Development of real-time, quantitative free fatty acid uptake assays for adipose tissue explants using fluorescence and microfluidics

Abstract

Free fatty acid (FFA) uptake by adipocytes in response to nutritional or hormonal stimuli is a key process in the complex metabolic cascade leading to diabetes and obesity. The work presented in this thesis is focused on developing analytical tools to quantify free fatty acid uptake by mouse adipocytes in real time. Two approaches were used to quantify the uptake kinetics of free fatty acids. Chapter 1 talks about general concepts of fatty acid uptake and previous approaches with their benefits and drawbacks. Chapter 2 introduces and examines the first approach in our project, which is quantifying free fatty acid uptake dynamics in primary adipocytes using customized micro-wells made with 3D-printed templates. In chapter 3 a more sensitive, simple, inexpensive fluorescence-based microfluidic assay was developed to achieve a real-time quantitative BODIPY-FFA uptake measurement. We employed a fluorescently labeled FFA analog, BODIPY-hexadecanoic acid (i.e. BODIPY-palmitic acid or BODIPY-FFA), to directly follow FFA uptake by adipocyte tissue explants under various treatments using a multi-channel microfluidic device. A five channel microfluidic chip was used, with two central channels to interrogate adipocyte tissue explant. At the same time, three calibration channels were analyzed as a novel method of real-time calibration. Explants were treated with BODIPY-FFA as the fluorescence intensity was measured downstream using a fluorescence microscope for quantification. Unlike traditional techniques like quencher based technique (QBT), flow cytometry, or radioactive fatty acids which rely on the end-point readout, our system allows us to flow the reagents continuously over the adipocyte tissue, therefore, mimicking the physiological environment. With this system, we were able to quantify free fatty acid uptake by adipocytes in real-time with minimal optical drift and without exposing cells to direct excitation light. We have also used the system to simultaneously measure BODIPY-FFAC12 and the glucose analogue 2-NBDG (2-(N-(7-Nitrobenz-2-oxa-1, 3-diazol-4-yl) Amino)-2-Deoxyglucose) uptake kinetics in adipose tissue explants. Finally, chapter 4 outlines future plans to fully automate the system using valving in order to allow rapid, real-time quantification of FFA uptake dynamics in adipose tissue explants.