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-磷酸酶催化亚基约50%相同，最近已被鉴定为1型糖尿病小鼠非肥胖糖尿病（NOD）模型中的主要自身抗原。在目的1中，我们提出了NOD小鼠和HRP敲除小鼠的杂交，以确定NOD背景中缺乏HRP基因是否足以预防1型糖尿病的发作。如果糖尿病被预防，我们将进行（i）NOD/LtJ IGRP-/-小鼠中免疫系统功能的详细分析，以评估在这些动物中是否存在<$RP反应性T细胞，以及对针对其它胰岛自身抗原的自身免疫应答的影响（ii）基因拯救实验，以确定是否以BAG或cDNA的形式重新引入<$RP恢复糖尿病易感性;只有前者将被剪接，初步数据表明，在胸腺和胰岛中的IGRPRNA的差异剪接可能解释了IGRP如何逃避中枢耐受。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，并（iv）产生NOD/LtJ组合的IGRP-/-和胰岛素I -/-小鼠，以确定缺乏IGRP表达与胰岛素表达减少的组合现在是否足以预防糖尿病的发展。初步数据显示，在小鼠中缺失IGRP基因仅导致轻度低血糖。在目标2中，我们建议调查这种观察的生理基础。由于<$RP催化葡萄糖-6-磷酸水解，并且仅在胰岛β细胞中表达，我们假设<$RP缺失改变了葡萄糖刺激的胰岛素分泌的Km。因此，将使用口服葡萄糖耐量试验和高血糖钳夹来比较在体内的HRP敲除小鼠和野生型同窝小鼠中的胰岛素分泌。此外，将原位比较来自野生型和HRP敲除小鼠灌注胰腺的胰岛素分泌。最后，将在体外比较来自分离的野生型和HRP敲除小鼠胰岛的胰岛素分泌。相关性：一种名为IGRP的蛋白质与1型糖尿病的发展有关。该项目将评估小鼠中缺乏IGRP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.