Characterisation of adipocyte-derived extracellular vesicle subtypes identifies distinct protein and lipid signatures for large and small extracellular vesicles

Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells by conveying signalling proteins and genomic material. The level of EVs in plasma is significantly increased in cardiometabolic diseases associated with obesity, suggesting their possible participation in the development of metabolic dysfunction. With regard to the poor definition of adipocyte-derived EVs, the purpose of this study was to characterise both qualitatively and quantitatively EVs subpopulations secreted by fat cells. Adipocyte-derived EVs were isolated by differential centrifugation of conditioned media collected from 3T3-L1 adipocytes cultured for 24 h in serum-free conditions. Based on morphological and biochemical properties, as well as quantification of secreted EVs, we distinguished two subpopulations of adipocyte-derived EVs, namely small extracellular vesicles (sEVs) and large extracellular vesicles (lEVs). Proteomic analyses revealed that lEVs and sEVs exhibit specific protein signatures, allowing us not only to define novel markers of each population, but also to predict their biological functions. Despite similar phospholipid patterns, the comparative lipidomic analysis performed on these EV subclasses revealed a specific cholesterol enrichment of the sEV population, whereas lEVs were characterised by high amounts of externalised phosphatidylserine. Enhanced secretion of lEVs and sEVs is achievable following exposure to different biological stimuli related to the chronic low-grade inflammation state associated with obesity. Finally, we demonstrate the ability of primary murine adipocytes to secrete sEVs and lEVs, which display physical and biological characteristics similar to those described for 3T3-L1. Our study provides additional information and elements to define EV subtypes based on the characterisation of adipocyte-derived EV populations. It also underscores the need to distinguish EV subpopulations, through a combination of multiple approaches and markers, since their specific composition may cause distinct metabolic responses in recipient cells and tissues

Characterization of extracellular vesicles in a context of obesity and monitoring of their evolution during the practice of physical activity in obese patients

Les microparticules (MP) et les exosomes (EXO) sont des sous-types de vésicules extracellulaires(VE) libérés par la plupart des types de cellules. Identifiées depuis peu comme intervenant dans lacommunication intercellulaire, ces VE participent activement à de nombreux processusbiologiques. Cependant, leurs taux circulants se sont avérés considérablement augmenter avecl’obésité et pourraient constituer de véritables vecteurs pathologiques de part les différentsfacteurs qu’ils transportent au cours de la maladie.Malheureusement, peu de données existent quand à la libération des VE par les cellules adipeuseset musculaires. La caractérisation desVE adipocytaires a permis, dans un premier temps, d’identifier un grand nombre d’adipokines, quileur sont associées, dont certaines connues pour leur implication dans divers processusmétaboliques. De plus, nous avons distingué des signatures protéiques différentes au sein de cesdeux sous-types de VE, suggérant des effets biologiques qui leur sont propres. Le phénotypage desVE circulantes de patients obèses a révélé l’association de la cytokine MIF (Macrophagesmigration inhibitor factor) aux MP. Cette cytokine, connue pour sa contribution aux processusinflammatoires, s’avère présenter des fonctions similaires en étant liée aux MP. Enfin, l’activitéphysique (AP), connue pour ses multiples effets bénéfiques sur l’obésité et les complications quilui sont associées, s’est avérée être un facteur diminuant les taux circulants des MP et augmentantceux des EXO, chez des patients obèses, soulignant ainsi des effets distinct de l’AP sur lasécrétion de ces 2 soustypes de VE.Ainsi, les VE apparaissent comme des vecteurs pathologiques dont les taux circulants totauxvarieraient avec l’AP.Microparticles (MP) and exosomes (EXO) are subtypes of extracellular vesicles (EV) released bymost cell types. Recently identified as intervening in intercellular communication, these EVactively participate in many biological processes. However, their circulating levels have beenshown to increase considerably with obesity and could constitute pathological vectors because ofthe different factors they carry during the disease.Unfortunately, there is few data available on the release of EV by fat and muscle cells. Thecharacterization of adipocyte EV made it possible to identify a large number of adipokinesassociated with them, some of which are known to be involved in various metabolic processes. Inaddition, we distinguished different protein signatures within these two EV subtypes, suggestingbiological effects that are unique to them. Phenotyping of circulating EV in obese patientsrevealed the association of cytokineMIF (Macrophage migration inhibitor factor) with MP. This cytokine, known for itscontribution to inflammatory processes, is found to have similar functions by being linkedto MP. Finally, physical activity (PA), known for its multiple beneficial effects on obesity andassociated complications, has been shown to be a factor decreasing the circulating levels ofMP and increasing those of EXO in obese patients, highlighting the distinct effects of PAon the secretion of these 2 EV subtypes.Thus, EV appear as pathological vectors whose total circulating levels would change with PA

Effects of docosahexaenoic acid on bovine granulosa cell proliferation and steroidogenesis

National audienc

Effects of docosahexaenoic acid on bovine granulosa cells in vitro: involvement of FFAR4 receptor

Effects of docosahexaenoic acid on bovine granulosa cells in vitro: involvement of FFAR4 receptor. 2. Journées du GdR 3606 Repr

Docosahexaenoic acid (DHA) effects on proliferation and steroidogenesis of bovine granulosa cells

International audienceBackground Docosahexaenoic acid (DHA) is a n-3 polyunsaturated fatty acid (PUFA) belonging to a family of biologically active fatty acids (FA), which are known to have numerous health benefits. N-3 PUFAs affect reproduction in cattle, and notably directly affect follicular cells. In terms of reproduction in cattle, n-3 PUFA-enriched diets lead to increased follicle size or numbers. Methods The objective of the present study was to analyze the effects of DHA (1, 10, 20 and 50 μM) on proliferation and steroidogenesis (parametric and/or non parametric (permutational) ANOVA) of bovine granulosa cells in vitro and mechanisms of action through protein expression (Kruskal-Wallis) and signaling pathways (non parametric ANOVA) and to investigate whether DHA could exert part of its action through the free fatty acid receptor 4 (FFAR4). Results DHA (10 and 50 μM) increased granulosa cell proliferation and DHA 10 μM led to a corresponding increase in proliferating cell nuclear antigen (PCNA) expression level. DHA also increased progesterone secretion at 1, 20 and 50 μM, and estradiol secretion at 1, 10 and 20 μM. Consistent increases in protein levels were also reported for the steroidogenic enzymes, cytochrome P450 family 11 subfamily A member 1 (CYP11A1) and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (HSD3B1), and of the cholesterol transporter steroidogenic acute regulatory protein (StAR), which are necessary for production of progesterone or androstenedione. FFAR4 was expressed in all cellular types of bovine ovarian follicles, and in granulosa cells it was localized close to the cellular membrane. TUG-891 treatment (1 and 50 μM), a FFAR4 agonist, increased granulosa cell proliferation and MAPK14 phosphorylation in a similar way to that observed with DHA treatment. However, TUG-891 treatment (1, 10 and 50 μM) showed no effect on progesterone or estradiol secretion. Conclusions These data show that DHA stimulated proliferation and steroidogenesis of bovine granulosa cells and led to MAPK14 phosphorylation. FFAR4 involvement in DHA effects requires further investigation, even if our data might suggest FFAR4 role in DHA effects on granulosa cell proliferation. Other mechanisms of DHA action should be investigated as the steroidogenic effects seemed to be independent of FFAR4 activation

La caractérisation des vésicules extracellulaires (VEs) adipeuses révèle une adsorption non spécifique de l’adiponectine sur ces vésicules

International audienc

La caractérisation des vésicules extracellulaires (VEs) adipeuses révèle une adsorption non spécifique de l’adiponectine sur ces vésicules

International audienc

Additional file 2: of Docosahexaenoic acid (DHA) effects on proliferation and steroidogenesis of bovine granulosa cells

Table S1. Characteristics of primary antibodies used for western blotting and / or immunohistochemistry or immunofluorescence. (DOCX 16 kb

MIF et MFG-E8 utilisent les vésicules extracellulaires comme voie de secretion.

International audienc

Additional file 1: of Docosahexaenoic acid (DHA) effects on proliferation and steroidogenesis of bovine granulosa cells

Figure S1. Control of customized free fatty acid receptor 4 (FFAR4) antibody specificity. Protein extracts from bovine lung tissue were separated by electrophoresis on 4–12% (w:v) SDS-polyacrylamide gel. After electrotransfer to nitrocellulose membranes, the proteins were probed with anti-FFAR4 antibody (0.95 μg/mL, customized FFAR4 rabbit antibody, Agro-Bio), which was pre-incubated for 15 min with different concentrations of the bovine specific peptide (Agro-Bio) used to produce the antibody (from 0 to 2.5 μg/mL). The blots were stripped and re-probed with antibodies against vinculin (VCL) used as the loading control. (TIF 93 kb