Periodic Reporting for period 1 - ExoDia (Cross-talk between skeletal muscle and pancreatic islets: impact of exosomes)
Période du rapport: 2018-01-02 au 2020-01-01
The worldwide explosion of obesity has resulted in an ever-increasing prevalence of type 2 diabetes (T2D), a noncommunicable disease that affects more than 370 million people worldwide. T2D is characterized by insulin resistance with a relative deficiency in insulin secretion. It is now recognized that there is decreased beta-cell function and mass in T2D but the precise underlying mechanism remains to be determined. Skeletal muscle accounts for >50% of the total glucose uptake in the post-prandial state, it is also the largest organ in non-obese individuals. Recent studies demonstrated that there is conversation between skeletal muscle and beta-cells, and that certain peptides (i.e. myokines) secreted by insulin-resistant skeletal muscle cells may impact negatively on beta-cell function, proliferation and survival in T2D vs myokines from healthy skeletal muscle cells which have a protective role. Myokines can modulate the function and survival of pancreatic beta cells depending on its nature and metabolic status. Nevertheless, other factors than myokines could also be involved in this inter-organ communication. A novel concept suggests that extracellular vesicles (EVs), such as exosomes and microparticles, as a new cell-to-cell communication mode. Exosomes and microparticles are small vesicles (30-100 nm and 100-200 nm of diameter, respectively) that contain large amount of information (proteins, genetic material, lipids). We have demonstrated (Guay C, Cell Met 2019) that exosomes containing specific miRNA can target specifically beta-cells triggering chemokine expression and beta-cell death. Blocking those specific miRNA transferred in β cells decreases diabetes incidence in a diabetic mouse model.
A major goal of this study is to provide further insight into the regulation of exosome secretion produced myotubes with different fiber composition (type I vs type II) and different insulin sensitivity, on the regulation of beta cell and islet function, with particular attention given to microRNA transfer from skeletal muscle cells to pancreatic beta cell and islet.
The findings may have important implications for understanding decreased functional beta-cell mass in diabetes, especially in T2D etiology, and therefore bring new insights into the development of innovative therapies.
A major goal of this study is to provide further insight into the regulation of exosome secretion produced myotubes with different fiber composition (type I vs type II) and different insulin sensitivity, on the regulation of beta cell and islet function, with particular attention given to microRNA transfer from skeletal muscle cells to pancreatic beta cell and islet.
The findings may have important implications for understanding decreased functional beta-cell mass in diabetes, especially in T2D etiology, and therefore bring new insights into the development of innovative therapies.
Human primary skeletal muscle cells coming from triceps (type II) or soleus (type I) were treated under control or TNFα condition for 24 h, and conditioned media was collected for exosomes isolation. Several months were required to reach the desired number of material needed (more than two hundred flasks for each condition), as primary human culture is very sensible to contamination and low proliferation rate. Some of the characterization approaches have been first validated in a rat muscle cell line (C2C12), because of it higher proliferation rate.
1. We have observed that soleus- and triceps-derived myotubes secrete EVs at the same concentration. However, EVs concentration released from soleus and triceps myotubes from healthy donnors are differentially affected by TNFα. There are significantly more exosomes in Triceps-TNFα samples vs Triceps-control, while Soleus-TNFα derived exosomes were undetectable (Figure 1).
2. SEM images showed vesicles of around 100 nm diameter in both control and TNFα-treated triceps-derived EVs (Figure 2). We can observe a positive expression of CD63 in control and TNFα-treated samples by TEM images (Figure 3). However, we observe cluster of CD63 in the control condition which are not observables in the TNFα condition. Thus, suggesting a different population of EVs in response to TNFα.
3. We have analysed the mRNA and miRNA expression on different muscle type (soleus vs triceps vs vastus lateralis) cells in the presence or absence of TNFα. We found that each muscle type has a unique expression signature for mRNA. Genes which are highly expressed (in red) in one muscle type had, on the contrary, a low expression (in green), showing a “mirror” expression pattern between muscle types (Figure 4A). When we compared the most differentially expressed genes between muscles, we obtained 22 genes that are different among them (Figure 4B and 4C). Those genes are implicated in different biological process, components and functions (Figure 4D). Furthermore, we obtained a different expression of miRNA among different muscle cell types (Figure 5A). At least 5 miRNA from the top differentially expressed miRNA were different the different muscle types. Those 5 miRNA are implicated in different metabolic functions (Figure 5B).
4. We studied the lipid concentration and distribution in MPs and exosomes obtained from C2C12 cells. Cholesterol concentration was lower in exosomes but higher in MPs isolated from TNFα-treated C2C12 cells in comparison to control (Figure 4). For ceramides, a diminution in TNFα-MPs and an increased in TNFα-exosomes was observed when compared to control (Figure 6). Regarding phospholipids (phosphatidylcholines [PC], phosphatidylethanolamines [PE] and phosphatidylserine [PS]), a decreased of PC in TNFα-exosomes and an increased in TNFα-MPs is observed when compared to control exosomes and MPs, respectively (Figure 6), while PE and PS the opposite was observed.
5. C2C12 treated with MPs from TNFα C2C12 showed a significant decrease of AKT phosphorylation compared to control-MPs treated cells (Figure 7B). However, when treated with exosomes from TNFα C2C12, no changes were observed in AKT phosphorylation in healthy cells (Figure 7A).
6. Beta-cells glucose-stimulated insulin secretion showed a tendency to decrease when treated with MPs from control and TNFα-treated C2C12 cells, when compared to untreated beta-cells (Figure 8A). However, when treated with exosomes from control and TNFα-treated C2C12 cells, we observed and increase in the basal insulin secretion compared to untreated beta-cells, indicating a potential effect on the insulin granules trafficking (Figire 8A). No effect was observed in cell death in any condition (Figure 8B and C)
Dissemination and publications
The ExoDia project has been disseminated at national and international congress and scientific publications, giving always visibility to the EU funding and logo. Furthermore, the laureate has participated in many abroad public events as diabetes screenings, running races, national science day and lab visits of high school students. Several General public Press articles related to the Marie Curie Fellowship were also published.
1. We have observed that soleus- and triceps-derived myotubes secrete EVs at the same concentration. However, EVs concentration released from soleus and triceps myotubes from healthy donnors are differentially affected by TNFα. There are significantly more exosomes in Triceps-TNFα samples vs Triceps-control, while Soleus-TNFα derived exosomes were undetectable (Figure 1).
2. SEM images showed vesicles of around 100 nm diameter in both control and TNFα-treated triceps-derived EVs (Figure 2). We can observe a positive expression of CD63 in control and TNFα-treated samples by TEM images (Figure 3). However, we observe cluster of CD63 in the control condition which are not observables in the TNFα condition. Thus, suggesting a different population of EVs in response to TNFα.
3. We have analysed the mRNA and miRNA expression on different muscle type (soleus vs triceps vs vastus lateralis) cells in the presence or absence of TNFα. We found that each muscle type has a unique expression signature for mRNA. Genes which are highly expressed (in red) in one muscle type had, on the contrary, a low expression (in green), showing a “mirror” expression pattern between muscle types (Figure 4A). When we compared the most differentially expressed genes between muscles, we obtained 22 genes that are different among them (Figure 4B and 4C). Those genes are implicated in different biological process, components and functions (Figure 4D). Furthermore, we obtained a different expression of miRNA among different muscle cell types (Figure 5A). At least 5 miRNA from the top differentially expressed miRNA were different the different muscle types. Those 5 miRNA are implicated in different metabolic functions (Figure 5B).
4. We studied the lipid concentration and distribution in MPs and exosomes obtained from C2C12 cells. Cholesterol concentration was lower in exosomes but higher in MPs isolated from TNFα-treated C2C12 cells in comparison to control (Figure 4). For ceramides, a diminution in TNFα-MPs and an increased in TNFα-exosomes was observed when compared to control (Figure 6). Regarding phospholipids (phosphatidylcholines [PC], phosphatidylethanolamines [PE] and phosphatidylserine [PS]), a decreased of PC in TNFα-exosomes and an increased in TNFα-MPs is observed when compared to control exosomes and MPs, respectively (Figure 6), while PE and PS the opposite was observed.
5. C2C12 treated with MPs from TNFα C2C12 showed a significant decrease of AKT phosphorylation compared to control-MPs treated cells (Figure 7B). However, when treated with exosomes from TNFα C2C12, no changes were observed in AKT phosphorylation in healthy cells (Figure 7A).
6. Beta-cells glucose-stimulated insulin secretion showed a tendency to decrease when treated with MPs from control and TNFα-treated C2C12 cells, when compared to untreated beta-cells (Figure 8A). However, when treated with exosomes from control and TNFα-treated C2C12 cells, we observed and increase in the basal insulin secretion compared to untreated beta-cells, indicating a potential effect on the insulin granules trafficking (Figire 8A). No effect was observed in cell death in any condition (Figure 8B and C)
Dissemination and publications
The ExoDia project has been disseminated at national and international congress and scientific publications, giving always visibility to the EU funding and logo. Furthermore, the laureate has participated in many abroad public events as diabetes screenings, running races, national science day and lab visits of high school students. Several General public Press articles related to the Marie Curie Fellowship were also published.
To the best of our knowledge, human primary skeletal muscle-derived EVs have not been described. We provide data showing that human skeletal muscle (soleus and triceps) secrete EVs having a diameter around 100 nm and expressing exosomes specific markers as CD63. We have also observed that insulin resistance (TNFα treatment) affects differently in triceps and soleus the concentration of EVs and the expression of CD63. These results may suggests that the content of EVs could be different from type I and type II muscle type, which could imply a different effect of EVs on muscle and/or beta cells. Taking together preliminary and actual results we consider that these findings open a new and exciting research field, offering news potential organ crosstalk pathways, still poorly studied. Potential impacts of these findings is to develop potential therapeutic treatments targeting one specific EVs population or miRNA.