Mapping cell metabolism in tissues: NADPH/NADP+ redox state in the regulation of cell dedifferentiation, proliferation, and survival.

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Metabolism critically governs cell differentiation, survival, and proliferation. To fully understand the links between cell metabolism and these varied responses, we need methods that measure metabolic flux with high temporal and spatial resolution. The Rocheleau Lab has a long-standing interest in combining quantitative fluorescence microscopy and microfluidic devices ("islet-on-a-chip") to measure the metabolism of individual beta-cells within living pancreatic islets. Pancreatic islets are a valuable model system of nutrient-stimulated insulin secretion; yet also offer the opportunity to study the plasticity and survival of primary cells within a tissue. Our previous NSERC funding allowed us to develop a spectrally tunable genetically encoded sensor (Apollo-NADP+) to measure cytoplasmic NADPH/NADP+ redox state. We also recently developed microfluidic devices for highly efficient adenoviral transduction (HEAT), which we can use to express sensor throughout islets. NADPH/NADP+ redox state has been implicated in modulating insulin secretion yet the dynamics of this response are unclear. NADPH/NADP+ redox state also supports proliferation through biosynthetic pathways, and cell survival through the scavenging of reactive oxygen species. To address these cellular responses within a tissue, we will exploit and further develop the Apollo-NADP+ sensor and islet-on-a-chip devices. In particular, we anticipate the spectrally versatile Apollo-NADP+ will allow multiparametric imaging with other genetically encoded sensors, and by further adaptation be used to measure the redox state of various organelles. Our long-term objective is to reveal how the metabolism of individual cells ultimately controls tissue phenotype including stimulus-coupled secretion, proliferation and survival. These studies have three short-term objectives focused on NADPH/NADP+ redox state:***1) Determine the role of NADPH/NADP+ redox state in regulating insulin secretion.***2) Determine whether NADPH/NADP+ redox state is a marker for proliferation.***3) Determine the role of NADPH/NADP+ redox state in the production and scavenging of reactive oxygen species.***This research program critically relies on our ability to transduce primary cells (i.e. cells with normal metabolism) throughout the tissue with a novel genetically encoded sensor. Our focus on islet biology will reveal new insight into the dynamics of NADPH/NADP+ metabolism with direct relevance to diabetes, yet the tools we develop will show facile translation to the study of other tissues and diseases. Finally, our research program will provide HQP advanced training in sensor design, cell biology, quantitative fluorescence microscopy, bioengineering, microfluidic device design, and biophysics, all at the forefront of modern research and highly sought in academic and industrial settings.**