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Beta cell extracellular vesicle miR-21-5p cargo is increased in response to inflammatory cytokines and serves as a biomarker of type 1 diabetes.
Diabetologia. 2018 05; 61(5):1124-1134.D

Abstract

AIMS/HYPOTHESIS

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.

METHODS

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.

RESULTS

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.

CONCLUSIONS/INTERPRETATION

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.

Links

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Authors+Show Affiliations

Lakhter AJ
Department of Pediatrics, Section of Pediatric Endocrinology and Diabetology, Indiana University School of Medicine, Indianapolis, IN, USA. Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, 635 Barnhill Dr., Rm 2031, Indianapolis, IN, 46202, USA. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA.
Pratt RE
Department of Pediatrics, Section of Pediatric Endocrinology and Diabetology, Indiana University School of Medicine, Indianapolis, IN, USA. Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, 635 Barnhill Dr., Rm 2031, Indianapolis, IN, 46202, USA. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA.
Moore RE
Department of Pediatrics, Section of Pediatric Endocrinology and Diabetology, Indiana University School of Medicine, Indianapolis, IN, USA. Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, 635 Barnhill Dr., Rm 2031, Indianapolis, IN, 46202, USA. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA.
Doucette KK
Department of Pediatrics, Section of Pediatric Endocrinology and Diabetology, Indiana University School of Medicine, Indianapolis, IN, USA. Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, 635 Barnhill Dr., Rm 2031, Indianapolis, IN, 46202, USA. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA.
Maier BF
Department of Pediatrics, Section of Pediatric Endocrinology and Diabetology, Indiana University School of Medicine, Indianapolis, IN, USA. Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, 635 Barnhill Dr., Rm 2031, Indianapolis, IN, 46202, USA.
DiMeglio LA
Department of Pediatrics, Section of Pediatric Endocrinology and Diabetology, Indiana University School of Medicine, Indianapolis, IN, USA. Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, 635 Barnhill Dr., Rm 2031, Indianapolis, IN, 46202, USA. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA.
Sims EK
Department of Pediatrics, Section of Pediatric Endocrinology and Diabetology, Indiana University School of Medicine, Indianapolis, IN, USA. eksims@iu.edu. Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, 635 Barnhill Dr., Rm 2031, Indianapolis, IN, 46202, USA. eksims@iu.edu. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA. eksims@iu.edu.

MeSH

AnimalsApoptosisBiomarkersCytokinesDiabetes Mellitus, Type 1Extracellular VesiclesFemaleGene Expression ProfilingHumansInflammationInsulin-Secreting CellsInterleukin-1betaMiceMice, Inbred NODMicroRNAsTumor Necrosis Factor-alpha

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

29445851

Citation

Lakhter, Alexander J., et al. "Beta Cell Extracellular Vesicle miR-21-5p Cargo Is Increased in Response to Inflammatory Cytokines and Serves as a Biomarker of Type 1 Diabetes." Diabetologia, vol. 61, no. 5, 2018, pp. 1124-1134.
Lakhter AJ, Pratt RE, Moore RE, et al. Beta cell extracellular vesicle miR-21-5p cargo is increased in response to inflammatory cytokines and serves as a biomarker of type 1 diabetes. Diabetologia. 2018;61(5):1124-1134.
Lakhter, A. J., Pratt, R. E., Moore, R. E., Doucette, K. K., Maier, B. F., DiMeglio, L. A., & Sims, E. K. (2018). Beta cell extracellular vesicle miR-21-5p cargo is increased in response to inflammatory cytokines and serves as a biomarker of type 1 diabetes. Diabetologia, 61(5), 1124-1134. https://doi.org/10.1007/s00125-018-4559-5
Lakhter AJ, et al. Beta Cell Extracellular Vesicle miR-21-5p Cargo Is Increased in Response to Inflammatory Cytokines and Serves as a Biomarker of Type 1 Diabetes. Diabetologia. 2018;61(5):1124-1134. PubMed PMID: 29445851.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Beta cell extracellular vesicle miR-21-5p cargo is increased in response to inflammatory cytokines and serves as a biomarker of type 1 diabetes. AU - Lakhter,Alexander J, AU - Pratt,Rachel E, AU - Moore,Rachel E, AU - Doucette,Kaitlin K, AU - Maier,Bernhard F, AU - DiMeglio,Linda A, AU - Sims,Emily K, Y1 - 2018/02/14/ PY - 2017/09/21/received PY - 2018/01/09/accepted PY - 2018/2/16/pubmed PY - 2019/1/29/medline PY - 2018/2/16/entrez KW - Beta cell signal transduction KW - Cell lines KW - Human KW - Prediction and prevention of type 1 diabetes SP - 1124 EP - 1134 JF - Diabetologia JO - Diabetologia VL - 61 IS - 5 N2 - AIMS/HYPOTHESIS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS/INTERPRETATION: 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. SN - 1432-0428 UR - https://www.unboundmedicine.com/medline/citation/29445851/Beta_cell_extracellular_vesicle_miR_21_5p_cargo_is_increased_in_response_to_inflammatory_cytokines_and_serves_as_a_biomarker_of_type_1_diabetes_ DB - PRIME DP - Unbound Medicine ER -