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.

Epigenetic modifications in obesity‐associated diseases

“…From a mechanistic standpoint, miRNAs released from skeletal muscle may be involved in the regulation of irisin expression 265 . Exercise can influence the miRNA expression profile in exosomes derived from myotubes and upregulate the expression of genes associated with anti‐inflammatory responses and appetite regulation 266 . The effects of exercise are comprehensive, and designing a tailored exercise plan based on individual conditions can enhance insulin sensitivity and cardiopulmonary health.…”

Non-Coding RNA in Salivary Extracellular Vesicles: A New Frontier in Sjögren’s Syndrome Diagnostics?

“…The size distribution and particle concentration of EVs in the pSS and control samples were examined by nanoparticle tracking analysis (NTA) using a Nanosight NS500 Instrument (Malvern Instruments Ltd., Amesbury, UK) [56], with detection threshold 3 and camera level 14.…”

“…Immunoaffinity capture and detection of EV-membrane protein CD9 were performed in conjunction with flow cytometry to identify EVs in the patient sample, as previously described [56]. EVs were captured with the Exosome-Human CD9 Flow Detection Reagent (cat.…”

“…Western blotting was performed as previously described [56]. In addition to the four salivary EV-isolated samples (SEC-and exoRNeasy isolated EVs), a recombinant Exosome standard (Merck Life Science, Oslo, Norway, Cat no SAE0193) and an SW480 cell lysate were used as controls.…”

Physical Exercise and Appetite Regulation: New Insights

“…Moreover, the elevation in plasma Lac-Phe seen in men after exercising points towards opportunities for improving weight management if the role of Lac-Phe in appetite and body weight regulation translates into humans. In this regard, recent work in humans (in vitro and in vivo, mimicking exercise conditions in muscle cells [ 33 , 34 ]) shows that acute exercise increases plasma Lac-Phe levels. Higher Lac-Phe plasma levels were related to a greater reduction in abdominal subcutaneous fat and to a smaller reduction in visceral adipose tissue during an eight-week supervised training intervention [ 34 ].…”