The Role of IGRP in the Pathogenesis of Type 1 Diabetes

描述（由申请人提供）：胰岛特异性葡萄糖-6-磷酸酶催化亚基相关蛋白（IGRP）在氨基酸水平上与葡萄糖-6-磷酸酶催化亚基大约相同50%，最近被确定为小鼠非肥胖糖尿病（NOD）模型中的主要自身抗原1型糖尿病。在目的1中，我们提出将NOD小鼠和IGRP敲除小鼠杂交，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)对NOD/LtJ IGRP-/-小鼠的免疫系统功能进行详细分析，以评估IGRP反应性t细胞是否在这些动物中持续存在，以及对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，以确定作为BAG或cDNA重新引入IGRP是否恢复糖尿病易感性；只有前者会被剪接，初步数据表明，胸腺和胰岛中IGRP RNA的不同剪接可能解释了IGRP如何逃脱中心耐受。或者，如果不能预防糖尿病，我们将（iii）对针对IGRP和其他胰岛自身抗原（尤其是胰岛素）的细胞和体液自身反应性进行详细分析；（iv）生成NOD/LtJ IGRP-/-和胰岛素I -/-联合小鼠，以确定IGRP表达缺失与胰岛素表达降低是否足以预防糖尿病的发展。初步数据显示，小鼠IGRP基因的缺失只会导致轻度低血糖。在目标2中，我们建议研究这一观察的生理基础。由于IGRP催化葡萄糖-6-磷酸水解并仅在胰岛β细胞中表达，我们假设IGRP缺失改变了葡萄糖刺激胰岛素分泌的Km。因此，我们将采用口服糖耐量试验和高血糖钳来比较IGRP基因敲除小鼠和野生型窝鼠体内的胰岛素分泌。此外，还将原位比较野生型和IGRP敲除小鼠灌注胰腺的胰岛素分泌情况。最后，比较野生型和IGRP敲除小鼠胰岛的胰岛素分泌情况。相关性：一种名为IGRP的蛋白质与1型糖尿病的发展有关。该项目将评估小鼠体内IGRP的缺乏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.