The Role of IGRP in the Pathogenesis of Type 1 Diabetes

描述(申请人提供)：胰岛特异性葡萄糖-6-磷酸酶催化亚单位相关蛋白(IGRP)在氨基酸水平上与葡萄糖-6-磷酸酶催化亚单位大约50%相同，最近被鉴定为1型糖尿病小鼠非肥胖糖尿病(NOD)模型中的主要自身抗原。在目标1中，我们提出了杂交NOD小鼠和IGRP基因敲除小鼠，以确定NOD背景中IGRP基因的缺失是否足以预防1型糖尿病的发生。如果糖尿病得到预防，我们将进行：(I)详细分析NOD/LTJ IGRP-/-小鼠的免疫系统功能，以评估IGRP反应性T细胞是否在这些动物中持续存在以及针对其他胰岛自身抗原的自身免疫反应的影响；(Ii)基因拯救实验，以确定是否将IGRP作为袋子或cDNA重新引入，以恢复糖尿病的易感性；只有前者将被剪接，并且初步数据表明，在胸腺和胰岛中对IGRP RNA进行差异剪接可能会解释IGRP如何逃避中枢耐受。或者，如果不能预防糖尿病，我们将(Iii)针对IGRP和其他胰岛自身抗原，特别是胰岛素，进行详细的细胞和体液自反应分析，(Iv)建立NOD/LTJ IGRP-/-和INS I-/-小鼠，以确定IGRP表达缺失和胰岛素表达减少现在是否足以防止糖尿病的发展。初步数据显示，小鼠IGRP基因的缺失只会导致轻微的低血糖。在目标2中，我们建议研究这一观察的生理学基础。由于IGRP催化葡萄糖-6-磷酸水解酶，并且仅在胰岛β细胞中表达，我们假设IGRP缺失改变了葡萄糖刺激的胰岛素分泌的Km。因此，口服葡萄糖耐量试验和高血糖钳夹试验将被用来比较IGRP基因敲除小鼠和野生型小鼠体内的胰岛素分泌。此外，还将对野生型和IGRP基因敲除小鼠灌流胰腺的胰岛素分泌进行原位比较。最后，对分离的野生型和IGRP基因敲除小鼠胰岛的胰岛素分泌进行体外比较。相关性：一种名为IGRP的蛋白质与1型糖尿病的发生有关。该项目将评估小鼠体内IGRP的缺失是否足以预防糖尿病的发生。

DESCRIPTION (provided by applicant): The islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is approximately 50% identical at the amino acid level to the glucose-6-phosphatase catalytic subunit and has recently been identified as a major autoantigen in the mouse Non-Obese Diabetic (NOD) model of type 1 diabetes. In Aim 1 we propose cross- breeding NOD mice and IGRP knockout mice to determine whether the absence of the IGRP gene in the NOD background is sufficient to prevent the onset of type 1 diabetes. If diabetes is prevented, we will perform (i) a detailed analysis of immune system function in the NOD/LtJ IGRP-/- mice to evaluate whether IGRP reactive T-cells persist in these animals and the impact on the autoimmune response directed at other islet autoantigens (ii) gene rescue experiments to determine whether re-introduction of IGRP as a BAG or cDNA restores diabetes susceptibility; only the former will be spliced and preliminary data suggest that differential splicing of IGRP RNA in thymus and islets may explain how IGRP escapes central tolerance. Alternatively, if diabetes is not prevented, we will (iii) perform a detailed analysis of cellular and humoral autoreactivity targeted at IGRP and other islet autoantigens, especially insulin and (iv) generate combined NOD/LtJ IGRP-/- and insulin I -/- mice to determine whether the absence of IGRP expression combined with a reduction in insulin expression is now sufficient to prevent the development of diabetes. Preliminary data show that deletion of the IGRP gene in mice only results in mild hypoglycemia. In Aim 2 we propose investigating the physiological basis for this observation. Since IGRP catalyzes glucose-6-phosphate hydrolysis and is expressed exclusively in pancreatic islet beta cells, we hypothesize that IGRP deletion alters the Km of glucose-stimulated insulin secretion. Therefore, oral glucose tolerance tests and hyperglycemic clamps will be used to compare insulin secretion in IGRP knockout mice and wild type littermates in vivo. In addition, insulin secretion from wild type and IGRP knockout mouse perfused pancreata will be compared in situ. Finally, insulin secretion from isolated wild type and IGRP knockout mouse islets will be compared in vitro. Relevance: A protein called IGRP has been implicated in the development of type 1 diabetes. This project will assess whether the absence of IGRP in mice is sufficient to preven the onset of diabetes.