SHORT-TERM REGULATION OF HEPATIC CHOLESTEROL METABOLISM (7ALPHA-HYDROXYLASE, ACYLTRANSFERASE, HMG - COENZYME A REDUCTASE)
Abstract
Studies evaluated the importance of different factors on the short-term regulation of hepatic cholesterol metabolism. Activities of microsomal enzymes controlling hepatic cholesterol balance were assayed under conditions of chylomicron remnant cholesterol uptake, changes in phosphorylation state and hormone treatment. Previous attempts to develop a reliable assay for cholesterol 7(alpha)-hydroxylase (7(alpha)-OHase) met with methodological problems due to inhibition by detergents used for solubilization of substrate. An improved assay for measurement of 7(alpha)-OHase using liposome solubilization of substrate was developed which prevented time dependent inactivation observed with detergent solubilization. Using this method, changes in 7(alpha)-OHase and HMG-CoA reductase (HMGR) in response to chylomicron remnant cholesterol were measured. Results indicated a time and concentration dependent decrease in both HMGR and 7(alpha)-OHase in response to remnant cholesterol. This decline in 7(alpha)-OHase supports the thesis that newly synthesized cholesterol is preferred as a substrate, while dietary cholesterol as remnants is not readily available for bile acid synthesis. Metabolism of remnant cholesterol via esterification by ACAT was then investigated. The assay procedure for ACAT was modified to use liposome solubilized substrate. With remnant treatment time and concentration dependent decreases in HMGR and 7(alpha)-OHase were observed, decreases not mediated by chylomicron cholesterol. Assay of ACAT activity revealed a time and concentration dependent decline as well, but this decline was not as great as that observed for 7(alpha)-OHase. These data suggest that ACAT is not as dependent on de novo cholesterol as 7(alpha)-OHase but also indicates that both ACAT and 7(alpha)-OHase can be appreciably down regulated by substrate supply on a short-term basis. Importance of conditions that could alter the phosphorylation state of 7(alpha)-OHase was also investigated. In vitro 7(alpha)-OHase was modulated to a variable extent by kinases and phosphatases. Addition of cAMP dependant protein kinase increased 7(alpha)-OHase to the greatest extent, while alkaline phosphatase caused the greatest reduction. When hormones known to alter the modulation state of HMGR were included in hepatocyte incubations, little difference in the activity of 7(alpha)-OHase was observed. In conclusion, activity of 7(alpha)-OHase appears to be principally regulated on a short-term basis by substrate supply and to a lesser extent by phosphorylation/dephosphorylation.
Degree
Ph.D.
Subject Area
Biochemistry
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