Title: Peroxisomal and Mitochondrial Fatty Acid β-Oxidation in Mice Nullizygous for Both Peroxisome Proliferator-activated Receptor α and Peroxisomal Fatty Acyl-CoA Oxidase
Abstract: Fatty acid β-oxidation occurs in both mitochondria and peroxisomes. Long chain fatty acids are also metabolized by the cytochrome P450 CYP4A ω-oxidation enzymes to toxic dicarboxylic acids (DCAs) that serve as substrates for peroxisomal β-oxidation. Synthetic peroxisome proliferators interact with peroxisome proliferator activated receptor α (PPARα) to transcriptionally activate genes that participate in peroxisomal, microsomal, and mitochondrial fatty acid oxidation. Mice lacking PPARα (PPARα−/−) fail to respond to the inductive effects of peroxisome proliferators, whereas those lacking fatty acyl-CoA oxidase (AOX−/−), the first enzyme of the peroxisomal β-oxidation system, exhibit extensive microvesicular steatohepatitis, leading to hepatocellular regeneration and massive peroxisome proliferation, implying sustained activation of PPARα by natural ligands. We now report that mice nullizygous for both PPARα and AOX (PPARα−/− AOX−/−) failed to exhibit spontaneous peroxisome proliferation and induction of PPARα-regulated genes by biological ligands unmetabolized in the absence of AOX. In AOX−/− mice, the hyperactivity of PPARα enhances the severity of steatosis by inducing CYP4A family proteins that generate DCAs and since they are not metabolized in the absence of peroxisomal β-oxidation, they damage mitochondria leading to steatosis. Blunting of microvesicular steatosis, which is restricted to few liver cells in periportal regions in PPARα−/−AOX−/− mice, suggests a role for PPARα-induced genes, especially members of CYP4A family, in determining the severity of steatosis in livers with defective peroxisomal β-oxidation. In age-matched PPARα−/− mice, a decrease in constitutive mitochondrial β-oxidation with intact constitutive peroxisomal β-oxidation system contributes to large droplet fatty change that is restricted to centrilobular hepatocytes. These data define a critical role for both PPARα and AOX in hepatic lipid metabolism and in the pathogenesis of specific fatty liver phenotype. Fatty acid β-oxidation occurs in both mitochondria and peroxisomes. Long chain fatty acids are also metabolized by the cytochrome P450 CYP4A ω-oxidation enzymes to toxic dicarboxylic acids (DCAs) that serve as substrates for peroxisomal β-oxidation. Synthetic peroxisome proliferators interact with peroxisome proliferator activated receptor α (PPARα) to transcriptionally activate genes that participate in peroxisomal, microsomal, and mitochondrial fatty acid oxidation. Mice lacking PPARα (PPARα−/−) fail to respond to the inductive effects of peroxisome proliferators, whereas those lacking fatty acyl-CoA oxidase (AOX−/−), the first enzyme of the peroxisomal β-oxidation system, exhibit extensive microvesicular steatohepatitis, leading to hepatocellular regeneration and massive peroxisome proliferation, implying sustained activation of PPARα by natural ligands. We now report that mice nullizygous for both PPARα and AOX (PPARα−/− AOX−/−) failed to exhibit spontaneous peroxisome proliferation and induction of PPARα-regulated genes by biological ligands unmetabolized in the absence of AOX. In AOX−/− mice, the hyperactivity of PPARα enhances the severity of steatosis by inducing CYP4A family proteins that generate DCAs and since they are not metabolized in the absence of peroxisomal β-oxidation, they damage mitochondria leading to steatosis. Blunting of microvesicular steatosis, which is restricted to few liver cells in periportal regions in PPARα−/−AOX−/− mice, suggests a role for PPARα-induced genes, especially members of CYP4A family, in determining the severity of steatosis in livers with defective peroxisomal β-oxidation. In age-matched PPARα−/− mice, a decrease in constitutive mitochondrial β-oxidation with intact constitutive peroxisomal β-oxidation system contributes to large droplet fatty change that is restricted to centrilobular hepatocytes. These data define a critical role for both PPARα and AOX in hepatic lipid metabolism and in the pathogenesis of specific fatty liver phenotype. peroxisome proliferator-activated receptor long chain fatty acid very long chain fatty acids dicarboxylic fatty acids straight chain fatty acyl-CoA oxidase enoyl-CoA hydratase/l-3-hydroxyacyl-CoA dehydrogenase bifunctional protein d-3-hydroxyacyl-CoA dehydratase/d-3-hydroxyacyl-CoA dehydrogenase bifunctional protein peroxisomal 3-ketoacyl-CoA thiolase sterol carrier protein x or 3-ketoacyl-CoA thiolase/sterol carrier protein 2 carnitine octanoyltransferase carnitine acetyltransferase peroxisomal membrane protein catalase urate oxidase palmitoyl-CoA synthetase very long chain acyl-CoA synthetase long chain acyl-CoA dehydrogenase very chain acyl-CoA dehydrogenase medium chain acyl-CoA dehydrogenase mitochondrial enoyl-CoA hydratase 3-hydroxyacyl-CoA dehydrogenase mitochondrial trifunctional protein mitochondrial 3-ketoacyl-CoA thiolase mitochondrial acetoacetyl-CoA-specific thiolase encode microsomal cytochrome P450 fatty acid ω-hydroxylases kilobase(s) double knock-out mice nullizygous for both PPAR and AOX In animal cells, mitochondria as well as peroxisomes oxidize fatty acids via β-oxidation, with long chain and very long chain fatty acids (LCFAs and VLCFAs)1being preferentially oxidized by peroxisomes (1Lazarow P.B. de Duve C. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2043-2046Crossref PubMed Scopus (1174) Google Scholar, 2Reddy J.K. Mannaerts G.P. Annu. Rev. Nutr. 1994; 14: 343-370Crossref PubMed Scopus (363) Google Scholar, 3Hashimoto T. Neurochem. Res. 1999; 24: 551-563Crossref PubMed Scopus (60) Google Scholar). Peroxisomal β-oxidation is carried out by two distinct groups of enzymes. The classical first group utilizes straight chain saturated fatty acyl-CoAs as substrates, whereas the second group acts on branched chain acyl-CoAs (3Hashimoto T. Neurochem. Res. 1999; 24: 551-563Crossref PubMed Scopus (60) Google Scholar, 4Baumgart E. Vanhooren J.C. Fransen M. Marynen P. Puype M. Vanderkerckhove J. Leunissen J.A. Fahimi H.D. Mannaerts G.P. van Veldhoven P.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13748-13753Crossref PubMed Scopus (76) Google Scholar). In the classical l-3-hydroxy-specific β-oxidation spiral, dehydrogenation of acyl-CoA esters to their corresponding trans-2-enoyl-CoAs is catalyzed by fatty acyl-CoA oxidase (AOX), whereas the second and third reactions, hydration and dehydrogenation of enoyl-CoA esters to 3-ketoacyl-CoA, are catalyzed by a single enzyme, enoyl-CoA hydratase/l-3-hydroxyacyl-CoA dehydrogenase (l-bifunctional enzyme (l-PBE)) (3Hashimoto T. Neurochem. Res. 1999; 24: 551-563Crossref PubMed Scopus (60) Google Scholar). The third enzyme of this classical system, 3-ketoacyl-CoA thiolase (PTL), cleaves 3-ketoacyl-CoAs to acetyl-CoA and an acyl-CoA that is two carbon atoms shorter than the original molecule, and it can re-enter the β-oxidation spiral (1Lazarow P.B. de Duve C. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2043-2046Crossref PubMed Scopus (1174) Google Scholar, 2Reddy J.K. Mannaerts G.P. Annu. Rev. Nutr. 1994; 14: 343-370Crossref PubMed Scopus (363) Google Scholar). In the secondd-3-hydroxy-specific β-oxidation pathway, dehydrogenation of acyl-CoA esters to their corresponding trans-2-enoyl-CoAs is catalyzed by the branched chain acyl-CoA oxidase (2Reddy J.K. Mannaerts G.P. Annu. Rev. Nutr. 1994; 14: 343-370Crossref PubMed Scopus (363) Google Scholar). The recently identified d-3-hydroxyacyl-CoA dehydratase/d-3-hydroxyacl-CoA dehydrogenase (d-bifunctional enzyme, (d-PBE)) then converts enoyl-CoAs to 3-ketoacyl-CoAs viad -3-hydroxyacyl-CoAs (3Hashimoto T. Neurochem. Res. 1999; 24: 551-563Crossref PubMed Scopus (60) Google Scholar). The third enzyme of this second system is designated as sterol carrier protein x (SCPx), which possesses 3-ketoacyl-CoA thiolase activity (5Seedorf U. Brysch P. Engel T. Schrage K. Assmann G. J. Biol. Chem. 1994; 269: 21277-21283Abstract Full Text PDF PubMed Google Scholar). The first step in mitochondrial β-oxidation is the α-β dehydrogenation of the acyl-CoA ester by a family of four chain-length-specific acyl-CoA dehydrogenases, which include very long chain, long chain, medium chain, and short chain acyl-CoA dehydrogenases (VLCAD, LCAD, MCAD, and SCAD respectively) (2Reddy J.K. Mannaerts G.P. Annu. Rev. Nutr. 1994; 14: 343-370Crossref PubMed Scopus (363) Google Scholar, 3Hashimoto T. Neurochem. Res. 1999; 24: 551-563Crossref PubMed Scopus (60) Google Scholar). The second, third, and fourth steps in the mitochondrial β-oxidation spiral are carried out by a 2-enoyl-CoA hydratase (MH), a 3-hydroxyacyl-CoA dehydrogenase (HADH), and a 3-ketoacyl-CoA thiolase (MTL1) (3Hashimoto T. Neurochem. Res. 1999; 24: 551-563Crossref PubMed Scopus (60) Google Scholar). The MH, HADH, and MTL activities lie within mitochondrial trifunctional protein (TFP) (3Hashimoto T. Neurochem. Res. 1999; 24: 551-563Crossref PubMed Scopus (60) Google Scholar). Of these two peroxisomal β-oxidation systems, the enzymes belonging to the classical group are markedly induced in conjunction with profound proliferation of peroxisomes in the liver of rats and mice by a group of structurally diverse agents designated as peroxisome proliferators (6Reddy J.K. Krishnakantha T.P. Science. 1975; 190: 787-789Crossref PubMed Scopus (293) Google Scholar). These synthetic peroxisome proliferators exert their pleiotropic effects in liver by activating a nuclear receptor called peroxisome proliferator-activated receptor α (PPARα) (7Issemann I. Green S. Nature. 1990; 347: 645-650Crossref PubMed Scopus (3027) Google Scholar). The induction of peroxisome proliferation is associated with transcriptional activation of genes encoding for the peroxisomal β-oxidation system and cytochrome P450 CYP 4A isoforms, CYP4A1 and CYP4A3, among others (8Reddy J.K. Goel S.K. Nemali M.R. Carrino J.J. Laffler T.G. Reddy M.K. Sperbeck S.J. Soumi T. Hashimoto S. Lalwani N.D. Rao M.S. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1747-1751Crossref PubMed Scopus (313) Google Scholar, 9Johnson E.F. Palmer C.N.A. Griffin K.J. Hsu M.-H. FASEB J. 1996; 10: 1241-1248Crossref PubMed Scopus (169) Google Scholar, 10Reddy J.K. Chu R. Ann. N. Y. Acad. Sci. 1996; 804: 176-201Crossref PubMed Scopus (99) Google Scholar, 11Lemberger T. Desvergne B. Wahli W. Annu. Rev. Cell Dev. Biol. 1996; 12: 335-363Crossref PubMed Scopus (633) Google Scholar). For this to occur, PPARα heterodimerizes with retinoid X receptor, and this PPAR-retinoid X receptor complex binds to PPAR response element, a region consisting of a degenerate direct repeat of the canonical AGGTCA sequence separated by 1 base pair (DR1), present in the 5′-flanking region of target genes (12Kliewer S.A. Umesono K. Noonan D.J. Heyman R.A. Evans R.M. Nature. 1992; 358: 771-774Crossref PubMed Scopus (1516) Google Scholar). The generation of PPARα−/− mice established that this receptor is essential for hepatic peroxisome proliferation and coordinate transcriptional activation of AOX, l-PBE, PTL, CYP4A,1 and CYP4A3 and other genes by structurally diverse synthetic peroxisome proliferators (13Lee S.S.-T. 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 (1494) Google Scholar). PPARα−/− mice display normal complement of peroxisomes in liver cells, but these mice remain nonresponsive to the inductive influence of synthetic peroxisome proliferators (13Lee S.S.-T. 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 (1494) Google Scholar). A mild degree of centrilobular fatty change develops in these mice that is attributed to reduction in the constitutive levels of mitochondrial fatty acid β-oxidation, because the constitutive or basal oxidation of VLCFAs by peroxisomal β-oxidation system appears unaffected by PPARα deficiency (14Aoyama T. Peters J.M. Iritani N. Nakajima T. Furihata K. Hashimoto T. Gonzalez F.J. J. Biol. Chem. 1998; 273: 5678-5684Abstract Full Text Full Text PDF PubMed Scopus (745) Google Scholar). We investigated the functional implications of disrupting the basal metabolism of VLCFAs by generating mice deficient in AOX, the first enzyme of inducible peroxisomal β-oxidation system, and found that AOX−/− mice develop severe microvesicular steatohepatitis and spontaneous peroxisome proliferation in liver cells (15Fan C.-Y. Pan J. Chu R. Lee D. Kluckman K.D. Usuda N. Singh I. Yeldandi A.V. Rao M.S. Maeda N. Reddy J.K. J. Biol. Chem. 1996; 271: 24698-24710Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 16Fan C.-Y. Pan J. Usuda N. Yeldandi A.V. Rao M.S. Reddy J.K. J. Biol. Chem. 1998; 273: 15639-15645Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). These results suggested that very long chain and long chain acyl-CoAs and other putative substrates for classical AOX play a role in triggering spontaneous peroxisome proliferation by functioning as PPARα ligands if they remain unmetabolized and in the development of microvesicular hepatic steatosis (16Fan C.-Y. Pan J. Usuda N. Yeldandi A.V. Rao M.S. Reddy J.K. J. Biol. Chem. 1998; 273: 15639-15645Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). The morphological and biochemical changes observed in AOX−/− mice suggested a sustained hyperfunction of PPARα because of biological ligands (16Fan C.-Y. Pan J. Usuda N. Yeldandi A.V. Rao M.S. Reddy J.K. J. Biol. Chem. 1998; 273: 15639-15645Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). Although PPARα is essential for the induction of pleiotropic responses by synthetic peroxisome proliferators as evidenced from studies in PPARα−/− mice (13Lee S.S.-T. 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 (1494) Google Scholar), it is uncertain if the spontaneous peroxisome proliferation induced by biological mediators in AOX-deficient mice is effected by PPARα or by some other mechanism. In the present study, we investigated the potential for in vivo compensatory functions by generating mice deficient in both PPARα and AOX. We report that these double nullizygous mice (PPARα−/− AOX−/−) do not show spontaneous hepatic peroxisome proliferation, implying that PPARα deficiency is not compensated by other transcription factors. Also pertinent is that the microvesicular steatosis was markedly diminished in PPARα−/− AOX−/− mice in comparison to severe steatosis observed in AOX−/− mice, suggesting that the presence of PPARα exaggerates steatosis developing in the absence of peroxisomal β-oxidation. The generation of PPARα nulls and AOX nulls has been described elsewhere (13Lee S.S.-T. 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 (1494) Google Scholar, 15Fan C.-Y. Pan J. Chu R. Lee D. Kluckman K.D. Usuda N. Singh I. Yeldandi A.V. Rao M.S. Maeda N. Reddy J.K. J. Biol. Chem. 1996; 271: 24698-24710Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Because of reduced fertility of homozygous AOX−/− males and females, homozygous PPARα−/− males and heterozygous AOX+/−females were mated to produce F1 progeny heterozygous for both genes. Sibs were intercrossed to produce progeny null for both PPARα and AOX (PPARα−/− AOX−/−). The genotypes of these progenies and subsequent generations were analyzed by Southern blot analysis of DNA (10 μg) isolated from tail tip of 2-week-old mice. Male and female mice nullizygous for both PPARα and AOX genes (PPARα−/− AOX−/−) were fertile. All animal procedures used in this study were approved by the Institutional Review Board for Animal Research of the Northwestern University. Wild type (C57BL/6J), AOX−/− (15Fan C.-Y. Pan J. Chu R. Lee D. Kluckman K.D. Usuda N. Singh I. Yeldandi A.V. Rao M.S. Maeda N. Reddy J.K. J. Biol. Chem. 1996; 271: 24698-24710Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar), PPARα−/− (13Lee S.S.-T. 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 (1494) Google Scholar), and PPARα−/−AOX−/− mice, 3 to 4 months of age, were used in these studies. They were fed powdered diet with or without ciprofibrate (0.0125% w/w), a peroxisome proliferator, for 2 weeks. For light microscopy, tissues were fixed in 10% neutral buffered formalin and embedded in paraffin. Histological analysis was carried out on 4-μm-thick sections stained with hematoxylin and eosin. Frozen sections of liver (5-μm thick) were used for Sudan black histochemical staining of lipids. For cytochemical localization of catalase (CTL), tissues were processed and examined as described elsewhere (17Reddy J.K. Lalwani N.D. Milman H.A. Weisburger E.K. Handbook of Carcinogen Testing. Noyes Publications, NJ1985: 482-500Google Scholar). Protein concentrations were determined by a protein assay kit (Bio-Rad) using bovine serum albumin as standard. Contents of β-oxidation enzymes and other proteins in liver were determined by immunoblot analysis of total liver proteins using polyclonal antibodies raised in rabbits against rat palmitoyl-CoA synthetase (PCS), AOX, l-PBE, PTL,d-PBE, carnitine octanoyltransferase (COT), very long-chain acyl-CoA synthetase, carnitine acetyltransferase (CAT), peroxisomal membrane protein (PMP) 70, PMP 26, PMP 22, VLCAD, LCAD, MCAD, and SCAD, electron transfer flavoprotein, MH, HADH, TFP, MTL1, mitochondrial acetoacetyl-CoA-specific thiolase (MTL2), urate oxidase (UOX), and glycolate oxidase as described previously (14Aoyama T. Peters J.M. Iritani N. Nakajima T. Furihata K. Hashimoto T. Gonzalez F.J. J. Biol. Chem. 1998; 273: 5678-5684Abstract Full Text Full Text PDF PubMed Scopus (745) Google Scholar, 18Qi C. Zhu Y. Pan J. Yeldandi V. Rao M.S. Maeda N. Subbarao V. Pulikuri S. Hashimoto T. Reddy J.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1585-1590Crossref PubMed Scopus (70) Google Scholar). Antibody against the amino-terminal part of SCPx expressed in Escherichia coli was a gift from Professor Yukio Fujiki. Total RNA was isolated from liver using Trizol reagent (Life Technologies, Inc.). After glyoxylation, samples were electrophoresed on 0.8% agarose gel, transferred to nylon membrane, and then hybridized at 42 °C in 50% formamide hybridization solution using 32P-labeled cDNA probes CTL, l-PBE, PTL, UOX, CYP4A1, CYP4A3, liver fatty acid-binding protein, and 28 S RNA as described previously (16Fan C.-Y. Pan J. Usuda N. Yeldandi A.V. Rao M.S. Reddy J.K. J. Biol. Chem. 1998; 273: 15639-15645Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). Changes in mRNA levels were estimated by densitometric scanning of autoradiograms. PPARα−/− mice (13Lee S.S.-T. 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 (1494) Google Scholar) and AOX+/−mice (15Fan C.-Y. Pan J. Chu R. Lee D. Kluckman K.D. Usuda N. Singh I. Yeldandi A.V. Rao M.S. Maeda N. Reddy J.K. J. Biol. Chem. 1996; 271: 24698-24710Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar) were cross-bred to generate mice nullizygous for both PPARα and AOX (PPARα−/− AOX−/−) used in this study. Southern analysis of tail tip DNA from double heterozygous (PPARα+/− AOX+/−) mice revealed two bands (8.0 and 13.4 kb) with AOX probe and two bands (6.3 and 7.5 kb) with PPARα probe (Fig. 1). The genomic DNA from double nullizygous (PPARα−/− AOX−/−) mice yielded a single 13.4-kb band with AOX probe and a single 7.5-kb band with PPARα probe (Fig. 1). In contrast, the DNA from a wild type mouse showed an 8.0-kb band with AOX probe and a 6.3-kb band with PPARα probe. The livers of mice nullizygous for both PPARα and AOX did not show the presence of AOX mRNA and protein when analyzed, respectively, by northern and Western blotting. Both male and female PPARα−/− AOX−/− mice were fertile and displayed no apparent gross phenotypic changes. These double nullizygous mice were grossly indistinguishable from PPARα−/− mice and from wild type mice, whereas growth retardation was common in pre-weaning and young AOX−/−mice (15Fan C.-Y. Pan J. Chu R. Lee D. Kluckman K.D. Usuda N. Singh I. Yeldandi A.V. Rao M.S. Maeda N. Reddy J.K. J. Biol. Chem. 1996; 271: 24698-24710Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). After about 3 months of age, AOX−/− mice began to gain body weight and by ∼6 months of age their body weight equalized to those of wild type mice. The absolute liver weight of PPARα−/−AOX−/− mice and of PPARα−/− mice was similar to that of age-matched wild type mice but lower in comparison to AOX−/− mice. In 3-month-old double nullizygous mice, liver weight accounted for ∼6% of body weight, whereas in the age-matched AOX−/−, PPARα−/−, and wild type, animal liver weight accounted for ∼10, ∼5, and ∼ 5%, respectively. As expected, wild type mice treated with ciprofibrate, a peroxisome proliferator, showed hepatomegaly with liver weight accounting for ∼15% of body weight. No increase in liver weight occurred in AOX−/−, PPARα−/−AOX−/−, and PPARα−/− mice treated with a peroxisome proliferator (data not shown). The lobular architecture of PPARα−/− AOX−/− mouse liver was normal except for the presence of hepatocytes with striking microvesicular vacuolation, scattered singly or in clusters in the periportal regions (Fig. 2, A, C, andD). These cells stained positively for fat when stained with Oil Red O or with Sudan black (Fig. 2 E). Few clusters of inflammatory aggregates, reminiscent of lipogranulomatous lesions found in AOX−/− mouse liver (16Fan C.-Y. Pan J. Usuda N. Yeldandi A.V. Rao M.S. Reddy J.K. J. Biol. Chem. 1998; 273: 15639-15645Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar), were also found among liver cells with microvesicular fatty change in PPARα−/−AOX−/− mouse liver (Fig. 2 D). The fatty change was generally restricted to periportal location, and even in this zone, a majority of hepatocytes appeared normal. Very few or no hepatocytes with microvesicular fatty change were present in midzonal and centrilobular regions (Fig. 2, A and C). In contrast, livers of age-matched PPARα−/− (Fig. 2,B and F) and AOX−/− (Fig. 2,G and H) mice revealed characteristically different histological phenotypes. A mild degree of centrilobular macrovesicular or large droplet fatty change occurred in PPARα−/− mice (Fig. 2, B and F), whereas younger AOX−/− mice displayed extensive microvesicular steatohepatitis, and with age, there was age-progressive hepatocellular regeneration commencing in periportal region and extending toward centrizonal region (Fig. 2, G andH). PPARα−/− mice revealed a normal complement of peroxisome profiles in liver parenchymal cells (Fig.3 A), and the number of these organelles was not increased when these mice were treated with a peroxisome proliferator (13Lee S.S.-T. 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 (1494) Google Scholar). As reported previously, liver cells with microvesicular steatosis contained few peroxisomes, whereas regenerated hepatocytes in mice deficient in AOX showed profound spontaneous peroxisome proliferation (Fig. 3, B and C), indicating a sustained activation of PPARα because of biological ligands of PPARα that require AOX for inactivation or metabolism (16Fan C.-Y. Pan J. Usuda N. Yeldandi A.V. Rao M.S. Reddy J.K. J. Biol. Chem. 1998; 273: 15639-15645Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). We surveyed for alterations in peroxisome population in livers of PPARα−/− AOX−/− mice to ascertain if natural/biological ligands that are not metabolized in the absence of AOX induce peroxisome proliferation in the absence of PPARα in these animals, possibly by activating a different transcription factor such as PPARβ. No spontaneous peroxisome proliferation was discerned in liver parenchymal cells of PPARα−/−AOX−/− mice, when sections processed for the cytochemical localization of peroxisomal catalase were examined at the light microscopic level (Fig. 3, D and E). Periportal liver cells with microvesicular fatty change in these double nullizygous mice contained few peroxisomes (Fig. 3 D); similar paucity of peroxisomes was also noted in hepatocytes with microvesicular steatosis in AOX−/− mice (15Fan C.-Y. Pan J. Chu R. Lee D. Kluckman K.D. Usuda N. Singh I. Yeldandi A.V. Rao M.S. Maeda N. Reddy J.K. J. Biol. Chem. 1996; 271: 24698-24710Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Liver cells of mice deficient in both PPARα and AOX also failed to respond to synthetic peroxisome proliferators in comparison to wild type mice (Fig. 3 F)). These observations clearly indicate that PPARα is the principal transcription factor responsible for the phenomenon of peroxisome proliferation induced by both natural/biological ligands, as well as synthetic peroxisome proliferators. VLCAS exhibits specificity toward very long chain fatty acids, whereas PCS catalyzes the activation of long chain fatty acids to the CoA esters (19Uchiyama A. Aoyama T. Kamijo K. Uchida Y. Kondo N. Orii T. Hashimoto T. J. Biol. Chem. 1996; 271: 30360-30365Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The content of hepatic VLCAS was not much different among AOX−/−, PPARα−/−, and PPARα−/− AOX−/− mice (TableI). The amount of PCS, an enzyme with tripartite (microsomal, mitochondrial, and peroxisomal) distribution (20Miyazawa S. Hashimoto T. Yokota S. J. Biochem. (Tokyo). 1985; 98: 723-733Crossref PubMed Scopus (120) Google Scholar), increased ∼2-fold in wild type mice treated with ciprofibrate (Table I). The hepatic PCS content in AOX−/− mice maintained on control diet was also elevated with no further increase occurring when these mice were treated with ciprofibrate (Table I). A slight increase in PCS content occurred in the livers of ciprofibrate-treated PPARα−/− mice and also in PPARα−/− AOX−/− mice, suggesting that the increase may represent A and B forms of this enzyme and not the PPARα-regulated C-form of PCS (21Suzuki H. Watanabe M. Fujino T. Yamamoto T. J. Biol. Chem. 1995; 270: 9676-9682Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar).Table IQuantitation of hepatic fatty acid activating enzymes and peroxisomal proteinsCiprofibrateWild typeAOX−/−PPARα−/−DKO−+−+−+−+VLCAS1.01.01.82.00.80.80.80.8PCS1.02.72.52.80.81.41.51.5COT1.0 ± 0.322 ± 421 ± 1.924 ± 81.4 ± 0.80.74 ± 0.705.1 ± 1.78.0 ± 1.1AOX1.0 ± 0.110 ± 2NDND0.53 ± 0.170.43 ± 0.16NDNDL-PBE1.0 ± 0.7120 ± 1620 ± 720 ± 80.17 ± 0.120.53 ± 0.130.28 ± 0.302.8 ± 0.5PTL1.0 ± 0.213 ± 56.8 ± 0.74.9 ± 1.00.38 ± 0.140.74 ± 0.060.73 ± 0.181.3 ± 0.6D-PBE1.0 ± 0.43.0 ± 0.32.0 ± 0.11.5 ± 0.20.68 ± 0.060.70 ± 0.420.63 ± 0.241.2 ± 0.2SCPx1.0 ± 0.10.9 ± 0.11.0 ± 0.20.83 ± 0.111.3 ± 0.21.3 ± 0.12.0 ± 0.12.5 ± 0.2CTL1.0 ± 0.21.3 ± 0.11.7 ± 0.10.90 ± 0.061.2 ± 0.31.2 ± 0.11.3 ± 0.21.4 ± 0.4UOX1.0 ± 0.11.1 ± 0.30.81 ± 0.060.026 ± 0.0451.0 ± 0.21.3 ± 0.11.4 ± 0.21.8 ± 0.2GOX1.0 ± 0.30.71 ± 0.31.1 ± 0.10.26 ± 0.030.90 ± 0.131.1 ± 0.11.1 ± 0.21.3 ± 0.1PMP 701.0 ± 0.23.8 ± 1.02.2 ± 0.41.3 ± 0.51.1 ± 0.11.4 ± 0.31.4 ± 0.42.1 ± 0.6PMP 261.0105.96.01.22.01.31.3PMP 221.0 ± 0.32.0 ± 0.30.58 ± 0.390.14 ± 0.040.91 ± 0.101.8 ± 0.40.86 ± 0.461.8 ± 0.4Total liver proteins were subjected to immunoblot analysis, and the signals were quantified by scanning densitometry. The values were normalized (= 1.0) to the signal intensities obtained with the wild type mice fed the control diet. The values are expressed by the mean ±S.D. (n = 3). DKO, double knock-out mice nullizygous for both PPARα and AOX; ND, not detected); GOX, glycolate oxidase. Open table in a new tab Total liver proteins were subjected to immunoblot analysis, and the signals were quantified by scanning densitometry. The values were normalized (= 1.0) to the signal intensities obtained with the wild type mice fed the control diet. The values are expressed by the mean ±S.D. (n = 3). DKO, double knock-out mice nullizygous for both PPARα and AOX; ND, not detected); GOX, glycolate oxidase. Table I and Fig.4 depict the relative amounts of peroxisomal proteins in wild type, PPARα−/−AOX−/−, PPARα−/−, and AOX−/− mouse livers. A 20-fold increase in COT content occurred in wild type mice treated with ciprofibrate, similar to that reported previously in rat liver with other peroxisome proliferators (22Ozasa H. Miyazawa S. Osumi T. J. Biochem. (Tokyo). 1983; 94: 543-549Crossref PubMed Scopus (19) Google Scholar). The hepatic COT content in AOX−/− mice was 20-fold higher than that of wild type controls; this increase was comparable with that observed in ciprofibrate-treated wild type mice. COT content in PPARα−/− mice and in PPARα−/−AOX−/− mice was similar to that present in wild type mice, and these nulls did not respond to ciprofibrate treatment, suggesting that COT gene transcription is regulated by PPARα. As expected, a marked increase in the amounts of AOX, l-PBE and PTL, three enzymes of the inducible classical peroxisomal β-oxidation system, occurred in the livers of wild type mice treated with a peroxisome proliferator (Table I; Fig. 4). Significant increases in AOX and l-PBE proteins occurred in wild type mice fed ciprofibrate. AOX was not detected in livers of AOX−/−and PPARα−/− AOX−/− mice (Table I).l-PBE level was 20-fold higher in AOX−/−mice, and the amount of this enzyme did not change following ciprofibrate treatment. Hepatic l-PBE levels in PPARα−/− AOX−/− and PPARα−/− mice were lower as compared with wild type mic