R-spondin-1 (Rspo1) is an intestinal growth factor known to exert its effects through activation of the canonical Wnt (cWnt) pathway, but its function in the β-cell had not been explored. In Chapter 2, Rspo1 mRNA was found to be expressed in murine islets and the murine MIN6 and βTC β-cell lines, and Rspo1 protein was detected in MIN6 β-cells. Rspo1 activated cWnt signaling and induced insulin mRNA expression in MIN6 β-cells. Analysis of MIN6 and mouse β-cell proliferation revealed that Rspo1 stimulated cell growth and significantly abolished cytokine-induced cellular apoptosis. Rspo1 also stimulated insulin secretion in a glucose-independent fashion. Chapter 2 further demonstrated that the glucagon-like peptide-1 receptor agonist, exendin-4 (EX4), stimulated Rspo1 mRNA transcript levels in MIN6 cells in a glucose-, time-, dose- and PI3-kinase-dependent fashion. Together, these studies demonstrate that Rspo1 is a novel β-cell growth factor and insulin secretagogue that is regulated by EX4. In Chapter 3, the role of Rspo1 in β-cells in vivo was explored using Rspo1 knock-out (Rspo1-/-) mice. Rspo1-/- mice had normal fasting glycemia but an improved glycemic control after an oral glucose challenge compared to Rspo1+/+ mice, with no difference in insulin sensitivity but an enhanced insulin response over 30 min; glucagon responses were normal. Rspo1 deficiency also resulted in an increase in β-cell mass in association with an increase in Ki67-positive β-cells, a marker of proliferation, relative to Rspo1+/+ mice. Rspo1-/- pancreatic tissues also demonstrated a significant increase in the number of insulin-positive ductal cells, suggestive of β-cell neogenesis. Rspo1-/- islets displayed no changes in glucose-induced insulin secretion but showed a complete absence of glucose-induced suppression glucagon secretion. Treatment of Rspo1-/- mice for 2 wk with EX4 resulted in a similar glycemic profile to EX4-treated Rspo1+/+ mice after an oral glucose challenge, with no changes in insulin sensitivity. Interestingly, EX4 administration to Rspo1-/- normalized β-cell mass to a level comparable to that in Rspo1+/+ mice. Although further studies are required, the findings in this thesis reveal a novel role for Rspo1 as a regulator of β-cell behaviour in vivo, and suggest novel roles for Rspo1 in both a- and ductal-cells.

R-spondin-1 (Rspo1) is an intestinal growth factor known to exert its effects through activation of the canonical Wnt (cWnt) pathway, but its function in the β-cell had not been explored. In Chapter 2, Rspo1 mRNA was found to be expressed in murine islets and the murine MIN6 and βTC β-cell lines, and Rspo1 protein was detected in MIN6 β-cells. Rspo1 activated cWnt signaling and induced insulin mRNA expression in MIN6 β-cells. Analysis of MIN6 and mouse β-cell proliferation revealed that Rspo1 stimulated cell growth and significantly abolished cytokine-induced cellular apoptosis. Rspo1 also stimulated insulin secretion in a glucose-independent fashion. Chapter 2 further demonstrated that the glucagon-like peptide-1 receptor agonist, exendin-4 (EX4), stimulated Rspo1 mRNA transcript levels in MIN6 cells in a glucose-, time-, dose- and PI3-kinase-dependent fashion. Together, these studies demonstrate that Rspo1 is a novel β-cell growth factor and insulin secretagogue that is regulated by EX4. In Chapter 3, the role of Rspo1 in β-cells in vivo was explored using Rspo1 knock-out (Rspo1-/-) mice. Rspo1-/- mice had normal fasting glycemia but an improved glycemic control after an oral glucose challenge compared to Rspo1+/+ mice, with no difference in insulin sensitivity but an enhanced insulin response over 30 min; glucagon responses were normal. Rspo1 deficiency also resulted in an increase in β-cell mass in association with an increase in Ki67-positive β-cells, a marker of proliferation, relative to Rspo1+/+ mice. Rspo1-/- pancreatic tissues also demonstrated a significant increase in the number of insulin-positive ductal cells, suggestive of β-cell neogenesis. Rspo1-/- islets displayed no changes in glucose-induced insulin secretion but showed a complete absence of glucose-induced suppression glucagon secretion. Treatment of Rspo1-/- mice for 2 wk with EX4 resulted in a similar glycemic profile to EX4-treated Rspo1+/+ mice after an oral glucose challenge, with no changes in insulin sensitivity. Interestingly, EX4 administration to Rspo1-/- normalized β-cell mass to a level comparable to that in Rspo1+/+ mice. Although further studies are required, the findings in this thesis reveal a novel role for Rspo1 as a regulator of β-cell behaviour in vivo, and suggest novel roles for Rspo1 in both a- and ductal-cells.

Pancreatic β-cells secrete insulin in response to glucose. Intracellular glucose metabolism drives a cascade of events, including ATP production, calcium influx, and insulin processing, culminating in insulin granule exocytosis. However, insulin secretory mechanisms are incompletely understood. β-cells have the capacity to flow pyruvate into the TCA cycle via the anaplerotic enzyme pyruvate carboxylase to engage one of several pathways of pyruvate recycling. Previous work demonstrated that pyruvate cycling was correlated with insulin secretion, and that NADPH may be involved in granule exocytosis. We hypothesized that NADPH-producing cytosolic enzymes isocitrate dehydrogenase (ICDc) and malic enzyme (MEc) may be involved in both pyruvate cycling and insulin secretion. ICDc expression was reduced using siRNA in the INS-1 derived cell line 832/13 and in isolated rat islets, which led decreased glucose-stimulated insulin secretion (GSIS), pyruvate cycling, and NADPH. Organic acid profiling revealed that decreased pyruvate cycling was compensated by an increase in lactate and stable pyruvate levels. This work established an important role for ICDc in maintaining GSIS through pyruvate-isocitrate cycling. MEc expression was reduced using siRNA in two β-cell lines, 832/13 and 832/3, as well as isolated rat islets. MEc suppression inhibited GSIS in the 832/13 cells only, and these effects were not due to changes in pyruvate cycling, NADPH, or the organic acid profile. This suggests that in normal β-cells, MEc does not participate in pyruvate cycling. Acetyl CoA carboxylase 1 (ACC1) is essential in de novo lipogenesis, which has been implicated in GSIS by other laboratories. Chronic, but not acute, inhibition of ACC1 via siRNA reduced insulin secretion independent of lipogenesis. ACC1 siRNA decreased glucose oxidation, pyruvate cycling, and ATP:ADP, due to an unexpected decrease in glucokinase protein. This work questions the use of ACC inhibitors in obesity and diabetes therapy. In summary, these studies on ICDc, MEc, and ACC1, coupled with concurrent work in our laboratory, eliminate two potential pyruvate cycling pathways (pyruvate-malate and pyruvate-citrate) and establish that pyruvate-isocitrate cycling is the critical pathway for control of GSIS. Future work will focus on identifying the signaling intermediate generated in the pyruvate-isocitrate pathway that links to insulin granule exocytosis. / Dissertation

Glucose metabolism in pancreatic α- and β-cells is believed to regulate secretion of glucagon and insulin, respectively. In β-cells, ATP links glucose metabolism to electrical activity and insulin secretion. In α-cells, ATP has been attributed various roles in glucose-regulated glucagon release, but the underlying mechanisms are poorly understood. Despite its importance in insulin and glucagon secretion little is known about ATP kinetics in α- and β-cells. In this thesis, the novel fluorescent ATP biosensor Perceval was used to monitor physiologically relevant ATP concentrations with little influence of ADP. Glucose stimulation of β-cells within mouse and human pancreatic islets induced pronounced rise of ATP with superimposed oscillations. Simultaneous measurements of the sub-plasma membrane ATP and Ca2+ concentrations revealed glucose-induced oscillations in opposite phase. ATP increased further and the oscillations ceased when voltage-dependent Ca2+ influx was prevented. In contrast, ATP promptly decreased in response to K+-depolarization-induced elevation of Ca2+. Also mobilization of Ca2+ from intracellular stores lowered ATP, but the negative effect was not due to increased ATP consumption by the sarco/endoplasmic reticulum Ca2+-ATPase. Store-operated Ca2+ entry alone had little effect but markedly elevated ATP when combined with muscarinic receptor activation. When comparing ATP and Ca2+ responses in α- and β-cells within the same islet, glucose-induced ATP generation was much less pronounced and the dose-response relationship left-shifted in the α-cells. At basal glucose, individual α-cells showed Ca2+ and concomitant ATP oscillations in opposite-phase with variable frequency. These oscillations largely cancelled out when averaging data from several α-cells. At high glucose, the Ca2+ and ATP oscillations in α-cells tended to synchronize with the corresponding β-cell oscillations. Since β-cell Ca2+ oscillations drive pulsatile insulin secretion, which is antiparallel to pulsatile glucagon secretion, there seems to be an inverse relationship between changes in α-cell Ca2+ and glucagon release. This paradox is attributed to paracrine inhibition overriding Ca2+ stimulation, since somatostatin receptor blockade potently stimulated glucagon release with little effect on α-cell Ca2+ signalling. The data indicate that complex ATP-Ca2+ interactions in α- and β-cells underlie cell-intrinsic regulation of glucagon and insulin secretion and that paracrine inhibition of glucagon release becomes important in hyperglycaemia.

The study of diabetes mellitus is vital in this day and age because its incidence is increasing at an alarming rate. Diabetes results in the loss of function of beta-cells within the pancreas. Insulin resistance contributes to diabetes but the human body can compensate in various ways such as increasing the islet cell mass, glucose disposal and insulin secretion, in order to prevent the onset of diabetes. Growth hormone (GH) and insulin-like growth factor-I (IGF-I) are two integral hormones important in both glucose homeostasis and islet cell growth. Early studies using cultured islet cells have demonstrated positive regulation of beta-cell growth by both GH and IGF-I. To evaluate their relevance on normal beta-cell growth, compensatory growth, as well as in insulin responsiveness, we have used two mouse models that represent opposite manipulations of the GH/IGF-I axis. Specifically, the growth hormone receptor gene deficient (GHR-/-) and the IGF-I overexpression (MT-IGF) mice, to help understand the role of glucose homeostasis and islet cell growth in the GH/IGF-I axis. GH is essential for somatic growth and development as well as maintaining metabolic homeostasis. It is known that GH stimulates normal islet cell growth. Moreover, GH may also participate in islet cell overgrowth and compensate for insulin resistance induced by obesity. To determine whether the islet cell overgrowth is dependent on GH signaling, we studied the response of GHR-/- mice to high-fat diet (HFD)-induced obesity. We also studied the insulin responsiveness in GHR-/- mice. On the other hand, IGF-I promotes embryonic development, postnatal growth and the maturation of various organ systems. The notion that IGF-I stimulates islet cell growth has been challenged in recent years by results from IGF-I and receptor gene targeted models. We have characterized MT-IGF mice which overexpress the IGF-I gene. / The results of our studies indicate that (1) GH is essential for normal islet cell growth, but not required for compensatory overgrowth of the islets in response to obesity, (2) GHR gene deficiency caused delayed insulin responsiveness in skeletal muscle; in contrast to elevated insulin sensitivity in the liver; (3) although overexpression does not stimulate islet cell growth, a chronic IGF-I elevation caused significant hypoglycemia, hypoinsulinemia, and improved glucose tolerance, (4) finally IGF-I overexpression mice are resistant to experimental diabetes.

The development of microarray technology has provided an unprecedented wealth of data on gene expression in various tissue and cell types. Few studies have, however, taken full advantage of these data by selecting and then extensively characterising the functions of particular genes chosen from these microarray datasets. In this study, after analysing differentially-regulated genes revealed by microarray analysis of human mast cells activated via Fc??RI cross-linking, we chose two promising gene candidates for further research, A20 and Gem. Our group??s extensive gene expression database of major leukocytes showed that both A20 and Gem were up-regulated in other leukocyte types, and yet neither of these genes has been extensively explored in mast cells or in the immune system prior to our study. In order to investigate the first of these genes selected for further study, A20, we utilised both A20-deficient mast cells and mast cells in which A20 was over-expressed. Our findings establish for the first time that A20 is an important regulator of mast cell inflammatory responses to both LPS and Fc??RI cross-linking, and that it plays a novel role in mast cell proliferation. Our study of the second gene chosen for investigation, Gem, was conducted in a Gemdeficient mouse model developed by our group. In this study, we investigated the effect of Gem deficiency in two key immune cell types, macrophages and T-cells, complementing the work of a previous group member who investigated Gem deficiency in mast cells. Our results clearly exclude a role for Gem in macrophage and T-cell effector responses, and further establish that Gem is dispensable for in vivo inflammatory responses in models of delayed-type hypersensitivity and allergic airway inflammation. In addition to these findings, and given that the physiological role of Gem was not yet understood prior to our study, we extended our investigation to explore a potential function for Gem in the metabolic system. Using Gem-deficient mice, we found that Gem is necessary for insulin secretion from pancreatic islets. These findings confirm the potential for microarray expression data to reveal excellent gene candidates for further research and functional characterisation.

Type 2 diabetes accounts for 85-90% of all people with diabetes and is currently estimated to affect more than 180 million people worldwide, a figure estimated to double by the year 2030. Thus understanding the basic biology of glucose homeostasis and how it is altered during disease progression is crucial to the development of safe and effective treatment regimes. The link between high dietary fat and the development of type Il diabetes is well established. Chronic treatment of pancreatic islets with the lipid palmitate induces defects in glucose stimulated insulin secretion (GSIS) akin to those seen in the development of type Il diabetes. Previous studies from our group have identified the lipid-activated kinase protein kinase C epsilon (PKCε) as a potential mediator of some of these effects. Deletion of PKCε in mice results in complete protection from high-fat diet induced glucose intolerance. This protection is associated with enhanced circulating insulin suggesting that PKCε may be involved in the regulation of insulin release from the pancreatic β-Cell. The data presented here suggests that PKCs plays an important role in the regulation of insulin secretion under both physiological and pathophysiological conditions. We demonstrate that PKCε can be activated by chronic lipid treatment and acute cholinergic stimulation. Under these conditions insulin secretion is enhanced by PKCε deletion or inhibition suggesting that PKCε is a negative regulator of insulin secretion. Mechanistically the PKCs mediated inhibition of insulin release by acute or chronic PKCε activation appears to be distinct. The effect of PKCε induced by palmitate pre-treatment appears to be distal to calcium influx. The pool of pre-docked vesicles is enhanced in palmitate pre-treated β-cells lacking PKCε suggesting that PKCε may be involved in the regulation of vesicle dynamics. In contrast, calcium dynamics induced by cholinergic stimulation are altered by PKCε deletion, suggesting an effect on either the calcium channels themselves or on the upstream signalling. Given the ability of PKCε to inhibit insulin secretion, inhibition of PKCε in the β-cells of people suffering from insulin resistance and (or) type II diabetes represents a novel target for the treatment of type II diabetes.

A via de amplificação da secreção de insulina estimulada por glicose (GSIS) é um fenômeno discutido na literatura, cujos componentes são amplamente debatidos. Evidências sugerem que a condutância a Cl- compõe parte desta via. Porém, o mecanismo pelo qual essa condutância desempenharia papel na via de amplificação ainda é debatido, e, além disso, as ferramentas farmacológicas para estudo dessas afeta o transporte de outros ânions, como bicarbonato (HCO3-). Buscamos neste trabalho compreender a contribuição do transporte desses ânions para a via de amplificação da GSIS levando em consideração a distribuição de Cl- e HCO3- extracelular em células INS-1E. Concluímos que o transporte de ânions nas células INS-1E não contribui para a via de amplificação da GSIS, porém essas células não expressaram os canais CFTR e Anoctamina 1 que foram relacionados com esse fenômeno. Acreditamos que em células secretoras de insulina que expressem esses canais, o transporte de ânions possua alguma relevância funcional. / The amplification pathway of glucose stimulated insulin secretion (GSIS) is a phenomenon discussed in the literature, which components are broadly debated. Evidence suggests that Cl- conductance composes part of this pathway. However, the mechanism that this conductance would play role on the amplification pathway still is debated, and, besides that, the pharmacological tools to study these affects transport of other anions, such as bicarbonate (HCO3-). We aimed in this study to understand the contribuition of anion transport for the amplification of GSIS considering the Cl- and HCO3- extracellular distribuition in INS-1E cells. We concluded that anion transport in INS-1E cell line do not contribute for the amplification pathway of GSIS, however those cells do not express CFTR and Anoctamin 1 channels which were related with this phenomenon. We believe that in insulin secretin cells that express those channels, the anion transport may have a functional relevance.

Orientador: Jose Roberto Bosqueiro / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-06T10:10:15Z (GMT). No. of bitstreams: 1 Roma_LeticiaPrates_M.pdf: 861159 bytes, checksum: 232359e1de6e654ce6bb13758ca58bd2 (MD5) Previous issue date: 2006 / Resumo: As células B pancreáticas possuem controle multifatorial que permite a secreção de insulina em quantidade e tempo adequados. Os glicocorticóides modulam a secreção de insulina dependendo do tempo e dose em que forem utilzados. Assim,o presente trabalho teve por objetivo analisar as alterações na expressão gênica e protéica de ilhotas de ratos tratados com dexametasona (1mg/kg, 5dias). Utilizando a técnica de cDNA Macroarray observamos que dos 1176 genes presentes na membrana, 66 tiveram sua expressão aumentada e 38 genes tiveram sua expressão diminuída. Os genes com expressão aumentada pertencem às vias de estresse celular (JNK1), inibidores do ciclo celular (p21) e vias de apoptose (Baxa e Fas). Os genes com expressão diminuída pertencem ao ciclo celular (ciclinas D1 e D2, CDK4) e vias de sinalização PI3K, AKT1 e P70. Demonstramos também aumento na expressão protéica da Bax a, redução na expressão da proteína anti-apoptótica Bcl-2, PI3K e P70. Animais tratados com dexametasona por 5 dias possuem níveis plasmáticos aumentados de insulina, triglicérides e ácidos graxos livres. Ilhotas isoladas de animais tratados com dexametasona por 5 e 10 dias apresentaram maior secreção de insulina em relação aos controles, em concentrações basais e estimulatórias de glicose e 40mM de potássio. Porém, o tratamento por 10 dias com dexametasona induziu diminuição na secreção de insulina quando comparado aos animais tratados por 5 dias. Nossos dados sugerem o tratamento com dexametasona pode modular (direta ou indiretamente) a expressão de diversos genes e proteínas envolvidas na apoptose e sobrevivência de células na ilhota pancreática. Essa modulação pode, em longo prazo, se refletir na secreção de insulina como visto nos animais tratados por 10 dias / Abstract: Insulin secretion from pancreatic B-cells is regulated by nutrients like glucose and amino acids and by neurotransmitters, and hormones. Since glucocorticoids modulate insulin secretion we investigated the effects of dexamethasone on gene and protein expression in pancreatic islets from rats treated with the glucocorticoid for 5 days (mg/kg/day). Using cDNA array analysis we found that, out of 1176 genes presented in the array, 66 were up-regulated and 38 down-regulated after dexamethasone treatment. RT-PCR confirmed the macroarray results for 4 genes whereas the expression of these transcripts was confirmed by Western blotting for the corresponding proteins. Many of the up-regulated genes are implicated in apoptosis (Bax a, Fas), cell cycle regulation (p21) and stress response (JNK1) whereas many of the down-regulated were involved in cell cycle progression (cyclins D1 and D2, and CDK4), and survival and proliferations pathways (PI3K, AKT, P70). The protein expression of Bax a was increased whereas Bcl-2, PI3K and P70 repressed. The rats treated with dexamethasone for 5 days showed higher insulin, triglicerides and free fatty acids plasma levels than controls. The insulin secretion, in response to glucose and high concentrations of K+, in islets isolated from dexamethasone-treated rats for 5 and 10 days was higher than control rats. However, after 10 days of treatment with dexamethasone the insulin secretion was lower than after 5 days, but still higher than controls. In conclusion, these data indicate that dexamethasone-treatment (directly or indirectly) modulates the expression of several genes and proteins involved in apoptosis and survival of pancreatic islet-cells, and could, thereafter, modulates the insulin secretion in rats treated for 10 days / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Orientador: Everardo Magalhães Carneiro / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T09:09:25Z (GMT). No. of bitstreams: 1 Ribeiro_RosaneAparecida_D.pdf: 2339564 bytes, checksum: 146ea5a2123d76628db5f4758417e020 (MD5) Previous issue date: 2009 / Resumo: Neste estudo, investigamos os efeitos da suplementação com taurina (TAU; 2% adicionada à água de beber) sobre a tolerância à glicose e a secreção de insulina frente a diferentes secretagogos em camundongos adultos. Camundongos suplementados apresentaram aumento da tolerância à glicose e da sensibilidade à insulina. Ilhotas isoladas destes animais secretaram mais insulina em resposta à glicose e L-leucina. A oxidação da L-leucina foi maior no grupo TAU, não havendo diferenças quanto ao consumo de glicose, concentrações de ATP e expressão do transportador da glicose (GLUT) 2 e da glicoquinase (GCK). A captação de Ca2+, na presença de glicose, e a expressão protéica da subunidade ß2 do canal de Ca2+ sensível à voltagem foi maior no grupo TAU comparado ao controle (CTL). Ainda, a expressão protéica da PL (fosfolipase) C ß 2 e da PK (proteína quinase) Aa, bem como a secreção de insulina em resposta a agentes potencializadores tais como carbacol (Cch) e IBMX, foi maior nas ilhotas TAU. A mobilização intracelular de Ca2+ induzida por Cch foi também maior em ilhotas deste grupo, e observamos que a inibição da PKA reduziu a captação de Ca2+ em resposta à glicose no grupo suplementado. Além disso, ilhotas TAU secretaram mais glucagon em relação a ilhotas CTL, quando em presença de baixa concentração de glicose. Concluindo, a suplementação com TAU melhora a homeostase glicêmica e aumenta a secreção de insulina de ilhotas isoladas e incubadas na presença de nutrientes e agentes potencializadores da secreção. Os efeitos sobre a secreção estão relacionados ao melhor manejo dos íons Ca2+ pelas células insulares provenientes dos animais suplementados com TAU. / Abstract: In this study, we investigated the effects of taurine (TAU)-supplementation (2% in the drinking water) on glucose tolerance and insulin secretion stimulated by different secretagogues in adult mice. TAU-supplemented mice showed enhanced glucose tolerance and insulin sensitivity when compared to controls (CTL). In addition, their islets secreted more insulin in response to high concentrations of glucose and L-leucine. L-[U-14C]leucine oxidation was higher in TAU islets compared with CTL islets, whereas D-[U-14C]glucose oxidation, ATP levels, and the protein expression of the glucose transporter (GLUT) 2 and of glucokinase (GCK) were similar. 45Ca uptake induced by high glucose concentrations was increased in TAU islets as well as the expression of the ß2 subunit of the L-type Ca2+ channel. In addition, the insulin secretion induced by carbachol (Cch) and IBMX, but not, by forskolin and PMA was higher in TAU-supplemented compared with CTL islets. The higher insulin secretion in the presence of Cch is accompanied by an increase in the expression of PL (phospholipase) C ß 2 protein and a higher intracellular Ca2+ mobilization. Besides, TAU-supplemented islets showed increased PK (protein kinase) Aa expression. Since the increase in Ca2+ uptake induced by glucose in TAU islets was minimized by the presence of the PKA inhibitor, H89, this kinase seems to be important for the better Ca2+ handling in these islets. TAUsupplementation also turns the a-cells more sensitivity since these cells secreted more glucagons compared with CTL islets. In conclusion, TAU supplementation enhances glucose tolerance and insulin sensitivity in mice and turns the islets more sensitive to nutrients and to potentiators of secretion. The effect on insulin secretion seems to be linked to a better Ca2+ handling by ß-cells. / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular