Die Herzinsuffizienz ist eine der führenden Todesursachen weltweit. Eine auf die neurohumorale Aktivierung zugeschnittene Therapie mit ACE-Hemmern bzw. Angiotensin-Rezeptorantagonisten, Betablockern, Aldosteronantagonisten und Diuretika verbessert zwar die Symptomatik und Prognose. Letztere ist bei Diagnosestellung jedoch immer noch schlechter als die vieler maligner Erkrankungen einzuschätzen. Ziel ist daher die Entwicklung von Medikamenten, die den Krankheitsverlauf des Syndroms Herzinsuffizienz aufhalten bzw. umkehren. Ein Ansatz ist dabei die Analyse sogenannter Kandidatengene, die im kranken Herzen differentiell exprimiert werden und potentiell medikamentös beeinflussbar sind. Im Rahmen der vorliegenden Arbeit wurden zwei solcher Kandidatengene charakterisiert. Mäuse mit kardialer Überexpression b1-adrenerger Rezeptoren entwickeln eine Herzinsuffizienz mit kardialer Hypertrophie und Fibrose. In Gen Arrays mit 21000 Maus-ESTs zeigten sich unter anderem die Gene des Uncoupling Protein 2 (UCP2) und der Diphosphoinositol-Polyphosphat-Phosphohydrolase 2 alpha (DIPP2a) aktiviert. Diese Befunde wurden zunächst mittels RNase Protection Assay und RT-PCR bestätigt. Auch andere murine Herzinsuffizienzmodelle wurden untersucht. So ließ sich ebenfalls im druckinduzierten Herzinsuffizienzmodell nach artifizieller Aortenstenose sowie im b2-AR überexprimierenden Herzen eine erhöhte Konzentration von UCP2- und DIPP2a-mRNA messen. Um zu prüfen, ob diese differentielle mRNA-Expression deletäre oder protektive Effekte vermittelt, wurden jeweils transgene Mauslinien mit herzspezifischer Überexpression von UCP2 und DIPP2a generiert. Die Linie UCP2-TG1 mit hoher Überexpression sowie ein Gründer-Tier der UCP2-transgenen Mäuse entwickelten eine Herzinsuffizienz mit vergrößertem Herzen, linksventrikulärem Pumpversagen, interstitieller Fibrose und typischen Veränderungen der molekularen Marker ANF und SERCA. Zudem fanden sich dilatierte Vorhöfe sowie eine bradykarde Herzrhythmusstörung. UCP2 ist ein Entkoppler der oxidativen Phosphorylierung im Mitochondrium. Im energieintensiven Stoffwechsel des Myokards könnte eine durch UCP2 reduzierte ATP-Synthese zu den genannten Veränderungen führen. Für UCP2 wurden auch protektive Eigenschaften durch das Abfangen freier Radikale beschrieben. Die Linie UCP2-TG3 mit niedrigerem Überexpressionsniveau und ohne kardialen Phänotyp wurde deshalb einem Aortic banding unterzogen, wo sich in der Überlebenskurve kein protektiver oder deletärer Effekt einer moderat vermehrten Entkopplung im Vergleich zum Wildtyp zeigte. In drei unabhängigen Linien transgener Mäuse mit herzspezifischer Überexpression von DIPP2a ließ sich morphometrisch eine kardiale Hypertrophie nachweisen. Die Linie DIPP2a-TG9 mit dem höchsten Überexpressionsniveau zeigte zudem eine kardiale Fibrose sowie unter Dobutamin eine verminderte kardiale Kontraktilitätsreserve im Vergleich zum Wildtypen. DIPP-Proteine hydrolysieren Inositolphosphate und Nukleosiddiphosphate und greifen so in zentrale Stoffwechselvorgänge ein, die im einzelnen noch nicht geklärt sind. Es konnte hier im Mausmodell gezeigt werden, dass UCP2 und DIPP2a zwei für die Entwicklung einer Herzinsuffizienz relevante Zielproteine darstellen. Geplant ist die weitere Aufklärung der beteiligten Mechanismen, um diese letztlich auch therapeutisch beeinflussen zu können. / The expression of uncoupling protein 2 (UCP2) and diphosphoinositol polyphosphate phosphohydrolase 2 alpha (DIPP2a) was shown to be upregulated in a murine model of chronic heart failure overexpressing beta1-adrenergic receptors and in other heart failure models. These candidate genes could play a protective or detrimental role in the development of chronic cardiac insufficiency. To test this hypothesis mice overexpressing UCP2 and DIPP2a, respectively, were generated. UCP2-transgenes with high expression level developed a chronic heart failure with interstitial fibrosis, cardiac hypertrophy and altered molecular markers of heart failure. They showed dysplastic atria and a bradyarrhythmia. DIPP2a-transgens with high expression level showed minor fibrotic changes in the myocardium in advanced age. Cardiac reserve was reduced as well. UCP2 and DIPP2a are two candidate genes which might be involved in the development of chronic heart failure.

The whole body UCP2 knockout (UCP2−/−) have enhanced insulin secretion and higher ATP content. However, these changes could be due to indirect effects of extra-pancreatic deletion and therefore, generating beta-cell specific knockout mice (UCP2BKO) is essential. A 90% knockdown of UCP2 protein was observed in beta-cells of UCP2BKO mice. No significant differences were observed in body weight accumulation, fasting blood glucose, plasma insulin or glucagon. UCP2BKO had impaired oral glucose tolerance with no differences in insulin secretion or sensitivity. Enhanced ROS accumulation was observed in the beta-cells of UCP2BKO and upregulation of antioxidant enzyme genes. Morphometric analysis showed an increased glucagon positive area in the pancreata of UCP2BKO mice. Results obtained from UCP2BKO were contrary to the phenotype observed in UCP2−/− mice. Overall, the characterization of UCP2BKO demonstrates that UCP2 in the beta-cell is involved in modulating ROS production.

The whole body UCP2 knockout (UCP2−/−) have enhanced insulin secretion and higher ATP content. However, these changes could be due to indirect effects of extra-pancreatic deletion and therefore, generating beta-cell specific knockout mice (UCP2BKO) is essential. A 90% knockdown of UCP2 protein was observed in beta-cells of UCP2BKO mice. No significant differences were observed in body weight accumulation, fasting blood glucose, plasma insulin or glucagon. UCP2BKO had impaired oral glucose tolerance with no differences in insulin secretion or sensitivity. Enhanced ROS accumulation was observed in the beta-cells of UCP2BKO and upregulation of antioxidant enzyme genes. Morphometric analysis showed an increased glucagon positive area in the pancreata of UCP2BKO mice. Results obtained from UCP2BKO were contrary to the phenotype observed in UCP2−/− mice. Overall, the characterization of UCP2BKO demonstrates that UCP2 in the beta-cell is involved in modulating ROS production.

The prototype uncoupling protein 1 (UCP1) mediates proton leak-dependent thermogenesis in mammals, but the physiological functions of the novel UCP2-5 are unclear. Nematodes only express one uncoupling protein that is most similar to UCP4 in the human brain, which is believed to be the most evolutionarily conserved of the uncoupling proteins. Consistent with reported UCP functions in mammals, we observed that ceUCP4-null nematodes had decreased metabolic rates and increased adiposity compared to wild type. Surprisingly, these phenotypes corresponded to decreased succinate-mediated mitochondrial respiration without apparent changes in mitochondrial uncoupling. ceUCP4-null mitochondria exhibited normal electron transport chain functions, but had a decreased capacity for succinate import. Supporting the functional importance of ceUCP4-dependent complex II regulation in vivo, ceUCP4 deficiency was demonstrated to result in a selectively lethal response to genetic and pharmacological inhibition of Complex I. Similarly, ceUCP4-deficiency significantly prolonged lifespan in the short-lived mev-1 mutant that generates deleterious complex II-derived reactive oxidants. These results define a new physiological function for the ancestral ceUCP4 in the regulation of complex II-mediated oxidative phosphorylation through an unexpected effect on mitochondrial succinate transport. The data described in this dissertation also describe a novel mechanism by which uncoupling proteins mediate mitochondrial bioenergetics. / text

La protéine UCP2 (UnCoupling Protein 2) appartient à la famille des transporteurs de la membrane interne de la mitochondrie. Son expression est restreinte à certains tissus comme la rate, l'estomac ou l'intestin. Au niveau cellulaire, UCP2 est particulièrement présente dans les macrophages où elle régule la production de radicaux libres (ROS). L'analyse des souris Ucp2-KO a montré qu'elles survivent mieux à une infection par le parasite Toxoplasma gondii que les animaux sauvages grâce à des macrophages superactifs en terme de production de ROS. Par ailleurs, dans le modèle murin de l'athérosclérose humaine, les souris Ucp2-KO développent des plaques athéromateuses plus instables et plus larges, présentant une forte accumulation de macrophages et des dégats importants liés au monoxyde d'azote (NO). <br />Au cours de ma thèse, nous avons cherché à approfondir les connaissances sur le rôle physiologique d'UCP2 ainsi que sur son activité biochimique.<br />Nous avons démontré que la diminution rapide d'UCP2 en réponse au LPS potentialise l'activation des MAPK dans les macrophages. La mitochondrie via UCP2 est ainsi au coeur d'une boucle d'amplification du signal impliquant la modulation des ROS mitochondriaux. Par conséquent, la signalisation et la vitesse d'activation des macrophages Ucp2-KO est accélérée, conduisant à une production accrue de NO et de cytokines.<br />La pertinance de ces résultats a ensuite été testée in vivo avec un volet infection et un volet auto-immunité. L'infection des souris par la bactérie Listeria monocytogenes a révélé une meilleure résistance des souris Ucp2-KO. Une production accrue de cytokines pro-inflammatoires chez les souris Ucp2-KO ainsi qu'un recrutement plus important de phagocytes au niveau de leur rate soulignent le rôle régulateur d'UCP2 sur l'immunité innée. En ce qui concerne, l'auto-immunité, l'induction expérimentale d'un diabète de type 1 est nettement accélérée chez les souris Ucp2-KO. L'analyse de ces souris montrent un rôle capital des macrophages dans le développement de la maladie grâce à leur forte capacité de production de cytokines et de NO.<br />L'activité biochimique d'UCP2, c'est-à-dire son activité de transport, a également été abordée. La glutamine est un inducteur spécifique de l'expression d'UCP2. Par conséquent, la comparaison du métabolisme de la glutamine dans les macrophages Ucp2-KO et Ucp2-WT a démontré que l'expression d'UCP2 est requise pour une oxydation correcte de la glutamine.<br />Enfin, grâce à la disponibilité de génomes complets de nombreuses espèces, l'étude phylogénomique des UCP a permis de tracer une histoire de l'évolution des UCP de mammifères et aviaire.<br />Nos études ont mis en évidence la participation d'UCP2 au métabolisme des macrophages. L'altération de celui-ci influe sur la signalisation et l'activité des cellules. Une meilleure compréhension de la fonction d'UCP2 et du métabolisme des cellules immunitaires pourrait ouvrir de nouvelles perspectives thérapeutiques.

Proteínas desacopladoras (UCPs) estão presentes na membrana mitocondrial interna e por meio do transporte de prótons do espaço intermembranas para a matriz mitocondrial desacoplam a oxidação dos substratos da síntese de ATP, dissipando a energia do potencial de membrana e, conseqüentemente, diminuindo a produção de ATP pela cadeia respiratória mitocondrial. O aumento na expressão de UCP2 está relacionado com um risco diminuído de obesidade e risco aumentado de desenvolvimento de diabetes melito. O polimorfismo - 866G/A na região promotora do gene da UCP2 está associado a um aumento da expressão do RNAm desta proteína. O objetivo do presente estudo foi avaliar o envolvimento do polimorfismo -866G/A no gene UCP2 nas respostas metabólicas ao exercício e na taxa metabólica basal em jovens saudáveis. Foram recrutados 27 homens com idades entre 20 e 35 anos, sem histórico de doenças e sem uso de medicamentos, eutróficos e que não estivessem envolvidos em nenhum tipo de treinamento físico. Os indivíduos foram alocados intencionalmente em três diferentes grupos, de acordo com o genótipo para o polimorfismo em questão: A/A(n=9), A/G (n=10) e G/G (n=8). Todos os indivíduos foram submetidos a uma análise de taxa metabólica basal e após uma refeição padrão realizavam 30 minutos de corrida em esteira em intensidade equivalente a 10% abaixo do 2º limiar ventilatório. Em jejum, antes do exercício, logo após realização do exercício, uma hora após e duas horas após o exercício foram realizadas coletas de sangue para a verificação do comportamento das concentrações de glicose, insulina e perfil lipídico. Não foram encontradas diferenças entre as respostas dos três grupos. Este estudo concluiu que isoladamente o polimorfismo -866G/A não exerce influência sobre a TMB e respostas de glicose, insulina e perfil lipídico ao exercício em jovens saudáveis. / Uncoupling proteins (UCPs) are present in the inner mitochondrial membrane and through the transport of protons from the intermembrane space to the mitochondrial matrix to uncouple oxidation of substrates for ATP synthesis, dissipating the energy of the membrane potential and, consequently, decreasing the production of ATP by the mitochondrial respiratory chain. The increased expression of UCP2 is associated with a decreased risk of obesity and increased risk of developing diabetes mellitus. The polymorphism -866G/A in the promoter region of the UCP2 gene is associated with an increased mRNA expression of this protein. The aim of this study was to evaluate the involvement of the polymorphism -866G/A UCP2 gene in the metabolic responses to exercise and basal metabolic rate (BMR) in healthy young adults. We recruited 27 men aged between 20 and 35 years without history of disease and drug treatment, eutrophic and that were not involved in any type of physical training. The individuals were placed intentionally in three different groups according to genotype for the polymorphism related above: A/A (n=9), A/G (n=10) and G/G (n=8). All subjects underwent an analysis of BMR and after a standard meal performed 30 minutes of treadmill running at an intensity equivalent to 10% below the 2nd ventilatory threshold. Fasting, before exercise, immediately after exercise, after one hour and two hours after exercise, blood samples were collected to verify the behavior of glucose, insulin and lipid profile. No differences were found between the responses of the three groups. This study concluded that the polymorphism -866G/A in an isolated way does not influence BMR and responses of glucose, insulin and lipid profile over exercise in young healthy men.

Proteínas desacopladoras (UCPs) estão presentes na membrana mitocondrial interna e por meio do transporte de prótons do espaço intermembranas para a matriz mitocondrial desacoplam a oxidação dos substratos da síntese de ATP, dissipando a energia do potencial de membrana e, conseqüentemente, diminuindo a produção de ATP pela cadeia respiratória mitocondrial. O aumento na expressão de UCP2 está relacionado com um risco diminuído de obesidade e risco aumentado de desenvolvimento de diabetes melito. O polimorfismo - 866G/A na região promotora do gene da UCP2 está associado a um aumento da expressão do RNAm desta proteína. O objetivo do presente estudo foi avaliar o envolvimento do polimorfismo -866G/A no gene UCP2 nas respostas metabólicas ao exercício e na taxa metabólica basal em jovens saudáveis. Foram recrutados 27 homens com idades entre 20 e 35 anos, sem histórico de doenças e sem uso de medicamentos, eutróficos e que não estivessem envolvidos em nenhum tipo de treinamento físico. Os indivíduos foram alocados intencionalmente em três diferentes grupos, de acordo com o genótipo para o polimorfismo em questão: A/A(n=9), A/G (n=10) e G/G (n=8). Todos os indivíduos foram submetidos a uma análise de taxa metabólica basal e após uma refeição padrão realizavam 30 minutos de corrida em esteira em intensidade equivalente a 10% abaixo do 2º limiar ventilatório. Em jejum, antes do exercício, logo após realização do exercício, uma hora após e duas horas após o exercício foram realizadas coletas de sangue para a verificação do comportamento das concentrações de glicose, insulina e perfil lipídico. Não foram encontradas diferenças entre as respostas dos três grupos. Este estudo concluiu que isoladamente o polimorfismo -866G/A não exerce influência sobre a TMB e respostas de glicose, insulina e perfil lipídico ao exercício em jovens saudáveis. / Uncoupling proteins (UCPs) are present in the inner mitochondrial membrane and through the transport of protons from the intermembrane space to the mitochondrial matrix to uncouple oxidation of substrates for ATP synthesis, dissipating the energy of the membrane potential and, consequently, decreasing the production of ATP by the mitochondrial respiratory chain. The increased expression of UCP2 is associated with a decreased risk of obesity and increased risk of developing diabetes mellitus. The polymorphism -866G/A in the promoter region of the UCP2 gene is associated with an increased mRNA expression of this protein. The aim of this study was to evaluate the involvement of the polymorphism -866G/A UCP2 gene in the metabolic responses to exercise and basal metabolic rate (BMR) in healthy young adults. We recruited 27 men aged between 20 and 35 years without history of disease and drug treatment, eutrophic and that were not involved in any type of physical training. The individuals were placed intentionally in three different groups according to genotype for the polymorphism related above: A/A (n=9), A/G (n=10) and G/G (n=8). All subjects underwent an analysis of BMR and after a standard meal performed 30 minutes of treadmill running at an intensity equivalent to 10% below the 2nd ventilatory threshold. Fasting, before exercise, immediately after exercise, after one hour and two hours after exercise, blood samples were collected to verify the behavior of glucose, insulin and lipid profile. No differences were found between the responses of the three groups. This study concluded that the polymorphism -866G/A in an isolated way does not influence BMR and responses of glucose, insulin and lipid profile over exercise in young healthy men.

Proteínas desacopladoras (UCPs) estão presentes na membrana mitocondrial interna e por meio do transporte de prótons do espaço intermembranas para a matriz mitocondrial desacoplam a oxidação dos substratos da síntese de ATP, dissipando a energia do potencial de membrana e, conseqüentemente, diminuindo a produção de ATP pela cadeia respiratória mitocondrial. O aumento na expressão de UCP2 está relacionado com um risco diminuído de obesidade e risco aumentado de desenvolvimento de diabetes melito. O polimorfismo - 866G/A na região promotora do gene da UCP2 está associado a um aumento da expressão do RNAm desta proteína. O objetivo do presente estudo foi avaliar o envolvimento do polimorfismo -866G/A no gene UCP2 nas respostas metabólicas ao exercício e na taxa metabólica basal em jovens saudáveis. Foram recrutados 27 homens com idades entre 20 e 35 anos, sem histórico de doenças e sem uso de medicamentos, eutróficos e que não estivessem envolvidos em nenhum tipo de treinamento físico. Os indivíduos foram alocados intencionalmente em três diferentes grupos, de acordo com o genótipo para o polimorfismo em questão: A/A(n=9), A/G (n=10) e G/G (n=8). Todos os indivíduos foram submetidos a uma análise de taxa metabólica basal e após uma refeição padrão realizavam 30 minutos de corrida em esteira em intensidade equivalente a 10% abaixo do 2º limiar ventilatório. Em jejum, antes do exercício, logo após realização do exercício, uma hora após e duas horas após o exercício foram realizadas coletas de sangue para a verificação do comportamento das concentrações de glicose, insulina e perfil lipídico. Não foram encontradas diferenças entre as respostas dos três grupos. Este estudo concluiu que isoladamente o polimorfismo -866G/A não exerce influência sobre a TMB e respostas de glicose, insulina e perfil lipídico ao exercício em jovens saudáveis. / Uncoupling proteins (UCPs) are present in the inner mitochondrial membrane and through the transport of protons from the intermembrane space to the mitochondrial matrix to uncouple oxidation of substrates for ATP synthesis, dissipating the energy of the membrane potential and, consequently, decreasing the production of ATP by the mitochondrial respiratory chain. The increased expression of UCP2 is associated with a decreased risk of obesity and increased risk of developing diabetes mellitus. The polymorphism -866G/A in the promoter region of the UCP2 gene is associated with an increased mRNA expression of this protein. The aim of this study was to evaluate the involvement of the polymorphism -866G/A UCP2 gene in the metabolic responses to exercise and basal metabolic rate (BMR) in healthy young adults. We recruited 27 men aged between 20 and 35 years without history of disease and drug treatment, eutrophic and that were not involved in any type of physical training. The individuals were placed intentionally in three different groups according to genotype for the polymorphism related above: A/A (n=9), A/G (n=10) and G/G (n=8). All subjects underwent an analysis of BMR and after a standard meal performed 30 minutes of treadmill running at an intensity equivalent to 10% below the 2nd ventilatory threshold. Fasting, before exercise, immediately after exercise, after one hour and two hours after exercise, blood samples were collected to verify the behavior of glucose, insulin and lipid profile. No differences were found between the responses of the three groups. This study concluded that the polymorphism -866G/A in an isolated way does not influence BMR and responses of glucose, insulin and lipid profile over exercise in young healthy men.

Mitochondrial uncoupling proteins (UCPs) belong to the superfamily of mitochondrial anion-carriers. The longest known is UCP1, predominantly expressed in brown adipose tissue, where it takes part in nonshivering thermogenesis. In the late 1990s were discovered other sequence homologs of UCP1 with tissue specific distribution. The Function of these "new" uncoupling proteins is still uncertain. It is assumed that each of the isoforms has a specific function depending on the type of tissue. This thesis showed differences in tissue transcription pattern between rat and mice using RT-PCR absolute quantification. Significant differences in pattern were found in lungs, brain and muscle. In each case UCP expression was higher in mice tissues. Mice lungs express mainly UCP2. The difference in mice brain is caused by ucp4 and ucp5 genes transcription and finally in muscle is highest content of UCP3 mRNA. We investigated whether any of ucp transcript can complement ucp2 transcripton in spleen or lungs of ucp2 -/- mice. We did not find any difference which can explain, that in isolated lung mitochondria of fasted ucp2-/- mice were uncoupled in state 4. In the last project, we found relationship between ucp2 transcription in insulinoma INS-1E cells and oxygen levels of the cultivation atmosphere.

<p>During development of type 1 and type 2 diabetes plasma insulin patterns are altered. Since the islet insulin release pattern has been implicated in this development, insulin secretion from single islets was studied and linked to the islet protein levels of uncoupling protein-2 (UCP2). Islets were isolated from NOD- and KKA^y- mice, GK- and GK-derived congenic rats, which are animal models of diabetes, and three human subjects with type 2 diabetes. At basal glucose (3 mM), insulin release from such islets was pulsatile and the amount released was comparable to that of control islets. When the glucose concentration was raised to 11 mM insulin release was essentially unchanged in islets isolated from older NOD- and KKA^y- mice, GK- and Niddm1i congenic rats, and NIDDM persons. In islets from Niddm1f congenic rats, younger NOD- and KKA^y-mice, control animals and normal human donors the secretion rate increased 2-9 fold when the glucose concentration was raised. This rise in secretion was manifested as increase of the amplitude of the insulin oscillations without affecting their frequency. Impaired glucose-induced insulin release was associated with reduction in glucose oxidation measured in NOD-islets, unaffected respiration measured in GK-islets and higher protein level of UCP2 measured in KKA^y-islets. When the UCP2 amounts in KKA^y-islets were reduced by culture to those of control islets, glucose-induced insulin secretion was essentially normalized. Our studies suggest that the deranged plasma insulin patterns observed in diabetes are related to decrease in the amplitude of insulin oscillations from the islets rather than loss of the oscillatory activity. This reduction of pulse amplitude may be related to impaired glucose metabolism and/or increased mitochondrial uncoupling. </p>