Introduction
Skeletal muscle (SkM)-secreted proteins have important roles in intercellular communications. Among them, a large number of soluble peptide hormones and cytokines (myokines) are capable of triggering adaptations of homeostasis in other peripheral organs or are involved in the process of myogenesis [
1]. Studies during the last 10 years have demonstrated that cells also release bioactive nanovesicles. A particularly important class of extracellular vesicle is the exosomes, which represent a discrete population of 50–120 nanometre-sized vesicles [
2]. Exosomes are formed in endocytic compartments called multivesicular bodies (MVBs) during endosome maturation, by inward budding of their limiting membrane. They are released from the cell into the microenvironment following the fusion of MVBs with the plasma membrane. Exosomes can transfer both functional protein and RNA species from one cell to another whereby an array of biological processes, including cell proliferation and differentiation, can be affected [
3]. It is believed that exosomes could have a much more potent influence on the physiology of the cells they encounter than single-molecule mediators because the numerous proteic, lipidic and nucleic acid components they carry can affect multiple signalling pathways inside target cells. Exosomes can modulate immune-regulatory processes, set up tumour escape mechanisms and mediate regenerative or degenerative processes [
4].
All insulin-sensitive tissues release exosomes [
5‐
8]. In this context, it has been demonstrated that adipose tissue-released exosomes in leptin-deficient (
ob/
ob) mice are taken up by peripheral blood monocytes and can stimulate the differentiation of monocytes into activated macrophages with increased secretion of TNF-α and IL-6 [
9]. This finding suggests that exosomes released from adipose tissue of obese animals could act as a mode of communication with macrophages. Moreover, when administered to wild-type mice, adipose tissue-released exosomes from
ob/
ob mice contributed to the development of insulin resistance (IR) [
9]. Other studies have shown that adipocyte-derived exosomes are involved in the control of lipid storage and cell size between adipocytes [
10] and they have been described as mediators of angiogenesis [
11].
Although communication between SkM and other tissues appears to be important in the relationship between obesity and diabetes [
12], the possibility that SkM-derived exosomes act as a mode of systemic communication has hitherto never been discussed. Until now, the roles of exosomes in the development of metabolic syndrome and IR are poorly understood and little is known about the connection between lipid exposure and exosome secretion from muscle cells.
Therefore, we investigated whether SkM cells might transmit specific signals via exosomes during lipid-induced IR. Especially, we examined whether exosomes from palmitate-treated myotubes could transfer the deleterious action of palmitate between muscle cells. In addition, we analysed in mice the biodistribution of labelled exosomes from control- or palmitate-exposed myotubes. We provide evidence that exosomes from muscle cells act both as ‘paracrine-like’ signals by locally perturbing muscle homeostasis during lipid-induced IR and as ‘endocrine-like’ signals by targeting other insulin-sensitive tissues in vivo.
Discussion
Palmitic acid is the most abundant systemic saturated NEFA of palm oil and has received considerable attention in investigations of metabolic consequences of dietary lipids since it is suspected to participate in the onset of IR in SkM [
25,
26]. In this context it has been demonstrated that palmitate-induced IR in C2C12 myotubes is associated with impaired expression of myokines (i.e. irisin, CTRP15 [family with sequence similarity 132, member B] and FGF21 [fibroblast growth factor 21]) known for their potential beneficial roles in metabolic diseases [
27]. In agreement, irisin in plasma is reduced in type 2 diabetes by 50% [
28]. These data indicated that under chronic elevation of plasma NEFA SkM may secrete specific signals that would act either as ‘paracrine-like’ signals by locally perturbing muscle homeostasis or as ‘endocrine-like’ signals by targeting other insulin-sensitive tissues. In this study, HP-fed mice consuming a standard diet enriched with 20% palm oil for 16 weeks presented marked obesity, hyperinsulinaemia and hyperglycaemia compared with SD-fed mice. Moreover, they displayed altered Akt phosphorylation in response to insulin and reduced expression of
Slc2a4 mRNA in SkM. Interestingly, HP-fed mice were also found to have reduced mRNA levels of
Myod1 and
Myog, two markers of muscle differentiation, and increased mRNA level of
Ccnd1, indicating that palm oil had a deep impact on muscle homeostasis in addition to IR.
The first finding of our study is that feeding mice with palm oil modified the rate of exosome secretion from SkM ex vivo. This was confirmed in vitro by showing that increasing the amount of palmitate in the medium of C2C12 cells induced an increase in exosome release. It is possible that de novo synthesis of ceramides from palmitate could contribute to an increase in intracellular exosome formation as exosome membranes are enriched in ceramides and intracellular ceramide levels are involved in exosome secretion by permitting the inward budding of MVBs [
29,
30]. Here, we did not quantify ceramides in exosomes but we found a marked enrichment in palmitic acid after treatment of C2C12 cells indicating that there was a modification of the lipid composition of exosomes. In addition, palmitate-induced exosome release could also occur through the palmitoylation of proteins involved in invagination of endosomal membranes (i.e. proteins involved in the ESCRT [endosomal sorting complex required for transport] machinery [
31]), possibly leading to modifications of the proteins’ subcellular localisation, stability, trafficking, translocation to lipid rafts, aggregation and interactions with new effectors [
32].
Although palmitate treatment does not reproduce exactly the in vivo situation where a mixture of fatty acids exists provided by the diet, these results support the notion that excessive concentration of circulating saturated fatty acids might modify exosome secretion from SkM in vivo.
We have previously shown that muscle-released exosomes are involved in the process of myogenesis, being able to transfer proteins and miRNAs from differentiated myotubes to proliferating myoblasts [
14,
15]. In this study we further demonstrated that exosomes can also transfer their protein contents between differentiated muscle cells. Thus we postulated that in response to palm oil, muscle cells might secrete exosomes able to transfer part of the deleterious effect of palm oil to neighbouring cells. Our data showed that palmitate-induced IR is not transferred between muscle cells through the exosomal route. However, EXO-Post Palm, as well as EXO-HP, was able to perturb the myotube phenotype (i.e. decrease in
Myog and
Myod1 mRNA levels) and to induce myoblast proliferation (i.e. increase in Akt protein and
Ccnd1 mRNA levels). Interestingly, these alterations in genes expressions were similar to those observed in vivo in muscles of HP-fed mice or in palmitate-treated C2C12 cells. These results indicate, therefore, that muscle-released exosomes can likely transfer the deleterious effect of palm oil between muscle cells by transferring lipids and thus may participate in vivo in the alterations of muscle homeostasis in high-fat diets.
EXO-Post Palm or EXO-HP modified the expression of genes encoding 20 secreted proteins in recipient myotubes (ESM Table
2). Among them,
Cxcl1 and
Cxcl5 have been found to be altered in the serum of patients with type 2 diabetes [
33,
34]. We validated the upregulation of
Il-
6, the gene encoding IL-6, one of the several pro-inflammatory cytokines associated with IR and type 2 diabetes [
35]. In vitro studies have suggested that IL-6 plays a part in myogenesis and muscle atrophy [
36,
37]. Thus these data support the concept that in addition to altering muscle homeostasis, muscle-exosomes are able to modulate the expression of several myokines, hence suggesting a potential role for these extracellular vesicles in whole-body homeostasis during lipid-induced IR.
Until now the mechanism of exosome uptake by target cells has been poorly understood. Integrins and tetraspanins [
38], as well as cell-surface heparan sulfate proteoglycans, have been implicated in exosome uptake and internalisation [
39]. However, despite the fact that exosomes can be used for the delivery of small RNAs and anti-inflammatory agents to target cells in vivo [
40,
41] it is not known whether exosomes have restricted tissue specificities. Indeed, in these previous studies exosomes were modified in order to target tissues of interest. We have shown recently that, in vitro, myotube-released exosomes can transfer siRNA and miRNA to myoblasts [
14]. Importantly, we found in the present study that fluorescently labelled muscle-released exosomes intravenously injected into mice were incorporated into various tissues in addition to SkM. Incorporation into the pancreas and liver might suggest that SkM could transfer specific signals through the exosomal route to key metabolic tissues in vivo. Alterations in this signalling system during a high-fat diet may eventually contribute to the development of IR and type 2 diabetes. Further studies are now required to demonstrate whether exosomes from insulin-resistant SkM can reach the blood circulation and regulate the homeostasis of the other insulin-sensitive tissues and to decipher the affected functions in these target tissues.
Acknowledgements
We thank E. Errazuriz (Centre Commun d’Imagerie de Laënnec, SFR Santé Lyon-Est, University of Lyon, France) for transmission electron microscopy images.