Obesity is a prevalent problem in modern society, which requires the upmost attention in the biomedical sciences. A leading cause of obesity related diseases is due to overeating, especially in industrialized countries. Leptin is the hormone that is secreted by fat cells responsible for communicating body nutritional status to the brain. Leptin interacts with other bodily systems such as the cognitive, digestive, neuronal, and endocrine systems. Leptin acts mainly on the Ob-Rb receptor in the arcuate nucleus of the hypothalamus and largely suppresses food intake and increases energy expenditure by activating Proopiomelanocortin and Cocaine- and amphetamine-regulated transcript (anorexigenic signals) neurons and by suppressing Neuropeptide Y and Agouti-related peptide (orexigenic signals) neurons, among other chemical signaling pathways. In both rodent and human studies, exogenous leptin administration resulted in elevated plasma leptin concentrations. When researchers tried to use leptin for weight reducing medical treatments in humans, the results show difficulty in establishing clinical efficacy. However, for diseases such as congenital leptin deficiency, obesity related leptin resistance, and lipodystrophy, medical treatments involving exogenous leptin have been relatively successful. The goal of this thesis is to give readers an understanding of leptin’s role in regulating appetite and the different leptin associated diseases. Leptin’s role is still continuing to be developed and more research is needed to fully utilize leptin for therapeutic benefit.

La ghréline est un peptide de 28 acides aminés, produit principalement par l’estomac et caractérisé par la présence d’un groupement octanoyl sur la sérine en position 3 (Kojima et al. 1999). La ghréline stimule la libération de l’hormone de croissance (GH) et régule la prise alimentaire et le métabolisme énergétique (Gualillo et al. 2003). Ces activités biologiques sont principalement médiées par le « growth hormone secretagogue receptor » (GHS-R). Deux sous-types de GHS-R, produits par épissage alternatif d’un même gène, ont été clonés : le GHS-R 1a, dont l’activation entraîne une libération de calcium via la formation d’inositol 1,4,5-trisphosphate (IP3), et le GHS-R 1b, qui ne semble pas lié à une activité biologique (Howard et al. 1996). La première partie de mon travail de thèse consistait en l’étude de la dégradation de la ghréline. La ghréline circule dans le sang principalement sous forme de des-acyl ghréline, une forme de ghréline dépourvue du groupement octanoyl qui ne se lie pas au GHS-R 1a. Peu d’études ont été réalisées sur le catabolisme de la ghréline. Les enzymes impliquées dans la dégradation de la ghréline étant des régulateurs importants de son activité biologique, le but de cette étude était d’identifier les sites de clivage et les enzymes impliquées dans la dégradation de la ghréline par du sérum, des sous-fractions plasmatiques et des homogénats de tissus. Nous avons montré qu’au contact de sérum humain et de rat, la ghréline est désoctanoylée, sans protéolyse. Dans le sérum humain, nous avons montré que la butyrylcholinestérase et la « platelet-activating factor acetylhydrolase » (PAF-AH), une phospholipase associée aux lipoprotéines de basse densité (LDL), sont impliquées dans ce phénomène (articles n°1 et n°2). En parallèle, nous avons montré que la ghréline peut être transportée dans la circulation sanguine par les lipoprotéines riches en triglycérides (TRL), les LDL, et les lipoprotéines de haute et de très haute densité (HDL et VHDL) (article n°2). Dans le sérum de rat, la désoctanoylation de la ghréline implique une carboxylestérase (article n°1). Au contact d’homogénats de tissus, la ghréline est dégradée à la fois par désoctanoylation et protéolyse N-terminale, suggérant la participation d’estérases et d’aminopeptidases. Nous avons identifié cinq sites de clivage dans la ghréline : entre les résidus Ser2-(acyl)Ser3 (dans l’estomac et le foie), (acyl ?)Ser3-Phe4 (dans l’estomac, le foie et le rein), Phe4-Leu5 (dans l’estomac et le rein), Leu5-Ser6 et Pro7-Glu8 (dans le rein) (article n°1). La deuxième partie de mon travail de thèse consistait à étudier l’expression et la production de ghréline par différentes lignées leucémiques (HEL, HL-60, THP-1, SupT1), par des leucocytes poly- et mononucléés et par des plaquettes sanguines, et à étudier l’effet de la ghréline sur la prolifération cellulaire. Pour cela, nous avons mis au point des dosages radioimmunologiques (RIA) permettant de quantifier et de distinguer les formes octanoylées et non octanoylées de la ghréline, et nous avons caractérisé en détail les anticorps SB801 et SB969 obtenus. Par HPLC en phase inverse suivie des RIAs, nous avons mis en évidence la présence de ghrélines octanoylée et non octanoylée dans chaque population de cellules. Plus de 80 % de la ghréline produite est octanoylée dans les cellules HEL, les leucocytes et les plaquettes. Nous avons montré que la ghréline endogène stimule la prolifération des cellules HEL de façon autocrine impliquant un récepteur encore non identifié, distinct du GHS-R 1a (article n°3). La ghréline et la des-acyl ghréline inhibent la prolifération des leucocytes mononucléés mais sont dépourvues d’effet sur les cellules HL-60, THP-1 et SupT1. Malgré la présence du GHS-R 1a dans les leucocytes mononucléés, cet effet pourrait être médié par un récepteur différent puisque la des-acyl ghréline exerce le même effet que la ghréline sur la prolifération (article n°4).

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010.

Chickens from lines that have been divergently selected for either low (LWS) or high (HWS) body weight at 56 days of age for more than 57 generations serve as unique models to study eating disorders. The LWS have different severities of anorexia while all HWS become obese. Over the past decade our groups has demonstrated that these lines have differential food intake threshold responses to a range of intracerebroventricular (ICV) injected neurotransmitters. The major brain region regulating homeostatic regulation of appetite is the hypothalamus, and hence this dissertation was focused on understanding how the hypothalamus is different between LWS and HWS lines. Experiments 1 and 2 were performed as follows: whole hypothalamus as well as individual hypothalamic nuclei, respectively, were collected from 5 day-old chicks that had been fasted for 180 min or had free access to food. The hypothalamic nuclei included those primarily associated with appetite including the lateral hypothalamus, paraventricular nucleus (PVN), ventromedial hypothalamus, dorsomedial nucleus, and arcuate nucleus (ARC). Total RNA was isolated, reverse transcribed, and real time PCR performed. Hypothalamic expression of anorexigenic factors was greater in LWS than HWS, those factors including calcitonin, corticotropin-releasing factor receptor 1, leptin receptor, neuropeptide S, melanocortin receptor 3 (MC3R), and mesotocin. The gene expression data from individual hypothalamic nuclei revealed that mesotocin from the PVN may play an important role in the inhibition of appetite in the LWS. Experiment 3 was then designed to evaluate the effects of stress on food intake: besides the differences in hypothalamic gene expression between the lines, they also have different feeding responses when stressed: ICV injection of neuropeptide Y (0.2 nmol, NPY) did not increase food intake in LWS on day 5 after stress exposure. Experiment 4 was thus designed to study the molecular mechanisms underlying conditional feeding responses to exogenous NPY after stress in the LWS. The melanocortin system (AgRP and MC3R) changed in the hypothalamus after stress in the LWS, and hence may be responsible for the loss of responsiveness to exogenous NPY in stressed LWS. Experiment 5 was designed to evaluate whether hypothalamic differences exist at the protein level: label-free liquid chromatography coupled to tandem-mass spectrometry was used to measure the abundance of proteins in the hypothalamus. Hypothalamus was obtained from fed and 180 minute-fasted 5 day-old male LWS and HWS chicks. Proteins involved in energy metabolism were different between the lines. Differences were also found in proteins involved in GABA synthesis and uptake as well as protein ubiquitination. In conclusion, these results suggest that different feeding behaviors of LWS and HWS may be due to differences in gene and protein expression in the hypothalamus. / Ph. D.

Energy homeostasis is essential for survival across all vertebrate species and involves a multitude of physiological systems that are regulated by both central and peripheral neural signaling. The hypothalamus is responsible for integrating and processing these signals and thus is regarded as the regulatory center for balancing energy homeostasis. Eating disorders, such as compulsive eating behavior associated with obesity, and anorexia, are significant public health concerns worldwide. Thus, studying appetite regulation is necessary to provide novel information for the design of solutions for health concerns that stem from altered energy intake. Such information is also relevant for improving chicken health and productivity in an agricultural setting. The objective of this dissertation research was to determine the hypothalamic mechanisms underlying appetite regulation in birds. In Experiment 1, the Virginia lines of chickens were used to elucidate the mechanisms underlying stress-induced anorexia. These chickens have been selected for low (LWS) or high (HWS) body weight at 56 days of age and have different severities of anorexia and obesity, respectively. Chicks were subjected to a combination of thermal and nutritional stress after hatch and hypothalamic nuclei, including the lateral hypothalamus (LH), paraventricular nucleus (PVN), ventromedial hypothalamus (VMH), and arcuate nucleus (ARC), were collected 5 days later. Real-time PCR was used to measure the mRNA abundance of appetite-associated neuropeptides and receptors in each nucleus. The results showed that the two lines displayed distinct gene expression profiles in response to stress. In particular, the PVN of the LWS was significantly affected by stress, and expression of several anorexigenic factors was up-regulated including corticotropin-releasing factor (CRF), CRF receptor sub-types 1 and 2 (CRFR1 and CRFR2, respectively), melanocortin receptor 4, and urocortin 3, suggesting that stress-induced anorexia in the LWS may result from overriding anorexigenic signaling in the PVN, primarily through CRF signaling. This CRF signaling-associated hypothesis was further supported by results showing that the original phenotypes were restored when the LWS chicks were treated with astressin (CRF receptor antagonist) before exposure to stress. In Experiments 2 and 3, we attempted to determine the mechanisms of CRF's anorexigenic effect in chickens and Japanese quail. We administered CRF by intracerebroventricular (ICV) injection and the hypothalamus was collected 1 hour later for molecular analyses. Results showed that CRF exerted a similar inhibitory effect on food intake in these two bird species, however the hypothalamic mechanisms underlying this anorexigenic effect were different. ICV injection of CRF increased c-Fos expression in the PVN, VMH, dorsomedial nucleus (DMN), and ARC in chicks while it only affected the PVN and LH in quail. Hypothalamic gene expression results suggested that CRF decreased neuropeptide Y receptor sub-type 1 (NPYR1) in chicks while it increased proopiomelanocortin (POMC), MC4R, CRF, and CRFR2 in quail. These results suggested that the anorexigenic effect of CRF may involve a dampened neuropeptide Y (NPY) system in chicks whereas it is associated with activated CRF and melanocortin systems in quail. At the nucleus level in chicks, CRF injection decreased NPY system-associated gene expression (ARC and DMN) and increased CRF (ARC and PVN) and mesotocin (MT) (VMH)-associated mRNAs, suggesting that orexigenic signaling through NPY was overridden by the heightened anorexigenic tone through CRF and MT, which led to the inhibition of food intake. In Experiments 4 and 5, we used the same experimental design as for CRF studies to determine the hypothalamic mechanisms of the anorexigenic effects of neuropeptide K (NPK) and adrenomedullin (AM) in Japanese quail. Results from Experiment 4 showed that NPK injection activated the ARC and PVN, which was associated with increased mRNAs for a group of anorexigenic factors including CRF, UCN3, cocaine and amphetamine-regulated transcript (CART), and POMC, and decreased expression of several orexigenic factors, such as NPY and agouti-related peptide (AgRP). In Experiment 5, ICV injection of AM activated the ARC, the nucleus in which POMC and CART mRNAs were increased. In conclusion, these experiments revealed novel hypothalamic mechanisms underlying stress or exogenous neuropeptide-induced anorexia in birds and may provide insights on understanding appetite regulation from evolutionary, agricultural, and biomedical perspectives. / Ph. D. / Appetite regulation is important for survival across all vertebrate species and the hypothalamus is the regulatory center for control of feeding behavior. Thus, studying the functions of the hypothalamus on appetite regulation provide novel insight into the eating disorders, such as obesity and anorexia, a worldwide health issue. Also, such information is relevant for improving productivity in the modern chicken industry. The objective of this dissertation research was to determine the hypothalamic mechanisms underlying appetite regulation in birds. In Experiment 1, the Virginia lines of chickens were used to elucidate the mechanisms underlying stress-induced anorexia. These chickens have been selected for low (LWS) or high (HWS) body weight at 56 days of age and have different severities of anorexia and obesity, respectively. Chicks were subjected to a combination of thermal and nutritional stress after hatch. The results suggested the two lines displayed distinct appetite-associated gene expression profiles in response to stress in the hypothalamus. In particular, stress-induced anorexia in the LWS may result from potent feeding-inhibitory factor corticotropin-releasing factor (CRF). Thus, in Experiments 2 and 3, we attempted to determine the mechanisms of CRF's inhibitory effect on food intake in chickens and Japanese quail. We administered CRF by intracerebroventricular (ICV) injection and the hypothalamus was collected 1 hour later for molecular analyses. Results showed that CRF exerted a similar inhibitory effect on food intake in these two bird species. However, the inhibitory effect of CRF was primarily associated with a dampened neuropeptide Y (NPY) system which is a potent stimulatory factor for feeding behavior in chickens, whereas it may involve activated CRF and melanocortin systems in quail. In Experiments 4 and 5, we used the same experimental design as for CRF studies to determine the hypothalamic mechanisms of the inhibitory effects of neuropeptide K (NPK) and adrenomedullin (AM) in Japanese quail. Results from Experiment 4 showed that the feeding-inhibitory effect of NPK was associated with a group of increased feeding-inhibitory factors such as CRF and cocaine and amphetamine-regulated transcript (CART) and decreased feeding-stimulatory factors, such as NPY and agouti-related peptide (AgRP) in the hypothalamus. In Experiment 5, AM increased gene expression of CART and proopiomelanocortin (POMC). Overall, these experiments suggested the roles of the hypothalamus in stress or exogenous neuropeptide-induced anorexia in birds and may provide insights on understanding appetite regulation from evolutionary, agricultural, and biomedical perspectives.

The purpose of this study was to investigate whether a cyclic feeding regime alters growth, lipid content and the expression of genes involved in appetite regulation and lipid metabolism in rainbow trout (Onchorynchus mykiss). Progeny from two lots of diallel crosses were raised under a daily fed control or a cyclic feeding regime containing alternating reduced and compensatory rations. Although the cyclic feeding regime did not improve overall growth, there was some degree of compensatory growth seen during re-alimentation, and the reduced ration led to sustained reductions in condition factor and muscle fat content. Appetite-regulating genes showed little or no differences in expression between feeding regimes. However, the lipid metabolism genes involved in fatty acid synthesis and muscle lipid uptake showed differences in expression during reduced and compensatory ration. This study helps to clarify optimal cyclic feeding regimes in order to enhance growth characteristics preferential to the fish culture industry. / NSERC Strategic Grant, OMAFRA

Orientador: Jose Barreto Campello Carvalheira / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-10T01:57:33Z (GMT). No. of bitstreams: 1 Fernandes_MariaFernandadeAndrade_M.pdf: 2919034 bytes, checksum: c3f7796b55fb5ad53b9eaf2ee58536d5 (MD5) Previous issue date: 2007 / Resumo: As proteínas AMPK e mTOR são as principais moduladoras do balanço energético intracelular, e exercem influência decisiva sobre a ação hipotalâmica da leptina. O exercício agudo, através da produção de IL-6, está associado ao aumento da sensibilidade à ação anorexigênica da leptina. Visando investigar a possível influência das vias AMPK e mTOR, neste aumento da sensibilidade hipotalâmica à leptina induzido pelo exercício agudo, ratos Wistar foram submetidos à natação e posteriormente receberam injeção intracerebroventricular de IL-6, ativadores e/ou inibidores da AMPK e mTOR. A IL-6 reduziu a ingestão alimentar dos animais não exercitados; no entanto, o pré-tratamento com ativador da AMPK ou inibidor da mTOR bloqueou esta ação da IL-6. Ativadores da AMPK aumentaram a ingestão alimentar de forma mais significativa nos animais não exercitados. Inibidores da AMPK reduziram a ingestão mais expressivamente nos animais exercitados. A injeção central de leptina reduziu a ingestão de ratos exercitados mais expressivamente do que foi observado nos animais controle. Tanto o pré-tratamento com inibidor da IL-6, como com ativador da AMPK ou inibidor da mTOR, reverteram esta ação da leptina. O exercício também está associado à redução da fosforilação da via da AMPK e à maior fosforilação da via da mTOR, no hipotálamo. A da resposta das vias em questão ao estímulo com leptina provavelmente seja um dos principais determinantes da modulação do set point hipotalâmico pelo exercício agudo / Abstract: AMP-activated protein kinase (AMPK) and mammalian Target of Rapamycin (mTOR) are key regulators of cellular energy balance and of the effects of leptin on food intake. Acute exerci se is associated with increased sensitivity to the effects of leptin on food intake in an IL-6-dependent manner. To determine whether exerci se ameliorates the AMPK and mTOR response to leptin in the hypothalamus in an IL-6-dependent manner, rats performed two 3-h exercise bouts, separated by one 45-min rest períod. Intracerebroventrícular IL-6 infusion reduced food intake and pretreatment with AMPK activators and mTOR inhibitors prevented IL-6-induced anorexia. Activators of AMPK increased food intake in control rats to a greater extent than that observed in exercised ones, whereas inhibitors of AMPK had the opposite effect. Exercise was associated with both reduced phosphorylation of the AMPK/ ACC signaling pathway and increased phosphorylation of proteins involved in mTOR signal transduction Ín the hypothalamus. The regulatory role of IL-6 in mediating the modulation in AMPK and mTOR pathways in the hypothalamus was also investigated. Treatment with leptin reduced food intake in exercised rats that were pretreated with vehicle, although no increase in sensitivity to leptin-induced anorexia after pretreatment with anti-IL6 antibody, AICAR or Rapamycin was detected. Improved responses of AMPK and mTOR to leptin may contribute to the appetite suppressive actions of exercise / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

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.

Diss. (sammanfattning) Stockholm : Karolinska institutet 2006. / Härtill 4 uppsatser.

Fundação de Amparo a Pesquisa do Estado de Alagoas / Estudos recentes sugerem que a técnica de estimulação elétrica transcraniana (ETCC) pode reduzir a vontade subjetiva de comer e desejo pelo alimento, mostrando-se uma possível ferramenta terapêutica no combate à obesidade. O exercício físico suscita efeitos positivos sobre a manutenção e perda do peso corporal total, balanço energético, regulação do apetite e neuroplasticidade, oxigenação e vascularização cerebral. Desse modo, o objetivo do presente estudo foi verificar o efeito da ETCC, associada à prática de exercício físico, sobre a variabilidade da freqüência cardíaca (VFC) e sensações subjetivas do apetite em humanos obesos. Participaram do estudo nove indivíduos de ambos os sexos. Os sujeitos foram submetidos a duas etapas de procedimentos experimentais. A primeira consistiu na realização de um teste cardiopulmonar máximo no cicloergômetro com protocolo do tipo rampa, a fim de determinar sua capacidade cardiorrespiratória (VO2MAX; FCMAX; CargaMAX). Na segunda etapa, os sujeitos realizaram duas sessões não consecutivas de testes, em que inicialmente permaneceram sentados por 10min, receberam ETCC anódica ou placebo (2mA por 20min), em ordem randomizada e, logo após, executaram sessões isocalóricas (~200kcal) a 70%VO2MAX. Em seguida, permaneceram em recuperação sentados em repouso por 30 minutos. Em todo o protocolo, escalas visuais analógicas para avaliação das sensações subjetivas de apetite, bem como a VFC, foram registradas. Os resultados mostraram que, para os escores médios das sensações de saciedade na condição de ETCC anódica, houve diminuição significativa entre os momentos 30 PÓS EXERCÍCIO vs. todos os momentos analisados (P<0.05). Houve aumento da sensação de fome, em ambas condições de ETCC, entre os momentos 30 PÓS EXERCÍCIO vs. PÓS EXERCÍCIO. No entanto, houve diminuição do desejo pelo alimento após a ETCC anódica (PRÉ ETCC vs. PÓS ETCC; p=0.04), mas não quando os indivíduos receberam ETCC placebo. Quando se associou a ETCC com o exercício, uma diminuição ainda maior foi evidenciada (PRÉ ETCC vs. PÓS EXERCÍCIO; p=0.05). Quanto à VFC, a variável low frequency (LFLOG) demonstrou, em todos os momentos e condições analisados, semelhança em seu comportamento. Por outro lado, as variáveis high frequency (HFLOG) e balanço simpatovagal (LF/HFLOG), apresentaram aumento e diminuição significativas, respectivamente, entre as condições analisadas (placebo e anódica) após a realização da sessão de exercício. Um maior HF e menor LF/HF foram mantidos até o trigésimo minuto de recuperação. Portanto, pode-se concluir que a aplicação da ETCC anódica associada à prática de exercício físico não foi capaz de modificar as respostas hedônicas de fome e saciedade. Porém, a ETCC foi capaz de diminuir o desejo pelo alimento, o que foi potencializado pela associação com o exercício físico. Por outro lado, a ETCC foi capaz de alterar o comportamento do sistema nervoso autonômico cardíaco, propiciando uma maior modulação da atividade parassimpática e diminuição no balanço simpatovagal durante todo o período de recuperação após exercício aeróbio.