Glucose transport in the diabetic outer retina.

 描述（由申请人提供）： 高血压是糖尿病视网膜病变的主要危险因素。严格控制血糖水平目前被视为减缓视网膜病变进展的主要手段，但葡萄糖进入视网膜的机制以及它们在糖尿病中如何受到干扰仍不清楚。因此，了解高血糖如何影响外视网膜（RPE和光感受器）的机制，并确定改善葡萄糖诱导的外视网膜功能障碍和糖尿病视网膜病变早期生物标志物的方法是重要的。最近，它已被证明，促进扩散葡萄糖转运蛋白，GLUT 1的系统性减少将降低视网膜中的葡萄糖水平;然而，这些研究没有提供一种机制或手段来控制葡萄糖进入特定的视网膜通过血视网膜屏障（BRB）。该提案的第一个目的是确定RPE内GLUT 1的减少是否导致RPE/外视网膜内视网膜葡萄糖积累减少，预防糖尿病小鼠模型中的RPE功能障碍和糖尿病视网膜病变的早期生物标志物。第二个目的是研究胰岛素对GLUT 1的调节机制，并确定在糖尿病小鼠模型中，废除RPE中的胰岛素信号转导是否可以通过调节GLUT 1的表达和定位来减轻异常的RPE功能。我们假设（1）高血糖诱导的RPE中GLUT 1表达和再分布的增加介导了糖尿病相关的RPE/外视网膜功能降低和视网膜病变的早期体征，（2）胰岛素也通过RPE中的GLUT 1调节葡萄糖转运，（3）RPE中胰岛素信号传导的阻断将加剧糖尿病小鼠模型中发现的视网膜病理生理学。为了检验这些假设，将采用1型糖尿病的小鼠模型来研究RPE的顶膜和基底膜内的GLUT 1表达和分布，并确定这是否伴随细胞内葡萄糖的积累。为了确定GLUT 1表达的减少是否能预防外视网膜功能障碍，将使用单倍型不足的Glut 1 +/-小鼠和Glut 1基因仅在RPE内特异性失活的小鼠。为了进一步评估胰岛素如何影响GLUT 1介导的葡萄糖进入和在视网膜中蓄积，将采用胰岛素受体（IR）的条件性失活来消除该受体的下游信号传导。培养的RPE细胞的体外分析也将用于确定胰岛素处理是否通过影响GLUT 1与结合伴侣GIPC和/或E3泛素连接酶Nedd 4 -2的结合来调节GLUT 1从而改变葡萄糖转运。这些实验将确定GLUT 1在高血糖导致的外视网膜功能障碍中的作用，阐明胰岛素如何参与GLUT 1调节，并确定可用于治疗干预的机制。

 DESCRIPTION (provided by applicant): Hyperglycemia is the major risk factor for development of diabetic retinopathy. Tight control of serum glucose levels is currently viewed as the primary means to slow progression to retinopathy, but the mechanisms by which glucose enters the retina and how they become perturbed in diabetes remains unclear. Therefore, understanding the mechanism of how hyperglycemia affects the outer retina (RPE and photoreceptors) and identifying methods to ameliorate glucose-induced outer retina dysfunction and early biomarkers of diabetic retinopathy is important. Recently, it has been shown that systemic reduction of the facilitative diffusion glucose transporter, GLUT1 will reduce glucose levels in the retina; however, these studies do not provide a mechanism or means to control glucose entry specifically to the retina via the blood retinal barrier (BRB). The first objective of this proposal is to determine if reductions in GLUT1 within the RPE leads to reduced retinal glucose accumulation within the RPE/outer retina, prevention of RPE dysfunction and early biomarkers of diabetic retinopathy in mouse models of diabetes. The second objective is to investigate mechanisms of GLUT1 modulation by insulin and to determine if abrogation of insulin signal transduction in the RPE can mitigate abnormal RPE function in mouse models of diabetes by regulation of GLUT1 expression and localization. We hypothesize that (1) hyperglycemia-induced increases in the expression and redistribution of GLUT1 in the RPE mediates diabetes-associated reductions in RPE/outer retina function and early signs of retinopathy, (2) that insulin also modulates glucose transport by GLUT1 in the RPE and (3) that blockade of insulin signaling in the RPE will exacerbate retinal pathophysiology found in a mouse model of diabetes. To test these hypotheses, a mouse model of type 1 diabetes will be employed to investigate GLUT1 expression and distribution within the apical and basolateral membranes of the RPE and determine if this is concomitant with an accumulation of intracellular glucose. To determine if reductions in GLUT1 expression prevents outer retina dysfunction, a haploinsufficient Glut1+/- mouse and mice in which the Glut1 gene is specifically inactivated only within the RPE will be utilized. To additionally assess how insulin affects GLUT1-mediated glucose entry and accumulation in the retina, conditional inactivation the insulin receptor (IR) will be employed to abrogate downstream signaling from this receptor. In vitro analysis of cultured RPE cells will also be used to determine if insulin treatment alters glucose transport through regulation of GLUT1 by affecting its association with a binding partner, GIPC and/or the E3 ubiquitin ligase, Nedd4-2. These experiments will determine the role that GLUT1 plays in outer retina dysfunction as a result of hyperglycemia, elucidate how insulin is involved in GLUT1 modulation, and identify a mechanism which can be targeted for therapeutic intervention.