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

Abstract

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.

Keywords: electrical pulse stimulation (EPS); extracellular vescicles; human myotubes; microRNA; morbid obesity; proteomic; transcriptomic; type 2 diabetes (T2D).

FIGURE 1

Study workflow and cell medium concentration of interleukin-6 (IL-6). Flow chart of the study from human biopsy sampling, skeletal muscle cell culturing, electrical pulse stimulation (EPS) of myotubes, isolation of extracellular vesicles (EVs) from conditioned medium, separation of exosomes from microvesicles (MV), characterization of EVs by nanoparticle tracking analysis (NTA), flow cytometry, Western blotting, transmission electron microscopy (TEM) to transcriptomic (RNA microarray) and proteomic analysis (A). Cultured human myotubes were exposed to EPS for 24 h, conditioned medium was harvested, and the concentration of IL-6 was detected with a human ELISA kit according to the producer’s protocol (SigmaAldrich). Data are given as mean (±SEM) (n = 6). *Significantly different from control (unstimulated cells) p < 0.05 (B).

FIGURE 2

Characterization of myotube derived extracellular vesicles (EVs). Human myotubes were exposed to electrical pulse stimulation (EPS) for 24 h, and cell derived EVs were collected for 24 h thereafter. Size (A) and concentration (C) of exosomes (EXO) and microvesicles (MV) were measured by nanoparticle tracking analysis (NTA). The presence of EV markers on exosomes and MV captured by anti-CD81-coated magnetic beads were detected with PE-conjugated CD81 (B) and CD63 (D) antibodies by flow-cytometry (BD Accuri C6 flow cytometer). Presence of CD9, calnexin, and heat shock protein 70 (Hsc/Hsp70) on exosomes were measured by Western blotting (E). Cell lysates of SW480 cells (SW) were used as control. Transmission electron microscopy (TEM) images of skeletal muscle cell derived EVs (F). 1. Freshly isolated EVs from conditioned media from human myotubes, scale bar 1 µm. 2. Close up of framed EVs in picture 1, scale bar 200 nm. Data are presented as mean ± SEM (n = 6 in each group). MFI = mean fluorescence intensity.

FIGURE 3

Content of non-coding RNA in exosomes (EXO), microvesicles (MV) and myotubes from unstimulated cells. Quality assessment of isolated RNA from exosomes, MV, and myotubes by Agilent BioAnalyzer with two representative samples of each (A). Small non-coding RNA in exosomes, MV, and myotubes were identified by microarray transcriptomics. Distribution of total non-coding RNA content of EXO, MV, and myotubes (B). The total number of microRNAs (miR) that were unique and common for EXO, MV, and myotubes (C).

FIGURE 4

Exosome miRs affected by electrical pulse stimulation (EPS). The number and distribution of miRs present in exosomes before and after EPS (A). Enrichment analysis of the miRs in exosomes after EPS (performed by FunRich) which shows the predicted molecular functions significantly associated (p < 0.05) with the miRs (B).

FIGURE 5

Protein content in unstimulated myotube derived extracellular vesicles. Extracellular vesicles were isolated from human myotube cell media after 24 h collection. The protein content of both exosomes (EXO) and microvesicles (MV) were detected by nanoLC-MS/MS and identified by MaxQuant against the Uniprot database. About 1000 proteins were detected in EXO and 2000 in MV, and most of these were found in Vesiclepedia (A). FunRich was used to predict which biological processes the detected proteins were significantly (p < 0.01) associated with in exosomes (B) and MV (C).

FIGURE 6

Panther classification and Ingenuity Pathway analysis (IPA) of extracellular vesicle proteins changed by EPS. Of the 73 proteins that were changed by EPS in exosomes, 60 were classified into protein classes by the Panther classification system (A). The top predicted canonical pathways of proteins regulated by EPS in exosomes assessed by IPA (B). Panther classification of 77 of the 95 proteins changed by EPS in MV (C), the top canonical pathways of the proteins changed by EPS in MV (D), and the proteins of the mitochondrial oxidative phosphorylation pathway activated by EPS in MV (E).

FIGURE 7

Possible effects of physical activity on skeletal muscle-derived extracellular vesicles on metabolically active cells and organs in obesity and type 2 diabetes (T2D). Skeletal muscles release extracellular vesicles (EVs) in response to exercise, and most likely will these EVs circulate and reach target organs. The EVs contain bioactive molecules, both microRNAs (miRs) and proteins, that might have a regulatory role on the metabolically active target cells and organs. Possibly, will EVs contribute to the health effects of exercise by reducing appetite, improve insulin sensitivity and reduce the low-grade inflammation in obesity and T2D. CNDP2 = cytosolic non-specific dipeptidase, ATP5B = ATP synthase subunit beta mitochondrial, GANAB = alpha-glucosidase AB, HSPA9 = stress-70 protein mitochondrial.