Fatty Acids are carboxylic acids with a long saturated or unsaturated aliphatic tail. Fatty acids are very important sources of energy because they produce a large amount of ATP when metabolized. To enable noninvasive, real-time and spatiotemporal quantitative imaging of fatty acid fluxes in animals, we developed a bioactivatable molecular imaging probe FFA-SS-Luc based on bioluminescence1.
The bioluminescent probe D-luciferin is attached to a long fatty acid chain via a disulfide bridge that is stable in the extracellular matrix but rapidly reduces by intracellular glutathione after cellular uptake. Alkylation of Luciferin on the phenolic oxygen prohibit the generation of light because they are not a substrate for Luciferase.2 After FFA-SS-Luc cell internalization, glutathione reduces the disulfide bridge and D-luciferin is generated instantaneous through cyclization of the linker. Free luciferin is then converted by luciferase to oxyluciferin and produce a photon of light that can be detected and quantify by CCD camera (Figure 1).3
The FFA-SS-Luc probe can be used for in vitro as well as in vivo to sensitively detect fatty acid uptake by bioluminescence.
Fatty-acid transporter inhibitor (FFAi) screening assay
The FFAi screening assay is a non-radioactive assay that can be used for screening of potential fatty-acid transporter inhibitors in vitro and in vivo. These robust assay uses bioluminescence as readout in high throughput. Below is the validation of the FFA-SS-Luc probe in vitro where the probe kinetic uptake has been monitored in two different cell lines. The probe show high specificity for Adipocytes (high FATP expression) compared to fibroblasts (Low expression of FATP).
FFA-SS-Luc for in vivo imaging
The FFA-SS-Luc probe can be used to localize and quantitate in real time lipid fluxes in living animals with or without treatment. Below is represented a live-bioluminescence imaging of FFA-SS-Luc uptake in full-luciferase expressing mouse overtime.
FFA-SS-luc probe enables the interrogation of FFA uptake in real time in vivo by bioluminescence imaging. Bioluminescence imaging allows spatiotemporal and quantitative measure of fatty acids uptake in vivo. This probe can also be used for drug screening to limit fatty acid uptake and can benefit understanding fundamental biological processes.