Human fatty acid transport protein 2a/very long chain Acyl-CoA synthetase 1 (FATP2a/Acsvl1) has a preference in mediating the channeling of exogenous n-3 fatty acids into phosphatidylinositol
ABSTRACT The trafficking of fatty acids across the membrane and into downstream metabolic pathways requires their activation to CoA thioesters. Members of the fatty acid transport protein/very long chain acyl-CoA synthetase (FATP/Acsvl) family are emerging as key players in the trafficking of exogenous fatty acids into the cell and in intracellular fatty acid homeostasis. We have expressed two naturally occurring splice variants of human FATP2 (Acsvl1) in yeast and 293T-REx cells and addressed their roles in fatty acid transport, activation, and intracellular trafficking. Although both forms (FATP2a (M(r) 70,000) and FATP2b (M(r) 65,000 and lacking exon3, which encodes part of the ATP binding site)) were functional in fatty acid import, only FATP2a had acyl-CoA synthetase activity, with an apparent preference toward very long chain fatty acids. To further address the roles of FATP2a or FATP2b in fatty acid uptake and activation, LC-MS/MS was used to separate and quantify different acyl-CoA species (C14-C24) and to monitor the trafficking of different classes of exogenous fatty acids into intracellular acyl-CoA pools in 293T-REx cells expressing either isoform. The use of stable isotopically labeled fatty acids demonstrated FATP2a is involved in the uptake and activation of exogenous fatty acids, with a preference toward n-3 fatty acids (C18:3 and C22:6). Using the same cells expressing FATP2a or FATP2b, electrospray ionization/MS was used to follow the trafficking of stable isotopically labeled n-3 fatty acids into phosphatidylcholine and phosphatidylinositol. The expression of FATP2a resulted in the trafficking of C18:3-CoA and C22:6-CoA into both phosphatidylcholine and phosphatidylinositol but with a distinct preference for phosphatidylinositol. Collectively these data demonstrate FATP2a functions in fatty acid transport and activation and provides specificity toward n-3 fatty acids in which the corresponding n-3 acyl-CoAs are preferentially trafficked into acyl-CoA pools destined for phosphatidylinositol incorporation.
- SourceAvailable from: sciencedirect.com
[Show abstract] [Hide abstract]
- "2.1. Cell growth, expression of FATP2, and incubation of different classes of fatty acids Stable T-REx HEK293 cells allowing the regulated expression of FATP2 or transformed with the vector (control) have been previously described . Cells were routinely maintained in DMEM medium containing 10% FBS; expression of FATP2 was accomplished by the addition of tetracycline to a final concentration of 2 lg/ml for 48 h; the cells are harvested and then subjected to analytical studies as detailed below. "
ABSTRACT: In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4hr. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The trafficking of exogenous C16:0 and C22:6 into PA was significant where there was 6.9- and 5.3-fold increased incorporation, respectively, over the control; C18:3 and C20:4 also trended to increase in the PA pool while there were no changes for C18:1 and C18:2. The trafficking of C18:3 into PC and PI trended higher and approached significance. In the case of C20:4, expression of FATP2 resulted in increases in all four classes of phospholipid, indicating little selectivity. In the case of C22:6, there were significant increases of this exogenous fatty acids being trafficking into PC and PI. Collectively, these data support the conclusion that FATP2 has a dual function in the pathways linking the transport and activation of exogenous fatty acids. We discuss the differential roles of FATP2 and its role in both fatty acid transport and fatty acid activation in the context of lipid homeostasis.Biochemical and Biophysical Research Communications 10/2013; 440(4). DOI:10.1016/j.bbrc.2013.09.137 · 2.28 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The plant cuticle is a lipid-based barrier on the aerial surfaces of plants that plays a variety of protective roles. The cuticle is comprised largely of long-chain and very-long-chain fatty acids and their derivatives. In Arabidopsis, LONG-CHAIN ACYL-COA SYNTHETASE1 (LACS1), LACS2, and LACS3 are known or suspected cuticle biosynthetic genes. Very-long-chain acyl-coenzyme A (CoA) synthetase activity has been demonstrated for LACS1 and LACS2, although the role for such an activity in cuticle biosynthesis is currently unclear. In yeast and mammalian systems, some very-long-chain acyl-CoA synthetases are also called fatty acid transport proteins (FATPs) due to a second function of mediating transmembrane movement of fatty acids. We sought to determine if LACS1-3 also have this dual functionality. A yeast fat1Δ mutant is deficient in both very-long-chain acyl-CoA synthetase activity and exogenous fatty acid uptake. We demonstrate that heterologous expression of LACS1, 2, or 3 is able to complement both of these deficiencies. Furthermore, expression of each LACS enzyme in yeast resulted in uptake of the long-chain fatty acid analogue, C(1)-BODIPY-C(12). Only expression of LACS1 resulted in uptake of the very-long-chain fatty acid analogue, BODIPY-C(16). These results demonstrate that LACS1, LACS2, and LACS3 have the dual functionality of yeast and mammalian FATP enzymes. These findings have implications in the transmembrane transport and intracellular trafficking of plant lipids destined for export to the cuticle.Plant Physiology and Biochemistry 02/2012; 51:31-9. DOI:10.1016/j.plaphy.2011.10.003 · 2.35 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Knowledge of the consequences of maternal obesity in human placental fatty acids (FA) transport and metabolism is limited. Animal studies suggest that placental uptake of maternal FA is altered by maternal overnutrition. We hypothesized that high maternal body mass index (BMI) affects human placental FA transport by modifying expression of key transporters. Full-term placentas were obtained by vaginal delivery from normal weight (BMI, 18.5-24.9 kg/m(2)) and obese (BMI > 30 kg/m(2)) women. Blood samples were collected from the mother at each trimester and from cord blood at delivery. mRNA and protein expression levels were evaluated with real-time RT-PCR and Western blotting. Lipoprotein lipase (LPL) activity was evaluated using enzyme fluorescence. In vitro linoleic acid transport was studied with isolated trophoblasts. Our results demonstrated that maternal obesity is associated with increased placental weight, decreased gestational age, decreased maternal high-density lipoprotein (HDL) levels during the first and third trimesters, increased maternal triglyceride levels during the second and third trimesters, and increased maternal T3 levels during all trimesters, and decreased maternal cholesterol (CHOL) and low-density lipoprotein (LDL) levels during the third trimester; and increased newborn CHOL, LDL, apolipoprotein B100, and T3 levels. Increases in placental CD36 mRNA and protein expression levels, decreased SLC27A4 and FABP1 mRNA and protein and FABP3 protein expression, and increased LPL activity and decreased villus cytotrophoblast linoleic acid transport were also observed. No changes were seen in expression of PPARA, PPARD, or PPARG mRNA and protein. Overall this study demonstrated that maternal obesity impacts placental FA uptake without affecting fetal growth. These changes, however, could modify the fetus metabolism and its predisposition to develop diseases later in life.Biology of Reproduction 05/2012; 87(1):14, 1-11. DOI:10.1095/biolreprod.111.098095 · 3.45 Impact Factor