Circulating Exosomal miR-20b-5p Is Elevated in Type2 Diabetes and Could Impair Insulin Action in HumanSkeletal MuscleMutsumi Katayama,1 Oscar P.B. Wiklander,2 Tomas Fritz,3 Kenneth Caidahl,3,4,5 Samir El-Andaloussi,2,6

Juleen R. Zierath,1,4 and Anna Krook1

Diabetes 2019;68:515–526 | https://doi.org/10.2337/db18-0470

miRNAs are noncoding RNAs representing an importantclass of gene expression modulators. Extracellular circu-lating miRNAs are both candidate biomarkers for diseasepathogenesis and mediators of cell-to-cell communica-tion. We examined the miRNA expression profile of totalserum and serum-derived exosome-enriched extracellu-lar vesicles in people with normal glucose tolerance ortype 2 diabetes. In contrast to total serum miRNA, whichdid not reveal any differences in miRNA expression, weidentified differentially abundant miRNAs in patients withtype 2 diabetes using miRNA expression profiles ofexosome RNA (exoRNA). To validate the role of these dif-ferentially abundant miRNAs on glucose metabolism,we transfected miR-20b-5p, a highly abundant exoRNA inpatients with type 2 diabetes, into primary human skeletalmuscle cells. miR-20b-5p overexpression increased basalglycogen synthesis in human skeletal muscle cells. Weidentified AKTIP and STAT3 as miR-20b-5p targets. miR-20b-5p overexpression reduced AKTIP abundance andinsulin-stimulated glycogen accumulation. In conclusion,exosome-derived extracellularmiR-20b-5p is a circulatingbiomarker associated with type 2 diabetes that plays anintracellular role in modulating insulin-stimulated glucosemetabolism via AKT signaling.

miRNAs are a class of small noncoding RNAs that functionas translational repressors by direct interaction with their

target mRNA (1,2). miRNAs function to negatively regu-late the abundance of specific proteins and in this wayexert control over numerous cellular and biological pro-cesses including metabolism (3,4). While miRNAs aretranscribed and exert many effects directly in the cell oforigin, miRNAs are also secreted and stable miRNAs can bedetected in plasma (5). Circulating miRNAs have beendetected in most biofluids including blood (serum/plasma),urine, breast milk, and cerebrospinal fluids and are pro-tected from degradation by a variety of mechanisms. Aproportion of circulating miRNAs are packaged in extra-cellular vesicles, such as “exosomes” (50- to 200-nmmembrane-coated vesicles) (6–9) that protect RNA cargofrom endogenous RNase activity (10). miRNAs can alsobind to various proteins including lipoproteins, namely,HDL, or argonaute (AGO) proteins, which are the keycomponents of the RNA-induced silencing complex, toform miRNA-protein complexes for transport (10–12).Exosomes in plasma/serum have been implicated in trans-fer of miRNA into target cells and thus play a role in cell-cell communication (11–15).

The presence of circulating miRNAs has promptedefforts to identify biomarkers for various pathologies,including cancer, and diseases affecting cardiovascular,neurological, metabolic, and immune function (16–22).Specific circulating miRNAs may be useful biomarkersfor diagnoses and management of progressive diseases

1Department of Physiology and Pharmacology, Karolinska Institute, Stockholm,Sweden2Department of Laboratory Medicine, Karolinska Institute, Huddinge, Sweden3Department of Clinical Physiology, Karolinska University Hospital, Stockholm,Sweden4Department of Molecular Medicine and Surgery, Karolinska Institute, Stockholm,Sweden5Department of Molecular and Clinical Medicine, Institute of Medicine, SahlgrenskaAcademy, University of Gothenburg, Gothenburg, Sweden6Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford,U.K.

Corresponding author: Anna Krook, anna.krook@ki.se

Received 24 April 2018 and accepted 1 December 2018

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db18-0470/-/DC1.

© 2018 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

Diabetes Volume 68, March 2019 515

METABOLISM

such as type 2 diabetes (23–25). Despite the fact thatpatients with type 2 diabetes are characterized by hyper-glycemia and elevated HbA1c levels, these changes inclinical chemistry are only detected once metabolic imbal-ance has occurred. Defects in multiple tissues controllingglucose homeostasis and insulin sensitivity are often pres-ent years prior to diagnosis (26). Given the complexpathophysiology and disease burden of type 2 diabetes,efforts have been focused on identifying circulating miR-NAs as novel prognostic biomarkers (27–29).

To date, there is little consensus on the precise natureof circulating miRNA biomarkers in different cohorts ofpatients with type 2 diabetes, and little is known regardingthe functional role(s) of the identified miRNAs in metabolicprocesses implicated in type 2 diabetes pathogenesis. Here,we determined total serum and exosomal miRNA expres-sion profiles in men with normal glucose tolerance or type2 diabetes and validated the effects of the differentiallyabundant miRNAs on metabolism in skeletal muscle.

RESEARCH DESIGN AND METHODS

Study Participants and Serum SamplesThe study was approved by the ethics committee ofKarolinska Institute. Informed written consent wasobtained from all volunteers. Twenty men with normalglucose tolerance, 16 men with impaired glucose tolerance,and 21 men with type 2 diabetes were recruited bynewspaper advertisement or from a primary health careclinic. The participants with type 2 diabetes, impairedglucose tolerance, and normal glucose tolerance werematched for age and BMI. Clinical characteristics of thestudy participants are presented in Supplementary Table 1.Blood samples were separated in serum and peripheralblood mononuclear cells.

Isolation of Exosome-Enriched Extracellular Vesiclesand Nanoparticle Tracking AnalysisExtracellular vesicles were obtained from serum witha miRCURY Exosome Isolation Kit – Serum and Plasma(Exiqon, Vedbaek, Denmark) according to the manufac-turer’s instructions. Isolated extracellular vesicle sampleswere analyzed using nanoparticle tracking analysis (NTA).Samples were loaded into the sample chamber of an NS500unit (NanoSight, Amesbury, U.K.), and five 1-min videos ofeach sample were recorded (threshold, 6 – multi; blur,auto; andminimum expected particle size, auto). Data anal-ysis was performed with NTA 2.3 software (NanoSight),and the size and concentration of particles included in theextracellular vesicle samples were calculated. An aliquot ofisolated exosome-enriched extracellular vesicles from se-rum was used for NTA, and the remaining isolated extra-cellular vesicles were used for exosome RNA (exoRNA)extraction.

RNA Extraction and Evaluation of miRNA ExpressionAmiRCURY RNA Isolation Kit (Exiqon) was used to extractexoRNA, together with an RNA Spike-in Template Kit

(Exiqon), using MS2 RNA (Roche, Basel, Switzerland) ascarrier RNA according to the manufacturer’s instructions.Each exoRNA elute was reverse transcribed using themiRCURY LNA Universal RT cDNA Synthesis Kit (Exiqon).For the first screening, human serum/plasma FocusmiRNA PCR panels (96-well [V43.AF]) (Exiqon) wereused in a quantitative (q)RT-PCR (qRT-PCR)-based ap-proach to determine levels of 179 human miRNAs. Thequantitative PCR (qPCR) was performed using a StepOnePlus (Applied Biosystems) with 40 amplification cycles,using the cycling parameters recommended by Exiqon.Raw data were processed using StepOne software version2.3 (Applied Biosystems) to assign the baseline and thresh-old for threshold cycle (Ct). For determination of thetechnical variation between the Exiqon serum/plasmaFocus miRNA PCR panel plates, the interplate calibrator(UniSp3) was analyzed. Ct values of the interplate calibra-tor were analyzed to be highly similar across all samples.The normalization and analysis of the PCR panel plateresults were provided by Exiqon GenEx software, version6. For validation of the results of the PCR panel plate ina second screening, we used a miRCURY LNA miRNA PCRSystem (Exiqon) to assess the expression level of individualmiRNAs. Gene expression levels were quantified using themiRNA-specific LNA PCR primer. Relative expression wascalculated using the comparative Ct method.

Primary Human Skeletal Muscle Culture and miRNATransfection ProtocolSatellite cells were isolated from vastus lateralis skeletalmuscle as previously described (30). Cell cultures weremaintained at 37°C under 7.5% CO2 as myoblast cells. Formeasurement of gene expression, myoblast cells weredifferentiated into myotubes as previously described(31). Myotube cells where fusion and multinucleation wereobserved at day 10 after initiation of differentiation wereused for total RNA extraction, protein detection, and cellassays. Cells were transfected using a double transfectionprotocol 48 h after differentiation (day 6) and 48 h later(day 8) with 10 nmol/L Ambion miRNA-20b-5p (ThermoFisher Scientific). Control cells were transfected witha scrambled miRNA mimic (10 nmol/L). Each transfectionwas performed for 5 h with transfection complexesformed in reduced serum media (OptiMEM; ThermoFisher Scientific) using Lipofectamine RNAiMAX trans-fection reagent according to the manufacturer’s protocol.RNA was isolated using an miRNeasy Kit (QIAGEN) at day10.

Culture of Human Embryonic Kidney (HEK293) andHuman Hepatocellular Carcinoma (HepG2) Cells andmiRNA Transfection ProtocolHEK293 and HepG2 cells were obtained from ATCC andcultured in high-glucose (4.5 g/L) DMEM supplementedwith 10% (vol/vol) FBS. miRNA-20b-5p or miRNA mimicwas transfected into those cells using Lipofectamine RNAi-MAX transfection reagent according to the manufacture’s

516 Exosomal miR-20b-5p in Type 2 Diabetes Diabetes Volume 68, March 2019

protocol, and RNA was isolated using the miRNeasy Kit(QIAGEN).

Microarray AnalysismRNA content from miR-20b–transfected cells was pro-filed by hybridizing biotinylated sense strand cDNA toGeneChip Human Gene 2.0 ST arrays (Thermo FisherScientific). Sense strand cDNA was synthesized from totalRNA and biotin labeled with the GeneChip WT PLUSReagent Kit (Thermo Fisher Scientific) before being hy-bridized to arrays. Gene arrays were washed, stained, andscanned as instructed by Affymetrix (Santa Clara, CA).Preprocessing of data was performed using a robust multi-array average with sketch quantile normalization by Ex-pression Console software (Affymetrix). Differentialexpression of transcripts was determined with a pairedclass comparison with a univariate test using a randomvariance model comparing gene expression of control(miRNA mimic–transfected cells) versus miR-20b-5p–transfected cells. Genes with a false discovery rateof ,10% were considered to be differentially regulated.The microarray data were submitted to the NationalCenter for Biotechnology Information Gene ExpressionOmnibus (GEO) and can be found under the GEO seriesaccession number GSE102295.

Gene Set Enrichment AnalysisGene Set Enrichment Analysis (GSEA) was used to linkgenes identified in a specific gene group with their occur-rence in biological pathways or processes. The rank genelist was further annotated using MSigDBv5.0 downloadedfrom the Broad Institute (http://www.broadinstitute.org/),which contains curated functional gene sets of variousbiological states.

Evaluation of miR-20b-5p TargetsPutative target sites were probed in silico by targetprediction algorithms (TargetScan). For validationexperiments, 500 ng total RNA from cells was reversetranscribed using a high-capacity cDNA reverse tran-scription kit (Thermo Fisher Scientific), and qRT-PCRwas performed to measure the expression level of sixgenes using SYBR Green Master Mix Kit (Thermo FisherScientific) and StepOnePlus (Bio-Rad) (primer list inSupplementary Table 2). For the stat3 gene only weused TaqMan Assay (Thermo Fisher Scientific). Weused the TBP and M2B genes as reference genes, andrelative quantification values were calculated using theequation 22DDCt.

Immunoblot AnalysisWestern blot analysis was performed as previously de-scribed (32). Protein content of the supernatants wasdetermined by BCA Protein Assay Kit (Pierce Biotechnol-ogy, Rockford, IL). Membranes were stained with PonceauS to confirm equal loading of samples and quality controlfor the transfer procedure. Membranes were incubated

with primary antibodies directed to glycogen synthase(number 3893; Cell Signaling Technology), phosphorylated(phospho)–glycogen synthase (3891; Cell SignalingTechnology), AKT (9272; Cell Signaling Technology),phospho-AKT (Thr308) (4056; Cell Signaling Technology),signal transducer and activator of transcription (STAT)3(9139; Cell Signaling Technology), and GAPDH (sc-25778;Santa Cruz Biotechnology, Dallas, TX). Membranes wereincubated with species-appropriate horseradish peroxidase–conjugated secondary antibody before proteins were vi-sualized by enhanced chemiluminescence (AmershamECL Western Blotting Detection Reagent, GE HealthcareLife Sciences, Little Chalfont, U.K.). When appropri-ate, protein content was quantified by densitometry(Quantity One; Bio-Rad). All quantifications were per-formed using a positive control to control for intergelvariability.

Luciferase Activity MeasurementThe luciferase reporter clone having the AKTIP 39 un-translated region (UTR) (HmiT088513-MT05) was pur-chased from GeneCopoeia (Rockville, MD). This cloneincluded two predicted miR-20b-5p target sites in AKTIP39UTR (Fig. 3H). Target search in microRNA.org (http://www.microrna.org) was used for prediction of miR-20b-5ptarget sites. Predicted miR-20b-5p binding sites weremutated using the QuikChange II XL Site-Directed Muta-genesis Kit (Agilent Technologies, Santa Clara, CA). Oli-goprimers used for mutagenesis of the AKTIP 39UTR were59-GATGGTGAATCTGGTGCACCATCCTGAAACCTGCTAG-ACTCTGGCCTAG-39, 59-CTAGGCCAGAGTCTAGCAGGTT-TCAGGATGGTGCACCAGATTCACCATC-39, 59-GAGAGCA-GGTTCCATAGCTCACCTGCGATAAGTGGAAGATCATTTG-AATCTC-39, and 59-GAGATTCAAATGATCTTCCACTTATC-GCAGGTGAGCTATGGAACCTGCTCTC-39. Cells were seededin 96-well plates 24 h before experimentation. 39UTR pro-moter plasmids (100 ng/well) was transfected into HEK293cells in 96-well plates using the transfection reagent Lipo-fectamine 2000 (Life Technologies) with 10 nmol/L miRNAmimic for miR-20b-5p. Control cells were transfected withappropriate scrambled miRNA mimic. After 24 h, the cellculture medium was collected and processed for luciferaseassay using the Secrete-Pair Luminescence Assay Kit(GeneCopoeia). Assays were read in the CLARIOstar (BMGLABTECH) and normalized with secreted alkaline phos-phatase signals.

Glucose Incorporation Into GlycogenInsulin-stimulated glucose incorporation into glycogenwas determined as previously described (30).

StatisticsData are presented as mean 6 SEM. Differences wereanalyzed using either paired or unpaired Student t test asappropriate. Relationships were evaluated by computationof Pearson correlation coefficients. Significance was set atP , 0.05.

diabetes.diabetesjournals.org Katayama and Associates 517

RESULTS

Exosome-Enriched Extracellular Vesicles IsolatedFrom Serum FromMenWith Normal Glucose Toleranceor Type 2 DiabetesWe used a commercial exosome isolation kit to isolateexosome-enriched extracellular vesicles rapidly from se-rum to determine miRNA expression profile of exosomes.Differential ultracentrifugation, the current gold stan-dard of isolation, was compared with the commer-cial isolation kit. While both methods yielded similarresults, the reduced requirement of serum for isolationdictated the use of the commercial kit. The resultantfractions were analyzed by immunoblotting for knownexosome markers and major protein components ofHDL particles in serum (Supplementary Fig. 1). Thisshowed that exosome marker proteins, ALIX and CD9,were more enriched in the isolated fraction comparedwith original serum, accompanied by a significant re-duction in the HDL marker protein, apolipoprotein A1(APOA1). Next, size distribution and concentration ofextracellular vesicles in serum from men with normalglucose tolerance, or type 2 diabetes, were analyzed byNTA. Patients with type 2 diabetes had altered serumlipid levels (Supplementary Table 1), which may influ-ence the presence of extracellular vesicles, includingexosomes, which contain lipids. However, particle size(Fig. 1A) and particle concentration (Fig. 1B) of theextracellular vesicle samples were unaltered betweenmen with normal glucose tolerance and men withtype 2 diabetes.

Differentially Expressed miRNAs in ExoRNA and TotalSerum RNA From Men With Normal Glucose Toleranceor Type 2 DiabetesExosomes are known to carry noncoding RNAs (6), such asmiRNA. We determined the miRNA expression profile of

exoRNA derived from serum from men with normalglucose tolerance or type 2 diabetes. For the initial screen-ing, exoRNA was extracted from four men with normalglucose tolerance and four men with type 2 diabetes, andmiRNA expression was profiled using a qPCR panel. Fourmen from each group were randomly selected, still match-ing for age and BMI. In this first screen, six exosomalmiRNAs (miR-20b-5p, miR-532-3p, miR-150-5p, miR-502-3p, miR-363-3p, and miR-30d-3p) were identified to be up-or downregulated among the 179 miRNAs included in theqPCR panel (P, 0.05) (Table 1). Investigating the miRNAexpression profiles of total serum RNA obtained from thesame individuals showed larger interindividual variationand did not reveal any significant differences of miRNAsbetween the men with normal glucose tolerance and menwith type 2 diabetes (data not shown), suggesting exoso-mal rather than serum-derived miRNAs are altered in type2 diabetes. Next, we validated the results of the qPCR panelusing individual miRNA assays in exoRNA isolated froma larger cohort. This analysis confirmed that expression ofboth miR-20b-5p and miR-150-5p was increased in serumexosome-enriched extracellular vesicles from men withtype 2 diabetes (Table 1). For determination of whetherchanges in miRNA content of extracellular vesicles arepresent in subjects with a high risk of developing diabetes,miRNA expression in extracellular vesicle RNA was de-termined in individuals with impaired glucose tolerance(N = 16) (Supplementary Table 1). Size distribution andconcentration of extracellular vesicles analyzed by NTA inserum from men with impaired glucose tolerance were notdifferent compared with subjects with diabetes or normalglucose tolerance (data not shown). The relative expressionof miR-20b-5p was 1.30 6 0.3 (P = 0.63) and miR-150-5p1.15 6 0.11 (P = 0.68) in subjects with impaired glucosetolerance compared with subjects with normal glucosetolerance. Although both miR-20b-5p and miR-150-5pcontent were slightly increased in subjects with impairedglucose tolerance, this was not significant.

Correlation of Clinical Parameters With ExosomemiRNA ContentmiRNAs have been proposed as progression biomarkersin various diseases (19,33). Thus, we determined whetherexpression of exosome miRNAs correlated with clinicalparameters in the study cohorts (Table 2). Exosome-enriched extracellular vesicle content of miR-150-5pwas not correlated with clinical features of the studycohorts (data not shown), while content of miR-20b-5pcorrelated with 2-h glucose, as well as with the percent fatmass, in the men with normal glucose tolerance (P ,0.05) (Table 2). Interestingly, these correlations were notsignificant in either the cohort with impaired glucosetolerance or the cohort with type 2 diabetes (r = 0.12, P =0.61). We noted an inverse, but nonsignificant, correla-tion between miR-150-5p and HOMA of insulin resis-tance in the men with normal glucose tolerance(r = 20.446, P = 0.091).

Figure 1—Quantitative determination of isolated exosome-enrichedserum fractions by NTA. Particle diameter size (A) and particleconcentration (B) of exosomes determined by NTA. Values representmean 6 SEM for n = 20 men with normal glucose tolerance (NGT)and n = 21 men with type 2 diabetes (T2DM). In all box plots, centerlines show the medians; box limits indicate the 25th and 75thpercentiles as determined by R software; whiskers extend 1.5 timesthe interquartile range from the 25th to 75th percentiles, and outliersare represented by dots. n = 20 and 21 sample points for controlsubjects and subjects with type 2 diabetes, respectively.

518 Exosomal miR-20b-5p in Type 2 Diabetes Diabetes Volume 68, March 2019

miR-20b-5p Reduces STAT3 in Human CellsOf the two miRNAs that were identified as more highlyexpressed in serum exosome-enriched extracellular vesi-cles from men with type 2 diabetes, miR-20b-5p displayedthe larger fold change in the qPCR panel data (Table 1),and miR-20b-5p content in serum exosome-enriched ex-tracellular vesicles from men with normal glucose tol-erance correlated with 2-h glucose values (Table 2). Thus,miR-20b-5p was transfected into three different humancell types to evaluate miR-20b-5p effects on gene expres-sion. miR-20b-5p was overexpressed in HEK293 (a kidney-derived cell line), HepG2 (a liver-derived cell line), andprimary human skeletal muscle cells. miR-20b-5p expres-sion increased significantly in all three cell types (data notshown). STAT3 is a direct target of miR-20b-5p in MCF-7breast cancer cells (34) (Supplementary Fig. 2A); thus, wedetermined the effect miR-20b-5p expression on mRNAand protein content of STAT3 in transfected cells. WhileSTAT3 mRNA was decreased by miR-20b-5p transfectionin both HepG2 and human skeletal muscle cells, STAT3

mRNA in HEK293 was not affected (Fig. 2A). Furthermore,STAT3 protein was reduced by miR-20b-5p transfectiononly in human skeletal muscle cells (Fig. 2B).

Expression Profile of Human Skeletal Muscle CellsTransfected With miR-20b-5pWe next evaluated the mRNA expression profile in humanskeletal muscle cells after miR-20b-5p transfection. GSEA,followed by miR-20b-5p overrepresentation analysis, iden-tified key cellular functions for each gene category (Table 3and Supplementary Fig. 3). Fourteen gene sets with aP value of , 0.05 and a false discovery rate of ,0.05 wereconsidered significant. Analysis revealed that five gene setswere downregulated by miR-20b-5p, all of which regulatedimmune response or immune function. Pathway analysisidentified interferon-a and interferon-g response, tumornecrosis factor-a, interleukin (IL)2-STAT5, and IL6–januskinase (JAK)–STAT3 signaling pathways. Nine gene setswere upregulated in cells overexpressing miR-20b-5p(Table 3). The upregulated pathways include several metabolic

Table 1—Differential expression analysis of miRNA abundance in exoRNA

miRNA

qPCR panel Individual miRNA assay relativeexpression level (control vs. diabetes)Direction of change Fold change from NGT P

hsa-miR-20b-5p Up 5.3 0.044 1.52 6 0.27*

hsa-miR-532-3p Up 3.2 0.008 1.29 6 0.15

hsa-miR-150-5p Up 1.5 0.024 1.65 6 0.19*

hsa-miR-502-3p Down 3.0 0.034 1.07 6 0.20

hsa-miR-363-3p Down 1.8 0.038 1.24 6 0.19

hsa-miR-30d-5p Down 1.2 0.043 1.23 6 0.11

Data from the PCR panel are reported as mean values of fold changes for men with type 2 diabetes vs. men with normal glucosetolerance (control) and each P value of n = 4 each group. Six miRNAs showing significantly altered expression in exoRNAs derivedfrom men with type 2 diabetes were confirmed by individual miRNA qRT-PCR assays. Shown are the relative levels (mean 6 SEMfor men with type 2 diabetes vs. normal glucose tolerance [control]) for n = 20 control subjects and n = 21 subjects with type 2diabetes. NGT, normal glucose tolerance. *P , 0.05 comparing control subjects vs. subjects with diabetes.

Table 2—Correlation between exosomal miR-20b-5p content and clinical characteristics

Control Prediabetes Type 2 diabetes

r P r P r P

Fasting glucose 20.04 0.88 0.28 0.29 20.22 0.36

2-h glucose 20.58 0.01 20.31 0.26 0.12 0.61

HbA1c 0.06 0.80 20.07 0.78 20.07 0.76

HOMA-IR 20.45 0.09 20.003 0.99 20.26 0.29

Insulin 20.43 0.07 20.03 0.91 20.28 0.24

Total cholesterol 0.01 0.95 20.04 0.89 20.16 0.49

HDL 0.02 0.94 0.10 0.71 20.37 0.12

LDL 0.11 0.64 20.03 0.91 20.05 0.83

TG 20.15 0.53 20.09 0.73 20.20 0.40

Fat mass (%) 20.62 0.007 20.003 0.99 20.35 0.16

The relationship between miR-20b-5p content in exosomes and clinical characteristics was investigated by Pearson correlationcoefficient test. HOMA-IR, HOMA of insulin resistance.

diabetes.diabetesjournals.org Katayama and Associates 519

pathways such as cholesterol homeostasis (P , 0.001),fatty acid metabolism (false discovery rate: q = 0.077), andmammalian target of rapamycin (mTOR [also known asmechanistic target of rapamycin]) signaling pathway (P ,0.001).

We next focused on validating the genes that weredownregulated after miR-20b-5p transfection. We identi-fied six genes (expression fold change .2.5, P val-ues .0.005) as possible direct miR-20b-5p targetcandidates based on a target scan identification of putativemiR-20b-5p target sites (Table 4). These six genes werecytochrome b reductase 1 (CYBRD1), TBC1 domain familymember 2 (TBC1D2), AKT (also known as PKB) interactingprotein (AKTIP), RNase/angiogenin inhibitor 1(RNH1),glycoprotein integral membrane 1 (GINM1), and musclecofilin 2 (CFL2). For confirmation of the microarray data ofthese six genes, individual qRT-PCR analysis was per-formed, and reduced expression of all targets, with theexception of RNH1, was validated (Table 4).

Protein Abundance and Insulin Signaling in SkeletalMuscle Cells Expressing miR-20b-5pWe determined the protein abundance and insulin-stimulated phosphorylation of proteins involved in glycogensynthesis. Total protein content of glycogen synthase wasreduced in skeletal muscle cells expressing miR-20b-5p,and levels were unaffected by 1 h exposure to insulin (Fig.3A). In control myotubes, insulin exposure reduced in-active phospho–glycogen synthase as expected. In myo-tubes expressing miR-20b-5p, phospho–glycogen synthasewas reduced under basal conditions, reflecting the reducedabundance of glycogen synthase. Furthermore, in miR-20b-5p–transfected cells, insulin did not alter total phospho–glycogen synthase content (Fig. 3B). The ratio of theinactive form of phospho-GSK3 to total GSK3 abundancewas increased in myotubes overexpressing miR-20b-5p,and this ratio was increased in response to insulin, al-though not to the same extent as in control cells (Fig. 3C).AKT is an upstream regulator of GSK3 in the insulinsignaling pathway. We found that AKTIP was downregu-lated by miR-20b-5p transfection (Table 4). Insulin in-creased AKT phosphorylation in control cells; however,this effect was attenuated by miR-20b-5p overexpression(Fig. 3D) (AKT phospho-Thr308). Similar results werenoted for AKT phospho-Ser473 (data not shown), whereastotal AKT protein was unaltered. GSEA identified thatmiR-20b-5p overexpression reduced STAT3 signalingpathway (Table 4). Total STAT3 protein content was de-creased by miR-20b-5p transfection (Fig. 3F).

miR-20b-5p Directly Targets the AKTIP GeneFor further validation of whether miR-20b-5p is directlyinvolved in the regulation of AKTIP, a protein identified tointeract with AKT1 and enhance its phosphorylation ofboth regulatory sites, we constructed luciferase reporterassays for the AKTIP 39UTR region containing the pre-dicted miR-20b-5p target sites (Supplementary Fig. 2B andFig. 3H). After overexpression of miR-20b-5p, luciferaseactivity for AKTIP 39UTR was decreased (SupplementaryFig. 4), whereas mutagenesis of the predicted target sitesof miR-20b-5p in the AKTIP 39UTR abolished the effects ofmiR-20b-5p overexpression on luciferase activity (Fig. 3H).

miR-20b-5p Alters Glycogen Synthesis in HumanSkeletal Muscle CellsWe determined whether miR-20b-5p transfection had di-rect effects on glucose or lipid metabolism. Palmitateoxidation, as well as basal and insulin-mediated glucoseuptake, was unaltered in human muscle cells transfectedwith miR-20b-5p (data not shown). In contrast, miR-20b-5p transfection increased basal (1.2-fold) (P , 0.05) andabsolute insulin-stimulated glucose incorporation intoglycogen (Fig. 4A). However, the insulin-stimulatedincrement above basal was reduced in cells expressingmiR-20b-5p, indicating that overexpression of miR-20b-5pattenuated the insulin response with respect to glucosemetabolism (Fig. 4B).

Figure 2—Effects of miR-20b-5p overexpression on STAT3 mRNAand protein abundance in human cells. Bar graph shows gene orprotein expression in relation to negative control (NC)-transfectedcell basal. Gene expression of stat3 (A) and protein expression ofSTAT3 (B) in miR-20b-5p (miR-20b)-overexpressed cells derivedfrom different human tissues. *P , 0.05, #P , 0.01 comparingmiR-20b-5p–transfected cells with mimic miRNA–transfected con-trol cells (NC). Hskm, primary human skeletal muscle cells.

520 Exosomal miR-20b-5p in Type 2 Diabetes Diabetes Volume 68, March 2019

DISCUSSION

We found that RNA abundance of miR-20b-5p and miR-150-5p is increased in exosome-enriched extracellularvesicles prepared from serum of patients with type 2 di-abetes, whereas total serum miRNA expression of thesemiRNA species is not significantly altered compared withglucose-tolerant men. To study the effects of miR-20b-5pon gene expression, signal transduction, and metabolism,we overexpressed miR-20b-5p miRNA in three differenthuman cell types. We found that miR-20b-5p overexpres-sion in primary human muscle cells suppressed expressionof AKTIP, STAT3, and glycogen synthase and impairedinsulin signaling in primary human skeletal muscle cells.Thus, peripheral expression ofmiR-20b-5p alters expression

of genes involved in pathways related to immune functionand impairs glucose metabolism.

miRNAs are present in biological fluids, including blood,urine, breast milk, and cerebrospinal fluids, and recentefforts have been focused on delineating the expressionprofiles of miRNAs for use as disease biomarkers (33,35).While serum and plasma are easily obtained by minimallyinvasive methods and offer sufficient volume for analysis,the presence of cell debris, proteins, and protein complexesmakes any analysis of miRNA profiles in biofluids techni-cally challenging. miRNAs in serum and plasma are foundin several contexts, including protein complexes such asAgo2-miRNA, exosomes, microvesicles, and lipid-proteincomplexes such as HDL-miRNA (36). While miRNAs in

Table 3—Summary of GSEA

Annotated cellular function Overlapping genes (n) P FDR (q)

Downregulated by miR-20b-5pInterferon_alpha_response 96 ,0.001 ,0.001Interferon_gamma_response 195 ,0.001 ,0.001TNFA_signaling_via_NFKB 198 ,0.001 ,0.001IL2_STAT5_signaling 194 ,0.001 ,0.001IL6_JAK_STAT3_signaling 83 ,0.05 ,0.05

Upregulated by miR-20b-5pMYC_targets_V1 195 ,0.001 ,0.01Cholesterol_homeostasis 74 ,0.001 ,0.001MTORC1_signaling 198 ,0.001 ,0.001G2M_checkpoint 196 ,0.001 ,0.001E2F_targets 192 ,0.001 ,0.001Unfolded_protein_response 111 ,0.001 ,0.01Androgen_response 98 ,0.001 ,0.01Estrogen_response_early 196 ,0.001 ,0.05MYC_targets_V2 54 ,0.05 ,0.05Fatty_acid_metabolism 153 ,0.05 0.077288PI3K_AKT_MTOR_signaling 104 0.079439 0.133628

GSEA analysis was performed on the ranked genes according to the ratios of transcripts from mimic control– and miR-20b-5p–transfected human skeletal muscle cells. Fourteen gene sets with a P value of ,0.05 and a false discovery rate (FDR) of ,0.05 wereconsidered significant comparing miR-20b-5p–transfected cells with mimic miRNA–transfected control cells. Of these, five genesets associate with the miR-20b-5p–induced downregulated genes and nine gene sets are associated with miR-20b-5p–inducedupregulated genes.

Table 4—miR-20b-5p–induced downregulated genes in human skeletal muscle cells

Gene symbol Gene name

Microarray

Predicted miR-20b-5ptarget site

Individual miRNA assayrelative expression level(transfected vs. NC)

Fold change(transfected vs. NC) P

CYBRD1 Cytochromeb reductase 1

4.1 0.0023 Yes 0.15 6 0.039**

TBC1D2 TBC1 domain family,member 2

3.0 0.0049 Yes 0.30 6 0.038**

AKTIP AKT interacting protein 2.7 0.0022 Yes 0.37 6 0.020**

RNH1 RNase/angiogenininhibitor 1

2.6 0.0028 Yes ND

GINM1 Glycoprotein integralmembrane 1

2.6 0.000013 Yes 0.37 6 0.0064**

CFL2 Cofilin 2 (muscle) 2.5 0.0016 Yes 0.41 6 0.084*

Data of relative expression level by individual miRNA assay are means 6 SEM of n = 3. The microRNA.org resource was used forprediction of miR-20b-5p target sites. NC, mimic miRNA–transfected; ND, no data. *P , 0.05; **P , 0.01 comparing miR-20b-5p–transfected cells with mimic miRNA–transfected control cells.

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protein complexes or extracellular vesicles are quite stablein blood, different miRNA carriers appear to have differentfunctions in cells (9). In comparison of serum and plasmaprepared from the same blood sample, higher miRNAconcentration was noted in serum compared with thecorresponding plasma, and the miRNA spectrum betweenserum and plasma differed (37). In the current study, wefocused on miRNA isolated from serum exosome-enrichedextracellular vesicles, as exosomes have been functionallyidentified as mediators of cell-to-cell miRNA transfer.

The particle characteristics of exosomes isolated from menwith normal glucose tolerance or type 2 diabetes in this studywere similar. A limitation of the current study is thatalthough subjects were matched for age and BMI, a numberof the subjects with type 2 diabetes were on antidiabetesmedication and as a group had a higher level of use of othermedications. Thus, we are unable to exclude potentialeffects of medication on the miRNA profiles of exosome-enriched extracellular vesicles. While total serum miRNAspecies did not differ between the cohorts, possibly

Figure 3—Effect of miR-20b-5p overexpression on protein abundance and insulin signaling and miR-20b-5p–regulated targets in humanskeletal muscle cells. Bar graph shows protein abundance in relation to negative control (NC)-transfected cell basal. A: Glycogen synthase(GS). B: phospho–glycogen synthase (P-GS). C: phospho (P)-GSK3–to–GSK3 ratio. D: phospho-AKT (P-AKT). E: Total AKT. F: STAT3. G:Representative Western blot of GS, phospho-GS, phospho-GSK3, GSK3a, GSK3b, AKT, phospho-AKT, STAT3, and GAPDH in humanskeletal muscle cells transfected with control mimic miRNA or miR-20b-5p incubated in the absence (0) or presence of 1.2 nmol/L(submaximal) or 120 nmol/L (maximal) insulin. H: Luciferase activity in HEK293 cells overexpressing the AKTIP 39UTR constructs andschematic of constructs used for luciferase assays after transfection with negative control or miR-20b-5p (miR-20b) (n = 3). The controlconstruct without 39UTR region (empty), construct including the AKTIP 39UTR region (AKTIP 39UTR), and construct with point mutations inthe putative miR-20b-5p binding positions of AKTIP 39UTR (Mut) were each transfected as were the AKTIP 39UTR constructs. The sche-matic of constructs shows the region of AKTIP 39UTR (1–2,525/2,525 bp) included in AKTIP 39UTR constructs used in this study(1,137–2,525/2,525 bp), the putative miR-20b-5p positions in the AKTIP 39UTR region, and positions of introduced mutations. Data aremeans 6 SEM. *P , 0.05 and #P , 0.01 as determined by paired Student t test comparing miR-20b-5p–transfected cells with mimicmiRNA–transfected control cells or mutated 39UTR plasmid to the same condition as indicated. †P, 0.05 for insulin response within onecondition.

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reflecting the more heterogeneous composition of totalserum miRNA, we found two miRNA species that wereincreased in exosome-enriched extracellular vesicles fromserum of men with type 2 diabetes. Neither miR-20b-5pnor miR-150-5p was significantly elevated in serum-derived exosome-enriched extracellular vesicles from in-dividuals with impaired glucose tolerance, which couldindicate that these miRNAs reflect a more advanced dis-ease stage. Thus, miRNA profiling of functional units suchas exosomes may increase the probability of identifyingdisease-relevant biomarkers.

miR-20b-5p and miR-150-5p content was increasedin serum-derived exosome-enriched extracellular vesiclesfrom men with type 2 diabetes. Circulating miR-150-5p inplasma has been proposed as a potential biomarker foracute myeloid leukemia (38) and has been implicated in the

promotion of angiogenesis by microvesicle-mediatedtransfer of miR-150-5p (39). Notably, miR-150-5p is spe-cifically upregulated in skeletal muscle from diabetic Goto-Kakizaki rats (40). Whether the elevation in miR-150-5pdirectly contributes to the insulin resistant phenotype inGoto-Kakizaki rats, or secondarily to impaired metabolichomeostasis, is not known. In the current study, we didnot observe any correlations between miR-150-5p exo-some content and type 2 diabetes–related metabolic traits.miR-20b-5p belongs to the miR-17 family and is part of thelarger family of highly similar miRNAs, including miR-106a-363, miR-17-192, andmiR-106b-25 cluster (41), thatmodulate VEGF expression by targeting HIF-1a andSTAT3 in MCF-7 breast cancer cells (34). While thesemiRNAs have been associated with metabolic disordersand type 2 diabetes, a detailed understanding of the

Figure 4—Glycogen synthesis in primary human muscle cells after miR-20b-5p overexpression. A: Glucose incorporation into glycogen inhuman skeletal muscle cells transfected with miR-20b-5p and (B) insulin-stimulated increment above basal in negative control (NC) mimicmiRNA–transfected human skeletal muscle cells or miR-20b-5p–transfected human skeletal muscle cells (miR-20b). Data are mean6 SEM(n = 3 independent experiments). Insulin concentrations as indicated (0, 1.2, and 120 nmol/L). *P , 0.05, #P , 0.01, relative to the samecondition comparing mir-20b-5p–transfected cells with mimic miRNA–transfected control cells. †P , 0.05 for insulin response within onecondition. C and D: Schematic model of proposed role of miR-20b-5p in the presence and absence of insulin. The insulin-AKT signalingpathway (arrows) has been well characterized and involves the insulin receptor, IRS1, PI3K, AKT, GSK3, and glycogen synthase (GS). C: Wepropose a model where in the absence of insulin, miR-20b-5p reduces abundance of glycogen synthase, most likely via an indirectmechanism. Phosphorylation of glycogen synthase renders it inactive. Reduced basal glycogen synthase phosphorylation in myotubesoverexpressing miR-20b-5p was coincident with increased basal rate of glycogen synthase (D). In the presence of insulin, miR-20b-5p–mediated direct targeting of AKTIP (reducing phospho-AKT stability) reduces the insulin signal from phosphorylation of AKT anddownstream. Red color indicates reduced protein expression, green color indicates metabolic end points, and blue arrows indicatephosphorylation events, denoted by p.

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physiological functions of miR-150-5p or miR-20b-5p iswarranted.

Type 2 diabetes is a multifactorial metabolic diseaseaffecting numerous tissues, including liver, skeletal mus-cle, adipose tissue, pancreas, and brain. To explore possiblephysiological functions of miR-20b-5p, we transfectedmiR-20b-5p into human liver, kidney, and skeletal musclecells and assessed STAT3 protein abundance. STAT3 pro-tein has previously been reported to be a direct target ofmiR-20b-5p (34). We noted that the miR-20b-5p over-expression led to the greatest reduction in STAT3 proteinabundance in skeletal muscle cells. Whether this is due totissue-specific differences or properties of the cultures(primary muscle cells as opposed to immortalized cell linesfor kidney and liver cells) remains to be further investi-gated. mRNA expression of genes relevant for severalpathways implicated in immune function was altered inmyotubes overexpressing miR-20b-5p. Given the growingappreciation that insulin resistance and type 2 diabetes areassociated with chronic low-grade inflammation, and pre-vious findings that miR-20b-5p may play a role in themodulation of some inflammatory signals (42), our resultsin men with type 2 diabetes and cultured myotubes arecompelling. miR-20b-5p targets the STAT3 gene in MCF-7breast cancer cells (34), and we confirm this association inour microarray data, as well as at the protein level.Collectively, these results suggest that miR-20b-5p directlytargets the STAT3 gene in human skeletal muscle cells.

Lifestyle intervention programs to increase physicalactivity and promote weight loss in adults at risk fortype 2 diabetes are associated with changes in circulatingmiRNAs, including reductions in miR-20b-5p (43). Inmouse models, miR-20a-5p promotes adipocyte differen-tiation (44). Nevertheless, a direct link between changes inmiRNA-20b-5p abundance and improvements in glucosehomeostasis is unknown. Here, we report that overexpres-sion of miR-20b-5p in human skeletal muscle cells im-pacted mRNA expression of genes involved in severalmetabolic pathways, including cholesterol homeostasis,fatty acid metabolism, and glycolysis. While genes involvedin fatty acid oxidation or glucose uptake were unaltered inmiR-20b-5p–transfected myotubes (data not shown), gly-cogen synthesis was affected. We found that basal glucoseincorporation into glycogen was increased in myotubesexpressing miR-20b-5p, and insulin action was blunted.mRNA content of several genes directly relevant to glyco-gen synthesis, as well as insulin signaling, was altered inmiR-20b-5p–transfected myotubes, which could partlyexplain the alterations in glucose metabolism. Althoughthere was no change in mRNA, myotubes expressing miR-20b-5p had reduced total glycogen synthase protein, likelyreflecting posttranslational downregulation. Phosphoryla-tion of glycogen synthase renders it inactive (45). Wefound that basal glycogen synthase phosphorylation wasreduced in myotubes overexpressing miR-20b-5p, which isconsistent with the increased basal rate of glycogen syn-thase noted in these cells. Upon insulin stimulation, the

level of phosphorylated glycogen synthase was signifi-cantly reduced in control cells as expected (coincidentwith increased glycogen synthesis), while in myotubesexpressing miR-20b-5p, insulin did not reduce glycogensynthase phosphorylation, and in miR-20b-5p–expressingcells there was an impaired insulin-stimulated increase inglycogen synthesis. The effects of miR-20b-5p on skeletalmuscle metabolism are likely to reflect changes in proteincontent from miR-20b-5p targets as well as second-ary consequences of these changes in protein expres-sion, which are evident at the level of insulin signaltransduction.

We identified AKTIP as a direct miR-20b-5p target.AKTIP, also known as mouse Ft1 orthologous (46), isreported to interact directly with AKT and modulatethreonine kinase AKT phosphorylation and activation byPDK1 (47). AKTIP facilitates telomeric DNA replication(48), but the functions of AKTIP in other AKT signalingpathways are ambiguous. For example, RNA interference–mediated reduction of AKTIP in primary human fibro-blasts leads to a profound proliferation defect arrested inlate S phase (48). We provide evidence that AKTIP is adirect target of miR-20b-5p. The reduced AKTIP mRNAin miR-20b-5p–overexpressing myotubes was coincidentwith a reduced insulin-stimulated AKT phosphoryla-tion. The glycogen synthase gene has a putative miRNA-20b target sequence in the 39UTR region, and although wedid not observe changes of glycogen synthase mRNA aftermiRNA-20b transfection, the total protein content of gly-cogen synthase was reduced. miRNAs exert effects bothby reducing message stability and by preventing transla-tion. A schematic overview of putative functional effectsof miR-20b-5p in skeletal muscle is presented in Fig. 4C andD, which are likely to reflect modification of both primary(direct miRNA-20b-5p targets) and indirect effects.

Exosomes are released from cells into the circulationand transported to target cells to deliver cargo, includingproteins and nucleic acids, such as various RNA species.Functional miRNA are delivered to target cells (11,49);however, little is known of the molecular machinery thatregulates this process, including the tissue of origin of theexosomes, the specific target tissues of exosomes, and themanner in which the cargo is delivered. Thus, we areunable to ascertain the cellular mechanism by which theexosomes derived from serum of patients with type 2 di-abetes are released or targeted and the specific cell or tissueinvolved, and this remains a limitation of the currentstudy. Downregulation of miR-20b-5p targets, includingSTAT3 and AKTIP, are likely to have tissue-specific effects.Further studies are required to understand the physiolog-ical role of increased serummiR-20b-5p in individuals withtype 2 diabetes.

Here, we provide evidence that miR-20b-5p is highlyexpressed in serum exosome-enriched extracellularvesicles isolated from patients with type 2 diabetes. More-over, when introduced into skeletal muscle cells, miR-20b-5palters cellular glucose metabolisms and the STAT3 and AKT

524 Exosomal miR-20b-5p in Type 2 Diabetes Diabetes Volume 68, March 2019

signaling pathway. Since serum exosomal miR-20b-5p iscorrelated with low 2-h glucose values as well as down-regulation of inflammatory pathways, it is possible thatmiR-20b-5p may not be an effector of impaired insulinsignaling and glycogen synthesis but, instead, may be partof a homeostatic mechanism attempting to improve meta-bolic control. Taken together, our results highlight poten-tial interactions between exosome-enriched extracellularvesicles and metabolic regulation.

Acknowledgments. The authors thank Dr. Jorge Ruas and Dr. LarsKetscher, both at Karolinska Institutet, for helpful discussion and input. The authorsalso acknowledge the core facility for Bioinformatics and Expression Analysis atNovum, where the gene arrays were performed. This facility is supported by the boardof research at the Karolinska Institute and the research committee at the KarolinskaUniversity Hospital.Funding. This work was supported by grants from the Strategic DiabetesProgram at Karolinska Institute, European Research Council Ideas Program(ICEBERG, ERC-2008-AdG23285), Vetenskapsrådet (Swedish Research Council)(2011-3550, 2012-1760, 2015-165), Swedish Diabetes Foundation (DIA2015-032, DIA2015-052), Stiftelsen för Strategisk Forskning (Swedish Foundation forStrategic Research) (SRL10-0027), Diabetes Wellness Sweden, Novo NordiskFoundation (NNF14OC0009941), Swedish Research Council for Sport Science(FO2016-0005), the Swedish Heart Lung Foundation (20150423), and StockholmLäns Landsting (Stockholm County Council).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. M.K. conceived and performed experiments,analyzed data, and wrote the manuscript. O.P.B.W. and S.E.-A. provided expertiseand feedback. T.F. and K.C. performed clinical analysis and provided feedback.J.R.Z. and A.K. conceived and planned the study, secured funding, and wrote themanuscript. M.K., O.P.B.W., T.F., K.C., S.E.-A., J.R.Z., and A.K. read and approvedthe final manuscript. M.K. and A.K. are the guarantors of this work and, as such,had full access to all the data in the study and take responsibility for the integrity ofthe data and the accuracy of the data analysis.

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