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A preliminary study: proteomic analysis of exosomes derived from thyroid-stimulating hormone-stimulated HepG2 cells

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Abstract

Background

Thyroid-stimulating hormone (TSH) plays an important role in the regulation of lipid metabolism. However, little is known about the role that exosomes play in the process of TSH-induced lipotoxicity in non-alcoholic fatty liver disease (NAFLD). As a preliminary step, the present study set out to investigate alterations in protein expression in exosomes derived from TSH-stimulated HepG2 cells.

Methods

HepG2 cells were treated with TSH, exosomes were collected, and proteins were identified by mass spectrometry (MS). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis were performed to analyze the identified proteins.

Results

TSH treatment significantly increased exosomal production and changed the exosomal proteomic profile in HepG2 cells. Among the 1728 proteins, 140 identified proteins were upregulated and seven proteins were downregulated. GO analysis and KEGG analysis revealed that these proteins were involved in multiple processes including metabolism, apoptosis, and inflammation.

Conclusion

Our preliminary study demonstrated that exosomes derived from TSH-stimulated hepatocytes were increased and showed a specific altered spectrum of proteins, many of which were involved in metabolism, signal transduction, apoptosis, and inflammation. This study offers new insights into the pathogenesis of TSH-induced lipotoxicity in NAFLD.

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References

  1. Min HK, Kapoor A, Fuchs M, Mirshahi F, Zhou H, Maher J, Kellum J, Warnick R, Contos MJ, Sanyal AJ (2012) Increased hepatic synthesis and dysregulation of cholesterol metabolism is associated with the severity of nonalcoholic fatty liver disease. Cell Metab 15(5):665–674

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Chung GE, Kim D, Kim W, Yim JY, Park MJ, Kim YJ, Yoon JH, Lee HS (2012) Non-alcoholic fatty liver disease across the spectrum of hypothyroidism. J Hepatol 57(1):150–156

    Article  CAS  PubMed  Google Scholar 

  3. Sert A, Pirgon O, Aypar E, Yilmaz H, Odabas D (2013) Subclinical hypothyroidism as a risk factor for the development of cardiovascular disease in obese adolescents with nonalcoholic fatty liver disease. Pediatr Cardiol 34(5):1166–1174

    Article  PubMed  Google Scholar 

  4. Liangpunsakul S, Chalasani N (2003) Is hypothyroidism a risk factor for non-alcoholic steatohepatitis? J Clin Gastroenterol 37(4):340–343

    Article  PubMed  Google Scholar 

  5. Kaltenbach TE, Graeter T, Oeztuerk S, Holzner D, Kratzer W, Wabitsch M, Denzer C (2017) Thyroid dysfunction and hepatic steatosis in overweight children and adolescents. Pediatr Obes 12(1):67–74

    Article  PubMed  Google Scholar 

  6. Guo Z, Li M, Han B, Qi X (2018) Association of non-alcoholic fatty liver disease with thyroid function: a systematic review and meta-analysis. Dig Liver Dis 50(11):1153–1162

    Article  CAS  PubMed  Google Scholar 

  7. Lemoinne S, Thabut D, Housset C, Moreau R, Valla D, Boulanger CM, Rautou PE (2014) The emerging roles of microvesicles in liver diseases. Nat Rev Gastroenterol Hepatol 11(6):350–361

    Article  CAS  PubMed  Google Scholar 

  8. Masyuk AI, Masyuk TV, Larusso NF (2013) Exosomes in the pathogenesis, diagnostics and therapeutics of liver diseases. J Hepatol 59(3):621–625

    Article  CAS  PubMed  Google Scholar 

  9. Huang-Doran I, Zhang CY, Vidal-Puig A (2017) Extracellular vesicles: novel mediators of cell communication in metabolic disease. Trends Endocrinol Metab 28(1):3–18

    Article  CAS  PubMed  Google Scholar 

  10. Ban LA, Shackel NA, McLennan SV (2016) Extracellular vesicles: a new frontier in biomarker discovery for non-alcoholic fatty liver disease. Int J Mol Sci 17(3):376

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Kogure T, Lin WL, Yan IK, Braconi C, Patel T (2011) Intercellular nanovesicle-mediated microRNA transfer: a mechanism of environmental modulation of hepatocellular cancer cell growth. Hepatology 54(4):1237–1248

    Article  CAS  PubMed  Google Scholar 

  12. Tamai K, Shiina M, Tanaka N, Nakano T, Yamamoto A, Kondo Y, Kakazu E, Inoue J, Fukushima K, Sano K, Ueno Y, Shimosegawa T, Sugamura K (2012) Regulation of hepatitis C virus secretion by the Hrs-dependent exosomal pathway. Virology 422(2):377–385

    Article  CAS  PubMed  Google Scholar 

  13. Deng ZB, Liu Y, Liu C, Xiang X, Wang J, Cheng Z, Shah SV, Zhang S, Zhang L, Zhuang X, Michalek S, Grizzle WE, Zhang HG (2009) Immature myeloid cells induced by a high-fat diet contribute to liver inflammation. Hepatology 50(5):1412–1420

    Article  CAS  PubMed  Google Scholar 

  14. Lee YS, Kim SY, Ko E, Lee JH, Yi HS, Yoo YJ, Je J, Suh SJ, Jung YK, Kim JH, Seo YS, Yim HJ, Jeong WI, Yeon JE, Um SH, Byun KS (2017) Exosomes derived from palmitic acid-treated hepatocytes induce fibrotic activation of hepatic stellate cells. Sci Rep 7(1):3710

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Ma S, Jing F, Xu C, Zhou L, Song Y, Yu C, Jiang D, Gao L, Li Y, Guan Q, Zhao J (2015) Thyrotropin and obesity: increased adipose triglyceride content through glycerol-3-phosphate acyltransferase 3. Sci Rep 5:7633

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Li Y, Wang L, Zhou L, Song Y, Ma S, Yu C, Zhao J, Xu C, Gao L (2017) Thyroid stimulating hormone increases hepatic gluconeogenesis via CRTC2. Mol Cell Endocrinol 446:70–80

    Article  CAS  PubMed  Google Scholar 

  17. Song Y, Xu C, Shao S, Liu J, Xing W, Xu J, Qin C, Li C, Hu B, Yi S, Xia X, Zhang H, Zhang X, Wang T, Pan W, Yu C, Wang Q, Lin X, Wang L, Gao L, Zhao J (2015) Thyroid-stimulating hormone regulates hepatic bile acid homeostasis via SREBP-2/HNF-4alpha/CYP7A1 axis. J Hepatol 62(5):1171–1179

    Article  CAS  PubMed  Google Scholar 

  18. Asvold BO, Vatten LJ, Nilsen TI, Bjoro T (2007) The association between TSH within the reference range and serum lipid concentrations in a population-based study. The HUNT Study. Eur J Endocrinol 156(2):181–186

    Article  PubMed  CAS  Google Scholar 

  19. Lee YK, Kim JE, Oh HJ, Park KS, Kim SK, Park SW, Kim MJ, Cho YW (2011) Serum TSH level in healthy Koreans and the association of TSH with serum lipid concentration and metabolic syndrome. Korean J Intern Med 26(4):432–439

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Kim D, Kim W, Joo SK, Bae JM, Kim JH, Ahmed A (2018) Subclinical hypothyroidism and low-normal thyroid function are associated with nonalcoholic steatohepatitis and fibrosis. Clin Gastroenterol Hepatol 16(1):123 e121–131 e121

    Article  CAS  Google Scholar 

  21. Thery C, Amigorena S, Raposo G, Clayton A (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol 3:22

    PubMed  Google Scholar 

  22. Gao L, Wang L, Dai T, Jin K, Zhang Z, Wang S, Xie F, Fang P, Yang B, Huang H, van Dam H, Zhou F, Zhang L (2018) Tumor-derived exosomes antagonize innate antiviral immunity. Nat Immunol 19(3):233–245

    Article  CAS  PubMed  Google Scholar 

  23. Huang DW, Sherman BT, Tan Q, Collins JR, Alvord WG, Roayaei J, Stephens R, Baseler MW, Lane HC, Lempicki RA (2007) The DAVID gene functional classification tool: a novel biological module-centric algorithm to functionally analyze large gene lists. Genome Biol 8(9):R183

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Povero D, Eguchi A, Li H, Johnson CD, Papouchado BG, Wree A, Messer K, Feldstein AE (2014) Circulating extracellular vesicles with specific proteome and liver microRNAs are potential biomarkers for liver injury in experimental fatty liver disease. PLoS ONE 9(12):e113651

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Visweswaraiah J, Hinnebusch AG (2017) Interface between 40S exit channel protein uS7/Rps5 and eIF2alpha modulates start codon recognition in vivo. Elife 20:6

    Google Scholar 

  26. Hetherington AM, Sawyez CG, Sutherland BG, Robson DL, Arya R, Kelly K, Jacobs RL, Borradaile NM (2016) Treatment with didemnin B, an elongation factor 1A inhibitor, improves hepatic lipotoxicity in obese mice. Physiol Rep 4:17

    Article  CAS  Google Scholar 

  27. Anczukow O, Rosenberg AZ, Akerman M, Das S, Zhan L, Karni R, Muthuswamy SK, Krainer AR (2012) The splicing factor SRSF1 regulates apoptosis and proliferation to promote mammary epithelial cell transformation. Nat Struct Mol Biol 19(2):220–228

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Tian L, Song Y, Xing M, Zhang W, Ning G, Li X, Yu C, Qin C, Liu J, Tian X, Sun X, Fu R, Zhang L, Zhang X, Lu Y, Zou J, Wang L, Guan Q, Gao L, Zhao J (2010) A novel role for thyroid-stimulating hormone: up-regulation of hepatic 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase expression through the cyclic adenosine monophosphate/protein kinase A/cyclic adenosine monophosphate-responsive element binding protein pathway. Hepatology 52(4):1401–1409

    Article  CAS  PubMed  Google Scholar 

  29. de Lloyd A, Bursell J, Gregory JW, Rees DA, Ludgate M (2010) TSH receptor activation and body composition. J Endocrinol 204(1):13–20

    Article  PubMed  CAS  Google Scholar 

  30. Friesema EC, Docter R, Moerings EP, Verrey F, Krenning EP, Hennemann G, Visser TJ (2001) Thyroid hormone transport by the heterodimeric human system L amino acid transporter. Endocrinology 142(10):4339–4348

    Article  CAS  PubMed  Google Scholar 

  31. Lee JW, Ryan F, Swaffield JC, Johnston SA, Moore DD (1995) Interaction of thyroid-hormone receptor with a conserved transcriptional mediator. Nature 374(6517):91–94

    Article  CAS  PubMed  Google Scholar 

  32. Kim DK, Kanai Y, Choi HW, Tangtrongsup S, Chairoungdua A, Babu E, Tachampa K, Anzai N, Iribe Y, Endou H (2002) Characterization of the system L amino acid transporter in T24 human bladder carcinoma cells. Biochim Biophys Acta 1565(1):112–121

    Article  CAS  PubMed  Google Scholar 

  33. Huang X, Charbeneau RA, Fu Y, Kaur K, Gerin I, MacDougald OA, Neubig RR (2008) Resistance to diet-induced obesity and improved insulin sensitivity in mice with a regulator of G protein signaling-insensitive G184S Gnai2 allele. Diabetes 57(1):77–85

    Article  CAS  PubMed  Google Scholar 

  34. Winzell MS, Ahren B (2007) G-protein-coupled receptors and islet function-implications for treatment of type 2 diabetes. Pharmacol Ther 116(3):437–448

    Article  CAS  PubMed  Google Scholar 

  35. Peterhoff M, Sieg A, Brede M, Chao CM, Hein L, Ullrich S (2003) Inhibition of insulin secretion via distinct signaling pathways in alpha2-adrenoceptor knockout mice. Eur J Endocrinol 149(4):343–350

    Article  CAS  PubMed  Google Scholar 

  36. Qian F, Huang P, Ma L, Kuznetsov A, Tamarina N, Philipson LH (2002) TRP genes: candidates for nonselective cation channels and store-operated channels in insulin-secreting cells. Diabetes 51(Suppl 1):S183–189

    Article  CAS  PubMed  Google Scholar 

  37. Cholankeril G, Perumpail RB, Pham EA, Ahmed A, Harrison SA (2016) Nonalcoholic fatty liver disease: epidemiology, natural history, and diagnostic challenges. Hepatology 64(3):954

    Article  PubMed  Google Scholar 

  38. Adams LA, Lymp JF, St Sauver J, Sanderson SO, Lindor KD, Feldstein A, Angulo P (2005) The natural history of nonalcoholic fatty liver disease: a population-based cohort study. Gastroenterology 129(1):113–121

    Article  PubMed  Google Scholar 

  39. Malhi H, Gores GJ, Lemasters JJ (2006) Apoptosis and necrosis in the liver: a tale of two deaths? Hepatology 43(2 Suppl 1):S31–44

    Article  CAS  PubMed  Google Scholar 

  40. Brunt EM, Kleiner DE, Wilson LA, Unalp A, Behling CE, Lavine JE, Appendix NCRNlomotNSCRNcbfit (2009) Portal chronic inflammation in nonalcoholic fatty liver disease (NAFLD): a histologic marker of advanced NAFLD-clinicopathologic correlations from the nonalcoholic steatohepatitis clinical research network. Hepatology 49(3):809–820

    Article  PubMed  Google Scholar 

  41. Cazanave SC, Gores GJ (2010) Mechanisms and clinical implications of hepatocyte lipoapoptosis. Clin Lipidol 5(1):71–85

    Article  PubMed  PubMed Central  Google Scholar 

  42. Michelotti GA, Machado MV, Diehl AM (2013) NAFLD, NASH and liver cancer. Nat Rev Gastroenterol Hepatol 10(11):656–665

    Article  CAS  PubMed  Google Scholar 

  43. Hoo RL, Lee IP, Zhou M, Wong JY, Hui X, Xu A, Lam KS (2013) Pharmacological inhibition of adipocyte fatty acid binding protein alleviates both acute liver injury and non-alcoholic steatohepatitis in mice. J Hepatol 58(2):358–364

    Article  CAS  PubMed  Google Scholar 

  44. Ohiro Y, Garkavtsev I, Kobayashi S, Sreekumar KR, Nantz R, Higashikubo BT, Duffy SL, Higashikubo R, Usheva A, Gius D, Kley N, Horikoshi N (2002) A novel p53-inducible apoptogenic gene, PRG3, encodes a homologue of the apoptosis-inducing factor (AIF). FEBS Lett 524(1–3):163–171

    Article  CAS  PubMed  Google Scholar 

  45. Hase T, Sato M, Yoshida K, Girard L, Takeyama Y, Horio M, Elshazley M, Oguri T, Sekido Y, Shames DS, Gazdar AF, Minna JD, Kondo M, Hasegawa Y (2011) Pivotal role of epithelial cell adhesion molecule in the survival of lung cancer cells. Cancer Sci 102(8):1493–1500

    Article  CAS  PubMed  Google Scholar 

  46. Arinze IJ, Kawai Y (2005) Transcriptional activation of the human Galphai2 gene promoter through nuclear factor-kappaB and antioxidant response elements. J Biol Chem 280(11):9786–9795

    Article  CAS  PubMed  Google Scholar 

  47. Li P, Neubig RR, Zingarelli B, Borg K, Halushka PV, Cook JA, Fan H (2012) Toll-like receptor-induced inflammatory cytokines are suppressed by gain of function or overexpression of Galpha(i2) protein. Inflammation 35(5):1611–1617

    Article  CAS  PubMed  Google Scholar 

  48. Wang H, Yu J, Zhang L, Xiong Y, Chen S, Xing H, Tian Z, Tang K, Wei H, Rao Q, Wang M, Wang J (2014) RPS27a promotes proliferation, regulates cell cycle progression and inhibits apoptosis of leukemia cells. Biochem Biophys Res Commun 446(4):1204–1210

    Article  CAS  PubMed  Google Scholar 

  49. Nosrati N, Kapoor NR, Kumar V (2015) DNA damage stress induces the expression of ribosomal protein S27a gene in a p53-dependent manner. Gene 559(1):44–51

    Article  CAS  PubMed  Google Scholar 

  50. Fararjeh AS, Chen LC, Ho YS, Cheng TC, Liu YR, Chang HL, Chang HW, Wu CH, Tu SH (2019) Proteasome 26S subunit, non-ATPase 3 (PSMD3) regulates breast cancer by stabilizing HER2 from degradation. Cancers (Basel) 11:4

    Article  CAS  Google Scholar 

  51. Gunn PJ, Green CJ, Pramfalk C, Hodson L (2017) In vitro cellular models of human hepatic fatty acid metabolism: differences between Huh7 and HepG2 cell lines in human and fetal bovine culturing serum. Physiol Rep. 5:24

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by the Natural Science Foundation of China (nos. 81230018, 81430020, 81500595, 81600604, 81900716), the Special Funds of Taishan Scholars Project of Shandong Province, Shandong Provincial Natural Science Foundation (ZR2019BH023), and the Medical and Health Technology Development Program in Shandong Province (2016WS0427).

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Authors and Affiliations

  1. Department of Endocrinology and Metabolism, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, 250021, China

    S. Ma & S. Shao

  2. Department of Endocrinology and Metabolism, Shandong Provincial Hospital Affiliated To Shandong University, Jinan, 250021, China

    S. Ma, S. Shao, C. Yang & Z. Yao

  3. Scientific Center, Shandong Provincial Hospital Affiliated to, Shandong First Medical University, 324 Jing 5 Road, Jinan, 250021, Shandong, China

    L. Gao & W. Chen

  4. Scientific Center, Shandong Provincial Hospital Affiliated To Shandong University, Jinan, 250021, China

    L. Gao & W. Chen

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  1. S. Ma
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  3. C. Yang
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  4. Z. Yao
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  5. L. Gao
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Correspondence to L. Gao or W. Chen.

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This study was approved by the Ethics Committee of Shandong Provincial Hospital (NO 2016-KY-102).

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Ma, S., Shao, S., Yang, C. et al. A preliminary study: proteomic analysis of exosomes derived from thyroid-stimulating hormone-stimulated HepG2 cells. J Endocrinol Invest 43, 1229–1238 (2020). https://doi.org/10.1007/s40618-020-01210-y

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