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Indiana University-Purdue University Indianapolis (IUPUI) / Primary cilia are solitary cellular appendages that function as signaling centers for cells in adult energy homeostasis. Here in chapter 1, I introduce cilia and how dysfunction of these conserved organelles results in ciliopathies, such as Bardet-Biedl Syndrome (BBS), which present with childhood obesity. Furthermore, conditional loss of primary cilia from neurons in the hypothalamus leads to hyperphagia and obesity in mouse models of ciliopathies. Classically, cilia coordinate signaling often through specific G-protein coupled receptors (GPCRs) as is the case in both vision and olfaction. In addition, neurons throughout the brain including hypothalamic neurons possess primary cilia whose dysfunction contributes to ciliopathy-associated obesity. How neuronal cilia regulate the signaling of GPCRs remains unclear and many fundamental cell biology questions remain about cilia mediated signaling. For example, how cilia coordinate signaling to influence neuronal activity is unknown. To begin to address some of these cell biology questions around neuronal cilia, chapter 2, describes the development and use of a system for primary neuronal cultures from the hypothalamus. Using this system, we found that activation of the cilia regulated hedgehog pathway, which is critical in development, influenced the ability of neurons to respond to GPCR ligands. This result highlights the role of the developmentally critical hedgehog pathway on terminally differentiated hypothalamic neurons. One challenge facing the cilia field is our ability to assess cilia in large numbers without potential bias. This is especially true in tissues like the brain, where cilia appear to have region-specific characteristics. Work included in Chapter 3 describes the use of a computer-assisted artificial intelligence (Ai) approach to analyze cilia composition and morphology in a less biased and high throughput manner. Cilia length and intensities are important parameters for evaluation of cilia signaling. Evidence suggests that activation of some ciliary GPCRs results in shortening of cilia whereas deviations from normal cilia length in mutant phenotypes affects normal physiological processes such as decreased mucociliary clearance. Therefore, to analyze a large number of cilia, we describe the use of the Ai module from in vitro and in vivo samples in a reproducible manner that minimizes user bias. Using this approach, we identified that Mchr1 expression is significantly stronger in the cilia of paraventricular nucleus than that in the arcuate nucleus of adult mice. Work in Chapter 4 continues to explore the integration between hedgehog pathway and ciliary GPCR signaling in the central nervous system, and its relevance with energy homeostasis. We evaluated the hedgehog ligand in the plasma of mice in acute and long-term metabolic changes and identified that the activity of the ligand changed under altered metabolic conditions. We also developed a genetic mouse model where hedgehog signaling was constitutively active in neuronal cilia. These mice become hyperphagic and obese. These results further emphasize the potential role of the hedgehog signaling pathway in regulation of feeding behavior in adult vertebrates. Overall, results from this work will provide a better understanding of the defects not only underlying ciliopathy-associated obesity but may also reveal more common mechanisms of centrally mediated obesity. In addition, the tools I have developed will help in understanding how neuronal cilia are used for intercellular communications and ultimately how they regulate behaviors like feeding.

No description available.

Obesity is a physiological consequence of dysregulated energy homeostasis. Energy homeostasis depends on energy intake and energy expenditure. Factors controlling the development of different adipose tissue deposits in the body and their distinct metabolic phenotypes are of considerable interest from both an agricultural and biomedical perspective. Following the literature review, the first chapter was devoted to studies designed to bridge the neural-adipose interface in understanding the relationship between appetite regulation and adipose tissue deposition in chickens, using chickens selected for low or high juvenile body weight as a model. Appetite regulation in the brain, particularly the hypothalamus, is the main factor governing food intake. Neuropeptide Y (NPY), known as a potent orexigenic factor, also promotes energy storage in fat in mammals and thus has a dual role in promoting energy intake via appetite regulation in the brain and energy storage/expenditure via direct effects on adipose tissue function. There have been no reports of the effects of NPY on adipose tissue function in any avian species. By exposing chicken preadipocytes to different concentration of NPY, we found that NPY enhances both proliferation and differentiation and thus appears to play a major role in chicken adipogenesis, an effect that has not yet been reported, to our knowledge. In the body weight selected chicken lines, we found that NPY and receptor sub-type expression was elevated in the abdominal fat of chickens from the high body weight chicken line and expression of these genes displayed heterosis in the reciprocal crosses of the parental lines as compared to both the high and low body weight selected lines. Intriguingly, expression of those same genes was greater in the low weight than high weight chickens in the hypothalamus. Hypothalamic transcriptomic profiling revealed that genes involved in serotonergic and dopaminergic systems may also play an important role in both appetite regulation and insulin-regulated energy homeostasis in the body weight chicken lines. Intracerebroventricular injection of serotonin in broiler chicks was associated with a dose and time dependent reduction in food intake that was coupled with the activation of the ventromedial hypothalamus and arcuate nucleus, as determined by c-fos immunoreactivity. The remainder of this dissertation project describes the effects of knocking down expression of a recently discovered transcription factor, ZBED6, on mouse preadipocyte proliferation and differentiation. The dissertation ends with a study using diet-induced porcine prepubertal obesity as a model to examine differences in adipokine gene expression between different fat depots from pigs that consumed diets that differed in carbohydrate composition. Overall, we conclude that both NPY and monoamines such as serotonin and dopamine are of importance in the regulation of energy balance in chickens. Moreover, we propose that NPY is a factor that mediates hypothalamus and adipose tissue crosstalk in chickens. An understanding of this system may provide a new avenue for the treatment of obesity and associated disease complications by re-orchestrating the neuronal outputs or adiposity inputs. This information may also be of value in developing strategies to improve feed conversion and meat yield in commercial broilers. / Ph. D.

Κύριος σκοπός τη παρούσης μελέτης ήταν η εκτίμηση της επίδρασης της οξείας και χρόνιας εντατικής άσκησης και του χρόνιου αρνητικού ενεργειακού ισοζυγίου στα επίπεδα αντιπονεκτίνης, ρεζιστίνης και βισφατίνης. Επιπλέον, μελετήθηκε η συσχέτιση των επιπέδων των ανωτέρω λιποκινών με τα επίπεδα κορτιζόλης και ινσουλίνης, καθώς και με τις παραμέτρους έντασης της άσκησης. Ως μοντέλο ενεργειακής και μεταβολικής ομοιοστασίας χρησιμοποιήθηκαν αθλήτριες Ρυθμικής Γυμναστικής (RGs) υψηλού επιπέδου πρωταθλητισμού, Οι RGs υποβάλλονται σε χρόνια έντονη ψυχολογική και σωματική καταπόνηση, ενώ, στην προσπάθεια για επίτευξη και διατήρηση λεπτού σωματότυπου, υιοθετούν αυστηρές διαιτητικές συνήθειες και χρόνιο αρνητικό ενεργειακό ισοζύγιο. Υλικό και Μέθοδοι Η αδυναμία συλλογής δειγμάτων αίματος από αθλητές κορυφαίου επιπέδου πρωταθλητισμού επέβαλε τη συλλογή και επεξεργασία σιέλου για την πραγματοποίηση των ορμονικών προσδιορισμών. Ως εκ τούτου, προτού ξεκινήσει η υλοποίηση του πρωτοκόλλου της μελέτης (διαμόρφωση πληθυσμού και συλλογή δειγμάτων), σχεδιάστηκε και εκπονήθηκε μια μελέτη με σκοπό την ανίχνευση και τη μέτρηση των συγκεντρώσεών των λιποκινών αντιπονεκτίνη, βισφατίνη και ρεζιστίνη στο σίελο και τη συσχέτιση των συγκεντρώσεων τους στο σίελο με τις αντίστοιχες συγκεντρώσεις στο αίμα. Προσδιορισμός των συγκεντρώσεων λιποκινών στο σίελο Τα επίπεδα των λιποκινών αντιπονεκτίνη, ρεζιστίνη και βισφατίνη προσδιορίσθηκαν στο σάλιο και τον ορρό 50 υγιών άτομα (33 γυναίκες και 17 άνδρες), με ταυτόχρονη μέτρηση των ανθρωπομετρικών χαρακτηριστικών (ύψος και βάρος σώματος, υπολογισμός ΒΜΙ, ποσοστό λιπώδους μάζας) . Η επίδραση της άσκησης και του αρνητικού ενεργειακού ισοζυγίου στην ορμονική ομοιοστασία του λιπώδους ιστού Στη μελέτη συμπεριελήφθησαν 51 RGs υψηλού επιπέδου από 8 Ευρωπαϊκές χώρες, οι οποίες συμμετείχαν στη διοργάνωση «Κύπελλο Καλαμάτας 2010» τον Απρίλιο του 2010 στην Καλαμάτα. Επίσης, η μελέτη συμπεριέλαβε 27 υγιείς γυναίκες, μη αθλήτριες και χωρίς σημαντική διαφορά ως προς την ηλικία σε σύγκριση με τον πληθυσμό των αθλητριών (matched for age) ως πληθυσμό ελέγχου (μάρτυρες). Το πρωτόκολλο της μελέτης περιελάμβανε μη επεμβατικές κλινικές και εργαστηριακές εξετάσεις, καθώς και τη συμπλήρωση ενός ερωτηματολογίου. Ειδικότερα, προσδιορίσθηκαν τα ανθρωπομετρικά χαρακτηριστικά αθλητριών και πληθυσμού ελέγχου, οι αθλήτριες συμπλήρωσαν ένα ερωτηματολόγιο που αφορούσε σε στοιχεία γυναικολογικού και αθλητικού ιστορικού, ενώ συλλέχθηκαν δείγματα σιέλου από τις αθλήτριες και τις μάρτυρες. Οι ορμονικοί προσδιορισμοί στο σίελο αφορούσαν σε μετρήσεις επιπέδων ρεζιστίνης, βισφατίνης, αντιπονεκτίνης, κορτιζόλης και ινσουλίνης σε κατάσταση ηρεμίας και μετά από άσκηση. Αποτελέσματα Προσδιορισμός των συγκεντρώσεων λιποκινών στο σίελο Οι λιποκίνες ρεζιστίνη και αντιπονεκτίνη ανιχνεύονται στο σίελο του ανθρώπου και τα επίπεδά τους συσχετίζονται με τα αντίστοιχα επίπεδα στον ορρό (r=0.441, p=0.003 και r=0.347, p=0.019 αντίστοιχα). Αντίθετα, η λιποκίνη βισφατίνη ανιχνεύεται στο σίελο του ανθρώπου, όμως τα επίπεδά της δε συσχετίζονται με τα αντίστοιχα επίπεδα στον ορρό (r=-0.128, p=0.4431). Η επίδραση της άσκησης και του αρνητικού ενεργειακού ισοζυγίου στην ορμονική ομοιοστασία του λιπώδους ιστού Παρατηρήθηκαν μειωμένα επίπεδα αντιπονεκτίνης (p<0.001) και αυξημένα επίπεδα βισφατίνης (p<0.05) στο σάλιο RGs, ενώ δεν παρατηρήθηκαν διαφορές στα επίπεδα ινσουλίνης, κορτιζόλης και ρεζιστίνης στο σάλιο μεταξύ αθλητριών και πληθυσμού ελέγχου. Στις RGs κορυφαίου επιπέδου η επίδραση της μικρής διάρκειας, έντονης αναερόβιας άσκησης του επίσημου αγώνα οδήγησε σε αύξηση των επιπέδων ινσουλίνης (p<0.001), μείωση των επιπέδων αντιπονεκτίνης και βισφατίνης (p<0.001και p<0.05 αντίστοιχα) και καμιά μεταβολή στα επίπεδα κορτιζόλης και ρεζιστίνης στο σάλιο. Επίσης, καταγράφηκε απουσία διημερήσιας διακύμανσης της κορτιζόλης. Τέλος, τα επίπεδα αντιπονεκτίνης ηρεμίας παρουσίασαν σημαντική συσχέτιση με τις παραμέτρους έντασης της άσκησης. Συμπεράσματα Προσδιορισμός των συγκεντρώσεων λιποκινών στο σίελο Οι λιποκίνες ρεζιστίνη, αντιπονεκτίνη και βισφατίνη ανιχνεύονται στο σίελο του ανθρώπου. Όμως, τα ορμονικά επίπεδα στο σίελο συσχετίζονται με τα αντίστοιχα επίπεδα στον ορρό μόνο για τη ρεζιστίνη και την αντιπονεκτίνη. Η επίδραση της άσκησης και του αρνητικού ενεργειακού ισοζυγίου στην ορμονική ομοιοστασία του λιπώδους ιστού Σε RGs κορυφαίου επιπέδου η χρόνια εντατική άσκηση και το χρόνιο αρνητικό ενεργειακό ισοζύγιο αυξάνουν τα επίπεδα αντιπονεκτίνης και μειώνουν τα επίπεδα βισφατίνης στο σάλιο, ενώ η μικρής διάρκειας, έντονη αναερόβια άσκηση καταστέλλει τα επίπεδα τόσο της αντιπονεκτίνης, όσο και της βισφατίνης στο σάλιο. Επιπλέον, τα επίπεδα αντιπονεκτίνης στο σίελο συσχετίζονται με τη συνήθη ένταση της άσκησης, αντανακλώντας, μάλλον, την επιδείνωση του ενεργειακού ισοζυγίου παρά την κλιμάκωση της έντασης της άσκησης καθαυτής, υποδεικνύοντας έναν πιθανός ρόλο των συγκεντρώσεων αντιπονεκτίνης στο σάλιο στην εκτίμηση της επιδείνωσης του ενεργειακού ισοζυγίου, με ενδεχόμενη χρησιμότητα στην κλινική έρευνα και την ιατρική της άσκησης. / Exercise represents a physical stress challenging homeostasis, disturbing the energy balance and leading to adaptive changes in central and peripheral regulatory mechanisms. The aim of the present study was to evaluate the effect of negative energy balance, acute and chronic exercise on adiponectin, resistin and visfatin levels, their interaction with salivary cortisol and insulin levels and the relationship of the specific adipokines with training frequency and intensity. Elite Rhythmic Gymnasts (RGs) were used as a model of energy homeostasis and metabolism. Elite RGs begin exercise at an early age, undergo physical and psychological stress and adopt negative energy balance to retain a lean physique. The main problem in studying hormonal responses in elite athletes on the field of competition lies on the difficulty in obtaining blood samples. The determination of salivary hormone levels provides a convenient, non-invasive and stress-free alternative to blood analysis. As part of the study design, we invested interest in introducing the methodology for detecting and measuring adipokine levels in saliva and evaluating their association with relative serum levels. Materials and Methods Detection and measurement of adipokine levels in human saliva Resistin, visfatin and adiponectin levels were measured in serum and saliva of 50 healthy adult volunteers (17 male and 33 female) using commercial enzyme immunoassay kits for plasma with minor modifications. The influence of acute and chronic intensive physical training and negative energy balance on salivary adipokine levels in elite RGs The study included 51(fifty one) elite female athletes of RG from 8 European countries, participating in the top level tournament of “Kalamata 2010 Rhythmic Gymnastics World Cup” in Kalamata, Greece on April 2010. 24 (twenty four) healthy pubertal girls not engaged in strenuous sports activities were used as controls. The study protocol included noninvasive clinical and laboratory investigations as well as the completion of a questionnaire. The athletes completed a questionnaire including personal data (age of training onset, usual weekly training intensity and number of participations in international championships per year). Baseline and post exercise salivary cortisol, insulin, adiponectin, resistin and visfatin levels were measured. Results Detection and measurement of adipokine levels in human saliva The present study documented the determination of resistin and adiponectin levels in saliva and the significant correlation of salivary with their respective serum levels (r=0.441, p<0.01 and r=0.347, p<0.05, respectively). Moreover, the identification of visfatin in saliva was achieved, but no significant correlation with serum visfatin levels was observed. The influence of acute and chronic intensive physical training and negative energy balance on salivary adipokine levels in elite RGs At baseline, a significant inverse correlation was documented between salivary insulin and adiponectin levels (r=-0.316, p<0.05), after controlling for the effect of age and BMI. Salivary adiponectin levels were higher (p<0.001) and visfatin lower (p<0.05) in RG’s compared with controls, while no significant changes were observed regarding salivary cortisol, insulin and resistin levels. In elite RG’s short term intensive anaerobic exercise led to increased salivary insulin levels (p<0.001), reduced salivary adiponectin (p<0.001) and visfatin levels (p<0.05) and no changes in salivary resistin levels. Moreover, diurnal variation of salivary cortisol was lost. In addition, salivary adiponectin levels are associated with the intensity of training. Conclusions Detection and measurement of adipokine levels in human saliva The adipokines resistin, visfatin and adiponectin can be detected in human saliva. However, significant correlation between salivary and their relative serum levels are documented only for resistin and visfatin, but not for visfatin. The influence of acute and chronic intensive physical training and negative energy balance on salivary adipokine levels in elite RGs. Chronic intensive physical training and negative energy balance up-regulate salivary adiponectin and down-regulate salivary visfatin levels, while acute glucoregulatory response to short term intensive anaerobic exercise down-regulates salivary adiponectin and visfatin levels. Moreover, salivary adiponectin levels are associated with the intensity of training, reflecting the deterioration of energy balance rather than the training stress. Thus, salivary adiponectin could be introduced as a possible marker of significant energy deprivation, with potential usefulness in clinical and sports medicine.

En dehors de son rôle dans la régulation de la balance hydrosodée, le récepteur minéralocorticoïde (MR) est un facteur de transcription hormono-dépendant qui exerce des effets pro-adipogéniques et anti-thermogéniques in vitro, mais son rôle dans la régulation du métabolisme énergétique in vivo n’a jamais été précisément étudié. Dans ce travail, nous avons montré que les souris surexprimant le MR humain (Tg) ont une résistance à l’obésité induite par le régime hyperlipidique. Ceci s’accompagne d’un défaut de développement de la masse adipeuse comme en témoignent des surfaces adipocytaires plus petites en histomoprhométrie et une diminution de l’expression de gènes impliqués dans l’adipogenèse tels que PPARγ2. Ce défaut d’adipogenèse n’est pas dû à une altération de la capacité intrinsèque des préadipocytes surexprimant le MR, isolés de la fraction stroma vasculaire, mais probablement à une modification de la polarisation macrophagique analysée par la technique du FACS. Ces résultats soulignent un impact immuno-métabolique de la surexpression du MR in vivo. Par ailleurs, dans notre modèle adipocytaire brun, nous démontrons que les corégulateurs du MR ont un profil d’expression différentiel pouvant rendre compte d’une coopération moléculaire au cours de la différenciation adipocytaire des cellules T37i. De plus, nous confirmons in vitro l’effet inhibiteur de l’aldostérone sur l’expression de UCP1 (Uncoupling protein 1). Enfin nous démontrons in vivo que la surexpression du MR dans le tissu adipeux brun des souris Tg induit une diminution de l’induction l’expression de UCP1 par une exposition au froid. L’ensemble de ces résultats apporte une meilleure compréhension du rôle du MR dans la régulation du métabolisme énergétique et devrait ouvrir des nouvelles perspectives thérapeutiques innovantes tels que l’utilisation de modulateurs sélectifs du MR dans le traitement des troubles métaboliques / Besides its role in the regulation of sodium homeostasis, the mineralocorticoid receptor (MR) is a hormone-dependent transcription factor that exerts pro-adipogenic and anti- thermogenic effects in vitro, but its role in vivo in the regulation of energy balance has never been precisely studied. In this study, we show that human MR overexpressing mice (Tg) were resistant to high fat diet-induced obesity. This was associated with a defect of fat mass as evidenced by smaller adipocyte size analyzed by histomorphometric study and a decreased expression of genes involved in adipogenesis such as PPARγ2. This alteration in adipogenesis was not related to a defect of the intrinsic capacity of MR overexpressing preadipocytes to differentiate into adipocytes, but probably to a change in macrophage polarization studied by FACS analysis. These results indicate an immuno-metabolic impact of MR overexpression in vivo. Moreover, in our brown adipocyte model, we demonstrate that MR coregulators have a differential expression profile, consistent with a coordinated and physiologically relevant cooperation occuring during brown adipogenesis. In addition, we confirm in vitro the inhibitory effect of aldosterone on UCP1 expression (Uncoupling protein 1). Finally, we demonstrate in vivo that MR overexpression in brown adipose tissue of Tg mice induced a decrease in the cold-induced UCP1 expression. Taken together, these results provide a better understanding of MR involvement in the regulation of energy metabolism and should open new therapeutic oportunities such as the use of selective MR modulators in the management of metabolic disorders.

Les maladies métaboliques telles que l’obésité ou le diabète de type 2 sont multifactorielles et multigéniques. Ces dernières décennies, la prévalence de ces pathologies notamment chez les enfants et les jeunes adultes a drastiquement augmenté, désignant ainsi l’environnement comme un élément-clé de ces pathologies. Ainsi, un environnement métabolique et/ou endocrinien déséquilibré pendant les périodes de gestation et/ou lactation prédispose la descendance à certaines pathologies à l’âge adulte. Ces phénotypes sont souvent associés, dans l’hypothalamus, structure impliquée dans la régulation de l’homéostasie énergétique, à des modifications de l’expression d’ARNm ou de protéines. Les microARNs (miARNs), régulateurs post-transcriptionnels majeurs, apparaissent donc comme des candidats intéressants pour l’exploration des mécanismes moléculaires sous-tendant les dysfonctionnements hypothalamiques. Le but du ce travail de thèse a été de caractériser le phénotype ainsi que le profil hypothalamique d’expression des miARNs chez des rats adultes nés et/ou allaités dans deux contextes différents de programmation métabolique. Dans un premier temps, nous avons étudié les conséquences à long-terme du blocage de la leptine postnatale, hormone contrôlant la mise en place du métabolisme et l’établissement de connexions hypothalamiques. Les rats traités avec un antagoniste de la leptine présentent un surpoids quel que soit le régime (normal ou hyperlipidique). Ces animaux présentent des signes de résistance à l’insuline dès le sevrage. Leur profil hypothalamique d’expression des microARNs est modifié notamment en ce qui concerne l’expression de certains miARNs associés à l’insulino-résistance périphérique. Dans un second temps nous avons étudié l’impact d’un régime maternel hyperlipidique. Ce régime maternel induit un moindre poids chez la descendance dès le deuxième jour postnatal. A l’âge adulte, les mâles présentent une sensibilité normale à l’insuline et à la leptine et ne sont pas prédisposés au surpoids lorsqu’ils sont soumis à un régime hyperlipidique. Les femelles présentent le même phénotype associé à une meilleure tolérance au glucose. Nous avons étudié le profil d’expression des miARNs dans les noyaux arqué et paraventriculaire de l’hypothalamus des mâles. L’expression de certains miARNs abondants est modulée chez les animaux nés de mères soumises au régime hyperlipidique. Les travaux de cette thèse ouvrent la voie à une étude systématique des profils hypothalamiques d’expression des miARNs dans un contexte de programmations métaboliques diverses. / Epidemiological studies have demonstrated that the incidence of metabolic diseases in adults such as hypertension, insulin resistance, obesity and the metabolic syndrome is markedly increased when maternal nutrition is altered at critical periods of foetal development. Numerous studies in humans and rodents have demonstrated the importance of the perinatal environment in metabolic programming. Thus, a metabolic or endocrine unbalanced environment predisposes offspring to various metabolic diseases in adulthood. These phenotypes are often associated with changes in mRNA or protein expression in the hypothalamus, a central structure involved in the regulation of energy homeostasis. In this context, microRNAs (miRNAs) appear as attractive candidates for exploration of the molecular mechanisms underlying hypothalamic dysfunction observed during metabolic programming. The aim of my project was to characterize the phenotype and the hypothalamic profile of miRNAs in the adult offspring in two contexts of metabolic programming. First, we studied the long-term consequences of early postnatal leptin blockade, a hormone described to be involved in the establishment of metabolism and hypothalamic connections. Rats treated with a leptin antagonist exhibit overweight (under chow and high-fat diet) and are also subjected to insulin resistance. As signs of insulin resistance appeared as soon as weaning, we analyzed at this age by microarray the hypothalamic miRNAs expression profile which reveals modification of hypothalamic miRNA expression pattern including miRNAs previously linked to peripheral insulin-résistance. In the second part of my thesis, we have demonstrated that a maternal high-fat diet induced a lower weight in the offspring from the second postnatal day. In adulthood, these animals exhibit similar insulin and leptin sensitivities as compared to controls and are not predisposed to overweight when exposed to a high fat diet. This phenotype is associated with changes in the miRNA expression profile in arcuate and paraventricular hypothalamic nuclei. In conclusion, we show for the first time that metabolic programming is associated with altered hypothalamic expression of miRNAs, which could contribute, at least partially, to the establishment of the offspring phenotype.

The prevalence of obesity is dramatically increasing and thus constitutes a major risk factor for developing chronic diseases such as type 2 diabetes, dyslipidemia, cardiovascular diseases, and certain forms of cancer. High-caloric nutrition and a lack of physical activity are the main contributing factors for this global epidemic. Estrogen receptors (ERs) are recognized to be involved in many processes related to the control of energy homeostasis. In my studies, I investigated the impact of estrogens (17beta-estradiol (E2)) on energy homeostasis. Special emphasis was given to the effects of two synthetic ER subtype-selective agonists, 16alpha-LE2 (Alpha) and 8beta-VE2 (Beta), to determine to what extend the two distinct ER subtypes are involved in the underlying molecular mechanisms. Because of its estrogenic activity and also its widespread use as a nutritional supplement the influence of the isoflavone genistein (Gen) was examined. For this purpose two different female rat models were used: Wistar rats with nutrition-induced obesity and leptin resistant Zucker diabetic fatty (ZDF) rats. In both experiments, the animals were ovariectomized (OVX) and treated with vehicle (untreated controls) or the estrogenic compounds. The most important finding was that treatment of OVX animals with Beta enlarges soleus muscle fiber sizes in both animal models compared to untreated OVX animals. This anabolic effect may in turn improve the muscle/fat ratio of the body that enhances muscular uptake and utilization of fuels. By contrast, in the gastrocnemius muscle of OVX ZDF rats substitution with Alpha increased expression and distribution of the insulin-dependent glucose transporter 4 (GLUT4). Consequently, systemic insulin sensitivity in both animal models was improved by treatment with estrogenic compounds compared to untreated OVX animals. The strongest effect was observed in E2-treated rats that indicate an additive effect through activation of both pathways. In all OVX rats, treatment with either ER subtype-selective agonist showed an anti-lipogenic effect in adipose tissue, liver, and skeletal muscle of nutrition-induced obese Wistar rats in comparison to OVX animals without treatment. Decreased visceral fat mass, adipocyte sizes, serum leptin levels, triglyceride accumulation in liver and muscle as well as mRNA expression of genes that are involved in lipo-/adipogenesis reflected this. Therefore, the lower visceral fat mass as well as decreased accumulation of triglycerides in non-adipose tissues such as liver and skeletal muscle most likely contributes to the improved insulin sensitivity in such treated animals. Gen exerted effects similar to those of the ER beta-selective agonist (except on adipose tissue in Wistar rats). Especially, the similar ability to induce anabolic activity in the soleus muscle might be highly relevant. Gen-treated animals might have a more effective utilization of fuels compared to untreated OVX animals because they showed a lower TG content in muscle and liver as well as improved glucose metabolism. In conclusion, because of my studies and the fact that ER beta signaling is not involved in proliferation of uterus and mammary gland, an effective way to treat obesity and co-morbidities in postmenopausal women might be substances that only activate ER beta. A combination with physical activity may support the therapy of obesity and co-morbidities. The isoflavone Gen is able to activate both ER-subtypes. This compound is already placed on the market for treatment of postmenopausal complaints, although adverse effects of Gen cannot be excluded so far (e.g., increased risk of breast cancer). However, Gen might be a natural alternative – not only to the conventional hormone replacement therapy, but also as a strategy for treatment of obesity and co-morbidities – that deserves further research with respect to these new data. / Die dramatisch zunehmende Prävalenz der Adipositas und das damit verbundene Risiko für Folgeerkrankungen wie Diabetes mellitus, Hypertonie, Dyslipidämie und koronare Herzkrankheiten stellt eine große Herausforderung für das Gesundheitswesen dar. Als Hauptursache wird ein chronisches Missverhältnis der Energiehomöostase aufgrund permanenter Überernährung und Bewegungsmangel postuliert. Estrogene beeinflussen den Glukose- und Lipidstoffwechsel und sind somit in die Regulation des Energiehaushaltes involviert. Estrogene vermitteln ihre Effekte über zwei Estrogenrezeptor (ER)-Subtypen, den ER alpha und den ER beta. Ziel der vorliegenden Arbeit war es mittels tierexperimentellen Studien den Einfluss von Estrogenen, speziell 17beta-Estradiol, auf den Energiehaushalt zu untersuchen. Um einen tieferen Einblick in die zugrundeliegenden molekularen Mechanismen zu erhalten, wurden zwei Subtyp-selektive ER-Agonisten, 16alpha-LE2 (Alpha) and 8beta-VE2 (Beta), synthetischer Herkunft eingesetzt. Aufgrund der estrogenen Aktivität und der Verfügbarkeit als Nahrungsergänzungsmittel wurde des Weiteren der Einfluss des Isoflavons Genistein untersucht. Für die Studien wurden zwei Tiermodelle genutzt: zum einen weibliche Wistar-Ratten mit ernährungsinduzierter Adipositas und zum anderen weibliche leptinresistente „Zucker diabetic fatty“ (ZDF)-Ratten. Die Tiere wurden ovarektomiert (OVX) und entweder mit einem Vehikel (unbehandelte Kontrolltiere) oder mit der entsprechenden estrogenen Substanz behandelt. Die interessanteste Erkenntnis war, dass im Vergleich zu unbehandelten OVX-Tieren beider Tiermodelle die Behandlung mit Beta zur Vergrößerung der Faserquerschnitte im Soleusmuskel führte. Dieser anabole Effekt könnte die muskuläre Aufnahme und Verwertung von Brennstoffmolekülen verbessern und sich insgesamt positiv auf die Körperzusammensetzung auswirken. Den stärksten Effekt hinsichtlich einer erhöhten Expression und Translokation des insulinabhängigen Glukosetransporters 4 (GLUT4) in die Zellmembran des Gastrocnemiusmuskels zeigte sich dagegen durch die Behandlung von OVX ZDF-Ratten mit Alpha. Im Endergebnis zeigten die Tiere beider Modelle durch die Behandlung mit estrogenen Substanzen eine verbesserte systemische Insulinsensitivität im Vergleich zu unbehandelten Kontrolltieren. E2-behandelte Tiere tolerierten die Glukose am besten und lassen einen additiven Effekt aufgrund der Aktivierung beider Signalwege vermuten. Im Vergleich zu unbehandelten OVX Wistar-Ratten führte die Behandlung mit E2 oder mit jeweils einem der beiden ER-Subtyp-selektiven Agonisten zu einer geringeren viszeralen Fettmasse, kleineren Fettzellen, niedrigeren Leptinspiegeln im Serum und geringeren Triglyzeridwerten in Leber und Muskel. Auf der Ebene der Genexpression waren zudem geringere mRNA-Spiegel von lipo- und adipogenen Genen messbar. Somit scheinen beide ER-Subtypen in die antilipogene Wirkung von E2 involviert zu sein. Sowohl die reduzierte viszerale Fettmasse als auch die geringere Anreicherung von Triglyzeriden in Leber und Muskel tragen sehr wahrscheinlich ebenfalls zur verbesserten Insulinsensitivität bei. Die Behandlung von OVX Tieren mit Gen führte zu ähnlichen Ergebnissen wie die Behandlung mit Beta. Eine alleinige Ausnahme stellte das Fettgewebe dar, da hier eine Gen-Behandlung keine antilipogenen/-adipogenen Effekte zeigte. Speziell die Fähigkeit von Gen ebenfalls anabol zu wirken, könnte die molekulare Grundlage sein, weshalb Gen-behandelte Tiere im Vergleich zu unbehandelten Tiere eine verbesserte Toleranz gegenüber Glukose und eine geringere Anreicherung von Triglyzeriden in Muskel und Leber zeigten. Der ER beta ist nicht in die estrogenvermittelte Proliferation von Uterus und Brustdrüse involviert. Vor diesem Hintergrund lassen meine Ergebnisse vermuten, dass eine Behandlung mit ER beta-selektiven Substanzen eine effektive Möglichkeit darstellt, um Adipositas und deren Folgeerkrankungen in postmenopausalen Frauen zu behandeln, ohne deren Risiko für estrogenabhängige Krebsformen zu erhöhen. Eine Kombination mit regelmäßiger körperlicher Aktivität könnte die Erfolge bei der Behandlung von Adipositas und deren Folgeerkrankungen noch maximieren bzw. eine geringere Dosierung der verwendeten Substanz bei gleichbleibendem Behandlungserfolg ermöglichen. Das Isoflavon Gen mit seiner Fähigkeit beide ERs zu aktivieren ist eine bereits auf dem Markt befindliche Substanz und wird zur Behandlung von postmenopausalen Beschwerden eingesetzt, obwohl mögliche negative Effekte (z.B. ein erhöhtes Brustkrebsrisiko) noch nicht abschließend geklärt sind. Falls diese Risiken von Gen ausgeräumt werden können, könnte diese Substanz eventuell eine kostengünstige Alternative darstellen, um sowohl postmenopausale Beschwerden als auch Adipositas und deren Folgekrankheiten zu behandeln.

The central nervous system (CNS) melanocortin signaling pathway plays a critical role in the regulation of metabolism. However, the regulatory effects of CNS melanocortin signaling on hepatic lipid metabolism and fatty liver disease have not been well established. Although the activity of the CNS melanocortin system is regulated by metabolic signals, the mechanism for this regulation is not fully understood. Variants of the FTO (fat mass and obesity-associated) gene are associated with obesity and FTO is expressed in the hypothalamic neurons including proopiomelanocortin (POMC) neurons. Therefore, it is hypothesized that hypothalamic FTO plays a role in the regulation of metabolism by mediating the effect of metabolic signals on hypothalamic melanocortinergic neurons, and that impairments in this regulation may cause metabolic impairments including obesity and fatty liver disease. Intracerebroventricular (i.c.v.) treatment with SHU9119, a melanocortin antagonist, increased hepatic lipid accumulation and the expression of genes encoding lipogenic enzymes in lean mice. Conversely, i.c.v. treatment with MTII, a melanocortin agonist, reduced the expression of hepatic lipogenic genes in association with reduction in body weight in ob/ob mice, a mouse model of fatty liver disease. Immunohistochemical analysis demonstrated that Fto is co-expressed in both POMC and agouti-related protein (AgRP) neurons in the mouse hypothalamus. Fto mRNA and protein expression was reduced by fasting and increased by glucose treatment in nutritionally important hypothalamic nuclei. Fasting-induced reduction in hypothalamic Fto expression was observed in both lean wild-type and obese ob/ob mice, while the stimulatory effect of glucose on hypothalamic Fto expression was absent in ob/ob mice. These findings support the hypothesis that central melanocortin signaling regulates hepatic lipid metabolism in part by regulating de novo lipogenesis. Impairments in the central melanocortin signaling lead to the development of hepatic steatosis, while enhanced melanocortin signaling may be beneficial in reversing abnormal hepatic lipid metabolism in fatty liver disease (Poritsanos et al., 2008). These findings also support the hypothesis that Fto is expressed in the hypothalamic melanocortinergic neurons and is regulated by metabolic signals involving changes in CNS glucose availability and/or glucose action. Impairments in this regulation may cause metabolic impairments including obesity and fatty liver disease.

The central nervous system (CNS) melanocortin signaling pathway plays a critical role in the regulation of metabolism. However, the regulatory effects of CNS melanocortin signaling on hepatic lipid metabolism and fatty liver disease have not been well established. Although the activity of the CNS melanocortin system is regulated by metabolic signals, the mechanism for this regulation is not fully understood. Variants of the FTO (fat mass and obesity-associated) gene are associated with obesity and FTO is expressed in the hypothalamic neurons including proopiomelanocortin (POMC) neurons. Therefore, it is hypothesized that hypothalamic FTO plays a role in the regulation of metabolism by mediating the effect of metabolic signals on hypothalamic melanocortinergic neurons, and that impairments in this regulation may cause metabolic impairments including obesity and fatty liver disease. Intracerebroventricular (i.c.v.) treatment with SHU9119, a melanocortin antagonist, increased hepatic lipid accumulation and the expression of genes encoding lipogenic enzymes in lean mice. Conversely, i.c.v. treatment with MTII, a melanocortin agonist, reduced the expression of hepatic lipogenic genes in association with reduction in body weight in ob/ob mice, a mouse model of fatty liver disease. Immunohistochemical analysis demonstrated that Fto is co-expressed in both POMC and agouti-related protein (AgRP) neurons in the mouse hypothalamus. Fto mRNA and protein expression was reduced by fasting and increased by glucose treatment in nutritionally important hypothalamic nuclei. Fasting-induced reduction in hypothalamic Fto expression was observed in both lean wild-type and obese ob/ob mice, while the stimulatory effect of glucose on hypothalamic Fto expression was absent in ob/ob mice. These findings support the hypothesis that central melanocortin signaling regulates hepatic lipid metabolism in part by regulating de novo lipogenesis. Impairments in the central melanocortin signaling lead to the development of hepatic steatosis, while enhanced melanocortin signaling may be beneficial in reversing abnormal hepatic lipid metabolism in fatty liver disease (Poritsanos et al., 2008). These findings also support the hypothesis that Fto is expressed in the hypothalamic melanocortinergic neurons and is regulated by metabolic signals involving changes in CNS glucose availability and/or glucose action. Impairments in this regulation may cause metabolic impairments including obesity and fatty liver disease.