Indiana University-Purdue University Indianapolis (IUPUI) / The epidemic of obesity and associated cardiovascular complications continues to grow at an alarming rate. Currently, obesity is thought to initiate a state of chronic inflammation, which if unresolved potentially causes cardiovascular dysfunction and disease. Although poorly understood, release of inflammatory mediators and other cytokines from adipose tissue (adipocytokines) has been proposed to be the molecular link between obesity and coronary artery disease. Furthermore, the anatomic location of adipose has been increasingly recognized as a potential contributor to vascular disease. Importantly, the development of coronary atherosclerosis, a key component of heart disease, is typically found in segments of coronary arteries surrounded by perivascular adipose tissue. Accordingly, the goal of this project was to determine how perivascular adipose tissue affects coronary artery function and elucidate the critical mechanisms involved. Initial studies assessing arterial function were conducted with and without perivascular adipose tissue. Preliminary results demonstrated that factors released by perivascular adipose tissue effectively impaired coronary endothelial function both in vitro and in vivo. This observation was determined to be caused by direct inhibition of nitric oxide synthase (NOS), a critical enzyme for the production nitric oxide. Attenuation of endothelium-dependent vasodilation was independent of changes in superoxide production, smooth muscle response, or peroxide-mediated vasodilation. Additional studies revealed that perivascular adipose-induced impairment of NOS was due to increased inhibitory regulation by the β isoform of protein kinase C (PKC-β). Specifically, perivascular adipose-derived factors caused site specific phosphorylation of nitric oxide synthase at Thr-495. Additional experiments investigated how perivascular adipose-derived factors contributed to coronary artery disease in an animal model of obesity. Results from these studies indicated that perivascular adipose-derived leptin markedly exacerbated underlying endothelial dysfunction, and significantly contributed to coronary endothelial dysfunction through a PKC-β dependent mechanism. Findings from this project confirm epicardial perivascular adipose tissue as a local source of harmful adipocytokines. In addition, perivascular adipose-derived leptin was demonstrated to be a critical mediator of coronary vascular dysfunction in obesity. Together, the results strongly suggest that perivascular adipose tissue is a key contributor to coronary artery disease in obesity.

Preterm birth is the number one cause of death in neonates, accounting for 35% of neonatal mortality. The preterm birth rate in the U.S. in 2021 was 10.5%, disproportionally affected by race and ethnicity. Obese women have an increased risk of preterm pregnancy, and if delivered before 37 weeks of gestation, the offspring have higher rates of complications that extend from the neonatal period into life-long metabolic and immune adverse outcomes. In the early months after delivery, preterm infants have higher rates of adverse health outcomes than term infants, including infections, extrauterine growth restrictions, respiratory, metabolic, and neurological complications, necrotizing enterocolitis (NEC), and bronchopulmonary dysplasia (BPD). Diet for the preterm infant is crucial for infection prevention, and maternal breast milk is most beneficial when given in the first few days after birth. Expectant and breastfeeding mothers should consume appropriate food and supplements to optimize their nutrition. In addition to nutrients, bioactive components, vitamins, and minerals found in breast milk (BM), there is evidence that microbes (microbiome) are a significant factor in infant development, contributing to protection against pathogens and playing a role in the development of the immune and nervous systems. Maternal BM composition and microbiome are affected by many factors, including maternal body mass index (BMI), maternal health, antibiotics use, mode of delivery, maternal parity, gestational age, and time and duration of lactation. Maternal body composition, however, and not maternal nutritional status, is associated with breast milk nutritional composition. Altogether, these maternal factors may modify the premature infant gut microbiome. We examined the role of maternal adiposity and how it impacts the composition of human breast milk, specifically hormones, nutrient composition, short and long chain fatty acids, and microbiome. We also examined the role of maternal adiposity and how it impacts the short-chain fatty acids and microbiome in infant fecal samples. We found that maternal adiposity affects breast milk hormones, potentially modulating infant metabolism. Additionally, we found that maternal adiposity does not alter the nutrient composition of breast milk; however, differences in both short and long chain fatty acids and maternal adiposity were detected. In our small cross-sectional cohort of preterm infants, we did not observe differences in short-chain fatty acids in the preterm infant stool samples compared to maternal adiposity. Concerning maternal adiposity and its impact on the microbiomes of breast milk and infants, we observed differences in phyla and genera between the maternal BMI groups on the outcome of breast milk and preterm infant microbiomes but no statistical significance in alpha and beta diversities between the groups. Thus, our results indicate that maternal adiposity impacts hormonal, microbial composition, and short-chain fatty acid profiles in breast milk, which tremendously influences infant growth and development.

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Το αξιόπιστο πειραματικό μοντέλο της ωοθηκεκτομής σε επίμυες εφαρμόστηκε για τη μελέτη των παθογενετικών μηχανισμών της οστεοπόρωσης. Σκοπός της παρούσας διδακτορικής διατριβής ήταν η διερεύνηση του ρόλου της λεπτίνης και των κυτοκινών RANKL και οστεοπροτεγερίνης (OPG) στην εξέλιξη της οστεοπόρωσης. Για την διεκπεραίωση της μελέτης χρησιμοποιήθηκαν 40 ενήλικοι θηλυκοί επίμυες ηλικίας 9 μηνών. Πριν την ωοθηκεκτομή στους επίμυες εφαρμόστηκε η τεχνική της ποσοτικής υπολογιστικής τομογραφίας (pQCT) παράλληλα με την πρωτοποριακή μη επεμβατική τεχνική του θερμοδυναμικού συντελεστή εσωτερικής απόσβεσης (MDF) ώστε να διαπιστωθεί η κατάσταση της οστικής πυκνότητας των πειραματόζωων. Σε χρονικά διαστήματα 20, 40 και 60 ημερών μετά την ωοθηκεκτομή πραγματοποιήθηκαν τυχαίες ομαδικές θυσίες με σκοπό τη συλλογή αίματος και την απομόνωση μηριαίων οστών και οστών κνήμης. Την 60η ημέρα πριν τη θυσία στην τελευταία ομάδα των ωοθηκεκτομήθέντων επίμυων επαναλήφθηκαν οι μετρήσεις pQCT και MDF για να διαπιστωθεί η εγκατάσταση σοβαρής οστεοπόρωσης με τη λήξη της πειραματικής πορείας. Οι οροί αίματος που συλλέχθηκαν από όλα τα χρονικά πειραματικά σημεία χρησιμοποιήθηκαν για τη διερεύνηση των επιπέδων έκφρασης των βιοχημικών δεικτών του οστικού μεταβολισμού NTx και οστεοκαλσίνης καθώς και της ελεύθερης λεπτίνης με την τεχνική της ενζυμικής ανοσοπροσρόφησης (ELISA). Στα οστά της κνήμης εφαρμόστηκε η τεχνική της ιστομορφομετρίας για τη μελέτη των μεταβολών της μικροαρχιτεκτονικής δομής των οστών κατά την εξέλιξη της οστεοπόρωσης. Επιπρόσθετα η τεχνική της ανοσοϊστοχημείας εφαρμόστηκε στα οστά της κνήμης για τη διερεύνηση των μεταβολών που προκαλεί η ωοθηκεκτομή στα επίπεδα έκφρασης του υποδοχέα της λεπτίνης και των κυτοκινών RANKL και οστεοπροτεγερίνης στους οστικούς κυτταρικούς πληθυσμούς. Στα μηριαία οστά εφαρμόστηκαν οι τεχνικές της ηλεκτροφόρησης σε πηκτή πολυακρυλαμιδίου (SDS-PAGE electrophoresis) και του ανοσοστυπώματος (Western Blot) για να συλλεχθούν επιπλέον πληροφορίες σχετικά με τις μεταβολές στα επίπεδα έκφρασης του υποδοχέα της λεπτίνης και των κυτοκινών RANKL και οστεοπροτεγερίνης κατά την εξέλιξη της οτεοπόρωσης. Τα συμπεράσματα που διεξάγονται από τα επιμέρους πειραματικά δεδομένα επιβεβαιώνουν την υπόθεση ότι η λεπτίνη κατέχει σημαντικό ρόλο στη ρύθμιση του οστικού μεταβολισμού και συμμετέχει ενεργά μέσω κάποιου άγνωστου μέχρι στιγμής μηχανισμού στη διαδικασία της οστικής ανακατασκευής στην οστεοπόρωση. Όσον αφορά τις ρυθμιστικές κυτοκίνες της οστεοκλαστογένεσης RANKL και OPG, διαπιστώθηκε ότι μεταβάλλονται σημαντικά κατά την εξέλιξη της οστεοπόρωσης στους ωοθηκεκτομηθέντες επίμυες, υποδηλώνοντας τη σημαντικότητα του ρόλου τους στον οστικό μεταβολισμό. / Ovariectomy in mature rats mimics the changes in bone metabolism observed in postmenopausal women and results in osteoporosis. The aim of this thesis was to investigate the role of leptin and the cytokine RANKL and Osteoprotegerin (OPG) in the progression of ovariectomy-induced osteoporosis. Nine–month-old female Wistar rats were bilaterally ovariectomized (n=40). Before the operation, pQCT and MDF technology were applied on rats in order to estimate the bone mineral density of the animals. On days 20, 40 and 60 after the operation the rats were randomly sacrificed and blood samples, dissected knees and femurs were collected. On day 60, pQCT and MDF techniques were applied in order to confirm the establishment of severe osteoporosis until the end of the experimental procedure. Leptin, and the biochemical markers of bone metabolism, osteocalcin and NTx, were measured in blood serum from all time points, by an ELISA method. Bone sections from the knees of the rats were examined by histomorphometric techniques in order to investigate the alterations in the micro-architectural structure of the skeleton caused by ovariectomy. Furthermore, immunohistochemistry was applied on knee sections and SDS-PAGE electrophoresis and western blot techniques were performed in femur homogenized tissueς, in order to investigate whether the expression levels of leptin receptor, RANKL and OPG were altered during the progression of osteoporosis. The results indicate that leptin is involved in the molecular mechanisms of bone remodeling in osteoporosis. The regulators of osteoclastogenesis, RANKL and OPG are also important players in the field of bone metabolism

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Neste estudo, foi avaliada a relação entre polimorfismos dos genes da leptina (LEP) e receptores da leptina (LEPR) e parâmetros antropométricos, leptinemia glicemia e lipídeos séricos, em indivíduos da população brasileira. Foram incluídos 238 indivíduos com idade entre 30 e 80 anos. Foram medidos o índice de massa corporal (IMC), a cintura abdominal (CA) e a razão cintura quadril (RCQ). Amostras de sangue periférico foram obtidas para análise do perfil bioquímico e extração de DNA. Os polimorfismos de nuleotideo único (SNPs) LEP G-2548A e LEPR Lys109Arg, Gln223Arg e Lys656Asn foram detectados por PCR-RFLP. Os SNPs LEPR Lys109Arg e Gln223Arg foram associados com obesidade e com IMC e CA aumentados (p<0.05). Estes polimorfismos também foram associados com leptina e glicose elevada (p<0,05). O perfil lipídico sérico foi influenciado pelo polimorfismo LEPR Lys109Arg (p<0.05). A relação entre os SNPs LEPR Lys109Arg e Gln223Arg e o perfil lipídico foi modificada pelo gênero. Os haplótipos LEP G-2548/ LEPR Lys109Arg foram relacionados com diferenças no IMC de obesos. Os haplotipos LEPR Lys109Arg/Gln223Arg foram associados com diferenças na CA e glicemia e lipídeos séricos. Em conclusão, os polimorfismos LEPR Lys109Arg e Gln223Arg estão associados com obesidade e alterações de leptina, glicose e lipídeos circulantes de forma dependente do gênero. / We have assessed the relationship between polymorphisms of the leptin (LEP) and the leptin receptor (LEPR) genes and anthropometric parameters, plasma leptin and glucose and serum lipids in individuals of the Brazilian population. We included 238 individuais with 30 to 80 years. Body mass index (BMI), abdominal circumference (AC) and waist-to-hip ratio (WHR) were measured. Peripheral blood samples were collected for analysis of the biochemical profile and DNA extraction. The single nucleotide polymorphisms (SNP) LEP G-2548A and LEPR Lys109Arg, Gln223Arg and Lys656Asn were detected by PCR-RFLP. The SNPs LEPR Lys109Arg and Gln223Arg were associated with obesity and with increased BMI and AC (p <0.05). These polymorphisms were also associated with increase leptin and glucose (p<0,05). The serum lipid profile was influenced by the LEPR Lys 1 09Arg (p<0.05). The relationship between the SNPs LEPR Lys 1 09Arg and Gln223Arg and the lipid profile was modified by gender. The haplotypes LEP G-2548A1 LEPR Lys109Arg were related with differences on BMI in obese group. The haplotypes LEPR Lys109Arg/Gln223Arg were associated with differences on AC, glucose and serum lipids. In conclusion, the LEPR Lys109Arg and Gln223Arg polymorphisms are associated with obesity and alterations in blood leptin, glucose and lipids in a gender-dependent manner.

INTRODUÇÃO: A cardiopatia chagásica crônica (CCC) apresenta características específicas, tais como: disfunção autonômica e atividade inflamatória exacerbada. Esta fisiopatologia sugere que alguns parâmetros metabólicos podem estar alterados em pacientes chagásicos. O objetivo deste estudo foi avaliar os parâmetros metabólicos e inflamatórios nas diversas formas evolutivas de doença de Chagas e sua correlação com medidas de avaliação do Sistema Nervoso Autônomo (SNA). MÉTODOS: Foram avaliados 60 indivíduos divididos em 4 grupos (n=15): Grupo controle (GC), Grupo FI - forma indeterminada, Grupo ECG- cardiopatia chagásica com alteração eletrocardiográfica sem disfunção ventricular e Grupo IC - cardiopatia chagásica com disfunção ventricular e insuficiência cardíaca. Todos os grupos foram pareados de acordo com sexo, idade e índice de massa corporal. Os pacientes realizaram dosagens sanguíneas de insulina, leptina, adiponectina, interleucina-6 (IL- 6) e fator de necrose tumoral-alfa (TNF-alfa) pelo método de ELISA. O SNA foi avaliado através da variabilidade da frequência cardíaca no holter 24 horas e no teste de inclinação postural. Os valores de RMSSD, pNN50 e do componente alta frequência (AF) foram utilizados como estimativa da atividade parassimpática. Os valores do componente de baixa frequência (BF) estimaram a atividade simpática. A análise estatística foi feita utilizando-se a ANOVA ou teste de Kruskal-Wallis para a comparação entre os grupos, o coeficiente de Spearman para a análise das correlações e a regressão linear múltipla para a análise multivariada. RESULTADOS: A leptina e insulina não apresentaram diferenças significativas entre os grupos [Leptina: GC=3,42 (7,43); FI=3,03 (6,53); ECG=5,56 (6,2); IC=2,86 (2,67) ng/ml; p=0,626. Insulina: GC=3,41 (1,98); FI=4,31 (2,85); ECG=4,30 (3,06); IC=4,58 (2,88) ng/ml; p=0,901] A adiponectina apresentou níveis maiores nos grupos ECG e IC [GC=4766,5 (5529,5); FI= 4003,5 (2482,5); ECG= 8376,5 (8388,5); IC= 8798 (4188) ng/ml; p < 0,001]. IL-6 e TNF-alfa foram maiores no Grupo IC [IL-6: GC=1,85 (6,41); FI=1,58 (1,91); ECG=1,0 (1,57); IC= 31,44 (72,19) pg/ml; p=0,001. TNF-?: GC=22,57 (88,2); FI=19,31 (33,16); ECG=12,45 (3,07); IC=75,15 (278,57) pg/ml; p=0,04]. A insulina, leptina e TNF-alfa não apresentaram correlações significativas com medidas de avaliação do SNA. A adiponectina apresentou correlação positiva com o componente AF (r= 0,336; p= 0,009) e correlação negativa com o componente BF (r= -0,336; p= 0,009). A interleucina-6 apresentou correlação positiva com o componente AF (r= 0,419; p=0,004) e correlação negativa com o componente BF (r= -0,393; p= 0,007). Porém, na análise multivariada apenas a adiponectina apresentou correlação significativa com medidas de função do SNA. CONCLUSÃO: A adiponectina foi maior nos grupos ECG e IC. A IL-6 e o TNF-alfa foram maiores no grupo IC. O aumento dos níveis de adiponectina esteve associado a diminuição da atividade simpática e predomínio da atividade parassimpática. / BACKGROUND: Chagas disease (CD) has specific characteristics such as autonomic dysfunction and increased inflammatory activity. This pathophysiology suggests that metabolic parameters can be altered in patients with CD. The aim of this study was to evaluate the metabolic and inflammatory parameters in different forms of CD and their correlation with Autonomic Nervous System (ANS) measures. METHODS: We evaluated 60 subjects divided into 4 groups (n=15): control group (CG), group IF (indeterminate form); group ECG (ECG abnormalities and normal left ventricular function in echocardiogram) and HF group (heart failure with left ventricular dysfunction). All groups were matched for age, sex and body mass index. The patients underwent insulin, adiponectin, leptin, interleukin-6 (IL-6) and tumor necrosis factor-alfa (TNF-alfa) measurements by ELISA. The Autonomic Nervous System was assessed by heart rate variability in 24-hour Holter and tilt test. RMSSD, pNN50 and High Frequency (HF) component values were used to estimate parasympathetic activity and low frequency (LF) components were used to estimate sympathetic activity. Statistical analyses were performed using ANOVA or Kruskal- Wallis tests to compare groups. Spearman coefficient was used for correlation analysis and linear regression for multivariate analysis. RESULTS: No significant differences were observed in leptin and insulin levels between groups. [Leptin: CG=3.42 (7.43); IF=3.03 (6.53); ECG=5.56 (6.2); HF=2.86 (2.67) ng/ml; p=0.626. Insulin: CG=3.41 (1.98); IF=4.31 (2.85); ECG=4.30 (3.06); HF=4.58 (2.88) ng/ml; p=0.901]. Adiponectin was higher in ECG and HF groups. [CG=4766.5 .(5529.5); IF= 4003.5 (2482.5); ECG= 8376.5 (8388.5); HF= 8798 (4188) ng/ml; p < 0.001)]. IL-6 and TNF-alfa were higher in HF group. [IL-6: CG=1.85 (6.41); IF=1.58 (1.91); ECG=1.0 (1.57); HF= 31.44 (72.19) pg/ml; p=0.001. TNF-alfa: CG=22.57 (88.2); IF=19.31 (33.16); ECG=12.45 (3.07); HF=75.15 (278.57) pg/ml; p=0.04]. Insulin, leptin and TNF-alfa did not correlate with autonomic dysfunction. Adiponectin correlated positively with HF component (r=0.336; p= 0.009) and inversely with LF component (r= -0.336; p=0.009). IL-6 correlated positively with HF component (r= 0.419; p=0.004) and inversely with LF component (r= -0.393; p= 0.007). However, in multivariate analysis only adiponectin correlated significantly with ANS measures. CONCLUSION: Adiponectin levels were higher in ECG and HF groups. IL-6 and TNF-alfa were higher in HF group. Higher levels of adiponectin were associated with reduced sympathetic activity and predominance of parasympathetic activity

Le maintien de l’homéostasie énergétique est placé sous le contrôle de cytokines qui agissent dans le système nerveux central, notamment au niveau de l’hypothalamus. En particulier, la leptine, cytokine produite par le tissu adipeux, diminue la prise alimentaire et stimule la perte de poids. L’obésité est une épidémie mondiale qui progresse de façon alarmante, notamment chez les enfants et souvent associée à des pathologies sévères et des désordres endocriniens comme la résistance à la leptine ou à l’insuline. Le CNTF (Ciliary Neurotrophic Factor) est une neurocytokine de la même famille que la leptine dont l’un des principaux avantages est qu’il stimule la perte de poids dans les cas de leptino-résistance en activant les mêmes voies de signalisation que la leptine (Benomar et al., 2009). Face à l’épidémie mondiale d’obésité,chez la population adulte comme enfantine, il apparaît nécessaire de décrypter les mécanismes impliqués dans la genèse de la maladie ainsi que les potentiels agents thérapeutiques.L’objectif premier de ce travail de thèse a été de caractériser l’impact d’une alimentation maternelle hyperlipidique (HF) sur les capacités de contrôle de l’homéostasie énergétique chez la descendance. En effet, le concept de « programmation métabolique » propose que des pertubations de l’environnement périnatal puissent influencer durablement la descendance, la rendant plus susceptible de développer une obésité dans un contexte nutrionnellement riche.Des études menées au sein du laboratoire ont montré qu’un régime maternel HF pouvait programmer l’acquisition de la leptino-résistance chez la descendance à l’âge adulte (Ferezou-Viala et al., 2007b). Nous avons donc testé la prédispostion de ces animaux à prendre du poids lorsqu’ils étaient nourris avec un régime hypercalorique (P). Nos données ont montréqu’étonnamment, le régime maternel HF protégeait la descendance contre le gain de poids induite par le régime P, induisait des modifications d’expression des marqueurs de l’homéostasie énergétique dans le foie et l’hypothalamus, ainsi que de profondes réorganisations cytoarchitectoniques dans le noyau arqué. Plus précisément, le régime maternel HF était associé à une réorganisation de la couverture astrocytaire périvasculaire dans le noyau arqué de la descendance qui persistait à l’âge adulte.Dans une seconde partie de la thèse, nous avons étudié les mécanismes d’action du CNTF. En effet, notre équipe a récemment mis en évidence que le CNTF endogène pourrait jouer un rôle dans la régulation de l’homéostasie énergétique. Les niveaux hypothalamiques de cette cytokine, présente dans les astrocytes et les neurones du noyau arqué, augmentent chez les animaux résistant à une alimentation hypercalorique. Cela pourrait suggérer un rôle protecteur du CNTF contre la prise de poids chez certains individus (Vacher et al., 2008). A ce jour, les mécanismes d’action du CNTF restent cependant mal compris car ce dernier ne possède pas de peptide signal et n’est donc pas sécrété selon des mécanismes d’exocytose classiques. Partant du constat que le CNTF et ses sous-unités réceptrices étaient distribuées de façon similaire dans les cellules du noyau arqué, nous avons émis l’hypothèse que le CNTF pourrait exercer une action intracellulaire sur les cellules de cette structure. Dans cette étude nous démontrons que le CNTF peut interagir directement avec ses récepteurs dans le noyau des neurones anorexigènes du noyau arqué, pour réguler leur activité transcriptionnelle. Ces données proposent ainsi un nouveau mécanisme à l’action anorexigène du CNTF / Obesity is a major health disease which involves numerous metabolic disorders. Increasing evidence suggests that the risk of developing the pathology in adulthood may be influenced through inappropriate perinatal nutrition. In our study, we first investigated the impact of a maternal high-fat (HF) diet, which is known to induce hypothalamic leptin resistance in adult offspring (Férézou-Viala et al., 2007), to develop obesity in a rich diet environment (P diet). Our results showed that surprisingly, HF maternal diet protected offspring against body weight gain induced by P diet. In a second part of the thesis, we studied mechanisms of action of CNTF, a neurocytokine which could protect some people against body weight gain induced by a P diet (Vacher et al., 2008). Results of this study showed that CNTF ant its subunits receptors could translocate to the hypothalamic cell nucleus to induced POMC transcription.