Hydrolysis of retinyl esters (RE) in the liver plays a key role in the body's metabolism of vitamin A. Dietary vitamin A enters the liver as RE in chylomicron remnants, which undergo hydrolysis and reesterification for storage. Prior to mobilization, RE are hydrolyzed and free retinol binds to retinol-binding protein for secretion into the plasma. Neutral and acid retinyl ester hydrolases (REHs) have been purified from rat liver and shown by sequence analysis to be carboxylesterases, ES-2 and ES-10. The goal of this project is to define the role of ES-2, ES-10, and the related ES-3 and ES-4, in the metabolism of vitamin A. Carboxylesterases have been studied with other substrates, but it is not known if they are important in vitamin A metabolism. Their efficiencies in catalyzing the hydrolysis of RE compared with other esters are also unknown. These gaps in knowledge will be explored with the following specific aims:
AIM 1 : Enzymoloqy of REHs. Enzyme kinetics will be used to study the substrate specificity of the carboxylesterases and to test the hypothesis that these enzymes differ in their intrinsic ability to catalyze the hydrolysis of RE and other lipid esters.
AIM 2 : Tissue distribution of carboxylesterases. Real-time PCR (for mRNA), immunodetection (for protein) and enzyme assay will be used to define the levels and distributions of enzymes in various tissues and to test the hypothesis that the carboxylesterases are present in peripheral tissues as well as in liver.
AIM 3 : Cell type distribution in liver. Laser Capture Microdissection and cell separations coupled with real-time PCR, immunodetection, and enzyme assays will be used to define the distribution of carboxylesterases between stellate cells & hepatocytes and to test the hypotheses that carboxylesterase ES-2 is localized exclusively in hepatocytes and that carboxylesterase ES-10 is localized in hepatocytes and stellate cells.
AIM 4 : Physiologic roles of the carboxylesterases in the metabolism of RE in cultured cells, cDNA transfection and RNA interference will be used to modulate carboxylesterase enzyme levels to test the hypothesis that the various carboxylesterases play a metabolic role in the hepatic uptake and/or metabolism of chylomicron RE, and to test the hypothesis that the carboxylesterases play a metabolic role in the turnover of intracellular RE and cholesteryl esters in cells that store them.
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