Patients with type II diabetes present alterations in glucose homeostasis due to insufficient amount of insulin (β-cell dysfunction) and inability to properly use the insulin that is secreted (insulin resistance). Combined genetical and environmental factors are believed to be responsible for these dysfunctions and the resulting impairment in glucose homeostasis. The pancreatic gland is composed of exocrine and endocrine tissues. The endocrine part of the organ couples glucose sensing to insulin release. Within this endocrine gland, also known as islets of Langerhans, the insulin secreting β-cell is the main player and therefore highly important for proper glucose metabolism. In this thesis, mice were developed in order to assess the role of BMP signaling molecule and Arsenite induced ATPase-1 (Asna1) for pancreas development and β-cell function. The mature β-cell responds to elevated glucose levels by secreting insulin in a tightly controlled manner. This physiological response of the β-cell to elevated blood glucose levels is critical for maintenance of normoglycaemia and impaired Glucose stimulated insulin secretion (GSIS) is a prominent feature of overt type 2 diabetes. Thus, the identification of signals and pathways that ensure and stimulate GSIS in β-cells is of great clinical interest. Here we show (Paper I) that BMPRIA and its high affinity ligand BMP4 are expressed in fetal and adult islets. We also provide evidence that BMPRIA signaling in adult β-cell is required for GSIS, and that both transgenic expression of Bmp4 in β-cells or systemic administration of BMP4 protein to mice enhances GSIS. Thus, BMP4-BMPRIA signaling in β-cells positively regulates the genetic machinery that ensures GSIS. Arsenite induced ATPase (Asna1), the homologue of the bacterial ArsA ATPase, is expressed in insulin producing cells of both mammals and the nematode Caenorhabditis elegans (C.elegans). Asna1 has been proposed to act as an evolutionary conserved regulator of insulin/insulin like factor signaling. In C.elegans, asna-1 has been shown to regulate growth in a non-cell autonomous and IGF-receptor dependent manner. Here we show that transgenic expression of ASNA1 in β-cells of mice leads to enhanced Aktactivity and β-cell hyperplasia (manuscript). ASNA1 transgenic mice develop, however, diabetes due to impaired insulin secretion. The expression of genes involved in secretion stimulus coupling and insulin exocytosis is perturbed in islets of these mice. These data suggest that activation of ASNA1, here mimicked by enhanced expression, positively influences β-cell mass but negatively affects insulin secretion.

Certain membrane lipids are involved in intracellular signalling processes, among them phosphoinositides and diacylglycerol (DAG). They mediate a variety of functions, including the effects of nutrients and neurohormonal stimuli on insulin secretion from pancreatic β-cells. To ensure specificity of the signal, their concentrations are maintained under tight spatial and temporal control. Here, live-cell imaging techniques were employed to investigate spatio-temporal aspects of lipid signalling in the plasma membrane of insulin-secreting β-cells. The concentration of phosphatidylinositol 4-phosphate [PtdIns(4)P] increased after stimulation with glucose or Gq protein-coupled receptor agonists. The glucose effect was Ca2+-dependent, whereas the receptor response was mediated by isoforms of novel protein kinase C (PKC). The increases in PtdIns(4)P were paralleled by lowerings of the phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] concentration. This relationship was not caused by conversion of PtdIns(4,5)P2 to PtdIns(4)P but rather reflected independent regulation of the two lipids. Stimulation of β-cells with glucose or a high K+ concentration induced pronounced, repetitive increases in plasma-membrane DAG concentration, which were locally restricted and lasted only for a few seconds. This pattern was caused by exocytotic release of ATP, which feedback-activates purinergic P2Y1-receptors and stimulates local phospholipase C-mediated DAG generation. Despite their short durations the DAG spikes triggered local activation of PKC. Novel PKCs were recruited to the plasma membrane both after glucose and muscarinic receptor stimulation. While the glucose-induced translocation was synchronized with DAG spiking, muscarinic stimulation induced sustained elevation of the DAG concentration and stable membrane association of the kinase. Also conventional PKCs translocated to the membrane after glucose and receptor stimulation. The glucose-induced response was complex with sustained membrane association mirroring the cytoplasmic Ca2+ concentration, and superimposed brief recurring translocations caused by DAG. Interruption of the purinergic feedback loop underlying DAG spiking suppressed insulin secretion. Since the DAG spikes reflected exocytosis events, a single-cell secretion assay was established, which allowed continuous recording of secretion dynamics from many cells in parallel over extended periods of time. With this approach it was possible to demonstrate that insulin exerts negative feedback on its own release via a phosphatidylinositol 3,4,5-trisphosphate-dependent mechanism.

Diet-induced obesity promotes type 2 diabetes (T2D). Drugs that inhibit the renin-angiotensin system (RAS) have been demonstrated in clinical trials to decrease the onset of T2D. Previously, we demonstrated that mice made obese from chronic consumption of a high-fat (HF) diet have marked elevations in systemic concentrations of angiotensin II (AngII). Pancreatic islets have been reported to possess components of the renin-angiotensin system (RAS), including angiotensin type 1a receptors (AT1aR), the primary receptor for AngII, and angiotensin converting-enzyme 2 (ACE2), which negatively regulates the RAS by catabolizing AngII to angiotensin-(1-7) (Ang-(1-7)). These two opposing proteins have been implicated in the regulation of β-cell function. We hypothesized that the RAS contributes to the decline of β-cell function during the development of T2D with obesity. To test this hypothesis we first examined the effects of whole-body deficiency of ACE2 in mice on β-cell function in vivo and in vitro during the development of T2D. Whole-body deficiency of ACE2 resulted in impaired β-cell adaptation to insulin resistance with HF-feeding and a reduction of in vivo glucose-stimulated insulin secretion (GSIS) associated with reduced β- cell mass and proliferation. These results demonstrate that ACE2 plays a role in the adaptive response to hyperinsulinemia with obesity. In islets from HF-fed mice, AngII inhibited GSIS. In mice with pancreatic-specific deletion of AT1aR, AngII-induced inhibition of GSIS in vitro from islets of HF-fed mice was abolished. However, there was no effect of pancreatic AT1aR-deficiency on glucose homeostasis in vivo in HF-fed mice exhibiting pronounced hyperinsulinemia. Notably, pancreatic weight, insulin content and basal and glucose-stimulated insulin secretion from islets were decreased in mice with pancreatic AT1aR deficiency. These results suggest that AT1aR may contribute to pancreatic cell development, and also contribute to AngII-induced reductions in GSIS from islets of HF-fed mice. Overall, these studies suggest a role for the RAS in the regulation of β-cell function in T2D.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23082号 / 医博第4709号 / 新制||医||1049(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 妹尾 浩, 教授 村川 泰裕 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p>Type 1 Diabetes Mellitus (T1D), a chronic disease affecting over 1.5 million Americans, is characterized by the autoimmune destruction of insulin-producing β-cells within pancreatic islets. Islet/β-cell replacement therapies, where replenishable β-cell sources are implanted within protective microenvironments, have the potential to provide a long-term solution for individuals with T1D by restoring glucose-sensitive, insulin release and overall glycemic control. However, most conventional encapsulation materials elicit an immune reaction, known as a foreign body response (FBR), which compromises β-cell health and function. In this dissertation, we designed and evaluated various formulations of a polymerizable collagen, namely type I oligomeric collagen (Oligomer), as encapsulation materials for minimally invasive, subcutaneous delivery of replacement β-cells. Preclinical validation in chemically-induced diabetic mice demonstrated rapid (within 24 hours) reversal of diabetes for beyond 90 days with no signs of rejection or FBR after subcutaneous delivery of both allogeneic and xenogeneic (rat) islets. To further define this uncommon mechanism of protection, the tissue response to Oligomer, in comparison to commercial synthetic and collagen-based materials, was evaluated following subcutaneous implantation within rats, a well-established biocompatibility model. Histological and transcriptomics analyses were used to define the immune response at both cellular and molecular levels. Interestingly, Oligomer showed minimal and transient activation of innate immune cells similar to the sham surgical control, with no evidence of foreign body giant cell formation, inflammatory-mediated bioresorption, or fibrosis. Overall, this work evaluates preclinical efficacy and demonstrates mechanistic understanding of immune tolerance for Oligomer materials for β-cell replacement therapy and other regenerative medicine applications.</p>

No / Ran is a small ras-related GTPase that controls the nucleocytoplasmic exchange of macromolecules across the nuclear envelope. It binds to chromatin early during nuclear formation and has important roles during the eukaryotic cell cycle, where it regulates mitotic spindle assembly, nuclear envelope formation and cell cycle checkpoint control. Like other GTPases, Ran relies on the cycling between GTP-bound and GDP-bound conformations to interact with effector proteins and regulate these processes. In nucleocytoplasmic transport, Ran shuttles across the nuclear envelope through nuclear pores. It is concentrated in the nucleus by an active import mechanism where it generates a high concentration of RanGTP by nucleotide exchange. It controls the assembly and disassembly of a range of complexes that are formed between Ran-binding proteins and cellular cargo to maintain rapid nuclear transport. Ran also has been identified as an essential protein in nuclear envelope formation in eukaryotes. This mechanism is dependent on importin-β, which regulates the assembly of further complexes important in this process, such as Nup107–Nup160. A strong body of evidence is emerging implicating Ran as a key protein in the metastatic progression of cancer. Ran is overexpressed in a range of tumors, such as breast and renal, and these perturbed levels are associated with local invasion, metastasis and reduced patient survival. Furthermore, tumors with oncogenic KRAS or PIK3CA mutations are addicted to Ran expression, which yields exciting future therapeutic opportunities.

Pour étudier l’impact de la signalisation de la prolactine (PRL), une hormone impliquée dans la proliférationcellulaire sur l’adipocyte et la cellule β pancréatique, deux types cellulaires impliqués dans la balanceénergétique, nous avons caractérisé le phénotype de souris déficientes en récepteur de la PRL (R PRL-/-) sousdifférentes conditions physiopathologiques. Dans un premier temps, nous avons étudié l’impact du R PRL sur ledéveloppement d’une obésité induite par un régime obésogène. Dans un deuxième temps, nous nous sommesintéressés à l’impact du R PRL sur l’ontogenèse des cellules β durant les adaptations périnatales. Nous avonsaussi évalué son rôle sur la sécrétion d’insuline à l’âge adulte.Notre première étude montre que les souris R PRL-/- sous régime obésogène ont une prise de poids réduite etune augmentation de la dépense énergétique comparées à celles des souris sauvages. Nous montrons que desadipocytes beiges, une nouvelle classe d’adipocytes thermoactifs récemment caractérisés et exprimant laprotéine découplante UCP1, émergent dans le tissu adipeux blanc périrénal des souris R PRL-/- soumises à unrégime gras. Nous avons démontré que le R PRL contribue à l’apparition des adipocytes beiges en modulant lavoie de signalisation pRb/FoxC2 permettant la résistance à l’obésité induite par le régime gras.Notre deuxième étude montre que la souris R PRL-/- et le rat GK, un modèle de diabète de type 2, ont un défautd’adaptation de la masse des cellules β en période périnatale. Cette altération est corrélée à un défautd’expression d’igf2 (Insulin-like Growth Factor 2), une cible de la PRL. A partir d’îlots de Langherans de sourisadultes, nous avons confirmé que le R PRL est essentiel à la sécrétion d’insuline.Les résultats obtenus ont permis de mieux comprendre le rôle de la PRL sur la balance énergétique. Ces travauxouvrent des perspectives nouvelles pour le développement de stratégies thérapeutiques dans la lutte contrel’obésité et le diabète de type II. / In order to study the impact of prolactin (PRL) signaling on pancreatic β-cell and adipocyte, two cell typesinvolved in energy balance, we characterized the phenotype of PRL receptor deficient mice (PRL R-/-) underdifferent physiopathological conditions. First, we studied the impact of PRL R on the development of obesityinduced by a high fat diet. Second, we investigated the impact of PRL R on β-cell ontogenesis during perinataladaptation and its role in insulin secretion during adulthood.Our first study shows that PRL R-/- mice under obesogenic diet have a reduced weight gain and an increase ofenergy expenditure as compared to those of wild-type mice. We showed that beige adipocytes, a new class ofthermogenic adipocytes recently characterized expressing uncoupling protein UCP1, emerged in the perirenalwhite adipose tissue of PRL R-/- mice challenged with a high fat diet. Altered expression of pRb/FoxC2 suggeststhat PRL R contributes to the development of beige adipocytes modulating this signaling pathway for resistanceto high fat diet induced obesity.Our second study shows that PRL R-/- mice do not adapt β-cell mass in perinatal period and this alteration isassociated with a lack of igf2 (Insulin-like Growth Factor 2) expression, a PRL target. We confirmed that R PRL isessential for insulin secretion using b islets in adult animals.These results lead to a better understanding of the PRL role on energy balance, and open new perspectives forthe development of therapeutic strategies in obesity and type II diabetes

<p>The mechanisms of β-cell destruction leading to type 1 diabetes are complex and not yet fully understood, but infiltration of the islets of Langerhans by autoreactive immune cells is believed to be important. Activated macrophages and T-cells may then secrete cytokines and free radicals, which could selectively damage the β-cells. Among the cytokines, IL-1β, IFN-γ and TNF-α can induce expression of inducible nitric synthase (iNOS) and cyclooxygenase-2. Subsequent nitric oxide (NO) and prostaglandin E₂ (PGE₂) formation may impair islet function.</p><p>In the present study, the ability of melatonin (an antioxidative and immunoregulatory hormone) to protect against β-cell damage induced by streptozotocin (STZ; a diabetogenic and free radical generating substance) or IL-1β exposure was examined. <i>In vitro</i>, melatonin counteracted STZ- but not IL-1β-induced islet suppression, indicating that the protective effect of melatonin is related to interference with free radical generation and DNA damage, rather than NO synthesis. <i>In vivo</i>, non-immune mediated diabetes induced by a single dose of STZ was prevented by melatonin.</p><p>Furthermore, the effects of proinflammatory cytokines were examined in islets obtained from mice with a targeted deletion of the iNOS gene (iNOS -/- mice) and wild-type controls. The <i>in vitro</i> data obtained show that exposure to IL-1β or (IL-1β + IFN-γ) induce disturbances in the insulin secretory pathway, which were independent of NO or PGE₂ production and cell death. Initially after addition, in particular IL-1β seems to be stimulatory for the insulin secretory machinery of iNOS –/- islets, whereas IL-1β acts inhibitory after a prolonged period. Separate experiments suggest that the stimulatory effect of IL-1β involves an increased gene expression of phospholipase D1a/b. In addition, the formation of new insulin molecules appears to be affected, since IL-1β and (IL-1β + IFN-γ) suppressed mRNA expression of both insulin convertase enzymes and insulin itself.</p>

The mechanisms of β-cell destruction leading to type 1 diabetes are complex and not yet fully understood, but infiltration of the islets of Langerhans by autoreactive immune cells is believed to be important. Activated macrophages and T-cells may then secrete cytokines and free radicals, which could selectively damage the β-cells. Among the cytokines, IL-1β, IFN-γ and TNF-α can induce expression of inducible nitric synthase (iNOS) and cyclooxygenase-2. Subsequent nitric oxide (NO) and prostaglandin E2 (PGE2) formation may impair islet function. In the present study, the ability of melatonin (an antioxidative and immunoregulatory hormone) to protect against β-cell damage induced by streptozotocin (STZ; a diabetogenic and free radical generating substance) or IL-1β exposure was examined. In vitro, melatonin counteracted STZ- but not IL-1β-induced islet suppression, indicating that the protective effect of melatonin is related to interference with free radical generation and DNA damage, rather than NO synthesis. In vivo, non-immune mediated diabetes induced by a single dose of STZ was prevented by melatonin. Furthermore, the effects of proinflammatory cytokines were examined in islets obtained from mice with a targeted deletion of the iNOS gene (iNOS -/- mice) and wild-type controls. The in vitro data obtained show that exposure to IL-1β or (IL-1β + IFN-γ) induce disturbances in the insulin secretory pathway, which were independent of NO or PGE2 production and cell death. Initially after addition, in particular IL-1β seems to be stimulatory for the insulin secretory machinery of iNOS –/- islets, whereas IL-1β acts inhibitory after a prolonged period. Separate experiments suggest that the stimulatory effect of IL-1β involves an increased gene expression of phospholipase D1a/b. In addition, the formation of new insulin molecules appears to be affected, since IL-1β and (IL-1β + IFN-γ) suppressed mRNA expression of both insulin convertase enzymes and insulin itself.

Insulin and glucagon are released in pulses from pancreatic β- and α-cells, respectively. Both cell types are electrically excitable, and elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i) due to depolarization with voltage-dependent entry of the cation is the main trigger of hormone secretion. Store-operated Ca2+ entry  (SOCE) also contributes to the [Ca2+]i elevation and this process has been suggested to be particularly important for glucagon secretion. cAMP is another important messenger that amplifies Ca2+-triggered secretion of both hormones, but little is known about cAMP dynamics in islet cells. In type-2 diabetes, there is deteriorated β-cell function associated with elevated concentrations of fatty acids, but the underlying mechanisms are largely unknown. To clarify the processes that regulate insulin and glucagon secretion, cAMP signalling and the store-operated pathway were investigated in β- and α-cells, primarily within their natural environment in intact mouse and human islets of Langerhans. Fluorescent biosensors and total internal reflection microscopy were used to investigate signalling specifically at the plasma membrane (PM). Adrenaline increased and decreased the sub-PM cAMP concentration ([cAMP]pm) in immuno-identified α-cells and β-cells, respectively, which facilitated cell identification. Glucagon elicited [cAMP]pm oscillations in α- and β-cells, demonstrating both auto- and paracrine effects of the hormone. Whereas glucagon-like peptide 1 (GLP-1) consistently elevated [cAMP]pm in β-cells, only few α-cells responded, indicating that GLP-1 regulates glucagon secretion without changes of α-cell [cAMP]pm. Both α- and β-cells responded to glucose with pronounced oscillations of [cAMP]pm that were partially Ca2+-dependent and synchronized among islet β-cells. The glucose-induced cAMP formation was mediated by plasma membrane-bound adenylyl cyclases. Several phosphodiesterases (PDEs), including the PDE1, -3, -4, and -8 families, were required for shaping the [cAMP]pm signals and pulsatile insulin secretion. Prolonged exposure of islets to the fatty acid palmitate deteriorated glucose-stimulated insulin secretion with loss of pulsatility. This defect was associated with impaired cAMP generation, while [Ca2+]i signalling was essentially unaffected. Stromal interacting molecule 1 (STIM1) is critical for activation of SOCE by sensing the Ca2+ concentration in the endoplasmic reticulum (ER). ER Ca2+ depletion caused STIM1 aggregation, co-clustering with the PM Ca2+ channel protein Orai1 and SOCE activation. Glucose, which inhibits SOCE by filling the ER with Ca2+, reversed the PM association of STIM1. Consistent with a role of the store-operated pathway in glucagon secretion, this effect was maximal at the low glucose concentrations that inhibit glucagon release, whereas considerably higher concentrations were required in β-cells. Adrenaline induced STIM1 translocation to the PM in α-cells and the reverse process in β-cells, partially reflecting the opposite effects of adrenaline on cAMP in the two cell types. However, cAMP-induced STIM1 aggregates did not co-cluster with Orai1 or activate SOCE, indicating that STIM1 translocation can occur independently of Orai1 clustering and SOCE.