THEME
Nonalcoholic Steatosis and Steatohepatitis
III. Peroxisomal beta -oxidation, PPARalpha , and steatohepatitis

Janardan K. Reddy

Department of Pathology, Northwestern University Medical School, Chicago, Illinois 60611-3008


    ABSTRACT
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ABSTRACT
INTRODUCTION
FA OXIDATION
ENZYMES OF THE PEROXISOMAL...
REGULATORY ROLE OF PPARalpha
PEROXISOMAL BIOGENESIS...
MOUSE MODELS OF PEROXISOMAL...
REFERENCES

Peroxisomes are involved in the beta -oxidation chain shortening of long-chain and very-long-chain fatty acyl-CoAs, long-chain dicarboxylyl-CoAs, the CoA esters of eicosanoids, 2-methyl-branched fatty acyl-CoAs, and the CoA esters of the bile acid intermediates, and in the process, they generate H2O2. There are two complete sets of beta -oxidation enzymes present in peroxisomes, with each set consisting of three distinct enzymes. The classic PPARalpha -regulated and inducible set participates in the beta -oxidation of straight-chain fatty acids, whereas the second noninducible set acts on branched-chain fatty acids. Long-chain and very-long-chain fatty acids are also metabolized by the cytochrome P-450 CYP4A omega -oxidation system to dicarboxylic acids that serve as substrates for peroxisomal beta -oxidation. Evidence derived from mouse models of PPARalpha and peroxisomal beta -oxidation deficiency highlights the critical importance of the defects in PPARalpha -inducible beta -oxidation in energy metabolism and in the development of steatohepatitis.

peroxisomes; fatty acid beta - and omega -oxidation; peroxisomal proliferator-activated receptor alpha ; peroxisomal biogenesis disorders


    INTRODUCTION
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ABSTRACT
INTRODUCTION
FA OXIDATION
ENZYMES OF THE PEROXISOMAL...
REGULATORY ROLE OF PPARalpha
PEROXISOMAL BIOGENESIS...
MOUSE MODELS OF PEROXISOMAL...
REFERENCES

FATTY ACIDS (FAs) are ubiquitous molecules that are pivotal for a variety of cellular processes including energy storage, synthesis of cellular membranes, and generation of lipid-containing messengers in signal transduction. Disturbances in FA metabolism and regulation, especially involving FA synthesis and oxidation, can contribute to hyperlipidemia, obesity, insulin resistance, atherosclerosis, and steatohepatitis in industrialized as well as in developing societies (30, 32). When energy (glucose) intake is abundant, FA synthesis is enhanced and FA oxidation is reduced to facilitate storage of excess energy in the form of energy- dense triacylglycerol in adipocytes, leading to obesity (30, 32). Under conditions of reduced energy supply, the synthesis of FAs is diminished, culminating in lipolysis and release of FAs from adipocytes, and the FAs reaching the liver then undergo oxidation there to generate ketone bodies. These are then exported out of liver to serve as fuels for extrahepatic tissues such as cardiac and skeletal muscle (10, 11, 16, 18). Thus FA oxidation plays a central role in energy homeostasis in that maintenance of high levels of FA oxidation can result in reduced fat accumulation despite excess energy consumption (1, 6); in contrast, reduced FA oxidation can contribute to the development of obesity as well as hepatic steatosis (Fig. 1).


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Fig. 1.   The 3 fatty acid oxidation systems in liver and the regulatory role of peroxisomal proliferator-activated receptor alpha  (PPARalpha ) in energy metabolism. PPARalpha -induced microsomal fatty acid omega -oxidation generates dicarboxylyic acids, and the dicarboxylyl-CoAs and all very-long-chain fatty acyl-CoAs are oxidized by PPARalpha -inducible peroxisomal beta -oxidation system. The chain-shortened fatty acyl-CoAs produced by the peroxisomal beta -oxidation system are shunted to the mitochondrial beta -oxidation pathway for completion of oxidation. Highly efficient PPARalpha -inducible fatty acid oxidation can reduce storage of energy (triacylglycerol) in liver and in adipose tissue. Decreased fatty acid oxidation, either due to inherent defects in the fatty acid oxidation systems or defects in PPARalpha , can lead to steatohepatitis and, in conjunction with increased triglyceride synthesis in liver, can also contribute to adiposity by stimulating PPARgamma -induced adipogenic response.


    FA OXIDATION
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ABSTRACT
INTRODUCTION
FA OXIDATION
ENZYMES OF THE PEROXISOMAL...
REGULATORY ROLE OF PPARalpha
PEROXISOMAL BIOGENESIS...
MOUSE MODELS OF PEROXISOMAL...
REFERENCES

Oxidation of FAs occurs in three subcellular organelles, with beta -oxidation confined to mitochondria and peroxisomes and the CYP4A catalyzed omega -oxidation occurring in the endoplasmic reticulum (14, 20, 21, 28). Some of the key enzymes of these three FA oxidation systems are transcriptionally regulated by peroxisome proliferator-activated receptor-alpha (PPARalpha ), a member of the nuclear hormone receptor superfamily (5, 12). Mitochondrial beta -oxidation is responsible for the oxidation of the bulk of the short (<C8)-, medium (C8-C12)-, and long (C12-C20)-chain FAs and, in the process, contributes to energy generation via ATP, producing oxidative phosphorylation (reviewed in Ref. 28). Long-chain FAs constitute the bulk of dietary fat and thus are the predominant source of energy under normal feeding conditions and also during fasting-induced lipolysis. FAs are completely oxidized to acetyl-CoA by mitochondrial beta -oxidation, and the acetyl-CoA subunits so produced can either condense to form ketone bodies (acetoacetate, acetone, and beta -hydroxybutyrate) or degrade to CO2 by the tricarboxylic acid cycle (28). Mitochondrial beta -oxidation is regulated by carnitine palmitoyltransferase I (CPTI), carnitine concentration, and also by malonyl-CoA, which inhibits CPTI (26, 28). CPTI is markedly induced by FAs/fatty acyl-CoAs and by several structurally different synthetic compounds known as peroxisome proliferators, which activate PPARalpha (9, 25, 28).

The peroxisomal beta -oxidation is responsible, almost exclusively, for the oxidation of very-long-chain FAs (>C20) as they cannot be processed by the mitochondrial beta -oxidation system due to the absence of very-long-chain fatty acyl-CoA synthetase (20, 28, 34). The peroxisomal beta -oxidation system also participates in the metabolism of long-chain dicarboxylic acids, eicosanoids, bile acid precursors, and the side chains of some xenobiotics (28). The role of peroxisomal beta -oxidation in the synthesis and metabolism of docosahexanoic acid (DHA) and retroconversion of DHA to eicosapentaenoic acid has been reviewed recently (28).

There are two complete sets of beta -oxidation enzymes present in peroxisomes, with each set consisting of three distinct enzymes (Fig. 2). Straight-chain acyl-CoA oxidase (AOX) is responsible for the initial oxidation of very-long-chain fatty acyl-CoAs in the PPARalpha -regulated and inducible classic beta -oxidation system, whereas branched-chain acyl-CoA oxidase (BOX) oxidizes branched-chain fatty acyl-CoAs in the noninducible second system (20, 28). The three enzymes of the classic peroxisomal beta -oxidation cycle, namely AOX, enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase [L-bifunctional protein (L-PBE)], and 3-ketoacyl-CoA thiolase, are transcriptionally activated by PPARalpha -ligands (28). In the noninducible branched-chain FA oxidation system, the enoyl-CoAs generated by BOX are dehydrated and then dehydrogenated by D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase [D-bifunctional enzyme (D-PBE)]. The third enzyme of the second system is designated sterol carrier protein x (SCPx), the NH2 terminal of which exerts thiolytic activity (20, 28). The separation between two peroxisomal beta -oxidation pathways is not rigid after the first desaturation step catalyzed by specific oxidase (AOX or BOX) as the L- and D-hydroxy intermediates (enoyl-CoA esters of both straight- and branched-chain FAs) generated in the two beta -oxidation systems can be metabolized by the same enzyme, namely the D-PBE, leaving uncertain the role of L-PBE of the straight-chain beta -oxidation system (3, 24, 28). The peroxisomal acyl-CoA oxidases, which are the first and rate-limiting enzymes of each of the two beta -oxidation pathways in peroxisomes, account for the basic difference between peroxisomal and mitochondrial oxidation pathways (20, 28).


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Fig. 2.   Cross-talk between the 2 sets of peroxisomal beta -oxidation systems. Straight-chain acyl-CoA oxidase (AOX) is responsible for the initial oxidation of very-long-chain fatty acyl-CoAs in the PPARalpha -regulated and inducible classic beta -oxidation system, whereas branched-chain acyl-CoA oxidase (BOX) oxidizes branched-chain fatty acyl-CoAs in the noninducible second system. The enoyl-CoAs generated in the classic system can be metabolized by both L-peroxisomal bifunctional enzyme (PBE) of the classic set and the D-PBE of the branched-chain system, but L-PBE does not appear to be effective in dealing with enoyl-CoAs generated by BOX in the noninducible system. SCPx, sterol carrier protein x.

The first step in peroxisomal FA oxidation is directly coupled to the molecular oxygen, resulting in the cyanide insensitivity of the system, in contrast to the cyanide sensitivity of the mitochondrial system in which the first step is directly coupled to an electron-transfer chain (20). Peroxisomal beta -oxidation generates H2O2, whereas mitochondrial beta -oxidation produces energy. Unlike the mitochondrial system, peroxisomal beta -oxidation is carnitine independent, and it does not go to completion as the chain-shortened acyl-CoAs (medium-chain acyl-CoAs) are exported to the mitochondria for the completion of beta -oxidation (20, 28).

The omega -oxidation of FAs by cytochrome P-450 CYP4A subfamily starts almost exclusively in smooth endoplasmic reticulum, with the initial omega -hydroxylation step generating a omega -hydroxy FA, which is then dehydrogenated to a dicarboxylic acid in the cytosol (14, 21, 28). Dicarboxylic acid is converted to its CoA derivative by an acyl-CoA synthetase present in the endoplasmic reticulum. In humans, long-chain dicarboxylyl-CoAs derived from omega -oxidation of FAs are almost exclusively metabolized by the classic peroxisomal beta -oxidation system, whereas in rats, a significant portion of dicarboxylyl-CoAs appears to be metabolized via the noninducible branched-chain oxidation system (28). Although microsomal omega -oxidation is considered a minor pathway for FA metabolism, significant quantities of dicarboxylic acids can be formed from omega -oxidation of long-chain monocarboxylic FAs under conditions of FA overload in the liver; for example, in diabetes mellitus and in situations in which mitochondrial beta -oxidation is impaired. omega -Oxidation of FAs can generate reactive oxygen species as the rate of NADPH consumption increases to reduce oxygen to superoxide and/or H2O2 (21). The reactive oxygen species generated during the omega -oxidation of FAs to dicarboxylic acids and when these dicarboxylyl-CoAs undergo beta -oxidation within peroxisomes can contribute to steatohepatitis under certain conditions (6, 13, 26).


    ENZYMES OF THE PEROXISOMAL beta -OXIDATION SYSTEM
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ABSTRACT
INTRODUCTION
FA OXIDATION
ENZYMES OF THE PEROXISOMAL...
REGULATORY ROLE OF PPARalpha
PEROXISOMAL BIOGENESIS...
MOUSE MODELS OF PEROXISOMAL...
REFERENCES

Before an FA can be beta -oxidized by peroxisomes, it must be activated to its CoA derivative, and this is accomplished by long-chain and very-long-chain acyl-CoA synthetases present in the peroxisomal membrane (20, 28). Long-chain acyl-CoA synthetase, which activates long-chain FAs, is also present in the mitochondrial outer membrane and in the endoplasmic reticulum, but very-long-chain acyl-CoA synthetase, which activates very-long-chain FAs, is present in peroxisomal and microsomal membranes but not in mitochondria, accounting for the exclusive beta -oxidation of these FAs within the peroxisome.

The first step of the peroxisomal beta -oxidation cycle is catalyzed by FAD-containing acyl-CoA oxidases that donate electrons directly to molecular oxygen, thereby generating H2O2 (20). In the classic PPARalpha -regulated and inducible beta -oxidation spiral, which deals with straight-chain acyl-CoAs, this first step is catalyzed by AOX, a single enzyme, in all species examined (22, 28). AOX is a dimeric protein with a molecular mass of 140 kDa, which oxidizes CoA esters of straight-chain FAs, dicarboxylyic acids, and eicosanoids. The human AOX gene, localized to chromosome band 17q25, is present as a single copy per haploid genome, and it is structurally similar to the rat gene (22, 28, 33). Both rat and human AOX proteins contain a Ser-Lys-Leu peroxisomal targeting signal (PTS1) in the COOH-terminal end (31, 33). In the second noninducible beta -oxidation set that acts on 2-methyl-branched FAs, such as pristanic acid, which is derived from phytol contained in food, and on the bile acid intermediates, di- and trihydroxycoprostanoic acids, which also contain a 2-methyl substitution in their side chain, the BOX functions as the first and rate-limiting step in humans (20, 28). Unlike in humans, the branched-chain beta -oxidation spiral in rats can be initiated by two different noninducible oxidases, namely pristanoyl-CoA oxidase, which acts on CoA esters of 2-methyl-branched FAs, and trihydroxycoprostanoyl-CoA oxidase, which uses the bile acid intermediates as substrates (15, 18).

The second and third steps of peroxisomal beta -oxidation are catalyzed by two different bifunctional proteins. The second step is the hydration of the enoyl-CoAs to 3-hydroxyacyl-CoAs, which are then dehydrogenated to generate 3-ketoacyl-CoAs in the third step. A single protein with both enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase activities, hence called peroxisomal bifunctional enzyme (PBE) catalyzes these two steps (20, 28). Two different bifunctional proteins exist in peroxisomal matrix: the first is the L-PBE of the classic PPARalpha -ligand inducible beta -oxidation system, and the second is the D-PBE of the branched-chain noninducible beta -oxidation system (20, 28). The substrate specificities of L-PBE and D-PBE differ in that both enzymes can metabolize straight-chain enoyl-CoAs as substrates, but they differ markedly with respect to 3alpha -, 7alpha -, and 12alpha -hydroxy-5beta -cholest-24-enoyl-CoA and with respect to 3alpha -, and 7alpha -dihydroxy-5beta -cholest-24-enoyl-CoA, which appear to be more desirable as substrates for D-PBE (28). Finally, the L-PBE and not the D-PBE is markedly induced by PPARalpha ligands in liver.

The last step of the peroxisomal beta -oxidation process, the thiolytical cleavage of 3-ketoacyl-CoA into a chain-shortened acyl-CoA and acetyl-CoA or propionyl-CoA (in case of two methyl-branched FAs), is catalyzed by two distinct enzymes. These two enzymes are the 3-ketoacyl-CoA thiolase of the classic straight-chain beta -oxidation system and the SCPx of the noninducible branched-chain beta -oxidation system (20, 28). The 3-ketoacyl-CoA thiolase and SCPx exhibit distinct substrate specificities: the former plays a principal but restricted role in the beta -oxidation of straight-chain 3-ketoacyl-CoAs, and the latter plays a broader role in the cleavage not only of branched-chain FAs and the bile acid intermediates but also of 3-ketoacyl-CoAs of straight-chain FAs (28).


    REGULATORY ROLE OF PPARalpha
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ABSTRACT
INTRODUCTION
FA OXIDATION
ENZYMES OF THE PEROXISOMAL...
REGULATORY ROLE OF PPARalpha
PEROXISOMAL BIOGENESIS...
MOUSE MODELS OF PEROXISOMAL...
REFERENCES

PPARalpha has been identified as the key regulator of the genes involved in peroxisomal, mitochondrial, and microsomal FA oxidation systems in liver (5, 10, 12). The induction of some of the critical enzymes of the beta - and omega -oxidation systems in liver by peroxisome proliferators is transcriptionally controlled by PPARalpha , because these effects are abrogated in PPARalpha -null mice (14, 17). The ligand-activated PPARalpha forms a heterodimer with the 9-cis-retinoic acid receptor RXR, and the PPAR/RXR heterodimers bind to DNA sequences, termed PPAR response elements (PPRE), composed of direct repeats of the hexanucleotide sequence AGGTCA separated by one nucleotide, known as DR-1 response elements, present in the 5'-flanking region of target genes (reviewed in Ref 5). PPARalpha is highly expressed in the liver and, to a modest extent, in the kidney and intestinal epithelium, and the levels of PPARalpha appear to reflect the sensitivity of various tissues to the induction of beta -oxidation by synthetic PPARalpha ligands (5). These synthetic ligands are structurally diverse peroxisome proliferators and include certain hypolipidemic drugs, phthalate ester plasticizers, herbicides, and leukotriene D4 receptor antagonists (9, 25, 27). These synthetic ligands may mimic FAs/fatty acyl-CoAs and other endogenous/biological ligands of PPARalpha (7). Although confirmation-based in vitro assays identified FAs and eicosanoids as biological PPARalpha ligands (7), observations in AOX-null mice strongly imply that substrates of AOX such as very-long-chain fatty acyl-CoAs are the most likely PPARalpha activators in vivo (6). Thus PPARalpha appears to be a xenosensor as well as a sensor of fluxes in certain lipid moieties in liver (7). In conditions associated with excess energy consumption, acetyl-CoA generated from glucose is used for FA synthesis, and its subsequent conversion to triglycerides leads to adiposity (30, 32). Evidence indicates that adipogenesis is facilitated by PPARgamma , a member of the PPAR subfamily (4, 26), and an increase in adipogenesis occurs to store triglycerides under conditions of decreased PPARalpha activity and reduction of FA oxidation, implying a cross-talk between these two receptors.

The molecular mechanisms by which PPARs and other nuclear receptors achieve transcriptional activation in a gene-, tissue-, and species-specific manner are the subjects of intense investigation in recent years (for review, see Ref. 8). The current models call for participation of a series of cofactors (accessory proteins) that associate with nuclear receptors in a ligand-dependent fashion (8). During the past 6 years, several "coactivator" proteins for nuclear receptors have been identified (8). Some of these possess histone acetyltransferase activity, and they also recruit other proteins with similar activity, indicating their role in chromatin modification (8). There is also emerging evidence suggesting a role for cofactor-mediated histone methylation and possibly RNA methylation in the transcriptional activation by nuclear receptors (4, 35). Elucidation of the tissue- and cell-specific mechanisms of transcriptional activation of genes involved in peroxisomal beta -oxidation is important for a greater appreciation of the importance of lipid homeostasis in health and disease. Such studies may lead to new insights into the modulation of FA oxidation and in developing measures to limit disease processes.


    PEROXISOMAL BIOGENESIS DISORDERS AND beta -OXIDATION DEFICIENCY
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INTRODUCTION
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ENZYMES OF THE PEROXISOMAL...
REGULATORY ROLE OF PPARalpha
PEROXISOMAL BIOGENESIS...
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REFERENCES

Peroxisomal disorders in humans manifesting as disturbances in lipid metabolism represent a group of inherited diseases in which there is an impairment of peroxisomal beta -oxidation resulting from defects in one or more enzymes (31, 34). These disorders, classified on the basis of biochemical defects, generally fall into three categories depending on whether there is a generalized (group A), multiple (group B), or single (group C) loss of peroxisomal functions (31, 34). Groups A and B are mostly related to defects in posttranslational import of peroxisomal matrix proteins due to mutations in PEX genes (PEX5, and PEX7), whose gene products, peroxins (PEX proteins), are involved in peroxisome biogenesis (30, 33). PEX5 and PEX7 serve as receptors for peroxisomal targeting signals PTS1 and PTS2, respectively, and mutations in these PEX genes will disrupt peroxisomal import of proteins, leading to serious defects in peroxisomal beta -oxidation (2, 31, 34). Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum's disease are examples of group A with generalized defects in peroxisome assembly, manifesting in the functional loss of many peroxisomal enzymes (34). Abnormalities in the peroxisomal beta -oxidation pathway in these disease states involve specifically the metabolism of very-long-chain FAs, long-chain dicarboxylic acids, pristanic acid, and certain prostaglandins among others (2, 28, 34). Furthermore, deficiency of the peroxisomal beta -oxidation system affects the biosynthetic pathway of DHA, and reduced levels of DHA affect structure of cell membranes, particularly of neuronal tissues, accounting for dysmyelination, retinopathy, and psychomotor retardation observed in Zellweger patients (34). Rhizomelic chondrodysplasia punctata with dysfunction of the PEX7 gene, which encodes PTS2 receptor, is an example of group B disease, and peroxisomes of these patients lack all proteins, which are imported via the PTS2 receptor. These patients manifest defects in ether phospholipid metabolism due to abnormalities in dihydroxyacetonephosphate acyltransferase and alkyldihydroxyacetonephosphate synthase (34).

Peroxisomal disorders, reflecting the loss of a single peroxisomal function due to loss/mutation in a single gene, represent group C. Diseases in this category representing peroxisomal beta -oxidation include x-linked adrenoleokodystrophy (X-ALD), straight-chain AOX deficiency, D-PBE deficiency, and thiolase deficiency (34). To date, no patients have been discovered manifesting deficiency of L-PBE, BOX, or SCPx. X-ALD, a most frequent peroxisomal genetic disorder, presents either as a lethal childhood form or as a mild "Addison's-only" form (34). In X-ALD, very-long-chain FAs accumulate due to impaired peroxisomal beta -oxidation in most part due to a defect in an 80-kDa peroxisomal membrane protein called adrenoleukodystrophy protein (ALDP), which belongs to the ATP-binding cassette transporter superfamily (34). This protein transports C26:0 CoA esters across the peroxisome membrane into peroxisome matrix. There is inadequate information regarding the pathological and pathogenetic manifestations in patients with defects in peroxisomal beta -oxidation. A major unresolved issue is how individuals with genetic defects in peroxisomal beta -oxidation or PPARalpha -inducible genes react to nutritional and environmental stress and sensors.


    MOUSE MODELS OF PEROXISOMAL beta -OXIDATION DEFICIENCY: STEATOHEPATITIS AND PPARalpha
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INTRODUCTION
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ENZYMES OF THE PEROXISOMAL...
REGULATORY ROLE OF PPARalpha
PEROXISOMAL BIOGENESIS...
MOUSE MODELS OF PEROXISOMAL...
REFERENCES

To gain a better understanding of the pathophysiology of peroxisomal deficiency disorders, several investigators have begun generating gene knockout mouse models. Sustained activation of PPARalpha and the induction of PPARalpha -responsive genes that participate in lipid catabolism in liver by synthetic or natural PPARalpha ligands manifest in hepatic peroxisome proliferation and liver tumor development in both rats and mice (6, 9, 25). Because available evidence suggests the refractoriness of human liver cells to peroxisome proliferation (9), it is implied that the carcinogenic risk to humans from exposure to synthetic PPARalpha ligands is somewhat negligible. The reasons for the nonresponsiveness of human liver cells to peroxisome proliferation are not clear but may be attributed, in part, to lower PPARalpha levels, among others (14, 23). The identity of PPARalpha target genes involved in hepatic lipid catabolism is well established. The use of gene targeting to disrupt PPARalpha and some of the genes involved in peroxisome biogenesis and in the peroxisomal beta -oxidation system has provided new insights about the role of peroxisomal beta -oxidation in energy metabolism (2, 3, 6, 15, 17, 19, 24, 29).

A knockout mouse model for Zellweger syndrome was generated by disrupting the PEX5 gene (3), which encodes Pex5p, the cytoplasmic shuttle receptor for the import of most of peroxisomal matrix proteins (31). PEX5-/- mice lack peroxisomes and manifest severe defects characteristic of Zellweger patients (3). A mouse model for X-ALD has been developed by disrupting X-ALD gene, which encodes ALDP, which is a peroxisomal membrane protein and transporter (19). The X-ALD mouse exhibits reduced beta -oxidation of very-long-chain FAs, culminating in significant accumulations of these insoluble FAs in many tissues (19). At the beta -oxidation level, the first two genes of the classic inducible beta -oxidation set, namely AOX and L-PBE, have been knocked out in mice using homologous recombination (6, 24). Likewise, mice lacking the second and third genes, namely D-PBE and SCPx, of the noninducible second beta -oxidation set have also been generated (3, 15, 29). Several recent reviews have described these models in detail and point to their usefulness as experimental systems for detailed investigations of the pathogenetic alterations during the lifespan of these genetically altered animals and for testing therapeutic interventions (2, 9, 26, 28).

Two major observations are worth noting here: 1) the disruption of AOX, the first and the rate-limiting enzyme of the classic beta -oxidation set, leads to dramatic activation of PPARalpha (6), and 2) the disruption of the peroxisomal beta -oxidation pathway, either proximal to or distal to classic AOX, fails to hyperactivate PPARalpha (19, 24). Mice with a disrupted AOX gene (AOX-/-) exhibit, during the first 2-4 mo of life, increased serum levels of very-long-chain FAs, growth retardation, hepatomegaly with severe microvesicular steatohepatitis, and lipogranulomatous reaction (6). AOX knockout mice reveal age-progressive hepatocellular regeneration commencing in the periportal region and extending toward the centrizonal region of the liver lobule (6). The regenerated hepatocytes reveal a profound degree of spontaneous peroxisome proliferation, implying that substrates of AOX left unmetabolized in the absence of AOX function as PPARalpha ligands and thus cause sustained hyperactivation of this transcription factor (6). The AOX null mouse model serves as a paradigm for several important pathophysiological processes (6). The microvesicular steatosis and steatohepatitis developing during early life in these AOX-null mice is most likely due to very-long-chain FA toxicity and to increased levels of dicarboxylyic acids produced as a result of highly induced CYP4A omega -oxidation enzymes resulting from PPARalpha activation (6, 10). In the absence of peroxisomal beta -oxidation due to AOX deficiency, these dicarboxylic acids cannot be metabolized, highlighting the importance of peroxisomal beta -oxidation in the pathogenesis of steatohepatitis under conditions of PPARalpha -mediated increased omega -oxidation (6). Support for the role of dicarboxylic acids and PPARalpha hyepractivation is also derived from the observation that mice deficient in both AOX and PPARalpha do not exhibit hepatic steatosis, implying that failure to induce CYP4A enzymes abrogates the production of these toxic derivatives. Mice lacking L-PBE, the second enzyme of this classic beta -oxidation set, do not develop steatohepatitis and do not show any evidence of sustained PPARalpha activation, implying that disruption of this beta -oxidation system distal to AOX will not result in spontaneous peroxisome proliferation (24).

Also of considerable significance is that mice deficient in PPARalpha and those deficient in both PPARalpha and AOX exhibit severe hepatic steatosis when subjected to fasting for 24-72 h, indicating that a defect in PPARalpha -inducible FA oxidation accounts for severe FA overload in liver, causing steatosis, in contrast to the wild-type mice, which respond by enhancing FA oxidation (10, 11, 16, 18). These models point to the criticality of PPARalpha -inducible FA oxidation systems in energy metabolism and in the development of hepatic steatosis. It remains to be established if abnormalities in PPARalpha levels and PPARalpha -inducible oxidation systems contribute to hepatic steatosis in humans. These observations suggest that maintenance of high levels of FA oxidation reduces fat accumulation in liver and most likely prevents unwanted energy storage in extrahepatic tissues. Thus pharmacological enhancement of FA oxidation can be accomplished by augmenting mitochondrial beta -oxidation directly or by judicious pharmacological inhibition of AOX, which may lead to transcriptional activation of PPARalpha and upregulation of the mitochondrial oxidation system, as seen in AOX-null mice.


    FOOTNOTES

Address for reprint requests and other correspondence: J. K. Reddy, Dept. of Pathology, Northwestern Univ. Medical School, 303 East Chicago Ave., Chicago, IL 60611-3008 (E-mail: jkreddy{at}northwestern.edu).


    REFERENCES
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ABSTRACT
INTRODUCTION
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ENZYMES OF THE PEROXISOMAL...
REGULATORY ROLE OF PPARalpha
PEROXISOMAL BIOGENESIS...
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REFERENCES

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