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Orientador: Licio Augusto Velloso Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: A proteína PGC1? é um co-ativador de transcrição gênica que desempenha papel importante na regulação de uma série de fenômenos metabólicos que compreendem desde o controle da termogênese e mitocondriogênese até a regulação da secreção de insulina e a produção hepática de glicose. Como vários dos fenômenos biológicos controlados direta ou indiretamente pela PGC1? tem importância vital, a regulação dos níveis de PGC1? nos tecidos deve ser finamente ajustada. Nos últimos anos, inúmeros estudos exploraram os mecanismos envolvidos com o controle da expressão gênica e tradução da PGC1?. Entretanto, apenas alguns poucos estudos avaliaram a degradação da mesma. Um dos mais importantes mecanismos envolvidos com a regulação funcional e da meia-vida de proteínas é a ubiquitinação, que pode direcionar proteínas alvo ao proteassoma para degradação ou a outras modificações pós-traducionais. O objetivo do presente estudo foi avaliar a participação de uma proteína com atividade deubiquitinase e ubiquitina ligase, a A20, na manutenção da homeostase do tecido adiposo de animais submetidos à dieta rica em gordura e voluntários humanos magros e obesos antes e após cirurgia de redução de peso. Foram utilizados o tecido adiposo branco visceral e subcutâneo e o tecido adiposo marrom de camundongos Swiss machos submetidos a 16 semanas de dieta hiperlipídica e o tecido adiposo subcutâneo de voluntários magros e obesos antes e após a cirurgia bariátrica. Esses tecidos foram avaliados quanto ao conteúdo protéico e expressão gênica da proteína A20, e sua associação com a PGC1? por imunoprecipitação e imunofluorescência, bem como a ubiquitinação desta última. Os resultados obtidos a partir do tecido adiposo de humanos mostram uma diminuição na expressão da proteína A20 nos pacientes antes e após a cirurgia bariátrica com relação aos voluntários magros. A PGC1? aparece mais ubiquitinada nos pacientes obesos em relação e a associação entre A20 e PGC1? parece aumentar com o ganho de peso na mesma proporção que o conteúdo protéico de PGC1? parece diminuir. No tecido adiposo subcutâneo de camundongos obesos, observamos uma diminuição de PGC1? bem como redução da marcação por cadeias de poliubiquitina desta proteína, associado a um aumento de A20 e aumento da associação de A20 com PGC1?. Camundongos obesos foram também tratados com um oligonucleotídeo antisense (ASO) para A20, resultando na redução de sua expressão gênica. Os animais tratados apresentaram uma piora na tolerância à glicose no teste de GTT o que ocorreu concomitantemente a redução de PGC1?. Nossos resultados indicam que, no tecido adiposo, a A20 se associa a PGC1? e a redução da sua expressão resulta em redução da expressão da PGC1? o que é acompanhando de uma piora no controle homeostático da glicose Abstract: Peroxisome proliferator-activated receptor ? coactivator 1 alpha (PGC-1?) plays an important role in whole body metabolism and, particularly in glucose homeostasis. Its expression is tightly regulated and, small variations in tissue levels can have a major impact in a number of physiological and pathological conditions. Recent studies have shown that the ubiquitin/proteasome system plays a role in the control of PGC-1? degradation. Here we evaluated the interaction of PGC-1? with the protein A20, which plays a dual-role in the control of the ubiquitin/proteasome system acting as a deubiquitinase and as an E3 ligase. We employed immunoprecipitation, quantitative real-time PCR and immunofluorescence staining to evaluate PGC-1?, A20, PPAR? and ubiquitin in the adipose tissue of humans and mice. Our results show that, in distinct sites of the adipose tissue A20 binds to PGC-1?. At least in the subcutaneous fat of humans and mice the levels of PGC-1? decrease during obesity, while its physical association with A20 increases. The inhibition of A20 leads to a reduction of PGC-1? and PPAR? expression, suggesting that A20 acts as a protective factor against PGC-1? disposal. Thus, we provide evidence that mechanisms regulating PGC-1? ubiquitination are potentially involved in the control of the function of this transcriptional co-activator Mestrado Fisiopatologia Médica Mestra em Ciências

Agradecimentos: The Laboratory of Cell Signaling is supported by funding provided by the Fundação de Amparo a Pesquisa do Estado de São Paulo, Conselho Nacional de Desenvolvimento Cientifico e Tecnologico. The Laboratories of Cell Signaling belongs to the Obesity and Comorbidities Research Center and the National Institute of Science and Technology - Neuroimmunomodulation Abstract: The characterization of the hypothalamic neuronal network, that controls food intake and energy expenditure, has provided great advances in the understanding of the pathophysiology of obesity. Most of the advances in this field were obtained thanks to the development of a number of genetic and nongenetic animal models that, at least in part, overtook the anatomical constraints that impair the study of the human hypothalamus. Despite the undisputed differences between human and rodent physiology, most seminal studies undertaken in rodents that have unveiled details of the neural regulation of energy homeostasis were eventually confirmed in humans; thus, placing experimental studies in the forefront of obesity research. During the last 15 years, researchers have provided extensive experimental proof that supports the existence of hypothalamic dysfunction, which leads to a progressive whole-body positive energy balance, and thus, to obesity. Here, we review the experimental work that unveiled the mechanisms behind hypothalamic dysfunction in obesity FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ Fechado

Evaluation of the impact of a high-fat diet on the expression of claudin-5 in the median eminence or mice. Six-week-old male Swiss mice were randomly divided to feed on chow or a high-fat diet for 1, 2, or 4 weeks; at the end of the respective experimental periods, the mice were used in experiments. Median eminence was eLaser microdissected for real-time PCR determination of claudin-5. Expression of target transcripts is presented as relative to paired controls fed chow (line in y = 1). In all conditions, n = 4; *p

Orientador: Licio Augusto Velloso Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: Obesidade é hoje o principal fator de risco para uma série de doenças e o aumento de sua prevalência é alarmante. A ativação de um processo inflamatório subclínico no hipotálamo desempenha papel central como desencadeador dos distúrbios associados ao ganho de peso, como a resistência à insulina. A ativação do processo inflamatório característico da obesidade decorre da complexa associação entre fatores genéticos e ambientais. O consumo de dietas ricas em ácidos graxos saturados é certamente um dos mais importantes fatores ambientais, levando à inflamação através da ativação de receptores TLR4. A participação do sistema imune inato nessa inflamação hipotalâmica tem sido extensivamente estudada. No entanto, o papel da imunidade adaptativa no curso da obesidade é ainda desconhecido. Em doenças neurodegenerativas, a inflamação do sistema nervoso central é responsável pela atração e ativação de linfócitos T, sendo este evento concomitante ao início da doença. Conhecendo o papel dos ácidos graxos nesse cenário imunológico, nós aventamos a hipótese de que o CD1, proteína que apresenta lipídios a células T, teria papel relevante na resposta imune adaptativa no hipotálamo de camundongos obesos. Neste trabalho, mostramos que com poucos dias de dieta, ocorre um aumento da expressão hipotalâmica de CD1 e modificação de sua distribuição anatômica. A redução da sua expressão utilizando um anticorpo imunoneutralizador acarreta em melhora do perfil glicêmico e redução do ganho de peso enquanto sua ativação no hipotálamo exacerba o fenótipo obeso dos animais, inclusive levando animais resistentes à obesidade a apresentarem um perfil de ganho de peso semelhante a animais propensos. A análise por bioinformártica revelou que a expressão de CD1 no hipotálamo se correlaciona a outros marcadores envolvidos no distúrbio. Dessa forma, o CD1 parece exercer papel relevante na inflamação hipotalâmica frente ao consumo de ácidos graxos saturados Abstract: Obesity is the main risk factor for many comorbidities and its prevalence is rising to astounding numbers. The activation of a subclinic and chronic inflammatory process in the hypothalamus has been shown to be the trigger event leading to several comorbidities, such as insulin resistence and diabetes. This process occurs due to a complex interaction between genetic and envirolmental factors. The consumptions of saturated fatty acid rich diets is one of the most important envirolmental factors, leading to inflammation due to activation of the innate immune receptor, TLR4. The role of innate immune response in the hypothalamic inflammation has been extensively studied. However, the putative role of the adaptive immune response in this scenario is still unknown. In neurodegenative diseases, the inflammation is responsible for the recruitment and activation of T lymphocytes to the brain, which contributes to the emergence of many syptoms of the diseases. As we know that fatty acids trigger an infammatory immune response that disrupts hypothalamic control of food intake and energy homeostases, we hypothesized that CD1, a lipid-presenting protein to lymphocytes, would play a central role in the hypothalamic immune response. In this work, we show that as early as one day of high fat diet, the CD1 expression is higher in the hypothalamus of mice and the distribution in this tissue is also disturbed. The deacrease in CD1 expression leads to improvement of glucose tolerance e reduced weight gain; wheras the increase of CD1 signaling exacerbates the obese phenotype. Bioinformatics analyses showed that the hypothalamic expression of CD1 correlates with other celular mechanisms involved in the pathophysiology of obesity. Therefore, CD1 seems to have a pivotal role in hypothalamic inflammation due to saturated fatty acids consumption Doutorado Fisiopatologia Médica Doutora em Ciências FAPESP 2013/25415-9

Quantification of immunofluorescence of Fig. 4. (PDF 67 kb)

Evaluation of the blood-brain barrier integrity. The protocol employed for evaluation of BBB integrity is shown in Fig. 2a. Confocal microscopy analysis was employed for determining FITC-dextran endogenous fluorescence in the regions of the vascular organ of lamina terminalis (OVLT), subfornical organ (SFO), and subcomissural organ (SCO); in all acquisitions, the same settings of the microscope were employed (laser 488, wavelength = 405, %laser = 20%, gain = 1015, offset = − 0.3799). The fluorescence in the distinct regions was determined using an ImageJ software and presented as relative to control. N = 4. (PDF 141 kb)

Immunofluorescence staining of markers of the median eminence blood-brain barrier and markers of glial cells. Immunofluorescence staining using primary antibodies against IGFBP2 (green), GFAP (red) (A), and IBA1 (red) (B). Specimens were obtained from bregma-anteroposterior − 2.06 to − 2.18. The images are representative of three independent experiments. 3v, third ventricle; IGFBP2, insulin-like growth factor-binding protein 2; GFAP, glial fibrillary acidic protein; IBA1, ionized calcium-binding adapter molecule-1. (PDF 17432 kb)

Quantification of immunofluorescence of Fig. 7d. (PDF 58 kb)

Quantification of immunofluorescence of Fig. 6. (PDF 59 kb)

Quantification of immunofluorescence of Fig. 5. (PDF 59 kb)

Evaluation of median eminence blood-brain barrier integrity in mice treated with an anti-BDNF immunoneutralizing antibody. The protocol employed for evaluation of BBB integrity is shown in Fig. 2a. Confocal microscopy analysis was employed for determining FITC-dextran endogenous fluorescence in the region of the median eminence in all acquisitions; the same settings of the microscope were employed (laser 488, wavelength = 405, %laser = 20%, gain = 1015, offset = − 0.3799). The fluorescence intensity was determined using an ImageJ software and presented as relative to control. N = 6. ABDNF, antibody-anti-BDNF; BDNF, brain-derived neurotrophic factor; CTR, control; HFD, high-fat diet; IGG, non-immune antiserum. (PDF 2780 kb)

Quantification of immunofluorescence of Fig. 7e. (PDF 59 kb)

Quantification of immunofluorescence of Fig. 8i. (PDF 68 kb)

The anatomical and cellular distribution of LIF and LIF receptor in the hypothalamus of mice. Hypothalamic 5.0 μm frozen sections were prepared from 5-week old mice fed on chow. Immunofluorescence staining was performed using antibodies against LIF (A), LIF receptor (LIFR) (B), alpha-MSH (aMSH) (A, B). The high magnification images depict details of cells (arrows). Nuclei was labeled using DAPI. Color code and magnifications are presented in the panels. Figures are representative of three independent experiments. Figure S2. The impact of LIF immunoneutralization on STAT3 phosphorylation in the hippocampus and hypothalamus. The expression of phosphor-STAT3 (pSTAT3) protein was evaluated by immunoblot in the hippocampus (A) and hypothalamus (B) of obesity-resistant mice treated with non-immune IgG or anti-LIF antibody for 15 days. N = 3–4; *p

Six-week-old, male Swiss mice were stereotaxically instrumented in a Stoelting stereotaxic apparatus to receive a cannula placed in the lateral hypothalamic ventricle, using the following stereotaxic coordinates: anteroposterior 0.34 mm, lateral 1.0 mm, and dorsoventral 2.2 mm. The correct position of the cannula was tested 5 days after surgery by evaluation of the thirst response elicited by intracerebroventricular (icv) angiotensin II (106 M). After 1 week, icv-cannulated mice were transferred to the high-fat diet and treated once a day for 3 days with 2 μL of saline (CTR) or 2 μL of DHA (5, 10 or 20 ng). Caloric intake and body mass were determined during the experimental period. At the end of the experimental period, the hypothalamus was obtained for real-time PCR quantitative determination of the transcripts encoding for PLA2 (A), 15-LOX (B), 5-LOX (C), GPR18 (D), IL6 (E) and IL10 (F). The methods for hypothalamic extraction and real-time PCR are described in the Methods section of the paper. (PDF 768 kb)

Agradecimentos: We thank E. Roman and G. Ferraz from the University of Campinas for technical assistance. Support for the study was provided by the Fundação de Amparo a Pesquisa do Estado de São Paulo, Conselho Nacional de Desenvolvimento Cientifico e Tecnologico and grants from the Trust in Science Initiative from Glaxo-Smithkline, UK. The Laboratories of Cell Signaling and Experimental Endocrinology belong to the Obesity and Comorbidities Research Center and the National Institute of Science and Technology–Diabetes and Obesity Abstract: BACKGROUND/OBJECTIVES: The identification of brown/beige adipose tissue in adult humans has motivated the search for methods aimed at increasing its thermogenic activity as an approach to treat obesity. In rodents, the brown adipose tissue is under the control of sympathetic signals originating in the hypothalamus. However, the putative connection between the depots of brown/beige adipocytes and the hypothalamus in humans has never been explored. The objective of this study was to evaluate the response of the hypothalamus and brown/beige adipose tissue to cold stimulus in obese subjects undergoing body mass reduction following gastric bypass. SUBJECTS/METHODS: We evaluated twelve obese, non-diabetic subjects undergoing Roux-in-Y gastric bypass and 12 lean controls. Obese subjects were evaluated before and approximately 8 months after gastric bypass. Lean subjects were evaluated only at admission. Subjects were evaluated for hypothalamic activity in response to cold by functional magnetic resonance, whereas brown/beige adipose tissue activity was evaluated using a (F 18) fluorodeoxyglucose positron emisson tomography/computed tomography scan and real-time PCR measurement of signature genes. RESULTS: Body mass reduction resulted in a significant increase in brown/beige adipose tissue activity in response to cold; however, no change in cold-induced hypothalamic activity was observed after body mass reduction. No correlation was found between brown/beige adipose tissue activation and hypothalamus activity in obese subjects or in lean controls. CONCLUSIONS: In humans, the increase in brown/beige adipose tissue activity related to body mass reduction occurs independently of changes in hypothalamic activity as determined by functional magnetic resonance FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ Fechado