Mo1041 Fatty Acid Translocase FAT/CD36 Interacts With the Intracellular Fatty Acid Activating Enzymes FATP4 and ACSL1 but Not With Cav-1 in Enhancing Oleate Uptake - A Quantitative Analysis

Abstract

Mechanisms of long chain fatty acid translocation across the plasma membrane are important targets in treatment of many human diseases like obesity or fatty liver disease. Translocation is achieved by a concert of co-existing mechnisms. Two mechanisms are suggested for cellular fatty acid uptake: passive diffusion across the plasmamembrane and protein facilitated uptake. FAT/CD36 is a transmembrane glycoprotein that directly mediates extracellular fatty acid uptake across the plasma membrane and has been described to interact with the structural protein Caveolin-1 (Cav-1). The intracellular acyl-CoA synthetases (ACS) FATP4 and ACSL1 indirectly drive fatty acid uptake by esterification of intracellular fatty acids with CoA (metabolic trapping). Within this study we analysed the role of CD36, FATP4 and ACSL1 in fatty acid uptake by quantification of absolute protein amounts and testing for functional cooperativity. Stable or transient protein overexpression in MDCK, HuH7 and HepG2 cells were achieved by retroviral and adenoviral infection. Immunofluorescence analysis confirmed a localization for CD36 at the plasma membrane, FATP4 at the endoplasmic reticulum and ACSL1 on mitochondria. Fatty acid uptake was enhanced upon overexpression of CD36, FATP4 and ACSL1 whenmeasured after treatment with radiolabeled oleate over three hours. Oleate uptake increased in correlation to rising protein amounts of CD36 and FATP4 as quantified by comparison to a recombinant protein standard during western blotting. Although protein quantities were strikingly lower for CD36 than for FATP4 oleate uptake was comparable for both proteins, indicating a higher potency for CD36 in mediating fatty acid uptake. The function of CD36 was independent from Cav-1 expression, as CD36 mediated fatty acid uptake was unaltered in Cav-1 knock down cells. Interestingly, co-expression of CD36 with either FATP4 or ACSL1 significantly increased fatty acid uptake more than overexpression for a single protein alone. Combined overexpression of the two intracellular localised FATP4 and ACSL1 resulted in only a minimal increase of oleate uptake, indicating that both proteins are restricted in increasing fatty acid uptake by the transport rate of fatty acids across the plasma membrane. We therefore conclude that the plasma membrane localized protein CD36 functionally interacts with the intracellular fatty acid activating enzymes FATP4 and ACSL1 in enhancing oleate uptake by efficient combination of facilitated fatty acid transport across the plasma membrane with intracellular fatty acid metabolism. Inhibition of this mechanism have to be considered as targets for the treatment of lipid disorders.