Mitogenic Signal Transduction in Pancreatic Beta Cells

描述（由申请人提供）：β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细胞质量背后的分子机制是复杂的，尚未得到很好的定义。由于有丝分裂、存活、大小和/或新生对成年a细胞的生长有不同的影响，因此它的生长更加复杂。近年来，IRS-2信号转导在β细胞质量的调控中起着关键作用。本研究旨在更好地将IRS-2信号机制与a细胞有丝分裂、存活和新生的调控联系起来。Aim-1将从葡萄糖/IGF-1诱导的IRS-2信号传导到胰岛β细胞的细胞周期控制成分，特别是通过形成cyclin-D/Cdk-4/p27KIP/p21CIP复合物，特异性增加cyclin-D2和激活cyclin-D依赖性激酶4 (Cdk-4)，这是诱导a细胞有丝分裂的关键。Aim-2将研究原代胰岛β细胞的存活机制，特别是IRS-2/PKB的下游。a细胞中PKB的激活对ffa诱导的凋亡具有保护作用，这表明PKB是β细胞存活的关键。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，我们将通过腺病毒介导的表达研究某些PKB底物（如GSK3、Foxo1、Mdm2和BAD），以保护β细胞免受ffa诱导的凋亡。Aim-3将在再生模型（nedh大鼠的可移植胰岛素瘤）中检测β细胞新生。当胰岛素瘤在皮下生长时，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细胞群恢复，主要是新生。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，局部生长因子和假定的β细胞祖细胞标记物（如Ngn3， Pdx-1和nestin）与胰岛素+细胞的早期出现之间的相关性。关键的候选IRS-2信号元件和因子将从该模型中出现，然后通过腺病毒在胰腺导管上皮细胞系中的表达来检查这些元件和因子，以确定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，这些将整合到目标4中，研究非糖尿病性肥胖和肥胖相关糖尿病体内模型中控制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、存活和新生对α细胞生长/存活的贡献，以及相对于IRS-2信号转导的控制，以及动物的年龄（新生儿到成年）和代谢稳态。我们将特别关注活跃的IRS-2信号，它与增加β细胞生长相补充，以适应肥胖相关的胰岛素抵抗，以及IRS-2信号的损伤，可能导致β细胞数量减少，这标志着肥胖相关的2型糖尿病的发生。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.