While adipose tissue (fat) was traditionally considered important only for energy storage, it is now recognized to be a complex, multicellular, endocrine organ with profound systemic effects, altering function in nearly all other organ systems. Despite its importance, there is a lack of information on the dynamic nature of lipolysis, adipokine secretion, and nutrient uptake, highlighting several unmet needs in methodology. Few techniques exist to interrogate small amounts of adipose tissue, and our understanding of dynamic function in adipose tissue is particularly limited, perhaps due to the belated perspective on its endocrine nature and the added culture and sampling challenges from cell buoyancy. It is clear that better, adipose-customized tools are needed for this purpose. As shown in our previous two funding periods, we propose that our microfluidic systems are ideal to meet these ongoing needs, permitting dynamic interrogation of tissue in ways not possible with standard techniques. Our long-term goal is to use expert insights in endocrine biology (Granneman, Judd) to drive the development of customized bioanalytical tools (Easley) and in vitro models of the endocrine system for applications in nutrition, metabolism, and drug discovery. Our short-term objective is to refine and further develop microfluidic and biosensing methods to answer pressing questions, e.g. lipolytic dynamics via the ABHD5/PLIN1 interaction pathway, questions that cannot be answered with current methods. The premise is that unmatched temporal resolution of our droplet-based microfluidic systems provide unique lenses into lipolytic efflux and protein dynamics. We expect these first-of-their-kind results on adipose function to better inform human physiology. Thus, the proposal is innovative in its technological and its biological approaches. Aim 1 of this proposal will multiplex quantification of both glycerol and non-esterified fatty acids (NEFA) from adipose tissue at high temporal resolution (<5 sec), achieved by integrating droplet-based microfluidic analog- to-digital circuits (µADC) with salt-water electrode mergers. In Aim 2, we will customize bioanalytical tools for adipose tissue signaling pathways. µADC devices will quantify secretions at high resolution under ABHD5 ligand treatment. Mix-and-read fluorescence assays will be customized for rapid (off-chip) quantification of PLIN1 and HSL phosphorylation, and for cAMP levels. Aim 3 will focus on using these novel tools for mechanistic analysis of substrate and protein efflux in white adipocytes. High-resolution microfluidics, used with genetically-encoded fluorescent protein sensors, will correlate protein trafficking and interactions with secretory output. Improved microfluidic digital-to-analog circuits (µDAC) will also be designed for rapid tissue stimulation during imaging. The rationale for this research is that custom tool development will provide novel information on adipose tissue dynamics, and we have ...