Reduced expression of peroxisomal proliferator activated receptor alpha in the early stage of Nonalcoholic fatty liver disease.

초록

Background: The importance of Nonalcoholic fatty liver disease (NAFLD) in public health is increasing in the context of certain portion of patients may progress into a chronic liver disease. Although the pathogenesis of NAFLD has remained poorly understood, metabolic syndrome associated with insulin resistance is the most reproducible factor in the development of NAFLD. Fat accumulation in hepatocyte results from imbalance of input, output and oxidation of fatty acid. Peroxisome proliferators-activated receptors alpha (PPAR-alpha), nuclear receptor family forms heterodimer with retinoid receptor X, regulate the transcriptional expression of enzyme which is involved in beta oxidation of fatty acid. The peroxisomal beta oxidation spirals of very long chain fatty acid and long chain fatty acid are composed of enzymes, the first of which, Acyl Co-A oxidase (AOX) initiate the beta oxidation spiral. It has been suggested that inadequate PPAR-mediated response may contribute to abnormal fatty acid metabolism in non alcoholic fatty liver disease. The aim of this study was to define the role of PPAR-alpha and AOX, namely peroxisomal beta oxidation enzyme, in the development of NAFLD using the Otsuka Long-Evans Tokushima Fatty (OLETF) rat model. Materials and Methods: Liver tissue from OLETF (n=12) and control (n=1O, Letto) rats at 12th, 28th, 40th week were processed to histopathology and western blotting analysis. 100ug of protein extracted from liver were used for immunoblotting with PPAR-alpha antibody (Santa Cruz, U.S.A). Total RNA was extracted using TriZol (Gibco BRL, U.S.A) according to the manufacturer's protocol and reverse transcribed using first strand cDNA synthesis kit (Roche, U.S.A). PPAR-alpha and AOX mRNA were measured using Real Time RT PCR (Opticon, MJ Research, U.S.A). From the threshold cycle of target gene and beta-actin, relative quantification of mRNA was calculated as 2-delta_upper;delta_upper;Ct. Results: At age 40h week, histological analysis of OLETF rat liver showed steatosis (~66%) and mild inflammation comparable to NAFLD. But, at 12th, 28th week, the liver of OLETF rat did not show definite evidence of steatosis or inflammation. The control liver did not show histological changes at any ages. We examined the sequential expression of important peroxisomal beta oxidation enzyme, namely AOX and PPAR-alpha in OLETF and control rats. In OLETF rats which have been shown comparable histological changes to NAFLD, AOX mRNA was 63% of control at the age of 12th week. Because the transcription of AOX is regulated by PPAR-alpha, we measured the PPAR-alpha mRNA expression at the age of 12 weeks. Messenger RNA expression of PPAR- alpha in OLETF was 3% at the age of 12th week Protein expression of PPAR-alpha was also significantly reduced 17% of control at 12& week But at the age of 28th, 40th weeks, the PPAR-alpha protein expression was gradually increased up to 75%, 78% of control. Along with the changes of protein expression, PPAR-alpha mRNA expression was increased up to 26% and 110% of control. AOX, regulated by PPAR-alpha, also increased 220% and 110% of controls. Conclusion: Reduced expression of PPAR-alpha and AOX even in the absence of definite histological changes was observed on OLETF rat model. Alteration of fatty acid metabolism in early stage of NAFLD may have an important role in the development of non-alcoholic fatty liver disease.