The objective of this research is to study the transfer of free fatty acids (ffa) in the intestine at the molecular level, in order to elucidate the mechanisms by which ffa assimilation occurs. The ultimate aim of these studies is to learn how to control lipid assimilation by regulation of rate limiting processes. The proposed studies will address the gaps in our knowledge concerning the regulation of ffa transfer, both within the intestinal lumen and within the cytoplasm of the enterocyte. The approach used will be an integrated one, combining the techniques of purified model lipid systems with reconstituted systems of cell membranes and cytoplasmic components. Recent work has shown that during the course of intestinal lipid digestion, two product phase are formed, a micelle phase and a unilamellar vesicle phase. The proposed studies will use a fluorescence assay recently developed by the applicant to compare the kinetics of ffa transfer from these two distinct lipid phases to acceptor membranes, and to determine the molecular mechanisms and rate limiting steps by which transfer from each of these phases occurs. The majority of ffa within the enterocyte cytoplasm are bound to fatty acid binding protein(s) (FABP). Our preliminary studies have shown that when ffa are bound to FABP, the fatty acyl chain is highly constrained within a hydrophobic pocket. The proposed studies will continue to investigate the physical nature of the FABP binding site of ffa, and will characterize and compare ffa binding to FABP v. ffa binding to membranes, in order to assess the relative roles of these interactions in cellular ffa assimilation. A further goal is to systemically investigate the manner in which FABP modulates intracellular ffa transport. The studies will examine and compare and the transfer kinetics of ffa from model membranes and subcellular membranes to FABP, and from FABP to membranes, and will elucidate the precise role of FABP in inter- membrane ffa transfer in the enterocyte. An understanding of ffa assimilation at the molecular level will enable more effective treatment of several pathophysiological and drug-induced malabsorption syndromes, and will support the continued development of oral drug delivery systems.
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