Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-10-17T11:03:10Z No. of bitstreams: 1 lilianesenapinheiro.pdf: 8108520 bytes, checksum: b03200480798ddb2cf88a9276e4c9d8d (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-10-22T13:09:54Z (GMT) No. of bitstreams: 1 lilianesenapinheiro.pdf: 8108520 bytes, checksum: b03200480798ddb2cf88a9276e4c9d8d (MD5) / Made available in DSpace on 2016-10-22T13:09:54Z (GMT). No. of bitstreams: 1 lilianesenapinheiro.pdf: 8108520 bytes, checksum: b03200480798ddb2cf88a9276e4c9d8d (MD5) Previous issue date: 2008-06-25 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O diabetes mellitus (DM) reduz a secreção de catecolaminas (CAs) das células cromafins adrenais, sendo esse um evento patofisiológico crítico por favorecer a ocorrência de episódios de hipoglicemia grave decorrentes do próprio tratamento da doença. Vários trabalhos relatam a participação de proteínas quinase C (PKCs) nas vias de síntese e secreção de CAs nas células cromafins. Os objetivos desse trabalho foram analisar o efeito do DM sobre a expressão das isoformas α, ε e ζ de PKC em células cromafins de ratos e avaliar se o controle glicêmico reverte os efeitos da doença. Foram utilizados ratos Wistar com DM induzido por estreptozotocina. Foram estabelecidos três grupos experimentais, ratos controles (C), diabéticos tratados com salina (DTS) ou com insulina (DTI). As análises foram feitas 15 dias após a indução. Utilizamos as técnicas de imunohistoquímica e Western Blot. A insulinoterapia foi estabelecida após estudos do comportamento alimentar e da variação dos níveis glicêmicos de ratos controles e doentes durante 24h consecutivas. Foi testada a eficácia de diferentes esquemas de tratamento com insulina. O tratamento estabelecido consistiu em injeções de insulina NPH, sendo 1U aplicada às 13h e 4U às 19h. Após os 15 dias de tratamento, o ganho médio de massa corporal dos ratos C (+37±3g) e DTI (+43±3g) foram similares enquanto os DTS emagreceram (-9±6g). A média da glicemia de jejum dos ratos C (74±1mg/dl) e dos DTI (93±6mg/dl) foram similares e dentro dos níveis normais, enquanto que a dos ratos DTS foi elevada (471±23mg/dl). A insulinoterapia restabeleceu os níveis plasmáticos do colesterol total, c-LDL e c-VLDL nos ratos DTI. O DM não alterou os níveis de c-HDL, triglicerídos e frutosamina. As análises da expressão de PKCs mostraram que a PKCα é a mais expressada seguida de ζ e depois de ε. O DM reduziu em 39,5% a expressão da PKCα, enquanto a de ζ foi aumentada em 74,2%. A expressão da PKCε não foi afetada pelo DM. O tratamento com insulina reverteu o efeito do DM sobre a expressão de PKCα, a expressão da PKCε continuou inalterada e a expressão da PKCζ permaneceu elevada (+32,6%) quando comparada aos ratos C. Concluímos que em células cromafins adrenais, o diabetes afeta a expressão de isoformas de PKCs de maneira diferenciada. Trabalhos realizados em nosso laboratório mostraram que o DM reduz o conteúdo total (21,1%), a secreção basal (-24,3%) e a estimulada por carbacol (-28,9%) e K+ (42,2%) de CAs. Como observado para PKCα, a insulinoterapia reverteu o efeito do DM sobre o conteúdo total. Já foi demonstrado que PKCα participa de uma via de sinalização que estimula a atividade de tirosina hidroxilase. Por outro lado, o tratamento não restabeleceu os processos secretórios, sugerindo que PKCζ possa estar envolvida nessa alteração. Há fortes evidências de que PKCζ regula canais de K+ retificadores, o que pode explicar o efeito da doença sobre o processo de secreção via despolarização da membrana. / The diabetes mellitus (DM) reduces the catecholamine (CAs) secretion of adrenal chromaffin cells, a critical pathophysiologic event that promotes the occurrence of serious hypoglycemia episodes, consequence of the disease treatment. Several papers report the participation of protein kinase C (PKC) on catecholamine synthesis signal pathways of adrenal chromaffin cells. The objectives of this work were to study the effect of DM on expression of PKC isoforms α, ε and ζ in rat chromaffin cells and to evaluate if the glicemic control revert the effect of the illness. Male Wistar rats with diabetes induced by streptozotocin were used. Three experimental groups were determined: Control (C), diabetic rats receiving saline solution (DS) and diabetic rats receiving insulin (DI). The analyses were made after 15 days of DM induction. Immunohistochemistry and western blotting techniques were done. The insulin therapy protocol was established after studying the feeding behavior and glycemic level variations during the whole 24h. The information made possible to establish the time of insulin applications. Several schemes of insulin treatments were tested to keep the diabetic rat as close as possible to normoglycemia path. The best results were found by using 1U at 1:00 PM and 4U at 7:00 PM of NPH insulin. After 15 days of treatment the acquired body weight was similar between C and DI rats, 37±3g and 43±3g, respectively. The DS rats emaciated 9±6g. The fasting glycemic levels were 74±1mg/dl, 93±6mg/dl and 471±23mg/dl to C, DI and DS rats, respectively. The insulin therapy reestablishes the plasmic levels of total cholesterol, c-LDL and c-VLDL on DI rats. The DM did not change the levels of c-HDL, triglycerides and frutosamine. The PKCα is the more expressed isoform in adrenal chromaffin cells, followed by ζ and ε. The DM reduced 39,5% the PKCα expression and, unlike, increased 74,2% the expression of PKCζ. The expression of PKCε was not affected by DM. The insulin treatment reverted the effect of DM on PKCα, the expression of PKCε remained unchanged and the expression of PKCζ remained higher than the control group (+32,6%). Studies of our laboratory show that the DM causes reduction on adrenal catecholamine content (21,1%), basal secretion (-24,3%) and catecholamine secretion stimulated by carbachol (-28,9%) and high K+ (-42,2%). The insulin therapy, in like manner as observed on PKCα, reverted the DM effect on adrenal catecholamine content. It was shown that PKCα participates on signal transduction pathway that stimulates the activity of tyrosine hydroxylase. Otherwise, the insulin treatment did not restore the secretory processes, suggesting that PKCζ could be involved in this process. There are strong evidences showing that PKCζ regulates the voltage-dependent delayed rectifier K (Kv) and its expression was not normalized by insulin therapy.

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<p> Exposure to chronic low oxygen (hypoxia) leads to a series of adaptive responses involving changes in gene expression that are critical for cell, tissue, and organismal survival. These changes are mediated by an important set of regulators belonging to the hypoxia inducible factor (HIF) family of transcription factors (e.g. HIF-lα, HIF-2α, HIF3α) which undergo rapid degradation during normal oxygen (normoxia) but are rapidly stabilized during hypoxia. While the role of HIF-1α has been extensively studied in many cell types, there have been relatively few studies on the role of HIF-2α, though recent evidence suggests its function maybe tissue specific. This thesis examined the hypothesis that HIF-2α plays a central role in the development and function of catecholaminergic cells of the sympathoadrenal (SA) lineage. The study was aided by use of an immortalized line of rat adrenomedullary chromaffin cells (i.e. MAH cells), derived from fetal SA progenitors, which express several hypoxia-sensitive properties characteristic of native cells in the adrenal gland. In Chapter 2, I investigated the potential contributions of mitochondrial reactive oxygen species (ROS) and 0 2 consumption to HIF-2α induction in MAH cells exposed to chronic hypoxia (2% O(2); 24 hr). In MAH cells, chronic hypoxia caused an increase in HIF-2α induction which was blocked by inhibition of any of the mitochondrial complexes using pharmacological agents, or by specific inhibition of complexes III and IV using RNAi techniques. It was found that in this 0 2-sensitive chromaffin cell line mitochondrial O(2) consumption, rather than changes in ROS, regulated HIF-2α induction during hypoxia. In Chapter 3, I investigated the hypothesized role of HIF-2α in the development of the catecholaminergic phenotype in cells of the SA lineage using the MAH cell line as a model. Mutant MAH cells, with depleted HIF-2α due to siRNA knock-down, showed dramatically lower levels of dopamine and noradrenaline compared to untransfected and scrambled control cells, regardless of whether the cells were cultured under normoxia or chronic hypoxia. This was correlated with a marked reduction in the expression of DOPA decarboxylase (DDC) and dopamine B hydroxylase (DBH), though the expression of tyrosine hydroxylase (TH) was unaffected. Moreover, HIF-2α was able to bind to a region of the DDC gene promoter which contains two putative hypoxia response elements (HREs). These data suggest that a basal level of HIF-2α function is required for the normal developmental expression of DDC and DBH in SA progenitor cells, and that loss of this function leads to impaired catecholamine (CA) biosynthesis. In Chapter 4, I investigated genes regulated by chronic hypoxia in MAH cells, with a focus on those involved in CA metabolism, storage, and secretion. Using microarray analysis combined with QPCR and RNAi knock-down methodology I uncovered several genes, involved in amine vesicular packaging, trafficking and secretion, which were upregulated during chronic hypoxia. One gene specifically, the adenosine A(2A) receptor (A(2A)R) gene, which appears to modulate CA secretion via autocrine or paracrine actions of extracellular adenosine, was dramatically upregulated in chronic hypoxia. Interestingly, this effect was completely abolished in HIF-2α knockdown MAH cells, suggesting a critical involvement of HIF-2α. Chromatin immunoprecipitation (ChIP) assays revealed that HIF-2α bound to the promoter region of the A(2A)R gene which contains a putative hypoxia response element (HRE) immediately upstream of exon 1. Ratiometric fluorescence measurements of intracellular Ca(2+) revealed that adenosine (50 μM) potentiated the high K(+)-evoked rise in [Ca(2+)]i in MAH cells. This effect of adenosine was further enhanced after chronic hypoxia, but was abolished in HIF-2α knock-down cells. In conclusion, these data suggest that HIF-2α is a key regulator of several genes involved in CA biosynthesis, and of others that mediate the facilitatory effects of chronic hypoxia on CA secretion in sympathoadrenal derivatives. / Thesis / Doctor of Philosophy (PhD)