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Class of 2007 Abstract / Objectives: To explore the effects of human immunodeficiency virus protease inhibitors (HPI) on insulin metabolism and protein degradation in HepG2 hepatocytes in vitro. Methods: To see if HIV-protease inhibitors affect insulin degradation in a dose-dependent manner, HepG2 cells were incubated with various concentrations of tipranavir, indinavir, or atazanavir. After 125I-insulin was added, its degradation was measured by precipitation with trichloroacetic acid (TCA). To see the effect of HPIs on protein degradation, HepG2 cells labeled overnight with 3H-leucine were incubated with 50 mM of an HPI, followed by another HPI incubation including concentrations of insulin ranging from 10-12 to 10-6 M. Cells were solubilized and proteins were precipitated using TCA. Degradation was quantified as percent TCA soluble, normalized, plotted, and then compared using student’s t-test or one- way ANOVA. Results: Cellular insulin degradation was inhibited only by tipranavir at the highest concentrations of 75 and 100 mM (12.06 ± 1.07%, p=0.047 and 9.35 ± 0.44%, p=0.024, respectively) when compared to the control (17.01 ± 1.37%; n=3). None of the concentrations of indinavir or atazanavir decreased insulin degradation significantly. From the protein degradation experiments, the log EC50 of the control (no HPI) insulin dose-response curve was not statistically different compared to those of the individual HPIs. Conclusions: Except for high concentrations of tipranavir, it appears that HPI does not inhibit the cellular degradation of insulin. HPIs do not appear to inhibit the role of insulin in the inhibition of protein degradation significantly.

A series of in vitro trials have been conducted to determine what relationship exists between zinc and the insulin mechanism. There were no significant differences of the zinc contents in total pancreases excised from the rats thirty minutes after the intraperitoneal glucose dose or from the non-dosed controls. When pancreases were cut into three pieces, and treated in three ways: Incubated without glucose (IWG) followed by isolation of the islets; incubated with glucose (IWG) followed by isolation of the islets; islets were isolated, then incubated with glucose (IWG). The zinc content in the islets was significantly higher (p < 0.01) in the first group than the others. The insulin release and zinc movement were studied in zinc-deficient status. In both Experiment 3 and 5 each pancreas was divided into two, one was treated IWG, one was treated IWOG. There were significant (p < 0.01) differences of zinc content in the islets between lWG and IWOG groups in zinc-supplemented pair-fed and ad lib. controls but not in zinc-deficient rats. In Experiment 5 the zinc contents in the islets of IWOG group were significantly (p < 0.01) lower in zinc-deficient rats than in zinc-supplemented pair-fed rats. Insulin release from the islets incubated with glucose was significantly (p < 0.01) less in zinc-deficient group than in zinc-supplemented pair-fed group. Glutathione contents of the pancreases were measured in Experiment 3, no significant difference was found between zinc-deficient group and both zinc-supplemented groups. Rats were intraperitoneally dosed with glucose or with saline in Experiment 6. After 30 minutes pancreases were excised and the isolated islets were treated with IWG or IWOG. There was no significant difference of zinc content in the islets treated IWOG in glucose-dosed and saline0dosed groups, but significant (p < 0.01) difference was found in the islets treated IWG. Insulin release from the islets treated IWG was significantly (p < 0.01) less in glucose-dosed group than in saline-dosed group. It is concluded that zinc is released from the islets as a component of insulin on glucose stimulation, zinc-deficiency results in an impairment of insulin release or synthesis, and insulin secretion was reduced in the isolated islets taken from animals prestimulated with glucose in vivo.

D.Litt et Phil. (Psychology) / Please refer to full text to view abstract

Ovine placental lactogen (oPL) is postulated to be involved in the repartitioning of maternal nutrients during pregnancy, through its effect on insulin metabolism. Ovine pancreatic insulin responses to exogenous glucose are depressed during pregnancy and this depression becomes more pronounced as gestation advances. In addition, under the hormonal environment of rising oPL and growth hormone (oGH) concentrations, maternal whole body glucose irreversible loss (GIL) increases. The percentage of GIL accounted for by uterine glucose uptake also increases with advancing gestation and increasing litter size. Regression analysis of oPL concentration with glucose uterine uptake as a percentage of GIL, accounts for 39% of variation. Maternal oPL concentrations which increase with gestational age, were significantly greater in multiple bearing ewes and ewes subjected to reduced metabolisable energy (ME) intakes. It is postulated that through actions on pancreatic sensitivity, oPL plays a major role as a homeorhetic control during pregnancy. Elevated oPL concentrations were strongly associated with continually depressed pancreatic insulin secretory ability. The reduction in pancreatic sensitivity to glucose was not as a result of elevation in GH or non-esterified fatty acid (NEFA) concentrations. Muscle insulin receptor number and affinity were found to increase with increasing litter size, suggesting that pregnancy associated insulin resistance occurs predominantly in adipose tissue. During ovine pregnancy there is a specific stimulation of maternal gluconeogenesis. As gestation advances, an increasingly greater proportion of this glucose is partitioned to the gravid uterus. The development of insulin resistance, together with the suppression of pancreatic activity, ensures the preferential uptake of glucose by non-insulin dependent tissues over insulin dependent tissues. These activities favour uterine glucose uptake, decrease adipose glucose uptake, and also promote adipose mobilisation and hepatic gluconeogenesis, so as to meet the increasing energy requirement of pregnancy. It is postulated that through these effects on insulin secretion and associated adipose tissue mobilisation factors, oPL plays a major role in homeorhesis during pregnancy. / Doctor of Philosophy (PhD)

Activation of the Unfolded Protein Response (UPR) following stress in the Endoplasmic Reticulum (ER) is an important mechanism by which obesity results in insulin resistance and type II diabetes. We uncovered a role for the adaptor protein Nck in modulating the UPR. In this study, we report that obese Nck1-/- mice, which show lower levels of UPR in liver and adipose tissue, present improved insulin signalling in these tissues. We established that the effect of Nck1 is cell autonomous by showing that HepG2 cells treated with Nck1 siRNA have reduced ER stress-induced UPR and Insulin Receptor Substrate-1 (IRS-1) serine phosphorylation. In these cells, we observed that the IRS-1 levels and activation of signalling components downstream of the insulin receptor were increased. This correlates with enhanced cell survival to stress and insulin stimulated glycogen synthesis. Overall, we demonstrated that Nck1 participates in ER-stress-induced insulin resistance and regulation of IRS-1-dependent signalling.

Orientador: Elinton Adami Chaim / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T03:30:35Z (GMT). No. of bitstreams: 1 Cazzo_Everton_M.pdf: 2625294 bytes, checksum: 2f57e795cce0f0c4c5a74d56bad92609 (MD5) Previous issue date: 2013 / Resumo: A Síndrome Metabólica é um conjunto de fatores interconectados que elevam diretamente o risco de doenças cardiovasculares e diabetes mellitus tipo II. Estes fatores são alterações no metabolismo glicídico, elevação da pressão arterial, níveis elevados de triglicerídeos e reduzidos de lipoproteína de alta densidade, associados à obesidade, especialmente sua forma central ou abdominal. Apresenta prevalência crescente nas últimas décadas, levando a importantes consequências socioeconômicas. Sua fisiopatologia é complexa e ainda não foi totalmente esclarecida, porém sabe-se que possui como elemento principal a resistência insulínica. Em decorrência, observa-se a ocorrência de hiperinsulinemia, disfunção endotelial e inflamação. Seu tratamento clínico consiste em mudanças de estilo de vida associadas à terapia farmacológica concomitante de seus fatores individuais. Com a realização cada vez mais frequente de procedimentos cirúrgicos bariátricos, tem-se observado relevantes resultados advindos de tais operações no controle da síndrome. O objetivo deste estudo é avaliar o impacto do bypass gastrojejunal em Y de Roux sobre a síndrome metabólica, seus componentes individuais e as alterações bioquímicas observadas após a cirurgia. Foi realizada uma coorte histórica envolvendo 96 indivíduos com obesidade grau II/III previamente portadores de síndrome metabólica que foram submetidos à cirurgia há pelo menos 12 meses, avaliando os parâmetros clínicos e bioquímicos dos mesmos antes e após a cirurgia. Observaram-se índices relevantes de resolução pós-operatória da síndrome metabólica (88,5%), diabetes mellitus tipo II (90,6%), hipertensão arterial (85,6%) e dislipidemias (54,2%). A resolução da síndrome metabólica esteve estatisticamente associada aos seguintes fatores: controle glicêmico pós-operatório; glicemia, trigliceridemia e hemoglobinemia glicada pós-operatórias; homeostasis model assessment (HOMA) pós-operatório; número de classes anti-hipertensivas pré e pós-operatórias; percentual de perda de peso. O número de medicações anti-hipertensivas reduziu-se inclusive em indivíduos que não obtiveram resolução completa da hipertensão. Houve redução significativa nos níveis séricos de glicose, insulina, hemoglobina glicada, colesterol total, triglicerídeos e lipoproteína de baixa densidade, e elevação da lipoproteína de alta densidade. Observou-se decréscimo importante da resistência insulínica avaliada através do HOMA. Os índices de resolução de síndrome metabólica e comorbidades relacionadas encontradas neste estudo foram consistentes com achados prévios na literatura. A melhora no perfil glicêmico, insulínico e lipídico foi comparável à descrita em outros estudos, assim como a significativa redução da resistência insulínica avaliada pelo HOMA. Dentro do grupo analisado, o bypass gastrojejunal em Y de Roux apresentou resultados benéficos significativos, constituindo, portanto, uma importante ferramenta no tratamento da síndrome metabólica, propiciando elevados índices de resolução da mesma e de seus componentes individuais, bem como melhora geral do perfil bioquímico relativo à patologia / Abstract: Metabolic syndrome is defined as a set of interconnected factors that increase risk for cardiovascular disease and type II diabetes. These factors are changes on glycemic metabolism, high blood pressure, high serum triglyceride levels and low high density lipoprotein serum levels, associated with obesity, specially the central or abdominal type. Its prevalence has raised on later decades leading to important socioeconomical consequences. It has a complex pathophysiology that has not been completely understood yet but it is known that its main element is insulin resistance. As consequence it is observed hyperinsulinemia, endothelial disfunction and inflammation. Clinical management is based on lifestyle changes associated to drug treatment of its individual components. As surgical treatment of obesity has become more frequent lately it has been seen great results on metabolic syndrome control after bariatric procedures. This study aims to determine the impact of Roux-en-Y gastric bypass on metabolic syndrome and its individual components as well as biochemical changes upon glycemic metabolism observed after surgery. A historic cohort was carried out evaluating 96 subjects with grade II/III obesity previously diagnosed with metabolic syndrome who underwent surgery and whose postoperative follow-up was at least 12 months. Clinical and biochemical parameters were analyzed before and after surgery. This study has shown high resolution rates of metabolic syndrome (88,5%), diabetes (90,6%), hypertension (85,6%) and dyslipidemias (54,2%). Metabolic syndrome resolution was statistically linked to these factors: postoperative glycemic control; postoperative glycemia, hemoglobin A1c and triglyceridemia; postoperative homoestasis model assessment (HOMA); pre and postoperative number of anti-hypertensive classes; percent weight loss. Number of anti-hypertensive classes decreased even in subjects who do not achieve complete hypertension resolution. There was significant decrease on serum levels of glucose, insulin, hemoglobin A1c, total cholesterol, triglycerides and low density lipoprotein, and increase on high density lipoprotein. It was observed relevant decrease on insulin resistance assessed through HOMA. Resolution rates for metabolic syndrome and related comorbidities found on this study were comparable to previous findings on medical literature. Improvement on glycemic, insulinic and lipid profiles was also similar to previous findings, as well as significant decrease on insulin resistance as evaluated through HOMA. Within this group of subjects Roux-en-Y gastric bypass has led to significant positive results proving to be an important therapeutical tool on metabolic syndrome management since it can bring high resolution rates of the syndrome and its individual components as well as general improvement on the biochemical profile related to this pathology / Mestrado / Fisiopatologia Cirúrgica / Mestre em Ciências

La liaison de l’insuline à son récepteur permet le recrutement de protéines adaptatrices, ce qui conduit notamment à l’activation de la voie mitogénique des MAPK et des voies impliquées dans le métabolisme du glucose. Deux voies complémentaires contribuent au recrutement du transporteur de glucose, GLUT4, à la membrane ;la voie de la PI3-kinase qui implique la formation du messager secondaire PtdIns(3,4,5)P3 conduisant à l’activation de la PKB et la voie de la petite protéine G, TC10, qui implique les protéines APS, CAP et c-Cbl. <p><p>La phosphatidylinositol 5-phosphatase 2 (SHIP2) contrôle négativement la voie des MAPK par interaction avec des acteurs de la cascade et la voie de la PI3-kinase en hydrolysant le PtIns(3,4,5)P3 en PtIns(3,4)P2. De plus, il a été montré dans notre laboratoire que SHIP2 peut interagir directement avec la protéine CAP ainsi que co-immunoprécipiter avec le récepteur de l’insuline et c-Cbl, participant ainsi à un complexe multiprotéique formé des protéines CAP et c-Cbl et du récepteur. Au cours de ce travail, nous avons tenté de mieux comprendre l’implication moléculaire de SHIP2 dans la cascade TC10. Comme APS est la première protéine de la cascade à être recrutée au récepteur suite à une stimulation par l’insuline et qu’elle peut interagir directement avec le récepteur et les protéines CAP et c-Cbl, nous avons étudié dans un premier temps le lien potentiel entre APS et SHIP2.<p><p>Nous avons montré que SHIP2 interagit de manière directe avec la protéine adaptatrice APS tant dans un système de sur-expression (CHO-IR) que dans un système endogène (3T3-L1). Bien qu’une stimulation par l’insuline ne semble pas modifier cette interaction, elle induit néanmoins le recrutement d’une fraction des protéines APS et SHIP2 du cytoplasme vers la membrane plasmique. L’étude des domaines d’interaction a montré que la région centrale de SHIP2 qui comprend le domaine catalytique est nécessaire pour cette association. <p><p>Nous avons ensuite montré que cette association entre APS et SHIP2 peut moduler certaines de leurs propriétés biochimiques. D’une part, bien que la sur-expression de SHIP2 n’influence pas le recrutement d’APS au récepteur, SHIP2 diminue, indépendamment de son activité enzymatique, la phosphorylation sur tyrosine d’APS induite par l’insuline et l’interaction entre APS et c-Cbl, qui sont deux étapes cruciales dans la cascade TC10. Ainsi, SHIP2 pourrait non seulement influencer la cascade de l’insuline par son activité enzymatique, mais également par interaction avec des acteurs de la cascade. D’autre part, APS augmente l’activité 5-phosphatase de SHIP2 dans un test in vitro. Elle pourrait ainsi, outre son rôle positif dans la cascade, participer au rétrocontrôle négatif de la voie de signalisation à l’insuline. Finalement, nous avons déterminé comment ces deux protéines influencent les cascades de l’insuline. Alors qu’APS n’influence pas l’activation de la PKB, ni le taux de PtdIns(3,4,5)P3, la sur-expression d’APS et de SHIP2 induit une inhibition plus forte de l’activation de la PKB comparée à celle provoquée par SHIP2 seul. De plus, une sur-expression de SHIP2 abolit l’augmentation induite par APS de la phosphorylation des MAPK. Cette activation des MAPK par APS semble dépendre de sa liaison au récepteur car les domaines PH et essentiellement SH2 sont indispensables pour cet effet positif.<p><p>En conclusion, nous avons mis en évidence l’existence d’une association entre APS et SHIP2. Cette interaction modifie certaines de leurs propriétés biochimiques et fournit un nouveau mécanisme d’action pour ces protéines dans le contrôle négatif de la voie de signalisation à l’insuline. <p><p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

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Les phosphoinositides (PI) sont impliqués dans de nombreux processus biologiques cellulaires, tels que la régulation du cytosquelette d'actine, le trafic vésiculaire membranaire et les voies de signalisation médiées par des récepteurs membranaires. Le métabolisme des phosphoinositides est un processus hautement régulé par un ensemble de PI-kinases et PI-phosphatases. Des mutations dans ces enzymes sont associées à des maladies tels que le syndrome de Lowe, le diabète de type 2, les désordres bipolaires et le cancer.<p>\ / Doctorat en Sciences / info:eu-repo/semantics/nonPublished