The Fatty Acyls Core (Core G) will identify and quantitate the levels of free fatty acids and fatty acid metabolites (FAMs). FAMs include, but are not restricted to, the prostanoids, hydroxyl- and hydroperoxyeicosaenoic acids, epoxyeicosatrienoic acids, leukotrienes, free fatty acids, and fatty acid amides. Resting cells contain and secrete very low levels of FAMs. These compounds are usually generated in response to a signal, are produced for a specific function, and are often potent second messengers. They are important components of the inflammatory response and play central roles in many homeostatic processes. As such they are crucial factors in many diseases and have proven to be important pharmacological targets. This Core will identify and quantitate FAMs present in and secreted by macrophages and other cells and tissues that are employed in the coordinated studies conducted by the LIPID MAPS Consortium. In addition, this core will conduct several detailed studies that will explore issues unique to the fatty acyls. First, we discovered that macrophages simultaneously activated with Kdo2-Lipid A and ATP, produce a synergistic response. Studies are now proposed that will determine the mechanism of this enhanced response. These studies will be carried out in collaboration with Core H. Second, time course studies showed that the levels of free arachidonic acid (AA), the precursor of eicosanoids, peak in the cytosol and medium long before significant levels of eicosanoids are produced. This result implies that the release of AA interacts with the various eicosanoid biosynthetic pathways in a complex manner. The first step in eicosanoid synthesis is the release of AA from lipids. The primary enzyme that carries out this reaction is phospholipase A2 (PLA2), a superfamily of fifteen different enzymes. All cells contain several types of PLA2 and each isozyme possesses different biochemical characteristics. One or all of the PLA2s could be responsible for eicosanoid biosynthesis. Studies are proposed to employ fluxomic measurements using stable-isotope labeled AA to ascertain how AA is released and how free AA release is coupled to eicosanoid generation. Third, similar studies will be carried out after supplementing cells with the predominant Q-3 fatty acids, EPA and DMA. Fourth, to extend our Kdo2-Lipid A/RAW264.7 cell studies to a clinically relevant disease model, we will apply the LIPID MAPS eicosanoid and fatty acid analysis to explore the role of FAMS in Lyme disease. Lav Summary Fatty acid metabolites, in particular the eicosanoids and related compounds, play important roles in normal physiological function and in many diseases. Determining how the levels of these compounds change during the course of disease and in response to various pharmacological interventions will increase our understanding of disease processes and enhance our ability to develop effective treatments.
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