Metabolic biosensor zebrafish transgenics
Hyperglycemic conditions in diabetic patients cause impaired cellular metabolism in many organs, leading to pathogenesis of complications including Diabetic Nephropathy (DN). Exact disease etiology, however, remains largely unknown due to lack of model systems that allow studies of cellular metabolic changes in vivo. Traditional biochemical measurements are invasive and thus do not always reflect in vivo conditions. Also, it is difficult to extrapolate data from in vitro cell culture systems since many cells change their metabolism when isolated from their native states. A rapidly growing number of genetically-encoded biosensors have been developed for live monitoring of cellular metabolites. These biosensors have so far been mainly characterized in in vitro systems and a relatively few studies have exploited them to visualize metabolic parameters in vivo. Here, taking advantage of the optical clarity of embryos combined with the ease of transgenesis, we propose using the zebrafish as an ideal system for intravital biosensor imaging. In this project, we will develop versatile transgenic zebrafish incorporating genetically-encoded fluorescent biosensors for critical metabolites in order to establish an in vivo imaging platform to investigate metabolism in vivo. We will generate and distribute transgenic zebrafish designed for tissue and cell-type specific expression (UAS-GAL4 system) of fluorescent biosensors for ATP/ADP ratio, intracellular and extracellular glucose, NADH/NAD ratio and other metabolites. Initial validation of these transgenic lines will be performed by analyzing changes in biosensor readouts in genetic mutants and/or under conditions of dysregulated metabolism associated with DN. Successful completion of this Pilot and Feasibility project will yield new, versatile transgenic models for linking in vivo physiology and energy status with interventions designed to ameliorate diabetes pathology.

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