Published: August 29, 2011r2011 American Chemical Society 4757dx.doi.org/10.1021/pr200553c | J. Proteome Res. 2011, 10, 4757–4768ARTICLEpubs.acs.org/jprProteome of Skeletal Muscle Lipid Droplet Reveals Association withMitochondria and Apolipoprotein A-IHuina Zhang, †,‡ Yang Wang, †,‡,§ Jing Li, †,‡,§ Jinhai Yu, †,§ Jing Pu, †,§ Linghai Li, † Hongchao Zhang, ||Shuyan Zhang, † Gong Peng, †,§ Fuquan Yang,* ,† and Pingsheng Liu* ,†† National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China§ Graduate University of Chinese Academy of Sciences, Beijing, 100049, China)Department of Cardiac Surgery, Airforce Genral Hospital, Beijing, 100142, ChinabS Supporting Information’ INTRODUCTIONNeutral lipids are contained in two structures in the body, thecytoplasmic lipid droplet (LD) and lipoproteins carried in thebloodstream. 1 Although these two structures differ with respecttotheirsize,functionandlocation,bothlipoproteinparticlesandLDs have similar forms and may even have the same origin fromthe endoplasmic reticulum (ER). 2?4 Moreover, traditional lipo-proteinspecificproteins,theapolipoproteinfamilymembersapoBandapoA-V,wererecentlyfoundassociatedwithlipiddroplets(LDs). 5,6 Thephysiologicrolesoflipoproteinparticleshavebeenstudied for almost 40 years, 7,8 while their intracellular counter-parts, lipid droplets, have received close attention only recently. 9LDs are an ubiquitous cytoplasmic structure found inthe cellsof organisms from bacteria to mammals. 1 They are composed ofa hydrophobic neutral lipid core surrounded by a protein-coatedphospholipid monolayer. 1,10 Much in contrast to their simplestructure, LDs have been proposed to have multiple functions, 11such as lipid metabolism, 12?16 signal transduction, 17,18 proteinstorage 19 and lipid trafficking. 20 More than 16 proteomic studieshave shown that a variety of proteins are either embedded in orassociated with LDs, facilitating the multiple functions of thisorganelle. In addition, based on the characteristics of theidentifiedLDproteins,LDsaresuspectedtointeractwithvariousother cellular compartments including the cytoskeleton, 21ER, 22,23 endosomes, 24 mitochondria 25 and peroxisomes. 26 How-ever, these studies have been conducted in different species andtissues, precluding a comprehensive view of the LD proteincomposition.In humans, LDs are most prominent in adipose cells andsteroidogeniccells,butalsoexistinothercelltypes.Animportantbut so far unstudied tissue in terms of LD protein compositionand LD function is skeletal muscle. Skeletal muscle is the maintissue responsible for insulin-induced glucose disposal in thehumanbody.Furthermore,insulinresistanceofskeletalmuscleisfundamental to the development of obesity and Type 2 DiabetesMellitus (T2DM). Accumulated evidence indicates that aberra-tion of intramuscular triglyceride (IMTG) storage in skeletalmuscle LDs is more closely linked with insulin resistance andT2DM than excessive storage of lipid in liver and adiposetissue. 27?30 Accordingly, it is important to study the skeletalmuscle LD proteome to provide clues to the mechanism linkingIMTG and T2DM.Inthis study,wecarried outproteomic analysestoidentifytheproteinsofLDspurifiedfromskeletalmuscleaswellasoleicacid-treated C2C12 myotubes. We found 324 proteins in skeletalmuscleLDsandalmost72%ofthemarealsoidentifiedinLDs ofC2C12 myotubes. Among these proteins, 20% (65 proteins)wereassociatedwithmitochondriaand,amongthem,57proteinswere involved in TCA cycle, suggesting close proximity betweenReceived: June 9, 2011ABSTRACT: The lipid droplet (LD) is a universal organelle governing the storageand turnover of neutral lipids. Mounting evidence indicates that elevated intramus-cular triglyceride (IMTG) in skeletal muscle LDs is closely associated with insulinresistance and Type 2 Diabetes Mellitus (T2DM). Therefore, the identification ofthe skeletal muscle LD proteome will provide some clues to dissect the mechanismconnectingIMTGwithT2DM.Inthepresentwork,weidentified324LD-associatedproteins in mouse skeletal muscle LDs through mass spectrometry analysis. Besideslipid metabolism and membrane traffic proteins, a remarkable number of mitochondrial proteins were observed in the skeletalmuscle LD proteome. Furthermore, imaging by fluorescence microscopy and transmission electronic microscopy (TEM) directlydemonstrated that mitochondria closely adhere to LDs in vivo. Moreover, our results revealed for the first time that apolipoproteinA-I (apo A-I), the principal apolipoprotein of high density lipoprotein (HDL) particles, was also localized on skeletal muscle LDs.Further studies verified that apo A-I was expressed endogenously by skeletal muscle cells. In conclusion, we report the proteincomposition and characterization of skeletal muscle LDs and describe a novel LD-associated protein, apo A-I.KEYWORDS: lipid droplet, skeletal muscle, mitochondria, apolipoprotein A-I
