Expression and antioxidant function of liver fatty acid binding protein in normal and bile-duct ligated rats
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.
Section snippets
Materials
Trizol LS Reagent was purchased from GIBCO/BRL (Burlington, ON). All other chemicals were purchased from Sigma-Aldrich Canada LTD (Oakville, ON). Rat L-FABP antibody was raised in our lab (Wang et al., 2004). Rabbit anti-rat IgG and the enhanced chemiluminescence Western blot kit were purchased from Amersham-Pharmacia Biotech Inc. (Baie d'Urfe, Quebec). Advantage RT-for-PCR Kit, Advantage cDNA PCR Kit and Polymerase Mix were purchased from Clontech Laboratories Inc. (Palo Alto, CA). Male
Liver injury following bile-duct ligation
As shown in Fig. 1, bile-duct proliferation and mononuclear cell infiltration were detected in the portal area of the bile-duct ligated rat liver sections. Macrovascular cytoplasmic alterations of hepatocytes and many lysed cell areas (arrows in Fig. 1) were observed in this group. Bile-duct ligation also was associated with significant proliferation of bile-duct epithelial cells, inflammation and altered liver structure (circle arrow in Fig. 1).
Liver fatty acid binding protein expression in bile-duct ligated rats
Liver fatty acid binding protein mRNA and protein
Discussion
Bile-duct ligation is a typical model of biliary disease in animals, which features proliferation of bile-duct epithelial cells, hepatocellular necrosis and apoptosis, stellate cell activation and eventually the formation of liver fibrosis and cirrhosis (Kountouras et al., 1984, Scobie and Summerskill, 1965). Bile-duct ligation has been associated with hepatic mitochondrial dysfunction that includes oxidative damage to mitochondrial proteins and lipids and cytotoxicity of bile components such
Acknowledgements
This work was supported by an operating grant from the Canadian Institute of Health Research Grant. GQ Wang gratefully acknowledges research award from CIHR/Rx&D.
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