<p>RanBPM is a conserved putative scaffold protein of unknown function. Loss-of-function in <em>RanBPM</em> leads to pleiotropic phenotypes such as reduced locomotion, decreased size and larval lethality in the <em>Drosophila melanogaster</em>.</p> <p><em>dRanBPM</em> mutants have decreased branching and boutons at the neuromuscular junction, which may contribute to their locomotory defect. To investigate if dRanBPM is involved in controlling synaptic architecture at the neuromuscular junction, levels of two cytoskeletal proteins, Futsch and profilin, were assessed in <em>dRanBPM</em> mutants.</p> <p>Due to time constraints, immunoblots for Futsch were not fully optimized for protein measurement. Immunoblots for profilin, on the other hand, were successfully carried out. However, results from the reproduction of a blot demonstrating the negative regulation of <em>Drosophila</em> FMRP on profilin did not agree with that of the literature. In addition, results from an epistatic experiment demonstrated that profilin levels were not affected in FMRP deficient flies when compared to those with additional decrease in dRanBPM function.</p> <p>Targeted expression of <em>dRanBPM</em> to neurosecretory cells is able to rescue size and lethality of <em>dRanBPM</em> mutants, suggesting a common pathway through which both phenotypes operate is disrupted in these mutants. Activation of the insulin signaling pathway was indeed found to be downregulated in <em>dRanBPM</em> mutants. A longevity assay was alternatively carried out to demonstrate decreased insulin pathway activation in <em>dRanBPM</em> mutants. Unfortunately, due to inappropriate controls used for this experiment, no conclusive points can be made. Together, these findings contribute to the knowledge that RanBPM plays and to designing future experiments to test for RanBPM function.</p> / Master of Science (MSc)

Compartmentalization of redox reactions is essential to all life forms. Protein activity can respond to changes in the local redox environment through the reversible oxidation of cysteine thiols. For the majority of cysteines in the proteome, this interaction takes place through equilibration with the glutathione pool; this raises the question whether this redox pool acts as a buffer, or instead as a sensitive media, transducing information from a local physiological state into protein function.

<p>In type 2 diabetes the concentration of glucose in the blood is increased, and tissues like fat and musclebecome less sensitive to insulin. These two phenomena are interrelated, but since the glucose-insulininterplay is highly complex, many aspects are still not understood. Here, a model-based approachmight help. Nevertheless, also a model-based approach has a limited impact, unless models for thesub-systems can be combined into a model for the whole-body regulation. Such a multi-level,module-based model is referred to as a hierarchical model, and this thesis is a proof-of-principle studyfor the future development of such models.</p><p>We have extended one of the best available models for the whole-body regulations, to include azoomable module for the fat tissue. The first step was to implement the whole-body model in thesoftware MathModelica, which support hierarchical modeling. Second, the originally mergedinsulin-responding module was sub-divided, so that a fat tissue was singled out. Third, a model for theinput-output profile for the fat tissue was developed by combining mechanistic knowledge withexisting and novel data from human fat cells. Finally, this detailed model was fitted to the profile of theoriginal fat model, and inserted in the whole-body model, with negligible effect on the whole-bodysimulations.</p><p>The resulting model has the ability to translate mechanistically oriented simulations on the biochemicallevel, which is the level were drugs act, to the whole-body level, which is of clinical interest. This is aquantum leap forward for modeling, and understanding, glucose homeostasis and type 2 diabetes.</p>

The aim of the present thesis was to further increase our understanding of mechanisms contributing to and maintaining cellular insulin resistance in type 2 diabetes (T2D). For this reason, the effects of high glucose and insulin levels on glucose transport capacity and insulin signaling, with emphasis on insulin receptor substrate 1 (IRS-1) were assessed in fat cells. Altered levels of IRS-1 have previously been observed in adipose tissue from insulin-resistant and T2D subjects. A high glucose level (≥15 mM) for 24 h exerted only a minor impairment on glucose transport capacity in human adipocytes, as opposed to rat adipocytes. However, when combined with a high insulin level (104 µU/ml), basal and insulin-stimulated glucose transport was significantly impaired in both human and rat adipocytes. This was associated with a depletion of IRS-1 and IRS-2 protein levels in rat adipocytes, as a result of post-translational changes and altered gene transcription, respectively. In human adipocytes was only IRS-1 protein levels reduced. The high glucose/high insulin setting achieved maximal impairment of glucose transport within 6 h. Subsequent incubations of rat adipocytes under physiological conditions could partially restore insulin sensitivity. Interestingly, in both human and rat fat cells, decreased levels of IRSs occurred after the establishment of impaired glucose transport, suggesting that the observed depletion of IRSs is a consequence rather than a cause of insulin resistance. Nonetheless, IRS depletion is likely to further aggravate insulin resistance. Tyrosine phosphorylation of IRS-1 upon insulin stimulation activates the signaling pathway that mediates glucose transport. Pre-treatment of human adipocytes with high glucose and insulin levels was not associated with any alterations in the total IRS-1 Tyr612 phosphorylation following 10 min insulin stimulation. However, a significant increase in basal Tyr612 phosphorylation was observed. Furthermore, a rise in basal IRS-1 Ser312 phosphorylation was found. This is associated with reduced IRS-1 function and is considered to target IRS-1 to degradation pathways, and thus could potentially explain the observed decrease in IRS-1 protein levels. Our results imply an enhanced activation of insulin’s negative-feedback control mechanism that inhibit IRS-1 function. This could potentially have contributed to the observed impairment of insulin action on glucose transport in these cells. Accordingly, we have also shown that the downstream activation of protein kinase B upon insulin-stimulation is significantly impaired in human adipocytes exposed to the high glucose/high insulin setting, indicating a defect in the signaling pathway mediating glucose transport. We also investigated whether there are humoral factors in the circulation of T2D patients that contribute to peripheral insulin resistance. Human adipocytes cultured for 24 h in medium supplemented with 25% serum from T2D subjects, as compared to serum from non-diabetic subjects, displayed significantly reduced insulin-stimulated glucose uptake capacity. The effect could neither be attributed to glucose, insulin, FFA, TNF-α or IL-6 levels in the serum, but other circulating factor(s) seem to be of importance. In conclusion, chronic conditions of elevated glucose and/or insulin levels all impair insulin action on glucose turnover, but to different extents. A clear distinction between rat and human fat cells in the response to these different milieus was also observed. Alterations in the function of the key insulin signaling protein IRS-1 might be involved in the mechanisms underlying the impaired glucose uptake capacity. IRS-1 reduction however, occurs after but probably aggravates the existing insulin resistance. The effects of high glucose and/or insulin levels may be of importance in T2D, but additional novel factors present in the circulation of T2D patients seem to contribute to cellular insulin resistance.

In type 2 diabetes the concentration of glucose in the blood is increased, and tissues like fat and musclebecome less sensitive to insulin. These two phenomena are interrelated, but since the glucose-insulininterplay is highly complex, many aspects are still not understood. Here, a model-based approachmight help. Nevertheless, also a model-based approach has a limited impact, unless models for thesub-systems can be combined into a model for the whole-body regulation. Such a multi-level,module-based model is referred to as a hierarchical model, and this thesis is a proof-of-principle studyfor the future development of such models. We have extended one of the best available models for the whole-body regulations, to include azoomable module for the fat tissue. The first step was to implement the whole-body model in thesoftware MathModelica, which support hierarchical modeling. Second, the originally mergedinsulin-responding module was sub-divided, so that a fat tissue was singled out. Third, a model for theinput-output profile for the fat tissue was developed by combining mechanistic knowledge withexisting and novel data from human fat cells. Finally, this detailed model was fitted to the profile of theoriginal fat model, and inserted in the whole-body model, with negligible effect on the whole-bodysimulations. The resulting model has the ability to translate mechanistically oriented simulations on the biochemicallevel, which is the level were drugs act, to the whole-body level, which is of clinical interest. This is aquantum leap forward for modeling, and understanding, glucose homeostasis and type 2 diabetes.

Cushing's syndrome and glucocorticoid therapy lead to central obesity, insulin resistance, and symptoms of altered energy regulation similar to those observed in the metabolic syndrome. We hypothesized that excess glucocorticoids alter energy sensing/signaling in skeletal muscle through mediation of the LKB1/AMPK signaling pathway. To test this hypothesis, three 100 mg pellets of corticosterone were implanted subcutaneously in each of nine rats for two weeks. Responses were compared with sham operated controls fed ad libitum or food restricted to produce the body weights similar to the treatment group rats. After the treatment period, animals were anesthetized and the right gastrocnemius-plantaris and soleus were removed for analysis. After tibial nerve stimulation for 5 min, the left gastrocnemius-plantaris and soleus were also removed. We assessed AMPK activity and subunit expression, as well as several metabolic indicators including ATP, creatine phosphate, creatine, glycogen, and malonyl-CoA levels in rested and stimulated gastrocnemius-plantaris and soleus muscles. We found that high levels of glucocorticoids decreased AMPKγ3 subunit expression in the gastrocnemius-plantaris. We also observed reduced AMPKα2 activity in the stimulated gastrocnemius-plantaris, but not the soleus; and that this decreased activity corresponded to a significant reduction in phosphorylated TBC1D1, a protein involved in signaling GLUT-4 translocation. Finally, in the gastrocnemius-plantaris, we also noted an increase in glycogen stores in the hypercorticosteronemic rats. Our data suggest that altered energy sensing/signaling associated with high levels of glucocorticoids may be due in part to inhibition of AMPKα2 activity and the high energy state produced by increased glycogen stores. We also conclude that high levels of glucocorticoids decrease the levels of AMPKγ3 and diminish insulin/contraction signaling through phosphorylated TBC1D1.

Résumé : Les podocytes, cellules épithéliales rénales, sont nécessaires au maintien de la structure et de la fonction de filtration des glomérules rénaux. La dédifférenciation et l’apoptose des podocytes sont des évènements précoces de la néphropathie diabétique. Des études ont rapporté que l’insuline est nécessaire à la survie des podocytes puisque la délétion du récepteur à l’insuline dans les podocytes de souris entraîne une pathologie glomérulaire semblable à la néphropathie. D’autres études ont montré que la protéine tyrosine phosphatase Src homology-2 domain-containing phosphatase-1 (SHP-1) inhibe les voies de signalisation de l’insuline au niveau du foie et du muscle en déphosphorylant la sous-unité bêta du récepteur à l’insuline (IRβ) et la kinase Phosphatidylinositide 3-kinase (PI3K). Il a récemment été démontré que l’expression de SHP-1 est élevée dans les cortex rénaux de souris diabétiques. Nous avons donc émis l’hypothèse que l’expression de SHP-1 induite par l’hyperglycémie altère les actions de l’insuline dans les podocytes. Nous avons premièrement utilisé un modèle in vivo de souris diabétiques de type 1 (Ins2+/C96Y; Akita). Comparées aux souris contrôles (Ins2+/+), les souris Akita présentaient une apoptose élevée des podocytes ainsi qu’une perte des pédicelles. La phosphorylation de la protéine kinase B (Akt) et de Extracellular signal-regulated kinase 1/2 (ERK1/2), suite à une injection systémique d’insuline, était également significativement diminuée dans les cortex rénaux des souris Akita. Cette diminution correspondant à une résistance à l’insuline corrélait avec une augmentation de deux fois de l’expression de SHP-1 dans les glomérules. Nous avons ensuite utilisé une lignée immortalisée de podocytes murins en culture et avons observé que l’exposition à des concentrations élevées de glucose (HG; 25 mM) pendant 96 h, entraînait l’augmentation de l’expression de marqueurs apoptotiques et de l’activité enzymatique de caspase-3/7 en comparaison aux concentrations normales de glucose (NG; 5,6 mM). L’exposition en HG a augmenté l’expression de l’ARNm et protéique de SHP-1, en plus de réduire la signalisation de l’insuline dans les podocytes. La surexpression de la forme dominante-négative de SHP-1 dans les podocytes a permis de renverser les effets de HG et de restaurer les actions de l’insuline. Finalement, l’augmentation de l’expression de SHP-1, tant in vivo qu’in vitro, a été directement corrélée à son association avec IRβ et à la diminution de la phosphorylation de IRβ, Akt et ERK1/2 suite à une stimulation à l’insuline. En conclusion, nous avons montré que l’expression élevée de SHP-1 dans les glomérules cause une résistance à l’insuline et la mort des podocytes contribuant ainsi à la néphropathie diabétique. // Abstract : Podocytes are epithelial renal cells required to preserve glomerular structure and filtration. Their dedifferentiation and apoptosis are early events of diabetic nephropathy progression. Previous studies have shown that insulin action is critical for podocyte survival since deletion of its receptor lead to a glomerular pathology similar to nephropathy. It has also been demonstrated that Src homology-2 domain-containing phosphatase-1 (SHP-1), a protein tyrosine phosphatase, inhibits insulin signaling pathway in liver and muscle by dephosphorylating tyrosine residues on insulin receptor beta-subunit (IRβ) and the Phosphatidylinositide 3-kinase (PI3K). A recent study concluded that SHP-1 is elevated in kidney cortex of type 1 diabetic mice. We hypothesized that hyperglycemia-induced SHP-1 expression may affect insulin actions in podocytes. To confirm this hypothesis, we used type 1 diabetic Akita mice (Ins2+/C96Y). Compared to control littermate mice (Ins2+/+), Akita mice developed elevated podocyte foot process effacement and podocyte apoptosis. In contrast to control mice, insulin-stimulated protein kinase B (Akt) and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation was remarkably reduced in renal podocytes of Akita mice. This phosphorylation diminution associated to a renal insulin resistance was correlated with a two-fold increase of SHP-1 expression in the glomeruli. We then used cultured murine podocytes cell line to confirm our in vivo results. Podocytes exposed to high glucose concentration (HG; 25 mM) for 96 h exhibited high levels of apoptotic markers and caspase-3/7 enzymatic activity as compared to normal glucose concentration (NG; 5,6 mM). HG exposure raised mRNA and protein levels of SHP-1 and reduced the insulin-signaling pathway in podocytes. Overexpression of dominant-negative SHP-1 in podocytes prevented HG effects and restored insulin actions. Finally, elevated SHP-1 expression induced by high glucose levels was directly correlated to an increased association with insulin receptor-β subunit (IRβ) in vitro and in vivo. This association is therefore leading to the reduction of both IRβ phosphorylation and insulin-stimulated Akt and ERK phosphorylation. In conclusion, our results showed that high levels of SHP-1 in glomeruli cause insulin resistance and podocyte loss, thereby contributing to diabetic nephropathy.

A restrição de sal na dieta está associada com resistência à ação da insulina e obesidade. O mecanismo molecular pelo qual a dieta hipossódica (HO) pode induzir resistência à insulina e obesidade não está totalmente compreendido. O objetivo do presente estudo foi avaliar a influência da ingestão crônica de sal sobre o peso corporal (PC), sinalização da insulina no fígado, músculo e tecido adiposo branco (TAB) e sua associação com adiposidade e resistência à insulina. Com esta finalidade, ratos Wistar foram alimentados com dieta HO, normossódica (NR) ou hipersódica (HR) desde o desmame. O PC foi avaliado desde o desmame. Ao completarem 12 semanas de vida, foram avaliados pressão arterial, balanço energético, consumo de ração, glicemia, angiotensina II (ANGIO II) plasmática e perfil hormonal. A atividade motora espontânea foi estudada em ratos com 8 e 12 semanas. A sensibilidade à insulina foi analisada pelo índice de HOMA. A expressão da proteína desacopladora mitocondrial 1 (UPC-1) foi quantificada no tecido adiposo marrom (TAM) e o conteúdo de ANGIO II no TAM, TAB e hipotálamo. As etapas iniciais da sinalização da insulina foram avaliadas por imunoprecipitação e immunoblotting das proteínas envolvidas como o receptor da insulina (IR), substrato 1 e 2 do IR (IRS-1 e IRS-2), enzima fosfatidilinositol 3 – quinase (PI-3q), proteína quinase B (Akt/PKB), ativação da proteína c-jun NH2-terminal quinase (JNK) e fosforilação em serina 307 do IRS-1. O PC no desmame foi semelhante entre os grupos de dieta. No entanto, na idade adulta os ratos em dieta HO apresentaram maior PC, adiposidade visceral, glicemia e insulinemia de jejum, concentração de ANGIO II plasmática e aumento do conteúdo de ANGIO II no TAM. Por outro lado, nestes mesmos animais a dieta HO diminuiu o consumo de ração, o gasto energético, a expressão da proteína UCP-1, adiponectina plasmática e o conteúdo de ANGIO II no TAB. A atividade motora não foi diferente entre os grupos estudados. A dieta HO diminuiu a via IR/PI-3q/Akt/Foxo1 de sinalização da insulina no fígado e músculo. Por outro lado, parte desta via (IRS-2/Akt/Foxo1) mostrou-se aumentada no TAB. No fígado e músculo houve um aumento da fosforilação da proteína JNK associada com maior fosforilação do IRS-1ser307 no grupo HO. Em conclusão, a restrição ou sobrecarga crônica de sal altera a evolução ponderal associada com modificações no balanço energético e no perfil hormonal na idade adulta. A resistência à insulina induzida pela dieta HO é tecido-específico e foi acompanhada por uma ativação da proteína JNK e um aumento da fosforilação dos resíduos de serina 307 do IRS-1. / Restriction of sodium chloride intake has been associated with insulin resistance (INS-R) and obesity. The molecular mechanisms by which the low salt diet (LSD) can induce INS-R and obesity have not yet been established.The aim of the present study was to evaluate the influences of salt intake on body weight (BW) and on insulin signaling in liver, muscle and white adipose tissue (WAT). Wistar rats were fed a LSD, normal (NSD), or high (HSD) salt diet since weaning. At 12 weeks of age, BW, blood pressure(BP),energy balance, food intake, plasma glucose and angiotesin II (ANGIO II), and hormonal profile were evaluated. Afterward, motor activity, HOMA index, uncoupling protein 1 expression (UCP-1) and tissue adipose ANGIO II content was determined. The early steps of insulin signaling (IR: insulin receptor, IRS-1 and IRS-2: IR substrate 1 and 2, PI-3K: phosphatidylinositol 3-kinase), Akt (protein kinase B) phosphorylation, JNK (c-jun NH2-terminal kinase) activation and IRS-1ser307 (serine 307 of IRS-1) phosphorylation were evaluated by immunoprecipitation and immunoblotting. LSD increased BW, visceral adiposity, blood glucose, insulin, leptin, plasma ANGIO II and its content in BAT. Otherwise, LSD decreased food intake, energy expenditure, UCP-1 expression, adiponectin and ANGIO II content in WAT. Motor activity was not influenced by the dietary salt content. In LSD, a decreasing in IR/PI-3K/Akt/Foxo1 was observed in liver and muscle and an increase in this pathway was showed in adipose tissue. JNK activity and IRS-1ser307 phosphorylation were higher in liver and muscle. In conclusion, LSD induced obesity and insulin resistance due to changes in energy expenditure, SRA and insulin signaling. The INS-R is tissuespecific and is accompanied by JNK activation and IRS-1ser307 phosphorylation.

A restrição calórica (RC) estende a expectativa de vida de muitos organismos por mecanismos ainda em estudo. Entre os vários efeitos fisiológicos da RC encontra-se o aumento na biogênese mitocondrial, dependente de óxido nítrico (NO&#8226;), sintetizado pela enzima óxido nítrico sintase endotelial (eNOS). Um dos indutores fisiológicos mais potentes da eNOS é a insulina, cujos níveis plasmáticos são consideravelmente reduzidos nos organismos em RC. O objetivo deste trabalho foi investigar os mecanismos associados ao aumento da sinalização por NO&#8226; durante a RC in vivo e in vitro, e as conseqüências celulares do aumento de massa mitocondrial no que diz respeito à longevidade e capacidade respiratória celulares. Submetemos camundongos Swiss fêmeas à RC de 40% e observamos um considerável aumento tecido-específico na fosforilação basal de Akt e eNOS em músculo esquelético, tecido adiposo visceral e cérebro, os quais também apresentaram maior massa mitocondrial. A associação entre a sinalização por insulina, NO&#8226; e biogênese mitocondrial foi adicionalmente confirmada em um grupo de camundongos tratados com o desacoplador mitocondrial dinitrofenol (DNP), que também reduz a insulinemia e aumenta a longevidade em camundongos. Para o estudo mecanístico deste fenômeno, usamos soros de ratos Sprague-Dawley submetidos à RC de 40% ou alimentados ad libitum (AL) em cultura celular de células vasculares da musculatura lisa (VSMC), reproduzindo um protocolo descrito para RC in vitro. O uso do soro RC aumentou a fosforilação do receptor de insulina e Akt, a expressão de eNOS e nNOS (forma neural da NOS) e a fosforilação de eNOS, o que se refletiu em maior liberação de nitrito (NO2) no meio de cultura. Inibindo-se a Akt, todos os efeitos promovidos pela RC na sinalização por NO&#8226; foram revertidos. Ao se imunoprecipitar do soro a adiponectina, citocina conhecida por aumentar a sensibilidade à insulina, aumentada durante a RC, os efeitos do soro RC na via de sinalização de insulina foram abolidos e, conseqüentemente, os efeitos na sinalização por &#8226;NO foram prevenidos. Neurônios de células granulosas de cerebelo, que não expressam eNOS, apenas nNOS, foram cultivados com os soros AL ou RC, e também apresentaram considerável aumento na sinalização por &#8226;NO. Estas alterações induziram a biogênese mitocondrial e capacidade respiratória, e foram associadas à maior longevidade celular. Os mesmos efeitos mitocondriais foram observados em células secretoras de insulina, INS1, entretanto a secreção de insulina em resposta à glicose tornou-se inibida, por um mecanismo desconhecido, porém associado a reduzidos níveis intracelulares de espécies oxidantes, moléculas-chave para a secreção de insulina; e à alteração da morfologia mitocondrial, provavelmente devido à maior expressão de mitofusina-2 (Mfn-2). Ao se nocautear a Mfn-2, houve um aumento na geração de EROs e as células em RC passaram a secretar insulina a níveis comparáveis aos das células controle. Concluímos que durante a RC a maior sensibilidade à insulina aumenta a atividade de eNOS, via Akt, associada à maior biogênese mitocondrial. A adiponectina é uma molécula-central nestes eventos. A expressão de nNOS também é afetada, por mecanismos desconhecidos. O aumento de biogênese mitocondrial eleva a capacidade respiratória celular e impacta positivamente a longevidade in vitro. A alteração da morfologia mitocondrial associa-se a alterações na produção de oxidantes intracelulares e mudanças na secreção de insulina. / Calorie restriction (RC) is known to extend the lifespan in many organisms, and its mechanisms of action are still under investigation. Enhanced mitochondrial biogenesis driven by nitric oxide (&#8226;NO), synthesized by the endothelial nitric oxide synthase (eNOS), is proposed to be a CR central effect. Insulin is one of the most potent physiological activators of eNOS. However, plasmatic insulin levels are dramatically reduced in organisms under CR. The goal of this work was uncover the mechanisms associated with enhanced &#8226;NO signaling during CR, in vivo and in vitro, as well as the cellular consequences of increased mitochondrial mass, regarding lifespan and reserve respiratory capability. Female Swiss mice were submitted to 40% of CR. A tissue-specific (skeletal muscle, abdominal adipose tissue and brain) increment in basal Akt and eNOS phosphorylation, which was related to enhanced mitochondrial biogenesis, was observed. Indeed, this association was also verified in tissues from mice treated with low doses of a mitochondrial uncoupler, dinitrophenol (DNP). To unveil the mechanism behind the insulin signaling effects on &#8226;NO levels, serum from Sprague-Dawley rats submmited to 40% of CR was used to culture in VSMC cells, an in vitro CR protocol. CR sera enhanced insulin receptor (IR) and Akt phosphorylation, as well as nitrite (NO2-) accumulation in the culture media, the expression of eNOS and nNOS (neural NOS isoform) and eNOS phosphorylation. The effects of CR sera were reversed by Akt inhibition. The immunoprecipitation of serum adiponectin, a cytokine known to improve peripheral insulin sensitivity, also reversed the CR serum effects on insulin and &#8226;NO signaling. Cerebellar neurons, which do not express eNOS, just nNOS, were also cultured with CR or AL serum and also presented striking increments in &#8226;NO signaling, associated with mitochondrial biogenesis, increased reserve respiratory capability and lifespan extension. The mitochondrial effects promoted by CR were also observed in insulin secreting cells (INS1). However, under the CR condition, insulin secretion stimulated by glucose was impaired. The likely explanations are reduced mitochondrial reactive oxygen species (ROS) generation, or the alteration in mitochondrial morphology, associated, in our model, with enhanced mitofusin-2 expression (Mfn-2). In cells which the Mfn-2 was knocked down, insulin secretion in CR and AL groups was responsive to glucose at the same level, and the intracellular oxidants levels were much higher. Overall, CR improves &#8226;NO signaling due to enhanced insulin sensitivity, through Akt, and results in mitochondrial biogenesis. Adiponectin is a key molecule in this phenomenon. Increments in mitochondrial mass enhance the cellular reserve respiratory capability and lifespan. Mitochondrial morphology alterations are associated with possible decreases in ROS generation and impaired insulin release, maintained the low levels of plasmatic insulin.

A administração de deidroepiandrosterona (DHEA) tem resultado em efeitos anti-diabetogênicos em animais de experimentação e no homem. Assim, o objetivo desse trabalho é avaliar o efeito do DHEA in vitro na expressão protéica do IR, do IRS-1, IRS-2, PI3-K, Akt, ERK-1/2; na expressão gênica do PDX-1, do PGC-1, da insulina, do GLUT-2 e da glicocinase; e avaliar a secreção estática de insulina de ilhotas pancreáticas de ratos. O cultivo das ilhotas por 24 horas com DHEA, não induziu nenhuma alteração tanto na expressão das proteínas quanto na secreção estática de insulina estimulada por glicose. Ocorreu aumento da fosforilação de ERK-1/2 e na expressão gênica do PGC-1. As células RINm5F, cultivadas por 72 horas com DHEA, apresentaram aumento da expressão total de IRS-1 e IRS-2. Concluímos, que 24 horas de cultura com ilhotas não é tempo suficiente para observar nenhuma alteração induzida pelo DHEA, na secreção de insulina, e na expressão das proteínas da via IRS/PI3-K/Akt. Células RINm5F podem ser um modelo alternativo para investigar os efeitos diretos do DHEA. / The dehydroepiandrosterone (DHEA) administration has resulted in reduction of abdominal fat and protection against insulin resistance from experimental animals and humans. So, the purpose of this project is measure the in vitro effects from DHEA: on protein expression of insulin receptor, the proteins IRS-1, IRS-2, PI3-K, Akt, and ERK-1/2; on gene expression of transcriptional factors PDX-1 and PGC-1, insulin, glucose transport GLUT-2 and glicocinase; and to measure the static insulin secretion, on cultured pancreatic islets of the rat. The culture of pancreatic islet for 24 hours with DHEA, did not induce nothing alteration on protein expression of the IR, IRS-1, IRS-2, PI3-K, Akt-1 and ERK-1/2, and static insulin secretion induced by glucose. However, happened increase ERK-1/2 phosphorylation and PGC-1 gene expression. The RINm5F cells, cultured by 72 hours, showed increase of the IRS-1 and IRS-2 expression. We conclude that 24 hours of the pancreatic islets culture are not sufficient time to look any alteration induced by DHEA, on insulin secretion, and on protein expression involved on IRS/PI3-K/Akt pathway. RINm5F cells can be an alternative model to research the direct effects from DHEA.