Análisis de resultados

detected in the ultracentrifuged pellet

MiRNAs have been found in several biological fluids incorporated into vesicles [188] and/or associated with vesicle-free systems such as Ago2 protein [375]. Ultracentrifugation is a technique widely employed to isolate vesicles from various types of fluidic sources [376]. To understand whether miRNAs secreted by adipocytes are encapsulated into vesicles and could be depleted from media by ultracentrifugation, miRNA content in serum-free conditioned media from differentiated brown adipocytes was analysed before and after ultracentrifugation at 120,000xg for 2 hours. The same number of differentiated cells was seeded for each biological replicate and kept in serum free media for 5 hours. Conditioned media was collected and processed in parallel with unconditioned media for miRNA analysis by Real Time qPCR. Figure 43 shows miRNA

abundance (2-ΔCt _{values) before and after vesicle depletion from the media. All miRNAs}

investigated were detected at lower levels in the supernatant after vesicle isolation. In particular, ultracentrifugation was able to significantly deplete 44.6% of miR-196a, 41.85% of miR-322 and 19.5% of miR-378 from the media. As expected no miRNAs were detectable in unconditioned media.

The pellet obtained by the ultracentrifugation was resuspended in 2.5 ml of unconditioned media and tested for miRNA content as well. As reported in figure 44 all miRNAs were present in the pellet from conditioned media, at relatively high level, while, no miRNAs were detectable in the pellet from unconditioned media. Because the pellet is obtained by concentrating the vesicles contained in the media, miRNA levels in the two types of samples are not directly comparable. However, miRNA presence in the pellet and depletion from supernatant suggest that all the microRNAs tested are at least partially encapsulated into vesicles and in part associated with vesicle-free systems.

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Figure 43 MiRNAs are depleted from conditioned media by ultracentrifugation.

The graphs show miRNA abundance before and after ultracentrifugation of serum free unconditioned and conditioned media samples from differentiated brown adipocytes. The same number of cells was seeded for each biological replicate. MiRNA levels were normalized to Unisp6 spiked in just prior to RNA extraction. Bars express the miRNA mean ± SEM of 2-ΔCt values of the biological triplicates. Student’s t- test was used to assess significance with cut-off of p-value<0.05(*). ** indicates p<0.01.

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Figure 44 MiRNAs detected in the pellet of ultracentrifuged media.

The graphs show 2-ΔCt _{values (mean of biological triplicates) of miRNAs in pellets obtained from}

unconditioned and conditioned media incubated with brown adipocyte for 5 hours. MiRNA levels were normalized to Unisp6 spiked in immediately prior to RNA extraction.

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  ß3-adrenergic activation affects the level of miRNA depletion in ultracentrifuged conditioned media

To investigate whether ß3-adrenergic activation has an effect on miRNA secretion via incorporation into vesicles or in association with vesicle-free systems, miRNA content

in serum-free media from differentiated brown adipocytes treated with CL (10 µM) for 5

hours was analysed before and after ultracentrifugation. The percentage of miRNA depletion observed in untreated and CL treated samples are summarized in table 19. The

first column of the table shows increment (­) or decrement (¯) of total miRNA in media

from BAT treated with CL compared to untreated from previous experiment (paragraph 4.1). Consistent with previous results, all the miRNAs were detected in the pellet (figure 46) and at least partially depleted from the media (figure 45). Little variations in the percentage of miRNA depletion were observed between CL-treated and untreated samples for miR-196a, miR-322 and miR-103. In contrast, miR-378 depletion from 19.5% in untreated samples became 42.83% in CL-treated, similar to that observed for miR-10b. In untreated brown adipocytes, 48% of miR-365 was depleted after centrifugation while its depletion in treated cells was only of 9.9%. These results suggest that CL favours miR-378 and miR-10b secretion via incorporation into vesicles while it promotes miR-365 release in association with vesicle-free systems.

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Figure 45 Treatment with CL affects the level of miRNA depletion in ultracentrifuged conditioned media

The graphs show miRNA abundance before and after ultracentrifugation of serum free unconditioned and conditioned media samples from differentiated interscapular brown adipocytes treated for 5 hours with CL (10µM). The same number of cells were seeded for each biological replicate. MiRNA levels were normalized to Unisp6 spiked in immediately prior to RNA extraction. Bars express the miRNA mean ± SEM of 2-ΔCt _{values of the biological triplicates. Student’s t-test was used to assess significance with cut-}

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Table 18 Percentage of miRNA depletion after ultracentrifugation of media from differentiated brown adipocytes untreated and treated with CL.

Tot miRNA

CLvsC miRName

UNTREATED CL TREATED

% depletion SEM % depletion SEM

­2_* miR-196a 44.60 ± 8.70 34.37 ± 48.9 ¯ miR-10b 18.60 ± 38.70 45.74 ± 35.0 ¯2_* miR-103 36.23 ± 75.39 40.41 ± 25.5 ­* miR-322 41.85 ± 8.00 32.95 ± 23.9 ­ miR-378 19.50 ± 4.40 42.83 ± 21.4 ­ miR-365 48.50 ± 30.00 9.90 ± 25.0

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Figure 46MiRNAs detected in the pellet of ultracentrifuged media from CL-treated adipocytes.

The graphics show 2-ΔCt _{values (mean of biological triplicates) of miRNAs in pellet obtained from}

unconditioned and conditioned media from brown adipocyte treated with CL for 5 hours (B). MiRNA levels were normalized to Unisp6 spiked in just prior RNA extraction

177

  Nanoparticle characterization

To investigate the nanoparticles secreted by adipocytes, media samples from mouse and human differentiated white and brown adipocyte cell lines were analysed through NanoSight technology. This system detects particles suspended in fluids determining their concentration and profiling their size distribution. To perform the experiment, an equal number of cells was seeded into each T-175 flask and incubated for 3 days in serum-free media. However, cell density visibly decreased at day 3, probably due to long time incubation in serum-free condition. Therefore, cells were counted and data normalised to cell count so that the graphs show size distribution (figure 47 and 48 left panels) and concentration (figure 47 and 48 right panels) of particles/ml secreted by

1x107 cells. Media samples were collected and processed. As the samples were analysed

at the University of Cardiff, the first steps of sample preparation (serial centrifugation and filtration) were performed prior to freezing at -80 °C, to avoid large particle breakdown into smaller particles and therefore false positive results. Media samples were then analysed using NanoSight technology. As shown in the right panel of figure 47, non-

conditioned media had a concentration of 1.6 (± 0.8) x108 particles per ml. As the media

from brown and white adipocytes had a significantly higher concentration of

nanoparticles per ml, 1.15 (± 0.5) x 1012 and 1.37 (± 0.3) x 1012 respectively, it is possible

to conclude that nanoparticles in conditioned media originated from adipocytes. The analysis revealed that there were no significant differences between white and brown adipocytes and that the majority of the nanoparticles had a size of 100 nm. This suggests that a large proportion of the vesicles secreted by adipocytes consist of exosomes, which are sized 50-200 nm. The different type of vesicles such as microvesicles and exosomes, differ not only in size but also in chemical composition. Exosomes are known to express on their membrane surface the tetraspanins CD9, CD81 and CD63, which have been established as exosomes markers [377, 378]. To characterize the composition of the vesicles analysed by nanosight and confirm that adipocytes secrete exosomes, the conditioned media from mouse and human adipocytes was ultracentrifuged at 120,000xg for 2 hours to isolate vesicles. The supernatant was discarded, the pellet resuspended in 2.5 ml of PBS and analysed for exosome marker detection through Europium-based ELISA-like assay. As reported in figure 47, bottom panel, mouse vesicles were positive for CD9, CD81 and CD63 tetraspanins. CD63 was expressed only in BAT. However, no

178 statistical analysis was performed on these data as the replicate number was lower than 3.

Figure 47 Mouse adipocytes secret exosome-like nanoparticles.

Media samples were collected after 3 days from mouse differentiated brown and white adipocyte cell lines. The analysis was performed at the Velindre Centre, University of Cardiff, with the assistance of Dr J. Webber. (A) Nanosight analysis of non-conditioned and conditioned media samples from differentiated brown and white adipocytes was adjusted for dilutions adopted when loading the samples. Left panel shows size distribution of nanoparticles. The curves represent the mean trend lines (period 11) of the 3 biological replicates of conditioned media samples for each group. The right panel shows mean concentration of nanoparticles and relative SEM. (B) Europium based ELISA-like assay. The graphic shows the mean fluorescence intensity for the exosomes markers (CD9, CD81 and CD63) on the surface of the nanoparticles isolated from the conditioned media samples normalised to the unconditioned media and non-specific signals for primary and secondary antibodies.

179 Concentration and size profiling of vesicles secreted by human adipocytes, through Nanosight analysis, revealed that, also in this case, particle concentration in conditioned media was significantly higher than in non-conditioned medium and very

similar between BAT and WAT, with 2.1(± 0.2) x1010 and 1.8 (± 0.4) x1010 particles per

ml respectively (figure 48, right panel). However, a different profile of particle size distribution between human BAT and WAT was observed. BAT presented a high concentration of particles with a diameter of about 100 nm as shown by the sharp peak of the curve in figure 48 top left panel, while WAT is represented by the curve with a smoother peak in the range of 100 and 250 nm which reflects a higher heterogeneity in the vesicle population.

When analysed for the exosome markers expression through the Europium-based ELISA-like assay, the isolated vesicles from both human brown and white adipocytes were positive for CD9, CD63 and CD81 and showed a similar trend of expression, even if the replicates were less than 3 and no statistical analysis were performed (figure 48 bottom panel).

In conclusion, all these results suggest that both mouse and human adipocytes secrete nanoparticles and that a large part of these may consist of exosomes.

180

Figure 48 Human adipocytes secret exosome-like nanoparticles.

Media samples were collected after 3 days from mouse differentiated brown and white adipocyte cell lines. The analysis was performed at the Velindre Centre, University of Cardiff, with the assistance of Dr J. Webber. (A) Nanosight analysis of non-conditioned and conditioned media samples from differentiated brown and white adipocytes. Left panel shows size distribution of nanoparticles. The curves represent the mean trend lines (grade 11) of the 3 biological replicates of conditioned media samples for each group. The right panel shows mean concentration of nanoparticles and relative SEM. (B) Europium based ELISA-like assay. The graph shows the mean fluorescence intensity for the exosomes markers (CD9, CD81 and CD63) on the surface of the nanoparticles isolated from the conditioned media samples normalised to the unconditioned media and non-specific signals for primary and secondary antibodies.

181