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Fatty acid transport proteins (FATPs) provide pivotal roles in fatty acid transport and activation and thus are crucial for overall fatty acid homeostasis. Peroxisome Proliferators Activated Receptors (PPARs) are important transcription factors, which control many genes that govern lipid metabolism.
Using 293 T-REx cell lines that stably express FATP1 or FATP4 from a tetracycline-inducible promoter, this work evaluated gene expression of key genes involved in fatty acid metabolism using QPCR, protein expression of FATP1 and FATP4 using Western blots, and fatty acid transport to address the roles of these two FATP isoforms in fatty acid homeostasis. This work found that the expression of FATP1 from the heterologous Tet promoter resulted in increased FATP4 expression from its native promoter; the expression of FATP4 from the Tet promoter did not, however, increase the expression of FATP1. These results identified an unexpected regulatory loop between FATP1 and FATP4, which is hypothesized to occur at the transcriptional or post transcriptional level. Western blots confirmed these relationships by evaluating the protein expression of FATP1 and FATP4. Studies monitoring the transport of fatty acids using the fluorescent long chain fatty acid C1-BODIPY-C12 demonstrated the expression of FATP1 resulted in higher levels of transport relative to FATP4 overexpression. To address whether this regulatory loop proceeds through PPARa or PPARg, the expression of these two genes along with PPARs target genes were monitored. The over expression of FATP1 and FATP4 from the Tet promoter did not increase PPARs gene expression; the expression of FATP1 from the Tet promoter did result in an increased expression of PPARa target genes. Collectively these results supported the conclusion that the enzymatic product of FATP1 resulted in increased expression of FATP4 through a PPARα-mediated process. To further address this question, fatty acid and fatty acyl CoA profiles were measured in 293 T-REx cells expressing FATP1 or FATP4. The acyl CoA profiles showed both C18:2-CoA and C20:4-CoA were elevated in FATP1 expressing cell lines, perhaps indicating a relationship in n-3 fatty acid activation including further downstream metabolism. Likely effectors in this regulatory loop are arachidonic acid metabolites, including prostaglandins.
Advisor: Paul N. Black