Impact of Lipofuscin in Retinal Pigment Epithelial Cells

项目总结/摘要 肥胖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- 在诱导肥胖（DIO）小鼠中，维生素A醛加合物（双维甲酸）以升高的水平积累。双维甲酸 构成视觉周期加合物的复杂混合物，这些加合物在光感受器外节中随机形成， 非酶促反应的反应。这些荧光团沉积在视网膜色素上皮细胞中 作为被吞噬的外节膜的成分，构成视网膜的脂褐素。众所周知的是 双视黄酸的形成受维生素A的可用性调节（即视觉周期动力学）。的毒性 这类双视黄酸至少部分归因于它们光生活性氧的倾向 物种和光分解成二羰基-（乙二醛，GO;甲基乙二醛，MG）和含硫醚 片段 在本申请中提出的研究的广泛目标是探索 有毒的双维甲酸和维生素A调节异常与肥胖相关。具体实验 目的1将进行高脂饮食，维生素A，视黄醇结合之间的关系的新的探索 蛋白4（Rbp 4）和增强的双视黄酸。我们的工作模型是，增加维生素A的供应， 在这些条件下，维生素A（视黄醇）对RPE的作用涉及Rbp 4和非Rbp 4机制。在喂食高脂肪的小鼠中， 在肥胖ob/ob小鼠中，我们将测量类维生素A和双类维生素A的眼部水平， 定量眼底自发荧光成像（qAF）用于双视黄酸的非侵入性测量。的作用 将通过测量血清Rbp 4和通过研究由于基因突变而导致Rbp 4-/-缺陷的小鼠来在小鼠中测试Rbp 4。 删除和药物治疗。光感受器细胞健康将通过外核的组织学分析来评估。 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体重超标的人类受试者： 使用非侵入性定量眼底自发荧光（qAF）测量双维甲酸。在实验中， 在具体目标3中，我们将挑战现有的概念，即控制双维甲酸程度的条件， 形成，损失的双视黄酸由于光氧化和因素决定的地形分布 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。我们将 通过比较在黑暗和周期性光照下饲养的小鼠中的水平来测量双维甲酸的损失。双视黄酸 脂褐素将通过色谱法和非侵入性qAF测量。这项工作将采用眼底成像 以及生物化学、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代谢和双维甲酸荧光团之间的联系以前未被认识。

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.