Overexpression of Peroxisome Proliferator-activated Receptor- (cid:1) (PPAR (cid:1) )-regulated Genes in Liver in the Absence of Peroxisome Proliferation in Mice Deficient in both L - and D -Forms of Enoyl-CoA Hydratase/Dehydrogenase Enzymes of Peroxisomal (cid:2) -Oxidation System*

Peroxisomal (cid:2) -oxidation system consists of peroxisome proliferator-activated receptor (cid:1) (PPAR (cid:1) )-induci-ble pathway capable of catalyzing straight-chain acyl-CoAs and a second noninducible pathway catalyzing the oxidation of 2-methyl-branched fatty acyl-CoAs. Disruption of the inducible (cid:2) -oxidation pathway in mice at the level of fatty acyl-CoA oxidase (AOX), the first and rate-limiting enzyme, results in spontaneous peroxisome proliferation and sustained activation of PPAR (cid:1) , leading to the development of liver tumors, whereas disrup-tions at the level of the second enzyme of this classical pathway or of the noninducible system had no such discernible effects. We now show that mice with

In mammals, fatty acid oxidation occurs in mitochondria, peroxisomes, and smooth endoplasmic reticulum (1). Whereas mitochondria and peroxisomes oxidize fatty acids via ␤-oxidation, the cytochrome P450 CYP4A subfamily of enzymes located in the smooth endoplasmic reticulum metabolizes fatty acids by -oxidation (1)(2)(3). Mitochondrial ␤-oxidation is responsible for the oxidation of the major portion of the short-(ϽC 8 ), medium-(C 8 -C 12 ), and long-(C 14 -C 20 ) chain fatty acids and, in the process, constitutes the primary source of energy derived from fatty acids. On the other hand, peroxisomal ␤-oxidation is responsible for the metabolism, almost exclusively, of very long straight-chain fatty acids (ϾC 20 ), 2-methyl-branched fatty acids (e.g. pristanic acid that is generated after ␣-oxidation of the 3-methyl-branched fatty acid phytanic acid), prostanoids, dicarboxylic acids, and the C 27 bile acid intermediates di-and trihydroxycoprostanoic acids (1,2,4). The importance of peroxisomes and peroxisomal fatty acid ␤-oxidation for human health is underscored by the existence of peroxisomal biogenesis disorders such as Zellweger syndrome and other genetic diseases affecting peroxisomal ␤-oxidation (1,2) and by the ability of many structurally diverse chemicals designated as peroxisome proliferators to induce peroxisome proliferation and increase fatty acid oxidation in liver cells, leading to the development of liver tumors in rodents (5)(6)(7).
Peroxisomal ␤-oxidation consists of four steps. Each metabolic conversion can be carried out by at least two different enzymes. The classical peroxisome proliferator-inducible pathway utilizes straight-chain acyl-CoA as substrates, whereas the second noninducible pathway catalyzes the oxidation of 2-methyl-branched fatty acyl-CoAs (1,2,4). In the classical inducible ␤-oxidation pathway, dehydrogenation of acyl-CoA esters to their corresponding trans-2-enoyl-CoAs is catalyzed by fatty acyl-CoA oxidase (AOX), 1 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-bi-/multifunctional enzyme (L-PBE/MFP1)) (1,2,4). The third enzyme of this inducible system, 3-ketoacyl-CoA thiolase (PTL), converts 3-ketoacyl-CoA to acetyl-CoA and an acyl-CoA that is two carbon atoms shorter than the original molecule, and the shortened acyl-CoA re-enters the ␤-oxidation cycle (1,2,4). In the noninducible ␤-oxidation pathway, dehydrogenation of branched fatty acyl-CoA esters to their corresponding trans-2-enoyl-CoAs is catalyzed by the branched chain acyl-CoA oxidase or by pristanoyl-CoA oxidase (4). These enzymes are also capable of desaturating straight-chain acyl-CoAs. The second and third reactions of this pathway are performed by D-3-hydroxyacyl-CoA dehydratase/D-3hydroxyacyl-CoA dehydrogenase (D-bi/multifunctional enzyme (D-PBE/MFP2)), and the resulting 3-ketoacyl-CoAs are cleaved by the third enzyme of this system, designated sterol carrier protein x (SCPx), which possesses thiolase activity (4,8). The gene encoding SCPx, a 58-kDa protein with 3-ketoacyl-CoA thiolase activity, also codes for a smaller 5.3-kDa protein designated sterol carrier protein 2 (SCP2 or nonspecific lipid transfer protein) (4,8). The three enzymes of the classical ␤-oxidation pathway are markedly induced in conjunction with peroxisome proliferation by peroxisome proliferators (9). These agents exert their pleiotropic effects in liver by activating a nuclear receptor designated as peroxisome proliferator-activated receptor ␣ (PPAR␣) (10). The PPAR subfamily consists of three members, ␣, ␤/␦, and ␥ (11), that heterodimerize with retinoid X receptor (12), and the PPAR/retinoid X receptor complex binds to peroxisome proliferator-response element, a region consisting of a degenerate direct repeat of the canonical AGGTCA sequence separated by 1 base pair (DR1), present in the 5Ј-flanking region of target genes (12,13). Sustained activation of PPAR␣ in liver by these synthetic ligands in rats and mice leads to the development of liver tumors (7,14). Activation of PPAR␣ by natural ligands in the liver of AOX Ϫ/Ϫ mice also leads to the development of liver cancer (15). In this sense, the functional integrity of the classical ␤-oxidation pathway at the level of the first enzyme AOX appears to be essential to keep PPAR␣ in check and prevent spontaneous peroxisome proliferation by effectively metabolizing AOX substrates, which appear to function as natural PPAR␣ ligands (15). Disruption of the gene encoding L-PBE, the second enzyme of this classical system, did not manifest either in spontaneous peroxisome proliferation or in the transcriptional activation of PPAR␣ target genes in liver, indicating that inactivation of classical ␤-oxidation system distal to AOX step does not interfere with the inactivation of endogenous ligands of PPAR␣ (15,16). The absence of appreciable defects in lipid metabolism in L-PBE Ϫ/Ϫ mice suggested that enoyl-CoAs, generated in the classical ␤-oxidation system, are diverted to the noninducible branched chain ␤-oxidation pathway for degradation by D-PBE (16). In this study, to investigate the functional implications of the disruption of the metabolism of enoyl-CoAs on lipid metabolism, peroxisome proliferation, and PPAR␣ activation in liver, we generated mice deficient in both L-PBE and D-PBE enzymes so that both peroxisomal ␤-oxidation pathways are effectively disrupted at the second enzyme level. Mice deficient in both L-PBE and D-PBE enzymes (L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ ) exhibited severe growth retardation and postnatal mortality. They revealed microvesicular hepatic steatosis, abnormalities in mitochondrial structure, and overexpression of PPAR␣-regulated genes in liver, despite the lack of morphological evidence of peroxisome proliferation. These findings establish that the induction of many of the PPAR␣ target genes can occur independently of peroxisome proliferation, further extending previous results (16), and that per-oxisome proliferation in rodent liver is highly correlatable with the induction mostly of the L-PBE gene.
Morphological Analysis, Catalase Cytochemistry, and AOX and L-PBE Immunohistochemistry-Wild-type (C57BL/6J), L-PBE Ϫ/Ϫ , and D-PBE Ϫ/Ϫ mice, 6 -8 weeks of age, were fed powdered diet with or without Wy-14,643 (0.0125% w/w), a peroxisome proliferator (5), for 2 weeks. L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ mice were with the dams that were maintained on a normal diet without Wy-14,643. AOX Ϫ/Ϫ mice were also on a normal diet without the drug. For light microscopy, tissues were fixed in 10% neutral buffered formalin and embedded in paraffin. Histological analysis was performed using 4-m-thick sections stained with hematoxylin and eosin. For cytochemical localization of catalase (CTL), the peroxisomal marker enzyme, tissues were incubated in alkaline 3,3-diaminobenzidine reaction mixture and processed for light and electron microscopy as described (15,17). Immunohistochemical localization of AOX and L-PBE proteins in liver fixed in 10% formalin or 70% ethanol was performed using monospecific polyclonal antibodies as described (16,17).
Northern Analysis-Total RNA extracted from liver with TRIzol reagent (Invitrogen) was glyoxylated, separated on 0.8% agarose gel, transferred to nylon membrane, and then hybridized at 42°C in 50% formamide hybridization solution using 32 P-labeled cDNA probes as described (17,19). Changes in mRNA levels were estimated by densitometric scanning of autoradiograms.
SDS-PAGE Analysis and Immunoblotting-Liver homogenates were separated by SDS-PAGE and stained with Coomassie Brilliant R Blue for assessing the induction of 78-kDa protein, which represents L-PBE, and was initially identified as peroxisome proliferation-associated polypeptide (20). Contents of ␤-oxidation enzymes AOX, L-PBE, D-PBE, PTL, and sterol carrier protein (SCPx) and of peroxisomal CTL and urate oxidase (UOX) in liver were determined by immunoblot analysis as described (16,21,22).
Fatty Acid Analysis-Serum was stored at Ϫ20°C until analysis. Sample preparation and analysis were according to the procedures described (23). (16) and D-PBE Ϫ/Ϫ (18) mice were crossbred to generate mice nullizygous for both L-PBE and D-PBE (L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ ) (Fig. 1A). Genotype analysis performed on litters during the late gestational age (embryonic day 17.5) or on postnatal day 1 revealed the expected Mendelian ratios from L-PBE Ϫ/Ϫ D-PBE ϩ/Ϫ animals, suggesting no intrauterine mortality (data not shown). L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ DKO litter revealed variable but striking growth retardation at birth as compared with L-PBE Ϫ/Ϫ D-PBE ϩ/Ϫ and L-PBE Ϫ/Ϫ D-PBE ϩ/ϩ littermates. Mice deficient in both L-PBE and D-PBE that died during the first 3 postnatal days were grossly undersized, underweight, and hypotonic with difficulties in suckling as evidenced by the reduced quantity of milk in their stomach. Some of the DKO mice that were stronger appeared to suckle adequately but showed marked retardation in weight gain and growth rate (Fig. 1B). Almost all of the L-PBE and D-PBE double null mice died before weaning, and a rare animal (Ͻ10% of double nulls) that survived up to 5 weeks was still with the dam suckling and did not consume solid diet. As described previously, mating of L-PBE ϩ/Ϫ mice yielded L-PBE Ϫ/Ϫ mice with the expected Mendelian inheritance ratio of 25%; likewise, like sequence; PDK-4, pyruvate dehydrogenase kinase protein; C3f, gene-rich cluster C3f; Ly-6D, lymphocyte antigen 6 complex locus D; mLipase, monoglyceride lipase. mating of D-PBE ϩ/Ϫ mice also generated D-PBE Ϫ/Ϫ mice at the expected ratio of ϳ25%, and these single nulls were indistinguishable at birth from their heterozygous and wild-type littermates (16,18). No detectable phenotype was noted in L-PBE Ϫ/Ϫ mice, which were viable and fertile (16), whereas, 30% of the D-PBE Ϫ/Ϫ mice died before postnatal day 12, and most of the remaining mice survived to adulthood (18). Thus, the failure of L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ mice to survive beyond 5 weeks of age is considered a reflection of the consequences of the total disruption of two ␤-oxidation pathways to metabolize the enoyl-CoAs generated by the first step of the peroxisomal ␤-oxidation cycle catalyzed by acyl-CoA oxidases (1). Increases in C 26 :0 (3.5-fold) and phytanic acid (4-fold) levels were noted in the serum of L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ mice. A 7-fold increase in C26:0/C22:0 was noted in L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ mice, as compared with wild-type mice. There was also a significant decrease in docosahexaenoic acid level (3.5-fold) in the serum of these double knockout mice. These observations indicate that in DKO mice, peroxisomal ␤-oxidation is abnormal.

Growth Retardation and Postnatal Mortality in L-PBE
Characterization of Liver Phenotype in L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ Mice That Survived beyond the Age of 3 Weeks-The histological architecture of liver and the morphologic appearance of hepatocytes in mice deficient in either L-PBE or D-PBE did not differ significantly from each other or from that of wild-type animals during the first 5 weeks of their postnatal development (Fig. 2, A-C). In contrast, the liver of mice deficient in both L-PBE and D-PBE revealed a mild degree of microvesicular fatty change between 3 and 5 weeks of age (Fig. 2D). The hepatic steatosis observed in L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ mice was considerably less pronounced as compared with that noted in AOX Ϫ/Ϫ mice (15,17). Survey for alterations in peroxisome population in liver cells of L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ mice using 0.5m-thick liver sections that were processed using the alkaline 3,3-diaminobenzidine cytochemical method to visualize peroxisomal marker enzyme catalase (15) revealed similar peroxisome density and numerical distribution in mice deficient in either L-PBE (Fig. 2F) or D-PBE (Fig. 2G) as compared with wild type (Fig. 2E). In contrast, L-PBE and D-PBE double knockout mouse livers did not reveal the presence of discrete brown dots in the hepatocyte cytoplasm that represent peroxi-somes (Fig. 2H). The hepatocyte cytoplasm and nuclei exhibited a diffuse brown reaction, suggesting the presence of soluble catalase in these locations instead of in the expected peroxisomal compartment.
At the ultrastructural level, peroxisomes in hepatocytes of wild-type mouse livers appeared as discrete single membrane limited particles with dense osmiophilic material in sections that were processed for the localization of catalase (Fig. 3A). Similar distribution of peroxisomes was also noted in L-PBE Ϫ/Ϫ or D-PBE Ϫ/Ϫ mouse livers (not illustrated). In contrast, the liver cells of mice deficient in both L-PBE and D-PBE revealed a marked reduction in peroxisome population, and when present, they were seen as microperoxisomes (Fig. 3B). Liver cells in DKO mice also revealed severe abnormalities in the appearance of mitochondrial structure (Fig. 3B). The mitochondrial abnormalities include condensations with curvilinear alterations of cristae, myelin-like rings, evaginations and invaginations of the outer membrane, and an occasional mitochondrial ghost. Microvesicular steatosis is also seen in the cytoplasm. Defects in mitochondrial structure have also been described in Zellweger syndrome patients and in mouse models of defective peroxisomal biogenesis (24).
Up-regulation of PPAR␣ Target Gene Expression in the Absence of Peroxisome Proliferation-Previously, we demonstrated that mice deficient in AOX, the first and rate-limiting enzyme of the inducible classical peroxisomal ␤-oxidation system, exhibit spontaneous peroxisome proliferation and transcriptional activation of PPAR␣ target genes in liver (15). These observations indicated that AOX substrates that remain unmetabolized by the branched-chain oxidase function as natural ligands of PPAR␣, leading to sustained activation of this receptor (15,25). Because no such changes occurred in the livers of mice deficient in L-PBE, the second enzyme of this inducible ␤-oxidation system, it appeared that D-PBE, the second enzyme of the noninducible peroxisomal ␤-oxidation pathway, would metabolize the substrates of L-PBE (16). The disruption of both L-PBE and D-PBE genes in the present study provided an opportunity to examine whether the induction of PPAR␣ target genes occurs in liver when the two ␤-oxidation pathways are interrupted at the level of the second enzyme, although there was no morphological evidence of spontaneous peroxisome proliferation (Fig. 2H). Northern analysis of liver RNA obtained from 4-week-old animals for changes in the levels of PPAR␣-regulated fatty acid oxidation system genes, namely AOX, L-PBE, PTL, CYP4A1, and CYP4A3, revealed marked increases in the mRNA levels of AOX, PTL, CYP4A1, and CYP4A3 in L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ mice with the expected absence of L-PBE and D-PBE mRNAs due to the gene disruption (Fig. 4A). We noted marked increase in the mRNA content of Riken clone W09719 in earlier cDNA microarrays, which was identified as a PPAR␣-regulated gene in liver (19,26). Recently, this Riken clone W09719 has been found identical to SCP2/SCPx (27,28), encoding for the second thiolase enzyme (1)(2)(3)(4). Increase in hepatic SCP2/SCPx mRNA level indicates (Fig. 4B) that this gene, like AOX, L-PBE, and PTL, is also regulated by PPAR␣. Modest increases in liver fatty acyl-CoA synthetase and fatty acid synthetase mRNA levels were noted in L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ and AOX Ϫ/Ϫ mice (Fig. 4A). These results strongly suggest spontaneous up-regulation of PPAR␣ target gene expression in liver in double knockout mice, similar to that seen in AOX Ϫ/Ϫ livers (Fig. 4A). It is of interest to note that modest increases in the mRNA levels of ACTE, PEX11␣, and PMP70 also occurred in the L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ and AOX Ϫ/Ϫ mouse livers. As expected, no perceptible alterations in UOX and CTL mRNA levels were noted (Fig. 4A). No significant changes in PPAR␣ and PPAR␥ mRNA levels were detected among various groups except that PPAR␣ levels appeared slightly increased in AOX Ϫ/Ϫ livers (Fig. 4A).
These observations clearly establish that induction of PPAR␣ target genes occurs in liver in the absence of peroxisome proliferation in L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ double null mice. The mRNA  lanes 7 and 8), and AOX Ϫ/Ϫ (lanes 9 and 10) mice (two mice for each group). Twenty g of total RNA was electrophoresed on a 0.8% agarose gel, blotted onto a nylon membrane, and probed with different random-primed 32  expression profiles are similar to that seen in livers with peroxisome proliferation occurring spontaneously as in AOX Ϫ/Ϫ mouse liver or as a result of induction in wild-type mice fed Wy-14,643 (Fig. 4B). Immunoblot analysis confirmed the absence of L-PBE and D-PBE proteins in L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ mouse liver and increases in the content of 72-kDa subunit A (but decrease or absence of 51-kDa subunit) of AOX protein (8,29,30) and in the amount of PTL (Fig. 5). Also of interest is the marked decrease in the amount of the 40-kDa subunit of SCPx (8) in L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ mouse liver (Fig. 5), although the SCP2/SCPx mRNA level is markedly elevated (Fig. 4B). Decreases in the amount of AOX subunit B (51 kDa) and of SCPx 40-kDa subunit strongly suggest that these proteins are not properly targeted and packaged in peroxisomes for posttranslational modification (8,29,30). Immunoblotting for UOX in liver samples revealed very little of this protein in DKO livers (Fig. 5) despite the presence of UOX mRNA (Fig. 4A). The relative lack of UOX protein in DKO livers is consistent with the paucity of peroxisomes and failure to see distinct peroxisomal UOX cores in these hepatocytes. These observations suggest possible defects in protein import into peroxisomes (31).

Induction of Peroxisome Proliferation Depends upon the Induction of L-PBE Gene, but Peroxisome Proliferation Is Not
Required for the Induction of PPAR␣ Target Genes-No peroxisome proliferation was observed in the hepatic parenchymal cells of L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ mice, whereas Northern analysis revealed the activation of PPAR␣-regulated genes similar to that noted in AOX Ϫ/Ϫ mouse liver and in wild-type mice fed Wy-14,643 (Fig. 4). To further establish the critical role of transcriptional up-regulation of L-PBE in the induction of the phenomenon of peroxisome proliferation, we analyzed the livers of wild type, L-PBE Ϫ/Ϫ , and D-PBE Ϫ/Ϫ with and without Wy-14,643-treatment to assess the amount of 78-kDa L-PBE protein (Fig. 6, A and B), which we originally identified as peroxisome proliferation-associated peptide (20). SDS-PAGE and Coomassie Blue staining (Fig. 6A) and immunoblotting (Fig. 6B) revealed a massive amount of L-PBE protein in the liver of Wy-14,643-treated wild-type and D-PBE Ϫ/Ϫ mice, which correlates well with peroxisome proliferation in liver cells. No appreciable increase in L-PBE protein was seen in the liver of L-PBE Ϫ/Ϫ mice fed Wy-14,643, as noted previously, and in these livers, the peroxisome proliferation was markedly subdued (16). Comparison of SDS-PAGE and immunoblot patterns revealed increases in L-PBE protein in AOX Ϫ/Ϫ liver, but no such increase occurred in L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ double knockout livers (Fig. 6), although both L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ and AOX Ϫ/Ϫ mouse livers exhibited induction of PPAR␣ target genes on Northern analysis (Fig. 5). Immunohistochemical staining of liver sections using antibodies against L-PBE (Fig. 7, A-D) revealed induction of L-PBE (Fig. 7, B and C) in wild-type mice treated with Wy-14,643 and in AOX Ϫ/Ϫ (Fig. 7C) mice. The L-PBE immunostaining revealed distinct cytoplasmic granular pattern consistent with peroxisome proliferation. As expected, L-PBE staining was absent in L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ mouse liver (Fig. 7D). Of interest is that AOX immunostaining (Fig. 7, E-H) showed increased staining of liver cells of wild-type mice fed Wy-14,643 (Fig. 7F) and in L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ mouse liver (Fig. 7H). The staining in Wy-14,6643-treated wild-type mouse liver is granular (Fig. 7F, inset), whereas in DKO mouse liver, the AOX staining was somewhat diffuse surrounding the lipid droplets (Fig. 7H, inset). These observations clearly establish that the induction of many of the PPAR␣ target genes can occur independently of peroxisome proliferation and that peroxisome proliferation in rodent liver is highly correlatable with the induction mostly of the L-PBE gene and to D-PBE gene to a lesser extent (Fig. 6). DISCUSSION Peroxisomes are single-membrane bound organelles that are present in all eukaryotic cells. These organelles contain many enzyme systems responsible for a wide variety of metabolic functions, including the ␤-oxidation of very long-chain fatty acids (1,2,4). Abnormalities of peroxisomal fatty acid ␤-oxidation and absence or reduction in the number of peroxisomes in cells play a significant role in the pathophysiological manifestations of Zellweger syndrome and other peroxisome biogenesis disorders (1,2,24,32,33). Also important is that the induction in liver of enzymes involved in the classical ␤-oxidation system and profound increases in peroxisome population in liver cells caused by structurally diverse peroxisome proliferators has been associated with the development of liver cancer in rats and mice, and these effects are regulated by the nuclear receptor PPAR␣ (1,6,7,10,14,34). Thus, increases and decreases in fatty acid oxidation and peroxisome population appear to be detrimental to health (1).
Evidence from gene knockout mouse models of peroxisomal proteins points to the role, in particular, of peroxisomal ␤-oxidation system in preventing the accumulation of toxic peroxisomal ␤-oxidation substrates that appear to function as PPAR␣ ligands. This scenario is vividly exhibited in the liver of AOX Ϫ/Ϫ mouse in that absence of AOX, the first and ratelimiting enzyme of the classical ␤-oxidation pathway, leads to sustained PPAR␣ activation with induction of PPAR␣-regulated target genes, spontaneous peroxisome proliferation, and the eventual development of hepatocellular carcinomas, implying that unmetabolized substrates of AOX function as PPAR␣ ligands (15,25). The mouse model of disruption of the L-PBE gene further supported the assumption that AOX substrates function as PPAR␣ ligands because L-PBE Ϫ/Ϫ mouse livers revealed no such increases in peroxisome number and no upregulation of PPAR␣ target genes in liver (16). However, there is uncertainty in that straight-chain enoyl-CoAs generated by AOX may crossover to be metabolically degraded by D-PBE, the second enzyme of the noninducible peroxisomal ␤-oxidation pathway (16). Although D-PBE Ϫ/Ϫ mouse corroborated this hypothesis to a certain extent (18), it appeared necessary to examine the role of disruption of the metabolism of enoyl-CoAs in the activation of PPAR␣ in liver by generating L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ DKO mice because these mice have a complete block in peroxisomal ␤-oxidation at the level of L-and D-bifunctional enzymes as these genes are disrupted, resulting in the absence of these mRNAs and proteins in their livers (Figs. 4,5). Mouse embryonic skin fibroblasts obtained from L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ mice showed defects in the degradation of lignoceric acid and 2-metyhlhexadecanoic acid, indicating that in these DKO mice, peroxisomal degradation is completely blocked (35). Although in the present study we did not directly assess the peroxisomal and mitochondrial ␤-oxidation activities in the livers of DKO mice, the accumulation of very long-chain fatty acids and the data on embryonic fibroblasts clearly establish the complete block of peroxisomal ␤-oxidation in these animals. Furthermore, in the mouse and human genome, only two bifunctional ␤-oxidation genes (L-and D-PBE) have been identified, and the disruption of both these genes in our DKO mice ensures blockage of peroxisomal fatty acid oxidation at the level of the second enzyme. The present study demonstrates that absence of both bifunctional enzymes in L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ mice leads to intrauterine and postnatal growth retardation with death occurring predominantly during early postnatal phase. Mice that survive beyond the first week of life develop severe hypotonia, fail to feed properly, become growth-retarded, and do not survive weaning. Thus, although knockout of either the L-PBE gene or the D-PBE gene is compatible with life, the disruption of both these genes in the same animal leads to lethal outcome by weaning age. Although these double knockout mice do not exhibit any anatomical defects in neuronal migration, the functional inactivity of nervous system caused by unmetabolized toxic substrates of ␤-oxidation cannot be excluded (35). Evidence clearly suggests that the interruption at the second step of the fatty acid oxidation catalyzed by the inducible and noninducible pathways leads to alterations in hepatic mitochondrial structure, reminiscent of changes occurring in Zellweger patients and some mouse models for disturbances in peroxisome biogenesis (24,36). In aggregate, microvesicular steatosis, paucity of peroxisomes and mitochondrial abnormalities encountered in L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ mouse liver mimic changes described in Zellweger patients, and this is most likely related to the combined bifunctional enzyme deficiency and total interruption of fatty acid oxidation at the enoyl-CoA level.
Of considerable interest is that this study presents evidence for the up-regulation of PPAR␣ target genes in liver in the absence of the morphological phenomenon of peroxisome proliferation. In L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ mouse liver, increases in the hepatic AOX, PTL, CYP4A1, CYP4A3, and ACTE mRNA levels ( Fig. 4A) were evident, and these are known PPAR␣ target genes (3,6,34,37,38). Increases in PEX11␣ and PMP70 mRNA levels were also observed. PEX11␣ has been shown to be dispensable for PPAR␣-mediated peroxisome proliferation, although overexpression of this peroxin is known to enhance peroxisome formation (32,33). Increases in PEX11␣ mRNA in the absence of peroxisome proliferation in L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ mouse liver may appear paradoxical, but it provides an indication of PPAR␣ activation. Overexpression of other putative PPAR␣-regulated genes such as macrophage scavenger receptor CD36 (37,39), Ly-6D (19,40), pyruvate dehydrogenase kinase protein (PDK4) (41), SCP2/SCPx (19,27,28,42), SHLR (uncharacterized endogenous virus-like sequence) (43), mLipase (44), and others (19) in the liver of these double nulls is further indication of PPAR␣ activation by natural ligands that are unmetabolized substrates of peroxisomal ␤-oxidation. Although fatty acids are generally considered to be PPAR␣ ligands (45,46), we entertain the possibility that very longchain acyl-CoAs (Ͼ20 C) or acyl-carnitines in AOX Ϫ/Ϫ mice and very-long-chain enoyl-CoAs in L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ mice can act as PPAR␣ ligands, and these possibilities should be properly evaluated. Since acyl-CoAs enter peroxisomes, they can also exit peroxisomes by diffusion or transported out as acyl-carnitines. Although the possibility that acyl-CoAs can be reconverted or hydrolyzed to free fatty acids by peroxisomal thioesterases (ACTE) (38) and that enoyl-CoAs can be used by 2-enoyl-CoA reductatse and be converted to acyl-CoAs (47) exists, this is considered futile because it wastes NADPH. It is possible that in these double null mice, under limiting peroxisomal NADPH concentration, there will be increases in the concentration of cellular enoyl-CoAs, which may diffuse out of peroxisomes to act as PPAR␣ ligands. Evidence also suggests that phytanic acid activates PPAR␣ in SCPx-deficient mice (48), and consistent with this is the increased levels of phytanic acid in the serum of L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ mice. These knockout models of peroxisomal ␤-oxidation raise the question that different endogenous ligands generated during very long-chain fatty acid oxidation can serve as PPAR␣ ligands. Additional studies are needed to analyze the amounts and composition of acyl-CoAs, acyl-carnitines, and enoyl-CoAs in AOX Ϫ/Ϫ and L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ mice. These studies also indicate that PPAR␣ target gene overexpression can occur in the absence of peroxisome proliferation and that these two events can be dissociable. Studies with a variety of structurally diverse synthetic peroxisome proliferators have unequivocally established a strong correlation between hepatic peroxisome proliferation, PPAR␣ target gene overexpression, and the development of liver cancer in rats and mice (5,6,7,14,49). Sustained activation of PPAR␣ and spontaneous peroxisome proliferation in the liver of AOX Ϫ/Ϫ mouse also lead to the development of liver tumors after 12 months of age (15). Although PPAR␣ activation was seen in L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ mouse livers, these mice did not survive long enough to develop liver tumors. It is argued that the carcinogenic risk to humans of chronic exposure to these PPAR␣ ligands is negligible to nonexistent because peroxisome proliferation is not profound in human hepatic cells studied mostly in cell culture (50 -53). The present study establishes unequivocally that absence of peroxisome proliferation does not necessarily imply the total negation of the global PPAR␣ target gene-activating effects of synthetic or endogenous peroxisome proliferators (endogenous/biological ligands of this receptor). Our data strongly suggest that in the mouse, the morphological parameter of peroxisome proliferation is a reflection mostly of the L-PBE gene transcriptional activity (13,54), and to a lesser extent of D-PBE induction (16,55). In the rodent, the peroxisome proliferator-response element of L-PBE gene binds one or two PPAR-retinoid X receptor heterodimers, providing the peroxisome poliferator signaling pathway with two levels of response (13,54). The paucity of peroxisomes in liver cells of L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ mice is reminiscent of that noted in AOX Ϫ/Ϫ hepatocytes with microvesicular steatosis (17). The excess accumulation of potentially toxic very long-chain fatty acid metabolites may interfere with import of proteins into the peroxisome matrix for organelle formation (17,31). The morphological abnormalities in liver of in L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ liver cells and marked reduction in the processing of AOX and of SCPx proteins (Fig. 5) are reminiscent of changes described in certain Zellweger syndrome patients (29,30,56). The near absence of UOX protein in DKO livers also suggests that peroxisomal proteins are not appropriately targeted to the peroxisome (17,31). Immunohistochemical staining for AOX shows increased staining in DKO livers, but unlike in wild-type mice fed Wy-14,643, the staining is not granular (peroxisomal) but somewhat diffuse in the cytoplasm. Because of the disruption of branched chain fatty acid oxidation system in the L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ mice, it is likely that these animals may have abnormalities in bile acid metabolism (57) in addition to defects in the metabolism of very long-chain fatty acids.