Proteomic Analysis Of The Spectrum Of Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) has emerged as one of the most common chronic liver diseases in recent years, partly due to the lifestyle changes, including physical inactivity and high daily fat consumption. NAFLD consists of a spectrum of liver disease, ranging from simple steatosis to steatohepatitis, fibrosis, cirrhosis and even hepatocellular carcinoma. NAFLD is strongly associated with obesity, type 2 diabetes and hyperdyslipemia, which are the main features of the recently characterized metabolic syndrome. While a high fat diet is one of the main causes that will induce obesity and its comorbidities. Accumulate evidence suggested that a higher intake of saturated fat can directly affect hepatic fatty infiltration and oxidative damage in different types of liver disease. However, the exact role of saturated fat to the development of NAFLD remains poorly understood. By feeding Sprague-Dawley (SD) rats a high-fat diet (HFD) reproduced many key features of human NAFLD. Current study adopted proteomic approaches to systematically analyze the liver proteomes during the development of HFD-induced NAFLD which may help clarify the pathogenesis of NAFLD.When SD rats were fed with a HFD for 4, 12 and 24 weeks, they successfully reproduced a spectrum of histological changes similar to human NAFLD: that was simple steatosis (SS) at 4 weeks, nonspecific inflammation (NSI) at 12 weeks, and steatohepatitis (NASH) at 24 weeks. Using two-dimensional difference gel electrophoresis (2-D DIGE) combined with MALDI-TOF/TOF analysis, ninety-five proteins exhibiting significant difference (ratio≥1.5 or≤-1.5, P<0.05) during the development of NAFLD were unambiguously identified. This included 53 proteins at SS stage, 42 proteins at NSI stage and 49 proteins at NASH stage. Biological functions of these proteins exhibited phase-specific characteristics during evolution of the disease: at steatosis stage, metabolic enzymes involved in lipid, carbohydrate and amino acid metabolisms were affected highlighted by depressed fatty acidβ-oxidation; by nonspecific inflammation stage, mitochondrial functions were impaired with decreasing proteins involved in mitochondrial respiratory chain and oxidative phosphorylation; up to steatohepatitis stage, cytoskeleton proteins were up-regulated, which may affect hepatocyte structural and functional integrity. Furthermore, a pathway analysis of these proteins identified PPARαand C/EBPαas key regulators that were subsequently verified by Western blot. The results showed that PPARa was decrease at all three stages of NAFLD, while C/EBPαwas increased at the earlier two stages of NAFLD. More importantly, we found that four ER chaperones, PDI, PDIA3, GRP78 and ERp29 were up-regulated especially at the more advanced stages of NAFLD. Up-regulation of ER chaperones is one of the important cellular markers of ER stress. Evidence suggested that ER stress was linked to the development of several diseases including obesity and type 2 diabetes, two major risk factors that strongly associated with NAFLD. Accordingly, we proposed that the disruption of ER function may contribute to the progression of NAFLD from steatosis to NASH. Together, these results help clarify the pathogenesis of NAFLD and identify potential targets for therapeutic interventions.