Type 1 diabetes mellitus (T1D) is an autoimmune disorder that targets the insulin-producing β-cells. The gut may play a role in the pathogenesis of T1D, as genetically-susceptible individuals and animal models of T1D exhibit increased intestinal permeability and improving gut barrier function can interfere with the onset of diabetes. Moreover gut-derived peptides are capable of modifying barrier function and regulate β-cell mass via effects on proliferation and apoptosis. I tested whether chronic administration of glucagon-like peptide-2 (GLP-2), a peptide which potently improves gut barrier function, modifies diabetes onset in a mouse model of T1D, the non obese diabetic (NOD) mouse. Although chronic treatment with a long-acting GLP-2 analogue was associated with improved intestinal barrier function, it failed to delay the onset of T1D. Once the autoimmune attack is initiated, pathogenic T-cells infiltrate the islets and trigger the death of β-cells. Studies in animal models have revealed that β-cells exhibit a compensatory response in the initial stages of the immune attack, which eventually fails, resulting in β-cell mass deficiency and onset of T1D. Glucagon-like peptide-1 (GLP-1) exerts both proliferative and anti-apoptotic actions on β-cells. I hypothesized that chronic activation of the GLP-1 receptor (GLP-1R) would delay or prevent the loss of functional β-cell mass in the NOD mouse. I have shown that chronic administration of the GLP-1R agonist exendin-4 significantly delayed the onset of diabetes and enhanced β-cell mass. Furthermore, GLP-1R activation was associated with a reduction of islet-infiltrating immune cells, as well as changes in lymphocyte subpopulations. Consequently, I addressed whether the GLP-1R has a role in the immune system of NOD and C57Bl/6 mice. GLP-1R mRNA transcripts were detectable in several immune subpopulations, and GLP-1R activation was associated with cAMP production in primary splenocytes and thymocytes. Furthermore I demonstrated that GLP-1R signaling controls proliferation of thymocytes and lymphocytes, and is required for maintaining peripheral regulatory T-cells. In summary, these studies establish that while GLP-2R activation is not sufficient to modify disease onset in a murine model of T1D, GLP-1R activation reduces the extent of diabetes development by exerting actions on β-cells and the immune system.

Submitted by PPG Biologia Celular e Molecular (bcm@pucrs.br) on 2017-07-18T12:45:18Z No. of bitstreams: 1 FERNANDA_CRISTINA_DE_MESQUITA_TES.pdf: 3705815 bytes, checksum: d4984b596a690199bbc567a00b9a5f63 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-07-28T18:46:03Z (GMT) No. of bitstreams: 1 FERNANDA_CRISTINA_DE_MESQUITA_TES.pdf: 3705815 bytes, checksum: d4984b596a690199bbc567a00b9a5f63 (MD5) / Made available in DSpace on 2017-07-28T18:54:15Z (GMT). No. of bitstreams: 1 FERNANDA_CRISTINA_DE_MESQUITA_TES.pdf: 3705815 bytes, checksum: d4984b596a690199bbc567a00b9a5f63 (MD5) Previous issue date: 2017-03-17 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Liver fibrosis is the wound healing response to repeated injury of the liver. This process begins with the damage of the parenchymal cells and subsequent inflammation, characterized by the rupture of the hepatic architecture associated to the increase of the expression of the components of the extracellular matrix. The development of hepatic fibrosis is based on the activation of hepatic stellate cells (HSC) that undergo phenotypic changes and are characterized by loss of vitamin A deposition and increased cell proliferation, triggering hepatic microcirculatory dysfunction and fibrogenesis in patients with chronic liver disease (CLD). Liraglutide is a GLP-1 agonist (glucagon-like peptide 1) well established as an antidiabetic drug, but also has anti-inflammatory properties, in addition to the effectiveness for NAFLD (non-alcoholic fatty liver disease). Therefore, the aim of this study was to evaluate the effects of liraglutide on the HSC phenotype and liver microvascular function using diverse pre-clinical models of CLD. The results obtained demonstrate that Liraglutide de-activated human and rat HSC phenotype through a GLP1-Rindependent mechanism. Liraglutide did not affect the HSC viability but decreased cell proliferation. CLD-rats receiving liraglutide exhibited significantly lower portal pressure (-20%) with a consequent reduction in intrahepatic vascular resistance. There was also a marked improvements in hepatic vascular function, fibrosis, HSC phenotype and sinusoidal endothelial phenotype. The anti-fibrotic effects of liraglutide were confirmed in human liver tissue. In conclusion, this study demonstrates for the first time that liraglutide improves hepatic sinusoidal endothelium in clinically relevant experimental models of cirrhosis, which leads to improvement in fibrosis and portal hypertension, and therefore is valid in the treatment of advanced chronic liver disease. / A fibrose hep?tica ? a resposta cicatricial do f?gado ? les?es repetidas. Este processo inicia com o dano das c?lulas parenquimatosas e consecutiva inflama??o, caracterizado pelo rompimento da arquitetura hep?tica associada ao aumento da express?o dos componentes da matriz extracelular. O desenvolvimento da fibrose hep?tica ? baseado na ativa??o das c?lulas hep?ticas estreladas (HSC) que sofrem mudan?as fenot?picas e se caracterizam pela perda do dep?sito de vitamina A e aumento da prolifera??o celular, desencadeando disfun??o microcirculat?ria hep?tica e fibrog?nese nos pacientes com doen?a hep?tica cr?nica (CLD). A liraglutida ? um an?lago do GLP-1 (glucagon-like peptide 1) bem estabelecido como f?rmaco antidiab?tico, mas que tamb?m possui propriedades antinflamat?rias, al?m da efetividade para NAFLD (doen?a hep?tica gordurosa n?o alco?lica). Por essa raz?o, o objetivo deste estudo foi avaliar os efeitos da liraglutida sobre o fen?tipo das HSC e a fun??o microvascular hep?tica utilizando diversos modelos pr?-cl?nicos de CLD. Os resultados obtidos demonstram que a liraglutida desativou o fen?tipo das HSC humanas e de ratos atrav?s de um mecanismo independente do receptor GLP1. A liraglutida n?o afetou a viabilidade das HSC mas diminuiu a prolifera??o celular. Os ratos com CLD que receberam liraglutida apresentaram press?o portal significativamente menor (-20%) com consequente redu??o da resist?ncia vascular intra-hep?tica. Houve tamb?m uma acentuada melhoria na fun??o vascular hep?tica, fibrose, fen?tipo das HSC e fen?tipo endotelial sinusoidal. Os efeitos anti-fibr?ticos da liraglutida tamb?m foram confirmados em tecido hep?tico humano. Como conclus?o, este estudo demonstra pela primeira vez que a liraglutida melhora o endotelio sinusoidal hep?tico em modelos experimentais clinicamente relevantes de cirrose, o que leva a melhora no quadro fibr?tico e na hipertens?o portal e, portanto, pode ser v?lido no tratamento da doen?a hep?tica cr?nica avan?ada.

Type 1 diabetes mellitus (T1D) is an autoimmune disorder that targets the insulin-producing β-cells. The gut may play a role in the pathogenesis of T1D, as genetically-susceptible individuals and animal models of T1D exhibit increased intestinal permeability and improving gut barrier function can interfere with the onset of diabetes. Moreover gut-derived peptides are capable of modifying barrier function and regulate β-cell mass via effects on proliferation and apoptosis. I tested whether chronic administration of glucagon-like peptide-2 (GLP-2), a peptide which potently improves gut barrier function, modifies diabetes onset in a mouse model of T1D, the non obese diabetic (NOD) mouse. Although chronic treatment with a long-acting GLP-2 analogue was associated with improved intestinal barrier function, it failed to delay the onset of T1D. Once the autoimmune attack is initiated, pathogenic T-cells infiltrate the islets and trigger the death of β-cells. Studies in animal models have revealed that β-cells exhibit a compensatory response in the initial stages of the immune attack, which eventually fails, resulting in β-cell mass deficiency and onset of T1D. Glucagon-like peptide-1 (GLP-1) exerts both proliferative and anti-apoptotic actions on β-cells. I hypothesized that chronic activation of the GLP-1 receptor (GLP-1R) would delay or prevent the loss of functional β-cell mass in the NOD mouse. I have shown that chronic administration of the GLP-1R agonist exendin-4 significantly delayed the onset of diabetes and enhanced β-cell mass. Furthermore, GLP-1R activation was associated with a reduction of islet-infiltrating immune cells, as well as changes in lymphocyte subpopulations. Consequently, I addressed whether the GLP-1R has a role in the immune system of NOD and C57Bl/6 mice. GLP-1R mRNA transcripts were detectable in several immune subpopulations, and GLP-1R activation was associated with cAMP production in primary splenocytes and thymocytes. Furthermore I demonstrated that GLP-1R signaling controls proliferation of thymocytes and lymphocytes, and is required for maintaining peripheral regulatory T-cells. In summary, these studies establish that while GLP-2R activation is not sufficient to modify disease onset in a murine model of T1D, GLP-1R activation reduces the extent of diabetes development by exerting actions on β-cells and the immune system.

No description available.

Thesis / Doctor of Philosophy (PhD)

In response to ingestion of nutrients, enteroendocrine L cells secrete the incretin hormone, glucagon-like peptide-1 (GLP-1), to enhance glucose-dependent insulin release. Therapies related to GLP-1 are approved for type 2 diabetes. The GLP-1 receptor (GLP-1R) is expressed in cells of the gastrointestinal tract and elsewhere. In pancreatic beta cells, GLP-1R are coupled to the Gs/cAMP/PKA pathway. The expression and function of GLP-1R in gastrointestinal smooth muscle are not known. Aim. To test the hypothesis that GLP-1 regulates smooth muscle function by acting on GLP-1R expressed on smooth muscle. Methods. Smooth muscle cells (SMC) were isolated and cultured. Expression of GLP-1R mRNA was measured by RT-PCR. Expression of GLP-1R protein was measured by western blot. The effect of GLP-1 (7-36) amide on Gαs activation, cAMP formation, and PKA activity was examined in cultured SMC. The effect of GLP-1 on basal activity and on acetylcholine-induced contraction was measured in intact colon via organ bath. Results. Amplification of GLP-1R mRNA suggested expression of GLP-1R mRNA in mucosal and non-mucosal colon cells, which was confirmed in pure SMC cultures. Similar patterns of protein expression were obtained with western blot. Addition of GLP-1 caused relaxation of phasic activity and agonist-induced tonic contractions in intact colon, suggesting a role of smooth muscle Gs-coupled GLP-1R in mediating relaxation. In SMC, GLP-1 (7-36) amide activated Gas, increased cAMP levels, and stimulated PKA activity. Conclusion. Colonic SMC express GLP-1R, and GLP-1 inhibits both basal and acetylcholine-induced contraction. The GLP1-R is coupled to the heterotrimeric G protein, Gas.