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Proteomic Analysis of Extracellular Vesicles Released by Adipocytes of Otsuka Long-Evans Tokushima Fatty (OLETF) Rats

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Abstract

Extracellular vesicles (EVs) such as exosomes are secretory vesicles that act as autocrine, paracrine, or endocrine messengers; mediate intercellular cross-talk; and carry a cargo of various proteins. Because EVs can be transported to recipient cells via circulation, many researchers have been studying EVs from immune cells or cancer cells. Adipocytes are also considered endocrine cells and secrete adipokines such as adiponectin, regulating a variety of intracellular signaling pathways. Expansion of adipose tissue in obesity alters adipokine secretion, thereby increasing the risk of metabolic diseases. Characterization of adipocyte-derived exosomes is necessary to explain the communication between adipocytes and other cell types. In the present study, to identify proteins associated with adipocyte-derived exosomes, we isolated exosomes from adipose tissue of obese diabetic and obese nondiabetic rats. We identified proteins by analyzing exosomes from obese rats with type 2 diabetes and their matched control littermates using nano-liquid chromatography with tandem mass spectrometry coupled with label-free relative quantification. We identified 509 proteins from adipocytes including 81 known adipokines; ~78 % of all the identified proteins were categorized as exosome-associated proteins. Among the protein profiles, we uncovered 128 upregulated and 72 downregulated proteins, which are differentially expressed in OLETF adipocyte-derived exosomes. This study seems to demonstrate for the first time hundreds of proteins in exosomes released by adipocytes in obese rats and rats with type 2 diabetes. Thus, protein profiles of exosomes from adipocytes possibly indicate the transmission of signals as part of cell–cell communication and should further our understanding of obesity- and diabetes-related diseases.

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Abbreviations

EVs:

Extracellular vesicles

LETO:

Long-Evans Tokushima Otsuka

OLETF:

Otsuka Long-Evans Tokushima fatty

UPLC:

Ultra-performance liquid chromatography

GPI:

Glycophosphatidylinositol

TEM:

Transmission electron microscope

NTA:

Nanoparticle tracking analysis

TEABC:

Triethylammonium bicarbonate

DDA:

Data-dependent analysis

References

  1. Skog J, Wurdinger T, van Rijn S, Meijer DH, Gainche L, Sena-Esteves M, Curry WT Jr, Carter BS, Krichevsky AM, Breakefield XO (2008) Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat Cell Biol 10(12):1470–1476. doi:10.1038/ncb1800

    Article  CAS  Google Scholar 

  2. Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO (2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 9(6):654–659. doi:10.1038/ncb1596

    Article  CAS  Google Scholar 

  3. Deng ZB, Poliakov A, Hardy RW, Clements R, Liu C, Liu Y, Wang J, Xiang X, Zhang S, Zhuang X, Shah SV, Sun D, Michalek S, Grizzle WE, Garvey T, Mobley J, Zhang HG (2009) Adipose tissue exosome-like vesicles mediate activation of macrophage-induced insulin resistance. Diabetes 58(11):2498–2505. doi:10.2337/db09-0216

    Article  CAS  Google Scholar 

  4. Gross JC, Chaudhary V, Bartscherer K, Boutros M (2012) Active Wnt proteins are secreted on exosomes. Nat Cell Biol 14(10):1036–1045. doi:10.1038/ncb2574

    Article  CAS  Google Scholar 

  5. Luga V, Zhang L, Viloria-Petit AM, Ogunjimi AA, Inanlou MR, Chiu E, Buchanan M, Hosein AN, Basik M, Wrana JL (2012) Exosomes mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell migration. Cell 151(7):1542–1556. doi:10.1016/j.cell.2012.11.024

    Article  CAS  Google Scholar 

  6. Peinado H, Aleckovic M, Lavotshkin S, Matei I, Costa-Silva B, Moreno-Bueno G, Hergueta-Redondo M, Williams C, Garcia-Santos G, Ghajar C, Nitadori-Hoshino A, Hoffman C, Badal K, Garcia BA, Callahan MK, Yuan J, Martins VR, Skog J, Kaplan RN, Brady MS, Wolchok JD, Chapman PB, Kang Y, Bromberg J, Lyden D (2012) Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET. Nat Med 18(6):883–891. doi:10.1038/nm.2753

    Article  CAS  Google Scholar 

  7. Moon PG, You S, Lee JE, Hwang D, Baek MC (2011) Urinary exosomes and proteomics. Mass Spectrom Rev 30(6):1185–1202. doi:10.1002/mas.20319

    Article  CAS  Google Scholar 

  8. Antonyak MA, Li B, Boroughs LK, Johnson JL, Druso JE, Bryant KL, Holowka DA, Cerione RA (2011) Cancer cell-derived microvesicles induce transformation by transferring tissue transglutaminase and fibronectin to recipient cells. Proc Natl Acad Sci USA 108(12):4852–4857. doi:10.1073/pnas.1017667108

    Article  CAS  Google Scholar 

  9. Sano S, Izumi Y, Yamaguchi T, Yamazaki T, Tanaka M, Shiota M, Osada-Oka M, Nakamura Y, Wei M, Wanibuchi H, Iwao H, Yoshiyama M (2014) Lipid synthesis is promoted by hypoxic adipocyte-derived exosomes in 3T3-L1 cells. Biochem Biophys Res Commun 445(2):327–333. doi:10.1016/j.bbrc.2014.01.183

    Article  CAS  Google Scholar 

  10. Muller G, Jung C, Straub J, Wied S, Kramer W (2009) Induced release of membrane vesicles from rat adipocytes containing glycosylphosphatidylinositol-anchored microdomain and lipid droplet signalling proteins. Cell Signal 21(2):324–338. doi:10.1016/j.cellsig.2008.10.021

    Article  Google Scholar 

  11. Muller G, Schneider M, Biemer-Daub G, Wied S (2011) Microvesicles released from rat adipocytes and harboring glucosylphosphatidylinositol-anchored proteins transfer RNA stimulating lipid synthesis. Cell Signal 23(7):1207–1223. doi:10.1016/j.cellsig.2011.03.013

    Article  Google Scholar 

  12. Phoonsawat W, Aoki-Yoshida A, Tsuruta T, Sonoyama K (2014) Adiponectin is partially associated with exosomes in mouse serum. Biochem Biophys Res Commun 448(3):261–266. doi:10.1016/j.bbrc.2014.04.114

    Article  CAS  Google Scholar 

  13. Wellen KE, Hotamisligil GS (2005) Inflammation, stress, and diabetes. J Clin Investig 115(5):1111–1119. doi:10.1172/JCI25102

    Article  CAS  Google Scholar 

  14. Kershaw EE, Flier JS (2004) Adipose tissue as an endocrine organ. The Journal of clinical endocrinology and metabolism 89(6):2548–2556. doi:10.1210/jc.2004-0395

    Article  CAS  Google Scholar 

  15. Scherer PE (2006) Adipose tissue: from lipid storage compartment to endocrine organ. Diabetes 55(6):1537–1545. doi:10.2337/db06-0263

    Article  CAS  Google Scholar 

  16. Kannel WB, Cupples LA, Ramaswami R, Stokes J 3rd, Kreger BE, Higgins M (1991) Regional obesity and risk of cardiovascular disease; the Framingham Study. J Clin Epidemiol 44(2):183–190

    Article  CAS  Google Scholar 

  17. Maeda K, Okubo K, Shimomura I, Mizuno K, Matsuzawa Y, Matsubara K (1997) Analysis of an expression profile of genes in the human adipose tissue. Gene 190(2):227–235

    Article  CAS  Google Scholar 

  18. Hotamisligil GS, Shargill NS, Spiegelman BM (1993) Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science 259(5091):87–91

    Article  CAS  Google Scholar 

  19. Kobayashi K (2005) Adipokines: therapeutic targets for metabolic syndrome. Curr Drug Targets 6(4):525–529

    Article  CAS  Google Scholar 

  20. Matsuzawa Y (2005) White adipose tissue and cardiovascular disease. Best Pract Res Clin Endocrinol Metabol 19(4):637–647. doi:10.1016/j.beem.2005.07.001

    Article  CAS  Google Scholar 

  21. Lehr S, Hartwig S, Lamers D, Famulla S, Muller S, Hanisch FG, Cuvelier C, Ruige J, Eckardt K, Ouwens DM, Sell H, Eckel J (2012) Identification and validation of novel adipokines released from primary human adipocytes. Mol Cell Proteomics : MCP 11(1):M111 010504. doi:10.1074/mcp.M111.010504

    Article  Google Scholar 

  22. Ishida K, Mizuno A, Min Z, Sano T, Shima K (1995) Which is the primary etiologic event in Otsuka Long-Evans Tokushima Fatty rats, a model of spontaneous non-insulin-dependent diabetes mellitus, insulin resistance, or impaired insulin secretion? Metab Clin Exp 44(7):940–945

    Article  CAS  Google Scholar 

  23. Kawano K, Hirashima T, Mori S, Saitoh Y, Kurosumi M, Natori T (1992) Spontaneous long-term hyperglycemic rat with diabetic complications. Otsuka Long-Evans Tokushima Fatty (OLETF) strain. Diabetes 41(11):1422–1428

    Article  CAS  Google Scholar 

  24. Kawano K, Hirashima T, Mori S, Natori T (1994) OLETF (Otsuka Long-Evans Tokushima Fatty) rat: a new NIDDM rat strain. Diabetes Res Clin Pract 24(Suppl):S317–S320

    Article  Google Scholar 

  25. Muller G, Ertl J, Gerl M, Preibisch G (1997) Leptin impairs metabolic actions of insulin in isolated rat adipocytes. J Biol Chem 272(16):10585–10593

    Article  CAS  Google Scholar 

  26. Cho YE, Singh TS, Lee HC, Moon PG, Lee JE, Lee MH, Choi EC, Chen YJ, Kim SH, Baek MC (2012) In-depth identification of pathways related to cisplatin-induced hepatotoxicity through an integrative method based on an informatics-assisted label-free protein quantitation and microarray gene expression approach. Mol Cell Proteomics MCP 11(1):M111 010884. doi:10.1074/mcp.M111.010884

    Article  Google Scholar 

  27. Lee JE, Park JH, Moon PG, Baek MC (2013) Identification of differentially expressed proteins by treatment with PUGNAc in 3T3-L1 adipocytes through analysis of ATP-binding proteome. Proteomics 13(20):2998–3012. doi:10.1002/pmic.201200549

    CAS  Google Scholar 

  28. Tsou CC, Tsai CF, Tsui YH, Sudhir PR, Wang YT, Chen YJ, Chen JY, Sung TY, Hsu WL (2010) IDEAL-Q, an automated tool for label-free quantitation analysis using an efficient peptide alignment approach and spectral data validation. Mol Cell Proteomics MCP 9(1):131–144. doi:10.1074/mcp.M900177-MCP200

    Article  CAS  Google Scholar 

  29. Moon PG, Kwack MH, Lee JE, Cho YE, Park JH, Hwang D, Kim MK, Kim JC, Sung YK, Baek MC (2013) Proteomic analysis of balding and non-balding mesenchyme-derived dermal papilla cells from androgenetic alopecia patients using on-line two-dimensional reversed phase-reversed phase LC-MS/MS. J Proteomics 85:174–191. doi:10.1016/j.jprot.2013.04.004

    Article  CAS  Google Scholar 

  30. Muller G, Jung C, Wied S, Biemer-Daub G (2009) Induced translocation of glycosylphosphatidylinositol-anchored proteins from lipid droplets to adiposomes in rat adipocytes. Br J Pharmacol 158(3):749–770. doi:10.1111/j.1476-5381.2009.00360.x

    Article  CAS  Google Scholar 

  31. Kim HS, Choi DY, Yun SJ, Choi SM, Kang JW, Jung JW, Hwang D, Kim KP, Kim DW (2012) Proteomic analysis of microvesicles derived from human mesenchymal stem cells. J Proteome Res 11(2):839–849. doi:10.1021/pr200682z

    Article  CAS  Google Scholar 

  32. Hwang HH, Moon PG, Lee JE, Kim JG, Lee W, Ryu SH, Baek MC (2011) Identification of the target proteins of rosiglitazone in 3T3-L1 adipocytes through proteomic analysis of cytosolic and secreted proteins. Mol Cells 31(3):239–246. doi:10.1007/s10059-011-0026-6

    Article  CAS  Google Scholar 

  33. Ertunc ME, Sikkeland J, Fenaroli F, Griffiths G, Daniels MP, Cao H, Saatcioglu F, Hotamisligil GS (2015) Secretion of fatty acid binding protein aP2 from adipocytes through a nonclassical pathway in response to adipocyte lipase activity. J Lipid Res 56(2):423–434. doi:10.1194/jlr.M055798

    Article  CAS  Google Scholar 

  34. Aoki N, Yokoyama R, Asai N, Ohki M, Ohki Y, Kusubata K, Heissig B, Hattori K, Nakagawa Y, Matsuda T (2010) Adipocyte-derived microvesicles are associated with multiple angiogenic factors and induce angiogenesis in vivo and in vitro. Endocrinology 151(6):2567–2576. doi:10.1210/en.2009-1023

    Article  CAS  Google Scholar 

  35. Muller G, Schneider M, Biemer-Daub G, Wied S (2011) Upregulation of lipid synthesis in small rat adipocytes by microvesicle-associated CD73 from large adipocytes. Obesity 19(8):1531–1544. doi:10.1038/oby.2011.29

    Article  Google Scholar 

  36. Koeck ES, Iordanskaia T, Sevilla S, Ferrante SC, Hubal MJ, Freishtat RJ, Nadler EP (2014) Adipocyte exosomes induce transforming growth factor beta pathway dysregulation in hepatocytes: a novel paradigm for obesity-related liver disease. J Surg Res 192(2):268–275. doi:10.1016/j.jss.2014.06.050

    Article  CAS  Google Scholar 

  37. Shono S, Kose H, Yamada T, Matsumoto K (2007) Proteomic analysis of a diabetic congenic rat identified age-dependent alteration of an acidic protein. J Med Investig JMI 54(3–4):289–294

    Article  Google Scholar 

  38. Nakatani S, Kakehashi A, Ishimura E, Yamano S, Mori K, Wei M, Inaba M, Wanibuchi H (2011) Targeted proteomics of isolated glomeruli from the kidneys of diabetic rats: sorbin and SH3 domain containing 2 is a novel protein associated with diabetic nephropathy. Exp Diabetes Res 2011:979354. doi:10.1155/2011/979354

    Article  Google Scholar 

  39. Oh-Ishi M, Satoh M, Maeda T (2000) Preparative two-dimensional gel electrophoresis with agarose gels in the first dimension for high molecular mass proteins. Electrophoresis 21(9):1653–1669. doi:10.1002/(SICI)1522-2683(20000501)21:9<1653:AID-ELPS1653>3.0.CO;2-9

    Article  CAS  Google Scholar 

  40. Catalan V, Gomez-Ambrosi J, Rodriguez A, Silva C, Rotellar F, Gil MJ, Cienfuegos JA, Salvador J, Fruhbeck G (2008) Expression of caveolin-1 in human adipose tissue is upregulated in obesity and obesity-associated type 2 diabetes mellitus and related to inflammation. Clin Endocrinol 68(2):213–219. doi:10.1111/j.1365-2265.2007.03021.x

    CAS  Google Scholar 

  41. Otsu K, Toya Y, Oshikawa J, Kurotani R, Yazawa T, Sato M, Yokoyama U, Umemura S, Minamisawa S, Okumura S, Ishikawa Y (2010) Caveolin gene transfer improves glucose metabolism in diabetic mice. Am J Physiol Cell Physiol 298(3):C450–C456. doi:10.1152/ajpcell.00077.2009

    Article  CAS  Google Scholar 

  42. Wang H, Eckel RH (2009) Lipoprotein lipase: from gene to obesity. Am J Physiol Endocrinol Metab 297(2):E271–E288. doi:10.1152/ajpendo.90920.2008

    Article  CAS  Google Scholar 

  43. Hida K, Wada J, Zhang H, Hiragushi K, Tsuchiyama Y, Shikata K, Makino H (2000) Identification of genes specifically expressed in the accumulated visceral adipose tissue of OLETF rats. J Lipid Res 41(10):1615–1622

    CAS  Google Scholar 

  44. Lee DH, Park DB, Lee YK, An CS, Oh YS, Kang JS, Kang SH, Chung MY (2005) The effects of thiazolidinedione treatment on the regulations of aquaglyceroporins and glycerol kinase in OLETF rats. Metab Clin Exp 54(10):1282–1289. doi:10.1016/j.metabol.2005.04.015

    Article  CAS  Google Scholar 

  45. Rodriguez A, Catalan V, Gomez-Ambrosi J, Fruhbeck G (2006) Role of aquaporin-7 in the pathophysiological control of fat accumulation in mice. FEBS Lett 580(20):4771–4776. doi:10.1016/j.febslet.2006.07.080

    Article  CAS  Google Scholar 

  46. Burkart A, Shi X, Chouinard M, Corvera S (2011) Adenylate kinase 2 links mitochondrial energy metabolism to the induction of the unfolded protein response. J Biol Chem 286(6):4081–4089. doi:10.1074/jbc.M110.134106

    Article  CAS  Google Scholar 

  47. Sugimoto K, Tsuruoka S, Fujimura A (2001) Effect of enalapril on diabetic nephropathy in OLETF rats: the role of an anti-oxidative action in its protective properties. Clin Exp Pharmacol Physiol 28(10):826–830

    Article  CAS  Google Scholar 

  48. Okuno Y, Matsuda M, Kobayashi H, Morita K, Suzuki E, Fukuhara A, Komuro R, Shimabukuro M, Shimomura I (2008) Adipose expression of catalase is regulated via a novel remote PPARgamma-responsive region. Biochem Biophys Res Commun 366(3):698–704. doi:10.1016/j.bbrc.2007.12.001

    Article  CAS  Google Scholar 

  49. Quiroga AD, Lian J, Lehner R (2012) Carboxylesterase1/Esterase-x regulates chylomicron production in mice. PLoS One 7(11):e49515. doi:10.1371/journal.pone.0049515

    Article  CAS  Google Scholar 

  50. Quiroga AD, Li L, Trotzmuller M, Nelson R, Proctor SD, Kofeler H, Lehner R (2012) Deficiency of carboxylesterase 1/esterase-x results in obesity, hepatic steatosis, and hyperlipidemia. Hepatology 56(6):2188–2198. doi:10.1002/hep.25961

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by the grant of Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0022811 to M.C.B.) and the National Research Foundation of Korea (NRF) grant funded by the Korea government (2014R1A5A2009242 to M.C.B.) and the National Research Foundation of Korea (NRF) grant (NRF-2013R1A6A3A01024597 to P.G.M.).

Conflict of interest

The authors declare that they have no conflict of interests.

Ethical approval

All procedures performed in studies involving animals were in accordance with protocols approved by Kyungpook National University (KNU) Institutional Animal Care and Use Committees (IACUCs).

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Authors and Affiliations

  1. Department Molecular Medicine, Cell and Matrix Research Institute, School of Medicine, Kyungpook National University, 101 Dongin-dong 2 Ga, Jung-Gu, Daegu, 700-422, Republic of Korea

    Jeong-Eun Lee, Pyong-Gon Moon & Moon-Chang Baek

  2. Department Internal Medicine, Kyungpook National University, Daegu, 700-422, Republic of Korea

    In-Kyu Lee

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  1. Jeong-Eun Lee
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Correspondence to Moon-Chang Baek.

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Lee, JE., Moon, PG., Lee, IK. et al. Proteomic Analysis of Extracellular Vesicles Released by Adipocytes of Otsuka Long-Evans Tokushima Fatty (OLETF) Rats. Protein J 34, 220–235 (2015). https://doi.org/10.1007/s10930-015-9616-z

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