O consumo de ácidos graxos n-3 tem sido associado à proteção contra a obesidade, inflamação e resistência à insulina. Os n-3 são ligantes fracos dos receptores nucleares PPARγ, e pela ativação deste podem exercer suas ações metabólicas e anti-inflamatórias. No presente trabalho, foi investigado se o aumento da disponibilidade dos n-3 geneticamente ou por dieta, via ativação de PPARγ, protege camundongos do desenvolvimento da obesidade, intolerância a glicose e inflamação do tecido adiposo. Foi visto em um modelo com camundongos fat-1 (elevados níveis endógenos de n-3) que dentre as ações dos ácidos graxos n-3, a proteção contra o aumento de peso/adiposidade associada à obesidade, bem como a melhora da intolerância à glicose são dependentes de PPARγ. Além disso, por meio da utilização de camundongos com deleção de PPARγ em hepatócitos os dados desse trabalho mostram que os PPARγ são, ao menos em parte, essenciais ao aumento da oxidação de ácidos graxos induzida pelos n-3 devido à modulação da expressão gênica e protéica de enzimas mitocondriais e peroxissomais. / The intake of n-3 fatty acids have been associated to the protection against obesity, inflammation and insulin resistance. The n-3 fatty acids are ligands of the nuclear receptor PPARγ, and by the activation of this receptor can promote their metabolic and anti-inflammatory effects. Herein, we investigated whether increasing body n-3 fatty acids levels either genetically or by a n-3 enriched diet protects mice from diet-induced obesity, glucose intolerance and adipose tissue inflammation through PPARγ activation. Fat-1 mice were protected from diet-induced obesity, glucose intolerance and adipose tissue inflammation. To better investigate PPARγ involvement in n-3 beneficial actions, mice with genetic deletion of PPARγ specifically in hepatocytes. In spite of the absence of changes in body weight and glucose homeostasis PPARγ deletion in hepatocytes completely abolished the increase in liver fatty acid oxidation and hepatic gene expression of genes associated to mitochondrial and peroxisomal activity.

Les sécrétines de l’hormone de croissance (GHRPs) sont de petits peptides synthétiques capables de stimuler la sécrétion de l’hormone de croissance à partir de l’hypophyse via leur liaison au récepteur de la ghréline GHS-R1a. Le GHRP hexaréline a été utilisé afin d’étudier la distribution tissulaire de GHS-R1a et son effet GH-indépendant. Ainsi, par cette approche, il a été déterminé que l’hexaréline était capable de se lier à un deuxième récepteur identifié comme étant le récepteur scavenger CD36. Ce récepteur possède une multitude de ligands dont les particules oxLDL et les acides gras à longue chaîne. CD36 est généralement reconnu pour son rôle dans l’athérogénèse et sa contribution à la formation de cellules spumeuses suite à l’internalisation des oxLDL dans les macrophages/monocytes. Auparavant, nous avions démontré que le traitement des macrophages avec l’hexaréline menait à l’activation de PPARƔ via sa liaison à GHS-R1a, mais aussi à CD36. De plus, une cascade d’activation impliquant LXRα et les transporteurs ABC provoquait également une augmentation de l’efflux du cholestérol. Une stimulation de la voie du transport inverse du cholestérol vers les particules HDL entraînait donc une diminution de l’engorgement des macrophages de lipides et la formation de cellules spumeuses. Puisque CD36 est exprimé dans de multiples tissus et qu’il est également responsable du captage des acides gras à longue chaîne, nous avons voulu étudier l’impact de l’hexaréline uniquement à travers sa liaison à CD36. Dans le but d’approfondir nos connaissances sur la régulation du métabolisme des lipides par CD36, nous avons choisi des types cellulaires jouant un rôle important dans l’homéostasie lipidique n’exprimant pas GHS-R1a, soient les adipocytes et les hépatocytes. L’ensemble de mes travaux démontre qu’en réponse à son interaction avec l’hexaréline, CD36 a le potentiel de réduire le contenu lipidique des adipocytes et des hépatocytes. Dans les cellules adipeuses, l'hexaréline augmente l’expression de plusieurs gènes impliqués dans la mobilisation et l’oxydation des acides gras, et induit également l’expression des marqueurs thermogéniques PGC-1α et UCP-1. De même, hexaréline augmente l’expression des gènes impliqués dans la biogenèse mitochondriale, un effet accompagné de changements morphologiques des mitochondries; des caractéristiques observées dans les types cellulaires ayant une grande capacité oxydative. Ces résultats démontrent que les adipocytes blancs traités avec hexaréline ont la capacité de se transformer en un phénotype similaire aux adipocytes bruns ayant l’habileté de brûler les acides gras plutôt que de les emmagasiner. Cet effet est également observé dans les tissus adipeux de souris et est dépendant de la présence de CD36. Dans les hépatocytes, nous avons démontré le potentiel de CD36 à moduler le métabolisme du cholestérol. En réponse au traitement des cellules avec hexaréline, une phosphorylation rapide de LKB1 et de l’AMPK est suivie d’une phosphorylation inhibitrice de l’HMG-CoA réductase (HMGR), l’enzyme clé dans la synthèse du cholestérol. De plus, la liaison d'hexaréline à CD36 provoque le recrutement d’insig-2 à HMGR, l’étape d’engagement dans sa dégradation. La dégradation de HMGR par hexaréline semble être dépendante de l’activité de PPARƔ et de l’AMPK. Dans le but d’élucider le mécanisme d’activation par hexaréline, nous avons démontré d’une part que sa liaison à CD36 provoque une déphosphorylation de Erk soulevant ainsi l’inhibition que celui-ci exerce sur PPARƔ et d’autre part, un recrutement de l’AMPK à PGC-1α expliquant ainsi une partie du mécanisme d’activation de PPARƔ par hexaréline. Les résultats générés dans cette thèse ont permis d’élucider de nouveaux mécanismes d’action de CD36 et d'approfondir nos connaissances de son influence dans la régulation du métabolisme des lipides. / Growth hormone releasing peptides (GHRPs) are small synthetic peptides aimed at stimulating GH release from the pituitary through their binding to ghrelin receptor known as growth hormone secretagogue receptor 1a (GHS-R1a). Using the GHRP, hexarelin to study tissue distribution of GHS-R1a and its GH-independent effect, it was observed that hexarelin was capable of binding to a second receptor identified as scavenger receptor CD36. While having multiple ligands, CD36 is mainly known for binding and internalizing oxLDL and long chain fatty acids. CD36 is thought to play a detrimental role in macrophage derived foam cell formation and development of atherosclerosis. Previously, we have shown that in macrophages, expressing both GHS-R1a and CD36, hexarelin promoted an activation of PPARƔ via GHS-R1a but also through its binding to CD36. This activation led to the induction of the LXRα-ABC transporters pathway and an increase in cholesterol efflux, reducing lipid-laden macrophage content. This positive effect on macrophages was reproduced in apolipoprotein E-null mice on a high fat diet treated with hexarelin. A significant reduction in the size of atherosclerotic lesions was observed while similar increases in the expression of PPARƔ, LXRα and ABC transporters occurred in isolated peritoneal macrophages. CD36 also plays a role in fatty acid uptake, and to further investigate the impact of the interaction of hexarelin with CD36, we aimed at evaluating the role of CD36 in regulating lipid metabolism in cells devoid of GHS-R1a such as adipocytes and hepatocytes. In the present thesis, we demonstrated through its interaction with hexarelin, the ability of CD36 to decrease intracellular lipid content in both adipocytes and hepatocytes. In adipocytes, hexarelin was able to increase the expression of several genes involved in fatty acid mobilization, fatty acid oxidation but also to induce the expression of the thermogenic markers, PGC-1α and UCP-1. In addition, hexarelin increased the expression of genes involved in mitochondrial biogenesis which was accompanied by mitochondrial morphological changes in agreement with what is usually seen in highly oxidative cells. In support of these findings, we also observed an increase in the activity of cytochrome c oxidase (a component of the respiratory chain) which could reflect an increase in oxidative phosphorylation. The results generated with cultured white adipocytes suggest the ability of hexarelin to promote changes toward a brown fat-like phenotype which also occurred in vivo and was dependent on the presence of CD36. In hepatocytes, CD36 was capable of regulating cholesterol metabolism by rapidly phosphorylating LKB1 and AMPK which subsequently resulted in the inactivating phosphorylation of HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis. Hexarelin via CD36 also induced the recruitment of insig-2 to HMGR, the committed step in HMGR degradation while lifting the exerted inhibitory effect of Erk on nuclear receptor PPARƔ activity, and promoting the recruitment of AMPK to PPARƔ coactivator PGC-1α, suggesting an enhanced transcriptional potential of PPARƔ. The results generated during my graduate studies represent unique and novel mechanisms by which CD36 is capable of regulating lipid metabolism.

Bien que le changement dans le choix des substrats énergétiques des acides gras (AGs) vers les glucides soit considéré comme bénéfique pour le cœur insuffisant, il n’est pas clair à savoir pourquoi les patients atteints de désordres de la β-oxydation (β-OX) des AGs à chaîne longue (AGCLs) développent des troubles du rythme et des cardiomyopathies. De plus, le traitement actuel ne permet pas de prévenir l’apparition du phénotype clinique chez tous les patients, spécifiquement en condition de jeûne ou de stress. Ainsi, plusieurs modèles de souris déficientes pour des enzymes impliquées dans l’oxydation des acides gras ont été développés de manière à améliorer les connaissances de la maladie ainsi que les traitements offerts aux patients. À cet égard, cette étude vise à évaluer le phénotype métabolique et fonctionnel des cœurs de souris déficientes pour le récepteur activé de la prolifération des peroxysomes-α (PPARα), un facteur de transcription des gènes impliqués notamment dans la β-OX des AGs, et pour la déshydrogénase des acyl-CoA à très longue chaîne (very-long chain acyl-CoA dehydrogenase, VLCAD), le déficit de l’oxydation des AGCLs le plus commun chez l’humain. L’approche expérimentale utilisée comprend plusieurs techniques dont (i) la perfusion ex vivo de cœur de souris au travail combinée à l’utilisation de substrats marqués au carbone 13 (13C) et à l’analyse par chromatographie gazeuse-spectrométrie de masse (GCMS), (ii) l’analyse de l’expression génique par qPCR et (iii) l’analyse de l’activité électrique du cœur in vivo par télémétrie. De manière inattendue, les résultats de cette étude menée chez la souris ont permis de mettre en évidence que des déficits pour des protéines impliquées dans l’oxydation des AGCLs sont associés à des altérations du métabolisme (i) des glucides, (ii) des AGs polyinsaturés (AGPIs), et (iii) mitochondrial, incluant l’anaplérose, en plus d’être liés à des désordres de la fonction électrique du cœur, à savoir une prolongation du segment QTc. Pris dans leur ensemble, les résultats de cette thèse pourraient servir à l’élaboration de nouvelles interventions métaboliques destinées à améliorer les traitements possibles et donc, la qualité de vie des patients atteints de désordres héréditaires de la β-OX des AGCLs. / While a shift from fatty acids to carbohydrate is considered beneficial for the failing heart, it is unclear why patients with fatty acid oxidation disorders present clinical manifestations such as cardiomyopathy, arrhythmia and conduction defects. Unfortunately, the current nutritional treatment for these patients is limited in its ability to prevent these symptoms, especially under fasting and stress conditions. Many mouse models of fatty acid oxidation deficiency have been developed to improve the knowledge of the disease and the treatment of these patients. In this regard, this study aims to characterize the metabolic and functional phenotype of hearts from mice that are deficient for the peroxisome proliferator-activated receptor α, a transcription factor for gene involved in fatty acid oxidation, and very long chain acyl-CoA dehydrogenase, the most common inherited long chain fatty acid oxidation disorder in human, under various conditions. In this study, numerous approaches have been used, which includes validated experimental paradigms, namely, (i) ex vivo heart perfusion in the working mode with concomitant evaluation of myocardial contractility and metabolic fluxes, employing 13C-labeled substrates combined with mass isotopomer analysis by gas chromatography coupled to mass spectrometry, (ii) gene expression analysis by qPCR and (iii) electrocardiogram monitoring in vivo by telemetry. Unexpectedly, results from the present thesis demonstrate that fatty acid oxidation disorders cause alterations in metabolism of (i) carbohydrates (ii) polyunsaturated fatty acids of the omega-3 type, specifically docosahaexanoic acid, and (iii) mitochondria including anaplerosis, in addition to lead to functional abnormalities, namely a prolongation of the QT interval. Altogether, results from this thesis could contribute to new metabolic therapy development to improve the quality of life of the patients with inherited long chain fatty acid oxidation disorder.

Submitted by Edineia Teixeira (edineia.teixeira@unioeste.br) on 2018-02-27T12:47:21Z No. of bitstreams: 2 Fernanda_Morita2017.pdf: 1916527 bytes, checksum: d53cab0d448357d03f1447766ef5948a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-02-27T12:47:21Z (GMT). No. of bitstreams: 2 Fernanda_Morita2017.pdf: 1916527 bytes, checksum: d53cab0d448357d03f1447766ef5948a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-08-25 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Objective: Here, we compared the effects of Roux-en-Y gastric bypass (RYGB) and sleeve gastrectomy (SG) on fat liver deposition and expression of hepatic enzymes involved in hepatic de novo (DN) lipogenesis and β-oxidation, in western diet (WD)-obese rats. Background: Bariatric operations are known to improve non-alcoholic fatty liver disease (NAFLD), often found in obese humans. However, comparative studies on the efficacy of different bariatric procedures against NAFLD are scarce. Methods: At two months after WD consumption, the rats were divided into three groups: WD sham operation (WD-Sham), WD-RYGB and WD-SG. Three months after procedures, hepatic steatosis and lipid metabolism were verified. Results: After 3 months, body weight and abdominal fat mass were lower in WD-RYGB, compared with WD-SG rats. Both bariatric operations enhanced glucose tolerance and decreased triglycerides (TG) serum levels. However, total serum cholesterol (CHOL) as well as, hepatic TG and CHOL contents were reduced only in liver of WD-RYGB rats. Hepatic steatosis was corrected in 83% of the WD-RYGB rats, whereas microvesicular steatosis occurred in 100% of the WD-SG livers. Reduction in FASN protein content was observed in both WD-RYGB and WD-SG rats. However, reduced hepatic ACC and enhanced pACC/ACC and CPT-1a protein levels were observed only in WD-RYGB rats. Conclusions: NAFLD is more marked reduced in obese rats that underwent RYGB than SG procedures. This RYGB effect may be associated with decreased hepatic DN lipogenesis, associated with enhancement in β-oxidation, which reduced TG and CHOL content in the liver and serum of WD rats. / Introdução: As operações bariátricas são conhecidas por melhorar a doença hepática gordurosa não alcoólica (DHGNA), frequentemente encontrada em indivíduos obesos. No entanto, estudos que comparam a eficácia de diferentes procedimentos bariátricos sobre a DHGNA são escassos. Objetivo: Nesse trabalho, são comparados os efeitos da Derivação Gástrica em Y de Roux (DGYR) e da Gastrectomia Vertical (GV) sobre a esteatose hepática e sobre a expressão de enzimas envolvidas na lipogênese de novo e β-oxidação no fígado de ratos obesos pela dieta de cafeteria. Métodos: Dois meses após o consumo de dieta de cafeteria, os ratos foram divididos aleatoriamente em três grupos: cafeteria pseudo-cirúrgico (CAF-PC), cafeteria derivação gástrica em Y Roux (CAF-DGYR) e cafeteria gastrectomia vertical (CAF-GV). Três meses após os procedimentos cirúrgicos, a esteatose hepática e o metabolismo lipídico foram avaliados. Resultados: Três meses após os procedimentos operatórios, os animais CAF-DGYR apresentaram menor peso corporal e gordura abdominal em comparação com os ratos CAF-PC e CAF-GV. Ambas as operações bariátricas aumentaram a tolerância à glicose e diminuíram a concentração de triglicerídeos (TG) plasmáticos. No entanto, o colesterol total (COL), bem como as concentrações de TG e COL hepáticos, foram reduzidos apenas no fígado dos ratos CAF-DGYR. A esteatose hepática foi corrigida em 83% dos ratos CAF-DGYR, enquanto que os animais CAF-GV apresentaram 100% de esteatose microvesicular. Os ratos CAF-DGYR e CAF-GV apresentaram redução na expressão proteica da FASN. No entanto, apenas os animais CAF-DGYR tiveram redução da proteína ACC e aumento da razão da pACC/ACC e CPT-1a. Conclusões: A operação de DGYR foi mais eficaz na redução da DHGNA em ratos obesos, em comparação com a GV. Esse efeito da DGYR pode estar associado à diminuição da lipogênese hepática de novo, associado ao aumento da β-oxidação, que levou à redução das concentrações de TG e COL no plasma e no fígado de ratos obesos por dieta de cafeteria.

O consumo de ácidos graxos n-3 tem sido associado à proteção contra a obesidade, inflamação e resistência à insulina. Os n-3 são ligantes fracos dos receptores nucleares PPARγ, e pela ativação deste podem exercer suas ações metabólicas e anti-inflamatórias. No presente trabalho, foi investigado se o aumento da disponibilidade dos n-3 geneticamente ou por dieta, via ativação de PPARγ, protege camundongos do desenvolvimento da obesidade, intolerância a glicose e inflamação do tecido adiposo. Foi visto em um modelo com camundongos fat-1 (elevados níveis endógenos de n-3) que dentre as ações dos ácidos graxos n-3, a proteção contra o aumento de peso/adiposidade associada à obesidade, bem como a melhora da intolerância à glicose são dependentes de PPARγ. Além disso, por meio da utilização de camundongos com deleção de PPARγ em hepatócitos os dados desse trabalho mostram que os PPARγ são, ao menos em parte, essenciais ao aumento da oxidação de ácidos graxos induzida pelos n-3 devido à modulação da expressão gênica e protéica de enzimas mitocondriais e peroxissomais. / The intake of n-3 fatty acids have been associated to the protection against obesity, inflammation and insulin resistance. The n-3 fatty acids are ligands of the nuclear receptor PPARγ, and by the activation of this receptor can promote their metabolic and anti-inflammatory effects. Herein, we investigated whether increasing body n-3 fatty acids levels either genetically or by a n-3 enriched diet protects mice from diet-induced obesity, glucose intolerance and adipose tissue inflammation through PPARγ activation. Fat-1 mice were protected from diet-induced obesity, glucose intolerance and adipose tissue inflammation. To better investigate PPARγ involvement in n-3 beneficial actions, mice with genetic deletion of PPARγ specifically in hepatocytes. In spite of the absence of changes in body weight and glucose homeostasis PPARγ deletion in hepatocytes completely abolished the increase in liver fatty acid oxidation and hepatic gene expression of genes associated to mitochondrial and peroxisomal activity.

Newborn screening programs measure analyte levels in neonatal blood spots to identify individuals at high risk of disease. Carnitine and acylcarnitine levels are primary markers used in the detection of fatty acid oxidation disorders. These analytes may be influenced by certain pre/perinatal or newborn screening related factors. The primary objective of this study was to explore the association between these characteristics and levels of blood carnitines and acylcarnitines in the newborn population. The study was composed of two parts: a systematic review and a clinical database analysis of existing newborn screening data. The systematic review results suggested considerable variability across studies in the presence and directionality of associations between analyte levels and birth weight, gestational age, age at time of blood spot collection, type of sample, and storage time. Sex was not significantly associated with carnitine or acylcarnitine levels in neonatal blood. We identified a need to more fully investigate a potential interaction between gestational age and birth weight in regard to analyte levels. The secondary data analyses indicated a statistically significant relationship between analyte levels and all perinatal / infant and newborn screening related factors of interest, but effect sizes were generally small. The interaction between gestational age and birth weight was significant in all models; when further explored through graphical analysis with conditional means, extremely premature neonates stood out as having distinct analyte patterns in relation to birth weight. Variation in the ratio of total acylcarnitine to free carnitine was better accounted for by the perinatal and newborn factors than was variation in any individual carnitine or acylcarnitine, indicating that proportions of carnitine and acylcarnitines may be more important in understanding an individual’s metabolic functioning than individual analyte levels. A low proportion of variation was explained in all multivariate models, supporting the use of universal algorithms in newborn screening and suggesting the need for further large scale empirical research targeted at previously unaccounted for perinatal factors such as birth stress.

Newborn screening programs measure analyte levels in neonatal blood spots to identify individuals at high risk of disease. Carnitine and acylcarnitine levels are primary markers used in the detection of fatty acid oxidation disorders. These analytes may be influenced by certain pre/perinatal or newborn screening related factors. The primary objective of this study was to explore the association between these characteristics and levels of blood carnitines and acylcarnitines in the newborn population. The study was composed of two parts: a systematic review and a clinical database analysis of existing newborn screening data. The systematic review results suggested considerable variability across studies in the presence and directionality of associations between analyte levels and birth weight, gestational age, age at time of blood spot collection, type of sample, and storage time. Sex was not significantly associated with carnitine or acylcarnitine levels in neonatal blood. We identified a need to more fully investigate a potential interaction between gestational age and birth weight in regard to analyte levels. The secondary data analyses indicated a statistically significant relationship between analyte levels and all perinatal / infant and newborn screening related factors of interest, but effect sizes were generally small. The interaction between gestational age and birth weight was significant in all models; when further explored through graphical analysis with conditional means, extremely premature neonates stood out as having distinct analyte patterns in relation to birth weight. Variation in the ratio of total acylcarnitine to free carnitine was better accounted for by the perinatal and newborn factors than was variation in any individual carnitine or acylcarnitine, indicating that proportions of carnitine and acylcarnitines may be more important in understanding an individual’s metabolic functioning than individual analyte levels. A low proportion of variation was explained in all multivariate models, supporting the use of universal algorithms in newborn screening and suggesting the need for further large scale empirical research targeted at previously unaccounted for perinatal factors such as birth stress.

Newborn screening programs measure analyte levels in neonatal blood spots to identify individuals at high risk of disease. Carnitine and acylcarnitine levels are primary markers used in the detection of fatty acid oxidation disorders. These analytes may be influenced by certain pre/perinatal or newborn screening related factors. The primary objective of this study was to explore the association between these characteristics and levels of blood carnitines and acylcarnitines in the newborn population. The study was composed of two parts: a systematic review and a clinical database analysis of existing newborn screening data. The systematic review results suggested considerable variability across studies in the presence and directionality of associations between analyte levels and birth weight, gestational age, age at time of blood spot collection, type of sample, and storage time. Sex was not significantly associated with carnitine or acylcarnitine levels in neonatal blood. We identified a need to more fully investigate a potential interaction between gestational age and birth weight in regard to analyte levels. The secondary data analyses indicated a statistically significant relationship between analyte levels and all perinatal / infant and newborn screening related factors of interest, but effect sizes were generally small. The interaction between gestational age and birth weight was significant in all models; when further explored through graphical analysis with conditional means, extremely premature neonates stood out as having distinct analyte patterns in relation to birth weight. Variation in the ratio of total acylcarnitine to free carnitine was better accounted for by the perinatal and newborn factors than was variation in any individual carnitine or acylcarnitine, indicating that proportions of carnitine and acylcarnitines may be more important in understanding an individual’s metabolic functioning than individual analyte levels. A low proportion of variation was explained in all multivariate models, supporting the use of universal algorithms in newborn screening and suggesting the need for further large scale empirical research targeted at previously unaccounted for perinatal factors such as birth stress.