Quantitative analysis of the murine lipid droplet-associated proteome during diet-induced hepatic steatosis

Abstract

Hepatic steatosis is characterized by the accumulation of lipid droplets (LDs), which are composed of a neutral lipid core surrounded by a phospholipid monolayer embedded with many proteins. Although the LD-associated proteome has been investigated in multiple tissues and organisms, the dynamic changes in the murine LD-associated proteome in response to obesity and hepatic steatosis have not been studied. We characterized the hepatic LD-associated proteome of C57BL/6J male mouse livers following high-fat feeding using isobaric tagging for relative and absolute quantification. Of the 1,520 proteins identified with a 5% local false discovery rate, we report a total of 48 proteins that were increased and 52 proteins that were decreased on LDs in response to high-fat feeding. Most notably, ribosomal and endoplasmic reticulum proteins were increased and extracellular and cytosolic proteins were decreased in response to high-fat feeding. Additionally, many proteins involved in fatty acid catabolism or xenobiotic metabolism were enriched in the LD fraction following high-fat feeding. In contrast, proteins involved in glucose metabolism and liver X receptor or retinoid X receptor activation were decreased on LDs of high-fat-fed mice. This study provides insights into unique biological functions of hepatic LDs under normal and steatotic conditions. Hepatic steatosis is characterized by the accumulation of lipid droplets (LDs), which are composed of a neutral lipid core surrounded by a phospholipid monolayer embedded with many proteins. Although the LD-associated proteome has been investigated in multiple tissues and organisms, the dynamic changes in the murine LD-associated proteome in response to obesity and hepatic steatosis have not been studied. We characterized the hepatic LD-associated proteome of C57BL/6J male mouse livers following high-fat feeding using isobaric tagging for relative and absolute quantification. Of the 1,520 proteins identified with a 5% local false discovery rate, we report a total of 48 proteins that were increased and 52 proteins that were decreased on LDs in response to high-fat feeding. Most notably, ribosomal and endoplasmic reticulum proteins were increased and extracellular and cytosolic proteins were decreased in response to high-fat feeding. Additionally, many proteins involved in fatty acid catabolism or xenobiotic metabolism were enriched in the LD fraction following high-fat feeding. In contrast, proteins involved in glucose metabolism and liver X receptor or retinoid X receptor activation were decreased on LDs of high-fat-fed mice. This study provides insights into unique biological functions of hepatic LDs under normal and steatotic conditions. Non-alcoholic fatty liver disease (NAFLD), which is characterized by hepatic steatosis, is considered the hepatic component of metabolic syndrome. NAFLD is the most common chronic liver disease in Western nations and commonly occurs with comorbidities such as obesity and type 2 diabetes (1.Milić S. Štimac D. Nonalcoholic fatty liver disease/steatohepatitis: epidemiology, pathogenesis, clinical presentation and treatment.Dig. Dis. 2012; 30: 158-162Crossref PubMed Scopus (148) Google Scholar, 2.Williams K.H. Shackel N.A. Gorrell M.D. McLennan S.V. Twigg S.M. Diabetes and nonalcoholic fatty liver disease: a pathogenic duo.Endocr. Rev. 2013; 34: 84-129Crossref PubMed Scopus (168) Google Scholar). In support of a causative role for NAFLD, subjects with NAFLD have an increased risk of developing type 2 diabetes, cardiovascular disease, and liver cancer (3.Duan X-Y. Zhang L. Fan J-G. Qiao L. NAFLD leads to liver cancer: do we have sufficient evidence?.Cancer Lett. 2014; 345: 230-234Crossref PubMed Scopus (46) Google Scholar, 4.Adams L.A. Waters O.R. Knuiman M.W. Elliott R.R. Olynyk J.K. NAFLD as a risk factor for the development of diabetes and the metabolic syndrome: an eleven-year follow-up study.Am. J. Gastroenterol. 2009; 104: 861-867Crossref PubMed Scopus (328) Google Scholar, 5.Bhatia L.S. Curzen N.P. Calder P.C. Byrne C.D. Non-alcoholic fatty liver disease: a new and important cardiovascular risk factor?.Eur. Heart J. 2012; 33: 1190-1200Crossref PubMed Scopus (338) Google Scholar). Thus, understanding the pathobiology of NAFLD is critical for the prevention and treatment of this disease and its comorbidities. During hepatic steatosis, excess lipids accumulate in lipid droplets (LDs), which are composed of a neutral lipid core of mainly TG and cholesterol esters surrounded by a phospholipid monolayer (6.Fujimoto T. Parton R.G. Not just fat: the structure and function of the lipid droplet.Cold Spring Harb. Perspect. Biol. 2011; 3: a004838Crossref PubMed Scopus (316) Google Scholar). Hepatic LDs are dynamic organelles that change in size and number in response to acute perturbations, such as fasting/feeding, and to chronic diseases, such as obesity. LDs play a role in protein quality control, protein storage, cell signaling, and viral repl