Contribution à l'étude de la régulation de l'expression du gène humain UCP3 dans le muscle squelettique de souris : étude expérimentale

UCP3 est une protéine découplante de la membrane interne mitochondriale exclusivement exprimée ou presque dans les muscles squelettiques chez l’Homme et potentiellement impliquée dans le métabolisme des acides gras. Ce travail concerne la régulation de l’expression du gène humain UCP3 chez la souris. Des animaux portant le gène UCP3 en entier ou en partie ont été créés par transgenèse additive dans le but de délimiter les séquences responsables de la spécificité musculaire d’expression et celles responsables de la réponse aux nutriments. Une séquence intronique de 600 pb confère l’expression musculaire in vivo. Le jeûne et le sevrage sont deux situations entraînant une induction de l’expression du transgène et de la forme murine d’UCP3. Ces manipulations nutritionnelles font intervenir les acides gras, modulateurs connus de l’expression d’UCP3. Cependant, d’autres facteurs de régulation pourraient être impliqués dans la régulation de l’expression du gène UCP3

Régulation de la protéine découplante UCP3 et inhibition génique ou pharmacologique de la lipase hormono-sensible

Ce manuscrit est premièrement axé sur la régulation de l'expression de la protéine découplante 3 (UCP3). UCP3 est une protéine découplante de la membrane interne mitochondriale exclusivement exprimée ou presque dans les muscles squelettiques chez l'Homme et potentiellement impliquée dans le métabolisme des acides gras. Des souris portant le gène UCP3 humain entier ou en partie ont été créés par transgenèse additive dans le but de délimiter les séquences responsables de la spécificité musculaire d'expression. Nous avons identifié une séquence intronique de 600 pb qui confère l'expression musculaire in vivo. Dans une deuxième partie, une étude des conséquences fonctionnelles d'une altération des capacités lipolytiques du tissu adipeux a été réalisée chez la souris. La lipolyse adipocytaire est en partie assurée par la lipase hormono-sensible (LHS). L'expression et l'activité de la LHS sont diminuées dans le tissu adipeux du sujet obèse et/ou insulino-résistant. Nous avons développé des modèles animaux d'inhibition génique et pharmacologique de la LHS chez lesquels nous avons caractérisé le métabolisme énergétique, l'insulino-sensibilité et l'inflammation du tissu adipeux. La diminution des capacités lipolytiques est accompagnée d'une amélioration de la sensibilité à l'insuline et d’une modification du métabolisme des acides gras dans ces deux modèles. L'inflammation du tissu adipeux, reconnue comme facteur modulateur de l'insulino-sensibilité, ne semble pas être impliquée dans ce phénotype.In a first part, this study focuses on the regulation of the expression of the uncoupling protein-3 (UCP3). UCP3 is an inner mitochondrial protein almost exclusively expressed in skeletal muscle in human which could be implicated in fatty acid metabolism. A transgenesis approach has been used to create animal bearing all or part of the UCP3 gene in order to delineate the sequences responsible for the muscle specific expression. A 600 bp sequence of the human UCP3 intron 1 gene has been identified which confers the muscular expression in vivo. The second part of this study is dedicated to the functional consequences of the alteration of lipolytic capacities in mouse adipose tissue. Adipocyte lipolysis is partly achieved by the hormone sensitive-lipase (HSL). HSL expression and activity is altered in adipose tissue of obese and/or insulin resistant subjects. We developed mouse models of genetic and pharmacological inhibition of HSL. Reduction of lipolytic capacity was associated with an improvement of insulin sensitivity and fatty acid metabolism in both animal models. Adipose tissue inflammation, that is a well known modulator of insulin sensitivity, did not seem to be involved in this phenotype

Lyplal1 is dispensable for normal fat deposition in mice.

Genome-wide association studies (GWAS) have detected association between variants in or near the Lysophospholipase-like 1 (LYPLAL1) locus and metabolic traits, including central obesity, fatty liver and waist-to-hip ratio. LYPLAL1 is also known to be upregulated in the adipose tissue of obese patients. However, the physiological role of LYPLAL1 is not understood. To investigate the function of Lyplal1 in vivo we investigated the phenotype of the Lyplal1tm1a(KOMP)Wtsi homozygous mouse. Body composition was unaltered in Lyplal1 knockout mice as assessed by dual-energy X-ray absorptiometry (DEXA) scanning, both on normal chow and on a high-fat diet. Adipose tissue distribution between visceral and subcutaneous fat depots was unaltered, with no change in adipocyte cell size. The response to both insulin and glucose dosing was normal in Lyplal1tm1a(KOMP)Wtsi homozygous mice, with normal fasting blood glucose concentrations. RNAseq analysis of liver, muscle and adipose tissue confirmed that Lyplal1 expression was ablated with minimal additional changes in gene expression. These results suggest that Lyplal1 is dispensable for normal mouse metabolic physiology and that despite having been maintained through evolution Lyplal1 is not an essential gene, suggesting possible functional redundancy. Further studies will be required to clarify its physiological role

Contribution à l'étude de la régulation de l'expression du gène humain UCP3 dans le muscle squelettique de souris (étude expérimentale)

UCP3 est une protéine découplante de la membrane interne mitochondriale exclusivement exprimée ou presque dans les muscles squelettiques chez l'Homme et potentiellement impliquée dans le métabolisme des acides gras. Ce travail concerne la régulation de l'expression du gène humain UCP3 chez la souris. Des animaux portant le gène UCP3 en entier ou en partie ont été créés par transgenèse additive dans le but de délimiter les séquences responsables de la spécificité musculaire d'expression et celles responsables de la réponse aux nutriments. Une séquence intronique de 600 pb confère l'expression musculaire in vivo. Le jeûne et le sevrage sont deux situations entraînant une induction de l'expression du transgène et de la forme murine d'UCP3. Ces manipulations nutritionnelles font intervenir les acides gras, modulateurs connus de l'expression d'UCP3. Cependant, d'autres facteurs de régulation pourraient être impliqués dans la régulation de l'expression du gène UCP3.TOULOUSE-EN Vétérinaire (315552301) / SudocSudocFranceF

Régulation de la protéine découplante UCP3 et inhibition génique ou pharmacologique de la lipase hormono-sensible

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Et si l’origine des progéniteurs fibro-adipeux contribuait à leur hétérogénéité dans le muscle ?

Les progéniteurs fibro-adipogéniques (FAPs), cellules stromales mésenchymateuses (CSMs) résidentes du muscle squelettique, jouent un rôle crucial dans l’homéostasie et la régénération musculaire via leur activité paracrine. Les avancées technologiques récentes dans le domaine du séquençage de l’ARN en cellule unique ont permis la description de l’hétérogénéité de cette population cellulaire. Dans cet article, nous présenterons les différentes sous-populations de FAPs en condition basale, lésionnelle ou de dégénérescence, ainsi que leurs fonctions associées chez la souris et l’homme. Nous discuterons ensuite de l’origine extra-musculaire possible d’une population de FAPs post-lésionnelle. En effet, nos travaux récents démontrent que des CSMs provenant du tissu adipeux et infiltrées dans le muscle pourraient participer à l’hétérogénéité des FAPs

Endogenous Mobilization of Mesenchymal Stromal Cells: A Pathway for Interorgan Communication?

International audienceTo coordinate specialized organs, inter-tissue communication appeared during evolution. Consequently, individual organs communicate their states via a vast interorgan communication network (ICN) made up of peptides, proteins, and metabolites that act between organs to coordinate cellular processes under homeostasis and stress. However, the nature of the interorgan signaling could be even more complex and involve mobilization mechanisms of unconventional cells that are still poorly described. Mesenchymal stem/stromal cells (MSCs) virtually reside in all tissues, though the biggest reservoir discovered so far is adipose tissue where they are named adipose stromal cells (ASCs). MSCs are thought to participate in tissue maintenance and repair since the administration of exogenous MSCs is well known to exert beneficial effects under several pathological conditions. However, the role of endogenous MSCs is barely understood. Though largely debated, the presence of circulating endogenous MSCs has been reported in multiple pathophysiological conditions, but the significance of such cell circulation is not known and therapeutically untapped. In this review, we discuss current knowledge on the circulation of native MSCs, and we highlight recent findings describing MSCs as putative key components of the ICN

Transcription of the human uncoupling protein 3 gene is governed by a complex interplay between the promoter and intronic sequences.

International audienceAIMS/HYPOTHESIS: Uncoupling protein (UCP) 3 is an inner mitochondrial membrane transporter mainly produced in skeletal muscle in humans. UCP3 plays a role in fatty acid metabolism and energy homeostasis and modulates insulin sensitivity. In humans, UCP3 content is higher in fast-twitch glycolytic muscle than in slow-twitch oxidative muscle and is dysregulated in type 2 diabetes. Here, we studied the molecular mechanisms determining human UCP3 levels in skeletal muscle and their regulation by fasting in transgenic mice. METHODS: We produced a series of transgenic lines with constructs bearing different putative regulatory regions of the human UCP3 gene, including promoter and intron sequences. UCP3 mRNA and reporter gene expression and activity were measured in different skeletal muscles and tissues. RESULTS: The profile of expression and the response to fasting and thyroid hormone of human UCP3 mRNA in transgenic mice with 16 kb of the human UCP3 gene were similar to that of the endogenous human gene. Various parts of the UCP3 promoter did not confer expression in transgenic lines. Inclusion of intron 1 resulted in an expression profile in skeletal muscle that was identical to that of human UCP3 mRNA. Further dissection of intron 1 revealed that distinct regions were involved in skeletal muscle expression, distribution among fibre types and response to fasting. CONCLUSIONS/INTERPRETATION: The control of human UCP3 transcription in skeletal muscle is not solely conferred by the promoter, but depends on several cis-acting elements in intron 1, suggesting a complex interplay between the promoter and intronic sequences

Surplus fat rapidly increases fat oxidation and insulin resistance in lipodystrophic mice.

OBJECTIVE: Surplus dietary fat cannot be converted into other macronutrient forms or excreted, so has to be stored or oxidized. Healthy mammals store excess energy in the form of triacylgycerol (TAG) in lipid droplets within adipocytes rather than oxidizing it, and thus ultimately gain weight. The 'overflow hypothesis' posits that the capacity to increase the size and number of adipocytes is finite and that when this limit is exceeded, fat accumulates in ectopic sites and leads to metabolic disease. METHODS: Here we studied the energetic and biochemical consequences of short-term (2-day) excess fat ingestion in a lipodystrophic (A-ZIP/F-1) mouse model in which adipose capacity is severely restricted. RESULTS: In wildtype littermates, this acute exposure to high fat diets resulted in excess energy intake and weight gain without any significant changes in macronutrient oxidation rates, glucose, TAG, or insulin levels. In contrast, hyperphagic lipodystrophic mice failed to gain weight; rather, they significantly increased hepatic steatosis and fat oxidation. This response was associated with a significant increase in hyperglycemia, hyperinsulinemia, glucosuria, hypertriglyceridemia, and worsening insulin tolerance. CONCLUSIONS: These data suggest that when adipose storage reserves are saturated, excess fat intake necessarily increases fat oxidation and induces oxidative substrate competition which exacerbates insulin resistance resolving any residual energy surplus through excretion of glucose

Adipose Stem Cells (ASCs) isolation by on-chip pre-treatment of biological samples

International audienceThis study presents the development of an original 2-steps Lab-on-chip that aims at isolating Adipose Stem Cells (ASCs), cells of considerable interest for regenerative medicine, from complex biological samples. Based on hydrodynamic filtration, the device pre-isolates ASCs by removing cells with a diameter below 10µm such as red blood cells or lymphocytes