Characterization of the biochemical activities associated with human FATP2 splice variants

Members of the fatty acid transport protein (FATP) family are involved in the trafficking of exogenous fatty acids into the cell and are also very long-chain acyl CoA synthetases (Acsvl) functioning in intracellular fatty acid homeostasis. Several FATP isoforms are proposed to function alone or in concert with a long chain acyl CoA synthetase (Acsl) in the import of exogenous fatty acids by the process of vectorial acylation. Splice variants of human FATP2 have been identified that differ in the ATP binding region, which is required for adenylate formation. FATP2a contains the entire ATP/AMP motif while FATP2b lacks exon 3 resulting in a protein lacking critical residues within this motif. When expressed in yeast or 293 T-REx cells, FATP2a (M r 70,000) and FATP2b (Mr 65,000) were fully functional in the long chain fatty acid import pathway. Most interestingly, when expressed in yeast or 293 T-REx cells, FATP2a provides significant Acsvl activity; while expression of FATP2b does not contribute to this activity. Stable 293 T-REx cells expressing FATP2a and FATP2b and the yeast counterparts demonstrated that the transport and very long chain activating activities could be distinguished. To explore these functional differences further, LC/ESI/MS was employed to define total acyl CoA pools and to monitor the trafficking of exogenous stable isotopically labeled fatty acids into the intracellular acyl CoA pools. The expression of FATP2a increased the steady state acyl CoA pools by 20% when compared to cells expressing FATP2b. LC/ESI/MS analysis of stable isotopically labeled acyl CoAs extracted from T-Rex cells expressing FATP2a or FATP2b showed that FATP2a is involved in the vectorial acylation of exogenous fatty acids, with preference towards n-3 fatty acids (C18:3 and C22:6) as well as very long chain saturated fatty acids (C24:0). These data support the hypothesis that the different FATP isoforms are likely to function in the transport and activation of specific classes of fatty acids. Additionally we addressed the role of these isoforms in trafficking specific fatty acids into phospholipid pools (PC, PE, PI, PS, PG and PA) by employing stable isotope labeling in combination with direct infusion ESI/MS analyses of phospholipids in 293 T-Rex cells expressing FATP2a or FATP2b. Of particular note is the DHA-CoA arising from vectorial acylation of exogenous DHA, which is preferentially trafficked into phosphatidylinositol in cells expressing FATP2a. While the precise role of FATP2b is unknown, these data demonstrate FATP2a is fully functional in the transport-coupled activation of exogenous fatty acids, with a preference towards n-3 fatty acids. These studies revealed novel insights into the role of FATP2 isoforms in providing selectivity and specificity of acyl CoA formation and subsequent trafficking into complex lipids.;This work also includes studies which investigate the effects of RNAi knockdown of FATP2 on fatty acid metabolism in Caco-2 and HepG2 cells. Recent evidence demonstrates that FATP2 is the predominant FATP expressed in both cell lines [1]. The physiological role of FATP2 in the intestine and liver or in cell culture model systems such as Caco-2 and HepG-2 cells is unknown. We employed shRNA technology to knockdown FATP2 expression and examine the effects on fatty acid transport, activation, trafficking, and downstream utilization. Stable isotope labeling in combination with mass spectrometry revealed a trend for depressed n-3 fatty acid acyl CoA formation as well as metabolic channeling into PC, PI, and PE phospholipid pools as a result of knocking down FATP2 in Caco2 cells. The impact of FATP2 knockdown on the metabolic phenotype of HepG2 cells was subtle. Taken together, overexpression and loss of function studies have expanded our understanding of how FATP2 isoforms function in fatty acid import and trafficking pathways.;[1] Please see dissertation for reference.