Transport Of Exogenous Fatty Acids Research Articles

The Lysophosphatidylcholine Transporter MFSD2A Is Essential for CD8+ Memory T Cell Maintenance and Secondary Response to Infection

Access to nutrients is critical for an effective T cell immune response to infection. Although transporters for sugars and amino acids have previously been described in the context of the CD8+ T cell immune response, the active transport of exogenous fatty acids has remained enigmatic. In this study, we discovered that the sodium-dependent lysophosphatidylcholine (LPC) transporter major facilitator superfamily domain containing 2A (MFSD2A) is upregulated on activated CD8+ T cells and is required for memory T cell maintenance. MFSD2A deficiency in mice resulted in decreased import of LPC esterified to long chain fatty acids into activated CD8+ T cells, and MFSD2A-deficient cells are at a competitive disadvantage resulting in reduced memory T cell formation and maintenance and reduced response to secondary infection. Mechanistically, import of LPCs was required to maintain T cell homeostatic turnover, which when lost resulted in a decreased memory T cell pool and thus a reduced secondary response to repeat infection.

The lysophosphatidylcholine transporter, MFSD2A, is essential for CD8+ memory T cell maintenance and secondary response to infection

Abstract Access to nutrients is critical for an effective T cell immune response to infection. Although transporters for sugars and amino acids have previously been described in the context of the CD8+ T cell immune response, the active transport of exogenous fatty acids has remained enigmatic. Here we discovered the sodium-dependent lysophosphatidylcholine transporter, Major Facilitator Super Family Domain Containing 2a (MFSD2A), is upregulated on activated CD8+ T cells and is required for memory T cell maintenance. MFSD2A deficiency in mice resulted in decreased import of lysophosphatidylcholine (LPC) esterified to long chain fatty acids (LCFAs) into activated CD8+ T cells, and MFSD2A deficient cells are at a competitive disadvantage resulting in reduced memory T cell formation and maintenance and reduced response to secondary infection. Mechanistically, import of LPCs was required to maintain T cell homeostatic turnover, that when lost resulted in a decreased memory T cell pool and thus a reduced secondary response to repeat infection.

Lipid distribution, composition and uptake in bovine articular cartilage studied using Raman micro-spectrometry and confocal microscopy.

The distribution and composition of endogenous lipids in articular cartilage and transport of exogenous fatty acids have been investigated on a microscopic scale in fresh bovine articular cartilage. To investigate the distribution and composition of the endogenous lipids, hyperspectral Raman maps were taken of chondrocytes and their surrounding matrix in both the deep and superficial zones. These revealed differences in both lipid distribution and composition between the two zones. Extracellular lipid was observed surrounding the cells in the superficial zone but not in the deep zone. Additionally, intracellular lipid droplets were observed that were larger and more numerous in the deep zone (P = 0.01). The extracellular lipid was primarily free saturated fatty acid, whereas the cellular lipid droplets contained triglycerides with unsaturated fatty acid chains. Fatty acid uptake and transport were investigated by incubating cartilage samples in Dulbecco's modified Eagle's medium containing fluorescently labelled palmitate for a range of times and temperatures. After incubation, the palmitate distribution was imaged using confocal microscopy. Palmitate accumulated preferentially in the territorial matrix only in the superficial zone where the concentration was up to 100‐fold greater than that in the bulk matrix (P = 0.001). Palmitate uptake by the chondrocytes in both zones showed differential temperature sensitivity (P = 0.05), which would support the idea that cells take up palmitate by both active and passive mechanisms. The study reveals large differences between chondrocytes in the superficial and deep zones in their lipid content, in their extracellular lipid environment and in their access to exogenous fatty acids.

Fatty acid transport proteins: targeting FATP2 as a gatekeeper involved in the transport of exogenous fatty acids

The fatty acid transport proteins (FATP) are classified as members of the Solute Carrier 27 (Slc27) family of proteins based on their ability to function in the transport of exogenous fatty acids. These proteins, when localized to the plasma membrane or at intracellular membrane junctions with the endoplasmic reticulum, function as a gate in the regulated transport of fatty acids and thus represent a therapeutic target to delimit the acquisition of fatty acids that contribute to disease as in the case of fatty acid overload. To date, FATP1, FATP2, and FATP4 have been used as targets in the selection of small molecule inhibitors with the goal of treating insulin resistance and attenuating dietary absorption of fatty acids. Several studies targeting FATP1 and FATP4 were based on the intrinsic acyl CoA synthetase activity of these proteins and not on transport directly. While several classes of compounds were identified as potential inhibitors of fatty acid transport, in vivo studies using a mouse model failed to provide evidence these compounds were effective in blocking or attenuating fatty acid transport. Studies targeting FATP2 employed a naturally occurring splice variant, FATP2b, which lacks intrinsic acyl CoA synthetase due to the deletion of exon 3, yet is fully functional in fatty acid transport. These studies identified two compounds, 5'-bromo-5-phenyl-spiro[3H-1,3,4-thiadiazole-2,3'-indoline]-2'-one), now referred to as Lipofermata, and 2-benzyl-3-(4-chlorophenyl)-5-(4-nitrophenyl)pyrazolo[1,5-a]pyrimidin-7(4H)-one, now called Grassofermata, that are effective fatty acid transport inhibitors both in vitro using a series of model cell lines and in vivo using a mouse model.

Characterization of the Biochemical Activities Associated with Splice Variants of Human FATP2

One mechanism governing the transport of exogenous fatty acids (FA) is vectorial acylation whereby the imported FA is converted into acyl CoA upon transport as a consequence of the activities of the fatty acid transport proteins (FATP) and long chain acyl CoA synthetases (Acsl). Splice variants of human FATP2 have been identified that differ in the ATP binding region. FATP2a contains the entire ATP/AMP motif, while FATP2b lacks exon 3 resulting in a protein lacking critical residues within the ATP/AMP motif. Previously we have shown both forms are proficient in FA transport, but only FATP2a functions in very long chain FA activation. These findings demonstrate that these naturally occurring splice variants of FATP2 have distinct and separable biochemical activities. In order to investigate these splice variants further, both were cloned into pcDNA4/TO/myc‐His‐A allowing tetracycline regulated expression of the target protein containing a polyhistidine and c‐myc epitope tag. 293 T‐REx cell lines stably expressing either FATP2a or FATP2b have been generated. Current experimental studies are [1] defining the intracellular localization of both FATP2a and FATP2b, [2] correlating those observations with changes in FA transport and activation activities and [3] addressing the roles of both variants in the trafficking of exogenous FA into lipid metabolic pools. Supported by NIH grant GM56850.

Functional domains of the fatty acid transport proteins: Studies using protein chimeras

Fatty acid transport proteins (FATP) function in fatty acid trafficking pathways, several of which have been shown to participate in the transport of exogenous fatty acids into the cell. Members of this protein family also function as acyl CoA synthetases with specificity towards very long chain fatty acids or bile acids. These proteins have two identifying sequence motifs: The ATP/AMP motif, an approximately 100 amino acid segment required for ATP binding and common to members of the adenylate-forming super family of proteins, and the FATP/VLACS motif that consists of approximately 50 amino acid residues and is restricted to members of the FATP family. This latter motif has been implicated in fatty acid transport in the yeast FATP orthologue Fat1p. In the present studies using a yeast strain containing deletions in FAT1 (encoding Fat1p) and FAA1 (encoding the major acyl CoA synthetase (Acsl) Faa1p) as an experimental platform, the phenotypic and functional properties of specific murine FATP1–FATP4 and FATP6–FATP4 protein chimeras were evaluated in order to define elements within these proteins that further distinguish the fatty acid transport and activation functions. As expected from previous work FATP1 and FATP4 were functional in the fatty acid transport pathway, while and FATP6 was not. All three isoforms were able to activate the very long chain fatty acids arachidonate (C 20:4) and lignocerate (C 24:0), but with distinguishing activities between saturated and highly unsaturated ligands. A 73 amino acid segment common to FATP1 and FATP4 and between the ATP/AMP and FATP/VLACS motifs was identified by studying the chimeras, which is hypothesized to contribute to the transport function.

Topology of the yeast fatty acid transport protein Fat1p: mechanistic implications for functional domains on the cytosolic surface of the plasma membrane

The fatty acid transport protein (FATP) Fat1p in the yeast Saccharomyces cerevisiae functions in concert with acyl-coenzyme A synthetase (ACSL; either Faa1p or Faa4p) in vectorial acylation, which couples the transport of exogenous fatty acids with activation to CoA thioesters. To further define the role of Fat1p in the transport of exogenous fatty acids, the topological orientation of two highly conserved motifs [ATP/AMP and FATP/very long chain acyl CoA synthetase (VLACS)], the carboxyl 124 amino acid residues, which bind the ACSL Faa1p, and the amino and carboxyl termini within the plasma membrane were defined. T7 or hemagglutinin epitope tags were engineered at both amino and carboxyl termini, as well as at multiple nonconserved, predicted random coil segments within the protein. Six different epitope-tagged chimeras of Fat1p were generated and expressed in yeast; the sidedness of the tags was tested using indirect immunofluorescence and protease protection by Western blotting. Plasma membrane localization of the tagged proteins was assessed by immunofluorescence. Fat1p appears to have at least two transmembrane domains resulting in a N(in)-C(in) topology. We propose that Fat1p has a third region, which binds to the membrane and separates the highly conserved residues comprising the two halves of the ATP/AMP motif. The N(in)-C(in) topology results in the placement of the ATP/AMP and FATP/VLACS domains of Fat1p on the inner face of the plasma membrane. The carboxyl-terminal region of Fat1p, which interacts with ACSL, is likewise positioned on the inner face of the plasma membrane. This topological orientation is consistent with the mechanistic roles of both Fat1p and Faa1p or Faa4p in the coupled transport/activation of exogenous fatty acids by vectorial acylation.

Mechanisms and Regulation of Protein-Mediated Cellular Fatty Acid Uptake: Molecular, Biochemical, and Physiological Evidence

![Figure][1] Arend Bonen Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada abonen{at}uoguelph.ca ![Figure][1] Adrian Chabowski Department of Physiology, Medical University of Bialystok, Bialystok, Poland ![

Transmembrane movement of exogenous long-chain fatty acids: proteins, enzymes, and vectorial esterification.

The processes that govern the regulated transport of long-chain fatty acids across the plasma membrane are quite distinct compared to counterparts involved in the transport of hydrophilic solutes such as sugars and amino acids. These differences stem from the unique physical and chemical properties of long-chain fatty acids. To date, several distinct classes of proteins have been shown to participate in the transport of exogenous long-chain fatty acids across the membrane. More recent work is consistent with the hypothesis that in addition to the role played by proteins in this process, there is a diffusional component which must also be considered. Central to the development of this hypothesis are the appropriate experimental systems, which can be manipulated using the tools of molecular genetics. Escherichia coli and Saccharomyces cerevisiae are ideally suited as model systems to study this process in that both (i) exhibit saturable long-chain fatty acid transport at low ligand concentrations, (ii) have specific membrane-bound and membrane-associated proteins that are components of the transport apparatus, and (iii) can be easily manipulated using the tools of molecular genetics. In both systems, central players in the process of fatty acid transport are fatty acid transport proteins (FadL or Fat1p) and fatty acyl coenzyme A (CoA) synthetase (FACS; fatty acid CoA ligase [AMP forming] [EC 6.2.1.3]). FACS appears to function in concert with FadL (bacteria) or Fat1p (yeast) in the conversion of the free fatty acid to CoA thioesters concomitant with transport, thereby rendering this process unidirectional. This process of trapping transported fatty acids represents one fundamental mechanism operational in the transport of exogenous fatty acids.