Impact of Lipofuscin in Retinal Pigment Epithelial Cells

项目摘要/摘要 肥胖症在美国人口中的流行是一个日益增长的健康风险。我们观察到，在高脂肪饮食中- 诱导肥胖(DIO)小鼠，维生素A醛加合物(双维甲酸)水平升高。双维A酸类化合物 构成在感光细胞外节中随机形成的视觉循环加合物的复杂混合物 与视黄醛的非酶反应有关。这些荧光团沉积在视网膜色素上皮细胞中。 作为吞噬外节膜的成分，构成视网膜的脂褐素。这是众所周知的 维生素A的可获得性(即视觉周期动力学)调节了类双维A的形成。它的毒性 这类双维A酸类化合物至少部分归因于它们光生活性氧的倾向。 物种和光解成二羰基-(乙二醛，GO；甲基乙二醛，MG)和含醛 碎片。 在本申请中提出的研究的广泛目标是探索 与肥胖相关的有毒的双维甲酸和维生素A调节失调。在具体建议中提出的实验 目标1将对高脂肪饮食、维生素A、视黄醇结合之间的关系进行新的探索 视网膜中的蛋白4(Rbp4)和增强型双视黄醇。我们的工作模式是增加维生素A的释放 (视黄醇)到RPE在这些条件下涉及Rbp4和非Rbp4机制。在喂食高脂肪的小鼠中 在肥胖的ob/ob小鼠中，我们将测量眼睛中维甲酸和双维甲酸的水平，我们将使用 定量眼底自体荧光成像(QAF)非侵入性测量双视黄醇。的作用 Rbp4将通过测量血清Rbp4和研究Rbp4-/-基因缺陷的小鼠在小鼠身上进行测试 基因缺失和药物治疗。光感受器细胞健康状况将通过外核的组织计量学分析进行评估 一层。在具体目标2中描述的研究中，我们将通过以下方式解决超重的人类受试者 使用非侵入性定量眼底自体荧光(QAF)测量双维A酸类化合物。在展示的实验中 在具体目标3中，我们将挑战现有的关于支配双维甲酸程度的条件的观念。 光氧化造成的双视黄酸的损失和决定其地形分布的因素 SW-AF。为此，我们将量化维生素A摄入量和双维甲酸形成之间的关系。我们会 通过比较在黑暗中饲养的小鼠和在周期光下饲养的小鼠的水平来测量双维A酸类化合物的损失。双维A酸类 脂褐素将通过色谱和非侵入性QAF进行测量。这项工作将使用眼底成像 以及生化、组织学和细胞分析。这项研究的完成将促进我们对 肥胖、维生素A新陈代谢和双维A类荧光团之间的联系以前并不为人所知。

Project Summary/Abstract The prevalence of obesity in the US population is a growing health risk. We have observed that in high fat diet- induced obese (DIO) mice, vitamin A aldehyde adducts (bisretinoids) accumulate at elevated levels. Bisretinoids constitute the complex mixture of visual cycle adducts that form randomly in photoreceptor outer segments due to non-enzymatic reactions of retinaldehyde. These fluorophores are deposited in retinal pigment epithelial cells as components of phagocytosed outer segment membrane and constitute the lipofuscin of retina. It is well known that bisretinoid formation is modulated by the availability of vitamin A (i.e. visual cycle kinetics). The toxicity of this family of bisretinoids is attributable, at least in part, to their propensity to photogenerate reactive oxygen species and photodecompose into dicarbonyl- (glyoxal, GO; methylglyoxal, MG) and aldehyde-bearing fragments. The broad objectives of the studies proposed in this application are to explore links between the formation of toxic bisretinoids and dysregulation of vitamin A in association with obesity. Experiments proposed in Specific Aim 1 will undertake the novel exploration of relationships amongst a high fat diet, vitamin A, retinol binding protein 4 (Rbp4) and augmented bisretinoid in retina. Our working model is that increased delivery of vitamin A (retinol) to RPE under these conditions involves both Rbp4 and non-Rbp4 mechanisms. In mice fed a high fat diet and in obese ob/ob mice we will measure ocular levels of retinoid and bisretinoid and we will employ quantitative fundus autofluorescence imaging (qAF) for non-invasive measurement of bisretinoid. The role of RBP4 will be tested in mice by measuring serum Rbp4 and by studying mice deficient in Rbp4-/- due to gene deletion and drug treatment. Photoreceptor cell health will be evaluated by histometric analysis of outer nuclear layer. In the studies described in Specific Aim 2, we will address human subjects having excess weight by measuring bisretinoids using non-invasive quantitative fundus autofluorescence (qAF). In experiments presented in Specific Aim 3 we will challenge existing notions as to the conditions that govern the the extent of bisretinoid formation, the loss of bisretinoid due to photooxidation and factors determining the topographic distribution of SW-AF. To this end, we will quantify the relationship between vitamin A intake and bisretinoid formation. We will measure loss of bisretinoid by comparing levels in mice reared in darkness versus cyclic light. Bisretinoid lipofuscin will be measured chromatographically and by non-invasive qAF. The work will employ fundus imaging and biochemical, histological and cellular assays. Completion of this research will advance our understanding of previously unrecognized links amongst obesity, vitamin A metabolism and bisretinoid fluorophores.