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Abstract

Fatty acids (FAs) are essential metabolites for membrane synthesis, energy generation and signalling. Most cells in higher organisms receive FAs circulating in the bloodstream only after their transport across endothelial cells. Many questions regarding this process have remained unresolved. Here, FA-CoA synthesis and metabolism, extracellular concentration of FAs and basolateral FA trapping were analysed in a time resolved manner for their effect on the efficiency of trans-endothelial FA transport. The model system were human umbilical vein endothelial cells (HUVECs) grown on transwell filters incubated with radiolabelled oleate. Uptake of radiolabelled oleic acid into endothelial cells was initially high but attenuated rapidly. During prolonged incubation, non-esterified FA (NEFA) levels remained relatively constant whereas labelled lipids increased over time. The assessed rate of transport was higher than the rate of metabolism. Results showed that the apical FA concentration drives FA uptake and upregulation of the putative fatty acid transport protein FATP4 has only a minor effect. Due to low FA metabolic capacity, endothelial cells retained a large pool of non-esterified FAs that is rapidly diminished for FA transport. It was remarkably that lipolysis of triacylglycerol (TG) contributed to the efflux of FAs. The lipolysis of TG for FA transport was reduced by inhibition of the adipose triglyceride lipase (ATGL). Increasing the extracellular apical FA concentration or the storage capacity in the basolateral compartment increased the transport of FAs. Increase in esterification rate due to FATP4 upregulation did not show any significant effect on trans-endothelial FA transport, but reduction in total ACS activity due to Acsl3 knockdown increased the efflux of FAs from endothelial cells in a time dependent manner. The lipid fraction of the basolateral compartment contained only fatty acids, as analyzed by thin-layer chromatography (TLC). This suggests that efflux of lipids consists only of NEFA, but not lipoproteins or complex lipids.