HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hashimoto, T. Articles by Reddy, J. K. Search for Related Content PubMed PubMed Citation Articles by Hashimoto, T. Articles by Reddy, J. K. Social Bookmarking                      What's this?

J Biol Chem, Vol. 274, Issue 27, 19228-19236, July 2, 1999

Peroxisomal and Mitochondrial Fatty Acid -Oxidation in Mice Nullizygous for Both Peroxisome Proliferator-activated Receptor  and Peroxisomal Fatty Acyl-CoA Oxidase
GENOTYPE CORRELATION WITH FATTY LIVER PHENOTYPE^*

Takashi Hashimoto, Tomoyuki Fujita, Nobuteru Usuda, William Cook, Chao Qi, Jeffrey M. Peters^§, Frank J. Gonzalez^§, Anjana V. Yeldandi, M. Sambasiva Rao, and Janardan K. Reddy^¶

From the  Department of Pathology, Northwestern University Medical School, Chicago, Illinois 60611-3008 and ^§ Laboratory of Metabolism, NCI, National Institutes of Health, Bethesda, Maryland 20892

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Fatty acid -oxidation occurs in both mitochondria and peroxisomes. Long chain fatty acids are also metabolized by the cytochrome P450 CYP4A -oxidation enzymes to toxic dicarboxylic acids (DCAs) that serve as substrates for peroxisomal -oxidation. Synthetic peroxisome proliferators interact with peroxisome proliferator activated receptor  (PPAR) to transcriptionally activate genes that participate in peroxisomal, microsomal, and mitochondrial fatty acid oxidation. Mice lacking PPAR (PPAR^/) fail to respond to the inductive effects of peroxisome proliferators, whereas those lacking fatty acyl-CoA oxidase (AOX^/), the first enzyme of the peroxisomal -oxidation system, exhibit extensive microvesicular steatohepatitis, leading to hepatocellular regeneration and massive peroxisome proliferation, implying sustained activation of PPAR by natural ligands. We now report that mice nullizygous for both PPAR and AOX (PPAR^/ AOX^/) failed to exhibit spontaneous peroxisome proliferation and induction of PPAR-regulated genes by biological ligands unmetabolized in the absence of AOX. In AOX^/ mice, the hyperactivity of PPAR enhances the severity of steatosis by inducing CYP4A family proteins that generate DCAs and since they are not metabolized in the absence of peroxisomal -oxidation, they damage mitochondria leading to steatosis. Blunting of microvesicular steatosis, which is restricted to few liver cells in periportal regions in PPAR^/ AOX^/ mice, suggests a role for PPAR-induced genes, especially members of CYP4A family, in determining the severity of steatosis in livers with defective peroxisomal -oxidation. In age-matched PPAR^/ mice, a decrease in constitutive mitochondrial -oxidation with intact constitutive peroxisomal -oxidation system contributes to large droplet fatty change that is restricted to centrilobular hepatocytes. These data define a critical role for both PPAR and AOX in hepatic lipid metabolism and in the pathogenesis of specific fatty liver phenotype.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In animal cells, mitochondria as well as peroxisomes oxidize fatty acids via -oxidation, with long chain and very long chain fatty acids (LCFAs and VLCFAs)¹ being preferentially oxidized by peroxisomes (1-3). Peroxisomal -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 (3, 4). In the classical L-3-hydroxy-specific -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)) (3). 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 -oxidation spiral (1, 2). In the second D-3-hydroxy-specific -oxidation pathway, dehydrogenation of acyl-CoA esters to their corresponding trans-2-enoyl-CoAs is catalyzed by the branched chain acyl-CoA oxidase (2). 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 via D-3-hydroxyacyl-CoAs (3). The third enzyme of this second system is designated as sterol carrier protein x (SCPx), which possesses 3-ketoacyl-CoA thiolase activity (5). The first step in mitochondrial -oxidation is the - 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) (2, 3). The second, third, and fourth steps in the mitochondrial -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) (3). The MH, HADH, and MTL activities lie within mitochondrial trifunctional protein (TFP) (3).

Of these two peroxisomal -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 (6). These synthetic peroxisome proliferators exert their pleiotropic effects in liver by activating a nuclear receptor called peroxisome proliferator-activated receptor  (PPAR) (7). The induction of peroxisome proliferation is associated with transcriptional activation of genes encoding for the peroxisomal -oxidation system and cytochrome P450 CYP 4A isoforms, CYP4A1 and CYP4A3, among others (8-11). For this to occur, PPAR 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 (12). The generation of PPAR^/ 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 (13). PPAR^/ mice display normal complement of peroxisomes in liver cells, but these mice remain nonresponsive to the inductive influence of synthetic peroxisome proliferators (13). A mild degree of centrilobular fatty change develops in these mice that is attributed to reduction in the constitutive levels of mitochondrial fatty acid -oxidation, because the constitutive or basal oxidation of VLCFAs by peroxisomal -oxidation system appears unaffected by PPAR deficiency (14). We investigated the functional implications of disrupting the basal metabolism of VLCFAs by generating mice deficient in AOX, the first enzyme of inducible peroxisomal -oxidation system, and found that AOX^/ mice develop severe microvesicular steatohepatitis and spontaneous peroxisome proliferation in liver cells (15, 16). 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 PPAR ligands if they remain unmetabolized and in the development of microvesicular hepatic steatosis (16). The morphological and biochemical changes observed in AOX^/ mice suggested a sustained hyperfunction of PPAR because of biological ligands (16). Although PPAR is essential for the induction of pleiotropic responses by synthetic peroxisome proliferators as evidenced from studies in PPAR^/ mice (13), it is uncertain if the spontaneous peroxisome proliferation induced by biological mediators in AOX-deficient mice is effected by PPAR or by some other mechanism.

In the present study, we investigated the potential for in vivo compensatory functions by generating mice deficient in both PPAR and AOX. We report that these double nullizygous mice (PPAR^/ AOX^/) do not show spontaneous hepatic peroxisome proliferation, implying that PPAR deficiency is not compensated by other transcription factors. Also pertinent is that the microvesicular steatosis was markedly diminished in PPAR^/ AOX^/ mice in comparison to severe steatosis observed in AOX^/ mice, suggesting that the presence of PPAR exaggerates steatosis developing in the absence of peroxisomal -oxidation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Generation of PPAR^/ AOX^/ Mice-- The generation of PPAR nulls and AOX nulls has been described elsewhere (13, 15). Because of reduced fertility of homozygous AOX^/ males and females, homozygous PPAR^/ males and heterozygous AOX^+/ females were mated to produce F1 progeny heterozygous for both genes. Sibs were intercrossed to produce progeny null for both PPAR and AOX (PPAR^/ 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 PPAR and AOX genes (PPAR^/ AOX^/) were fertile. All animal procedures used in this study were approved by the Institutional Review Board for Animal Research of the Northwestern University.

Treatment with Peroxisome Proliferators and Morphological Studies-- Wild type (C57BL/6J), AOX^/ (15), PPAR^/ (13), and PPAR^/ 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 (17).

Western Blot Analysis and Quantitation of Protein-- Protein concentrations were determined by a protein assay kit (Bio-Rad) using bovine serum albumin as standard. Contents of -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 (14, 18). Antibody against the amino-terminal part of SCPx expressed in Escherichia coli was a gift from Professor Yukio Fujiki.

Northern Hybridization-- 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 (16). Changes in mRNA levels were estimated by densitometric scanning of autoradiograms.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Generation of PPAR^/ AOX^/ Mice-- PPAR^/ mice (13) and AOX^+/ mice (15) were cross-bred to generate mice nullizygous for both PPAR and AOX (PPAR^/ AOX^/) used in this study. Southern analysis of tail tip DNA from double heterozygous (PPAR^+/ AOX^+/) mice revealed two bands (8.0 and 13.4 kb) with AOX probe and two bands (6.3 and 7.5 kb) with PPAR probe (Fig. 1). The genomic DNA from double nullizygous (PPAR^/ AOX^/) mice yielded a single 13.4-kb band with AOX probe and a single 7.5-kb band with PPAR 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 PPAR probe. The livers of mice nullizygous for both PPAR and AOX did not show the presence of AOX mRNA and protein when analyzed, respectively, by northern and Western blotting. Both male and female PPAR^/ AOX^/ mice were fertile and displayed no apparent gross phenotypic changes. These double nullizygous mice were grossly indistinguishable from PPAR^/ mice and from wild type mice, whereas growth retardation was common in pre-weaning and young AOX^/ mice (15). 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.

View larger version (40K): [in this window] [in a new window]   Fig. 1.   Southern blot analysis of genomic DNA. Genomic DNA (5 µg) isolated from tail tips was digested with HincII and probed with PPAR probe (panel A) (13) or digested with SstI and probed with AOX probe (panel B) (15). Mice nullizygous for both AOX and PPAR are indicated as / and show a single 7.5-kb band with PPAR probe (A) and a single 13.4-kb band with AOX (B). Heterozygous for both PPAR and AOX are indicated as +/. Wild type are shown as +/+.

Characterization of Liver Phenotype in PPAR^/ AOX^/ Mice and Comparison with Mice Deficient in AOX or PPAR-- The absolute liver weight of PPAR^/ AOX^/ mice and of PPAR^/ 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^/, PPAR^/, 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^/, PPAR^/ AOX^/, and PPAR^/ mice treated with a peroxisome proliferator (data not shown). The lobular architecture of PPAR^/ 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, and D). These cells stained positively for fat when stained with Oil Red O or with Sudan black (Fig. 2E). Few clusters of inflammatory aggregates, reminiscent of lipogranulomatous lesions found in AOX^/ mouse liver (16), were also found among liver cells with microvesicular fatty change in PPAR^/ AOX^/ mouse liver (Fig. 2D). 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 PPAR^/ (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 PPAR^/ 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 and H).

View larger version (129K): [in this window] [in a new window]   Fig. 2.   Liver morphology in mice nullizygous for both PPAR and AOX and comparison with mice deficient in PPAR or AOX. Low magnification survey of liver of PPAR/ AOX/ mouse and PPAR mouse (panels A and B, respectively). In PPAR/ AOX/ mouse liver, microvesicular steatosis is seen in few liver cells scattered in periportal (P) and not in centrilobular (C) areas (panels A, C, and D), and these cells stain positively for fat with Sudan black (panel E). Arrow heads in panels D and E point to cells with microvesicular steatosis in periportal areas and also present are few lipogranulomas (arrows in panel D). In PPAR-deficient mouse, fatty change is in centrilobular hepatocytes (C) and not in periportal (P) regions (panels B and F). Panels G and H represent low and high magnification view of liver of AOX-deficient mice. Note the presence of extensive microvesicular steatosis (L) and the emergence of regenerating fat free hepatocytes in periportal (P) region.

PPAR^/ mice revealed a normal complement of peroxisome profiles in liver parenchymal cells (Fig. 3A), and the number of these organelles was not increased when these mice were treated with a peroxisome proliferator (13). 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 PPAR because of biological ligands of PPAR that require AOX for inactivation or metabolism (16). We surveyed for alterations in peroxisome population in livers of PPAR^/ AOX^/ mice to ascertain if natural/biological ligands that are not metabolized in the absence of AOX induce peroxisome proliferation in the absence of PPAR in these animals, possibly by activating a different transcription factor such as PPAR. No spontaneous peroxisome proliferation was discerned in liver parenchymal cells of PPAR^/ 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. 3D); similar paucity of peroxisomes was also noted in hepatocytes with microvesicular steatosis in AOX^/ mice (15). Liver cells of mice deficient in both PPAR and AOX also failed to respond to synthetic peroxisome proliferators in comparison to wild type mice (Fig. 3F)). These observations clearly indicate that PPAR is the principal transcription factor responsible for the phenomenon of peroxisome proliferation induced by both natural/biological ligands, as well as synthetic peroxisome proliferators.

View larger version (186K): [in this window] [in a new window]   Fig. 3.   Peroxisomes in the livers of PPAR/, AOX/, and PPAR/ AOX/ mice. In sections of liver, processed for the cytochemical localization of peroxisomal catalase (17), peroxisomes appear as black dots in these black and white photographs of 0.5-µm thick sections visualized by light microscope. PPAR/ (A) and PPAR/ AOX/ (D and E) mouse livers show few peroxisomes in hepatocytes. Hepatocytes with microvesicular steatosis in periportal regions (see Fig. 2, C and D) of PPAR/ AOX/ mouse liver show few or no identifiable peroxisomes (panel E). PPAR/ AOX/ mice treated with a peroxisome proliferator showed no significant increase in peroxisome population (panel F). Panels B and C represent sections of liver of AOX-deficient mouse. In these livers, cells with fatty change (L) show few or no peroxisomes (panel B), and regenerated hepatocytes (arrows in panels B and C) (such as those shown in Fig. 2H) reveal extensive spontaneous peroxisome proliferation (see numerous black dots).

Fatty Acid-activating Enzymes-- VLCAS exhibits specificity toward very long chain fatty acids, whereas PCS catalyzes the activation of long chain fatty acids to the CoA esters (19). The content of hepatic VLCAS was not much different among AOX^/, PPAR^/, and PPAR^/ AOX^/ mice (Table I). The amount of PCS, an enzyme with tripartite (microsomal, mitochondrial, and peroxisomal) distribution (20), 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 PPAR^/ mice and also in PPAR^/ AOX^/ mice, suggesting that the increase may represent A and B forms of this enzyme and not the PPAR-regulated C-form of PCS (21).

View this table: [in this window] [in a new window]   Table I Quantitation of hepatic fatty acid activating enzymes and peroxisomal proteins 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 PPAR and AOX; ND, not detected); GOX, glycolate oxidase.

Peroxisomal Proteins-- Table I and Fig. 4 depict the relative amounts of peroxisomal proteins in wild type, PPAR^/ AOX^/, PPAR^/, 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 (22). 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 PPAR^/ mice and in PPAR^/ 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 PPAR. As expected, a marked increase in the amounts of AOX, L-PBE and PTL, three enzymes of the inducible classical peroxisomal -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 PPAR^/ 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 PPAR^/ AOX^/ and PPAR^/ mice were lower as compared with wild type mice, and no significant increase occurred following ciprofibrate treatment.

View larger version (48K): [in this window] [in a new window]   Fig. 4.   Western blot analysis of selected proteins involved in peroxisomal fatty acid metabolism. Liver homogenates from wild type (wild), PPAR/ AOX/ (DKO), PPAR/, and AOX/ mice fed either a control () or ciprofibrate (cipro) (+)-containing diet were subjected to SDS/polyacrylamide gel electrophoresis and immunoblotting (three mice for each group). 1 and 2, COT (10 µg); 3, AOX (polypeptides A and B are shown) (20 µg); 4, AOX 2 µg (wild), 20 µg (DKO, PPAR/, AOX/); 5, L-PBE 20 µg (wild, DKO, PPAR/) and 5 µg (AOX/); 6, L-PBE 2 µg (wild), 20 µg (DKO, PPAR/), and 5 µg (AOX/); 7, PTL 20 µg (wild, DKO, PPAR/) and 5 µg (AOX/); 8, PTL 2 µg (wild), 20 µg DKO, PPAR/, and 5 µg (AOX/); 9 and 10, D-PBE (20 µg); 11 and 12, SCPx (20 µg); 13 and 14, CTL (5 µg). Lanes 1-3 are mice 1-3.

In wild type mice, hepatic PTL content increased after the administration of ciprofibrate, and this increase was similar to that seen in AOX^/ mice on control diet (Table I). Two isozymes of PTL (constitutive and inducible) are present in rats, but on SDS-polyacrylamide gel electrophoresis they appear as one band. The products of two PTL genes are synthesized as larger precursors; the amino-terminal pre-piece is then cleaved upon translocation of the enzyme into peroxisomes (23, 24). Two PTL bands were seen in AOX^/ mouse liver but not in any other groups (Fig. 4). The main band appears to be the mature subunit, and the slower moving band is assumed to be immature PTL (Fig. 4). When the antibody was absorbed with purified PTL, both bands disappeared, indicating that these two bands specifically represent PTL and that in AOX^/ liver cells the proteolytic processing of PTL is not complete, similar to that reported in patients with rhizomelic chondrodysplasia punctata (25-27). Whether this processing is defective in cells with microvesicular steatosis or this defect is present in all hepatocytes in AOX^/ liver remains unclear.

D-PBE and SCPx are two enzymes participating in the second D-3-hydroxy-specific -oxidation pathway (3, 4). These enzymes catalyze the conversion of straight chain as well as 2-methyl-branched chain fatty acids and bile acid precursors (3, 5). A marginal increase in D-PBE occurred in wild type mice treated with ciprofibrate but not in PPAR^/ AOX^/ and PPAR^/ mice (Fig. 4). SCPx content was essentially similar among all groups (Table I and Fig. 4).

We assessed the changes in the levels of three peroxisomal matrix proteins (CTL, UOX, and glycolate oxidase) (28, 29) and three peroxisomal membrane proteins (PMP 70, PMP 26, and PMP 22) (30) in wild type, PPAR^/, AOX^/, and PPAR^/ AOX^/ mice (Table I). CTL levels increased marginally (<2-fold) in wild type mice fed ciprofibrate and in mice with AOX gene disruption. No change in the content of UOX and glycolate oxidase occurred in these animals. PMP 70 level increased ~4-fold in wild type mice treated with ciprofibrate and in AOX null mice on control diet, possibly reflecting spontaneous peroxisome proliferation (Table I).

Mitochondrial Enzymes-- Carnitine palmitoyltransferase I catalyzes the transfer of a long chain fatty acyl group from coenzyme A to carnitine. Carnitine palmitoyltransferase II, an enzyme located in the inner mitochondrial membrane, then converts fatty acylcarnitines into CoA esters, which then undergo -oxidation (31, 32). The expression of carnitine palmitoyltransferase II increased ~3-fold in the livers of AOX^/ mice on control diet and in ciprofibrate-treated wild type mice Table II; Fig. 5). Carnitine palmitoyltransferase II level did not change in PPAR^/ AOX^/ and in PPAR^/ mice treated with ciprofibrate.

View larger version (54K): [in this window] [in a new window]   Fig. 5.   Western blot analysis of selected proteins involved in mitochondrial fatty acid metabolism. Liver homogenates from wild type (wild), PPAR/ AOX/ (DKO), PPAR/, and AOX/ mice fed either control () or ciprofibrate (cipro) (+)-containing diet were subjected to SDS/polyacrylamide gel electrophoresis and immunoblotting (three mice for each group). 1, PCS (10 µg); 2, CPTII (20 µg); 3, CAT (30 µg); 4, VLCAD (20 µg); 5, LCAD (40 µg); 6, MCAD (20 µg); 7, SCAD (20 µg); 8, HADH (20 µg); 9, MTL1 (1.5 µg); 10, MTL2 (20 µg);11, TFP- (20 µg); 12, TFP- (20 µg).

Immunoblot analysis for CAT revealed three signal bands (Fig. 5), and these were confirmed as CAT by competing out the antibodies with pigeon breast muscle CAT (data not shown). CAT content in the livers of AOX^/ mice was ~10-fold higher than that found in wild type mice, and this increase was comparable with that seen in ciprofibrate-treated wild type mice. No increase in CAT content occurred in the livers of PPAR^/ AOX^/ and PPAR^/ mice fed ciprofibrate (Table II).

Four acyl-CoA dehydrogenases, namely VLCAD, LCAD, MCAD, and SCAD, participate in mitochondrial fatty acid -oxidation (33, 34). VLCAD content in wild type mice increased only slightly with ciprofibrate treatment, and its content was also higher in AOX^/ mice when compared with wild type mice (Table II and Fig. 5). Increases in the quantities of the remaining three classical acyl-CoA dehydrogenases, LCAD, MCAD, and SCAD, also occurred in wild type mice treated with ciprofibrate (Table II, Fig. 5). Levels of these three enzymes in AOX^/ mice were higher than that in wild type mice, and ciprofibrate did not cause additional increases in AOX^/ mice (Table II, Fig. 5). In PPAR^/ and double nullizygous mice, the levels of these proteins were comparable with that present in wild type mice. No increases in the quantities of these enzymes occurred in these animals following ciprofibrate administration.

The hepatic levels of electron transfer flavoprotein and MH remained essentially the same in all groups. The content of HADH was higher in ciprofibrate-treated wild type mice and in mice with AOX deficiency. The quantity of - and -subunits of TFP (35, 36) increased in AOX^/ mouse liver (Table II and Fig. 5), and these levels were comparable with that observed in ciprofibrate-treated wild type mice. PPAR^/ and PPAR^/ AOX^/ mice treated with ciprofibrate failed to show any increase in liver TFP content. No change in MTL1 level was detected among different groups. The content of MTL2, which participates in ketone metabolism, increased in wild type mice treated with ciprofibrate. The level of this enzyme was higher in AOX^/ mice, but the amount did not change following ciprofibrate administration.

Induction of mRNAs-- We have used Northern blotting to assess the levels of AOX, L-PBE, PTL, CYP4A1, CYP4A3, fatty acid-binding protein, CTL, and UOX mRNAs in the livers of PPAR^/ AOX^/, PPAR^/, and AOX^/ mice and compared with wild type mice (Fig. 6). AOX mRNA was not detected in AOX^/ and PPAR^/ AOX^/ mice. The mRNA levels of L-PBE, PTL, CYP4A1, and CYP4A3 were markedly elevated in AOX^/ maintained on normal diet and in wild type mice fed ciprofibrate (Fig. 6). There was no increase in the levels of these mRNAs in PPAR^/ AOX^/ (Fig. 6) and PPAR^/ (data not shown) mice fed ciprofibrate. A slight decrease in fatty acid-binding protein mRNA occurred in PPAR^/ and in double nullizygous mice. No appreciable change in UOX and CTL mRNA levels was discerned among different groups.

View larger version (97K): [in this window] [in a new window]   Fig. 6.   Northern blot analysis of total RNA extracted from the liver of wild type (wild), PPAR/, AOX/, and PPAR/ AOX/ (DKO) mice. Lanes 1 and 2 represent wild type mice on control diet, and lanes 3 and 4 represent wild type mice on ciprofibrate (cipro)-containing diet. Lanes 5 and 6 represent PPAR/ mice on control diet, and lanes 7 and 8 represent AOX/ mice also on control diet. Lanes 9 and 10 represent mice nullizygous for both PPAR and AOX on control diet. Lanes 11 and 12 represent PPAR/ AOX/ (DKO) mice on ciprofibrate-containing diet. Twenty µg of total RNA was electrophoresed on a 0.8% agarose gel, blotted onto a nylon membrane, and probed with different random-primed 32P-labeled cDNA probes as shown. The 28 S RNA is for loading control. FABP, fatty acid-binding protein.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In animals, fatty acids stored as triacylglycerols and formed from de novo synthesis constitute the main fuel sources. These are metabolized at the intracellular level mostly via the mitochondrial and peroxisomal -oxidation enzyme systems (2, 37, 38). The substrate spectra of these two fatty acid oxidation systems partly overlap, with oxidation of the major portion of medium and long chain fatty acids occurring in mitochondria and that of the VLCFAs taking place preferentially in peroxisomes (2, 3). LCFAs and VLCFAs are also metabolized by the microsomal CYP4A1 and CYP4A3 fatty acid -oxidases, resulting in the formation of DCAs that are further degraded by peroxisomal -oxidation system (39, 40). LCFAs constitute the bulk of fatty acids in animals, and their abundance makes them the only significant source of metabolic fuel for mitochondrial -oxidation system (2). Under normal physiologic conditions, mitochondrial -oxidation is the dominant metabolic pathway, whereas the extramitochondrial fatty acid oxidation occurring within peroxisomes and endoplasmic reticulum assumes a minor role (2, 3). Accordingly, decreased mitochondrial fatty acid -oxidation has been considered one of the major mechanisms underlying the disturbances in lipid metabolism in liver leading to steatosis (41-43). Oxidation of fatty acids by mitochondria depends not only upon the -oxidation enzymes and substrate concentration but is also influenced by other factors such as changes in carnitine content and oxidative phosphorylation (41). The extramitochondrial oxidation (peroxisomal -oxidation and microsomal -oxidation) of LCFAs and VLCFAs, on the other hand, is not constrained by confounding events and remains a reflection of enzyme-substrate availability (2, 3). In view of some overlap of substrate spectra (especially LCFAs) between mitochondrial and extramitochondrial oxidation pathways (2, 3), delineation of cross-talk between these metabolic systems in the pathogenesis of disturbances of lipid metabolism and fatty liver phenotypes becomes important. Here we have described the generation and initial characterization of a mouse nullizygous for both PPAR and peroxisomal AOX. Comparison of changes in the levels of several proteins involved in mitochondrial and extramitochondrial fatty acid oxidation and profiles of morphologic alterations in livers of mice deficient in AOX^/, PPAR^/, and PPAR^/ AOX^/ in this study provided new insights regarding the role of VLCFA metabolism by peroxisomal -oxidation system vis à vis AOX, the role of PPAR in the acuity of steatosis developing in mice deficient in AOX possibly by inducing CYP4A family enzymes, and the dominant role of constitutive mitochondrial oxidation in the blunting of hepatic steatosis in mice deficient in both PPAR and AOX.

The striking findings in mice nullizygous for both PPAR and AOX were the absence of spontaneous peroxisome proliferation, the marked attenuation of the extent of microvesicular steatosis in liver, and the lack of liver cell proliferation, in contradistinction to that observed in AOX-deficient genotype. As described previously, mice deficient in AOX develop extensive microvesicular steatosis in liver, which is followed by hepatocellular regeneration and profound spontaneous peroxisome proliferation presumably because of sustained activation of PPAR by natural ligands that require AOX for metabolism (16). In these mice, several genes encoding for peroxisomal, mitochondrial, and microsomal proteins such as L-PBE, PTL, MCAD CYP4A1, and CYP4A3 were up-regulated in liver, suggestive of spontaneous sustained PPAR hyperactivity analogous to that seen in the livers of wild type mice exposed to synthetic peroxisome proliferators (16). We proposed that LCFAs and VLCFAs or their acyl-CoAs and other putative substrates for AOX serve as natural ligands for PPAR and that AOX is indispensable for the physiological regulation of PPAR by keeping the natural ligands in check (16). Mice deficient in L-PBE (the second enzyme of this classical peroxisomal -oxidation spiral) do not show hepatic steatosis and spontaneous peroxisome proliferation, further confirming that disruption of -oxidation pathway distal to AOX does not affect the metabolism of natural ligands of PPAR (18). In PPAR^/AOX^/ mice, we did not observe spontaneous peroxisome proliferation and found no evidence of overexpression of L-PBE, PTL, CYP4A1, CYP4A3, and other genes unlike that found in the livers of mice deficient in AOX (16). These observations establish unequivocally that PPAR is responsible for spontaneous peroxisome proliferation induced by natural ligands whose metabolism depends on intact AOX activity (16, 18). Thus, in double nulls, even though natural ligands are expected to be abundant, the absence of PPAR in these mice abrogates spontaneous peroxisome proliferation, indicating that PPAR is not redundant. The loss of this receptor is not compensated by the other PPAR isotypes, namely PPAR and PPAR, or by any other factor(s). Accordingly, natural ligands that require AOX for inactivation as well as synthetic peroxisome proliferators exert their pleiotropic effects by activating PPAR. This assumption is further supported by the finding that administration of synthetic peroxisome proliferators to AOX^/ mice does not result in an additive induction of proteins in liver, suggesting that natural ligands essentially saturate PPAR in AOX^/ livers. If natural ligands were acting on a different transcription factor, such as PPAR, to induce spontaneous peroxisome proliferation in AOX^/ mice, then administration of a peroxisome proliferator that activates PPAR would result in additional increases in the gene activity.

Young AOX^/ mice develop extensive microvesicular steatosis affecting the entire liver lobule, but little is known about the molecular mechanisms of hepatic steatosis and its disappearance with the emergence of regenerated hepatocytes in these mutant mice (15, 16). The results obtained from PPAR^/ AOX^/ mice, in which fatty liver is remarkably blunted with few scattered cells in periportal regions, strongly suggest that the absence of peroxisomal -oxidation of fatty acids imposes a major toxic burden on hepatocytes provided the PPAR function is intact. LCFAs and VLCFAs and their CoA esters or some other AOX substrate not metabolized because of AOX deficiency may be particularly toxic to hepatocyte plasma or mitochondrial membranes, leading to injury and dysfunction (44). The functional PPAR leads to the transcriptional activation of CYP4A1 and CYP4A3 genes in AOX^/ mouse liver by natural PPAR ligands, such as VLCFAs or their acyl-CoA esters that remain unmetabolized in the absence of AOX (16). The increased levels of CYP4A1 and CYP4A3 in AOX^/ mouse liver will then metabolize LCFAs into DCAs that are toxic to mitochondria, as they can uncouple oxidative phosphorylation (45). The absence of PPAR in PPAR^/ AOX^/ mice most likely limits the generation of toxic DCAs by this extramitochondrial fatty acid oxidation pathway and minimizes mitochondrial damage, thereby reducing steatosis. Basal levels of DCAs produced in these double nulls cannot be further degraded because of AOX deficiency and possibly account for the presence of steatotic cells scattered in periportal regions (Fig. 2, C-E). This limited microvesicular steatosis in double nullizygous mice is most likely because of constitutive or basal expression of CYP4A1 and CYP4A3 and lack of inducibility of these enzymes in the absence of PPAR. An intriguing possibility is that in AOX^/ mice, unmetabolized PPAR ligands also activate PPAR to induce hepatocellular proliferation and that dividing cells may be more susceptible to steatosis in AOX deficiency. However, once the cell division is complete, regenerated hepatocytes generally exhibit resistance to toxic insults, and this reasoning is consistent with the absence of steatosis in older AOX^/ mice (16) and also in chronic alcoholic liver disease (46). Absence of hepatocellular regeneration in PPAR^/ AOX^/ mice because of deficiency of PPAR can contribute to the blunting of microvesicular steatosis and the appearance of periportal fatty phenotype in these animals. In mice deficient in PPAR, hepatic steatosis was mild with large droplet fatty change involving few hepatocytes in the centrilobular regions (Fig. 2). We propose that in PPAR^/ genotype, constitutive levels of expression of extramitochondrial fatty acid oxidation systems (peroxisomal -oxidation and microsomal -oxidation) are able to metabolize LCFAs and VLCFAs and thus prevent or minimize their toxic manifestations. The mild degree of fatty change is attributed to slight but sustained reduction in constitutive mitochondrial -oxidation in PPAR null genotype (14). As these PPAR^/ mice get older, they become obese, and centrilobular steatosis becomes somewhat more pronounced (47).

The studies with these genetically altered mice underscore the importance of PPAR and peroxisomal -oxidation, in particular the critical role of AOX in hepatic lipid metabolism (Fig. 7). As proposed in Fig. 7, PPAR deficiency alone can down-regulate mitochondrial -oxidation, but the overall short term consequences appear insignificant as long as the constitutive extramitochondrial fatty acid oxidation is maintained. On the other hand, chronic PPAR deficiency vis à vis sustained reduction in basal mitochondrial -oxidation leads to obesity and a mild to moderate degree of centrilobular fatty change in liver (47). In contrast, inhibition of peroxisomal -oxidation at the level of AOX in animals with intact PPAR exerts severe abnormalities of lipid metabolism similar to that observed in children with Reye's syndrome (45). If PPAR is also deficient along with AOX, there is a marked attenuation of this disturbance in lipid metabolism as evidenced in mice nullizygous for both PPAR and AOX. We attribute this to the ability of PPAR to up-regulate CYP4A family genes and the resultant generation of toxic DCAs in AOX^/ mice and not in mice deficient in both PPAR and AOX. Accordingly, it would be important to assess the relative levels of PPAR and of peroxisomal -oxidation in human liver and their role in lipid homeostasis (48, 49). Alterations in PPAR and AOX can affect lipid homeostasis, and changes in the extramitochondrial lipid metabolism may be as important, if not more important than the mitochondrial oxidation in the pathogenesis of fatty liver.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Schematic representation of the role of mitochondrial and extramitochondrial fatty acid oxidation and PPAR in the pathogenesis of fatty liver. PPAR/, AOX/, and PPAR/ AOX/ represent the mouse genotypes. In PPAR/ mice, constitutive mitochondrial -oxidation is reduced and leads to slow progression of large droplet centrilobular steatosis in liver. The constitutive extramitochondrial fatty acid metabolism (peroxisomal and CYP4A metabolism of LCFAs and VLCFAs) is not adversely unaffected in the absence of PPAR. In AOX/ mice, the sustained hyperactivity of PPAR because of ligands that are not metabolized because of AOX deficiency results in overexpression of CYP4A enzymes, and the LCFAs and VLCFAs not metabolized by peroxisomal -oxidation because of lack of AOX are converted to DCAs by CYP4A -oxidation. Extensive microvesicular steatosis is considered a reflection DCA induced inhibition of mitochondrial -oxidation. In mice nullizygous for both PPAR and AOX, the absence of PPAR and lack of CYP4A induction resulted in the blunting of microvesicular steatosis.

	ACKNOWLEDGEMENTS

We thank Sujata Pulikuri, Yuzhi Jia, and V. Subbarao for excellent technical assistance.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant GM 23750 (to J. K. R.), Veterans Affairs Merit Review grants (to A. V. Y. and M. S. R.), and the Joseph L. Mayberry, Sr. Endowment Fund.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Pathology, Northwestern University Medical School, 303 East Chicago Ave., Chicago, IL 60611-3008. Tel.: 312-503-8144; Fax: 312-503-8249; E-mail: jkreddy{at}nwu.edu.

	ABBREVIATIONS

The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; LCFA, long chain fatty acid; VLCFA, very long chain fatty acids; DCA, dicarboxylic fatty acids; AOX, straight chain fatty acyl-CoA oxidase; L-PBE, enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase bifunctional protein; D-PBE, D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein; PTL, peroxisomal 3-ketoacyl-CoA thiolase; SCPx, sterol carrier protein x or 3-ketoacyl-CoA thiolase/sterol carrier protein 2; COT, carnitine octanoyltransferase; CAT, carnitine acetyltransferase; PMP, peroxisomal membrane protein; CTL, catalase; UOX, urate oxidase; PCS, palmitoyl-CoA synthetase; VLCAS, very long chain acyl-CoA synthetase; LCAD, long chain acyl-CoA dehydrogenase; VLCAD, very chain acyl-CoA dehydrogenase; MCAD, medium chain acyl-CoA dehydrogenase; MH, mitochondrial enoyl-CoA hydratase; HADH, 3-hydroxyacyl-CoA dehydrogenase; TFP, mitochondrial trifunctional protein; MTL1, mitochondrial 3-ketoacyl-CoA thiolase; MTL2, mitochondrial acetoacetyl-CoA-specific thiolase; CYP4A1 and CYP4A3, encode microsomal cytochrome P450 fatty acid -hydroxylases; kb, kilobase(s); DKO, double knock-out mice nullizygous for both PPAR and AOX.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

1. Lazarow, P. B., and de Duve, C. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 2043-2046[Abstract/Free Full Text]
2. Reddy, J. K., and Mannaerts, G. P. (1994) Annu. Rev. Nutr. 14, 343-370[CrossRef][Medline] [Order article via Infotrieve]
3. Hashimoto, T. (1999) Neurochem. Res. 24, 551-563[CrossRef][Medline] [Order article via Infotrieve]
4. Baumgart, E., Vanhooren, J. C., Fransen, M., Marynen, P., Puype, M., Vanderkerckhove, J., Leunissen, J. A., Fahimi, H. D., Mannaerts, G. P., and van Veldhoven, P. P. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13748-13753[Abstract/Free Full Text]
5. Seedorf, U., Brysch, P., Engel, T., Schrage, K., and Assmann, G. (1994) J. Biol. Chem. 269, 21277-21283[Abstract/Free Full Text]
6. Reddy, J. K., and Krishnakantha, T. P. (1975) Science 190, 787-789[Abstract/Free Full Text]
7. Issemann, I., and Green, S. (1990) Nature 347, 645-650[CrossRef][Medline] [Order article via Infotrieve]
8. Reddy, 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., and Rao, M. S. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 1747-1751[Abstract/Free Full Text]
9. Johnson, E. F., Palmer, C. N. A., Griffin, K. J., and Hsu, M.-H. (1996) FASEB J. 10, 1241-1248[Abstract]
10. Reddy, J. K., and Chu, R. (1996) Ann. N. Y. Acad. Sci. 804, 176-201[Medline] [Order article via Infotrieve]
11. Lemberger, T., Desvergne, B., and Wahli, W. (1996) Annu. Rev. Cell Dev. Biol. 12, 335-363[CrossRef][Medline] [Order article via Infotrieve]
12. Kliewer, S. A., Umesono, K., Noonan, D. J., Heyman, R. A., and Evans, R. M. (1992) Nature 358, 771-774[CrossRef][Medline] [Order article via Infotrieve]
13. Lee, S. S.-T., Pineau, T., Drago, J., Lee, E. J., Owens, J. W., Kroetz, D. L., Fernandez-Salguero, P. M., Westphal, H., and Gonzalez, F. J. (1995) Mol. Cell. Biol. 15, 3012-3022[Abstract]
14. Aoyama, T., Peters, J. M., Iritani, N., Nakajima, T., Furihata, K., Hashimoto, T., and Gonzalez, F. J. (1998) J. Biol. Chem. 273, 5678-5684[Abstract/Free Full Text]
15. Fan, C.-Y., Pan, J., Chu, R., Lee, D., Kluckman, K. D., Usuda, N., Singh, I., Yeldandi, A. V., Rao, M. S., Maeda, N., and Reddy, J. K. (1996) J. Biol. Chem. 271, 24698-24710[Abstract/Free Full Text]
16. Fan, C.-Y., Pan, J., Usuda, N., Yeldandi, A. V., Rao, M. S., and Reddy, J. K. (1998) J. Biol. Chem. 273, 15639-15645[Abstract/Free Full Text]
17. Reddy, J. K., and Lalwani, N. D. (1985) in Handbook of Carcinogen Testing (Milman, H. A. , and Weisburger, E. K., eds) , pp. 482-500, Noyes Publications, NJ
18. Qi, C., Zhu, Y., Pan, J., Yeldandi, V., Rao, M. S., Maeda, N., Subbarao, V., Pulikuri, S., Hashimoto, T., and Reddy, J. K. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 1585-1590[Abstract/Free Full Text]
19. Uchiyama, A., Aoyama, T., Kamijo, K., Uchida, Y., Kondo, N., Orii, T., and Hashimoto, T. (1996) J. Biol. Chem. 271, 30360-30365[Abstract/Free Full Text]
20. Miyazawa, S., Hashimoto, T., and Yokota, S. (1985) J. Biochem. (Tokyo) 98, 723-733[Abstract/Free Full Text]
21. Suzuki, H., Watanabe, M., Fujino, T., and Yamamoto, T. (1995) J. Biol. Chem. 270, 9676-9682[Abstract/Free Full Text]
22. Ozasa, H., Miyazawa, S., and Osumi, T. (1983) J. Biochem. (Tokyo) 94, 543-549[Abstract/Free Full Text]
23. Osumi, T., Tsukamoto, S., Hata, S., Yokota, S., Miura, S., Fujiki, Y., Hijikata, M., Miyazawa, S., and Hashimoto, T. (1991) Biochem. Biophys. Res. Commun. 181, 947-954[CrossRef][Medline] [Order article via Infotrieve]
24. Swinkels, B. W., Gould, S. J., Bodnar, A. G., Rachubinski, R. A., and Subramani, S. (1991) EMBO J. 10, 3255-3262[Medline] [Order article via Infotrieve]
25. Braverman, N., Steel, G., Obie, C., Moser, A., Moser, H., Gould, S. J., and Valle, D. (1997) Nat. Genet. 15, 369-376[CrossRef][Medline] [Order article via Infotrieve]
26. Purdue, P. E., Zhang, J. W., Skoneczy, M., and Lazarow, P. B. (1997) Nat. Genet. 15, 381-384[CrossRef][Medline] [Order article via Infotrieve]
27. Authier, F., Bergeron, J. J., Ou, W. J., Rachubinski, R. A., Posner, B. I., and Walton, P. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3859-3863[Abstract/Free Full Text]
28. Usuda, N., Reddy, M. K., Hashimoto, T., Rao, M. S., and Reddy, J. K. (1988) Lab. Invest. 58, 100-111[Medline] [Order article via Infotrieve]
29. Yokota, S., Ichikawa, K., and Hashimoto, T. (1985) Histochemistry 82, 25-32[CrossRef][Medline] [Order article via Infotrieve]
30. Hashimoto, T., Kuwabara, T., Usuda, N., and Nagata, T. (1986) J. Biochem. (Tokyo) 100, 301-310[Abstract/Free Full Text]
31. Esser, V., Brown, N. F., Cowan, A. T., Foster, D. W., and McGarry, J. D. (1996) J. Biol. Chem. 271, 6972-6977[Abstract/Free Full Text]
32. Brandt, J. M., Djouadi, A., and Kelly, D. P. (1998) J. Biol. Chem. 273, 23786-23792[Abstract/Free Full Text]
33. Furuta, S., Miyazawa, S., and Hashimoto, T. (1981) J. Biochem. (Tokyo) 90, 1739-1750[Abstract/Free Full Text]
34. Izai, K., Uchida, Y., Orii, T., Yamamoto, S., and Hashimoto, T. (1992) J. Biol. Chem. 267, 1027-1033[Abstract/Free Full Text]
35. Uchida, Y., Izai, K., Orii, T., and Hashimoto, T. (1992) J. Biol. Chem. 267, 1034-1041[Abstract/Free Full Text]
36. Kamijo, T., Aoyama, T., Miyazaki, S., and Hashimoto, T. (1993) J. Biol. Chem. 268, 26452-26460[Abstract/Free Full Text]
37. Mannaerts, G. P., and Debeer, L. J. (1982) Ann. N. Y. Acad. Sci. 386, 30-39[Medline] [Order article via Infotrieve]
38. Kurtz, D. M., Rinaldo, P., Rhead, W. J., Tian, L., Millington, D. S., Vockley, J., Hamm, D. A., Brux, A. E., Lindsey, J. R., Pinkert, C. A., O'Brien, W. E., and Wood, P. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 15592-15597[Abstract/Free Full Text]
39. Kimura, S., Hardwick, J. P., Kozak, C. A., and Gonzalez, F. J. (1989) DNA 8, 517-525[Medline] [Order article via Infotrieve]
40. Muerhoff, A. S., Griffin, K. J., and Johnson, E. F. (1992) J. Biol. Chem. 267, 19051-19053[Abstract/Free Full Text]
41. Ockner, R. K., Kaikaus, R., and Bass, N. M. (1993) Hepatology 18, 669-676[CrossRef][Medline] [Order article via Infotrieve]
42. Bacon, B. R., Farahvash, M. J., Janney, C. G., and Neuschwander-Tetri, B. A. (1994) Gastroenterology 107, 1103-1109[Medline] [Order article via Infotrieve]
43. Formenty, B., and Pessayre, D. (1995) Pharmacol. Ther. 67, 101-154[CrossRef][Medline] [Order article via Infotrieve]
44. Ho, J. K., Moser, H., Kishimoto, Y., and Hamilton, M. A. (1995) J. Clin. Invest. 96, 1455-1463
45. Tonsgard, J. H., and Getz, G. S. (1985) J. Clin. Invest. 76, 816-825
46. Sherlock, S. (1995) Lancet 345, 227-229[CrossRef][Medline] [Order article via Infotrieve]
47. Costet, P., Legendre, C., More, J., Edger, A., Galtier, P., and Pineau, T. (1998) J. Biol. Chem. 273, 29577-29585[Abstract/Free Full Text]
48. Palmer, C. N. A., Hsu, M-H., Griffin, K. J., Raucy, J. L., and Johnson, E. F. (1998) Mol. Pharmacol. 53, 14-22[Abstract/Free Full Text]
49. Unger, R. H., Zhou, Y.-T., and Orci, L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 2327-2332[Abstract/Free Full Text]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				Z. El Kebbaj, P. Andreoletti, D. Mountassif, M. Kabine, H. Schohn, M. Dauca, N. Latruffe, M. S. El Kebbaj, and M. Cherkaoui-Malki Differential Regulation of Peroxisome Proliferator-Activated Receptor (PPAR)-{alpha}1 and Truncated PPAR{alpha}2 as an Adaptive Response to Fasting in the Control of Hepatic Peroxisomal Fatty Acid {beta}-Oxidation in the Hibernating Mammal Endocrinology, March 1, 2009; 150(3): 1192 - 1201. [Abstract] [Full Text] [PDF]

				N. Anderson and J. Borlak Molecular Mechanisms and Therapeutic Targets in Steatosis and Steatohepatitis Pharmacol. Rev., September 1, 2008; 60(3): 311 - 357. [Abstract] [Full Text] [PDF]

				A. Brolinson, S. Fourcade, A. Jakobsson, A. Pujol, and A. Jacobsson Steroid Hormones Control Circadian Elovl3 Expression in Mouse Liver Endocrinology, June 1, 2008; 149(6): 3158 - 3166. [Abstract] [Full Text] [PDF]

				Y. Tanaka, L. M. Aleksunes, R. L. Yeager, M. A. Gyamfi, N. Esterly, G. L. Guo, and C. D. Klaassen NF-E2-Related Factor 2 Inhibits Lipid Accumulation and Oxidative Stress in Mice Fed a High-Fat Diet J. Pharmacol. Exp. Ther., May 1, 2008; 325(2): 655 - 664. [Abstract] [Full Text] [PDF]

				A. R. Sheets, P. Fulop, Z. Derdak, A. Kassai, E. Sabo, N. M. Mark, G. Paragh, J. R. Wands, and G. Baffy Uncoupling protein-2 modulates the lipid metabolic response to fasting in mice Am J Physiol Gastrointest Liver Physiol, April 1, 2008; 294(4): G1017 - G1024. [Abstract] [Full Text] [PDF]

				A. Gatica, M. C. Aguilera, D. Contador, G. Loyola, C. O. Pinto, L. Amigo, J. E. Tichauer, S. Zanlungo, and M. Bronfman P450 CYP2C epoxygenase and CYP4A {omega}-hydroxylase mediate ciprofibrate-induced PPAR{alpha}-dependent peroxisomal proliferation J. Lipid Res., April 1, 2007; 48(4): 924 - 934. [Abstract] [Full Text] [PDF]

				S. Neschen, K. Morino, J. Dong, Y. Wang-Fischer, G. W. Cline, A. J. Romanelli, J. C. Rossbacher, I. K. Moore, W. Regittnig, D. S. Munoz, et al. n-3 Fatty Acids Preserve Insulin Sensitivity In Vivo in a Peroxisome Proliferator-Activated Receptor-{alpha}-Dependent Manner Diabetes, April 1, 2007; 56(4): 1034 - 1041. [Abstract] [Full Text] [PDF]

				S. Huyghe, H. Schmalbruch, K. De Gendt, G. Verhoeven, F. Guillou, P. P. Van Veldhoven, and M. Baes Peroxisomal Multifunctional Protein 2 Is Essential for Lipid Homeostasis in Sertoli Cells and Male Fertility in Mice Endocrinology, May 1, 2006; 147(5): 2228 - 2236. [Abstract] [Full Text] [PDF]

				S. Neschen, K. Morino, J. C. Rossbacher, R. L. Pongratz, G. W. Cline, S. Sono, M. Gillum, and G. I. Shulman Fish Oil Regulates Adiponectin Secretion by a Peroxisome Proliferator-Activated Receptor-{gamma}-Dependent Mechanism in Mice. Diabetes, April 1, 2006; 55(4): 924 - 928. [Abstract] [Full Text] [PDF]

				M. J. Santos, R. A. Quintanilla, A. Toro, R. Grandy, M. C. Dinamarca, J. A. Godoy, and N. C. Inestrosa Peroxisomal Proliferation Protects from {beta}-Amyloid Neurodegeneration J. Biol. Chem., December 9, 2005; 280(49): 41057 - 41068. [Abstract] [Full Text] [PDF]

				M. B. Sandberg, M. Bloksgaard, D. Duran-Sandoval, C. Duval, B. Staels, and S. Mandrup The Gene Encoding Acyl-CoA-binding Protein Is Subject to Metabolic Regulation by Both Sterol Regulatory Element-binding Protein and Peroxisome Proliferator-activated Receptor {alpha} in Hepatocytes J. Biol. Chem., February 18, 2005; 280(7): 5258 - 5266. [Abstract] [Full Text] [PDF]

				J. Zhang, R. M. Lewis, C. Wang, N. Hales, and C. D. Byrne Maternal dietary iron restriction modulates hepatic lipid metabolism in the fetuses Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2005; 288(1): R104 - R111. [Abstract] [Full Text] [PDF]

				F. M. Rausa III, D. E. Hughes, and R. H. Costa Stability of the Hepatocyte Nuclear Factor 6 Transcription Factor Requires Acetylation by the CREB-binding Protein Coactivator J. Biol. Chem., October 8, 2004; 279(41): 43070 - 43076. [Abstract] [Full Text] [PDF]

				R. Chu, H. Lim, L. Brumfield, H. Liu, C. Herring, P. Ulintz, J. K. Reddy, and M. Davison Protein Profiling of Mouse Livers with Peroxisome Proliferator-Activated Receptor {alpha} Activation Mol. Cell. Biol., July 15, 2004; 24(14): 6288 - 6297. [Abstract] [Full Text] [PDF]

				J. K. Reddy Peroxisome Proliferators and Peroxisome Proliferator-Activated Receptor {alpha}: Biotic and Xenobiotic Sensing Am. J. Pathol., June 1, 2004; 164(6): 2305 - 2321. [Full Text] [PDF]

				Y. Jia, C. Qi, Z. Zhang, T. Hashimoto, M. S. Rao, S. Huyghe, Y. Suzuki, P. P. Van Veldhoven, M. Baes, and J. K. Reddy Overexpression of Peroxisome Proliferator-activated Receptor-{alpha} (PPAR{alpha})-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 {beta}-Oxidation System J. Biol. Chem., November 21, 2003; 278(47): 47232 - 47239. [Abstract] [Full Text] [PDF]

				S. Yu, K. Matsusue, P. Kashireddy, W.-Q. Cao, V. Yeldandi, A. V. Yeldandi, M. S. Rao, F. J. Gonzalez, and J. K. Reddy Adipocyte-specific Gene Expression and Adipogenic Steatosis in the Mouse Liver Due to Peroxisome Proliferator-activated Receptor gamma 1 (PPARgamma 1) Overexpression J. Biol. Chem., January 3, 2003; 278(1): 498 - 505. [Abstract] [Full Text] [PDF]

				L. A. Cowart, S. Wei, M.-H. Hsu, E. F. Johnson, M. U. Krishna, J. R. Falck, and J. H. Capdevila The CYP4A Isoforms Hydroxylate Epoxyeicosatrienoic Acids to Form High Affinity Peroxisome Proliferator-activated Receptor Ligands J. Biol. Chem., September 13, 2002; 277(38): 35105 - 35112. [Abstract] [Full Text] [PDF]

				H. Huang, O. Starodub, A. McIntosh, A. B. Kier, and F. Schroeder Liver Fatty Acid-binding Protein Targets Fatty Acids to the Nucleus. REAL TIME CONFOCAL AND MULTIPHOTON FLUORESCENCE IMAGING IN LIVING CELLS J. Biol. Chem., August 2, 2002; 277(32): 29139 - 29151. [Abstract] [Full Text] [PDF]

				J. K. Reddy Nonalcoholic Steatosis and Steatohepatitis: III. Peroxisomal beta -oxidation, PPARalpha , and steatohepatitis Am J Physiol Gastrointest Liver Physiol, December 1, 2001; 281(6): G1333 - G1339. [Abstract] [Full Text] [PDF]

				S. Yu, W.-Q. Cao, P. Kashireddy, K. Meyer, Y. Jia, D. E. Hughes, Y. Tan, J. Feng, A. V. Yeldandi, M. S. Rao, et al. Human Peroxisome Proliferator-activated Receptor alpha (PPARalpha ) Supports the Induction of Peroxisome Proliferation in PPARalpha -deficient Mouse Liver J. Biol. Chem., November 2, 2001; 276(45): 42485 - 42491. [Abstract] [Full Text] [PDF]

				G. Robertson, I. Leclercq, and G. C. Farrell Nonalcoholic Steatosis and Steatohepatitis: II. Cytochrome P-450 enzymes and oxidative stress Am J Physiol Gastrointest Liver Physiol, November 1, 2001; 281(5): G1135 - G1139. [Abstract] [Full Text] [PDF]

				L. Carlsson, D. Linden, M. Jalouli, and J. Oscarsson Effects of fatty acids and growth hormone on liver fatty acid binding protein and PPAR{alpha} in rat liver Am J Physiol Endocrinol Metab, October 1, 2001; 281(4): E772 - E781. [Abstract] [Full Text] [PDF]

				E. Baumgart, I. Vanhorebeek, M. Grabenbauer, M. Borgers, P. E. Declercq, H. D. Fahimi, and M. Baes Mitochondrial Alterations Caused by Defective Peroxisomal Biogenesis in a Mouse Model for Zellweger Syndrome (PEX5 Knockout Mouse) Am. J. Pathol., October 1, 2001; 159(4): 1477 - 1494. [Abstract] [Full Text]

				C. Wolfrum, C. M. Borrmann, T. Börchers, and F. Spener Fatty acids and hypolipidemic drugs regulate peroxisome proliferator-activated receptors alpha - and gamma -mediated gene expression via liver fatty acid binding protein: A signaling path to the nucleus PNAS, February 15, 2001; (2001) 51619898. [Abstract] [Full Text]

				C. S. Elangbam, R. D. Tyler, and R. M. Lightfoot Peroxisome Proliferator-activated Receptors in Atherosclerosis and Inflammation--An Update Toxicol Pathol, February 1, 2001; 29(2): 224 - 231. [Abstract] [PDF]

				W. Wang, W. Li, Y. Ikeda, J.-I. Miyagawa, M. Taniguchi, E. Miyoshi, Y. Sheng, A. Ekuni, J. H. Ko, Y. Yamamoto, et al. Ectopic expression of {{alpha}}1,6 fucosyltransferase in mice causes steatosis in the liver and kidney accompanied by a modification of lysosomal acid lipase Glycobiology, February 1, 2001; 11(2): 165 - 174. [Abstract] [Full Text] [PDF]

				P. She, M. Shiota, K. D. Shelton, R. Chalkley, C. Postic, and M. A. Magnuson Phosphoenolpyruvate Carboxykinase Is Necessary for the Integration of Hepatic Energy Metabolism Mol. Cell. Biol., September 1, 2000; 20(17): 6508 - 6517. [Abstract] [Full Text]

				J. H. Capdevila, J. R. Falck, and R. C. Harris Cytochrome P450 and arachidonic acid bioactivation: molecular and functional properties of the arachidonate monooxygenase J. Lipid Res., February 1, 2000; 41(2): 163 - 181. [Abstract] [Full Text]

				M. Baes, S. Huyghe, P. Carmeliet, P. E. Declercq, D. Collen, G. P. Mannaerts, and P. P. Van Veldhoven Inactivation of the Peroxisomal Multifunctional Protein-2 in Mice Impedes the Degradation of Not Only 2-Methyl-branched Fatty Acids and Bile Acid Intermediates but Also of Very Long Chain Fatty Acids J. Biol. Chem., May 19, 2000; 275(21): 16329 - 16336. [Abstract] [Full Text] [PDF]

				T. E. Akiyama, J. M. Ward, and F. J. Gonzalez Regulation of the Liver Fatty Acid-binding Protein Gene by Hepatocyte Nuclear Factor 1alpha (HNF1alpha ). ALTERATIONS IN FATTY ACID HOMEOSTASIS IN HNF1alpha -DEFICIENT MICE J. Biol. Chem., August 25, 2000; 275(35): 27117 - 27122. [Abstract] [Full Text] [PDF]

				T. Hashimoto, W. S. Cook, C. Qi, A. V. Yeldandi, J. K. Reddy, and M. S. Rao Defect in Peroxisome Proliferator-activated Receptor alpha -inducible Fatty Acid Oxidation Determines the Severity of Hepatic Steatosis in Response to Fasting J. Biol. Chem., September 8, 2000; 275(37): 28918 - 28928. [Abstract] [Full Text] [PDF]

				C. Wolfrum, C. M. Borrmann, T. Borchers, and F. Spener Fatty acids and hypolipidemic drugs regulate peroxisome proliferator-activated receptors alpha - and gamma -mediated gene expression via liver fatty acid binding protein: A signaling path to the nucleus PNAS, February 27, 2001; 98(5): 2323 - 2328. [Abstract] [Full Text] [PDF]

				F. M. Gregoire, Q. Zhang, S. J. Smith, C. Tong, D. Ross, H. Lopez, and D. B. West Diet-induced obesity and hepatic gene expression alterations in C57BL/6J and ICAM-1-deficient mice Am J Physiol Endocrinol Metab, March 1, 2002; 282(3): E703 - E713. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hashimoto, T. Articles by Reddy, J. K. Search for Related Content PubMed PubMed Citation Articles by Hashimoto, T. Articles by Reddy, J. K. Social Bookmarking                      What's this?