Role of GDNF in the regulation of pancreatic beta cell mass

描述(由申请人提供)： 糖尿病--退伍军人的一个主要健康问题--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)发生和发展的基础，了解2细胞质量的调节对于预防和治疗都是必不可少的。尽管对2-细胞质量的调控机制知之甚少，但胶质细胞系衍生神经营养因子(GDNF)可能起着重要作用。我们之前已经证明了GDNF转基因小鼠(GDNF-TG，在胶质细胞中过表达GDNF)增加了2个细胞质量，并抵抗了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-细胞质量的药物的基础，我们首先需要更好地了解GDNF对2-细胞的作用机制。我们的假设是，GDNF通过促进2-细胞分化促进2-细胞质量增加，并通过增加2-细胞质量、存活和再生来预防糖尿病。此外，GDNF通过提高2-细胞存活率来改善2-细胞(胰岛)移植。在特定的目标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存在的情况下，GDNF介导的2-TC-6细胞和HIT细胞的分化减少。利用体外2-细胞分化模型(2-TC-6细胞和HIT细胞)，结合特异性siRNA、启动子分析和DNA结合分析，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。在特定目的2中，我们将使用体外(thapsigargin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，在SOX9 siRNA存在的情况下，GDNF诱导的2-细胞增殖被抑制。利用神经元/神经胶质细胞与2-细胞共培养模型，我们将确定肠上皮神经元与2-细胞相互作用的机制。在具体目标3中，我们将评估GDNF对小鼠和猪胰岛移植后存活的影响。初步数据显示，预先培养了GDNF的小鼠胰岛移植后存活率增加。猪胰岛可以用于人体移植。我们将用赋形剂或GDNF对小鼠和猪胰岛进行预处理，并对糖尿病小鼠进行肝内门静脉移植。GDNF和肠神经元/神经胶质细胞对胰岛移植后存活的影响将通过评估胰岛细胞的质量和功能来评估。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，发病率还在上升。这项研究的结果可能有助于我们确定新的靶点，以提高β细胞质量，以帮助预防或治疗糖尿病。糖尿病新疗法的确定将直接使退伍军人受益。 公共卫生相关性： 糖尿病--退伍军人的一个主要健康问题--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)发生和发展的基础，了解2细胞质量的调节对于预防和治疗都是必不可少的。尽管对2-细胞质量的调控机制知之甚少，但胶质细胞系衍生神经营养因子(GDNF)可能起着重要作用。这项提案中概述的实验将研究GDNF如何调节2-细胞质量并导致2-细胞在受伤后再生的机制。这些实验不仅有助于了解2-细胞团的调节机制，还可能为糖尿病的预防和治疗带来新的治疗靶点。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，发病率还在上升。这项研究的结果可能有助于我们确定新的靶点，以提高β细胞质量，以帮助预防或治疗糖尿病。糖尿病新疗法的确定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.