Elsevier

Hepatology Research

Volume 19, Issue 2, February 2001, Pages 117-130
Hepatology Research

Regulation of peroxisome proliferator activated receptor α-mediated pathways in alcohol fed cytochrome P450 2E1 deficient mice

https://doi.org/10.1016/S1386-6346(00)00089-9Get rights and content

Abstract

Fatty acids are substrates and inducers for cytochrome P450 2E1 (CYP2E1) and peroxisome proliferator activated receptor α (PPARα). Previously, we have shown that the ethanol-induced CYP2E1 expression in rat is accompanied by the inhibition of the expression of the PPARα gene and the reduction in polyunsaturated fatty acid content. To further analyze the effect of CYP2E1 and ethanol in PPARα-mediated fatty acid homeostasis, the expression of PPARα and retinoid x receptor α (RXRα) and their target genes was examined in ethanol fed CYP2E1 deficient mice. Our data demonstrated that the expression of PPARα and RXRα genes was activated in the livers of CYP2E1-null mice suggesting a compensatory effect for the absence of CYP2El. In addition, the expression of PPARα target genes, which included the liver fatty acid-binding protein, malic enzyme, and CYP4A1 genes, was induced indicating the activation of PPARα-mediated pathways in CYP2E1 deficient mice. Ethanol inhibited the expression of some of the PPARα target genes in wild-type mouse livers, and the inhibitory effect of ethanol was particularly prominent in the CYP2E1-null mice. Morphologically, centrilobular fat accumulation was detected in the ethanol fed CYP2E1-null mouse livers suggesting that inhibition of PPARα-mediated pathways might be responsible for the ethanol-induced fatty liver in CYP2El-null mice. In addition, the expression of CYP2E1 was not changed in the PPARα-null mice. These data suggest that CYP2E1 and ethanol can regulate PPARα-mediated fatty acid homeostasis. CYP2E1-induced lipid peroxidation might play a major role in lipid metabolism, PPARα only becomes important when the CYP2E1 level is low and polyunsaturated fatty acids increase.

Introduction

Cytochrome P450 2E1 (CYP2E1) is constitutively expressed in liver. CYP2E1 is effectively induced in the liver and other tissues by a diverse set of chemicals having different structures (for reviews see [1]). CYP2E1 expression is regulated by several xenobiotics, many of which are substrates and thus they induce their own metabolism. Ethanol, an inducer of CYP2E1 in rodent models, is also metabolized by CYP2E1 [2], [3]. Although, CYP2El-mediated ethanol metabolism is secondary to alcohol dehydrogenase, it may play an important role in alcoholic liver disease, since P450-mediated reactions produce activated oxygen species [3], [4]. The pathogenesis of alcoholic liver disease may be due to the induction of CYP2E1 which could transform other drugs to toxic metabolites and the generation of free radicals resulting in cell death.

Besides xenobiotics, the expression of hepatic CYP2E1 is also strongly influenced by nutritional factors such as high-fat-diet. Like the xenobiotics, fatty acids such as arachidonic acid is an inducer, as well as, a substrate of CYP2E1 [1], [5]. Fatty acids increase the expression of CYP2E1 and it induces lipid peroxidation [1]. Administration of ethanol induces CYP2E1 and lipid peroxidation and decreases liver arachidonic acid content in alcoholic liver disease in rats [6], [7]. Therefore, ethanol intake causes a significant change in the utilization of fatty acids [8]. It induces CYP2E1 in the centrilobular region of the liver, an area subjected to increased lipid peroxidation after ethanol treatment [9].

CYP2E1 is one of the most well conserved P450s. However, the CYP2El-null mouse does not demonstrate any obvious phenotype or pathological abnormalities thus, suggesting that CYP2E1 is not essential for normal mammalian development or physiological homeostasis [10].

Hepatic fatty acid metabolism is also mediated through peroxisomal and mitochondria β-oxidation and microsomal ω-oxidation. The enzymes involved in β-oxidation of fatty acids are acyl coenzyme A (acyl-CoA) oxidase, bifunctional enzyme, and 3-ketoacyl CoA thiolase located in the peroxisomal matrix. The ω-oxidation is mediated through CYP4A1. Peroxisome proliferators such as hypolipidemic drugs are the inducers of these enzymes (for reviews see [11], [12], [13]). Administration of peroxisome proliferators to rodent results in a marked increase in peroxisome proliferation. Morphologically, the rodent liver exhibits hepatomegaly [14]. Biochemically, besides the above mentioned enzymes, cytosolic liver fatty acid binding protein and lipogenic malic enzyme are all induced [11], [12], [13]. Then, the expression of apolipoproteins AI, AII, and CIII are reduced [15], [16]. As a result, the plasma triglyceride and cholesterol levels in the treated animals decrease [15], [16].

Peroxisome proliferators exert their responses via peroxisome proliferator activated receptor α (PPARα), a member of the nuclear receptor superfamily. Two other related receptors have been described: PPARβ (also called δ or NUC-1) and γ [17], [18], [19], [20], [21]. PPARα is predominantly expressed in tissues capable of oxidizing fatty acids, such as liver, kidney, and heart [22]. Fatty acid metabolites are potential ligands for PPARα in vivo [23], [24]. Arachidonate levels are positively correlated to PPARα expression suggesting that arachidonate or a metabolite regulate PPARα activity [25]. Absence of PPARα expression in knockout mice prevents the inducibility of fatty acid synthesizing enzymes and β-oxidizing enzymes by Wy14,643, the most potent peroxisome proliferator [26], [27], [28]. In addition, PPARα deficiency leads to steatosis in aging mice [29].

Since fatty acids are substrates and ligand for CYP2E1 and PPARα, respectively, there may be a cross-talk between them. Indeed, we found that alcohol-induced CYP2E1 expression is accompanied by down-regulation of PPARα mRNA in the rat liver, suggesting a reciprocal relationship between these two fatty acid metabolic systems [25]. In addition, decreased PPARα gene expression in ethanol-fed rats might result from a reduction in the content of polyunsaturated fatty acid in the liver [25]. To further analyze the interaction between CYP2E1 and PPARα and the role of CYP2E1 in the PPARα mediated pathways, CYP2E1-null mice were used as a model to study the PPARα-mediated pathways. In the absence of CYP2El, the PPARα gene and PPARα-mediated pathways are spontaneously activated in order to perhaps maintain fatty acid homeostasis. Furthermore, in contrast to CYP2El, which is inducible by ethanol, ethanol intake inhibits PPARα-mediated pathways in CYP2E1 deficiency mice and this could account in part for ethanol induced fatty liver.

Section snippets

Animals

The CYP2E1 and PPARα null mice have been described earlier [10], [26]. Male mice weighing 25 g were used. A permanent intragastric cannula connected to a swivel to allow free movement was implanted into mice under thiopental sodium (2 mg/kg, ip) and ketamine hydrochloride (6 mg/kg, ip) anesthesia. Mice were maintained according to the Guidelines of Animal Care as described by the National Academy of Sciences and published by the National Institutes of Health (NIH publication number 86–23,

Hepatocyte fat accumulation occurs in alcohol fed CYP2El-null mice

Using immunohistochemistry, we first analyzed the tissue distribution of CYP2E1 in wild-type and knockout mice fed control and alcohol diet in order to confirm the phenotype of the mice. The wild-type mouse livers demonstrated extensive distribution of CYP2E1 in the centrilobular region of the liver (Fig. 1A). The alcohol-treated wild-type mouse livers also showed extensive CYP2E1 staining (Fig. 1B). Feeding the wild-type mice with alcohol for 21 days did not cause any obvious induction of

Discussion

We have demonstrated that the expression of the CYP2E1 gene affected the expression of the PPARα and RXRα genes and PPARα-mediated pathways. When the CYP2E1 gene is inactivated, the PPARα and RXRα genes are induced which in turn enhance the expression of PPARα-target genes. When the CYP2E1 gene is induced by alcohol in the wild-type mouse, some of the PPARα target genes are also down-regulated. Therefore, there is a negative correlation between the expression of CYP2E1 and PPARα-mediated

Acknowledgments

This work was supported by NIH grant CA53596.

References (40)

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