Fatty acids (ffa) and monoacylglycerol (MG) are the major digestive products of dietary lipid. The intestine presents a barrier through which the ffa and MG must pass, yet the mechanisms by which they are transported remain largely unresolved. The objective of this research plan is to continue to address the gaps in our knowledge of the molecular mechanisms of ffa transfer a) from the two lipid phases present in the intestinal lumen, micelles and vesicles, to the enterocyte; b) across the microvillus membrane (MVM) of the intestinal cell; and c) within the enterocyte cytoplasm. The latter studies will focus on the functions of intestinal and liver fatty acid binding proteins (I- and L-FABP). These are thought to play a role in intracellular ffa transport, but their precise functions remain unknown. The proposed studies will also begin to analyze the kinetics and mechanism of MG absorption. The experimental approach will combine the use of purified model lipid systems, reconstituted systems of cell membranes and cytoplasmic components, and the Caco-2 intestinal cell culture system. Two product phases are now known to be formed during the course of intestinal lipid digestion, a micelle phase and a vesicle phase. The kinetics of ffa and MG transfer from the two phases will be studied using fluorescent ffa and MG derivatives, and a resonance energy transfer assay. Studies will continue to define the mechanisms of transfer from each of these phases, their relative contributions to lipid uptake, and their regulation by structural and physiologically important solution variables. The mechanism by which ffa cross the MVM is presently controversial, and little is known about intestinal MG uptake. To determine the role of plasma membrane protein vs. lipid diffusion pathways, studies will use fluorescence microscopy to directly monitor the transmembrane transport of ffa and MG at the level of the single Caco-2 cell. In order to define the role that I- and L-FABP play in ffa assimilation, the proposed studies will use the protein fluorescence of the FABP and a series of fluorescent ffa derivatives, to compare the structural interactions with ffa for I-FABP vs. L-FABP. Interactions with MG will also be examined for the first time. The proposed studies will also systematically analyze the mechanisms of ffa and MG transfer from I- vs. L-FABP to model and subcellular membranes, and the role of these FABP in intermembrane ffa traffic in the enterocyte. Both fluorescent and natural ligands will be used for these investigations. A better understanding of the molecular mechanisms underlying ffa and MG assimilation in the intestine may enable more effective treatment of malabsorption syndromes, the continued development of enteral drug delivery systems, and the regulation of dietary energy utilization.
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