Circulating Exosomal Mir-20B-5P Is Elevated in Type 2 Diabetes and Could Impair Insulin Action in Human Skeletal Muscle

Diabetes Volume 68, March 2019 515

Circulating Exosomal miR-20b-5p Is Elevated in Type 2 Diabetes and Could Impair Insulin Action in Human Skeletal Muscle

Mutsumi 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 important target mRNA (1,2). miRNAs function to negatively regu- class of gene expression modulators. Extracellular circu- late the abundance of specific proteins and in this way lating miRNAs are both candidate biomarkers for disease exert control over numerous cellular and biological pro- pathogenesis and mediators of cell-to-cell communica- cesses including metabolism (3,4). While miRNAs are tion. We examined the miRNA expression profile of total transcribed and exert many effects directly in the cell of serum and serum-derived exosome-enriched extracellu- origin, miRNAs are also secreted and stable miRNAs can be lar vesicles in people with normal glucose tolerance or detected in plasma (5). Circulating miRNAs have been METABOLISM type 2 diabetes. In contrast to total serum miRNA, which detected in most biofluids including blood (serum/plasma), did not reveal any differences in miRNA expression, we urine, breast milk, and cerebrospinal fluids and are pro- identified differentially abundant miRNAs in patients with tected from degradation by a variety of mechanisms. A type 2 diabetes using miRNA expression profiles of proportion of circulating miRNAs are packaged in extra- exosome RNA (exoRNA). To validate the role of these dif- cellular vesicles, such as “exosomes” (50- to 200-nm ferentially abundant miRNAs on glucose metabolism, – we transfected miR-20b-5p, a highly abundant exoRNA in membrane-coated vesicles) (6 9) that protect RNA cargo patients with type 2 diabetes, into primary human skeletal from endogenous RNase activity (10). miRNAs can also muscle cells. miR-20b-5p overexpression increased basal bind to various proteins including lipoproteins, namely, glycogen synthesis in human skeletal muscle cells. We HDL, or argonaute (AGO) proteins, which are the key identified AKTIP and STAT3 as miR-20b-5p targets. miR- components of the RNA-induced silencing complex, to 20b-5p overexpression reduced AKTIP abundance and form miRNA-protein complexes for transport (10–12). insulin-stimulated glycogen accumulation. In conclusion, Exosomes in plasma/serum have been implicated in trans- exosome-derived extracellular miR-20b-5p is a circulating fer of miRNA into target cells and thus play a role in cell- biomarker associated with type 2 diabetes that plays an cell communication (11–15). intracellular role in modulating insulin-stimulated glucose The presence of circulating miRNAs has prompted metabolism via AKT signaling. efforts to identify biomarkers for various pathologies, including cancer, and diseases affecting cardiovascular, neurological, metabolic, and immune function (16–22). miRNAs are a class of small noncoding RNAs that function Specific circulating miRNAs may be useful biomarkers as translational repressors by direct interaction with their for diagnoses and management of progressive diseases

1Department of Physiology and Pharmacology, Karolinska Institute, Stockholm, Corresponding author: Anna Krook, [email protected] Sweden Received 24 April 2018 and accepted 1 December 2018 2Department of Laboratory Medicine, Karolinska Institute, Huddinge, Sweden This article contains Supplementary Data online at http://diabetes 3Department of Clinical Physiology, Karolinska University Hospital, Stockholm, .diabetesjournals.org/lookup/suppl/doi:10.2337/db18-0470/-/DC1. Sweden 4Department of Molecular Medicine and Surgery, Karolinska Institute, Stockholm, © 2018 by the American Diabetes Association. Readers may use this article as Sweden long as the work is properly cited, the use is educational and not for profit, and the 5Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska work is not altered. More information is available at http://www.diabetesjournals Academy, University of Gothenburg, Gothenburg, Sweden .org/content/license. 6Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, U.K. 516 Exosomal miR-20b-5p in Type 2 Diabetes Diabetes Volume 68, March 2019 such as type 2 diabetes (23–25). Despite the fact that (Exiqon), using MS2 RNA (Roche, Basel, Switzerland) as patients with type 2 diabetes are characterized by hyper- carrier RNA according to the manufacturer’s instructions. glycemia and elevated HbA1c levels, these changes in Each exoRNA elute was reverse transcribed using the clinical chemistry are only detected once metabolic imbal- miRCURY LNA Universal RT cDNA Synthesis Kit (Exiqon). ance has occurred. Defects in multiple tissues controlling For the first screening, human serum/plasma Focus glucose homeostasis and insulin sensitivity are often pres- miRNA PCR panels (96-well [V43.AF]) (Exiqon) were ent years prior to diagnosis (26). Given the complex used in a quantitative (q)RT-PCR (qRT-PCR)-based ap- pathophysiology and disease burden of type 2 diabetes, proach to determine levels of 179 human miRNAs. The efforts have been focused on identifying circulating miR- quantitative PCR (qPCR) was performed using a StepOne NAs as novel prognostic biomarkers (27–29). Plus (Applied Biosystems) with 40 amplification cycles, To date, there is little consensus on the precise nature using the cycling parameters recommended by Exiqon. of circulating miRNA biomarkers in different cohorts of Raw data were processed using StepOne software version patients with type 2 diabetes, and little is known regarding 2.3 (Applied Biosystems) to assign the baseline and thresh- the functional role(s) of the identified miRNAs in metabolic old for threshold cycle (Ct). For determination of the processes implicated in type 2 diabetes pathogenesis. Here, technical variation between the Exiqon serum/plasma we determined total serum and exosomal miRNA expres- Focus miRNA PCR panel plates, the interplate calibrator sion profiles in men with normal glucose tolerance or type (UniSp3) was analyzed. Ct values of the interplate calibra- 2 diabetes and validated the effects of the differentially tor were analyzed to be highly similar across all samples. abundant miRNAs on metabolism in skeletal muscle. The normalization and analysis of the PCR panel plate results were provided by Exiqon GenEx software, version RESEARCH DESIGN AND METHODS 6. For validation of the results of the PCR panel plate in Study Participants and Serum Samples a second screening, we used a miRCURY LNA miRNA PCR System (Exiqon) to assess the expression level of individual The study was approved by the ethics committee of miRNAs. Gene expression levels were quantified using the Karolinska Institute. Informed written consent was fi obtained from all volunteers. Twenty men with normal miRNA-speci c LNA PCR primer. Relative expression was calculated using the comparative Ct method. glucose tolerance, 16 men with impaired glucose tolerance, and 21 men with type 2 diabetes were recruited by newspaper advertisement or from a primary health care Primary Human Skeletal Muscle Culture and miRNA clinic. The participants with type 2 diabetes, impaired Transfection Protocol glucose tolerance, and normal glucose tolerance were Satellite cells were isolated from vastus lateralis skeletal matched for age and BMI. Clinical characteristics of the muscle as previously described (30). Cell cultures were study participants are presented in Supplementary Table 1. maintained at 37°C under 7.5% CO2 as myoblast cells. For Blood samples were separated in serum and peripheral measurement of gene expression, myoblast cells were blood mononuclear cells. differentiated into myotubes as previously described (31). Myotube cells where fusion and multinucleation were Isolation of Exosome-Enriched Extracellular Vesicles observed at day 10 after initiation of differentiation were and Nanoparticle Tracking Analysis used for total RNA extraction, protein detection, and cell Extracellular vesicles were obtained from serum with assays. Cells were transfected using a double transfection a miRCURY Exosome Isolation Kit – Serum and Plasma protocol 48 h after differentiation (day 6) and 48 h later (Exiqon, Vedbaek, Denmark) according to the manufac- (day 8) with 10 nmol/L Ambion miRNA-20b-5p (Thermo turer’s instructions. Isolated extracellular vesicle samples Fisher Scientific). Control cells were transfected with were analyzed using nanoparticle tracking analysis (NTA). a scrambled miRNA mimic (10 nmol/L). Each transfection Samples were loaded into the sample chamber of an NS500 was performed for 5 h with transfection complexes unit (NanoSight, Amesbury, U.K.), and five 1-min videos of formed in reduced serum media (OptiMEM; Thermo each sample were recorded (threshold, 6 – multi; blur, Fisher Scientific) using Lipofectamine RNAiMAX trans- auto; and minimum expected particle size, auto). Data anal- fection reagent according to the manufacturer’s protocol. ysis was performed with NTA 2.3 software (NanoSight), RNA was isolated using an miRNeasy Kit (QIAGEN) at day and the size and concentration of particles included in the 10. extracellular vesicle samples were calculated. An aliquot of isolated exosome-enriched extracellular vesicles from se- Culture of Human Embryonic Kidney (HEK293) and rum was used for NTA, and the remaining isolated extra- Human Hepatocellular Carcinoma (HepG2) Cells and cellular vesicles were used for exosome RNA (exoRNA) miRNA Transfection Protocol extraction. HEK293 and HepG2 cells were obtained from ATCC and cultured in high-glucose (4.5 g/L) DMEM supplemented RNA Extraction and Evaluation of miRNA Expression with 10% (vol/vol) FBS. miRNA-20b-5p or miRNA mimic A miRCURY RNA Isolation Kit (Exiqon) was used to extract was transfected into those cells using Lipofectamine RNAi- exoRNA, together with an RNA Spike-in Template Kit MAX transfection reagent according to the manufacture’s diabetes.diabetesjournals.org Katayama and Associates 517 protocol, and RNA was isolated using the miRNeasy Kit with primary antibodies directed to glycogen synthase (QIAGEN). (number 3893; Cell Signaling Technology), phosphorylated (phospho)–glycogen synthase (3891; Cell Signaling Microarray Analysis Technology), AKT (9272; Cell Signaling Technology), mRNA content from miR-20b–transfected cells was pro- phospho-AKT (Thr308) (4056; Cell Signaling Technology), filed by hybridizing biotinylated sense strand cDNA to signal transducer and activator of transcription (STAT)3 GeneChip Human Gene 2.0 ST arrays (Thermo Fisher (9139; Cell Signaling Technology), and GAPDH (sc-25778; Scientific). Sense strand cDNA was synthesized from total Santa Cruz Biotechnology, Dallas, TX). Membranes were RNA and biotin labeled with the GeneChip WT PLUS incubated with species-appropriate horseradish peroxidase– Reagent Kit (Thermo Fisher Scientific) before being hy- conjugated secondary antibody before proteins were vi- bridized to arrays. Gene arrays were washed, stained, and sualized by enhanced chemiluminescence (Amersham scanned as instructed by Affymetrix (Santa Clara, CA). ECL Western Blotting Detection Reagent, GE Healthcare Preprocessing of data was performed using a robust multi- Life Sciences, Little Chalfont, U.K.). When appropri- array average with sketch quantile normalization by Ex- ate, protein content was quantified by densitometry pression Console software (Affymetrix). Differential (Quantity One; Bio-Rad). All quantifications were per- expression of transcripts was determined with a paired formed using a positive control to control for intergel class comparison with a univariate test using a random variability. variance model comparing gene expression of control (miRNA mimic–transfected cells) versus miR-20b-5p– Luciferase Activity Measurement transfected cells. Genes with a false discovery rate The luciferase reporter clone having the AKTIP 39 un- of ,10% were considered to be differentially regulated. translated region (UTR) (HmiT088513-MT05) was pur- The microarray data were submitted to the National chased from GeneCopoeia (Rockville, MD). This clone Center for Biotechnology Information Gene Expression included two predicted miR-20b-5p target sites in AKTIP Omnibus (GEO) and can be found under the GEO series 39UTR (Fig. 3H). Target search in microRNA.org (http:// accession number GSE102295. www.microrna.org) was used for prediction of miR-20b-5p target sites. Predicted miR-20b-5p binding sites were Gene Set Enrichment Analysis mutated using the QuikChange II XL Site-Directed Muta- Gene Set Enrichment Analysis (GSEA) was used to link genesis Kit (Agilent Technologies, Santa Clara, CA). Oli- genes identified in a specific gene group with their occur- goprimers used for mutagenesis of the AKTIP 39UTR were rence in biological pathways or processes. The rank gene 59-GATGGTGAATCTGGTGCACCATCCTGAAACCTGCTAG- list was further annotated using MSigDBv5.0 downloaded ACTCTGGCCTAG-39,59-CTAGGCCAGAGTCTAGCAGGTT- from the Broad Institute (http://www.broadinstitute.org/), TCAGGATGGTGCACCAGATTCACCATC-39,59-GAGAGCA- which contains curated functional gene sets of various GGTTCCATAGCTCACCTGCGATAAGTGGAAGATCATTTG- biological states. AATCTC-39,and59-GAGATTCAAATGATCTTCCACTTATC- GCAGGTGAGCTATGGAACCTGCTCTC-39. Cells were seeded Evaluation of miR-20b-5p Targets in 96-well plates 24 h before experimentation. 39UTR pro- Putative target sites were probed in silico by target moter plasmids (100 ng/well) was transfected into HEK293 prediction algorithms (TargetScan). For validation cells in 96-well plates using the transfection reagent Lipo- experiments, 500 ng total RNA from cells was reverse fectamine2000(LifeTechnologies)with10nmol/LmiRNA transcribed using a high-capacity cDNA reverse tran- mimic for miR-20b-5p. Control cells were transfected with scription kit (Thermo Fisher Scientific), and qRT-PCR appropriate scrambled miRNA mimic. After 24 h, the cell was performed to measure the expression level of six culture medium was collected and processed for luciferase genes using SYBR Green Master Mix Kit (Thermo Fisher assay using the Secrete-Pair Luminescence Assay Kit Scientific) and StepOnePlus (Bio-Rad) (primer list in (GeneCopoeia). Assays were read in the CLARIOstar (BMG Supplementary Table 2). For the stat3 gene only we LABTECH) and normalized with secreted alkaline phos- used TaqMan Assay (Thermo Fisher Scientific). We phatase signals. used the TBP and M2B genes as reference genes, and relative quantification values were calculated using the Glucose Incorporation Into Glycogen 2DDCt equation 2 . Insulin-stimulated glucose incorporation into glycogen was determined as previously described (30). Immunoblot Analysis Western blot analysis was performed as previously de- Statistics scribed (32). Protein content of the supernatants was Data are presented as mean 6 SEM. Differences were determined by BCA Protein Assay Kit (Pierce Biotechnol- analyzed using either paired or unpaired Student t test as ogy, Rockford, IL). Membranes were stained with Ponceau appropriate. Relationships were evaluated by computation S to confirm equal loading of samples and quality control of Pearson correlation coefficients. Significance was set at for the transfer procedure. Membranes were incubated P , 0.05. 518 Exosomal miR-20b-5p in Type 2 Diabetes Diabetes Volume 68, March 2019

RESULTS exoRNA derived from serum from men with normal Exosome-Enriched Extracellular Vesicles Isolated glucose tolerance or type 2 diabetes. For the initial screen- From Serum From Men With Normal Glucose Tolerance ing, exoRNA was extracted from four men with normal or Type 2 Diabetes glucose tolerance and four men with type 2 diabetes, and We used a commercial exosome isolation kit to isolate miRNA expression was profiled using a qPCR panel. Four exosome-enriched extracellular vesicles rapidly from se- men from each group were randomly selected, still match- rum to determine miRNA expression profile of exosomes. ing for age and BMI. In this first screen, six exosomal Differential ultracentrifugation, the current gold stan- miRNAs (miR-20b-5p, miR-532-3p, miR-150-5p, miR-502- dard of isolation, was compared with the commer- 3p, miR-363-3p, and miR-30d-3p) were identified to be up- cial isolation kit. While both methods yielded similar or downregulated among the 179 miRNAs included in the results, the reduced requirement of serum for isolation qPCR panel (P , 0.05) (Table 1). Investigating the miRNA dictated the use of the commercial kit. The resultant expression profiles of total serum RNA obtained from the fractions were analyzed by immunoblotting for known same individuals showed larger interindividual variation exosome markers and major protein components of and did not reveal any significant differences of miRNAs HDL particles in serum (Supplementary Fig. 1). This between the men with normal glucose tolerance and men showed that exosome marker proteins, ALIX and CD9, with type 2 diabetes (data not shown), suggesting exoso- were more enriched in the isolated fraction compared mal rather than serum-derived miRNAs are altered in type with original serum, accompanied by a significant re- 2 diabetes. Next, we validated the results of the qPCR panel duction in the HDL marker protein, apolipoprotein A1 using individual miRNA assays in exoRNA isolated from (APOA1). Next, size distribution and concentration of a larger cohort. This analysis confirmed that expression of extracellular vesicles in serum from men with normal both miR-20b-5p and miR-150-5p was increased in serum glucose tolerance, or type 2 diabetes, were analyzed by exosome-enriched extracellular vesicles from men with NTA. Patients with type 2 diabetes had altered serum type 2 diabetes (Table 1). For determination of whether lipid levels (Supplementary Table 1), which may influ- changes in miRNA content of extracellular vesicles are ence the presence of extracellular vesicles, including present in subjects with a high risk of developing diabetes, exosomes, which contain lipids. However, particle size miRNA expression in extracellular vesicle RNA was de- (Fig. 1A) and particle concentration (Fig. 1B)ofthe termined in individuals with impaired glucose tolerance extracellular vesicle samples were unaltered between (N = 16) (Supplementary Table 1). Size distribution and men with normal glucose tolerance and men with concentration of extracellular vesicles analyzed by NTA in type 2 diabetes. serum from men with impaired glucose tolerance were not different compared with subjects with diabetes or normal Differentially Expressed miRNAs in ExoRNA and Total glucose tolerance (data not shown). The relative expression Serum RNA From Men With Normal Glucose Tolerance of miR-20b-5p was 1.30 6 0.3 (P = 0.63) and miR-150-5p or Type 2 Diabetes 1.15 6 0.11 (P = 0.68) in subjects with impaired glucose Exosomes are known to carry noncoding RNAs (6), such as tolerance compared with subjects with normal glucose fi miRNA. We determined the miRNA expression pro le of tolerance. Although both miR-20b-5p and miR-150-5p content were slightly increased in subjects with impaired glucose tolerance, this was not significant.

Correlation of Clinical Parameters With Exosome miRNA Content miRNAs have been proposed as progression biomarkers in various diseases (19,33). Thus, we determined whether expression of exosome miRNAs correlated with clinical parameters in the study cohorts (Table 2). Exosome- enriched extracellular vesicle content of miR-150-5p was not correlated with clinical features of the study cohorts (data not shown), while content of miR-20b-5p correlated with 2-h glucose, as well as with the percent fat Figure 1—Quantitative determination of isolated exosome-enriched mass, in the men with normal glucose tolerance (P , serum fractions by NTA. Particle diameter size (A) and particle B 0.05) (Table 2). Interestingly, these correlations were not concentration ( ) of exosomes determined by NTA. Values represent ﬁ mean 6 SEM for n = 20 men with normal glucose tolerance (NGT) signi cant in either the cohort with impaired glucose and n = 21 men with type 2 diabetes (T2DM). In all box plots, center tolerance or the cohort with type 2 diabetes (r =0.12,P = lines show the medians; box limits indicate the 25th and 75th 0.61). We noted an inverse, but nonsigniﬁcant, correla- percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th to 75th percentiles, and outliers tion between miR-150-5p and HOMA of insulin resis- are represented by dots. n = 20 and 21 sample points for control tance in the men with normal glucose tolerance subjects and subjects with type 2 diabetes, respectively. (r = 20.446, P = 0.091). diabetes.diabetesjournals.org Katayama and Associates 519

Table 1—Differential expression analysis of miRNA abundance in exoRNA qPCR panel Individual miRNA assay relative miRNA Direction of change Fold change from NGT P expression level (control vs. diabetes) 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 glucose tolerance (control) and each P value of n = 4 each group. Six miRNAs showing signiﬁcantly altered expression in exoRNAs derived from men with type 2 diabetes were conﬁrmed by individual miRNA qRT-PCR assays. Shown are the relative levels (mean 6 SEM for men with type 2 diabetes vs. normal glucose tolerance [control]) for n = 20 control subjects and n = 21 subjects with type 2 diabetes. NGT, normal glucose tolerance. *P , 0.05 comparing control subjects vs. subjects with diabetes.

miR-20b-5p Reduces STAT3 in Human Cells mRNA in HEK293 was not affected (Fig. 2A). Furthermore, Of the two miRNAs that were identified as more highly STAT3 protein was reduced by miR-20b-5p transfection expressed in serum exosome-enriched extracellular vesi- only in human skeletal muscle cells (Fig. 2B). cles from men with type 2 diabetes, miR-20b-5p displayed the larger fold change in the qPCR panel data (Table 1), Expression Profile of Human Skeletal Muscle Cells and miR-20b-5p content in serum exosome-enriched ex- Transfected With miR-20b-5p tracellular vesicles from men with normal glucose tol- We next evaluated the mRNA expression profile in human erance correlated with 2-h glucose values (Table 2). Thus, skeletal muscle cells after miR-20b-5p transfection. GSEA, miR-20b-5p was transfected into three different human followed by miR-20b-5p overrepresentation analysis, iden- cell types to evaluate miR-20b-5p effects on gene expres- tified key cellular functions for each gene category (Table 3 sion. miR-20b-5p was overexpressed in HEK293 (a kidney- and Supplementary Fig. 3). Fourteen gene sets with a derived cell line), HepG2 (a liver-derived cell line), and P value of , 0.05 and a false discovery rate of ,0.05 were primary human skeletal muscle cells. miR-20b-5p expres- considered significant. Analysis revealed that five gene sets sion increased significantly in all three cell types (data not were downregulated by miR-20b-5p, all of which regulated shown). STAT3 is a direct target of miR-20b-5p in MCF-7 immune response or immune function. Pathway analysis breast cancer cells (34) (Supplementary Fig. 2A); thus, we identified interferon-a and interferon-g response, tumor determined the effect miR-20b-5p expression on mRNA necrosis factor-a, interleukin (IL)2-STAT5, and IL6–janus and protein content of STAT3 in transfected cells. While kinase (JAK)–STAT3 signaling pathways. Nine gene sets STAT3 mRNA was decreased by miR-20b-5p transfection were upregulated in cells overexpressing miR-20b-5p in both HepG2 and human skeletal muscle cells, STAT3 (Table 3). The upregulated pathways include several metabolic

Table 2—Correlation between exosomal miR-20b-5p content and clinical characteristics Control Prediabetes Type 2 diabetes rP rP 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 correlation coefﬁcient test. HOMA-IR, HOMA of insulin resistance. 520 Exosomal miR-20b-5p in Type 2 Diabetes Diabetes Volume 68, March 2019

Protein Abundance and Insulin Signaling in Skeletal Muscle Cells Expressing miR-20b-5p We determined the protein abundance and insulin- stimulated phosphorylation of proteins involved in glycogen synthesis. Total protein content of glycogen synthase was reduced 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 inactive phospho–glycogen synthase as expected. In myotubes expressing miR-20b-5p, phospho–glycogen synthase was reduced under basal conditions, reflecting the reduced abundance of glycogen synthase. Furthermore, in miR- 20b-5p–transfected cells, insulin did not alter total phospho– glycogen synthase content (Fig. 3B). The ratio of the inactive form of phospho-GSK3 to total GSK3 abundance was increased in myotubes overexpressing miR-20b-5p, and this ratio was increased in response to insulin, although not to the same extent as in control cells (Fig. 3C). AKT is an upstream regulator of GSK3 in the insulin signaling pathway. We found that AKTIP was downregulated by miR-20b-5p transfection (Table 4). Insulin increased AKT phosphorylation in control cells; however, this effect was attenuated by miR-20b-5p overexpression (Fig. 3D) (AKT phospho-Thr308). Similar results were noted for AKT phospho-Ser473 (data not shown), whereas total AKT protein was unaltered. GSEA identified that miR-20b-5p overexpression reduced STAT3 signaling pathway (Table 4). Total STAT3 protein content was decreased by miR-20b-5p transfection (Fig. 3F). Figure 2—Effects of miR-20b-5p overexpression on STAT3 mRNA and protein abundance in human cells. Bar graph shows gene or protein expression in relation to negative control (NC)-transfected miR-20b-5p Directly Targets the AKTIP Gene cell basal. Gene expression of stat3 (A) and protein expression of For further validation of whether miR-20b-5p is directly STAT3 (B) in miR-20b-5p (miR-20b)-overexpressed cells derived involved in the regulation of AKTIP, a protein identified to from different human tissues. *P , 0.05, #P , 0.01 comparing miR-20b-5p–transfected cells with mimic miRNA–transfected con- interact with AKT1 and enhance its phosphorylation of trol cells (NC). Hskm, primary human skeletal muscle cells. both regulatory sites, we constructed luciferase reporter assays for the AKTIP 39UTR region containing the predicted miR-20b-5p target sites (Supplementary Fig. 2B and Fig. 3H). After overexpression of miR-20b-5p, luciferase pathways such as cholesterol homeostasis (P , 0.001), activity for AKTIP 39UTR was decreased (Supplementary fatty acid metabolism (false discovery rate: q = 0.077), and Fig. 4), whereas mutagenesis of the predicted target sites mammalian target of rapamycin (mTOR [also known as of miR-20b-5p in the AKTIP 39UTR abolished the effects of mechanistic target of rapamycin]) signaling pathway (P , miR-20b-5p overexpression on luciferase activity (Fig. 3H). 0.001). We next focused on validating the genes that were miR-20b-5p Alters Glycogen Synthesis in Human downregulated after miR-20b-5p transfection. We identi- Skeletal Muscle Cells fied six genes (expression fold change .2.5, P val- We determined whether miR-20b-5p transfection had di- ues .0.005) as possible direct miR-20b-5p target rect effects on glucose or lipid metabolism. Palmitate candidates based on a target scan identification of putative oxidation, as well as basal and insulin-mediated glucose miR-20b-5p target sites (Table 4). These six genes were uptake, was unaltered in human muscle cells transfected cytochrome b reductase 1 (CYBRD1), TBC1 domain family with miR-20b-5p (data not shown). In contrast, miR-20b- member 2 (TBC1D2), AKT (also known as PKB) interacting 5p transfection increased basal (1.2-fold) (P , 0.05) and protein (AKTIP), RNase/angiogenin inhibitor 1(RNH1), absolute insulin-stimulated glucose incorporation into glycoprotein integral membrane 1 (GINM1), and muscle glycogen (Fig. 4A). However, the insulin-stimulated cofilin 2 (CFL2). For confirmation of the microarray data of increment above basal was reduced in cells expressing these six genes, individual qRT-PCR analysis was per- miR-20b-5p, indicating that overexpression of miR-20b-5p formed, and reduced expression of all targets, with the attenuated the insulin response with respect to glucose exception of RNH1, was validated (Table 4). metabolism (Fig. 4B). diabetes.diabetesjournals.org Katayama and Associates 521

Table 3—Summary of GSEA Annotated cellular function Overlapping genes (n) P FDR (q) Downregulated by miR-20b-5p Interferon_alpha_response 96 ,0.001 ,0.001 Interferon_gamma_response 195 ,0.001 ,0.001 TNFA_signaling_via_NFKB 198 ,0.001 ,0.001 IL2_STAT5_signaling 194 ,0.001 ,0.001 IL6_JAK_STAT3_signaling 83 ,0.05 ,0.05 Upregulated by miR-20b-5p MYC_targets_V1 195 ,0.001 ,0.01 Cholesterol_homeostasis 74 ,0.001 ,0.001 MTORC1_signaling 198 ,0.001 ,0.001 G2M_checkpoint 196 ,0.001 ,0.001 E2F_targets 192 ,0.001 ,0.001 Unfolded_protein_response 111 ,0.001 ,0.01 Androgen_response 98 ,0.001 ,0.01 Estrogen_response_early 196 ,0.001 ,0.05 MYC_targets_V2 54 ,0.05 ,0.05 Fatty_acid_metabolism 153 ,0.05 0.077288 PI3K_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 were considered signiﬁcant comparing miR-20b-5p–transfected cells with mimic miRNA–transfected control cells. Of these, ﬁve gene sets associate with the miR-20b-5p–induced downregulated genes and nine gene sets are associated with miR-20b-5p–induced upregulated genes.

DISCUSSION of genes involved in pathways related to immune function We found that RNA abundance of miR-20b-5p and miR- and impairs glucose metabolism. fl 150-5p is increased in exosome-enriched extracellular miRNAs are present in biological uids, including blood, fl vesicles prepared from serum of patients with type 2 di- urine, breast milk, and cerebrospinal uids, and recent abetes, whereas total serum miRNA expression of these efforts have been focused on delineating the expression miRNA species is not significantly altered compared with profiles of miRNAs for use as disease biomarkers (33,35). glucose-tolerant men. To study the effects of miR-20b-5p While serum and plasma are easily obtained by minimally on gene expression, signal transduction, and metabolism, invasive methods and offer sufficient volume for analysis, we overexpressed miR-20b-5p miRNA in three different the presence of cell debris, proteins, and protein complexes human cell types. We found that miR-20b-5p overexpres- makes any analysis of miRNA profiles in biofluids techni- sion in primary human muscle cells suppressed expression cally challenging. miRNAs in serum and plasma are found of AKTIP, STAT3, and glycogen synthase and impaired in several contexts, including protein complexes such as insulin signaling in primary human skeletal muscle cells. Ago2-miRNA, exosomes, microvesicles, and lipid-protein Thus, peripheral expression of miR-20b-5p alters expression complexes such as HDL-miRNA (36). While miRNAs in

Table 4—miR-20b-5p–induced downregulated genes in human skeletal muscle cells Microarray Individual miRNA assay Fold change Predicted miR-20b-5p relative expression level Gene symbol Gene name (transfected vs. NC) P target site (transfected vs. NC) CYBRD1 Cytochrome 4.1 0.0023 Yes 0.15 6 0.039** b reductase 1 TBC1D2 TBC1 domain family, 3.0 0.0049 Yes 0.30 6 0.038** member 2 AKTIP AKT interacting protein 2.7 0.0022 Yes 0.37 6 0.020** RNH1 RNase/angiogenin 2.6 0.0028 Yes ND inhibitor 1 GINM1 Glycoprotein integral 2.6 0.000013 Yes 0.37 6 0.0064** membrane 1 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 for prediction 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. 522 Exosomal miR-20b-5p in Type 2 Diabetes Diabetes Volume 68, March 2019

Figure 3—Effect of miR-20b-5p overexpression on protein abundance and insulin signaling and miR-20b-5p–regulated targets in human skeletal 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 human skeletal 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 and schematic of constructs used for luciferase assays after transfection with negative control or miR-20b-5p (miR-20b) (n = 3). The control construct without 39UTR region (empty), construct including the AKTIP 39UTR region (AKTIP 39UTR), and construct with point mutations in the putative miR-20b-5p binding positions of AKTIP 39UTR (Mut) were each transfected as were the AKTIP 39UTR constructs. The schematic 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 are means 6 SEM. *P , 0.05 and #P , 0.01 as determined by paired Student t test comparing miR-20b-5p–transfected cells with mimic miRNA–transfected control cells or mutated 39UTR plasmid to the same condition as indicated. †P , 0.05 for insulin response within one condition.

protein complexes or extracellular vesicles are quite stable The particle characteristics of exosomes isolated from men in blood, different miRNA carriers appear to have different with normal glucose tolerance or type 2 diabetes in this study functions in cells (9). In comparison of serum and plasma were similar. A limitation of the current study is that prepared from the same blood sample, higher miRNA although subjects were matched for age and BMI, a number concentration was noted in serum compared with the ofthesubjectswithtype2diabeteswereonantidiabetes corresponding plasma, and the miRNA spectrum between medication and as a group had a higher level of use of other serum and plasma differed (37). In the current study, we medications. Thus, we are unable to exclude potential focused on miRNA isolated from serum exosome-enriched effects of medication on the miRNA proﬁles of exosome- extracellular vesicles, as exosomes have been functionally enriched extracellular vesicles. While total serum miRNA identiﬁed as mediators of cell-to-cell miRNA transfer. species did not differ between the cohorts, possibly diabetes.diabetesjournals.org Katayama and Associates 523

Figure 4—Glycogen synthesis in primary human muscle cells after miR-20b-5p overexpression. A: Glucose incorporation into glycogen in human skeletal muscle cells transfected with miR-20b-5p and (B) insulin-stimulated increment above basal in negative control (NC) mimic miRNA–transfected human skeletal muscle cells or miR-20b-5p–transfected human skeletal muscle cells (miR-20b). Data are mean 6 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 same condition comparing mir-20b-5p–transfected cells with mimic miRNA–transfected control cells. †P , 0.05 for insulin response within one condition. C and D: Schematic model of proposed role of miR-20b-5p in the presence and absence of insulin. The insulin-AKT signaling pathway (arrows) has been well characterized and involves the insulin receptor, IRS1, PI3K, AKT, GSK3, and glycogen synthase (GS). C:We propose a model where in the absence of insulin, miR-20b-5p reduces abundance of glycogen synthase, most likely via an indirect mechanism. Phosphorylation of glycogen synthase renders it inactive. Reduced basal glycogen synthase phosphorylation in myotubes overexpressing 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 and downstream. Red color indicates reduced protein expression, green color indicates metabolic end points, and blue arrows indicate phosphorylation events, denoted by p.

reflecting the more heterogeneous composition of total promotion of angiogenesis by microvesicle-mediated serum miRNA, we found two miRNA species that were transfer of miR-150-5p (39). Notably, miR-150-5p is spe- increased in exosome-enriched extracellular vesicles from cifically upregulated in skeletal muscle from diabetic Goto- serum of men with type 2 diabetes. Neither miR-20b-5p Kakizaki rats (40). Whether the elevation in miR-150-5p nor miR-150-5p was significantly elevated in serum- directly contributes to the insulin resistant phenotype in derived exosome-enriched extracellular vesicles from in- Goto-Kakizaki rats, or secondarily to impaired metabolic dividuals with impaired glucose tolerance, which could homeostasis, is not known. In the current study, we did indicate that these miRNAs reflect a more advanced dis- not observe any correlations between miR-150-5p exo- ease stage. Thus, miRNA profiling of functional units such some content and type 2 diabetes–related metabolic traits. as exosomes may increase the probability of identifying miR-20b-5p belongs to the miR-17 family and is part of the disease-relevant biomarkers. larger family of highly similar miRNAs, including miR- miR-20b-5p and miR-150-5p content was increased 106a-363, miR-17-192, and miR-106b-25 cluster (41), that in serum-derived exosome-enriched extracellular vesicles modulate VEGF expression by targeting HIF-1a and from men with type 2 diabetes. Circulating miR-150-5p in STAT3 in MCF-7 breast cancer cells (34). While these plasma has been proposed as a potential biomarker for miRNAs have been associated with metabolic disorders acute myeloid leukemia (38) and has been implicated in the and type 2 diabetes, a detailed understanding of the 524 Exosomal miR-20b-5p in Type 2 Diabetes Diabetes Volume 68, March 2019 physiological functions of miR-150-5p or miR-20b-5p is level of phosphorylated glycogen synthase was signifi- warranted. cantly reduced in control cells as expected (coincident Type 2 diabetes is a multifactorial metabolic disease with increased glycogen synthesis), while in myotubes affecting numerous tissues, including liver, skeletal mus- expressing miR-20b-5p, insulin did not reduce glycogen cle, adipose tissue, pancreas, and brain. To explore possible synthase phosphorylation, and in miR-20b-5p–expressing physiological functions of miR-20b-5p, we transfected cells there was an impaired insulin-stimulated increase in miR-20b-5p into human liver, kidney, and skeletal muscle glycogen synthesis. The effects of miR-20b-5p on skeletal cells and assessed STAT3 protein abundance. STAT3 pro- muscle metabolism are likely to reflect changes in protein tein has previously been reported to be a direct target of content from miR-20b-5p targets as well as second- miR-20b-5p (34). We noted that the miR-20b-5p over- ary consequences of these changes in protein expres- expression led to the greatest reduction in STAT3 protein sion, which are evident at the level of insulin signal abundance in skeletal muscle cells. Whether this is due to transduction. tissue-specific differences or properties of the cultures We identified AKTIP as a direct miR-20b-5p target. (primary muscle cells as opposed to immortalized cell lines AKTIP, also known as mouse Ft1 orthologous (46), is for kidney and liver cells) remains to be further investi- reported to interact directly with AKT and modulate gated. mRNA expression of genes relevant for several threonine kinase AKT phosphorylation and activation by pathways implicated in immune function was altered in PDK1 (47). AKTIP facilitates telomeric DNA replication myotubes overexpressing miR-20b-5p. Given the growing (48), but the functions of AKTIP in other AKT signaling appreciation that insulin resistance and type 2 diabetes are pathways are ambiguous. For example, RNA interference– associated with chronic low-grade inflammation, and pre- mediated reduction of AKTIP in primary human fibro- vious findings that miR-20b-5p may play a role in the blasts leads to a profound proliferation defect arrested in modulation of some inflammatory signals (42), our results late S phase (48). We provide evidence that AKTIP is a in men with type 2 diabetes and cultured myotubes are direct target of miR-20b-5p. The reduced AKTIP mRNA compelling. miR-20b-5p targets the STAT3 gene in MCF-7 in miR-20b-5p–overexpressing myotubes was coincident breast cancer cells (34), and we confirm this association in with a reduced insulin-stimulated AKT phosphoryla- our microarray data, as well as at the protein level. tion. The glycogen synthase gene has a putative miRNA- Collectively, these results suggest that miR-20b-5p directly 20b target sequence in the 39UTR region, and although we targets the STAT3 gene in human skeletal muscle cells. did not observe changes of glycogen synthase mRNA after Lifestyle intervention programs to increase physical miRNA-20b transfection, the total protein content of gly- activity and promote weight loss in adults at risk for cogen synthase was reduced. miRNAs exert effects both type 2 diabetes are associated with changes in circulating by reducing message stability and by preventing transla- miRNAs, including reductions in miR-20b-5p (43). In tion. A schematic overview of putative functional effects mouse models, miR-20a-5p promotes adipocyte differen- of miR-20b-5p in skeletal muscle is presented in Fig. 4C and tiation (44). Nevertheless, a direct link between changes in D, which are likely to reflect modification of both primary miRNA-20b-5p abundance and improvements in glucose (direct miRNA-20b-5p targets) and indirect effects. homeostasis is unknown. Here, we report that overexpres- Exosomes are released from cells into the circulation sion of miR-20b-5p in human skeletal muscle cells im- and transported to target cells to deliver cargo, including pacted mRNA expression of genes involved in several proteins and nucleic acids, such as various RNA species. metabolic pathways, including cholesterol homeostasis, Functional miRNA are delivered to target cells (11,49); fatty acid metabolism, and glycolysis. While genes involved however, little is known of the molecular machinery that in fatty acid oxidation or glucose uptake were unaltered in regulates this process, including the tissue of origin of the miR-20b-5p–transfected myotubes (data not shown), gly- exosomes, the specific target tissues of exosomes, and the cogen synthesis was affected. We found that basal glucose manner in which the cargo is delivered. Thus, we are incorporation into glycogen was increased in myotubes unable to ascertain the cellular mechanism by which the expressing miR-20b-5p, and insulin action was blunted. exosomes derived from serum of patients with type 2 di- mRNA content of several genes directly relevant to glyco- abetes are released or targeted and the specific cell or tissue gen synthesis, as well as insulin signaling, was altered in involved, and this remains a limitation of the current miR-20b-5p–transfected myotubes, which could partly study. Downregulation of miR-20b-5p targets, including explain the alterations in glucose metabolism. Although STAT3 and AKTIP, are likely to have tissue-specific effects. there was no change in mRNA, myotubes expressing miR- Further studies are required to understand the physiolog- 20b-5p had reduced total glycogen synthase protein, likely ical role of increased serum miR-20b-5p in individuals with reflecting posttranslational downregulation. Phosphoryla- type 2 diabetes. tion of glycogen synthase renders it inactive (45). We Here, we provide evidence that miR-20b-5p is highly found that basal glycogen synthase phosphorylation was expressed in serum exosome-enriched extracellular reduced in myotubes overexpressing miR-20b-5p, which is vesicles isolated from patients with type 2 diabetes. More- consistent with the increased basal rate of glycogen syn- over, when introduced into skeletal muscle cells, miR-20b-5p thase noted in these cells. Upon insulin stimulation, the alters cellular glucose metabolisms and the STAT3 and AKT diabetes.diabetesjournals.org Katayama and Associates 525 signaling pathway. Since serumexosomalmiR-20b-5pis 10. Redis RS, Calin S, Yang Y, You MJ, Calin GA. Cell-to-cell miRNA transfer: from correlatedwithlow2-hglucosevaluesaswellasdown- body homeostasis to therapy. Pharmacol Ther 2012;136:169–174 regulation of inflammatory pathways, it is possible that 11. Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Exosome- miR-20b-5p may not be an effector of impaired insulin mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 2007;9:654–659 signaling and glycogen synthesis but, instead, may be part 12. Vickers KC, Palmisano BT, Shoucri BM, Shamburek RD, Remaley AT. of a homeostatic mechanism attempting to improve meta- MicroRNAs are transported in plasma and delivered to recipient cells by high- bolic control. Taken together, our results highlight poten- density lipoproteins. Nat Cell Biol 2011;13:423–433 tial interactions between exosome-enriched extracellular 13. Chen X, Liang H, Zhang J, Zen K, Zhang CY. Secreted microRNAs: a new form vesicles and metabolic regulation. of intercellular communication. Trends Cell Biol 2012;22:125–132 14. Pegtel DM, Cosmopoulos K, Thorley-Lawson DA, et al. Functional delivery of viral miRNAs via exosomes. Proc Natl Acad Sci U S A 2010;107:6328–6333 Acknowledgments. The authors thank Dr. Jorge Ruas and Dr. Lars 15. Thomou T, Mori MA, Dreyfuss JM, et al. Adipose-derived circulating miRNAs – Ketscher, both at Karolinska Institutet, for helpful discussion and input. The authors regulate gene expression in other tissues. Nature 2017;542:450 455 ņš ņ also acknowledge the core facility for Bioinformatics and Expression Analysis at 16. Endzeli E, Melne V, Kalni a Z, et al. Diagnostic, prognostic and predictive Novum, where the gene arrays were performed. This facility is supported by the board value of cell-free miRNAs in prostate cancer: a systematic review. Mol Cancer of research at the Karolinska Institute and the research committee at the Karolinska 2016;15:41 University Hospital. 17. Hosseini M, Khatamianfar S, Hassanian SM, et al. Exosome-encapsulated Funding. This work was supported by grants from the Strategic Diabetes microRNAs as potential circulating biomarkers in colon cancer. Curr Pharm Des – Program at Karolinska Institute, European Research Council Ideas Program 2017;23:1705 1709 (ICEBERG, ERC-2008-AdG23285), Vetenskapsrådet (Swedish Research Council) 18. Joyce DP, Kerin MJ, Dwyer RM. Exosome-encapsulated microRNAs as – (2011-3550, 2012-1760, 2015-165), Swedish Diabetes Foundation (DIA2015- circulating biomarkers for breast cancer. Int J Cancer 2016;139:1443 1448 032, DIA2015-052), Stiftelsen för Strategisk Forskning (Swedish Foundation for 19. Kumar S, Reddy PH. Are circulating microRNAs peripheral biomarkers for ’ – Strategic Research) (SRL10-0027), Diabetes Wellness Sweden, Novo Nordisk Alzheimer s disease? Biochim Biophys Acta 2016;1862:1617 1627 Foundation (NNF14OC0009941), Swedish Research Council for Sport Science 20. Mirzaei H, Gholamin S, Shahidsales S, et al. MicroRNAs as potential di- – (FO2016-0005), the Swedish Heart Lung Foundation (20150423), and Stockholm agnostic and prognostic biomarkers in melanoma. Eur J Cancer 2016;53:25 32 Läns Landsting (Stockholm County Council). 21. Ramanathan S, Ajit SK. MicroRNA-based biomarkers in pain. Adv Pharmacol – Duality of Interest. No potential conflicts of interest relevant to this article 2016;75:35 62 were reported. 22. Wang J, Chen J, Sen S. MicroRNA as biomarkers and diagnostics. J Cell – Author Contributions. M.K. conceived and performed experiments, Physiol 2016;231:25 30 fl analyzed data, and wrote the manuscript. O.P.B.W. and S.E.-A. provided expertise 23. Goodpaster BH, Sparks LM. Metabolic exibility in health and disease. Cell – and feedback. T.F. and K.C. performed clinical analysis and provided feedback. Metab 2017;25:1027 1036 J.R.Z. and A.K. conceived and planned the study, secured funding, and wrote the 24. Leahy JL. Pathogenesis of type 2 diabetes mellitus. Arch Med Res 2005;36: – manuscript. M.K., O.P.B.W., T.F., K.C., S.E.-A., J.R.Z., and A.K. read and approved 197 209 the final manuscript. M.K. and A.K. are the guarantors of this work and, as such, 25. Willeit P, Skroblin P, Moschen AR, et al. Circulating microRNA-122 is as- had full access to all the data in the study and take responsibility for the integrity of sociated with the risk of new-onset metabolic syndrome and type 2 diabetes. – the data and the accuracy of the data analysis. Diabetes 2017;66:347 357 26. Eriksson J, Franssila-Kallunki A, Ekstrand A, et al. Early metabolic defects in References persons at increased risk for non-insulin-dependent diabetes mellitus. N Engl J 1. Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell 2009; Med 1989;321:337–343 136:215–233 27. Bhatia P, Raina S, Chugh J, Sharma S. miRNAs: early prognostic biomarkers 2. Pillai RS, Bhattacharyya SN, Filipowicz W. Repression of protein synthe- for Type 2 diabetes mellitus? Biomarkers Med 2015;9:1025–1040 sis by miRNAs: how many mechanisms? Trends Cell Biol 2007;17:118– 28. Guay C, Regazzi R. Circulating microRNAs as novel biomarkers for diabetes 126 mellitus. Nat Rev Endocrinol 2013;9:513–521 3. Kloosterman WP, Plasterk RH. The diverse functions of microRNAs in animal 29. Raffort J, Hinault C, Dumortier O, Van Obberghen E. Circulating microRNAs development and disease. Dev Cell 2006;11:441–450 and diabetes: potential applications in medical practice. Diabetologia 2015;58: 4. Massart J, Katayama M, Krook A. microManaging glucose and lipid me- 1978–1992 tabolism in skeletal muscle: role of microRNAs. Biochim Biophys Acta 2016;1861: 30. Al-Khalili L, Chibalin AV, Kannisto K, et al. Insulin action in cultured human 2130–2138 skeletal muscle cells during differentiation: assessment of cell surface GLUT4 and 5. Mitchell PS, Parkin RK, Kroh EM, et al. Circulating microRNAs as stable GLUT1 content. Cell Mol Life Sci 2003;60:991–998 blood-based markers for cancer detection. Proc Natl Acad Sci U S A 2008;105: 31. Mudry JM, Lassiter DG, Nylén C, et al. Insulin and glucose alter Death- 10513–10518 Associated Protein Kinase 3 (DAPK3) DNA methylation in human skeletal muscle. 6. EL Andaloussi S, Mäger I, Breakefield XO, Wood MJA. Extracellular vesicles: Diabetes 2017;66:651–662 biology and emerging therapeutic opportunities. Nat Rev Drug Discov 2013;12: 32. Kulkarni SS, Karlsson HK, Szekeres F, Chibalin AV, Krook A, Zierath JR. 347–357 Suppression of 59-nucleotidase enzymes promotes AMP-activated protein kinase 7. Huang-Doran I, Zhang CY, Vidal-Puig A. Extracellular vesicles: novel me- (AMPK) phosphorylation and metabolism in human and mouse skeletal muscle. J diators of cell communication in metabolic disease. Trends Endocrinol Metab Biol Chem 2011;286:34567–34574 2017;28:3–18 33. Correia CN, Nalpas NC, McLoughlin KE, et al. Circulating microRNAs as 8. Simpson RJ, Lim JW, Moritz RL, Mathivanan S. Exosomes: proteomic in- potential biomarkers of infectious disease. Front Immunol 2017;8:118 sights and diagnostic potential. Expert Rev Proteomics 2009;6:267–283 34. Cascio S, D’Andrea A, Ferla R, et al. miR-20b modulates VEGF expression by 9. Turchinovich A, Samatov TR, Tonevitsky AG, Burwinkel B. Circulating targeting HIF-1 alpha and STAT3 in MCF-7 breast cancer cells. J Cell Physiol 2010; miRNAs: cell-cell communication function? Front Genet 2013;4:119 224:242–249 526 Exosomal miR-20b-5p in Type 2 Diabetes Diabetes Volume 68, March 2019

35. Busch A, Eken SM, Maegdefessel L. Prospective and therapeutic screening value of 43. Flowers E, Kanaya AM, Fukuoka Y, Allen IE, Cooper B, Aouizerat BE. Pre- non-coding RNA as biomarkers in cardiovascular disease. Ann Transl Med 2016;4:236 liminary evidence supports circulating microRNAs as prognostic biomarkers for 36. Sebastiani G, Nigi L, Grieco GE, Mancarella F, Ventriglia G, Dotta F. Circulating type 2 diabetes. Obes Sci Pract 2017;3:446–452 microRNAs and diabetes mellitus: a novel tool for disease prediction, diagnosis, 44. Zhu E, Zhang J, Zhou J, Yuan H, Zhao W, Wang B. miR-20a-5p promotes and staging? J Endocrinol Invest 2017;40:591–610 adipogenic differentiation of murine bone marrow stromal cells via targeting 37. Wang K, Yuan Y, Cho JH, McClarty S, Baxter D, Galas DJ. Comparing the Kruppel-like factor 3. J Mol Endocrinol 2018;60:225–237 MicroRNA spectrum between serum and plasma. PLoS One 2012;7:e41561 45. Embi N, Rylatt DB, Cohen P. Glycogen synthase kinase-3 from rabbit skeletal 38. Fayyad-Kazan H, Bitar N, Najar M, et al. Circulating miR-150 and miR-342 in muscle. Separation from cyclic-AMP-dependent protein kinase and phosphorylase plasma are novel potential biomarkers for acute myeloid leukemia. J Transl Med kinase. Eur J Biochem 1980;107:519–527 2013;11:31 46. Lesche R, Peetz A, van der Hoeven F, Rüther U. Ft1, a novel gene related to 39. Li J, Zhang Y, Liu Y, et al. Microvesicle-mediated transfer of microRNA-150 ubiquitin-conjugating enzymes, is deleted in the Fused toes mouse mutation. from monocytes to endothelial cells promotes angiogenesis. J Biol Chem 2013; Mamm Genome 1997;8:879–883 288:23586–23596 47. Remy I, Michnick SW. Regulation of apoptosis by the Ft1 protein, a new 40. He A, Zhu L, Gupta N, Chang Y, Fang F. Overexpression of micro ribonucleic modulator of protein kinase B/Akt. Mol Cell Biol 2004;24:1493–1504 acid 29, highly up-regulated in diabetic rats, leads to insulin resistance in 3T3-L1 48. Burla R, Carcuro M, Raffa GD, et al. AKTIP/Ft1, a new shelterin- adipocytes. Mol Endocrinol 2007;21:2785–2794 interacting factor required for telomere maintenance. PLoS Genet 2015;11: 41. Tanzer A, Stadler PF. Molecular evolution of a microRNA cluster. J Mol Biol e1005167 2004;339:327–335 49. Montecalvo A, Larregina AT, Shufesky WJ, et al. Mechanism of transfer of 42. Donath MY, Shoelson SE. Type 2 diabetes as an inﬂammatory disease. Nat functional microRNAs between mouse dendritic cells via exosomes. Blood 2012; Rev Immunol 2011;11:98–107 119:756–766