Mitogenic Signal Transduction in Pancreatic Beta Cells

描述(由申请人提供)：控制β细胞的生长和存活对1型和2型糖尿病都有影响，但维持最佳β细胞质量背后的分子机制是复杂的，还没有很好的确定。成人a细胞的生长更加复杂，因为它可以通过有丝分裂、存活、大小和/或新生来不同地促进。近年来，IRS-2信号转导通路在调节胰岛β细胞质量中的作用日益明显。这项拟议的研究旨在更好地将IRS-2信号机制与a细胞有丝分裂、存活和新生的控制联系起来。AIM-1通过形成细胞周期蛋白-D/CDK-4/p27KIP/p21CIP复合体，实现了葡萄糖/IGF-1诱导的IRS-2信号与胰岛细胞周期调控成分的机械性联系，尤其是细胞周期蛋白-D2的特异性增加和细胞周期蛋白-D依赖的激酶-4(CDK-4)的激活。AIM-2将研究原代胰岛β细胞的存活机制，特别是IRS-2/PKB下游的机制。激活a细胞中的PKB对FFA诱导的细胞凋亡具有保护作用，这意味着PKB是β细胞生存的关键。然而，PKB有过多的底物，并不是所有的底物都是抗凋亡的。因此，为了获得PKB介导的预防a细胞凋亡的特异性，将通过腺病毒介导的表达来研究某些PKB底物(如GSK3、Foxo1、MDM2和BAD)来保护β细胞免受FFA诱导的凋亡。AIM-3将在NEDH大鼠的可移植胰岛素瘤复活模型中检测β细胞新生。随着胰岛素瘤的皮下生长，内源性胰岛β细胞萎缩，但在手术切除胰岛素瘤后，内源性胰岛细胞数量恢复，主要是通过新生。有趣的是，在这个β细胞新生过程中，IRS-2+细胞和胰岛素+细胞同时出现在增殖的导管组织中。这个模型将被用来描述IRS-2信号、局部生长因子和早期出现的假定的β细胞前体标记物(如Ngn3、PDX-1和Nestin)与胰岛素+细胞之间的相关性。关键的候选IRS-2信号元件和因子将从这个模型中出现，然后通过腺病毒在胰腺导管上皮细胞系中的表达来检测这些元件和因子，看看是否可以在体外产生胰岛素+细胞。从AIMS 1-3中学到的教训，这些将被整合到AIM-4中，研究非糖尿病肥胖和肥胖相关糖尿病体内模型中a细胞有丝分裂、大小、存活率和新生的控制机制。这些研究将确定与IRS-2信号转导以及年龄(从新生儿到成人)和动物的代谢稳态相比，β细胞有丝分裂、大小、存活和新生对a细胞生长/存活的贡献是什么。将特别关注活跃的IRS-2信号，补充增加β细胞生长以适应肥胖相关的胰岛素抵抗，以及IRS-2信号的损害，这可能有助于减少标志着肥胖相关的2型糖尿病发病的β细胞质量。总而言之，这项拟议的研究将使人们对β细胞生长的控制有一个更机械、更全面的理解，这可能对开发治疗糖尿病的新疗法具有洞察力。

DESCRIPTION (provided by applicant): Control of betaa-cell growth and survival has implications for both type-1 and -2 diabetes, but the molecular mechanisms behind the maintenance of an optimal beta-cell mass are complex and not well defined. Adult a-cell growth is further complicated in that it can be differentially contributed to by mitogenesis, survival, size, and/or neogenesis. Recently, it has become evident that IRS-2 signal transduction is key for regulation of beta-cell mass. This proposed research intends to better link IRS-2 signaling mechanisms to control of a-cell mitogenesis, survival and neogenesis. Aim-1 will develop a mechanistic connection from glucose/IGF-1 induced IRS-2 signaling to components of cell cycle control in islet beta-cells, especially in regard to a specific increase in cyclin-D2 and activation of cyclin-D dependent kinase-4 (Cdk-4) via formation of a cyclin-D/Cdk-4/p27KIP/p21CIP complex which is key to inducing a-cell mitogenesis. Aim-2 will investigate survival mechanisms in primary islet beta-cells, especially downstream of IRS-2/PKB. Activation of PKB in a-cells is protective against FFA-induced apoptosis, implicating PKB as key to beta-cell survival. However, PKB has a plethora of substrates, and not all of which are anti-apoptotic. Thus, to gain specificity of PKB-mediated prevention of a-cell apoptosis, certain PKB substrates (e.g. GSK3, Foxo1, Mdm2 and BAD) will be investigated, via adenoviral-mediated expression, for protection of beta-cells from FFA-induced apoptosis. Aim-3 will examine beta -cell neogenesis in a resurrected model, the transplantable insulinoma in the NEDH-rat. As the insulinoma grows subcutaneously, the endogenous pancreatic beta-cells atrophy but on surgical removal of the insulinoma the endogenous beta-cell population recovers, mostly by neogenesis. Intriguingly, there is a parallel emergence of IRS-2+ and insulin+ cells from proliferating ductal tissue during this beta-cell neogenesis. This model will be used to characterize a correlation between IRS-2 signaling, local growth factors and the early appearance of putative beta-cell progenitor markers (e.g. Ngn3, Pdx-1 & nestin) with insulin+ cells. Key candidate IRS-2 signaling elements and factors will emerge from this model, and these will then be examined, via adenoviral expression in pancreatic ductal epithelial cell lines, to see if insulin+ cells can be generated in vitro. From the lessons learned in aims 1-3, these will be integrated into aim-4 that examines the mechanisms for controlling a-cell mitogenesis, size, survival, and neogenesis in in vivo models of non-diabetic obesity and obesity-linked diabetes. These studies will ascertain what contribution beta-cell mitogenesis, size, survival, and neogenesis makes to a-cell growth/survival relative to control by IRS-2 signal transduction, as well as age (neonate to adult) and metabolic homeostasis of the animal. Particular attention will be paid to active IRS-2 signaling complementary to increasing beta-cell growth for adaptation to obesity-associated insulin resistance, as well as impairment of IRS-2 signaling that could contribute to a decrease in beta-cell mass that marks the onset of obesity-linked type-2 diabetes. All in all, the proposed research will give a more mechanistic and comprehensive understanding of the control of beta-cell growth that may prove insightful for generating novel therapies to treat diabetes.