Thesis
Reference
Implications du métabolisme mitochondrial sur le diabète et l'obésité avant et après perte de poids
GASTALDI, Giacomo Gianni Giuseppe
Abstract
L'homéostasie énergétique dépend de l'équilibre entre les apports et la dépense énergétique quotidiennes qui se subdivisent en trois composantes majeures : 1. le métabolisme basal, 2.
l'activité physique et 3. la thermogenèse induite. Les caractéristiques de l'homéostasie énergétique découlent de l'application de la première loi de la thermodynamique à des systèmes biologiques. Le métabolisme basal dépend du rendement énergétique cellulaire, équivalent au nombre d'ATP produit à partir des substrats énergétiques disponibles. En effet, l'oxydation de ces derniers permet la génération d'équivalents réducteurs (NADH ; H+), utilisés par la chaîne respiratoire à la formation d'un gradient de protons à travers la membrane interne de la mitochondrie. C'est ce gradient qui fournira l'énergie nécessaire à la synthèse d'ATP. Le couplage, entre substrat énergétique disponible et taux d'ATP produit peut varier selon les conditions ce qui résultera une plus ou moins grande dissipation d'énergie sous forme de chaleur, en accords avec la deuxième loi physique de la thermodynamique (« dans un [...]
GASTALDI, Giacomo Gianni Giuseppe. Implications du métabolisme mitochondrial sur le diabète et l'obésité avant et après perte de poids. Thèse de doctorat : Univ. Genève, 2009, no. Méd. 10577
URN : urn:nbn:ch:unige-20622
DOI : 10.13097/archive-ouverte/unige:2062
Available at:
http://archive-ouverte.unige.ch/unige:2062
Disclaimer: layout of this document may differ from the published version.
UNIVERSITE DE GENEVE FACULTE DE MEDECINE
Section de médecine Fondamentale Département de Physiologie cellulaire et métabolisme
Thèse préparée sous la direction du Professeur Jean-Paul Giacobino
Implications du métabolisme mitochondrial sur le diabète et l’obésité avant et après
perte de poids
Thèse
présentée à la Faculté de Médecine de l'Université de Genève
pour obtenir le grade de Docteur en médecine par
Giacomo Gianni Giuseppe GASTALDI
de Genève (GE)
Thèse n°10577 Genève
2009
UNIVERSITE DE GENEVE FACULTE DE MEDECINE
Section de médecine Fondamentale Département de Physiologie cellulaire et métabolisme
Thèse préparée sous la direction du Professeur Jean-Paul Giacobino
Implications du métabolisme mitochondrial sur le diabète et l’obésité avant et après
perte de poids
Thèse
présentée à la Faculté de Médecine de l'Université de Genève
pour obtenir le grade de Docteur en médecine par
Giacomo Gianni Giuseppe GASTALDI
de Genève (GE)
Thèse n°10577 Genève
2009
Remerciements
Cette thèse est le résultat d’une collaboration mémorable entre le monde de la recherche fondamentale et celui de la recherche clinique. Pour m’avoir donné la possibilité de participer à une telle expérience, je tiens à remercier :
Monsieur le professeur Jean-Paul GIACOBINO qui a dirigé cette thèse dans la continuité de mon passage dans son laboratoire jusqu’à bien après. Il m’a guidé dans ce travail avec une disponibilité unique. Sa passion, sa rigueur scientifique et son sens de la critique constructive m’ont beaucoup apporté tant dans la rédaction de cette thèse que sur un plan plus personnel.
Pour toutes ces qualités et un talent inégalé à organiser, pour 003, des moments d’intense partage scientifique comme sportif, artistique ou citadin, je lui adresse mes plus vifs et sincères remerciements.
Le Docteur Aaron Russell qui m’a enseigné les rudiments du travail de laboratoire et encadré pour l’ensemble des analyses moléculaires de ce projet avec un dynamisme et une bonne humeur contaminante. Sa vision scientifique, ses relectures attentives et les sympathiques moments partagés à jogger ensemble ont grandement contribué à la réalisation de ce travail. Pour tout cela il mérite un remerciement tout particulier.
Madame la doctoresse, PD, Elisabetta Bobbioni-Harsch qui m’a permis de découvrir la recherche clinique du lit du patient à l’examen des données. Les nombreux moments de réflexion et d’analyse passés en sa compagnie m’ont permis d’appréhender la complexité du métabolisme du glucose et bien au delà.
Pour la richesse de ces échanges, derrière son vieux coucou, pendant le sacro- saint café ou autour d’un repas, je tiens à la remercier tout spécialement.
Monsieur le professeur Alain Golay qui m’a guidé dans mes premières expériences cliniques, sans manquer de m’insuffler le virus de l’ouverture scientifique avec toujours une oreille attentive dédiée au vécu du patient. Je ne saurais en outre le remercier assez pour son aide administrative déterminante dans la construction de ce projet. Pour tout cela, je le remercie vivement.
Un grand merci à l’ensemble de l’équipe 003. A tous les moments passés ensemble au travail et en dehors. A l’accueil chaleureux que vous m’avez donné dés mon arrivée et jusqu’à bien après mon départ. Pour tous ces moments, je vous exprime toute mon amitié.
Enfin, mes remerciements les plus sincères au soutien reçu de la part de la Fondation pour l’encouragement de la recherche sur la nutrition en Suisse, la Fondation Barbour et la Fondation Ousseimi sans qui ce travail et ces rencontres n’auraient pas vu le jour.
Toute ma reconnaissance va bien sûr à Fabienne et Lorenzo pour leur patient soutien.
Résumé
L’homéostasie énergétique dépend de l’équilibre entre les apports et la dépense énergétique quotidiennes qui se subdivisent en trois composantes majeures : 1. le métabolisme basal, 2. l’activité physique et 3. la thermogenèse induite. Les caractéristiques de l’homéostasie énergétique découlent de l’application de la première loi de la thermodynamique à des systèmes biologiques.
Le métabolisme basal dépend du rendement énergétique cellulaire, équivalent au nombre d’ATP produit à partir des substrats énergétiques disponibles. En effet, l’oxydation de ces derniers permet la génération d’équivalents réducteurs (NADH ; H+), utilisés par la chaîne respiratoire à la formation d’un gradient de protons à travers la membrane interne de la mitochondrie. C’est ce gradient qui fournira l’énergie nécessaire à la synthèse d’ATP [1]. Le couplage, entre substrat énergétique disponible et taux d’ATP produit peut varier selon les conditions ce qui résultera une plus ou moins grande dissipation d’énergie sous forme de chaleur, en accords avec la deuxième loi physique de la thermodynamique (« dans un système fermé, tout processus physique engendre une dissipation d’énergie sous la forme de chaleur »).
L’augmentation de la production de chaleur systématiquement observée, chez les mammifères, après ingestion d’aliments ou lors d’une exposition au froid a été dénommée la thermogenèse induite [2].
Les modulations de la dissipation d’énergie peuvent influencer la capacité de stocker efficacement de l’énergie. Elles constituent un élément essentiel à la survie d’une espèce [3], tout particulièrement lorsque la quantité de nourriture est amenée à varier de façon considérable. Le tissu adipeux blanc est l’organe dévolu à la création de réserves. Il se remplit de triglycérides en périodes de pléthore alimentaire et les relâche sous forme d’acides gras libres en cas de jeune prolongé ou d’activité physique [4][5]. Dans le premier cas une élévation du métabolisme via une activation de la thermogenèse et ou d’autres systèmes de régulation empêche un stockage excessif. Dans le second cas, en l’occurrence, le jeune prolongé ou l’activité physique, les réserves de triglycérides permettent le fonctionnement de l’organisme.
Conjointement celui-ci diminue son métabolisme de base et intensifie les signaux stimulant spécifiquement les centres de l’appétit. Le but étant de vider les stocks lentement et de retarder au maximum l’utilisation de constituants structurels comme substrats énergétiques. Un tel fonctionnement implique donc un minimum de trois niveaux de contrôle [6]: 1. des signaux libérés par la périphérie et fonctionnant comme indicateur de l’état des stocks. 2. un centre d’intégration, localisé dans les noyaux hypothalamiques, qui reçoit les afférences périphériques et centrales et d’où partent ensuite des efférences influençant les effecteurs périphériques et le comportement. 3. des effecteurs périphériques permettant de moduler la dépense énergétique de l’organisme, dont les plus connus sont les hormones thyroïdiennes (T3, T4), le système nerveux autonome via les récepteurs β3-adrénergiques et certaines protéines mitochondriales.
L’obésité qui touche en Suisse bientôt une personne adulte sur cinq est une situation caractéristique d’un déséquilibre de l’homéostasie énergétique. L’obésité, une fois déclarée [7], il est extrêmement difficile de démêler les facteurs étiologiques des conséquences de l’accumulation de graisses. Toutefois, l’observation de l’augmentation de la prévalence de l’obésité dans le monde [8], montre que le système de contrôle, tel que nous venons de le décrire, n’est plus autant garant d’un
poids similaire entre individus d’une même espèce. Quels sont donc les facteurs permettant d’expliquer ces différences ? Proviennent-ils de l’environnement [9] [10], des pressions sociales [11] [12], des ressources alimentaires [13], du degré d’activité physique [14], des co-morbidités psychiatriques [15] ou apparaissent-ils dans le patrimoine génétique [16]? Vraisemblablement, l’obésité est la résultante d’une association de facteurs dont l’importance individuelle reste à déterminer.
Nous avons, dans ce travail, focalisé notre attention sur les facteurs contrôlant la dépense énergétique en périphérie et plus particulièrement sur les protéines mitochondriales impliquées dans le métabolisme. En effet, c’est dans les mitochondries du tissu adipeux brun qu’à été découverte la protéine découplante-1 (UnCoupling Protein-1,UCP1) permettant la thermogenèse. Chez le rongeur, l’activation d’UCP1 par le froid ou une alimentation riche en graisse, induit une dissipation du gradient de protons nécessaires à la génération d’ATP par la chaîne respiratoire [15]. Il en résulte une augmentation compensatoire du catabolisme des acides gras associée à une production de chaleur. L’homme adulte n’ayant plus de tissu adipeux brun, la découverte de la protéine UCP3 dans le muscle, possédant une homologie d’environ 55% en acides aminés avec UCP1 fit penser que l’on avait affaire à une protéine découplante [17]. L’expression d’UCP3 dans le muscle est fibre dépendante, et par ordre décroissant, majoritairement exprimé dans les fibres IIx, essentiellement glycolytique, suivie des fibres IIa puis des fibres de type I, fonctionnant sur un mode oxydatif [18].
Différents polymorphismes du gène d’UCP3 semblent impliqués dans les régulations du métabolisme basal. En effet, le polymorphisme c55t du gène d’UCP3 est associé à une protection contre le développement de l’obésité [19], alors qu’un polymorphisme du promoteur est quant à lui associé à une élévation de l’expression d’UCP3 négativement corrélée au BMI et positivement au métabolisme basal pendant le sommeil [20].
Enfin, UCP3 est positivement corrélé au taux d’acide gras libres circulant [21] et son expression est augmentée en situation de jeune [22]. Ces différents éléments plaident en faveur d’une action d’UCP3 sur le contrôle pondéral et/ou dans la régulation de la dépense énergétique et du métabolisme des acides gras. D’autres groupes de recherche ont postulé que l’élévation d’UCP3 permet de limiter les dommages liés à la production des radicaux libres dans la mitochondrie. Toutefois, à ce jour, malgré un grand nombre de travaux scientifiques, le rôle et la fonction d’UCP3 ne sont que partiellement élucidés.
De nombreuses publications montrent le rôle de perturbations du métabolisme mitochondrial, dans l’émergence de la résistance à l’insuline, du diabète et des maladies cardio-vasculaires [23, 24]. Par exemple, l’insulino-résistance observée avec l’avancement de l’âge, serait liée à un défaut de la phosphorylation oxydative résultant en une diminution de l’oxydation totale des substrats énergétiques et de la synthèse d’ATP, tant au niveau du muscle que du foie[25]. Il a aussi été montré que l’accumulation intramyocellulaire de lipides (acyl-CoA et diacylglycérol) perturbe les voies de signalisation à l’insuline et entraîne une insulino-résistance secondaire liée à un effet inhibiteur direct ces lipides sur les transporteurs au glucose insulino- dépendants [26] [27] Des données similaires ont été obtenues chez les descendants de parents diabétiques de type 2, qui présentent un risque accru de diabète [27] et plusieurs autres travaux semblent d’ailleurs s’accorder sur l’influence de la graisse intramyocellulaires dans. D’autres recherches montrent chez les patients obèses et diabétiques, une diminution de l’activité enzymatique dédiée à l'oxydation des acides
gras par comparaison à celle de patients obèses non intolérants au glucose [28].
Enfin, des publications attestent que ces défauts d’oxydation des acides gras sont le résultat d’une diminution du nombre de mitochondries dans les tissus concernés [29].
Toutefois, l’hypothèse d’un défaut secondaire, est aussi envisageable. En effet, Asmann et al. constatent qu’une glycémie élevée chez des patients diabétiques de type 2 entraîne une oxydation moins efficace des acides gras [30].
Le co-activateur PGC-1 alpha qui favorise l’action de facteurs de transcriptions impliqués dans la réplication de l’ADN mitochondrial et la transcription de médiateurs clés de la fusion et de la biogenèse mitochondriale, tels que la mitofusine-2 (MFN2) ou le facteur respiratoire nucléaire NRF[31], joue, à ce titre, un rôle clé dans le métabolisme oxydatif. Sans compter qu’il est lui-même sous l’influence de stimuli provenant de l’environnement ou de facteurs hormonaux, comme les catécholamines, reconnus aujourd’hui comme ayant des implications sur la survenue de l’obésité ou du diabète. Rabol et al. [32] rapportent d’ailleurs des défauts dans l’expression de ces facteurs de transcription associés à la présence d’une résistance à l’insuline.
L’avancement de l’âge et le style de vie semblent aussi influencer les capacités oxydatives des mitochondries, vraisemblablement suite à des atteintes de l’ADN mitochondrial, elles mêmes dues à l’exposition à des radicaux libres (ROS) générés lors de la phosphorylation oxydative[33]. L’ADN mitochondrial étant dépourvu d’histone et de système de réparation alors qu’il code pour des protéines nécessaires à la phosphorylation oxydative, est effectivement plus vulnérable [34].
Les mitochondries disposent cependant de la possibilité de fusionner entre elles et d'échanger leur matériel génétique, créant des réseaux étendus et interconnectés de filaments mitochondriaux qui assurent un meilleur apport énergétique. Ces réseaux sont d’ailleurs particulièrement présents dans les muscles et les tissus où la demande en ATP est élevée et, par conséquent, l’ADN mitochondrial plus exposés aux ROS. Les propriétés de fissions de la mitochondrie, servent, quant à elles, à séparer les réseaux, à partager les mitochondries lors des divisions cellulaires et à initier l’apoptose cellulaire[35] [36]. La régulation de ces deux processus, fusion et fission, étant essentielle au bon contrôle de l’homéostasie énergétique des cellules, il est particulièrement intéressant de retrouver un défaut d’expression de la protéine MFN2, reconnue pour son rôle prépondérant dans la fusion mitochondriale, chez des patients diabétiques de type 2 et dans la survenue de la maladie de Charcot Marie Tooth 2A, une neuropathie periphérique axonale [37].
Tous ces exemples montrant l’implication de la phosphorylation oxydative et des régulations de la mitochondriogenèse dans la réponse à l’insuline nous ont incités à étudier les changements survenant dans les mitochondries musculaires lors d’un processus de perte pondérale massif, comme celui pouvant être observé après un bypass gastrique de type Roux-en-Y. Cette procédure de chirurgie bariatrique restrictive et malabsorptive est reconnue pour son action durable sur la courbe pondérale et une amélioration de la sensibilité à l’insuline, au point qu’un certain nombre de patients parvient même à interrompre l’insulino-thérapie débutée avant l’intervention [38].
Nous avons décidé d’observer deux périodes, l’une à 3 mois et l’autre à 12 mois post-opératoire, qui sont représentatives de deux type de perte pondérale : la phase
rapide entre 0-3 mois et la phase lente entre 3-12 mois. A chaque phase correspond une diminution de 50% de la masse pondérale totale [39].
Pour ce faire, nous avons suivis 17 patientes souffrant d’une obésité morbide (BMI : 45.9 +/-4 kg/m2) avant un bypass grastrique et 3 puis 12 mois après. Nous avons mesuré la sensibilité à l’insuline par un clamp euglycémique hyperinsulinémique et la dépense énergétique et l’oxydation des substrats par une calorimétrie indirecte.
Nous avons également mesuré l’expression des ARNm de PGC-1α, MFN2 et UCP3 par PCR quantitative sur des prélèvements effectués par biopsie musculaire.
Les patientes suivies ont toutes perdu une quantité de poids significative et amélioré leurs paramètres métaboliques. A 3 mois post-opératoire, PCG-1α était significativement augmenté (p=0.02) tout comme à 12 mois (p=0.03) ainsi que l’était MFN2 (p=0.008 et p=0.03 à 3 et 12 mois respectivement). L’UCP3, quant à lui, n’était diminué qu’à 12 mois post-opératoire (p=0.03). Les expressions de PGC1 et de MFN2 étaient très fortement corrélées (p<0.0001). La sensibilité à l’insuline qui était très significativement améliorée en post-opératoire (p=0.002 à 3 mois et p=0.003 à 12 mois) était aussi corrélée de manière significative avec PCG-1α et MFN2. Toutefois, après une analyse de régression multiple, seulement MFN2 restait associée à l’amélioration de la résistance à l’insuline. La dépense énergétique était aussi significativement réduite à 3 mois post-opératoire (p=0.001), mais restait inchangée jusqu’à 12 mois. Les modifications de la dépense énergétique n’étaient pas corrélées avec UCP3.
Ces résultats montrent que la perte de poids est associée à une augmentation de l’expression de PGC-1α qui est responsable de plus de 50% de l’augmentation de MFN2. Ces 2 facteurs contribuent de manière significative et indépendante à l’amélioration de la sensibilité à l’insuline. Les co-expressions de PCG-1α et de MFN2 avaient déjà été observées après l’effort physique et confirmées par des résultats cellulaires [40]. Elle est démontrée ici pour la première fois dans une situation de perte de poids.
L’amélioration de la sensibilité à l’insuline est évidente de par l’amélioration des valeurs d’oxydation du glucose elle même confirmée par les changements du quotient respiratoire tant à 3 qu’à 12 mois post-opératoire. Cette amélioration, classique après un bypass gastrique, est, d’après nos analyses de régressions multiples, pour plus de 50%, liée aux deux facteurs indépendants que sont le BMI et l’expression de MFN2.
La diminution significative d’UCP3 observée à 12 mois après le bypass n’est pas en lien avec une diminution de la dépense énergétique. Cette observation est en faveur des hypothèses mettant en lien l’expression d’UCP3 avec l’utilisation des acides gras et l’accumulation de lipides intramyocellulaires.
Nos données confirment que l’influence positive d’une perte de poids sur la sensibilité à l’insuline pourrait être médiée par PGC-1 et MFN2, deux facteurs, reconnus pour améliorer la phosphorylation oxydative. Elles indiquent par ailleurs la persistance d’une plasticité métabolique même chez des patients souffrant d’une obésité morbide et doivent nous amener à réfléchir sur les moyens de stimuler la fonction mitochondriale pour traiter la survenue de la résistance à l’insuline et des dysfonctions métaboliques associées à l’obésité.
Il est, à cet égard, intéressant d’observer qu’une grande partie des traitements proposés aux patients souffrant d’obésité et des facteurs de risques cardio- vasculaires associés participent à l’amélioration de la phosphorylation oxydative. En effet, les régimes hypocaloriques et l’activité physique sont favorables à la biogenèse mitochondriale [41]. Quel que soit l’âge, que le diabète soit avéré ou non, la pratique de l’exercice physique stimule la phosphorylation oxydative [42]. Cet effet semblerait être lié à la prolifération des mitochondries sous-sarcolemales du muscle. Cette fraction mitochondriale est impliquée dans l’oxydation des acides gras et dans une production d’énergie permettant la pénétration de substrats énergétiques dans le muscle [43]. Nos résultats et l’observation que chez des patientes souffrant d’une obésité morbide, la perte de poids et l’amélioration de la résistance à l’insuline sont associés à une augmentation de l’expression des co-activateurs impliqués dans la biogenèse mitochondriale [44] et d’une diminution d’UCP3 méritent des recherches ultérieures pour mieux déterminer les mécanismes moléculaires contrôlant l’oxydation de substrats intra-musculaire et espérer développer de nouvelles thérapies contre le diabète et l’obésité.
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Abbreviations... 9
Introduction... 10
1.1. Thermodynamic laws ... 10
1.2. The adipose tissue ... 10
1.2.1. Its function ... 10
1.2.2. Fat and lean ... 11
1.2.3. Body mass index (BMI)... 11
1.3. Energy balance... 11
1.3.1. Definition ... 11
1.3.2. Food intake... 12
1.3.3. Energy expenditure ... 14
Basal thermogenesis... 14
Exercise induced thermogenesis ... 14
Adaptive thermogenesis... 14
1.3.4. Modulations of adaptive thermogenesis... 15
Cold-induced thermogenesis... 15
Diet-induced thermogenesis... 15
1.4. Obesity ... 15
1.4.1. Definition ... 15
1.4.2. The thermogenic side: uncoupling protein 1... 16
1.4.3 UCP3... 17
Structure of UCP3... 17
Genetic of UCP3: linkage, associations and variants... 18
UCP 3 and skeletal muscle... 18
Modulation of UCP3 expression... 18
Possible functions of UCP 3 and role in obesity ... 19
1.5. Diabetes Mellitus... 21
1.5.1. Definition and diagnosis... 21
1.5.2. Physiopathology... 21
Type 1 ... 21
Type 2 ... 21
The metabolic syndrome ... 22
1.6. Link between obesity and diabetes... 22
1.7. Mitochondriogenic factors ... 23
1.7.1. PGC-1 α ... 23
PGC-1α gene family... 23
Functions of PGC-1α ... 23
Genetics and PCG-1 α expression... 24
Modulation of PCG-1 α expression... 24
1.7.2. Mitofusion-2... 25
Structure and function of Mfn-2... 25
Modulation of Mfn-2 expression... 25
1.8. Bypass and weight loss ... 26
1.9. Aim of the thesis... 26
Publication ... 27
Supplementary results... 35
Modification in insulin sensitivity : ... 35
Energy balance... 35
Skeletal muscle mRNA expression ... 36
Discussion about the difference between normoglycemic and diabetic patient ... 42
Summary... 44
References ... 45
Abbreviations
BAT: brown adipose tissue
CPT1: carnitine palmitoyltransferase-1 IMTG: intramyocellular triglycerides IFG: impaired fasting glucose
LBM, lean body mass KO : knock out
NFG: normal fasting glucose MFN2: mitofusin-2
PGC1: peroxisome proliferator-activated receptor gamma coactivator RER, respiratory exchange rate
RYGB : Roux-en-Y gastric bypass UCP: uncoupling protein
UCP-tg : UCP-transgenic
O2
V , oxygen consumption
CO2
V , carbon dioxide production
Introduction
Body weight is controlled by energy intake on one hand and expenditure on the other. Obesity is a common condition resulting of an imbalance between energy intake and energy expenditure. It leads to an accumulation of fat that as a significant adverse effect on health, like diabetes, hypertension, sleep apnea, cardio-vascular diseases and cancers [1]. It has been defined by the WHO as a body mass index (BMI) > 30kg/m2 as this is the limit above which the risk of obesity-related diseases start to rise exponentially [2]. Investigators confronted to this condition, reaching today worldwide epidemic proportion, haven’t yet answer to the fundamental question: is obesity a disease or a symptom? The debate is still open.
1.1. Thermodynamic laws
Fluctuations in body weight, like the melting of an ice cube to water can be explained by physics. The theories of well-known scientists of the 18th century like Hermann von Helmhotz or Julius Robert Mayer who, for the first time, attempted to explain physiological phenomena in terms of physics and chemistry are still actually the fundaments of our understanding of obesity.
Herman von Helmhotz was the first to propose that the first law of thermodynamic, which states that the amount of stored energy equals the difference between energy intake and work, is uniformly applicable to biological systems [3]. In other words, if energy intake is higher than energy spending, it will be stored in the body as fat.
Julius Mayer proposed that the second law of thermodynamic which states that in a closed system, you end up any real physical process with less useful energy than you started with (energy dissipation as heath) is also applicable to biological systems [4].
1.2. The adipose tissue
1.2.1. Its function
The assimilation, storage and use of energy from nutrients constitute a homeostatic system essential for human life. In mammals, the ability to store energy in a large amount is made possible by lipid storage. Lipids are stored under the form of triglycerides in specialised cells: the adipocytes and will allow survival during possible periods of food deprivation. Triglycerides are very efficient energy storage molecules because being totally hydrophobic; their storage is exempted of water molecules and necessitate a reduced space. Hence, 1kg of fat tissue contain 9000 Kcal [5]. To use the triglycerides energy lipolysis is necessary. Three main organs hydrolyse triglycerides and release them into the circulation: white adipose tissue (WAT), the intestine and the liver. But only WAT releases non esterified (free) fatty acids (FFA).
Distinct lipolytic states are also documented [6] [7]. FFA can be released from the adipocytes with a low level of circulating insulin and catecholamine during prolonged fasting. In acute stress, brief and catecholamine triggered lipolysis occurred and in obesity despite high circulating levels of glucose and insulin lipolysis is not inhibited (insulin resistance). In mammals lipolysis is mainly a cAMP/protein-kinase A (PKA)- regulated metabolic pathway under the control of catecholamine (stimulation of
lipolysis) and insulin (inhibition of lipolysis). Catecholamines are of major importance for the regulation of lipid mobilization in human adipose tissue and for the increase of non-esterified fatty acid supply to the working muscle even though other hormones and metabolites can influence lipolytic rate [6]. Recently Lafontan added atrial natriuretic peptide (ANP) to the list of the regulators of lipolysis. ANP plays a important role in conjunction with catecholamines in the control of exercise-induced lipid mobilization via the activation of cyclic GMP dependent protein kinase-I (cGK-I) [8]
1.2.2. Fat and lean
Body mass and composition is a function of where energy is channelled. Kleiber was the first to demonstrate in animals of different sizes that energy expenditure is proportional to the body free fat mass [9]. Since this observation, mammalian body is considered to be composed of two distinct compartments: fat mass and lean body mass (or fat free mass). The former, fat mass, includes for its main part stored triglycerides and the latter essentially skeletal muscle but also all the organs and bones. Two main depots of triglycerides are defined, located respectively under the skin (subcutaneous fat) or in the abdominal cavity (visceral fat) [10] [11]. However it is also important to take into account non-adipose tissue store like intramyocellular (IMTG) or intrahepatic fat droplets for their metabolic implications [12][13].
1.2.3. Body mass index (BMI)
In human, BMI is defined by the weight in kilograms divided by the square of the height in meters. It is easy to calculated, well correlated with direct measures of body fatness and clearly associated with increased mortality at a value above 28 [14].
Leanness, a BMI ranging between 19-26 kg m-2, is a positive factor in life expectancy [15]. It was also found that it is important to maintain during life a stable BMI. A weight gain of 10 kg or more between the age of 18 years and mid adulthood was associated with increased mortality [15].
In the clinical assessment of obesity and overweight it is important to consider not only BMI but also fat distribution [16]. The waist hip ratio, a measure of the waist vs abdominal circumference, allows to define fat distribution. When fat depots are located predominantly around the waist obesity is called “gynoïde” and the ratio is <1.
If fat depots are located predominantly in the abdominal area, showing a central pattern, obesity will be defined android and the ratio will be > 1. Fat distribution is a better index of morbidity’s risk than the absolute fat mass [17]. It has indeed been shown that obese visceral fat is a stronger predictor than subcutaneous fat of insulin resistance [18] and cardiovascular diseases.
1.3. Energy balance
1.3.1. Definition
As mentioned above, from a thermodynamic point of view, keeping a stable body weight implies energy balance between food intake and energy expenditure. To maintain the internal environment constant, the body uses a control system that limits the changes in body state. The control of stability is defined homeostasis. The Central Nervous System (CNS) whose function is the integration of numerous signals
coming from the periphery (endocrine, neuro-endocrine signal and sensory) and the cerebral cortex (emotion, wishes, curiosity, etc.) modulates energy balance to reach homeostasis through three different mechanisms: 1. Control of behaviour (feeding and physical activity) 2. Control of the neuroendocrine system (secretion of numerous hormones: growth hormones, cortisol, thyroid, insulin, sex steroids) and 3.
Control of the autonomous nervous system activity, which regulates energy expenditure and other aspects of metabolism. These three kinds of effects can occur concomitantly or independently but are also influenced by the peculiar metabolic situation especially in obesity.
1.3.2. Food intake
Feeding behaviour lies at the interface of physiology and psychology and therefore depends of many intricate factors. We will not describe in this thesis the aspects related to alimentary disorders like anorexia observed in young female or athletes particularly runners or dancers [19], or bulimia nervosa. Several studies show a higher prevalence of psychiatric disorders in obesity and many physicians recognise overeating as an efficient way to calm anxiety [20]. Moreover alimentary disorders lead to physiological changes potentially influencing further body weight fluctuations [21]. For example, in obesity, the severity of adiposity probably contributes to the amount of daily motion [22] and to feeding habits. The degree of stress or thymia felt while eating can also modify the capacities to integrate the inputs coming from the
“feed forward” sensor in taste that contributes to the selection of foods and palatability [23]. Indeed human cortex involves a centre where the pleasantness of food (palatability) is measured. Not only taste but also the vision and the smell are emotionally recorded [24] and influence what and how we eat [25]. Eating behaviour is therefore under the influence of three distinct chronological signals: those involved in the initiation of food intake, those that maintain feeding once eating as started and those mediating meal termination [26]. The latter seems to be clearly deficient in several monogenic form of obesity characterised by hyperphagia [27], [28], [29]. The CNS and particularly the cerebral cortex, the integrative centre where physiological and psychological informations interact, is then responsible for the conscious and motivated behavioural responses to the availability of food. The challenge in analysing eating behaviour is to better characterise the physiological steps involved in the control of food intake and concomitantly to integrate which steps are also influenced by psychological and or emotional inputs.
Physiologically, the food intake system is organised on three levels [30]. The first level implies signals coming from the periphery. Such signals can be eating-elicited stimuli (sensory, mechanical and chemical stimuli) which can activate peripheral receptors and modulate energy intake [31] [32]. Hormonal signalling released from enteroendocrine cells and mostly mediated via vagal afferents to the midbrain and the hypothalamus that reports information about the nutritional level. Grehlin for example is an orexigenic peptide secreted by the stomach, whose level is increased by fasting and decreased by gastric wall distension following the ingestion of food [33]. Cholecystokin (CCK) is an other peptide, secreted by the upper intestinal mucosa which activates satiety mechanisms through its action on CCK1 receptors on the vagal nerve (Hunger satiety and nausea represent different steps on the same physiological spectrum) [34]. Not to mention the neuroendocrine signals secreted primarily by adipocytes. Leptin is probably the most studied. It is a multifunctional protein expressed in proportion of the amount of energy stored and playing a pivotal
role in regulating food intake, energy expenditure and neuroendocrine functions.
Besides its inhibitory effect on food intake and its stimulating effect on thermogenesis, leptin was found to stimulate fatty acids oxidation and glucose uptake in muscle and adipose tissue and to prevent lipid accumulation in non-adipose tissues, the so called “lipotoxicity” [35].
The second level is an integrative centre of the SNC, receiving the neural and hormonal signals from the periphery. The brainstem and sevral hypothalamus nuclei have the essential function, mediated by neuromessengers such as NPY, AgRP, POMC or CART [36], to deliver an integrative message to higher behavioural centers and to the periphery.
The third and final level is located in periphery and its activation can modulate the energy expenditure. Its major players which are the β3 adrenoceptor, the UCPs, thyroid hormones and mitochondrial activity are will be a major focus of this thesis.
Figure 1 [30]
1.3.3. Energy expenditure
Total body energy expenditure results from the catabolic reactions which are exothermic and from the work on the environment. At equilibrium, food energy intake should be equal to heat released by biological combustions plus work performed on the environment. Energy expenditure at rest, i.e. in the absence of any work performed on the environment, can be measured directly as heat produced. The latter can be quantified either in a calorimeter room (direct calorimetry) or by measuring the amount of oxygen consumed and CO2 produced (indirect calorimetry) [37]. Total energy expenditure in human consists in basal, physical activity-induced and adaptive energy expenditure (figure 2).
Basal thermogenesis
Basal thermogenesis, called also obligatory thermogenesis or basal metabolic rate (BMR) is the heat produced by the functioning of metabolic pathways at rest. It includes the digestive process, i.e. the transformation and absorption of food. BMR depends on several factors: including body composition (% lean body mass), age, sex, ethnicity, activity and glucose tolerance [38].
Exercise induced thermogenesis
Exercise-induced thermogenesis is the heat produced in response to any physical activity. Indeed exercise will increase the energy intake/expenditure of the body because it involves the activation of numerous metabolic pathways at a cellular and organic level. So any muscle contraction necessitates a higher amount of energy consumption resulting in a higher energy expenditure.
Adaptive thermogenesis
Finally adaptive thermogenesis is the heat produced in response to changes in environmental temperature or food intake, with the aim of protecting the organism against cold exposure or over-eating.
Figure 2 [39]
Total Energy expenditure can be subdivided in 3 categories: 1 Obligatory energy expenditure or basal thermogenesis 2 Exercise induced thermogenesis 3 Adaptive thermogenesis
1.3.4. Modulations of adaptive thermogenesis Cold-induced thermogenesis
To study cold-induced adaptive thermogenesis rodents have been exposed to a 4°temperature. The results show an increase in oxygen consumption between 2-4 fold in rodents after both acute and chronic cold exposure (4°C). The acute response is partly due to shivering. The latter then disappears and other mechanisms become prominent, including adaptive thermogenesis in brown adipose tissue. The effect of cold exposure on energy metabolism is called cold-induced thermogenesis. In human wearing identical clothes Dauncey and al showed an increase of 7% in heat production if the room temperature was decreased from 28 to 22°C [40]. These data indicate that in adult humans who lack brown adipose tissue cold induced thermogenesis is still present.
Diet-induced thermogenesis
Studies in rodents and humans show also an increase of 25% to 40% in total energy expenditure after a meal or upon a chronic high fat diet[41]. The effect of food on energy metabolism is called diet-induced thermogenesis. The modulation of diet- induced thermogenesis is complex, involving insulin, leptin, thyroid hormones, the autonomous nervous system and mitochondrial metabolism.
1.4. Obesity
1.4.1. Definition
Obesity is the consequence of an imbalance between energy intake and energy expenditure. It is defined as a state of increased body weight due to an abnormal or excessive triglycerides accumulation in adipose tissue predisposing to adverse health consequences. During the development of obesity, the adipocytes become first hypertrophic then this state seems to activate a process of recruitment leading to an hyperplasia of the adipose tissue [42]. Hypertrophy is reversible whereas hyperplasia is not. Therefore, when hyperplasia has occurred obesity is only partially reversible. Following an evolutionary point of view, pregnancy is an interesting situation. Indeed pregnant women are supposed to easily accumulate fat and they tend to do it around the hips but not in the abdomen, possibly because of the increase in circulating oestrogens. This tendency contributes to complete the pregnancy without the adverse effects of visceral lipotoxicity. Interestingly however post-pregnancy is associated with a 2.7 kg body weight increase [43] which seems to confirm the relative irreversibility of this form of obesity.
Why certain people are more prone to obesity is a very debated question. More than 50 genes [44], environment [45] [46], social network [47, 48], physical exercise [49]
food quality [50] and psychiatric co morbidities [20] are possible contributing factors to the development of obesity. Interestingly there are also molecular candidates that have a role at the interplay between environment and genes like several proteins involved in the mitochondrial regulatory pathway. Such candidates participate in the regulation of energy expenditure and therefore could have a significant role in the development of obesity and diabetes.
1.4.2. The thermogenic side: uncoupling protein 1
The uncoupling protein 1 (UCP1), expressed in the mitochondria of brown adipose tissue, catalyses a regulated proton leak across the inner mitochondrial membrane and thus diverts energy from ATP synthesis to thermogenesis. Indeed, the chemiosmotic hypothesis of Mitchell proposes that substrate oxidation is coupled in the electron transport chain to the pumping of protons from the matrix to the inter- membrane space of the mitochondria. This creates a gradient that is dissipated by the re-entry of protons into the mitochondrial matrix through the ATP synthase. The energy thus released allows the phosphorylation of ADP into ATP. Protons can enter the matrix only when ADP is available. In the absence of ADP the increased proton gradient will inhibit the electron transport chain and the reduction of NADHH+ and FADH2. As a result, substrate oxidation will decrease. This control by ADP is accounted for by the theory of Mitchell [51]. However in mitochondria some oxygen is used in the absence of ADP revealing the existence of a basal proton leak and that the coupling of substrate oxidation to ATP synthesis is imperfect. This is called state IV respiration. One should distinguish between the basal proton leak present in all mitochondria and the much larger proton leak due to the presence of the uncoupling proteins (UCPs) in the mitochondria.
Figure 3:
In mitochondria, UCP1 proteins decrease the membrane potential through a proton leak responsible of an imperfect coupling of substrate oxidation to ATP synthesis. This uncoupling seems to take place in condition of low ADP availability and divert the proton flux from the ATP synthase creating a futile cycle.
The thermogenic capacity of brown adipose tissue is stimulate by thyroid hormone [52] and by norepinephrine released from the sympathetic nervous system (SNS) [53]. Cold environment or diet via activation of the SNS and through a selective BAT β3-adreno-receptor stimulates the expression and activation of UCP1 in BAT resulting in increased thermogenesis [54]. The SNS-BAT-UCP1 axis is markedly increased in response to cold environment and modulated by diet composition [55].
Its activation can result in a 10 fold increase in BAT thermogenesis and plays an important role in the control of heat dissipation in animals [56]. In human the Trp64Arg mutation of the gene for the β3-adrenoceptor is related to an increased capacity to gain weight [57]. This finding is intriguing since the amount of BAT is negligible in human adults. It supports the hypothesis that, in a still unidentified location, some β3-adrenoceptor-mediated thermogenesis persists in human.
1.4.3 UCP3
Therefore, in 1997, the discovery of UCP3, a novel UCP selectively expressed in skeletal muscle was considered a breakthrough and an intense interest has developed concerning this protein. UCP3 is a member of the mitochondrial anion carrier super family. Based upon its high homology with UCP1, UCP3 was proposed to be an uncoupling protein [58]. However despite all the research made the role and function of UCP3 is still not clear. Its tissue distribution restricted to skeletal muscle and brown adipose tissue suggested that it could play an important role in the regulation of energy expenditure and in the control of body weight. Other postulated roles for UCP3 include regulation of fatty acid metabolism and prevention of reactive oxygen species (ROS) formation.
Figure 4
Structure of UCP3
UCP3 is a carrier monomer of ~300 amino acid long and has a tripartite structure (3 times 100 amino acid–long repeats). Each repeat posses a mitochondrial carrier signature motif. The latter is thought to be important in targeting carriers by way of the Tim10 / Tim12 / Tim22 pathway to the mitochondrial inner membrane [59]. Each monomer is thought to consist of six transmembrane domains, and functional carriers are thought to be homodimers [60]. The members of the mitonchondrial carrier family show significantly higher levels of sequence similarity to each other than to other proteins.
The respiratory chain is composed of four complexes : I (NADH-ubiquinone reductase), II (succinate- ubiquinone reductase), III (ubiquinol-cytochrome C reductase) IV (cytochrome oxidase) Ubiquinone shuttles electrons from complexes I and II to III. Cytochrome C shuttles electrons from complex III to IV Electrons are transfered through the electron transport chain to the final acceptor 02. The electron energy transfer through redox potential is used by complex I, III and IV to pump out H+ and generate the elecrogradient necessary to the production of ATP.
Genetic of UCP3: linkage, associations and variants
The UCP3 gene was shown to lie in humans in the region 11q13 between markers D11S916 and D11S911 or D11S3966 [61]. The result of most studies suggest that genetic variation of the coding region of the UCP3 gene is not a major cause of body weight abnormalities [62]. However, many variants of the non-coding regions of UCP3 more or less correlated with obesity have been described. Genetic markers close to the UCP3 locus have been shown to be linked to resting metabolic rate;
percent body fat and fat mass. A genetic variation in the 5' flanking region of the UCP3 gene is associated with body mass index in humans in interaction with physical activity [63]. Halsall and al. showed in a Caucasian population a protective effect of the UCP3 c-55t polymorphism on the development of obesity [64]. Clement and al. observed an additive effect of A-->G (-3826) variant of the uncoupling protein gene and of a β3-adrenoceptor gene mutation on weight gain in morbid obesity [65].
Several polymorphisms of the UCP3 promoter have also been found and in various ethnicities. In Pima Indians, Schrauwen et al. [66] reported a polymorphism of the proximal UCP3 promoter region which was associated with increased skeletal muscle UCP3 mRNA levels. Its expression was negatively correlated with BMI and positively correlated with the metabolic rate during sleep. Although none of these studies can support conclusively a role of UCP3 in energy expenditure, they suggest that UCP3 could play a role in metabolic efficiency in humans and therefore contribute to body weight control.
UCP 3 and skeletal muscle
Skeletal muscle fibers are subdivided in 3 types due to how the myofibrillar ATPase was histochemicaly stained and/or by determination of oxidative phosphorylation and glycolysis enzyme activities (type I: slow-twitch oxidative; type IIa: fast-twitch oxidative-glycolytic; and type IIx: fast-twitch glycolytic) [67]. Type I fibers are fatigue resistant as long as fatty acid beta-oxidation and oxidative phosphorylation can be maintained and, in contrast IIx fibers are susceptible to fatigue. UCP3 expression in skeletal muscle has been shown to be fiber-type dependent. Hesselink and al.
showed that UCP3 is mostly expressed in type IIx, less in type IIa and only moderatly in type I fibre [68].
However it is important not to forget that skeletal muscle fiber type expression is under the control of the metabolic condition and that a change in fiber type composition could in turn modify the content of UCP3. Gaster and al. showed that the fraction of type I fibers was higher (0.51 +- 0.02) in lean than in obese (0.44 +- 0.03) and in type 2 diabetic patients (0.38 +- 0.05) patients [69]. In COPD patients, whose muscle is characterized by increased fatty ascid oxidation and decreased intramyocytic triglycerides (IMTG), a shift from fiber type I to type II with selective atrophy of IIx fibers was observed [70].
Modulation of UCP3 expression
UCP3 mRNA expression studies show a very broad spectre of regulations described in more than 600 publications. We will focus our attention mainly on humans. In the latter, the mRNA expression of UCP3 is positively and linearly correlated with circulating FFA [71]. Fatty acid loading by infusion up regulates skeletal muscle UCP3 mRNA in vivo [72]. In both human and rats, UCP3 mRNA is increased in response to fasting and a subsequent decrease is observed when refeeding occurs
[73] [74]. The increase in UCP3 during short-term fasting in human is paralleled by an increase in several other factors involved in lipid metabolism (PDK4, LPL, CPT I) in skeletal muscle. Moreover, during refeeding variations in the regulation of UCP3 are observed depending on the type of meal consumed. Indeed UCP3 remained elevated in subjects refed with low carbohydrate (FAT trial) and decreased to basal level after a high-carbohydrate meal [73].
In COPD patients, UCP3 mRNA expression is 44% lower than in controls. UCP3 protein content is significantly decreased by 89% and 83% and the IMTG content by 64% and 54%, respectively, in types I and IIa fibers. Both UCP3 protein and IMTG are unchanged in IIx fibers [75].
The lipid storage myopathy, riboflavin-responsive, multiple acylcoenzyme A dehydrogenase deficiency (RR-MAD) is characterized by, among others, a decrease in fatty acid (FA) beta-oxidation capacity due to a reduced amount of intramitochondrial flavin adenine dinucleotide. The consequences of the decreased FA beta-oxidation is an increased IMTG which can fortunately be cured by the administration of riboflavin. In this peculiar situation, we showed that UCP3 content is increased before the treatment compared with healthy subjects and can be restored to control value after riboflavin administration [76].
All these data support the hypothesis that UCP3 regulation is somehow related to fatty acid metabolism and particularly to IMTG, an hypothesis strengthened by the results of Mingrone and al. who showed that IMTG level represents the most powerful independent variable for predicting skeletal muscle UCP3 variation [77].
Interestingly some investigators didn’t find an up regulation of UCP3 after a high fat diet [78]. The kind of fatty acids ingested could be incriminated as evidenced by a study using polyunsaturated fatty acids which shows that particularly the fatty acids of the n-3 family upregulate the transcription of UCP3 and concomitantly that of enzymes of fatty acid oxidation (carnitine palmitoyltransferase and acyl-CoA oxidase) [79].
Upon body weight loss variations in UCP3 mRNA expression seem to depend on the duration and the kind of diet. After a very low calorie diet resulting in significant weight loss and decrease in energy expenditure, skeletal muscle UCP3 mRNA levels were either found to remain unchanged in lean and obese individuals [80] or to be significantly reduced in lean and obese individuals [81] as well as in type 2 diabetic patients [82]. In morbidly obese patients, two years after a bilio-pancreatic diversion and an impressive body weight loss, UCP3 expression was also down regulated [77].
Interestingly, UCP3 mRNA in skeletal muscle is similar in lean and obese individual, although obesity is well known for high level of circulating free fatty acids [81]. An other condition, associated with increased circulating level of free fatty acids is physical activity [6]. In endurance trained athlete Russell and al. [...] found that UCP3 protein content in type I, IIa and IIx muscle fibers is significantly reduced by 54, 29 and 16 % respectively whereas in sprint trained athlete it is reduced by 24, 31 and 26% respectively [83]. According to these results it is tempting to speculate for a protective effect of UCP3 from the toxic effect of accumulating nonesterified fatty acids in skeletal muscles in situations where fatty acid delivery exceeds oxidation.
Possible functions of UCP 3 and role in obesity
The hypothesis that a dysfunction of uncoupling proteins might predispose to obesity is based on the finding that a chemical uncoupling of the mitochondrial membrane
mice an over expression of UCP3 in skeletal muscle induces an increase in oxygen consumption, a decrease in body weight, a better tolerance to glucose and no change in food intake [85]. The impact of the physiological proton leak on the basal metabolic rate has been estimated through computations to account for 20% of the BMR at rest and for 15% of it during exercise in contracting skeletal muscle [86.].
UCP3 being expressed almost exclusively in skeletal muscle, which makes up to 40% of the body mass and is responsible for the majority of the basal metabolic rate in lean individuals has been considered as an effector of energy expenditure.
However UCP3-KO mice were similar to their wild-type littermates and didn’t show any change in cold- and diet-induced thermogenesis [87]. Moreover the expression of UCP3 after body weight loss in human was never found to correlate with BMR variations. Altogether, these findings don’t support the hypothesis that UCP3 is involved in the control of energy balance.
In UCP3KO mice, Brand and al. reported increased production of superoxide anions and of mitochondrial aconitase in vitro, suggesting that one of the functions of UCP3 could be the prevention of excessive oxidative stress[88]. UCP3’s proton leak would decrease the mitochondrial membrane potential, thus lowering the probability for electrons to interact with oxygen [87]. In line with a protective role of UCP3 against oxidative stress, Echtay et al. showed that exogenous superoxides, in the presence of coenzymeQ, induced an uncoupling of skeletal muscle mitochondria. They also showed a 2-fold increase of the effect of superoxides if UCP3 levels were doubled by fasting and a lack of effect of superoxides in mitochondria isolated from UCP3KO mice [89].
Another hypothesis, based on the observation that UCP3 expression is closely related to fatty acid metabolism is that UCP3 could be involved in the exit of fatty acids outside the mitochondrial membrane [74]. Indeed, stimuli like cold exposure, thyroïd hormones and adrenergic agonists not only stimulate thermogenesis but also enhance the utilisation of lipids as fuel substrate. Therefore the function of UCP3 would be to export fatty acids out of the mitochondria when fatty acid oxidation predominates [90]. UCP3 would therefore be implicated in the control of lipid oxidation or prevention of lipotoxicity.
Finally, several studies show that changes in UCP3 expression are accompanied by changes in glucose metabolism. UCP3-tg mice showed reduced fasting plasma glucose levels, improved glucose tolerance after an oral glucose load, and reduced fasting plasma insulin. Schrauwen and al. show that UCP3 protein content is reduced in prediabetic subjects and type 2 diabetic patients and is restored by a rosiglitazone treatment in parallel with a significant increase in insulin sensitivity [91]. Moreover, Choi and al. observed that UCP3-tg mice were completely protected against fat- induced insulin-resistance. They hypothesized that this protection was mediated by conversion of IMTG into thermal energy[92]. None of these results, however, provide evidence for a functional role of UCP3 in glucose metabolism.
