Orientador: Antonio Carlos Boschiero / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T05:10:32Z (GMT). No. of bitstreams: 1 Ferreira_SandraMara_D.pdf: 2032504 bytes, checksum: da4ca0c7e913b98f9a0ec91b1a14ba82 (MD5) Previous issue date: 2015 / Resumo: A ARHGAP21 é uma proteína da família das RhoGAPs (Proteínas ativadoras de GTPases Rho) que, em diferentes tipos de células, controla múltiplas funções tais como: migração, proliferação, diferenciação e tráfego intracelular de vesículas. Em ilhotas de camundongo Swiss neonato o knockdown da ARHGAP21 aumentou a secreção de insulina estimulada por glicose (GSIS), por mecanismos ainda desconhecidos. Contudo, não se conhece os efeitos da ARHGAP21 sobre a secreção de insulina e sobre a homeostase glicêmica em camundongos adultos. Assim, o objetivo do trabalho foi avaliar: a) em ilhotas pancreáticas de camundongo Swiss neonato knockdown para ARHGAP21, a expressão de genes envolvidos com a proliferação, maturação e extrusão de grânulos de insulina, bem como o rearranjo do citoesqueleto de actina; b) em camundongos C57BL/6 adultos, o efeito do knockdown da ARHGAP21 sobre GSIS e sobre alguns genes que codificam proteínas envolvidas na função secretória (maturação e extrusão) e sobre a homeostase glicêmica. Os neonatos foram tratados (i.p.) com 1 nmol de antisense anti-ARHGAP21 (neonato AS) ou mismatch (CTL) por dois dias (redução da expressão de 60%). Os adultos foram tratados (i.p.) com 1,5 nmol/g de antisense (adulto AS) ou mismatch (adulto CTL) por 3 dias consecutivos (redução de 50%). A secreção de insulina foi avaliada na presença de concentrações crescentes de glicose (2,8 - 22,2 mM). A F-actina (polímero) foi medida através da marcação com faloidina. A expressão gênica foi avaliada por PCR em tempo real. Tolerância à glicose e ao Piruvato foi medida através de ipGTT e ipPTT, respectivamente, e a sensibilidade à insulina através do ipITT, clamp hiperinsulinêmico-euglicêmico e, AKT fosforilada. Como já descrito, ilhotas de neonatos AS apresentaram maior secreção basal de insulina (2,8 mM) bem como menor presença de F-actina. Observamos também maior expressão dos genes da VAMP2 e SNAP25. Ilhotas de adultos AS apresentaram maior secreção de insulina apenas na presença de 22,2 mM. Contudo, o aumento do [Ca2+]i, induzido por glicose, foi similar ao CTL. Observou-se também aumento da expressão dos genes da SYT VII (SYNAPTOTAGMINA VII) e CX 36 (CONEXINA 36). Adultos AS apresentaram intolerância à glicose e aumento da insulinemia durante o ipGTT, acompanhado de resistência à insulina específica no músculo, além de menor produção de glicose pelo fígado. Concluímos que a ARHGAP21 modula negativamente a secreção de insulina em neonato provavelmente através do rearranjo da actina e da redução da expressão de VAMP2 e SNAP25. Em ilhotas isoladas de camundongos adultos, a ARHGAP21 modula negativamente a expressão de genes envolvidos na sensibilidade ao cálcio e resposta secretória (SYT VII e CX 36). Apesar da melhora na secreção de insulina, os adultos AS apresentaram intolerância à glicose com discreta resistência à insulina no músculo que parece ser compensada pela menor liberação de glicose pelo fígado / Abstract: ARHGAP21 is a protein of the RhoGAPs (Rho GTPases activating proteins) family that exerts several functions such as: migration, proliferation, differentiation and intracellular traffic of vesicles, in multiple cell types. In islets from Swiss mice knockdown for ARHGAP21 the glucose-induced insulin secretion (GSIS) was significantly increased, by mechanisms not yet elucidated. Although, it is still unknown the effects of ARHGAP21 knockdown on the insulin secretion and glucose homeostasis in adult mice. Here, we evaluated: a) in islets from ARHGAP21 knockdown mice the expression of genes involved in proliferation, maturation, and extrusion of the insulin containing granules, as well as on the rearrangement of the actin cytoskeleton, and b) the effect of ARHGAP21 knockdown on GSIS and on the expression of genes that encode proteins involved with the secretory function (maturation and extrusion), as well as on the glucose homeostasis in adult C57BL/6 mice. Neonatal Swiss mice received (i.p.) 1 nmol of anti-ARHGAP21 anti-sense (neonate AS) (reduction of 60%) or mismatch (neonate CTL), subcutaneously, for two days. Adult C57BL/6 mice received (i.p.) 1.5 nmol/g of anti-ARHGAP21 anti-sense (adult AS) (50% reduction) or mismatch (adult CTL) for three consecutive days. Insulin secretion was measured in the presence of increasing concentrations of glucose (2.8 ¿ 22.2 mM). F-actin (polymer) was measured using phalloidin. Gene expression was assessed by Real Time PCR. Glucose and Piruvate tolerance were measured by ipGTT and ipPTT, respectively, and insulin sensitivity by ipITT, hyperinsulinemic-euglycemic clamp, and AKT phosphorylation. Islets from neonate AS displayed higher insulin secretion at 2.8 mM glucose, and lower expression of F-actin. Higher expression of VAMP2 and SNAP25 genes was also observed. Islets from adult AS showed higher insulin secretion at 22.2 mM glucose. However, glucose-induced increase in [Ca2+]i was not different from CTL. The expression of SYT VII and CX 36 genes was also increased. Adult AS mice displayed glucose intolerance and higher insulinemia during ipGTT, accompanied by insulin resistance specifically in skeletal muscle, and a lower hepatic glucose production. In conclusion, ARHGAP21 negatively modulates insulin secretion in neonatal mice through the rearrangement of the actin cytoskeleton and reduction of the expression of VAMP2 e SNAP25 genes. ARHGAP21 also may negatively modulate the expression of genes involved in the Ca2+ sensitivity and secretory response (SYT VII and CX 36, respectively) in adult mice. Despite the higher insulin secretion, adult AS mice showed glucose intolerance associated with a mild insulin resistance in the skeletal muscle, which may be compensated by a lower glucose production in the liver / Doutorado / Fisiologia / Doutora em Biologia Funcional e Molecular

No description available.

The dual-specificity protein tyrosine phosphatases (PTPs) play integral roles in the regulation of cell signaling. There is a need for new tools to study these phosphatases, and the identification of inhibitors potentially affords not only new means for their study, but also possible therapeutics for the treatment of diseases caused by their dysregulation. However, the identification of selective inhibitors of the protein phosphatases has proven somewhat difficult. PTP localized to mitochondrion 1 (PTPMT1) is a recently discovered dual-specificity phosphatase that has been implicated in the regulation of insulin secretion. Screening of a commercially available small-molecule library yielded alexidine dihydrochloride, a dibiguanide compound, as an effective and selective inhibitor of PTPMT1 with an in vitro concentration that inhibits response by 50% of 1.08 microM. A related dibiguanide analog, chlorhexidine dihydrochloride, also significantly inhibited PTPMT1, albeit with lower potency, while a monobiguanide analog showed very weak inhibition. Treatment of isolated rat pancreatic islets with alexidine dihydrochloride resulted in a dose-dependent increase in insulin secretion, whereas treatment of a pancreatic beta-cell line with the drug affected the phosphorylation of mitochondrial proteins in a manner similar to genetic inhibition of PTPMT1. Furthermore, knockdown of PTPMT1 in rat islets rendered them insensitive to alexidine dihydrochloride treatment, providing evidence for mechanism-based activity of the inhibitor. Taken together, these studies establish alexidine dihydrochloride as an effective inhibitor of PTPMT1, both in vitro and in cells, and support the notion that PTPMT1 could serve as a pharmacological target in the treatment of type II diabetes. / Dissertation

A secreção de insulina é estimulada pela glicose, porém os ácidos graxos (AG) podem influenciar o processo secretório. A oxidação de AG é importante para a estimulação da secreção de insulina por aumentar o ATP, porém, existem vias dependentes e independentes de ATP. Os AG <font face=\"Symbol\">w-3 interferem em processos fisiológicos e na composição e função da membrana plasmática, promovendo potente ação anti-inflamatória. Considerando a importante relação da NAD(P)H oxidase com a secreção de insulina, o estudo das alterações induzidas pela suplementação com AG <font face=\"Symbol\">w-3 sobre o conteúdo de superóxido (O2<font face=\"Symbol\">&#183;) e a expressão da NAD(P)H oxidase, é importante para a compreensão da fisiologia das células <font face=\"Symbol\">b-pancreáticas. Neste estudo, o grupo suplementado apresentou redução do conteúdo de O2<font face=\"Symbol\">&#183;, redução da expressão das subunidades da NAD(P)H oxidase e aumento na expressão da superóxido dismutase (SOD1 e 2), quando comparado ao grupo controle. Embora desconhecido o mecanismo, este dado é relevante, pois pressupõe melhor regulação do estado redox durante a secreção de insulina. / Insulin secretion is stimulated by glucose (GSIS), but fatty acid (FA) may influence the secretory process. The oxidation of FA is important for the stimulation of insulin secretion by increasing the ATP, although there are dependent and independent ATP pathways. The <font face=\"Symbol\">w-3 FA change physiological processes, and affect the composition and function of the plasma membrane, promote potent anti-inflammatory action. Considering the important relationship of NAD(P)H oxidase with insulin secretion, the study of changes induced by supplementation with <font face=\"Symbol\">w-3 FA on the superoxide (O2<font face=\"Symbol\">&#183;) content, and expression of NAD(P)H oxidase, becomes of great importance for understanding the pancreatic <font face=\"Symbol\">b cells physiology. In this study, the group supplemented with <font face=\"Symbol\">w-3 FA showed a reduction of the O2<font face=\"Symbol\">&#183; content, reduced expression of NAD(P)H oxidase subunits, and increased the expression of the enzyme superoxide dismutase (SOD 1 and 2), compared to control. Although unknown the mechanism, this data is relevant, because it represents better regulation of the redox state during GSIS .

O estresse oxidativo e a enzima NADPH oxidase-2 (NOX2) estão associados com a diminuição da massa funcional de células-&#946; em pacientes com diabetes do tipo 2 (DT2). Neste estudo, testamos o papel da NOX2 sobre a glicotoxicidade em células-&#946;. Ilhotas de camundongo C57BL/6J nocautes ou não para NOX2 (NOX2-KO e WT, respectivamente) foram isoladas e cultivadas por até 3 semanas em 10 ou 30 mmol/l de glucose (G10 e G30, respectivamente). A secreção de insulina foi maior nas ilhotas NOX2-KO vs. WT sem apresentar diferenças metabólicas ou do potencial redox da glutationa citosólica (EGSH). O cultivo de ilhotas em G30 aumenta a concentração de H2O2 e a oxidação de tióis no compartimento citosólico, seguido por aumento de apoptose de células-&#946;, mas, preservando a reposta máxima secretória. Estas respostas foram quase idênticas em ambos os tipos de ilhotas. Em conclusão, a NOX2 regula negativamente a secreção de insulina em ilhotas de camundongos C57BL/6J, mas não é um componente crítico para a sobrevivência de células &#946; em um modelo in vitro de glicotoxicidade. / Oxidative stress and NADPH oxidase-2 (NOX2) enzyme are associated to the decline of the functional &#946;-cell mass in type 2 diabetes (T2D). Here, we tested the role of NOX2 on &#946;-cell glucotoxicity. NOX2 knockout (NOX2 KO) and wild type (WT) C57BL/6J mice islets were isolated and cultured up to 3 weeks at 10 or 30 mmol/l glucose concentrations (G10 and G30, respectively). The insulin secretion was higher in NOX2-KO vs. WT islets despite similar metabolic and cytosolic glutathione-redox potential (EGSH) changes. The prolonged culture at G30 increases the H2O2 concentration and cytosolic thiol oxidation, followed by increased &#946;cell apoptosis but preserving maximal secretory response. These responses were almost identical in both types of islets. In conclusion, NOX2 is a negative regulator of insulin secretion in C57BL/6J mouse islets, but is not a critical component for &#946;-cell survival in a model of glucotoxicity in vitro.

O receptor ativado do peroxissomo &#947; (PPAR-&#947;) é regulador do metabolismo e diferenciação do tecido adiposo, sendo um alvo conhecido das tiazolidinedionas (TZD), utilizadas para o tratamento do diabetes tipo 2 (DM2). As TZD agem como um agente sensibilizador da ação da insulina nos tecidos periféricos e tem sido especulado que as TZDs podem ter um papel na função da célula , prevenindo perda de massa e melhorando a sua viabilidade a longo prazo. Este efeito seria supostamente mediado pela transcrição de genes que favoreceriam a lipólise, diminuindo o conteúdo intracelular de triglicérides e, portanto, diminuindo a lipotoxicidade. Entretanto, alguns estudos também mostraram efeito nulo ou mesmo deletério das TZDs sobre as ilhotas pancreáticas. Na realidade, o papel de genes-alvo para o PPAR- nas ilhotas pancreáticas é ainda pouco conhecido. Estudamos o perfil de expressão gênica induzido pelo tratamento com Pioglitazona (Pio), uma TZD aprovada e disponível para uso clínico no tratamento do DM2, em ilhotas pancreáticas murídeas em cultura primária, com concentrações normal e suprafisiológica de glicose no meio de cultura. As ilhotas foram obtidas de ratos wistar machos de dois meses de idade e isoladas pelo método do gradiente de Ficoll e então cultivadas em 5,6 mM ou 23 mM de glicose por 24h, sendo tratadas com Pio 10 M ou DMSO 0,1% (veículo). A Pioglitazona foi cedida pela Takeda Farmacêutica, Osaka, Japão. O RNA foi extraído com Trizol e purificado com o kit RNeasy (Qiagen). As amostras foram marcadas e hibridizadas no microarranjo de cDNA Mouse Panchip 13k, usando-se cinco replicatas biológicas diferentes para cada condição. A análise estatística dos dados do microarranjo foi feita com o uso do programa significance analysis of microarrays (SAM) com uso de taxa de descobrimento falso (FDR) de 20%. A análise das vias acometidas foi feita com o Ingenuity Pathway Analysis (www.ingenuity.com). Os resultados de expressão gênica foram confirmados por RT-qPCR. Em concentração de 5,6 mM de glicose no meio de cultura, 101 genes foram modulados pela Pio, sendo 49 regulados para cima, com aumento de sua expressão na presença da droga e 52 genes regulados para baixo. Em 23 mM de glicose, 1.235 genes foram afetados, sendo 621 para cima e 623 para baixo. A comparação entre as duas condições revelou 74 genes que foram modulados em ambas as concentrações de glicose. A análise das vias biológicas alteradas mostrou que genes relacionados ao metabolismo de lípides foram modulados em ambas as concentrações de glicose. Em 23 mM foi ainda significativo o grupo de genes relacionados a ciclo celular e morte celular que tiveram sua expressão modificada pela presença da droga na cultura. Este dado demonstrou que além de seus efeitos conhecidos nos adipócitos, o sensibilizador de insulina Pioglitazona modula a expressão de genes nas ilhotas pancreáticas, especialmente na presença de concentrações suprafisiológicas de glicose, afetando notadamente genes relacionados ao metabolismo lipídico, sendo vários deles ligados a lipogênese, como Srebf1, Scd2 e Fabp4 cujas expressões aumentaram em ambas as concentrações de glicose. Além disso foi observado aumento na expressão de genes com atividade pró-apoptótica como Tnf, Bad, Bax, Caspase4, Fadd e Myc. A Pioglitazona parece induzir um perfil gênico desfavorável em ilhotas pancreáticas mantidas em cultura em concentrações suprafisiológicas de glicose. / Peroxisome proliferator-activator receptor-&#947; (PPAR-&#947;) is a target for thiazolidinedione (TZD) antidiabetic drugs and a regulator of adipose tissue differentiation and metabolism. TZD act as an insulin sensitizing agent on peripheral tissues. It has been speculated that TZD could play a role on beta-cell function, preventing loss and improving viability in the long-term. This effect is supposed to be mediated through a potential benefit against lipotoxicity, favouring lypolisis and decreasing intracellular tryglicerides content. Nevertheless some studies also showed a lack or even a potential deleterious effect of TZD on islets. The role of PPAR-&#947; target genes in pancreatic islets is actually still largely unclear. We studied the gene expression profile induced by the treatment with Pioglitazone (Pio), an approved TZD for T2DM therapy, on rat pancreatic islets primary culture both at normal and supraphysiological glucose medium concentrations. Islets were obtained from 2 month-old, male, wistar rats and isolated through the Ficoll gradient method and then cultured with 5.6 mM or 23 mM of glucose concentration for 24h, being treated with Pio 10 µM or DMSO 0.1% (vehicle). Pioglitazone was provided by Takeda Pharmaceuticals, Osaka, Japan. RNA was extracted with Trizol (Sigma) and purified with RNeasy kit (Qiagen). Samples were labeled and then hybridized on the Mouse PanChip 13k cDNA microarray, using 5 different biological replicates for each test condition. Statistical Analysis of the microarray data was performed using significance analysis of microarrays (SAM) with a false discovery rate of 20%. Pathways assessment was performed through Ingenuity Pathway Analysis (www.ingenuity.com). Gene expression results were confirmed through RT-qPCR. At 5.6 mM glucose 101 genes were modulated by Pio, 49 upregulated and 52 downregulated. At 23 mM, 1,235 genes were affected, 612 upregulated and 623 downregulated. Comparison between both conditions revealed 74 genes that were similarly modulated at both glucose concentrations. Pathway analysis of perturbed genes revealed biologically relevant networks related to lipid metabolism at both glucose medium concentrations. At 23 mM, cell cycle and cell death pathways were significant modulated as well. These data demonstrates that in addition to known effect in adipocytes, the insulin sensitizing agent Pioglitazone modulates gene expression in pancreatic islets, especially in the presence of supraphysiological glucose concentrations, affecting especially lipid metabolism and mechanisms of cell death and cell cycle. Considering the ontology of modulated genes it seems to be a trend towards lypogenesis (increased Srebf1, Scd2 and Fabp4 RNA expressions) with Pio treatment also enhancing the abundance of some genes considered to be pro apoptotic like Tnf, Bad, Bax, Caspase4, Fadd and Myc. Pioglitazone seems to induce a negative gene expression profile in islets cultured at high glucose concentrations.

Poon, Chui Wa Christina. / "August 2011." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 123-131). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 論文摘要 --- p.vi / ACKNOWLEDGEMENTS --- p.ix / PUBLICATIONS --- p.x / Abstracts --- p.x / ABBREVIATIONS --- p.xii / Chapter 1. --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1. --- Diabetes --- p.1 / Chapter 1.1.1. --- Overview --- p.1 / Chapter 1.1.2. --- Diagnostic Criteria of Type-2 Diabetes --- p.2 / Chapter 1.1.3. --- Type-2 Diabetes (T2DM) --- p.3 / Chapter 1.1.3.1. --- Impaired Insulin Synthesis and Insulin Secretory Defects in Type-2 Diabetes --- p.3 / Chapter 1.1.3.2. --- β-Cell Dysfunction --- p.5 / Chapter 1.1.3.3. --- Insulin Resistance --- p.5 / Chapter 1.1.4. --- Glucose Toxicity --- p.6 / Chapter 1.1.4.1. --- Fasting Hyperglycemia --- p.8 / Chapter 1.1.4.2. --- Postprandial Hyperglycemia --- p.8 / Chapter 1.2. --- Oxidative Stress --- p.8 / Chapter 1.2.1. --- ROS and Mitochondria --- p.8 / Chapter 1.2.2. --- ROS Production by Mitochondria --- p.9 / Chapter 1.2.3. --- The Relationship of Glucose Recognition by β-cells and Oxidative Stress --- p.11 / Chapter 1.2.4. --- Important Roles of Glutathione in Pancreatic β-cells and Glutathione Synthesis --- p.14 / Chapter 1.2.5. --- N-acetyl-L-cysteine - A Potential Drug Treatment for Type-2 Diabetes? --- p.17 / Chapter 1.3. --- Role of F-actin Cytoskeleton on Glucose-induced Insulin Secretion --- p.18 / Chapter 1.4. --- Current Clinical Treatments for Type-2 Diabetes Mellitus --- p.21 / Chapter 1.4.1. --- Metformin --- p.22 / Chapter 1.4.2. --- Sulfonylureas --- p.22 / Chapter 1.4.3. --- Thiazolidinediones --- p.23 / Chapter 1.4.4. --- Glinides (Meglitinide Analogues) --- p.23 / Chapter 1.4.5. --- α-Glucosidase (AG) Inhibitors --- p.24 / Chapter 1.4.6. --- Dipeptidyl Peptidase-4 (DPP-4) Inhibitors --- p.24 / Chapter 1.4.7. --- (Clinical) Antioxidant Treatment --- p.24 / Chapter 1.5. --- Animal Models Used in Type-2 Diabetes Research --- p.25 / Chapter 1.6. --- Aims of Study --- p.27 / Chapter 2. --- RESEARCH DESIGN & METHODS --- p.28 / Chapter 2.1. --- Materials --- p.28 / Table 1. Sources and concentrations of drugs tested in this study: --- p.28 / Culture Medium - --- p.29 / General Reagents --- p.29 / Chapter 2.2. --- Isolation of Islets of Langerhans and Single Pancreatic β-Cells --- p.31 / Chapter 2.3. --- Measurement of Mitochondrial ROS Levels --- p.32 / Chapter 2.4. --- Measurement of Islets Insulin Release and Insulin Content --- p.34 / Chapter 2.4.1. --- Preparation of Samples --- p.34 / Chapter 2.4.2. --- Enzyme-Link Immunosorbent Assay (ELISA) --- p.35 / Chapter 2.5. --- Immunocytochemistry --- p.35 / Chapter 2.6. --- Data and Statistical Analysis --- p.37 / Chapter 3. --- RESULTS --- p.38 / Chapter 3.1. --- "Effects of L-NAC, Various Oxidative Stress Inducers/Reducers and Actin Polymerisation/Depolymerisation Inducers on Releasable Insulin Levels and Insulin Contents in Response to Low Glucose (5 mM) and High Glucose (15 mM) of Isolated Pancreatic Islets of (db+/m+) and (db+/db+) Mice" --- p.38 / Chapter 3.1.1. --- Effect of L-NAC on Insulin Secretion and Insulin Contents --- p.38 / Chapter 3.1.2. --- Effect of Cytochalasin B on Insulin Secretion and Insulin Contents --- p.39 / Chapter 3.1.3. --- Effect of 4-Phenyl Butyric Acid on Insulin Secretion and Insulin Contents --- p.43 / Chapter 3.1.4. --- Effect of Ursodeoxycholic Acid on Insulin Secretion and Insulin Contents --- p.46 / Chapter 3.1.5. --- Effect of Hydrogen Peroxide on Insulin Secretion and Insulin Contents --- p.49 / Chapter 3.1.6. --- Effect of Jasplakinolide on Insulin Secretion and Insulin Contents --- p.53 / Chapter 3.1.7. --- Effect of Thapsigargin on Insulin Secretion and Insulin Contents --- p.57 / Chapter 3.1.8. --- Effect of BSO on Insulin Secretion and Insulin Contents --- p.61 / Chapter 3.2. --- "Effects of L-NAC, Various Oxidative Stress Inducers/Reducers and Actin Polymerisation/Depolymerisation Inducers on Mitochondrial ROS Levels in Response to High Glucose (15 mM) Challenge in Isolated Single Pancreatic β-Cells of (db +/m+) and (db +/db +) Mice" --- p.65 / Chapter 3.2.1. --- "Effects of L-NAC (20 mM), 4-Phenyl Butyric Acid (4-PBA) (1 mM), Ursodeoxycholic Acid (UA) (500 μg/ml), H202 (200 μM), Thapsigargin (0.5 μM) and DL-Buthionine-[S,R]-Sulfoximine (BSO) (0.1 μM) Pre-treatments on Mitochondrial ROS Level in Response to High Glucose (15 mM) Challenge" --- p.65 / Chapter 3.2.2. --- "Effects of L-NAC (20 mM), Cytochalasin B (10 μM) and Jasplakinolide (5 μM) Pre-treatments on Mitochondrial ROS Level in Response to High Glucose (15 mM) Challenge_" --- p.76 / Chapter 3.3. --- "Effects of L-NAC, Various Oxidative Stress Inducers/Reducers and Actin Polymerisation/Depolymerisation Inducers on F-actin Cytoskeleton Levels Incubated in Low Glucose (5 mM) and High Glucose (15 mM) Medium in Single Pancreatic β-Cells of Non-Diabetic (db +/m+) and Diabetic (db +/db +) Mice" --- p.81 / Chapter 4. --- DISCUSSION --- p.100 / Chapter 4.1. --- General Discussion --- p.100 / Chapter 5. --- SUMMARY --- p.120 / Chapter 6. --- FUTURE PERSPECTIVES --- p.121 / Chapter 7. --- REFERENCES --- p.123

In the first study, I aimed to compare the angiotensin II type 1 receptor (AT1R) expression levels of the isolated pancreatic islets from normal and mouse model of T2DM. In addition, 4-week-old diabetic mice were orally treated with AT1R antagonist losartan for 8 weeks. It is found that AT1R mRNA was upregulated markedly in diabetic islets and double-immunolabeling confirmed that AT1R was localized to beta-cells. Losartan selectively improved glucose-induced insulin release and (pro)insulin biosynthesis in diabetic islets. Oral losartan treatment delayed the onset of diabetes, and reduced hyperglycemia and glucose intolerance in diabetic mice. These data indicate that AT1R antagonism improves beta-cell function and glucose tolerance in young T2DM mice. / In the second study, I aimed to examine how the upregulated RAS could impair beta-cell function, where oxidative stress is the potential mediator. Meanwhile, T2DM results in oxidative stress-mediated activation of uncoupling protein 2 (UCP2), a negative regulator of islet function. Thus, it was postulated that some of the protective effects of AT1R antagonism might be mediated through interference with this pathway and tested this hypothesis in a mouse model of T2DM. In order to achieve this, losartan was given to 4-week-old diabetic mice for 8 weeks. UCP2-driven oxidative damage and apoptosis were analyzed in isolated islets. Results showed that losartan selectively inhibited oxidative stress via NADPH oxidase downregulation; this in turn suppressed UCP2 expression, thus improving beta-cell insulin secretion while decreasing apoptosis-induced beta-cell mass loss in diabetic mice islets. These data indicate that islet AT1R activation in young diabetic mice can lead to progressive islet beta-cell failure through UCP2-driven oxidative damage and apoptosis. / The mechanisms by which chronic hyperglycemia associated with glucotoxicity causes beta-cell dysfunction and apoptosis remain ambiguous. Voltage-gated outward potassium (Kv) current, which mediates beta-cell membrane potential and limits insulin secretion, could play a role in glucotoxicity. Meanwhile the RAS has been shown to be upregulated by prolonged exposure to high glucose. In the third part of my study, I therefore investigated the effects of prolonged exposure to high glucose and angiotensin II (Ang II) on the expression and activity of Kv channels in mouse pancreatic beta-cell. Dissociated mice beta-cells, incubated in 5.6 mM or 28 mM glucose for 3-5 days, were used for electrophysiological study; while isolated islets cultured for 1-7 days were proceeded for gene/protein expression analysis. Both Kv channel expression and current were markedly increased by prolonged glucose incubation. Simultaneously, Ang II reduced Kv current under normal glucose condition, while high glucose incubation abolished the effect of Ang II. Moreover, the ability of Ang II on Kv current reduction was eliminated by inhibiting AT2R but not AT1R. These data indicated that Ang II reduced Kv current via AT2R, which was abolished by prolonged high glucose incubation. On the other hand, high glucose increased Kv channel expression and current, which might alter the ability of insulin secretion in beta-cell. (Abstract shortened by UMI.) / Chu, Kwan Yi. / Adviser: P. S. Leung. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3246. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 163-188). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

BACKGROUND AND OBJECTIVES: Type 2 diabetes mellitus (T2DM) is a complex disease with genetic predisposition and histopathological characterization. Pancreatic islet amyloidosis, hyaline arteriolosclerosis, and diabetic glomerulopathy are histopathological hallmarks of T2DM at autopsy examination. The associations of genetic variants with diabetic amyloidosis, arteriosclerosis and glomerulopathy have not been fully elucidated. Several candidate genes including apolipoprotein E (ApoE), insulin degrading-enzyme (IDE) and glucose transporter-1 ( GLUT1) have been reported to increase risk of T2DM in human studies although results are not always consistent. Capitalizing on the pathological hallmarks of T2DM, I used autopsy specimens to investigate the risk associations of polymorphisms of ApoE (rs429358 and rs7412), IDE (rs6583813) and GLUT1 (rs710218) genes with clinical features and specific pathological changes in diabetic kidney and pancreas. I further explored the mechanisms of these associations by evaluating the histopathological changes and protein expression in pancreas and kidney. / CONCLUSIONS: These findings suggest that genetic factors have important effects in the development of tissue-specific changes and chronic complications in T2DM. Islet amyloidosis, arteriosclerosis and glomerulosclerosis in T2DM may share common pathogenetic processes as suggested by the coexistence of chaperone proteins, amyloid P and ApoE. Genetic--pathologic correlation studies are useful in advancing our understanding of the mechanisms of complex diseases such as T2DM. / METHODS AND MATERIALS: Genomic DNA was extracted from white blood cell-concentrated paraffin embedded formalin fixed spleen tissues. Genotyping for ApoE, IDE and GLUT1 polymorphisms was determined by polymerase chain reaction (PCR) and ligase detection reaction (LDR). The pathological changes were blindly assessed in pancreatic and kidney tissues of autopsy specimens. Protein expression of these genes was examined by immunostaining and quantified by using Metamorph image analysis system. / RESULTS: In a consecutive study population of 3693 autopsy specimens containing 328 T2DM and 209 control cases, the respective frequencies of genotypes were as follows: 1) TT of GLUT1 rs710218: 11.2% vs. 11.3%; 2) ApoE epsilon2: 19.4% vs. 10.9%; 3) ApoE epsilon4: 12.1% vs. 9.1% and 4) C carriers of IDE rs6583813: 51.2% vs. 47.9%. The key genotype-phenotype correlations were as follows. 1) In the T2DM cases, GLUT1 rs710218 IT genotype carriers (0% in TT genotype vs. 59.1% in AA genotype, P=0.0407) were less likely but ApoE epsilon 2 allele carriers (57.1% in epsilon2 allele carriers vs. 23.5% in epsilon3 allele carriers P=0.0382) were more likely to have diabetic glomerular hypertrophy than referential group. ApoE epsilon2 carriers showed increased glomerular ApoE protein expression with the immunoreactivity found mainly in the mesangial regions and nodular lesions. On the other hand, ApoE epsilon 3/epsilon4 cases had diffuse ApoE expression in glomerular capillaries. 2) ApoE epsilon4 carriers were more likely to have islet amyloidosis than non-carriers (62.5% in epsilon4 allele carriers vs. 23.6% in epsilon 3 allele carriers P=0.0232). There was immunolocalization of the chaperone proteins, amyloid P and ApoE in both islet amyloid deposits and arterial walls with hyaline arteriolosclerosis. 3) In T2DM cases, IDE rs6583813 C allele carriers had higher prevalence of vascular disorders [hypertension (67.4% vs. 43.6%, P=0.0332), death due to cardiovascular disease (58.1% vs. 25.6%, P=0.0479) and cerebral vascular accident (CVA) (20.9% vs. 2.4%, P=0.0412)1 than T allele carriers. / Guan, Jing. / Adviser: Chan Chung Ngor Juliana. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 175-192). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Les cellules β des îlots de Langerhans jouent un rôle central dans l’homéostasie glucidique car elles seules sécrètent l’insuline, unique hormone hypoglycémiante de l’organisme. La cellule β est un détecteur du glucose qui couple sa réponse sécrétoire et son expression génique aux niveaux ambiants de glucose. Le couplage entre le métabolisme du glucose et l’exocytose des granules d’insuline implique la génération d’une activité électrique. Son étude est importante pour déchiffrer la façon dont la cellule β encode la demande en insuline de l’organisme. Afin de contourner les limites des approches électrophysiologiques classiques incompatibles avec les études à long-terme, les enregistrements extracellulaires par matrice de microélectrodes (MEA) ont été mis en place.L’objectif de ma thèse était de mieux comprendre les signaux complexes enregistrés par MEAs. Cette étude a révélé l’existence d’une nouvelle signature électrique des cellules des îlots, les slow potentials (SP), qui reflète la fonction de couplage des cellules β. Les SP jouent un rôle important dans l’homéostasie du glucose et représentent un biomarqueur de la fonction normale des îlots. La réponse en hystérèse des îlots au glucose suggère l’existence d’un algorithme d’encodage de la demande en insuline intégrée au niveau du micro-organe. De plus, ce nouveau signal a été exploité pour le phénotypage d’îlots de souris invalidées pour le gène GluK2, que nous avons utilisées comme modèle d’interaction entre les cellules α et β. La caractérisation de ce nouveau type de signal constitue aussi une avancée importante pour le développement d’un biocapteur destiné à être intégré dans le futur à un pancréas artificiel. / Pancreatic β cells are central to glucose homeostasis because they are the only cell that secretes insulin, the sole hypoglycemic hormone in the organism. The β cell is a glucose sensor that regulates its secretory response and gene expression according to ambient glucose levels. The coupling between glucose metabolism and insulin granule exocytosis involves the generation of electrical activity. An investigation of this activity is important to decipher how β cells encode the organism’s insulin demand. In order to overcome the limits of classically used electrophysiological approaches that are not compatible with long-term studies, extracellular recordings using multielectrode arrays (MEA) have been set-up.My thesis aim was to better understand the complex signals recorded with MEA. This study revealed the existence of a new electrical signature of islet cells: slow potentials (SP) that reflect the coupling function of β cells. SP play an important role in glucose homeostasis and represent a biomarker of normal functioning of islets. The observed hysteretic response of islets to glucose suggests the existence of an algorithm encoding the insulin demand embedded at the microorgan level. Moreover, this new signal was used for the phenotyping of GluK2 deficient mouse islets that were employed as an α-to-β cell interaction model. The characterization of this new signal is an important progress in the development of a biosensor intended to be integrated in an artificial pancreas in the future.