Maria Pini

Sothea Touch

Hélène Poirier

Elise Dalmas

Isabelle Niot

Christine Rouault

Céline Druart

Nathalie Delzenne

Karine Clément

Sébastien André

Michèle Guerre- Millo

Adipose

Hedi Soussi

Sophie Reggio

Rohia Alili

Cecilia Prado

Sonia Mutel

Isabelle Dugail

de l'acide linoléique engage des macrophages anti-inflammatoires

issues in research, as in life, with the right distance. At the end, I thank all my friends near and far for their presence, support and good humor in my daily life.

I would like to thank especially Dr. Michèle Guerre-Millo, the director of the exposed projects, for having shared with me her critical approach toward qualitative scientific research and her endless love for science. I thank her very much for the confidence she granted me, her support and her mentorship over these years of scientific work together. Thanks to her I have learned also some good French expressions, that I will remember with a smile, now that I know their meaning, like "tout c'est passé comme sur des roulettes" after one of our experiments. I deeply thank also my EPHE thesis director Dr. Sophie Thenet for her kindness, availability and assistance during all the critical steps that brought me to the end of this unique itinerary.

I am sincerely grateful to Pr. Karine Clément, Director of the Institute of Cardiometabolism and Nutrition (ICAN), for allowing me to do my EPHE PhD in her team UMR S 1166, assisting the realization of this work with the necessary technical and financial supports and giving me the constant opportunity to learn from her team research interests. I extend my sincere thanks to those who contribute to the achievement of the different projects, contributing to this PhD work, making it primarily a team work. In particular I would like to thank Dr. Sébastien Andre and his PhD student Sothea Touch for their expertise in the field of immunology. A big thank to Jean-Charles Lafarge, the former PhD student of Dr.Michèle Guerre-Millo, with which I have worked on my first project in the team of K. Clément, the cathepsin S study. I thank him for his encouragement, his great sense of humor and even more for his English level, all of which helped me to pass unscathed through the transition from USA to France. A special thank to Dr. Christine Rouault for her availability, her listening and technical assistance. I thank also all the Nutromique members, past and present, in particular Dr. Daniele Lacasa for her discreet guidance and the discussions about art.

Many thanks to our collaborators from the Institute National Supérieur AgroSup in Dijon, Dr. Helene Poirier and Dr. Isabelle Niot, for their help in the study with conjugated linoleic acid. For the same study, I thank also Pr. Nathalie Delzenne and her former PhD student Celine Druart from Louvain University for their warm reception in Bruxelles and their technical competence in analyzing fatty acids.

I also want to express gratitude to Dr. Hana Koutnikova-Rousselin, Dr. Peggy Garault, Dr. Timothy Swartz, Dr. Aurélie Coutillard and Dr. Ludovic Le Chat for the interesting discussions during the preparation and finalization of Study II.

I finally want to warmly thank all the Equipe 6 members (past and present), in particular the ones working in the Centre de Recherche des Cordeliers (CRC), for sharing with me good time, but also the not so good one. A special thought to Sophie Reggio, who showed me how we can face small and big

It's Not How Fat You Are, It's What You

Do with It that Counts.

by Samuel Virtue and Antonio Vidal-Puig

RÉSUMÉ FRANÇAIS

A. Contexte du projet

Mon projet de thèse, intitulé « Impact de l'inflammation sur les altérations métaboliques associées à l'obésité: dialogues entre le tissu adipeux et l'intestin » a été effectué dans le laboratoire du Pr Karine Clément ( INSERM UMRS 1166 Nutriomics Eq 6 ) sous la direction du Dr Michèle Guerre-Millo (INSERM) et du Dr Sophie Thenet (EPHE).

Ce projet s'inscrit dans le domaine des maladies métaboliques avec changement de masse grasse telles que l'obésité et les syndromes lipoathrophiques. Bien qu'opposées en terme de modification de la masse adipeuse, ces deux pathologies ont des co-morbidités communes, dont l'insulino-résistance et le diabète de type II. Il est maintenant reconnu que le tissu adipeux est au coeur des mécanismes impliqués dans ces co-morbidités, étant à la fois cible et acteur des dérégulations métaboliques. Par ailleurs, les altérations du microbiote et de la barrière intestinale, longtemps sousestimées, sont actuellement sur le devant de la scène pour leur participation au développement des maladies métaboliques.

Les altérations du tissu adipeux dans les maladies métaboliques

Dans les pathologies chroniques s'accompagnant de modification de la masse adipeuse, la physiologie du tissu adipeux est profondément altérée. La principale fonction du tissu adipeux est de stocker l'énergie de l'organisme sous forme de triglycérides dans des cellules hautement spécialisées, les adipocytes. Les substrats précurseurs sont le glucose pour la voie de la lipogenèse de novo et les lipides circulants qui subissent une hydrolyse et une ré-estérification dans les adipocytes. Ces cellules possèdent également l'équipement enzymatique nécessaire pour permettre, en situation de déficit énergétique, la libération d'acides gras via le processus de « lipolyse ». Le stockage des lipides dans les adipocytes et leur mobilisation sont deux voies métaboliques finement régulées par l'état nutritionnel, avec un rôle primordial de l'insuline, qui stimule le stockage et inhibe la lipolyse. Si la capacité de stockage des adipocytes est dépassée, soit à cause d'un excès de substrats lipoformateurs (obésité), soit à cause d'une réduction des capacités d'expansion des cellules (lipoathrophie), les acides gras peuvent s'accumuler dans les organes périphériques, tels que les muscles squelettiques et le foie et constituer des dépôts de graisse « ectopique ». Les cellules de ces derniers tissus ne sont pas capables de stocker de grandes quantités de lipides et cette surcharge lipidique entraine une toxicité cellulaire ou « lipotoxicité » et, à terme, l'altération des fonctions biologiques des tissus. L'une des conséquences majeures est la diminution de l'efficacité de l'insuline, qui conduit à un état d'insulino-résistance précurseur du diabète de type II.

Il est maintenant reconnu que l'inflammation contribue à la pathologie du tissu adipeux et favorise la résistance à l'insuline. Ainsi, de nombreuses études précliniques et cliniques ont établi que des cellules immunitaires sont recrutées dans le tissu adipeux de souris rendues obèses par un régime hyperlipidique et chez les sujets obèses. Localement, l'inflammation est aggravée et entretenue notamment par des macrophages pro-inflammatoires qui s'accumulent dans le tissu. Les interactions entre adipocytes et cellules immunitaires conduisent à des changements dans la libération d'adipokines, de cytokines et d'acides gras non estérifiés contribuant à la mise en place d'un environnement inflammatoire. Il faut noter que des altération identiques ont été décrites dans le tissu adipeux atrophique chez des souris génétiquement modifiées et chez les patients atteints de lipodystrophie associée au VIH.

En lien avec l'accumulation de cellules immunitaires, le remodelage de la matrice extra cellulaire et l'accumulation de fibrose sont considérées comme une nouvelle signature de la pathologie du tissu adipeux associée à l'obésité. Il est remarquable qu'une accumulation de fibrose est également observée dans le tissu adipeux des sujets positifs pour le VIH. L'équipe du Prof Karine Clément a été l'une des premières à mettre en évidence l'accumulation pathologique de matrice extracellulaire dans le tissu adipeux de sujets obèses. Par la suite, des études in vitro ont révélé le rôle crucial des interactions entre macrophages et pré-adipocytes dans le développement et le maintien de la fibrose dans le tissu adipeux. Cependant, l'importance physiopathologique de ces remaniements matriciels, plus spécifiquement en lien avec le statut glycémique et la résistance à l'insuline, n'est pas totalement élucidée.

Les altérations de l'intestin et du microbiote intestinal dans les malacies métaboliques

Les choix alimentaires, en particulier les régimes riches en matières grasses, conduisent à des changements du microbiote et à l'altération de la fonction de la barrière intestinale. Chez les rongeurs, l'une des conséquences d'un régime riche en graisse est l'augmentation de la perméabilité paracellulaire dans l'iléon, facilitant le passage de produits bactériens, en particulier les lipopolysaccharides (LPS), de la lumière intestinale dans la circulation. L'élévation des quantités de LPS circulantes, qualifiée d'«endotoxémie », contribue à l'inflammation et potentiellement aussi à l'insulino-résistance systémique. Chez l'homme, les liens entre régime alimentaire, microbiote intestinal et dérégulation métabolique font l'objet d'une recherche intense, essentiellement dans le cadre de l'obésité. Fait intéressant, une partie des altérations pathologiques liées au phénotype d'obésité peut être améliorée par des interventions nutritionnelles ou la chirurgie bariatrique, en relation avec une modification de la composition du microbiote vers un profil plus sain. D'autre part, des interventions nutritionnelles, telles que l'administration de probiotiques ou de prébiotiques, sont explorées en tant que nouvelles stratégies pour rétablir l'équilibre de la microflore intestinale et améliorer le statut métabolique de l'hôte. La modification de la microflore intestinale via l'administration de probiotiques pourrait alléger les altérations métaboliques à la lumière de plusieurs mécanismes, y compris une éventuelle augmentation de la capacité du microbiome à gérer le défi nutritionnel représenté par une diète riche en gras.

B. Objectifs et principaux résultats

Notre équipe s'intéresse aux liens entre dysfonctionnement du tissu adipeux, altérations métaboliques et microbiote intestinal, avec comme objectif global d'élaborer des stratégies thérapeutiques pour prévenir et/ou traiter les désordres métaboliques associés à l'obésité.

Mes contributions principales, basées sur l'utilisation de différents modèles de souris, sont la démonstration a) que le tissu adipeux a la capacité de s'adapter à un stress nutritionnel aigu, reposant sur le recrutement de macrophages anti-inflammatoires (Etude I et Publication 1) et b) que l'administration de probiotique n'est pas suffisante pour atténuer l'obésité et la résistance à l'insuline induite par un régime riche en graisses chez la souris (Etude II).

J'ai également contribué à trois études dans lesquelles des modèles animaux (souris génétiquement modifiées rendues obèses par un régime riche en graisse) et cellulaires (culture de d'adipocytes et de pré-adipocytes) ont été utilisés pour déterminer l'implication de nouvelles cibles moléculaires dans la régulation de la masse grasse et l'homéostasie métabolique (Publications 2, 3 et 4 présentées en annexe). Ces cibles potentielles ont été récemment identifiées dans l'équipe du Prof Karine Clément par des études d'expression génique comparant le tissu adipeux de sujets non-obèses à celui de sujet massivement obèses. Ce sont la protéase Cathepsine S, le facteur de transcription proinflammatoire IRF5 et DAPK2, une kinase régulatrice de l'autophagie.

Le schéma général ci-dessous place les différentes études inclues dans ce rapport dans le contexte de la contribution de différents organes (tissu adipeux, foie et intestin) à l'insulino-résistance systémique associée à l'obésité ou aux syndromes lipoatrophiques:

Résistance à l'insuline

Introduction

Il est largement admis que l'accumulation de macrophages pro-inflammatoires est une composante clé de la réponse du tissu adipeux à une surcharge en nutriments. Grâce à la libération de chimiokines et cytokines pro-inflammatoires les macrophages perpétuent et propagent l'inflammation dans le tissu adipeux, conduisant finalement à un état pathologique chronique. Bien qu'opposées en terme de modification de la masse grasse, les lipodystrophies sont associées à des altérations du tissu adipeux identiques à celles décrites dans l'obésité, dont l'inflammation et l'infiltration de macrophages. La présence de dépôts de matrice extracellulaire et de fibrose est une signature supplémentaire de la pathologie du tissu adipeux commune aux deux conditions. Ces altérations locales sont accompagnées d'inflammation systémique et de résistance à l'insuline, qui représentent les conséquences délétères les plus courantes de la dysfonction du tissu adipeux associée à l'obésité et aux lipodystrophies.

Le temps nécessaire à l'accumulation desmacrophage dans le tissu adipeux et au développement de la résistance à l'insuline est généralement long, allant de quelques mois chez les souris nourries avec un régime riche en graisses à des années dans l'obésité humaine. Dans la situation inverse de restriction calorique, l'inflammation du tissu adipeux et le statut glycémique sont améliorés, également après de longues périodes de réduction ou de stabilisation du poids corporel. Ces observations soulignent la plasticité du tissu adipeux en réponse à des modifications à long terme du bilan énergétique, en lien avec les modifications de l'état métabolique.

Les acides linoléiques conjugués (CLA) sont un groupe de dérivés de l'acide linoléique comportant plusieurs isomères positionnels et géométriques. Parmi ceux-ci, le trans10, cis12 CLA (t10, c12-CLA) exerce un effet puissant et rapide de réduction du poids corporel chez la souris, ce qui a conduit à proposer son utilisation en tant qu'agent « anti-obésité ». Mais, contrairement à la restriction calorique, l'administration de t10, c12-CLA n'améliore pas le statut métabolique, mais provoque au contraire une hyperinsulinémie marquée et une résistance systémique à l'insuline. Dans ce modèle, ce phénotype d'accompagne d'un recrutement de macrophages étonnamment rapide dans le tissu adipeux. La présence de cellules positives pour le marqueur macrophagique F4/80 est détectable dans le tissu adipeux après seulement 3 jours de traitement des souris par le t10, c12-CLA. Ces observations suggèrent que, en plus d'une plasticité à long-terme, le tissu adipeux a la capacité de produire une réponse immunitaire rapide à un stress nutritionnel.

Dans ce travail, nous avons utilisé le t10, c12-CLA comme un outil pour explorer, chez la souris, les composants et la dynamique des altérations produites par ce lipide dans le tissu adipeux d'une part, et, d'autre part, au niveau systémique et dans l'intestin. Une étude cinétique a été réalisée sur deux périodes consécutives de 7 jours d'administration de t10,c12-CLA par gavage journalier suivie de 7 jours d'interruption du traitement.

Résultats

Le t10, c12-CLA provoque des altérations métaboliques réversibles dans le tissu adipeux et au niveau systémique.

Comme attendu, le poids corporel a diminué régulièrement pendant l'administration t10, c12-CLA sans changement majeur dans la prise alimentaire. A l'arrêt du traitement, le poids corporel est revenu rapidement au niveau initial. Reflétant une lipoatrophie généralisée, le pourcentage de masse grasse est réduit après 3 jours et reste diminué après 7 jours de t10, c12-CLA. La réduction de la masse grasse a été plus prononcée dans le tissu adipeux sous-cutané (sWAT) que dans le tissu adipeux périutéral (pWAT). Après 7 jours sans t10, c12-CLA, la lipoatrophie et la masse du pWAT ont été normalisées, alors que sWAT est resté diminué.

L'insuline plasmatique a augmenté progressivement, sans changement significatif de la glycémie, pour atteindre des valeurs 8 fois plus élevées que chez les souris témoins après 7 jours de t10, c12, CLA. Cette situation reflète le développement rapide d'un état de résistance à l'insuline en réponse au t10, c12-CLA, en accord avec des études précédentes. A l'interruption du traitement, l'hyperinsulinémie a diminué et retrouvé des valeurs semblables à celles du groupe témoin, établissant un lien direct entre résistance à l'insuline et administration du t10, c12-CLA.

Nous montrons que le t10, c12-CLA administré par gavage journalier s'accumule dans le tissu adipeux, où les concentrations ont atteint des valeurs 8 à 20 fois plus élevées que dans l'intestin ou le foie. Cette différence souligne que le tissu adipeux est le tissu métabolique principalement ciblé par cet isomère. Après 7 jours de t10, c12-CLA, l'expression de plusieurs « adipokines » produites dans tissu adipeux est considérablement réduite et ce de manière strictement réversible. Parmi elles, l'adiponectine suit le même schéma de variation au niveau génique et dans la circulation. En revanche, les niveaux d'ARNm et plasmatiques de la leptine sont très peu modifiés. Ces données suggèrent que le t10, c12-CLA modifie le sécrétome du tissu adipeux en ciblant les niveaux de transcription des facteurs sécrétés, tout en laissant intactes les voies de sécrétion.

Plusieurs gènes codant pour des enzymes clé des voies métaboliques de la lipogenèse (FAS, ACC) et de la lipolyse (HSL, ATGL, PLN1) dans le tissu adipeux étaient nettement régulés à la baisse après 1 semaine de t10, c12-CLA. Malgré ces dérégulations métaboliques majeures, la taille des adipocytes, qui reflètent le niveau de stockage des lipides dans la cellule, n'était que légèrement diminuée, ce qui indique que la majorité des adipocytes ont survécu au traitement par le t10, c12-CLA. Conformément, le niveau d'expression des gènes métaboliques est quasi normalisé à l'arrêt du traitement. Il est notable que les gènes impliqués dans le processus de « lipophagie », tels que le facteur de transcription TFEB, sa cible, la lipase acide lysosomale (Lipa), et la protéine mitochondriale UCP2, sont régulés à la hausse et de manière réversible en réponse au t10, c12-CLA. Ces observations suggèrent que les adipocytes utilisent une voie métabolique alternative permettant l'utilisation d'une partie des lipides stockés comme substrat énergétique en réponse au stress métabolique produit par le t10, c12-CLA.

Le t10, c12-CLA provoque une accumulation réversible de macrophages dans le tissu adipeux.

Des analyses immunohistochimiques montrent que les cellules positives pour le marqueur macrophagique CD68 s'accumulent dans le tissu adipeux après 3 jours, continuent d'augmenter après 7 jours de t10, c12-CLA et chutent brusquement à l'arrêt des gavages. L'accumulation réversible de macrophages a été confirmée par l'expression du gène CD68 dans le tissu adipeux et par le nombre des cellules F4/80 + évalué en cytométrie, qui suivent des profils similaires. Dans ce modèle, l'abondance des macrophages est corrélée négativement avec la masse adipeuse, en opposition à la relation inverse trouvée dans l'obésité. Cependant, comme dans l'obésité, l'insulinémie est positivement associée à l'accumulation macrophagique.

Le t10, c12-CLA provoque une accumulation réversible de matrice extracellulaire dans le tissu adipeux.

Le marquage des coupes de tissu adipeux au rouge de picrosirius a révélé que t10, c12-CLA provoque des dépôts de collagène, avec un effet plus prononcé dans le sWAT que dans pWAT. A l'interruption des gavages, l'abondance du collagène a diminué dans les deux dépôts, mais n'a pas été entièrement normalisée dans le sWAT. Une analyse d'expression génique a permis d'identifier la fibronectine et le collagène 1 comme les composants de la matrice extracellulaire sélectivement augmentés dans les deux types de tissu adipeux, avec une contribution additionnelle du collagène 3 dans le sWAT. Malgré l'augmentation des dépôts de collagène, les enzymes de pontage telles que Lox, (lysyl oxidase) ou Loxl2 (lysyl oxidase-like 2) sont peu modifiées en réponse au t10, c12-CLA. De même, l'expression de plusieurs facteurs profibrotiques, tels que le TGF-β1, l'inhibine βA et le CTGF, n'est que marginalement affectée, rendant peu probable l'induction d'un phénotype fibrotique dans les fibroblastes. Ces résultats montrent que, en réponse au t10, c12-CLA, il existe un remodelage de la matrice extracellulaire au sein du tissu adipeux, mais sans constitution d'un réseau rigide de fibrose. L'étonnante rapidité de constitution et la réversibilité de ces altérations matricielles, qui suivent temporellement l'accumulation des macrophages, révèle une plasticité structurale remarquable du tissu adipeux.

Les macrophages du tissu adipeux induit par le t10, c12-CLA ont un profil anti-inflammatoire « M2 ».

Nous avons déterminé le phénotype des macrophages induits par le t10, c12-CLA par la quantification des marqueurs de surface CD11c et CD206 dans les cellules F4/80 + . Les cellules CD206 + CD11c -sont la population cellulaire dominante chez les souris témoins. En réponse au t10, c12-CLA, cette population a augmenté de façon importante, ainsi qu'un sous groupe moins abondant de cellules CD206 + CD11c + , qui était pratiquement indétectable dans le groupe témoin. La régulation à la hausse d'une série de marqueurs géniques associés au phénotype anti-inflammatoire « M2 » confirme la prédominance des macrophages M2 anti-inflammatoires dans le tissu adipeux des souris traitées au t10, c12-CLA. Tous les marqueurs M2 ont fortement diminué au retrait du t10, c12-CLA. Les niveaux d'ARNm des cytokines anti-inflammatoires IL-10 et IL-1ra ont considérablement augmenté en réponse à t10, c12-CLA et démontré une réversibilité quasi-totale à l'interruption des gavages. Ces données révèlent que le tissu adipeux réagit au t10, c12-CLA en engageant une réponse anti-inflammatoire marquée, spécifique et réversible.

Le t10, c12-CLA modifie le profil immunitaire inné et adaptatif dans le tissu adipeux.

A côté de l'abondante population de macrophages, d'autres cellules immunitaires innées, dont les cellules NK (CD3 -NK1.1 + ) et les cellules dendritiques (F4/80 -CD11c + ) contribuent à l' infiltrat induit par le t10, c12-CLA. En outre, les cellules NKT (CD3 + NK1.1 + ), les cellules T et les cellules B sont également plus fréquentes dans le tissu adipeux des souris recevant le t10, c12-CLA, tout en restant moins nombreuses que les cellules immunitaires innées. Une petite population de cellules T γδ a augmenté, alors que l'abondance de cellules Treg Foxp3 + n'a pas été significativement modifiée lors de l'administration t10, c12-CLA. Ces résultats montrent que l'administration de t10, c12-CLA provoque des modifications substantielles du profil immunitaire du tissu adipeux.

Le t10, c12-CLA modifie le profil des chimiokines dans le tissu adipeux.

L'ampleur de l'infiltrat de macrophages induit par le t10, c12-CLA soulève la question de leur origine, à partir soit d'un recrutement de précurseurs monocytaires, soit d'une prolifération in situ ou des deux. Les analyses d'expression génique montrent que le t10, c12-CLA induit une régulation spécifique de plusieurs chimiokines, avec une augmentation marquée de CCL7, CCL2/MCP1, de l'ostéopontine (OPN) et CXCL10. Ces données suggèrent que t10, c12-CLA génère un environnement favorisant le recrutement de cellules sanguines dans le tissu adipeux. Cependant, la prolifération in situ des macrophages ne peut être exclue. En effet, lorsque nous avons étudié le marqueur de prolifération cellulaire Ki-67, son niveau d'ARNm a augmenté en proportion avec celui du marqueur de macrophage CD68. Fait intéressant, l'expression de Ki-67 est restée élevée après arrêt du traitement quand l'expression du gène CD68 est normalisée. Ceci suggère que des cellules non-immunitaires pourraient proliférer dans tissu adipeux pendant le traitement au t10, c12-CLA et également pendant la période de récupération. La prolifération de précurseurs adipocytaires pourrait contribuer à la récupération de la masse adipeuse post-t10, c12-CLA.

Résultats complémentaires

Le t10, c12-CLA modifie le profil d'expression génique dans le jéjunum.

Les effets du t10, c12-CLA au niveau du tractus intestinal ont été évalués par une étude d'expression génique dans le jéjunum et le colon des souris après 7 jours de traitement.

Contrairement aux effets produits dans le tissu adipeux, l'administration de t10, c12-CLA pendant 7 jours n'a pas entrainé d'effet majeur sur l'expression des gènes CD68 (marqueur de macrophage) et CD3 (marqueur de lymphocyte T). Bien que nous ne puissions pas exclure un effet sur d'autres types de cellules immunitaires, ces observations ne militent pas en faveur d'une modification majeure du profil immunitaire.

En revanche, le t10, c12-CLA a modifié l'expression de deux récepteurs « Toll-like », TLR4 et TLR5, qui tend à être augmentée dans le jéjunum. TLR induisent la production de cytokines proinflammatoires en réponse à des éléments d'origine bactérienne, comme le LPS. L'activation des ces TLR en réponse au t10, c12-CLA est suggérée par l'augmentation marquée et significative (x 5) de l'expression du TNFα dans le jéjunum. Les ligands reconnus par les TLR sont variés. Bien que le LPS soit considéré comme le principal ligand de TLR4, l'activation par le t10, c12-CLA n'est pas exclue.

Une série de gènes impliqués dans le métabolisme des lipides au niveau intestinal a également été évaluée. Ce sont la protéine de liaison des acide gras FABP2, connue pour être exprimée exclusivement dans les entérocytes de l'intestin grêle, l'histone désacétylase 1 (HDAC1), importante pour l'homéostasie des cellules épithéliales intestinales, le récepteur SR-BI, impliqué dans l'absorption du cholestérol, ainsi que d'autres gènes du métabolisme des lipoprotéines, telles que l'apolipoprotéine B (apoB), l'apolipoprotéine A-IV (Apoa4), acétylglucosaminyltransférase mannoside 2 (MGAT2), la protéine de transfert de triglycéride microsomique (MTTP). Aucune modification majeure n'a été observée dans les niveaux d'expression de ces gènes après 7 jours de gavage par le t10, c12-CLA ni dans le jéjunum ni dans le côlon des souris traitées par le t10, c12-CLA.

Nous avons pu détecter une diminution d'environ 30 % de l'expression de gènes codant pour trois protéines des jonctions serrées (claudine, occludine et « zonula occludens-1 » ZO-1) dans le jéjunum des souris traitées par le t10, c12-CLA. Des études ultérieures devront confirmer si ces modifications d'expression génique se traduisent par une diminution des protéines et une altération de la perméabilité intestinale. Si tel était le cas, une partie des effets du t10,c12-CLA sur le tissu adipeux pourrait être relayé indirectement par des produits issus du microbiote intestinal tel que le LPS. A l'encontre de cette hypothèse, nous avons mesuré les niveaux circulants de LPS chez des souris ayant reçu du t10, c12-CLA pendant 1 mois, sans détecter de différence significative avec les souris témoins.

Le t10, c12-CLA altère le microbiote intestinal.

Les effets du t10, c12-CLA sur le microbiote intestinal a été analysé, afin de rechercher la contribution d'éventuels changements de composition de la microflore dans les perturbations métaboliques induites par ce lipide chez la souris.

Dans cette étude, les espèces microbiennes ont été organisées en utilisant le concept de « d'entérotypes », regroupant les souris suivant la prédominance des bactéries de différentes espèces dans les fèces. Trois entérotypes principaux (M1, M2 et M3) ont été trouvés dans le groupe de souris étudié : M1 = prédominance de Barnesiella et Alistipes M2 = prédominance de Barnesiella, Alistipes, Clostridium et Lachnospiracées M3 = prédominance de Bacteroides et Barnesiella Environ un tiers des souris étaient réparties dans chaque groupe au début des gavages. L'entérotype M3 enrichi en Bacteriodes est le moins riche en variété de bactéries. Au cours des gavages, la proportion de souris avec un entérotype M3 a progressivement diminué dans le groupe traité avec le t10, c12-CLA, mais est resté stable dans le groupe contrôle. Après 7 jours d'administration du t10, c12-CLA, plus aucune souris ne présentait un entérotype M3.

Conclusion

Nos résultats montrent que t10,c12-CLA induit une dérégulation des gènes métaboliques et l'accumulation de matrice extracellulaire (ECM) dans différents dépôts de tissu adipeux sous-cutané et 13 profond. De plus, le profil immunologique du tissu adipeux est profondément perturbé en réponse au t10,c12-CLA, avec comme caractéristique majeure l'accumulation de macrophages M2 antiinflammatoires. Ces altérations métaboliques, immunitaires et structurelles sont réversées après 7 jours de récupération, mais pas toujours normalisées en particulier dans le tissu adipeux sous-cutané qui reste réduit en masse. Cette réversibilité indique que la réponse du tissu adipeux est plus adaptative que pathologique. Elle reflète la capacité de ce tissu à s'adapter rapidement et efficacement à un environnement métabolique altéré, avec, cependant, une meilleure réactivité des tissus profonds par rapport aux tissus sous-cutanés. A ce stade, il est difficile d'attribuer un rôle spécifique aux différents types cellulaires dont l'abondance est modifiée par le traitement. Certaines cellules immunitaires sont considérées comme « délétères » (macrophages inflammatoires, cellules NK, B et Tγδ) et d'autres comme « protectrices » (macrophages M2, cellules NKT et Treg) pour les fonctions du tissu adipeux. La réversibilité des altérations induites par le t10, c12-CLA milite en faveur d'un effet protecteur des modifications complexes du profil immunitaire du tissu adipeux observées dans cette étude. Malgré cela, les anomalies métaboliques, cellulaires et structurales provoquées dans le tissu adipeux par le t10, c12-CLA sont temporellement associées avec le développement d'une hyperinsulinémie marquée et réversible. Ces observations renforcent l'idée que le dysfonctionnement du tissu adipeux est un facteur majeur de dérégulation de l'homéostasie glucidique.

Au niveau intestinal, l'administration par gavage du t10, c12-CLA ne semble pas modifier l'abondance des cellules de l'immunité, à l'inverse de la réponse immunitaire de grand ampleur observée dans le tissu adipeux. Ces résultats basés uniquement sur une étude d'expression génique devront être confirmés par des analyses en immunohistochimie et en cytométrie. Cependant, nos données suggèrent que le t10, c12-CLA initie une réponse inflammatoire dans le jéjunum via l'activation de TLR4 et la production de TNFα. Il est donc possible que des altérations du statut immunitaire interviennent dans l'intestin après une exposition de plus longue durée. De plus, il n'est pas exclu que le t10, c12-CLA modifie la perméabilité paracellulaire au niveau de l'épithélium intestinal. L'analyse du microbiote intestinal, bien que préliminaire, suggère que le t10, c12-CLA altère la composition en bactéries, avec une diminution de l'espèce bactéroides. Des travaux de notre équipe ont montré que cette espèce est peu abondante chez le sujet obèse et que son abondance augmente après chirurgie bariatrique et perte de poids. L'importance de ces altérations dans les complications métaboliques de l'obésité n'est pas clairement établie. Dans ce contexte, le modèle de souris traitées par le t10, c12-CLA pourrait permettre d'apporter de nouveaux éléments sur le rôle de cette bactérie commensale dans le métabolisme de l'hôte.

2.

Etude II: Effet de deux souches de probiotiques sur la prise de poids et le statut glycémique chez la souris rendue obèse par un régime hyperlipidique.

L'étude II a été conçue sur la base de l'hypothèse que la modification de la microflore intestinale via l'administration quotidienne de probiotiques pourrait atténuer les altérations métaboliques associées à un régime obésogène chez la souris. L'effet de deux souches de probiotiques (identifiées par X et Y pour des raisons de confidentialité) a été évalué sur le poids corporel, l'adiposité, la glycémie, la sensibilité à l'insuline, la tolérance au glucose et sur des variables métaboliques. Un traitement de 12 semaines a été réalisé au cours duquel 3 groupes de souris (contrôle (C), X et Y) ont été nourries avec un régime contenant 60 % de calories lipidiques. Les groupes X et Y ont reçu des probiotiques dans l'eau de boisson à une concentration permettant l'absorption de 10 9 bactéries vivantes par jour par souris.

Le gain de poids corporel ainsi que le pourcentage de masse grasse et l'index d'adiposité (poids des tissus adipeux/poids corporel) étaient identiques entre les groupes C et X, mais systématiquement plus élevé dans le group Y. Confirmant ce phénotype, la leptinémie était augmentée dans le groupe Y. Ce groupe a démontré une augmentation de l'insulinémie et une réponse glycémique altérée dans un test de sensibilité à l'insuline, bien que les différences n'aient pas atteint la significativité statistique. Enfin, les triglycérides hépatiques étaient augmentés dans le groupe Y et inchangés dans le groupe X par rapport au contrôle.

Ces résultats montrent qu'une des souches de probiotiques testée dans cette étude a généré un phénotype défavorable, avec gain de poids et augmentation de la masse grasse, en association avec une élévation de la leptine circulante et des triglycérides du foie et une tendance à la résistance à l'insuline. L'étude des mécanismes cellulaires et moléculaires à l'origine du phénotype observé est actuellement en cours. A terme, si des altérations dans le métabolisme des lipides dans le tractus gastro-intestinal et/ou le tissu adipeux blanc sont détectées, ces données seront informatives pour aider à détecter des effets néfastes potentiels de certaines souches de probiotique.

Conclusion générale

En résumé, ces deux séries de travaux ont permis d'étudier les relations entre l'inflammation et le métabolisme dans le contexte d'un dialogue indirect entre le tissu adipeux et l'intestin. Compte-tenu du fait que l'ampleur des changements produits par les facteurs alimentaires est faible et cumulative, l'administration de t10, c12-CLA peut être considérée comme un stress aigu. Le régime riche en graisse, au contraire représente un stress nutritionnel chronique. En concentrant notre attention sur le tissu adipeux, nous montrons qu'en réponse à un stress nutritionnel aigu, le tissu adipeux regagne rapidement son homéostasie, grâce à la mise en oeuvre d'une réponse immunitaire appropriée. Lorsque la contrainte est plus chronique, comme dans l'obésité, la plupart des interventions, y compris l'administration de probiotiques, ne parvient pas à inverser l'état dysfonctionnel du tissu adipeux et les séquelles métaboliques qui en découlent. Le grand défi dans les années à venir reste donc de découvrir des stratégies thérapeutiques à long terme pour les maladies métaboliques. La cathepsine S appartient à une famille de protéases qui ont été impliquées dans de nombreuses pathologies, dont l'athérosclérose. Nous avons identifié la cathepsine S comme une protéine fortement surexprimée dans le tissu adipeux des personnes obèses. Après perte de poids et amélioration du statut glycémique suite à une chirurgie de gastrique, l'expression de la cathepsine S est substantiellement diminuée dans le tissu adipeux. Ces observations nous ont conduits à émettre l'hypothèse d'un rôle de cette protéase dans la pathogenèse du diabète de type 2. Cette hypothèse a été testée dans un modèle de souris déficientes pour la cathepsine S Des souris déficientes et de type sauvage, traitées ou non avec des inhibiteurs de cathepsine S actifs par voie orale, ont été nourries en régime standard ou enrichi en matière grasse. Le phénotype métabolique des ces différents groupes de souris a été déterminé, avec un focus particulier sur l'homéostasie glucidique.

C. Contribution à des publications présentées en annexe

Nos résultats montrent que la délétion du gène ou l'inhibition pharmacologique de la cathepsine S produit une réduction robuste de la glycémie, qui est conservée dans l'obésité induite par le régime gras et au cours du vieillissement. Une série de tests in vivo de tolérance au glucose, de sensibilité à l'insuline et de réponse glycémique à des substrats gluconéogéniques a permis d'établir que la suppression de l'activité cathepsine S réduit la production hépatique de glucose, sans modifier la sensibilité à l'insuline. Ce phénotype a été confirmé par la régulation négative de l'expression de gènes gluconéogéniques dans le foie. De plus, une diminution de la respiration hépatocellulaire a pu être mise en évidence chez les souris déficientes en cathepsine S. D'un point de vue mécanistique, nous avons constaté que REDD1, qui est un facteur potentiellement impliqué dans la réduction de l'activité de la chaîne respiratoire, était surexprimé dans le foie de souris chez qui la cathepsine S était absente ou inhibée pharmacologiquement.

Cette étude a révélé un effet métabolique inattendu de la cathepsine S, qui pourrait contribuer à favoriser un état pré-diabétique en augmentant l'activité de la gluconéogenèse dans le foie. Des inhibiteurs de cathepsine S sont actuellement proposés pour le traitement de maladies autoimmunes. Nos résultats militent en faveur d'une extension des indications thérapeutiques aux maladies métaboliques. Ces molécules pourraient aider à réduire la production de glucose hépatique chez les personnes obèses à risque de diabète de type 2. L'accumulation de tissu adipeux viscéral est systématiquement associée à une élévation de l'inflammation et à un risque accru de maladies métaboliques. Toutefois, on connait peu les mécanismes moléculaires qui contrôlent son expansion pathologique. Le facteur de transcription IRF5 (Interferon regulatory factor 5) a été impliqué dans la polarisation des macrophages vers un phénotype inflammatoire.

Publication 3 Dalmas E, Toubal

Nous démontrons dans cette étude que les souris déficientes en IRF5 ne montrent aucune différence dans la croissance de leur tissu adipeux épididymaire, mais que leur tissu adipeux souscutané est plus développé que celui des souris de type sauvage en réponse à une alimentation riche en matière grasse. Le tissu adipeux épididymaire des souris déficientes est caractérisée par l'accumulation de macrophages de type « M2 » et l'accumulation de collagène. Les souris déficientes en IRF5 présentent des adipocytes de taille plus faible dans le tissu épididymaire et une meilleure sensibilité à l'insuline que les souris de type sauvage.

A côté de ces travaux chez la souris, nous avons réalisé une étude chez l'homme, montrant que, chez les sujets obèses, l'expression d'IRF5 est associée négativement à la sensibilité à l'insuline et à l'abondance des dépôts de collagène dans le tissu adipeux viscéral. L'analyse du génome des macrophages du tissu adipeux a permis d'identifier le gène pro-fibrotique TGFB1 comme une cible directe d'IRF5.

Cette étude révèle une nouvelle fonction pour IRF5 dans le contrôle de la masse relative des différents dépôts de tissus adipeux et de la sensibilité à l'insuline. De plus, nos résultats suggèrent que l'inhibition d'IRF5 permettrait de promouvoir un état métabolique plus sain chez le sujet obèse.

LIST OF FIGURES

Figure 1: Features of the gut microbiota that promote obesity and insulin resistance [START_REF] Tremaroli | Functional interactions between the gut microbiota and host metabolism[END_REF] [START_REF] Flegal | Overweight and obesity in the United States: prevalence and trends, 1960-1994[END_REF]

LIST OF TABLES

INTRODUCTION

A. Metabolic diseases

Metabolic syndrome

The term metabolic syndrome was first used in 1977 by Dr. Hermann Haller, at the time Head of the Department of Medicine in Medical Academy Dresden, Germany, to describe the interconnected relationship of obesity, diabetes, high levels of fats in blood, and other factors. Nowadays a part of scientific literature tends to neglect this concept, even if aware that those factors do not cluster together by chance. Moreover, thanks to its ability to simplify a complex health issue, the concept of the metabolic syndrome (MetS) represents a useful tool for scientific divulgation and awareness of the associated health risks for general population. The metabolic syndrome is a collection of risk factors, such as high blood pressure, high glucose and cholesterol levels, together with abdominal obesity, which associates with a two-fold increase in the risk of having a heart attack or stroke, and a five-fold increased risk of developing type 2 diabetes [START_REF] Stern | Does the metabolic syndrome improve identification of individuals at risk of type 2 diabetes and/or cardiovascular disease?[END_REF]), [START_REF] Reaven | The metabolic syndrome: is this diagnosis necessary?[END_REF]) (Figure 1). Importantly, insulin resistance (IR) has been identified as the basis of most if not all of the features of this syndrome [START_REF] Dandona | The anti-inflammatory and potential anti-atherogenic effect of insulin: a new paradigm[END_REF]. Based on the International Diabetes Federation (IDF) definition, 20-25% of the world's population has MetS [START_REF] Alberti | Metabolic syndrome--a new world-wide definition. A Consensus Statement from the International Diabetes Federation[END_REF]. This Federation defines MetS as central obesity (defined as waist circumference ≥ 94cm for men and ≥ 80cm for European women, with ethnicity specific values for other groups) plus any two of the following: elevated plasma triglyceride (TG) levels (≥150 mg/dl), reduced high-density lipoproteins (HDL) (<40 mg/dl for men and <50 mg/dl for women), increased blood pressure (≥130 mmHg systolic or ≥85 mmHg diastolic), or increased fasting plasma glucose (≥100 mg/dl). In 2005 the American Heart Association (AHA) and the National Heart, Lung, and Blood Institute (NHLBI) released a scientific about MetS [START_REF] Grundy | Diagnosis and management of the metabolic syndrome: an American Heart Association/National Heart, Lung, and Blood Institute Scientific Statement[END_REF]. Retrieved from the cited report, Table 1 intends to provide up-to-date guidance for professionals on the diagnosis and management of the MetS in adults. Insulin resistance/hyperinsulinemia, early identified as the principal cause of MetS [START_REF] Reaven | Banting lecture 1988. Role of insulin resistance in human disease[END_REF]), [START_REF] Ferrannini | Hyperinsulinaemia: the key feature of a cardiovascular and metabolic syndrome[END_REF] once acquired, would prompt individuals with a genetic predisposition to develop all the other aspects of the disorder. Obesity, hypertension, dyslipidemia, polycystic ovarian syndrome, fatty liver disease, pre-diabetes, sleep and breathing disorder, certain cancers, and cognitive impairment represent diagnostic criteria that help to identify the syndrome. Due to its complexity, MetS has been criticized for lack of precision in the definition. However, over time, MetS has been strongly defended, especially since it is a powerful predictor for T2D and a major risk factor for cardiovascular morbidity and mortality [START_REF] Isomaa | Cardiovascular morbidity and mortality associated with the metabolic syndrome[END_REF], [START_REF] Gotto | The metabolic syndrome: a call to action[END_REF]. The role of the brain in the development of MetS will not be developed in this Thesis. However it is important to mention here its critical role in modulating lipid and carbohydrate metabolism and above all in perpetuating the unhealthy behaviors, such as deregulated eating behaviors reported in pathologies as obesity (overfeeding) [START_REF] Kenny | Epigenetics, microRNA, and addiction[END_REF]. The Thesis will focus on white adipose tissue, on the whole, extremely important as well in the MetS-associated alterations. This is particularly true for central visceral adiposity, now widely recognized as carrying greater health risk factors then other organs in various pathologies, including MetS.

Overweight and obesity

According to World Health Organization (WHO), overweight and obesity are important risks of death worldwide. Around 3.4 million adults die each year as a result of being overweight or obese. In addition, 44% of the diabetes burden, 23% of the ischemic heart disease burden and between 7% and 41% of certain cancer burdens is attributable to overweight and obesity. In 1972, in an article published by Ancel Keys in the Journal of Chronic Diseases, the new term "body mass index" (BMI) was coined as the best proxy for body fat percentage, a parameter to measure in prosperous Western societies with increasing obesity. The BMI is defined as a person's weight divided by the square of height -with the value universally being given in units of kg/m2. Based on this simple formula, the worldwide prevalence of obesity has nearly doubled between 1980 and 2008, in particular within high income countries, but without exclusion of low income ones. The World Health Organization (WHO) makes these global observations using BMI to classify underweight, overweight and obesity in adults, assuming an average body composition (Table 2).

Table 2. Definition and classification of obesity based on Body Mass Index (BMI) (modified from (Flegal, Carroll et al. 1998)).

However BMI alone cannot differentiate between fat and fat-free body mass, so measurements of waist circumference can be used as a supplementary factor for diagnosis of obesity (obesity threshold defined as 102 cm for men and 88 cm for women). Other methods, more expensive and time consuming, such as bioelectrical impedance, densitometry, X-ray absorptiometry (DXA, dual energy Xray Absorptiometry) and imaging methods (magnetic resonance imaging (MRI) and computed tomography) can measure adipose tissue distribution. Used as a prognostic factor to evaluate potential cardiovascular and metabolic complications, fat distribution can be influenced by age, sex and ethnicity. Central obesity, the body type also known as "apple shaped" in relation to the waist circumference, is one of the key features of the MetS. Central obesity is associated with dyslipidemia, a lipid disorder that may manifest as lipoprotein overproduction or deficiency. In developed countries, most dyslipidemias are hyperlipidemias, as in obesity, and the lipoprotein pattern can help predict cardiovascular risks.

Reported in

Diabetes mellitus type II

Type 2 diabetes mellitus (T2D) is a chronic metabolic disorder, described first as a component of MetS in 1988 [START_REF] Patlak | Combating diabetes[END_REF]. About 80% of T2D patients are diagnosed with the MetS [START_REF] Isomaa | Cardiovascular morbidity and mortality associated with the metabolic syndrome[END_REF]. T2D is the most common form of diabetes characterized by hyperglycemia, insulin resistance, and relative insulin deficiency. T2D is primarily resulting from the interactions between lifestyle, environmental factors and genetics. Lack of physical activity, sedentary lifestyle, cigarette smoking and important consumption of alcohol are behaviors associated with increased risk of developing T2D [START_REF] Hu | Diet, lifestyle, and the risk of type 2 diabetes mellitus in women[END_REF]. Overweight and obesity have been found to strongly contribute to the development of glucose intolerance [START_REF] Ding | Influenza vaccination coverage among pregnant women--United States, 2013-14 influenza season[END_REF] As a consequence, dysfunctional adipose tissue and the associated ectopic fat deposition (muscle, liver and pancreas) have been receiving full attention when studying the relationship between insulin resistance and beta-cell dysfunction in T2D. In fact, in order to predict diabetes risk more than the amount of adipose tissue mass, the ability of the tissue to act as an energy buffer has clearly more importance.

Lypodistrophic syndromes

Lipodystrophies are rare disorders in general connected with MetS. Lipodystrophy is a clinical condition identified by an inherent failure in adipose tissue development and/or function. The loss of adipose tissue results in metabolic abnormalities that are typically more severe than those associated with obesity [START_REF] Chan | Clinical classification and treatment of congenital and acquired lipodystrophy[END_REF]. Individuals with lipodystrophic disorders are extremely lean, yet they often suffer from diabetes, dyslipidemia, and other metabolic complications [START_REF] Virtue | It's not how fat you are, it's what you do with it that counts[END_REF]. The severity of the metabolic phenotype is approximately proportional to the degree of adipose tissue loss and therefore tends to be worse in generalized lipodystrophy. Two major categories classify lipodystrophies: familial and acquired. Familial lipodystrophies are congenital generalized lipodistrophy, Beradinelli-Seip syndrome, characterized by near complete lack of metabolically active adipose tissue from birth, and Dunnigan familial partial lipodystrophy, characterized by loss of subcutaneous fat from the lower part of the body during adolescence and excess fat accumulation in the face and neck.

Between generalized and partial lipodystrophies the degree of metabolic abnormalities can vary. The severity of metabolic abnormalities and/or the serious co-morbidities in generalized syndromes is associated with substantial morbidity and mortality. Both varieties occur approximately three times more often in women. Acquired lipodystrophy happens, for example, in patients infected with the human immunodeficiency virus (HIV), receiving highly active retroviral therapy. These patients are subject to loss of subcutaneous fat from the extremities and face but excess fat deposition in the neck and trunk. These patients in particular present abnormal blood lipid levels (dyslipidemia) due to therapies. Other drugs or unknown mechanisms can cause localized lipodystrophies. Biological similarities between lipodystrophic and obese patients include severe insulin resistance, hyperglycemia, hypertriglyceridemia, dyslipidaemia, hepatic steatosis and low HDL-cholesterol [START_REF] Huang-Doran | Lipodystrophy: metabolic insights from a rare disorder[END_REF]. Diabetes frequently occurs over the course of the disease and the risk of cardiovascular complications is very high. Few treatments have proven effective for lipodystrophic syndromes, necessarily focusing on limiting hyperlipidemia and diabetic complications. Few oral antidiabetic or lipidlowering medications have been useful, e.g. thiazolidinediones (TZDs), metformin, and fibrates {Agarwal, 2006 #903). Diabetes frequently occurs over the course of the disease and the risk of cardiovascular complications is very high. Few treatments have proven effective for lipodystrophic syndromes, necessarily focusing on limiting hyperlipidemia and diabetic complications. TZDs, peroxisome proliferator-activator receptor gamma (PPAR-γ) agonists, regulate adipocyte differentiation.

In fact these compounds induce adipogenesis in cell culture models and increase weight gain in rodents and humans [START_REF] Mukherjee | A selective peroxisome proliferator-activated receptorgamma (PPARgamma) modulator blocks adipocyte differentiation but stimulates glucose uptake in 3T3-L1 adipocytes[END_REF]. In 2002 Rosiglitazone, another TZD, has been shown to be able to induce adipocyte differentiation in preadipocytes from congenital generalized lipodystrophic patients [START_REF] Fischer | Induction of adipocyte differentiation by a thiazolidinedione in cultured, subepidermal, fibroblast-like cells of an infant with congenital generalized lipodystrophy[END_REF]. However when tried in patients with different types of lipodystrophies, in randomized double-blind controlled studies, this treatment, with few exception [START_REF] Victoria | Reduced adipogenic gene expression in fibroblasts from a patient with type 2 congenital generalized lipodystrophy[END_REF], was not of proven efficacy, put aside the potential negative effects [START_REF] Sutinen | Rosiglitazone in the treatment of HAART-associated lipodystrophy--a randomized double-blind placebo-controlled study[END_REF]) [START_REF] Walker | Improvement of acquired partial lipodystrophy with rosiglitazone despite ongoing complement activation[END_REF]) [START_REF] Sleilati | Efficacy and safety of pioglitazone in treatment of a patient with an atypical partial lipodystrophy syndrome[END_REF]. [START_REF] Schindler | The effect of rosiglitazone on insulin sensitivity, beta cell function, bone mineral density, and body composition in hiv-positive patients on highly-active antiretroviral therapy (HAART)[END_REF]), [START_REF] Raboud | A meta-analysis of six placebo-controlled trials of thiazolidinedione therapy for HIV lipoatrophy[END_REF]. More recently, depending on the form of lipodydrophy, a recombinant analogue of human leptin, Metreleptin, has been approved to treat the associated metabolic alterations [START_REF] Chan | Clinical effects of long-term metreleptin treatment in patients with lipodystrophy[END_REF]. This drug has been shown to decrease TG and other lipid intermediates and reduces ectopic accumulation in tissues such as liver and muscle, resulting in increased insulin sensitivity, thereby improving hypertriglyceridemia and hyperglycemia. Gastric bypass surgery could be also taken into consideration to control metabolic alterations associated with lipodystrophies [START_REF] Utzschneider | Effectiveness of gastric bypass surgery in a patient with familial partial lipodystrophy[END_REF].

B. Metabolic diseases: the available models

In order to understand the disease process and the biological factors that participate in particular human pathologies, non-human species can be extensively studied without run into ethical and technical limitation. Moreover, animal models are important in the development process of potential treatments and cures for human diseases, despite the intrinsic limit that biological activity in a model organism does not ensure an effect in humans [START_REF] Chakraborty | Zebrafish: a complete animal model for in vivo drug discovery and development[END_REF]). In the past decades, humanized mouse models have been used as preclinical tool to bypass this limit and this advancement allowed for example the study of human-specific infectious agents without putting patients at risk [START_REF] Brehm | Humanized mouse models to study human diseases[END_REF]. The mouse (Mus musculus) is the classic model vertebrate currently used. This animal presents several favorable characteristics to be selected over other organisms as a model: short generation time, accessibility, easy manipulation, genetics and conservation of mechanisms relatively comparable to humans and in addition easy genetic selection. Mice genetically engineered are called transgenic and it means that through gene knock-out and knock-in technologies they can be used to mimic the genetic and biological evolution of certain diseases, such as cancer, or simply to study the gene function itself.

Most of the evidence about obesity-associated health problems has been obtained from epidemiological analyses of human subjects. The use of animal models can help to explore the precise molecular mechanisms of obesity-associated deleterious conditions. Studies on body and fat mass can include: high fat or high fat/high sugar (Cafeteria) diets, bariatric surgery and dietary restriction, impact of candidate pharmaceuticals or natural compounds. The right animal model must be selected carefully, depending on the degree of fatness and the characteristics important for the planned study. Compared to human studies, using animal models we can investigate in detail the different cellular components of adipose tissue, in physiological or pathological state, i.e. mimicking the adipocyte dysfunctions driven by fat expansion in obesity. In addition though transgenic mice, the cellular responses of adipose tissue, for example, can be completed exploring the multiple molecular activities that a specific gene (inserting, removing, activating or inactivating, inducing a mutation in a time-controlled manner, over-expressing, humanizing through replacing the mouse gene by a portion or the entire human gene) can have in the whole body or in specific tissues/cells. In animal research it is important to consider gender differences. However, most studies continue to be performed only in one gender, using male mice, for complexity and cost reasons. An additional reason is linked to estrous cycle that creates a tremendous amount of variability in female mice. However, this knowledge gap in animal research reflects limits in human research that only recently has started evaluating sex differences in the obesity context.

It is important to underline here that all laboratory experiments involving living animals take into account, when planned, the recommendations of specific committee (Comité National de Réflexion Ethique sur l'Expérimentation Animale, CNREEA) and they need to be approved by the French Ministry of Agriculture and Fisheries. It is the same Institution that gives the animal experimentation authorization (renewable every 5 years) to the staff members which followed the obligatory national training programs. Planned projects must prove the potential for benefit to human health, minimization of pain and distress, humane euthanasia, and the described protocols must follow the principles of the 3 R "Replacement, Reduction and Refinement".

Mouse models of obesity

There are several rodent and non-rodent models of obesity, but we will focus our attention on the mouse models mainly used in experimental research. Two primary models are used to study obesity, the monogenic and polygenic. The most well-characterized monogenic mouse models have either the ob or db gene mutation for obesity, whereas the most commonly used polygenic obesity model is the C57BL/6 inbred mouse fed with high fat diet (diet-induced obese (DIO) mice). Despite being very useful models for obesity, they differ from the human associated complications in many different aspects. Naturally, cut-off points such as waist circumference cannot be transferred from humans to mice. In addition, MetS is characterized by a low HDL in association with an elevated triglyceride concentration. In mice, normal lipoprotein profiles have primarily atheroprotective HDL, whereas normal human lipoprotein profiles contain primarily atherogenic low-density lipoproteins (LDL). The relationship between obesity and hypertension, well established in children and adults, is not reflected in obese mice [START_REF] Kennedy | Mouse models of the metabolic syndrome[END_REF]). On the other hand, despite the limits, mouse models reflect many of the characteristics of human obese phenotype, including hyperinsulinemia, hyperglycemia, IR, hypertriglyceridemia and hepatic steatosis, and they keep providing valuable insights into the pathophysiology of these metabolically altered conditions.

Leptin-deficient mice

The leptin-deficient or ob/ob mouse model reflects single gene mutation that has arisen spontaneously in rodent colonies and discovered by chance in 1949 [START_REF] Ingalls | Obese, a new mutation in the house mouse[END_REF] The identification of the gene responsible for the obese phenotype observed took nearly 50 years [START_REF] Zhang | Positional cloning of the mouse obese gene and its human homologue[END_REF]. Responsible mutation gene is named as obese (ob) gene. The ob/ob mutation is recessive and mutant mice gain weight rapidly throughout their lives, reaching a weight that can arrive to be three times that of wild-type control mice. The obesity is characterized by an increase in both adipocyte number and size, associated with reduced energy expenditure (hypometabolic state). The genetic background is an extremely important factor to consider when choosing ob/ob mice to study MetS. Whereas leptin deficiency results in hyperphagia and extreme obesity on all backgrounds, a diabeteslike syndrome of hyperglycemia, glucose intolerance and elevated plasma insulin will appear or not depending on genetic background [START_REF] Kennedy | Mouse models of the metabolic syndrome[END_REF]. Specifically ob/ob mice on the C57BL/6J background have mild and transient hyperglycemia, whereas when on the C57BL/KsJ background, ob/ob mice develop hyperglycemia and diabetes, with maximum weight reached at 3-4 months of age, after which they gradually lose weight and often die by 6 months of age. More recently ob/ob mice on the BALB/cJ background have been shown to have a 35-40% reduction in weight gain with elevations in fed insulin and plasma TG levels, whereas ob/ob mice on the FVB/N background have exacerbated IR compared with those on the C57BL/6J background. The model, as a whole, presents some limitations. First of all the absence of leptin, a hormone extremely important for central and neuroendocrine signaling, is influencing not only food intake and energy expenditure, but also immune system development and angiogensis [START_REF] Fantuzzi | Leptin in the regulation of immunity, inflammation, and hematopoiesis[END_REF] [START_REF] Busso | Leptin signaling deficiency impairs humoral and cellular immune responses and attenuates experimental arthritis[END_REF]. Leptin-deficient ob/ob mice, as leptin receptor-deficient db/db, display in fact a marked thymic atrophy and exhibit defective immune responses. Moreover, ob/ob mice short life span, and the male mice reduced fertility, make this model rather costly.

Leptin receptor-deficient mice

Leptin receptor also known as LEP-R is a protein that functions as a receptor for the fat cell-specific hormone leptin. The db/db mouse is a model of obesity, diabetes, and dyslipidemia wherein leptin receptor activity is deficient because the mice have a single gene mutation in the long isoform of leptin receptor gene. As for ob/ob mice, the obese and insulin-resistant phenotype of db/db mice depends upon their genetic background [START_REF] Kennedy | Mouse models of the metabolic syndrome[END_REF]. Specifically mice on the C57BL/KsJ background develop diabetes and mice on the C57BL/6J background only become insulin resistant. At 2 weeks of age db/db mice already present elevated plasma insulin. After 4 weeks of age they start expressing the obese phenotype, associated with fasting hyperglycemia (Harlan Laboratories). Although a useful model to study the pathogenesis and treatment of diabetic dyslipidemia [START_REF] Kobayashi | The db/db mouse, a model for diabetic dyslipidemia: molecular characterization and effects of Western diet feeding[END_REF], these mice, as ob/ob mice, require special care and handling because they are relatively immunocompromised. Indeed, expression of the leptin receptor in the environment has been demonstrated to be important for T cell development [START_REF] Palmer | Indirect effects of leptin receptor deficiency on lymphocyte populations and immune response in db/db mice[END_REF]. As for leptindeficient mice, this model is also rather expensive.

High fat diet-induced obesity

Being diet one of the main environmental factors contributing to obesity and diabetes [START_REF] Rolls | The influence of dietary fat on food intake and body weight[END_REF], [START_REF] Swinburn | Energy balance or fat balance?[END_REF], [START_REF] Marshall | Dietary fat and the development of type 2 diabetes[END_REF], the mouse model of diet-induced obesity (DIO) can be a useful tool to study metabolic dysfunctions [START_REF] Buettner | High-fat diets: modeling the metabolic disorders of human obesity in rodents[END_REF]).

Though chronic excessive energy intake, in particular by means of dietary fat, can gradually reproduce the development of the deleterious sequels of obesity. High fat consumption leads to an increase in body fatness in mice and in parallel it may cause IR through the excessive plasma free fatty acids (FFA) availability [START_REF] Boden | Obesity and free fatty acids[END_REF]. The HFD increases the supply of triacylglycerols (TAGs) that accumulate in the major insulin target organs (skeletal muscle and liver) and increased FFA produce the characteristic lowgrade inflammation also in mouse adipose tissue, liver, muscles. Cafeteria diet, rich in sugar, salt, fat, is meant to simulate the human Western diet. However nowadays it is not the first choice for scientific research, being difficult to replicate results obtained with it [START_REF] Moore | The cafeteria diet--an inappropriate tool for studies of thermogenesis[END_REF]. High-fat diets (HFD) used in laboratory research, instead, present a well-defined purified composition that allows accurate replications for future studies. The HFD typically contain about 32 to 60% of calories from fat. Nutritionally, 60 kcal% fats is considerable extreme in human diet. However this fat percentage is commonly chosen to induce obesity, because mice tend to gain more weight more quickly [START_REF] Johnston | Intake compensates for resting metabolic rate variation in female C57BL/6J mice fed high-fat diets[END_REF]). The other frequent choice is 45 kcal% fat, that induced a milder obese phenotype, allowing to study the effects of external interventions (i.e. drug or probiotics) in the prevention or reversion of the deleterious alterations associated to obesity. It is important also to note that not all dietary fats have the same effects on the onset of obesity [START_REF] Bell | Voluntary exercise and monounsaturated canola oil reduce fat gain in mice fed diets high in fat[END_REF]. Omega-3 fatty acids-fed mice, for example, did not gain as much weight as those fed with comparable amounts of more saturated fat, such as lard or coconut oil [START_REF] Ikemoto | High-fat diet-induced hyperglycemia and obesity in mice: differential effects of dietary oils[END_REF]) [START_REF] Wang | Effects of dietary fat types on body fatness, leptin, and ARC leptin receptor, NPY, and AgRP mRNA expression[END_REF]. However the health benefits reported for unsaturated fatty acids [START_REF] Ikemoto | High-fat diet-induced hyperglycemia and obesity in mice: differential effects of dietary oils[END_REF] are controversial and the effects of different dietary fats are under current investigation in both humans and mice. Finally, fatty acids and lipid mediators derived from fatty acids can strongly modulate the immune response (de Jong, [START_REF] De Jong | Fatty acids, lipid mediators, and T-cell function[END_REF], so the diet-induced obese model can be used to evaluate the immune response during the development of different inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease, and asthma, known to be exacerbated by obesity.

Lipodystrophic mouse models

There are multiple mouse models reproducing lipodystrophy features that can be used for scientific research [START_REF] Kennedy | Mouse models of the metabolic syndrome[END_REF]). These animal models have been developed with a focus on disruption of adipocyte functions, since this cell is at the base of lipodystrophy-associated metabolic complications, including ectopic lipid accumulation and severe insulin resistance. Independently by the etiology of lipodystrophy, dysfunctional adipocytes present diminished storage capacity and altered adipokine secretion. These local alterations affect the metabolism of the organism, possibly through distinct pathways, and animal models allow focusing on single mechanisms. In lipodystrophy, as in obesity, progression of IR is linked to increased circulation of lipids, stored in insulin-sensitive organs such as the liver or muscle, and inflammatory adipokines. However, as opposed to obesity, in lipodystrophy, IR, hyperglycemia, hyperinsulinemia are provoked by low leptin due to the lack of adipose tissue [START_REF] Shimomura | Decreased IRS-2 and increased SREBP-1c lead to mixed insulin resistance and sensitivity in livers of lipodystrophic and ob/ob mice[END_REF]. Low leptin in lipodystrophic patients is perceived at the brain level as a starvation signal. However the excess calories consumed due to hyperphagia cannot be stored in the dysfunctional adipocytes. It has been exactly in this field of research that the causal link between insulin sensitivity and adipose tissue function has been demonstrated using the mouse models of lipodystrophy [START_REF] Shimomura | Insulin resistance and diabetes mellitus in transgenic mice expressing nuclear SREBP-1c in adipose tissue: model for congenital generalized lipodystrophy[END_REF]) [START_REF] Reitman | Transgenic mice lacking white fat: models for understanding human lipoatrophic diabetes[END_REF]). The big limit in using these models is that the majority of type 2 diabetic individuals are not lipodystrophic, but obese. Indeed the question about the role of reduced or exhausted adipose tissue expansion in the development and exacerbation of metabolic alterations in an obese state remains open. To elucidate the involvement of adipose tissue expandability in the development of IR in obesity, a recent study elegantly demonstrated that when mice have a mutation equivalent to lipodystrophic patients (dominant-negative mutations in PPARgamma) on a hyperphagic ob/ob background, their adipose tissue expandability is insufficient compared to the excessive energy availability and this exacerbate IR and the metabolic complications associated with leptin deficiency [START_REF] Gray | Leptin deficiency unmasks the deleterious effects of impaired peroxisome proliferator-activated receptor gamma function (P465L PPARgamma) in mice[END_REF]). This study, as many other ones, proves that the use of animal models is important to decipher the different molecular mechanisms underlying regulation of body fat distribution and IR. Examples of lipodystophic mice generated through genic manipulation are described below.

PPARγ-deficient mice

PPARγ belongs to the superfamily of nuclear hormone receptors and is a primary pharmacological target in the treatment of type II diabetes, because of its role in the control of glucose homeostasis and insulin sensitivity. This gene promotes macrophage differentiation [START_REF] Tontonoz | PPARgamma promotes monocyte/macrophage differentiation and uptake of oxidized LDL[END_REF], inflammation, and carcinogenesis, but also adipogenesis [START_REF] Tontonoz | Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipidactivated transcription factor[END_REF]. Due to this important function, different kinds of mutations in PPARγ have been shown to cause lipodystrophic phenotypes or metabolic symptoms [START_REF] Ludtke | New PPARG mutation leads to lipodystrophy and loss of protein function that is partially restored by a synthetic ligand[END_REF]). Similar phenotypes have been described in mice [START_REF] Barak | PPAR gamma is required for placental, cardiac, and adipose tissue development[END_REF]) (Wang, Mullican et al. 2013), in which the absence of PPARγcompromises the induction of genes mediating fatty acid uptake, metabolism, and storage. It was in 2003 using the lipodystrophic model of PPARγ hypomorphic mice (PPARγ hyp/hyp) that it was unequivocally demonstrated the importance of PPARγ as the master regulator of adipogenesis [START_REF] Koutnikova | Compensation by the muscle limits the metabolic consequences of lipodystrophy in PPAR gamma hypomorphic mice[END_REF]. These specific PPARγ hyp/hyp mice also showed that glucose homeostasis can be relatively well maintained in the absence of white adipose [START_REF] Koutnikova | Compensation by the muscle limits the metabolic consequences of lipodystrophy in PPAR gamma hypomorphic mice[END_REF]. Three splice variants of PPAR gamma are known: PPAR gamma1, PPAR gamma2, and PPAR-gamma3. For example PPARg2 is the nutritionally regulated isoform of PPARg and it has been proved that selective disrupting this specific variant is sufficient to impair the development and the expansion of adipose tissue (Medina-Gomez, [START_REF] Medina-Gomez | PPAR gamma 2 prevents lipotoxicity by controlling adipose tissue expandability and peripheral lipid metabolism[END_REF].

Transgenic aP2-SREBP-1c mice

The adipose-nSREBP1c transgenic mouse was created in 1998. In this mouse, SREBP1c, a gene required for glucose metabolism and fatty acid and lipid biosynthesis, is constitutively activated. These mice present adipogenesis impairment that results in a lipodystrophic animal. Mice nSREBP1c lack white adipose tissue and recapitulate the metabolic abnormalities observed in humans with lipodystrophy.

Besides reduced body weight and the associated very low levels of adipokines (leptin and adiponectin), these mice present elevated plasma TG, glucose and insulin levels. It was using this model that it was demonstrated that functional adipose tissue is necessary to maintain normal carbohydrate and lipid metabolism [START_REF] Shimomura | Insulin resistance and diabetes mellitus in transgenic mice expressing nuclear SREBP-1c in adipose tissue: model for congenital generalized lipodystrophy[END_REF]. However, more recently, it has been demonstrated that adipocyte fatty-acid-binding protein (aP2) is not exclusively specific to adipocytes; it can be expressed also in macrophages and in the central nervous system [START_REF] Makowski | Lack of macrophage fatty-acid-binding protein aP2 protects mice deficient in apolipoprotein E against atherosclerosis[END_REF]) [START_REF] Martens | Ectopic recombination in the central and peripheral nervous system by aP2/FABP4-Cre mice: implications for metabolism research[END_REF]). As a consequence, results obtained targeting the promoter aP2 need to be carefully interpreted.

A-ZIP/F-1 mice

The transgenic mouse (named A-ZIP/F-1) was created in 1998 as a model for understanding human lipoatrophic diabetes and presents the same limitation as the aP2-SREBP-1c model. This mouse, express a dominant-negative protein, termed A-ZIP/F, under the control of the aP2 promoter, consequently not targeting exclusively adipocytes. However this dominant-negative protein inhibits the DNA binding and function of B-ZIP proteins in both the C/EBP and AP-1 families of transcription factors, inactivating adipocyte growth and differentiation, resulting in a mouse with no white adipose tissue and dramatically reduced amounts of brown adipose tissue, that is inactive [START_REF] Moitra | Life without white fat: a transgenic mouse[END_REF]) [START_REF] Reitman | Transgenic mice lacking white fat: models for understanding human lipoatrophic diabetes[END_REF]. Having no white fat tissue brings severe physiological consequences, such as diabetes, with reduced leptin (20-fold) and elevated serum glucose (3-fold), insulin (50-to 400-fold), free fatty acids (2fold), and triglycerides (3-to 5-fold). The liver and the other organs are enlarged due to lipid accumulation. This phenotype clearly presents similarities with humans with severe lipoatrophic diabetes: IR, hyperglycemia, hyperlipidemia and fatty liver.

C. The metabolic organs

The research goal of the present Doctoral project is to use animal models to understand the signaling from three critical metabolic organs in the body, adipose tissue, liver and intestine, during metabolic altered conditions mimicking human MetS diseases. The potential interaction between gut microbiota and host metabolism is also considered, when possible [START_REF] Martens | Ectopic recombination in the central and peripheral nervous system by aP2/FABP4-Cre mice: implications for metabolism research[END_REF]) (Figure 1).

Adipose tissue: functions and dysfunctions

White adipose tissue (WAT) fills up space between organs and tissues, providing structural support, protecting them against trauma (in particular visceral depot) and functioning as a natural thermal insulator. The quantity of body fat varies widely in mammals, ranging from 2% to >50% of body mass, typically from 10% to 20% in mice and humans. Much of this variability depends on both an individual's genetic background and environmental factors, such as dietary and physical activity behaviors. In humans, adipose tissue is found in specific locations, referred to as adipose depots. Depending on the localization, recently new profiling technologies have shown that WAT displays depot-specific differences in the metabolic profiles, indicating that WAT is not a homogeneous organ. Located beneath the skin (subcutaneous fat), around internal organs (visceral fat), in bone marrow and in the breast tissue, adipose tissue is now fully recognized as an important multidepot metabolic organ. In experimental animal models, this distinction between WAT depending on the different location is maintained. In particular in the mouse, the classical model, WAT collected under the skin around the inguinal area corresponds to the human subcutaneous fat. What in humans is visceral fat, in a mouse corresponds to WAT collected inside the peritoneal cavity, but it can have different names depending on the precise place where it is collected (perigonadal or depending on the sex of the mice, peri-uteral for females and epidydimal for males = WAT located around the reproductive system; retroperitoneal = WAT behind the kidneys; mesenteric = WAT located around intestinal tractus).

The primary function of WAT is to store excess energy in the form of triglycerides and to release it as free fatty acids (FFA) or glycerol in time of fasting or starvation. The process is mediated by key lipogenic and lipolytic enzymes. Concurrently normal WAT regulates a wide array of homeostatic processes, such as glucose metabolism, whole-body insulin sensitivity and energy homeostasis, through active secretion of multiple bioactive factors called adipokines (endocrine function). However, in conditions of excess or lack of WAT, adipose tissue becomes dysfunctional, affecting other organs of the body through insulin resistance, dyslipidemia and associated metabolic complications. The excess of WAT in obesity corresponds to abnormally increased triglyceride storage as a result from a chronic, positive imbalance between energy intake and energy expenditure. In obese patients, excess lipid accumulation by large adipocytes increases basal lipolysis, a process in which triglycerides (TG) are hydrolyzed into glycerol and FFA. The net release of FFA into the bloodstream is increased and since the deregulated adipocyte cannot re-esterify them, FFA travel and accumulate to other tissues (ectopic fat deposition), such as liver, pancreas, heart and muscles. For example for skeletal muscles, the obesity-associated excessive free fatty acid production is believed to contribute to IR. In obesity, molecular mechanisms characterizing dysfunctional hypertrophic adipose tissue are abnormal production of adipokines and dysregulation of adipose tissue expandability. Similar failures in WAT functions are reflected in lipodystrophies. Metabolic complications, such as IR, diabetes mellitus, hypertriglyceridemia, and fatty liver, increase in severity with the degree of fat loss. The mechanisms that contribute to adipose tissue dysfunctions and consequently regulate altered metabolic pathways are currently under investigation in these metabolic diseases, obesity and lipodysdrophy.

Metabolic and endocrine functions

Besides the efficient storage capacity, WAT has multiple metabolic roles in regulating whole-body physiology. The typical WAT cells are adipocytes that contain lipid droplets that fill the middle of the cell and are anchored by collagen fibers. The importance of adipocytes for health was clearly demonstrated with the use of a mouse model of lipoatrophic diabetes [START_REF] Gavrilova | Surgical implantation of adipose tissue reverses diabetes in lipoatrophic mice[END_REF].

Transplantation of WAT from healthy mice into A-ZIP/F-1 mice reversed their characteristic phenotype, a severe form of lipoatrophic diabetes. Hyperglycemia and hyperlipidemia were decreased, insulin sensitivity improved, serum triacylglycerol decreased, and the amount of fat deposited in muscle and liver was decreased thanks to reconstitution with normal WAT. The complexity of endocrine hormones secreted by healthy adipose tissue has allowed increasingly recognition for this tissue as an endocrine organ. In fact, through the secretion of active molecules (adipokines), WAT influences the regulation of important physiological functions [START_REF] Cao | Adipocytokines in obesity and metabolic disease[END_REF]. Appetite, satiety, energy expenditure, insulin sensitivity, glucose and lipid metabolism, fat distribution are some of the metabolic parameters partially or fully controlled by WAT (FFA, leptin, adiponectin, resistin, retinol binding protein-4-RBP4) (Bluher and Mantzoros 2015) (Table 4). In recent years, pangenomic and proteomic approaches have identified new adipose tissue-secreted factors whose functions still need to be characterized [START_REF] Lehr | Adipokines: a treasure trove for the discovery of biomarkers for metabolic disorders[END_REF]. White adipose tissue functions and how substrate utilization could regulate cell signaling attract special attention in metabolic and cardiovascular physiology, immunology, and cancer biology.

Adipokines with a prominent role in auto-/paracrine crosstalk within the adipose tissue such as adiponectin and RBP4 are of particular interest in conditions of metabolic diseases in accordance with their direct effects on skeletal muscle insulin resistance, cardiovascular function and β-cell activity [START_REF] Romacho | Adipose tissue and its role in organ crosstalk[END_REF]. The two most abundant depots, visceral and subcutaneous adipose tissues, produce unique profiles of adipokines [START_REF] Samaras | Subcutaneous and visceral adipose tissue gene expression of serum adipokines that predict type 2 diabetes[END_REF]. Adiponectin, discovered specifically expressed in WAT in 1995 [START_REF] Scherer | A novel serum protein similar to C1q, produced exclusively in adipocytes[END_REF], at the same time of leptin [START_REF] Rosen | Direct demonstration of an intramolecular SH2phosphotyrosine interaction in the Crk protein[END_REF], is more abundantly expressed in subcutaneous WAT and its main function is to directly sensitize the body to insulin and influence adipose tissue growth and function. Aside from autocrine effects on adipocytes themselves, adiponectin exerts many functional effects on peripheral tissues, such as heart, vasculature, skeletal muscle, lung, bones and not least liver [START_REF] Dadson | Adiponectin action: a combination of endocrine and autocrine/paracrine effects[END_REF]. Modulating a number of metabolic processes, recently, it has been proposed as a starvation gene in the course of evolution, enabling fat storage in case of adiposity loss [START_REF] Kadowaki | The physiological and pathophysiological role of adiponectin and adiponectin receptors in the peripheral tissues and CNS[END_REF]. Systemic adiponectin, and especially adiponectin HMW levels, are paradoxically reduced in obese and type 2 diabetes mellitus patients [START_REF] Dadson | Adiponectin action: a combination of endocrine and autocrine/paracrine effects[END_REF]. Moreover, recent data from obese individuals confirm that visceral adiposity, more than global, influences adiponectin levels, whom production is potentially inhibited by WAT associated chronic inflammation in a self-perpetuating loop [START_REF] Guenther | Adiposity distribution influences circulating adiponectin levels[END_REF]) [START_REF] Fantuzzi | Adiponectin and inflammation: consensus and controversy[END_REF]. The decrease activity of this specific adipokine, principally caused by increased proinflammatory cytokines, in particular tumor necrosis factor α (TNFα) and monocyte chemoattractant protein-1 (MCP-1), forms a vicious adipokine network causing insulin resistance and metabolic sundrome [START_REF] Kadowaki | Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome[END_REF]) [START_REF] Yamauchi | Physiological and pathophysiological roles of adiponectin and adiponectin receptors in the integrated regulation of metabolic and cardiovascular diseases[END_REF]. Bariatric surgery has been shown to increase circulating adiponectin, in association with improved hyperglycemia and insulin sensitivity [START_REF] Sjostrom | Bariatric surgery and long-term cardiovascular events[END_REF]. Adiponectin concentration can be affected by pharmacological and notably dietary interventions as well [START_REF] Swarbrick | Physiological, pharmacological, and nutritional regulation of circulating adiponectin concentrations in humans[END_REF]. For example, in a recent review with meta-analysis of randomized controlled trials, it was underlined how lipid intake can change circulating adiponectin levels [START_REF] Von Frankenberg | Effect of dietary lipids on circulating adiponectin: a systematic review with meta-analysis of randomised controlled trials[END_REF]. In particular, interesting for the purpose of this project, conjugated linoleic acid supplementation showed significant adiponectin reduction compared to unsaturated fat supplementation in several studies [START_REF] Von Frankenberg | Effect of dietary lipids on circulating adiponectin: a systematic review with meta-analysis of randomised controlled trials[END_REF]). In contrast with adiponectin, circulating RBP4, a retinol transporter, synthetized in the liver and adipose tissue, is significantly increased in obesity, in association with IR in mice and humans. The profile of RBP4 in MetS patients, without cardiovascular diseases or diabetes, follows the profile of other circulating biomarkers of inflammation, known to be biologically relevant in the pathogenesis of MetS: leptin, CRP, serum amyloid A, plasminogen activator inhibitor-1, IL-1, IL-6, IL-8, and monocyte chemotactic protein-1 (MCP-1) [START_REF] Bremer | Adipose tissue dysregulation in patients with metabolic syndrome[END_REF]). However, when looking at RBP4 mRNA expression, it is important to take into consideration that the regulation of RBP4 is gender-specific, similarly to adiponectin, leptin and other AT-derived proteins. Very interesting are the recent results demonstrating the role of RBP4 in causing IR in association with the activation of adipose antigen-presenting cells, such as macrophages, and the induction of proinflammatory cytokines [START_REF] Norseen | Retinol-binding protein 4 inhibits insulin signaling in adipocytes by inducing proinflammatory cytokines in macrophages through a c-Jun N-terminal kinase-and toll-like receptor 4-dependent and retinol-independent mechanism[END_REF][START_REF] Moraes-Vieira | RBP4 activates antigen-presenting cells, leading to adipose tissue inflammation and systemic insulin resistance[END_REF]. In obesity, the role of these adipocytokines in affecting lipid metabolism/flux and thereby contributing to altered fat distribution is fully recognized. Consequently their implication in regulating WAT expandability connects them with obesity-related IR. It is noteworthy to underline here the recognized causal link between visceral WAT expansion, macrophage accumulation and IR [START_REF] Patsouris | Ablation of CD11c-positive cells normalizes insulin sensitivity in obese insulin resistant animals[END_REF]. Visceral WAT expansion is in fact highly correlated with co-morbidities, due to the higher inflammatory potential in comparison to subcutaneous WAT in terms of increased production of pro-inflammatory adipocytokines and reduced expression of the protective adiponectin [START_REF] Fantuzzi | Adipose tissue, adipokines, and inflammation[END_REF]. Complementary to the more classic adipokines linked to metabolic functions, WAT secretes other bioactive factors that, in an auto-/paracrine manner, regulate other tasks, like endothelial and cardiovascular function (angiotensinogen and plasminogen activator inhibitor-1,PAI-1), neuroendocrine function (sex steroids, various growth factors) and immunity (complement factors, haptoglobin). To conclude, the beneficial effects of having a "healthy" adipose tissue are receiving increasing attention, in particular in search of MetS treatment [START_REF] Gunawardana | Benefits of healthy adipose tissue in the treatment of diabetes[END_REF].

Adipose tissue plasticity

Adipose tissue plasticity can be reflected by multiple aspects that protected the functionality of this tissue during the course of evolution. It is of recent interest the great heterogeneity among the cells types that can become adipocytes, as well as the natural metabolic plasticity of mature cells. The concept of AT plasticity has been extended to two very different adipose present in mammals, different for anatomical location and functions, brown and white adipose tissue. Beige fat, as suggested by its name, is an intermediate between them. Beige adipocytes, with their newly described biology, well represents AT plasticity as well as an interesting potential for new therapies against metabolic diseases [START_REF] Gunawardana | Benefits of healthy adipose tissue in the treatment of diabetes[END_REF]. Brown adipose tissue (BAT) is a highly oxidative tissue containing multilocular fat cells packed with mitochondria that contain uncoupling protein-1 (UCP1) for FFA oxidation. White adipose tissue (WAT) has been historically defined by anatomical location and the presence of parenchymal cells containing a single large lipid droplet and sparse mitochondria lacking UCP1. However, clusters of UCP1-expressing adipocytes with more oxidative capacity can develop in white adipose tissue (WAT) in response to various physiological and pharmacological conditions [START_REF] Vitali | The adipose organ of obesity-prone C57BL/6J mice is composed of mixed white and brown adipocytes[END_REF]). These adipocytes have been named beige, 'brite' (brown in white), iBAT (induced BAT), recruitable BAT and wBAT (white adipose BAT) and resemble to adipocytes in BAT for the presence of multilocular lipid droplet cells rich in mitochondria containing UCP1, but they possess a distinct gene signature [START_REF] Wu | Beige adipocytes are a distinct type of thermogenic fat cell in mouse and human[END_REF]. However the distinct functionality of beige cells from brown cells need to be further explored. It is unknown, for example, if beige adipocytes may secrete certain factors that affect WAT function, systemic metabolism or both. In formed WAT, mature unilocular white adipocytes transform into beige adipocytes in response to cold or β3-adrenergic agonist [START_REF] Vitali | The adipose organ of obesity-prone C57BL/6J mice is composed of mixed white and brown adipocytes[END_REF]. However the question about transdifferentiation from pre-existing adipocytes [START_REF] Himms-Hagen | Multilocular fat cells in WAT of CL-316243-treated rats derive directly from white adipocytes[END_REF] or de novo differentiation [START_REF] Wang | Tracking adipogenesis during white adipose tissue development, expansion and regeneration[END_REF]) still needs to be answered for the different fat depots (Figure 2). Beige cell population arises predominantly from de novo adipogenesis rather than dedifferentiation. After cold exposure or β3 agonist treatment of subcutaneous WAT, most beige adipocytes are induced by differentiating from precursors rather than existing mature adipocytes.

Most of these findings are related to mouse biology. Although the existence of human precursor cells capable of expressing UCP1 and other brown and/or beige characteristics in WAT is known [START_REF] Elabd | Human multipotent adipose-derived stem cells differentiate into functional brown adipocytes[END_REF], the prevalence of beige adipocytes and their developmental process within different human WAT depots has not been carefully evaluated. And yet to come is a clear picture about the role of these "flexible" adipocytes in regulating metabolism, being a potential beneficial resource, especially for obese individuals, like brown adipocytes. That explains why studies on "browning" stimuli to "re-program" pre-existing adipocytes and/or to "program" fat progenitor cells have been increasing exponentially in recent years [START_REF] Gesta | Developmental origin of fat: tracking obesity to its source[END_REF]) [START_REF] Esteve | Human White and Brite Adipogenesis is Supported by MSCA1 and is Impaired by Immune Cells[END_REF]) [START_REF] Lee | Activated type 2 innate lymphoid cells regulate beige fat biogenesis[END_REF]. In addition, adipose progenitor cells, preadipocytes, resident within WAT stromal compartment, represent an increasing area of research lately. Their potential role in influencing how adipose tissue remodels during expansion (obesity) or reduction (drastic fat mass loss) of WAT is highly studied [START_REF] Jeffery | Rapid depot-specific activation of adipocyte precursor cells at the onset of obesity[END_REF]. The concept of adipose tissue hyperplasia (increased adipocyte number) versus hypertrophy (increased adipocyte size) was proposed in obesity, during the 1960s [START_REF] Hirsch | The fat cell[END_REF]. Recent studies have shown that, during fat accumulation, preadipocytes, and their interaction with WAT immune cells, can influence the balance toward a dysfunctional hypertrophic state against a hyperplastic process in which mature adipocytes remain relatively responsive [START_REF] Sorisky | Macrophage-induced adipose tissue dysfunction and the preadipocyte: should I stay (and differentiate) or should I go?[END_REF]). However, not all AT expansion is necessarily associated with pathological alterations. The adipogenic capacity of preadipocytes could influence the "healthy" AT expansion as well in physiological condition. This is when the generation of healthy functional smaller adipocytes arises from responsive and abundant preadipocytes, increasing the overall storage capacity while maintaining healthy adipose tissue function [START_REF] Sun | Adipose tissue remodeling and obesity[END_REF]. The described hyperplastic growth clearly supports the concept of "metabolically healthy obese" state [START_REF] Samocha-Bonet | Insulin-sensitive obesity in humans -a 'favorable fat' phenotype?[END_REF]). The number and, even more importantly, the functionality of preadipocytes assure the constant adipocytes turnover present in adults [START_REF] Spalding | Dynamics of fat cell turnover in humans[END_REF] and consequently the WAT "healthy" expansion in the context of excess caloric intake [START_REF] Wernstedt Asterholm | Adipocyte inflammation is essential for healthy adipose tissue expansion and remodeling[END_REF].

The dynamics of WAT growth definitely reflects its plasticity. In fact, adipose tissue is the only organ that has the potential of growing unlimited during the whole life. Adipogenesis is a multistep process during which fibroblast like preadipocytes developed into mature adipocytes [START_REF] Kirkland | Age, anatomic site, and the replication and differentiation of adipocyte precursors[END_REF]).

During this process, at various levels influenced by age, gender and life style, numerous transcription factors needs to be sequentially activated, including the CCAAT/enhancer-binding protein (C/EBP) gene family and peroxisome proliferator activated receptor-γ (PPAR-γ). Several factors facilitate the maturation of preadipocytes, including insulin-like growth factor I (IGF-l), macrophage colony stimulating factor, fatty acids, prostaglandins, glucocorticoids [START_REF] Ali | Adipocyte and adipogenesis[END_REF]) and newly explored pathway, such as phosphoinositide 3-kinase (PI3K)-AKT2 [START_REF] Jeffery | Rapid depot-specific activation of adipocyte precursor cells at the onset of obesity[END_REF]. Whereas, there are factors that inhibit the maturation process, such as glycoproteins, like zinc-α2-glycoprotein (ZAG), transforming growth factor-β (TGF-β), inflammatory cytokines and growth hormones [START_REF] Ali | Adipocyte and adipogenesis[END_REF]. In response to positive energy balance, WAT responds through an increase in number and size of adipocytes. This is when adipogenesis leads to central obesity if it occurs in the abdominal fat depot and peripheral obesity if it occurs in subcutaneous tissue. However analysis of adipocyte turnover using carbon-14 dating has demonstrated that new adipocytes form constantly to replace lost adipocytes through a dynamic and highly regulated process, particularly active in the human subcutaneous depot, where approximately 50% of adipocytes are replaced every 8 years [START_REF] Spalding | Dynamics of fat cell turnover in humans[END_REF]. However fat accumulation is not due only to adipogenesis, but also to a very different process, lipogenesis. The balance between fat synthesis (lipogenesis) and fat breakdown (lipolysis/fatty acid oxidation) contributes to the balance between lipid storage and mobilization and it is subjected to a complex regulation from various factors, such as glucose, insulin and leptin [START_REF] Kersten | Mechanisms of nutritional and hormonal regulation of lipogenesis[END_REF]) (Figure 3). In the last years, new data have explained different mechanisms of regulation of lipogenesis that, apart from hormones can be regulated by nutrients. For example polyunsaturated fatty acids, conjugated linoleic acid included, down-regulate expression of lipogenic genes, i.e. the peroxisome proliferatoractivated receptors (PPARs), that are important lipid sensor and play a critical role in the regulation of cellular uptake, activation, and oxidation of FFA [START_REF] Xu | Molecular recognition of fatty acids by peroxisome proliferatoractivated receptors[END_REF]) [START_REF] Berger | The mechanisms of action of PPARs[END_REF].

Between the three nuclear receptor isoforms, PPAR gamma, PPAR alpha, and PPAR delta, PPARγ is the transcription factor mainly involved in the regulation of both adipogenesis and lipogenesis in WAT PPARγ is necessary and sufficient to differentiate adipocytes [START_REF] Tontonoz | Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipidactivated transcription factor[END_REF]), but also important in controlling adipose tissue expansion, as it is the case for the PPARγ2 isoform (Medina-Gomez, [START_REF] Medina-Gomez | PPAR gamma 2 prevents lipotoxicity by controlling adipose tissue expandability and peripheral lipid metabolism[END_REF]). This process, traditionally considered as passive adaptation, it is now well accepted as a dynamic nutritional adaptation. In conditions as obesity and MetS, in response to variations in energy supply, WAT gradually expands and avoids the lipotoxic side effects exerted by excess lipid and fatty acid (FA) deposition in cells other than adipocytes. Different cell types possess different degree of TG storage capacity. Hepatocytes, enterocytes and macrophages can store lipids in cellular lipid droplets, but only adipocytes have that extraordinary plasticity, both in white and brown adipose tissue (Medina-Gomez, [START_REF] Medina-Gomez | PPAR gamma 2 prevents lipotoxicity by controlling adipose tissue expandability and peripheral lipid metabolism[END_REF]). However WAT expandability is challenged, due to altered lipid storage capacity for genetic defects, i.e. in the most severe form of congenital lipodystrophy -mutations of Berardinelli-Seip 2 gene (BSCL2/SEIPIN) [START_REF] Cartwright | Seipin: from human disease to molecular mechanism[END_REF] or due to external stressors, i.e. excess calories, we observe the alteration of an important balance for human health, the balance of fat. In the Figure 4, it is possible to visualize human genes linked to these extremes of body fat storage, in condition of lipodystrophy or cachexia and on the opposite side of neutral lipid storage disease (NLSD), metabolic syndrome (obesity in primis), non-alcoholic fatty liver disease (NAFLD) and atherosclerosis. These are human genes linked to these extremes of body fat and LD storage, on one side conditions of deficiency in forming or maintaining fat stores (lipodystrophy and cachexia) and on the opposite side neutral lipid storage disease (NLSD), metabolic syndrome (obesity in primis), non-alcoholic fatty liver disease (NAFLD) and atherosclerosis.

During physiological weight gain, induced by an overfeeding program in humans, it was shown how WAT expansion is associated with the induction of a concerted transcriptional program [START_REF] Alligier | Subcutaneous adipose tissue remodeling during the initial phase of weight gain induced by overfeeding in humans[END_REF]. The primary response of WAT is the upregulation of genes related to lipid metabolism and lipid synthesis, whereas over time genes involved in angiogenesis and extracellular matrix remodeling appear [START_REF] Alligier | Subcutaneous adipose tissue remodeling during the initial phase of weight gain induced by overfeeding in humans[END_REF]) (Figure 5). It is worthwhile to notice that similar transcriptional program involving genes related to lipid metabolism and lipid synthesis is enrolled during lipodystrophies, either congenital [START_REF] Liu | Adipose-specific knockout of SEIPIN/BSCL2 results in progressive lipodystrophy[END_REF] or induced by antiretroviral therapy for treatment of HIV infection [START_REF] Mallon | In vivo, nucleoside reverse-transcriptase inhibitors alter expression of both mitochondrial and lipid metabolism genes in the absence of depletion of mitochondrial DNA[END_REF].

In 2009, the group of Prof Scherer demonstrated that during states of positive energy balance, such as obesity, the extracellular constituents are globally upregulated and this up-regulation, with a particular contribution of collagen VI, restricts further expansion of individual fat cells, increasing rigidity of adipose tissue (see Chapter e) [START_REF] Khan | Metabolic dysregulation and adipose tissue fibrosis: role of collagen VI[END_REF]). However the adipose tissue plasticity in conditions with energy deficit, such as anorexia nervosa, or rapid weight loss, such as after gastric bypass, has been less explored. Nutrient deficit could represent the other face of the coin for the effects that metabolic stress could exert on the extracellular matrix. The importance of the impact of dysfunctional WAT state, in the broadest sense, on deregulated adipose plasticity and vice versa is reflected by the current research unrest about functions like cellular turnover and autophagy.

Adipose tissue immunity and inflammation

Among the multiple bioactive factors secreted by adipocytes, there are a various range of cytokines and chemokines [START_REF] Makki | Adipose tissue in obesity-related inflammation and insulin resistance: cells, cytokines, and chemokines[END_REF]. Anti-inflammatory adipokines such as adiponectin, transforming growth factor beta (TGFβ), interleukin (IL)-10, IL-4, IL-13, IL-1 receptor antagonist (IL-1Ra), and apelin identify the preferential secretion from "healthy and lean" adipose tissue. While "deregulated and obese" adipose tissue essentially secretes proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin IL-1alpha, IL-1beta IL-6, IL-8, macrophage inflammatory protein (MIP), serum amyloid A (SAA), leptin, visfatin, resistin, angiotensin II, and plasminogen activator inhibitor 1 [START_REF] Samaras | Subcutaneous and visceral adipose tissue gene expression of serum adipokines that predict type 2 diabetes[END_REF]) [START_REF] Ouchi | Adipokines in inflammation and metabolic disease[END_REF]. The inflammatory response promoted by the hypertrophic adipocytes in an obese state activates innate and adaptive immune cells resisdent within the tissue. The activation of these cells induces an increased recruitment of other immune cells, associated with autocrine and paracrine mechanisms of release of cytokines which exacerbate local inflammation. These complex alterations in WAT secretion (cytokines, adipokines, and chemokines) and immune cell composition characterize the adipose tissue-related pathologies in general. In obesity, in particular, white adipocyte overexpansion carries the above described stress state that ultimately can lead to cellular death. The NLRP3 (NLR family, pyrin domain containing 3)-dependent caspase-1 activation associated to the cellular death has been hypothesized to happen through a proinflammatory programmed cell death, pyroptosis [START_REF] Giordano | Obese adipocytes show ultrastructural features of stressed cells and die of pyroptosis[END_REF]. When adipocytes die, the intracellular content dispersed in the extracellular space triggers a multitude of cell and tissue changes, for example the enhanced secretion of chemokines, such as MCP-1, an insulin-resistance factor. It is in the context of insulin resistance that the concept of lipotoxicity has been introduced.

When WAT expandability becomes a limiting factor as it is the case in obesity, nutrients cannot be stored safely in the adipocyte being the storage capacity exceeded. These nutrients are then transported to peripheral organs, such as muscle, liver, heart, kidney or pancreas. Despite their nutrient buffer capacity, these tissues are not designed to store large amounts of lipids and therefore a toxic state is determined by this nutritional insult. Different tissues respond differently to these potentially toxic species and different lipids can provoke different grade of toxicity, inflammation and the associated local insulin resistance (Medina-Gomez, [START_REF] Medina-Gomez | PPAR gamma 2 prevents lipotoxicity by controlling adipose tissue expandability and peripheral lipid metabolism[END_REF].

It is in the context of insulin resistance that the concept of lipotoxicity has been introduced. When WAT expandability becomes a limiting factor as it is the case in obesity, nutrients cannot be stored safely in the adipocyte being the storage capacity exceeded. These nutrients are then transported to peripheral organs, such as muscle, liver, heart, kidney or pancreas. Despite their nutrient buffer capacity, these tissues are not designed to store large amounts of lipids and therefore a toxic state is determined by this nutritional insult. Different tissues respond differently to these potentially toxic species and different lipids can provoke different grade of toxicity, inflammation and the associated local insulin resistance. If in obesity ectopic lipid accumulation is due to saturation of expansion capacity of WAT, in lipodystrophies WAT is partially or completely unable to expand. Therefore, in lipodystrophies different mechanisms lead to the same result, ectopic lipid accumulation in periphery organs, inflammation and insulin resistance (Figure 6). An altered metabolic state is surely represented by fat expansion, but it is now clear that fat reduction challenges the local adipocyte physiology as well [START_REF] Cao | Adipocytokines in obesity and metabolic disease[END_REF]. Indeed, as in obesity, common inflammatory pathways are activated in lipoatrophic patients, presenting similar alterations in lipid droplet proteins (reduced expression of PLIN1, CIDEC, or CIDEA) in adipocytes (Figure 7). The role of lipid droplets in metabolic diseases is emerging as increasingly important due to their function of "organizing centers" for triglyceride metabolism and the deleterious effects of inefficient fat storage in the promotion of local IR and inflammation [START_REF] Mcdonough | Quantification of lipid droplets and associated proteins in cellular models of obesity via high-content/high-throughput microscopy and automated image analysis[END_REF]) [START_REF] Boden | Obesity, insulin resistance and free fatty acids[END_REF].

In states of metabolic diseases, the local IR through the participation of proinflammatory adipokines becomes systemic and it is maintained either directly affecting the insulin signaling pathway or indirectly via stimulation of inflammatory pathways, mostly involving TNF-α, IL-1β, IL-6, MCP1, resistin and PAI-1 generation. These mechanisms have been demonstrated recently in vivo through the high-fat diet (HFD) model [START_REF] Lee | Inflammation is necessary for long-term but not short-term high-fat dietinduced insulin resistance[END_REF]. Adipose tissue appears to be the first tissue responding to HFD and initiate inflammation in this obesity model. In fact three days of HFD were sufficient to deregulate adipocytokine production in C57BL/6J male mice and, continuing HFD, the inability of WAT to properly accumulate TGs gives rise to lipid storage in other tissues and tissue lipotoxicity. This process is now known to lead to the recruitment and activation of immune cells in key metabolic tissues, such as liver, muscles, and pancreas. Adipose tissue is not excluded by this immune-inflammatory response. Indeed, the active immune engagement was first demonstrated through transcription profile analysis of perigonadal adipose tissue from mice in which adiposity varied due to sex, diet, and genetic background [START_REF] Weisberg | Obesity is associated with macrophage accumulation in adipose tissue[END_REF]. The connection between the chronic low-grade inflammatory response of WAT and the associated immune and metabolic alterations can be l illustrated by the concept of "metainflammation" [START_REF] Lumeng | Inflammatory links between obesity and metabolic disease[END_REF]. Local and systemic proinflammatory and proatherogenic mediators, produced by adipocytes and endothelial cells respectively, enter the circulation to promote IR [START_REF] Shoelson | Obesity, inflammation, and insulin resistance[END_REF]) (Figure 8). The adipocyte-, immune cell-, and endothelial cell-derived substances create an inflammatory environment that promotes IR locally and entering the circulation systemically.

However, recently the negative paradigm on WAT inflammation causing insulin resistance has been challenged by an intriguing study showing that adipocyte inflammation is essential for healthy adipose tissue expandability and consequently reduced ectopic lipid deposition (Wernstedt Asterholm, Tao et al. 2014). In conditions of excess nutrients, inflammation would then be an adaptive response that enables WAT to store correctly the lipids, positively affecting metabolic profile. Extremely important for the inflammatory responses provoked by excess of nutrients, lipids in particular, in WAT are adipocyte surface receptors, in particular toll like receptors (TLRs) and G-protein-coupled receptors, which will be developed in Chapter f.

Liver: functions and dysfunctions

Metabolic functions

A healthy liver has the main function of regulating blood composition for the amounts of glucose (stored as glycogen), fat and protein entering the bloodstream from digested food. Processing most of the nutrients absorbed by the intestine during digestion, but also many medicines and alcohol, the liver helps the body to use vitamins and minerals as well as to eliminate poisons and toxins from the blood (i.e. bilirubin). Essential is also its role in producing proteins that help the blood to clot.

The traditional view of nutrient processing has been that after carbohydrate ingestion, insulin is secreted by the beta cells of the pancreas, increasing portal insulin concentrations and resulting in stimulation of glucose utilization and direct suppression of endogenous glucose production. Glucose production occurs in liver and kidney as well. Gluconeogenesis is the metabolic process through which organisms are capable of producing glucose from non-carbohydrate precursors, such as pyruvate, lactate, glycerol, and glucogenic amino acids. In glycolysis, the process occurs in reverse. In both processes, hepatic glucose metabolism is highly regulated at multiple levels. Through transcription, genes like cyclic AMP (cAMP) response element binding (CREB) [START_REF] Herzig | CREB regulates hepatic gluconeogenesis through the coactivator PGC-1[END_REF] or FoxOs [START_REF] Zhang | FoxO1 regulates multiple metabolic pathways in the liver: effects on gluconeogenic, glycolytic, and lipogenic gene expression[END_REF]) [START_REF] Haeusler | FoxOs function synergistically to promote glucose production[END_REF]) together with phosphoenol pyruvate carboxykinase (PEPCK) and glucose 6 phosphatase catalytic subunit (G6Pase), or on the other side glucokinase (GK)-the rate-limiting enzyme for hepatic glycolysis-contribute to the maintenance of glucose homeostasis (Figure. 9).

Figure 9. Diagram showing glycolytic and gluconeogenic pathways.

Since glycogen is the main storage form of glucose in the body, the process of its synthesis (glycogenesis) and breakdown (glycogenolysis) is carefully regulated as well. The fundamental form of control is the varied phosphorylation of glycogen synthase and glycogen phosphorylase by protein kinase A (PKA). In obesity and type II diabetes, defects in the regulation of glycogenolysis as well as gluconeogenesis have been shown to cause hepatic IR [START_REF] Basu | Obesity and type 2 diabetes impair insulin-induced suppression of glycogenolysis as well as gluconeogenesis[END_REF]. When carbohydrates are not converted into glycogen, they can be converted into fats and liver is the major organ responsible for converting excess dietary carbohydrate to lipid for storage. Lipogenesis is the process by which fatty acids, i.e. palmitate, are synthesized endogenously. Hepatocytes, under basal conditions, minimally -less than 5%-contribute to hepatic fat through the process of de novo lipogenesis [START_REF] Diraison | Role of human liver lipogenesis and reesterification in triglycerides secretion and in FFA reesterification[END_REF]. Under metabolically altered conditions, elevated plasma glucose (hyperglycemia) combined with high insulin concentrations (hyperinsulinemia) promote lipogenesis and impairs β-oxidation, thereby contributing to the development of hepatic steatosis. Moreover the proximity of the liver to intra-abdominal fat results in greater exposure to FFA than in peripheral tissues, explaining why IR can often develop sooner in this organ. In WAT the altered activity of important lipogenesis enzymes, such as ACC (acetyl-CoA carboxylase) FAS (fatty acid synthase), participate to the increased hepatic lipogenesis, enhancing liver fat deposition up to 30% of the organ [START_REF] Postic | Contribution of de novo fatty acid synthesis to hepatic steatosis and insulin resistance: lessons from genetically engineered mice[END_REF]. Conditions belonging to MetS, such as nonalcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) (see below), characterized by excessive lipid deposition, clearly illustrate how much de novo lipogenesis can be detrimental in liver. Transcription factors such as SREBP-1c and ChREBP, that coordinate the transcriptional regulation of enzymes directing the glycolytic end-products into lipogenesis, are of particular interest in those metabolic conditions, since they are at the interface between glucose and lipid metabolism [START_REF] Postic | Contribution of de novo fatty acid synthesis to hepatic steatosis and insulin resistance: lessons from genetically engineered mice[END_REF]. In summary, the above mentioned set of hepatic functions presents abnormalities in patients with IR, altered lipid metabolism and deregulated adipokines (e.g. adiponectin and leptin), features of metabolic disorders included in MetS.

Non-alcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFDL) is the major hepatic disorder in patients with MetS [START_REF] Watanabe | Liver diseases and metabolic syndrome[END_REF]. In conditions of positive energy balance (obesity) or reduced or absent fat storage capacity (drastic weight loss or lipodystrophy), IR leads to inefficient control of lipolysis in WAT.

As a consequence levels of lipids and lipoprotein variables are high in the bloodstream of those patients.

Liver processes them and a deregulated triglyceride (TG) deposition is observed in insulin-sensitive tissues, such as liver and muscle, that are not intended to be organs of fuel storage other than for glycogen. In general, cells displaying an abnormally large number of lipid droplets are often impaired in function and associated with disease pathologies. Both alcoholic and non-alcoholic fatty liver diseases present this abnormality within hepatocytes (Figure 10). Different sources of fatty acids contribute to the development of fatty liver. 1) fatty acids can reach the liver as circulating FFA, when derived from lipolysis in white adipose tissue. 2) Dietary fatty acids are taken up by the liver through the uptake of intestinally derived chylomicron (CM). 3) In liver, after the esterification step, TG can then be stored as lipid droplets or secreted into the blood as VLDL. Although obesity and alcoholism are two very different health issues, their impact on the liver, on the long run, can be quite similar, with comparable complications over time, such as gastrointestinal bleeding, cirrhosis and liver failure [START_REF] Marchesini | Obesity-associated liver disease[END_REF]. In both contexts, the development of liver damage is critically influenced by host factors including age, gender, ethnicity, dietary choices, presence of diabetes, genetic polymorphisms and more recently by the gut microbiome [START_REF] Zivkovic | Comparative review of diets for the metabolic syndrome: implications for nonalcoholic fatty liver disease[END_REF]) [START_REF] Neuman | Alcoholic and non-alcoholic steatohepatitis[END_REF]) [START_REF] Browning | Prevalence of hepatic steatosis in an urban population in the United States: impact of ethnicity[END_REF]) [START_REF] Bellentani | Prevalence of and risk factors for hepatic steatosis in Northern Italy[END_REF]. However, in the last cited Italian study, steatosis was more strongly associated with obesity than with alcohol consumption, suggesting a greater impact of body weight excess in the hepatic accumulation of fat. In fact, in developed countries the increase in Body Mass Index in the last decades goes in concert with NAFDL diagnosis. The reported prevalence of obesity with NAFLD varies between 30 and 100%, whereas the prevalence of NAFLD within Type 2 diabetes varies between 10 and 75%. Visceral adiposity, hyperglycemia, type II diabetes mellitus and hypertriglyceridemia are all metabolic risk factors that clearly identify NAFLD as the hepatic component of the metabolic syndrome. In the context of obesity, increased FFA and their metabolites, oxidative stress and altered profiles of adipocytokines follow the progression of liver pathology from steatosis alone, through the necroinflammatory disorder of non-alcoholic steatohepatitis (NASH) up to cirrhosis and liver cancer. Hepatic steatosis can be a noninflammatory condition, with no adverse consequences, for decades. NASH represents a shift from simple steatosis to an inflammatory condition characterized by marked hepatomegaly and cellular injury [START_REF] Bugianesi | Insulin resistance in nonalcoholic fatty liver disease[END_REF]. The steatohepatitis can rapidly progress to cirrhosis. It is, in fact, one of the most common causes of cirrhosis in lipodystrophic patients [START_REF] El-Zayadi | Hepatic steatosis: a benign disease or a silent killer[END_REF]. In order to investigate the etiology, pathogenesis and potential treatments for NAFLD, it has been inevitable to study the potential of proxy markers for the disease, due to the difficulty/impossibility of liver biopsy use. Among the richness of these biomarkers, more or less reliable, I will focus only on factors in line with the purpose of this Thesis.

NEFA concentrations have been proposed as a biochemical marker for NAFLD [START_REF] Byrne | Metabolic disturbances in non-alcoholic fatty liver disease[END_REF], since the initial accumulation of fat in liver is critical to the disease development [START_REF] Bugianesi | Insulin resistance in nonalcoholic fatty liver disease[END_REF]. Moreover, increased NEFA concentrations are associated with NAFLD independently of classic measures of insulin resistance. In fact, elevated NEFA concentrations have been associated with impaired glucose metabolism and impaired insulin secretion [START_REF] Pirro | Plasma free fatty acid levels and the risk of ischemic heart disease in men: prospective results from the Quebec Cardiovascular Study[END_REF]. However, it is important to consider the level variation throughout the day, when using NEFA concentrations as a biochemical marker for NAFLD. Another proposed biochemical marker for NAFLD is adiponectin, since high circulating adiponectin have been reported as protective against both alcoholic fatty liver disease and NAFLD. The decreased adiponectin level in NAFLD patients may represent a pathogenic mechanism in the disease [START_REF] Musso | Interactions among bone, liver, and adipose tissue predisposing to diabesity and fatty liver[END_REF]. Eventually measuring circulating triglycerides, in case of overnutrition, might be a good marker for liver status, since the degree of excess nutrient will reflect the severity of the lipotoxic insult, mediated by TNFα [START_REF] Feldstein | Free fatty acids promote hepatic lipotoxicity by stimulating TNF-alpha expression via a lysosomal pathway[END_REF]). The role of TNFα in mediating insulin resistance is long known in rodents [START_REF] Hotamisligil | Mechanisms of TNF-alpha-induced insulin resistance[END_REF].

Whereas in humans, increased TNFα (in association with increased presence of endotoxins in the blood = endotoxemia) in liver has been shown to have a role in the development of hepatic steatosis [START_REF] Ruiz | Lipopolysaccharide-binding protein plasma levels and liver TNFalpha gene expression in obese patients: evidence for the potential role of endotoxin in the pathogenesis of non-alcoholic steatohepatitis[END_REF]), but the precise role of TNF in human insulin signaling still requires extensive investigation [START_REF] Li | Adipokines and hepatic insulin resistance[END_REF]. Knowing that IR is often associated with chronic low-grade inflammation, it is clear how numerous mediators released from immune cells and adipocytes may contribute liver damage and liver disease progression. In fact, in the whole wide spectrum of liverdamage severity presented by NAFDL patients, insulin resistance is always present and therefore is considered the pathophysiological hallmark of NAFDL [START_REF] Abdelmalek | Nonalcoholic fatty liver disease as a complication of insulin resistance[END_REF]. Since IR possibly causes and worsens NAFLD, thiazolidinediones, a class of oral antidiabetic drugs that improve insulin sensitivity, were suggested as a potential therapeutical option, being able to reduce hepatic and peripheral IR, decrease hepatic steatosis, and attenuate the inflammatory response. However, in human trials, TZDs still need to prove their effectiveness [START_REF] Mahady | The role of thiazolidinediones in non-alcoholic steatohepatitis -a systematic review and meta analysis[END_REF]).

Intestine: functions and dysfunctions

The intestine, together with the adipose tissue, is the largest endocrine organ. The intestines include the small intestine, large intestine and rectum. The small intestine is a tubular structure divided into the duodenum, jejunum, and ileum. Thanks to its large inner surface area, small intestine conducts its main function of absorbing most of the nutrients from what we eat and drink, through simple passive diffusion (i.e. lipids) or active transport (i.e. glucose) across the epithelial cells of the gut into the blood stream. It is important to note that this organ has its own defense system. Aggregates of lymphoid follicles are spread throughout the small intestine, mostly within the ileum, where they are called Peyer's patches. Located in the lamina propria layer of the mucosa, their full function has yet to be defined. However they have the important role of monitoring intestinal bacteria populations and preventing the growth of pathogenic bacteria in the intestines [START_REF] Gao | Intestinal dysbacteriosis induces changes of T lymphocyte subpopulations in Peyer's patches of mice and orients the immune response towards humoral immunity[END_REF].

The large intestine (colon) performs the vital functions of converting food into feces, absorbing essential vitamins produced by gut bacteria, and reclaiming water from waste. Interestingly, due to its anatomical localization, the small intestine has a serial relationship with the liver relative to the absorption. This has been clearly observed for orally administered drugs that can reach the systemic circulation in reduced amounts due to both intestinal and hepatic metabolism. The intestinal and hepatic local environments, influenced by dietary choices, weigh changes, drugs, are studied with increasing attention in pharmacology as well as in pathophysiology due to their potential in affecting periphery organs and consequently systemic homeostasis.

Since the intestine has the rare property to be renewed within a few days, its capacity to adapt to its nutritional environment is currently under exploration. Research on how diet can affect the intestinal functions and as a consequence obesity-induced metabolic disorders is progressing. For example, using HFD models it is possible to study the associations between dietary lipid amounts and the magnitude of endotoxemia, inflammation and the physiological alterations developed [START_REF] Mani | Dietary oil composition differentially modulates intestinal endotoxin transport and postprandial endotoxemia[END_REF].

Dietary choices, such as a a high-fat meal, have been shown to increase plasma endotoxin in humans 4 hours after administration [START_REF] Erridge | A high-fat meal induces low-grade endotoxemia: evidence of a novel mechanism of postprandial inflammation[END_REF], suggesting a potential contribution to inflammation through leukocyte and endothelial cell activation when consumed regularly. When a diet rich in fat is administered for several weeks in rats, it was demonstrated to be associated with gut microbiota alteration, increased activation of TLR4 in the gut wall, increased epithelial inflammation and permeability, followed by increased in plasma lipopolysaccharide (LPS) (de La Serre, [START_REF] De La Serre | Propensity to high-fat diet-induced obesity in rats is associated with changes in the gut microbiota and gut inflammation[END_REF].

Similarly in BALB/c mice it was demonstrated that an in situ duodenal administration of butter rich in saturated fat was sufficient to markedly increase the production of TNF-α by lamina propria macrophages [START_REF] Fujiyama | Butter feeding enhances TNF-alpha production from macrophages and lymphocyte adherence in murine small intestinal microvessels[END_REF]). However, using a model of acute phase inflammation in the intestine of mice (bacterial administration, able to initiate the TLR2/4 signaling cascade) it was demonstrated that TLR and an increased immune response (DCs) could mediated an amelioration of disease [START_REF] Wittmann | TLR Signaling-induced CD103-expressing Cells Protect Against Intestinal Inflammation[END_REF]. These results suggest a positive role for the activation of certain inflammatory pathways in different contexts. Indeed, it has been hypothesized that the increased expression of TLRs in patients with inflammatory bowel disease might result in counter-regulation of inflammation and serve in preventing the disease.

Several studies from the group of P. Cani have clearly shown the relevance of gut microbiota in affecting host metabolism [START_REF] Cani | Metabolic endotoxemia initiates obesity and insulin resistance[END_REF][START_REF] Cani | Changes in gut microbiota control inflammation in obese mice through a mechanism involving GLP-2-driven improvement of gut permeability[END_REF][START_REF] Cani | Involvement of gut microbiota in the development of low-grade inflammation and type 2 diabetes associated with obesity[END_REF]. A recent study reported that gut microbiota changes corresponded to a metabolic adaptation to high fat diet [START_REF] Serino | Metabolic adaptation to a high-fat diet is associated with a change in the gut microbiota[END_REF]. Moreover it was shown that the increased paracellular permeability in the ileum and cecum of the HFD-fed mice facilitated the local (adipose cell size and stroma vascular fraction cell number) and sytemic (increased circulating adipokines) effects exerted by the different microbial community [START_REF] Serino | Metabolic adaptation to a high-fat diet is associated with a change in the gut microbiota[END_REF]. However, the precise mechanism by which intestine with this low but chronic increase in plasma LPS leads to insulin resistance [START_REF] Cani | Metabolic endotoxemia initiates obesity and insulin resistance[END_REF]) [START_REF] Naito | Beneficial effect of oral administration of Lactobacillus casei strain Shirota on insulin resistance in diet-induced obesity mice[END_REF]) deserves further characterization in murine models of obesity. Whereas in humans, the connection between dietary choices, gut microbiota and host metabolism will need to be explored and validated in physiological as well as pathological conditions. In particular, in the context of lipodystrophy, the potential link between development of IR, microbial communities, pattern recognition receptors and potential bacteria-derived inflammation, waits to be uncovered.

Muscle: functions and dysfunctions

In skeletal muscles the excess glucose residues are converted for storage as glycogen. Glycogen/glucose levels are finely regulated by degradation (glycogen phosphorylase) and accumulation (glucose transport rate and glycogen synthase activity). Given their critical role in the control of cellular energy metabolic pathways, PGC1-α (PPARGC1A), a transcriptional coactivator of nuclear receptors regulating mitochondrial genes involved in oxidative metabolism, and peroxisome proliferator-activated receptor δ (PPARδ)-mediated transcriptional pathway are implicated in the signaling network that controls skeletal muscles as well. Fiber-type transformation and metabolic profiles are regulated by PGC1-α and PPARδ, that protect muscles and other tissues against IR and obesity (Finck and Kelly 2006) [START_REF] Wang | Peroxisome-proliferator-activated receptor delta activates fat metabolism to prevent obesity[END_REF]. As for liver, the chronic increase of glucose and lipid availability, in the case of obesity, for example, may result in a metabolic conflict in metabolically sensitive organs like skeletal muscle. Plasma FFA levels are elevated in most obese and lipodystophic subjects. At the muscle level, FFA influence energy balance as well as metabolism, in particular insulin sensitivity [START_REF] Boden | Role of fatty acids in the pathogenesis of insulin resistance and NIDDM[END_REF]. In heart and muscle, FFA are primarily utilized as a source of energy for ATP synthesis through β-oxidation. However they can also regulate glucose utilization in skeletal muscles, in addition to insulin action [START_REF] Bergman | Non-esterified fatty acids and the liver: why is insulin secreted into the portal vein?[END_REF]. In fact, when FFA concentration increases, muscle glucose utilization can be considerably inhibited, possibly by interfering with insulin signaling and glucose transport, or phosphorylation or both [START_REF] Dresner | Effects of free fatty acids on glucose transport and IRS-1associated phosphatidylinositol 3-kinase activity[END_REF].

Obese patients can be exposed to permanent muscle weakness due to severe increased muscle lipid content. However diagnosis can be difficult, because lipid metabolism disorders are inconstantly leading to a muscle lipidosis and because of subjective evaluation of lipid content by pathologists [START_REF] Laforet | Disorders of muscle lipid metabolism: diagnostic and therapeutic challenges[END_REF]. Considering that the average lipid fraction of the fiber volume is estimated to less than 0.2% (Di Donato S, Taroni F. Disorders of lipid metabolism. In: Engel A, editor. Myology. New York: McGraw-Hill Professional Publishing;2004. p. 1587-615.), the magnitude of the lipotoxic insult depends on the duration of the excess of nutrients and on the oxidative capacity of the organ. The muscle is one of the primary organs affected by defects in cellular energy metabolism. Increased NEFA at the muscle level inhibit insulin sensitivity and insulin-stimulated glucose uptake. Consequently there are three main forms of metabolic myopathies linked to glycogen storage diseases, fatty acid oxidation defects, and mitochondrial disorders due to respiratory chain impairment [START_REF] Volpi | Metabolic myopathies: functional evaluation by different exercise testing approaches[END_REF]. Patients with a diagnosis in metabolic myopathies can be subjected to cardiac complications, such as cardiomyopathy. Being heart disease is the leading cause of death in patients with diabetes, studying muscle, in general, but heart, in particular, dysfunctions in human and in animal models will allow better discerning the pathophysiologic link between diabetes and heart disease in settings of disrupted insulin signaling or lipid homeostasis [START_REF] Matsuzawa | Peripheral endothelial function and cardiovascular events in high-risk patients[END_REF]).

D. Metabolic diseases: Innate and adaptive immunity

The immunometabolism is an exciting new field emerged from the awareness of the immune system contribution to metabolic processes [START_REF] Mathis | Immunometabolism: an emerging frontier[END_REF]. In recent years studies on immunometabolism have been growing exponentially based on the hypothesis that obesity is a complex immune-mediated disease. The multilevel interactions between metabolism and immune system involve the innate and adaptive immune cells in multiple metabolic organs. Although the critical role of the immune system in surveillance and modulation of metabolism in those tissues is recognized, adipose is definitely the most studied tissue [START_REF] Huh | Crosstalk between adipocytes and immune cells in adipose tissue inflammation and metabolic dysregulation in obesity[END_REF]. It is in fact in the obesity context that the field of immunometabolism truly blossomed, starting from the concept of "adipose tissue inflammation" with the interaction between adipocytes and macrophages. The complexity of adipose tissue is represented by the variety of cell types that peacefully coexist in it. Next to adipocytes, preadipocytes, fibroblasts, endothelial cells and multipotent stem cells, adipose tissue contains immune cells. Details on the adipose tissue unique immune response have been emerging only recently. Resident WAT immune cells belong to both innate (neutrophils, macrophages, mast cells, eosinophils) and adaptive (regulatory T-cells, type 1 helper T-cells, CD8 T-cells, B cells) immunity [START_REF] Ferrante | The immune cells in adipose tissue[END_REF]. Immune cells, able to induce lypolysis, when they need to fuel the immune response, collaborate with each other to promote immunity and control inflammation and tissue homeostasis. In adipose tissue disorders, such as those encountered in obesity and lipodystrophy, alterations in WAT distribution and function are associated with broad effects, from cytokine, chemokine, and hormone expression changes to basal systemic inflammation [START_REF] Makki | Adipose tissue in obesity-related inflammation and insulin resistance: cells, cytokines, and chemokines[END_REF]. Clinical and preclinical studies in these two extreme and opposite ends of the adiposity spectrum, have shown that cells of the myeloid and lymphoid lineage are recruited and may play a causal role in the associated downstream complications, from local inflammation to systemic IR. The different immune-cell populations (innate, bridging and adaptive) that reside in the adipose tissue will be presented in the next pages, with a particular focus on obesityassociated alterations, the most studied condition (Figure.11).

Innate immunity

The innate immune system is composed of a diverse array of evolutionarily ancient haematopoietic cell types, including dendritic cells, monocytes, macrophages, mastocytes and granulocytes. Unlike the adaptive immune system, present only in vertebrates, innate immune system is the first line of defense in plants, fungi, insects, and primitive organisms as well, such as bacteria. The major functions of the vertebrate innate immune system include: recognition, recruitment, removal, and not least activation of the adaptive immune system (antigen presentation). In order to perform the recognition task, innate immune cells present receptors, contained on the surface or within the cell, named pattern recognition receptors (PRRs), including Toll-like receptors and cytoplasmic receptors, functionally there to recognize molecules that are broadly shared by pathogens, referred to as pathogen-associated molecular patterns (PAMPs) [START_REF] Akira | Pathogen recognition and innate immunity[END_REF]. In a broader view, innate immunity evolved towards discrimination of self from nonself, meaning not only molecular patterns defining infectious nonself, but also normal and abnormal self [START_REF] Medzhitov | Decoding the patterns of self and nonself by the innate immune system[END_REF]) [START_REF] Coers | Self and non-self discrimination of intracellular membranes by the innate immune system[END_REF]). In fact innate immune cells can recognize endogenous stress signals released during cell damage, so called damageassociated molecular patterns (DAMPs). Both PAMPs and DAMPs serve as a starting signal (signal zero) in response to environmental stress, preceeding the later signals that will trigger the adaptive immune response (Janeway 1989) (Figure 12). These same signals have been shown to serve to protect the cell itself in response to cellular stress. Through promotion of autophagy, a conserved lysosomal degradation pathway, the cell can control the degradation of self-constituents, i.e. cellular damage control, or the degradation of foreign invaders, called xenophagy [START_REF] Tang | PAMPs and DAMPs: signal 0s that spur autophagy and immunity[END_REF]. In this sense, autophagy represents an innate defense mechanism thanks to its role as earliest host defense strategies [START_REF] Gutierrez | Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages[END_REF]) [START_REF] Tang | PAMPs and DAMPs: signal 0s that spur autophagy and immunity[END_REF].

Figure 12. Initial signals play critical roles in autophagy and immunity (Tang, Kang et al. 2012).

Innate immune pathways have evolved not only to recognize pathogens, but also to assess the nutrient environment. An example of DAMPs, important for the context of this Thesis, is represented by lipids. Lipids, found in the adipocyte surrounding when an adipocyte dies (apoptosis, necrosis, pyroptosis), could be the endogenous ligands to which the immune system responds (this part will be discussed more in detail in the Chapter d). FFA can activate innate cells via the signaling pathway of TLR4 that can sense their presence [START_REF] Nguyen | A subpopulation of macrophages infiltrates hypertrophic adipose tissue and is activated by free fatty acids via Toll-like receptors 2 and 4 and JNK-dependent pathways[END_REF]. Another example of nutrient sensing is represented by inflammasome that can be activated by ATP [START_REF] Mariathasan | Cryopyrin activates the inflammasome in response to toxins and ATP[END_REF]. Once one, or more, of similar receptors recognize a PAMP or a DAMP, the inflammatory and innate response is organized rapidly, right at the onset of an infection, burn, or injury (i.e. local lipotoxic event). All the innate immune cells can participate to inflammation, balancing a variety of potential responses, from cytokine production to tissue repair, depending by the context and the organ involved.

Mast cells

Mast cells are a type of innate immune cell that reside in connective tissue and in the mucous membranes. However they also have a role as effector cells in adaptive immunity [START_REF] Galli | Mast cells in the development of adaptive immune responses[END_REF]. They are intimately associated with wound healing and defense against pathogens, but are also often associated with allergy and anaphylaxis. Many inflammatory diseases involve mast cells in crosstalk with T-cells, such as atopic dermatitis, psoriasis and multiple sclerosis [START_REF] Voehringer | Protective and pathological roles of mast cells and basophils[END_REF]. When activated, mast cells rapidly release characteristic granules, rich in histamine and heparin, along with various hormonal mediators. Tryptases and chymases are the major proteins stored and secreted by mast cells and depending on mast cell subtype, tissue, and mammal of origin can modulate inflammation, matrix destruction, and tissue remodeling [START_REF] Caughey | Mast cell tryptases and chymases in inflammation and host defense[END_REF]. Mast cells play also a role in host defense, releasing chemotactic molecules into the environment that recruit other innate cells, such as neutrophils and macrophages [START_REF] Conti | Impact of Rantes and MCP-1 chemokines on in vivo basophilic cell recruitment in rat skin injection model and their role in modifying the protein and mRNA levels for histidine decarboxylase[END_REF]. Mast cells can be triggered by a variety of physiological factors, including auto-secreted molecules, and environmental factors, such as stimulants of Toll-like receptors [START_REF] Sandig | TLR signaling in mast cells: common and unique features[END_REF]. Depending on the trigger, their location and stage of maturation, mast cells express different types and levels of surface antigens and are able to produce multiple effector molecules, i.g. pro-or anti-inflammatory cytokines [START_REF] Bischoff | Interleukin-4 induces a switch of human intestinal mast cells from proinflammatory cells to Th2-type cells[END_REF]. For example at the intestinal level, TLRs, well known for their role in lipids sensing, could be involved in the activation of mucosal mast cells observed when an infusion of polyunsaturated fatty acid-rich lipid emulsion was administered inside the duodenal tract [START_REF] Ji | Nutrient-induced inflammation in the intestine[END_REF]. Possibly activated through similar pathways, these innate cells have been implicated in the pathogenesis of diet-induced metabolic disease. In this study, our group showed using genetic and pharmacologic approaches that when mast cells are absent, mice were protected from obesity-induced metabolic syndrome [START_REF] Liu | Genetic deficiency and pharmacological stabilization of mast cells reduce diet-induced obesity and diabetes in mice[END_REF]. As for a majority of immune cells their implication in WAT inflammation was recently linked to tissue remodeling, the process characteristic of obese adipose tissue, in mice [START_REF] Liu | Genetic deficiency and pharmacological stabilization of mast cells reduce diet-induced obesity and diabetes in mice[END_REF]) [START_REF] Hirai | Involvement of mast cells in adipose tissue fibrosis[END_REF]) and humans as well (Divoux, Tordjman et al. 2010). However, mast cells have been observed promoting fibroblast growth and collagen synthesis also in other tissues, such as liver [START_REF] Armbrust | Mast cells distribution in human liver disease and experimental rat liver fibrosis. Indications for mast cell participation in development of liver fibrosis[END_REF]) [START_REF] Shen | A target role for mast cell in the prevention and therapy of hepatic fibrosis[END_REF]) and lung [START_REF] Edwards | c-Kit immunophenotyping and metalloproteinase expression profiles of mast cells in interstitial lung diseases[END_REF].

Neutrophils

Belonging to the innate immune system, neutrophils are the leukocyte subpopulation arriving first in case of acute inflammation, with the primary function of producing MCP-1 and other chemokines to promote the recruitment of inflammatory monocytes [START_REF] Soehnlein | Neutrophil secretion products pave the way for inflammatory monocytes[END_REF]. As mast cells, neutrophils also accumulate in WAT during obesity [START_REF] Rouault | Roles of chemokine ligand-2 (CXCL2) and neutrophils in influencing endothelial cell function and inflammation of human adipose tissue[END_REF] and it is possible that they have a close modus operandi. Their transient infiltration is associated with the weight gain and IR caused by high-fat feeding (Elgazar-Carmon, Rudich et al. 2008). Neutrophils accumulate already 1 hour after a high caloric meal, rich in glucose or fat, in association with macrophages, and the expression of their specific activation markers was observed elevated [START_REF] Mohanty | Glucose challenge stimulates reactive oxygen species (ROS) generation by leucocytes[END_REF][START_REF] Van Oostrom | Activation of leukocytes by postprandial lipemia in healthy volunteers[END_REF]). Neutrophils accumulate already 1 hour after a high caloric meal, rich in glucose or fat, in association with macrophages, and the expression of their specific activation markers was observed elevated [START_REF] Mohanty | Macrophages are targeted by rotavirus in experimental biliary atresia and induce neutrophil chemotaxis by Mip2/Cxcl2[END_REF]) [START_REF] Kim | Neuroproteomics: expression profiling of the brain's proteomes in health and disease[END_REF]).

In WAT and liver of diet-induced obese mice, the activity of neutrophil-specific elastase represents a good marker of the amount of infiltrated neutrophils. Notably, neutrophil elastase can degrade the insulin receptor substrate 1 and reduce insulin-induced Akt phosphorylation in adipocytes [START_REF] Talukdar | Neutrophils mediate insulin resistance in mice fed a high-fat diet through secreted elastase[END_REF]. Through this mechanism neutrophils could participate in obesity-associated IR, in tandem with activation of the TLR-4-dependent AT inflammation. Moreover neutrophils were shown to be involved in tissue damage via the production of MMP-9 and reactive oxygen species [START_REF] Mantovani | Neutrophils in the activation and regulation of innate and adaptive immunity[END_REF]). More recently, our team, using a cocultures of adipocytes and endothelial cells in a 3D Setting, proved that conditioned media from neutrophils from obese subjects have increased angiogenic capacities, possibly though the release of multiple angiogenic molecules ( IL-8, CXCL4, MMP-8, MMP-9, TIMP1) [START_REF] Rouault | Roles of chemokine ligand-2 (CXCL2) and neutrophils in influencing endothelial cell function and inflammation of human adipose tissue[END_REF]. However, despite this progress, certainly more mechanisms remain to be discovered in order to elucidate the apparent contradiction of elastase deficiency in obesity [START_REF] Chmelar | The role of innate immune cells in obese adipose tissue inflammation and development of insulin resistance[END_REF].

Macrophages

Macrophages are a heterogeneous population of myeloid-derived mononuclear cells that are a critical component of the innate immune response [START_REF] Gordon | Monocyte and macrophage heterogeneity[END_REF]. They are resident in all tissues of the body, being particularly augmented in tissues that are frequently exposed to exogenous and endogenous antigens and toxins, such as the lungs and liver. Their high plasticity allows these monocyte-derived cells to adapt to the environment and the different bioactive molecules they are exposed to [START_REF] Dalmas | Adipose tissue, a new playground for immune cells[END_REF]. Once abandoned circulation, macrophages can entry to normal tissues (M2-like macrophages, "resident") or be recruited to inflammatory sites (infections, injury/cell death and exposure to toxic substances, such as FFA in obesity) returning to basal state when the inflammation is resolved (M1-like macrophages, "effector") [START_REF] Italiani | From Monocytes to M1/M2 Macrophages: Phenotypical vs. Functional Differentiation[END_REF]. Notably, when inflammation becomes chronic, as observed in the pathological conditions comprised in MetS, macrophages and immune system in general steadily participate in the pathophysiology of the associated metabolic abnormalities. However, it is important to consider that the definition of M1 and M2 macrophage immune activation (classic vs. alternative, also M1 and M2, respectively) originated from the pre-genomic era, when few markers could be used to establish differences and similarities in macrophage responses to stimuli [START_REF] Martinez | The M1 and M2 paradigm of macrophage activation: time for reassessment[END_REF]. At present, we know that precisely defining in vivo a functional phenotype of macrophages based on this dichotomy is quite hard. Knowing that M1 and M2 stimuli do not exist alone in tissues and that, depending on micro-environmental signal complexity, macrophages assume and/or dynamically change their phenotype, the many and apparently opposed macrophage functions have to be acknowledged [START_REF] Martinez | The M1 and M2 paradigm of macrophage activation: time for reassessment[END_REF]. Currently, this over-simplified view of the two functionally polarized states M1 and M2 based on selected macrophage markers is still found useful to characterize stimuli effects in specific contexts [START_REF] Mantovani | The chemokine system in diverse forms of macrophage activation and polarization[END_REF]) (Figure 13). For example, in the context of specific pathologies, such as autoimmunity, sepsis, cellular stress, or fibrosis, macrophages are defined M1-like, since they respond producing a panel of M1 (Th-1) proinflammatory cytokines, the prototypical ones being TNFα, IFNγ and IL1β [START_REF] Huang | Depletion of liver Kupffer cells prevents the development of diet-induced hepatic steatosis and insulin resistance[END_REF]. However in vivo the macrophage populations are much more dynamic, as demonstrated by the mixed M1/M2 phenotype observed for ATMs in obesity and in lipodystrophy, although with very different characteristics (Herrero, Shapiro et al. 2010).

Adipose tissue represents a lipid-rich environment, where macrophages develop and differentiate, most probably adapting to it. In conditions where lipid homeostasis is altered, excess of saturated fatty acids affect the adipose tissue at multiple cellular levels, in particular for macrophages affecting their number, activation state and function [START_REF] Hill | A decade of progress in adipose tissue macrophage biology[END_REF]). Nevertheless, the developmental program and tissue-specific functions of ATMs, i.g. lipid metabolism, have not been deeply explored yet, either in normal or pathological settings. Cellular functions of ATMs are possibly regulated by adiposity and conversely ATMs have been shown to participate in local lipid turnover [START_REF] Xu | Obesity activates a program of lysosomal-dependent lipid metabolism in adipose tissue macrophages independently of classic activation[END_REF]. In metabolically altered conditions, macrophage number is increased in WAT, due to the induction of a precise transcriptional program of chemokines, such as CC chemokine ligand 2 (CCL2), CCL5, and CCL3, as well as their receptors, CC chemokine receptor 2 (CCR2), and CCR5. However, scientific literature shows conflicting results about these mechanisms of recruitment [START_REF] Xu | Obesity activates a program of lysosomal-dependent lipid metabolism in adipose tissue macrophages independently of classic activation[END_REF]. Aside from blood monocyte recruitment possibly due to adipocyte death, chemokine or FFA release, macrophage number can be influenced by recruitment-independent mechanisms such as impaired apoptosis, decreased tissue egress (migration) and increased in situ proliferation [START_REF] Amano | Local proliferation of macrophages contributes to obesityassociated adipose tissue inflammation[END_REF].

In obesity, next to systemic adiposity, insulin sensitivity is tightly correlated with macrophage number in WAT. The role of macrophages in influencing insulin sensitivity has been demonstrated also in liver and muscles. Liver-specific macrophages, named Kupffer cells (KCs), have a role in high fat or high sucrose diet-induced alterations in hepatic lipid metabolism and insulin sensitivity. Interestingly, in vitro stimulation of KCs with saturated fatty acids alone was sufficient to induce decreases in hepatocyte fatty acid oxidation and increases in fatty acid esterification, although with no alteration of triglyceride levels [START_REF] Huang | Depletion of liver Kupffer cells prevents the development of diet-induced hepatic steatosis and insulin resistance[END_REF]. Similarly, in a mouse model of hepatic steatosis and lipoatrophy (A-ZIP/F-1 transgenic mice), lipid accumulation in the liver leads to an increased macrophage recruitment, as a consequence of the high local and circulating level of CCL2, in association with a proinflammatory state [START_REF] Yang | Inhibition of the chemokine (C-C motif) ligand 2/chemokine (C-C motif) receptor 2 pathway attenuates hyperglycaemia and inflammation in a mouse model of hepatic steatosis and lipoatrophy[END_REF]). The association between circulating FFA and inflammation has been demonstrated also in skeletal muscle from obese subjects, in which an higher macrophage infiltrate was reported [START_REF] Varma | Muscle inflammatory response and insulin resistance: synergistic interaction between macrophages and fatty acids leads to impaired insulin action[END_REF]). Various studies have proposed a potential contribution of lipid intermediate accumulation and the associated immuneinflammatory response in muscles to the reduction of insulin sensitivity [START_REF] Kraegen | Muscle insulin resistance: a case of fat overconsumption, not mitochondrial dysfunction[END_REF].

Finally, though aware of the primary role of these cells, i.e. antigen presentation, it is important to follow closely recent evidence about their metabolic role. Indeed macrophages are able to substantially participate to iron homeostasis regulation in different settings and with specific functions [START_REF] Recalcati | Differential regulation of iron homeostasis during human macrophage polarized activation[END_REF]) [START_REF] Ganz | Macrophages and systemic iron homeostasis[END_REF]) [START_REF] Jung | Macrophage iron homeostasis and polarization in the context of cancer[END_REF]. Moreover macrophages could influence insulin sensitivity, behind their participation to tissue inflammation [START_REF] Lee | Macrophage metalloelastase (MMP12) regulates adipose tissue expansion, insulin sensitivity, and expression of inducible nitric oxide synthase[END_REF])

Dendritic cells

Following over time the research that worked on defining and characterizing different populations of dendritic cells (DCs) the complexity of the DC family is evident. At the present we know that, these sentinels of the immune system [START_REF] Banchereau | Dendritic cells and the control of immunity[END_REF], whose main goal is to survey the tissues and instruct the adaptive immune system in response to peripheral cues, can be found in lymphoid organs but also in most non-lymphoid tissues [START_REF] Merad | The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting[END_REF]). An additional complexity is that DCs, contrary to macrophages, do not have phenotypic markers that can specifically identify them. Thanks to their superior ability to migrate toward lymph nodes [START_REF] Forster | Lymph node homing of T cells and dendritic cells via afferent lymphatics[END_REF], DCs are extremely effective cells for processing and presenting phagocytosed antigens to T lymphocytes. Their role in inducing both protective immunity as well as immunological tolerance is well represented in case of tissue injuries (inflamed state) and skin immunosurveillance (steady state) [START_REF] Ginhoux | Blood-derived dermal langerin+ dendritic cells survey the skin in the steady state[END_REF]. Depending on the nature of the stimuli and the tissues in which they arise, DCs phenotype will change in response to inflammation. The regulation of differentiation and function of DCs has been mostly explored in tissues such as skin, lung and intestine, whereas interest on IR resistance, concerning DCs involvement in the chronic low inflammatory grade characterizing WAT, are not that numerous. Obese mice and humans present the accumulation of these innate cells possibly contributing to insulin resistance ( [START_REF] Wu | CD11c expression in adipose tissue and blood and its role in dietinduced obesity[END_REF]) [START_REF] Yekollu | Targeting curcusomes to inflammatory dendritic cells inhibits NF-kappaB and improves insulin resistance in obese mice[END_REF]. More recently, a specific set of DCs has been shown in obese WAT intervening in the complex process of obesityassociated IR [START_REF] Bertola | Identification of adipose tissue dendritic cells correlated with obesity-associated insulin-resistance and inducing Th17 responses in mice and patients[END_REF]. Those DCs take part in the regulation of tissue inflammation, influencing the induction of a Th17 cell response that could substantially contribute IR [START_REF] Bertola | Identification of adipose tissue dendritic cells correlated with obesity-associated insulin-resistance and inducing Th17 responses in mice and patients[END_REF]. Once again FFA, elevated in the dysfunctional WAT environment, i.e. obesity and lipodystrophy, are possibly the starting point of the local inflammation causing IR, as for macrophages [START_REF] Nguyen | A subpopulation of macrophages infiltrates hypertrophic adipose tissue and is activated by free fatty acids via Toll-like receptors 2 and 4 and JNK-dependent pathways[END_REF].

Natural killer cells

As for the other innate immune cells, natural killer cells (NK) can be found in a variety of tissues with the primary functions of controlling inflammation and maintaining immune homeostasis [START_REF] Fu | Subsets of human natural killer cells and their regulatory effects[END_REF]. Depending on the microenvironment, NK cells can have distinct functions, as NK(tolerant) , NK(cytotoxic) and NK(regulatory) cells, inhibiting or enhancing immune reactions. Mainly known for their regulatory proprieties in various disease contexts, such as rheumatoid arthritis joints [START_REF] Pridgeon | Natural killer cells in the synovial fluid of rheumatoid arthritis patients exhibit a CD56bright,CD94bright,CD158negative phenotype[END_REF] or hepatitis B therapy [START_REF] Micco | Differential boosting of innate and adaptive antiviral responses during pegylated-interferon-alpha therapy of chronic hepatitis B[END_REF]), many aspects and functions of NK cells remain to be unveiled, especially their phenotype and function in adipose tissue [START_REF] Caspar-Bauguil | Adipose tissues as an ancestral immune organ: site-specific change in obesity[END_REF]) (O'Rourke, Gaston et al. 2013) [START_REF] Chung | Role of NKG2D in obesity-induced adipose tissue inflammation and insulin resistance[END_REF]. The detailed study of NK cells infiltrating adipose tissue of obese subjects has revealed that these cells, known to be able to recognize lipid antigens, present a specific activated phenotype, prone to promote IR and WAT inflammation in obesity (O'Rourke, [START_REF] O'rourke | Adipose tissue NK cells manifest an activated phenotype in human obesity[END_REF]. And even more recently the potential mechanisms through which these cells promote visceral adipose tissue inflammation and insulin resistance in obesity were elucidated: a phenotypically distinct population of tissue-resident natural killer (NK) cells was described to be able to proliferate in situ and through interferon-γ (IFN-γ) production to induce adipose stress and pro-inflammatory macrophage differentiation in obesity (Wensveen, Jelencic et al. 2015).

Adaptive immunity

The adaptive immune system, in collaboration with innate cell and non-haematopoietic cell populations, has the complex role of promoting immunity and regulating tolerance at the same time. Recent findings have clearly demonstrated the presence of adaptive lymphocytes, such as T and B cells, in adipose tissue, even if their function there has not be well established yet [START_REF] Chatzigeorgiou | Lymphocytes in obesity-related adipose tissue inflammation[END_REF].

For the purpose of this Thesis the alterations related to the state and function of adaptive cells will focus on the metainflammatory response to obesity, therefore on adipose tissue.

Lymphocytes

Compared to secondary lymphoid tissues adipose tissue contains few naïve T-cells and a high amount of effector/memory type CD4+ T-cells, with the main role of regulating adaptive immunity. During obesity chronic inflammation involves altered lymphocyte function, in addition to the previously described macrophage accumulation. In particular T-cells with a prevalent pro-inflammatory role accumulate in WAT, contributing to the overall inflammatory state associated to the development of metabolic abnormalities. Meanwhile resident T-cells, known for their anti-inflammatory profile, are reduced in obesity, often depleted [START_REF] Chatzigeorgiou | Lymphocytes in obesity-related adipose tissue inflammation[END_REF]. A clear example is given by regulatory Tcells, whose presence may represent a crucial factor to preserve insulin sensitivity. In abdominal WAT of normal mice an abundant population of CD4(+) Foxp3(+) T regulatory (T(reg)) cells was found, but their amount was significantly decreased in insulin-resistant models of obesity [START_REF] Feuerer | Lean, but not obese, fat is enriched for a unique population of regulatory T cells that affect metabolic parameters[END_REF].

All the different subpopulations of lymphocytes, such as Th1 lymphocytes (CD4+), cytotoxic lymphocytes (CD8+) or B cells have been implicated in AT inflammation [START_REF] Chatzigeorgiou | Lymphocytes in obesity-related adipose tissue inflammation[END_REF]), [START_REF] Nishimura | CD8+ effector T cells contribute to macrophage recruitment and adipose tissue inflammation in obesity[END_REF]). In the case specific of T-cells, using the high fat diet-induced obesity model, it has been shown that different sets of T-cells increase in number and activation state in WAT ( [START_REF] Xu | Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance[END_REF], [START_REF] Wu | T-cell accumulation and regulated on activation, normal T cell expressed and secreted upregulation in adipose tissue in obesity[END_REF]). Obese adipose tissue has been hypothesized as a unique playground where the dominating ATMs interact with T-cells to establish a crucial network that perpetuates metainflammation [START_REF] Cho | An MHC II-dependent activation loop between adipose tissue macrophages and CD4+ T cells controls obesity-induced inflammation[END_REF]. Interestingly, a T-cell accumulation, similar to the one described in obese adipose tissue, has been recently reported in a mouse model of weight cycling [START_REF] Anderson | Weight cycling increases T-cell accumulation in adipose tissue and impairs systemic glucose tolerance[END_REF] In this study, mice were cycled between HFD and low-fat diet (LFD) for three 9-week diet periods and their metabolic and immunological profile was evaluated compared with mice that gain weight under HFD for the duration of the study (27 weeks). The increased accumulation of CD4(+) and CD8(+) in WAT of weight cycling mice demonstrate that an altered local adaptive immunity can participate to worsen metabolic dysfunction, not only during obesity but also when weight loss alternates with weight regain (weight cycling) [START_REF] Anderson | Weight cycling increases T-cell accumulation in adipose tissue and impairs systemic glucose tolerance[END_REF]. In obesity, the migration of these pro-inflammatory T-cells in WAT is coordinated by RANTES/CCL5, a chemokine specifically increased in obese WAT [START_REF] Wu | T-cell accumulation and regulated on activation, normal T cell expressed and secreted upregulation in adipose tissue in obesity[END_REF] and not involved in T-cells accumulation during weight cycling. The pathophysiological significance of the increased T-cells in AT in obesity is under current investigation. Ballantyne and colleagues hypothesized that T-cells reduce the ability of accumulating TG in WAT, as observed in vitro during adipocyte differentiation, and this could increase ectopic TG deposition in liver and skeletal muscle, and thus cause IR in these organs [START_REF] Wu | T-cell accumulation and regulated on activation, normal T cell expressed and secreted upregulation in adipose tissue in obesity[END_REF]). More recently Lumeng and colleagues demonstrated the importance of MHC Class II (major histocompatibility complex class II) in activating and maintaining the loop between adipose tissue resident CD11c-, T-cells, and CD11c+ responsible for metainflammation [START_REF] Cho | An MHC II-dependent activation loop between adipose tissue macrophages and CD4+ T cells controls obesity-induced inflammation[END_REF].

Finally, despite the well-established demonstration that adipokines have a role in modulating the function of immune cells, in particular T-cells [START_REF] Fantuzzi | Defining the role of T cell-derived leptin in the modulation of hepatic or intestinal inflammation in mice[END_REF], the interactions between lymphocytes and adipocytes has received the full attention only recently.

Natural killer T-cells

Natural killer T-cells (NKT) are a heterogeneous group of T-cells that share properties of both Tcells and natural killer cells. They have previously been associated with obesity for their potential regulatory phenotype [START_REF] Caspar-Bauguil | Adipose tissues as an ancestral immune organ: site-specific change in obesity[END_REF]. Similarly to NK cells, NKT cells have been shown to contribute to inflammation in obesity [START_REF] Sintes | Cutting edge: Ly9 (CD229), a SLAM family receptor, negatively regulates the development of thymic innate memory-like CD8+ T and invariant NKT cells[END_REF]). In the obesity context, the abundance of self and foreign lipid antigens in AT works well with the lipid-sensitive and antigenpresentation functions carried out by these cells (lipid/CD1d complex binding). Similar mechanisms have been proposed for another family of NKT cells, invariant natural killer T (iNKT), shown to be involved in multiple metabolic diseases [START_REF] Hammond | alpha/beta-T cell receptor (TCR)+CD4-CD8-(NKT) thymocytes prevent insulin-dependent diabetes mellitus in nonobese diabetic (NOD)/Lt mice by the influence of interleukin (IL)-4 and/or IL-10[END_REF]) [START_REF] Hong | The natural killer T-cell ligand alpha-galactosylceramide prevents autoimmune diabetes in non-obese diabetic mice[END_REF]) [START_REF] Lee | Testing the NKT cell hypothesis of human IDDM pathogenesis[END_REF]) [START_REF] Matzinger | Tissue-based class control: the other side of tolerance[END_REF]. As for the above mentioned T-regs, iNKT cells are an example of anti-inflammatory cells greatly reduced in obesity [START_REF] Lynch | Adipose tissue invariant NKT cells protect against diet-induced obesity and metabolic disorder through regulatory cytokine production[END_REF]). This evolutionarily conserved innate NK subpopulation represents a large proportion of lymphocytes in human and murine adipose tissue, and their number can be rescued after weight loss. The ability of iNKT cells to decrease body fat, circulating TG and consequently fatty liver, and improve insulin sensitivity in mice offers an interesting potential prospective for the management of human obesity [START_REF] Lynch | Adipose tissue invariant NKT cells protect against diet-induced obesity and metabolic disorder through regulatory cytokine production[END_REF].

Helper T-cells

Helper T-cells (Th) are probably the most important cells in adaptive immunity, as they are required for almost all adaptive immune responses. Through the release of T-cell cytokines, these cells help suppress or regulate immune responses of other immune cells, not only B-and T-cells, but also macrophages (at site of infection). Th effector cells stimulate an inflammatory response by recruiting more phagocytic cells into the infected site. They can secrete cytokines that act on the bone marrow to increase the production of monocytes and neutrophils, they can secrete chemokines and increase adhesion capacity of endothelial cells to allow more efficient recruitment of innate cells from the blood into the site of infection. In obesity, as regulatory T-cells, the number of Th effector cells has been shown to diminish.

In HFD-fed mice CD4(+) helper cells decrease in epididymal adipose tissue [START_REF] Nishimura | CD8+ effector T cells contribute to macrophage recruitment and adipose tissue inflammation in obesity[END_REF]. However in humans the balance of Th1/Th2 has been shown to be modulated by leptin cells toward a Th1 phenotype and the pro-inflammatory profile of the secreted cytokines may participate in the in obesity-associated inflammation [START_REF] Pacifico | Increased T-helper interferon-gamma-secreting cells in obese children[END_REF].

Gamma delta T-cells

Gamma delta+ T (γδ T)-cells represent a small population of overall T-lymphocytes (0.5-5%) and have variable tissue distribution in the body. The functions of γδ T-cells have been studied mostly in relation to epithelial damage in obesity. When γδ T-cells reside in the epidermis, they are also referred to as dendritic epidermal T-cells. These cells are impaired in number and activity in the skin of obese mice, compromising their ability to resolve tissue damage [START_REF] Taylor | Gammadelta T cells are reduced and rendered unresponsive by hyperglycemia and chronic TNFalpha in mouse models of obesity and metabolic disease[END_REF]. In obesity, metabolic disease, and type 2 diabetes, these epidermal cells become dysfunctional due the chronic inflammatory environment. Their negative influence on keratinocyte homeostasis is one of the mechanisms involved in the commonly associated skin pathologies [START_REF] Taylor | Dysfunctional gammadelta T cells contribute to impaired keratinocyte homeostasis in mouse models of obesity[END_REF]. FACS analysis of the stromavascular fraction of white adipose tissues demonstrated that the regulation of T cells in adipose tissue could be depot dependent in obesity [START_REF] Caspar-Bauguil | Adipose tissues as an ancestral immune organ: site-specific change in obesity[END_REF]. In fact, using a diet rich in fat, it was shown that lymphocytes in epididymal WAT display an "ancestral" immune system phenotype, with up to 70% of natural killer (NK), γδ T-and NKT cells among all lymphocytes. Moreover, obesity decreased the percentage of NK cells in epidydymal fat, whereas increased γδ T-cells in subcutaneous fat. However, only more recently the potential role of this particular subpopulation of adaptive cells has been investigated in the context of metabolic disorders. In 2015, research conducted on at Baylor College of Medicine in Houston (Texas) suggested that γδ T-cells could be used to treat and prevent type 2 diabetes caused by obesity (Mehta, Nuotio-Antar et al. 2015). Mice lacking γδ T-cells had reduced inflammation in the liver, skeletal muscle and adipose tissue, and a consequent reduced systemic IR, under a Western-type diet, rich saturated fats and sugar (Mehta, Nuotio-Antar et al. 2015).

Consequently, the phenotypic and functional plasticity of γδ T-cells, in particular in the cross-talk with other immune cells, is driving increasing attention not only in the field of immunology [START_REF] Paul | Role of gamma-delta (gammadelta) T cells in autoimmunity[END_REF] but also immunometabolism.

B lymphocytes

Recently a specific B cell population has been described within adipose tissue, regulatory B (Breg) cells. Breg cells are identified as cells able to produce interleukin-10 constitutively in WAT [START_REF] Seida | Clinical review: Effect of vitamin D3 supplementation on improving glucose homeostasis and preventing diabetes: a systematic review and meta-analysis[END_REF]. In this study the role of IL10 specifically produced by Breg cells in protecting mice from the deleterious effects of high fat diet was demonstrated. Breg cells could have role in controlling the progression of adipose tissue inflammation in obesity. During onset of obesity, following reverse mechanisms, Breg cells decrease, while CD8 effector T-cells increase in number [START_REF] Nishimura | CD8+ effector T cells contribute to macrophage recruitment and adipose tissue inflammation in obesity[END_REF]. Whether the recruitment of CD8 T-cells to the adipose tissue occurs through antigendependent or -independent processes is not clear yet. However, cytotoxic CD8 (+) T-cells infiltrating the WAT may contribute to the recruitment, differentiation and activation of macrophages and consequently to WAT inflammation. For instance, B cells WAT infiltration is a very early response to high-fat feeding and can even precede the accumulation of macrophages [START_REF] Nishimura | CD8+ effector T cells contribute to macrophage recruitment and adipose tissue inflammation in obesity[END_REF]. Their role in affecting systemic IR and worsening glucose metabolism through modulation of Tcells and producing pathogenic antibodies has been demonstrated [START_REF] Nishimura | CD8+ effector T cells contribute to macrophage recruitment and adipose tissue inflammation in obesity[END_REF]) [START_REF] Winer | B Lymphocytes in obesity-related adipose tissue inflammation and insulin resistance[END_REF]. Unable to produce an unique profile of pathogenic IgG autoantibody, high fat diet-induced obese mice lacking B cells are protected from IR despite weight gain (Winer, Winer et al. 2011). The vicious cycle of interactions between CD8(+) T-cells, macrophages and adipocytes clearly indicates how these interactions are critical for the initiation and propagation of adipose inflammation in obesity.

In addition to the above described cell population, adipose tissue is enriched with innate lymphoid type 2 cells (ILC2), known to support the activity of Treg and contribute to insulin sensitivity in lean individuals.

To summarize, the innate and the adaptive immune cells represent a highly dynamic system in WAT, particularly studied and characterized during the transition from a lean to an obese state (Figure 14).

Immunological memory

An important role of adaptive immunity is immune surveillance [START_REF] Sallusto | Two subsets of memory T lymphocytes with distinct homing potentials and effector functions[END_REF]. Effector (TEM) and memory (TCM) cells are T-cells that have encountered and responded to antigens (also called antigen-experienced T-cells). They are thought to be distributed to all tissues in the body. In humans, isoforms of CD45 (a phosphatase involved in cell signalling) is used to identify these cells, when analyzed in opposition to CCR7; whereas, in the mouse, the specific markers to identify memory cells are CD44 versus CD62L. To clarify with a pratical example, in our experimental setting (flow cytometry on the stromal vascular fraction from WAT of linoleic and conjugated linoleic acid-treated mice), we used the above mentioned mouse markers to divide antigen-experienced T-cells in: central memory (TCM, CD44+CD62L+), effector memory (TEM, CD44+CD62L-) and naive T-(CD44+CD62L+) cells. On one hand TEM cells, as the name says, are able to migrate to inflamed tissue and display immediate effector function. On the other hand TCM cells express lymph-node homing receptors and can differentiate into effector cells upon secondary stimulation only. These cells are not restricted to lymphoid organs but can be present in significant numbers in nonlymphoid tissues. Maintaining functional memory responses is essential to respond effectively to infections and other potential endogenous and exogenous danger signals (lipids?). Obesity has been linked to numerous health problems and chronic diseases, including increased infection susceptibility; as a consequence it is critical to understand how the immune cell responsiveness can be altered by an altered microenvironment.

Immunity and inflammation: chemokines and other recruitment factors

The localization and dynamic distribution of the above described immune cells is controlled by various factors, first of all, chemokines and their receptors. The recruitment of leukocytes inside the tissues happens from the endothelium through sequential steps, such as integrin-dependent arrest and transmigration. This Chapter will focus on the specialized roles of chemokines during this process, looking specifically at cell infiltration inside metabolically compromised tissues. The focus will be in particular on macrophages, knowing that they have an impact on metabolism and, vice versa, tissue metabolic microenvironment affects their acquired functional phenotype [START_REF] Biswas | Orchestration of metabolism by macrophages[END_REF] and also because it is the main population recruited in WAT in obesity and in the lipoatrophy model we used for our experiments (Poirier, Shapiro et al. 2006). Indeed in obese subjects WAT macrophages accumulation can arrive to such a magnitude that almost half of all cells in a fat depot are macrophages [START_REF] Weisberg | Obesity is associated with macrophage accumulation in adipose tissue[END_REF]. Strategies to control leukocyte mobilization and accumulation in tissues are needed today not only when thinking about potential new therapies in obesity, but also in other contexts, such as cancer immunotherapy [START_REF] Vela | Chemokine receptor-specific antibodies in cancer immunotherapy: achievements and challenges[END_REF]) [START_REF] Kufareva | Chemokine and chemokine receptor structure and interactions: implications for therapeutic strategies[END_REF]. Modulating the activity of chemokines and/or their receptors would allow increase migration of cells critical for their high endocytic and phagocytic capacity, such as DCs or macrophages, in tissues that would require their action, such as in tumors. Alternatively, reduction of macrophage infiltrating obese WAT could be a therapeutic option conducted to improve inflammation and the associated deleterious metabolic alterations [START_REF] Kanda | MCP-1 contributes to macrophage infiltration into adipose tissue, insulin resistance, and hepatic steatosis in obesity[END_REF].

Adipochemokines

Chemokines are potent chemoattractants for all types of leukocytes, including hematopoietic precursors, mature leukocytes of the innate immune system, as well as naive, memory, and effector lymphocytes. Most of the inflammatory chemokines are produced by WAT macrophages and their upregulation precedes systemic IR. In particular several studies have reported the increased expression of chemokines and their receptors in obesity in mice [START_REF] Xu | Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance[END_REF] as well as in humans [START_REF] Dahlman | A unique role of monocyte chemoattractant protein 1 among chemokines in adipose tissue of obese subjects[END_REF]) [START_REF] Huber | CC chemokine and CC chemokine receptor profiles in visceral and subcutaneous adipose tissue are altered in human obesity[END_REF]. The typical inflammatory chemokines linked to inflammation and obesity include: CCL2, CCL3 and CCL5, CXCL1, CXCL2 and CXCL8 [START_REF] Weisberg | CCR2 modulates inflammatory and metabolic effects of highfat feeding[END_REF]) [START_REF] Xu | Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance[END_REF]) [START_REF] Curat | Macrophages in human visceral adipose tissue: increased accumulation in obesity and a source of resistin and visfatin[END_REF]. In particular obese humans display elevation of certain specific chemokines and their coupled receptors, including CCL2/MCP-1, CCL3/MIP1α, CCL4/MIP-1β, CXCL2/MIP-2α, and CXCL3/MIP-2β in both adipocytes and PBMCs, elevation that could is possibly mediated by certain dietary choices [START_REF] Munoz | Nutritionally mediated oxidative stress and inflammation[END_REF]. It is important to underline how different families of chemokines present positive feed-back mechanisms between each other, surely accelerating the progression of inflammation. Controlling chemokines has been demonstrated to have therapeutic potential. In tissues other than WAT, there are studies showing how neutralization of specific chemokines, i.e. CCL3 and CCL2/MCP1, can be effective in reducing fibrosis development [START_REF] Smith | Production and function of murine macrophage inflammatory protein-1 alpha in bleomycin-induced lung injury[END_REF], [START_REF] Belperio | Critical role for the chemokine MCP-1/CCR2 in the pathogenesis of bronchiolitis obliterans syndrome[END_REF]).

CCL2/CCR2

The most studied couple of chemokine/receptor in the context of obesity and IR is represented undoubtedly by CCL2 (MCP1/ Monocyte Chemoattractant Protein 1) and its receptor CCR2.

Despite the multiple studies showing the increased mRNA expression of CCL2 in the context of obesity (Yu et al., 2006), its role in contributing to WAT inflammation is still under debate [START_REF] Kirk | Monocyte chemoattractant protein deficiency fails to restrain macrophage infiltration into adipose tissue [corrected][END_REF].

Research in diet-induced obese mice has demonstrated that CCR2 is also critical in the process of macrophage accumulation in WAT [START_REF] Weisberg | Obesity is associated with macrophage accumulation in adipose tissue[END_REF]) [START_REF] Ito | Role of CC chemokine receptor 2 in bone marrow cells in the recruitment of macrophages into obese adipose tissue[END_REF]. In particular, during diet-induced obesity, CCR2 is overexpressed in recruited macrophages and not in resident tissue macrophages, indicating the important role of this receptor for the recruitment of inflammatory macrophages [START_REF] Lumeng | Increased inflammatory properties of adipose tissue macrophages recruited during diet-induced obesity[END_REF]. Considering that this is a receptor common to other chemokines, including CCL7, CCL12, CCL13, it is plausible that other chemokines compensate the absence of CCL2 activity, inhibited or genetically modified, explaining the conflicting results obtained in mice.

CCL5/CCR1-3-5

CCL5 (RANTES, Regulated upon Activation, Normal T cell Expressed and Secreted) plays an important role in immune cell recruitment. Studied in particular in the pathology of atherosclerosis [START_REF] Eriksson | Mechanisms of leukocyte recruitment to atherosclerotic lesions: future prospects[END_REF]), [START_REF] Zernecke | Chemokines in atherosclerosis: an update[END_REF], opposite effects of the couple CCL5/CCR5 (pro-atherogenic) and CCL5/CCR1 (atheroprotector) have been reported in mouse models, underling complexity and specificity of functions. In HFD-fed mice this chemokine and its receptors have been observed increased at the mRNA and protein level in WAT [START_REF] Wu | T-cell accumulation and regulated on activation, normal T cell expressed and secreted upregulation in adipose tissue in obesity[END_REF]) and their function has been related to macrophage and lymphocyte recruitment. The team of K. Clément has demonstrated that circulating CCL5 is increased in obese subjects, in line with other studies [START_REF] Huber | CC chemokine and CC chemokine receptor profiles in visceral and subcutaneous adipose tissue are altered in human obesity[END_REF]). The main cellular component that expressed CCL5 in the stroma vascular fraction from obese patients was represented by macrophages and CCL5 mRNA positively correlated with M1 markers, such as CD11b and CD11c, and protected these cells from apoptosis [START_REF] Keophiphath | CCL5 promotes macrophage recruitment and survival in human adipose tissue[END_REF]. This chemokine can be sensed by three different receptors, CCR1, CCR3 and CCR5, in common with other chemokines, such as CCL3 /MIP1 and CCL7/MCP-3 known to be increased in WAT of obese subjects [START_REF] Huber | CC chemokine and CC chemokine receptor profiles in visceral and subcutaneous adipose tissue are altered in human obesity[END_REF].

CXCLs/CXCR2

Other chemokines have been shown to be implicated in obesity, such as CXCL1, CXCL2, CXCL5 and CXCL8 [START_REF] Nunemaker | Increased serum CXCL1 and CXCL5 are linked to obesity, hyperglycemia, and impaired islet function[END_REF]) [START_REF] Rouault | Roles of chemokine ligand-2 (CXCL2) and neutrophils in influencing endothelial cell function and inflammation of human adipose tissue[END_REF]) [START_REF] Chavey | CXC ligand 5 is an adipose-tissue derived factor that links obesity to insulin resistance[END_REF]) (Bruun, Lihn et al. 2004) that can be produced by circulating monocytes, macrophages, but also non immune cells. In common these chemokines bind to the same receptor, CXCR2, together with other CXCL-family members [START_REF] Neels | Keratinocyte-derived chemokine in obesity: expression, regulation, and role in adipose macrophage infiltration and glucose homeostasis[END_REF]). The therapeutic potential of this receptor has been evaluated in a variety of diseases, in animal models and clinical trials, and promising results have been obtained [START_REF] Stadtmann | CXCR2: From Bench to Bedside[END_REF]. In the context of obesity, CXCR2-deficient mice challenged with HFD showed a reduced infiltration of macrophages in WAT, associated with an improvement in insulin sensitivity [START_REF] Neels | Keratinocyte-derived chemokine in obesity: expression, regulation, and role in adipose macrophage infiltration and glucose homeostasis[END_REF]). Studies of CXCR2 inhibition, with the aim of determining the efficiency in blocking immune cell recruitment showed a good tolerance of the specific inhibitor in short term time course, whereas over long term, some toxic effects were noticed [START_REF] Lazaar | SB-656933, a novel CXCR2 selective antagonist, inhibits ex vivo neutrophil activation and ozone-induced airway inflammation in humans[END_REF], [START_REF] Day | Regulation of neutrophil trafficking from the bone marrow[END_REF]. More recently blocking CXCR2 has been shown to reduce neutrophil accumulation in pancreas, dampen the diabetogenic T-cell response taking apart the later development of the autoimmune type 1 diabetes, opening a potential therapeutic avenue for this disease as well [START_REF] Diana | Macrophages and beta-cells are responsible for CXCR2-mediated neutrophil infiltration of the pancreas during autoimmune diabetes[END_REF]. Between the chemokines interacting with this receptor, CXCL1 and CXCL5 are the most studies in metabolic altered contexts. CXCL1 was recently shown to attenuate HFD-induced obesity and the associated IR [START_REF] Pedersen | Muscle-derived expression of the chemokine CXCL1 attenuates diet-induced obesity and improves fatty acid oxidation in the muscle[END_REF]. When overexpressed in skeletal muscle CXCL1, possibly through improvement in tissue fatty acid oxidation, reduced visceral and subcutaneous fat mass of HFD-fed mice, without affecting their food intake [START_REF] Pedersen | Muscle-derived expression of the chemokine CXCL1 attenuates diet-induced obesity and improves fatty acid oxidation in the muscle[END_REF]. Whereas CXCL1 has been shown to be important at the muscle level, CXCL5 is mostly found as an adipose tissue derived factor [START_REF] Chavey | CXC ligand 5 is an adipose-tissue derived factor that links obesity to insulin resistance[END_REF]. CXCL5 is increased in ob/ob and db/db and HFD-fed mice and macrophages were the main population expressing this chemokine within WAT. Inhibition of CXCL5 through antibody reduced the HFD-induced insulin resistance. However it is important to note that blocking its receptor, CXCR2, is more efficient than neutralizing one of its numerous chemokines, suggesting the compensatory role that other recruitment factors can adopt. In human obesity, the systemic level of CXCL5 is increased and recently it has been indicated as a new marker of atherosclerosis [START_REF] Chen | Serum CXC ligand 5 is a new marker of subclinical atherosclerosis in type 2 diabetes[END_REF].

CXCL10/CXCR3

CXCL10 (IP-10 / Interferon gamma-induced protein 10) is a chemokine produced by human mature adipocytes. Its secretion positively correlates with obesity and type 2 diabetes, conditions in which can be induced by interferon gamma (IFN) [START_REF] Herder | Constitutive and regulated expression and secretion of interferongamma-inducible protein 10 (IP-10/CXCL10) in human adipocytes[END_REF]). In massively obese patients, candidate for bariatric surgery, our team has recently shown a significant increase in systemic levels of CXCL10 compared to lean subjects [START_REF] Dalmas | Variations in circulating inflammatory factors are related to changes in calorie and carbohydrate intakes early in the course of surgery-induced weight reduction[END_REF]). However, unlike CCL2, CXCL8 and CCL5, serum level of CXCL10 does not decrease after a drastic weight loss induced by surgery [START_REF] Rouault | Roles of chemokine ligand-2 (CXCL2) and neutrophils in influencing endothelial cell function and inflammation of human adipose tissue[END_REF]. The critical role of this chemokine in tissue inflammation has been demonstrated also in liver pathologies. Indeed due to its pathogenic role in the development of steatohepatitis in mice, CXCL10 has been proposed as potential non-invasive biomarker for NASH patients [START_REF] Zhang | CXCL10 plays a key role as an inflammatory mediator and a noninvasive biomarker of non-alcoholic steatohepatitis[END_REF].

CX3CL1/CX3CR1

By functional genomics experiments, Shah and his team have shown that CX3CL1 (fractalkine) is upregulated in human mature adipocyte during inflammation induced by LPS. Secretion of CX3CL1 is increased in obese subjects, being particularly elevated in visceral adipose tissue more than in the subcutaneous WAT. In addition, CX3CL1 favors the adherence of a cell line of monocytes (THP-1) on human adipocytes. A receptor polymorphism, CX3CR1, is associated with metabolic disorders (insulin resistance index HOMA, circulating leptin and hip circumference). Blood levels of CX3CL1 are higher among type 2diabetic subjects than in the control subjects [START_REF] Shah | Neutrophil infiltration and systemic vascular inflammation in obese women[END_REF].

In conclusion, many chemokines and their receptors have been detected in adipose tissue in recent years and that their production and function, undoubtedly redundant, are influenced by obesity.

Cathepsins

However, in addition to chemokines, there are other factors important for leukocyte migration to the site of inflammation, including cathepsins. Cathepsins are proteases with different structure and actions depending on the protein to degrade. Their chemotactic role has been mostly studied in atherosclerosis development. The expression of these proteases can be high in the cells of immune system, it is the case of Cathepsin X, and consequently they can potentially participate to the processes of inflammatory and immune responses in a variety of conditions. Cathepsin X, also known as cathepsin Z, regulates processes such as adhesion, T lymphocyte activation and phagocytosis through its interaction with beta2 integrins [START_REF] Zhang | CXCL10 plays a key role as an inflammatory mediator and a noninvasive biomarker of non-alcoholic steatohepatitis[END_REF]. Contrary to other cathepsins, cathepsin X is not widely expressed in cells and tissues, but is restricted to immune system cells, mostly monocytes, macrophages and dendritic cells [START_REF] Zhang | CXCL10 plays a key role as an inflammatory mediator and a noninvasive biomarker of non-alcoholic steatohepatitis[END_REF]. Interestingly, its upregulation has also been detected in the brain of patients with Alzheimer disease [START_REF] Wendt | Differential expression of cathepsin X in aging and pathological central nervous system of mice[END_REF]) and in gastric mucosa [START_REF] Buhling | Cathepsins K, L, B, X and W are differentially expressed in normal and chronically inflamed gastric mucosa[END_REF]) [START_REF] Krueger | Up-regulation of cathepsin X in Helicobacter pylori gastritis and gastric cancer[END_REF]. Using two-and three-dimensional models that mimicked the extra cellular matrix, a recent study demonstrated that cathepsin-X-overexpressing T lymphocytes displayed an increased migratory activity and aggregation capacity [START_REF] Jevnikar | The role of cathepsin X in the migration and invasiveness of T lymphocytes[END_REF].

Other Cathepsins are abundantly expressed in macrophages, i.e. isolated from human atheroma [START_REF] Sukhova | Expression of the elastolytic cathepsins S and K in human atheroma and regulation of their production in smooth muscle cells[END_REF], or in other pathologic contexts, such as infections [START_REF] Muller | The endolysosomal cysteine cathepsins L and K are involved in macrophage-mediated clearance of Staphylococcus aureus and the concomitant cytokine induction[END_REF], bone remodeling [START_REF] Buhling | Cathepsin K--a marker of macrophage differentiation?[END_REF], and cancer [START_REF] Shree | Macrophages and cathepsin proteases blunt chemotherapeutic response in breast cancer[END_REF]), [START_REF] Gocheva | IL-4 induces cathepsin protease activity in tumor-associated macrophages to promote cancer growth and invasion[END_REF]. However cathepsins can be involved not only in inflammatory responses from the innate immunity, through TLR9 signaling in particular, but also adaptive immunity, like antigen presentation (i.e. CD4+ T-cells). Recent in vitro experiments showed that Cathepsin S-deficient monocytes exhibit less migration activity through smooth muscle cell layer than wild-type monocytes. Moreover, during the last decade, these proteases have been recognized as critical in the degradation of extracellular proteins [START_REF] Fonovic | Cysteine cathepsins and extracellular matrix degradation[END_REF]. Macrophages, in particular, which are able to secrete these proteases, once activated, use them during the degradation process. Considered their involvement in a number of ECM-related diseases, such as osteoporosis, cancer and cardiovascular diseases, and their role in innate immune responses (mediated TLR9 in particular), further research is required also in the context of obesity.

Galectins

Galectins are a large family of animal lectins defined by their binding specificity for β-galactoside sugars. They present a broad variety of functions including mediation of cell-cell interactions, cellmatrix adhesion and transmembrane signaling. They are differentially expressed by various immune cells and their expression levels depend on cell differentiation and activation, in particular by macrophage and fibroblast. Galectins can play important roles in inflammation and immunologic responses, since they have chemotactic proprieties [START_REF] Liu | Regulatory roles of galectins in the immune response[END_REF]. A total of 15 galectins have been identified. Galectin-3 (Lgal3) is the most widely studied member of this protein family. Lgal3 has been shown to actively participate to persistence of the inflammation, affecting virtually all inflammatory cells, including neutrophils and macrophages. Obese humans and mice present markedly elevated levels of Lgal3, systemically and locally (liver, subcutaneous and intra-abdominal WAT). Current data suggest that FFA and IL-6 increase Lgal3 in 3T3-L1 adipocytes, identifying one of the mechanisms possibly contributing to higher Lgal3 levels in obesity [START_REF] Krautbauer | Free fatty acids and IL-6 induce adipocyte galectin-3 which is increased in white and brown adipose tissues of obese mice[END_REF]. Several studies have shown upregulation of Lgals3 in association with the concomitant activation of macrophages in different contexts. In particular, in recent years, Lgals3 has gained prominence as a central regulator of chronic inflammation and fibrogenesis (In Galectins and Disease Implications for Targeted Therapeutics; Klyosov, A. et al., ACS Symposium Series; American Chemical Society: Washington, DC, 2012.). In particular, Lgals3 ihas been shown nvolved in the development of organ fibrogenesis and scarring in kidney (Henderson, Mackinnon et al. 2008), in liver (Henderson, Mackinnon et al. 2006) and more recently in heart [START_REF] Liu | N-acetyl-seryl-aspartyl-lysyl-proline prevents cardiac remodeling and dysfunction induced by galectin-3, a mammalian adhesion/growth-regulatory lectin[END_REF]) (Figure 15). In liver, for example, Lgal3 was demonstrated to affect tissue remodeling through macrophage activation. Macrophages in turn activate and differentiate fibroblasts into myofibroblasts, promoting collagen synthesis (Henderson, Mackinnon et al. 2006).

Despite the association of Lgal3 with macrophage accumulation and fibrosis, in metabolic diseases this protein has been suggested to have a protective role against inflammation regulating adiposity and glucose homeostasis [START_REF] Pejnovic | Galectin-3 is a regulator of metaflammation in adipose tissue and pancreatic islets[END_REF]. In conclusion, targeted immunomodulatory interventions on recruitment factors or specific cell subpopulations, in particular, could provide interesting therapeutic options towards amelioration of metabolic altered conditions, such as obesity.

E. Adipose tissue: cellular and structural alterations in obesity 19). Adipocyte highly specialized activity is regulated at the transcriptional level by a program of enzymes involved in triacylglycerol synthesis and degradation (FAS, ACC, PLIN, HSL, ATGL and more), in synthesis of adipokine (leptin, adipsin, resistin, adiponectin), glucose transporters (GLUT4) and insulin receptors (IRS). All the mentioned components allow flexibility to the adipocyte in adapting to its nutrient environment, preserving its metabolic homeostasis (Figure 16). Local adipocyte physiology, including the WAT adipokine secretion [START_REF] Cao | Adipocytokines in obesity and metabolic disease[END_REF], is thus transferred at the systemic level with potentially beneficial or detrimental effects.

Extra cellular matrix: composition and pathophysiology

The protein composition and dynamics of the extracellular matrix (ECM) is crucial for the functioning of adipocytes. Adipocytes, as endothelial cells, are surrounded by a thick ECM referred to as basal lamina [START_REF] Pierleoni | Fibronectins and basal lamina molecules expression in human subcutaneous white adipose tissue[END_REF]. While a strong ECM is the principal function of bone and cartilage, the basal lamina in adipocytes may be more important for cell survival. ECM components of adipocytes have been studied mostly by immunohistochemistry and RT-PCR. These components are represented by different types of collagens and collagen-related proteins, such as cell surface integrins, fibronectin, elastin, laminin, proteoglycans, tenascin-C, several matrix metalloproteinases (MMP), tissue inhibitors of matrix metalloproteinases (TIMP) and some more. It is important to note that some of these proteins are not produced only by the adipocytes, but also by cells of the stromal vascular fraction containing preadipocytes, capillary endothelial cells, infiltrated monocytes/macrophages, and a population of multipotent stem cells [START_REF] Mariman | Adipocyte extracellular matrix composition, dynamics and role in obesity[END_REF]. More than 30 collagens are known, but the most present in adipose tissue Col 1, 4 and 6 are also the best characterized [START_REF] Nakajima | Adipose tissue extracellular matrix: newly organized by adipocytes during differentiation[END_REF]) [START_REF] Khan | Metabolic dysregulation and adipose tissue fibrosis: role of collagen VI[END_REF]. Specific collagens and their regulators have a role during the physiological process of adipogenesis, when the adipocyte changes the cell shape from fibroblastic to spherical shape, changing in level and type, in parallel with level of cytoskeletal components [START_REF] Pierleoni | Fibronectins and basal lamina molecules expression in human subcutaneous white adipose tissue[END_REF], [START_REF] Spiegelman | Fibronectin modulation of cell shape and lipogenic gene expression in 3T3-adipocytes[END_REF]. Obviously, ECM components intervene during the adaptive response of adipose to cellular alterations due to expansion/reduction as well. In obesity, in both rodents and humans, expanding AT displays increases in components of the ECM, including collagen VI and thrombospondin, parallel to considerable tissue remodeling and increase in inflammatory macrophages [START_REF] Varma | Thrombospondin-1 is an adipokine associated with obesity, adipose inflammation, and insulin resistance[END_REF]) [START_REF] Spencer | Adipose tissue macrophages in insulin-resistant subjects are associated with collagen VI and fibrosis and demonstrate alternative activation[END_REF]) [START_REF] Pasarica | Adipose tissue collagen VI in obesity[END_REF]) (Divoux, Tordjman et al. 2010) (Sun, Tordjman et al. 2013). ECM composition and ECM regulatory proteins, such as glycoconjugates, important in influencing the rate of assembly, size, and structure of collagen fibrils, are currently studied in conditions of broad biomedical importance from arthritis to tissue repair, from tumor invasion to cardiovascular disease [START_REF] Kadler | Collagen fibrillogenesis: fibronectin, integrins, and minor collagens as organizers and nucleators[END_REF]).

Extra cellular matrix: a dynamic structure

Next to protein composition of ECM, its dynamics is of crucial importance for adipocyte functioning. ECM is in fact under constant turnover. During ECM remodeling, physiological or pathological, proteins have their specific roles of mediating the balanced result between constructive and destructive enzymes together with their enhancers and inhibitors. An example of this complemented balance can be given by lumican and decorin. Despite the fact that both are collagen cross-linking proteins, these two enzymes have been found to have opposing roles in fibrosis [START_REF] Pasarica | Adipose tissue collagen VI in obesity[END_REF]) [START_REF] Khan | Metabolic dysregulation and adipose tissue fibrosis: role of collagen VI[END_REF]. Another classical example important for ECM regulation is represented by the balance between MMPs and TIMPs. In vivo research on MMPs provides contradicting results, indicating the complexity of MMP activity that depends on relative concentrations of different MMPs, their substrate specificity, and the activity of their inhibitors (TIMPs), (TIMPs), and the fibrinolytic system comprising plasminogen/plasmin and their activators (tissue-type plasminogen activator and urokinasetype activator [START_REF] Christiaens | Role of proteolysis in development of murine adipose tissue[END_REF]) [START_REF] Demeulemeester | Effect of matrix metalloproteinase inhibition on adipose tissue development[END_REF]) [START_REF] Lijnen | Matrix metalloproteinase inhibition impairs adipose tissue development in mice[END_REF]. Another interesting ECM component is a membrane-anchored metalloproteinase MMP14, which was proved to be important for a proper WAT development using null mice (MT1-MMP). Using null mice, MT1-MMP was proved to be important for a proper WAT development [START_REF] Chun | A pericellular collagenase directs the 3-dimensional development of white adipose tissue[END_REF]. Null mice were in fact lipodystrophic and in a 3-dimensional (3-D) ECM context, during adipocyte maturation, MT1-MMP was required to modulate pericellular collagen rigidity. However, besides the physiological activity of ECM, there are pathological conditions in which ECM accumulation is deregulated, as in obese WAT. In that case the same protease, MT1-MMP, cannot properly control the formation of a rigid network of collagen fibrils and consequently contribute to restrain adipocyte expansion. Other ECM components can participate to pathological remodeling and tissue stiffening. For example lysyl oxidase (Lox) and the LOX family of secreted amine oxidases have been observed increased in many fibrotic diseases due to their role in rigid cross-linking of elastin and collagens [START_REF] Sivakumar | Upregulation of lysyl oxidase and MMPs during cardiac remodeling in human dilated cardiomyopathy[END_REF]) [START_REF] Kagan | Lysyl oxidase: properties, specificity, and biological roles inside and outside of the cell[END_REF]) [START_REF] Csiszar | Lysyl oxidases: a novel multifunctional amine oxidase family[END_REF]) [START_REF] Smith-Mungo | Lysyl oxidase: properties, regulation and multiple functions in biology[END_REF].

Finally, it is important to acknowledge the role of proteases in contributing to tissue remodeling through a variety of different mechanisms [START_REF] Cheng | Role for cysteine protease cathepsins in heart disease: focus on biology and mechanisms with clinical implication[END_REF]). For example it has been shown that cathepsins can be secreted within the extracellular spaces and directly affect collagen turnover and degradation, as it is the case for cathepsin S [START_REF] Shi | Molecular cloning and expression of human alveolar macrophage cathepsin S, an elastinolytic cysteine protease[END_REF]). The cathepsin biology and their significance in ECM remodeling in the development and progression of various pathological conditions continue receiving scientific attention in search of successful therapeuctics.

Adipose tissue fibrosis in obesity and after drastic weight loss

Fibrosis is the common pathological outcome of many chronic inflammatory diseases in the context of the liver, lung, kidney and several other tissues. The term fibrosis has been defined as the formation of fibrous tissue as a reparative or reactive process. In the "healthy" adipose tissue, remodeling is a complex process characterized by dynamic changes in cellular composition and function [START_REF] Itoh | Adipose tissue remodeling as homeostatic inflammation[END_REF]. Instead until now fibrosis in WAT has been explained as the initial response to adipocyte hypertrophy. In particular, our team demonstrated the ECM accumulation in human obesity through the method of picrosirius red staining of WAT [START_REF] Henegar | Adipose tissue transcriptomic signature highlights the pathological relevance of extracellular matrix in human obesity[END_REF], and also the importance of macrophage-preadipocyte interactions in WAT fibrosis development or maintenance [START_REF] Keophiphath | Macrophage-secreted factors promote a profibrotic phenotype in human preadipocytes[END_REF]). As the collagen content increases, the overall rigidity of adipose tissue also increase. This rigidity in WAT, as in other tissues, may impair angiogenesis, so that adipocytes surrounded by fibrotic tissue cannot be adequately oxygenated, becoming more susceptible to necrosis [START_REF] Trayhurn | Hypoxia and the endocrine and signalling role of white adipose tissue[END_REF]. Signals emerging from hypertophic (dysfunctional) or dying adipocytes could influence the formation of fibrous structure around the cells. Fibrosis in WAT has been presented as the hallmark response of metabolically challenged adipocytes and in particular collagen 6 has been shown to have a key role in limiting WAT expansion and consequently influencing insulin sensitivity [START_REF] Pasarica | Adipose tissue collagen VI in obesity[END_REF]) [START_REF] Khan | Metabolic dysregulation and adipose tissue fibrosis: role of collagen VI[END_REF]. Adipose tissue inflammation and immune cell accumulation have been observed in obesity as well as during dynamic weight loss (due to caloric restriction or fasting), in relation with increased lipolytic activity (Kosteli, Sugaru et al. 2010). However, differently from obesity, adipose tissue remodeling and the potential contribution of WAT macrophages in the process has not been fully investigated after drastic weight loss (Kosteli, Sugaru et al. 2010). In 2010, our team showed that human WAT fibrosis, in particular peri-cellular fibrosis, could be a process able to limit the lipolytic activity of adipocytes, possibly a mechanism preventing an excess weight loss after bariatric surgery (Divoux, Tordjman et al. 2010). During drastic weight loss, dysfunction or death in adipocytes is also found, as suggested by perilipin-negative adipocytes engulfed in fibrosis, similarly to what has been reported in obesity for the adipocytes surrounded by crown-like structures of macrophages [START_REF] Cinti | Adipocyte death defines macrophage localization and function in adipose tissue of obese mice and humans[END_REF]. Consequently WAT fibrosis could be a response to signals emerging from adipocytes within the specific inflammatory milieu. In this view, fibrosis is considered a pathologically deregulated healing response to inflammation-induced destruction and injury of tissue, as summarized in Figure 18. During obesity, dead adipocytes as identified by perilipin-negative staining are found surrounded by macrophages in crown-like structures [START_REF] Cinti | Adipocyte death defines macrophage localization and function in adipose tissue of obese mice and humans[END_REF]. After drastic weight loss, perilipin-negative adipocytes are also found engulfed in fibrosis (Divoux, Tordjman et al. 2010). Consequently WAT fibrosis could be a response to signals emerging from dead adipocytes. During the fibrotic process, stimulated myofibroblasts and epithelial/endothelial cells also produce matrix metalloproteinases (MMPs) that disrupt the basement membrane, allowing the efficient recruitment of cells to sites of injury. Activated macrophages and neutrophils use their scavenger function to 'clean-up' tissue debris and dead cells, releasing at the same time cytokines and chemokines that recruit and activate other cells, including T-cells and fibroblasts. Failure to adequately contain or eliminate the provoking factors can exacerbate the inflammatory response, leading to chronic inflammation, excessive immune cell accumulation (as observed in obese WAT) and chronic repair that causes an excessive accumulation of ECM components. Recovering the delicate regulation between collagen synthesis and collagen degradation (MMPs and their inhibitors TIMPs) [START_REF] Liu | Mechanical stretch promotes matrix metalloproteinase-2 and prolyl-4hydroxylase alpha1 production in human aortic smooth muscle cells via Akt-p38 MAPK-JNK signaling[END_REF]) is fundamental to control the process and regain homeostasis. Although ATMs accumulation is classically associated to inflammation and inflammation seems necessary for the initiation of the ECM remodeling, some observations suggest that the mechanisms promoting fibrosis can be distinct and different to those that impact inflammation. This has been shown in pulmonary, liver, kidney and intestinal (IBD) fibrosis, where molecules, classically identified as pro-inflammatory (i.e. IFNγ, IL12), were shown to reduce collagen deposition [START_REF] Wynn | Fibrotic disease and the T(H)1/T(H)2 paradigm[END_REF]). Moreover, lasting of chronic pro-fibrotic injury may compromise tissue repair (Figure 19). Resident and extravasating peripheral macrophages play an important role in the early stages of muscle repair after acute injury, with pro-inflammatory (M1) macrophages first acting to clear the damage, and anti-inflammatory (M2c) macrophages and alternatively activated macrophages (M2a), implicated in the subsequent resumption of inflammation, extracellular matrix (ECM) deposition and tissue repair. M2c and M2a macrophages release anti-inflammatory cytokines and pro-fibrotic molecules such as transforming growth factor (TGF)-β, which in turn activate fibroblasts in a regulated manner to produce ECM components and ECM-remodeling factors, including autocrine production of TGFβ, collagen, fibronectin, serine proteases (such as uPA/plasmin), and metalloproteinases (MMPs) and their inhibitors (TIMPs). However, during chronic tissue damage, as in muscular dystrophies, the increased and persistent presence of macrophages modify the intensity, duration and interactions of these released factors, leading to excessive ECM accumulation and replacement of muscle with fibrotic tissue.Studying adipocyte dysfunction in opposed pathological settings can further complicate the situation, since the same process could have contrasting functions. In obesity, fibrosis could serve to limit the excessive expansion of WAT, whereas in lipodystrophy, it could have the role of limiting the excessive loss of adipocyte lipid content. Research will need to further explore the regulation of these delicate mechanisms, in the context of obesity and, even less explored, weight loss, in order to possibly design new therapies.

F. A cross-road between metabolism and immunity

In our body there are "organs" considered key for their plastic endocrine functions, like the above presented adipose tissue and the more recently identified gut microbiota [START_REF] Brown | The gut microbial endocrine organ: bacterially derived signals driving cardiometabolic diseases[END_REF].

Both systems are exposed to nutritional fluctuation and participate in maintaining body homeostasis converting dietary molecules into hormonal (WAT) and hormone-like (metabolites from microbiota) signals. Excess or defects of energy flux can dysregulate the delicate balance of interactions between metabolism and immunity in both "organs" (Exley, Hand et al. 2014) [START_REF] Kau | Human nutrition, the gut microbiome and the immune system[END_REF]).

In the context of metabolic disorders, adipose tissue has been particularly studied, being at the junction of immune and metabolic co-regulation. WAT efficiently monitors metabolic homeostasis through the release of adipokines. These adipocyte-specific secretory proteins are now recognized participants in the regulation of immune responses through their own pro-and anti-inflammatory activities [START_REF] Ouchi | Adipokines in inflammation and metabolic disease[END_REF]). In addition, the intrinsic presence of various immune cells, innate and adaptive, tightens up the immune-metabolic role of adipose tissue [START_REF] Makki | Adipose tissue in obesity-related inflammation and insulin resistance: cells, cytokines, and chemokines[END_REF]). However, the interest in gut microbial communities has been rapidly increasing recently due to the impact that microbial metabolites can have in physiological and/or pathological processes. Adipose tissue and host receptors have the primary function of establishing the connection between metabolic and immune systems.

Nutrient-sensing and metabolism

Nutrient homeostasis is critical for living organisms, from the unicellular to the multicellular ones [START_REF] Efeyan | Nutrient-sensing mechanisms and pathways[END_REF]. Nutrient fluctuations in the environment can be directly sensed by unicellular organisms, whereas in multicellular organisms those fluctuations are internal and more limited. Energy homeostasis in the body is regulated in the different metabolic tissues by a coordinated management of cellular nutrients, such as lipids, amino acids, glucose and related sugars. Fully understanding the role of nutrient-sensing mechanisms could allow us to know how nutrients are sensed and maintained both at the intracellular and circulating levels in physiological and pathological conditions. In general, mechanisms that sense and manage nutrients integrate with immune and inflammatory pathways in a complex system evolved to protect the body. In the case of the defense system, for example, it is critical to acknowledge that the immune system efficiency strictly depends on highly energy-demanding cells, for which glucose is the main source of fuel [START_REF] Wolowczuk | Feeding our immune system: impact on metabolism[END_REF]. This close functional and molecular integration of the immune and metabolic systems is of particular interests in metabolic diseases in which sensing mechanisms become dysfunctional [START_REF] Hotamisligil | Nutrient sensing and inflammation in metabolic diseases[END_REF].

For the interest of this project, simply lipid sensing will be developed. Lipids are a diverse set of nutrients, like for example fatty acids and cholesterol, characterized by a hydrophobic nature. For this reason, lipids are normally packaged into lipoproteins and chylomicrons or bound by albumin in the serum [START_REF] Richieri | Unbound free fatty acid levels in human serum[END_REF]. Our knowledge of lipid metabolism is represented by a certain body of literature on the almost ubiquitous peroxisome proliferator-activated receptors (PPARs), with its three subtypes (α, δ, and γ), and their well-tuned role in metabolic organs, such as muscle (PPARα and PPARγ), liver (PPARα) and adipose tissues (PPARγ). Furthermore lipid-sensing mechanisms have been definitely less explored and our knowledge about them, with few exceptions, is quite limited [START_REF] Efeyan | Nutrient-sensing mechanisms and pathways[END_REF]. In particular, an interesting area of research receiving a new wave of attention is represented by the co-evolution of metabolic and immune responses. For mounting a potent immune response to defend the host against infectious agents, it is necessary to have stored and handle energy efficiently using mechanisms for nutrient sensing that have been selected under the pressure of food deprivation. Starvation and malnutrition can impair immune function. For example, in mice, a total reduction in body fat has been shown to result in a decrease in the energy that is available for immune responses [START_REF] Demas | Reductions in total body fat decrease humoral immunity[END_REF]. On the opposite side, energy surplus, characteristic of obesity, can also impair immune responses and induce chronic inflammation, as described above in this manuscript.

Upon immune stress, energy demands can become excessive and in tissues like liver and adipose tissue immune effector cells, such as Kupffer cells and macrophages, can represent a competitive advantage over other tissues. The local immune response is as more efficient as nutrients and soluble mediators are provided quickly. It is the case of lymph nodes embedded within adipose tissue. Focusing in particular on WAT immune cells, it is the case of the now recognized lipolytic capacity of immune cells over adipose tissue, one of the mechanisms that allow the cells to fuel a proper immune response.

Nutrient-sensing is one of the multiple common signaling pathways between adipocytes (included preadipocytes) and macrophages which need to be further deciphered [START_REF] Charriere | Preadipocyte conversion to macrophage. Evidence of plasticity[END_REF]. The association between metabolism and inflammation is particularly clear in obesity, during which insulin action has a critical role. In conditions where metabolic homeostasis is altered, including lipodystrophy next to obesity, understanding what is the primary insulting factor for the induction of insulin resistance is very important [START_REF] Medzhitov | Origin and physiological roles of inflammation[END_REF]. The intricate connections between metabolic homeostasis and inflammatory responses can converge at many levels, including at the level of cell-surface receptors, intracellular chaperones or nuclear receptors. In the present manuscript only two examples of receptors involved in the interplay between nutrient-sensing pathways, FFA in particular, and immune responses will be given: Toll like receptors (TLRs) and G-protein coupled receptors (GPRs).

In summary, the exploration of the general mechanisms that integrate nutritional status, immune response and systemic metabolic homeostasis is of future interest in order to understand and possibly therapeutically use these pathways in time of metabolic alterations, such as MetS diseases.

Toll like receptors (TLRs)

Toll-like receptors (TLRs) are membrane-bound pattern-recognition receptors, which can be found on immune cells, intestinal cells, and adipocytes, and are viewed as critical in ensuring bodily immune and metabolic health [START_REF] Wolowczuk | Feeding our immune system: impact on metabolism[END_REF].

These receptors play a central role in microbial pattern recognition in mammals. Besides this primary function, new evidence indicates that TLRs can also respond to different triggers. Of particular interest for metabolic disorders, is their capacity to sense abnormal levels of dietary lipids [START_REF] Wolowczuk | Feeding our immune system: impact on metabolism[END_REF]. Nutrients, such as saturated fatty acids and also elevated glucose levels, have been demonstrated to activate TLRs in adipocytes and macrophages, initiating an inflammatory cascade in WAT [START_REF] Shi | TLR4 links innate immunity and fatty acid-induced insulin resistance[END_REF]. The activation of TLRs can be differentially modulated depending on the types of ligands, as it is the case for dietary fatty acids [START_REF] Lee | Saturated fatty acid activates but polyunsaturated fatty acid inhibits Toll-like receptor 2 dimerized with Toll-like receptor 6 or 1[END_REF]), but also on the cellular context [START_REF] Mamedova | Toll-like receptor 4 signaling is required for induction of gluconeogenic gene expression by palmitate in human hepatic carcinoma cells[END_REF]. Similarly, after activation, the downstream signaling cascades, common or specific to single TLR (mainly represented by Toll/IL-1 receptor (TIR) domain, with the first characterized MyD88) vary depending on the contexts. The link between nutritional lipids and TLRs could reveal their potential role in the pathogenesis of obesity-associated insulin resistance, as it is the case of TLR4 [START_REF] Tsukumo | Loss-of-function mutation in Toll-like receptor 4 prevents diet-induced obesity and insulin resistance[END_REF]). Research on TLRs and saturated FFA has been mainly conducted in relation to adipocytes. However, the recent identification of TLR4 on hepatocytes [START_REF] Galloway | Activation of hepatocytes by extracellular heat shock protein 72[END_REF]) [START_REF] Li | Nuclear factor high-mobility group box1 mediating the activation of Toll-like receptor 4 signaling in hepatocytes in the early stage of nonalcoholic fatty liver disease in mice[END_REF]) encouraged research on potential mechanisms linking FFA to altered liver function. Using a human hepatocellular carcinoma cell line, the critical role of TLR4 in linking elevation of FFA and increased hepatic gluconeogenesis pointed to one of the mechanisms of FFA implication in the progression of obesity to type II diabetes [START_REF] Collins | p38 Mitogen-activated protein kinase mediates free fatty acidinduced gluconeogenesis in hepatocytes[END_REF]. As for adipose tissue inflammatory response, the classical signaling cascades consequent to TLRs binding are linked to activation of nuclear factor kB (NFkB), which in turn drives transcription of proinflammatory cytokines and AMPs [START_REF] Ronald | Plant and animal sensors of conserved microbial signatures[END_REF]. Considering the role of TLRs in triggering multiple responses, based on stimuli, and macrophage activation, the contribution of specific signaling pathways needs to be evaluated to understand and possibly predict metabolic outcomes (Figure 20). Toll-like receptor 4 (TLR4) is expressed on a variety of cell types, besides macrophages and adipocytes, and it is able to recognize a variety of endogenous ligands, such as lipopolysaccharide (LPS), a major constituent of the Gram-negative bacteria outer membrane, and FFA. The observation that TLR4-deficiency protected against insulin resistance in obesity induced by a high fat diet suggested that TLR4 could be the link between diet excess and insulin resistance [START_REF] Shi | TLR4 links innate immunity and fatty acid-induced insulin resistance[END_REF]. Moreover, aware of the causality link between inflammation and insulin resistance, the pathogenic role of increased TLR4 signalling in obesity-associated inflammation has also been demonstrated [START_REF] Brun | Increased intestinal permeability in obese mice: new evidence in the pathogenesis of nonalcoholic steatohepatitis[END_REF]) [START_REF] Cani | Metabolic endotoxemia initiates obesity and insulin resistance[END_REF]) [START_REF] Velloso | TLR4 at the crossroads of nutrients, gut microbiota and metabolic inflammation[END_REF]. Studies using C3H/HeJ mice with a loss of function mutation in TLR4 further proved the importance of this receptor in influencing WAT inflammation [START_REF] Suganami | Attenuation of obesity-induced adipose tissue inflammation in C3H/HeJ mice carrying a Toll-like receptor 4 mutation[END_REF]) and insulin resistance [START_REF] Poggi | C3H/HeJ mice carrying a toll-like receptor 4 mutation are protected against the development of insulin resistance in white adipose tissue in response to a high-fat diet[END_REF][START_REF] Tsukumo | Loss-of-function mutation in Toll-like receptor 4 prevents diet-induced obesity and insulin resistance[END_REF]). The role of a diet rich in saturated fatty acids, in particular oleate and palmitate, in inducing adipose-tissue inflammation was linked to TLR4 in monocyte/macrophage activation [START_REF] Lee | Saturated fatty acids, but not unsaturated fatty acids, induce the expression of cyclooxygenase-2 mediated through Toll-like receptor 4[END_REF]). Co-culture experiments with peritoneal macrophages and 3T3-L1 hypertrophied adipocytes highlighted the paracrine vicious cycle that most probably augments inflammation in obese WAT. In this cellular interplay, the importance of a functional TLR4 was demonstrated in anti-inflammatory adiponectin mRNA downregulation, suggesting a mechanism for the reduced adiponectin level observed in obese patients [START_REF] Suganami | A paracrine loop between adipocytes and macrophages aggravates inflammatory changes: role of free fatty acids and tumor necrosis factor alpha[END_REF]. In obesity, increased circulating FFA, released from WAT, and also influxes of LPS, attributed to increased intestinal permeability [START_REF] Brun | Increased intestinal permeability in obese mice: new evidence in the pathogenesis of nonalcoholic steatohepatitis[END_REF]) [START_REF] Creely | Lipopolysaccharide activates an innate immune system response in human adipose tissue in obesity and type 2 diabetes[END_REF], have explained some of the characteristic alterations, including infiltration of macrophages into WAT, insulin resistance and steatosis. In an elegant set of experiments, Cani et al. have shown that the chronic subcutaneous infusion of low doses of LPS in lean mice was sufficient to reproduce the dysfunctional metabolism of HFD-fed mice: higher glycemia and insulinemia, and whole-body, liver, and adipose tissue weight gain [START_REF] Cani | Metabolic endotoxemia initiates obesity and insulin resistance[END_REF]).

Despite all these progresses, the role of TLRs in the dialogue between adipocytes and macrophages in abnormal conditions such as obesity remains to be elucidated. In particular the physiological and pathological implication of macrophages in binding, internalizing and degrading, or transferring lipid components, including endotoxin [START_REF] Hampton | Recognition and plasma clearance of endotoxin by scavenger receptors[END_REF], to other cellular partners (i.e. adipocytes) needs to be further clarified.

In addition, TLRs are widely distributed in the body, including the brain. Glial cells [START_REF] Olson | Microglia initiate central nervous system innate and adaptive immune responses through multiple TLRs[END_REF], neurons [START_REF] Tang | Pivotal role for neuronal Toll-like receptors in ischemic brain injury and functional deficits[END_REF]) and endothelial cells forming the vessels that irrigate the brain [START_REF] Constantin | Neisseria meningitidis-induced death of cerebrovascular endothelium: mechanisms triggering transcriptional activation of inducible nitric oxide synthase[END_REF]) express functional TLRs. Better understanding the role of these receptors in the brain, in particular in regions mostly dedicated to food intake and body weight control, such as hypothalamus, will be fundamental in relation to central control of energy homeostasis in physiological and pathological conditions.

G-protein coupled receptors (GCPRs)

G protein-coupled receptor (GPCR) superfamily represents the largest protein family in the human genome. Identified also as GPRs, some of them are called free fatty acid receptors (FFARs) due to their role in free fatty acid sensing. Only recently, several studies have provided evidence that GPRs are dietary sensors that connect metabolic and immune systems [START_REF] Ichimura | Free fatty acid receptors as therapeutic targets for the treatment of diabetes[END_REF]).

Nutrient sensing does not implicate only sensing extracellular nutrients (intra-organism), but also extraorganismal sensing by taste receptors, important for food source evaluation. GPRs have this dual role. In fact, taste receptors present in the oral epithelium and at the intestinal level belong to the T1R and T2R families of G-protein coupled receptors [START_REF] Wu | Expression of bitter taste receptors of the T2R family in the gastrointestinal tract and enteroendocrine STC-1 cells[END_REF]. In addition specific GPRs can mediate the preference for specific fatty acids, as shown in mice deficient for GPR40 and GPR120 [START_REF] Cartoni | Taste preference for fatty acids is mediated by GPR40 and GPR120[END_REF]. Moreover, next to taste FFA, in the past half decade, deorphanization of several GPCRs has revealed that GPR40, GPR41, GPR43, GPR84 and GPR120 can sense concentration of extracellular FFA with various carbon chain lengths at various levels [START_REF] Yonezawa | Free fatty acids-sensing G protein-coupled receptors in drug targeting and therapeutics[END_REF]. Specifically, GPR40 and GPR120 are activated by medium-and long-chain FFA. GPR84 is activated by medium-chain, but not long-chain FFA [START_REF] Nagasaki | Inflammatory changes in adipose tissue enhance expression of GPR84, a medium-chain fatty acid receptor: TNFalpha enhances GPR84 expression in adipocytes[END_REF]). GPR41 (FFR3) and GPR43 (FFR2) are activated by short-chain FFA. Our interest in these specific receptors is due to the two animal models we used during the Doctorate, since in Study 1 we used two fatty acids (linoleic acid and conjugated linoleic acids) and in Study 2 we used a model of high fat diet-induced obesity.

For all these receptors the physiological roles are multiple and they are summarized in Figure 21. Several GPRs are found in the intestinal tract and one of their roles is to regulate energy homeostasis. For example, GPR40 (FFR1), GPR41 (FFR3), GPR43 (FFR2) and GPR120 (FFR4) have been demonstrated expressed in glucagon-like peptide-1 (GLP-1) and peptide YY, acting as sensors for short-chain fatty acids in enteroendocrine cells. Another metabolic tissue in which these receptors are well expressed is WAT. In this tissue, mostly GPR41, but also GPR43 and GPR120, have the role of sensing short-chain fatty acids and stimulating leptin production [START_REF] Xiong | Short-chain fatty acids stimulate leptin production in adipocytes through the G protein-coupled receptor GPR41[END_REF]), among others. However, in addition to their role in metabolism, GPRs have been found expressed on immune cells. For example, GPR43 in leukocytes (neutrophils, B-lymphocyte) of the lamina propria, but also in peripheral blood mononuclear cells (PBMC) or GPR84 in leukocytes and monocyte/macrophage, whereas GPR41 in leukocytes, although mostly in pancreatic β-cells [START_REF] Yonezawa | Free fatty acids-sensing G protein-coupled receptors in drug targeting and therapeutics[END_REF]) [START_REF] Ichimura | Free fatty acid receptors as therapeutic targets for the treatment of diabetes[END_REF].

In general, GPR40 and GPR120 are the two main studied receptors in the process of detecting long chain unsaturated FFA. Recent studies have shown how an increase in one particular nutrient (FFA) anticipates a response to the imminent increase in another nutrient (glucose), consequently stimulating insulin release by pancreatic beta cells [START_REF] Itoh | Free fatty acids regulate insulin secretion from pancreatic beta cells through GPR40[END_REF]) [START_REF] Hirasawa | Free fatty acids regulate gut incretin glucagon-like peptide-1 secretion through GPR120[END_REF]. GPR120 has been implicated in adipocyte differentiation and lipid accumulation [START_REF] Gotoh | The regulation of adipogenesis through GPR120[END_REF]). Indeed GPR120 is highly expressed in WAT, in particular at the plasma membrane of adipocytes more than in stromal-vascular cells [START_REF] Gotoh | The regulation of adipogenesis through GPR120[END_REF]. In adipocytes, it was shown to mediate insulin sensitizing effects, promoting PI3K/AKT activation [START_REF] Oh | GPR120 is an omega-3 fatty acid receptor mediating potent antiinflammatory and insulin-sensitizing effects[END_REF]. Studying its role in the context of obesity, in which GPR120 function is disrupted, this receptor has been recognized as key in the contribution to the systemic control of nutrient homeostasis and inflammation [START_REF] Ichimura | Dysfunction of lipid sensor GPR120 leads to obesity in both mouse and human[END_REF], (Oh da, Walenta et al. 2014). On the opposite, GPR43 (FFAR2) involvement in adipocyte differentiation is more controversial, holding this role in vitro (3T3-L1 preadipocytes) and in vivo (mice), but possibly not in humans [START_REF] Dewulf | Evaluation of the relationship between GPR43 and adiposity in human[END_REF]. In summary, due to the importance of FFA signaling in diverse metabolic processes, these dietary sensors have started to be studied in the context of inflammation and metabolic disorders.

Despite the fact that the precise mechanisms of action and regulation of GPRs in tissues are still unclear in physiological status, recent research has started to show their involvement in pathological conditions. In particular, GPR40 (FFA1) has been recently shown to be critical in the inflammatory response of microvascular endothelial cells to LPS [START_REF] Lu | GPR40/FFA1 and neutral sphingomyelinase are involved in palmitate-boosted inflammatory response of microvascular endothelial cells to LPS[END_REF]. Using human cardiac ECs, it was shown that palmitic acid, one of the saturated fatty acids mostly increased in T2D, increased IL-6 expression when alone and in combination with LPS, both at transcriptional and post-transcriptional level [START_REF] Lu | GPR40/FFA1 and neutral sphingomyelinase are involved in palmitate-boosted inflammatory response of microvascular endothelial cells to LPS[END_REF]. This same membrane receptor is activated by both CLA isomers (c9,t11-and t10,c12-CLA) in vitro; through this activation, glucose-stimulated insulin secretion is increased [START_REF] Schmidt | Conjugated linoleic acids mediate insulin release through islet G protein-coupled receptor FFA1/GPR40[END_REF]. This mechanism was used to explain the increased insulin-releasing capacity of pancreatic islets reported by Poirier et al. in CLA-fed mice (Diabetologia 2005, Biochimie 2005). However, the implication of GPR40 in the potential deleterious effects of fatty acids on β-cell function [START_REF] Schmidt | Conjugated linoleic acids mediate insulin release through islet G protein-coupled receptor FFA1/GPR40[END_REF] was not evaluated in this study. Another example of regulation of inflammatory responses by GPRs is given by GPR43 (FFAR2). It has been demonstrated that short chain fatty acids bind the GPR43 and through those interactions profoundly affect inflammatory responses, favoring resolution in murine models of colitis, arthritis and asthma [START_REF] Maslowski | Regulation of inflammatory responses by gut microbiota and chemoattractant receptor GPR43[END_REF]. In particular in a model of intestinal inflammation, GPR43 was found essential to conduct an effective immune response (neutrophil recruitment) in restraining? intestinal bacterial translocation in the acute setting [START_REF] Sina | G protein-coupled receptor 43 is essential for neutrophil recruitment during intestinal inflammation[END_REF], even though if in the chronic setting, GPR43 deficient mice were protected against inflammatory tissue destruction induced by DSS [START_REF] Sina | G protein-coupled receptor 43 is essential for neutrophil recruitment during intestinal inflammation[END_REF]. Concerning GPR120, it was suggested that this receptor has a critical role in the pathogenesis of obesity and type 2 diabetes, being highly upregulated in omental and subcutaneous adipose tissue of obese subjects [START_REF] Cao | Identification of a lipokine, a lipid hormone linking adipose tissue to systemic metabolism[END_REF]. In 2010, Olefsky and his colleagues demonstrated its anti-inflammatory properties.

Upon PUFA-activation, GPR120 is able to suppress LPS-induced inflammatory response (TNFα and IL-6) through beta-arrestin 2, a downstream molecule of GPR120, involved in toll-like receptor (TLR) and TNF pathway in macrophage, but not in adipocyte [START_REF] Oh | GPR120 is an omega-3 fatty acid receptor mediating potent antiinflammatory and insulin-sensitizing effects[END_REF]). More recently this antiinflammatory activity of GPR120 was further explained using mouse bone marrow-derived macrophages and a differentiated human monocyte cell line. This study showed that DHA, an omega 3 FFA suppresses inflammasome activation in macrophage predominantly using GPR120 (FFAR4) (Williams-Bey, Boularan et al. 2014). However its precise role in human pathologies needs to be further explored, as exemplified by GPR120 overexpression in correlation with TNFα in the inflamed ileal mucosa of Crohn disease patients [START_REF] Tsukahara | Tumor necrosis factor alpha decreases glucagon-like peptide-2 expression by up-regulating G-protein-coupled receptor 120 in Crohn disease[END_REF]. The dysfunction of this receptor has been causally linked to obesity in humans and rodents [START_REF] Ichimura | Dysfunction of lipid sensor GPR120 leads to obesity in both mouse and human[END_REF]. In particular, GPR120-deficient mice fed with a high-fat diet developed obesity, impaired glucose metabolism and insulin resistance, in association with increased WAT inflammation [START_REF] Ichimura | Dysfunction of lipid sensor GPR120 leads to obesity in both mouse and human[END_REF].

In summary, the role of GPRs has been most studied in the gastrointestinal tract, where the receptor activities are linked to their strategic location for sensing and transducing information to the host about nutrients and microbiome metabolic activity [START_REF] Little | Characterization of duodenal expression and localization of fatty acid-sensing receptors in humans: relationships with body mass index[END_REF]. Instead, in WAT, their regulation and function still lack an integrative view, in particular concerning the systemic control of nutrient homeostasis in physiological and pathological conditions, such as obesity.

G. Conjugated fatty acids: the t10,c12-CLA model

Fatty acids can be divided into four general categories: saturated, monounsaturated, polyunsaturated, and trans fats. The effects of FFA have been studied mostly in relation to coronary heart disease, with saturated fatty acids and trans fats increasing the risks of CVD and monounsaturated and polyunsaturated fatty acids decreasing them. However, careful attention must be given to the fact that these associations are not consistent in the literature.

Fatty acids are long-chain hydrocarbons. In saturated fatty acids, the carbon chain has the maximum number of hydrogen atoms attached to every carbon atom, whereas in unsaturated fatty acids a pair of hydrogen atoms (mono-) or more (poly-unsaturated) is missing because of a double bond between two carbon atoms. This carbon-carbon double bond can exist in the cis or trans configuration. When the two hydrogen atoms are on opposite sides of the double bond, the configuration is called trans. When the hydrogen atoms are on the same side of the double bond, the configuration is called cis More than 20 types of fatty acids are found in foods. They are extremely important for multiple functions, from energy production and storage to phospholipid membrane formation and signaling pathways. However due to their harmful effects in CVD, the Nutrition Committee of the American Heart Association has recommended to limit them in our diet. In particular it was recommended no more than 30 percent of a person's daily calories come from fat, with less than 7 percent of total calories from saturated fatty acids, and less than 1 percent from trans-fatty acids [START_REF] Lichtenstein | Diet and lifestyle recommendations revision 2006: a scientific statement from the American Heart Association Nutrition Committee[END_REF].

Once ingested, dietary lipids (usually triglycerides) need to be first degraded in order to be absorbed through the intestine. Salivary, gastric and pancreatic lipases are important enzymes that have the function to break down triglycerides into FFA and to make them soluble (emulsification) to aqueous intestinal environment. In enterocytes, which are the absorptive cells of the intestinal epithelium, they are reformed into triglycerides and packaged into chylomicrons or lipoproteins, which are released into the capillaries of the lymph system and then into the blood. Depending on their type, circulating FFA will have differential effects on the organism. This is a current area of investigation for very different aspects, such as for example how fatty acids differentially modulate the phagocytic activities of macrophages.

Only two fatty acids are known to be essential for humans: alpha-linolenic acid (an omega-3 fatty acid) and linoleic acid (an omega-6 fatty acid). This thesis will focus on the conjugated forms of Linoleic acid.

Conjugated fatty acids

Conjugated fatty acids are a mixture of positional and geometric isomers of polyunsaturated fatty acids with conjugated double bonds. Several studies indicate that this group of fatty acids has potent beneficial health effects, depending on different isomers [START_REF] Yuan | Conjugated linolenic acids and their bioactivities: a review[END_REF]). More detailed evaluations of the physiological bioactivities of conjugated fatty acids isomers on lifestyle-related diseases in humans and animals continue to be of great interest.

Conjugated linoleic acid (CLA)

Conjugated linoleic acids (CLA) refer to a group of dienoic derivatives of linoleic acid (LA) produced during biological or industrial hydrogenation of linoleic acid. CLA are long chain fatty acids with 18 carbon atoms, in which at least one pair of double bonds are separated by only one single bond in 14 possible positions and with 4 geometric combinations (cis/cis, cis/trans, trans/cis and trans/trans). CLA are mainly found in ruminant meats and dairy products [START_REF] Demizieux | Conjugated linoleic acid isomers in mitochondria: evidence for an alteration of fatty acid oxidation[END_REF], because they, are produced by microbial fermentation of linoleic acid (rumen bacteria Butyrivibrio fibrisolven - [START_REF] Kritchevsky | Antimutagenic and some other effects of conjugated linoleic acid[END_REF] in the rumen of ruminant animals and 9 desaturation of vaccenic acid in the mammary gland [START_REF] Griinari | Conjugated linoleic acid is synthesized endogenously in lactating dairy cows by Delta(9)-desaturase[END_REF]. Up to now only 20 of these polyunsaturated fatty acids have been identified, but two in particular have been the most studied due to their biologically active properties [START_REF] Pariza | The biologically active isomers of conjugated linoleic acid[END_REF]): rumenic acid (cis 9, trans 11-CLA) and trans 10, cis 12-CLA, the isomer of interest for the present study (Figure 22). Humans can have access to CLA from two sources, diet (essentially c9, t11-CLA -90% in human nutrition) and synthetic dietary supplements, often on the market as weight loss supplements, which commonly contain equal proportions of the two isomers, 40% of c9t11 and 44% of t10c12 [START_REF] Pariza | The biologically active isomers of conjugated linoleic acid[END_REF]) [START_REF] Gaullier | Clinical trial results support a preference for using CLA preparations enriched with two isomers rather than four isomers in human studies[END_REF]) [START_REF] Wang | Conjugated linoleic acid and obesity control: efficacy and mechanisms[END_REF], (House, Cassady et al. 2005) (Figure 22). Recent studies have reported the average of CLA consumed by non-vegetarian men and women from US and France, respectively 210 and 180 mg/day, representing less than 1% of the total energy intake [START_REF] Ritzenthaler | Estimation of conjugated linoleic acid intake by written dietary assessment methodologies underestimates actual intake evaluated by food duplicate methodology[END_REF].

CLA isomers: experimental effects

Different CLA isomers have been shown to exhibit a variety of unique properties such as anti-cancer (breast, lung, skin, stomach and colorectal), anti-atherogenic and immune response [START_REF] Ha | Anticarcinogens from fried ground beef: heat-altered derivatives of linoleic acid[END_REF], [START_REF] Bhattacharya | Biological effects of conjugated linoleic acids in health and disease[END_REF]. In particular enhancing effects in controlling body fat gain were reported in animal models [START_REF] Park | Effect of conjugated linoleic acid on body composition in mice[END_REF]) [START_REF] Wang | Conjugated linoleic acid and obesity control: efficacy and mechanisms[END_REF]) [START_REF] Pariza | Perspective on the safety and effectiveness of conjugated linoleic acid[END_REF]). Because technically CLA is a trans fat and there are numerous studies showing that industrial trans fats are harmful, research on the claimed health benefits of CLA has been followed carefully. Customers in general, but US customers in particular, are particularly attentive on the positive or negative press linking dietary choices to health. For example US consumers are increasingly demanding grass-fed products and also meat products designed to promote health [START_REF] Young | Novel aspects of health promoting compounds in meat[END_REF]). One of the reasons is that grass-fed beef and the derived raw dairy products have three to five times more cis-9 trans-11 CLA (seen as a potential anti-cancer and weight loss treatment) levels than those fed grainbased diet, together with lower total fat, higher minerals and vitamins.

Park et al. were the first to demonstrate that CLA modulated body composition, reducing body fat mass of 60% in both male and female mice [START_REF] Park | Effect of conjugated linoleic acid on body composition in mice[END_REF]). The "anti-obesity" effects of CLA, have been linked to its capacity to bind and activate PPARα, potentially affecting lipid metabolism through transcriptional events [START_REF] Moya-Camarena | Conjugated linoleic acid is a potent naturally occurring ligand and activator of PPARalpha[END_REF]), but a precise mechanism has yet to be defined. Despite differences in body composition changes in response to CLA, most of the animal studies report effects on food/energy intake and total body fat content reduction in mice [START_REF] Delany | Conjugated linoleic acid rapidly reduces body fat content in mice without affecting energy intake[END_REF]) [START_REF] Belury | Dietary conjugated linoleic acid in health: physiological effects and mechanisms of action[END_REF]) [START_REF] Wang | Conjugated linoleic acid and obesity control: efficacy and mechanisms[END_REF]) rats and chickens [START_REF] Park | Conjugated linoleic acid (CLA) prevents body fat accumulation and weight gain in an animal model[END_REF]) [START_REF] Choi | Effects of three different conjugated linoleic acid preparations on insulin signalling, fat oxidation and mitochondrial function in rats fed a high-fat diet[END_REF]). However, conversely to fat mass, liver weight has been shown to increase, due at least in part to lipid spilling accumulation. The t10c12 isomer may be the form responsible for the effects on weight loss and body fat deposition in animals [START_REF] Park | Evidence that the trans-10,cis-12 isomer of conjugated linoleic acid induces body composition changes in mice[END_REF]) [START_REF] Ryder | Isomer-specific antidiabetic properties of conjugated linoleic acid. Improved glucose tolerance, skeletal muscle insulin action, and UCP-2 gene expression[END_REF]). However, in human studies, results on CLA are controversial, with studies showing effects on fat mass, serum total lipids, decrease whole body glucose uptake [START_REF] Blankson | Conjugated linoleic acid reduces body fat mass in overweight and obese humans[END_REF]) [START_REF] Raff | Conjugated linoleic acids reduce body fat in healthy postmenopausal women[END_REF]) and other studies contradicting the beneficial effects [START_REF] Dhurandhar | Initial weight loss as a predictor of response to obesity drugs[END_REF]) [START_REF] Zambell | Conjugated linoleic acid supplementation in humans: effects on body composition and energy expenditure[END_REF], [START_REF] Moloney | Conjugated linoleic acid supplementation, insulin sensitivity, and lipoprotein metabolism in patients with type 2 diabetes mellitus[END_REF]). The dosage, the length of the treatment and the degree of obesity are factors that can affect the consistency of clinical results (Kennedy, Martinez et al. 2010).

A murine model of lypoatrophic syndrome: t10, c12 CLA

In 2005, Poirier et al. demonstrated that CLA-mediated lipoatrophic syndrome is strictly dependent on the t10, c12-CLA isomer (Clement, Poirier et al. 2002). The reduction of fat mass is especially dramatic in the mouse, in which WAT inflammation is associated with severe hyperinsulinemia, IR and massive liver steatosis in response to dietary supplementation (mixture of 50% t10,c12-CLA and 50% c9,t11-CLA) (Poirier, Niot et al. 2005) or daily gavage with t10,c12-CLA (Poirier, Shapiro et al. 2006). In Figure 23, the effects of dietry supplementation with linoleic acid, C9,t11-CLA and t10,c12-CLA for 28 days are reported for periuteral WAT, liver and circulating insulin levels. This is the animal model that we decided to use for our experiments thanks to a successful collaboration between Dr M Guerre-Millo, from the team of K Clément, and Dr H Poirier and Dr I Niot from AgroSup Dijon (Dijon, France). In the next pages the current concepts about the effects of trans-10, cis-12 CLA (CLA) on metabolic organs will be described, from adipose tissue lipodystrophy to hyperinsulinemia and hepatic steatosis in particular. It will be a single organ review of the literature about CLA effects, although aware that the effects of CLA are clearly the result of the interconnection between the different organs.

Adipose tissue and t10, c12 CLA

Most of the studies performed in mice demonstrated that t10,c12 CLA reduces the fat mass [START_REF] Bhattacharya | Biological effects of conjugated linoleic acids in health and disease[END_REF]. In particular the adult females C57BL6 are a hyper-sensible model, as shown in a study in which a diet with 1% CLA (compared to body mass) was administered for four weeks caused a lipoatrophic syndrome (Tsuboyama-Kasaoka, Takahashi et al. 2000). The rapid and massive changes in body composition in the mouse are not reflected only by fat store decrease, but also by an increase in lean body mass [START_REF] Park | Effect of conjugated linoleic acid on body composition in mice[END_REF]) [START_REF] West | Effects of conjugated linoleic acid on body fat and energy metabolism in the mouse[END_REF] (Poirier, Shapiro et al. 2006) and these effects were shown in obese mice as well (House, Cassady et al. 2005). In line with the increased proinflammatory cytokines (TNFα, IL6 and IL8 in particular), a drastic infiltration of macrophages was observed in WAT already after 3 days of CLA administration (daily gavage) (Poirier, Shapiro et al. 2006). The observed inflammatory response to CLA has been shown to be linked to the activation of NF-kB pathway [START_REF] Liu | Combined effects of rosiglitazone and conjugated linoleic acid on adiposity, insulin sensitivity, and hepatic steatosis in high-fat-fed mice[END_REF]).

The potential mechanisms explaining the effects of CLA supplementation on fat mass are multiple: CLA leads to an increase in energy expenditure [START_REF] West | Effects of conjugated linoleic acid on body fat and energy metabolism in the mouse[END_REF]) [START_REF] West | Conjugated linoleic acid persistently increases total energy expenditure in AKR/J mice without increasing uncoupling protein gene expression[END_REF], that could be secondary to a stimulation of sympathetic nervous activity [START_REF] Ohnuki | A single oral administration of conjugated linoleic acid enhanced energy metabolism in mice[END_REF]. CLA reduces lipid uptake and storage of adipocytes by inhibiting lipoprotein lipase, as demonstrated in vitro through 3T3-L1 adipocytes [START_REF] Park | Changes in body composition in mice during feeding and withdrawal of conjugated linoleic acid[END_REF]. CLA reduces lipogenesis by decreasing expression of SREBP-1c and PPAR-γ and their downstream genes, ACC, FAS, and SCD1 (as for adipocytes decreased size and lipid content) (House, Cassady et al. 2005). CLA reduces adipose tissue mass through an apoptotic mechanism linked to an increase in tumor necrosis factor production (as for adipocytes decrease in number) (Kennedy, Martinez et al. 2010).

All these mechanisms contribute to the higher circulatory levels of free FA (FFA) and the consequent effects on liver. In association with the adipocyte alterations described above, only one study previously reported extracellular matrix accumulation in the mouse mammary fat pad (Russell, McGee et al. 2007). One week of t10,c12-CLA enriched diet was sufficient to induced tissue remodeling and concurrent leukocyte accumulation in this depot. In relation to the experimental setting that we chose for the Study I presented in the Research Project section of the Thesis, it is important to mention one last study that focused on adipose tissue depletion and the associated development of IR [START_REF] Purushotham | Maintenance of adiponectin attenuates insulin resistance induced by dietary conjugated linoleic acid in mice[END_REF]. In this latter study the effects of CLA on hepatic steatosis and IR were observed in C57BL6 male mice after 30 days of diet containing 1.5% CLA, along with drastic reduction of epidydimal adipose mass and circulating adiponectin. To my knowledge, this is the only study that reported the reversibility of the effects of CLA: body weight, adipose tissue mass, in line with adiponectin levels, and hepatic steatosis were attenuated after switching the mice to a diet without CLA for the following 30 days.

Liver and t10, c12 CLA

Liver is an important target for CLA effects. In fact another consequence of dietary CLA supplementation in mice is massive liver enlargement (Tsuboyama-Kasaoka, Takahashi et al. 2000) (DeLany, Blohm et al. 1999) (DeLany and West 2000) [START_REF] West | Conjugated linoleic acid persistently increases total energy expenditure in AKR/J mice without increasing uncoupling protein gene expression[END_REF], concurrent to decrease in body adiposity and development of insulin resistance. The intensity of lipid accumulation varies depending on the level of CLA in the diet, duration of feeding, physiological condition, and animal species.

Our collaborators in Dijon demonstrated that fatty liver and hyperinsulinemia, were associated to lipoatrophy specifically induced by the t10,c12-isomer of CLA in mice, whereas 4 weeks of a diet enriched in in c9,t11-CLA and LA had no significant effect (Poirier, Niot et al. 2005). This observation suggests that hepatic lipid accumulation is the result of mobilized fatty acids from adipose tissue. One of the mechanisms suggested is that liver in this condition is probably not able to increase FFA oxidation and export of synthesized triglycerides sufficiently [START_REF] Vyas | Dietary conjugated linoleic Acid and hepatic steatosis: speciesspecific effects on liver and adipose lipid metabolism and gene expression[END_REF]. Adipocyte changes are certainly linked to steatosis. In fact re-establishing the levels of leptin through external supplementation or adiponectin maintenance using rosiglitazone is sufficient to attenuate hepatic steatotic condition and normalize the insulin levels in CLA-fed mice (Tsuboyama-Kasaoka, Takahashi et al. 2000) (Tsuboyama-Kasaoka, Miyazaki et al. 2003) [START_REF] Wendel | Conjugated linoleic acid fails to worsen insulin resistance but induces hepatic steatosis in the presence of leptin in ob/ob mice[END_REF]). However it is interesting to underline that leptin has no effect when infused into A-ZIP/F-1 fatless mice, suggesting that leptin insufficiency alone is not the cause of IR provoked by CLA [START_REF] Gavrilova | Surgical implantation of adipose tissue reverses diabetes in lipoatrophic mice[END_REF]. Not only re-establishing adipokine level, but also dietary FA or oil supplementation with higher n-3 and n-6 PUFA administered along with CLA were proved to ameliorate liver steatosis [START_REF] Oikawa | Arachidonic acid prevents fatty liver induced by conjugated linoleic acid in mice[END_REF], demonstrating that changes in hepatic FA composition could play an important role in progression of hepatic steatosis [START_REF] Vyas | Dietary conjugated linoleic Acid and hepatic steatosis: speciesspecific effects on liver and adipose lipid metabolism and gene expression[END_REF]. Another hypothesis proposed to explain CLA-induced liver steatosis is that it is the result of increased hepatic lipogenesis stimulated by high insulin levels. However, noteworthy is one study that showed CLA-induced hepatic steatosis in streptozotocin (STZ)-injected mice, with low insulin levels, indicating that next to hepatic lipogenesis induced by hyperinsulinemia, other regulatory mechanisms affect hepatic lipid accumulation [START_REF] Jourdan | Liver carbohydrate and lipid metabolism of insulin-deficient mice is altered by trans-10, cis-12 conjugated linoleic acid[END_REF].

Several transcription factors, including PPARα, liver-X-receptors (LXRs) and sterol responsive elementbinding protein 1 (SREBP1), have been evaluated for their role in hepatic lipid metabolism in an attempt to explain the mechanisms behind the massive hepatic steatosis observed in response to CLA [START_REF] Roche | Isomer-dependent metabolic effects of conjugated linoleic acid: insights from molecular markers sterol regulatory element-binding protein-1c and LXRalpha[END_REF]) [START_REF] Vyas | Dietary conjugated linoleic Acid and hepatic steatosis: speciesspecific effects on liver and adipose lipid metabolism and gene expression[END_REF]. For example, in mice, during hepatic steatosis, dietary CLA was associated with upregulation of genes associated with fatty acid uptake, such as FAT/CD36 and fat transporters (FATP5, FABP-1, FABP-4, and FABP-5). Finally, experiments in vitro, with Hep G2 cells, also contributed to clarify mechanisms of action of conjugated linoleic acids. For example, t10,c12 CLA in vitro reduces triglyceride secretion [START_REF] Lin | Conjugated linoleic acid isomers have differential effects on triglyceride secretion in Hep G2 cells[END_REF]) and acts on stearoyl-CoA desaturase-I regulation [START_REF] Choi | Regulation of stearoyl-CoA desaturase activity by the trans-10,cis-12 isomer of conjugated linoleic acid in HepG2 cells[END_REF].

Intestine and t10, c12 CLA

Due to its properties, conjugated linoleic acids have been proposed as important pharmaco-nutrients for modulating mucosal immunity and therapeutic responses in patients with inflammatory bowel disease (IBD)(Bassaganya-Riera and Hontecillas 2006). In this context, a mixture of 50:50 CLA isomers (c9,t11 and t10,c12-CLA) has been shown to contribute to delaying the onset of experimental IBD [START_REF] Evans | Conjugated linoleic acid ameliorates inflammation-induced colorectal cancer in mice through activation of PPARgamma[END_REF]. Moreover, a mixture of the two conjugated linoleic acid isomers appeared promising as a chemopreventive agent against colorectal cancer in mice, in part through a PPARγdependent mechanism [START_REF] Borniquel | Dietary conjugated linoleic acid activates PPARgamma and the intestinal trefoil factor in SW480 cells and mice with dextran sulfate sodium-induced colitis[END_REF]. However this proposition has been undermined by other studies reporting different effects. When used in vitro on human enterocyte-like Caco-2 cells, CLA altered global gene expression in an isomer-specific manner and increase paracellular epithelial permeability [START_REF] Roche | Fatty acids and epithelial permeability: effect of conjugated linoleic acid in Caco-2 cells[END_REF], as indicated by an altered distribution of occluding and ZO-1, structural components of the tight junctions regulating epithelium integrity. More recently, a microarray-based study further underlined the exclusive effect of t10,c12-CLA over c9,t11-CLA, in the intestinal model of Caco-2 cells [START_REF] Murphy | Conjugated linoleic acid alters global gene expression in human intestinal-like Caco-2 cells in an isomer-specific manner[END_REF]). In the specific study, t10,c12-CLA modulates processes inherently related to carcinogenesis, such as cell cycle, cell proliferation, and DNA metabolism, and transepithelial Ca transport (including claudin 2 and 4). However, the authors pointed out how the exact role of the individual claudin proteins within the functionality of the tight junction and consequently the physiological meaning of their alteration by t10,c12-CLA has still to be elucidated [START_REF] Murphy | Conjugated linoleic acid alters global gene expression in human intestinal-like Caco-2 cells in an isomer-specific manner[END_REF]). Finally, it is important to consider that CLA is introduced in our body through the diet, but also it can be produced and/or converted by bacteria inside the gastro-intestinal tract [START_REF] Lee | Human originated bacteria, Lactobacillus rhamnosus PL60, produce conjugated linoleic acid and show anti-obesity effects in diet-induced obese mice[END_REF]), [START_REF] Murphy | Conjugated linoleic acid alters global gene expression in human intestinal-like Caco-2 cells in an isomer-specific manner[END_REF]), (Druart, Neyrinck et al. 2014). Although most probably confined at the intestinal level, the biological role of the endogenously produced CLA could have effects on the intestinal epithelial barrier, as it has been recently demonstrated for a gut microbial metabolite of linoleic acid [START_REF] Miyamoto | A gut microbial metabolite of linoleic acid, 10-hydroxy-cis-12octadecenoic acid, ameliorates intestinal epithelial barrier impairment partially via GPR40-MEK-ERK pathway[END_REF].

Since in obesity intestinal permeability is increased [START_REF] Brun | Increased intestinal permeability in obese mice: new evidence in the pathogenesis of nonalcoholic steatohepatitis[END_REF]) and has been linked to endotoxemia and WAT inflammation, it is surprising that CLA-induced WAT inflammation has not been carefully explored yet in relationship to an organ so critical for metainflammation.

Pancreas and t10, c12 CLA

In humans the association between increased FFA concentration (from approximately 50 to approximately 750 microM) and increased IR was demonstrated using healthy volunteers during euglycemic-hyperinsulinemic clamping [START_REF] Boden | Mechanisms of fatty acid-induced inhibition of glucose uptake[END_REF]. In several studies the use of CLA was associated with hyperinsulinemia, with no hypoglycemia either in fed or fasted state. No alteration of blood glucose concentration were found after oral glucose tolerance test, whereas insulin sensitivity was markedly compromised in C57Bl/6J females fed for several weeks with a mixture of CLA isomers (Tsuboyama-Kasaoka, Takahashi et al. 2000). In 2005, Poirier et al demonstrated that CLA in vivo administration provokes changes in the secretory capacities of pancreas (Poirier, Rouault et al. 2005).

They studied the islets of Langerhans from healthy C57BL6 mice fed for 4 weeks with a CLA mixture and stimulated with different dose of glucose (2.8mM, basal dose and 16.7mM, high dose) and they observed non only that CLA increased insulin secretion, but also the size of islets was significantly increased (hyperplasia) (Poirier, Rouault et al. 2005). The precise mechanisms that are at the origin of such massive hyperinsulinemia in response to CLA are yet to be identified, even if the drastic reduction of adipokines, in particular adiponectin, has been called into question possibly as initial trigger.

Muscle and t10, c12 CLA

Up to now, only few studies have investigated the impact of CLA (mixture or single isomers) on insulin sensitivity of the muscle. In one study, in which CLA was supplemented 28 days, a slight up-regulation of Glut4 (1.5 fold) was found in gastrocnemius, contrasting with a massive down regulation in WAT (Tsuboyama-Kasaoka, Takahashi et al. 2000). In 2003, He et al showed that mice invalidated for PPARγ specifically in WAT presented lipoatrophy and insulin resistance in adipose tissue and liver, but not in muscles [START_REF] He | Adipose-specific peroxisome proliferator-activated receptor gamma knockout causes insulin resistance in fat and liver but not in muscle[END_REF]. More recently another study reported that CLA-fed mice do not accumulate excess lipid in their muscle, but preferentially in liver [START_REF] Kanosky | Mice do not accumulate muscle lipid in response to dietary conjugated linoleic acid[END_REF], suggesting that muscle probably remain insulin-sensitive during treatment. Indeed CLA does not appear to be a good model for lipid accumulation in muscles in this last study [START_REF] Scalerandi | Effect of conjugated linoleic acid mixtures and different edible oils in body composition and lipid regulation in mice[END_REF]. Lipid metabolism in muscles was decreased by CLA (mix-CLA) in growing mice, compared to mice fed with diets containing olive, maize and rapeseed oils supplemented with rumenic acid (c9,t11-CLA) for 30 days [START_REF] Scalerandi | Effect of conjugated linoleic acid mixtures and different edible oils in body composition and lipid regulation in mice[END_REF].

Immunity and t10,c12-CLA

Shortly after CLA administration a massive amount of macrophages infiltrate WAT of mice (Poirier, Shapiro et al. 2006) [START_REF] Liu | Combined effects of rosiglitazone and conjugated linoleic acid on adiposity, insulin sensitivity, and hepatic steatosis in high-fat-fed mice[END_REF]. CLA was also reported to induce mast cell recruitment in adipose tissue of murine mammary gland, in a study on CLA as a dietary chemopreventive agent in cancer; the effect was specific to the t10,c12 isomer (Russell, McGee et al. 2007).

Few human studies have reported a significant effect of CLA on leukocyte number [START_REF] Gaullier | Conjugated linoleic acid supplementation for 1 y reduces body fat mass in healthy overweight humans[END_REF]) [START_REF] Gaullier | Supplementation with conjugated linoleic acid for 24 months is well tolerated by and reduces body fat mass in healthy, overweight humans[END_REF]) [START_REF] Larsen | Conjugated linoleic acid supplementation for 1 y does not prevent weight or body fat regain[END_REF]; in one study no effects were observed [START_REF] Whigham | Safety profile of conjugated linoleic acid in a 12-month trial in obese humans[END_REF]). The significant increase observed at the circulatory level in humans has not been further explored by flow cytometry, so details about different subpopulations and tissue specific-infiltration are missing. In completely different experimental settings, CLA has been shown to affect monocyte migration [START_REF] Mcclelland | Conjugated linoleic acid suppresses the migratory and inflammatory phenotype of the monocyte/macrophage cell[END_REF], to increase chemotaxis of PMNs [START_REF] Paek | Trans-10, cis-12 conjugated linoleic acid directly enhances the chemotactic activity of porcine peripheral blood polymorphonuclear neutrophillic leukocytes by activating F-actin polymerization in vitro[END_REF] or to modulate macrophage phagocytic activity and phenotype [START_REF] Stachowska | Conjugated linoleic acids can change phagocytosis of human monocytes/macrophages by reduction in Cox-2 expression[END_REF]) [START_REF] Song | Upregulation of tumor necrosis factor-alpha expression by trans10cis12 conjugated linoleic acid enhances phagocytosis of RAW macrophages via a peroxisome proliferator-activated receptor gamma-dependent pathway[END_REF][START_REF] De Gaetano | Conjugated linoleic acid induces an atheroprotective macrophage MPhi2 phenotype and limits foam cell formation[END_REF].

In metabolic disorders, macrophages in particular uniquely possess a dual functionality, regulating and sustaining the chronic inflammatory response and regulating lipid accumulation and metabolism (Chinetti-Gbaguidi and Staels 2011). For these reasons, the effects of CLA on function and activity of this particular immune population deserve further investigations in vivo and in vitro.

Finally, conjugated linoleic acids have been and continue to be used as an interesting research tool [START_REF] Reynolds | Conjugated linoleic acid and inflammatory cell signalling[END_REF]. However the relevance of their systemic and local effects has yet to be clarified within the context of human health.

H. Intestinal microflora and metabolic diseases

Microbiota composition: functions and dysfunctions

In human adults, the gut contains between 1,000 and 1,500 bacterial species (commensal and symbiotic microbes), of which at least 160 species shared among individuals, forming a real organ with its proper metabolism and functions [START_REF] Qin | A human gut microbial gene catalogue established by metagenomic sequencing[END_REF]. Each person has a different microbiota in terms of type and quantity of species and of functional composition [START_REF] Arumugam | Enterotypes of the human gut microbiome[END_REF]) and its composition changes with age and diet, among other factors. In healthy humans, most of the bacterial cells belong to 4 phyla namely Firmicutes (65%), Bacteroidetes (16%), Proteobacteria (9%) and Actinobacteria (5%) [START_REF] Eckburg | Diversity of the human intestinal microbial flora[END_REF]. The figure below (Figure 26) shows slightly differences for humans, since it has been adapted from a more recent study [START_REF] Arumugam | Enterotypes of the human gut microbiome[END_REF], together with studies for the other organisms [START_REF] Brinkman | Caspase deficiency alters the murine gut microbiome[END_REF]) [START_REF] Chandler | Bacterial communities of diverse Drosophila species: ecological context of a host-microbe model system[END_REF]) [START_REF] Roeselers | Evidence for a core gut microbiota in the zebrafish[END_REF]) (Figure 24). Different phyla are represented by different color and the relative abundance of the lower taxonomic levels is indicated by font size. Looking at the phylum level, it is clear that mouse and human microbiota are similar, but at the genus level they differ -99% of wild-type mouse bacterial cells belong to two divisions only: Firmicutes and Bacteroidetes [START_REF] Ley | Obesity alters gut microbial ecology[END_REF]. Moreover rodents for their anatomy (i.e. gastrointestinal tract compartmentalization) and behavior (e.g., coprophagia) clearly differ significantly from humans. However, despite the differences, mice can still be a fundamental tool for unraveling mechanisms of host-microbiota interactions relevant to humans. Environmental factors can affect this immensely diverse ecosystem. Diet is considered the strongest determining factor. For example our gastrointestinal tract is colonized differently in early life, depending on dietary imputs. Human milk favors colonization by bifidobacteria (Firmicutes), whereas Bacteroides are dominant in formula-fed infants. Or another example is given by humans, who eat plenty of protein and animal fats typical of Western diet, displaying predominantly Bacteroides species in their gut, while for those who consume more carbohydrates, especially fibers, the Prevotella species dominate [START_REF] Wu | Linking long-term dietary patterns with gut microbial enterotypes[END_REF]. Or another example can be the gut microbiota of vegetarians and vegans, who are unable to metabolize L-carnitine, a nutrient present in red meat [START_REF] Koeth | Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis[END_REF]. Interestingly the combined genomes of the gut microbiota complement the human genome with 150-fold more genes [START_REF] Qin | A human gut microbial gene catalogue established by metagenomic sequencing[END_REF]) and these genes contribute significantly to the host physiology and metabolism [START_REF] Sommer | The gut microbiota--masters of host development and physiology[END_REF], [START_REF] Tremaroli | Functional interactions between the gut microbiota and host metabolism[END_REF]. In contrast to humans, gene richness of microbiota is much more plastic and alterations in gene presence and/or composition have been associated with several diseases. This association has been demonstrated in digestive diseases, such as inflammatory bowel diseases (IBD), but interestingly also in systemic diseases, including cancer and Parkinson. Metabolic diseases are obviously not excluded. An accumulating body of literature suggests that gut microbiota is an additional contributing factor to the pathophysiology of diseases such as obesity, anorexia and type II diabetes [START_REF] Turnbaugh | An obesity-associated gut microbiome with increased capacity for energy harvest[END_REF]) [START_REF] Rosser | Regulatory B cells are induced by gut microbiota-driven interleukin-1beta and interleukin-6 production[END_REF]. This is a rapidly-evolving field, where gut microbiota composition and gene richness are studied in relation to the altered metabolism of the host in order to find potential therapeutic. In 2007, it was demonstrated that consumption of a high-fat diet induces changes in the gut microbiota [START_REF] Erridge | A high-fat meal induces low-grade endotoxemia: evidence of a novel mechanism of postprandial inflammation[END_REF], whereas in 2008 the other side of the coin was revealed, showing that modulation of gut microbiota could be sufficient to control obesityassociated metabolic alterations [START_REF] Cani | Changes in gut microbiota control inflammation in obese mice through a mechanism involving GLP-2-driven improvement of gut permeability[END_REF]. Right after, a key study from the group of JI Gordon presented an effective model to test the effects of environmental and genetic factors on the gut microbiota and host physiology, the humanized gnotobiotic mice [START_REF] Turnbaugh | The effect of diet on the human gut microbiome: a metagenomic analysis in humanized gnotobiotic mice[END_REF]. In these mice, fresh or frozen adult human fecal microbial communities were transplanted into germ-free C57BL/6J mice, overcoming possible confounding factors like human environmental exposures, microbial ecology and genotype. This study not only demonstrated how gut microbiota adapt in composition and function in relation to dietary changes in short time, but also that a specific human metabolic phenotype (metabotype) can be transferred via the gut microbiota [START_REF] Turnbaugh | The effect of diet on the human gut microbiome: a metagenomic analysis in humanized gnotobiotic mice[END_REF]). This same approach helped exploring the potential causal relationship between altered gut microbiota and obesity in a study where microbiota from a human female twin discordant for the phenotype of obesity was transferred in germ free mice [START_REF] Walker | Microbiology. Fighting obesity with bacteria[END_REF] as illustrated in Figure 25. Germ-free mice inoculated with microbiota from the obese twin (red outline) had an increase in adiposity, whereas those receiving the lean microbiota (blue outline) remained lean [START_REF] Walker | Microbiology. Fighting obesity with bacteria[END_REF]. This elegant study proved for the first time that the microbiota from lean or obese humans induces similar phenotypes in mice. And even more remarkably this study substantiated that, when microbiota from lean donors was inoculated in the obese-recipient mice, the lean phenotype is transmissible and modifiable if the diet is appropriate (obese mice showed reduce adiposity gain). Moreover, recently, the temporal relationships between food intake, gut microbiota and metabolic and inflammatory phenotypes were investigated by our team in diet-induced weight-loss and weightstabilization interventions [START_REF] Cotillard | Dietary intervention impact on gut microbial gene richness[END_REF]. This approach revealed that obese people are more likely to have a low microbial gene richness (on average 379,436 genes -well below the threshold of 480,000 genes) that is associated with a greater gain of weight resistance and more marked inflammatory phenotype in general (increased serum leptin and triglycerides, decreased serum adiponectin and HDL-cholesterol) than healthy people [START_REF] Cotillard | Dietary intervention impact on gut microbial gene richness[END_REF]. The reduced microbial gene richness (40%) in association with these deleterious characteristics could bring obese individuals to increased risk of comorbidities such as pre-diabetes, type-2-diabetes and cardiovascular disorders (Le [START_REF] Chatelier | Richness of human gut microbiome correlates with metabolic markers[END_REF]). This study, together with a concomitant one [START_REF] Kong | Gut microbiota after gastric bypass in human obesity: increased richness and associations of bacterial genera with adipose tissue genes[END_REF], remarkably observed that part of the pathological alterations linked to the obese phenotype can be rescued by dietary interventions or bariatric surgery, including the modification of microbiota composition toward a more healthy profile. However, despite the recent progress, more research is needed to clarify the precise mechanisms by which the gut microbiota modulates obesity. In fact, little is known apart from a potential role in energy harvest from the diet and lipid metabolism [START_REF] Backhed | The gut microbiota as an environmental factor that regulates fat storage[END_REF]), [START_REF] Turnbaugh | An obesity-associated gut microbiome with increased capacity for energy harvest[END_REF]) [START_REF] Backhed | Mechanisms underlying the resistance to diet-induced obesity in germ-free mice[END_REF]) [START_REF] Tremaroli | Functional interactions between the gut microbiota and host metabolism[END_REF]. Moreover colonized mice consume less food than their germ-free counterparts and so increased food intake cannot explain the obese phenotype in colonized mice [START_REF] Backhed | The gut microbiota as an environmental factor that regulates fat storage[END_REF]. The use of genetically modified germ-free mice may facilitate to delineate the molecular mechanisms by which specific microbes or communities affect host metabolism.

Symbiosis between the host and the microbiota

Since each individual has his own microbiota, it is hard to evaluate the role and the involvement of the different bacterial strains at the local and systemic level. It was in 2011 that the concept of enterotypes was introduced [START_REF] Arumugam | Enterotypes of the human gut microbiome[END_REF]) as a way to stratify human individuals based on their gut microbiome. This study published by the MetaHIT consortium combined 22 newly sequenced faecal metagenomes of individuals from four countries with previously published data sets and identified three robust clusters (referred to as enterotypes) that are not nation or continent specific [START_REF] Arumugam | Enterotypes of the human gut microbiome[END_REF]. Precise computational procedures revealed that gut microbiota variation generally stratified, indicating the existence of a limited number of well-balanced host-microbial symbiotic states that might respond differently to diet and drug intake [START_REF] Arumugam | Enterotypes of the human gut microbiome[END_REF]. At the core of this classification there is the symbiotic relationship between host and microbiota, a relationship in which both entities benefit. In fact the host provides an ecologic niche and nutrient source for the microbiota, and, in turn, the commensal organisms help maintaining a healthy homeostasis [START_REF] Kostic | Exploring host-microbiota interactions in animal models and humans[END_REF], [START_REF] Chow | Host-bacterial symbiosis in health and disease[END_REF]. The gut microbiota has the capacity to contribute to host physiology within and outside the gut. Within the gut, microbiota is essential for normal development and homeostasis of the immune system in the gut, protects against pathogenic bacteria, and modulates proper development of villus architecture and angiogenesis within the intestine [START_REF] Sommer | The gut microbiota--masters of host development and physiology[END_REF]) [START_REF] Tremaroli | Functional interactions between the gut microbiota and host metabolism[END_REF]. Outside the gut, microbiota affects bone mineral density, several host metabolic functions, even behavior [START_REF] Greiner | The gut microbiota modulates glycaemic control and serum metabolite profiles in non-obese diabetic mice[END_REF]) [START_REF] Sommer | The gut microbiota--masters of host development and physiology[END_REF]) [START_REF] Tremaroli | Functional interactions between the gut microbiota and host metabolism[END_REF], and potentially systemic immunity [START_REF] Surana | Deciphering the tete-a-tete between the microbiota and the immune system[END_REF]. All the potential effects of gut microbiota on the host are the result of the delicate harmony of the symbiotic relashionship. If, however, this delicately balanced system is perturbed, a state termed "dysbiosis" may result and consequently an expansion of harmful ("proinflammatory" pathobionts) bacteria could occur (Round and Mazmanian 2009) (Figure 26). The causes for this deregulation are not completely clear, but genetic predisposition and civilization effects have been suggested. A perfect example is given by inflammatory bowel disease in link with the "leaky gut" syndrome (due to the resulting increase in the intestinal permeability) [START_REF] Tamboli | Dysbiosis in inflammatory bowel disease[END_REF]) [START_REF] Paolella | Gut-liver axis and probiotics: their role in non-alcoholic fatty liver disease[END_REF]. In this case microflora role has been studied with more interest in Europe compared to US [START_REF] Kaur | Intestinal dysbiosis in inflammatory bowel disease[END_REF]. In IBD, but also in the metabolic syndrome context, the associations between specific bacterial populations and the disease process (independently of the studied pathology) are typically correlative as they examine differences in the microbiota of patients with or without a given disease (case-control approach). Consequently, in order to find specific candidate organisms that prevent or exacerbate inflammatory conditions and/or metabolic alterations, broad prospective investigations will be surely welcomed in the next future.

Microflora modulation: potential therapeutics

Very few therapeutic solutions are available for patients at early stages of obesity; drug treatments or/and lifestyle interventions are their main options. These solutions are yet limited in terms of long term health improvements, resulting ineffective for subjects at advanced stage of obesity (BMI > 35). Through selective criteria, patients who can medically benefit from bariatric surgery are identified, with the necessary careful evaluation of the potential complications. Effective alternatives are urgently needed and interventions to maintain or repair the dysregulated equilibrium of intestinal microflora in relation to the host health status could be promising options.

Improvement of human health by modifying the intestinal flora was proposed long ago (Metchnikoff E. 1908. In "The Prolongation Of Life: Optimistic Studies"). More recently there has been renewed interest in the use of fecal transplants for various disease states (i.e. colitis), based on the idea that the healthier transplanted flora will replace the disease-promoting microbiota [START_REF] Pamer | Fecal microbiota transplantation: effectiveness, complexities, and lingering concerns[END_REF]. Another therapeutic option that has shown potential in improving the host inflammatory and consequently metabolic status could be oral intake of probiotics to modify the metabolically unfavorable ecosystem [START_REF] Naito | Beneficial effect of oral administration of Lactobacillus casei strain Shirota on insulin resistance in diet-induced obesity mice[END_REF]) [START_REF] Yadav | Beneficial metabolic effects of a probiotic via butyrate-induced GLP-1 hormone secretion[END_REF]) [START_REF] Yoo | Probiotics L. plantarum and L. curvatus in combination alter hepatic lipid metabolism and suppress diet-induced obesity[END_REF]) [START_REF] Stenman | Potential probiotic Bifidobacterium animalis ssp. lactis 420 prevents weight gain and glucose intolerance in diet-induced obese mice[END_REF]) [START_REF] Chen | Incorporation of therapeutically modified bacteria into gut microbiota inhibits obesity[END_REF]) [START_REF] Wang | Modulation of gut microbiota during probiotic-mediated attenuation of metabolic syndrome in high fat diet-fed mice[END_REF]. The clinical world in general, but customers in particular, are now aware that in the late 2000s the benefic effects of probiotics have been claimed by the industry without being based on high quality scientific research (Francisca Joly Gomez, Edition Marabout 2014). Besides the missing approved health claims for some probiotic-containing food, the term has been clearly misused, for example, on products such as shampoos, disinfectants and aftershave, for which maintenance of viability and efficacy of the microbes used are not established. Moreover, even when rigorous scientific results prove probiotic value in a specific setting, further investigations are needed before presenting any conclusion on the effectiveness of one single administered organism. This is the case for Faecalibacterium prausnitzii in the context of colitis [START_REF] Sokol | Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients[END_REF] or other potentially beneficial external interventions to modify gut microbiota and impact human health [START_REF] Surana | Deciphering the tete-a-tete between the microbiota and the immune system[END_REF]. Finally, going back to the concept of enterotype, future research will most probably need to look into patient stratification and the degree of success in microflora modulation interventions.

Effectiveness of probiotic and prebiotic interventions

When considering interventions, it is necessary to make a distinction between prebiotic and probiotic.

Based on the World Health Organization, the definition of probiotic is (any) "live microorganisms which, when administered in adequate amounts, confer a health benefit on the host" (May 2002, FAO/WHO Guidelines for the evaluation of probiotics in food) [START_REF] Reid | The importance of guidelines in the development and application of probiotics[END_REF]. This vague definition opens interpretations and misuses of probiotic term. Last year, an Expert Meeting was organized by the International Scientific Association for Probiotics and Prebiotics (ISAPP) in order to refine precisely the term probiotic. All the stakeholders involved in the probiotic field worked together performing their respective duties with the final goal to benefit society from scientific advancement in the field [START_REF] Hill | Expert consensus document. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic[END_REF]).

Together they proposed a set of benchmarks useful to guide clinicians and consumers in differentiating the diverse probiotic products on the market. Recommendations for definition of probiotic included the microbial species that have a reasonable expectation of delivering benefits for the wellbeing of the host (as reported in properly controlled trials), used only on products that deliver live micro-organisms with a suitable viable count of well-defined strains (dead micro-organisms, microbial products, microbial components do not come under the probiotic classification). All probiotics must be safe for their intended use. Any specific claim beyond "contains probiotics" must be further substantiated. Undefined faecal microbiota transplants need to be kept outside the probiotic framework Being live bacteria, probiotics are definitely more delicate than prebiotics. Prebiotic is a specialized plant fiber that beneficially nourishes the "good" bacteria already in the gut. As for the concept of probiotic, a group of both academic and industry experts (in the ILSI Europe Prebiotic Expert Group and Prebiotic Task Force, respectively) met to define the prebiotics [START_REF] Roberfroid | Prebiotic effects: metabolic and health benefits[END_REF]. In 2010, a new definition was published for the prebiotic effect: "the selective stimulation of growth and/or activity(ies) of one, or a limited number of microbial genus(era)/species in the gut microbiota that confer(s) health benefits to the host". No food products were clearly classified as prebiotic during that symposium, but the prebiotic concept was established as a scientific fact. Nutritional elements, such as plant fibers, which the body is not able to digest, can act as a fertilizer to promote the growth of many of the good bacteria in large bowel or colon. Usually the dietary fibers contained in prebiotic supplements are inulin and/or oligofructose. The use of prebiotics has been shown to improve insulin sensitivity either directly because their fermentation at the colonic level modulate systemic short chain fatty acids levels or indirectly through bacteria which, in turn, provide health benefits. Research on prebiotics has demonstrated that the important health benefits provided can extend from lower gut health to overall well-being [START_REF] Roberfroid | Prebiotic effects: metabolic and health benefits[END_REF]. Several studies have been conducted using prebiotics in the context of a variety of diseases, including obesity-associated disorders. Beneficial effects on the metabolic phenotype, usually body weight evaluation, energy homeostasis, energy intake, adipogenic effects, inflammatory markers, are evaluated in response to the microbiota modulation, using probiotics, but also prebiotics and food (da Silva, dos Santos et al. 2013) [START_REF] De | Microbiota-generated metabolites promote metabolic benefits via gut-brain neural circuits[END_REF]) [START_REF] Everard | Gut microbiota and GLP-1[END_REF]. However, further studies are required to prove and optimize the potential effectiveness of this type of nutritional intervention.

Since probiotics are taken orally, this confers increased value to the benefits provided by bacterial supplementation. Products that cause a selective modification of gut microbiota, favoring normobiosis or reducing the risk of dysbiosis and associated pathologies, have been demonstrated promising in some preliminary studies. For example the beneficial effect of certain probiotics could be explained through the regulation of GPR120 at the intestinal epithelial level [START_REF] Fredborg | G protein-coupled receptor120 (GPR120) transcription in intestinal epithelial cells is significantly affected by bacteria belonging to the Bacteroides, Proteobacteria, and Firmicutes phyla[END_REF]. For these reasons, at present, health and food industries are engaging in huge financial tasks towards scientific research in order to find and guarantee a bacterial strain property and use.

RESEARCH PROJET: PURPOSE AND RESULTS

A. Hypothesis and models used

Obesity, insulin resistance and the associated metabolic complications are among the greatest causes of morbidity and premature mortality today. To understand the appropriate functioning of adipose tissue is essential for human health. In chronic pathologies, such as fat excess (obesity) or fat loss (lipodystrophy), the physiology of adipose tissue is deeply altered. Studying the capacity of adipose tissue in maintaining or restoring physiological homeostasis, in the context of such functional disturbances, can reveal new common molecular and genetic pathways implicated in the MetS and help to conceive new therapeutic strategies for the associated metabolic disturbances.

For a decade now, obesity has been seen as an inflammatory disease characterized by a chronic lowgrade inflammatory state. Despite the proved association between inflammation and metabolism, the source and type of triggering factors activating the immune system at the level of the different metabolic organs remain unknown. In addition, the beneficial potential of dietary interventions to prevent and/or attenuate the severity of liver and adipose tissue inflammation and the associated metabolic alterations remains to be determined.

The overall purpose of our investigations was to explore the capacity of an organism to respond to a nutritional challenge and to evaluate the consequences at local and systemic level, consequences that could be positive or negative for the organism health. The complexity of this approach requires a global vision of the body physiology, linking nutrition (functional foods) to the complex of metabolic organs and activities. Because the immune system, with the classical first response represented by inflammation, is central to "self defense" in case of external attacks, from viruses to nutrients (excess), and because it is now recognized that it monitors the metabolic state of tissues, we focused on its response in metabolic altered conditions. In particular we ought to answer to those broad questions directing our attention specifically on white adipose tissue and the intestinal tract, two large reservoirs of immune cells, to explore tissue plasticity in response to nutritional intervention.

In the present project, two animal models were employed.

 Study I: a lipoatrophic mouse model induced by t10,c12-Conjugated Linoleic Acid administration (CLA).

 Study II: a mouse model of high fat diet (HFD)-induced obesity, with or without bacterial administration.

The study of these animal models is integrated in the context of adipose tissue alterations, in which inflammation, immune cells, angiogenesis and tissue remodeling have a role. The interactions of the different stromal components, such as preadipocytes, fibroblast-like, endothelial cells, vascular smooth muscle cells, neurons, and immune cells, with adipocytes have not been fully elucidated, not even during the most studied obesity. As described in the Introduction part, adipocyte dysfunction is due to hypertrophy in obesity, whereas after drastic fat mass loss, e.g. following HIV-infection, or in lipodistrophy, adipocyte dysfunction is due to lipoatrophy. Despite the contrasting alterations, these two pathologies present similar complications, in particular insulin resistance. The direct or indirect effects of adipocyte dysfunction on the metabolic profile of the animals represented the common question we aimed to answer in both studies.

In the specific context of Study I, through the acute administration of t10,c12-CLA, previously reported to induced a lipoatrophic state in mice (Poirier, Shapiro et al. 2006), we evaluated the ability of WAT to cope with this nutritional external stressor, dealing with local (molecular, cellular, structural) and systemic (plasma parameters) changes associated with the drastic fat mass loss induced by this isomer.

Adipose tissue plasticity was also evaluated after treatment disruption in relation to insulin alterations.

In particular the immune response of WAT was characterized by the analysis of the adipose tissue stromal fraction in two different depots, subcutaneous (inguinal -sWAT) and visceral (peri-uteral -pWAT) WAT. The actions of innate and adaptive immune responses were characterized by flow cytometry analysis in CLA-treated mice in comparison with the control group (LA-treated). Since infiltrating immune cells are known to have a profound effect on adipocytes, we decided to look for correlations between immune cell quantity (and phenotype) and WAT structural alterations. We focused in particular on macrophages (ATMs), the most represented population in WAT of CLA-treated mice, to identify their potential involvement in the ECM accumulation and/or tissue repair/resolution [START_REF] Olefsky | Macrophages, inflammation, and insulin resistance[END_REF]. Finally the hypothesis that the gastrointestinal tract and its microflora could be affected by CLA administration (as dietary stress) was tested, in order to consider potential endotoxemia in influencing local (WAT) and systemic perturbations.

In the specific context of Study II, using the HFD-induced model of obesity, we analyzed the metabolic consequences of WAT hypertophy in a positive energy balance setting, that mimics the chronic lowgrade inflammation associated to human obesity. In addition, through the concomitant administration of specific probiotics, we investigated the potential beneficial effects of gut microbiota changes (direct) or metabolites (indirect) on adiposity and metabolic perturbations associated to obesity.

In both studies, open questions were addressed:

 How adipose tissue copes with a chronic or acute nutritional stress?  What are the systemic consequences of adipose tissue response to a nutritional stress?  Are local and/or systemic effects of a nutritional stress mediated, at least in part, through gut microbiota derived signals?

In obesity, the altered interactions between adipocytes and the stromal components, in particular the immune cells, lead to changes in the release of adipokines, cytokines as well as FFA setting up an inflammatory environment [START_REF] Rosen | Adipocytes as regulators of energy balance and glucose homeostasis[END_REF], [START_REF] Ouchi | Adipokines in inflammation and metabolic disease[END_REF]. In both visceral and subcutaneous adipose tissues, macrophages are viewed as important actors of the inflammatory process taking place in obesity [START_REF] Lumeng | Increased inflammatory properties of adipose tissue macrophages recruited during diet-induced obesity[END_REF]), [START_REF] Zeyda | Human adipose tissue macrophages are of an anti-inflammatory phenotype but capable of excessive pro-inflammatory mediator production[END_REF]), [START_REF] Weisberg | CCR2 modulates inflammatory and metabolic effects of highfat feeding[END_REF]). Directly and/or indirectly affected by the deregulated adipokine milieu, macrophages released locally cytokines known to have a profound effect on adipocytes and the other stromal components [START_REF] Lumeng | Obesity induces a phenotypic switch in adipose tissue macrophage polarization[END_REF]), [START_REF] Zeyda | Human adipose tissue macrophages are of an anti-inflammatory phenotype but capable of excessive pro-inflammatory mediator production[END_REF]. In both studies the levels of these circulating factors were evaluated to better understand the effects of the two different nutritional interventions at the systemic level. Glucose and insulin levels were also considered.

It has been reported that the structure of consumed fat (emulsion vs. free oil) changes the extent of endotoxemia [START_REF] Mani | Dietary oil composition differentially modulates intestinal endotoxin transport and postprandial endotoxemia[END_REF]. Factors like diets inappropriately high in saturated fat and also bacterial infections have been associated with insulin resistance. Indeed the direct administration of lipopolysaccharide (LPS), a wall component of Gram-negative bacteria and well-known toll-like receptor 4 (TLR4) agonist, was shown to cause insulin resistance, recapitulating metabolic changes associated with high fat feeding [START_REF] Cani | Metabolic endotoxemia initiates obesity and insulin resistance[END_REF]. Accumulating evidence indicates that the gut microbiota might be involved in the inflammatory and metabolic perturbations linked to obesity (Ley, Turnbaugh et al. 2006), [START_REF] Turnbaugh | An obesity-associated gut microbiome with increased capacity for energy harvest[END_REF]) and even diabetes [START_REF] Larsen | Gut microbiota in human adults with type 2 diabetes differs from non-diabetic adults[END_REF]) [START_REF] Cani | Changes in gut microbiota control inflammation in obese mice through a mechanism involving GLP-2-driven improvement of gut permeability[END_REF]) [START_REF] Cani | Involvement of gut microbiota in the development of low-grade inflammation and type 2 diabetes associated with obesity[END_REF]. A diet rich in fat causes changes in the composition of the gut microbiota, possibly weakening the gut barrier function and consequently this might affects the host immune response [START_REF] Pendyala | A high-fat diet is associated with endotoxemia that originates from the gut[END_REF]. Chronic endotoxemia (the presence of endotoxins such as LPS in blood) occurs in obesity and the complex of CD14 and TLR4 on the surface of innate immune cells is engaged in the recognition of circulating gut-originated antigens, mainly LPS.

The hypothesis that acute administration of the specific lipids we used (LA and CLA) could affect gut microbiota and directly or indirectly affect gut permeability was explored through preliminary screening of microbiota and RT-PCR analysis of gut tissues. On the other hand, the hypothesis that the administration of specific probiotics could positively influence the microbiota composition and consequently improve the obesity-associated metabolic alterations was investigated. In two experimental settings, the potential anti-obesity effects of CLA (Study I) and probiotics (Study II) were studied in relation to inflammation and metabolism in the context of adipose tissue and intestine crosstalk.

B. Study I: Adipose tissue plasticity in response to short-term trans 10, cis12 conjugated linoleic acid (CLA) adimistration in mice

The objectives of the present study were:

 To determine the effects of a short-term administration of t10,c12-CLA on body weight, fat mass and circulating metabolic variables in C57Bl/6 female mice;

 To evaluate WAT cellular and molecular responses to t10,c12-CLA, with a particular focus on the dynamic of the WAT immune infiltrate;

 To evaluate the reversibility of the integrated response of the different WAT components after t10,c12-CLA withdrawal;

 To investigate potential intestinal alterations induced by t10,c12-CLA administration.

In this study, the previously characterized mouse model of lipoatrophic state induced by trans-10,cis-12 isomer conjugated linoleic acid (CLA) was employed to evaluate the ability of WAT to control the alterations induced by an acute nutritional stressor. Female mice are known to present higher susceptibility to CLA than male mice, although the mechanisms accounting for this disparity remain unknown. Moreover, in humans, because healthy women have more adipose tissue than men, they are more profoundly affected by lipodystrophy, with greater metabolic dysregulation [START_REF] Huang-Doran | Lipodystrophy: metabolic insights from a rare disorder[END_REF]. For these reasons, female C57Bl6 mice were employed for the study. The model has been developed by our team in collaboration with INSERM U866 NUTOX (Dijon, France). The experimental design is presented in the publication and synthesized in Figure 27.

INTRODUCTION

It is broadly admitted that accumulation of pro-inflammatory macrophages is a key component of adipose tissue (WAT) response to nutrient overload in obesity. Through the release of an array of pro-inflammatory chemokines and cytokines, macrophages are thought to perpetuate and propagate inflammation in WAT, leading eventually to a chronic pathological state. Although opposite in terms of excess versus deficiency of fat mass, obesity and lipodystrophy share common WAT alterations, including inflammation and macrophage infiltration as shown in several lipodystrophic mouse models [START_REF] Pajvani | Fat apoptosis through targeted activation of caspase 8: a new mouse model of inducible and reversible lipoatrophy[END_REF][START_REF] Herrero | Inflammation and adipose tissue macrophages in lipodystrophic mice[END_REF][START_REF] Wang | Lipoatrophy and severe metabolic disturbance in mice with fat-specific deletion of PPARgamma[END_REF] and in patients with HIVassociated lipodystrophy [START_REF] Jan | Altered fat differentiation and adipocytokine expression are inter-related and linked to morphological changes and insulin resistance in HIV-1-infected lipodystrophic patients[END_REF][START_REF] Caron-Debarle | HIVassociated lipodystrophy: from fat injury to premature aging[END_REF][START_REF] Giralt | Adipose tissue biology and HIVinfection[END_REF]. Enhanced extracellular matrix deposition and fibrosis are additional signatures of WAT pathology in both conditions [START_REF] Jan | Altered fat differentiation and adipocytokine expression are inter-related and linked to morphological changes and insulin resistance in HIV-1-infected lipodystrophic patients[END_REF][START_REF] Sun | Fibrosis and adipose tissue dysfunction[END_REF][START_REF] Vila | Immune cell Tolllike receptor 4 mediates the development of obesity-and endotoxemia-associated adipose tissue fibrosis[END_REF]. These local alterations are accompanied by systemic inflammation and insulin resistance, the most prevalent outcomes of WAT dysfunction common to obesity and lipodystrophy [START_REF] Villarroya | Lipodystrophy in HIV 1-infected patients: lessons for obesity research[END_REF].

The time frame required to mount WAT macrophage accumulation and insulin resistance is usually long, ranging from months in mice fed a high fat diet to years in human obesity.

Conversely, caloric restriction attenuates WAT inflammation and improves glycemic status in human and mouse obesity after prolonged periods of weight reduction or stabilization [START_REF] Clement | Weight loss regulates inflammation-related genes in white adipose tissue of obese subjects[END_REF][START_REF] Cancello | Reduction of macrophage infiltration and chemoattractant gene expression changes in white adipose tissue of morbidly obese subjects after surgery-induced weight loss[END_REF][START_REF] Divoux | Fibrosis in human adipose tissue: composition, distribution, and link with lipid metabolism and fat mass loss[END_REF][START_REF] Kovacikova | Dietary intervention-induced weight loss decreases macrophage content in adipose tissue of obese women[END_REF][START_REF] Kosteli | Weight loss and lipolysis promote a dynamic immune response in murine adipose tissue[END_REF].

These observations enlighten WAT plasticity in response to long-term modifications of energy balance and its close link with coordinated changes in metabolic status.

Conjugated linoleic acids (CLA) collectively refer to a group of linoleic acid derivatives with several positional and geometric isomers. Among those, the trans10,cis12 CLA (t10,c12-CLA) exerts a potent and rapid body weight and fat mass reducing effect in mice [START_REF] Tsuboyama-Kasaoka | Conjugated linoleic acid supplementation reduces adipose tissue by apoptosis and develops lipodystrophy in mice[END_REF][START_REF] Clement | Dietary trans-10,cis-12 conjugated linoleic acid induces hyperinsulinemia and fatty liver in the mouse[END_REF].

Contrary to caloric restriction, t10,c12-CLA administration does not improve metabolic status, but provokes hyperinsulinemia and systemic insulin resistance [START_REF] Tsuboyama-Kasaoka | Conjugated linoleic acid supplementation reduces adipose tissue by apoptosis and develops lipodystrophy in mice[END_REF][START_REF] Clement | Dietary trans-10,cis-12 conjugated linoleic acid induces hyperinsulinemia and fatty liver in the mouse[END_REF][START_REF] Poirier | Hyperinsulinaemia triggered by dietary conjugated linoleic acid is associated with a decrease in leptin and adiponectin plasma levels and pancreatic beta cell hyperplasia in the mouse[END_REF][START_REF] Poirier | Nutritional supplementation with trans-10, cis-12-conjugated linoleic acid induces inflammation of white adipose tissue[END_REF]. We previously showed that this phenotype is temporally linked with marked macrophage infiltration in peri-uteral WAT in lean female mice [START_REF] Poirier | Nutritional supplementation with trans-10, cis-12-conjugated linoleic acid induces inflammation of white adipose tissue[END_REF]. In this lipoathrophic model, macrophage recruitment was unexpectedly rapid, since the presence of cells positive for the macrophage marker F4/80 was detected in WAT after 3 days of t10,c12-CLA administration. A similar rapid macrophage infiltration was reported after 3 days of caloric restriction in obese mice, preceding a progressive reduction in macrophage number over a 2 month-period [START_REF] Kosteli | Weight loss and lipolysis promote a dynamic immune response in murine adipose tissue[END_REF]. These concordant observations suggest that WAT has the capacity to engage an innate immune response over a short period of nutritional stress.

Since t10,c12-CLA leads to WAT macrophage infiltration and insulin resistance as in obesity and lipodystrophy, we ought to use this compound as a tool to explore the dynamics of these interrelated local and systemic responses and to test their potential reversibility in a shortterm time frame.

MATERIALS AND METHODS

Animals and treatment. Female 10 week-old C57BL/6J mice (Charles River Laboratory, L'Arbresle, France) were housed under 12-h light/dark cycles (lights on at 7:00 am) with ambient temperature at 23°C. Gavage was performed daily with 0.1 g sunflower oil, whose main component is linoleic acid (LA), or 0.1 g of a mixture with 0.08 g sunflower oil and 0.02 g triglycerides containing 79% of t10,c12-CLA (total CLA isomers 90.4% (79% t10,c12; 7% c9,t11; and 3.1% t/t), oleic acid 2.5%, linoleic acid 0.2%, saturated fatty acids 6.6%, Loders Croklaan, Wormerveer, Holland). For evaluation of body weight changes and daily food intake, mice were single-housed and monitored longitudinally. Total body fat mass was assessed with Eco-MRI body composition analyzer (Echo Medical System, Houston, TX, USA). The morning after the last gavage, blood was collected from orbital sinus in anesthetized mice, which were killed by cervical dislocation to collect subcutaneous inguinal (sWAT) and peri-uteral (pWAT) adipose tissue, liver, jejunum and colon. Pieces of WAT were snap frozen in liquid nitrogen before being stored at -80°C, fixed in paraformaldehyde or submitted to collagenase digestion.

Care of animals was in accordance with institutional animal care committee guidelines (University of Bourgogne, Dijon, France n°7812).

Immunohistochemistry. Fixed WAT sections of 5 µm were mounted on glass slides and processed according to standard procedures. Immunohistochemical staining was performed with an antibody targeted to CD68 (MCA1957; Abd Serotec, Oxford, UK). Section images were taken by a Zeiss 20 Axiostar Plus microscope, acquired by a TRI CCD camera and processed using Perfect Image V7.7, as previously described [START_REF] Cancello | Increased infiltration of macrophages in omental adipose tissue is associated with marked hepatic lesions in morbid human obesity[END_REF].

Picrosirius red staining. Slides of WAT biopsies were stained with picrosirius red. Collagen deposition was measured by histomorphometry using an Alphelys platform (Histolab Software, Plaisir, France). Quantification of collagen deposition was expressed as the mean ratio of fibrous tissue area stained with picrosirius red/total tissue surface in 10 random fields at ×10 magnification for each biopsy.

Hydroxyproline assay. Hydroxyproline was evaluated with a colorimetric assay Kit (BioVision, Inc., Milpitas, CA, USA), with some modifications. Frozen WAT biopsies were homogenized in 37°C 6N HCl at a concentration of 0.1 mg/µl and heated at 95°C for 20 hrs. The samples were left for 1 hour at room temperature and centrifuged to collect 10 µl of supernatant. After evaporation, samples were resuspended in 10 µl distilled water before addition of chloramine T reagent. An equal volume of DMAB reagent was added and incubated at 60°C for 90 min.

Absorbance was read at 560nm.

RNA isolation and quantitative RT-PCR analysis.

Total RNA was isolated with RNeasy kits (Qiagen, Valencia, CA, USA). Gene expression was determined by SYBR Green real-time PCR on 25 ng cDNA. Target gene expression was normalized to the geometric mean of three housekeeping genes (S18, cyclophyllin and RPLP19) and then expressed as fold induction by the comparative CT method using LA-treated control group as reference. Primers for mouse genes are shown in Table S1.

Flow cytometry analysis. Cells of the stroma vascular fraction (SVF) of sWAT and pWAT were obtained by collagenase digestion [START_REF] Dalmas | T cell-derived IL-22 amplifies IL-1beta-driven inflammation in human adipose tissue: relevance to obesity and type 2 diabetes[END_REF]. Cells were incubated in erythrocyte lysis buffer and resuspended in endotoxin-free PBS supplemented with 3 % FCS and 2mM EDTA (FACS buffer). SVF cells were incubated with anti-CD16/CD32 (eBioscience, San Diego, CA, USA) for 15 minutes to block Fc receptors and stained with appropriate monoclonal antibodies for minutes at 4°C in the dark. APC-eFluor780-labeled anti-CD45 (30-F11), PE-Cy7-labeled anti-F4/80 (BM8), eFluor450-labeled anti-CD11c (N418), FITC-labeled anti-CD206 (C068C2), FITC-labeled anti-CD3 (17-A2), PerCP-Cy5.5-labeled anti-CD4 (RM4-5), eFluor450-labeled anti-CD8 (53-6.7), PE-labeled anti-TCRγδ (GL-3), Alexa-Fluor700-labeled anti-NK1.1 (PK136) and APC-labeled anti-B220 (RA3-GB2) (all from eBioscience) were used to characterize SVF cell surface markers. To characterize intracellular expression of the transcription factor Foxp3, SVF cells were first stained for CD45, CD3, CD4 and CD8 surface markers, fixed and permeabilized with Foxp3 fixation/permeabilization kit for 20 minutes and incubated for minutes with PE-labeled anti-Foxp3 (FJK-16s, eBioscience). After staining, cells were washed with FACS buffer and acquired on a FACS LSRII flow cytometer using FACS Diva software (BD Biosciences, San Jose, CA, USA). Data analysis was performed with FlowJo 10.7 software (Tree Star, Ashland, OR, USA).

Blood variables.

Glucose was evaluated on peripheral EDTA-treated blood with a commercially available kit (Cayman Biochemical, Ann Arbor, MI, USA). Plasma insulin was measured with the ALPCO ultrasensitive kit (Alpco, Salem, NH, USA). Adiponectin and leptin were quantified by ELISA (R&D Systems, Minneapolis, MN, USA).

Fatty acid profile analysis. 20 mg of sWAT and pWAT, 50 mg of intestinal tissue (jejunum and colon) and 100 mg of liver were homogenized in methanol-chloroform (1/2 v/v). Homogenates were subjected to alkaline hydrolysis followed by methylation, as previously described [START_REF] Druart | Role of the lower and upper intestine in the production and absorption of gut microbiotaderived PUFA metabolites[END_REF].

Fatty acid methyl esters were quantified by gas-liquid chromatography (Focus GC, Thermo-Finnigan, Interscience, Belgium). Identification and quantification of each peak were made by comparison of retention times with standards (Alltech Associates; except CLAisomers from Nu-Chek Prep).

Statistical analysis.

Results are expressed as mean ± standard error of the mean (SEM). Normal distribution of data was tested with Shapiro-Wilk test. Differences between groups were determined using unpaired Student't test or Mann-Whitney test for variables normally or not normally distributed, respectively. Relationships between variables were assessed using Spearman correlation. All statistical analyses were performed with GraphPad Prism 5.0 Software (GraphPad Software, Inc, La Jolla, CA, USA). A p value < 0.05 was considered statistically significant.

RESULTS

Local and systemic reversible metabolic alterations.

Body weight decreased steadily during t10,c12-CLA administration (Fig 1A ) with no major change in food intake (Fig 1B), and returned rapidly to pre-treatment values upon t10,c12-CLA withdrawal. Reflecting lipoatrophy, the percentage of MRI-evaluated fat mass was reduced after 3 days of t10,c12-CLA administration and remained low thereafter (Table 1). Accordingly, the weight of WAT pads decreased, with a more pronounced reduction of subcutaneous inguinal (sWAT) than deep peri-uteral (pWAT) WAT depot (Table 1). After 7 days without t10,c12-CLA, pWAT mass was normalized to the values found in LA-treated control mice, while sWAT remained reduced. Adipocyte diameters were modestly affected by t10,c12-CLA administration or removal (Table 1).

At systemic level, insulin concentrations increased progressively (Table 1). At day 7, insulin circulating levels were 8-fold higher than in control mice and sharply decreased back to normal values upon t10,c12-CLA withdrawal. These data establish a direct link between hyperinsulinemia and t10,c12-CLA administration. No concomitant change in glycemia was consistent with the development of systemic insulin resistance.

Fatty acid profiling revealed that t10,c12-CLA prominently accumulated in WAT, where its concentrations per gram of tissue were 8 to 20 fold higher than in intestine or liver (Fig 1C). This tissue-specific difference pointed out WAT as the main metabolic tissue targeted by the CLA isomer. After 7 days of t10,c12-CLA, several adipokines were markedly down-regulated in pWAT and returned to normal levels of expression upon t10,c12-CLA removal (Fig 1D ). A similar pattern of regulation was found in sWAT (data not shown). Adiponectin followed the same variations at gene level and in the circulation (Fig 1D ,Table 1). Unlike other adipokines, however, leptin mRNA and plasma levels were not significantly changed upon t10,c12-CLA manipulation. This suggests that t10,c12-CLA dictates adipocyte secretome mostly by targeting transcript levels, leaving secretion pathways virtually unaltered.

Besides adipokines, genes functionally involved in de novo lipogenesis and lipolysis were profoundly down-regulated by t10,c12-CLA (Fig 1E). Upon t10,c12-CLA withdrawal, mRNA levels rebounded, although not always back to the values of LA-treated mice. The mRNAs of two lipogenic enzymes, Fatty acid synthase (FAS) and Acetyl-CoA-carboxylase (ACC), were normalized (FAS) and even up-regulated (ACC) in pWAT, but remained decreased in sWAT in line with less efficient mass recovery in this subcutaneous fat depot. In sharp contrast with the marked down-regulation of lipogenic and lipolytic genes, genes implicated in lipophagy, including the transcription factor Tfeb, its target, the lysosomal acid lipase (Lipa) and the mitochondrial protein UCP2, were up-regulated in both sWAT and pWAT in response to t10,c12-CLA (Fig 1E). The reversibility of this regulation identified t10,c12-CLA as the trigger of lipophagic genes overexpression in WAT.

Reversible WAT macrophage accumulation.

Immunohistochemistry analysis of sWAT and pWAT showed that cells labeled by the pan-macrophage marker CD68 accumulated after 3 days and continued to increase after 7 days of t10,c12-CLA (Fig 2A ,B). In pWAT, the number of CD68 + cells dropped abruptly upon t10,c12-CLA disruption, back to the low level found in LA treated mice. In sWAT, however, residual CD68 immunolabeled cells persisted. Coordinated changes in CD68 gene expression and CD68 + cell number were found in both WAT depots (Fig 2C).

To substantiate these observations, we analyzed cells contained in the stroma vascular fraction (SVF) of WAT by flow cytometry. The number of CD45 + cells in this non-adipose cellular fraction increased markedly in response to 7 days of t10,c12-CLA administration (Fig 2D). Roughly 50% of these cells were positive for the F4/80 macrophage marker and this proportion was not significantly affected by t10,c12-CLA, supporting a large infiltration of macrophages in both adipose depots (Fig 2E).

In this lipoatrophic model, as opposed to the inverse relationship found in obesity, macrophage abundance was negatively correlated with WAT mass (Table 2). Nevertheless, as in obesity, adiponectinemia and insulinemia were respectively negatively and positively associated with WAT macrophage accumulation (Table 2). Of note, these relationships were independent of subcutaneous or deep WAT localization.

Reversible collagen deposition.

Picrosirius red labeling revealed that t10,c12-CLA induced collagen deposition in WAT.

This response was more pronounced in sWAT than in pWAT (Fig 3A ,B). Increased collagens mostly in sWAT was confirmed by hydroxyproline accumulation (Fig 3C). Upon t10,c12-CLA interruption, collagen labeling decreased in both depots, although less efficiently in sWAT than in pWAT. Gene expression analysis identified fibronectin and collagen 1 as the ECM components selectively increased in both adipose depots, with the additional contribution of collagen 3 in sWAT (Fig 3D). In line with histological data, ECM gene expression decreased sharply upon t10,c12-CLA withdrawal. Several ECM remodeling related genes followed a similar pattern of regulation, including the proteases Mmp12 and cathepsin S, the protease inhibitor Timp-1 and galectin 3 (Fig 3E). Despite increased collagen deposition, collagen fibril bridging enzymes were not found overexpressed but rather down-regulated (Lox, lysyl oxidase) or unchanged (Loxl2, lysyl oxidase-like 2) in response to t10, c12-CLA (Fig 3F). The expression of profibrotic factors, such as TGF-β1, inhibin beta-A and Ctgf, was modestly changed (Fig 3G).

Although TGF-β1 mRNA level slightly increased in sWAT, this transcriptional profile makes unlikely the induction of a fibrotic phenotype in fibroblasts and potentially other cell types in WAT. Supporting this possibility, Acta2 gene expression, a marker of fibrotic activation, did not correlate with fibronectin gene expression (Fig 3H). By contrast, strong correlations between fibronectin and Cd68 mRNA were found in both adipose depots, suggesting implication of macrophages in the ECM remodeling response to t10,c12-CLA (Fig 3H).

Alternatively-activated M2 profile of WAT macrophages.

We determined the phenotype of t10,c12-CLA-induced WAT macrophages through flow cytometry analysis of the cell surface markers CD11c and CD206 in the F4/80 + cell population.

In both WAT depots, over 80 % of the F4/80 + cells were CD206 + CD11c -in LA treated mice (Fig 4A-C). In response to t10,c12-CLA, this proportion only slightly decreased, coincident with the appearance of a minor subpopulation of CD206 + CD11c + cells. Both cell types markedly increased in number par gram of WAT, in line with F4/80 + cell accumulation with t10,c12-CLA (Fig 4D). Whatever the fat depot, CD206 + CD11c -cells represented the prominent macrophage subset in t10,c12-CLA treated mice. Up-regulation of a series of M2-associated gene markers confirmed the prevalence of alternatively activated macrophages in WAT (Fig 4E). All M2 markers decreased sharply upon t10,c12-CLA withdrawal. In sharp contrast, M1 markers were virtually not affected by t10,c12-CLA manipulation. The M2/M1 ratio estimated through Arg1/iNOS mRNA increased 3-fold in sWAT and nearly 20-fold in pWAT and was normalized with t10,c12-CLA disruption (Fig 4F). Anti-inflammatory cytokine IL-1ra and Il-10 mRNA levels dramatically increased in response to t10,c12-CLA, overshadowing slight elevation of proinflammatory Tnfα and Il-1β gene expression in both WAT depots (Fig 4G). Similar to M2 macrophage markers, anti-inflammatory cytokine gene expression dropped abruptly upon treatment interruption, demonstrating striking dependency on t10,c12-CLA administration.

These data show that WAT reacts to t10,c12-CLA by engaging a marked and reversible M2-like anti-inflammatory response.

Innate and adaptive immune cell profile in WAT.

Beside macrophages, other innate immune cells, including NK cells (CD3 -NK1.1 + ) and dendritic cells (F4/80 -CD11c + ) contributed to the t10,c12-CLA-induced CD45 + cell infiltrate in WAT (Fig 5A), along with less abundant NKT cells (CD3 + NK1.1 + ), T cells and B cells (Fig 5B). In pWAT, the proportions of CD8 + and CD4 -CD8 -cells inside the T cell population slightly increased, while the frequency of CD4 + cells decreased in response to t10,c12-CLA administration (Fig 5C ). A similar profile was found in sWAT (data not shown). When expressed per gram of WAT, the number of each cell subset tended to increase, reaching statistical significance only for CD8 + and CD8 -CD4 -cells in pWAT (Fig 5D). The frequency of Foxp3 + Tregs inside the CD4 + T cell subset was unchanged by t10,c12-CLA, amounting 8.2 ± 1.8 and 4.9 ± 0.64 % (n = 3) in the LA group and 8.8 ± 1.2 and 6.1 ± 1.4 % (n = 5, ns) in the t10, c12-CLA group in sWAT and pWAT, respectively. Finally, a small population of TCRγδ + CD3 + T cells was enhanced in both WAT depots (Fig 5E ,F).

Specific chemokine profile and cell proliferation in WAT.

The large t10,c12-CLA-induced infiltrate of macrophages raised the question of their origin, either from increased recruitment of circulating monocyte precursors, in situ proliferation or both. Gene expression analysis revealed that t10,c12-CLA induced a specific chemokine regulation, with marked increase of Ccl7, Ccl2, osteopontin (Opn) and Cxcl10 mRNA and lower stimulation of Ccl5, Cxcl2 and Cxcl3 genes in both WAT depots (Fig 6A ,C). Changes in chemokine gene expression were driven by t10,c12-CLA, as shown by their normalization with treatment interruption. These data suggest that t10,c12-CLA promotes an environment favoring blood cell recruitment in WAT. However, in situ proliferation cannot be excluded. Indeed, when we investigated Ki-67 gene expression as a marker of cell proliferation, its mRNA level increased in proportion with Cd68 mRNA in both sWAT and pWAT (Fig 6 B,D). Interestingly, Ki-67 gene expression was still observed enhanced after t10,c12-CLA removal, when Cd68 gene expression was normalized. This suggests that SVF non-immune cells might actually proliferate in WAT during t10,c12-CLA treatment and recovery period.

DISCUSSION

Here, whereas t10,c12-CLA administration in lean mice provokes a lipoatrophic syndrome along with macrophage accumulation and collagen deposition in WAT, we demonstrate that these alterations are reversible, at least in the short term. The results highlight an appropriate extracellular matrix remodeling with alternatively activated M2 macrophage accumulation in WAT. This reversible phenomenon reflects the ability of WAT to adequately adapt to a metabolically altered environment. We suggest that WAT M2 macrophage accumulation represents an early and adaptive response to environmental insult. Under persistent metabolic stress, M2-macrophages could become outnumbered by M1-macrophages, leading to chronic inflammation and insulin resistance.

As previously reported and confirmed here, mice receiving t10,c12-CLA developed marked hyperinsulinemia with no change in glycemia, reflecting a state of systemic insulin resistance [START_REF] Tsuboyama-Kasaoka | Conjugated linoleic acid supplementation reduces adipose tissue by apoptosis and develops lipodystrophy in mice[END_REF][START_REF] Clement | Dietary trans-10,cis-12 conjugated linoleic acid induces hyperinsulinemia and fatty liver in the mouse[END_REF][START_REF] Poirier | Hyperinsulinaemia triggered by dietary conjugated linoleic acid is associated with a decrease in leptin and adiponectin plasma levels and pancreatic beta cell hyperplasia in the mouse[END_REF][START_REF] Poirier | Nutritional supplementation with trans-10, cis-12-conjugated linoleic acid induces inflammation of white adipose tissue[END_REF]. In this model, as opposed to obesity, altered glucose homeostasis occurred in face of M2 macrophage accumulation and high M2/M1 ratio. Our data do not support a protective role for WAT M2 macrophages against metabolic deregulation occurring at this early stage. This situation is apparently at odds with studies showing that several procedures increasing the M2/M1 ratio in WAT, through thiazolidinediones [START_REF] Fujisaka | Regulatory mechanisms for adipose tissue M1 and M2 macrophages in diet-induced obese mice[END_REF][START_REF] Prieur | Differential lipid partitioning between adipocytes and tissue macrophages modulates macrophage lipotoxicity and m2/m1 polarization in obese mice[END_REF], dietary -3 fatty acids administration [START_REF] Oh Da | A Gpr120-selective agonist improves insulin resistance and chronic inflammation in obese mice[END_REF] or in response to nematode parasitic infection [START_REF] Yang | Parasitic nematode-induced modulation of body weight and associated metabolic dysfunction in mouse models of obesity[END_REF] [START_REF] Hussaarts | Chronic helminth infection and helminth-derived egg antigens promote adipose tissue M2 macrophages and improve insulin sensitivity in obese mice[END_REF] for example, improved glycemic status in obese mice. In these latter conditions, reduction of M1 proinflammatory macrophages rather than M2 macrophage accumulation might be the prominent trigger of metabolic improvement.

M2 macrophages are thought to be implicated in ECM remodeling and collagen turnover required for tissue repair in response to injury (27)[START_REF] Pesce | Arginase-1-expressing macrophages suppress Th2 cytokine-driven inflammation and fibrosis[END_REF][START_REF] Madsen | M2-like macrophages are responsible for collagen degradation through a mannose receptor-mediated pathway[END_REF]. In the t10,c12-CLA model, ECM deposition was associated with alternatively activated macrophage accumulation and dramatic up-regulation of the macrophage elastase MMP12. This appeared to be a rapid and adaptive response as opposed to pathological fibrosis. First, and most importantly, collagen accumulation and enhanced ECM-related gene expression were reversible, as soon as 7 days after we stop t10,c12-CLA administration. Second, enzymes involved in collagen cross-linking were not upregulated, suggesting that their relative deficiency limited the formation of stable fibrils. Third, there was no major induction of several well established pro-fibrotic factors and no evidence of pro-fibrotic activation of fibroblasts in WAT. Surprisingly, this occurred despite marked upregulation of Lgals3, a β-galactoside-binding lectin, which has been implicated in myofibroblast activation and fibrosis in kidney and liver [START_REF] Henderson | Galectin-3 regulates myofibroblast activation and hepatic fibrosis[END_REF][START_REF] Henderson | Galectin-3 expression and secretion links macrophages to the promotion of renal fibrosis[END_REF], but could play a distinct role in WAT. It was previously reported that feeding mice a t10,c12-CLA enriched diet induced ECM deposition in mammary fat pad [START_REF] Russell | Conjugated linoleic acid induces mast cell recruitment during mouse mammary gland stromal remodeling[END_REF]. Our data additionally revealed depot selectivity in collagen deposition, which was more pronounced and long-lasting in subcutaneous sWAT than in visceral pWAT.

Importantly, the persistence of increased ECM deposition in sWAT was associated with delayed tissue mass regain after t10,c12-CLA removal. This gives further support to the notion that abnormal collagen deposition in WAT contributes to adipocyte dysfunction [START_REF] Vila | Immune cell Tolllike receptor 4 mediates the development of obesity-and endotoxemia-associated adipose tissue fibrosis[END_REF][START_REF] Pellegrinelli | Human adipocyte function is impacted by mechanical cues[END_REF].

Recent studies have established that a specific immune profile prevails in WAT of lean mice and that obesity induces marked deregulation of cell frequency and/or phenotype [START_REF] Winer | The adaptive immune system as a fundamental regulator of adipose tissue inflammation and insulin resistance[END_REF]35).

How WAT immune cells interact with each other and influence the biology of non immune cells, [START_REF] Lee | Identification of an adipogenic niche for adipose tissue remodeling and restoration[END_REF]. In this study, PDGFRα + progenitor cells expressing the osteopontin receptor CD44 were recruited and proliferated in response to M2 macrophage-derived osteopontin. This suggests that the M2 macrophage accumulation and marked overexpression of ostepontin that we observed in response to t10,c11-CLA might contribute to progenitor proliferation, particularly in pWAT.

Increased cell apoptosis [START_REF] Tsuboyama-Kasaoka | Conjugated linoleic acid supplementation reduces adipose tissue by apoptosis and develops lipodystrophy in mice[END_REF][START_REF] Poirier | Nutritional supplementation with trans-10, cis-12-conjugated linoleic acid induces inflammation of white adipose tissue[END_REF] could support this process and be part of the triggering events for M2 macrophage recruitment [START_REF] Fischer-Posovszky | Targeted deletion of adipocytes by apoptosis leads to adipose tissue recruitment of alternatively activated M2 macrophages[END_REF].

To conclude, this study revealed WAT ability to mount a rapid and reversible response to a short-term nutritional insult, relying primarily on induction of an anti-inflammatory program and alternatively-activated M2 macrophage recruitment. The components of this response were strikingly distinct from WAT alterations reported in obesity, which comprise inflammation, M1 macrophage accumulation and fibrosis. Further studies will help to assess whether the capacity of WAT to adapt to a disturbed metabolic environment, enlightened in t10,c12-CLA treated mice, is lost under chronic and persistent metabolic constrain leading to adipocyte dysfunction in obesity or lipodystrophy. Nevertheless, despite WAT resilience, t10,c12-CLA induced metabolic, structural and immune alterations were associated with rapid increased of insulinemia, adding support to the view that WAT dysfunctional state is a crucial to trigger glucose homeostasis alteration. The systematic effort to identify the potential link between metabolism and immunity in the effects caused by CLA is reported in Publication 1. Additional findings will be reported in this paragraph, aimed at exploring the capacity of WAT to biologically respond to t10,c12-CLA nutritional challenge through regulation of genes known to mediate the interplay between nutrient-sensing pathways and immune system. Results for Toll like receptor (TLR) and G-protein coupled receptor (GPR) expression will be presented in the different tissues of mice treated for 7 days with t10,c12-CLA (daily gavage = G7). As shown in Figure 28, CLA, as other saturated fatty acids, upregulated the expression of specific TLR-family members (TLR1, TLR2, TLR9) in both WAT depots. These results are not surprising, considering that TLR9 is expressed in most of the tissues at high levels and together with TLR1 and TLR2 is mostly expressed on immune cells or immune cell rich tissues, such as bone marrow, lymph nodes and spleen. However, it was surprising that the classically FFA-activated TLR4 was totally not affected by CLA. Certainly these observations at the mRNA level would need to be further supported by protein measurements. Because FFA have been demonstrated to act as ligands of several GPCRs, we evaluated the effect of CLA on their tissue expression, although aware that their functions are still largely unknown. In particular, we evaluated GPR43 that is activated by short-chain FFA. Obesity increases GPR43 expression in the subcutaneous adipose tissue of HFD-fed mice (4 weeks) and inulin-type fructans (nondigestible/fermentable carbohydrates) counteract the HFD effects through microbiota changes [START_REF] Dewulf | Inulin-type fructans with prebiotic properties counteract GPR43 overexpression and PPARgamma-related adipogenesis in the white adipose tissue of high-fat diet-fed mice[END_REF]). GPR43-deficiency was shown protective toward HFD-induced obesity [START_REF] Bjursell | Improved glucose control and reduced body fat mass in free fatty acid receptor 2-deficient mice fed a high-fat diet[END_REF]. We evaluated GPR109A and GPR81, adipocyte cell surface receptors that enable the cells to "sense" metabolic changes in the environment and respond through lipolytic regulation and release of free fatty acids and pro-or anti-inflammatory adipokines. Chronic obesity (60% high fat diet for 11 weeks [START_REF] Wanders | Effects of high fat diet on GPR109A and GPR81 gene expression[END_REF]) and ob/ob mice [START_REF] Feingold | Inflammation inhibits GPR81 expression in adipose tissue[END_REF] significantly reduced GPR109A and GPR81 gene expression in WAT. Moreover, acute inflammation (in vivo injections of LPS, zymosan, turpentine) decreased GPR81 expression in WAT of C57BL/6 mice [START_REF] Feingold | Inflammation inhibits GPR81 expression in adipose tissue[END_REF]). We evaluated GPR116, which was shown to have a role in controlling adipocyte biology, in a WAT specific conditional GPR116 knockout mouse [START_REF] Nie | Adipose tissue deletion of Gpr116 impairs insulin sensitivity through modulation of adipose function[END_REF]. In this study, GPR116 WAT deletion caused a pronounced glucose intolerance and insulin resistance in mice, especially when challenged with a high fat diet [START_REF] Nie | Adipose tissue deletion of Gpr116 impairs insulin sensitivity through modulation of adipose function[END_REF]. Finally, we evaluated GPR120 that encodes an n-3 PUFA receptor/sensor and is activated by medium-and long-chain FFA. As presented in the introduction, recent studies indicated that GPR120 has a key role in sensing dietary fat in WAT and dysfunction of GPR120 is associated with obesity and insulin resistance in both humans and rodents [START_REF] Ichimura | Regulation of Energy Homeostasis via GPR120[END_REF]. Interestingly, we discovered that the expression of all the different GPRs was coordinately down-regulated in response to CLA in the three adipose depots: inguinal (sWAT), periuteral (pWAT) and retroperitoneal (rWAT) (Figure29). Currently, the local consequences of this striking and consistent GPRs down-regulation is difficult to interpret, given the lack of knwoledge of their specific implication in adipose tissue normal or altered biolgy. The reduction we observed for GPR120 at the mRNA level in WAT was unexpected. In particular, in relation to the anti-inflammatory environment observed in WAT in response to CLA (upregulation of IL-1ra and Il10 mRNAs), we had anticipated that CLA could have an anti-inflammatory effect through GPR120 activation, possibly like ω3 fatty acids (DHA). In a study from Olefsky's team, GPR120, expressed in macrophages and mature adipocytes, was shown to inhibit TLR2, TLR4 and TNFR-mediated inflammation though DHA [START_REF] Talukdar | Targeting GPR120 and other fatty acid-sensing GPCRs ameliorates insulin resistance and inflammatory diseases[END_REF]. The precise mechanism of GPR120 action pointed to the exclusively dependency on β-arrestin 2 (β-arr2) [START_REF] Talukdar | Targeting GPR120 and other fatty acid-sensing GPCRs ameliorates insulin resistance and inflammatory diseases[END_REF].

F n 1 C o l1 a 1 C o l3 a 1 C o l6 a 1 C o l4 a 1 F n 1 C o l1 a 1 C o l3 a 1 C o l6 a 1 C o l4
Consequently, the expression of β-arr2 was evaluated in our experimental model. Interestingly mRNA level of β-arr2 was found upregulated in response to CLA (from 2 to 6 fold in pWAT and sWAT, respectively). Figure 30 shows that classical pro-inflammatory cascades are largely mediated by TNF-α and TLR4 receptors and the downstream inflammatory effects require activation of NF-κB and JNK, via the interaction between TGF-beta activated kinase 1 (TAK1) and TGF-beta activated kinase 1 binding protein 1 (TAB1) [START_REF] Talukdar | Targeting GPR120 and other fatty acid-sensing GPCRs ameliorates insulin resistance and inflammatory diseases[END_REF]. It would be interesting to explore if some of the effects of t10, c12-CLA could be mediated by similar mechanisms. The activation of GPR120 by CLA (instead of DHA) could recruit β-arr 2 to the cytosolic putative binding sites on GPR120, and the GPR120-β-arr 2 complex would internalize and through TAB1 sequestration could participate to the inhibition of inflammation (represented by the strong up-regulation of Il10 and IL-1ra, in association with the M2 macrophage accumulation, in WAT of CLA-treated mice). Of note, when CLA treatment was dismissed, the expression of the GPCRs cell membrane receptors fully moved or trended back to levels found in LA-treated mice in most of the cases, with some depot specific peculiarity. Gene expression analysis of WAT in earlier time points then after 7 gavages would allow understanding if the changes at the mRNA level reported in the above article and as complementary data preceed and influence the depot specific response in term of fat mass reduction and fat mass recovery when the CLA treatment is dismissed, if WAT is analyzed before than after 7 days of treatment interruption.

These findings remain simple observations at this stage and they will require further exploration through full protein analysis, since it is known that there is not a strict correlation between gene expression and protein level for these factors. Moreover only few GPCRs are currently well characterized, so the potential role of these receptors remains to be explored in different organs in physiological and pathological conditions.

WAT plasticity in response to CLA

About the capacity of WAT to properly respond to environmental metabolic changes, it is important to underline the differences between depots. In sWAT the magnitude of collagen accumulation indicated a more intense ECM remodeling compared to deep adipose depots. This indicated that tissue homeostasis was not fully restored in sWAT after CLA disruption (7 days without treatment after 7 gavages), consistently with the unrecovered fat mass. Instead both pWAT and rWAT fully resolved the CLAinduced alterations. One of the differences between inguinal and deep WAT is that the inguinal depots enclose the inguinal group of lymph nodes (LN) (Figure 31). One possible hypothesis is that inguinal lymphoid cells, nestled in the "fibrofatty" tissue, and their (possible) cytokine secretion, could influence the lipolysis [START_REF] Pond | Interactions between adipose tissue around lymph nodes and lymphoid cells in vitro[END_REF] and consequently the fibrotic process in response. One interesting observation we made during the inguinal WAT collection was that the size of LN was drastically reduced by CLA treatment. In order to avoid interference from leukocytes present in LN during FACS analysis, LN were carefully excised and removed from sWAT before preparation of stromal-vascular fraction. After 7 gavages it was almost not possible to identify sWAT LN, that looked "emtly", compared to LN from LA-treated animals. This observation suggests that LN could possibly supply the massive WAT immune cell accumulation observed at 7G. Unfortunatly, we did not quantify LN cellularity counting the cells, so this hypothesis remains to be checked. Gene expression analysis of RNA isolated from the collected LN could possibly reveal more about LN content and potential signals at various time points (3G, 7G, 7G7R).

Another factor extremely important to recover "healthy" fat is oxygenation. The role of hypoxia in WAT fibrosis associated to obesity is well documented (Sun, Tordjman et al. 2013). The senescence of endothelial cells, along with decreased vessel density [START_REF] Pasarica | Adipose tissue collagen VI in obesity[END_REF], has been suggested as a possible cause of the modified oxygen tension and inflammation observed in hypertrophied WAT [START_REF] Rouault | Roles of chemokine ligand-2 (CXCL2) and neutrophils in influencing endothelial cell function and inflammation of human adipose tissue[END_REF]. Certainly adipose tissue vasculature has a critical role in ensuring that the tissue has appropriate access to oxygen. In fact, during adipose tissue expansion, proangiogenic activity proves to be beneficial for adipose tissue functions [START_REF] Sun | Dichotomous effects of VEGF-A on adipose tissue dysfunction[END_REF]. Overexpressing vascular endothelial growth factor (VEGF) in adipose tissue of HFD-fed mice was shown to be protective against obesity and insulin resistance [START_REF] Elias | Adipose tissue overexpression of vascular endothelial growth factor protects against diet-induced obesity and insulin resistance[END_REF]. In particular, transgenic mice presented increased M2 anti-inflammatory accumulation, with fewer M1 proinflammatory macrophages compared to control group, suggesting that the antiinflammatory milieu could contribute to mantain insulin sensitivity [START_REF] Elias | Adipose tissue overexpression of vascular endothelial growth factor protects against diet-induced obesity and insulin resistance[END_REF]). In our study, the administration of CLA caused WAT ECM accumulation, in association with early and drastic M2-like macrophages infiltration (see figure 3 in Publication 1). After CLA treatment disruption, the profibrotic transcriptional program engaged during CLA administration was dismissed, with all the genes involved in ECM remodeling and macrophage accumulation declining back to the control level (LA-treated group) (see figure 3 in Publication 1). Together with those genes, VEGF-A, a potent angiogenic factors, and CD31, also known as platelet endothelial cell adhesion molecule (PECAM-1), involved in blood vessel formation, were evaluated (Figure 32). The expression of these angiogenesis markers was unchanged or reduced during CLA treatment (Figure 32), indicating that the M2-like macrophage accumulation was independent from VEGF-A [START_REF] Elias | New insights into adipose tissue VEGF-A actions in the control of obesity and insulin resistance[END_REF]). However, interestingly, after CLA removal both VEGF-A and PECAM-1 showed upregulation exclusively in deep adipose depots (Figure 32). Although slight, this depot-specific increase suggests that new vessel formation possibly accompanies the recovery in fat mass observed at the visceral level. It would be interesting to complete these observations by visualization of vascular network in different adipose depots.

The precise mechanisms inducing the lipoatrophic state caused by CLA have not been elucidated. The rate of cellular stress and cell death induced by CLA (apoptosis, necrosis and/or pyroptosis has not been fully explored in vivo (Kennedy, Martinez et al. 2010) and it is possibly elevated as in obesity [START_REF] Cinti | Adipocyte death defines macrophage localization and function in adipose tissue of obese mice and humans[END_REF]. Temporary local hypoxia could be induced by CLA in association with adipocyte dysfunction. Consequently we investigated hypoxia-inducible ATF3 in comparison with other members of the ATF family of transcription factors, known to be activated by the stress. An integrated stress response specifically induced by t10,c12-CLA was highlighted through microarray analysis of 3T3-L1 preadipocytes treated for 4 and 24 h (LaRosa, Riethoven et al. 2007) and supported this hypothesis. Interestingly we found that ATF3 was the only ATF member markedly upregulated, compared to ATF4 and ATF6 (Figure 33). A role in adipocyte hypoxia-mediated mitochondria dysfunction in obesity has been shown recently for ATF3 [START_REF] Jang | ATF3 plays a role in adipocyte hypoxia-mediated mitochondria dysfunction in obesity[END_REF]. In this latter study HIF-1α and ATF3 were increased in WAT of HFD-induced obese mice compared with control lean mice, whereas mitochondria-related genes were significantly reduced (i.g. NRF-1, PGC-1α, COX1). Similarly, in WAT of CLA-treated mice, PGC-1α and COX1 were unchanged or reduced by the treatment (Figure 33), although HIF1α was unchanged (not shown). The increased mRNA level of ATF3 suggested that ATF3 may contribute to the inhibition of adipocyte differentiation as reported in hypoxic conditions in 3T3-L1 cells, in which lipid staining with oil red staining was decreased and adipogenic genes reduced [START_REF] Jang | ATF3 inhibits adipocyte differentiation of 3T3-L1 cells[END_REF], similarly to ACC and FAS in response to CLA. After 7 days of CLA treatment distruption, ATF3 remained upregulated only in sWAT. Subcutaneus WAT is the adipose depot that does not recover fat mass after CLA suspension, possibly due to unfavorable regulation of adipogenesis.

Recently it has been demonstrated using 3T3-L1 cells that ATF3 interacts with PPARγ and represses PPARγ-mediated transactivation, possibly playing a role in inhibition of adipocyte differentiation [START_REF] Jang | ATF3 inhibits PPARgamma-stimulated transactivation in adipocyte cells[END_REF]. Our results in vivo could be in line with those in vitro findings, since the expression of PPARγ remained highly down-regulated in sWAT (7G -54% and 7G7R -65% reduction compated to LAtreated mice), but not in pWAT (7G -58% and 7G7R -8% reduction compared to LA group). Therefore, in our model, the inhibition of PPARγ-regulated adipocyte differentiation by ATF3 (Jang and Jung 2015) could be one of the possible mechanisms explaining the inability of sWAT to recover fat mass after CLA disruption, in contrast to the two other WAT depots. Moreover, since ATF3 has been recently linked to macrophage lipid body formation [START_REF] Gold | ATF3 protects against atherosclerosis by suppressing 25hydroxycholesterol-induced lipid body formation[END_REF], the upregulation of this gene could represent a potential intersection point between lipid metabolism and inflammatory pathways in these cells highly infiltrating WAT after CLA administration. It would be interesting to further explore the role of this factor in WAT, in particular in light of a very recent study reporting a key role for ATF3 in experimental models of fibrosis [START_REF] Gold | ATF3 protects against atherosclerosis by suppressing 25hydroxycholesterol-induced lipid body formation[END_REF]).

Effects of CLA adminsitration on transcription factors of the IRF family

The family of interferon regulatory factors (IRFs) has been implicated as critical transcription factors that sense a variety of environmental stresses [START_REF] Sato | The interferon system and interferon regulatory factor transcription factors --studies from gene knockout mice[END_REF]. In particular, IRF5 has a key role in the activation of genes encoding pro-inflammatory cytokines in antiviral and immune response, being generally involved downstream of the TLR-MyD88 signalling pathway [START_REF] Takaoka | Integral role of IRF-5 in the gene induction programme activated by Toll-like receptors[END_REF], [START_REF] Krausgruber | IRF5 is required for late-phase TNF secretion by human dendritic cells[END_REF]). IRF5 has been shown highly expressed in human M1 macrophages, with a crucial role for conversion from M2 to M1 macrophages in response to LPS [START_REF] Krausgruber | IRF5 promotes inflammatory macrophage polarization and TH1-TH17 responses[END_REF]. In human obesity and diet-induced obese mice, WAT, mostly infiltrated by M1 macrophages displays elevated expression of IRF5 (Publication 3). In mice, this factor was shown to alter adipose tissue immune profile, promoting M1 macrophage accumulation, and to contribute to extracellular matrix deregulation specifically in deep adipose depots. For the reported implication of this specific IRF member in WAT immune and structural alterations, the expression of IRF5 and other IRFs family members was evaluated in WAT of CLA treated mice (Figure 34). Of note IRF5 and IRF7 were specifically up-regulated factor, in a coordinate manner in the three explored WAT depots. Recently, using two murine models of obesity, IRF7 has been shown to be implicated in the deterioration of glucose and lipid metabolism [START_REF] Wang | Interferon regulatory factor 7 deficiency prevents diet-induced obesity and insulin resistance[END_REF]. Being both IRF5 and IRF7 implicated primarily in innate immunity, their increased expression in WAT could represent the increased innate infiltrate observed in response to CLA. However, the metabolic role of these factors in conditions of WAT dysfunctions, such as the lipoatrophic state provoked by CLA administration, deserves further exploration.

Effects of CLA administration on the intestine: barrier function

Complementary studies were performed to evaluate the effects of CLA on gut immunity and permeability, not to exclude a potential action of dietary fat on gut leakiness and consequently on WAT alterations. Gastrointestinal tract is the most common route of food and drug administration and that means it is generally the first organ to be exposed to external attacks. The diet is known to modulate the production of pro-and anti-oxidant species, reactive nitrogen species and radicals, potentially contributing to toxicity level [START_REF] Lobo | Free radicals, antioxidants and functional foods: Impact on human health[END_REF]. All these diet-derived components are known to favor local inflammation and potentially alter the local immune functions [START_REF] Chamulitrat | Activation of the superoxide-generating NADPH oxidase of intestinal lymphocytes produces highly reactive free radicals from sulfite[END_REF]. High fat diet in particular is reported to activate immune cells, mast cells in particular, and Toll-like receptors [START_REF] Ji | Nutrient-induced inflammation in the intestine[END_REF], [START_REF] Kim | High fat diet-induced gut microbiota exacerbates inflammation and obesity in mice via the TLR4 signaling pathway[END_REF]. Along with this, sufficient evidence has proved that lipids induce inflammation in the course of obesity development.

To explore the relationship between nutrient absorption and intestinal inflammation, we made a preliminary evaluation of the health status of enterocytes and the intestinal immune cell infiltration in response to CLA administration by RT-PCR. The hypothesis was that, being a lipid, CLA would possibly induce acutely gut inflammatory responses similar to the one reported in response to chronic high-fat feeding [START_REF] Cani | Changes in gut microbiota control inflammation in obese mice through a mechanism involving GLP-2-driven improvement of gut permeability[END_REF]. Gene expression analysis of immune cell markers used to evaluate WAT revealed that CLA did not induced a more sustained lymphocyte (CD3) and macrophage (CD68) accumulation compared to LA, in intestinal tissues (Figure 35). However, CLA clearly modulated members of the toll-like receptor family in intestine and colon with a quite distinct profile (Figure 36) compared to adipose tissue (Figure 28). In jejunum, after 7G, CLA tended to increase the expression of TLR4 and TLR5, not surprisingly for this last receptor, since it is mainly expressed in intestinal epithelial cells. Whereas, the response in the colon was driven by TLR9, suggesting that CLA could possibly favor an intestinal epithelial inflammatory response, possibly due to increased pathogenic bacteria DNA recognition [START_REF] Ewaschuk | Surface expression of Toll-like receptor 9 is upregulated on intestinal epithelial cells in response to pathogenic bacterial DNA[END_REF]) [START_REF] Kruis | Bacterial translocation -impact on the adipocyte compartment[END_REF]. The activation of both TLR4 and TLR5 mobilize the nuclear factor NF-κB and stimulates tumor necrosis factor-alpha production, a pathway known to be critical in activating the inflammatory response (de La Serre, Ellis et al. 2010), [START_REF] Shi | TLR4 links innate immunity and fatty acid-induced insulin resistance[END_REF]). In the intestine of the CLA-treated mice both molecular actors TLR4 and TNF-α were engaged at the mRNA level. In fact, coupled with a 4-fold increase of TLR4, a significant upregulation of TNFα (5x) was observed in jejunum, in association with reduced expression of genes coding for tight junction proteins, claudin2 (CLDN2 gene), occluding (OCLN gene) and zonula occludens 1/ZO-1 (TJP1 gene) (Figure 37). These data suggest that, despite the modality of administration (short-time frame and transient exposure), CLA could possibly initiate an inflammatory response by binding to TLR4 and increasing TNF-α expression in the jejunum. This very powerful and potentially destructive inflammatory cytokine (TNFα) could participate in altering intestinal barrier on a longer time course, similarly to what occurs in Chrown-like pathology [START_REF] Roulis | Intestinal epithelial cells as producers but not targets of chronic TNF suffice to cause murine Crohn-like pathology[END_REF] or in response to a high fat diet [START_REF] Lam | Increased gut permeability and microbiota change associate with mesenteric fat inflammation and metabolic dysfunction in diet-induced obese mice[END_REF]) [START_REF] Suzuki | Dietary fat and bile juice, but not obesity, are responsible for the increase in small intestinal permeability induced through the suppression of tight junction protein expression in LETO and OLETF rats[END_REF]. Interestingly, the dramatic reduction of circulating adiponectin induced by CLA could be also associated to a ''leaky gut'' phenotype, since the anti-inflammatory role of adiponectin has been demonstrated in intestinal inflammation in other settings, together with different metabolic dysfunctions [START_REF] Yamamoto | Production of adiponectin, an anti-inflammatory protein, in mesenteric adipose tissue in Crohn's disease[END_REF]) [START_REF] Saxena | Mucus and adiponectin deficiency: role in chronic inflammationinduced colon cancer[END_REF]. Moreover, it is important to consider mesenteric WAT, the visceral fat depot located in the mesenterium of the intestine. Investigate the effects of CLA on this intestinal fat tissue compartment for activation, secretion and the infiltrating immune cells would be important, since it is known to play an important role for proper intestinal barrier function.

Unfortunately, this has not been possible due to the drastic reduction of WAT and near absence of mesenteric WAT caused by CLA after 7 gavages, when mRNA tight junction proteins were observed reduced. In contrast with the chronic administration of HFD in the murine model of obesity, the stressful effect of this fatty acid on the gut is limited due to the short time frame and possibly due to the administration modality as well. A longer time course would possibly increase the risk of an inflammatory immune engagement in the gut wall, which over time could result in epithelial damage (leaky gut). Long-chain PUFA are known for their propensity to easily oxidize and generate lipid peroxidation byproducts, so longer administration of CLA could determine the same risk of inducing leaky gut and endotoxemia observed in other contexts (de La Serre, Ellis et al. 2010) [START_REF] Erridge | A high-fat meal induces low-grade endotoxemia: evidence of a novel mechanism of postprandial inflammation[END_REF]) [START_REF] Cani | Metabolic endotoxemia initiates obesity and insulin resistance[END_REF]). The ligands recognized by TLRs are varied. Although the bacterial component LPS is considered the main ligand of TLR4, followed by production of proinflammatory cytokines, the activation by the t10, c12 -CLA is not excluded.

Some genes known to regulate lipid metabolism at the intestinal level were evaluated. In particular, fatty acid binding protein 2 (Fabp2), known to be expressed exclusively in enterocytes in the small intestine, histone deacetylase 1 (Hdac1), important for intestinal epithelial cell homeostasis, scavenger receptor class B, member 1 (SR-BI), suggested to be involved in the absorption of dietary cholesterol in rodent, plus other genes specific for lipoprotein metabolism, such as apolipoprotein B (Apob), apolipoprotein A-IV (Apoa4), mannoside acetylglucosaminyltransferase 2 (Mgat2), microsomal triglyceride transfer protein (Mttp), were studied. No major modifications were observed at the mRNA level at the time point examined (7G) for these genes in jejunum and colon of CLA-treated mice.

Effects of CLA administration on the intestine: microbiota

Through 16S rRNA pyrosequencing, the microbiota of mice receiving LA and CLA was assessed at different time points, following the hypothesis that fatty acid administration, even when short, could alter it. The association of gut microbiota with a disease does not mean causation. However understanding the potential role of microbiota, depending on the composition, could open up new avenues to treat or mitigate the disease itself [START_REF] Nguyen | How informative is the mouse for human gut microbiota research?[END_REF]. Diet components, in particular fatty acids, have been shown to impact the microflora richness and functions. The effect of lipids in the lumen or through direct action on the enterocytes and through activation of mast cells has been reported to provoke pro-inflammatory cytokine release. However our understanding on the onset of inflammation at the intestinal level is not complete without taking into account the gut microbiota. This point has been highlighted by the use of germ-free mice, that, despite a diet rich in fat, do not show upregulation of inflammatory markers, such as TNF-α and nuclear factor-kappa B.

During the course of the CLA project, fecal samples were collected from individual mice treated by LA and CLA at different time points in multiple experiments. Samples were stored at -80°C until metagenomics analysis was performed in INRA, thanks to the collaboration with MICALIS (Unit for food and gut microbiology for human health). The metagenomics analysis was based on microbial 16S rRNA gene sequencing to determine bacterial composition in fecal samples of 88 mice. Because the number of mice per group was low (n=4), we decided to pool the data from close days, 1-2G (=baseline), 3-4G, 6-7G (=7G) and 7G5-9R (=7G7R) for the two treatments LA versus CLA, to gain statistical power. The statistical analysis was performed by our collaborator Julien Tap (INRA) based on the pre-defined objectives of the study.

Microbial species were organized using the concept of enterotypes, a way of analyzing the ecological organization of gut microflora in mice (distinction between resistant and non-resistent microbiota). Three main clusters were found, with more or less 30% of the mice within each enterotype (Figure 38):

 M1 = prédominance de Barnesiella et Alistipes  M2 = prédominance de Barnesiella, Alistipes, Clostridium et Lachnospiracées  M3 = prédominance de Bacteroides et Barnesiella Each column identifies the microbial species of one mouse, with the average for each enterotype group indicated on the right (black rectangles).

Interestingly, the enterotype M3-Bacteroides, which was found the less rich in bacterial species, was particularly low in the CLA-treated mice, as indicated by the absence of red (M3) dots (CLA) (Figure 39). Bacteroidetes is a genus of Gram-negative, obligate anaerobic bacteria, found in lower amount in obese subjects, even if with some contradictory findings (Aron-Wisnewsky and Clement 2014). Our preliminary results could suggest a similarity between the effects of short-term t10,c12-CLA administration and obesity (i.e. chronic high fat consumption) on gut microbiota composition. However the sharp differences between human and animal environment and behavior, above all, clearly downsize this observation.

M1 M2 M3

Figure 39. Number of mice with a specific enterotype in three expreiments.

A limitation of the analysis is that no feces were collected before starting the treatment; also, microbiota changes are not correspondent to the same animals followed over time. However, we tried to distinguish a potential effect of CLA on microflora, making a distinction between mice that received only 1 gavage (1G), that we considered "baseline" (= 0 gavage), and mice in the groups 3G and 7G (Figure 40). The p values reported on top of the Figure 40 represent the independence test khi2, showing that there is dependence between enterotypes and gavages in CLA-treated mice, but not for LA. This figure shows that CLA treatment modified gut microbiota composition in mice, with a clear absence of the M3 enterotype (Bacteroides-Barnesiella) after 7 gavages. Other differences between time points within the same treatment or between treatments were not significant. These results could indicate that the microbiota progressively change in response to CLA, becoming more enriched in Barnisiella and Alistipes (M1 enterotype) and loosing Bacteroides (M3 enterotype). These are preliminary results and further studies will be needed to assess the possible consequences of gut microbiota changes on the animal metabolic phenotype. Gut microbiota changes would need to be confirmed through further exploration and characterization. For example, to obtain a more acurate picture about what happens at the enterotype level in response to CLA, the same metagenomics analysis would be necessary over time following the same mouse to avoid any confounding effect. This would allow associating the microbiota composition with the mouse immuno-metabolic phenotype with or with no CLA (from fat mass reduction or ECM remodeling to insulin level or WAT altered immunity).

Reversibility: permeability and intestinal ecology

The potential effects of CLA administration on intestinal barrier integrity suggested by mRNA changes observed at 7G were completely reversed 7 days after CLA removal (7G7R). Tight junction proteins recovered levels observed in the LA-treated group, whereas TLR4 and TNF-α upregulations were abolished (data not shown).

The importance of the intestinal epithelial barrier is emphasized by the alterations in paracellular permeability and tight junction functions observed in inflammatory bowel disease (IBD) and colon cancer. In future experiments, it would be important to support these observation during and after CLA treatment, evaluating paracellular permeability through more specific methods, i.e. FITCdextran administration and quantification at the circulating level 4 hours after gavage [START_REF] Gupta | Dual function of p38alpha MAPK in colon cancer: suppression of colitis-associated tumor initiation but requirement for cancer cell survival[END_REF] or measuring paracellular flux mounting intestinal biopsy specimens in Ussing chambers [START_REF] Clarke | A guide to Ussing chamber studies of mouse intestine[END_REF]. Moreover it could be relevant to study how the host immune system could influence gut microbiota composition, known that the opposite process has long been recognized [START_REF] Zhang | Host adaptive immunity alters gut microbiota[END_REF]. This approach could be possible evaluating microbiota richness in immunodeficient mice, such as Rag1 mice (no mature B and T-cells) or in highly immunocompromised SCID mice, for example, treated with LA and CLA mice. Finally, to conclude, the specific variations of intestinal ecology depending on treatment or time frame could be further enlighten through stratification of the mice based on the CLA-induced alterations (i.e. low versus high insulin level or low versus high percentage of fat mass loss), similarly to patient stratification in human metabolic diseases (i.e. clinical parameters, such as BMI or HOMA-IR, homeostatic model assessment for insulin resistance).

Effects of CLA administration on the liver

Alterations of liver homeostasis were also considered in response to CLA, but only marginally. No changes were observed by gross examination of the organ, in particular no steatotic appearance drawn our attention. Gene expression analysis was performed in liver of CLA-treated mice and compared to LA group. No alterations were observed at the level of immune infiltrate. However, Mcp1 and IL1β were upregulated (5.4-and 6-fold, respectively) in association with the lipogenic genes Acc and Fas (5.4-and 4.5-fold, respectively). These preliminary results indicate that even such a short-term administration of CLA is able to initiate an inflammatory response in liver, possibly due to the concomitant hepatic lipid accumulation, as demonstrated by tissue fatty acid analysis (see Figure 1C in the publication).

Are the CLA effects on WAT indirectly mediated by macrophages?

The direct action of CLA on human and murine adipocytes has been demonstrated by several studies, the majority of which conducted by the research group headed by Prof M McIntosh, in Emory University (Atlanta, GA, USA) [START_REF] Brown | Trans-10, cis-12, but not cis-9, trans-11, conjugated linoleic acid attenuates lipogenesis in primary cultures of stromal vascular cells from human adipose tissue[END_REF][START_REF] Den Hartigh | 10E,12Z-conjugated linoleic acid impairs adipocyte triglyceride storage by enhancing fatty acid oxidation, lipolysis, and mitochondrial reactive oxygen species[END_REF]. Although aware that WAT is the primary target of CLA in vivo, we decided to perform complementary in vitro experiments using macrophages, the most rapidly engaged and dominant immune cell population in WAT of CLAtreated mice. The hypothesis behind these experiments was that CLA could be directly "sensed" by macrophages and possibly affects the activation state of these cells, potentially elucidating one of the mechanisms of CLA-induced alterations in WAT. We hypothesized that macrophages could autonomously respond to LA or CLA stimulation by changing their polarity from M0 to M1 or M2 status. We also explored the possibility that LA or CLA has an anti-inflammatory effect on LPS-primed macrophages.

Methods: Linoleic acid and conjugated linoleic acid isomers were obtained from Matreya, LLC, PA, USA. Fatty acids were solubilized in ethanol and stored in aliquots at -20°C. Prior to each experiment, LA and CLA were conjugated to fatty acid-and endotoxin-free albumin (BSA) at a molar ratio of 10:1 (fatty acid : albumin) in serum-poor medium (1mM of BSA fatty acid free). Fatty acid-albumin complexes were freshly prepared at the physiological concentration of 50 μM (Kennedy, Martinez et al. 2010).

The effects of LA or t10, c12-CLA were investigated in vitro on wild-type bone marrow-derived macrophages (BMDM) from wild-type mice. The bone marrow-derived macrophages were selected by cell adherence. BM-derived cells were plated at the concentration of 1x106/ml in 12 wells plates. After 2 hours all the floating cells (in particular lymphocytes) were eliminated, by replacing the medium by a differentiation medium (30% L929 medium, 10% horse serum, 1% antifungal and 1% antibiotics). The differentiation medium was changed every 2-3 days, until when macrophages were confirmed fully differentiated by microspopic examination (10-12 days). Macrophages were then stimulated with LA or CLA (50 μM solution in 1mM BSA) alone or together with lipopolysaccharides (LPS 10 ng/ml) in a 1% serum-enriched medium. After 24h of stimulation, the supernatants were collected in order to evaluate protein secretion and macrophages on the bottom were resuspended in RLT buffer with 1% βmercaptoethanol and conserved at -80°C for RNA extraction. Gene expression analysis was performed in order to evaluate the potential effects of CLA on macrophage polarization.

The experiment was perfomed in 5 WT mice, irrespectively of sex, in separate trials and both hypotheses were evaluated by RT qPCR. The expression patterns for genes associated with classical (M1) and alternative (M2) activation was analyzed using the same markers selected to characterized mouse WAT sorted macrophages (Herrero, Shapiro et al. 2010). For the M1 phenotype: iNOS, IL-1β and CCL2/MCP1; for the M2 phenotype: CD206, arginase 1 (Arg1), IL-1Ra and IL-10, were the used markers.

Results: An average of 1.8-fold increase was observed in CLA treated BMDM over the untreated cells set to 1, whatever the gene of interest, whereas for LA there was no change. Both LA and CLA failed to polarize macrophages toward the M1 or M2 activation state. These results were somehow expected.

First of all because it is known that the primary target of CLA is the adipocyte, then because the macrophage expression of the above listed M1-M2 genes is tightly regulated by exogenous stimuli, such as Th1 cytokine IFNγ (for M1) and Th2 cytokines IL-4 and IL-13 (for M2). None of those cytokines was either found upregulated in WAT after CLA or secreted in the ex-vivo WAT explant medium (24h-48h culture of WAT from CLA-treated mice -data obtained by Luminex, not shown). Moreover, IL6 and IL1Ra, chosen as representative of pro-and anti-inflammatory cytokines, respectively, were measured by ELISA in the medium of stimulated BMDM and levels were in the lowest range of the standard curve.

In parallel to polarization, the effects of LA and CLA stimulation were evaluated on receptors potentially able to "sense" lipids, such as TLRs (TLR2, TLR4) and GPCRs (GPR120, GPR116, GPR109A) and on chemokines (CCL2, CCL5) able to recruit other immune cells. Once again, the expression of these genes was minimally altered by CLA, being CCL5 the most affected facter (2-fold increase compared to untreated cells). On the other hand, macrophages strongly responded to LPS stimulation, showing for example for CCL5 a 220-fold increase, independently of the presence of LA/CLA. No specific effects of LA or CLA were observed when LPS was used as activating factor, possibly due to the overwhelming effects of LPS on macrophages (data not shown).

Since no major effects were observed using "naïve" macrophages, we decided to evaluate the effects of CLA on pre-polarized macrophages, trying to mimic a more physiological situation, with a possible predominance of M1 or M2 macrophages depending on the pathophysiological state of the tissue. This experimental setting (in vitro polarization) was performed one time only using 1 WT male mouse, so these results are preliminary observations. Bone marrow-derived macrophages were differentiated as described above. After macrophages were fully differentiated, BMDM were stimulated for 24h to activate the specific phenotype with no stimulus for control group (M0), IFNγ (10 ng/ml) for M1 phenotype and IL4 (5 ng/ml) for M2 phenotype After 24h, 200 µl of medium was collected for further analysis and replaced with the same amount of LA or CLA at the usual concentration (50 μM). Following 24h stimulation with LA or CLA, medium was collected and macrophages were processed as described above. The gene expression analysis reported for this experiment is the result of one single experiment (from one wild-type mouse bone marrow). In Figure 39, the key markers for the M1 phenotype (iNOS, IL1β) and for the M2 phenotype (Cd206, arginase 1) indicated that macrophages were efficiently polarized by the recombinant cytokines used. No significant effects of CLA were observed in unpolarized macrophages, M0. Looking at inducible nitric oxide synthase (iNOS) expression, cytokine-polarized M1 cells showed a 2-fold increase of iNOS when stimulated with CLA, whereas in cytokine-polarized M2 cells iNOS was undetermined. Similarly IL1β expression trended higher in M1 cells stimulated with CLA (1.4-fold increase). On the contrary, in the M2 cells IL1β expression was decreased (4-fold reduction) by CLA. When the classical M2 markers, mannose receptor CD206 and arginase 1 (Arg1) were analyzed, we found them expressed only in M2 cells. However, CLA stimulation could possibly differentially affect their regulation (Figure 41). Based on these preliminary observations at the mRNA level, it is tempting to assume that t10,c12-CLA is sensed by macrophages, possibly affecting their transcriptional profile depending on their activation state within the tissue. However these results will need to be confirmed by other experiments in vitro, in order to exclude or not a direct action of t10, c12-CLA in vitro and in vivo. Moreover the cytokine secretion of the different macrophage phenotype in the presence or absence of LA and CLA stimulus will need to be analyzed for this and other in vitro experiments. The main limitation of these in vitro studies is that typically differentiated bone marrow macrophages respond differently than WAT-infiltrating macrophages to high concentrations of lipids and FFA. Tissue macrophages are in fact a group of cells that share common progenitors and that develop in that specific lipid-rich context [START_REF] Xu | Obesity activates a program of lysosomal-dependent lipid metabolism in adipose tissue macrophages independently of classic activation[END_REF]). However, since in the CLA in vivo experiments most of the macrophage accumulation in WAT was due to infiltration (proved by marked WAT upregulation of recruiting factors -see above publication), working with BMDM seemed a reasonable model to answer to the question about the potential role of macrophages to mediate the effects of CLA in WAT. Finally, despite these results were not obtained from macrophages isolated directly from different WAT depots, t10, c12-CLA does not seem to differentially affect the transcriptional profile of macrophages compared to linoleic acid, suggesting that macrophage polarization is most probably not the primary mechanism affecting the local response of WAT.

E. Conclusions

The magnitude of changes produced by dietary factors is known to be low and cumulative. CLA administration can be considered a diet-induced metabolic insult, since 7 gavages are sufficient to provoke a fat mass decrease, associated with a drastic reduction in circulating adiponectin and massive insulin secretion. Our results demonstrated that CLA caused alterations with great differences among different cell types, mainly targeting adipose tissue. Despite a series of pathways critical for WAT homeostasis (adiponectin, resistin, RBP4, Plin1, CD36, ATGL, AHL, PPARγ) were completely or almost completely downregulated in response to CLA, adipocytes survived potentially owing on upregulation of lysosomal acid lipase (Lipa) and transcription factor EB (TFEB), known to control a process called lipophagy. The regulation of one single transcription factor of multiple steps of lipid catabolism may be used by adipocytes to tightly control the lipid flow [START_REF] Settembre | TFEB controls cellular lipid metabolism through a starvationinduced autoregulatory loop[END_REF]. It is tempting to speculate that the process of lipophagy allowed mantaining energetic homeostasis during CLA treatment through mitochondrial oxidation of fatty acids released through Lipa action, given upregulation of the uncoupling protein-2 (UCP2).

The CLA-induced rapid and massive immune cell accumulation in WAT was possibly driven by endogenous signals linked to stress (ATF3) or hypoxia/apoptosis (Tsuboyama-Kasaoka, Takahashi et al. 2000). Complementary cell culture studies with bone marrow-derived macrophages allowed us to exclude a direct action of CLA on the most represented cell population infiltrating WAT after CLA administration. The interesting observation is that WAT immune cell recruitment, in particular M2-like activated macrophages, positively correlates with ECM accumulation. This correlation suggests an active role for these innate immune cells in what is a WAT adaptive response, possibly participating to homeostasis restoration. The almost total reversibility of the deleterious metabolic alterations, in particular insulin resistance, provoked by this CLA isomer revealed an unexpected resilience of WAT to an acute nutritional stressor. The results concerning the intestinal tract revealed that the acute administration of CLA provoked a local inflammatory response, as indicated by TLR4 and TNF-α upregulation, although with unchanged immune gene (Cd3 and Cd68) expression level. Intestinal inflammation was associated with a reduced expression of tight junction proteins in the jejunal tract. These preliminary observations suggest that part of the effect of CLA in WAT could be mediated by the potentially increased intestinal permeability. Further investigations could evaluate the degree of "leaky gut" measuring potential increased bacterial translocation (blood and tissue endotoxin level) and its effects on local and peripheral immune cell activation in association to WAT alterations (depending on depot localization). Finally, through metagenomics analysis, microbiota modifications in response to CLA could be analyzed over time following single animals in order to better define a microbial phenotypic signature induced by CLA treatment. The link between microbiota composition and the local and systemic immunity could be explored in relation to the animal metabolic state.

To conclude, dietary fatty acids can modulate adipose tissue immunity and remodeling, inducing transient modifications in an acute experimental setting. Once again the cellular plasticity of WAT in response to a varying nutrient environment allows this tissue to quickly adapt, effectively maintaining the control over glucose homeostasis.

STUDY II: PROBIOTIC ADMINISTRATION IN DIET-INDUCED OBESE MICE: EFFECT OF BODY WEIGHT GAIN AND GLYCEMIC STATUS.

A. Purpose

This study was designed in the context of prevention and treatment of obesity and the associated metabolic alterations. Beneficially altering gut microflora, through daily administration of specific probiotics, might provide long-term beneficial effects against obesity [START_REF] Stenman | Potential probiotic Bifidobacterium animalis ssp. lactis 420 prevents weight gain and glucose intolerance in diet-induced obese mice[END_REF]), [START_REF] Chen | Incorporation of therapeutically modified bacteria into gut microbiota inhibits obesity[END_REF]. This hypothesis gave us the opportunity to investigate the role of the indirect crosstalk between adipose tissue and intestine, and in particular the impact of local and systemic inflammation on the metabolic alterations associated with obesity.

The objectives of the presented study were to evaluate the effects of probiotics in HFD-fed mice on:

 Body weight gain and adiposity over a 12 weeks time course;  Glycemic status: glycemia, insulinemia, insulin sensitivity and glucose tolerance;  Systemic metabolic variables and triglyceride concentration in plasma and liver.

B. The experimental model

In the present study, the experimental model used is the murine high fat diet-induced obesity model.

The study design implicated the administration of specific probiotics concomitant to HFD (60% calories derived from fat). The strategy of daily administration of specific strain of probiotics at 109 CFU in daily drinking water was tested in male wild-type mice (C57BL/6J) under HFD for 12 weeks. The effects of the probiotic supplementation were evaluated over time for the different variables examined in comparison to HFD-fed age and sex-matched mice.

Methods

Probiotics:

Frozen dry pellets of probiotics were reconstituted immediately before use. Every day, bacterial pellets (probiotic X and Y) were thawed 10 minutes in ice and 10 minutes at room temperature prior the addition of a specific drinking water volume in order to have a resulting concentration of 109 cfu in 100 ml inside the biberon. Control group (CTL) received drinking water alone.

Animals: Thirty male 8 weeks old C57BL/6J mice were housed 5/cage (8 cages) with free access to food and water (12-h daylight cycle with light on at 7:00 am) in ICAN animal facility. Mice were given a high fat diet containing 60% Kcal from fat (Research Diets, Inc. New Brunswick, NJ, USA). After 2 week of acclimation, mice received Water (control) or bacterial strains resuspended in Water for 12 weeks. Water suspensions were prepared fresh daily. Daily water intake was recorded daily for the first two weeks and thereafter once a week, to make sure bacterial suspension did not change drinking habits. Mice were assigned to 3 treatment groups, CTL, probiotic X (10 9 cfu/per day/mouse), probiotic Y (10 9 cfu/per day/mouse).

Study design: Body weight and food intake were measured weekly. Food intake was determined per cage by weighing the food left in the food container after 1 week. In addition, the weight of food consumed per cage was followed daily during one week. On weeks 0, 2, 4, 6 and 8, non-fasted blood samples were drawn from the tail between 9:00 and 10:00 am and analyzed for glucose concentration using a glucometer (Roche, Basel, Switzerland). Body composition was analyzed by NMR using EchoMRI-100TM equipment (Echo Medical Systems, Houston, TX, USA) on week 11.

Insulin and glucose tolerance tests: Insulin (ITT) and oral glucose (OGTT) tolerance tests were performed at week 10 and 11, respectively in unanesthetized mice. For insulin tolerance tests, insulin (Actrapid, 100UI/ml; Novo Nordisk, Copenhagen, Denmark) was administered i.p. (0.5 U/kg) after 5h of food removal. For oral glucose tolerance test, glucose (10 mmol/kg) was administered by gavage after 16h of fasting. Glycemia was determined with a glucometer (Roche, Basel, Switzerland) from blood obtained at the tail 15, 30, 60 and 90 min after insulin or glucose administration.

Tissue collection:

For tissue and blood collection, mice were anesthetized with isoflurane and killed by cervical elongation after 3h of food deprivation the day after the last probiotic administration. Mice from each treatment were equally separated in 3 groups and sacrificed on consecutive days. Inguinal, epidydimal and retroperitoneal adipose pads were weighted to calculate an adiposity index (ratio of adipose tissue weight to body weight in %). Liver was also weighted and reported to body weight. Intestinal (caecum) content was collected, together with a part of intestine (jejunum) and colon, and stored at -80°C. Plasma samples prepared from orbital sinus blood samples, collected using EDTA capillary tubes, were stored at -20°C before use. Insulin, leptin and adiponectin concentrations were measured using immunoassay kits (R&D Systems, Minneapolis, MN, USA). Triglycerides levels were determined in plasma and liver lysate using a commercial kit (Sigma-Aldrich, St. Louis, MO, USA).

Statistical analysis:

Comparison between groups was performed by one-way ANOVA using JMP software (SAS, Cary, NC, USA). When this analysis revealed statistically significant difference (p < 0.05), groups were compared by Student't test.

Effects of probiotics on body weight and adiposity

Before starting the experiment, mice were randomized based on body weight (BW) in the 3 different groups, not to present any difference at baseline. In Figure 42, BW increase over the 12 weeks of HFD is reported in grams and percentage. As expected, the mice in the control (CTL) group developed obesity and presented values of body weight and weight gain expected for similar duration of the specific HFD.

The mice receiving probiotic X (from here identified as X) had significantly higher BW compared to the CTL group in weeks 5, 7, 8 and 10. Whereas mice receiving probiotic Y (from here identified as Y) had significantly higher BW compared to the CTL group from week 4 to week 12 (+1.80g to 6.40g). Percentage of fat mass was significantly higher in the Y group compared to the CTL group (7.21% of difference in means). The body weight difference of group Y was confirmed when body composition was analyzed by quantitative magnetic resonance system (Echo Medical System, Houston, TX, USA -EcoMRI) at week 11 of HFD. In fact, as shown in Figure 43, percentage of lean mass was significantly lower in the Y. group (-8.41% of difference in means) and percentage of fat mass significantly higher (7.21% of difference in means) in the Y group compared to the CTL group. Values of lean and fat mass for the mice in the CTL group were consistent with previous reports after 12 weeks of HFD feeding. At the sacrifice, WAT from different depots was carefully excised and weighted, as shown in Figure 44. Inguinal pad weight was significantly higher in the Y group compared to the CTL group, whereas no difference was observed in the other adipose depots. This observation suggested that the higher inguinal WAT weight might explain alone the increased percentage in fat mass in the Y group quantified by EcoMRI, also indicated by total WAT, as a result of the sum of the three adipose depots (Figure 44). Adiposity index, calculated as total WAT (gram) divided by body weight (gram) of each animal did not show any differences between groups (data not shown).

Effects of probiotics on glycemic status

Proonged high fat diet feeding is known to induce insulin resistance and to promote hyperglycemia. Control group mice presented both these metabolic alterations induced by HFD in expected range. Blood glucose was measure every two weeks in fed-state mice and no differences were observed between treatment groups (data not shown). To evaluate more directly whether IR developed, metabolic tests (ITT and OGTT) were performed in the 3 groups at the end of the HFD time frame. The mice in the control group presented expected responses to ITT and OGTT tests for mice under HFD. Insulin (ITT) tolerance test was performed also at week 10. Overall there was no significant difference between groups. However a trend for lower insulin sensitivity in the Y group was observed and confirmed by the area under the curve over time (Figure 45). Oral glucose (OGTT) tolerance test was performed at week 11. Glucose response curves were not statistically different between groups (Figure 46).

Effects of probiotics on plasma variables

Circulating factors were measured in HFD-fed mice and mice either treated with probiotic X or Y or untreated (control group). Insulin levels were quite variable, despite the use of an ultrasensitive kit (Figure 47). Few mice in the probiotic groups presented very high values (1 in group X and 4 in group Y). The high values found for those mice are acceptable, in particular after 12 weeks of HFD, so the insulin graph in Figure 47 shows the groups X and Y with all the mice. However no statistical significance was found compared to the CTL group due to the high standard deviations. Equal amounts of circulating adiponectin were found in the 3 groups, whereas plasma leptin was significantly higher only in the Y group (Figure 47), in accordance with higher BW and fat mass. Plasma triglyceride levels did not show any difference between the different groups, whereas liver presented significantly higher TG levels in the Y group compared to the CTL group (5.15 mol/g of difference in means) (Figure 48).

C. Conclusions

The current study shows that probiotics, the Y strain in particular, failed to promote a favorable metabolic phenotype in high fat diet-fed mice. Administration of X and Y probiotic strains in drinking water resulted in increased weight gain as compared to the control group starting after week 4 of HFD. Increased percentage of fat mass was found in the Y group, in association with higher inguinal white adipose tissue weight and circulating leptin levels. Additionally, liver triglyceride content was increased in the Y group, although without significant alteration of plasma triglycerides. No differences were observed for systemic adiponectic levels. Bacterial supplementation did not affect glucose homeostasis in any of the two groups. Glucose tolerance was not different between groups. However, a trend toward systemic insulin resistance was found in the Y group. Although drinking water supplementation is a less aversive method compared to the often reported probiotic administration by daily gavage, measurement of probiotic viability by flow cytometry revealed that probiotics were increasingly stressed (about 1-3%) or dead (6-30%) over time, also in relation to the specificity of the strain used. Finally, further examinations of cellular and molecular mechanisms behind the observed phenotype, such as lipid metabolism within the gastro-intestinal tract and white adipose tissue, will surely help to clarify and avoid those detrimental effects in the presence of certain probiotics. To conclude, research on beneficial effects of specific probiotics on metabolic parameters, such as body weight and glucose homeostasis in HFD-induced obese mice, will surely require further investigation. Phenotypic characterization of Cathepsin S deficient mice reported in Publication 2 revealed that mice displayed improved glycemic status and remained glucose tolerant when fed a high fat diet. However, contrary to our initial hypothesis, this was not related to adipose tissue dysfunction or immune alteration. By contrast, a clear liver phenotype was discovered, in which reduced hepatic glucose production was linked to reduced hepatocellular respiration. Although the mechanisms relying this effect were not deciphered, this study identified inhibition of Cathespsin S as a potential mean to improve glycemic status in obese subjects.

The transcription factor IRF5 was found increased in the adipose tissue of obese mice and humans, specifically in visceral depots. In mice, this factor was shown to alter adipose tissue immune profile, promoting M1 macrophage accumulation, and to contribute to extracellular matrix deregulation (Publication 3). IRF5 was up-regulated in adipose tissue of CLA treated mice, although we did not detected any site-specific difference in this model. Further investigations of the effect of CLA on adipose tissue in IRF5 deleted mice are likely to provide new information regarding the role of this transcription factor on adipose tissue dysfunction and ensuing metabolic deregulation.

I contributed to show that DAPK2 gene expression is actually reduced in the adipose tissue of obese mice, as found in human obesity (Publication 4). The role of this kinase in adipocyte was investigated, showing that it could be implicated in attenuation of autophagy in the adipocytes of obese subjects. We did not detect any change in DAPK2 gene expression in our model of CLA treated mice, potentially due to a short-term administration. The role of autopahgy in adipose tissue cellular remodeling in obesity and lipodystrophy is not firmly established. The CLA model used in the PhD program could be useful to address this specific question.

B. Adipose tissue plasticity and metabolic consequences

A wealth of studies using distinct mode of CLA administration, i.e. as mixture or isolated isomers, in the food or by gavage and at different doses, have helped to establish a general scheme of CLA-induced systemic and local alterations. All studies converge to the view that adipose tissue represents the main metabolic tissue targeted by CLA and that the t10,c12-CLA isomer is the main culprit in the deleterious effects of these compounds. The table below (Figure 50) summarizes the current knowledge regarding the pleiotropic effects of this specific isomer. In study I, we showed that most of these alterations, with the exception of liver steatosis, were acquired quickly in response to high doses of the t10,c12-CLA isomer and were strikingly reversible after shortterm interruption of the treatment.

This work as a whole allowed us to better recognize the central role of white adipose tissue in the regulation of glucose homeostasis. In particular, Study I pointed out the integrated involvement of several processes highly tuned to protect WAT homeostasis. Processes intrinsic to the cell itself (adipocyte), including adipokine secretion, mitochondrial fatty acid oxidation and lipolysis, or extrinsic,

BACTERIAL COMPOSITION

•Reduced Firmicutes •Increased Bacteroidetes such as immune cell dynamics, allow WAT to effectively adapt to metabolic changes. Through this study, we discovered that, in response to an acute nutritional stressor, WAT and glucose homeostasis could be rapidly regained. When the stress is more chronic, as in obesity, certain intrinsic and extrinsic processes, so tightly correlated with metabolism, might become irretrievably out of tune and the dysfunctional state of WAT difficult, if not impossible, to reverse.

During this thesis project, we used the t10, c12-CLA isomer to cause a lipoatrophic syndrome in mice (Clement, Poirier et al. 2002) and evaluate the ability of WAT to control the local and systemic changes provoked by this external stressor. To answer to the question, we analyzed the reversibility of the previously reported WAT alterations caused by the specific isomer, macrophage accumulation and tissue remodeling (Clement, Poirier et al. 2002) (Russell, McGee et al. 2007). We focused our attention in particular on WAT immunological response, using flow cytometry to analyze the dynamics and the profile of different immune sub-populations. We also evaluated the potential association between WAT cellular and molecular changes with the insulin resistance induced by CLA (Poirier, Shapiro et al. 2006).

The changes in body composition and adipose depot weight were the result of effects of CLA on adipocytes. Through fatty acid analysis we demonstrated that t10,c12-CLA targets mainly adipose tissue. Indeed a marked accumulation of this isomer was found in WAT compared to other peripheral organs, including liver and intestine. Several in vitro studies have described the effects of CLA on adipocyte functions, such as reduction of lipid uptake and storage capacity inhibiting lipoprotein lipase [START_REF] Park | Changes in body composition in mice during feeding and withdrawal of conjugated linoleic acid[END_REF], reduction of lipogenesis by decreasing expression of SREBP-1c and PPAR-γ and their downstream genes, ACC, FAS, and SCD1 (House, Cassady et al. 2005), increase in apoptosis (Kennedy, Martinez et al. 2010). Microarray profiling of epidydimal WAT of mice fed t10c12-CLA for 14 days indicated a significant reductionof mRNAs for major adipocyte regulatory factors, PPAR, SREBP-1, lipin 1 in line with the reduced transcript levels for key metabolic pathways in the WAT (LaRosa, Miner et al. 2006). Our results mostly confirmed those metabolic alterations (ACC, FAS, Plin1 severely downregulated). The broad chemokine upregulation reported by LaRosa and collegues for CXC family members in particular (LaRosa, Miner et al. 2006) was also confirmed by our RT-PCR results in the three adipose depots. This increased local chemotaxis was associated with a rapid and massive WAT macrophage accumulation, combined with fat mass reduction, changes in body composition and reduced circulating adipokines [START_REF] Park | Effect of conjugated linoleic acid on body composition in mice[END_REF]) [START_REF] West | Effects of conjugated linoleic acid on body fat and energy metabolism in the mouse[END_REF], (Poirier, Shapiro et al. 2006). However the infiltration of macrophages we observed as early as after 3 days of CLA administration was not associated with WAT inflammation, in sharp contrast with the previously reported increase of proinflammatory cytokines at the protein level (Poirier, Shapiro et al. 2006) and at the transcriptional level (2-to 100-fold induction of many cytokine transcripts, i.e. IL-6, IL-1, TNF ligands (LaRosa, Miner et al. 2006), and the reported activation of NF-kB pathway in response to CLA [START_REF] Liu | Combined effects of rosiglitazone and conjugated linoleic acid on adiposity, insulin sensitivity, and hepatic steatosis in high-fat-fed mice[END_REF]). In fact, in our experimental setting, gene expression analysis of WAT after CLA administration indicated a marked upregulation of the classical anti-inflammatory cytokines, IL-1ra and Il10, in the three distinct adipose depots analyzed, subcutaneous, peri-uteral and retroperitoneal WAT.

Macrophages have been reported accumulating in WAT in response to lipodystrophy (Jan, Cervera et al. 2004) and obesity (Cancello, Henegar et al. 2005). In the response induced by t10,c12-CLA, to further characterized the WAT massive accumulation of macrophages shown by immunolabeling, we used flow cytometry analysis. The stromal vascular fraction infiltrating WAT was analyzed, with a special focus on macrophage phenotype. As presented in the introduction, once these monocyte-derived cells arrive in the tissue, they can be found in a rich spectrum of polarization state (phenotype). The so called M2 macrophages represent the alternatively-activated phenotype and they are usually "resident" in the tissue. At the completely opposite side of the spectrum we have the M1 macrophages, also called "effector" because they are recruited to resolve inflammation. A precise definition of the functional phenotype of macrophages in vivo is complicated to achieve. However, through flow cytometry analysis, the phenotype of t10,c12-CLA-induced WAT macrophages was evaluated, using the cell surface markers CD11c and CD206 in F4/80+ cells. This exploration revealed that the majority of the F4/80+ cells infiltrating WAT during CLA administration were CD206+ CD11c-(M2-like macrophages) compared to LA control mice. In response to t10,c12-CLA, this population increased markedly, along with a less abundant subset of CD206+ CD11c+ (M1-like macrophages), which was virtually undetectable in the LA group. These data indicate that CLA did not induce any "phenotype switch" from M2 to M1 state over time, despite the development of insulin resistance in response to the treatment, in opposition to the model proposed by Lumeng for WAT macrophages in obesity [START_REF] Lumeng | Obesity induces a phenotypic switch in adipose tissue macrophage polarization[END_REF]). Moreover, gene expression analysis confirmed these findings with a clear predominance of M2 marker upregulation, well represented by the ratio Arg1/iNOS (3-fold increase in subcutaneous WAT and 20-fold increase in peri-uteral WAT. Lower elevation of pro-inflammatory Tnfα and Il-1β gene expression was observed in WAT in response to CLA, probable outcome of the slight increase of M1 macrophages in WAT during treatment. However their mRNA levels were boldly weighted down by the marked enhancement of IL-1ra and Il-10, the anti-inflammatory cytokines classically expressed in M2 macrophages, in, indicating that WAT favors an anti-inflammatory response to the short-term metabolic stress-induced by CLA. The increased M2 macrophage accumulation was combined with increase of other innate immune cells, including NK cells and dendritic cells, although to a lower extent. In parallel cells of the adaptive immunity, such as NKT cells, CD3+ T cells and B cells were also increased in both WAT depots, but remained less abundant than innate immune cells, whereas Tregs remained unaffected by t10,c12-CLA administration. We could have expected a reduction in Tregs abundance, given the link established between Tregs reduction and insulin resistance in mice and humans [START_REF] Deiuliis | Visceral adipose inflammation in obesity is associated with critical alterations in tregulatory cell numbers[END_REF]. It is possible that longer exposure to a nutritional stress is required for these cells to be altered in WAT.

The marked prevalence of M2-like macrophage accumulation in response to CLA-induced WAT alterations, including apoptosis (Kennedy, Martinez et al. 2010), is consistent with their scavenger function (Lee, Petkova et al. 2013). That such immune cell accumulation happened in the absence of local inflammation during CLA administration, is supported by the findings of one study in which dietary supplementation of t10,c12-CLA was used for one week in mice (Russell, McGee et al. 2007). They reported leukocyte accumulation in association with tissue remodeling in the mouse mammary fat pad (Russell, McGee et al. 2007). In addition, they suggested the dispensability of inflammation during the tissue remodeling process. Interestingly, using genetically modified mice, the authors showed that CLA induced the same response in wild-type and mice lacking TNF-α or functional mast cells (Russell, McGee et al. 2007). Since one week of dietary supplementation of t10,c12-CLA was sufficient to induce collagen deposition in mammary WAT of mice (Russell, McGee et al. 2007), this prompted us to evaluate WAT extracellular matrix accumulation in the three adipose depots during and after CLA treatment. In our experimental setting, daily gavage with the same CLA isomer induced a gradually increasing fibrillar collagen deposition in WAT, observed by picrosiruis red staining already after 3 gavages and confirmed at 7 gavages also by hydroxyproline content determination.

In obesity, WAT fibrosis is associated to tissue expansion and adipocyte dysregulation [START_REF] Berry | Weighing in on adipocyte precursors[END_REF], possibly to limit excessive adipocyte enlargement. Instead when fat mass decreases drastically as in lipodystrophy (due to genetic abnormalities, autoimmunity, or retroviral agents in HIV) or too rapidly as in certain cases after bariatric surgery, WAT remodeling intervenes possibly to safeguard adipocyte structure, to avoid excessive loss of adipocyte lipid content. A beneficial role of extracellular matrix remodeling has been proposed in IRF5 deficient mice, which displayed increased collagen deposition associated with limited visceral WAT expansion and improved insulin sensitivity (Publication 3). In 2010, the team of Karine Clément showed that human WAT fibrosis, in particular peri-cellular fibrosis, is a process with the potential of limiting the lipolytic activity of adipocytes after bariatric surgery (Divoux, Tordjman et al. 2010). This concept was further supported by a more recent study from our group, in which Pellegrinelli et al. have shown that human adipocytes decrease lipolysis and adipokine secretion in a 3D system modeling the fibrotic environment characteristic of obese WAT (Pellegrinelli, Heuvingh et al. 2014). In our model, collagen accumulation was found correlated with mRNA Cd68, suggesting that the prevalent M2-like macrophages could have implicated in this process.

The decreased expression of Lox and Loxl2, two transcriptional targets of HIF1α, known to be involved in pathological fibrosis [START_REF] Cox | Remodeling and homeostasis of the extracellular matrix: implications for fibrotic diseases and cancer[END_REF], and above all the reversibility of WAT cellular and molecular alterations indicate that WAT response to CLA is a physiological adaptation to metabolic changes. Further explorations will certainly allow better understanding the functions of these innate cells in response to WAT alterations, i.e. lipid engulfment or collagen turnover (Madsen, Leonard et al. 2013) or tissue repair (Lee, Petkova et al. 2013).

The role of macrophages in the pathological process of other fibrotic tissues can help to decipher which function these cells could exert in WAT remodeling. For example, in fibrotic liver pathology, in mice in mice infected with Schistosoma mansoni, IL-4-activated M2 macrophages have been shown to control collagen synthesis and tissue remodeling, expressing high levels of Arg1 that catalyzes L-polyamine production necessary for myofibroblast activity [START_REF] Hesse | Differential regulation of nitric oxide synthase-2 and arginase-1 by type 1/type 2 cytokines in vivo: granulomatous pathology is shaped by the pattern of L-arginine metabolism[END_REF]. However, another study using the same human pathology model (schistosomiasis infection) showed that Arg1-expressing M2 cells are required for the suppression and resolution of fibrosis (Pesce, Ramalingam et al. 2009). Despite this phenotype over-simplification, the role of these innate cells in exacerbating or resolving WAT fibrogenesis could depend on their respective associated molecular mediators. Indeed, evaluation of the transcriptomic profile and the secretion (cytokines, matrix remodeling enzymes and growth factors) of freshly isolated macrophages from WAT during and after t10,c12-CLA administration would allow to draw a well-defined course of macrophage functionality during tissue remodeling.

Finally, whether macrophages accumulate in WAT through infiltration and/or in situ proliferation, depending on the specific experimental context remains an interesting question to further explore. In addition, the localization of the CLA-increased M2-like macrophages (around dying cells or within the cytoplasm of WAT) could also help to clarify their role in CLA-induced WAT alterations.

Tissue homeostasis is controlled by an evolutionarily conserved tissue damage response. Studies in flies, mice, and human models have provided evidence that these highly conserved mechanisms are represented by a secretory program that regulates the tissue repair process when cells are damaged [START_REF] Neves | Of flies, mice, and men: evolutionarily conserved tissue damage responses and aging[END_REF]. In the context of chronic progressive diseases, such as obesity and aging, the degeneration of tissue homeostasis (due to diet or age-related decline) becomes chronic and as a consequence the adaptive immune response during tissue injury is amplified and grows uncontrolled.

The WAT alterations that we observed in response to t10,c12-CLA present few similarities to what happens after injury, an acute tissue degeneration. During would healing, damaged cells release growth factors and chemokines that stimulate the proliferation and recruitment of leukocytes that produce profibrotic cytokines, such as interleukin-13 and transforming growth factor-β (TGF-β). In our model, IL-13 had very low mRNA expression levels in WAT and the pro-fibrotic factors, inhibin beta-A and Ctgf were modestly increased in WAT. Only in subcutaneous WAT, TGF-β was significantly increased (3-fold over the LA-treated mice) in association with the extra cellular matrix accumulation observed after 7 gavages with CLA. However in common with the acute wound healing, the magnitude of chemokine upregulation in response to CLA was present and it was disproportionate compared to TGF-β, suggesting that immune cell recruitment overpassed the tissue remodeling process. The WAT alterations we observed in response to CLA possibly preceded ECM accumulation, which was still limited, as was limited in term of time and intensity the injury caused by CLA (7 gavages). We attempted to answer to this hypothesis with a small trial where RNA was isolated from WAT of mice treated for 3 gavages. Gene expression analysis of the three WAT, subcutaneous, peri-uteral and retroperinoneal, was performed. The most affected tissue, therefore possibly the first one, was subcutaneous WAT. Three gavages with the t10,c12-CLA were sufficient to induce transcriptional modifications, as reported by LaRosa et al (LaRosa, Miner et al. 2006). Our analysis showed that in sWAT the most affected genes were the chemokines Ccl7 and Ccl2, highly induced (30 to 10-fold induction, respectively), in association with the pan-macrophage marker Cd68 (8-fold), whereas the two collagens (Col1 and Col3) significantly upregulated in response to 7 gavages with CLA (Publication 1) were unchanged in the earlier time point (3 gavages, data not shown).

Interestingly, these data suggest that chemokine and consequently macrophage accumulation preceded ECM remodeling, leaving open the question about the effects of CLA during a longer time course.

It is worth to underline that subcutaneous WAT failed to completely recover fat mass after CLA disruption. This was associated with ECM accumulation and fibronectin gene expression still significant in the reversion CLA-treated group (7G 7R), whereas all the other factors, including chemokines, returned to control level. Another observation made through RT-PCR analysis is that this depot did not present increase of VEGF-A and PECAM-1 after CLA, in contrast with peri-uteral and retroperitoneal WAT, suggesting absence of angiogenesis in line with failure in fat mass recovery. Neovascularization is the process during which stromovascular network expands. During the development as well as expansion of adipose tissue, next to adipocyte growth and proliferation, the concomitance of this process is extremely important [START_REF] Christiaens | Angiogenesis and development of adipose tissue[END_REF]. Neovascularization has been deeply explored in the context of breast cancer, in particular, in association with extracellular matrix environment on adipose tissue plasticity [START_REF] Iyengar | Adipocyte-secreted factors synergistically promote mammary tumorigenesis through induction of anti-apoptotic transcriptional programs and proto-oncogene stabilization[END_REF]) [START_REF] Iyengar | Adipocyte-derived collagen VI affects early mammary tumor progression in vivo, demonstrating a critical interaction in the tumor/stroma microenvironment[END_REF]. In this context, the ability of WAT to secrete protein and lipid derivatives highly proangiogenic can affect the preexisting vasculature, contributing to a unique growth microenvironment. It would certainly be of interest to performe similar explorations in the t10,c12-CLA model, i.e. visualization of vascular network in the different adipose depots during and after treatment.

Concomitant or alternative mechanisms possibly implicated in the poor mass recovery in sWAT could rely on reduced fat cell renewal. The inhibition of PPARγ-regulated adipocyte differentiation by ATF3 [START_REF] Jang | ATF3 inhibits PPARgamma-stimulated transactivation in adipocyte cells[END_REF] could contribute to inability of sWAT to recover fat mass after CLA disruption, in contrast to the two other WAT depots. Whether the recruitment and proliferation of adipose progenitor cells (Lee, Petkova et al. 2013) [START_REF] Jeffery | Rapid depot-specific activation of adipocyte precursor cells at the onset of obesity[END_REF] is implicated remains to be determined. Interestingly, increased expression of Ki-67, a proliferation-associated marker, is still upregulated in the 7G7R condition, despite the complete regression of infiltrating immune cells in pWAT but not in sWAT. Different approaches could help to further support this preliminary observation. Quantification of preadipocyte replication is possible through adipose tissue in situ immunolabeling with Ki-67 antibody or through determination of BrdU incorporation rate in perilipin-positive adipocytes, to assay the rate of formation of new adipocytes [START_REF] Rigamonti | Rapid cellular turnover in adipose tissue[END_REF]). However, unfortunately, the signals triggering an influx of preadipocytes and enhancing their recruitment towards adipogenesis are only beginning to be deciphered, so it is difficult to identify the specific factors that enable WAT to successfully cope with CLA or other external stressors. It is only recently that the scientific community increased the attention level on adipose tissue centrality, including extracellular constituents, in relation to the systemic metabolic state. Adipose tissue dysfunction is now recognized as important contributor to the adverse outcomes associated to metabolic diseases. Both lipodystrophy and obesity are characterized by fat mass disturbancies and insulin resistance. The results obtained with the t10,c12-CLA-induced lipoatrophic syndrome model also suggest primacy of WAT functional integrity for glucose homeostasis. Moreover our data underline a potential common outcome between CLA-and diet induced-WAT and systemic alterations, i.e. adiponectin low levels. In another study insulin resistance was associated with drastic reduction of circulating adiponectin [START_REF] Purushotham | Maintenance of adiponectin attenuates insulin resistance induced by dietary conjugated linoleic acid in mice[END_REF]). To my knowledge, this is the only study that reported the reversibility of the effects of CLA on WAT mass and hepatic steatosis improvement, when mice were switched to a diet without CLA for the following 30 days. In this experimental design, insulin resistance was abolished concomitantly with adiponectin restoration, suggesting a causal link between those two parameters. This isomer has certain experimental advantages for investigating this link between WAT functionality and systemic metabolism: a rapid time course, the proved reversibility, the control of the stimulus and the specificity, since WAT is its main target.

Finally, the analysis of the microbial composition by 16S rRNA pyrosequencing suggested that even such a short-term administration of t10,c12-CLA possibly alter microbiota in mice, as recently shown [START_REF] Marques | Dietary trans-10, cis-12-conjugated linoleic acid alters fatty acid metabolism and microbiota composition in mice[END_REF]. The upregulation of the pro-inflammatory TNFα observed in the jejunal compartment could represent the starting point of the intestinal perturbation, caused by CLA directly or indirectly. Unknown endogenous factors could contribute to the microbiota composition changes observed in response to CLA. The initial inflammatory response potentially provoked by a shortadministration of this isomer could be amplified if followed by recruitment and/or activation of resident immune cells during longer exposure. This study suggests that mechanisms of t10,c12-CLA action on lipid metabolism might be mediated in part by alterations in gut microbiota composition and functionality. Dietary t10,c12-CLA supplementation for 8 weeks induced a decrease in the proportion of Firmicutes and an increase of Bacteroidetes. In our preliminary results, the enterotype M3, mostly represented by Bacteroides, the less rich in bacterial species, was particularly low in the CLA-treated mice (Figure 40), indicating that longer periods of treatment could change the experimental outcome. Carefull planning of future experiements will be necessary to better evaluate the potential changes at the microbiota level and possibly correlate them with metabolic parameters and WAT cellular and molecular alterations induced by CLA.

C. The future of probiotics to improve metabolic status?

It is now well established that consumption of a high-fat diet and obesity induce changes in the gut microbiota [START_REF] Erridge | A high-fat meal induces low-grade endotoxemia: evidence of a novel mechanism of postprandial inflammation[END_REF] [START_REF] Cotillard | Dietary intervention impact on gut microbial gene richness[END_REF] [START_REF] Kong | Gut microbiota after gastric bypass in human obesity: increased richness and associations of bacterial genera with adipose tissue genes[END_REF]. Whether modulation of gut microbiota by prebiotics or probiotics could beneficially affect the deleterious metabolic alterations induced by obesity is still controversial. In rodents challenged by a high fat diet, lactic acid bacteria and bifidobacteria supplementation results generally in reduced body weight gain and adiposity, reduced liver steatosis, improved glycemic status and in anti-inflammatory effects [START_REF] Amar | Intestinal mucosal adherence and translocation of commensal bacteria at the early onset of type 2 diabetes: molecular mechanisms and probiotic treatment[END_REF] [START_REF] Hamad | Milk fermented by Lactobacillus gasseri SBT2055 influences adipocyte size via inhibition of dietary fat absorption in Zucker rats[END_REF] [START_REF] Naito | Beneficial effect of oral administration of Lactobacillus casei strain Shirota on insulin resistance in diet-induced obesity mice[END_REF] [START_REF] Cano | Bifidobacterium CECT 7765 improves metabolic and immunological alterations associated with obesity in high-fat diet-fed mice[END_REF]) [START_REF] Miyoshi | Anti-obesity effect of Lactobacillus gasseri SBT2055 accompanied by inhibition of pro-inflammatory gene expression in the visceral adipose tissue in diet-induced obese mice[END_REF] [START_REF] Stenman | Potential probiotic Bifidobacterium animalis ssp. lactis 420 prevents weight gain and glucose intolerance in diet-induced obese mice[END_REF] [START_REF] Poutahidis | Microbial reprogramming inhibits Western dietassociated obesity[END_REF]) [START_REF] Wang | Modulation of gut microbiota during probiotic-mediated attenuation of metabolic syndrome in high fat diet-fed mice[END_REF]. However, some conflicting reports have been published showing increased weight gain and liver enlargement, increased inflammation and/or hyperlipidemia in mice receiving specific Lactobacillus strains [START_REF] Aronsson | Decreased fat storage by Lactobacillus paracasei is associated with increased levels of angiopoietin-like 4 protein (ANGPTL4)[END_REF] [START_REF] Angelakis | An evaluation of the effects of Lactobacillus ingluviei on body weight, the intestinal microbiome and metabolism in mice[END_REF] [START_REF] Cani | Changes in gut microbiota control inflammation in obese mice through a mechanism involving GLP-2-driven improvement of gut permeability[END_REF].

In Study II, using a high fat diet-induced obesity model (12 weeks), we found that under chronic nutritional stress, the resulting altered metabolic profile is difficult to be reversed. The gut microflora modulation through daily administration of two strains of probiotic was not able to attenuate WAT expansion, hyperglycemia and insulin resistance of HFD-fed mice. The potential beneficial contribution of microbiota to mouse metabolic phenotype could be due to multiple mechanisms, such as increased fatty acid oxidation at the peripheric level, or food intake reduction and/or delayed gastric emptying. In our experimental setting, no difference in food consumption was observed over time, so it is possible that the selected probiotics (X and Y) did not induce this positive effect. In case of mechanisms of action at the tissue level, gene expression analysis of key genes involved in lipid and glucose metabolism will be explored in the main metabolic organs (adipose tissue, liver, intestine). Further exploration, including pyrosequencing of the feces collected over time (before starting probiotic administration and after 6 and 12 weeks of treatment) will allow identifying the impact of the specific probiotic strain used on the intestinal ecology. In particular, one strain over two promoted an unfavorable phenotype, with increased weight gain and fat mass, in association with elevated circulating leptin and liver triglycerides. A limitation of the presented study was that one of the two strains used was supposed to have beneficial effects on the metabolic profile of the HFD-induced obese mice, since it was shown to be "protective" in a previous experimental setting. For this reason, for the studies planned in the next future the use of prebiotics will be introduced as a positive control. Prebiotics have been shown to have consistent beneficial effects on body weight gain, energy homeostasis, energy intake and inflammation, through direct or indirect effects [START_REF] Everard | Gut microbiota and GLP-1[END_REF]Cani 2014) (da Silva, dos Santos et al. 2013) [START_REF] De | Microbiota-generated metabolites promote metabolic benefits via gut-brain neural circuits[END_REF]. The potential effectiveness of this type of nutritional intervention will be tested in parallel to specific probiotic supplementation. Our results indicate that the potential benefits provided by probiotic supplementation need to be further explored in relation to human metabolic diseases, systemically, combining multiple experimental approaches.

D. Conclusions

To conclude, the results obtained with the presented experimental models will need to be reconducted to humans. As far as human consumption is concerned, a definite conclusion for conjugated linoleic acids safety has not been reached yet [START_REF] Benjamin | Conjugated linoleic acids as functional food: an insight into their health benefits[END_REF]. Type of CLA isomer and/or their combination with other polyunsaturated fatty acids, mode of administration, daily dose and duration of consumption are all parameters that remain to be carefully evaluated in relation to gender, age, or ethnic and geographical backgrounds. Similar concerns need to be taken into account for interventions of microflora modulation, including probiotic supplementation. Understanding how different nutritional

Abstract:

Accumulation of visceral adipose tissue correlates with elevated inflammation and increased risk of metabolic diseases. However, little is known about the molecular mechanisms that control its pathological expansion. Transcription factor interferon regulatory factor 5 (IRF5) has been implicated in polarizing macrophages towards an inflammatory phenotype. Here we demonstrate that mice lacking Irf5 show no difference in growth of their epididymal white adipose tissue (epiWAT), but expansion of their subcutaneous white adipose tissue, when placed on a high-fat diet compared to wild-type mice on the same diet. EpiWAT from Irf5-deficient mice is marked by accumulation of alternatively activated macrophages, higher collagen deposition that restricts adipocyte size and enhanced insulin sensitivity compared to wild-type mice. In obese subjects, IRF5 expression is negatively associated with insulin sensitivity and collagen deposition in visceral adipose tissue. Genome-wide analysis of gene expression in adipose tissue macrophages highlights TGFB1 gene itself as a direct target of IRF5 inhibition. This study uncovers a novel function for IRF5 in controlling the relative mass of different adipose tissue depots and thus insulin sensitivity during obesity, while suggesting that inhibition of IRF5 would promote a healthy metabolic state during this condition.

My contribution to this complex study is related to the IRF5 full body KO mice in comparison to WT mice. I was in charge of characterizing the mRNA profile of inguinal, epidydimal, retroperitoneal and mesenteric adipose tissue, liver, intestine (duodenum) and colon. I performed measurements of plasma parameters by ELISAs (glucose, adiponectin, leptin) and Millipore multiplex assays for a panel of cytokines/chemokines. I participated to the immunohistochemistry analysis of WAT infiltrating macrophages.

C. Publication 4

Hedi Soussi, Sophie Reggio, Rohia Allili, Cecilia Prado, Maria Pini, Christine Rouault, Karine Clément, Isabelle Dugail. DAPK2 down-regulation reversibly associates with deficient adipocyte autophagic clearance in human morbid obesity. Diabetes (Under second review).

Abstract:

Adipose tissue dysfunction in obesity has been linked to low-grade inflammation causing insulin resistance. Transcriptomics identified DAPK2 among the most strongly down-regulated adipose tissue gene in human obesity, but the role of this kinase is unknown. We show that mature adipocytes and not the stroma-vascular cells in adipose tissue mainly expressed DAPK2, and that DAPK2 mRNA in obese patients gradually recovered after bariatric surgery-induced weight loss. DAPK2 mRNA is also downregulated in high fat diet-induced obese mice. Adenoviral-mediated DAPK2 overexpression in 3T3-L1 adipocytes did not affected lipid droplet size or cell viability, but increased autophagic clearance in nutrient-rich conditions, dependent on protein kinase activity. Conversely, DAPK2 inhibition in human preadipocytes by siRNA decreased LC3-II accumulation rates with lysosome inhibitors. This led us to assess autophagic clearance in adipocytes freshly isolated from subcutaneous adipose tissue of obese patients. Severe reduction in autophagic flux was observed in obese adipocytes compared to controls, inversely correlated to fat cell lipids. Following bariatric surgery, adipocyte autophagic clearance partially recovered, proportional to fat cell size reduction extent. This study links adipocyte expression of an autophagy-regulating kinase, lysosome-mediated clearance and fat cell lipid accumulation, demonstrates obesity-related attenuated autophagy in adipocytes and identifies DAPK2 dependence in this regulation.

My contribution to the study is relative to the gene expression of DAPK family members in the adipose tissue of chow-and HFD-fed mice. Body weight and blood parameters were evaluated to describe the metabolic profile of those mice. Le tissu adipeux blanc (TA) joue un rôle central dans la physiopathologie de l'obésité et la résistance à l'insuline. Durant ma thèse, j'ai étudié la réponse cellulaire et moléculaire du TA au stress métabolique induit chez la souris par 1) l'administration d'un isomère de l'acide linoléique (CLA) et 2) un régime riche en graisses (HFD) associé à l'administration de probiotiques. Dans l'étude 1, nous avons utilisé l'isomère trans10,cis12 de l'acide linoléique (t10,c12-CLA) pour promouvoir un syndrome lipoatrophique chez la souris C57BL/6J. La dynamique de cette réponse a été explorée dans le TA, l'intestin et au niveau systémique, sur deux périodes consécutives de 7 jours d'administration de t10,c12-CLA puis d'interruption du traitement. Nos résultats montrent que t10,c12-CLA induit une dérégulation des gènes métaboliques et un dépôt de matrice extracellulaire (ECM) dans le TA. Le profil immunologique est perturbé, avec l'accumulation de macrophages M2 anti-inflammatoires. Ces altérations métaboliques, immunitaires et structurelles sont inversées, mais pas toujours normalisées, après 7 jours de récupération. La réversibilité des modifications observées dans le TA indique que cette réponse est plus adaptative que pathologique. Cependant, les altérations induites par t10, c12-CLA dans le TA sont temporellement liées à l'hyperinsulinémie, ce qui renforce l'idée que l'intégrité du TA est cruciale pour l'homéostasie glucidique. Bien qu'aucune réponse immunitaire n'ait été observée dans l'intestin, l'expression des gènes codant pour des protéines de jonctions serrées (claudine et occludine) est diminuée. Une partie des effets de t10,c12-CLA pourrait être relayée par l'augmentation de la perméabilité intestinale. L'étude 2 a été conçue sur la base de l'hypothèse que la modification de la microflore intestinale via l'administration quotidienne de probiotiques pourrait alléger altérations métaboliques associées à un régime obésogène chez la souris. L'effet de 2 souches de probiotique a été évalué sur le poids corporel, l'adiposité, la glycémie, la sensibilité à l'insuline,la tolérance au glucose et sur des variables métaboliques. Nos résultats montrent qu'une des souches a généré un phénotype défavorable, avec gain de poids et augmentation de la masse grasse, en association avec une élévation de la leptine circulante et des triglycérides du foie et une tendance à la résistance à l'insuline. Aucune des deux souches n'a pu atténuer l'expansion du TA ni la détérioration glycémique induite par le régime gras. Ces études montrent que, en réponse à un stress nutritionnel de courte durée, le TA regagne rapidement son homéostasie. Lorsque la contrainte est plus chronique, comme dans l'obésité, la plupart des interventions, y compris l'administration de probiotiques, ne parviennent pas à inverser l'état dysfonctionnel du TA. Le grand défi dans les années à venir reste de trouver des stratégies thérapeutiques à long terme dans les maladies métaboliques.

Mots clés: Tissu adipeux, Intestin, Macrophages, Fibrose, Métabolisme, Isomères conjugués de l'acide linoléique Title: Impact of inflammation on the metabolic changes associated with obesity : dialogues between adipose tissue and the intestine.

White adipose tissue (WAT) plays a central role in the physiopathology of obesity and insulin resistance. During my PhD, I studied the cellular and molecular response of WAT to metabolic stress induced in mice by 1) administration of an isomer of linoleic acid (CLA) and 2) a high fat diet (HFD) associated with specific probiotics. In Study I, we used trans10,cis12 isomer of linoleic acid (t10,c12-CLA) as a tool to promote a lipoatrophic syndrome in C57BL/6J lean female mice. The short-term dynamics of this response was explored in WAT, gut and at systemic level, over two consecutive 7-day periods of t10,c12-CLA administration and withdrawal. Our results show that t10,C12-CLA-induced alterations in WAT included metabolic gene deregulation and extracellular matrix deposition. The immunological profile of WAT was markedly disturbed, with antiinflammatory M2-polarized macrophage accumulation. Metabolic, immune and structural alteration were fully revered, but not always normalized after 7 days without treatment. The reversibility of WAT alterations indicates a more adaptive than pathological response to t10,C12-CLA. However, t10,c12-CLA-induced WAT deregulation is temporally linked to hyperinsulinemia supporting the primacy of WAT integrity for glucose homeostasis. No immune alteration was observed in response to t10,C12-CLA in the gut, where genes encoding tight junction proteins (claudin and occludin) were down regulated. Thus, part of the effects of t10,c12-CLA could be mediated by increased intestinal permeability. Study II was designed based on the hypothesis that altering gut microflora through daily administration of probiotic in C57BL/6J obese mice could alleviate metabolic alterations. The respective effects of two specific strains of probiotic were evaluated over time on body weight, adiposity, blood glucose, insulin sensitivity and glucose tolerance and after sacrifice on circulating metabolic variables. Our results show that one strain over two promoted an unfavorable phenotype, with increased weight gain and fat mass, in association with elevated circulating leptin and liver triglycerides. Moreover, a trend toward systemic insulin resistance was found. None of the two strains tested was able to attenuate WAT expansion and glycemic deterioration induced by HFD. These studies show that in response to an acute nutritional stressor, WAT and glucose homeostasis could be rapidly regained. When the stress is more chronic, as in obesity, most of the interventions, including probiotic supplementation, fail to reverse WAT dysfunctional state, underlining the big challenge in the years ahead of finding long-term therapeutic strategies in metabolic diseases.

Key words : Adipose tissue, Intestin, Macophages, Fibrosis, Metabolism, Conjugated linoleic acids

  lowers blood glucose levels in mice. L'inhibition de la cathepsine S diminue les niveaux de glucose circulant chez la souris. Diabetologia 2014 57:1674-83

  in macrophages promotes beneficial adipose tissue expansion and insulin sensitivity during obesity in mice.La délétion d'IrF5 dans les macrophages favorise l'expansion bénéfique du tissu adipeux et la sensibilité à l'insuline chez la souris.Nature Medecine (NMED-A67879D).

  Figure 1: Features of the gut microbiota that promote obesity and insulin resistance (Tremaroli and Backhed 2012). ............................................................................................................................................................................

Figure 2 :

 2 Figure 2: Generation of beige adipocytes (Wang, Tao et al. 2013). ...........................................................................

Figure 3 :

 3 Figure 3: Homeostasis of lipid metabolism in adipocytes (Sethi and Vidal-Puig 2007). ..............................................

Figure 4 .

 4 Figure 4. Equilibrium of body fat is important for human health (Krahmer, Farese et al. 2013). ...............................

Figure 5 .

 5 Figure 5. Evolution of the gene expression pattern in human subcutaneus adipose tissue during overfeeding (Alligier, Meugnier et al. 2012). ..........................................................................................................................

Figure 6 .

 6 Figure 6. Adipocyte storage capacity in obesity and lipodystrophy: lipotoxicity (Tchernof and Despres 2013). ........

Figure 7 .

 7 Figure 7. Alterations in lipid droplet proteins in adipocytes result in increased lipolysis and promote insulin resistance in lipoatrophic and obese humans (Greenberg, Coleman et al. 2011). .............................................

Figure 8 .

 8 Figure 8. Potential mechanisms for obesity-induced inflammation (Shoelson, Herrero et al. 2007). ........................

Figure 9 .

 9 Figure 9. Diagram showing glycolytic and gluconeogenic pathways. ..........................................................................

Figure 10 .

 10 Figure 10. Contribution of metabolic alterations to hepatic steatosis (Postic and Girard 2008). ...............................

Figure 11 .

 11 Figure 11. Immune-cell populations in adipose tissue (Schipper, Prakken et al. 2012). .............................................

Figure 12 .

 12 Figure 12. Initial signals play critical roles in autophagy and immunity (Tang, Kang et al. 2012). ..............................

Figure 13 .

 13 Figure 13. Inducers of macrophage polarization: M1 and M2 phenotypes[START_REF] Mantovani | The chemokine system in diverse forms of macrophage activation and polarization[END_REF])..................

Figure 14 .

 14 Figure 14. Leukocyte subsets in human adipose tissue (Feng, Ji et al. 2013). .............................................................

Figure 15 .

 15 Figure 15. Galectin-3 regulates tissue fibrosis in multiple organs. ..............................................................................

Figure 16 .

 16 Figure 16. Adipocytes are flexible cells (Rutkowski, Stern et al. 2015). ......................................................................

Figure 17 .

 17 Figure 17. Adipocyte stress in the obese and insulin-resistant state (Gregor and Hotamisligil 2007). .......................

Figure 18 .

 18 Figure 18. The pathogenesis of fibrosis (Wynn 2004). ................................................................................................

Figure 19 .

 19 Figure 19. Acute injury versus chronic remodeling process (Mann, Perdiguero et al. 2011). .....................................

Figure 20 .

 20 Figure 20. Signaling Pathways Connecting Macrophage Polarization to Metabolic Outcomes (Biswas and Mantovani 2012). ..................................................................................................................................................................

Figure 21 .

 21 Figure 21. Physiological functions of free fatty acid receptors (FFARs) (Ichimura, Hasegawa et al. 2014). ................

Figure 22 .

 22 Figure 22. The chemical structure of LA, c9,t11-CLA and t10.c12-CLA. .......................................................................

Figure 23 .

 23 Figure 23. Isomer-specific effect of CLA on adipose tissue, liver metabolic and isulinemia (Poirier, Niot et al. 2005). ............................................................................................................................................................................

Figure 24 .

 24 Figure 24. The composition of the microbiota across species (Kostic, Howitt et al. 2013). ........................................

Figure 25 .

 25 Figure 25. Human microbiota transfer into mice induces transfer of phenotypic characteristics (Walker and Parkhill 2013). ..................................................................................................................................................................

Figure 26 .

 26 Figure 26. Gut dysbiosis: immunological disequilibrium (Round and Mazmanian 2009). ..........................................

Figure 27 .

 27 Figure 27. Study I: Experimental design. ...................................................................................................................

Figure 28 .

 28 Figure 28. Adipose tissue expression of genes encoding TLRs after 7 CLA gavages. .................................................

Figure 29 .

 29 Figure 29. Tissue expression of genes encoding GPRs after 7 CLA gavages. .............................................................

Figure 30 .

 30 Figure 30. Mechanism by which activated GPR120 inhibits inflammation in the macrophage (Talukdar, Olefsky et al. 2011). ................................................................................................................................................................

Figure 31 .

 31 Figure 31. Scheme reporting localization of the lymphatic system in a mouse. .......................................................

Figure 32 .

 32 Figure 32. Tissue expression of genes encoding Vegf-a and Pecam-1/CD31. ...........................................................

Figure 33 .

 33 Figure 33. Tissue expression of stress-related (ATFs) and mitochondrial-related (Pgc1α, Cox1) genes. ..................

Figure 34 .

 34 Figure 34. Tissue expression of IRF family members. ................................................................................................

Figure 35 :

 35 Figure 35:Tissue expression of lymphocyte (CD3) and macrophage (CD68) markers. ..............................................

Figure 36 :

 36 Figure 36: Intestinal expression of genes encoding TLRs after 7 CLA gavages. .........................................................

Figure 37 :

 37 Figure 37: Tissue expression of TNF and TJ protein markers (claudin2, occludin, ZO-1) in jejunum of mice treated with CLA for 7 days. ..........................................................................................................................................

Figure 38 .

 38 Figure 38. Microbial species organized in three distinct enterotypes designed M1, M2 and M3. ...........................

Figure 39 .

 39 Figure 39. Number of mice with a specific enterotype in three expreiments. ..........................................................

Figure 40 .

 40 Figure 40. Proportions of the three enterotypes in mice gavaged with either CLA or LA. ........................................

Figure 41 .

 41 Figure 41. Gene expression analysis in vitro polarized BMDM stimulated with LA or t10, c12-CLA. ........................

Figure 42 .

 42 Figure 42. Body weight gain during probiotic treatment in diet-induced obese mice. .............................................

Figure 43 .

 43 Figure 43. Body composition analysis (%) was measured with EchoMRI. .................................................................

Figure 44 .

 44 Figure 44. White adipose tissue depot weight ..........................................................................................................

Figure 45 .

 45 Figure 45. Insulin tolerance test. ...............................................................................................................................

Figure 46 .

 46 Figure 46. Oral glucose tolerance test. ......................................................................................................................

Figure 47 .

 47 Figure 47. Plasma variables. ......................................................................................................................................

Figure 48 .

 48 Figure 48. Triglycerides concentration in plasma and liver lysate. ............................................................................

Figure 49 .

 49 Figure 49. Obesity-and lipodystrophy-induced insulin resistance. ...........................................................................

Figure 50 .

 50 Figure 50. Pleiotropic effect of the t10,c12-CLA isomer in mice. ..............................................................................

Figure 51 .

 51 Figure 51.Potential biological processes implicated in the poor recovery of subcutaneous WAT after an external injury. ................................................................................................................................................................

  ). ............................................................................................................................................................ Table 3. Reference value for Diagnostic Lipid Profile in adults (modified from ATPIII). .............................................. Table 4. Tissue-specific effects of adipokines on lipid metabolism and flux (Sethi and Vidal-Puig 2007). .................. Tableau 5:List of PCR primers (Complementaty results) ...........................................................................................

Figure 1 :

 1 Figure 1: Features of the gut microbiota that promote obesity and insulin resistance (Tremaroli and Backhed 2012).

Figure 2 :

 2 Figure 2: Generation of beige adipocytes (Wang, Tao et al. 2013).

Figure 3 :

 3 Figure 3: Homeostasis of lipid metabolism in adipocytes (Sethi and Vidal-Puig 2007).

Figure 4 .

 4 Figure 4. Equilibrium of body fat is important for human health (Krahmer, Farese et al. 2013).

Figure 5 .

 5 Figure 5. Evolution of the gene expression pattern in human subcutaneus adipose tissue during overfeeding (Alligier, Meugnier et al. 2012). Bars represent the number of genes realted to a given function or biological pathway after 14 days (D14, dark bars) and 56 days (D56, white bars) of overfeeding. Genes commonly regulated at D14 and D56 are indicated in gray.

Figure 6 .

 6 Figure 6. Adipocyte storage capacity in obesity and lipodystrophy: lipotoxicity (Tchernof and Despres 2013).

Figure 7 .

 7 Figure 7. Alterations in lipid droplet proteins in adipocytes result in increased lipolysis and promote insulin resistance in lipoatrophic and obese humans (Greenberg, Coleman et al. 2011).

Figure 8 .

 8 Figure 8. Potential mechanisms for obesity-induced inflammation (Shoelson, Herrero et al. 2007).

Figure 10 .

 10 Figure 10. Contribution of metabolic alterations to hepatic steatosis (Postic and Girard 2008).

Figure 11 .

 11 Figure 11. Immune-cell populations in adipose tissue (Schipper, Prakken et al. 2012).

Figure 13 .

 13 Figure 13. Inducers of macrophage polarization: M1 and M2 phenotypes (Mantovani, Sica et al. 2004).The M1-M2 dichotomy has been used by Lumeng and colleagues to describe what happens in WAT during obesity. The proposed "phenotype switch" model represents the effects of obesity on adipose tissue macrophage (ATMs) recruitment, with increase of M1-like macrophages that shift the noninflammatory millieu maintained by M2-like resident macrophages toward a proinflammatory state(Fujisaka, Usui et al. 2009)[START_REF] Lumeng | Increased inflammatory properties of adipose tissue macrophages recruited during diet-induced obesity[END_REF])[START_REF] Weisberg | CCR2 modulates inflammatory and metabolic effects of highfat feeding[END_REF])[START_REF] Kamei | Overexpression of monocyte chemoattractant protein-1 in adipose tissues causes macrophage recruitment and insulin resistance[END_REF]). The concept that obesity polarizes ATMs toward a classically activated state has been recently questioned[START_REF] Xu | Obesity activates a program of lysosomal-dependent lipid metabolism in adipose tissue macrophages independently of classic activation[END_REF], arguing in favor of the magnitude of macrophage accumulation in obesity. In light of these observations, for the purpose of this Thesis it is important focusing on the process of macrophage infiltration and eventually polarization in WAT in relation to fat mass changes. In states of dysfunctional lipid metabolism, such as obesity and lipodystrophy, the number and activity of macrophages in WAT are increased in rodents[START_REF] Weisberg | Obesity is associated with macrophage accumulation in adipose tissue[END_REF] [START_REF] Xu | Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance[END_REF])[START_REF] Briand | Distinct roles of endothelial and adipocyte caveolin-1 in macrophage infiltration and adipose tissue metabolic activity[END_REF] (Poirier, Shapiro et al. 2006) and humans(Cancello, Tordjman et al. 2006[START_REF] Tordjman | Human adipose tissue macrophages: m1 and m2 cell surface markers in subcutaneous and omental depots and after weight loss[END_REF]. Local environment clearly influence ATMs polarizing capacity.

Figure 14 .

 14 Figure 14. Leukocyte subsets in human adipose tissue (Feng, Ji et al. 2013).

Figure 15 .

 15 Figure 15. Galectin-3 regulates tissue fibrosis in multiple organs.

Figure 16 .

 16 Figure 16. Adipocytes are flexible cells (Rutkowski, Stern et al. 2015).The concept of lipotoxicity[START_REF] Unger | Gluttony, sloth and the metabolic syndrome: a roadmap to lipotoxicity[END_REF] in face of the nutritional pressures of obesity has been presented earlier in the Thesis. A nutritionally challenged microenvironment, besides mitochondrial dysfunction, inflammation and cell death, can submit adipocytes also to structural perturbations and modifications. For example it has been shown how increased concentrations of FFA the integrity of biological membranes, impairing their function and inducing endoplasmic reticulum (ER) stress. Endoplasmatic reticulum, a continuous membrane extending in the cytoplasm of most types of eukaryotic cells, has been proposed to have a role in dysregulation of key metabolic tissues, including liver and adipose tissue. Its functions can vary depending on ER and cell type[START_REF] Schroder | The mammalian unfolded protein response[END_REF]. In liver, in mice, it was suggested that ER can sense nutritional excess and translate that excess into metabolic and inflammatory responses, contributing to insulin resistance[START_REF] Ozcan | Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes[END_REF])[START_REF] Boden | Infusion of glucose and lipids at physiological rates causes acute endoplasmic reticulum stress in rat liver[END_REF] [START_REF] Kim | Fibroblast growth factor 21 participates in adaptation to endoplasmic reticulum stress and attenuates obesity-induced hepatic metabolic stress[END_REF]. Other studies confirmed this link between ER stress and obesity, IR, and inflammation in animal and human tissues[START_REF] Nakatani | Involvement of endoplasmic reticulum stress in insulin resistance and diabetes[END_REF] [START_REF] Boden | Increase in endoplasmic reticulum stress-related proteins and genes in adipose tissue of obese, insulin-resistant individuals[END_REF]. However the physiopathological role of ER in cellular adaptation to external signals, such as systemic insulin[START_REF] Boden | Insulin regulates the unfolded protein response in human adipose tissue[END_REF] or lipids[START_REF] Nivala | Fatty acid-mediated endoplasmic reticulum stress in vivo: differential response to the infusion of Soybean and Lard Oil in rats[END_REF]) needs further investigation. In particular research on molecules participating to the signaling pathways termed the unfolded protein response, the response to ER stress, is currently revealing the complexity of the

Figure 17 .

 17 Figure 17. Adipocyte stress in the obese and insulin-resistant state (Gregor and Hotamisligil 2007).

Figure 18 .

 18 Figure 18. The pathogenesis of fibrosis (Wynn 2004).

Figure 19 .

 19 Figure 19. Acute injury versus chronic remodeling process (Mann, Perdiguero et al. 2011).

Figure 20 .

 20 Figure 20. Signaling Pathways Connecting Macrophage Polarization to Metabolic Outcomes (Biswas and Mantovani 2012).

Figure 21 .

 21 Figure 21. Physiological functions of free fatty acid receptors (FFARs) (Ichimura, Hasegawa et al. 2014).

Figure 22 .

 22 Figure 22. The chemical structure of LA, c9,t11-CLA and t10.c12-CLA.

Figure 23 .

 23 Figure 23. Isomer-specific effect of CLA on adipose tissue, liver metabolic and isulinemia (Poirier, Niot et al. 2005).

Figure 24 .

 24 Figure 24. The composition of the microbiota across species (Kostic, Howitt et al. 2013).

Figure 25 .

 25 Figure 25. Human microbiota transfer into mice induces transfer of phenotypic characteristics (Walker and Parkhill 2013).

Figure 26 .

 26 Figure 26. Gut dysbiosis: immunological disequilibrium (Round and Mazmanian 2009).

Figure 27 .

 27 Figure 27. Study I: Experimental design. G = LA or CLA daily gavage -R = no treatment

Figure legend FIG 1 .FIG 2 .FIG 3 .

 legend123 Figure legend

  Fig 1

1 .

 1 Fig 4

Figure 28 .

 28 Figure 28. Adipose tissue expression of genes encoding TLRs after 7 CLA gavages. Data are shown as fold change over LA values set to 1. pWAT: peri-uteral adipose tissue; sWAT = inguinal adipose tissue. All data are means (n=4-5/group) and t-bars indicate SEM. *p < 0.05 is significant compared to LA control group.

Figure 29 .

 29 Figure 29. Tissue expression of genes encoding GPRs after 7 CLA gavages. Data are shown as fold change over LA values set to 1. pWAT: peri-uteral adipose tissue; sWAT = inguinal adipose tissue. All data are means (n=6-10 / group) and t-bars indicate SEM. *p < 0.05 is significant compared to LA control group.

Figure 30 .

 30 Figure 30. Mechanism by which activated GPR120 inhibits inflammation in the macrophage (Talukdar, Olefsky et al. 2011).

Figure 31 .

 31 Figure 31. Scheme reporting localization of the lymphatic system in a mouse.

Figure 32 .

 32 Figure 32. Tissue expression of genes encoding Vegf-a and Pecam-1/CD31. Data are shown after 7 gavages with CLA (black bars) and after 7 gavages followed by 7 days of CLA dismissal (grey bars) as fold change over LA set to 1. sWAT = inguinal adipose tissue-pWAT = peri-uteral-rWAT = retroperitoneal adipose tissue. All data are means (n=7-10 / group) and t-bars indicate SEM. *p < 0.05 is significant compared to LA control group.

Figure 33 .

 33 Figure 33. Tissue expression of stress-related (ATFs) and mitochondrial-related (Pgc1α, Cox1) genes. Data are shown after 7 gavages with CLA (black bars) and after 7 gavages followed by 7 days of CLA dismissal (grey bars) as fold change over LA set to 1. sWAT = inguinal adipose tissue-pWAT = peri-uteral-rWAT = retroperitoneal adipose tissue. All data are means (n=7-10/group) and t-bars indicate SEM. *p < 0.05 is significant compared to LA control group.

Figure 34 .

 34 Figure 34. Tissue expression of IRF family members. Data are shown after 7 gavages with CLA (black bars) and after 7 gavages followed by 7 days of CLA dismissal (grey bars) as fold change over LA set to 1. sWAT = inguinal adipose tissue-pWAT = peri-uteral-rWAT = retroperitoneal adipose tissue. All data are means (n=7-10/group) and t-bars indicate SEM. *p < 0.05 is significant compared to LA control group.

Figure 35 :

 35 Figure 35:Tissue expression of lymphocyte (CD3) and macrophage (CD68) markers. Data are shown after 7 gavages with CLA as fold change over LA set to 1. All data are means (n=5/group) and tbars indicate SEM.

Figure 36 :

 36 Figure 36: Intestinal expression of genes encoding TLRs after 7 CLA gavages. Data are shown after 7 gavages with CLA as fold change over LA set to 1. All data are means (n=5/group) and tbars indicate SEM. *p < 0.05 is significant compared to LA control group.

Figure 37 :

 37 Figure 37: Tissue expression of TNF and TJ protein markers (claudin2, occludin, ZO-1) in jejunum of mice treated with CLA for 7 days. Data are shown after 7 gavages with CLA as fold change over LA set to 1. All data are means (n=5/group) and tbars indicate SEM. *p < 0.05 is significant compared to LA control group.

Figure 38 .

 38 Figure 38. Microbial species organized in three distinct enterotypes designed M1, M2 and M3.

Figure 40 .

 40 Figure 40. Proportions of the three enterotypes in mice gavaged with either CLA or LA. 0 gavage: n=16; 3 gavages: n=11; 7 gavages: n=16. Enterotype proportions are significantly affected in the CLA group (Chi2 analysis).

Figure 41 .

 41 Figure 41. Gene expression analysis in vitro polarized BMDM stimulated with LA or t10, c12-CLA.

Figure 42 .

 42 Figure 42. Body weight gain during probiotic treatment in diet-induced obese mice. Obese mice were fed a high-fat diet (HFD) with 60 energy % fat. All data are means (n=10/group) and t-bars indicate SEM. *p < 0.05 when X (red *) and Y (green *) treatment groups are significant compared to control group.

Figure 43 .

 43 Figure 43. Body composition analysis (%) was measured with EchoMRI.

Figure 44 .

 44 Figure 44. White adipose tissue depot weight Weight of different WAT depots (Ing = inguinal, Epi = epidydimal and retroperitoneal = Retro) from HFD-fed mice without (CTL) or with probiotic treatment (X or Y). All data are means (n=10/group) and t-bars indicate SEM. *p < 0.05 is significant compared to control group.

Figure 45 .

 45 Figure 45. Insulin tolerance test. Curve of insulin tolerance test and areas under the curve (AUC) of ITT are presented. All data are means (n=10/group) and t-bars indicate SEM.

Figure 46 .

 46 Figure 46. Oral glucose tolerance test.

Figure 47 .

 47 Figure 47. Plasma variables.

Figure 48 .

 48 Figure 48. Triglycerides concentration in plasma and liver lysate.

Figure 50 .

 50 Figure 50. Pleiotropic effect of the t10,c12-CLA isomer in mice.

Figure 51 Figure 51 .

 5151 Figure 51 recapitulates the different cellular and molecular mechanisms potentillay implicated in WAT depot distinct recovery from CLA-induced injury.

B

  in macrophages promotes beneficial adipose tissue expansion and insulin sensitivity during obesity in mice. Nature Medecine (Accepted NMED-A67879D).

  sur les altérations métaboliques associées à l'obésité: dialogues entre le tissu adipeux et l'intestin

Table 1 :

 1 Criteria for Clinical Diagnosis of the Metabolic Syndrome (Grundy, Cleeman et al. 2005) ...........................

Table 2 .

 2 Definition and classification of obesity based on Body Mass Index (BMI) (modified from

Table 6 :

 6 List of PCR primers (Publication 1) ...............................................................................................................

	DXA	Dual Energy Xray Absorptiometry
	ECM	Extra cellular matrix
	FAS	Fatty acid synthase
	FFA	Free fatty acids
	FoxO	Forkhead box protein O
	GK	Glucokinase
	G6Pase	Glucose 6 phosphatase catalytic subunit
	GPR40 (FFR1)	G-protein-coupled receptor 40

Table 1 : Criteria for Clinical Diagnosis of the Metabolic Syndrome (Grundy, Cleeman et al. 2005)

 1

  Table 3 are the desirable lipid levels in adults and the values defining dyslipidemia for the US National Cholesterol Education Program III guidelines (2001).

Table 3 . Reference value for Diagnostic Lipid Profile in adults (modified from ATPIII).

 3

	Lipid	Desiderable Level	Dyslipidemia Level
		(mg/dL of blood)	(mg/dL of blood)
	Total cholesterol	Less than 200 mg/dL	> or = 200 mg/dL
	Low-density lipoprotein (LDL)	Less than 100 mg/dL	> or = 130 mg/dL
	cholesterol
	High-density lipoprotein (HDL)	More than 40 mg/dL	< 40 mg/dL
	cholesterol
	Triglycerides	Less than 150 mg/dL	> or = 150 mg/dL

Table 4 . Tissue-specific effects of adipokines on lipid metabolism and flux (Sethi and Vidal-Puig 2007).

 4

	Adipokine	Food Intake (Energy Intake)	Fat Utilization, β-Oxidation, Thermogenesis (Energy Expenditure)	Fat Storage, Adipogenesis, Nonadipose Lipogenesis Capacity) (Excess Energy Storage	Levels in Common Forms of Obesity
			Enhances β-oxidation via AMPK	Antiadipogenic in vitro,
	Leptin	Anorexigenic	activation in muscle and liver; reduces intracellular lipids;	promotes adipose lipolysis in vivo via the sympathetic	High (but evidence of leptin resistance)
			promotes thermogenesis in BAT	nervous system
		No effect on food intake, but
	Adiponectin	intracerebroventricular injection leads to increased energy expenditure; present in human	Enhances via AMPK activation in muscle and liver; reduces intracellular lipids	Promotes lipogenesis in adipocytes in vitro	Low
		cerebrospinal fluid
			Inhibits muscle AMPK;	Antiadipogenic and
	TNF-α	Anorexigenic	increased FA incorporation into diacylglycerol, and increase in intracellular ceramide levels; reduces BAT function and	stimulates lipolysis via TNFR1-mediated transcriptional signals; increases hepatic de novo synthesis of fatty acid	High (but with autocrine/paracrine activity)
			promotes BAT atrophy	and triglycerides
				Stimulates adipose tissue
	IL-6	Anorexigenic	Stimulates fat oxidation via AMPK activation	lipolysis; increases hepatic de novo synthesis of fatty acid	High
				and cholesterol
	Resistin	Potentially anorexigenic; present in human cerebrospinal fluid	Decreases fatty acid uptake and metabolism in muscle in vitro	Antiadipogenic and adipocytes stimulates lipolysis in	High
	RBP4	Unknown	Unknown	Promotes liver steatosis	High
	PBEF/visfatin	Unknown	Unknown	Promotes lipogenesis in adipocytes	Controversial: may be higher in visceral fat
	Omentin (humans, not mice)	Unknown	Unknown	Unknown	Increased in human visceral fat

  Of clear relevance for adipose tissue functionality is the diverse collection of proteins and sugars that surrounds cells in the tissue, representing the extra-cellular matrix (ECM). Only recently research started focusing on a thorough characterization of the metabolic impact of ECM on the altered adipocyte during obesity or during other metabolic conditions.FFA are efficiently stored without harmful side effects in the form of glycerol. The resulting TGs are relatively inert and can be efficiently deposited in lipid droplets in the cytoplasm of essentially all cell types. The most efficient cellular depot of TGs in mammals is represented by the adipocyte. Adipocyte structure is adapted to store big quantities of TGs that can be hydrolyzed (mobilized) as FFA and glycerol when needed in other tissues. All oxidative tissues, but WAT itself also, can use FFA to generate energy through a process called β-oxidation. In WAT, β-oxidative capacity increases during adipocyte differentiation, as indicated by increased expression of PGC1α, a regulator of mitochondrial biogenesis[START_REF] Wilson-Fritch | Mitochondrial remodeling in adipose tissue associated with obesity and treatment with rosiglitazone[END_REF]). Whereas, in obesity and diabetes, adipocytes lose oxidative capacity, together with insulin sensitivity[START_REF] Choo | Mitochondria are impaired in the adipocytes of type 2 diabetic mice[END_REF] [START_REF] Keller | Physiological insights gained from gene expression analysis in obesity and diabetes[END_REF]. Increasing the oxidative potential of the adipocyte has demonstrated efficacy in treating type 2 diabetes, as in case of thiazolidinedione drugs[START_REF] Wilson-Fritch | Mitochondrial remodeling in adipose tissue associated with obesity and treatment with rosiglitazone[END_REF]). In response to metabolic challenging environments, adipocytes can adapt[START_REF] Rutkowski | The cell biology of fat expansion[END_REF] (Figure

1. Adipocytes: functions and dysfunctions

  . Many transcriptional factors have been involved in CLA-induced adipose tissue modifications, including SREBP-1c and PPARγ, as shown in particular by the team of Dr McIntosh (Nord Carolina University, USA)[START_REF] Kennedy | Conjugated linoleic acid-mediated inflammation and insulin resistance in human adipocytes are attenuated by resveratrol[END_REF] Kennedy, Martinez et al. 2010). In 2006, Poirier et al. demonstrated that the drastic fat reduction was associated with WAT inflammation and reduced circulating adipokines in lean mice

Table 1 :

 1 Morphometric and blood metabolic variables in mice receiving linoleic acid (L)A or t10,c12-CLA by daily gavage for 3 or 7 days, followed by 7 days of recovery without treatment.

		Linoleic acid (LA)			t10,c12-CLA
	3 gavages	7 gavages	7 gavages + recovery	3 gavages	7 gavages	7 gavages + recovery

Table 2 :

 2 Spearman's correlation of macrophages accumulation with adipose tissue weight and

			F4/80 + cells		CD68 + cells
			(n / g WAT)		(n / 100 adipocytes)
			sWAT	pWAT	sWAT	pWAT
	Adipose tissue weight	r	-0.77	-0.55	-0.52	-0.41
		P	< 0.001	< 0.001	< 0.001	0.006
	Plasma adiponectin	r	-0.51	-0.43	-0.84	-0.74
		P	0.002	0.007	< 0.001	< 0.001
	Plasma insulin	r	0.72	0.63	0.61	0.53
		P	< 0.001	< 0.001	<0.001	0.002
	biomarker of glycemic status

  Curve of oral glucose tolerance test and areas under the curve (AUC) of OGTT are presented. All data are means (n=10/group) and t-bars indicate SEM.

  Triglyceride levels were measured in plasma and liver lysate from HFD-fed mice without (CTL) or with probiotic treatment (X or Y). All data are means (n=10/group) and t-bars indicate SD. *p < 0.05 is significant compared to control group.

Pleiotropic effects of t10,c12-CLA nutritional administration in mice

	Whole body	Adipose tissue	Intestine
	Lipoatrophic syndrome
	ENERGY BALANCE •Minor reduction of food intake •Increased energy expenditure	DIFFERENTIATION •Reduced rate of differentiation (3T3-L1 cells)	BARRIER FUNCTION •Increased paracellular permeability (CaCo2 cells)
	and energy loss in the excreta	•Beiging of adipocytes •Cell apoptosis	CELL PROLIFERATION
	BODY WEIGHT AND •Reduced body weight COMPOSITION	•Reduced rate of lipogenesis METABOLISM	•Increased cell proliferation (CaCo2 cells)
	•Marked fat mass reduction with no change in lean mass	•Reduced glucose intake (GLUT4) •Increased lipolysis
	GLUCOSE HOMEOSTASIS •Hyperinsulinemia	ADIPOKINES •Marked reduction of adiponectin	Gut microbiota
	•Insulin resistance (ITT)	•Moderate reduction of leptin
	•Increased pancreatic islet size	INFLAMMATION	SHORT CHAIN FATTY ACID •Increased production of
	LIPID HOMEOSTASIS	•Increased pro-inflammatory	acetate, propionate and
	•Liver steatosis	cytokines (TNF, IL1, IL6, IL8)	isobutyrate
		•Activation of NFκB pathway
		• Endoplasmique reticulum stress
		(ATF3, TRIB3)
		• Macrophages, mast cells
		recruitment
		EXTRACELLULAR MATRIX
		•Increased collagen deposition
		(mammary gland fat depot)

Tableau 5:List of PCR primers (Complementaty results)

	Gene symbol	Gene Name	Left	Right
	Atf3	activating transcription factor 3	gctggagtcagttaccgtcaa	cgcctccttttcctctcat
	Atf4	activating transcription factor 4	atgatggcttggccagtg	ccattttctccaacatccaatc
	Atf6	activating transcription factor 6	ggacgaggtggtgtcagag	gacagctcttcgctttggac
	Cldn2	claudin 2	tgaacacggaccactgaaag	ttagcaggaagctgggtcag
	Cox1	prostaglandin-endoperoxide synthase 1 (Ptgs1)	cctctttccaggagctcaca	tcgatgtcaccgtacagctc
	Gpr43	free fatty acid receptor 2 (Ffar2)	tgctctgaagaagccaatca	ttctcctctggtccagtgct
	Gpr81	G protein-coupled receptor 81	ggctgagaaaagcggtatga	tcgttaactctctccgagctaga
	Gpr109A	niacin receptor 1	ctgctcaggcaggatcatct	ccctcttgatcttggcatgt
	Gpr116	G protein-coupled receptor 116	cctcaaccatacttacaaaatggac acagtgaatttcgtctcattgc
	Gpr120	G protein-coupled receptor 120	ttggtgttgagcgtcgtg	ccagcagtgagacgacaaag
	Pecam1	platelet/endothelial cell adhesion molecule 1	cggtgttcagcgagatcc	actcgacaggatggaaatcac
	Pgc1a	PPAR Gamma Coactivator-1	gaaagggccaaacagagaga	gtaaatcacacggcgctctt
	OCLN	occludin	gtccgtgaggccttttga	ggtgcataatgattgggtttg
	Tlr1	toll-like receptor 1	ctgagggtcctgataatgtcct	tccagctctgtgttgaatttga
	Tlr2	toll-like receptor 2	ggggcttcacttctctgctt	agcatcctctgagatttgacg
	Tlr4	toll-like receptor 4	ggactctgatcatggcactg	ctgatccatgcattggtaggt
	Tlr5	toll-like receptor 5	ctggagccgagtgaggtc	cggcaagcattgttctcc
	Tlr9	toll-like receptor 9	gagaatcctccatctcccaac	ccagagtctcagccagcac
	Vegf-a	vascular endothelial growth factor A	aaaaacgaaagcgcaagaaa	tttctccgctctgaacaagg
	ZO-1	zonula occludens 1/tight junction protein 1	atgcagacccagcaaagg	tggttttgtctcatcatttcttca

Table 6 : List of PCR primers (Publication 1)

 6

	Gene symbol	Gene Name	Left	Right
	Acc1	acetyl-CoA carboxylase 1

Pour conclure, contrairement à notre hypothèse de départ, l'administration de probiotiques dans ce contexte expérimental n'a pas atténué l'expansion du tissu adipeux ni la détérioration glycémique induite par le régime gras. Il est clair que la recherche sur les effets des probiotiques sur les paramètres métaboliques et l'homéostasie glucidique va nécessiter des études plus approfondies et sans doute des paradigmes différents d'administration des probiotiques, avec des souches spécifiques isolées, en combinaison de plusieurs souches et/ou en présence de prébiotiques.

ACKNOWLEDGEMENTS

I address my first thanks to Professor Bernard Lacour for giving me the honor to accept to be the chair of my thesis jury, as well as Pr. Jocelyne Magre and Dr. Jean-Philippe Bastard for their availability and critical review of the manuscript. I also thank Prof. Helene Roche for having agreed to participate to the critical examination of this thesis.

ACKNOWLEDGEMENTS

The authors thank Remi Selleslagh and Sonia Mutel for expert technical assistance.

Ws also thank

Isabelle Dugail, Danièle Lacasa and Charles Caër for helpful discussion. This study was funded by the National Agency of Research (Adipofib to KCs' team), French Foundation for Medical Research (Equipe FRM DEQ20120323701 to KC's team) and by a grant from Groupe Lipides Nutrition (GLN) to MGM. This work has benefited from a French government grant managed by National Agency of Research (Institute of Cardiometabolism and Nutrition, ICAN, reference ANR-10-IHU). PhD fellowships were allocated to ST by the EU-Metacardis FP7 program. We thank Association Française d'Etude et de Recherche sur l'Obésité (AFERO) for financial support to MP. The work performed by ND and CD was supported upon granting by the Fonds National de la Recherche Scientifique in Belgium (FNRS Crédit aux Chercheurs 1.5121.12F). CD benefited from a grant from Institut Danone Belgique as a salary.

No potential conflicts of interest relevant to this article were reported.

RÉSUMÉ FRANÇAIS -

 -------------------------------------------------------------------------------------A. Contexte du projet - ----------------------------------------------------------------------------------------------------Les altérations du tissu adipeux dans les maladies métaboliques ----------------------2. Les altérations de l'intestin et du microbiote intestinal dans les malacies métaboliques ---------

List of nonstandard abbreviations

LA: linoleic acid t10,c12-CLA: trans10,cis12 isomer of linoleic acid ECM: extracellular matrix WAT: white adipose tissue sWAT: subcutaneous inguinal white adipose tissue pWAT: peri-uteral white adipose tissue SVF: stroma vascular fraction

Abstract

In mice, nutritional supplementation with the trans10, cis12 isomer of linoleic acid (t10,c12-CLA) promotes lipoatrophy, hyperinsulinemia and macrophage infiltration in white adipose tissue (WAT). We explored the dynamics of these interrelated responses over two consecutive 7day periods of t10,c12-CLA administration and withdrawal. t10,c12-CLA down-regulated lipogenic and lipolytic gene expression and increased collagen deposition, although with no evidence of cross-linking. An abundant CD45 + cell infiltrate, comprising prominently CD206 + CD11c -macrophages, was found in WAT in association with an anti-inflammatory gene signature. Infiltration of NK and dendritic cells contributed to WAT innate immune response to t10,c12-CLA. Less abundant adaptive immune cells colonized WAT, including B, NK T, γδ T and αβ T cells. By contrast, Treg abundance was not affected. Interruption of treatment allowed recovery of WAT mass and normalization of insulinemia, coincident with regain of WAT homeostasis owing to a coordinated reversion of genic, structural and immune deregulations.

These data revealed a striking resilience of WAT after a short-term metabolic injury induced by t10,c12-CLA, which relies on alternatively activated M2 macrophage engagement. Additionally, the temporal links between variations in WAT alterations and insulinemia upon t10,c12-CLA manipulation strengthen the view that WAT dysfunctional status is critically involved in altered glucose homeostasis. Key words: lipoathrophy; hyperinsulinemia; fibrosis; lipophagy. including adipocytes, is not firmly established. Through gain and loss of function mouse studies, it has been proposed that some cells, including NK cells [START_REF] Wensveen | NK cells link obesity-induced adipose stress to inflammation and insulin resistance[END_REF], B cells [START_REF] Winer | B cells promote insulin resistance through modulation of T cells and production of pathogenic IgG antibodies[END_REF][START_REF] Defuria | B cells promote inflammation in obesity and type 2 diabetes through regulation of T-cell function and an inflammatory cytokine profile[END_REF] and γδ T cells [START_REF] Mehta | gammadelta T cells promote inflammation and insulin resistance during high fat diet-induced obesity in mice[END_REF] promote WAT dysfunction and metabolic disturbances, while others, such as NKT cells [START_REF] Lynch | Adipose tissue invariant NKT cells protect against diet-induced obesity and metabolic disorder through regulatory cytokine production[END_REF] and Tregs [START_REF] Winer | Normalization of obesity-associated insulin resistance through immunotherapy[END_REF][START_REF] Ilan | Induction of regulatory T cells decreases adipose inflammation and alleviates insulin resistance in ob/ob mice[END_REF] contribute to maintain WAT homeostasis and improved metabolic status. In our model, the immunological profile of WAT was markedly and specifically modified in response to t10,c12-CLA-induced injury. The prominent response was the primary recruitment of innate immune cells. As a whole, the immune cell infiltrate driven by t10,c12-CLA contained a mix of cells shown to be protective or deleterious in mice models. Although not tested, the contribution of neutrophils, which transiently colonize WAT in the early phases of diet-induced obesity [START_REF] Elgazar-Carmon | Neutrophils transiently infiltrate intra-abdominal fat early in the course of high-fat feeding[END_REF] and/or eosinophils, which sustain WAT macrophage M2 activation [START_REF] Wu | Eosinophils sustain adipose alternatively activated macrophages associated with glucose homeostasis[END_REF] might add further complexity to WAT immune response to t10,c12-CLA. Future exploration will specify the implication of each cell types in WAT dysfunctional state and ensuing glycemic deregulation induced by t10,c12-CLA administration.

Although it is well established and confirmed in the present study that t10,c12-CLA accumulates selectively in WAT, it is currently not known how this CLA isomer impinges upon transcriptional programs in adipocytes. In our experimental setting, genes functionally implicated in both lipogenic and lipolytic metabolic pathways were severely down-regulated after 7 days of t10,c12-CLA administration. The reversibility of these alterations indicates that metabolic functions were restored in a majority of adipocytes when t10,c12-CLA was removed. Our data suggest that induction of lipophagy through overexpression of Lipa [START_REF] Lettieri Barbato | FoxO1 controls lysosomal acid lipase in adipocytes: implication of lipophagy during nutrient restriction and metformin treatment[END_REF], is part of a physiological response to t10,c12-CLA-induced catabolic phenotype in adipose cells. Fatty acids released through Lipa then could serve as alternative energy substrates, owing to t10,c12-CLAinduced overexpression of UCP2 [START_REF] Pecqueur | Uncoupling protein-2 controls proliferation by promoting fatty acid oxidation and limiting glycolysis-derived pyruvate utilization[END_REF][START_REF] Vozza | UCP2 transports C4 metabolites out of mitochondria, regulating glucose and glutamine oxidation[END_REF]. In line with our current observations, a coordinated and marked increase in Lipa and UCP2 gene expression was previously found through WAT transcriptomic analysis [START_REF] Larosa | Trans-10, cis-12 conjugated linoleic acid causes inflammation and delipidation of white adipose tissue in mice: a microarray and histological analysis[END_REF]. Strikingly, this up-regulation was detected after only 2 days of t10,c12-CLA oral supplementation in mice, indicating that induction of lipophagy might represent one of the earliest metabolic switch induced by t10, c12-CLA.

Whether adipogenic precursors contribute to tissue resilience after t10,c112-CLA injury remains to be assessed. We observed that high levels of Ki-67 gene expression persisted in pWAT after t10,c12-CLA withdrawal, when immune cells were not detected any more in WAT.

GENERAL DISCUSSION AND PERSPECTIVES

A. Contribution of adipose tissue to metabolic deregulation

During my PhD project, I performed and contributed to a series of mice studies conceived and executed in the framework of scientific focus of the Nutriomics team headed by Prof Karine Clément. In this research group, there is an active interest in linking adipose tissue dysfunction, metabolic alterations and gut microbiota, with the overall goal of finding potential therapeutic targets for preventing and/or treating obesity-associated disorders. My prominent contribution is the demonstration of an unexpected capacity of adipose tissue to adapt to an acute nutritional stress, with a major contribution of M2 anti-inflammatory macrophage recruitment (Publication 1, discussed below). This study strengthened the primacy of adipose tissue integrity for glucose homeostasis and enlightened the role of adipose tissue innate immune profile to control inflammation. In a second study, contrary to our expectation, we were unable to demonstrate a beneficial role of probiotic administration to alleviate body weight gain and insulin resistance induced by a high fat diet (Study II, discussed below). Beside, several potential targets were recently identified in adipose tissue by Karine Clément's team. This includes the matrix protease Cathepsin S, the pro-inflammatory transcription factor IRF5 and DAPK2, a kinase regulating autophagy. In this context, I contributed to studies in which high fat diet obese mice were used to substantiate the implication of these targets in fat mass regulation and metabolic homeostasis. A general scheme shown below (Figure 49) recapitulates these contributions, in the context of obesity and lipodystrophy-linked insulin resistance.

Abstract:

AIMS/HYPOTHESIS: Cathepsin S (CatS) belongs to a family of proteases that have been implicated in several disease processes. We previously identified CatS as a protein that is markedly overexpressed in adipose tissue of obese individuals and downregulated after weight loss and amelioration of glycaemic status induced by gastric bypass surgery. This prompted us to test whether the protease contributes to the pathogenesis of type 2 diabetes using mouse models with CatS inactivation. METHODS:CatS knockout mice and wild-type mice treated with orally active small-molecule CatS inhibitors were fed chow or high-fat diets and explored for change in glycaemic status. RESULTS:CatS deletion induced a robust reduction in blood glucose, which was preserved in dietinduced obesity and with ageing and was recapitulated with CatS inhibition in obese mice. In vivo testing of glucose tolerance, insulin sensitivity and glycaemic response to gluconeogenic substrates revealed that CatS suppression reduced hepatic glucose production despite there being no improvement in insulin sensitivity. This phenotype relied on downregulation of gluconeogenic gene expression in liver and a lower rate of hepatocellular respiration. Mechanistically, we found that the protein 'regulated in development and DNA damage response 1' (REDD1), a factor potentially implicated in reduction of respiratory chain activity, was overexpressed in the liver of mice with CatS deficiency. CONCLUSIONS/INTERPRETATION: Our results revealed an unexpected metabolic effect of CatS in promoting pro-diabetic alterations in the liver. CatS inhibitors currently proposed for treatment of autoimmune diseases could help to lower hepatic glucose output in obese individuals at risk for type 2 diabetes.

My implication in this study has been important as proved by the co-first authorship. * (JCL et MP, 1st author mentioned in the original article). My contribution is related to the experimental and the reviewing part. I contributed to the preparation of the paper through critical discussion.