Дисертації з теми "Beta cell insulin secretion"

cAMP and Ca2+ are key regulators of exocytosis in many cells, including insulin-secreting pancreatic β-cells. Glucose-stimulated insulin secretion from β-cells is pulsatile and driven by oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i), but little is known about the kinetics of cAMP signaling and the mechanisms of cAMP action. Evanescent wave microscopy and fluorescent translocation biosensors were used to monitor plasma membrane-related signaling events in single MIN6-cells and primary mouse β-cells. Glucose stimulation of insulin secretion resulted in pronounced oscillations of the membrane phospholipid PIP3 caused by autocrine activation of insulin receptors. Glucose also triggered oscillations of the sub-plasma membrane cAMP concentration ([cAMP]pm). These oscillations were preceded and enhanced by elevations of [Ca2+]i, but conditions raising cytoplasmic ATP triggered [cAMP]pm elevations without accompanying changes in [Ca2+]i. The [cAMP]pm oscillations were also synchronized with PIP3 oscillations and both signals were suppressed after inhibition of adenylyl cyclases. Protein kinase A (PKA) was important for promoting concomitant initial elevations of [cAMP]pm and [Ca2+]i, and PKA inhibitors diminished the PIP3 response when applied before glucose stimulation, but did not affect already manifested PIP3 oscillations. The glucose-induced PIP3 oscillations were markedly suppressed in cells treated with siRNA against the cAMP-dependent guanine nucleotide exchange factor Epac2. Pharmacological activation of Epac restored PIP3 responses after adenylyl cyclase or PKA inhibition. Glucose and other cAMP-elevating stimuli induced redistribution of fluorescence-tagged Epac2 from the cytoplasm to the plasma membrane. This translocation was modulated by [Ca2+]i and depended on intact cyclic nucleotide-binding and Ras-association domains. In conclusion, glucose generates cAMP oscillations in β-cells via a concerted action of Ca2+ and metabolically generated ATP. The oscillations are important for the magnitude and kinetics of insulin secretion. While both protein kinase A and Epac is required for initiation of insulin secretion the cAMP-dependence of established pulsatility is mediated by Epac2.

Las procianidinas son compuestos bioactivos presentes en frutas y vegetales. Aunque se conocen los efectos beneficiosos de estos compuestos en la homeostasis de la glucosa, su acción en la funcionalidad de la célula β no es clara. La presente tesis doctoral se ha centrado en describir los efectos de las procianidinas en la síntesis y secreción de insulina. Nuestros resultados muestran la capacidad de las procianidinas de modificar la funcionalidad de la célula β aumentando la relación insulina plasmática/mRNA, aunque la efectividad del tratamiento depende de la situación fisiológica. En situaciones no patológicas, las procianidinas afectan la insulinemia modificando la síntesis, secreción y/o degradación de la insulina. En situaciones de resistencia a la insulina, el tratamiento crónico con procianidinas disminuye la síntesis y secreción de insulina gracias a su acción limitando el acúmulo de lípidos. En cambio, en un modelo más dañado (obesidad genética), las procianidinas ejercen efectos similares pero no son capaces de mejorar la hipersinulinemia. En conclusión, las procianidinas, en las dosis ensayadas, pueden utilizarse únicamente como compuestos bioactivos limitando la disfuncionalidad de la célula β en sus estados iniciales.
Les procianidines són compostos bioactius presents en fruites i vegetals. Tot i que es coneixen els efectes beneficiosos d’aquests compostos en l’homeòstasi de la glucosa, la seva acció en la funcionalitat de la cèl•lulaβ no és clara. La present tesi doctoral s’ha centrat en descriureels efectes de les procianidines en la síntesi i secreció d’insulina. Els nostres resultats mostren la capacitat de les procianidines de modificar la funcionalitat de la cèl•lula β augmentant la relació insulina plasmàtica/mRNA, tot i que l’efectivitat del tractamentdepèn de la situaciófisiològica. En situacions no patològiques, les procianidines afecten la insulinèmia modificant la síntesi, secreciói/o degradació d’insulina. En situacions de resistència a la insulina, el tractamentcrònicamb procianidines disminueix la síntesi i secreció d’insulina gràcies a la seva acció limitant l’acumulació de lípids. En canvi, en un model més danyat (obesitat genètica), les procianidines exerceixen efectes similars però no son capaces de millorar la hiperinsulinèmia. En conclusió, les procianidines, en les dosis assajades, podenutilitzar-seúnicament coma compostos bioactiuslimitant la disfuncionalitat de la cèl•lula β en els seus estats inicials.
Procyanidins are bioactive compounds found in fruits and vegetables widely consumed. It has been reported that procyanidins show some beneficial effects on glucose homeostasis, although their effects on β-cell functionality remain unresolved. This doctoral thesis is focus on describing the effects of procyanidins on insulin synthesis and secretion. Our results showed that procyanidins modify β-cell functionality through increasing the plasma insulin/mRNA ratio, although the effectiveness of the treatment depends on the physiological situation. Under non-pathological situation, procyanidins affected insulinaemia by modifying insulin synthesis, secretion and/or degradation activity. Under insulin-resistance situation, chronic procyanidins administration decreased insulin synthesis and secretion, thanks to its lipid-lowering effect. Otherwise in a more damaged model, Zucker fatty rat, procyanidins treatment is not able to reduce insulin plasma levels although they repress insulin expression. In conclusion, procyanidins could be used as bioactive compound to limit β-cell dysfunctions under high-palatable diets, but at the assayed doses, it is not enough to counteract a strong metabolic disruption.

Loss of insulin secretion is a recognized characteristic of diabetes. It is therefore an important goal to understand how insulin secretion is controlled and what goes wrong in disease. The discovery of the spatial orientation of insulin granule fusion towards the vasculature adds more complexity to the regulation of insulin secretion. The underlying mechanisms however remain unknown. I therefore have investigated interactions of beta cells with the surrounding microenvironment in order to identify possible factors that regulate the vascular-orientated insulin granule fusion. In Chapter 3, I first investigate the beta cell-vascular basement membrane interaction by culturing dispersed beta cells on surfaces coated with 3 different basement membrane proteins: Laminin 511, Collagen IV or Fibronectin. Poly-l-lysine coated surface is used as the control. 3D two photon live cell imaging showed that insulin granule fusion is targeted towards the cell-protein interface in beta cells cultured on surface coated with either one of the proteins but not the control. The targeting of insulin granule fusion can be further segregated by culturing on surfaces micropatterned with Fibronectin and E-cadherin Fc which represent the beta cell-vasculature and beta cell-beta cell interfaces respectively. Using an integrin beta 1 blocking antibody and focal adhesion kinase inhibitor, I show that the basement membrane-induced targeting of insulin granule fusion is integrin beta 1 and focal adhesion complex dependent. I therefore conclude that the vascular basement membrane proteins and subsequent membrane protein-induced focal adhesion complex formation are crucial factors in establishing and maintaining the spatial targeting of insulin granule fusion. In Chapter 4 and 5, I further studied the polar organisation of beta cells in regulating insulin secretion. In Chapter 4, I first characterised the organisation of beta cells using a pancreatic slice preparation that better retains the native structural organisation of the beta cells than the isolated islets. 3D imaging and analysis of polarity-related proteins reveal that beta cells exhibit 3 distinct polarity domains: the apical, lateral and basal polarity domains. Such polarity organisation is present in single beta cell as well as throughout the whole islet. One of the basolateral polarity determinants, Scribble, was further investigated in Chapter 5 using Scrib+/- mice and rodent beta cell line MIN6. Scrib+/- mice exhibit better blood glucose homeostasis and bigger islet as well as an increase in basal insulin secretion. No differences are found in the spatial orientation of insulin granule fusion and overall glucose-stimulated insulin secretion in the Scrib+/- islets. The Scrib KD MIN6 on the other hand reduced glucose-stimulated insulin secretion. Such reduction of glucose-stimulated insulin secretion in Scrib KD MIN6 is possibly betaPIX independent as the betaPIX and Scribble staining exhibit limited overlapping and the betaPIX KD MIN6 had no effect on the glucose-stimulated insulin secretion. I therefore conclude that beta cells are polarised cell type and suggest that Scribble possibly control beta cell mass and regulate insulin secretion in a betaPIX independent manner. In summary, in this thesis, I showed that the vascular basement membrane is one of the factors orientating the insulin granule fusion towards the vasculature. I have also show that beta cells should be viewed as polarised cell type with distinct polarity domains expressing typical polarity proteins. One of the polarity determinants, Scribble possibly regulates beta cell mass and insulin secretion.

INTRODUCTION Carnitine palmitoyltransferase I, which is expressed in the pancreas as the liver isoform (LCPTI), catalyzes the rate-limiting step in the transport of fatty acids into the mitochondria for their oxidation. To directly examine whether the availability of long-chain fatty acyl-CoA affects the regulation of insulin secretion in the Beta-cell, we infected INS(832/13) cells and rat islets with an adenovirus encoding a mutant form of LCPTI (Ad-LCPTI M593S) that is insensitive to its inhibitor malonyl-CoA. C75 is described as a potential drug for treatment of obesity and type 2 diabetes. First known as a synthetic inhibitor of fatty acid synthase, it has been also described as an activator of CPTI, increasing peripheral energy utilization and fatty acid oxidation in mice. To further investigate the C75/CPTI interaction, we have characterized the effects of C75 on CPTI in vitro and in vivo.

OBJECTIVES 1) Study of the malonyl-CoA/CPTI interaction in the pancreatic Beta-cell and its involvement in glucose-stimulated insulin secretion (GSIS). 2) Construction of an INS stable cell line overexpressing LCPTI wt and LCPTI M593S. 3) Determine the effect of C75 on the CPTI activity and palmitate oxidation in pancreatic Beta-cells.

RESULTS. In Ad-LCPTI M593S infected INS(832/13) cells LCPTI activity increased six-fold. This was associated with enhanced fatty acid oxidation, at any glucose concentration, and a 60% suppression of GSIS. In isolated rat islets in which LCPTI M593S was overexpressed, GSIS decreased 40%. At high glucose concentration, overexpression of LCPTI M593S reduced partitioning of exogenous palmitate into lipid esterification products, and decreased PKC activation. Moreover, LCPTI M593S expression impaired KATP channel-independent GSIS in INS(832/13) cells.
INS-1 stable clones of LCPTIwt and LCPTImut were constructed, however none of them resulted in an increase in LCPTI protein expression compared to endogenous LCPTI nor in CPTI activity. Therefore, slight basal overexpression of LCPTI could probably be toxic for the cells, as a result of which only those cells that do not contain the LCPTI plasmids survived throughout cell passages.
When INS(823/13) cells are incubated with C75, CPTI activity is inhibited, as is fatty acid oxidation. In vivo, a single intraperitoneal injection of C75 to mice produces a short-term inhibition of CPTI activity in mitochondria from liver and pancreas.

DISCUSSION. The results with LCPTImut provide direct support for the hypothesis proposing that the malonyl-CoA/CPTI interaction is a component of a metabolic signalling network that controls insulin secretion. Overall, the findings with C75 provide compelling evidence that the drug is a potent inhibitor of CPTI.

Hyperglycaemia is a significant pathophysiological feature of diabetes mellitus. It has been considered to be a cause as well as a consequence of impaired pancreatic p-cell function and insulin action. The detrimental effects of a hyperglycaemic environment encourage the non-enzymatic glycation of regulatory and functional proteins. This thesis investigated the effects of pro insulin C-peptide and other important islet peptides such as insulin, somatostatin, islet amyloid polypeptide (IAPP) as well as the gut hormone gastric inhibitory polypeptide (GIP) in both native and glycated forms on insulin release and beta cell function in vitro using clonal pancreatic p-ce1ls and isolated mouse islets and in vivo, using Swiss TO lean mice. Pancreatic beta-cells not only secrete insulin into the bloodstream, but also release equimolar amounts of C-peptide. During the last decade, studies have provided definite evidence of C-peptide's active role in physiology and pathophysiology. However very little information currently exists on the direct effects of C-peptide on pancreatic beta cells. This study provides evidence that C-peptide is a biologically active endogenous peptide hormone that exerts tonic inhibitory effects on pancreatic beta cell function and these effects may be partially mediated through intracellular signalling pathways. Suppression of insulin secretion by C-peptide was observed at basal and stimulatory glucose concentrations and was shown to be concentration dependent. The ability of alanine and IBMX to potentiate glucose-induced insulin secretion was severely impaired in the pancreatic cell line and isolated islets in the presence of C-peptide. This inhibition of glucose stimulated insulin secretion may be associated with the mechanism by which cAMP and KATP channel potentiates insulin release. In vivo studies with the administration of C-peptide resulted in a decrease in plasma insulin levels and increase in plasma glucose concentrations. As proinsulin C-peptide clearly exhibited an inhibitory effect on insulin secretion, it was of interest to study the autocrine effect of the other important pancreatic p-cell peptide, insulin. Concentration dependent inhibitory effects of insulin on alanine- and IBMX-stimulated insulin secretion were observed in isolated islets. Validation of this result was confirmed in vivo using Swiss TO mice, where plasma C-peptide levels were decreased following insulin administration. In mechanistic studies, insulin not only inhibited cAMP production at stimulatory glucose concentration, but also decreased GLP-1 and forskolin-stimulated cAMP production in clonal pancreatic cells. Moreover, insulin exerted a negative effect on insulin biosynthesis by decreasing the alanine-stimulated insulin mRNA expression at basal and stimulatory glucose concentration. As expected, inhibitory effects on insulin secretion were also observed for the pancreatic peptides, somatostatin-14 and IAPP whilst the gut incretin hormone GIP exerted insulinotropic activity. Following incubation with glucose in vitro, glycated forms of C-peptide, insulin, somatostain-14 and GIP were all readily detectable by reverse-phase HPLC and MALDI- TOF mass spectrometry. The extent of glycation was shown to be time and glucose concentration dependent. G Iycated human C-peptide exerted an inhibitory effect on insulin secretion, but to a lesser extent than non-glycated C-peptide. Glycated human C- peptide in the presence of alanine, GIP and tolbutamide resulted in 22%, 12% and 27% inhibition, respectively compared to 50%, 52% and 42% inhibition with native human C- peptide. A similar trend was obtained when comparing glycated human insulin and non- glycated human insulin. Suppression of alanine- and IBMX-stimulated C-peptide release was observed with glycated insulin. Glycated insulin reduced cAMP production at stimulatory glucose concentration, GLP-l and forskolin-stimulated cAMP production. Both glycated C-peptide and glycated insulin exhibited a less potent inhibitory effect on insulin secretion. Contrastingly, glycation of somatostatin-14 increased its ability to inhibit insulin secretion. Likewise, glycated GIP was more insulinotropic than its native peptide. These studies indicate that C-peptide may be one of several players in the multifactorial regulation of the pancreatic beta cell. Insulin secretion is an important physiological process which is regulated by multiple regulatory mechanisms. C-peptide may have a compensatory regulatory effect through the fine control of insulin release. Collectively, these studies indicate the need for continuing investigation of the circulating levels of glycated peptides and their effects on the ability to modulate insulin secretion. Further investigations are thus necessary to find out the possible role of glycated peptides in the pathophysiology of type 2 diabetes.

Type 1 diabetes is an autoimmune disease in which patients' insulin-secreting beta cells in pancreatic islets are destroyed by their own immune system, leading to unregulated blood glucose levels and severe complications. Its only treatment is intensive insulin therapy, which carries the risk of hypoglycemic episodes and can result in seizures, coma, and even death. Islet transplantation has recently become an alternative, albeit experimental, treatment for type 1 diabetes patients. More than one donor graft is usually required to render recipients insulin independent, making the shortage of donor tissue an extremely important challenge in islet transplantation. Identifying the cell type that has the ability to differentiate into islet-like tissue is an important area of study. In this study, I hypothesized that insulin secreting human islet-like clusters could be generated from pancreatic ductal cells, a potential pancreatic progenitor cell type. Islet-like clusters were generated using crude exocrine tissue from human cadaveric donors. This crude exocrine tissue contained a large number of ductal cells, as well as other pancreatic cell types. To evaluate insulin secretion by human islet-like clusters, a static incubation system was set up and tested using Min6 cells, a known insulin-secreting cell line. Using static incubation, significant increases in insulin secretion by islet-like clusters were observed when the clusters were exposed to higher glucose levels and GLP-1, a known insulin secretagogue. Presence of corresponding C-peptide secretion demonstrated that de novo insulin secretion occurred. Furthermore, basal insulin secretion increased as culture stages progressed. An attempt was made to generate islet-like clusters using ductal cells purified by fluorescent activated cell sorting or magnetic activated cell sorting. Nevertheless, it was difficult to ensure survival and proliferation of purified ductal cells. Further studies will be necessary to confirm the role of ductal cells in the generation of islet-like clusters using the crude exocrine tissue, as well as to identify factors that can promote ductal cells proliferation after cell sorting.

A thorough understanding of beta-cell proliferation, function, death and regeneration under normal condition as well as in the progression of diabetes is crucial to the conquest of this disease. The work presented in this thesis aimed to investigate the expression and role of a novel transcription factor, Zinc finger BED domain-containing protein 6 (ZBED6), in beta-cells. ZBED6 was present in mouse βTC-6 cells and human islets as a double nuclear band at 115/120 kDa and as a single cytoplasmic band at 95-100 kDa, which lacked N-terminal nuclear localization signals. Lentiviral shRNA-mediated stable silencing of ZBED6 in βTC-6 cells resulted in altered morphology, decreased proliferation, a partial S/G2 cell cycle arrest, increased expression of beta-cell specific genes, and higher rates of apoptosis. ChIP sequencing of human islets showed that ZBED6 binding was preferentially to genes that control transcription, macromolecule biosynthesis and apoptosis. We proposed that ZBED6 supported proliferation and survival of beta-cells, possibly at the expense of specialized beta-cell function, i.e. insulin production. To further investigate the role of ZBED6 in beta-cells, ChIP sequencing and whole transcriptome analysis were performed using MIN6 cells. More than 4000 putative target genes of ZBED6 were identified, including Pdx1, MafA and Nkx6.1. ZBED6-silencing resulted in differential expression of more than 700 genes, which was paralleled by an increase in the content and release of insulin in response to a high glucose concentration. Altered morphology/growth patterns as indicated by increased cell clustering were observed in ZBED6 silenced cells. We found also that ZBED6 decreased the ratio between N- and E-cadherin. A lower N- to E-cadherin ratio may hamper the formation of three-dimensional beta-cell clusters and cell-to-cell junctions with neural crest stem cells, and instead promote efficient attachment to a laminin support and monolayer growth. Thus, by controlling beta-cell adhesion and cell-to-cell junctions, ZBED6 might play an important role in beta-cell differentiation, proliferation and survival.

Proper functioning of endocrine cells is crucial for organismal homeostasis. The underlying mechanisms that fine-tune the amount, and the timing of hormone secretion are not clear. JIP1 / MAPK8IP1 (JNK interacting protein 1) is a scaffold protein that mediates cellular stress response, and is highly expressed in endocrine cells, including insulin secreting b-cells in pancreas islets. However, the role of JIP1 in b-cells is unclear. This study demonstrates that b-cell specific Jip1 ablation results in decreased glucose-induced insulin secretion, without a change in Insulin1 and Insulin2 gene expression. Inhibition of both JIP1-kinesin interaction, and JIP1-JNK interaction by genetic mutations also resulted in decreased insulin secretion, suggesting that JIP1 may mediate insulin vesicle trafficking through interacting with kinesin and JNK. Autophagy is a cellular recycling mechanism and implicated in the b-cell function. Both JIP1 and JNK are proposed to regulate autophagy pathway. However, it is unclear whether JNK plays a role in the promotion or suppression of autophagy. The findings of this study show that JNK is not essential for autophagy induction, but can regulate autophagy in a cell and context specific manner. The results in this thesis implies a mechanism that link cellular trafficking and stress signaling pathways in the regulated hormone secretion. In addition to the known role of JIP1 in metabolism and insulin resistance, this finding may also be relevant to endocrine pathologies.

In 2011, Diabetes and prediabetes affected 9 million Canadians and 366 million people worldwide (Canadian Diabetes Website). The underlying pathophysiology of diabetes is beta cell dysfunction leading to loss of appropriate insulin secretion and resulting in hyperglycemia. I have focused on identifying critical molecular regulators of beta cell function and insulin secretion. The CRTC2-CREB pathway is required for maintaining beta cell mass and insulin secretion. I propose that identifying kinases that regulate CRTC-CREB activity will identify other important regulators of pancreatic beta cell survival and function. First, I have identified several AMP kinases as inhibitors of CRTC2-CREB that are activated by an upstream kinase, LKB1. I then went on to generate mice with a beta cell-specific deletion of LKB1 during adulthood. Loss of LKB1 increased insulin secretion and glucose clearance through enhanced beta cell mass and proliferation. The increased insulin secretion was largely the result of loss of AMPK activity and consequent constitutive mTor activity. AMPK is activated under starvation conditions and as such is thought to be a critical regulator of beta cell function. However, the decrease of AMPK activity in high glucose has been a strong argument against it being a critical effector of insulin secretion. I provide genetic evidence supporting the idea that AMPK activity attenuates insulin secretion. During periods of starvation where AMPK activity is high there is a chronic dampening effect on events that prepare beta cells for the next round of insulin secretion. Surprisingly, another downstream kinase of LKB1, SIK2, has opposing functions in the beta cell. I present evidence that the LKB1-AMPK axis attenuates beta cell functions and that targeting this pathway in beta cells may be of therapeutic benefit for T2D.

Orientador: Kleber Luiz de Araujo e Souza
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo:Introdução/Objetivos. A produção de espécies reativas de oxigênio (EROs) faz parte de diversos processos fisiológicos. Nos últimos anos, o aumento de EROs têm sido associado ao desenvolvimento de diversas doenças, dentre elas o Diabetes Mellitus Tipo 2. As células beta pancreáticas são notadamente mais suscetíveis ao estresse oxidativo devido a sua baixa capacidade antioxidativa, resultado da menor expressão e atividade de enzimas antioxidantes como superóxido dismutase e peroxidases. A dexametasona, um glicocorticóide sintético, tem efeitos diabetogênicos e citotóxicos em células produtoras de insulina e ilhotas pancreáticas. Entretanto, os mecanismos pelos quais a dexametasona atua sobre as células-alvo não estão bem esclarecidos. Dessa forma, nosso objetivo foi analisar se a dexametasona induz estresse oxidativo em células produtoras de insulina RINm5F e ilhotas pancreáticas. Utilizamos três modelos: 1) células RINm5F controle, que são extremamente sensíveis ao estresse oxidativo; 2) células RINm5F superexpressando a enzima catalase (RINm5F.Cat), que são resistente ao estresse oxidativo e 3) ilhotas de ratos adultos cultivadas por 72 h com dexametasona (Dexa) e ilhotas tratadas concomitantemente com dexametasona e o antioxidante N-acetilcisteína (Dexa+NAC). Resultados: Aumento na produção de EROs foi observado em células RINm5F tratadas com dexametasona. O tratamento com dexametasona aumentou a atividade/clivagem da caspase-3 e apoptose em células RINm5F após 3 dias de cultura. Expressão protéica e atividade de Cu/ZnSOD estava aumentada após o tratamento com dexametasona, enquanto que a expressão/atividade de MnSOD não foi modulada pelo corticóide. A superexpressão da catalase em linhagens de célula beta previniu todos os efeitos citotóxicos da dexametasona, inclusive a morte celular. Elevados níveis de Cu/ZnSOD podem favorecer o aumento na geração de EROs e conseqüentemente, apoptose. Da mesma forma, ilhotas tratadas com dexametasona apresentaramaumento na produção de EROs, efeito que foi revertido quando as ilhotas foram tratadas concomitantemente com dexametasona e NAC. Redução na secreção de insulina estimulada por glicose foi observada em ilhotas cultivadas com dexametasona. O tratamento com dexametasona e NAC restaurou a secreção de insulina a níveis próximos aos controles. Uma menor produção deNAD(P)H no grupo Dexa foi observado, sendo que o grupo Dexa+NAC mostrou níveis semelhantes ao grupo controle. Não ocorreram diferenças nas concentrações intracelulares de cálcio estimulado por glicose em nenhum dos grupos. A dexametasona reduziu a expressão gênica da sinaptotagmina VII, enquanto no grupo Dexa+NAC houve um aumento da expressão desse gene em ilhotas pancreáticas. Interessantemente, o tratamento com NAC diminuiu a expressão gênica da Cu/ZnSOD. Conclusões: Nossos resultados indicam que as ações da dexametasona em células produtoras de insulina e ilhotas pancreáticas são mediadas através do aumento do estresse oxidativo, sendo a Cu/ZnSOD importante nesse processo. A superexpressão da catalase e o uso do antioxidante n-acetilcisteína previnem contra os efeitos citotóxicos do glicocorticóide.
Abstract: Introduction/Aims: Reactive oxygen species (ROS) play a dual role on living organisms, being involved in many physiological processes and also being linked to the development of several pathologies, including the type 2 diabetes mellitus. Pancreatic beta cells are very sensitive to oxidative stress because of their low antioxidant capacity, wich results from their low expression and activity of antioxidant enzymes, especially peroxidases. Dexamethasone is a synthetic diabetogenic glucocorticoid that induces cytotoxic effects on pancreatic beta cells. However, the precise mechanisms of dexamethasone toxicity on target cells are not fully understood. The aim of the present study was to analyzed whether dexamethasone induces oxidative stress in insulinproducing cells and pancreatic islets. Experimental design: The experiments were performed using 3 models: 1) RINm5F control cells, extremely sensitive to oxidative stress; 2) RINm5F cells overexpressing the enzyme catalase (RINm5F.Cat), very resistant to oxidative stress and 3) rat pancreatic islets cultured for 72 h with dexamethasone (Dexa) or cultured concomitantly with dexamethasone and the antioxidant N-acetylcysteine (Dexa+NAC). Results: An increased generation of reative oxygen species (ROS) was observed in dexamethasone-treated insulinproducing cells together with an increase in caspase-3 activity and apoptosis rate. Interestingly, exposure to dexamethasone increased the cytosolic superoxide dismutase Cu/ZnSOD protein expression and activity, while the mitochondrial MnSOD isoform was not affected by the glucocorticoid. Overexpression of catalase in insulin-producing cells prevented all the cytotoxic effects of dexamethasone. Pancreatic islets cultured in the presence of dexamethasone (Dexa) for 72 h showed increased ROS production. Glucose-stimulated insulin secretion was decreased after Dexa treatment. Intracellular ROS levels were decreased and the insulin secretion capacitywas recovered by concomitant treatment with Dexa+NAC. The total insulin content and intracellular Ca+2 levels were not modulated in either Dexa or Dexa+NAC groups. There was a decrease in the NAD(P)H production rate, used as an indicator of viability, after dexamethasone treatment. Concomitant incubation with NAC returned viability to control levels. Dexamethasone also decreased SYT VII gene expression; in contrast, the Dexa+NAC group showed increased expression of SYT VII compared to controls. Surprisingly, treatment with NAC decreased the gene expression of the antioxidant enzyme, Cu/ZnSOD. Conclusions: The cytotoxic effects of dexamethasone in RINm5F insulin-producing cells and pancreatic islets are primarily ROS-mediated. High levels of expression and activity of the Cu/ZnSOD might favour the generation of ROS. The overexpression of catalase and the use of the antioxidant Nacetylcysteine counteract the cytotoxic effects of dexamethasone.
Doutorado
Fisiologia
Doutor em Biologia Funcional e Molecular

The sustained component of the sodium current (INaL) has been recently found enhanced in several cardiac pathologies. INaL enhancement has been highlighted as a major cause of cellular dysfunction, caused by INaL-dependent electrical instability (arrythmias) and alterations in intracellular ions (Na and Ca) homeostasis. Recent evidence of beneficial effects of the INaL blocker ranolazine (RAN) in diabetes motivates interest in the role of the INaL in glucose-induced insulin secretion (GSIS). The aim of the present thesis is to characterize INaL, identified as steady-state current blocked by RAN (IRAN) or TTX (ITTX), and its function in rat INS-1E cells and human islet cells (HIc). Veratridine (VERA) was used as INaL enhancer. Baseline INaL was similar between INS-1E and HIc. In INS-1E cells, tolbutamide-triggered action potentials were suppressed by TTX, but not by RAN. VERA caused depolarization, countered by INaL blockade. ITTX and IRAN reversal potentials (EREV) were negative to Na+ equilibrium one, but they approached it when K+-channels were blocked. This revealed INaL coupling to Na+-activated K+ current (IKNa); expression of IKNa channels (Slo2.1/2.2) was confirmed by transcript analysis. Concomitant activation of IKNa might blunt the effects of INaL on membrane potential, but at the same time it might increase the impact of INaL on intracellular Ca2+. Consistently, INaL blockade (by TTX) blunted cytosolic Ca2+ response to depolarization, with the highest effect in the intermediate potentials. Long-term exposure to 33 mM glucose (CHG) enhanced INaL. Whereas acute INaL enhancement (VERA) increased GSIS, chronic one (CHG or VERA) depressed GSIS, which was partially restored by RAN. Conclusions: 1) INaL is expressed in insulin-secreting cells, is coupled to IKNa, affects Ca2+ signalling and, when enhanced acutely, increases GSIS 2) sustained hyperglycemic stress enhances INaL and this contributes to GSIS depression instead. Overall, INaL enhancement may represent a common mechanism contributing to disease progression in cardiac and secretion disorders.

The development of type 2 diabetes over time involves defects in insulin action and insulin secretion. Defects in insulin action alone can be compensated with appropriate hyperinsulinemia. However, the progressive loss of pancreatic beta-cell function leads eventually to the development of persistent hyperglycemia that characterizes type 2 diabetes. Insulin secretion patterns reflect two phases when beta-cells are exposed to acute and sustained glucose stimulation. Through the study and understanding of the roles of these two phases in the regulation of glucose homeostasis, it is clear that insulin must not only be secreted in sufficient amounts, but also at the right time. In type 2 diabetes, the timing and magnitude of insulin secretion are altered, and an abnormal first-phase release initiates before the onset of the disease. Only a few pharmacokinetic/pharmacodynamic (PK/PD) models have considered the biphasic nature of insulin secretion. This study is aimed at describing the biphasic dynamics of insulin secretion through developing a PK/PD model based on current knowledge of the cellular mechanism of biphasic insulin secretion. The objectives of this work are to 1) evaluate the insulin-glucose kinetics using nonparametric analysis, 2) develop a physiologically based mechanistic PK/PD model to dynamically describe the biphasic insulin secretion, 3) evaluate the impact of ethnicity on insulin secretion kinetics following an intravenous glucose administration using population analysis and 4) extend the proposed model to oral glucose administration and utilize the co-secretion kinetics of insulin and C-peptide in a population PK/PD analysis of the prehepatic insulin secretion. Population analysis was done using a nonlinear mixed-effects model combined with the proposed PK/PD model to estimate population parameters and their variations between- and within-subjects and the covariates' effects on model parameters. The proposed model describes biphasic insulin behavior, accounts for first-phase insulin secretion, and also applies to oral glucose administration for estimating prehepatic insulin secretion in vivo and in liver extraction. This is done by an analysis that simultaneously uses plasma insulin and C-peptide concentrations. A significant higher first-phase insulin secretion was identified in healthy youths of African-American compared to Caucasians. The analysis showed no significant differences in the clearance of insulin from the plasma and the liver extraction of insulin between subjects with various levels of glucose tolerance. Obesity leads to a higher insulin production rate and lower elimination rate from the plasma than normal weight subjects. Also, type 2 diabetes and impaired glucose tolerance were found to reduce insulin production rate and resulted in a delayed insulin secretion from the beta-cells.

Orientadores: Antonio Carlos Boschiero, Luiz Fernando de Rezende
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Introdução: A Cálcio-Calmodulina quinase II (CaMKII) atua tanto na regulação da secreção de insulina com de neurotransmissores pela mesma via de sinalização. Além disso, a CaMKII é conhecida por ser a "molécula da memória", pois sua atividade é fundamental em sua formação. Portanto, hipotetizamos que células ß pancreática tem a capacidade de adquirir e estocar informações contidas em pulsos de cálcio, formando uma memória metabólica. Métodos: Para comprovar nossa hipótese, desenvolvemos um novo paradigma de exposição de células ? a pulsos de 30 mM de glicose, seguido de uma período de consolidação (24 hrs) para excluir qualquer efeito agudo do metabolismo da glicose. Após esse período analizamos a secreção de insulina (RIA), expressão proteica (Western blot), a resposta secretória frente a uma "rampa de glicose" e o Ca2+ citoplasmático induzido por glicose. Resultados: Células ß expostas a pulsos de glicose (30 mM) mostraram maior secreção de insulina estimulada por glucose, evidenciando a memória metabólica a qual foi totalmente dependente a CaMKII. Esse fenômeno foi refletido na expressão proteica de proteínas importantes na sinalização do cálcio e na secreção de insulina. Além disso, células expostas ao regime de pulsos de glucose apresentaram maior expressão do MAFA, um fator de transcrição chave para a função da célula ß. Conclusão: Em suma, assim como neurônios, células ß tumorais (MIN6), ilhotas de camundongos e de humanos são capazes de adquirir, estocar e evocar informações
Abstract: Backgroun: Ca2+/calmodulin-dependent protein kinase II (CaMKII) functions both in regulation of insulin secretion and neurotransmitter release through common downstream mediators. Memory is the ability to acquire, to store and to evocate any kind of information. In CNS, the process behind this phenomenon in the Long-Term Potentiation (LTP) and is known that it requires Ca2+ to occur. In additional, CaMKII is necessary to store information during LTP. In pancreatic ß-cells, CaMKII plays pivotal role during GSIS process. Therefore, we hypothesized that pancreatic ß-cells acquire and store the information contained in Ca2+ pulses as a form of "metabolic memory", just as neurons store cognitive information. Methods: To test this hypothesis, we developed a novel paradigm of pulsed exposure of mice and human ß-cells to intervals of high glucose, followed by a 24-hour consolidation period to eliminate any acute metabolic effects. After this period, we analyzed insulin secretion (by RIA), protein expression (by Western blot), response to a glucose-ramp and the glucose-induced Ca2+ influx. Results: Strikingly, ß-cells exposed to this high-glucose pulse paradigm exhibited significantly stronger insulin secretion. This metabolic memory was entirely dependent on CaMKII. We also observed, in pulse group, an increase in Ca2+ influx induced by glucose. In additional, metabolic memory was reflected on the protein level by increased expression of proteins involved in GSIS and Ca2+-dependent vesicle secretion, such as GCK, Cav1.2, SNAP25, pCaMKII and pSynapsin. Finally, we observed in human islet elevated levels of the key ß cell transcription factor MAFA. Discussion: Based on or findings we conclude that pancreatic ß cells, either from mice or humans, have the ability to acquire, store and retrieve information. This process is CaMKII-dependent and is due to modifications in the glucose-sensing machinery of the cell, since we observed an increase in GSIS and Ca2+ influx together with an increase in several proteins involved in this process. Our findings suggests that MAFA is the key effector in this memory, since (a) it is a potent activator of insulin gene, (b)is activated by CaMKII and (c) its expression is increased even 24 hours after the last pulse. Conclusion: In summary, like neurons, human and mouse ß-cells are able to acquire and retrieve information
Doutorado
Fisiologia
Doutor em Biologia Funcional e Molecular

Impaired glucose metabolism is frequently described in cirrhotic patients. The pathogenesis of diabetes mellitus (DM) in this population is complex and not precisely known. Insulin resistance (IR) plays a central role in the glucose metabolism disturbance and it has been speculated that genetic and environmental factors and some etiologic agents in liver disease impair insulin secretion. Aim of the study: evaluate β cell secretion and insulin sensitivity in a cohort of cirrhotic patients undergoing liver transplantation (LT). 107 cirrhotic patients (31 female e 76 male) were evaluated before LT. The patients who underwent LT were evaluated 3, 6, 12 months after surgery. To evaluate insulin resistance HOMA-IR was used. To assess the β cell secretion, a state-of-art modelling of glucose/C-peptide curves during OGTT was used. Two outputs were provided: dynamic evaluation (1st phase) and proportional evaluation (2nd phase). Before LT the prevalence of DM and prediabetes (pre DM) were 50.5% and 31.8 % respectively. DM patients showed a lower insulin secretion (both 1st and 2nd phase) and tended to have higher HOMA-IR when compared to pre DM and non DM subjects. After LT glucose metabolism improved (impaired glucose metabolism: 6 months 61.1%, 12 months 65.6%). HOMA-IR was decreased (pre LT: 5.48±5.14, 3 months post LT: 2.17±1.62, 6 months post LT: 2.28±1.44, 12 months post LT: 2.36±1.73 P=0.0017). No differences in β cell secretion was found. When the population who underwent LT was divided in 2 groups according to the improvement or not of glucose metabolism, higher insulin secretion was found in improved subjects (2nd phase). No differences in HOMA-IR, age, duration of hepatic disease, family history of type 2 DM, immunosuppressive therapy were observed. In conclusion IR and reduced insulin secretion was observed in cirrhotic diabetic patients. After LT an improved glucose metabolism was observed. Insulin sensitivity was increased in all patients and higher β cell secretion was observed only in subjects with improved glucose metabolism.

BACKGROUND & AIMS: Diabetes occurring as a direct consequence of loss of liver function is usually characterized by non-diabetic fasting plasma glucose (FPG) and haemoglobin A1c (HbA1c) levels and should regress after orthotopic liver transplantation (OLT). This observational, longitudinal study investigated the relationship between the time-courses of changes in all 3 direct determinants of glucose regulation, i.e., β-cell function, insulin clearance and insulin sensitivity, and diabetes regression after OLT. METHODS: Eighty cirrhotic patients with non-diabetic FPG and HbA1c levels underwent an extended oral glucose tolerance test (OGTT) before and 3, 6, 12 and 24 months after OLT. The OGTT data were analysed with a mathematical model to estimate derivative control (DC) and proportional control (PC) of β-cell function and insulin clearance (which determine insulin bioavailability), and with the Oral Glucose Insulin Sensitivity (OGIS)-2 h index to estimate insulin sensitivity. RESULTS: At baseline, 36 patients were diabetic (45%) and 44 were non-diabetic (55%). Over the 2-year follow-up, 23 diabetic patients (63.9%) regressed to non-diabetic glucose regulation, whereas 13 did not (36.1%); moreover, 4 non-diabetic individuals progressed to diabetes (9.1%), whereas 40 did not (90.9%). Both DC and PC increased in regressors (from month 3 and 24, respectively) and decreased in progressors, whereas they remained stable in non-regressors and only PC decreased in non-progressors. Insulin clearance increased in all groups, apart from progressors. Likewise, OGIS-2 h improved at month 3 in all groups, but thereafter it continued to improve only in regressors, whereas it returned to baseline values in the other groups. CONCLUSIONS: Increased insulin bioavailability driven by improved β-cell function plays a central role in favouring diabetes regression after OLT, in the presence of a sustained improvement of insulin sensitivity.

In healthy subjects, glucose concentration is tightly kept in a limited range around its basal value thanks to complex regulatory mechanisms. Impairment of this regulatory system is the cause of several metabolic disorders, such as diabetes, characterized by chronic hyperglycemia, which leads to severe micro- and macrovascular complications. Different hormones are involved in this regulation, with insulin being one of the most important and well studied. It is physiologically secreted at every meal by pancreatic beta-cells in response to increased blood glucose levels, in order to lower glucose concentration. Other substances can stimulate insulin secretion, for example glucagon-like peptide-1 (GLP-1) is an insulinotropic hormone released from intestinal L-cells in response to food ingestion. It is, together with other hormones, responsible for the so-called incretin effect, i.e., the fact that glucose ingested orally elicits a greater insulin response than glucose administered intravenously, even when glucose concentrations in plasma are matched. In type-2 diabetes, both insulin and GLP-1 secretion is impaired. In this context, a combination of experimental data and mathematical modeling could help in getting a deeper insight into the cellular mechanisms leading to the secretion of both hormones. In most of the excitable cells, the steps leading to secretion are quite similar: a trigger initiates the electrical activity in the cell, which leads to the opening of voltage gated calcium channels and a subsequent calcium influx inside the cell; the increase in calcium levels allows the vesicles to fuse with the plasma membrane and to release their content outside the cell. In this work, the different steps leading to secretion will be analyzed by means of a combination of both experimental data and mathematical modeling, with reference to the intestinal L-cells and the pancreatic beta-cells. Regarding the intestinal L-cells, a mathematical model of electrical activity was built to investigate the stimulus-secretion pathway, which is still poorly understood. However, two glucose-sensing mechanisms are known to contribute in the sensing of luminal glucose: the sodium-glucose cotransporters (SGLT) and ATP-sensitive K+-channels (K(ATP)-channels). The results showed how the two glucose-sensing mechanisms interact, and suggested that the depolarizing effect of SGLT currents is modulated by K(ATP)-channels activity. On the other hand, the stimulus-secretion pathway in pancreatic beta-cells is well established. SK-channels and calcium dynamics were included in a previous mathematical model of electrical activity in human beta-cells to investigate the heterogeneous and non-intuitive electrophysiological responses to ion channel antagonists. By using our model we also studied paracrine signals, and simulated slow oscillations by adding a glycolytic oscillatory component to the electrophysiological model. The model was further developed by including Kir channels, which play a critical role in the cardiac cells, by determining the shape of cardiac action potential. The inclusion of the Kir2.1 current in the model resulted in a clear improvement of the model behavior, by slowing down the spiking dynamics, thanks to the small outward Kir2.1 current, which tends to stabilize the inter-spike membrane potential. As a result of the beta-cell electrical depolarization, calcium channels open, leading to an influx and a subsequent diffusion of calcium inside the cell, which in turn triggers exocytosis. Hence, from the electrical activity analysis, we moved to the investigation of the relationship between insulin granule exocytosis, calcium levels, distance from calcium channels and channel clustering in beta-cells. This subproject is based on Total Internal Reflection Fluorescence (TIRF) microscopy, consisting in simultaneous visualization of two different fluorophores. The first fluorophore is used to label insulin-containing vesicles, and to differentiate them into two groups: the ones that undergo exocytosis in response to depolarization, and the ones that do not. The second fluorophore, a genetically encoded calcium indicator (R-GECO), is attached to the plasma membrane and permits visualizing calcium levels during the stimulus. Simulations were performed using the modeling program CalC, which implements calcium diffusion and buffering. Simulated calcium levels and the corresponding R-GECO signal were evaluated at different distances from the channel. The comparison of the simulations to the TIRF microscopy data allowed estimating the average distance from the channel of the granules that undergo exocytosis. Calcium diffusion simulations were coupled to a simple model for insulin granule exocytosis to investigate different pools of granules, in terms of vicinity to the calcium channel and calcium affinity. Furthermore, the fusion probability was evaluated both in a single channel, and in a cluster-of-channels context. Simulations confirmed that the hypothesis of a cluster significantly increases the fusion probability and a certain dependence between the channels in the cluster is functional advantageous. So far we analyzed cellular mechanisms, which translate in insulin secretion. Hence, the natural step was to move from a cellular point of view to a bigger scale considering the whole pancreas. In this context, the so called minimal model approach might become useful, by allowing the determination of indexes to assess beta-cell function in different experimental groups. A minimal model specific for the perfused pancreas experimental setting was built adapting the C-peptide minimal model previously applied to the intravenous glucose tolerance test. The model was initially applied to untreated pancreata and afterward used for the assessment of pharmacologically relevant agents (GLP-1, the GLP-1 receptor agonist lixisenatide, and a GPR40/FFAR1 agonist, SAR1) to quantify and differentiate their effect on insulin secretion. Model application showed that lixisenatide reaches improvement of beta-cell function similarly to GLP-1 and demonstrated that SAR1 leads to an additional improvement of beta-cell function in the presence of postprandial GLP-1 levels. In conclusion, in this work different aspects of GLP-1 and insulin secretion were investigated by means of a combination of experimental data and mathematical models. Starting from the modeling of electrical activity in both L-cells and beta-cells, we moved to the calcium diffusion and exocytosis of insulin vesicles, concluding with a minimal model of insulin secretion.
In soggetti sani, il controllo della glicemia si basa su un complesso sistema di regolazione che permette di mantenere il livello di glucosio nel sangue all'interno di un range ristretto che oscilla attorno al suo valore basale. Malfunzionamenti in questo sistema di regolazione causano diversi disordini metabolici, tra cui il diabete, caratterizzato da una iperglicemia cronica, che può portare a gravi complicanze micro- e macro-vascolari. Diversi ormoni fanno parte di questo sistema di regolazione. L'insulina, che è uno dei più importanti e maggiormente studiati, è fisiologicamente secreta ad ogni pasto dalle beta-cellule pancreatiche, a seguito dell'aumento dei livelli di glucosio per diminuirli. Altre sostanze stimolano la secrezione di insulina, ad esempio il Glucagon-like Peptide-1 (GLP-1) è un ormone insulinotropico rilasciato dalle L-cellule intestinali in risposta all'assunzione di cibo. Assieme ad altri ormoni, è responsabile del cosiddetto effetto incretinico, ovvere la risposta insulinica maggiore per l’assunzione di glucosio orale rispetto alla somministrazione di glucosio endovenoso, anche nel caso in cui le concentrazioni nel plasma siano le stesse. Nel diabete di tipo 2 è ridotta sia la secrezione di insulina che di GLP-1. In questo contesto, i modelli matematici in combinazione con i dati sperimentali posso aiutare nell'ottenere una visione più approfondita dei meccanismi cellulari che portano alla secrezione di entrambi gli ormoni. Nella maggior parte delle cellule eccitabili, le fasi che portano alla secrezione sono piuttosto simili: l'attività elettrica, indotta da uno stimolo esterno, fa aprire i canali di calcio voltaggio dipendenti e causa un influsso di calcio all'interno della cellula; l'aumento dei livelli di calcio permette ai granuli di fondersi con la membrana plasmatica e rilasciare il loro contenuto all'esterno della cellula. In questo lavoro, le diverse fasi che portano alla secrezione verranno analizzate attraverso una combinazione di dati sperimentali e modelli matematici, in riferimento alle L-cellule intestinali e le beta-cellule pancreatiche. Riguardo alle L-cellule intestinali, è stato sviluppato un modello matematico dell'attività elettrica per indagare il pathway stimolo-secrezione, che è ancora poco compreso. Tuttavia, è noto che due sono i meccanismi ritenuti responsabili della percezione del glucosio intestinale: il cotrasportatore sodio-glucosio (SGLT) e i canali potassio sensibili all'ATP (canali-K(ATP)). I risultati ottenuti hanno mostrato come i due meccanismi di percezione del glucosio interagiscono e suggeriscono che l'effetto depolarizzante delle correnti dovute ad SGLT è modulato dall'attività dei canali-K(ATP). Invece, il pathway stimolo-secrezione delle beta-cellule pancreatiche è noto. Per investigare alcune risposte elettrofisiologiche eterogenee e non intuitive ad antagonisti dei canali ionici, in un precedente modello matematico dell'attività elettrica nelle beta-cellule umane sono stati inseriti i canali SK e la dinamica del calcio. Utilizzando il modello sono stati studiati anche i segnali paracrini e, aggiungendo un oscillatore glicolitico al modello elettrofisiologico, sono state simulate anche le oscillazioni lente. Il modello è stato ulteriormente sviluppato includendo i canali Kir2.1, che svolgono un ruolo fondamentale nelle cellule cardiache, determinandone la forma dei potenziali d'azione. L'inserimento di questa corrente nel modello ne ha migliorato il comportamento rallentandone la dinamica. A seguito della depolarizzazione elettrica delle beta-cellule i canali di calcio si aprono permettendo l'influsso e la diffusione del calcio all'interno della cellula, che a sua volta causa l'esocitosi dei granuli di insulina. Per questo motivo, dall'analisi dell'attività elettrica, siamo passati allo studio nelle beta-cellule della relazione tra l'esocitosi dei granuli di insulina, i livelli di calcio, la distanza dai canali di calcio ed il clustering dei canali. Questo sotto-progetto è basato su dati di microscopia per fluorescenza a riflessione interna totale (TIRF), che consiste nella visualizzazione simultanea di due differenti fluorofori. Il primo è utilizzato per differenziare i granuli contenenti insulina in due gruppi, quelli che vanno incontro ad esocitosi e quelli che non lo fanno. Il secondo fluoroforo, un indicatore del calcio geneticamente codificato (R-GECO), è connesso con la membrana plasmatica e permette di visualizzare i livelli di calcio in quell'area durante lo stimolo. Le simulazioni sono state effettuate utilizzando il programma CalC, che permette di implementare la diffusione ed il buffering del calcio. Il confronto delle simulazioni con i dati di microscopia TIRF ha permesso di stimare la distanza media dai canali di calcio a cui si trovano i granuli che vanno incontro ad esocitosi. Le simulazioni di diffusione del calcio sono state accoppiate ad un modello dell'esocitosi per studiare i differenti gruppi di granuli, in termini di vicinanza ai canali di calcio ed affinità al calcio stesso. In questo contesto, la cinetica dei canali di calcio durante la depolarizzazione è stata simulata attraverso un sequenza stocastica ed un setup a canale singolo è stato confrontato con un setup di tre canali clusterizzati indipendenti o sincronizzati. Le simulazioni hanno confermato che l'ipotesi di un cluster di canali aumenta in modo significativo la probabilità di fusione e una certa dipendenza tra i canali risulterebbe funzionalmente vantaggiosa. Fino a questo momento, sono stati analizzati meccanismi cellulari, che si traduco nella secrezione di insulina. Quindi, un ulteriore passo è stato quello di spostarsi ad una scala maggiore considerando l'intero pancreas. In questo contesto, i cosiddetti modelli minimi possono essere un utile approccio, in quanto permettono di determinare degli indici per valutare la funzionalità beta-cellulare in differenti gruppi sperimentali. A partire dal modello minimo del C-peptide precedentemente applicato al test endovenoso di tolleranza al glucosio, è stato sviluppato un modello minimo specifico per il setup del pancreas perfuso. Il modello è stato inizialmente applicato a pancreas non trattati e, successivamente, utilizzato per la valutazione di importanti agenti farmacologici (GLP-1, lixisenatide, un agonista del recettore del GLP-1, e SAR1, un agonista del recettore GPR40/FFAR1), quantificando e differenziando il loro effetto sulla secrezione di insulina. I risultati hanno mostrato che lixisenatide ottiene un miglioramento della funzione beta-cellulare simile al GLP-1 e che SAR1 porta ad un miglioramento ulteriore della funzionalità beta-cellulare in presenza di livelli post-prandiali di GLP-1. In conclusione, in questo lavoro diversi aspetti della secrezione di GLP-1 e di insulina sono stati studiati con una combinazione di dati sperimentali e modelli matematici. Iniziando dai modelli dell'attività elettrica sia nella L-cellule che nelle beta-cellule, siamo passati alla diffusione del calcio e all'esocitosi dei granuli di insulina, concludendo con un modello minimo della secrezione di insulina.

Glucose regulation, in healthy subjects, relies on a complex control system that keeps blood glucose level within a narrow range around its basal value. Impairment of the glucose regulatory system is the cause of several metabolic derangements, including diabetes, which is characterized by chronic hyperglycemia which leads to severe micro and macro-vascular complications. Diabetes is generally classified into two categories, type 1 and type 2 diabetes. Both arise from complex interactions between genes and the environment, and are characterized by an absolute deficiency of insulin production (type 1) or a relative deficiency of the pancreas to produce insulin in amounts sufficient to meet the body needs (type 2). The prevalence of diabetes is increasing dramatically in populations of the world, and its global incidence has been increasing steadily in the past several years. Traditional medications for type 2 diabetes, including insulin, sulfonylureas, glitinides, acarbose, metformin, and thiazolidinediones, lower blood glucose through diverse mechanisms of action. However, many of the oral hypoglycemic agents lose their efficacy over time, resulting in progressive deterioration in β-cell function and loss of glycemic control due to progressive loss of β-cell mass. Consequently, there is an increasing interest in developing therapeutic agents that preserve or restore functional β-cells mass such as the incretin hormone Glucagon-Like Peptide-1 (GLP-1). It not only acutely lowers blood glucose by promoting insulin secretion and inhibiting glucagon release, but also engages signaling pathways in the islet β-cells that leads to stimulation of β-cells proliferation and neo-genesis and inhibition of β-cell apoptosis. Impairment of insulin secretion and glucagon suppression suggests that decreased β-cells responsiveness to GLP-1 is part of the pathogenesis of type 2 diabetes. Thus the ability to measure the effect of GLP-1 on insulin secretion can be useful to understand the pathogenesis of type 2 diabetes. Moreover it can be employed to optimized GLP-1 based therapy by determining those individuals who may benefit more from such therapy. However, a mechanistic model enabling direct quantitation of pancreatic response to GLP-1 has never been developed. In this contribution a mathematical model which describes the mechanism of GLP-1 action on insulin secretion is proposed. It provides a direct measure of the β-cells responsivity indexes to glucose and GLP-1. Three databases were used to develop, test and validate the model. Data of 88 healthy individuals, who underwent a hyperglycemic clamp with a concomitant GLP-1 intravenous infusion, were used for model formulation. A set of models of increasing complexity describing GLP-1 action on insulin secretion were tested. All models share the common assumption that insulin secretion is made up of two components, one proportional to glucose rate of change through dynamic responsivity, Φd, and one proportional to glucose through static responsivity, Φs, but differ in the modality of GLP-1 control on β-cells. For each model potentiation index П was derived representing the percent increase in secretion due to 1 pmol/l of circulating GLP-1. All the models fit the data well, as confirmed by the run test, which supported randomness of residuals in 70% of the subjects and provide precise estimate of model parameters. Model selection was tackled using standard criteria (e.g. ability to describe the data, precision of parameter estimates, model parsimony, residual independence). The most parsimonious model in most subjects assumes that above-basal insulin secretion depends linearly on GLP-1 concentration and its rate of change. However, the hyperglycemic clamp with concomitant intravenous infusion of GLP-1, is not physiological and easy to perfume in large scale studies. Thus data of 22 impairing fasting glucose (IFG) subjects, studied twice with a mixed meal, were used to test the model performance in a more physiological condition. We found that during an oral test, a simpler model is sufficient to describe the data. Validation of the model was performed using both simulations and real data of 10 healthy subjects studied with an OGTT and matched intravenous glucose challenge (I-IVG). The protocol allows to calculate a model-independent index (PI) from the comparison of insulin secretion rate estimated in these two occasions. The comparison between model-derived Π and incretin potentiation index PI shows that they are very similar (П = 6.55, CV = 65%; PI = 6.15 % per pmol/l). In addition in silico validation proved the ability of the model to single out the effect of GLP-1 on insulin secretion since it correctly estimated П in the 93 ± 1% of the simulations.
La regolazione della glicemia in soggetti sani, si basa su un complesso sistema di controllo che permette di mantenere il livello di glucosio nel sangue all’interno di un range ristretto che oscilla attorno al suo valore basale. Il mal funzionamento di tale sistema è la causa di patologie metaboliche, ad esempio il diabete. Questa patologia è caratterizzata da iperglicemia cronica che, se non curata, a lungo termine comporta gravi complicanze micro e marco vascolari. Il diabete è comunemente classificato in tipo 1 e tipo 2. Entrambi derivano da complesse interazioni tra ambente e geni, e sono caratterizzati da una totale mancanza di produzione di insulina, nel tipo 1, o da una carenza da parte del pancreas nel produrre insulina in quantità sufficiente per soddisfare le necessità dell’organismo, nel tipo 2. La prevalenza del diabete è in costante aumento in tutto il mondo, così come la sua incidenza è in costante crescita negli ultimi anni. I farmaci tradizionali per la terapia del diabete di tipo 2, come l’insulina, sulfaniluree, metformina e tiazolidinedioni, riducono la glicemia attraverso diversi meccanismi di azione. Tuttavia, molti degli agenti ipoglicemizzanti assunti per via orale, perdono di efficacia con il tempo causando un progressivo deterioramento della funzionalità e riduzione della massa delle β-cellule con conseguente riduzione del controllo glicemico. Di conseguenza vi è un crescente interesse nello sviluppo di nuovi agenti terapeutici che preservino la massa e ripristino la funzionalità delle β-cellule. Uno di questi è l’ormone Glucagon-Like Peptide-1 (GLP-1), che non solo riduce la glicemia aumentando la secrezione di insulina, ma agisce anche nel signaling nelle isole di Langherans stimolando la proliferazione e la neo-genesi delle β-cellule e inibendone l’apoptosi. La ridotta secrezione di insulina e la mancata soppressione del glucagone inducono ad ipotizzare che la diminuita risposata delle β-cellule al GLP-1 possa essere parte della patogenesi del diabete di tipo 2. Pertanto la capacità di misurare l’effetto del GLP-1 sulla secrezione dell’insulina è utile per studiare la patogenesi della malattia ed ottimizzare valutare l’efficacia delle terapie basate sul GLP-1. Infatti è cruciale determinare quali soggetti possono beneficiare maggiormente di tale terapia per ottimizzare le risorse. Tuttavia, non è ancora disponibile un modello che descriva l’azione del GLP-1 sulla secrezione di insulina e permetta di quantificarne l’entità. In questo lavoro viene proposto un modello matematico che descrive i meccanismi di azione del GLP-1 sulla secrezione di insulina, fornendo una misura diretta dell’aumento della secrezione dell’insulina dovuto all’effetto del GLP-1. Sono stati utilizzati tre database per sviluppare, testare e validare i modelli proposti. I dati di 88 soggetti sani sottoposti ad un clamp iperglicemico con contemporanea infusione intravenosa di GLP-1, sono stati utilizzati per lo sviluppo del modello. Sono stati testati una serie di modelli dell’azione del GLP-1 sulla secrezione di insulina di complessità crescente. Tutti i modelli si basano sulla comune assunzione che la secrezione di insulina è costituita da due componenti, una proporzionale alla concentrazione ed una alla velocità di variazione del glucosio plasmatico, modulate rispettivamente dalla responsività statica Φs e dalla responsività dinamica Φd. Ogni modello differisce dagli altri nella descrizione della modalità di azione del GLP-1. Per ciascun modello è stato derivato un indice di potenziamento, П, che rappresenta l’aumento percentuale della secrezione di insulina dovuta ad 1 pmol/l di GLP-1. I modelli predicono bene i dati (infatti il run test conferma la casualità dei residui nel 70% dei soggetti) e forniscono stime precise dei parametri . La selezione del modello ottimo è stata affrontata confrontando le prestazioni dei modelli sulla base di criteri standard (capacità di descrivere i dati, la precisione della stima dei parametri, la parsimonia, la casualità dei residui). Il modello più parsimonioso ipotizza che la secrezione sopra basale di insulina dipenda linearmente sia dalla concentrazione di GLP-1 sia dalla sua variazione. Tuttavia le condizioni sperimentali di tale protocollo non sono fisiologiche e applicabili su larga scala. Pertanto, i dati di 22 soggetti IFG (Impaired Fasting Glucose), studiati due volte con un pasto misto, sono stati utilizzati per testare il modello in una condizione sperimentale più vicina alla fisiologia. I risultati dimostrano che per descrivere i dati di un test orale, è sufficiente un modello più semplice. La validazione del modello è stata effettuata sia in simulazione sia utilizzando i dati reali di 10 soggetti, studiati due volte: una prima volta utilizzando un test orale di tolleranza al glucosio (OGTT) e successivamente un test intravenoso di tolleranza al glucosio durante il quale il glucosio è stato infuso in modo tale da riprodurre la glicemia osservata durante l’OGTT. Questo protocollo permette di calcolare un indice di potenziamento (PI) modello-indipendente dal confronto tra la secrezione di insulina stimata nelle due occasioni. Il confronto tra il potenziamento stimato con il modello, П, e l’indice di potenziamento PI mostra che i due indici sono molto simili (П = 6.55, CV = 65%; PI = 6.15 % per pmol/l). Inoltre nel 93 ± 1% delle simulazioni effettuate il modello è in grado di quantificare correttamente l’effetto del GLP-1 sulla secrezione di insulina.

Orientador: Carla Beatriz Collares-Buzato
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: As junções intercelulares são especializações da membrana plasmática através das quais células dentro de um tecido podem interagir e aderirem-se umas às outras. Nas ilhotas pancreáticas, as diferentes células endócrinas se interconectam por meio das junções de oclusão, comunicante, aderente e desmossomos. Tais contatos intercelulares parecem ser cruciais para o perfeito funcionamento deste órgão, porém, pouco se sabe sobre a função fisiopatológica e composição das junções intercelulares no pâncreas endócrino. O objetivo geral desta dissertação foi estudar a importância funcional da adesão e reconhecimento celular mediados pelas junções de oclusão (JO) e de adesão (JA) nos processos de maturação e disfunção da célula beta do pâncreas endócrino, ao longo do desenvolvimento do animal e na patogênese da diabetes tipo 2, respectivamente. Nossos dados mostram que as ilhotas de fetos e recém-nascidos de ratos Wistar, cuja resposta secretora de insulina à glicose é significativamente menor, apresentam uma morfologia menos organizada, caracterizada por um formato menos definido e uma associação mais freqüente com ductos, quando comparadas às ilhotas de jovens e adultos, que são responsivas à esse secretagogo. Na ausência de contato intercelular, quando as células das ilhotas de adultos foram dispersas, a resposta secretora de insulina foi completamente inibida, porém parcialmente restabelecida quando as células foram reagregadas. Quando comparado às ilhotas de adultos, o impacto da ausência de contatos intercelulares sobre a resposta secretora de insulina à glicose foi consideravelmente menor no caso das ilhotas de ratos recém-nascidos. A imunofluorescência para NCAM e pan-caderina revelou uma distribuição diferencial destas moléculas de adesão somente nas células das ilhotas pancreáticas de jovens e adultos, o que pode estar relacionada com a citoarquitetura típica (células beta, ocupando a região central e os outros tipos celulares na periferia da ilhota) adquirida no período pré-natal. Das proteínas associadas à JA e JO estudadas, ?- e ?-cateninas e ZO-1 (mas não a ocludina) foram expressas nas células endócrinas das ilhotas de todos os grupos experimentais. De forma geral, a imunofluorescência para tais proteínas revelou uma marcação intercelular menos definida nas células beta de ilhotas de recém-nascidos e fetos em comparação com os animais jovens e adultos. Isto pode indicar uma menor interação/adesão celular, que por sua vez, pode estar relacionada com a resposta secretora deficitária de insulina das ilhotas pancreáticas verificada na fase perinatal. Como modelo de diabetes do tipo 2, utilizamos camundongos C57, machos, alimentados com dieta hiperlipídica por 8 meses desde os 21 dias de idade. Após este período em dieta, os animais tornaram-se obesos e pré-diabéticos, apresentando moderada hiperglicemia e significativa hiperinsulinemia ao final desta dieta. Não observamos diferenças morfológicas marcantes entre os grupos e nem alterações significativas na distribuição intercelular da ZO-1, ?- e ?-cateninas nas ilhotas do grupo tratado em comparação ao grupo controle. Entretanto, verificou-se uma maior marcação intercelular para Ecaderina e uma maior associação de F-actina à membrana na região de contato intercelular nas células endócrinas das ilhotas do grupo pré-diabético em relação ao controle. Em conclusão, os contatos intercelulares mediados pelas proteínas associadas à JA e à JO parecem desempenhar um papel no processo de maturação do pâncreas do endócrino durante o desenvolvimento animal e na patogênese da diabetes do tipo 2
Abstract: Intercellular junctions are specializations of the plasma membrane that allow cells within a tissue to interact and adhere to each other. In endocrine pancreas, the different endocrine cells are interconnected by tight, gap and adherens-type junctions. Such intercellular contacts seem to be crucial for the function of this organ, however, little is known about the pathophysiological role and biochemistry of intercellular junctions in the endocrine pancreas. The aim of this thesis was to study the importance of cell-cell recognition and adhesion mediated by proteins associated to tight and adherens junctions in pancreatic islets, with emphasis on the process of insulin secretion, along the animal development and in pathogenesis of type 2 diabetes. Pancreatic islets of foetuses (F) and newborn (N) Wistar rats, that display a relatively poor insulin secretory response to glucose, present an immature morphology and a less defined cytoarchitecture when compared to islets from young (Y) and adult (A) rats, that are responsive to glucose. The immunofluorescence for N-CAM and pan-cadherin, adhesion molecules that are important in cell segregation, revealed a differential distribution of these proteins only in cells of the islets from Y and A. A lower junctional content of ?-and ?-catenins and ZO-1 in islet cells was seen in F and N in comparison with Y and A. In addition, we found that in the absence of intercellular contact, the glucose-stimulated insulin secretion was completely blocked in A islets. In contrast, the impact of the disruption of cell-cell adhesion on insulin secretory response of N islets was relatively small. As a model of type 2 diabetes, we employed male C57 mice, fed on high-fat (HF) diet for 8 months since they were 21 days old. After HF diet, these mice became obese and pre-diabetic (displaying moderate hyperglycemia and marked hyperinsulinaemia). We did not observe marked differences either in morphology either in the intercellular distribution of ZO-1, ?- and ?-catenins in islets between the HF-treated and control groups. However, there was a stronger immunoreaction for Ecadherin at the cell-cell contact site and an increased association of F-actin to the plasma membrane of islet endocrine cells from pre-diabetic mice as compared to control ones. In conclusion, the intercellular contacts mediated by adherens and tight junction and their constitutive proteins seem to play a role in the developmental maturation of the endocrine pancreas and in the pathogenesis of type 2 diabetes
Mestrado
Histologia
Mestre em Biologia Celular e Estrutural

Orientador: Carla Beatriz Collares Buzato
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Tem havido um grande interesse na determinação das vias envolvidas na proliferação e função/disfunção da célula beta e a aplicação deste conhecimento em terapias moleculares e celulares da diabetes. A patogênese da diabetes melito tipo 2 (T2DM) é complexa, mas frequentemente está associada com obesidade e distúrbios do metabolismo de lipídios (hipercolesterolemia e hipertrigliceridemia). A T2DM envolve o desenvolvimento de um quadro de resistência periférica à insulina parcialmente compensada por hiperinsulinemia e hiperplasia da célula beta pancreática, resultando em intolerância à glicose e hiperglicemia. Os mecanismos interligando os estados de obesidade/hipercolesterolemia e resistência à insulina ao fenômeno da hiperplasia da célula beta não são completamente conhecidos. A presente dissertação teve como objetivos: 1) caracterizar um modelo animal adequado para se estudar a proliferação e disfunção da célula beta pancreática, e 2) avaliar, no pâncreas endócrino desses animais, a possível ativação da via de sinalização Wnt/beta-catenina, conhecida por estar envolvida no processo de proliferação celular em outros tecidos/órgãos. Para tal, foram empregados camundongos C57BL/6, wild-type (WT) e knockout para receptor de lipoproteína LDL (LDLr-/-), os quais foram submetidos à dieta hiperlipídica (HF) por 60 dias. Após a dieta HF, os animais WT tornaram-se obesos e hipercolesterolêmicos, bem como moderadamente hiperglicêmicos, hiperinsulinêmicos, intolerantes à glicose e resistentes à insulina, caracterizando-os como pré-diabéticos. Além disso, os animais alimentados com dieta HF apresentaram uma diminuição significativa na resposta secretora das células beta à glicose. De modo geral, os animais LDLr-/- apresentaram uma susceptibilidade relativamente mais alta à dieta HF, sugerida pela acentuada hipercolesterolemia, intolerância à glicose, e reduzida secreção de insulina estimulada por glicose observadas nestes animais. No entanto, a dieta HF induziu, de forma semelhante em animais WT e LDLr-/-, uma diminuição significativa no conteúdo celular de Cx36, uma proteína associada à junção comunicante e um marcador de diferenciação terminal da célula beta. Ambos os grupos WT e LDLr-/- alimentados com dieta HF mostraram aumento na proliferação de células beta, como avaliada pela imunomarcação das ilhotas para a proteína Ki67, mas apenas os animais WT exibiram alterações morfométricas indicativas de hiperplasia do pâncreas endócrino, tais como aumento na massa total de ilhotas e de células beta. Uma vez estabelecido que camundongos WT alimentados com dieta HF por 60 dias consistiam em um modelo adequado para a segunda etapa deste estudo, fomos investigar a possível ativação da via Wnt/beta-catenina nas ilhotas pancreáticas desses animais, avaliando-se a distribuição e expressão celular das proteínas beta-catenina total, beta-catenina ativada, c-Myc e ciclina D. A análise por imunofluorescência para beta-catenina não mostrou acúmulo citoplasmático ou translocação para o núcleo desta proteína em ilhotas pancreáticas, que poderia indicar ativação da via Wnt/beta-catenina no nosso modelo de hiperplasia do pâncreas endócrino. No entanto, a análise por Western Blot revelou um aumento significativo na expressão de beta-catenina ativada e ciclina D em ilhotas de animais alimentados com dieta HF em relação ao grupo controle. Concluindo, a dieta HF por 60 dias induz alterações metabólicas típicas da pré-diabetes em animais WT e LDLr-/-. O estado de pré-diabetes está associado a uma diminuição da expressão de Cx36 nas células beta pancreáticas, sugerindo um possível papel da comunicação intercelular mediada pelas junções comunicantes na patogênese da T2DM. A maior susceptibilidade metabólica à dieta HF apresentada por camundongos LDLr-/-, em relação aos WT, pode ser explicada pela maior deficiência na secreção de insulina em resposta à glicose e ausência de hiperplasia compensatória do pâncreas endócrino. Ainda, a análise preliminar de expressão protéica de algumas proteínas da via Wnt/beta-catenina sugere que esta via parece estar ativada durante o processo de hiperplasia do pâncreas endócrino observada no nosso modelo animal
Abstract: The pathogenesis of type 2 diabetes mellitus (T2DM) is often associated with obesity and dyslipidemia (hypercholesterolemia and hypertriglyceridemia). T2DM involves intolerance to glucose and insulin resistance partially compensated by hyperinsulinemia and pancreatic beta cell hyperplasia. The mechanisms linking obesity/hypercholesterolemia and insulin resistance to beta cell hyperplasia are not fully known. The Wnt/beta-catenin signaling pathway has been reported to be involved in cell growth and differentiation in several tissues/organs but its role in endocrine pancreas development and function is still unclear. This work aimed at: 1) establishing an appropriate animal model of T2DM to study pancreatic beta cell proliferation and dysfunction and, 2) investigating a putative involvement of the Wnt/beta-catenin signaling pathway in the beta cell hyperplasia in this model. To this end, we employed C57BL/6 wild-type (WT) and LDL lipoprotein receptor knockout (LDLr-/-) mice, fed a high fat (HF) diet for 60 days. After feeding a HF diet, WT mice became obese, hypercholesterolemic and moderately hyperglycemic, hyperinsulinemic, glucose intolerant and insulin resistant, characterizing them as pre-diabetics. Moreover, animals fed a HF diet showed a significant decrease in beta-cell secretory response to glucose. In general, LDLr-/- animals showed a relatively higher susceptibility to HF diet, as suggested by a marked hypercholesterolemia, glucose intolerance and reduced insulin secretion stimulated by glucose observed in these animals as compared to the control ones. However, HF diet induced similarly in both WT and LDLr-/- mice a significant decrease in cellular content of Cx36, a gap junctional protein and marker of terminally differentiated beta cell. Both WT and LDLr-/- fed a HF diet showed increased proliferation of beta cells, as assessed by Ki67 immunostaining, but only WT mice exhibited morphometric changes indicative of endocrine pancreas hyperplasia, such as increased total islet and beta cell masses. After we investigated a possible activation of Wnt/beta-catenin signaling pathway in these hyperplasic pancreatic islets of WT animals fed a HF diet. This was done by assessing the distribution and cellular protein expression of some proteins associated to this pathway (i.e., total and activated beta-catenin, c-Myc and cyclin D) in islets of our animal model. Beta-catenin immunofluorescence showed no cytoplasmic accumulation or translocation into the nucleus of beta cells in HF-fed mice. However, immunoblotting revealed a significant increase of unphosphorylated beta-catenin (activated) and cyclin D expression in islets of HF diet-fed animals when compared to its control group. In conclusion, a HF diet for 60d induced pre-diabetes state in both WT and LDLr-/- mice. The pre-diabetes state is associated with a decreased expression of Cx36 in pancreatic beta cells, suggesting a possible role of intercellular communication mediated by gap junctions in the pathogenesis of T2DM. The relatively high metabolic susceptibility to the HF diet showed by LDLr-/- mice, as compared to WT, may be explained by a marked impairment of glucosestimulated insulin secretion and a lack of compensatory hyperplasia of the endocrine pancreas. In addition, the protein expression analysis suggests that the Wnt/beta-catenin pathway may be activated during the islet hyperplasia process in our animal model
Mestrado
Histologia
Mestre em Biologia Celular e Estrutural

Précédemment, nous avons montré que la surexpression de l'échangeur Na/Ca NCX1), une protéine responsable de la sortie de calcium (Ca2+) des cellules, augmentait la mort cellulaire programmée ou « apoptose » et réduisait la prolifération des cellules β. Afin d’étudier plus en profondeur le rôle de l’échangeur dans les cellules β in vivo, nous avons développé et caractérisé des souris présentant une inactivation de NCX1.

Des méthodes biologiques et morphologiques (imagerie du Ca2+, capture de Ca2+, métabolisme du glucose, sécrétion d'insuline et morphométrie par comptage de points) ont été utilisées pour évaluer la fonction de la cellule β in vitro. Les taux de glucose et d'insuline dans le sang ont été mesurés afin de déterminer le métabolisme du glucose et la sensibilité à l’insuline in vivo. Des îlots ont été transplantés sous la capsule rénale pour évaluer leur capacité à corriger le diabète chez les souris rendues diabétiques par l’alloxane.

L'inactivation hétérozygote de Ncx1 chez les souris provoque une augmentation de la sécrétion d’insuline induite par le glucose avec un renforcement important à la fois de la première et de la deuxième phase. Ces résultats s’accompagnent d’une augmentation de la masse et de la prolifération des cellules β. La mutation augmente également le contenu en insuline, l’immunomarquage de la proinsuline, la capture de Ca2+ induite par le glucose et la résistance à l'hypoxie des cellules β. En outre, les îlots de souris Ncx1+/- montrent une capacité à compenser le diabète 2 à 4 fois plus élevé que les îlots de souris Ncx1+/+ lorsque transplantés chez des souris diabétiques.

En conclusion, l’inactivation de l'échangeur Na/Ca conduit à une augmentation de la fonction de la cellule β, de sa prolifération, de sa masse et de sa résistance au stress physiologique, à savoir à divers changements de fonction des cellules β opposés aux principales anomalies rencontrées dans le diabète de type 2 (Type 2 Diabetes Mellitus,T2DM). Ceci nous procure un modèle unique pour la prévention et le traitement du dysfonctionnement des cellules β dans le T2DM et pour la transplantation d'îlots.

Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

Le diabète de type 2, défini par une hyperglycémie chronique, résulte d'un déficit de la sécrétion d'insuline et d'une insulinorésistance. Durant le prédiabète qui précède la maladie, la cellule ß pancréatique est capable d'établir une hyperactivité sécrétoire compensatrice de l'insulinorésistance. Les NO synthases neuronales (nNOS) pancréatique et musculaire contrôlent respectivement la sécrétion d'insuline induite par le glucose dans la cellule ß et la force contractile, la captation et l'utilisation du glucose dans les myocytes. Dans le modèle génétique du rat obèse Zucker fa/fa mimant l'état prédiabétique associant un hyperinsulinisme et une insulinorésistance, nous avons retrouvé au niveau de la cellule ß une forte augmentation du complexe entre la nNOS et son inhibiteur endogène PIN (Protein Inhibitor of Neuronal NOS) au niveau des granules de sécrétion d'insuline. Ce complexe, grâce à une interaction accrue avec la myosine V, participe à l'hyperactivité sécrétoire de la cellule ß pancréatique. En effet, des molécules inhibant spécifiquement l'interaction nNOS-PIN permettent de rétablir, chez le rat fa/fa, une sécrétion d'insuline normale. Au niveau musculaire, nous avons observé, dans ce modèle animal, une diminution d'expression de la nNOS sans variation du taux d'ARNm, traduisant une protéolyse accrue de la protéine. L'inhibition de la dégradation protéasomale permet de restaurer l'expression et l'activité catalytique de la nNOS dans le muscle squelettique. Cette perte de fonctionnalité de l'enzyme participerait à l'installation de l'insulinorésistance. Ces travaux ont permis de valider la nNOS comme une cible potentielle pour la prévention du diabète de type 2
Type 2 diabetes is a chronic disorder defined by chronic hyperglycemia resulting from a deficiency of insulin secretion and an insulin resistance in peripheral tissues and liver. A long lasting silent phase, called prediabetes, precedes the disease and in which pancreatic ß cell is able to improve insulin secretion to compensate for the insulin resistance. The pancreatic and muscular neuronal nitric oxide synthases (nNOS) control respectively glucose-induced insulin secretion in pancreatic ß cell and glucose uptake and utilization in myocytes. In the genetic model of obese Zucker fa/fa rat mimicking the prediabetic state characterized by hyperinsulinemia and insulin resistance, we found a high increase in the amount of the complex between nNOS and its endogenous inhibitor PIN (Protein Inhibitor of Neuronal NOS) at the level of insulin secretory granules within the ß cell. This complex, through an increased interaction with myosin V, participates in the secretory hyperactivity of the pancreatic ß cell, observed in this model of prediabetes. Indeed, molecules that specifically inhibit nNOS-PIN interaction allow to restore a normal insulin secretion in fa/fa rat. In skeletal muscle of this model, we observed a decreased expression of nNOS protein with no change in mRNA levels, suggesting an increased proteolysis of the protein. Inhibition of proteasomal degradation restores the expression and the catalytic activity of nNOS in skeletal muscle. Thus, this loss of functionality of the enzyme could participate in the installation of insulin resistance. This work therefore validated nNOS as a potential target for the prevention of type 2 diabetes

Type 2 diabetes is characterized by peripheral insulin resistance and insufficient insulin release from pancreatic islet β cells. However, the role and sequence of β cell dysfunction and mass loss for reduced insulin levels in type 2 diabetes pathogenesis are unclear. Here, we exploit freshly explanted pancreas specimens from metabolically phenotyped surgical patients using an in situ tissue slice technology. This approach allows assessment of β cell volume and function within pancreas samples of metabolically stratified individuals. We show that, in tissue of pre-diabetic, impaired glucose-tolerant subjects, β cell volume is unchanged, but function significantly deteriorates, exhibiting increased basal release and loss of first-phase insulin secretion. In individuals with type 2 diabetes, function within the sustained β cell volume further declines. These results indicate that dysfunction of persisting β cells is a key factor in the early development and progression of type 2 diabetes, representing a major target for diabetes prevention and therapy.

The trafficking of insulin secretory granules(SGs) of pancreatic b-cells is a tightly controlled complex network. Increasing evidence indicates that the cortical actin cytoskeleton modulates the mobility and exocytosis of SGs,yet the mechanisms anchoring SGs to the cytoskeleton is not completely understood.It has been shown by Ort et al.(2000,2001) that the cytoplasmic tail of an intrinsic membrane protein of the SGs named ICA512/IA-2 binds the PDZ domain of b2-syntrophin,which in turn binds to the F-actin-binding protein utrophin. These data also indicate that stimulation of SG exocytosis affects the phosphorylation of b2-syntrophin,hence altering its binding to ICA512.Therefore a model was proposed whereby SGs are anchored to the actin cytoskeleton through the ICA512/b2-syntrophin complex, whose dynamics are regulated by phosphorylation.To test this model GFP-b2-syntrophin stable INS-1 cell clones were generated.GFP-b2-syntrophin expression and localization pattern were similar to those of the endogenous protein. Electron microscopy showed that in GFP-b2-syntrophin INS-1 cells the number of SGs with a pear-like shape was increased relative to control cells. Insulin content and stimulated secretion were increased in three GFP-â2-syntrophin INS-1 cell clones,compared to non-transfected INS-1 cells and INS-1 cells expressing GFP. These increments correlated with the different expression levels of GFP-b2-syntrophin in the three GFP-b2-syntrophin INS-1 cell clones. These findings support the hypothesis that b2-syntrophin regulates the trafficking and exocytosis of SGs by modulating their tethering to the actin cytoskeleton.In order to confirm the proposed model, the phosphorylation of b2-syntrophin was investigated in more detail. Similar to endogenous b2-syntrophin,GFP-b2-syntrophin underwent Ca2+-dependent and okadaic acid-sensitive dephosphorylation upon stimulation of insulin secretion. Stimulation-dependent dephosphorylation was confirmed by immunoprecipitation of 32P-labeled GFP-b2-syntrophin.Mass spectrometry of immunoprecipitated GFP-b2-syntrophin allowed the identification of four serine-phosphorylation sites (S75,S90,S213,S373) that could affect the binding to ICA512.Mutants,in which all four phosphoserines, were replaced by either asp or ala to mimic(S/D) or prevent(S/A) phosphorylation were expressed in INS-1 cells. All S/D mutants retained a cortical localization,but by immunoblotting the pattern of the S75D allele differed from wild type and all other S/D alleles.Conversely, all S/A alleles were diffused cytosolically, except S213A,which was still restricted to the cortex. Finally, pull down assays showed increased binding of ICA512 to the S75A and S90D alleles compared to wild type b2-syntrophin,while the opposite was observed with the S75D and S90A mutants.Additionally,both the S75 and the S213 allele conform a consensus for phosphorylation by Cdk5,which is known to modulate insulin secretion. The phosphorylation of GFP-b2-syntrophin and particularly the S75 allele by Cdk5 was exhibited with pharmacological inhibitors,by in vitro phosphorylation and by RNAi. Taken together, these findings are consistent with the model by which phosphorylation of b2-syntrophin modulates the tethering of SGs to the cytoskeleton, and thereby their mobility and exocytosis. Specifically, the data of this thesis suggest that Cdk5-dependent phosphorylation of the S75 site of GFP-b2-syntrophin facilitates insulin secretion by reducing the interaction of b2-syntrophin with ICA512,thereby decreasing the actin cytoskeleton constrain on SG mobility. This process could occur in combination with the phosphatase-dependent dephosphorylation of b2-syntrophin at phosphosites other than S75
Der Transport Insulin-gefüllter sekretorische Granula(SG) ist ein streng kontrollierter komplexer Prozess.Es gibt vermehrt Beweise,dass das kortikale Actinzytoskelett die Ausschüttung der SGs beeinflusst.Bisher ist der Mechanismus der Verankerung von SGs am Zytoskelett noch nicht vollständig aufgeklärt.Ort et al.(2000,2001) haben gezeigt,daß der zytosoplasmatische Teil des trans-membranen SG-Proteins ICA512 mit der PDZ-Domäne von b2-Syntrophin interagiert.Dieses Protein bindet das F-Actin-Bindeprotein Utrophin.Die Ergebnisse zeigen außerdem,daß durch Stimulation der SG-Exozytose der Phosphorilierungsstatus von b2-Syntrophin beeinflusst wird,woraus ein verändertes Bindungsvermögen zu ICA512 resultiert.Es wurde ein Funktionsmodel vorgestellt,in dem sich SGs durch die Interaktion des ICA512/b2-Syntrophin Komplexes an das Actinzytoskelett binden.Dabei wird die Bindedynamik durch Phosphorilierung reguliert.Um dieses Model zu etablieren,wurden stabile GFP-b2-Syntrophin produzierende INS-1-Zellklone erzeugt.Die zelluläre Lokalisation und das Expressionsmuster von GFP-b2-Syntrophin stimmen mit dem des endogenen Proteins überein.Elektronenmikroskopie zeigte eine größe Anzahl oval-verformter SGs in GFP-b2-Syntrophin INS-1-Zellen im Vergleich zu Kontrollzellen.Verglichen mit nicht-transfizierten INS-1 Zellen waren in drei GFP-b2-Syntrophin INS-1-Zellklonen der Insulingehalt der Zellen und die stimulierte Insulinsekretion erhöht.Die Werte korrelierten mit den unterschiedlichen GFP-b2-Syntrophin Expressionsmengen der Klone.Diese Ergebnisse untermauern die Hypothese,daß b2-Syntrophin den Transport und die Sekretion der SGs durch Modulation ihres Bindevermögens an Actin reguliert.Um das postulierte Model genauer zu prüfen,wurde die Phosphorilierung von b2-Syntrophin detaillierter untersucht.Das GFP-Protein wurde,ähnlich dem endogenen b2-Syntrophin,durch Stimulation der Insulinausschüttung dephosphoriliert.Diese Dephosphorilierung ist Ca2+-abhängig und Okadeinsäuresensitiv.Die stimulationsabhängige Dephosphorilierung wurde durch Immunoprezipitation von 32P-markiertem GFP-b2-Syntrophin bestätigt.Massenspektrometrie des präzipitierten Proteins ermöglichte die Identifikation von vier Serin-Phosphorilierungsstellen(S75,S90,S213,S373),welche die Bindung zu ICA512 beeinflussen könnten.Mutanten,in denen die vier Phosphoserine durch Asp beziehungsweise Ala ersetzt wurden,um entweder eine Phosphorilierung(S/D) oder Dephosphorilierung(S/A) nachzuahmen,wurden in INS-1-Zellen exprimiert.Alle S/D Mutanten blieben kortikal lokalisiert.Das Expressionsmuster des S75D Allels unterschied sich jedoch von denen des Wild-Typs(wt).Im Gegensatz dazu waren alle S/A Allele zytosolisch verteilt.Eine Ausnahme bildete S213A,das an der Zellkortex lokalisiert blieb.Im Vergleich zu wt b2-Syntrophin zeigten PullDown-Assays eine erhöhte Bindung von ICA512 zu den S75A und S90D Allelen.Das Gegenteil konnte für die S75D und S90A Mutanten nachgewiesen werden.S75,S90 und S213 sind in einer Konsensussequenz für Cdk5-Phosphorilierung enthalten.Diese Kinase kann die Insulinsekretion regulieren.Die Phosphorilierung von b2-Syntrophin,insbesondere des S75 Allels durch Cdk5 wurde durch pharmakologische Inhibitoren,in vitro-Phosphorilierung und RNAi demonstriert.Zusammenfassend stimmen diese Erkenntnisse mit dem Model überein,daß die Phosphorilierung von b2-Syntrophin die Vernetzung von SGs mit Actin und dadurch deren Mobilität und Exozytose moduliert.Im Speziellen postulieren die Ergebnisse dieser Arbeit eine Cdk5-abhängige Phosphorilierung der S75 Stelle des b2-Syntrophins.Durch eine verminderte Interaktion von b2-Syntrophin und ICA512 erleichtert diese Mutante vermutlich die Insulinsekretion,da der Einfluss des Actinzytoskeletts auf die Granulamobilität vermindert ist.Dieser Prozess ereignet sich möglicherweise in Kombination mit einer Dephosphorilierung des b2-Syntrophins.in Kombination mit einer Dephosphorilierung des b2-Syntrophins

The trafficking of insulin secretory granules(SGs) of pancreatic b-cells is a tightly controlled complex network. Increasing evidence indicates that the cortical actin cytoskeleton modulates the mobility and exocytosis of SGs,yet the mechanisms anchoring SGs to the cytoskeleton is not completely understood.It has been shown by Ort et al.(2000,2001) that the cytoplasmic tail of an intrinsic membrane protein of the SGs named ICA512/IA-2 binds the PDZ domain of b2-syntrophin,which in turn binds to the F-actin-binding protein utrophin. These data also indicate that stimulation of SG exocytosis affects the phosphorylation of b2-syntrophin,hence altering its binding to ICA512.Therefore a model was proposed whereby SGs are anchored to the actin cytoskeleton through the ICA512/b2-syntrophin complex, whose dynamics are regulated by phosphorylation.To test this model GFP-b2-syntrophin stable INS-1 cell clones were generated.GFP-b2-syntrophin expression and localization pattern were similar to those of the endogenous protein. Electron microscopy showed that in GFP-b2-syntrophin INS-1 cells the number of SGs with a pear-like shape was increased relative to control cells. Insulin content and stimulated secretion were increased in three GFP-â2-syntrophin INS-1 cell clones,compared to non-transfected INS-1 cells and INS-1 cells expressing GFP. These increments correlated with the different expression levels of GFP-b2-syntrophin in the three GFP-b2-syntrophin INS-1 cell clones. These findings support the hypothesis that b2-syntrophin regulates the trafficking and exocytosis of SGs by modulating their tethering to the actin cytoskeleton.In order to confirm the proposed model, the phosphorylation of b2-syntrophin was investigated in more detail. Similar to endogenous b2-syntrophin,GFP-b2-syntrophin underwent Ca2+-dependent and okadaic acid-sensitive dephosphorylation upon stimulation of insulin secretion. Stimulation-dependent dephosphorylation was confirmed by immunoprecipitation of 32P-labeled GFP-b2-syntrophin.Mass spectrometry of immunoprecipitated GFP-b2-syntrophin allowed the identification of four serine-phosphorylation sites (S75,S90,S213,S373) that could affect the binding to ICA512.Mutants,in which all four phosphoserines, were replaced by either asp or ala to mimic(S/D) or prevent(S/A) phosphorylation were expressed in INS-1 cells. All S/D mutants retained a cortical localization,but by immunoblotting the pattern of the S75D allele differed from wild type and all other S/D alleles.Conversely, all S/A alleles were diffused cytosolically, except S213A,which was still restricted to the cortex. Finally, pull down assays showed increased binding of ICA512 to the S75A and S90D alleles compared to wild type b2-syntrophin,while the opposite was observed with the S75D and S90A mutants.Additionally,both the S75 and the S213 allele conform a consensus for phosphorylation by Cdk5,which is known to modulate insulin secretion. The phosphorylation of GFP-b2-syntrophin and particularly the S75 allele by Cdk5 was exhibited with pharmacological inhibitors,by in vitro phosphorylation and by RNAi. Taken together, these findings are consistent with the model by which phosphorylation of b2-syntrophin modulates the tethering of SGs to the cytoskeleton, and thereby their mobility and exocytosis. Specifically, the data of this thesis suggest that Cdk5-dependent phosphorylation of the S75 site of GFP-b2-syntrophin facilitates insulin secretion by reducing the interaction of b2-syntrophin with ICA512,thereby decreasing the actin cytoskeleton constrain on SG mobility. This process could occur in combination with the phosphatase-dependent dephosphorylation of b2-syntrophin at phosphosites other than S75.
Der Transport Insulin-gefüllter sekretorische Granula(SG) ist ein streng kontrollierter komplexer Prozess.Es gibt vermehrt Beweise,dass das kortikale Actinzytoskelett die Ausschüttung der SGs beeinflusst.Bisher ist der Mechanismus der Verankerung von SGs am Zytoskelett noch nicht vollständig aufgeklärt.Ort et al.(2000,2001) haben gezeigt,daß der zytosoplasmatische Teil des trans-membranen SG-Proteins ICA512 mit der PDZ-Domäne von b2-Syntrophin interagiert.Dieses Protein bindet das F-Actin-Bindeprotein Utrophin.Die Ergebnisse zeigen außerdem,daß durch Stimulation der SG-Exozytose der Phosphorilierungsstatus von b2-Syntrophin beeinflusst wird,woraus ein verändertes Bindungsvermögen zu ICA512 resultiert.Es wurde ein Funktionsmodel vorgestellt,in dem sich SGs durch die Interaktion des ICA512/b2-Syntrophin Komplexes an das Actinzytoskelett binden.Dabei wird die Bindedynamik durch Phosphorilierung reguliert.Um dieses Model zu etablieren,wurden stabile GFP-b2-Syntrophin produzierende INS-1-Zellklone erzeugt.Die zelluläre Lokalisation und das Expressionsmuster von GFP-b2-Syntrophin stimmen mit dem des endogenen Proteins überein.Elektronenmikroskopie zeigte eine größe Anzahl oval-verformter SGs in GFP-b2-Syntrophin INS-1-Zellen im Vergleich zu Kontrollzellen.Verglichen mit nicht-transfizierten INS-1 Zellen waren in drei GFP-b2-Syntrophin INS-1-Zellklonen der Insulingehalt der Zellen und die stimulierte Insulinsekretion erhöht.Die Werte korrelierten mit den unterschiedlichen GFP-b2-Syntrophin Expressionsmengen der Klone.Diese Ergebnisse untermauern die Hypothese,daß b2-Syntrophin den Transport und die Sekretion der SGs durch Modulation ihres Bindevermögens an Actin reguliert.Um das postulierte Model genauer zu prüfen,wurde die Phosphorilierung von b2-Syntrophin detaillierter untersucht.Das GFP-Protein wurde,ähnlich dem endogenen b2-Syntrophin,durch Stimulation der Insulinausschüttung dephosphoriliert.Diese Dephosphorilierung ist Ca2+-abhängig und Okadeinsäuresensitiv.Die stimulationsabhängige Dephosphorilierung wurde durch Immunoprezipitation von 32P-markiertem GFP-b2-Syntrophin bestätigt.Massenspektrometrie des präzipitierten Proteins ermöglichte die Identifikation von vier Serin-Phosphorilierungsstellen(S75,S90,S213,S373),welche die Bindung zu ICA512 beeinflussen könnten.Mutanten,in denen die vier Phosphoserine durch Asp beziehungsweise Ala ersetzt wurden,um entweder eine Phosphorilierung(S/D) oder Dephosphorilierung(S/A) nachzuahmen,wurden in INS-1-Zellen exprimiert.Alle S/D Mutanten blieben kortikal lokalisiert.Das Expressionsmuster des S75D Allels unterschied sich jedoch von denen des Wild-Typs(wt).Im Gegensatz dazu waren alle S/A Allele zytosolisch verteilt.Eine Ausnahme bildete S213A,das an der Zellkortex lokalisiert blieb.Im Vergleich zu wt b2-Syntrophin zeigten PullDown-Assays eine erhöhte Bindung von ICA512 zu den S75A und S90D Allelen.Das Gegenteil konnte für die S75D und S90A Mutanten nachgewiesen werden.S75,S90 und S213 sind in einer Konsensussequenz für Cdk5-Phosphorilierung enthalten.Diese Kinase kann die Insulinsekretion regulieren.Die Phosphorilierung von b2-Syntrophin,insbesondere des S75 Allels durch Cdk5 wurde durch pharmakologische Inhibitoren,in vitro-Phosphorilierung und RNAi demonstriert.Zusammenfassend stimmen diese Erkenntnisse mit dem Model überein,daß die Phosphorilierung von b2-Syntrophin die Vernetzung von SGs mit Actin und dadurch deren Mobilität und Exozytose moduliert.Im Speziellen postulieren die Ergebnisse dieser Arbeit eine Cdk5-abhängige Phosphorilierung der S75 Stelle des b2-Syntrophins.Durch eine verminderte Interaktion von b2-Syntrophin und ICA512 erleichtert diese Mutante vermutlich die Insulinsekretion,da der Einfluss des Actinzytoskeletts auf die Granulamobilität vermindert ist.Dieser Prozess ereignet sich möglicherweise in Kombination mit einer Dephosphorilierung des b2-Syntrophins.in Kombination mit einer Dephosphorilierung des b2-Syntrophins.

Dans le diabète de type 2 établi, l'hyperglycémie chronique, un taux élevé d'acides gras libres et l'inflammation induisent un stress oxydatif (SO) au niveau de la cellule beta. Le SO, qui apparaît dès le stade de pré-diabète, peut induire un dysfonctionnement précoce de cette cellule. Ainsi, la protection de la cellule β par des molécules anti-oxydantes pourrait ralentir la progression du pré-diabète au diabète.La quercétine, un flavonoïde, a présenté des propriétés antidiabétiques dans plusieurs études in vivo. Cependant, très peu de données traitent de son mécanisme d'action directement au niveau de la cellule beta. Dans ce contexte, nous avons étudié les effets de la quercétine au niveau de la cellule beta dans des conditions physiologiques et des conditions de SO.Nos résultats montrent qu'en présence de concentrations stimulantes de sécrétagogue, la quercétine potentialise la sécrétion d'insuline par un mécanisme impliquant l'augmentation de calcium intracellulaire et la potentialisation de ERK1/2 via l'activation des voies de la PKA et de la CaMK II. De plus, la quercétine protège la cellule beta du SO en sur-activant ERK1/2. Le resvératrol et la NAC, deux antioxydants de référence, sont inactifs dans ces conditions expérimentales.En absence de concentrations stimulantes de sécrétagogue, la quercétine induit une sécrétion d'insuline modérée en augmentant le calcium intracellulaire suite à une activation directe des CaV de type L. Dans ces conditions, l'activation de ERK1/2 induite par la quercétine, qui est indépendante de l'activation des voies de la PKA et de la CaMK II, ne serait pas impliquée dans le mécanisme sécrétoire. Nos résultats indiquent que le mécanisme d'action de la quercétine au niveau de la cellule β ne repose pas uniquement sur ses capacités anti-oxydantes mais fait intervenir des cibles pharmacologiques et la régulation de voies de signalisation intracellulaires
In type 2 diabetes, chronic hyperglycaemia, elevated free fatty acids and inflammation induce oxidative stress (OS) in pancreatic β cell. SO, which appears at the stage of pre-diabetes, may induce early dysfunction of this cell. Thus, the β cell protection by antioxidant molecules could slow the progression of pre-diabetes to diabetes.Quercetin, a flavonoid, has shown antidiabetic properties in several in vivo studies. However, very few data address its mechanism of action directly at the β cell. In this context, we studied the effects of quercetin at the β cell under physiological conditions and conditions of OS.Our results show that in the presence of stimulating concentrations of secretagogue, quercetin potentiates insulin secretion by a mechanism involving increased intracellular calcium and potentiation of ERK1 / 2 via activation of the PKA and the CaMK II pathways. In addition, quercetin protects beta cell from OS via a suractivation of ERK1/2. Resveratrol and NAC, two antioxidants of reference are inactive under these experimental conditions.In the absence of stimulating concentration of secretagogue, quercetin induced moderate insulin secretion by increasing the intracellular calcium via a direct activation of L-type CaV Under these conditions, the activation of ERK1/2 induced by quercetin, which is independent of the activation pathways of PKA and CaMK II to, would not be involved in the secretory mechanism.Our results indicate that the mechanism of action of quercetin at the β cell not only based on its antioxidant capacity but involves pharmacological targets and the regulation of intracellular signaling pathways

Pancreatic islet beta cell failure causes type 2 diabetes in humans. To identify transcriptomic changes in type 2 diabetic islets, the Innovative Medicines Initiative for Diabetes: Improving beta-cell function and identification of diagnostic biomarkers for treatment monitoring in Diabetes (IMIDIA) consortium (www.imidia.org) established a comprehensive, unique multicentre biobank of human islets and pancreas tissues from organ donors and metabolically phenotyped pancreatectomised patients (PPP).

Orientador: Carla Beatriz Collares Buzato
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O objetivo central dessa tese foi estudar o papel das junções comunicantes (GJs) no processo de maturação da célula B e na patogênese da diabetes tipo 2. Para isso, foram utilizados dois modelos animais. No modelo in vivo de maturação da célula B e do processo secretório de insulina foram empregados ratos em vários estágios do desenvolvimento (fetal, neonatal, jovem e adulto). No modelo de diabetes tipo 2 foram utilizados camundongos C57BL/6, wild-type e knockout para o gene do receptor de LDL (LDLR -/-), alimentados com dieta hiperlipídica. No primeiro modelo de estudo, observamos, por técnicas morfológicas (imunocitoquímica) e de biologia molecular (Western Blot, RT-PCR semi-quantitativo e qPCR) que a expressão gênica e protéica de Cx36 e Cx43, se altera durante o desenvolvimento. Os dados de microinjeção intracelular de traçadores confirmam tais resultados que sugerem que ilhotas de ratos recém-nascidos apresentam menor número de canais intercelulares associados à membrana e, conseqüentemente, um menor acoplamento intercelular mediado pelas junções comunicantes em comparação às ilhotas de adultos. Esses resultados estão de acordo com a observação de que a célula B durante o período perinatal mostra uma menor resposta secretória à glicose e sugerem a participação da comunicação intercelular mediada pelas junções comunicantes no processo de maturação da célula B. No caso do modelo in vivo de diabetes tipo 2, o estudo iniciou com a caracterização do quadro metabólico e de aspectos da morfologia e morfometria do pâncreas endócrino de animais submetidos ao tratamento com a dieta hiperlipídica. Os dados obtidos demonstram que os animais submetidos a essa dieta por um curto período de tempo já apresentam alterações metabólicas típicas da fase inicial da diabetes (hiperglicemia moderada, hiperinsulinemia, intolerância á glicose e resistência à insulina), bem como modificações de parâmetros da morfometria do pâncreas endócrino. Posteriormente, foi analisada a expressão e localização celular das proteínas juncionais (Cx36, Cx43 e ZO-1) e o aspecto funcional dos canais formados pela GJ, através da microinjeção de marcadores intracelulares. Observamos que os animais tratados com dieta hiperlipídica apresentam tendência de diminuição de expressão de Cx36 acompanhada por alteração no padrão de marcação para essa proteína. Esses dados estão de acordo os de microinjeção que revelam uma pequena diminuição do grau de acoplamento celular após o tratamento com a dieta hiperlipídica. As alterações observadas para Cx36 foram acompanhadas por modificações no grau de expressão e de localização celular de ZO-1, proteína envolvida em processos como a organização dos canais intercelulares da GJ na membrana e no turnover de vários subtipos de conexina. Esses resultados indicam que a comunicação intercelular, particularmente a mediada por canais formados pela Cx36 parecem desempenhar importante papel na patogênese da diabetes tipo 2, desde sua fase inicial.
Abstract: Intercellular communication and adhesion among pancreatic B-cells are crucial for a proper insulin biosynthesis and secretion. The aim of this work was to investigate the role of gap junction (GJ) mediated-intercellular communication in the processes of maturation and dysfunction of B-cells. It is well known that fetal and neonatal rat pancreatic B-cells exhibit a reduced insulin secretory response to glucose and to other secretagogues as compared to adult ones. Based on this finding, we have used Wistar rats at different stages of development (fetal, neonatal, young and adult) as a model of B-cell maturation. As a model of type 2 diabetes, C57BL/6 mice (wild-type) and LDL receptor-deficient mice were fed a high fat diet up to 60 days. The cellular expression of GJ connexin (Cx) subtypes found in the endocrine pancreas (Cx36, Cx43 and Cx45) was assessed by immunohistochemistry, Western Blot, quantitative and semi-quantitative RTPCR. Our results indicate that young and adult endocrine pancreas express relatively high levels of Cx36 on B-cells in comparison with the other groups. Meanwhile, fetal and neonatal islets express predominantly Cx43 on non B-cells located at the islet periphery. Islet microinjection of GJ tracers revealed that neonatal B-cells display lower intercellular exchange of the cationic molecule Ethidium Bromide (EB) in comparison with the adult ones, which is in accordance with their differences in Cx36 expression. No significant differences were found in expression and localization of Cx45 among all animal groups. Regarding the animal type 2 diabetic model, a metabolic characterization showed that the high-fat diet for 60 days induces glucose intolerance, insulin resistance, hyperinsulinemia and moderate hyperglycemia in both mice groups (LDLr knockout and wild-type groups). High-fat fed mice showed a subtle decrease in Cx36 islet expression and in the B-cell intercellular exchange of EB in comparison with the chow-fed group (control). We also observed an alteration in the pattern of Cx36 labeling in immunohistochemistry assays. The high-fat diet induced a redistribution of Cx36 within the islets from a disorganized pattern (more commonly seen in control mice) to a sub domain-pattern where Cx36 plaques demarcate groups of B-cells. These changes in Cx36 islet distribution observed in high-fat fed mice were accompanied by a similar pattern of immunolabeling for ZO-1, but an increase in its islet expression, a tight junctional protein that seem to be involved in Cx turnover and membrane organization of Cx-made channels. Taken all together, these findings suggest that cell-cell coupling mediated by GJ may play an important role in the pancreatic B-cell maturation observed during endocrine pancreas development as well as in the early stages of type 2 diabetes pathogenesis.
Doutorado
Histologia
Mestre em Biologia Celular e Estrutural

Diabetes mellitus comprises a cohort of genetic and metabolic diseases which are characterized by the hallmark symptom of hyperglycemia. Diabetic subtypes are based on their pathogenetic origins: the most prevalent subtypes are the autoimmune-mediated type 1 diabetes mellitus (T1DM) and the metabolic disease of type 2 diabetes mellitus (T2DM). Genetic factors are major contributory aspects to diabetes development, particularly in T2DM where there is close to 80% concordance rates between monozygotic twins. However, the functional state of the pancreatic β cell is of paramount importance to the development of diabetes. Perturbations that lead to β cell dysfunction impair insulin production and secretion and precede diabetes onset. The endoplasmic reticulum (ER) is a subcellular organelle network of tubes and cisternae with multifaceted roles in cellular metabolism. Alterations to ER function such as those begotten by the accumulation of misfolded and unfolded ER client proteins upset the ER homeostatic balance, leading to a condition termed ER stress. Subsequent sensing of ER stress by three ER transmembrane proteins, initiates an adaptive reaction to alleviate ER stress: this is known as the unfolded protein response (UPR). Divergent cascades of the UPR attempt to mitigate ER stress and restore ER homeostasis: Failing that, the UPR initiates pro-apoptotic pathways. The demand of insulin production on the β cell necessitates the presence of a highly functional ER. However, the consequence of dependence on the ER for insulin synthesis and secretion portends disaster for the functional state of the β cell. Disturbances to the ER that elicit ER stress and UPR activation causes β cell dysfunction and may lead to apoptosis. There are numerous well-characterized models of ER stress-mediated diabetes, including genetic mutations in UPR transducers and insulin. Recently, polymorphisms in Wolfram syndrome 1 (WFS1), an ER transmembrane protein involved in the UPR, were suggested to contribute to T2DM risk. In this thesis, one of the highlighted WFS1 polymorphism, H611R, was examined to identify its contribution to β cell function and viability, and hence, diabetes risk. It was revealed that augmentation of WFS1 expression increased insulin secretion and cellular content. In addition, WFS1 protected β cells against ER stress-mediated dysfunction, with a more pronounced effect in the WFS1-R611 protective allele. Subsequent gene expression analysis identified netrin-1 as a WFS1-induced survival factor. As a contributory factor to diabetes progression, ER stress and UPR are potential drug and biomarker targets. In this dissertation, a novel UPR-regulating microRNA (miRNA) family was uncovered in ER stressed, WFS1-deficient islets. These miRNAs, the miR-29 family, are induced in WFS1 -/- islets as a possible adaptive alteration to chronic ER stress conditions, and indirectly decreases the expression of UPR transducers, while directly targeting downstream ER stress-related pro-apoptotic factors. Collectively, this work extends the function of WFS1 as a protective factor in the pancreatic β cell through the induction of netrin-1 signaling. Additionally, it further strengthens the role of miRNA as regulatory members of the UPR which contribute to cell survival.

Le bypass gastrique (RYGB) a démontré un effet bénéfique sur le diabète de type 2. L’EGA (entéro-gastro-anastomose) est une procédure de bypass gastrique adaptée au modèle murin. Chez les souris ob-ob (déficientes pour la leptine), l’EGA améliore la tolérance au glucose en augmentant le contenu pancréatique en insuline et la sécrétion d’insuline induite par le glucose in vivo en l’absence de perte de poids, de restriction alimentaire, de modification de composition corporelle, sans modifier le nombre ni dans la taille des ilots. L’expression du gène de l’insuline, la prolifération beta cellulaire et l’infiltration immunitaire insulaire sont également inchangés. De plus, la chirurgie bariatrique régule l’expression de 193 gènes et 27 miRs intra-ilots. Certaines anomalies moléculaires observées dans les îlots pancréatiques des souris ob-ob (régulation négative du canal ionique TRPM5, du transporteur GLUT2, de la glucokinase, de la connexine 36) et fonctionnelles (sensibilité élevée des îlots à de faibles taux de glucose) ont été modulées par la chirurgie bariatrique. La chirurgie a favorisé l’enrichissement de 227 processus biologiques, notamment 21 gènes impliqués dans le transport hormonal et 20 gènes impliqués dans la sécrétion hormonale. Sept des 27 miRs (324-3p, 380-3p, 671-5p, 1927, 6904-5p, 6918-5p et 7682-3p) sont des noyaux dans le réseau de prédiction d’interaction gène-miRs. Globalement, nos données ont mis en évidence de nouveaux mécanismes moléculaires dans la résolution du diabète après une chirurgie bariatrique. De manière importante, notre modèle démontre que la résolution du diabète après la chirurgie bariatrique peut être indépendante de la perte de poids
EGA (entero-gastro-anastomosis) is a gastric bypass procedure adapted to rodent. EGA in leptin deficient ob/ob mice improves glucose tolerance by increasing pancreatic insulin content and glucose stimulated insulin secretion in vivo without persistent body weight loss dietary restriction, modification of body composition / energy expenditure. We do not observe differences in islets ‘number or size after EGA. Insulin gene expression, beta-cell proliferation (Ki67 index) and insular immune infiltration are also unchanged. Transcriptomic analysis of pancreatic islets showed that bariatric surgery differentially regulated 193 genes and 27 miRs. Interestingly, the surgery normalized molecular defects (down regulation of TRPM5, GLUT2, GCK, connexin 36) and functional alteration (high sensitivity of islets to low glucose levels) observed in diabetic ob pancreatic islets. In addition, the surgery promoted the enrichment of 227 biological process, composed of genes with known or undetermined beta cell function, especially 21 genes are involved in the hormone transport and 20 genes in the hormone secretion biological process. Computational analysis predicted that 7 of 27 miRs (324-3p, 380-3p, 671-5p, 1927, 6904-5p, 6918-5p and 7682-3p) are hubs in the miRs-gene interaction network. Altogether, our data highlighted novel molecular mechanisms in the resolution of diabetes after bariatric surgery. Overall, diabetes resolution in our model appears to be totally independent of body weight