Esterification of long-chain fatty acids to cholesterol and ethanol : role of acyltransferases and carboxylesterases

Abstract: The aim of the present thesis was to investigate esterification of long-chain fatty acids to cholesterol and ethanol. The esterification products, cholesteryl esters and ethyl esters, may under certain circumstances accumulate in some cell types and tissues and cause pathological changes. Accumulation of cholesteryl esters in macrophages and formation of fatty streaks may lead to atherosclerotic lesions. Accumulation of fatty acid ethyl esters (FAEEs) in some organs has been implicated in organ damage in alcoholics. The role of microsomal acyltransferases and carboxylesterases in the synthesis of cholesteryl- and ethyl- esters has been evaluated in the present study. Microsomal carboxylesterase ES-10 has been suggested to be responsible for acyl-CoA:cholesterol acyltransferase (ACAT) activity. We have shown in the present study, by using selective inhibitors of ACAT and carboxylesterases, that ACAT activity is not due to microsomal carboxylesterase ES-10. FAEEs syntesis is catalyzed by acyl-CoA:ethanol acyltransferase (AEAT) and FAEE synthase. Characterization of these two activities in rat liver microsomes demonstrated that AEAT is of major importance for the formation of FAEEs. Purified carboxylesterases ES-4 and ES-10 have no FAEE synthase activity, but carboxylesterase ES-4 shows some AEAT activity and high FAEE hydrolyzing activity. This enzyme is also capable of hydrolyzing acyl-CoA's (e.g. palmitoylCoA) which are substrates for AEAT. Accurate measurement of AEAT capacity thus requires inhibition of carboxylesterase ES-4 by e.g. bis(4-nitrophenyl) phosphate (BNPP). Measurement of FAEE synthesis in homogenates from human tissues revealed that AEAT activity is of major importance in intestine, adipose tissue, gallbladder, liver, lung, and heart, being several-fold higher than FAEE synthase. Pancreas is the only tissue among the tissues examined in this study where FAAE synthase and AEAT activities are similar. Intestinal mucosa contains high AEAT activity and low FAEE hydrolyzing activity, suggesting that FAEEs synthesized by intestinal mucosa may be the source of FAEEs that accumulate in other tissues. As AEAT activity seems to be important for formation of FAEEs in humans it is of general interest to identify this activity. In the present study we used an unconventional approach involving covalent labeling of AEAT protein with [3 H]DFP which is a potent serine esterase inhibitor. Partially purified labeled AEAT corresponds to a protein of about 110 kDa as judged by SDS-PAGE. Expression of carboxylesterase ES-4 in rat is selectively regulated by thyroid hormone, probably at the transcriptional level. The possible mechanism for this regulation may involve binding of thyroid hormone to its receptor with subsequent activation of transciption of the ES-4 gene. The regulation by thyroid hormone is consistant with a possible role for carboxylesterase ES-4 in lipid synthesis.