, Volume 61, Issue 5, pp 1124–1134 | Cite as

Beta cell extracellular vesicle miR-21-5p cargo is increased in response to inflammatory cytokines and serves as a biomarker of type 1 diabetes

  • Alexander J. Lakhter
  • Rachel E. Pratt
  • Rachel E. Moore
  • Kaitlin K. Doucette
  • Bernhard F. Maier
  • Linda A. DiMeglio
  • Emily K. Sims



Improved biomarkers are acutely needed for the detection of developing type 1 diabetes, prior to critical loss of beta cell mass. We previously demonstrated that elevated beta cell microRNA 21-5p (miR-21-5p) in rodent and human models of type 1 diabetes increased beta cell apoptosis. We hypothesised that the inflammatory milieu of developing diabetes may also increase miR-21-5p in beta cell extracellular vesicle (EV) cargo and that circulating EV miR-21-5p would be increased during type 1 diabetes development.


MIN6 and EndoC-βH1 beta cell lines and human islets were treated with IL-1β, IFN-γ and TNF-α to mimic the inflammatory milieu of early type 1 diabetes. Serum was collected weekly from 8-week-old female NOD mice until diabetes onset. Sera from a cross-section of 19 children at the time of type 1 diabetes diagnosis and 16 healthy children were also analysed. EVs were isolated from cell culture media or serum using sequential ultracentrifugation or ExoQuick precipitation and EV miRNAs were assayed.


Cytokine treatment in beta cell lines and human islets resulted in a 1.5- to threefold increase in miR-21-5p. However, corresponding EVs were further enriched for this miRNA, with a three- to sixfold EV miR-21-5p increase in response to cytokine treatment. This difference was only partially reduced by pre-treatment of beta cells with Z-VAD-FMK to inhibit cytokine-induced caspase activity. Nanoparticle tracking analysis showed cytokines to have no effect on the number of EVs, implicating specific changes within EV cargo as being responsible for the increase in beta cell EV miR-21-5p. Sequential ultracentrifugation to separate EVs by size suggested that this effect was mostly due to cytokine-induced increases in exosome miR-21-5p. Longitudinal serum collections from NOD mice showed that EVs displayed progressive increases in miR-21-5p beginning 3 weeks prior to diabetes onset. To validate the relevance to human diabetes, we assayed serum from children with new-onset type 1 diabetes compared with healthy children. While total serum miR-21-5p and total serum EVs were reduced in diabetic participants, serum EV miR-21-5p was increased threefold compared with non-diabetic individuals. By contrast, both serum and EV miR-375-5p were increased in parallel among diabetic participants.


We propose that circulating EV miR-21-5p may be a promising marker of developing type 1 diabetes. Additionally, our findings highlight that, for certain miRNAs, total circulating miRNA levels are distinct from circulating EV miRNA content.


Beta cell signal transduction Cell lines Human Prediction and prevention of type 1 diabetes 





Extracellular vesicle


microRNA 21-5p


Non-obese diabetes-resistant


Nanoparticle tracking analysis


Quantitative real-time PCR


Transmission electron microscopy



We thank C. Evans-Molina and R. Mirmira (Departments of Medicine and Pediatrics, Indiana University School of Medicine) for their support in developing this project. We thank the Indiana University Islet and Physiology core for assistance with serum collection and islet isolation and the University of Nebraska College of Medicine Electron Microscopy Core for assistance with TEM imaging. This work has been partially presented in oral abstract form and in poster presentations at the 2015–2017 American Diabetes Association Scientific Sessions, 2017 Human Islet Research Network, Pediatric Endocrine Society, Endocrine Society, 2015–2016 Midwest Islet Club, 2016 Central Society for Clinical and Translational Research, 2017 Extracellular RNA Communication Consortium and the 2017 International Society for Extracellular Vesicles meeting.

Contribution statement

AJL, REP, REM, and KKD performed the experiments, acquired the data and revised the manuscript. BFM made substantial contributions to study design and analysis of data and revised the manuscript. LAD made substantial contributions to acquisition of data and revised the manuscript. AJL and EKS designed the experiments, acquired and interpreted data, and drafted the manuscript. EKS is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors provided final approval of the version to be published.


This work was supported by NIDDK K08DK103983 (EKS), Pediatric Endocrine Society Clinical Scholar Award (EKS), a pilot and feasibility award within the Center for Diabetes and Metabolic Diseases NIH/NIDDK grant number P30 DK097512, funding by Indiana University Health and the Indiana Clinical and Translational Sciences Institute (EKS), Grant no. UL1TR001108, a Ralph W. and Grace M. Showalter research award (EKS), JDRF Pioneer Award and Strategic Research Agreement (LAD) and NIH grant no. 32DK064466 (AJL). This study utilised core services provided by the Diabetes Research Center grant P30 DK097512 to Indiana University School of Medicine. Human pancreatic islets were provided by the NIDDK-funded Integrated Islet Distribution Program (IIDP) at City of Hope, NIH grant no. 2UC4DK098085-02.

Duality of interest

The authors declare that there is no duality of interest associated with this manuscript.

Supplementary material

125_2018_4559_MOESM1_ESM.pdf (158 kb)
ESM Figs (PDF 158 kb)


  1. 1.
    Dabelea D, Mayer-Davis EJ, Saydah S et al (2014) Prevalence of type 1 and type 2 diabetes among children and adolescents from 2001 to 2009. JAMA 311:1778–1786CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Maahs DM, West NA, Lawrence JM, Mayer-Davis EJ (2010) Chapter 1: epidemiology of type 1 diabetes. Endocrinol Metab Clin N Am 39:481–497CrossRefGoogle Scholar
  3. 3.
    Eisenbarth GS (1986) Type I diabetes mellitus. A chronic autoimmune disease. N Engl J Med 314:1360–1368CrossRefPubMedGoogle Scholar
  4. 4.
    Atkinson MA, Eisenbarth GS, Michels AW (2014) Type 1 diabetes. Lancet 383:69–82CrossRefPubMedGoogle Scholar
  5. 5.
    Heninger A-K, Eugster A, Kuehn D, et al. (2017) A divergent population of autoantigen-responsive CD4+ T cells in infants prior to β cell autoimmunity. Science Translational Medicine 9Google Scholar
  6. 6.
    Cianciaruso C, Phelps EA, Pasquier M et al (2017) Primary human and rat beta-cells release the intracellular autoantigens GAD65, IA-2, and proinsulin in exosomes together with cytokine-induced enhancers of immunity. Diabetes 66:460–473CrossRefPubMedGoogle Scholar
  7. 7.
    Sims EK, Lakhter AJ, Anderson-Baucum E, Kono T, Tong X, Evans-Molina C (2017) MicroRNA 21 targets BCL2 mRNA to increase apoptosis in rat and human beta cells. Diabetologia 60:1057–1065CrossRefPubMedGoogle Scholar
  8. 8.
    Gould SJ, Raposo G (2013) As we wait: coping with an imperfect nomenclature for extracellular vesicles. J Extracell Vesicles.
  9. 9.
    Yanez-Mo M, Siljander PR, Andreu Z et al (2015) Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles 4:27066CrossRefPubMedGoogle Scholar
  10. 10.
    Kowal J, Arras G, Colombo M et al (2016) Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. Proc Natl Acad Sci U S A 113:E968–E977CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Willms E, Johansson HJ, Mäger I et al (2016) Cells release subpopulations of exosomes with distinct molecular and biological properties. Sci Rep 6:22519CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Chen Y, Pfeifer A (2017) Brown fat-derived exosomes: small vesicles with big impact. Cell Metab 25:759–760CrossRefPubMedGoogle Scholar
  13. 13.
    Rahman MJ, Regn D, Bashratyan R, Dai YD (2014) Exosomes released by islet-derived mesenchymal stem cells trigger autoimmune responses in NOD mice. Diabetes 63:1008–1020CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Pitt JM, Kroemer G, Zitvogel L (2016) Extracellular vesicles: masters of intercellular communication and potential clinical interventions. J Clin Invest 126:1139–1143CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Garcia-Contreras M, Brooks RW, Boccuzzi L, Robbins PD, Ricordi C (2017) Exosomes as biomarkers and therapeutic tools for type 1 diabetes mellitus. Eur Rev Med Pharmacol Sci 21:2940–2956PubMedGoogle Scholar
  16. 16.
    Ruan Q, Wang T, Kameswaran V et al (2011) The microRNA-21−PDCD4 axis prevents type 1 diabetes by blocking pancreatic β cell death. Proc Natl Acad Sci 108:12030–12035CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Roggli E, Britan A, Gattesco S et al (2010) Involvement of MicroRNAs in the cytotoxic effects exerted by proinflammatory cytokines on pancreatic β-Cells. Diabetes 59:978–986CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Roggli E, Gattesco S, Caille D et al (2012) Changes in MicroRNA expression contribute to pancreatic β-cell dysfunction in prediabetic NOD mice. Diabetes 61:1742–1751CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Osipova J, Fischer D-C, Dangwal S et al (2014) Diabetes-associated microRNAs in pediatric patients with type 1 diabetes mellitus: a cross-sectional cohort study. J Clin Endocrinol Metab 99:E1661–E1665CrossRefPubMedGoogle Scholar
  20. 20.
    Seyhan AA, Nunez Lopez YO, Xie H et al (2016) Pancreas-enriched miRNAs are altered in the circulation of subjects with diabetes: a pilot cross-sectional study. Sci Rep 6:31479CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Hatanaka M, Anderson-Baucum E, Lakhter A et al (2017) Chronic high fat feeding restricts islet mRNA translation initiation independently of ER stress via DNA damage and p53 activation. Sci Rep 7:3758CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Scharfmann R, Pechberty S, Hazhouz Y et al (2014) Development of a conditionally immortalized human pancreatic β cell line. J Clin Invest 124:2087–2098CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Clancy JW, Sedgwick A, Rosse C et al (2015) Regulated delivery of molecular cargo to invasive tumour-derived microvesicles. Nat Commun 6:6919CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    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 3:3.22.1–3.22.29Google Scholar
  25. 25.
    Fisher MM, Watkins RA, Blum J et al (2015) Elevations in circulating methylated and unmethylated preproinsulin DNA in new-onset type 1 diabetes. Diabetes 64:3867–3872CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Prochazka M, Serreze DV, Frankel WN, Leiter EH (1992) NOR/Lt mice: MHC-matched diabetes-resistant control strain for NOD mice. Diabetes 41:98–106CrossRefPubMedGoogle Scholar
  27. 27.
    Stull ND, Breite A, McCarthy R, Tersey SA, Mirmira RG (2012) Mouse islet of Langerhans isolation using a combination of purified collagenase and neutral protease. J Vis Exp 67:4137Google Scholar
  28. 28.
    Crescitelli R, Lasser C, Szabo TG et al (2013) Distinct RNA profiles in subpopulations of extracellular vesicles: apoptotic bodies, microvesicles and exosomes. J Extracell Vesicles.
  29. 29.
    Ha M, Kim VN (2014) Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol 15:509–524CrossRefPubMedGoogle Scholar
  30. 30.
    Snowhite IV, Allende G, Sosenko J, Pastori RL, Messinger Cayetano S, Pugliese A (2017) Association of serum microRNAs with islet autoimmunity, disease progression and metabolic impairment in relatives at risk of type 1 diabetes. Diabetologia 60:1409–1422CrossRefPubMedGoogle Scholar
  31. 31.
    Erener S, Mojibian M, Fox JK, Denroche HC, Kieffer TJ (2013) Circulating miR-375 as a biomarker of β-cell death and diabetes in mice. Endocrinology 154:603–608CrossRefPubMedGoogle Scholar
  32. 32.
    Song I, Roels S, Martens GA, Bouwens L (2017) Circulating microRNA-375 as biomarker of pancreatic beta cell death and protection of beta cell mass by cytoprotective compounds. PLoS One 12:e0186480CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Guay C, Menoud V, Rome S, Regazzi R (2015) Horizontal transfer of exosomal microRNAs transduce apoptotic signals between pancreatic beta-cells. Cell Commun Signal 13:17CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Sheng H, Hassanali S, Nugent C et al (2011) Insulinoma-released exosomes or microparticles are immunostimulatory and can activate autoreactive T cells spontaneously developed in nonobese diabetic mice. J Immunol 187:1591–1600CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Cantaluppi V, Biancone L, Figliolini F et al (2012) Microvesicles derived from endothelial progenitor cells enhance neoangiogenesis of human pancreatic islets. Cell Transplant 21:1305–1320CrossRefPubMedGoogle Scholar
  36. 36.
    Palmisano G, Jensen SS, Le Bihan M-C et al (2012) Characterization of membrane-shed microvesicles from cytokine-stimulated β-cells using proteomics strategies. Mol Cell Proteomics 11:230–243CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Zhu Q, Kang J, Miao H et al (2014) Low-dose cytokine-induced neutral ceramidase secretion from INS-1 cells via exosomes and its anti-apoptotic effect. FEBS J 281:2861–2870CrossRefPubMedGoogle Scholar
  38. 38.
    Vallabhajosyula P, Korutla L, Habertheuer A et al (2017) Tissue-specific exosome biomarkers for noninvasively monitoring immunologic rejection of transplanted tissue. J Clin Invest 127:1375–1391CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Hasilo CP, Negi S, Allaeys I et al (2017) Presence of diabetes autoantigens in extracellular vesicles derived from human islets. Sci Rep 7:5000CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Garcia-Contreras M, Shah SH, Tamayo A et al (2017) Plasma-derived exosome characterization reveals a distinct microRNA signature in long duration type 1 diabetes. Sci Rep 7:5998CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Turchinovich A, Samatov TR, Tonevitsky AG, Burwinkel B (2013) Circulating miRNAs: cell-cell communication function? Front Genet 4:119CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Erener S, Marwaha A, Tan R, Panagiotopoulos C, Kieffer TJ (2017) Profiling of circulating microRNAs in children with recent onset of type 1 diabetes. JCI Insight 2:e89656CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Nielsen LB, Wang C, Sørensen K et al (2012) Circulating levels of microRNA from children with newly diagnosed type 1 diabetes and healthy controls: evidence that miR-25 associates to residual beta-cell function and glycaemic control during disease progression. Exp Diabetes Res 2012:896362PubMedPubMedCentralGoogle Scholar
  44. 44.
    Akirav EM, Lebastchi J, Galvan EM et al (2011) Detection of β cell death in diabetes using differentially methylated circulating DNA. Proc Natl Acad Sci U S A 108:19018–19023CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Pesenacker AM, Wang AY, Singh A et al (2016) A regulatory T cell gene signature is a specific and sensitive biomarker to identify children with new-onset type 1 diabetes. Diabetes 65:1031CrossRefPubMedGoogle Scholar
  46. 46.
    Krichevsky AM, Gabriely G (2009) miR-21: a small multi-faceted RNA. J Cell Mol Med 13:39–53CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Alexander J. Lakhter
    • 1
    • 2
    • 3
  • Rachel E. Pratt
    • 1
    • 2
    • 3
  • Rachel E. Moore
    • 1
    • 2
    • 3
  • Kaitlin K. Doucette
    • 1
    • 2
    • 3
  • Bernhard F. Maier
    • 1
    • 2
  • Linda A. DiMeglio
    • 1
    • 2
    • 3
  • Emily K. Sims
    • 1
    • 2
    • 3
  1. 1.Department of Pediatrics, Section of Pediatric Endocrinology and DiabetologyIndiana University School of MedicineIndianapolisUSA
  2. 2.Center for Diabetes and Metabolic DiseasesIndiana University School of MedicineIndianapolisUSA
  3. 3.Wells Center for Pediatric ResearchIndiana University School of MedicineIndianapolisUSA

Personalised recommendations