Dataset Information

Distinct Adaptations of Mitochondrial Dynamics to Electrical Pulse Stimulation in Lean and Severely Obese Primary Myotubes.

ABSTRACT:

Background

Skeletal muscle from lean and obese subjects elicits differential adaptations in response to exercise/muscle contractions. In order to determine whether obesity alters the adaptations in mitochondrial dynamics in response to exercise/muscle contractions and whether any of these distinct adaptations are linked to alterations in insulin sensitivity, we compared the effects of electrical pulse stimulation (EPS) on mitochondrial network structure and regulatory proteins in mitochondrial dynamics in myotubes from lean humans and humans with severe obesity and evaluated the correlations between these regulatory proteins and insulin signaling.

Methods

Myotubes from human skeletal muscle cells obtained from lean humans (body mass index, 23.8 ± 1.67 kg·m-2) and humans with severer obesity (45.5 ± 2.26 kg·m-2; n = 8 per group) were electrically stimulated for 24 h. Four hours after EPS, mitochondrial network structure, protein markers of insulin signaling, and mitochondrial dynamics were assessed.

Results

EPS enhanced insulin-stimulated AktSer473 phosphorylation, reduced the number of nonnetworked individual mitochondria, and increased the mitochondrial network size in both groups (P < 0.05). Mitochondrial fusion marker mitofusin 2 was significantly increased in myotubes from the lean subjects (P < 0.05) but reduced in subjects with severe obesity (P < 0.05). In contrast, fission marker dynamin-related protein 1 (Drp1Ser616) was reduced in myotubes from subjects with severe obesity (P < 0.05) but remained unchanged in lean subjects. Reductions in DrpSer616 phosphorylation were correlated with improvements in insulin-stimulated AktSer473 phosphorylation after EPS (r = -0.679, P = 0.004).

Conclusions

Our data demonstrated that EPS induces more fused mitochondrial networks, which are associated with differential adaptations in mitochondrial dynamic processes in myotubes from lean humans and human with severe obesity. It also suggests that improved insulin signaling after muscle contractions may be linked to the reduction in Drp1 activity.

SUBMITTER: Kugler BA

PROVIDER: S-EPMC8656367 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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 6 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 4 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.