Type-1 Diabetes: Zn2+ Potentiated Beta-Cell Death By Sirtuin-Mediated NAD+ Loss

描述(申请人提供)：1型糖尿病是一种自身免疫性疾病，由T淋巴细胞产生的ROS介导的破坏产生胰岛素的β细胞引起。线粒体和糖酵解酶功能障碍，吡啶辅因子和磷酸丙糖失衡，以及多聚腺苷二磷酸核糖聚合酶(PARP)的激活都与此有关，这表明代谢介质，如NAD+/NADH比率可能受到影响。我们认为锌(Zn~(2+))增强了其中的一些功能障碍。锌离子普遍存在于胰腺中，在那里它从β细胞胰岛素分泌颗粒中释放出来，无论是游离的还是与胰岛素结合的，并被带回邻近的β细胞。在体外和体内，锌离子毒性与急性链脲佐菌素(STZ)暴露引起的胰岛细胞死亡有关。我们发现，烟酰胺、丙酮酸、NAD+和水飞蓟醇在体外通过对抗锌离子诱导的NAD+水平的下降(体内工作亚组)来阻止锌离子对神经元和β细胞的毒性。烟酰胺被证明是一种有效的预防人类和动物1型糖尿病的药物。最近，我们证明了NOD动物随着年龄的增长，胰岛的锌离子染色异常强烈，丙酮酸可以减轻NOD模型中的β细胞死亡和糖尿病。我们认为，自身免疫反应给β细胞带来压力，而β细胞又释放出有毒浓度的锌离子。这会导致邻近的β细胞因NAD+耗尽而死亡。升高[NAD+]i或降低[Zn2+]i的操作将是保护性的。烟酰胺是一种NAD+分解代谢抑制剂，也是NAD+的前体，丙酮酸转化为乳酸的作用是恢复NAD+水平和代谢酶，从而减少死亡。Sirtinol是sirtuin途径的一种特异性抑制剂，sirtuin途径由NAD+依赖的蛋白去乙酰基酶组成。在特定的目标1)中，我们将1)测量由锌离子、细胞因子和链脲佐菌素暴露引起的胰岛内[锌离子]i和65锌离子的积累；2)确定这些暴露对β细胞代谢的影响，以及丙酮酸、烟酰胺和水飞蓟醇恢复代谢的能力；3)利用慢病毒或基因操作NAD+的合成和分解代谢，以探索锌2+介导的NAD+丢失和β细胞死亡的具体途径。在具体目标2中，我们将确定丙酮酸和烟酰胺在NOD模型中减少代谢紊乱和糖尿病发生率的能力。锌转运蛋白5(ZnT5)可将锌离子装载到胰岛β细胞的分泌囊泡中。Sirt-1是sirtuin途径的创始成员，sirtuin途径与神经元和β细胞的锌毒性有关，而Wlds过度表达NAD+合成酶。在目标3中，我们将研究在MLDS链脲佐菌素暴露后，糖尿病发病率、β细胞死亡、锌染色和胰岛素释放方面的锌锌染色、+/+、Wlds和SIRT-1-/-动物的特征。这些实验将测试新的治疗化合物(丙酮酸、sirtinol)和机制(NAD+丢失、sirtuins)，这些机制涉及锌离子介导1型糖尿病患者的β细胞死亡。

DESCRIPTION (provided by applicant): Type 1 diabetes is an autoimmune disease caused by T-lymphocyte generated ROS-mediated destruction of the insulin-producing beta cells. Mitochondrial and glycolytic enzyme dysfunction, pyridine cofactor and triosephosphate imbalance, and poly-ADP ribose polymerase (PARP) activation have each been implicated, suggesting that a metabolic mediator such as the NAD+/NADH ratio may be affected. We propose that zinc (Zn2+) potentiates some of these dysfunctions. Zn2+ is prevalent in the pancreas where it is released from beta-cell insulin secretory granules, both free and bound to insulin, and is taken back up into neighboring beta-cells. Zn2+ toxicity was recently implicated in islet-cell death that occurs secondarily to acute streptozotocin (STZ) exposure in vitro and in vivo. We show that niacinamide, pyruvate, NAD+, and sirtinol prevent Zn2+toxicity in neurons and beta-cells by counteracting the Zn2+-induced decrease in NAD+ levels in vitro (subset work in vivo). Niacinamide was shown to be an effective prophylactic for human, and animal type-1 diabetes if given before onset. Recently, we demonstrated that NOD animals have abnormally strong Zn2+ staining of islets as they age, and pyruvate attenuates beta-cell death, and diabetes in the NOD model. We propose that the autoimmune reaction stresses beta-cells which in turn release toxic concentrations of Zn2+. This causes death of adjoining beta-cells by NAD+depletion. Manipulations which raise [NAD+]i or lower [Zn2+]i will be protective. Niacinamide is an NAD+catabolism inhibitor and the precursor of NAD+, and the conversion of pyruvate to lactate acts to restore NAD+levels and metabolic enzymes, thereby reducing death. Sirtinol is a specific inhibitor of the sirtuin pathway which consists of NAD+dependent protein deacetylases. In specific aim 1, we will 1) measure [Zn2+]i, and 65Zn2+accumulation in isolated islets induced by Zn2+, cytokine, and streptozotocin exposures; 2) Determine the effects of these exposures on beta-cell metabolism, and the ability of pyruvate, niacinamide, and sirtinol to restore metabolism; 3) Lentiviral or genetic manipulation of NAD+ synthesis and catabolism will be used to explore the specific pathways involved in Zn2+ mediated NAD+ loss and beta-cell death. In specific aim 2, we will determine the ability of pyruvate, and niacinamide to reduce the metabolic derangements and diabetic incidence in the NOD model. Zinc transporter 5 (ZnT5) was suggested to load Zn2+ into secretory vesicles in beta-cells. SIRT-1 is the founding member of the sirtuin pathway which was implicated in neuronal and beta-cell Zn2+ toxicity, and Wlds overexpresses an NAD+synthetic enzyme. In aim 3, we will characterize ZnT5 -/-, +/+, Wlds, and SIRT-1-/-animals for diabetes incidence, beta-cell death, Zn2+ staining, and insulin release after MLDS streptozotocin-exposure. These experiments will test novel therapeutic compounds (pyruvate, sirtinol) and mechanisms (NAD+loss, sirtuins) involved in Zn2+ mediation of beta-cell death in type-1 diabetes.