Made available in DSpace on 2017-07-21T19:59:56Z (GMT). No. of bitstreams: 1 Priscila Costa Estabile.pdf: 1735178 bytes, checksum: b258b31793d5d5954531a50cbe4206cc (MD5) Previous issue date: 2012-02-13 / Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Paraná / The Type 2 Diabetes Mellitus, as well as the metabolic syndrome (MS), is a multifactorial and metabolic disorder that now presents itself as a worldwide pandemic with effects on morbidity and mortality, possibly as a result of the mismatch between biological and cultural evolution of man. Was the object of research of this study to analyze the tissue expression of incretin hormones glucagon Pepitide Like-1 (GLP1) and Pepitide YY (PYY3-36), to identify and quantify L cells along the gastrointestinal tract in patients with DM2 subjected to adaptative gastroenteromentectomia with intestinal bipartition (Partial Duodenal Switch - DSP). The study was approved by the Ethics Committee of UEPG and patients informed and educated about the research objectives. The volunteer group consisted of 7 patients aged between 35 and 65 years, body mass index> 25 kg/m2 with T2DM on dietary treatment and medication for a minimum of 2 years and with difficulty on glycemic control and hypertriglyceridemia associated. Samples were obtained from the intestinal mucosa (jejunum and ileum) of DSP in patients undergoing preoperative and postoperative condition of fasting for 12 hours (three and twelve months respectively), through incisional biopsy. These biopsies were designed to test immunohistochemistry, qRT-PCR (Quantitative Real Time PCR) and Western Blott. The results were consistent and indicate a very significant differential expression between the state of pre-and postoperative tests for qRT-PCR (p = 0.1669) and Western Blot (p = 0.1569). Immunohistochemistry also showed low significance (p = 0.0043) of immune marked L cells for the same patients under the same conditions. These data can be interpreted in light of the fast imposed on patients. In addition the results are unprecedented for the immunostaining of L-cells of the human gastrointestinal tract, the data indicate that these cells have basal secretion for GLP-1, even after 12 hours without feed stimulation. In addition, the patients showed normalization of blood glucose levels in the post-surgery, suggesting metabolic improvement. It was also found that the number of L cells marked increases in density along the gastrointestinal tract toward the distal portion of the ileum (p = 0.0409). With these results it was possible to identify, locate and investigate different levels of expression and secretion from intestinal L cells in patients with DM2 and subjected to surgical control. / Diabetes mellitus do tipo 2 (DM2), assim como a Síndrome Metabólica (SM), é uma desordem metabólica e multifatorial que atualmente se apresenta como pandemia mundial com reflexos na morbimortalidade, possivelmente em decorrência do descompasso entre a evolução biológica e cultural do homem. Foi objeto de investigação do presente estudo analisar a expressão tecidual dos hormônios incretínicos Glucagon Like Pepitide-1 (GLP1) e Pepitide YY (PYY3-36), visando identificar e quantificar células L ao longo do trato gastrointestinal, de pacientes portadores de DM2 submetidos a gastroenteromentectomia adaptativa com bipartição intestinal (Duodenal Switch Parcial - DSP). O trabalho foi aprovado pelo Comitê de Ética da UEPG e os pacientes informados e esclarecidos sobre os objetivos da pesquisa. O grupo de voluntários foi composto de 7 pacientes com idade entre 35 e 65 anos, Índice de Massa Corporal > 25 Kg/m2, com DM2 em tratamento dietético e medicamentoso por um período mínimo de 2 anos e com dificuldade de controle glicêmico e hipertrigliceridemia associada. Foram obtidas amostras da mucosa intestinal (jejuno e íleo) dos pacientes submetidos à DSP no pré-operatório e no pós-operatório em condição de jejum de 12 horas (três e doze meses, respectivamente), através de biópsia incisional. Estas biópsias foram destinadas aos ensaios de Imunohistoquímica, qRT-PCR (Quantitative Real Time PCR) e Western Blott. Os resultados obtidos foram congruentes e apontam uma expressão diferencial pouco significativa entre o estado de pré- e pós-operatório para os ensaios de qRT-PCR (p=0,1669) e Western Blott (p=0,1569). A imunohistoquímica mostrou também baixa significância (p=0,0043) de células L imuno marcadas para os mesmos pacientes, nas mesmas condições. Estes dados podem ser interpretados a luz do jejum imposto aos pacientes. Além dos resultados serem inéditos para a imunomarcação de células L do trato gastrointestinal humano, os dados obtidos indicam que estas células apresentam secreção basal para GLP-1, mesmo após 12 horas sem estímulo alimentar. Em adição, os pacientes apresentaram normalização dos níveis de glicemia no estado pós-operado, sugerindo melhora metabólica. Foi ainda constatado que o número de células L marcadas aumenta em densidade ao longo do trato gastrointestinal em direção à porção mais distal do íleo (p=0,0409). Com estes resultados foi possível identificar, localizar e investigar diferentes níveis de expressão e secreção das células L intestinais em pacientes portadores de DM2 e submetidos a controle cirúrgico.

Diabetes mellitus is a complex disease characterised by chronic hyperglycaemia due to compromised insulin synthesis and secretion, or decreased tissue sensitivity to insulin, if not all three conditions. Endothelial dysfunction is a common complication in diabetes in which endothelium-dependent vasodilation is impaired. The aim of this study was to examine the involvement of TRPV4 in diabetes endothelial dysfunction. Male Charles River Wistar rats (350-450 g) were injected with 65mg/kg streptozotocin (STZ) intraperitoneally. STZ-injected rats were compared with naïve rats (not injected with STZ) or control rats (injected with 10ml/kg of 20mM citrate buffer, pH 4.0-4.5), if not both. Rats with blood glucose concentrations greater than 16mmol/L were considered to be diabetic. As the results revealed, STZ-diabetic rats showed significant endothelial dysfunction characterised by impaired muscarinic-induced vasodilation, as well as significant impairment in TRPV4-induced vasodilation in aortic rings and mesenteric arteries. Furthermore, STZ-diabetic primary aortic endothelial cells (ECs) showed a significant reduction in TRPV4-induced intracellular calcium ([Ca2+]i) elevation. TRPV4, endothelial nitric oxide synthase (eNOS), and caveolin-1 (CAV-1) were also significantly downregulated in STZ-diabetic primary aortic ECs and were later significantly restored by in vitro insulin treatment. Methylglyoxal (MGO) was significantly elevated in STZ-diabetic rat serum, and nondiabetic aortic rings incubated with MGO (100μM) for 12 hours showed significant endothelial dysfunction. Moreover, nondiabetic primary aortic ECs treated with MGO (100μM) for 5 days showed significant TRPV4 downregulation and significant suppression of 4-α-PDD-induced [Ca2+]i elevation, which was later restored by L-arginine (100μM) co-incubation. Incubating nondiabetic aortic rings with MGO (100μM) for 2 hours induced a spontaneous loss of noradrenaline-induced contractility persistence. Moreover, MGO induced significant [Ca2+]i elevation in Chinese hamster ovary cells expressing rat TRPM8 channels (rTRPM8), which was significantly inhibited by AMTB (1-5μM). Taken together, TRPV4, CAV-1, and eNOS can form a functional complex that is downregulated in STZ-diabetic aortic ECs and restored by insulin treatment. MGO elevation might furthermore contribute to diabetes endothelial dysfunction and TRPV4 downregulation. By contrast, MGO induced the loss of contractility persistence, possibly due to MGO's acting as a TRPM8 agonist.