Role of GDNF in the regulation of pancreatic beta cell mass

描述（由申请人提供）： 糖尿病-退伍军人的主要健康问题-是由于2细胞质量和功能的丧失。由于2细胞质量减少是1型和2型糖尿病（DM）发展和进展的基础，因此了解2细胞质量的调节对于预防和治疗都是必要的。虽然调节2-细胞质量的机制知之甚少，胶质细胞源性神经营养因子（GDNF）可能发挥重要作用。我们之前已经证明GDNF转基因小鼠（GDNF-tg，经改造以在神经胶质中过表达GDNF）增加了2个细胞质量并抵抗链脲佐菌素诱导的高血糖。为了指导GDNF、其受体激动剂或其信号转导靶点作为开发促进2-细胞团的药物的基础，我们首先需要更好地理解GDNF对2-细胞的作用机制。我们的假设是GDNF通过促进2-细胞分化来促进2-细胞质量的增加，并通过增强2-细胞质量、存活和再生来预防糖尿病。此外，GDNF通过提高2-细胞存活来改善2-细胞（胰岛）移植。在具体目标1中，我们将确定Pdx-1在GDNF诱导的胰腺2细胞分化中的作用。初步数据表明，在Pdx-1 siRNA存在下，GDNF介导的2-TC-6细胞和HIT细胞的分化减少。使用2-细胞分化的体外模型（2-TC-6细胞和HIT细胞）结合特异性siRNA、启动子测定和DNA结合测定，将确定Pdx-1在调节GDNF诱导的2-细胞分化中的必要性和充分性。在具体目标2中，我们将使用体外（毒胡萝卜素诱导2-TC-6细胞损伤）结合RNAi和体内（链脲佐菌素处理的GDFN-tg和WT小鼠）模型来确定Sox-9和Pdx-1在GDNF诱导的2-细胞再生中的作用。初步数据显示GDNF诱导的2-细胞增殖在SOX 9 siRNA存在下减少。利用神经元/神经胶质细胞在2-细胞上的共培养模型，我们将确定肠神经元和2-细胞相互作用的机制。在具体目标3中，我们将评估GDNF对鼠和猪胰岛移植后存活的影响。初步数据显示用GDNF预培养的小鼠胰岛的移植后存活增加。猪胰岛可用于人类移植。我们将用载体或GDNF预处理鼠和猪胰岛，并在糖尿病小鼠中进行肝内门静脉移植。GDNF和肠神经元/胶质细胞对胰岛移植后存活的影响将通过评估β细胞质量和功能来评估。对退伍军人医疗保健的影响：糖尿病在退伍军人中非常普遍，发病率正在上升。这项研究的结果可能有助于我们确定新的目标，以改善β细胞群，以帮助预防或治疗糖尿病。确定糖尿病的新疗法将直接使退伍军人受益。 公共卫生相关性： 糖尿病-退伍军人的主要健康问题-是由于2细胞质量和功能的丧失。由于2细胞质量减少是1型和2型糖尿病（DM）发展和进展的基础，因此了解2细胞质量的调节对于预防和治疗都是必要的。虽然调节2-细胞质量的机制知之甚少，胶质细胞源性神经营养因子（GDNF）可能发挥重要作用。在这个建议中概述的实验将研究GDNF如何调节2-细胞质量和2-细胞损伤后再生的机制。这些实验不仅有助于理解2-细胞质量的调节机制，还可能为预防和治疗糖尿病带来新的治疗靶点。对退伍军人医疗保健的影响：糖尿病在退伍军人中非常普遍，发病率正在上升。这项研究的结果可能有助于我们确定新的目标，以改善β细胞群，以帮助预防或治疗糖尿病。确定糖尿病的新疗法将直接使退伍军人受益。

DESCRIPTION (provided by applicant): Diabetes - a major health problem for veterans - is due to loss of 2-cell mass and function. Since decreased 2-cell mass underlies the development and progression of both Type 1 and Type 2 diabetes (DM), understanding the regulation of 2-cell mass is imperative for both prevention and treatment. Although the mechanisms regulating 2-cell mass are poorly understood, glial cell line-derived neurotrophic factor (GDNF) may play an important role. We have previously shown that GDNF transgenic mice (GDNF-tg, engineered to over-express GDNF in glia) have increased 2 cell mass and resist streptozotocin-induced hyperglycemia. In order to guide the use of GDNF, its receptor agonists, or its signal transduction targets as a basis for development of agents to promote 2-cell mass, we first need better understanding of mechanism of GDNF action on the 2-cell. Our hypothesis is that GDNF promotes increased 2-cell mass by promoting 2-cell differentiation and protects against diabetes by enhancing 2- cell mass, survival and regeneration. Further, GDNF improves 2-cell (islet) transplantation by enhancing 2-cell survival. In specific Aim 1 we will determine the role of Pdx-1 in GDNF-induced pancreatic 2-cell differentiation. Preliminary data demonstrates that GDNF-mediated differentiation of 2-TC-6 cells and HIT cells is reduced in the presence of Pdx-1 siRNA. Using in vitro models of 2-cell differentiation (2-TC-6 cells and HIT cells) in conjunction with specific siRNA, promoter assays and DNA binding assays, the necessity and sufficiency of Pdx-1 in modulating GDNF-induced 2-cell differentiation will be determined. In specific aim 2 we will determine the role of Sox-9 and Pdx-1 in GDNF induced 2-cell regeneration using in vitro (thapsigargin induce 2-TC-6 cell injury) in conjunction with RNAi and in vivo (streptozotocin treated GDFN-tg and WT mice) models. Preliminary data shows GDNF induced 2-cell proliferation is reduced in the presence of SOX9 siRNA. Using a co-culture model of neurons/glia on 2-cells we will determine the mechanism of interaction of enteric neurons and 2-cells. In Specific Aim 3 we will assess the effect of GDNF on murine and porcine islet post- transplantation survival. Preliminary data shows increased post transplantation survival of mouse islets pre-cultured with GDNF. Porcine islets can be used for human transplantation. We will pre-treat murine and porcine islets with vehicle or GDNF and perform intrahepatic portal vein transplantation in diabetic mice. The efect of GDNF and enteric neurons/glia on post transplantation survival of islets wil be assessed by evaluating beta cell mass and function. Impact on Veterans Health Care: Diabetes is highly prevalent in the Veteran population and the incidence is rising. Results from this study may help us identify new targets to improve beta cell mass to help in the prevention or treatment of diabetes. Identification of new therapies for diabetes will directly benefit the veteran population. PUBLIC HEALTH RELEVANCE: Project Narrative Diabetes - a major health problem for veterans - is due to loss of 2-cell mass and function. Since decreased 2-cell mass underlies the development and progression of both Type 1 and Type 2 diabetes (DM), understanding the regulation of 2-cell mass is imperative for both prevention and treatment. Although the mechanisms regulating 2- cell mass are poorly understood, glial cell line-derived neurotrophic factor (GDNF) may play an important role. Experiments outlined in this proposal will study the mechanism of how GDNF regulates 2-cell mass and results in regeneration of 2-cells after they have been injured. These experiments will not only contribute to the understanding of the mechanisms of regulation of 2-cell mass, but may also lead to new therapeutic targets for the prevention and treatment of diabetes. Impact on Veterans Health Care: Diabetes is highly prevalent in the Veteran population and the incidence is rising. Results from this study may help us identify new targets to improve beta cell mass to help in the prevention or treatment of diabetes. Identification of new therapies for diabetes will directly benefit the veteran population.