gender: female age: 43 disease state: type 2 diabetes, morbidly obese treatment: metformin + glimepiride cells: microvesicles, secreted from mature myotubes, in vitro model from skeletal muscle
Treatment protocol
Electrical pulse stimulation of myotubes for 24 h and collecting microvesicles after 24 h in serum free medium Human myotubes grown in 6-well EMC coated Costar plates (Corning®) were stimulated via carbon electrodes (C-dish, Ionoptics, Ireland) by applying continuous, low-frequency electrical pulse stimulation (EPS) (single, bipolar pulses of 2 ms, 30 V, 1 Hz) for 24 h from day 6 to 7 of the differentiation period, as described previously [34]. Electrical pulses were generated by a self-designed pulse generator (the Electronics Lab, Institute of Chemistry, University of Oslo, Norway), and the effect was investigated by measuring IL-6 concentration in the supernatants before and after EPS
Growth protocol
Cultures of multinucleated human myotubes were established by activation and proliferation of satellite cells isolated from muscle biopsy samples from m. obliquus internus abdominis. The biopsies were taken from 6 donors during gastric bypass surgery. All donors were female, average age 49 (± 12) years, BMI 45 (± 5) kg/m2, and all were diagnosed with T2D and treated with peroral antidiabetics or insulin. Average HbA1c was 6.7 (± 1.2) %, and fasting plasma glucose was 6.8 (± 0.9) mmol/L in blood samples collected the day before surgery. Satellite cells were isolated from biopsy samples by enzymatic digestion [35]. Briefly, the muscle biopsies were cut into small pieces, rinsed for adipose tissue, and trypsinized 3 times. The supernatants were collected, and the cells seeded in SkGMTM-2 (Skeletal Muscle Cell Growth Medium-2 BulletKit, (Lonza, Basel, Switzerland). The cells were split twice before storage in liquid nitrogen. For proliferation of myoblasts, DMEM-GlutamaxTM (5.5 mM glucose) medium supplemented with FBS (2 %) and Ultroser G (2 %) was used. At approximately 80 % confluence, the culture medium was changed to DMEM-GlutamaxTM (5.5 mM glucose) supplemented with FBS (2 %) and insulin (25 pM) to initiate differentiation into multinucleated myotubes. Both cell media contained gentamicin and amphotericin B. The cells were grown in 6-well plates pre-coated with extracellular matrix gel (ECM). The cells were allowed to differentiate for seven days. During the culturing process muscle cells were incubated in a humidified CO2 (5 %) atmosphere at 37°C, and medium was changed every 2nd to 3rd day. Exosome and microvesicle isolation After 24 h of EPS, cell medium was changed to serum free DMEM-GlutamaxTM (5.5 mM glucose) with 25 pM insulin, gentamicin, and amphotericin B, 1 ml/well for collecting EVs. The cells were incubated for another 24 h for synthesis and secretion of EVs and then harvested. EV-containing media were collected, and both exosomes and microvesicles (MV) were isolated. Briefly, media from parallel 6-well plates were combined, remnant cells and cell debris were removed by centrifugation at 4500 g for 5 min, and the final cell free supernatants were stored at 80 C. Supernatants were thawed at 37 C, and MV were isolated by centrifugation at 17000 g for 30 min using a fixed-angle Sorvall SS-34 rotor (Kendro Laboratory Products, Newtown, CT, USA). The remaining supernatant was transferred to a new vial for exosome isolation. MV pellets were thoroughly dissolved in the residual supernatant (2 ml) and concentrated using Amicon Ultra-4 100 kDa Centrifugal Filter Devices (Merck Millipore, Cork, Ireland). To exclude particles greater than 220 nm in the exosome preparation, the 17000 g supernatant was gently filtered through a 0.22 m filter (Merk Millipore) before exosomes were isolated by Centricon-70 Plus 100 kDa Centrifugal Filter Columns (Merk Millipore). Column units were centrifuged at 3500 g for 15 min, filters washed with PBS and further centrifuged at 3500 g for 10 min. Finally, the filters were turned upside down, centrifuged at 1000 g for 2 min, and the exosome concentrates were collected. All centrifugations were performed at room temperature (RT), and all samples were stored in aliquots at 80 C.
Extracted molecule
total RNA
Extraction protocol
Total RNA from the exosomes and MV suspensions were isolated using miRNeasy Micro kit (Qiagen, Hilden, Germany) following the manufacturer´s protocol. In short, up to 150 µL EV-solution was transferred to an Eppendorf tube containing 700 µL Qiazol, incubated in RT, added chloroform, and shaken vigorously, before the tube was incubated for 3 min at RT and centrifuged at 12000 g for 15 min at 4 oC. The upper phase was transferred to a new tube, mixed thoroughly with 1.5 volume 100% ethanol, and filled into a RNeasy Min Elute column before a short centrifugation step at 8000 g. Flow-through was discarded, the column washed with buffers RWT and RPE, centrifuged at 8000 g before 80 % ethanol was added, and the column centrifuged dry with open lid. Finally, RNA was eluted in 14 µl nuclease free water and stored at -80 oC until analyzed.
Label
biotin
Label protocol
2.5 ng of total RNA was subjected to the Affymetrix® FlashTag™ Biotin HSR RNA Labeling Kit, following the manufacturer’s protocol (Affymetrix).
Hybridization protocol
Labeled RNA was hybridized to the Affymetrix GeneChip Multispecies miRNA-4_0 Array (Affymetrix, Santa Clara, CA). The arrays were washed and stained using FS-450 fluidics station (Affymetrix)
Scan protocol
Fluorescent images were scanned by Hewlett Packard Gene Array Scanner 3000 7G (Hewlett Packard, Palo Alto, CA).The scanned images were then analyzed using Affymetrix GeneChip Command Console
Data processing
The CEL files were imported into Partek Genomics Suite software (Partek, Inc., St. Louis, MO). Robust microarray analysis (RMA) was applied for normalization.
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 [EPS_MV]
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
Data table header descriptions
ID_REF
VALUE
RMA signal estimated from Partek Genomics Suite software, Log2 values
