During the last funding cycle, progress was achieved in establishing the role of FAT, the adipose CD36 homologue, in fatty acid (FA) transport. Expression of the cDNA in the sense and antisense orientations induced the expected increase/decreases in FA transport. Like many proteins of FA metabolism, CD36 expression in pre-adipocytes was induced by FA and is a strong marker for adipocyte differentiation. Tissue distribution and regulation of CD36 expression strongly supported function. Pure CD36, isolated from adipocytes and from insect cells following Baculovirus expression, bound long-chain FA. Alignment of the predicted secondary sequence, with the crystal structure of muscle FA- binding domain. Finally, mice with muscle CD36 over-expression (MCK-CD36) exhibited lower blood FA, triglycerides and cholesterol and higher glucose, consistent with increased muscle FA uptake and consequent sparring of glucose.
The aims of the current proposal are 1) to define the FA-binding domain of CD36. Deletion or addition of nucleotide sequences and point mutagenesis will be coupled with functional expression in COS-7 and S2 cells. Native and mutate proteins will be expressed in inset cells, purified and assayed for FA binding; 2) to evaluate the role of CD36 in the uptake of lipoprotein FA and the relevance of lipoprotein binding to the process; 3) to assess the physiological significance of alterations of CD36 expression in vivo. MCK-CD36 mice will be studied for lipid and glucose metabolism and again at a later stage for the adaptive responses to exercise and high fat feeding. FA is the major energy substrates in humans. They are precursors of membrane phospholipids and they regulate many aspects of cell function, as modulators or mediators of membrane channels, receptors and enzymes. Uptake of long-chain FA is relevant to the organism in both health and disease.
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