Quantitative analysis of the murine lipid droplet-associated proteome during diet-induced hepatic steatosis
2015; Elsevier BV; Volume: 56; Issue: 12 Linguagem: Inglês
10.1194/jlr.m056812
ISSN1539-7262
AutoresSalmaan Khan, Edith E. Wollaston‐Hayden, Todd W. Markowski, LeeAnn Higgins, Douglas G. Mashek,
Tópico(s)Pancreatic function and diabetes
ResumoHepatic 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. 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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 replication, in addition to their major role as lipid metabolism mediators (7.Walther T.C. Farese R.V. Lipid droplets and cellular lipid metabolism.Annu. Rev. Biochem. 2012; 81: 687-714Crossref PubMed Scopus (975) Google Scholar). The dynamic nature of protein association with LDs is not fully understood (8.Hodges B.D.M. Wu C.C. Proteomic insights into an expanded cellular role for cytoplasmic lipid droplets.J. Lipid Res. 2010; 51: 262-273Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Known LD proteins have diverse targeting mechanisms, from hydrophobic protein segments to protein-protein interactions to covalent lipid modifications (7.Walther T.C. Farese R.V. Lipid droplets and cellular lipid metabolism.Annu. Rev. Biochem. 2012; 81: 687-714Crossref PubMed Scopus (975) Google Scholar) and some proteins dynamically shuttle to and from LDs (9.Bartz R. Zehmer J.K. Zhu M. Chen Y. Serrero G. Zhao Y. Liu P. Dynamic activity of lipid droplets: protein phosphorylation and GTP-mediated protein translocation.J. Proteome Res. 2007; 6: 3256-3265Crossref PubMed Scopus (245) Google Scholar, 10.Egan J.J. Greenberg A.S. Chang M.K. Wek S.A. Moos M.C.J. Londos C. Mechanism of hormone-stimulated lipolysis in adipocytes: translocation of hormone-sensitive lipase to the lipid storage droplet.Proc. Natl. Acad. Sci. USA. 1992; 89: 8537-8541Crossref PubMed Scopus (346) Google Scholar, 11.Turró S. Ingelmo-Torres M. Estanyol J.M. Tebar F. Fernández M.A. Albor C.V. Gaus K. Grewal T. Enrich C. Pol A. Identification and characterization of associated with lipid droplet protein 1: a novel membrane-associated protein that resides on hepatic lipid droplets.Traffic. 2006; 7: 1254-1269Crossref PubMed Scopus (160) Google Scholar). Additionally, LDs directly interact with numerous organelles and in some cases these interactions can result in protein transfer (12.Yang L. Ding Y. Chen Y. Zhang S. Huo C. Wang Y. Yu J. Zhang P. Na H. Zhang H. et al.The proteomics of lipid droplets: structure, dynamics, and functions of the organelle conserved from bacteria to humans.J. Lipid Res. 2012; 53: 1245-1253Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 13.Wilfling F. Wang H. Haas J.T. Krahmer N. Gould T.J. Uchida A. Cheng J-X. Graham M. Christiano R. Fröhlich F. et al.Triacylglycerol synthesis enzymes mediate lipid droplet growth by relocalizing from the ER to lipid droplets.Dev. Cell. 2013; 24: 384-399Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar). Recently, the human hepatic LD proteome has been described for the first time in NAFLD subjects (14.Su W. Wang Y. Jia X. Wu W. Li L. Tian X. Li S. Wang C. Xu H. Cao J. et al.Comparative proteomic study reveals 17β-HSD13 as a pathogenic protein in nonalcoholic fatty liver disease.Proc. Natl. Acad. Sci. USA. 2014; 111: 11437-11442Crossref PubMed Scopus (124) Google Scholar). A few studies have examined the murine hepatic LD proteome (11.Turró S. Ingelmo-Torres M. Estanyol J.M. Tebar F. Fernández M.A. Albor C.V. Gaus K. Grewal T. Enrich C. Pol A. Identification and characterization of associated with lipid droplet protein 1: a novel membrane-associated protein that resides on hepatic lipid droplets.Traffic. 2006; 7: 1254-1269Crossref PubMed Scopus (160) Google Scholar, 15.Crunk A.E. Monks J. Murakami A. Jackman M. MacLean P.S. Ladinsky M. Bales E.S. Cain S. Orlicky D.J. McManaman J.L. Dynamic regulation of hepatic lipid droplet properties by diet.PLoS One. 2013; 8: e67631Crossref PubMed Scopus (56) Google Scholar); however, changes in the LD proteome have not been quantified in a chronic animal model of steatosis. In the present study, we have quantified the hepatic LD-associated proteome and characterized its changes in response to diet-induced development of hepatic steatosis. The findings from this study further our understanding of LD biology and the dynamic nature of the LD-associated proteome. Male C57BL/6J weaned mice (n = 3) were fed a 60% fat diet [high-fat diet (HFD), F3282; Bio-Serv] or a normal fat diet [normal diet (ND), F4031; Bio-Serv] for 9 weeks. The liver was excised from fed anesthetized mice at approximately 9:00 AM and a portion was reserved for microscopy prior to LD isolation. All procedures were approved by the University of Minnesota Institutional Animal Care and Use Committee. LDs were isolated from freshly excised livers using the method of Zhang et al. (16.Zhang H. Wang Y. Li J. Yu J. Pu J. Li L. Zhang H. Zhang S. Peng G. Yang F. et al.Proteome of skeletal muscle lipid droplet reveals association with mitochondria and apolipoprotein A-I.J. Proteome Res. 2011; 10: 4757-4768Crossref PubMed Scopus (142) Google Scholar) with the following modifications. The minced liver was resuspended in 4 ml buffer A [25 mM tricene (pH 7.6), 250 mM sucrose, and protease inhibitor cocktail] and dounce homogenized 30 times on ice. Cells were homogenized using a needle and syringe instead of a nitrogen bomb and then centrifuged for 10 min at 1,000 g. Following addition of 4 ml of buffer A, the postnuclear supernatant was divided into two ultracentrifuge tubes, overlaid with 3 ml buffer B [20 mM HEPES (pH 7.4), 100 mM KCl, 2 mM MgCl2, and protease inhibitor cocktail] and centrifuged at 300,000 g for 1 h at 4°C. The LD-containing band was transferred to a fresh tube and centrifuged at 20,000 g for 20 min at 4°C. The underlying liquid was carefully removed and the LD fraction was washed six times with 200 μl buffer B to remove copurifying membranes (16.Zhang H. Wang Y. Li J. Yu J. Pu J. Li L. Zhang H. Zhang S. Peng G. Yang F. et al.Proteome of skeletal muscle lipid droplet reveals association with mitochondria and apolipoprotein A-I.J. Proteome Res. 2011; 10: 4757-4768Crossref PubMed Scopus (142) Google Scholar, 17.Ding Y. Zhang S. Yang L. Na H. Zhang P. Zhang H. Wang Y. Chen Y. Yu J. Huo C. et al.Isolating lipid droplets from multiple species.Nat. Protoc. 2013; 8: 43-51Crossref PubMed Scopus (115) Google Scholar) and acetone precipitated overnight. The LD precipitate was reconstituted with 65 μl of protein solubilization buffer [7 M urea, 2 M thiourea, 0.4 M triethylammonium bicarbonate (pH 8.5), 20% methanol, and 4 mM tris(2-carboxyethyl)phosphine]. The samples were bath sonicated for 2 min. The samples were then transferred to a pressure cycling technology tube with a 50 μl cap for the Barocycler NEP2320 (Pressure Biosciences, Inc.) and cycled between 35 kpsi for 30 s and 0 kpsi for 15 s for 40 cycles at 37°C. Two hundred millimoles of methyl methanethiosulfonate were added to a final concentration of 8 mM. Protein concentration was determined by Bradford assay. A 28 μg aliquot of each sample was transferred to a new 1.5 ml microfuge tube and brought to the same volume with protein solubilization buffer plus 8 mM methyl methanethiosulfonate. All samples were diluted 4-fold with 80% ultra-pure water; 20% methanol and trypsin (Promega) were added in a 1:35 ratio of trypsin to total protein. Samples were incubated overnight for 16 h at 37°C after which they were frozen at −80°C for 0.5 h and dried in a vacuum centrifuge. Subsequently, samples were cleaned with a 4 ml Extract Clean™ C18 SPE cartridge (Grace-Davidson) and eluates were vacuum dried and resuspended in 0.5 M triethylammonium bicarbonate (pH 8.5) to a final 1 μg/μl concentration. Twenty-six micrograms of each sample were labeled with isobaric tagging for relative and absolute quantification (iTRAQ)® 8-plex reagent (AB Sciex, Foster City, CA). After labeling, the samples were multiplexed together and dried in vacuo. The multiplexed sample was cleaned with a 4 ml Extract Clean™ C18 SPE cartridge, and the eluate was dried in vacuo. The iTRAQ®-labeled sample was resuspended in buffer A [10 mM ammonium formate (pH 10) in 98:2 water:acetonitrile] and fractionated offline by high pH C18 reversed-phase chromatography. A MAGIC 2002 HPLC system (Michrom BioResources, Inc.) was used with a C18 Gemini-NX column [150 mm × 2 mm internal diameter, 5 μm particle, 110 Å pore size (Phenomenex)]. The flow rate was 150 μl/min with a gradient from 0 to 35% buffer B [10 mM ammonium formate (pH 10) in 10:90 water:acetonitrile] over 60 min, followed by 35–60% over 5 min. Fractions were collected every 2 min and UV absorbances were monitored at 215 nm and 280 nm. Peptide-containing fractions were divided into two equal numbered groups, “early” and “late”. The first early fraction was concatenated with the first late fraction, and so on (18.Yang F. Shen Y. Camp D.G. Smith R.D. High pH reversed-phase chromatography with fraction concatenation as an alternative to strong-cation exchange chromatography for two-dimensional proteomic analysis.Expert Rev. Proteomics. 2012; 9: 129-134Crossref PubMed Scopus (199) Google Scholar). Concatenated samples were dried in vacuo, resuspended in load solvent (98:2:0.01, water:acetonitrile:formic acid), and 1.5 μg aliquots were run on a Velos Orbitrap mass spectrometer (Thermo Fisher Scientific, Inc.) as described previously, with the exception that the activation energy was 40 ms (19.Lin-Moshier Y. Sebastian P.J. Higgins L. Sampson N.D. Hewitt J.E. Marchant J.S. Re-evaluation of the role of calcium homeostasis endoplasmic reticulum protein (CHERP) in cellular calcium signaling.J. Biol. Chem. 2013; 288: 355-367Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The mass spectrometer RAW data (ProteoWizard files) were converted to mzXML using MSconvert software and to MGF files using TINT RAW-to-MGF converter (http://github.com/jmchilton/tint). ProteinPilot 4.5 (AB Sciex) searches were performed against the NCBI reference sequence Mus musculus (taxon 10090; April 21, 2012 version) protein FASTA database with canonical and isoform sequences (85,763 proteins), to which a contaminant database (http://www.thegpm.org/crap/) was appended. Search parameters were as follows: cysteine MMTS; iTRAQ 8plex (peptide labeled); trypsin; instrument Orbi MS (1–3 ppm) Orbi MS/MS; biological modifications ID focus; thorough search effort; and false discovery rate analysis (with reversed database). Proteins were accepted if they were identified by two or more unique peptides and fell within the 5% local false discovery rate cutoff in all of the biological replicates. Relative quantification of each protein was performed using ProteinPilot 4.5 (AB Sciex) with bias correction. Each ND sample (tag 113–115) was used as a denominator and protein summary results were exported to Microsoft Excel. The HFD/ND ratios and the corresponding P values from each report were combined, yielding nine total HFD/ND ratios. A subset of proteins was generated with the following criteria: Protein Pilot P values <0.05 for six out of nine protein ratios and a log fold change of ≥0.55 or less than or equal to −0.55, and two times the standard deviation of the nine ratios was not greater than the average log fold change. Formalin-fixed paraffin-embedded liver sections were processed for immunohistochemistry using standard methodologies. Slides were examined using a Nikon A1 spectral confocal microscope and NIS Elements imaging software. The nucleus was stained with DAPI and perilipin 2 (PLIN2) was immunolabeled with chicken polyclonal primary antibody (Abcam, Cambridge, MA; ab37516) and Rhodamine Red-X-conjugated donkey anti-chicken secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). Acyl-CoA synthetase 1 (ACSL1) was immunolabeled with goat primary antibody (Santa Cruz Biotechnology) and Alexa-Fluor 647-conjugated donkey anti-goat secondary antibody (Jackson ImmunoResearch Laboratories). Similarly, carnitine palmitoyltransferase 2 (CPT2), glucose phosphate isomerase (GPI), malic enzyme (ME), and ribosomal protein large P0 (RPLP0) were labeled with specific primary antibodies (One World Lab, San Diego, CA) and Alexa-Fluor 488 anti-rabbit secondary antibody (Cell Signaling). Voltage-dependent anion channel was labeled with a specific primary antibody (Cell Signaling) and Alexa-Fluor 488 anti-rabbit secondary antibody. Images were obtained with a 60× magnification under oil immersion. For immunoblotting, equal amounts of protein, verified by Ponceau staining, were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes for Western blot analysis. Primary antibodies used were as follows: anti-ATP-citrate lyase (ATPCL), anti-binding immunoglobulin protein (BIP), anti-cyclooxygenase IV (COX IV) (Cell Signaling Technology), anti-β-actin (LI-COR Biosciences), anti- PLIN2 (kindly provided by Dr. Andrew Greenberg), anti-early endosomal antigen 1 (Bethel Laboratories), and anti-catalase and anti-anexin-2 (One World Lab). For electron microscopy, livers of mice fed ND or HFD were perfused with 2.5% glutaraldehyde in 0.1 M sodium cacodylate. Tissues were subsequently washed, sectioned, and stained using standard methods (20.Gerbens F. Jansen A. van Erp A.J. Harders F. Meuwissen T.H. Rettenberger G. Veerkamp J.H. te Pas M.F. The adipocyte fatty acid-binding protein locus: characterization and association with intramuscular fat content in pigs.Mamm. Genome. 1998; 9: 1022-1026Crossref PubMed Scopus (110) Google Scholar). Sections were observed under a JEOL 1200 EX II transmission electron microscope (JEOL Ltd., Tokyo, Japan). Images were obtained using a Veleta 2K×2K camera with iTEM software (Olympus SIS, Munster, Germany). For quantitative analysis, images from 20 cells were randomly selected from each mouse (two mice per dietary group). LDs, mitochondria, and their physical interactions were subsequently quantified. Ingenuity Pathway Analysis (IPA) (QIAGEN) was used to assign enriched biological function and canonical pathway categories. The analysis settings were restricted to experimentally observed and hepatoma cell lines or liver. Suspension trapping (STRAP) was used for cellular component Gene Ontology (GO) term protein annotation (21.Bhatia V.N. Perlman D.H. Costello C.E. McComb M.E. Software tool for researching annotations of proteins (STRAP): open-source protein annotation software with data visualization.Anal. Chem. 2009; 81: 9819-9823Crossref PubMed Scopus (191) Google Scholar). Other GO term annotation was retrieved using QuickGO from the UniProt Consortium GO annotation dataset (22.Binns D. 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Postnuclear supernatant and LD fractions were evaluated for purity based on the presence of common copurifying organelles using Western blotting of marker proteins (Fig. 1B, supplementary Fig. 1). These data show that the LD fraction is highly enriched for the LD marker, PLIN2, while we were unable to detect common endoplasmic reticulum (ER) (BIP), mitochondrial (COX IV), cytosolic (ATPCL), plasma membrane (annexin-2), and endosome (early endosomal antigen 1) contaminating proteins. However, the peroxisomal marker protein, catalase, was not depleted in the LD fraction. Although the LD fraction is largely free of contaminant proteins, the sucrose centrifugation method to isolate LD does yield some contaminant proteins, as has been reported in other LD proteomic studies (14.Su W. Wang Y. Jia X. Wu W. Li L. Tian X. Li S. Wang C. Xu H. Cao J. et al.Comparative proteomic study reveals 17β-HSD13 as a pathogenic protein in nonalcoholic fatty liver disease.Proc. Natl. Acad. Sci. USA. 2014; 111: 11437-11442Crossref PubMed Scopus (124) Google Scholar, 15.Crunk A.E. Monks J. Murakami A. Jackman M. MacLean P.S. Ladinsky M. Bales E.S. Cain S. Orlicky D.J. McManaman J.L. Dynamic regulation of hepatic lipid droplet properties by diet.PLoS One. 2013; 8: e67631Crossref PubMed Scopus (56) Google Scholar, 16.Zhang H. Wang Y. Li J. Yu J. Pu J. Li L. Zhang H. Zhang S. Peng G. Yang F. et al.Proteome of skeletal muscle lipid droplet reveals association with mitochondria and apolipoprotein A-I.J. Proteome Res. 2011; 10: 4757-4768Crossref PubMed Scopus (142) Google Scholar). Following LC-MS/MS analysis, we identified 1,520 LD-associated proteins (supplementary Table 1). The proteins identified through this method are proteins that are associated with the LD fraction and are not necessarily bona-fide LD proteins confirmed to reside primarily or exclusively on the LD and, thus, will be referred to as LD-associated proteins. For comparison, a list of the LD-associated proteins identified by previous proteomic studies of mammalian cells and tissues was compiled (10.Egan J.J. Greenberg A.S. Chang M.K. Wek S.A. Moos M.C.J. Londos C. Mechanism of hormone-stimulated lipolysis in adipocytes: translocation of hormone-sensitive lipase to the lipid storage droplet.Proc. Natl. Acad. Sci. USA. 1992; 89: 8537-8541Crossref PubMed Scopus (346) Google Scholar, 11.Turró S. Ingelmo-Torres M. Estanyol J.M. Tebar F. Fernández M.A. Albor C.V. Gaus K. Grewal T. Enrich C. Pol A. Identification and characterization of associated with lipid droplet protein 1: a novel membrane-associated protein that resides on hepatic lipid droplets.Traffic. 2006; 7: 1254-1269Crossref PubMed Scopus (160) Google Scholar, 15.Crunk A.E. Monks J. Murakami A. Jackman M. MacLean P.S. Ladinsky M. Bales E.S. Cain S. Orlicky D.J. McManaman J.L. 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From this analysis, we determined that 671 out of 1,520 proteins identified have been reported in previous mammalian LD proteomics studies. In addition, 72 and 95% of the proteins found in previous studies on human (14.Su W. Wang Y. Jia X. Wu W. Li L. Tian X. Li S. Wang C. Xu H. Cao J. et al.Comparative proteomic study reveals 17β-HSD13 as a pathogenic protein in nonalcoholic fatty liver disease.Proc. Natl. Acad. Sci. USA. 2014; 111: 11437-11442Crossref PubMed Scopus (124) Google Scholar) and mouse (15.Crunk A.E. Monks J. Murakami A. Jackman M. MacLean P.S. Ladinsky M. Bales E.S. Cain S. Orlicky D.J. McManaman J.L. Dynamic regulation of hepatic lipid droplet properties by diet.PLoS One. 2013; 8: e67631Crossref PubMed Scopus (56) Google Scholar) hepatic LDs, respectively, were identified in our analysis (supplementary Table 1). Using confocal microscopy, we show that three of these proteins, RPLP0, GPI, and ME1, show partial colocalization with the LD marker protein, PLIN2, in mouse liver tissue sections (supplementary Fig. 2). It should be noted that these proteins do not primarily localize to LDs and the small amount of colocalization would suggest that these, and perhaps many other proteins identified, are not abundant LD-associated proteins. We next performed functional annotation of the hepatic LD-associated proteome.
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