Quantification of Endothelial Fatty Acid Uptake using Fluorescent Fatty Acid Analogs

Bloodborne nutrients, like fatty acids, must cross a capillary endothelial barrier to enter metabolic tissue via a poorly understood mechanisms. Clarifying these processes could reveal new therapeutic targets for treating metabolic diseases, such as type 2 diabetes. So we found that scalpel muscle and adipose tissue both regulate endothelial lipid uptake and transport using paracrine metabolites, partly controlled by the endocrine system as well.

And we've also found that mitochondrial ATP unexpectedly contribute to endothelial fat uptake despite their known reliance on glycolytic ATP. Using the assay outlined in this protocol, we plan to probe deeper into the molecular mechanisms underlying endothelial fatty acid uptake and transport, including how it might be regulated by other tissue. To begin, prepare a 0.1%gelatin solution by adding 25 milliliters of 2%gelatin stock to 475 milliliters of PBS.

Mix the gelatin solution thoroughly and filter sterilize it using a 0.2 micrometer vacuum filter or autoclave. Using a pipette, add 100 microliters of the 0.1%gelatin solution into each well of a black clear bottom 96-well plate. Place the plate in a 37 degree Celsius incubator for at least 30 minutes or overnight.

After incubation, remove extra gelatin solution from the pre-coated 96-well plate. Wash the plate with PBS once. Then, pipette 100 microliters of the cell suspension into each well using a seeding density that allows the cells to reach confluence by the next day.

Prepare 20 millimolar solutions of 3-hydroxyisobutyrate and lactate as positive controls. For the negative control, use one micromolar niclosamide. Then, pipette the solutions into a separate 96-well plate.

Wash the endothelial cells once with pre-warmed divalent PBS, pipetting at a steep angle to avoid disrupting the plated cells. Add 50 microliters per well of the appropriate treatment reagent and incubate the plate at 37 degrees Celsius for 30 minutes or 60 minutes. To prepare the BODIPY fatty acid BSA complex, mix a 2 micromolar BODIPY fatty acid solution with 1 micromolar fatty acid-free BSA in PBS.

Incubate the mixture at room temperature in the dark for 10 minutes before use. Next, add 50 microliters of the prepared BODIPY fatty acid BSA complex to each treated well and incubate the plate at 37 degrees Celsius for five minutes. Prepare a 1 micromolar solution of fatty acid-free BSA in PBS as a wash buffer and pre-warm it to 37 degrees Celsius.

Remove the BODIPY fatty acid BSA complex from the wells and wash the entire plate twice with 50 microliters of the pre-warmed wash buffer, performing each wash for 1.5 minutes. Then, add 50 microliters of 0.08%trypan blue to each well to quench extracellular fluorescence. Using a microplate reader, immediately measure intracellular fluorescence.

After removing the trypan blue solution from the wells, gently wash the cells with PBS. Add 4 micrograms per milliliter of Hoechst dye diluted in 10%media to each well. Incubate the plate at 37 degrees Celsius for 30 minutes.

Then, wash the plate once with PBS to remove excess dye. After that, add fresh PBS to each well and measure Hoechst fluorescence using a microplate reader. In both HUVEC and EA.hy926 cells, intracellular BODIPY-C12 signal increased significantly with higher BODIPY fatty acid concentrations at one minute, five minutes and 10 minutes of incubation.

Lactate treatment for one hour increased BODIPY-C12 uptake in a dose-dependent manner at 5 millimolar and 20 millimolar concentrations. Treatment with 3-hydroxyisobutyrate also significantly enhanced BODIPY-C12 uptake in a dose-dependent fashion with the highest uptake observed at 20 millimolar. Treatment with 1 micromolar niclosamide for 30 minutes led to a significant reduction in BODIPY-C12 uptake compared to untreated DMSO control.

Following five minute incubation with BODIPY-C16 in HUVEC, lactate treatment at 10 millimolar and 20 millimolar significantly increased uptake in a dose-dependent manner. Treatment with 1 micromolar niclosamide significantly decreased BODIPY-C16 uptake compared to the untreated DMSO control.