Altered Constitutive Expression of Fatty Acid-metabolizing Enzymes in Mice Lacking the Peroxisome Proliferator-activated Receptor alpha  (PPARalpha )*

Toshifumi AoyamaDagger §, Jeffrey M. Peters, Nobuko Iritanipar , Tamie Nakajima**, Kenichi FurihataDagger Dagger , Takashi HashimotoDagger , and Frank J. Gonzalez

From the Dagger  Department of Biochemistry, Shinshu University School of Medicine, Matsumoto, Nagano 390, Japan,  Laboratory of Metabolism, NCI, National Institutes of Health, Bethesda Maryland 20892, par  Tezukayama Gakuin College, Sakai, Osaka 590, Japan, ** Department of Hygiene and Medical Genetics, Shinshu University School of Medicine, Matsumoto, Nagano 390, Japan, and Dagger Dagger  Department of Laboratory Medicine, Shinshu University School of Medicine, Matsumoto, Nagano 390, Japan

    ABSTRACT
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Procedures
Results
Discussion
References

Peroxisome proliferator-activated receptor alpha  (PPARalpha ) is a member of the steroid/nuclear receptor superfamily and mediates the biological and toxicological effects of peroxisome proliferators. To determine the physiological role of PPARalpha in fatty acid metabolism, levels of peroxisomal and mitochondrial fatty acid metabolizing enzymes were determined in the PPARalpha null mouse. Constitutive liver beta -oxidation of the long chain fatty acid, palmitic acid, was lower in the PPARalpha null mice as compared with wild type mice, indicating defective mitochondrial fatty acid catabolism. In contrast, constitutive oxidation of the very long chain fatty acid, lignoceric acid, was not different between wild type and PPARalpha null mice, suggesting that constitutive expression of enzymes involved in peroxisomal beta -oxidation is independent of PPARalpha . Indeed, the PPARalpha null mice had normal levels of the peroxisomal acyl-CoA oxidase, bifunctional protein (hydratase + 3-hydroxyacyl-CoA dehydrogenase), and thiolase but lower constitutive expression of the D-type bifunctional protein (hydratase + 3-hydroxyacyl-CoA dehydrogenase). Several mitochondrial fatty acid metabolizing enzymes including very long chain acyl-CoA dehydrogenase, long chain acyl-CoA dehydrogenase, short chain-specific 3-ketoacyl-CoA thiolase, and long chain acyl-CoA synthetase are also expressed at lower levels in the untreated PPARalpha null mice, whereas other fatty acid metabolizing enzymes were not different between the untreated null mice and wild type mice. A lower constitutive expression of mRNAs encoding these enzymes was also found, suggesting that the effect was due to altered gene expression. In wild type mice, both peroxisomal and mitochondrial enzymes were induced by the peroxisome proliferator Wy-14,643; induction was not observed in the PPARalpha null animals. These data indicate that PPARalpha modulates constitutive expression of genes encoding several mitochondrial fatty acid-catabolizing enzymes in addition to mediating inducible mitochondrial and peroxisomal fatty acid beta -oxidation, thus establishing a role for the receptor in fatty acid homeostasis.

    INTRODUCTION
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Peroxisomes are single membrane-bound subcellular organelles that contain a variety of enzymes involved in a number of metabolic processes (1). The most well characterized reactions carried out by peroxisomes are those that catalyze in fatty acid beta -oxidation. Since plants lack mitochondria, peroxisomes are solely responsible for their fatty acid beta -oxidation. The peroxisomal fatty acid beta -oxidation pathway produces hydrogen peroxide through the activity of acyl-CoA oxidase, thus historically accounting for the name "peroxisomes." Typically, H2O2 is decomposed to molecular oxygen and water by catalase and glutathione peroxidase. Human genetic deficiencies in peroxisome biogenesis and individual peroxisomal enzymes have been described that result in accumulation of long chain fatty acids (2). The most severe of the peroxisome deficiencies causes neurological and anatomical abnormalities.

In addition to fatty acid oxidation, peroxisomes also carry out beta -oxidation of the cholesterol side chain during the synthesis of bile acids and participate in the biosynthesis of cholesterol (3), ether glycolipids, and dolichols. Catabolism of purines, polyamines, glyoxylate and certain amino acids have been attributed to peroxisome-localized enzymes. Thus, peroxisomes are essential organelles for maintaining cellular and organismal homeostasis.

The number of peroxisomes is increased in rodents by treatment with high fat diets, cold temperature, starvation, ACTH, and certain chemicals generically termed peroxisome proliferators (1). Peroxisome proliferators include a structurally diverse group of chemicals that include 1) hypolipidemic drugs (clofibrate, gemfibrozil, fenofibrate, benzofibrate, etofibrate, and Wy-14,643), 2) the azole antifungal compounds such as bifenazole, 3) leukotriene D4 antagonists, 4) herbicides, 5) pesticides, 6) phthalate esters used in the plastics industry (di-[2-ethylhexyl] phthalate), 7) simple solvents including trichloroethylene, and 8) natural chemicals such as phenyl acetate and the steroid dehydroepiandrosterone sulfate. Among them, the most potent peroxisome proliferator is Wy-14,643.

Peroxisome proliferation is most pronounced in liver, kidney, and heart. In liver, the number of peroxisomes increases from about 500-600/cell to >5,000/cell after exposure to peroxisome proliferators (1). This is accompanied by an increase in cell volume and cell number, resulting in hepatomegaly. Coincident with an increase in the number of peroxisomes, several peroxisomal enzymes are induced by transcriptional activation (4). Transcription of genes encoding the key beta -oxidation enzymes acyl-CoA oxidase, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (bifunctional enzyme), and thiolase are markedly elevated as a result of treatment with peroxisome proliferators (5). Genes encoding the microsomal cytochrome P450 in the CYP4A family are also activated by these agents (6). Changes in peroxisomal and microsomal gene expression induced by peroxisome proliferators are mediated by the peroxisome proliferator-activated receptor (PPAR),1 a member of the nuclear receptor superfamily. Three distinct PPARs have been found, designated PPARalpha , delta  (also called NUC-1 and beta ), and gamma . Tissue distribution of each receptor is different, suggesting that each has unique functions. In rodents, PPARalpha is abundant in the liver, kidney, and heart, all of which display peroxisome proliferation in response to PPARalpha activators and have high rates of lipid metabolism (7). Expression of PPARdelta is ubiquitous and is highly expressed in the central nervous system (7). The role of PPARdelta is not known. PPARgamma and PPARgamma 2, resulting from differential mRNA splicing, are present predominantly in adipose tissue and spleen. PPARgamma is responsible in part for adipocyte differentiation and regulation of adipocyte-specific genes (8). It is also the target for the thiazolidinedione drugs that increase insulin sensitivity of target tissues (9).

To determine the function of PPARalpha and its role in peroxisome proliferation and hepatocarcinogenesis, a PPARalpha null mouse was generated (10). These animals exhibit a normal phenotype and normal basal levels of hepatic peroxisomes. However, the PPARalpha null mouse is nonresponsive to peroxisome proliferation. Compared with wild type mice, administration of peroxisome proliferators to PPARalpha null mice does not cause an increase in the number of peroxisomes, hepatomegaly, nor increases in mRNA encoded by target genes. Furthermore, these mice do not display physiological, toxicological, or carcinogenic responses induced by peroxisome proliferators (11-13). Interestingly, abnormal hepatic lipid accumulation was initially reported in the PPARalpha null mice, suggesting an alteration in lipid metabolism (10). To investigate the biochemical basis for altered lipid metabolism, constitutive levels of peroxisomal and mitochondrial fatty acid-metabolizing enzymes were examined in the PPARalpha null mouse.

    EXPERIMENTAL PROCEDURES
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Procedures
Results
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References

Materials-- Sodium 2-[5-(4-chlorophenyl)pentyl]-oxirane-2-carboxylate (POCA) was purchased from Byk Gulden Pharmazeutika (Konstanz, Germany). [1-14C]lauric acid (55 mCi/mmol), [1-14C]palmitic acid (54 mCi/mmol), and [1-14C]lignoceric acid (47 mCi/mmol) were from American Radiolabeled Chemicals (St. Louis, MO).

Animals and Wy-14,643 Treatment-- PPARalpha null mice on an Sv/129 genetic background were produced as described (10). Wild type Sv/129 were used as controls in all experiments. Mice were fed either a control diet or one containing 0.1% Wy-14,643 for 2 weeks.

Fatty Acid beta -Oxidation Activity-- Fatty acid beta -oxidation activity was measured by the method of Shindo et al., (14). Briefly, unfrozen livers were homogenized in four volumes of 0.25 M sucrose containing 1 mM EDTA in a Potter-Elvehjem homogenizer using a tight-fitting teflon pestle. Approximately 500 µg of homogenate was incubated with the assay medium in 0.2 ml of 150 mM potassium chloride, 10 mM HEPES, pH 7.2, 0.1 mM EDTA, 1 mM potassium phosphate buffer, pH 7.2, 5 mM Tris malonate, 10 mM magnesium chloride, 1 mM carnitine, 0.15% bovine serum albumin, 5 mM ATP, and 50 µM each fatty acid (5.0 × 104 cpm of radioactive substrate). The reaction was run for 30 min at 25 °C and stopped by the addition of 0.2 ml of 0.6 N perchloric acid. The mixture was centrifuged at 2,000 × g for 10 min, and the unreacted fatty acid in the supernatant was removed with 2 ml of n-hexane using three extractions. Radioactive degradation products in the water phase were counted. In some experiments, 20 µM POCA or 2 mM potassium cyanide was added to the incubation mixture to inhibit mitochondrial beta -oxidation activity. Fatty acid beta -oxidation activity was expressed as nmol/min/liver.

Analysis of Fatty Acid Synthesizing Enzymes-- Fatty acid synthetase (15), malic enzyme (ME) (16), ATP-citrate lyase (17), acetyl-CoA carboxylase (18), and glucose-6-phosphate dehydrogenase (19) were measured as described previously.

Analysis of Fatty Acid beta -Oxidizing Enzymes-- Liver extracts were subjected to 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were incubated with the primary antibody followed by alkaline phosphatase-conjugated goat anti-rabbit IgG. Immunoblotting was performed using rabbit polyclonal antibodies against rat acyl-CoA oxidase (AOX) (20), short chain-specific 3-ketoacyl-CoA thiolase (T1) (21), acetoacetyl-CoA thiolase (T2) (22, 23), cytosolic thioesterase (CTE II) (24), short chain acyl-CoA dehydrogenase (SCAD) (25), medium chain acyl-CoA dehydrogenase (MCAD) (25), long chain acyl-CoA dehydrogenase (LCAD) (25), very long chain acyl-CoA dehydrogenase (VLCAD) (26), long chain acyl-CoA synthetase (LACS) (27), very long chain acyl-CoA synthetase (VLACS) (28), peroxisomal thiolase (PT) (21), carnitine palmitoyl-CoA transferase (CPT II) (29), short chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) (30), peroxisomal bifunctional protein (PH) (30), mitochondrial short chain specific hydratase (MH) (31), mitochondrial trifunctional protein alpha  and beta  subunit (TPalpha and TPbeta ) (32), mitochondrial thioesterase I (MTEI) (33), and peroxisomal D-type bifunctional protein (DBF) (34).

mRNA Analysis-- mRNA analysis was performed by Northern blotting. Total liver RNA was extracted, electrophoresed on 1.1 M formaldehyde-containing 1% agarose gels, and transferred to nylon membranes (23). The membranes were incubated with 32P-labeled cDNA probes and analyzed on a Fuji system analyzer (Fuji Photo Film Co., Tokyo, Japan). The cDNA probes used were for VLCAD (35), LCAD (36), LACS (37), ME (38), and SCHAD (39).

    RESULTS
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Analysis of Fatty Acid-metabolizing Enzymes-- To identify specific fatty acid-metabolizing enzymes that were influenced by PPARalpha , antibodies were used to measure protein levels on immunoblots (Fig. 1, Table I). Constitutive expression of several enzymes (VLCAD, LCAD, LACS, and T1) were lower by 30-60% in untreated PPARalpha null mice as compared with untreated wild type mice. Curiously, constitutive expression of the SCHAD was higher by about 4-fold in the PPARalpha null mouse liver as compared with wild type mice. Other mitochondrial enzymes examined were expressed at similar levels in untreated PPARalpha null and wild type mice. The expression of all mitochondrial, microsomal, and cytosolic fatty acid-metabolizing enzymes except for MH were increased in wild type mice fed Wy-14,643 compared with controls, with levels of induction ranging from 1.7-fold for T2 to 4.7-fold for SCHAD. The expression of CTE II was totally dependent on Wy-14,643 treatment in wild type mice. In the PPARalpha null animals, there was no increase in expression of these enzymes after feeding Wy-14,643 for 2 weeks (Fig. 1, Table I).


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Fig. 1.   Immunoblot analysis of selected fatty acid-metabolizing enzymes. Three lanes form a group of mice. Diet, age, and genotype are indicated. Liver cell lysate (8 µg) was subjected to electrophoresis and Western immunoblotting. The blots were stained with antibodies against VLCAD (panel A), LCAD (panel B), SCHAD (panel C), T1 (panel D), LACS (panel E), DBF (panel F), and CTE II (panel G).

                              
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Table I
Western immunoblot quantitation of hepatic fatty acid-metabolizing enzymes in wild type (+/+) and PPARalpha -null (-/-) mice
Mice were fed either a control diet or 0.1% Wy-14,643 for 14 days. Total liver cell extract was subjected to electrophoresis, and proteins were transferred to nitrocellulose membranes and screened with specific antibodies. The signals were quantified by scanning densitometry, and the values from (+/+) mice fed control diets were assigned the number 1.0. Results are the means ± S.D. of three determinations. ND, not detected.

Constitutive levels of hepatic peroxisomal fatty acid beta -oxidation enzyme expression of AOX, PH, PT, and VLACS was not significantly different between wild type and PPARalpha null mice (Fig. 1, Table I), although DBF was 36% lower in the PPARalpha null animals. All of the peroxisomal enzymes were induced from 3-7-fold in the wild type mice, and this effect was not observed in the PPARalpha null mice.

Basal activities of cytosolic enzymes involved in fatty acid synthesis including fatty acid synthetase, acetyl-CoA carboxylase, glucose-6-phosphate dehydrogenase, and ATP-citrate lyase were similar in the untreated wild type and PPARalpha null mice except for ME, which was present at only 50% of the level in the PPARalpha null mouse (Table II). ME and glucose-6-phosphate dehydrogenase were marginally induced (4-fold and 2-fold, respectively) by Wy-14,643 feeding in the wild type mice, and these effects were not observed in the PPARalpha null mouse treated with Wy-14,643.

                              
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Table II
Activities of enzymes involved in fatty acid synthesis in wild type (+/+) and PPARalpha -null (-/-) mice
Mice were fed either a control diet or 0.1% Wy-14,643 for 14 days. Activities are expressed in milliunits/mg of cell lysate protein. Results are the means ± S.D. of three determinations.

Expression of mRNAs-- To determine whether the lower expression of fatty acid-metabolizing enzymes and ME is due to altered gene expression, hepatic mRNA levels were analyzed by Northern blots (Fig. 2). Constitutive levels of VLCAD, LCAD, ME, and LACS mRNA were lower in the PPARalpha null mice compared with controls, consistent with the protein measurements. It is noteworthy that hepatic levels of mRNA for SCHAD were not different between untreated PPARalpha null and wild type mice even though the protein levels were increased by 4-fold in the null mouse. Levels of the mRNAs encoding, LCAD, and ME were significantly induced in wild type mice by Wy-14,643. These data are also consistent with the Western blot analysis. In contrast, the mRNAs encoding LACS and SCHAD were not significantly increased by the drug. In the PPARalpha null mice, there was no difference in mRNA levels for any of the enzymes after treatment with Wy-14,643.


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Fig. 2.   Northern analysis of hepatic mRNAs. Representative samples from three separate mice were used. Diet and genotype are indicated. Total RNA (5.4 µg) from three representative mice from each group were electrophoresed on a denaturing gel and probed using cDNAs for VLCAD (panel A), LCAD (panel B), ME (panel C), LACS (panel D), and SCHAD (panel E). The blots were exposed to autoradiographic film for 5 days (panels A-D) and 1 day (panel E).

Analysis of Overall Fatty Acid beta -Oxidation Activity-- As shown in Table I, six enzymes involved in fatty acid beta -oxidation had lower constitutive expression in the PPARalpha null mice. Four of the six (VLCAD, LCAD, LACS, and DBF) have highest catalytic activities with long chain fatty acid substrates (25-27, 34), whereas the other two (SCHAD and T1) are more active with short and medium chain fatty acids (21, 30). To evaluate the significance of the altered fatty acid beta -oxidation enzymes, total hepatic beta -oxidation was measured using lauric acid (C-12), palmitic acid (C-16), and lignoceric acid (C-24). Compared with wild type controls, the PPARalpha null mice basal levels of total fatty acid beta -oxidation was lower with palmitic acid as a substrate; there was no difference in metabolism of lauric acid and lignoceric acid (Fig. 3). Wy-14,643 feeding caused a significant increase in metabolism of all three fatty acids in wild type animals. No induction was observed in PPARalpha null mice, consistent with the results found with the enzymes levels (Fig. 1 and Table I). Results were identical whether the data were calculated per liver protein or per liver. These data provide evidence that the lower constitutive expression of several long chain-specific fatty acid beta -oxidation enzymes in the PPARalpha null mice compared with wild type mice (Table I) significantly affects long chain fatty acid oxidation. The lack of a difference in constitutive oxidation of the very long chain-specific fatty acid, lignoceric acid, carried out by peroxisomal enzymes supports the finding of no effect of PPARalpha on expression of these enzymes except DBF in untreated null mice (Table I).


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Fig. 3.   Hepatic fatty acid beta -oxidation in control and Wy-14,643-fed wild type (+/+) and PPARalpha null (-/-) mice. Total fatty acid beta -oxidation of lauric acid (panel A), palmitic acid (panel B), and lignoceric acid (panel C). Values are expressed as nmol/min/liver. Solid bars and open bars are from wild type and PPARalpha null mice fed control diet and a diet containing 0.1% Wy-14,643, respectively. Statistical analysis was done by means of two-way ANOVA. a, between (+/+) control diet and (-/-) control diet; b, between (+/+) Wy-14,643 and (-/-) Wy-14,643 diet; c, between (+/+) control diet and (+/+) Wy-14,643 diet. A significant difference was not found between (-/-) control diet and (-/-) Wy-14,643 diet. *** indicates p <= 0.001 between the two values.

To confirm that palmitic acid is preferentially metabolized by mitochondrial enzymes, the inhibitors KCN, an inhibitor of the mitochondrial respiratory chain, and POCA, a potent inhibitor of carnitine palmitoyl-CoA transferase I, were employed (Table III). Both compounds inhibited palmitic acid oxidation by 70-93% in either mouse line, irrespective of Wy-14,643 administration. These data confirm that the contribution of peroxisomal enzymes to palmitic acid beta -oxidation was minimal.

                              
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Table III
Contribution of the mitochondrial beta -oxidation system to long chain fatty acid oxidation
Activities of palmitic acid beta -oxidation were determined in the presence and absence of inhibitor. Results are the means ± S.D. of three determinations.

Time Course of Induction-- The kinetics of VLCAD and ME mRNA and protein expression were determined after administration of Wy-14,643. VLCAD mRNA and protein were rapidly induced within 1 day after Wy-14,643 treatment in wild type mice (Fig. 4A). No induction of VLCAD was found in PPARalpha null mice after 14 days of feeding Wy-14,643. Levels of VLCAD mRNA and protein decreased to about 2-fold after 5 days of feeding yet remained elevated up to 14 days of feeding. A similar time course of induction was also observed in protein levels of several mitochondrial fatty acid beta -oxidation enzymes (LCAD, SCAD, TPalpha , and TPbeta ). Induction of ME mRNA and enzyme activity reached maximal levels of 4-fold after 7 days of Wy-14,643 feeding in the wild type mice; neither mRNA nor activity were induced in the PPARalpha null animal (Fig. 4B). Similar time course of induction was observed in protein levels of several other enzymes (LACS, AOX, PH, PT, DBF, VLACS, MCAD, and CTE II). Thus, the kinetics of the increase in ME expression is slower than that of VLCAD. The reason for this differential induction is not presently known.


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Fig. 4.   Expression of VLCAD (panel A) and ME (panel B) protein and mRNA as a function of time after initiation of Wy-14,643-treatment in wild type and PPARalpha null mice. VLCAD protein or ME activity levels in wild type (black-square) and PPARalpha null (black-diamond ) mice. VLCAD or ME mRNA levels in wild type (black-triangle) and PPARalpha null (black-down-triangle ) mice are shown.

    DISCUSSION
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Constitutive expression of VLCAD, LCAD, SCHAD, T1, LACS, DBF, and ME is regulated by PPARalpha since their abundance was significantly altered in the absence of PPARalpha as compared with wild type controls. With the exception of SCHAD, which was up-regulated in the PPARalpha null mice, all of these proteins were found at lower levels in the PPARalpha null mice. This shows that PPARalpha has an important role in regulating basal levels of these enzymes involved in fatty acid beta -oxidation and ME that participates in fatty acid synthesis. Although the mechanism for this peroxisome proliferator-independent mechanism is not known, it may be a result of altered gene expression since PPARalpha is known to control transcription through interaction with peroxisome proliferator response elements (4). The down-regulation is selective since constitutive expression of other enzymes including MCAD, SCAD, TPalpha , TPbeta , MH, T2, CPT II, and MTE I appear to be unaffected by loss of the receptor. This is similar to the peroxisomal enzymes AOX, PH, PT, and VLACS, where there was no difference in expression levels between the untreated wild type and PPARalpha null mice. The data on enzyme levels are supported by the results of total fatty acid metabolism in liver where oxidation of long chain fatty acid, palmitate, which is reflective of mitochondrial metabolism, was lower in the PPARalpha null mice, whereas oxidation of the very long chain fatty acid, lignocerate, was not different between the wild type and PPARalpha null mice. The lack of difference in metabolism of this very long chain fatty acid is almost certainly due to similar levels of the peroxisomal enzymes in the two genotypes. Lower constitutive expression of fatty acid-metabolizing enzymes and mitochondrial palmitic acid beta -oxidation suggests that PPARalpha controls gene expression in the absence of exogenous ligands for the receptor, and mice lacking PPARalpha have an impaired ability to metabolize lipids.

To further elucidate the role of PPARalpha in lipid metabolism, the effect of the prototypical peroxisome proliferator Wy-14,643 in PPARalpha null mice was investigated. Indeed, PPARalpha was shown to transactivate genes in the presence of peroxisome proliferators (40-42). In addition to the peroxisomal beta -oxidation enzymes and microsomal fatty acid hydroxylase P450, liver fatty acid binding protein and the genes encoding MCAD (43), 3-hydroxy-3-methylglutaryl-CoA synthase (44), and ME (45) are also activated by PPARalpha as indicated by transactivation assays. The present study extends these observations by confirming that expression of VLCAD, LCAD, SCAD, TPalpha , TPbeta , MTE I, CTE II, SCHAD, T1, T2, LACS, and CPT II are all higher as a result of Wy-14,643 feeding in wild type mice but not in PPARalpha null mice. Northern analysis of mRNA encoding some of these enzymes revealed that induction is most likely due to increases in mRNA. These observations demonstrate that changes in gene expression of proteins involved in lipid metabolism are mediated by PPARalpha after exposure to peroxisome proliferators. Indeed, peroxisome proliferator response elements have been found and shown to be functionally active in the MCAD (43) and LACS (46) genes. A peroxisome proliferator response element has not been found in the LCAD (47), even though it is induced in wild type mice by Wy-14,643 feeding. Expression of ME is also elevated at the mRNA and protein level in agreement with a role of PPARalpha in its regulation (45).

The fibrate class of drugs can also lead to suppression of gene expression of numerous proteins involved in lipid metabolism (4). Levels of apolipoprotein A-I, apolipoprotein C-III, apolipoprotein A-IV, hepatic lipase, and lecithin cholesterol acyltransferase are all lowered by treatment of mice with fibrate drugs (4, 48, 49). The alterations of these proteins in addition to altered gene expression of peroxisomal fatty acid beta -oxidizing enzymes are thought to contribute to the lipid-lowering effects of hypolipidemic drugs (4). In addition, it was recently shown that the down-regulation of apolipoprotein C-III mRNA and protein that contributes to the triglyceride-lowering effect of Wy-14,643 is mediated by PPARalpha (50). The results presented here extend these observations by demonstrating that the peroxisome proliferator Wy-14,643 induces significant changes in many fatty acid beta -oxidizing enzymes and total hepatic beta -oxidation, which in turn are likely to further contribute to the triglyceride-lowering effect of the fibrate class of hypolipidemic drugs. Combined, these results establish that PPARalpha functions in the control of lipid homeostasis in mice by regulating constitutive and inducible expression of fatty acid catabolism. Since nuclear receptors usually require a ligand for gene activation, the constitutive control of the enzymes would suggest that an endogenous ligand exists in liver.

The lower expression of mRNAs encoding several fatty acid-metabolizing enzymes in PPARalpha null mice suggests either the presence of an endogenous ligand that preferentially controls genes encoding fatty acid-metabolizing enzymes and ME or that a ligand-independent mechanism is involved. Indeed, phosphorylation of a Ser-112 in PPARgamma through mitogen-activated protein kinase has been shown to modulate its activity (51, 52). This kinase recognition site is conserved between PPARgamma and PPARalpha . Irrespective of the mechanism of PPARalpha activation, these results suggest that it differentially activates genes in the absence of exogenous ligands.

The identification of endogenous ligands has recently been addressed. It was shown that PPARalpha participates in the control of the inflammatory response involving leukotriene B4 (53). These studies also established that leukotriene B4 can directly bind to recombinant PPARalpha . Possible direct binding of peroxisome proliferators was shown by induced conformational changes as detected by protease sensitivity of in vitro translated PPARalpha (54). Other indirect transactivation experiments suggest that PPARalpha may mediate the action of 8(S)-hydroxyeicosatetraenoic acid (55). It is likely that other ligands for PPARalpha exist, including fatty acid metabolites, as first suggested by the ability of fatty acids to transactivate the receptor (56, 57). Evidence exists for the presence of endogenous PPARalpha activators in cultured cells used for transactivation studies. High background levels of reporter gene activation are usually found in these types of studies (41, 58). Further support for the existence of endogenous ligands was provided by the demonstration that unsaturated fatty acids can bind to PPARalpha (59). Thus, constitutive regulation of genes encoding fatty acid-metabolizing enzymes and ME may be mediated by levels of one or more fatty acid metabolites. These metabolites may be important endogenous ligands that function in the control of fatty acid metabolism. Support for this idea was provide by the observation that dietary polyunsaturated fatty acids induce AOX and CYP4A P450 in a PPARalpha -dependent mechanism (11). Taken together, these studies suggest that PPARalpha may have several endogenous ligands, which upon binding to the receptor result in the activation of fatty acid catabolism including the oxidative degradation of leukotrienes, arachidonic acid epoxides, and other fatty acid derivatives.

Among the important issues that need to be addressed is the species differences in response to peroxisome proliferators (60). Mice and rats are highly susceptible to peroxisome proliferation and hepatocarcinogenesis, whereas nonhuman primates and humans appear to be resistant. The mechanism of this species differences is not presently known, but it might be due to lower hepatic levels of PPARalpha (61, 62). Despite the lack of demonstratable peroxisome proliferation, the fibrate drugs are highly effective lipid-lowering agents in humans (63). Thus, PPARalpha may differentially regulate expression of genes that cause peroxisome proliferation and genes encoding enzymes that are responsible for fatty acid mobilization, transport, and catabolism. For example, high cellular levels of receptor may activate genes encoding peroxisomal, mitochondrial, and microsomal fatty acid metabolism in addition to genes that directly or indirectly control the cell cycle and peroxisome proliferation.

    FOOTNOTES

* 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.

§ Dept. of Biochemistry, Shinshu University School of Medicine, Asahi, Matsumoto, Nagano, Japan 390. Tel.: 81-263-37-2602; Fax: 81-263-37-2604; E-mail: toshifu{at}gipac.shinshu-u.ac.jp.

1 The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; VLCAD, very long chain acyl-CoA dehydrogenase; LCAD, long chain acyl-CoA dehydrogenase; MCAD, medium chain acyl-CoA dehydrogenase; SCAD, short chain acyl-CoA dehydrogenase; TPalpha , trifunctional protein alpha  subunit (long chain- specific hydratase + long chain-specific 3-hydroxyacyl-CoA dehydrogenase); TPbeta , trifunctional protein beta  subunit (long chain- specific 3-ketoacyl-CoA thiolase); MH, mitochondrial (short chain-specific) hydratase; SCHAD, short chain 3-hydroxyacyl-CoA dehydrogenase; T1, short chain-specific 3-ketoacyl-CoA thiolase; T2, acetoacetyl-CoA thiolase; LACS, long chain acyl-CoA synthetase; CPT II, carnitine palmitoyl-CoA transferase; MTE I, mitochondrial thioesterase I; CTE II, cytosolic thioesterase II; AOX, acyl-CoA oxidase; PH, peroxisomal bifunctional protein (hydratase + 3-hydroxyacyl-CoA dehydrogenase); DBF, D-type (peroxisomal) bifunctional protein (hydratase + 3-hydroxyacyl-CoA dehydrogenase) and key enzyme of bile acid synthesis from cholesterol; PT, peroxisomal thiolase; VLACS, very long chain acyl-CoA synthetase; ME, malic enzyme; POCA, sodium 2-[5-(4-chlorophenyl)pentyl]-oxirane-2-carboxylate.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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