PNNL

Abstract

Background: Alcohol abuse is a major cause for the development of alcohol-associated liver disease (ALD). The earliest manifestation of ALD is steatosis characterized by deposition of fat in specialized organelles called lipid droplets (LDs). While alcohol administration causes a rise in LD numbers in the hepatocytes, little is known regarding their characteristics that allow their accumulation and size increase. The aim of the present study is to gain insights of underlying pathophysiological mechanisms by investigating the ethanol-induced changes in hepatic LD proteome as a function of LD size. Methods: Adult Wistar rats were fed with ethanol liquid diet for 6 weeks. At sacrifice, the different hepatic LD subpopulations (LD1-LD3) based on size were isolated and subjected to morphological analysis, MS proteomics and Western blot. Results: Morphological analysis of LD1-LD3 fractions of ethanol-fed rats clearly demonstrated LD1 contained larger LDs compared with LD2 and LD3 fractions. Our preliminary results from principal component analysis (PCA) showed that the proteome of hepatic LD1 (largest-sized), LD2 (medium-sized) and LD3 (smaller-sized) fractions were distinctly different. Proteomic data analysis identified over 2000 proteins in each LD fraction with significant alterations in protein abundance among the three LD fractions. Out of the significantly altered proteins, 11 proteins are down-regulated, and 1687 proteins upregulated in LD1 fraction isolated from ethanol-fed rat livers compared with LD2 fraction. Similarly, 367 proteins are down regulated, and 274 proteins upregulated in LD2 fraction compared with that of LD3 fraction. There are 7 proteins down regulated, and 1595 proteins upregulated in LD1 fraction compared with that of LD3. Among this altered protein, several were related to fat metabolism, including synthesis, incorporation of fatty acid and lipolysis. Ingenuity Pathway analysis (IPA) revealed increased fatty acid synthesis, fatty acid incorporation, LD fusion and reduced lipolysis in larger sized LDs (LD1) compared to the smaller sized LDs (LD3). Overall, the proteomic findings indicate that the increased level of protein that facilitates fusion of LDs combined with an increased association of negative-regulators of lipolysis dictates the generation of larger-sized LDs during the development of alcohol-associated hepatic steatosis. Conclusion: Several significantly altered proteins were identified in different sized LDs isolated from livers of ethanol-fed rats. These proteins were involved in LDs metabolism and hepatocyte function and could be potential targets for treating alcohol-associated fatty liver disease.