As stanniocalcinas (STC1 e STC2) são hormônios glicoproteicos identificados primeiramente em peixes ósseos, relacionados com o metabolismo do cálcio e fosfato. Recentes descobertas evidenciaram que as funções das STCs foram mantidas em mamíferos. A descoberta da localização do receptor para STC1 na membrana mitocondrial de roedores e sua ação como desacopladora da respiração celular sinalizam fortemente um importante papel desse hormônio no metabolismo intermediário de mamíferos. A STC estimula a lipogênese no tecido hepático e muscular de ratos, porém, suas ações sobre o metabolismo do tecido adiposo ainda não foram esclarecidas. No presente trabalho, estudamos o efeito, in vitro, da STC1 e da STC2 humanas, nas concentrações (A: 0,01ng/mL; B: 0,1ng/mL; C: 10ng/mL) no tecido adiposo branco (TAB) e no tecido adiposo marrom (TAM) em Rattus norvegicus machos, 300 ± 50g, alimentados ad libitum e submetidos ao jejum de 24 e 48 horas (n=61). Os resultados obtidos demonstram que, no TAB, a STC1 não apresentou efeito. Contudo, a STC2, nas concentrações A e C, diminuiu a formação de 14CO2 em animais em jejum de 24 horas e nas concentrações A e B, aumentou a incorporação de [214C] piruvato em 14C-glicerol, em animais alimentados (controle). No TAM, a STC1 na concentração B, diminuiu a formação de 14CO2 em animais alimentados (controle). Na mesma concentração, aumentou a incorporação de [214C] piruvato em 14C-glicerol em animais em jejum de 24 horas e aumentou a incorporação de 14C-ácido graxo em animais alimentados (controle). A STC2, na concentração B, diminuiu a formação de 14CO2 em animais alimentados (controle) e aumentou a formação de 14CO2 nos animais em jejum de 24 horas. A STC2 não alterou a via gliceroneogênica neste tecido. A dosagem plasmática do hormônio leptina apresentou acentuada diminuição no grupo jejum 48 horas. As STCs 1 e 2, nas concentrações utilizadas, não alteraram a atividade da enzima PEPCKc nos tecidos estudados (TAB e TAM), em animais alimentados (controle). Em quase todos os tratamentos observamos diferenças marcantes entre animais alimentados e jejuados. Este é o primeiro estudo que mostra as diferentes concentrações das STCs no metabolismo de lipídios. / The stanniocalcins (STC1 and STC2) are glycoprotein hormones related to the metabolism of calcium and phosphate first identified in fish bone. Recent findings showed that the functions of STCs were maintained in mammals. The discovery of the location of the receptor for STC1 in the mitochondrial membrane, as well as its uncoupling action of cellular respiration in rodents, strongly indicate an important role of this hormone in the intermediary metabolism of mammals. The STC stimulates lipogenesis in the liver and muscle tissue of rats. However, its action on the metabolism of adipose tissue has not yet been clarified. In this work, we studied the effect in vitro of human STC1 and STC2 concentrations (A: 0,01 ng/ml, B: 0,1 ng/ml, C: 10ng/mL) in white adipose tissue (WAT) and in brown adipose tissue (BAT) in male Rattus norvegicus , 300 ± 50 g , fed ad libitum and fasted for 24 and 48 hours (n= 61). The results show that in the WAT, the STC1 had no effect. However, STC2 at concentrations A and C decreased the formation of 14CO2 in fasted rats for 24 hours and the concentrations A and B increased the incorporation of [214C] into 14C-glycerol pyruvate in fed animals (control). In BAT, STC1 concentration B, decreased the formation of 14CO2 in fed animals (control). At the same concentration, increased the incorporation of [2 14C] pyruvate into 14C-glycerol in fasted rats for 24 hours and increased the incorporation of 14C-fatty acid in fed animals (control). The STC2 concentration B decreased the formation of 14CO2 in fed animals (control) and increased the formation of 14CO2 in animals fasted for 24 hours. The STC2 did not alter the glyceroneogenesis pathway in this tissue. The serum levels of leptin showed marked decrease in the group fasting for 48 hours. The STCs 1 and 2, in the concentrations used, did not alter the PEPCKc enzyme activity in the tissues studied (WAT and BAT) in fed animals (control). In almost all treatments we observed striking differences between fed and fasted animals. This is the first study that shows the effect of different concentrations of STCs in the metabolism of lipids.

Orientadores: Mario José Abdalla Saad, Alexandre Gabarra de Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T23:51:50Z (GMT). No. of bitstreams: 1 Leite_JulianaPaula_M.pdf: 4915396 bytes, checksum: 09ea43792fb47457eab9ac0df46c3416 (MD5) Previous issue date: 2014 / Resumo: A obesidade representa o maior fator de risco para o desenvolvimento de doenças como diabetes tipo 2, dislipidemia, esteatose hepática, doenças vasculares e alguns tipos de cânceres. Embora o aumento do tecido adiposo seja a principal característica da obesidade, nem todo depósito de gordura é prejudicial. Isso se deve ao fato do tecido adiposo ser subdividido em dois tipos: branco e marrom, os quais apresentam características bem distintas. O tecido adiposo branco estoca energia na forma de triglicerídeos, produz a secreção de vários tipos de citocinas inflamatórias e o seu aumento está associado a um estado inflamatório subclínico do organismo. Já o tecido adiposo marrom é especializado na dissipação da energia em forma de calor, estudos vêm mostrando a sua associação com a melhora da resistência à insulina e menor índice de massa corporal, por isso, o seu aumento pode ser potencial alvo para o tratamento de síndromes metabólicas. Paralelamente, estudos comprovaram que o exercício físico, quando praticado de forma crônica, pode exercer importante efeito anti-inflamatório nos obesos. Este efeito está associado à redução da massa de tecido adiposo branco e estudos comprovaram que o exercício também é capaz de promover o aumento da massa do tecido adiposo marrom. No entanto, ainda não está claro quais os mecanismos envolvidos para tais benefícios. Diante disso, o objetivo do nosso trabalho foi avaliar o efeito profilático do exercício crônico sobre a massa do tecido adiposo (branco e marrom), marcadores inflamatórios e resistência à insulina em ratos alimentados com dieta hiperlipídica. Além de verificar os mecanismos pelos quais o exercício é capaz de aumentar a atividade termogênica do tecido adiposo marrom. Para isso, utilizamos ratos Wistar, divididos em 3 grupos: animais alimentados com dieta padrão para roedores (CTL), animais alimentados com dieta hiperlipídica (HFD) e animais alimentados com dieta hiperlipídica e submetidos ao treinamento de natação (EXE). O protocolo de treinamento utilizado foi de 8 semanas. Nossos resultados mostraram que o exercício crônico de 8 semanas foi capaz de atenuar o desenvolvimento da massa de gordura e a expressão das proteínas de formação do tecido adiposo branco (TAB); apresentou efeito protetor contra a intolerância à glicose e RI. Fora também observado a redução dos circulantes de LPS, TNF-? e AGLs, além da expressão das serinas quinases JNK E IKK. Além disso, os dados demonstram ainda o efeito positivo do exercício na via de sinalização da insulina; aumento da massa do tecido adiposo marrom (TAM) e da expressão de proteínas envolvidas no processo de termogênese. Por último, verificamos que o exercício crônico foi capaz de atenuar a infiltração de macrófagos no TAM e promover maior polarização de macrófagos do tipo M2 no TAM. A partir dessas análises, podemos entender em parte que o exercício físico, quando aplicado antes do estabelecimento da obesidade, é capaz de atenuar o quadro de resistência à insulina e os efeitos deletérios da inflamação causada pela dieta hiperlipídica. Além de contribuir para a maior atividade do TAM através de um mecanismo orquestrado pela ativação alternativa de macrófagos / Abstract: Obesity is a major risk factor for the development of diseases such as type 2 diabetes, dyslipidemia, fatty liver, vascular disease and some cancers. Although the increase in adipose tissue is a hallmark of obesity, not every deposit of fat is harmful. This is because adipose tissue is subdivided into two types: white and brown, which have very different characteristics. White adipose tissue stocks energy in the form of triglycerides, and it is responsible to the secretion of various cytokines and their increase is associated with a proinflammatory state of the organism. As brown adipose tissue is specialized in the dissipation of energy as heat, studies have shown its association with improved insulin resistance and lower body mass index. Therefore, its increase may be a potential target for the treatment of metabolic syndromes. In parallel, studies have shown that chronic exercise may have an important anti-inflammatory effect on obesity due to a reduction mass of white adipose tissue and the capacity to promote the increased mass of brown adipose tissue. However, it remains unclear which mechanisms are involved for such benefits. Therefore, the aim of our study was to evaluate the prophylactic effect of chronic exercise on the mass of adipose tissue (white and brown), inflammatory markers and insulin resistance in high-fat diet rats. Besides, our study verified the mechanisms by which exercise can increase the thermogenic activity of brown adipose tissue. For this, we used 6 weeks male Wistar rats, which were divided into 3 groups as follows: animals fed with standard rodent diet (CTL) animals fed with high fat diet (HFD) and animals fed with high fat diet and submitted to swimming training (EXE). The training protocol used was 8 weeks. Our results showed that 8 weeks of chronic exercise was able to attenuate the development of fat mass and protein expression formation of white adipose tissue (WAT). It was also shown a protective effect against glucose intolerance and insulin and observed a reduction of circulating LPS, TNF-? and FFA, in addition to the expression of serine kinases JNK and IKK. The results also demonstrate the positive effect of exercise on insulin signaling pathway, increasing the mass of brown adipose tissue (BAT) and the expression of proteins involved in the thermogenesis process, which resulted in higher accumulation of fat in the adipocytes of TAM. Finally, we found that chronic exercise was able to attenuate the infiltration of macrophages in the TAM and promote greater polarization of the type M2 macrophages in TAM. From these analyses, we can understand in part that exercise, when applied before the onset of obesity, is able to attenuate the context of insulin resistance and the deleterious effects of inflammation caused by high-fat diet, contributing to the higher activity of TAM through an alternative orchestrated macrophage activation mechanism / Mestrado / Fisiopatologia Médica / Mestra em Ciências

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Adipose tissue, also known as fat tissue are the lipid filled cells with various functions such as, shock absorber, energy storage, and thermal insulation. In this study, the impact of compressive stress at the microstructural level on the porcine abdominal adipose tissue is analyzed under different strain rates of 0.1%, 1%, and 10%. The compressed tissues were fixed in formalin followed by hematoxylin and eosin staining, which were further analyzed at the cellular level using an optical microscope. The preliminary results showed a linear relationship between stress and strain up to 20% strain amplitude. Additionally, the stiffness of the fat tissue was observed to increase with the increase in loading rate. The non-linearity behavior of the adipose tissue and its effects on the cellular level will be studied to understand the mechanical response of fat tissue and its ability to mitigate and absorb energy from low to high strain rate.

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Largely as a consequence of the ongoing obesity epidemic, research into adipose tissue biology has increased substantially in recent years. Worldwide, the number of people classed as overweight or obese is growing, and this represents a major public health concern. Adipose tissue is broadly divided into two types; white and brown. Whilst white adipose tissue (WAT) functions to store and mobilise triglycerides, brown adipose tissue burns chemical energy to generate heat. WAT is further divided into visceral “bad” fat and subcutaneous “good” fat depots, and it is an increase in the former that is linked to obesity-associated diseases. As well as adipocytes, several other cell types including haematopoietic and endothelial are found within adipose tissue, and comprise the stromal vascular fraction (SVF). Adipocyte precursor cells (APCs) also reside within the SVF and are essential for the maintenance and expansion of adipose tissue. The protein encoded by the Wilms’ tumour 1 (Wt1) gene is predominantly known to function as a transcription factor, but also has a role in post-transcriptional processing. Deletion of Wt1 in adult mice results in a considerable loss of fat tissue. Moreover, recent work has revealed that a proportion of the APCs from all visceral WAT depots express Wt1, therefore revealing heterogeneity within the APC population. Additionally, visceral WAT depots are encapsulated by a WT1 expressing mesothelial layer, which has its origins in the lateral plate mesoderm (LPM), and can give rise to mature adipocytes. Lineage tracing has demonstrated that a significant proportion of the mature adipocytes in all adult visceral WAT depots (but not subcutaneous) are derived from cells that express Wt1 in late gestation. These findings uncovered key ontogenetic differences between visceral and subcutaneous WAT and led us to ask whether Wt1 functions in visceral adipose tissue biology. Preliminary work has shown that adipocytes derived from Wt1 expressing (Wt1+) precursor cells have fewer, larger lipid droplets than those derived from non-Wt1 expressing (Wt1-) precursors. In this thesis, this heterogeneity is explored further using a Wt1GFP/+ knock-in mouse. When Wt1+ and Wt1- APCs are cultured separately, the Wt1+ population differentiate into adipocytes more readily. Moreover, the Wt1+ APCs are more proliferative than the Wt1-. Preliminary results also suggest that the Wt1+ APCs may secrete a factor(s) that causes the Wt1- APCs to exhibit improved adipogenic differentiation, a result that is supported by data from comparative transcriptomic analysis. Finally, the percentage of APCs decreases when mice are fed a high fat diet. Interestingly, this decrease is more pronounced for the Wt1+ population. Therefore, it appears that as well as exhibiting differing behaviours in vitro, the Wt1+ and Wt1- populations respond differently to physiologically relevant conditions in vivo. Whilst the LPM is a major source of visceral WAT, the origin of subcutaneous WAT is currently unknown. Here, the Prx1-Cre and Prx1-CreERT2 mouse lines are used to investigate this. It is shown that the majority of subcutaneous WAT adipocytes and APCs are labelled by Prx1-Cre, however this is not the case for most of the visceral WAT depots. The exception to this is the pericardial (heart fat) depot, in which approximately 70% of the adipocytes and 40% of the APCs are labelled. Moreover, a proportion of the Prx1-Cre labelled pericardial APCs also express Wt1, therefore suggesting additional heterogeneity. Preliminary results show that this heterogeneity may have functional consequences, at least in vitro. Additionally, lineage tracing studies suggest that the somatic LPM may be one source of subcutaneous WAT and pericardial visceral WAT Finally, it is shown that the conditional deletion of Wt1 in the Prx1-Cre lineage results in abnormal diaphragm development. Congenital diaphragmatic hernia (CDH) is severe birth defect, the etiology of which is not well understood. Here, a new model of CDH has been developed, and the cellular and molecular mechanisms responsible for the defect in this model are investigated.

Obesity is a grave disease that is increasing in global prevalence. Aberrant neuronal DNA methylation patterns have been implicated in the promotion of obesity development, but the role of neuronal DNA methyltransferases (Dnmts; enzymes that catalyze DNA methylation) in energy balance remains poorly understood. We investigated the role of neuronal Dnmt1 in normal energy regulation and obesity development using a novel Dnmt1 knockout mouse model, Dnmt1fl/fl Synapsin1Cre (ND1KO), which specifically deletes Dnmt1 in neurons. ND1KO and fl/fl control littermates were fedeither a standard chow diet or a high fat diet (HFD). We conducted a deep analysis to characterize both peripheral and central aspects of the ND1KO phenotype. We found that neuronal Dnmt1 deficiency reduced adiposity in chow-fed mice and attenuated obesity in HFD-fed male mice. ND1KO male mice had reduced food intake and increased energy expenditure on the HFD. Furthermore, these mice had improved insulin sensitivity as measured by an insulin tolerance test. HFD-fed ND1KO mice had smaller fat pads and an upregulation of thermogenic genes in brown adipose tissue. These data suggest that neuronal Dnmt1 deletion increased diet-induced thermogenesis, which may explain the lean phenotype in HFD-fed ND1KO mice. Interestingly, we found that ND1KO male mice had elevated estrogen receptor-α gene expression in the hypothalamus, which previously has been shown to control body weight. Immunohistochemistry experiments revealed that estrogen receptor-α (ERα) protein expression was upregulated in the dorsomedial region of the VMH (VMHdm), a region which may mediate the central effect of leptin. Finally, we tested whether ND1KO mice had reduced methylation of the ERα gene promoter, which might explain the ERα upregulation. Neuronal Dnmt1 deficiency decreased methylation at two CpG sites on Exon A in chow-fed mice. Collectively, these data suggest that neuronal Dnmt1 regulates energy homeostasis through pathways controlling food intake and energy expenditure, and that ERα in the VMHdm may mediate these effects.