Background
With obesity rates escalating and an ageing population, the incidence of individuals who are co-morbid with sarcopenia and obesity (termed sarcopenic obesity) is becoming more prevalent. Critically, obesity worsens sarcopenia[1], impairing muscle metabolic function and exacerbating the age-related impairment in the anabolic response of muscle to exercise or nutrition[2, 3]. Currently, the pathogenesis of sarcopenic obesity is poorly elucidated. However, intercellular communication between adipose and muscle appears pivotal. Of significance, we previously reported that in older humans, the secretome from obese (but not lean) adipose tissue impairs myotube thickness and the fusion of muscle cells into multinucleated fibres[4], supporting the notion that cellular cross-talk between adipose and muscle is pathological with age and obesity.
Significantly, extracellular vesicles (EVs) have emerged as mediators of intercellular cross-talk, capable of transporting biomolecules between cell types[5], and mediating multiple biological processes implicated in the development of age-related chronic diseases. We hypothesised therefore that with obesity, adipose-derived EVs drive pathological adipose-muscle cross-talk, accelerating muscle ageing (sarcopenia). The aim of this study was to characterise EVs released from obese and lean human adipose tissue, and determine their effect on the transcriptome of human myotubes.
Methods
Adipose conditioned media (ACM) was generated over 24h from subcutaneous adipose tissue collected from patients undergoing orthopaedic surgery who were either lean (n=4), over-weight (n=5) or obese (n=7) (NRES 16/SS/0172). EVs were isolated from ACM by ultracentrifugation and characterised by nanoparticle tracking analysis (NTA), ExoView and their RNA cargo profiled by small RNA-sequencing. Primary human myoblasts were differentiated into multinucleated myotubes, and either untreated (n=5) or treated for 24h with either obese (n=5), over-weight (n=3) or lean (n=3) EVs, before being analysed by RNA sequencing and qPCR.
Results
NTA and ExoView analysis confirmed the presence of EVs in the resuspended ultracentrifuged ACM pellets. The concentration of EVs was significantly (p<0.05) greater from the ACM of lean individuals, compared to ACM from non-lean (BMI>25). RNAseq analysis revealed that human myotubes treated for 24h with obese EVs exhibited differential expression of 129 genes (86 upregulated, 43 down-regulated), compared to untreated myotubes. In contrast, myotubes treated with over-weight EVs exhibited differential expression of only 6 genes, and no significant differentially expressed genes were exhibited by myotubes treated with normal-weight EVs, compared to untreated (FDR=0.2). IPA and Quaternary Dot Product Scoring Statistic confirmed an atrophic, inflammatory effect of EV treatment, which was exacerbated with increasing BMI, with IL1β a key upstream regulator and p38 mediated MAPK signalling implicated. Investigating this further, we found via qPCR analysis that stimulation of myotubes with obese EVs induced the expression of the muscle atrophic genes (MAFBx, FOXO3) and the pro-inflammatory genes IL-6 and IL-1β.
Finally, in attempting to identify potential EV mechanisms, EV cargo analysis using small RNA-sequencing detected a total of 629 miRNAs, of which 14 were differentially expressed in non-lean EVs, compared to lean EVs, including miRNAs implicated in musculoskeletal remodelling and ageing (p<0.05, fold-change>1.5).
Conclusion
EVs from non-lean adipose tissue transport miRNA cargo implicated in musculoskeletal ageing and differentially affect the transcriptomic profile of human myotubes in vitro.