Expression and antioxidant function of liver fatty acid binding protein in normal and bile-duct ligated rats

Abstract

Liver fatty acid binding protein has recently been shown to possess antioxidant properties but its role in liver disease, such as cholestasis, is not known. Since oxidative stress has been recognized as an important contributing factor in liver disease, we investigated the expression and antioxidative function of this protein using the bile-duct ligated model of cholestasis. Rats were divided into 3 groups: sham, bile-duct ligated and bile-duct ligated plus clofibrate. Animals were sacrificed at various time points after bile-duct ligation. RT-PCR and Western blot were used to analyze liver fatty acid binding protein expression. Cellular lipid peroxidation products were assessed by measuring thiobarbituric acid-reactive substances. Liver function was evaluated by measuring serum total bilirubin, alanine aminotransferase and ammonia. Liver fatty acid binding protein mRNA and protein levels were reduced to 51% and 20% of sham, respectively at 2 weeks following bile-duct ligation (p < 0.05). The decreased liver fatty acid binding protein was associated with a statistical increase in hepatic lipid peroxidation products (224%) and decrease in hepatic function. Clofibrate treatment restored protein level and improved hepatic function. Clofibrate treatment also reduced hepatic lipid peroxidation products by 68% as compared with the bile-duct ligated group (p < 0.05). Liver fatty acid binding protein likely has important antioxidant function during hepatocellular oxidative stress.

Introduction

Fatty acid binding proteins (FABP) are lipid-binding proteins that play an important role in the trafficking of intracellular ligands, metabolism, cell proliferation and signal transduction (Glatz and van der Vusse, 1996, Storch and Thumser, 2000, Wang et al., 2004, Zimmerman and Veerkamp, 2002). Some tissue-specific isoforms of fatty acid binding protein include heart (H-FABP), liver (L-FABP), intestine (I-FABP), brain (B-FABP). Although they are similar in protein structure and function, they are encoded by different genes located on different chromosomes (Zimmerman and Veerkamp, 2002).

Liver fatty acid binding protein is a 14 kDa protein that accounts for 3–5% of the total cytosolic protein pool (Burnett et al., 1979). It contains seven methionine and one cysteine group in its amino acid sequence (Thompson et al., 1999). It has been postulated that this protein functions as an intracellular buffer of long chain fatty acids and their CoA and carnitine esters thus maintaining a low concentration of their unbound form. Liver fatty acid binding protein also has been suggested to trap or scavenge cytotoxins and superoxide species, thus protecting cells from reactive oxygen species (Ek-Von Mentzer et al., 2001, Kaikaus et al., 1993, Khan and Sorof, 1990, Luebker et al., 2002). Because liver fatty acid binding protein forms a large portion of the intracellular protein pool and contains a large number of methionines and cysteine, it may have an important function as a cytoprotectant (Levine et al., 1999, Thomas et al., 1995). We previously reported that Chang liver cells were devoid of liver fatty acid binding protein. Transfecting those cells with liver fatty acid binding protein cDNA produced a new stably transfected cell line. Inducing oxidative stress in the liver fatty acid binding protein cDNA transfected and vector transfected cells, we reported that the liver fatty acid binding protein cDNA transfected cells were associated with lower reactive oxygen species levels than the same cells transfected with the vector (Wang et al., 2005), suggesting that the protein indeed has important intracellular antioxidative properties.

In this report we examined the role of liver fatty acid binding protein in a cholestatic liver disease model. The mechanism of cholestatic liver disease is not well understood and several hypotheses have been proposed including the involvement of oxidative stress (Aboutwerat et al., 2003, Ljubuncic et al., 2000). According to the oxidative stress hypothesis, endogenous antioxidant systems could prevent liver damage during cholestatic liver disease progression. The experimental model widely used to study cholestatic liver disease is the bile-duct ligation model (Kountouras et al., 1984) which is associated with decreased antioxidant activities of hepatic catalase, superoxide dismutase and glutathione peroxidase (Orellana et al., 2000). Moreover, liver mitochondria antioxidative capacity and glutathione are decreased in bile-duct ligated rats (Krahenbuhl et al., 1995). While exogenous antioxidants, vitamin E (lipophilic) and Trolox (hydrophilic) improved lipid peroxidation and oxidation of glutathione in bile-duct ligated rats, it had no effect on liver injury (Baron and Muriel, 1999). Whether other endogenous antioxidant systems are available within the liver to improve liver function is not clear. Interestingly, liver fatty acid binding protein levels are known to be reduced in steatosis (Hung et al., 2005). Since liver fatty acid binding protein has been thought to function as an effective antioxidant, it may play an important role in the prevention of cholestatic liver disease. In this report we demonstrated the expression and antioxidative function of liver fatty acid binding protein in an animal model of cholestatic liver disease induced by bile-duct ligation.