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.