Lipid Droplet Proteome in the Mouse Liver

QUIROGA, ARIEL DARIO; KRAMER, DAVID; FAHLMAN, RICHARD; LEHNER, RICHARD

Congreso; FASEB - Lipid Droplets: Metabolic Consequences of the Storage of Neutral Lipids; 2012

Lipid Droplet Proteome in the Mouse Liver Ariel D. Quiroga1,2, David Kramer3, Richard Fahlman3, and Richard Lehner1,2,4   1Department of Pediatrics, University of Alberta, Edmonton, AB, Canada 2Group on Molecular and Cell Biology of Lipids, University of Alberta, Edmonton, AB, Canada 3Department of Biochemistry, University of Alberta, Edmonton, AB, Canada 4Department of Cell Biology, University of Alberta, Edmonton, AB, Canada   Lipid droplets (LDs) are very dynamic organelles. During fasting, the liver increases lipid storage as a mean to reserve and provide energy for vital cellular functions. After re-feeding, hepatocytes rapidly decrease the number and size of LD. Little is known about the liver LD proteome and changes in the proteome during the fasting/re-feeding transition. This study aimed to investigate the hepatic LD proteome in fasted and re-fed conditions in the mouse. For this purpose we utilized 4 month old male C57BL/6 mice. Mice were randomly split into two groups: fasted (24 h fast) and re-fed (24 h fast followed by 6 h re-feeding). At the end of each period mice were sacrificed by cardiac puncture, livers were harvested, rinsed in ice-cold PBS and immediately subjected to homogenization in hypotonic lysis medium (HLM), and LD isolated according to Brasaemle and Wolins, 2006, with some modifications. The isolated LD proteins were resolved in 10% SDS-PAGE and analyzed by LC-MS/MS using a Thermo Easy nLC-II and an LTQ Orbitrap XL. We found a total of 425 proteins associated with LD in the fasting state, while 346 proteins were found in the LD during the fad state. While 292 proteins were common for both energetic states, 133 LD-associated proteins were unique for fasting and 45 were unique for re-feeding state. Some of the shared LD proteins in different feeding states were the canonical LD markers perilipin 2, perilipin 3, CTP:phosphocholine cytidylyltransferase A (CCT-á), lipases, acyl-CoA ligases and acyltransferases. Several endoplasmic reticulum markers have also been found, including calnexin and proteins normally localized to the lumen of this organelle. Among fasting unique proteins we found perilipin 5, ABHD5/CGI-58 and long-chain-fatty-acid-CoA ligase 1 (ACSL-1) and several mitochondrial and peroxisomal marker proteins, supporting the role of LDs in the provision of substrates for fatty acid oxidation. Re-feeding unique proteins included AGPAT2, S-adenosylmethionine synthase 1 and Steroid 17-alpha-hydroxylase among others. These results indicate the dynamic nature of hepatic LD proteome according to the energetic requirements of the cell.