early reports for this mouse line used mice with a mixed genetic background (C57BL/6N × Sv/129) (3Lee S.S. Pineau T. Drago J. Lee E.J. Owens J.W. Kroetz D.L. Fernandez-Salguero P.M. Westphal H. Gonzalez F.J. Mol. Cell. Biol. 1995; 15: 3012-3022Crossref PubMed Scopus (1498) Google Scholar, 4Peters J.M. Hennuyer N. Staels B. Fruchart J.C. Fievet C. Gonzalez F.J. Auwerx J. J. Biol. Chem. 1997; 272: 27307-27312Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 9Ren B. Thelen A.P. Peters J.M. Gonzalez F.J. Jump D.B. J. Biol. 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Iritani N. Nasu-Nakajima T. Furihata K. Hashimoto T. Gonzalez F.J. J. Biol. Chem. 1998; 273: 5678-5684Abstract Full Text Full Text PDF PubMed Scopus (750) Google Scholar, 6Leone T.C. Weinheimer C.J. Kelly D.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7473-7478Crossref PubMed Scopus (819) Google Scholar, 13Anderson S.P. Cattley R.C. Corton J.C. Mol. Carcinog. 1999; 26: 226-238Crossref PubMed Scopus (35) Google Scholar, 14Barclay T.B. Peters J.M. Sewer M.B. Ferrari L. Gonzalez F.J. Morgan E.T. J. Pharmacol. Exp. Ther. 1999; 290: 1250-1257PubMed Google Scholar, 15Belury M.A. Moya-Camarena S.Y. Sun H. Snyder E. Davis J.W. Cunningham M.L. Vanden Heuvel J.P. Toxicol. Appl. Pharmacol. 1998; 151: 254-261Crossref PubMed Scopus (33) Google Scholar, 16Brandt J.M. Djouadi F. Kelly D.P. J. Biol. Chem. 1998; 273: 23786-23792Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 17Corton J.C. Fan L.Q. Brown S. Anderson S.P. Bocos C. Cattley R.C. Mode A. Gustafsson J.A. Mol. 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J. Biol. Chem. 2000; 275: 28488-28493Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar, 36Hanley K. Komuves L.G. Ng D.C. Schoonjans K. He S.S. Lau P. Bikle D.D. Williams M.L. Elias P.M. Auwerx J. Feingold K.R. J. Biol. Chem. 2000; 275: 11484-11491Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 37Watanabe K. Fujii H. Takahashi T. Kodama M. Aizawa Y. Ohta Y. Ono T. Hasegawa G. Naito M. Nakajima T. Kamijo Y. Gonzalez F.J. Aoyama T. J. Biol. Chem. 2000; 275: 22293-22299Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 38Patel D.D. Knight B.L. Wiggins D. Humphreys S.M. Gibbons G.F. J. Lipid Res. 2001; 42: 328-337Abstract Full Text Full Text PDF PubMed Google Scholar). There are a number of recent studies (8Kroetz D.L. Yook P. Costet P. Bianchi P. Pineau T. J. Biol. Chem. 1998; 273: 31581-31589Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 39Gueraud F. Alary J. Costet P. Debrauwer L. Dolo L. Pineau T. Paris A. J. 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Pineau T. Girard J. Pegorier J.P. Biochem. J. 2001; 354: 189-197Crossref PubMed Scopus (138) Google Scholar, 46Fourcade S. Savary S. Albet S. Gauthe D. Gondcaille C. Pineau T. Bellenger J. Bentejac M. Holzinger A. Berger J. Bugaut M. Eur. J. Biochem. 2001; 268: 3490-3500Crossref PubMed Scopus (58) Google Scholar) that used PPARα-null mice on a C57BL/6 background that were generated from several rounds of backcrossing with an unidentified substrain of the C57BL/6 mouse line to the original mixed genetic background PPARα-null mice in an independent laboratory (42Costet P. Legendre C. More J. Edgar A. Galtier P. Pineau T. J. Biol. Chem. 1998; 273: 29577-29585Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). However, due to the strategy used to generate PPARα-null mice (3Lee S.S. Pineau T. Drago J. Lee E.J. Owens J.W. Kroetz D.L. Fernandez-Salguero P.M. Westphal H. Gonzalez F.J. Mol. Cell. Biol. 1995; 15: 3012-3022Crossref PubMed Scopus (1498) Google Scholar), backcrossing to the C57BL/6N background requires backcrossing mice at least 10 generations to obtain a fully congenic mouse line (47Papaioannou V. Johnson R. Joyner A.L. Gene Targeting: A Practical Approach. Oxford University Press, New York1993: 106-146Google Scholar). The construction of the PPARα-null mouse used recombinant DNA and cells from two strains of mice, Sv/129 Jae and C57BL/6N (3Lee S.S. Pineau T. Drago J. Lee E.J. Owens J.W. Kroetz D.L. Fernandez-Salguero P.M. Westphal H. Gonzalez F.J. Mol. Cell. Biol. 1995; 15: 3012-3022Crossref PubMed Scopus (1498) Google Scholar). For the PPARα-null mouse line, the Sv/129 mouse was the source of the genomic DNA library used to construct a targeting vector and the embryonic stem cells used for transfection of a targeting vector, whereas the C57BL/6N mouse (NIH substrain) was the source of donor blastocysts used for microinjecting the heterozygous embryonic stem cells. Thus, the F1 offspring from mating the chimeric mice generated by this approach were not congenic, but contained the genetic background of both Sv/129 and C57BL/6N mice. Although many published phenotypes for the PPARα-null mouse have been reported that have significant influence on lipid metabolism, many of these reports focused on mice that were either of mixed genetic background or congenic Sv/129 mice. In this work, the phenotypic characterization of lipid metabolism in wild-type or PPARα-null mice on either a pure Sv/129 or C57BL/6N genetic background was performed in both male and female mice to determine if the phenotype is consistent between congenic mouse lines. Male chimeric mice for the targeted PPARα allele (3Lee S.S. Pineau T. Drago J. Lee E.J. Owens J.W. Kroetz D.L. Fernandez-Salguero P.M. Westphal H. Gonzalez F.J. Mol. Cell. Biol. 1995; 15: 3012-3022Crossref PubMed Scopus (1498) Google Scholar) were bred with Sv/129 Jae females since this line is the same genotype as the embryonic stem cells used to generate the chimeric mice. The heterozygous F1agoutioffspring from this breeding were subsequently crossed using brother-sister matings to obtain F2 purebred wild-type or PPARα-null mice. The homozygous F2 wild-type or PPARα-null mice were used to generate F3 homozygotes, which were then randomly assigned to breeding cages to establish a larger colony of mice to perform experiments. The Sv/129 mice used for this work were from the F6 generation of mice from this colony. The male chimeras described above were mated with purebred C57BL/6N females to obtain F1offspring. The heterozygous F1agouti offspring from this breeding were then backcrossed with purebred C57BL/6N mice (either heterozygous male × wild-type female or heterozygous female × wild-type male). The heterozygous F2offspring with black coat color were then removed and backcrossed with either male or female wild-type mice, and this process was continued until the F10 generation of mice was obtained. Heterozygous F10 mice were then crossed to produce homozygous wild-type or PPARα-null mice, and the homozygous F11 mice were randomly distributed to make a breeding colony of mice to obtain F12 mice for phenotypic analysis. 4-Chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (WY-14643) was purchased commercially (ChemSyn Science Laboratories, Lenexa, KS). Pelleted mouse chow containing either 0.0 (control) or 0.1% WY-14643 (Bioserv, Frenchtown, NJ) was prepared and provided to mice ad libitum. 6–8-week-old male or female PPARα+/+ or PPARα−/− mice on either a C57BL/6N (F12generation) or an Sv/129 (F6 generation) background were housed four to five animals per cage in a temperature- and light-controlled environment (T = 25 °C, 12-h light/12-h dark cycle). Mice were weighed every month for 9 months. Cohorts of mice were killed at the age of 12–14 weeks or 9 months by overexposure to carbon dioxide. Blood was collected by cardiac puncture for isolation of serum. Serum analysis of lipids and lipoproteins was performed as described below. Liver and gonadal fat pads were removed, weighed, snap-frozen, and stored at −80 °C until further analysis. An additional section of liver was fixed in phosphate-buffered formaldehyde for analysis of liver lipid accumulation as previously described (48Sinal C.J. Tohkin M. Miyata M. Ward J.M. Lambert G. Gonzalez F.J. Cell. 2000; 102: 731-744Abstract Full Text Full Text PDF PubMed Scopus (1409) Google Scholar). 10–12-week-old male or female PPARα+/+ or PPARα−/− mice on either a pure C57BL/6N (F12 generation) or an Sv/129 (F6 generation) background were housed three to five animals per cage as described above. Mice from both strains were fed either a control diet or one containing 0.1% WY-14643 for 7 days. Mice were killed by overexposure to carbon dioxide, and livers were removed, weighed, and snap-frozen until further use. Serum was obtained from whole blood collected from individual mice and used fresh for analysis of serum lipids and lipoproteins. Gonadal adipose was removed, and the weight was recorded for each mouse. Serum lipids (cholesterol and triglycerides) and high density lipoprotein cholesterol were measured as previously described (48Sinal C.J. Tohkin M. Miyata M. Ward J.M. Lambert G. Gonzalez F.J. Cell. 2000; 102: 731-744Abstract Full Text Full Text PDF PubMed Scopus (1409) Google Scholar). Total RNA was prepared from liver using the Trizol method (Life Technologies, Inc.) and quantified using standard spectrophotometric methods. 10 cDNA probes were used for sequential Northern blot analysis as previously described (3Lee S.S. Pineau T. Drago J. Lee E.J. Owens J.W. Kroetz D.L. Fernandez-Salguero P.M. Westphal H. Gonzalez F.J. Mol. Cell. Biol. 1995; 15: 3012-3022Crossref PubMed Scopus (1498) Google Scholar, 4Peters J.M. Hennuyer N. Staels B. Fruchart J.C. Fievet C. Gonzalez F.J. Auwerx J. J. Biol. Chem. 1997; 272: 27307-27312Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 5Aoyama A. Peters J.M. Iritani N. Nasu-Nakajima T. Furihata K. Hashimoto T. Gonzalez F.J. J. Biol. Chem. 1998; 273: 5678-5684Abstract Full Text Full Text PDF PubMed Scopus (750) Google Scholar), including peroxisomal acyl-CoA oxidase, peroxisomal bifunctional enzyme, peroxisomal 3-ketoacyl-CoA thiolase, cytochrome P450 4A1, mitochondrial very long chain acyl-CoA dehydrogenase, mitochondrial long chain acyl-CoA dehydrogenase, mitochondrial medium chain acyl-CoA dehydrogenase, PPARγ, apoC-III, and β-actin as a loading control. Monthly body weight measurements revealed small differences in average body weight between wild-type and PPARα-null mice on either the Sv/129 or C57BL/6N background (Fig.1). Body weight was significantly higher in male PPARα-null mice on an Sv/129 background compared with the respective wild-type controls at 3–4 months of age (Fig. 1). Although average body weight tended to be higher in male and female PPARα-null mice on both genetic backgrounds, these differences were not statistically different (Fig. 1). Liver weights were similar between PPARα-null and wild-type mice of both sexes compared with the respective controls (Tables I and II). Although liver weights were not significantly different between genotypes, hepatic accumulation of lipids was considerably higher in the livers of male PPARα-null mice of both strains after 6 months (Fig. 2). Similar results were observed with female mice (data not shown). PPARα-null mice had significantly larger gonadal adipose stores than the respective wild-type controls, and this effect was slightly more pronounced in female PPARα-null mice compared with male mice (Tables I and II). Although internal adipose stores were significantly greater in PPARα-null mice than in controls, the overall sizes of 7–8-month-old male and female wild-type and PPARα-null mice were not markedly different on either an Sv/129 or a C57BL/6N background (Fig.3).Table IBody, liver, and gonadal adipose weights in male or female wild-type (+/+) or PPARα-null (−/−) mice on either an Sv/129 or a C57BL/6N genetic backgroundGenotypeSexTreatment groupnBWLiverAdiposegggC57BL/6N+/+MControl1024 ± 11.1 ± 0.10.43 ± 0.04(4.5 ± 0.1%)(1.8 ± 0.1%)+/+MWY621 ± 11-aSignificantly different from wild-type controls (p < 0.05).2.1 ± 0.11-aSignificantly different from wild-type controls (p < 0.05).0.17 ± 0.011-aSignificantly different from wild-type controls (p < 0.05).(9.9 ± 0.5%)1-aSignificantly different from wild-type controls (p < 0.05).(0.8 ± 0.1%)1-aSignificantly different from wild-type controls (p < 0.05).−/−MControl1025 ± 11.1 ± 0.10.67 ± 0.031-bSignificantly different from control and WY-14643-treated wild-type controls (p < 0.05).(4.4 ± 0.1%)(2.7 ± 0.1%)1-bSignificantly different from control and WY-14643-treated wild-type controls (p < 0.05).−/−MWY525 ± 11.2 ± 0.10.72 ± 0.051-bSignificantly different from control and WY-14643-treated wild-type controls (p < 0.05).(4.5 ± 0.2%)(2.8 ± 0.2%)1-bSignificantly different from control and WY-14643-treated wild-type controls (p < 0.05).+/+FControl920 ± 10.99 ± 0.10.24 ± 0.03(4.9 ± 0.2%)(1.2 ± 0.1%)+/+FWY617 ± 11-aSignificantly different from wild-type controls (p < 0.05).1.8 ± 0.11-aSignificantly different from wild-type controls (p < 0.05).0.07 ± 0.021-aSignificantly different from wild-type controls (p < 0.05).(10.4 ± 0.4%)1-aSignificantly different from wild-type controls (p < 0.05).(0.4 ± 0.1%)1-aSignificantly different from wild-type controls (p < 0.05).−/−FControl1022 ± 11.1 ± 0.10.67 ± 0.031-bSignificantly different from control and WY-14643-treated wild-type controls (p < 0.05).(5.1 ± 0.2%)(3.1 ± 0.2%)1-bSignificantly different from control and WY-14643-treated wild-type controls (p < 0.05).−/−FWY521 ± 11.1 ± 0.10.58 ± 0.101-bSignificantly different from control and WY-14643-treated wild-type controls (p < 0.05).(5.1 ± 0.1%)(2.8 ± 0.4%)1-bSignificantly different from control and WY-14643-treated wild-type controls (p < 0.05).Sv/129+/+MControl1026 ± 11.1 ± 0.10.50 ± 0.03(4.3 ± 0.1%)(1.9 ± 0.1%)+/+MWY1022 ± 11-aSignificantly different from wild-type controls (p < 0.05).1.9 ± 0.11-aSignificantly different from wild-type controls (p < 0.05).0.14 ± 0.031-aSignificantly different from wild-type controls (p < 0.05).(8.7 ± 0.3%)1-aSignificantly different from wild-type controls (p < 0.05).(0.7 ± 0.1%)1-aSignificantly different from wild-type controls (p < 0.05).−/−MControl1027 ± 11.2 ± 0.10.65 ± 0.041-bSignificantly different from control and WY-14643-treated wild-type controls (p < 0.05).(4.2 ± 0.1%)(2.4 ± 0.1%)1-bSignificantly different from control and WY-14643-treated wild-type controls (p < 0.05).−/−MWY1028 ± 11.2 ± 0.10.60 ± 0.051-bSignificantly different from control and WY-14643-treated wild-type controls (p < 0.05).(4.3 ± 0.2%)(2.2 ± 0.1%)1-bSignificantly different from control and WY-14643-treated wild-type controls (p < 0.05).+/+FControl522 ± 11.2 ± 0.10.32 ± 0.02(5.2 ± 0.2%)(1.5 ± 0.1%)+/+FWY520 ± 12.0 ± 0.11-aSignificantly different from wild-type controls (p < 0.05).0.09 ± 0.041-aSignificantly different from wild-type controls (p < 0.05).(10.8 ± 0.6%)1-aSignificantly different from wild-type controls (p < 0.05).(0.5 ± 0.2%)1-aSignificantly different from wild-type controls (p < 0.05).−/−FControl523 ± 10.9 ± 0.10.41 ± 0.031-bSignificantly different from control and WY-14643-treated wild-type controls (p < 0.05).(3.9 ± 0.3%)(1.8 ± 0.1%)1-bSignificantly different from control and WY-14643-treated wild-type controls (p < 0.05).−/−FWY522 ± 10.7 ± 0.10.43 ± 0.061-bSignificantly different from control and WY-14643-treated wild-type controls (p < 0.05).(3.3 ± 0.1%)(1.9 ± 0.2%)1-bSignificantly different from control and WY-14643-treated wild-type controls (p < 0.05).Treatment group indicates a control or WY-14643 (WY) diet.n, number of mice examined; BW, body weight. Liver and gonadal adipose weights are expressed as grams and relative to body weight ((grams of adipose/g of body weight) × 100).1-a Significantly different from wild-type controls (p < 0.05).1-b Significantly different from control and WY-14643-treated wild-type controls (p < 0.05). Open table in a new tab Table IIBody, liver, and adipose weights and serum lipids in 9-month-old male or female C57BL/6N or Sv/129 wild-type (+/+) or PPARα-null (−/−) miceGenotypeSexnBWLiverAdiposeTGTCHDLgggmg/dlmg/dlmg/dlC57BL/6N+/+M1330 ± 11.4 ± 0.11.0 ± 0.183 ± 878 ± 269 ± 2(3.7 ± 0.1%)(2.7 ± 0.2%)+/+F1325 ± 10.9 ± 0.10.5 ± 0.168 ± 1071 ± 362 ± 3(3.9 ± 0.1%)(1.6 ± 0.2%)−/−M1129 ± 21.3 ± 0.11.5 ± 0.22-aSignificantly different from wild-type male (p < 0.05).110 ± 132-aSignificantly different from wild-type male (p < 0.05).84 ± 269 ± 4(3.5 ± 0.1%)(3.9 ± 0.3%)2-aSignificantly different from wild-type male (p < 0.05).−/−F1428 ± 21.2 ± 0.11.4 ± 0.22-bSignificantly different from wild-type female (p < 0.05).85 ± 102-bSignificantly different from wild-type female (p < 0.05).69 ± 259 ± 3(3.6 ± 0.2%)(4.3 ± 0.5%)2-bSignificantly different from wild-type female (p < 0.05).Sv/129+/+M1132 ± 11.1 ± 0.10.8 ± 0.1111 ± 9124 ± 4107 ± 7(3.8 ± 0.1%)(2.5 ± 0.3%)+/+F1226 ± 10.9 ± 0.10.5 ± 0.199 ± 8106 ± 593 ± 3(3.6 ± 0.2%)(1.9 ± 0.4%)−/−M1332 ± 11.0 ± 0.11.2 ± 0.22-aSignificantly different from wild-type male (p < 0.05).94 ± 10142 ± 52-aSignificantly different from wild-type male (p < 0.05).118 ± 4(3.3 ± 0.1%)(3.5 ± 0.3%)2-aSignificantly different from wild-type male (p < 0.05).−/−F1328 ± 10.8 ± 0.10.9 ± 0.22-bSignificantly different from wild-type female (p < 0.05).95 ± 11122 ± 82-bSignificantly different from wild-type female (p < 0.05).108 ± 32-bSignificantly different from wild-type female (p < 0.05).(2.9 ± 0.1%)(3.4 ± 0.1%)2-bSignificantly different from wild-type female (p < 0.05).n, number of mice examined; BW, body weight; TG, serum triglycerides; TC, serum total cholesterol; HDL, high density lipoprotein cholesterol. Liver and gonadal adipose weights are expressed as grams and relative to body weight ((grams of adipose/g of body weight) × 100).2-a Significantly different from wild-type male (p < 0.05).2-b Significantly different from wild-type female (p < 0.05). Open table in a new tab Figure 2Hepatic accumulation of lipids in male PPAR α-null mice. Shown are representative hematoxylin- and eosin-stained sections of livers (magnification × 300) from wild-type (+/+) and PPARα-null (−/−) mice on either an Sv/129 or a C57BL/6N genetic background.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3PPAR α-null mice are not overtly obese. Shown are representative 7–8-month-old male and female wild-type (+/+) or PPARα-null (−/−) mice on either an Sv/129 or a C57BL/6N genetic background. Bar = 1 inch.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Treatment group indicates a control or WY-14643 (WY) diet.n, number of mice examined; BW, body weight. Liver and gonadal adipose weights are expressed as grams and relative to body weight ((grams of adipose/g of body weight) × 100). n, number of mice examined; BW, body weight; TG, serum triglycerides; TC, serum total cholesterol; HDL, high density lipoprotein cholesterol. Liver and gonadal adipose weights are expressed as grams and relative to body weight ((grams of adipose/g of body weight) × 100). Serum concentrations of cholesterol and high density lipoprotein cholesterol were significantly higher in 9-month-old purebred Sv/129 PPARα-null mice than in wild-type controls (TableII). This effect was observed in both male and female mice, with no apparent difference in the magnitude of these effects (Table II). Serum levels of triglycerides were similar in Sv/129 PPARα-null and wild-type mice (Table II). Serum concentrations of cholesterol and high density lipoprotein cholesterol were similar in 9-month-old purebred C57BL/6N PPARα-null mice and wild-type controls (Table II). This was observed in both male and female mice (Table II). In contrast to Sv/129 mice, serum levels of triglycerides were significantly higher in both male and female PPARα-null mice compared with the respective wild-type controls (Table II). Constitutive hepatic levels of mRNAs encoding mitochondrial fatty acid-metabolizing enzymes (very long chain and long chain acyl-CoA dehydrogenases) were significantly lower in both C57BL/6N and Sv/129 PPARα-null mice of both sexes compared with wild-type controls (Fig.4), consistent with previous results (5Aoyama A. Peters J.M. Iritani N. Nasu-Nakajima T. Furihata K. Hashimoto T. Gonzalez F.J. J. Biol. Chem. 1998; 273: 5678-5684Abstract Full Text Full Text PDF PubMed Scopus (750) Google Scholar). Constitutive hepatic levels of apoC-III were not different between genotypes or sexes in either the C57BL/6N or Sv/129 mouse strain (Fig.4). Similarly, constitutive hepatic levels of mRNA encoding PPARγ were not different between either genotype in both strains and sexes of mice (Fig. 4). Liver weight was significantly higher in male and female wild-type mice fed WY-14643 compared with controls in both C57BL/6N and Sv/129 mice, and this effect was not different between strains (Table I). In contrast, liver weight was not different between male and female null mice compared with controls, and again there was no difference in this effect between C57BL/6N and Sv/129 mice (Table I). Consistent with previous studies, gonadal adipose stores were significantly lower in male and female wild-type mice fed WY-14643 for 1 week compared with controls, and this effect was not found in either strain of PPARα-null mice fed WY-14643 (TableI). Administration of WY-14643 to mice caused a significant decrease in serum triglycerides in both strains of purebred wild-type mice compared with untreated controls (Table II). Hepatic levels of mRNAs encoding acyl-CoA oxidase; bifunctional enzyme; 3-ketoacyl-CoA thiolase; cytochrome P450 4A1; and very long chain, long chain, and medium chain acyl-CoA dehydrogenases were higher in wild-type mice fed WY-14643 than in controls, and these effects were not different between wild-type C57BL/6N and Sv/129 mice of both sexes (Fig. 4). The PPARα-null mice were refractory to increased levels of these mRNAs, and there was no difference in this effect between strains (Fig. 4). Hepatic mRNA for apoC-III was reduced in wild-type mice fed WY-14643 compared with controls (Fig. 4), and this effect was absent in both strains and sexes of the PPARα-null mice. The original phenotypic assessment of PPARα-null mice on a mixed genetic background (C57BL/6N × Sv/129) provided strong in vivo evidence that PPARα mediates the pleiotropic response to peroxisome proliferators, including hepatomegaly, peroxisome proliferation, and induction of genes encoding peroxisomal and microsomal lipid-metabolizing enzymes (3Lee S.S. Pineau T. Drago J. Lee E.J. Owens J.W. Kroetz D.L. Fernandez-Salguero P.M. Westphal H. Gonzalez F.J. Mol. Cell. Biol. 1995; 15: 3012-3022Crossref PubMed Scopus (1498) Google Scholar). Although constitutive expression of peroxisomal and microsomal lipid-metabolizing enzymes was not influenced by targeted disruption of the PPARα gene, hepatic accumulation of lipids was described in PPARα-null mice, suggesting that constitutive lipid homeostasis is altered in the absence of a functional PPARα (3Lee S.S. Pineau T. Drago J. Lee E.J. Owens J.W. Kroetz D.L. Fernandez-Salguero P.M. Westphal H. Gonzalez F.J. Mol. Cell. Biol. 1995; 15: 3012-3022Crossref PubMed Scopus (1498) Google Scholar). Evidence that constitutive gene expression is altered in PPARα-null mice on an Sv/129 background was provided by the report that mRNAs encoding mitochondrial fatty acid-metabolizing enzymes are reduced compared with wild-type mice, whereas constitutive expression of mRNAs encoding peroxisomal and microsomal fatty acid-metabolizing enzymes is unaffected (5Aoyama A. Peters J.M. Iritani N. Nasu-Nakajima T. Furihata K. Hashimoto T. Gonzalez F.J. J. Biol. Chem. 1998; 273: 5678-5684Abstract Full Text Full Text PDF PubMed Scopus (750) Google Scholar). This study also confirmed that many of the observations made in mixed background PPARα-null mice are consistently found in purebred Sv/129 mice, including an absence of peroxisome proliferator-induced hepatomegaly and induction of mRNAs encoding peroxisomal and microsomal lipid-metabolizing enzymes (5Aoyama A. Peters J.M. Iritani N. Nasu-Nakajima T. Furihata K. Hashimoto T. Gonzalez F.J. J. Biol. Chem. 1998; 273: 5678-5684Abstract Full Text Full Text PDF PubMed Scopus (750) Google Scholar). This suggests that hepatic lipid accumulation found in PPARα-null mice may be the result of reduced mitochondrial fatty acid oxidation. Results from the present study confirm and extend this characterization by demonstrating that male and female C57BL/6N PPARα-null mice are refractory to the pleiotropic response induced by peroxisome proliferators and that constitutive hepatic lipid accumulation occurs as previously described. Furthermore, this work demonstrates that this response is similar between male and female PPARα-null mice on either a pure Sv/129 or C57BL/6N genetic background. Serum lipids in mixed background PPARα-null mice were also reported to be altered compared with wild-type controls. PPARα-null mice on a mixed genetic background exhibit significantly higher serum levels of cholesterol, in particular high density lipoprotein cholesterol, compared with wild-type controls (4Peters J.M. Hennuyer N. Staels B. Fruchart J.C. Fievet C. Gonzalez F.J. Auwerx J. J. Biol. Chem. 1997; 272: 27307-27312Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar). Similar results were found in this study in both male and female PPARα-null mice on a pure Sv/129 genetic background, consistent with the observations made in mixed background mice. In contrast, higher levels of serum cholesterol were not found, whereas serum levels of triglycerides were significantly higher than controls in both male and female PPARα-null mice on a C57BL/6N background. These results suggest that the genetic background of the PPARα-null mouse can significantly influence serum lipid biochemistry, likely through interactions with other genes. The mechanisms underlying this difference are unclear. Nevertheless, purebred Sv/129 and C57BL/6N PPARα-null mice provide unique tools for studies investigating the role of altered serum cholesterol and triglycerides in the etiology of atherosclerosis. The C57BL/6 mouse strain is better suited for evaluating the mechanisms contributing to atherosclerosis since atherosclerotic plaques can be induced by feeding a high fat diet (49Rubin E.M. Krauss R.M. Spangler E.A. Verstuyft J.G. Clift S.M. Nature. 1991; 353: 265-267Crossref PubMed Scopus (860) Google Scholar, 50Shimano H. Ohsuga J. Shimada M. Namba Y. Gotoda T. Harada K. Katsuki M. Yazaki Y. Yamada N. J. Clin. Invest. 1995; 95: 469-476Crossref PubMed Scopus (128) Google Scholar). Thus, the PPARα-null mouse line on a C57BL/6N genetic background may be well suited for this purpose since constitutively higher levels of lipids are a known risk factor for this disease (51Assmann G. Cullen P. Jossa F. Lewis B. Mancini M. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1819-1824Crossref PubMed Scopus (109) Google Scholar). As PPARα-null mice exhibit significant lipid accumulation that may be due in part to impaired mitochondrial oxidation of fatty acids, it is not surprising that adipose stores are significantly greater in this mouse line as well. Although it is clear from these results that purebred PPARα-null mice on a pure Sv/129 or C57BL/6N genetic background have larger stores of adipose and accumulate lipids in the liver, differences in body weight are not of sufficient magnitude to be indicative of an obese phenotype. In the original mixed background PPARα-null mouse line, it was noted that adipose stores were significantly greater than controls with little difference in overall body weight (52Gonzalez F.J. Adv. Exp. Med. Biol. 1997; 422: 109-125Crossref PubMed Scopus (52) Google Scholar). Similar reports of PPARα-null mice on an Sv/129 background are consistent with this observation in that large differences in body weight were not found even in male mice that are >1-year-old (26Nakajima T. Kamijo Y. Usuda N. Liang Y. Fukushima Y. Kametani K. Gonzalez F.J. Aoyama T. Carcinogenesis. 2000; 21: 677-682Crossref PubMed Scopus (48) Google Scholar, 27Peters J.M. Cattley R.C. Gonzalez F.J. Carcinogenesis. 1997; 18: 2029-2033Crossref PubMed Scopus (426) Google Scholar). Conflicting reports suggest that this phenotype may be influenced by other factors, including diet and genetics. Costet et al. (42Costet P. Legendre C. More J. Edgar A. Galtier P. Pineau T. J. Biol. Chem. 1998; 273: 29577-29585Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar) provided evidence suggesting that the PPARα-null mouse may be a useful model to study obesity and that this phenotype is more prevalent in female mice than in male mice. In contrast to results presented in the present study, these investigators reported that body weight of PPARα-null mice is significantly greater than controls in both sexes after 7 months of age. Consistent with previous work (4Peters J.M. Hennuyer N. Staels B. Fruchart J.C. Fievet C. Gonzalez F.J. Auwerx J. J. Biol. Chem. 1997; 272: 27307-27312Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar) and the present study, alterations in serum lipids, adipose stores, and hepatic lipid accumulation were also detected in PPARα-null mice compared with controls (42Costet P. Legendre C. More J. Edgar A. Galtier P. Pineau T. J. Biol. Chem. 1998; 273: 29577-29585Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). The difference in body weight between male and female PPARα-null mice was attributed in part to differences in hepatic PPARγ expression and differences in hepatic lipid accumulation (42Costet P. Legendre C. More J. Edgar A. Galtier P. Pineau T. J. Biol. Chem. 1998; 273: 29577-29585Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar); however, these changes were not detected in the present study. It is critical to emphasize that the genetic background of the PPARα-null mice used for the analysis performed by Costet et al. (42Costet P. Legendre C. More J. Edgar A. Galtier P. Pineau T. J. Biol. Chem. 1998; 273: 29577-29585Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar) is unclear, as the substrain of the C57BL/6 mouse used for backcrossing was not identified, and the extent of backcrossing described (<10 generations) theoretically would not result in a congenic line of mice. Thus, the congenic control C57BL/6 mice of unknown substrain used for controls are likely inappropriate and may have resulted in incorrect comparisons. Indeed, significant differences in the functional properties of another xenobiotic receptor (aryl hydrocarbon receptor) are known to exist between C57BL/6N and C57BL/6J mouse lines (53Poland A. Palen D. Glover E. Mol. Pharmacol. 1994; 46: 915-921PubMed Google Scholar, 54Poland A. Glover E. Mol. Pharmacol. 1990; 38: 306-312PubMed Google Scholar), demonstrating the importance of backcrossing mice with the identical line used for blastocyst transfer in this case. Differences in control mouse chow may also have contributed to the difference in body weight observed in PPARα-null mice between the present study and that of Costet et al. (42Costet P. Legendre C. More J. Edgar A. Galtier P. Pineau T. J. Biol. Chem. 1998; 273: 29577-29585Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar), although the percentage of fat was similar (4.5%), suggesting that the genetic background is more likely a confounding variable in this work. That dietary fatty acids may influence the phenotype of PPARα-null mice is also suggested by another report showing that purebred Sv/129 PPARα-null mice have larger adipose stores than controls (29Poynter M.E. Daynes R.A. J. Biol. Chem. 1998; 273: 32833-32841Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar). In contrast to data presented in this study and that of Costet et al. (42Costet P. Legendre C. More J. Edgar A. Galtier P. Pineau T. J. Biol. Chem. 1998; 273: 29577-29585Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar), these investigators reported that gonadal adipose stores and average body weight were greater in male PPARα-null mice compared with female PPARα-null mice (29Poynter M.E. Daynes R.A. J. Biol. Chem. 1998; 273: 32833-32841Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar). Although increased adipose stores and body weight in PPARα-null mice are consistent with this work, the fact that male PPARα-null mice on an Sv/129 background were reported to have larger adipose stores than female mice (29Poynter M.E. Daynes R.A. J. Biol. Chem. 1998; 273: 32833-32841Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar) illustrates how significant variation can occur between laboratories using an identical mouse line. The most likely explanation for this difference is the source of fat used for the control diet, which can significantly influence lipid metabolism in these mice (7Kersten S. Seydoux J. Peters J.M. Gonzalez F.J. Desvergne B. Wahli W. J. Clin. Invest. 1999; 103: 1489-1498Crossref PubMed Scopus (1360) Google Scholar). Given the conflicting accounts of phenotypes for the PPARα-null mouse lines with respect to obesity, it is critical that investigators indicate the source of fat used for control and experimental diets in the future and the strain of congenic mouse used for analysis. This study provides details of the backcrossing performed at the National Institutes of Health with the original line of mice, which to date has been the sole source for distribution of PPARα-null mice to independent investigators.