Rôle des récepteurs nucléaires PPAR gamma et PPAR alpha dans la conversion d'adipocytes blancs humains en adipocytes bruns/brites

Chez les mammifères, deux types de tissu adipeux (TA) sont présents: le TA blanc, qui est l'organe de stockage et de libération des lipides, et le TA brun, qui est un organe spécialisé dans la production de chaleur grâce à l'expression de la protéine découplante mitochondriale UCP1.Chez l'homme, la présence d'un TA brun métaboliquement actif est inversement corrélée à l'obésité et au diabète de type 2. Ce TA brun est composé de deux types distincts de cellules thermogéniques, les adipocytes bruns classiques présents dans des dépôts spécifiques et les adipocytes "brite " (brown-in-white). Chez la souris, les adipocytes " brite " apparaissent dans le TA blanc lors d'une exposition au froid et sont protecteurs contre l'insulinorésistance induite par l'obésité. Ainsi le "brunissement " du TA blanc ouvre la voie à de nouvelles approches thérapeutiques pour lutter contre les pathologies associées à l'obésité. Toutefois, la capacité des adipocytes blancs humains à acquérir un métabolisme brun/brite reste méconnue. Notre étude cherche donc à identifier les changements moléculaires et métaboliques associés à la conversion d'adipocytes blancs humains différenciés en adipocytes " brite ", après un traitement par des agonistes des récepteurs nucléaires PPARgamma ou PPARalpha. Dans un premier temps, nous avons montré in vitro que les cellules hMADS(adipocytes humains dérivés des cellules souches mésenchymateuses), différenciées en adipocytes blancs sont convertis en adipocytes " brites " par les agonistes PPARgamma et PPARalpha. Ces adipocytes brites ont une activité mitochondriale élevée et expriment la protéine découplante UCP1. Dans un deuxième temps, nous avons mis en évidence que le brunissement s'accompagne d'un profond changement métabolique. La mise en place d'un cycle futile lipolyse/ré-estérification couplé à une augmentation de l'oxydation des acides gras permet de fournir les substrats nécessaires à la thermogenèse mitochondriale. A l'inverse, le transport et l'oxydation du glucose sont diminués notamment suite à l'inhibition de la pyruvate déshydrogénase par la protéine PDK4. A la place le glucose va être dirigé vers la voie de la glycéronéogenèse pour fournir le glycérol-3-phosphate nécessaire à la synthèse des triglycérides. Ainsi, l'ensemble du métabolisme des adipocytes " brite " est réorganisé vers l'utilisation des acides gras comme source principale d'énergie. Enfin, nous avons validé l'implication de PPARalpha dans les mécanismes de brunissement in vivo grâce à l'utilisation de souris dont le gène codant pour PPARalpha a été invalidé. L'induction du brunissement par un agoniste betea3-adrénergique nous a permis de confirmer que PPARalpha est nécessaire à l'expression optimale des gènes thermogéniques et au brunissement du tissu adipeux blanc. L'ensemble de ces données permet d'affirmer que chez l'homme, les adipocytes blancs sont capables de se convertir en adipocytes " brite ". Cette conversion s'accompagne de changements métaboliques qui favorisent l'utilisation intracellulaire des acides gras, ce qui pourrait diminuer leur niveau plasmatique limitant ainsi leur stockage ectopique dans les tissus insulinosensibles.Two types of adipose tissue are present in mammals, white and brown adipose tissue. White adipose tissue (WAT) is specialized in storage and release of lipids, and brown adipose tissue (BAT) is specialized in energy dissipation as heat through the mitochondrial uncoupling protein 1 (UCP1).In humans,thermogenically-competent brown adipose tissue is negatively associated with body mass index and diabetes. BAT is composed of two distinct types of thermogenic cells, classical brown adipocytes located in specific depots and brite adipocytes (brown-in white). In mice, these cells appear in WAT upon cold exposure and are protective against obesity-induced insulin resistance. Therefore, fighting obesity through "browning" of white adipose tissue emerges as a promising strategy. However, the ability of human white adipocytes to acquire a brown/brite phenotype is not yet understood. Here, we aimed at identifying the molecular and metabolic changes associated with the white-to-brown conversion of human mesenchymal adipose-derived stem (hMADS) cells following treatment by agonists of the nuclear receptor PPARgamma or PPARalpha. First, we demonstrated in vitro that PPARgamma or PPARalpha agonists similarly induce white-to-brown conversion of hMADS cells into brite adipocytes that possess high mitochondrial content and express UCP1. Second, we showed that browning is associated with profound metabolic changes. The lipolytic machinery and fatty acid re-esterification was stimulated by the two treatments, resulting in a futile cycle. These adaptations combined with an increase of fatty acid betaoxidation provide substrates to sustain the high level of mitochondrial thermogenesis. In contrast, glucose uptake and oxidation are decreased through inactivation of the pyruvate dehydrogenase by PDK4. Consequently, glucose-carbons are redirected towards glyceroneogenesis to provide the glycerol-3-phosphate backbone necessary for triglyceride esterification. Thus, brite adipocyte metabolism is modified to promote fatty acid utilization as the main energy source. In order to confirm the involvement of PPARalpha in inducing browning in vivo, we treated mice with inactivation of the PPARalpha gene with a beta3-adrenergic agonist. In subcutaneous WAT, expression of BAT- and brite-specific markers was lower in PPARalpha knock out than in wild type mice, confirming that PPARa is required for WAT browning. Altogether, this study shows that PPARgamma and PPARalpha activation in human white adipocytes promotes browning associated with an increase in fatty acid utilization without enhancement of glucose metabolism. These metabolic changes favor intra-adipose fatty acid utilization and thus could diminish plasma fatty flux for ectopic storage into insulin-sensitive tissues

Exosomes and miRNA-Loaded Biomimetic Nanovehicles, a Focus on Their Potentials Preventing Type-2 Diabetes Linked to Metabolic Syndrome

Exosomes are small membrane vesicles of 30–150 nm, members of the extracellular vesicle family and secreted by various cell types. Different studies describe specific microRNA (miRNA) with altered expression in serum and/or plasma of patients suffering from diabetes or metabolic syndrome. Diabetic cardiomyocyte-derived exosomes loaded with miRNAs like miR-320-3p (or 320a) have been shown regulating angiogenesis on endothelial cell cultures. Insufficient myocardial angiogenesis is the major manifestation of diabetes-caused ischemic cardiovascular disease. Studies on transfer of functional microRNAs between mouse dendritic cells via exosomes have shown that some miRNAs (miR-320-3p, 29b-3p, 7a-5p) are distributed in immature and mature exosomes. Among these miRNAs, miR-320-3p is better known in epigenetics for silencing polr3d gene by binding to its promoter in Human Embryonic Kidney-293 cells. Moreover, quantitative and stoichiometric analysis of the microRNA content of exosomes highlights the lack of reliable natural source of such particles loaded with miRNA opening the need for tailoring exosomes or nanoparticles delivering efficiently miRNA intimately linked to immunity, metabolism and epigenetics in target cells. However, loading of extracellular mature miRNA into recipient cells comes with a cost by at least impeding dynamic localization of miRNAs in nucleoli or inefficient miRNA delivery due to rapid recycling by exonucleases. All these works are calling for the design of new biomimetic vehicles and in vivo assessment of miRNA functionality when delivered by natural or biomimetic nanoparticles in order to control metabolic diseases from infancy to adulthood

Role of nuclear receptors PPAR gamma and PPAR alpha in white-to-brown conversion of human white adipocytes

Chez les mammifères, deux types de tissu adipeux (TA) sont présents: le TA blanc, qui est l'organe de stockage et de libération des lipides, et le TA brun, qui est un organe spécialisé dans la production de chaleur grâce à l'expression de la protéine découplante mitochondriale UCP1.Chez l'homme, la présence d'un TA brun métaboliquement actif est inversement corrélée à l'obésité et au diabète de type 2. Ce TA brun est composé de deux types distincts de cellules thermogéniques, les adipocytes bruns classiques présents dans des dépôts spécifiques et les adipocytes "brite " (brown-in-white). Chez la souris, les adipocytes " brite " apparaissent dans le TA blanc lors d'une exposition au froid et sont protecteurs contre l'insulinorésistance induite par l'obésité. Ainsi le "brunissement " du TA blanc ouvre la voie à de nouvelles approches thérapeutiques pour lutter contre les pathologies associées à l'obésité. Toutefois, la capacité des adipocytes blancs humains à acquérir un métabolisme brun/brite reste méconnue. Notre étude cherche donc à identifier les changements moléculaires et métaboliques associés à la conversion d'adipocytes blancs humains différenciés en adipocytes " brite ", après un traitement par des agonistes des récepteurs nucléaires PPARgamma ou PPARalpha. Dans un premier temps, nous avons montré in vitro que les cellules hMADS(adipocytes humains dérivés des cellules souches mésenchymateuses), différenciées en adipocytes blancs sont convertis en adipocytes " brites " par les agonistes PPARgamma et PPARalpha. Ces adipocytes brites ont une activité mitochondriale élevée et expriment la protéine découplante UCP1. Dans un deuxième temps, nous avons mis en évidence que le brunissement s'accompagne d'un profond changement métabolique. La mise en place d'un cycle futile lipolyse/ré-estérification couplé à une augmentation de l'oxydation des acides gras permet de fournir les substrats nécessaires à la thermogenèse mitochondriale. A l'inverse, le transport et l'oxydation du glucose sont diminués notamment suite à l'inhibition de la pyruvate déshydrogénase par la protéine PDK4. A la place le glucose va être dirigé vers la voie de la glycéronéogenèse pour fournir le glycérol-3-phosphate nécessaire à la synthèse des triglycérides. Ainsi, l'ensemble du métabolisme des adipocytes " brite " est réorganisé vers l'utilisation des acides gras comme source principale d'énergie. Enfin, nous avons validé l'implication de PPARalpha dans les mécanismes de brunissement in vivo grâce à l'utilisation de souris dont le gène codant pour PPARalpha a été invalidé. L'induction du brunissement par un agoniste betea3-adrénergique nous a permis de confirmer que PPARalpha est nécessaire à l'expression optimale des gènes thermogéniques et au brunissement du tissu adipeux blanc. L'ensemble de ces données permet d'affirmer que chez l'homme, les adipocytes blancs sont capables de se convertir en adipocytes " brite ". Cette conversion s'accompagne de changements métaboliques qui favorisent l'utilisation intracellulaire des acides gras, ce qui pourrait diminuer leur niveau plasmatique limitant ainsi leur stockage ectopique dans les tissus insulinosensibles.Two types of adipose tissue are present in mammals, white and brown adipose tissue. White adipose tissue (WAT) is specialized in storage and release of lipids, and brown adipose tissue (BAT) is specialized in energy dissipation as heat through the mitochondrial uncoupling protein 1 (UCP1).In humans,thermogenically-competent brown adipose tissue is negatively associated with body mass index and diabetes. BAT is composed of two distinct types of thermogenic cells, classical brown adipocytes located in specific depots and brite adipocytes (brown-in white). In mice, these cells appear in WAT upon cold exposure and are protective against obesity-induced insulin resistance. Therefore, fighting obesity through "browning" of white adipose tissue emerges as a promising strategy. However, the ability of human white adipocytes to acquire a brown/brite phenotype is not yet understood. Here, we aimed at identifying the molecular and metabolic changes associated with the white-to-brown conversion of human mesenchymal adipose-derived stem (hMADS) cells following treatment by agonists of the nuclear receptor PPARgamma or PPARalpha. First, we demonstrated in vitro that PPARgamma or PPARalpha agonists similarly induce white-to-brown conversion of hMADS cells into brite adipocytes that possess high mitochondrial content and express UCP1. Second, we showed that browning is associated with profound metabolic changes. The lipolytic machinery and fatty acid re-esterification was stimulated by the two treatments, resulting in a futile cycle. These adaptations combined with an increase of fatty acid betaoxidation provide substrates to sustain the high level of mitochondrial thermogenesis. In contrast, glucose uptake and oxidation are decreased through inactivation of the pyruvate dehydrogenase by PDK4. Consequently, glucose-carbons are redirected towards glyceroneogenesis to provide the glycerol-3-phosphate backbone necessary for triglyceride esterification. Thus, brite adipocyte metabolism is modified to promote fatty acid utilization as the main energy source. In order to confirm the involvement of PPARalpha in inducing browning in vivo, we treated mice with inactivation of the PPARalpha gene with a beta3-adrenergic agonist. In subcutaneous WAT, expression of BAT- and brite-specific markers was lower in PPARalpha knock out than in wild type mice, confirming that PPARa is required for WAT browning. Altogether, this study shows that PPARgamma and PPARalpha activation in human white adipocytes promotes browning associated with an increase in fatty acid utilization without enhancement of glucose metabolism. These metabolic changes favor intra-adipose fatty acid utilization and thus could diminish plasma fatty flux for ectopic storage into insulin-sensitive tissues

Exosomes and miRNA-Loaded biomimetic nanovehicles, a focus on their potentials preventing type-2 diabetes linked to metabolic syndrome

Exosomes are small membrane vesicles of 30–150 nm, members of the extracellular vesicle family and secreted by various cell types. Different studies describe specific microRNA (miRNA) with altered expression in serum and/or plasma of patients suffering from diabetes or metabolic syndrome. Diabetic cardiomyocyte-derived exosomes loaded with miRNAs like miR-320-3p (or 320a) have been shown regulating angiogenesis on endothelial cell cultures. Insufficient myocardial angiogenesis is the major manifestation of diabetes-caused ischemic cardiovascular disease. Studies on transfer of functional microRNAs between mouse dendritic cells via exosomes have shown that some miRNAs (miR-320-3p, 29b-3p, 7a-5p) are distributed in immature and mature exosomes. Among these miRNAs, miR-320-3p is better known in epigenetics for silencing polr3d gene by binding to its promoter in Human Embryonic Kidney-293 cells. Moreover, quantitative and stoichiometric analysis of the microRNA content of exosomes highlights the lack of reliable natural source of such particles loaded with miRNA opening the need for tailoring exosomes or nanoparticles delivering efficiently miRNA intimately linked to immunity, metabolism and epigenetics in target cells. However, loading of extracellular mature miRNA into recipient cells comes with a cost by at least impeding dynamic localization of miRNAs in nucleoli or inefficient miRNA delivery due to rapid recycling by exonucleases. All these works are calling for the design of new biomimetic vehicles and in vivo assessment of miRNA functionality when delivered by natural or biomimetic nanoparticles in order to control metabolic diseases from infancy to adulthood

Oral delivery of mirna with lipidic aminoglycoside derivatives in the breastfed rat.

Context: Specific targeting of endogenous miRNAs which are involved in epigenetics, may help understanding homeostasis with therapeutic benefits. We use new biologically inspired vehicles consisting of lipoaminoglycosides to deliver in vivo mir-320-3p, a known human breast milk exosomal miRNA, or its antagomiR. Materials and Methods: Four lipoaminoglycosides were screened for cytotoxicity and their biophysical properties. 1-h breast-restricted rats received single-oral treatment of either the lipoaminoglycoside Dioleyl-Succinyl Paromomycin (DOSP) complexed with miRNA or antagomiR, or of control medium at the light on (ZeitGeber Time: ZT-0H) or off (ZT-12H). Glycemia, triglycerides, cholesterol, free-fatty acid were assayed at 0, 4, 8, and 12 h post-treatment. In the stomach, small intestine, liver, plasma, adipose tissue, plexus choroid, and cortex, relevant miRNA with precursors and mRNA (polr3d, hspb6, c-myc, stat1, clock, bmal1, per1, npas2, sirt1-6, and cyclinD1) were quantified by q-PCR. Expression of POLR3D and HSPB6 proteins were analyzed in stomach and liver by Western blot. Immunoprecipitations with anti-AGO1 and 2 were performed on nuclear and cytoplasmic fractions of gastric cells along with detection of miRNA-320-3p in nucleoli. Chromatin ImmunoPrecipitation with anti-Trimethyl-histone-3-Lys-4 and Lys-27 detecting the polr3d promoter and miR-320-3p, were performed for all groups. Results: Selected DOSP (diameter: 80–200 nm) did not alter gastric extracellular vesicle secretion a few hours after intake. The miR-320-3p was mainly found in gastric or small intestinal cells, reaching the blood and liver in low amount. We have found significant up-regulation of polr3d mRNA (ANOVA, p < 0.0001) at ZT-20H for the miR-320-3p-supplemented group and a higher expression of POLR3D for antagomiR group (ANOVA, p < 0.05). We had a low accumulation of miR-320-3p at ZT-20H in nucleoli, without stat1 evolution. Delivering a high amount of miRNA or antagomiR disrupts RNA-Induced Silencing Complexes in cytoplasm triggering some transfer of extracellular molecules into nuclei with alteration of immune complexes on the polr3d promoter (with a higher amount found in the K4 histone-3-me3 immune complexes at ZT-20H). Conclusion: Extracellular miRNAs embedded in DOSP have a rapid impact on RNAi and on nuclear chromatin complexes depending on the daily rhythm. An integrative view of the impact of extracellular miRNA on physiology will improve assaying epigenetic manipulations following nutritional stress

Oral Delivery of miRNA With Lipidic Aminoglycoside Derivatives in the Breastfed Rat

International audienceIntegrative Physiology, a section of the journal Frontiers in Physiology Context: Specific targeting of endogenous miRNAs which are involved in epigenetics, may help understanding homeostasis with therapeutic benefits. We use new biologically inspired vehicles consisting of lipoaminoglycosides to deliver in vivo mir-320-3p, a known human breast milk exosomal miRNA, or its antagomiR. Materials and Methods: Four lipoaminoglycosides were screened for cytotoxicity and their biophysical properties. 1-h breast-restricted rats received single-oral treatment of either the lipoaminoglycoside Dioleyl-Succinyl Paromomycin (DOSP) complexed with miRNA or antagomiR, or of control medium at the light on (ZeitGeber Time: ZT-0H) or off (ZT-12H). Glycemia, triglycerides, cholesterol, free-fatty acid were assayed at 0, 4, 8, and 12 h post-treatment. In the stomach, small intestine, liver, plasma, adipose tissue, plexus choroid, and cortex, relevant miRNA with precursors and mRNA (polr3d, hspb6, c-myc, stat1, clock, bmal1, per1, npas2, sirt1-6, and cyclinD1) were quantified by q-PCR. Expression of POLR3D and HSPB6 proteins were analyzed in stomach and liver by Western blot. Immunoprecipitations with anti-AGO1 and 2 were performed on nuclear and cytoplasmic fractions of gastric cells along with detection of miRNA-320-3p in nucleoli. Chromatin ImmunoPrecipitation with anti-Trimethyl-histone-3-Lys-4 and Lys-27 detecting the polr3d promoter and miR-320-3p, were performed for all groups. Results: Selected DOSP (diameter: 80-200 nm) did not alter gastric extracellular vesicle secretion a few hours after intake. The miR-320-3p was mainly found in gastric or small intestinal cells, reaching the blood and liver in low amount. We have found significant up-regulation of polr3d mRNA (ANOVA, p < 0.0001) at ZT-20H for the miR-320-3p-supplemented group and a higher expression of POLR3D for antagomiR group (ANOVA, p < 0.05). We had a low accumulation of miR-320-3p at ZT-20H in nucleoli, without stat1 evolution. Delivering a high amount of miRNA or antagomiR disrupts RNA-Induced Silencing Complexes in cytoplasm triggering some transfer of extracellular molecules into nuclei with alteration of immune complexes on the polr3d promoter (with a higher amount found in the K4 histone-3-me3 immune complexes at ZT-20H). Conclusion: Extracellular miRNAs embedded in DOSP have a rapid impact on RNAi and on nuclear chromatin complexes depending on the daily rhythm. An integrative view of the impact of extracellular miRNA on physiology will improve assaying epigenetic manipulations following nutritional stress

Embedding miRNA in biologically-inspired delivery vehicles : biophysical analysis and bioassay on the physiology of baby cells

National audienc

Embedding miRNA in biologically-inspired delivery vehicles : biophysical analysis and bioassay on the physiology of baby cells

National audienc

Testing the transfer of miRNA by biologically-inspired delivery vehicles on the physiology of baby cells

National audienc

Temperature of storage and cryoconservation procedure: consequences on Extracellular Vesicles in muscle cell culture supernatants.

International audienc