Mitogenic Signal Transduction in Pancreatic Beta Cells

描述（由申请人提供）：β α细胞生长和存活的控制对1型和2型糖尿病都有影响，但维持最佳β细胞群背后的分子机制很复杂，尚未明确。成体α-细胞生长进一步复杂化，因为它可以通过有丝分裂、存活、大小和/或新生而有差异地贡献。最近，研究表明IRS-2信号传导是调节β细胞质量的关键。这项拟议的研究旨在更好地将IRS-2信号传导机制与控制细胞有丝分裂、存活和新生联系起来。Aim-1将开发葡萄糖/IGF-1诱导的IRS-2信号传导与胰岛β细胞中细胞周期控制组分的机制联系，特别是关于细胞周期蛋白-D2的特异性增加和细胞周期蛋白-D依赖性激酶-4（Cdk-4）的活化，其通过形成细胞周期蛋白-D/Cdk-4/p27 KIP/p21 CIP复合物来诱导α细胞有丝分裂。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。α-细胞中PKB的活化对FFA诱导的细胞凋亡具有保护作用，暗示PKB是β-细胞存活的关键。然而，PKB具有过多的底物，并且并非所有底物都是抗凋亡的。因此，为了获得PKB介导的α细胞凋亡预防的特异性，将通过腺病毒介导的表达研究某些PKB底物（例如GSK 3、Foxo 1、Mdm 2和BAD）以保护β细胞免于FFA诱导的凋亡。目的-3将检查在复活模型中的β细胞新生，NEDH-大鼠中的可移植胰岛素瘤。随着胰岛素瘤皮下生长，内源性胰腺β细胞萎缩，但在手术切除胰岛素瘤后，内源性β细胞群主要通过新生恢复。有趣的是，在β细胞新生过程中，IRS-2+和胰岛素+细胞从增殖的导管组织中平行出现。该模型将用于表征IRS-2信号传导、局部生长因子和推定的β细胞祖细胞标志物（例如Ngn 3、Pdx-1和巢蛋白）与胰岛素+细胞的早期出现之间的相关性。关键的候选IRS-2信号传导元件和因子将从该模型中出现，然后将通过胰腺导管上皮细胞系中的腺病毒表达来检查这些元件和因子，以观察是否可以在体外产生胰岛素+细胞。从目标1-3中吸取的经验教训，这些将被整合到目标4中，目标4研究在非糖尿病肥胖和肥胖相关糖尿病的体内模型中控制a细胞有丝分裂、大小、存活和新生的机制。这些研究将确定β-细胞有丝分裂、大小、存活和新生相对于IRS-2信号转导的控制对α-细胞生长/存活的贡献，以及动物的年龄（新生儿至成年）和代谢稳态。将特别关注与增加β细胞生长以适应肥胖相关胰岛素抵抗的活性IRS-2信号传导，以及可能导致β细胞质量减少的IRS-2信号传导受损，这标志着肥胖相关2型糖尿病的发病。总而言之，拟议的研究将对β细胞生长的控制提供更机械和全面的理解，这可能对产生治疗糖尿病的新疗法具有深刻的见解。

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.