Extracellular vesicles and microRNAs are altered in response to exercise, insulin sensitivity and overweight.
Author(s): Doncheva AI, Romero S, Ramirez-Garrastacho M, Lee S, Kolnes KJ, Tangen DS, Olsen T, Drevon CA, Llorente A, Dalen KT, Hjorth M
Publication: Acta Physiol (Oxf), 2022, Vol. 236, Page e13862
PubMed ID: 36377504 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to compare miRNA profiles of extracellular vesicles (EVs) isolated from plasma and gene expression in adipose biopsies collected from sedentary men with and without dysglycemia before and after acute and long-term (12 weeks) exercise. Two different EV isolation methods were also compared.
Conclusion of Paper
Particle counts were comparable in plasma collected from men regardless of health status before and after exercise. Size exclusion chromatography (SEC) of EV isolates revealed protein expression of CD63 and CD81 in all individuals, while CD9 expression was only observed in EVs from one of the three participants. In contrast, CD9 protein expression was observed in all specimens when EVs were purified by the membrane affinity method (MEM). EVs isolated using both methods also contained CD41 and ApoA1 protein expression, indicating the presence of platelet-derived particles and high density lipoproteins in these samples. In membrane affinity isolated EVs, CD63 protein levels increased after acute exercise (P=0.04). The average expression levels of the 86 miRNAs identified in all specimens increased by 70% after acute exercise, and principal component analysis (PCA) of miRNA profiles clustered specimens collected before and after acute exercise separately. In the MEM and SEC purified EVs, 13 and 5 miRNAs, respectively, significantly increased following acute exercise. However, only exercise-induced increases in miR-30a-5p and miR-140-3p remained significant after normalization to mean miRNA levels and correction for multiple testing. Of the 12 miRNAs that were quantified in specimens from all 19 men at all timepoints, 8 miRNAs were increased after acute exercise but returned to baseline 2 h after and the 12 weeks of training had no effects on any of the 12miRNAs. Although initial analysis revealed five miRNAs with differential expression between normoglycemic and dysglycemic patients, only miR-652-3p remained significant after correction for multiple testing. Further, PCA clustered specimens from normoglycemic and dysglycemic patients together. The authors found several miRNAs that were associated with insulin resistance and expression in adipose tissue biopsies. miR-32-5p levels in EVs isolated from plasma were positively correlated and EV miR-339 levels were negatively correlated with macrophage markers in adipose tissue. Conversely, miR-32-5p levels in EVs were negatively correlated and miR-339-3p positively correlated with mitochondrial DNA levels in adipose tissues.
Studies
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Study Purpose
The purpose of this study was to compare miRNA profiles of EVs isolated from plasma and gene expression in adipose biopsies collected from sedentary men with and without dysglycemia before and after acute and long-term (12 weeks) exercise. Two different EV isolation methods were also compared. Blood was collected from twenty-six participants before and after acute exercise (45 min of cycling at 75% VO2 Max) and from 13 participants again after 12 weeks of training (two sessions each per week of strength training and interval training). Participants were normoglycemic (BMI 21.2–25.4 kg/m2) or dysglycemic (fasting glucose ≥5.6 mmol/L or 2 h glucose ≥7.8 mmol/L, BMI 26.5-32.3 kg/m2) without known hypertension or liver, kidney or chronic inflammatory disease. Blood was collected in EDTA tubes and plasma was separated by centrifugation at 1400 g for 10 min at 4°C. Plasma was snap frozen in liquid nitrogen and stored at -80°C. EVs were purified from plasma by membrane affinity columns (exoEasy Midi spin columns from the exoRNeasy Midi Kit) and concentrated with micron Ultra-2 Centrifugal Filter Devices and by size exclusion chromatography (qEV single 35 nm SEC columns) and concentrated with Amicon Ultra-2 Centrifugal Filter Devices. EVs were analyze by nanoparticle tracking analysis and protein levels were confirmed by Western blot with antibodies against CD9, CD41, CD63, CD81, ApoA1. RNA was isolated from EVs using exoRNeasy midi kit and levels of 183 miRNAs were quantified by real-time PCR using the miRCURY LNA miRNA PCR serum/plasma focus panel. Subcutaneous adipose tissue biopsies were collected at each timepoint from the peri-umbilical region 30–60 min after cycling. RNA was extracted from biopsies using the miRNeasy Mini RNeasy Lipid Tissue Mini Kit. RNA integrity was evaluated using a bioanalyzer and sequenced using a Illumina HiSeq 2000 system.
Summary of Findings:
Particle counts were comparable in plasma collected from men regardless of health status before and after exercise. Size exclusion chromatography of EV isolates revealed protein expression of CD63 and CD81 in samples from all individuals, while CD9 expression was only observed in EVs from one of the three participants. In contrast, CD9 protein expression was observed in all specimens when EVs were purified by the membrane affinity method. EVs isolated using both methods also contained CD41 and ApoA1 protein expression, indicating the presence of platelet-derived particles and high-density lipoproteins. In membrane affinity isolated EVs, protein content was high, which likely indicates contamination with plasma proteins. In membrane affinity EVs, an increase in CD63 was noted after acute exercise (P=0.04). Of the 178 miRNAs analyzed, in membrane affinity purified EVS, 143 were detected with 86 detected in all specimens. The average expression levels of the 86 miRNAs detectable in all individuals increased by 70% after acute exercise, and PCA of miRNA profiles clustered specimens collected before and after acute exercise separately. In MEM- and SEC-purified EVs, 13 miRNAs (miR-30a-5p, miR-140- 3p, let-7b-5p, miR-99a-5p, miR-126-5p, miR-10b-5p, miR-424-5p, miR-338-3p, miR-29c-3p, miR-222-3p, miR-126-3p, miR-145-5p, and miR-192-5p) and 5 miRNAs (miR-10b-5p, miR-222-3p, miR-30a-5p, miR-339- 3p, and miR-99a-5p), respectively, increased significantly after acute exercise. Cell ontology analysis revealed that these miRNAs are associated with immune cells, endothelial cells, epithelial cells, and muscle cells. However, after normalization to the mean miRNA levels and correction for multiple testing, miR-30a-5p and miR-140-3p alone displayed a significant increase. Of the 12 miRNAs that were quantified in specimens from all 19 men at all timepoints, 8 miRNAs (miR-10b-5p, miR-222-3p, miR-23a-3p, miR30a-5p, miR484, miR-652-3p, miR-92a-3p, and miR991-5p) were increased after acute exercise but returned to baseline 2 h after. Further, levels of these miRNAs were unaffected after 12 weeks of training. Although initial analysis identified five miRNAs with differential expression when normoglycemic and dysglycemic patients were compared, only miR-652-3p (downregulated in dysglycemic patients) remained significant after correction for multiple testing. Further, PCA clustered specimens from normoglycemic and dysglycemic patients together. Levels of miR-652-3p, miR-423-3p, miR-23a-3p, miR-23b-3p, and miR-27b-3p were correlated with insulin sensitivity. The authors also identified several miRNAs that were associated with gene expression in adipose tissue, as miR-32-5p levels were positively correlated and miR-339-3p levels were negatively correlated with macrophage markers in adipose tissue (r=0.74, P<0.5 and r=-0.80, P<0.01, respectively). Conversely, miR-32-5p levels were negatively correlated and miR-339-3p levels were positively correlated with mitochondrial DNA levels (r=-0.78 P<0.01 and r=0.56 P=0.07, respectively).
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform RNA Next generation sequencing Cell count/volume Light scattering Protein Western blot RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Time of biospecimen collection Before exercise
After acute exercise
2 h after acute exercise
After 12 weeks of training
Analyte Extraction and Purification Analyte isolation method Membrane affinity column
Size exclusion chromatography
Biospecimen Acquisition Biospecimen location Adipose tissue
Plasma