Distinct microRNA and protein profiles of extracellular vesicles secreted from myotubes from morbidly obese donors with type 2 diabetes in response to electrical pulse stimulation

Lifestyle disorders like obesity, type 2 diabetes (T2D), and cardiovascular diseases can be prevented and treated by regular physical activity. During exercise, skeletal muscles release signaling factors that communicate with other organs and mediate beneficial effects of exercise. These factors include myokines, metabolites, and extracellular vesicles (EVs). In the present study, we have examined how electrical pulse stimulation (EPS) of myotubes, a model of exercise, affects the cargo of released EVs. Chronic low frequency EPS was applied for 24 h to human myotubes isolated and differentiated from biopsy samples from six morbidly obese females with T2D, and EVs, both exosomes and microvesicles (MV), were isolated from cell media 24 h thereafter. Size and concentration of EV subtypes were characterized by nanoparticle tracking analysis, surface markers were examined by flow cytometry and Western blotting, and morphology was confirmed by transmission electron microscopy. Protein content was assessed by high-resolution proteomic analysis (LC-MS/MS), non-coding RNA was quantified by Affymetrix microarray, and selected microRNAs (miRs) validated by real time RT-qPCR. The size and concentration of exosomes and MV were unaffected by EPS. Of the 400 miRs identified in the EVs, EPS significantly changed the level of 15 exosome miRs, of which miR-1233-5p showed the highest fold change. The miR pattern of MV was unaffected by EPS. Totally, about 1000 proteins were identified in exosomes and 2000 in MV. EPS changed the content of 73 proteins in exosomes, 97 in MVs, and of these four were changed in both exosomes and MV (GANAB, HSPA9, CNDP2, and ATP5B). By matching the EPS-changed miRs and proteins in exosomes, 31 targets were identified, and among these several promising signaling factors. Of particular interest were CNDP2, an enzyme that generates the appetite regulatory metabolite Lac-Phe, and miR-4433b-3p, which targets CNDP2. Several of the regulated miRs, such as miR-92b-5p, miR-320b, and miR-1233-5p might also mediate interesting signaling functions. In conclusion, we have used a combined transcriptome-proteome approach to describe how EPS affected the cargo of EVs derived from myotubes from morbidly obese patients with T2D, and revealed several new factors, both miRs and proteins, that might act as exercise factors

Change in content of extracellular vesicles from human skeletal muscle cells after electrical pulse stimulation with focus on microRNA.

Background and Aim Extracellular vesicles (EVs) are nanoparticles with a double membrane postulated to be released by cells of all types and can be detected in all body fluids. They are classified in three groups exosomes, microvesicles and apoptotic bodies and encompasses lipids, proteins, and nucleic acids (RNA and DNA). In a previous master thesis, studies were done on electrically stimulated skeletal muscle cell derived EVs. In that study, it was illustrated that, skeletal muscles release EVs. These EVs were found to contain proteins on which electrical pulse stimulation (EPS) had significant effects. In addition, a pilot experiment found that the EVs encompassed mircroRNAs, whose functions were not fully understood. EVs have been suggested to be mediators of cell-to-cell communication and regular physical activity has been proven to prevent T2DM and improve blood sugar levels. It was of interest to find out whether electrical pulse stimulation could have any effects on microRNA encompassed in EVs derived from extremely obese patients with type 2 diabetes mellitus (T2DM), since it had had significant effects on proteins. Methods Primary human skeletal muscle cells isolated from abdominal muscle biopsies were cultured and allowed to differentiate into myotubes. Half of the cells were stimulated using a low-frequency EPS for 24 hours. The media were collected and used for interleukin-6 (IL-6) concentration measurement. After incubation of the cells in serum free media for 24 hours, the media was harvested, and used for EVs (exosomes and microvesicles) isolation, and the EVs were characterized using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA and western blotting. Exosome content of microRNA was accessed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Affymetrix microarray technology. Results The interleukin-6 concentrations in the cell media tended to be increased by 37% after EPS, although not significant (p=0.11). TEM pictures showed a spherical morphology and a cup shaped structure of EVs, typical characteristics. The mean size of unstimulated exosomes was 139 nm and 141 nm after EPS. Microvesicles were significantly larger, unstimulated 158 nm and stimulated 164 nm. In the identification of EVs surface biomarkers, the presence of CD63 and Hsc70/Hsp70 was shown, whereas CD9 was not present. RT-qPCR illustrated that skeletal muscle cell derived EVs were enriched with microRNA, the three myo-miRs, miR-1-3p, miR-133a and miR-206 were detected. Affymetrix microarray analysis performed on three donors were loaded in Ingenuity Pathway analysis software (IPA). A total of 271 transcripts were detected and out of the total, 219 were miR. Analysis of Affymetrix microarray data in IPA showed significant effects of EPS on 12 miRs. This was based on filtering the miRs by p1.5-fold change and “signal value” above 5. Pathway analysis showed their involvement in diseases and biological functions, and these included neurological diseases, psychological disorders, post-translational modification, connective tissue development and function, cardiovascular system development and function. A prediction of top upstream regulators and their interaction network suggested AGO2 (argonaute RISC catalytic component 2), MET (MET Proto-Oncogene, Receptor Tyrosine Kinase), TGS1 (Trimethylguanosine synthase 1), XPO1 (Exportin 1) and TP53 (Tumor protein p53) among others. Database search for targets of the miRs identified several possible targets of the regulated miRs. Conclusion Electrical pulse stimulation had a noticeable effect on miR content in exosomes, with a significant difference in 12 specific miRs between unstimulated and stimulated (EPS) myotubes. Characterization of EVs illustrated that EPS had no effect on EVs concentrations and sizes. The changes caused in EVs´ miR content by EPS might play an important role in numerous biological functions of mediating beneficial effects of exercise