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O mecanismo pelo qual a sinalização da leptina no hipotálamo permite o início da puberdade ainda não foi esclarecido. Um possível mecanismo para a ação molecular da leptina no eixo reprodutivo é constituído por uma alteração na sinalização do NPY. Objetivou-se neste estudo foi verificar se novilhas precoces Bos taurus indicus possuem a expressão modificada de genes hipotalâmicos relacionadas à sinalização da leptina. Dentre uma população de 500 novilhas entre 20 e 25 meses de idade, 100 novilhas foram selecionadas com base nas características da raça (Nelore), mês de nascimento e peso corpóreo (290 kg). Estas 100 novilhas foram classificadas de acordo com a presença ou não de um corpo lúteo (CL) notável. Dez novilhas sem um CL e dez novilhas com CL notável receberam uma injeção de prostaglandina, e de acordo com a observação visual de cio e palpação retal, 6 novilhas precoces e 6 novilhas não precoces foram selecionadas para o experimento. Estas 12 novilhas foram abatidas e amostras de tecido do hipotálamo foram coletadas e congeladas em nitrogênio liquido. A expressão de SOCS-3, NPY, NPY-Y1 e NPY-Y4 no hipotálamo foi quantificada por PCR em tempo real usando uma proteína ribossomal RP-L19 como um gene de referência. A expressão hipotalâmica de SOCS-3 ou NPY não foi diferente entre os grupos de novilhas (P > 0,50). Acreditava-se que as novilhas ciclando poderiam ser resistentes à leptina devido a um aumento na expressão do SOCS-3 no hipotálamo. Houve uma tendência (P = 0,10) de redução na expressão dos receptores do NPY, NPY-Y1 e NPY-Y4 em novilhas que atingiram a puberdade precocemente. A expressão do NPY-Y1 foi 8.3 vezes menor e a expressão do NPY-Y4 foi 14.3 vezes menor em novilhas precoces. Quando analisados em conjunto, houve uma redução de 11 vezes na expressão dos receptores de NPY em novilhas precoces, e este efeito foi estatisticamente significante (P = 0,03). Estes resultados sugerem que, a menor expressão dos receptores de NPY pelo hipotálamo de novilhas precoces pode torná-lo menos sensível à inibição do NPY, e permitir a obtenção da puberdade com maior peso vivo e níveis menores de leptina circulante. Em conclusão, não houve uma correlação entre a expressão do gene NPY e SOCS-3 e a precocidade sexual das novilhas Nelore, porém houve uma tendência significativa de redução da expressão dos receptores de NPY-Y1 e NPY-Y4 no hipotálamo das novilhas precoces. / The molecular mechanism by which leptin signaling in the hypothalamus might permit the initiation of puberty has not been elucidated. One possible mechanism for leptin molecular action on the reproductive axis is affecting NPY signaling. It was our objective to test whether early-maturing Bos indicus heifers have altered expression of hypothalamic genes related to leptin signaling. Among a population of 500 heifers between 20 and 25 months of age, 100 heifers were selected base on breed attributes (Nelore), month of birth, and body weight (290 kg). These 100 heifers were scored as prepubertal or pubertal according to the presence or not of a noticeable corpus luteum (CL). Ten heifers without a CL and ten heifers with noticeable CL received a prostaglandin injection, and according to visual observation of heat and rectal palpation, 6 prepubertal and 6 pubertal heifers were selected for the experiment. These 12 heifers were slaughtered and samples of hypothalamus were collected and frozen in liquid nitrogen. Expression of SOCS-3, NPY, NPY-Y1 and NPY-Y4 at the hypothalamus was quantified by real-time PCR using the ribosomal protein RP-L19 as a reference gene. Hypothalamic expression of SOCS-3 or NPY was not different between groups of heifers (P > 0, 50). It was thought that late-maturing heifers could be resistant to leptin due to an increased expression of SOCS-3 at the hypothalamus. However, there was a tendency for NPY-Y1 and NPY-Y4 expression to be reduced in heifers that reached puberty earlier (P = 0,10). Expression of NPY-Y1 was 8.3-folds lower and NPY-Y4 expression was 14.3-folds lower in early-maturing heifers. When analyzed together, there was an 11-fold reduction in NPY receptors expression in early-maturing heifers, and this effect was statistically significant (P = 0,03). These results suggest that, because of the lower expression of NPY receptors, the hypothalamus of early-maturing heifers could be less sensitive to NPY inhibition, and therefore reach puberty with lower levels of circulating leptin. In conclusion, there was no effect between the expression of NPY and SOCS-3 and sexual precocity of Nelore heifers, but there was a significant tendency of reduction in NPY-Y1 e NPY-Y4 receptors expression in the hypothalamus of sexually precocious heifers.

O mecanismo pelo qual a sinalização da leptina no hipotálamo permite o início da puberdade ainda não foi esclarecido. Um possível mecanismo para a ação molecular da leptina no eixo reprodutivo é constituído por uma alteração na sinalização do NPY. Objetivou-se neste estudo foi verificar se novilhas precoces Bos taurus indicus possuem a expressão modificada de genes hipotalâmicos relacionadas à sinalização da leptina. Dentre uma população de 500 novilhas entre 20 e 25 meses de idade, 100 novilhas foram selecionadas com base nas características da raça (Nelore), mês de nascimento e peso corpóreo (290 kg). Estas 100 novilhas foram classificadas de acordo com a presença ou não de um corpo lúteo (CL) notável. Dez novilhas sem um CL e dez novilhas com CL notável receberam uma injeção de prostaglandina, e de acordo com a observação visual de cio e palpação retal, 6 novilhas precoces e 6 novilhas não precoces foram selecionadas para o experimento. Estas 12 novilhas foram abatidas e amostras de tecido do hipotálamo foram coletadas e congeladas em nitrogênio liquido. A expressão de SOCS-3, NPY, NPY-Y1 e NPY-Y4 no hipotálamo foi quantificada por PCR em tempo real usando uma proteína ribossomal RP-L19 como um gene de referência. A expressão hipotalâmica de SOCS-3 ou NPY não foi diferente entre os grupos de novilhas (P > 0,50). Acreditava-se que as novilhas ciclando poderiam ser resistentes à leptina devido a um aumento na expressão do SOCS-3 no hipotálamo. Houve uma tendência (P = 0,10) de redução na expressão dos receptores do NPY, NPY-Y1 e NPY-Y4 em novilhas que atingiram a puberdade precocemente. A expressão do NPY-Y1 foi 8.3 vezes menor e a expressão do NPY-Y4 foi 14.3 vezes menor em novilhas precoces. Quando analisados em conjunto, houve uma redução de 11 vezes na expressão dos receptores de NPY em novilhas precoces, e este efeito foi estatisticamente significante (P = 0,03). Estes resultados sugerem que, a menor expressão dos receptores de NPY pelo hipotálamo de novilhas precoces pode torná-lo menos sensível à inibição do NPY, e permitir a obtenção da puberdade com maior peso vivo e níveis menores de leptina circulante. Em conclusão, não houve uma correlação entre a expressão do gene NPY e SOCS-3 e a precocidade sexual das novilhas Nelore, porém houve uma tendência significativa de redução da expressão dos receptores de NPY-Y1 e NPY-Y4 no hipotálamo das novilhas precoces. / The molecular mechanism by which leptin signaling in the hypothalamus might permit the initiation of puberty has not been elucidated. One possible mechanism for leptin molecular action on the reproductive axis is affecting NPY signaling. It was our objective to test whether early-maturing Bos indicus heifers have altered expression of hypothalamic genes related to leptin signaling. Among a population of 500 heifers between 20 and 25 months of age, 100 heifers were selected base on breed attributes (Nelore), month of birth, and body weight (290 kg). These 100 heifers were scored as prepubertal or pubertal according to the presence or not of a noticeable corpus luteum (CL). Ten heifers without a CL and ten heifers with noticeable CL received a prostaglandin injection, and according to visual observation of heat and rectal palpation, 6 prepubertal and 6 pubertal heifers were selected for the experiment. These 12 heifers were slaughtered and samples of hypothalamus were collected and frozen in liquid nitrogen. Expression of SOCS-3, NPY, NPY-Y1 and NPY-Y4 at the hypothalamus was quantified by real-time PCR using the ribosomal protein RP-L19 as a reference gene. Hypothalamic expression of SOCS-3 or NPY was not different between groups of heifers (P > 0, 50). It was thought that late-maturing heifers could be resistant to leptin due to an increased expression of SOCS-3 at the hypothalamus. However, there was a tendency for NPY-Y1 and NPY-Y4 expression to be reduced in heifers that reached puberty earlier (P = 0,10). Expression of NPY-Y1 was 8.3-folds lower and NPY-Y4 expression was 14.3-folds lower in early-maturing heifers. When analyzed together, there was an 11-fold reduction in NPY receptors expression in early-maturing heifers, and this effect was statistically significant (P = 0,03). These results suggest that, because of the lower expression of NPY receptors, the hypothalamus of early-maturing heifers could be less sensitive to NPY inhibition, and therefore reach puberty with lower levels of circulating leptin. In conclusion, there was no effect between the expression of NPY and SOCS-3 and sexual precocity of Nelore heifers, but there was a significant tendency of reduction in NPY-Y1 e NPY-Y4 receptors expression in the hypothalamus of sexually precocious heifers.

<p>The blood-brain barrier regulates the transport into the brain and protects the central nerve system (CNS) from toxics substances. However some areas of the brain, called circumventricular organs (CVO), lack the blood-brain barrier. One of these is area postrema (AP), which is located in the brainstem immediately adjacent to the nucleus tractus solitarius (NTS). These two areas together regulate autonomic behaviours such as food intake, and also make up the vomiting center.</p><p>The hormones leptin and ghrelin, which regulate food intake, are too big to pass the blood-brain barrier, but have receptors in NTS.</p><p>In this study we used immunohistochemistry to obtain a detailed map of the different components of the blood-brain barrier in AP and NTS.</p><p>The results suggest that there is a barrier that prevents diffusion of substances from AP into NTS. However, there seems to be some vessels in NTS that have a weaker or no barrier characteristics. These vessels could provide an entrance for peripheral substances to neurons in NTS.</p>

The blood-brain barrier regulates the transport into the brain and protects the central nerve system (CNS) from toxics substances. However some areas of the brain, called circumventricular organs (CVO), lack the blood-brain barrier. One of these is area postrema (AP), which is located in the brainstem immediately adjacent to the nucleus tractus solitarius (NTS). These two areas together regulate autonomic behaviours such as food intake, and also make up the vomiting center. The hormones leptin and ghrelin, which regulate food intake, are too big to pass the blood-brain barrier, but have receptors in NTS. In this study we used immunohistochemistry to obtain a detailed map of the different components of the blood-brain barrier in AP and NTS. The results suggest that there is a barrier that prevents diffusion of substances from AP into NTS. However, there seems to be some vessels in NTS that have a weaker or no barrier characteristics. These vessels could provide an entrance for peripheral substances to neurons in NTS.

Made available in DSpace on 2014-06-12T15:55:07Z (GMT). No. of bitstreams: 2 arquivo6204_1.pdf: 2527750 bytes, checksum: 95a408435ab58011c139c275dfbfbbcb (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2003 / Estudos sobre os efeitos da má-nutrição na organização temporal do comportamento têm indicado alterações na expressão da ritmicidade circadiana por comprometer a sincronização dos ritmos endógenos ao ciclo claro-escuro ambiental. O presente trabalho investigou em ratos jovens, os efeitos da má-nutrição crônica e severa sobre o desenvolvimento de componentes do sistema de temporização circadiana: retina, núcleo supraquiasmático do hipotálamo (NSQ), folheto intergeniculado do tálamo (FIG) e tratos retinohipotalâmico (TRH) e genículohipotalâmico (TGH). Ratos do grupo controle foram alimentados com uma dieta padrão contendo 22% de proteína desde a gestação e ratos mal nutridos com uma dieta multideficiente desde a gestação (grupo GAA) ou do aleitamento (grupo AA) e mantidos sob um ciclo claro-escuro de 12:12 h sendo analisados aos 27, 30 e 60 dias. Secções coronais diencefálicas do hipotálamo e tálamo foram processadas por imunohistoquímica para visualizar neurônios do TRH e TGH. A retina e o NSQ foram analisados pelo método de Nissl. Comparados ao grupo controle, animais malnutridos exibiram uma redução significativa na área retiniana e na distribuição de células ganglionares; modificações nas dimensões do NSQ e na área ocupada pelos terminais do TRH, mas não na densidade destes terminais. O desenvolvimento do FIG e TGH não foi afetado pela má-nutrição. Os resultados sugerem uma vulnerabilidade diferenciada das regiões estudadas aos efeitos da má-nutrição severa instalada no período crítico do desenvolvimento do cérebro

The objective of this dissertation research was to investigate the effect of dietary macronutrient composition on neuropeptide Y (NPY)-mediated regulation of adipose tissue physiology in broiler chickens during the early post-hatch period. A high-carbohydrate (HC), high-fat (HF) or high-protein (HP) diet was fed to broiler chicks in all experiments and various facets of physiology were evaluated at day 4 post-hatch, including diet-, fasting-, and neuropeptide Y-induced effects on gene expression, cellular morphology, and lipid metabolism. Experiment 1 was designed to study the effects of diet on molecular changes in different adipose tissue depots (subcutaneous, clavicular and abdominal) after 3 hours of fasting and 1 hour of refeeding. Adipose tissue weights were decreased in chicks that consumed the HP diet, whereas adipocyte diameter was increased in response to the HF diet. There was greater expression of mRNAs encoding fatty acid binding protein 4 (FABP4) and monoglyceride lipase in chicks fed the HC and HF diets than the HP diet in all three adipose tissue depots. Fasting increased plasma non-esterified fatty acid concentrations in chicks fed the HC and HP diets. Results suggest that the heavier fat depots and larger adipocytes in chicks fed the HF diet are explained by greater rates of hypertrophy, whereas the HP diet led to a decrease in adipose tissue deposition, likely as a result of decreased rates of adipogenesis. Experiments 2 and 3 were designed to investigate how dietary macronutrient composition affects the effect of centrally or peripherally administered NPY, respectively, on lipid metabolism-associated factor mRNAs in adipose tissue. In experiment 2, vehicle or 0.2 nmol of NPY was injected intracerebroventricularly (ICV) and abdominal and subcutaneous fat samples were collected at 1 hour post-injection. In the subcutaneous fat, ICV NPY injection decreased peroxisome proliferator-activated receptor gamma (PPAR gamma) and sterol regulatory element-binding transcription factor 1 (SREBP1) mRNAs in chicks fed the HF diet, whereas there was an increase in SREBP1 expression in chicks fed the HF diet after NPY injection. Expression of PPAR gamma and FABP4 mRNAs increased in the abdominal fat of HF diet-fed chicks after NPY injection. Thus, HF diet consumption may have enhanced the sensitivity of chick adipose tissue to the effect of centrally-injected NPY on gene expression of adipogenesis-associated factors. In experiment 3, vehicle, 60, or 120 micrograms/kg BW of NPY was injected intraperitoneally (IP), and subcutaneous, clavicular, and abdominal fat was collected at 1 and 3 hours post-injection. Food intake and plasma NEFA concentrations were not different among chicks fed the HC, HF or HP diet after IP NPY injection, indicating that the effects of NPY on adipogenesis were independent of secondary effects due to altered energy intake. In response to the lower dose of NPY, the expression of NPY receptor sub-type 2 mRNA was increased at 1 hour post-injection in the subcutaneous fat of chicks fed the HP diet, whereas there was less 1-acylglycerol-3-phosphate O-acyltransferase 2 mRNA in the subcutaneous fat of chicks fed the HC diet. The higher dose of NPY was associated with greater AGPAT2 mRNA in the clavicular fat of chicks that consumed the HP diet and less CCAAT/enhancer-binding protein alpha in the abdominal fat of chicks that were provided the HF diet. However, there was also a decrease in the expression of some of these factors, although mechanisms are unclear. In conclusion, dietary macronutrient composition influenced the response of adipose tissue to the adipogenic effects of NPY and metabolic effects of short-term fasting and refeeding during the first week post-hatch. Collectively, this research may provide insights on understanding NPY's effects on the development of adipose tissue during the early life period and mechanisms underlying diet-dependent and depot-dependent differences in adipose tissue physiology across species. / Ph. D. / Neuropetide Y (NPY) is a 36 amino-acid peptide that increases hunger and fat deposition. The objective of this dissertation research was to elucidate how dietary fat/protein affect NPY’s effect on fat tissue physiology in broiler chicks during the early post-hatch period. Three diets that were formulated to be high-carbohydrate (HC), high-fat (HF) or high-protein (HP) were fed to broiler chicks from day of hatch to day 4 post-hatch. In experiment 1, chicks were fasted for 3 hours and refed for 1 hour after 3 hours of fasting. Adipose tissue weight was decreased in chicks fed the HP diet and the diameter of fat cells was greater in chicks that consumed the HF diet. In the adipose tissue of chicks fed the HP diet there was reduced gene expression of factors associated with lipid synthesis and fat cell development. Fasting increased plasma free fatty acid concentrations in chicks fed the HC and HP diets. Results suggest that HP diet-induced decreases in fat deposition might be explained by a decrease in rates of fat cell development/maturation. However, chicks fed the HF diet had more fat deposition and larger fat cells, likely as a result of hypertrophy (growth in cell size). Experiment 2 was designed to investigate how NPY administration in the central nervous system affects adipose tissue physiology after feeding the three diets. Subcutaneous, clavicular and abdominal fat samples were collected at 1 hour post-injection. The injection of NPY increased the gene expression of factors associated with fat cell development and maturation in the abdominal fat of chicks fed the HF diet. Thus, HF diet feeding might have sensitized chicks to the effect of centrally-injected NPY on adipose tissue to deposit more fat and increase the number/size of fat cells. In experiment 3, NPY was injected into the peritoneum of chicks fed the HC, HF and HP diets. Although NPY injection increased the gene expression of factors involved in lipid synthesis and fat cell development/maturation, there was also a decrease in the expression of these factors, yet the mechanisms are unknown. Food intake and plasma free fatty acid concentrations were not affected in response to NPY injection at 1 or 3 hours post-injection, indicating that the effect of NPY on fat cell development and lipid synthesis is independent of secondary effects due to altered energy intake. Overall, this research may provide insights on understanding the effect of NPY on fat cell development and has implications for improving animal production efficiency by increasing feed conversion into muscle instead of fat and minimizing excess fat deposition during certain stages of growth.

Das Ziel der vorliegenden Arbeit war es, zu untersuchen, ob der Neurotransmitter Neuropeptid Y (NPY) Einfluss auf das Schwellungsverhalten retinaler Gliazellen der Ratte in hypoosmolarem Medium hat. Des Weiteren war von besonderem Interesse, welche Rezeptortypen und welche intrazellulären Signalwege in die Wirkung von NPY involviert sein könnten. Verwendet wurden 50 adulte Long-Evants-Ratten. Zuerst wurde bei einem Teil der Ratten eine transiente retinale Ischämie in einem Auge der Ratten induziert. Das andere Auge blieb unbehandelt und diente zur Kontrolle. Drei Tage post op wurden die Ratten euthanasiert. Nach Enukleation der Bulbi wurde die Netzhaut auf einen Membranfilter aufgebracht und Schnitte (1 mm) angefertigt. Um die Müllerzellen der vitalen Retina mit Hilfe des Laser Scanning-Mikroskops darstellen zu können, wurde der Farbstoff Mitotracker Orange verwendet (UCKERMANN et al. 2004). Erst kürzlich konnte gezeigt werden, dass die Müllerzellen der postischämischen Retina der Ratte in hypotonem Medium schwellen (PANNICKE et al. 2004). Dazu wurden die akut isolierten retinalen Schnitte einer hypotonen Lösung ausgesetzt (60 % der Kontrollosmolarität). Im Rahmen dieser Arbeit konnte gezeigt werden, dass NPY die Müllerzellschwellung in hypotonem Medium in der postischämischen Netzhaut verhindert. Die pharmakologische Untersuchung des durch NPY aktivierten Signalweges erfolgte an gesunden Netzhäuten. Hier führt ein hypotones Medium bei gleichzeitiger Blockade der Kaliumkanäle (K+-Kanäle) durch Ba2+ zu einer Gliazellschwellung, die mit derjenigen in der postischämischen Retina vergleichbar ist (PANNICKE et al. 2004). NPY hemmt konzentrationsabhängig das Schwellen der Gliazellen der gesunden Netzhaut in hypoosmolarem Medium in Anwesenheit von Ba2+. Die gleiche Wirkung konnte mit dem selektiven Y1-Rezeptoragonisten hervorgerufen werden, während die Y2- und Y5-Rezeptoragonisten keine Wirkung zeigten. Außerdem hatte NPY in der Anwesenheit des selektiven Y1-Rezeptoragonisten BIBP3226 keine Wirkung. Inkubation mit dem membranpermeablen Ca2+-Chelator BAPTA-AM kehrte die Wirkung des NPY um, ebenso wie die Inkubation mit den Proteinkinase C (PKC)-Inhibitoren Staurosporin und Gö6976. Die Neurotransmitter Glutamat und Adenosin zeigten eine dem NPY vergleichbare hemmende Wirkung auf das Schwellen der Müllerzellsomata. Außerdem konnte eine Stimulierung von metabotropen Glutamatrezeptoren (mGlu) und Adenosin A1-Rezeptoren (A1R) nachgewiesen werden. Jedoch hob der selektiven Na+-Kanalblocker Tetrodotoxin die hemmende Wirkung von NPY auf. Die erzielten Ergebnisse deuten darauf hin, dass NPY einen neuronalen Y1-Rezeptor aktiviert, was zu einer Mobilisierung von Ca2+ aus intrazellulären Speichern und zur Aktivierung der Proteinkinase C (PKC) führt. Weiterhin erfolgt eine von neuronaler Aktivität und Ca2+ abhängige Freisetzung von Glutamat und die Aktivierung von (glialen) mGlu. Letztendlich kommt es vermutlich zur Aktivierung des A1R. Resümierend könnten diese Ergebnisse wichtig sein für die Entwicklung neuer therapeutischer Strategien zur Vermeidung von postischämischen und posttraumatischen Gliazellschwellungen. / The aim of the present study was to determine wether neuropeptide Y (NPY) has an effect on hypotonic glia cell swelling from the retina of the rat. Furthermore, the special interest was to determine which receptor subtypes and which intracellular pathways are involved in the effect of NPY. 50 adult Long-Evants-rats were taken. Transient retinal ischemia was induced in one eye of the rats, while the other eye remained untreated and served as control. Three days after reperfusion, the animals were killed. After enucleation of the bulbi, the isolated retina was fixed on a membrane filter and 1 mm thick slices were produced. The acutely isolated slices were loaded with the vital dye Mitotracker Orange in order to selectively stain Müller glial cells (UCKERMANN et al. 2004). The slices were examined using a confocal laser scanning microscope. As shown recently (PANNICKE et al. 2004), the somata in postischemic retinas corresponded with swelling after changing the extracellular perfusate into a hypotonic solution which contained 60 % of the control ionic strength. NPY significantly decreased the hypotonic glia cell swelling in postischemic retinas. The following experiments for the pharmacological examination of the NPY pathway where made with untreated rats. Cell somata in control retinas showed an increase of their volume during hypotonic stress when the K+-channel blocker Ba2+ was present in the extracellular solution; this swelling is comparable with the swelling of glia cells in postischemic retina (PANNICKE et al. 2004). Cell somata in control retinas showed an increase of their volume during hypotonic stress when the K+-channel blocker Ba2+ was present in the extracellular solution (PANNICKE et al. 2004). NPY significantly decreased the hypotonic glia cell swelling in control retinas in the presence of Ba2+. NPY displayed a dose-dependent swelling effect. The Y1-receptor agonist inhibited dose-dependently the hypotonic glial cell swelling, while agonists for Y2- and Y5-receptors were largely ineffective. Incubation with the membrane permeable Ca2+-chelator BAPTA-AM reversed the swelling inhibiting effect of NPY, just as incubation with PKC-inhibitors staurosporine and Gö6976 did. A dependence of the NPY effect on release of Ca2+ from intracellular stores is also suggested by the effect of thimerosal. Glutamate and adenosine also decreased the hypotonic glia cell swelling in control retinas in the presence of Ba2+. In addition, glutamate stimulates metabotropic glutamte receptors (mGluR) and adenosin activates purinergic receptors. However, the selective Na+-canal blocker tetrodotoxin (TTX) reversed the inhibiting effect of NPY on swelling, but not of glutamate and adenosine. The data suggest that NPY inhibits hypotonic glia cell swelling by activation of neuronal Y1-receptors via Ca2+-dependent release of glutamate. This effect is mediated by subsequent stimulation of glial glutamergic and purinergic receptors in Müller cells. The results may have importance for the development of new therapeutic strategies for inhibition of postischemic and posttraumatic glial cell swelling.

<p>The incidence of obesity and associated diseases such as type 2-diabetes and hypertension has reached epidemic portions worldwide and attracted increased interest to understand the mechanisms that are responsible for these diseases. Obesity can result from excessive energy intake, and increasing evidence has emphasized the role of the central nervous system, especially the hypothalamus, in regulating food intake. White adipose, as a direct target of obesity and an important endocrine organ, also has long been a subject of scientific inquiry. AMPK, a conserved energy sensor, has been shown to play important roles in both the hypothalamus and adipose. Recently, CaMKK2 was shown to function as an AMPK kinase. I used intracerebroventricular cannulation as a means to acutely inhibit hypothalamic CaMKK2 with STO-609 and characterize the appetite change associated with loss of CaMKK2 function. Infusion of STO-609 in wild-type mice, but not CaMKK2-null mice, inhibited appetite and promoted weight loss consistent with reduced NPY and AgRP mRNA. Furthermore, intraperitoneal injection of ghrelin increased food intake in wild-type but not CaMKK2-null mice, and 2-DG increased appetite in both types of mice, indicating that CaMKK2 functions downstream of ghrelin to activate AMPK and upregulate appetite. As CaMKK2-null mice were protected from high-fat diet-induced obesity and diabetes, I performed a pair feeding experiment using a high-fat diet and demonstrated that protection of CaMKK2-null mice did not require reduced food consumption. Analysis of brown adipose tissue and metabolic analysis indicated that CaMKK2-null mice did not expend more energy than WT mice. Interestingly, we were surprised to find that CaMKK2-null mice had more adipose than wild-type mice when fed standard chow (5001). By real-time PCR and immunoblot, I identified CaMKK2 expression in preadipocytes and showed that it decreased during adipogenesis. I used STO-609 or shRNA to block CaMKK2 activity in preadipocytes, which resulted in enhanced adipogenesis and increased mRNA of adipogenic genes. I also identified AMPK as the relevant downstream target of CaMKK2 involved in inhibiting adipogenesis via a pathway that maintained Pref-1 mRNA. Consistent with the in vitro data, we further demonstrated that CaMKK2-null mice have more adipocytes but fewer preadipocytes, which supports our hypothesis that loss of CaMKK2 enhances adipogenesis by depleting the preadipocyte pool. Together the data presented herein contribute to our understanding of distinct mechanisms by which CaMKK2 contributes to feeding behavior and adipogenesis.</p> / Dissertation

No description available.