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RXRα-regulated liver SAMe and GSH levels influence susceptibility to alcohol-induced hepatotoxicity

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Abstract

Retinoids influence the pathogenesis of alcohol liver disease (ALD). To analyze the impact of retinoid X receptor alpha (RXRα) on ALD, alcohol-induced hepatotoxicity was studied using mice fed ethanol intragastrically for 25 days. Alcohol-induced microvesicular fat around the central vein and drug-induced morphological changes (loss of rough endoplasmic reticulum, pinkish cytoplasm, and enlarged hepatocyte) in the pericentral area were observed in the liver of wild-type mice. In the hepatocyte RXRα-deficient mouse liver, alcohol induced fat accumulation, mitosis, acute inflammation, and necrosis. The histology score after alcohol treatment was significantly higher in mutant mice than in wild-type mice. However, drug-induced morphological changes were not apparent in alcohol-treated hepatocyte RXRα-deficient mice. Northern analysis showed that the basal and alcohol-induced CYP2B, CYP2A, and CYP3A mRNA levels were lower in hepatocyte RXRα-deficient mice than in wild-type mice, which in turn may protect mutant mice from morphological changes. Compared with wild-type mice, hepatocyte RXRα-deficient mice have significant lower levels of S-adenosylmethionine and glutathione, which is further reduced after alcohol treatment, and that may account for severe liver injury induced by alcohol. The overall result suggests an important role of RXRα in preventing alcohol-induced liver injury.

Introduction

During hepatic fibrosis, hepatic stellate cells gradually lose their vitamin A content, which is concomitant with their increased proliferation and collagen synthesis, and transformation into myofibroblast-like cells (Mak et al., 1984). Congruent with these observations, using the intragastric tube-feeding model, a reduction of vitamin A storage in the liver of alcohol-fed rats was observed (French, 1993). In addition, the level of vitamin A is inversely correlated with the degree of fatty liver, inflammation, necrosis, fibrosis, and Ito cell activation found in the liver of rats fed corn oil and alcohol (Takahashi et al., 1991). Reduced levels of vitamin A have been found in the serum of alcoholic patients with and without liver disease and in liver biopsies from alcoholics (Leo and Liever, 1982). Conversely, hepatic stellate cells treated in vitro with vitamin A have shown suppressed proliferation (Davis et al., 1990), decreased collagen synthesis (Shiratori et al., 1986), and an amelioration of liver fibrosis (Mak et al., 1984). These studies imply that the maintenance of hepatic retinoid levels in stellate cells blocks the fibrogenic response and that retinoids may serve as inhibitors of proliferation and collagen synthesis of stellate cells.

It has been suggested that vitamin A supplements might provide protection against alcohol-related liver damage (Wang, 1999). However, clinical applications along these lines have been disappointing, as an excess of vitamin A is also known to be hepatotoxic Farrell et al., 1977, Russell et al., 1974. The smallest daily supplement of vitamin A reported to be associated with liver cirrhosis is 7500 μg retinol equivalents (25,000 IU) taken for 6 years (Geubel et al., 1991). The potentiation of the hepatotoxicity effect of vitamin A by ethanol also has been reported (Leo and Liever, 1982). Similar results were found in patients treated with 10,000 IU of vitamin A per day for sexual dysfunction attributable to excess alcohol consumption. Severe liver damage was seen in the liver of patients with hypervitaminosis (Minuk et al., 1988).

Thus, it appears that vitamin A deficiency and excess result in similar detrimental effect. The therapeutic window of using vitamin A to treat alcohol-related damage is very narrow, rendering vitamin A supplementation a problematic strategy in relation to alcoholic liver disease (ALD). The actions of retinoids are mediated through retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Among these receptors, RXRα is highly expressed in the liver and has been shown to dimerize with constitutive androstane receptor beta (CARβ), pregnane X receptor (PXR), peroxisome proliferator activated receptors (PPARs), and many other nuclear receptors. These heterodimer nuclear receptors play pivotal roles in various metabolic pathways within the liver Wan et al., 2000a, Wan et al., 2000b. In order to understand the impact of RXRα in ALD, we have studied the development of ALD in hepatocyte RXRα-deficient mice.

S-Adenosylmethionine (SAMe) is an obligatory intermediate in the transsulfuration pathway, a major pathway for the conversion of methionine to cysteine. The pathway, in its entirety, is found only in the liver, but major parts of this pathway also exist in the brain, kidney, spleen, and other tissues. The conversion of methionine to SAMe is catalyzed by methionine adenosyltransferase (MAT) and involves the transfer of the adenosyl moiety from ATP to mathionine. The adult human produces 6 to 8 g of SAMe everyday, most of it in the liver, where it is also used in a variety of reactions. SAMe is a precursor for the synthesis of glutathione (GSH). GSH plays a key role in protection against hydrogen peroxide and is a major hepatoprotective agent against liver injury, including lipid peroxidation. In this report, we document that hepatocyte RXRα-deficient mice have reduced SAMe and GSH levels and are more susceptible to alcohol-induced liver injury.

Section snippets

Mice

Mice with hepatocyte-specific RXRα deficiency were obtained by crossing Alb-cre transgene against the transgenic mice carrying RXRαflox/flox allele, which has been described previously Wan et al., 2000a, Wan et al., 2000b. Age-matched male animals were kept, in two or three, in plastic microisolator cages at 22°C with a 12-h light/12-h dark cycle. Free access to food and water was provided throughout the entire feeding period before the intragastric infusion.

Mouse intragastric ethanol infusion model

Mice at the age of 2–3 months were

Morphological changes induced by alcohol are different in wild-type and hepatocyte RXRα-deficient mice

Male wild-type and hepatocyte RXRα-deficient mice (5/group, 3 months old) were infused with either ethanol or isocaloric glucose for 25 days. At the end of the treatment, blood alcohol levels were around 150 mg/dL for the experimental groups (Table 1). Mice were sacrificed and their livers were harvested for measuring ratios of liver to body weight and morphological analysis. Alcohol induced hepatomegaly in both wild-type and hepatocyte RXRα-deficient mice. In addition, between alcohol-treated

Discussion

We have previously shown that RXRα is essential for cholesterol, fatty acid, carbohydrate, and xenobiotic metabolism in the liver Wan et al., 2000a, Wan et al., 2000b, Staudinger et al., 2003. In this article, we demonstrate that RXRα also plays a crucial role in ALD. In the absence of hepatocyte RXRα, fatty change induced by alcohol becomes more pronounced, which might be due to the compromise of RXRα/PPARα-mediated fatty acid metabolism in hepatocyte RXRα-deficient mice (Wan et al., 2000b).

Acknowledgements

This work is supported by National Institutes of Health Grants CA53596 and AA14147 (to Y.-J.Y.W.), DK45334 and AA12677 (to S.C.L.), and the Animal and Morphology Cores of the USC-UCLA Research Center for Alcoholic Liver and Pancreatic Diseases (P50AA11999).

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