Intestinal cholesterol esterase: intracellular enzyme

The location of cholesterol esterase in rabbit intestine was re-evaluated. In three different experiments that were designed to eliminate contaminating mucus and pancreatic enzymes from the lumen of the small intestine, it was observed that the activities of cholesterol esterase and amylase in intestinal cytosol and whole homogenate decreased in parallel fashion. After the mucus was carefully wiped from the intestinal mucosa prior to the preparation of cytosol, amylase and cholesterol esterase activities decreased sevenfold. The recovery of the total activity of both enzymes in the cytosol was approximately 15%. When the lumen of the small intestine was filled with phosphate buffer and incubated at 37OC for 20 min, cholesterol esterase and amylase activities in the cytosol prepared from this segment were further decreased. Moreover, the activities of amylase and cholesterol esterase were completely recovered from the lumen. Amylase and cholesterol esterase activities in the cytosol were eliminated if dithiothreitol was used as a mucolytic agent to prepare intestinal mucosa for the isolation of intestinal cells. In whole homogenates prepared from these intestinal segments, approximately 1015% of the total cholesterol esterase activity remained. This activity, which could not be accounted for by pancreatic contamination, was associated with intestinal nuclei and cellular debris. Progesterone, ethinyl estradiol, and 25-hydroxycholesterol regulated microsomal acyl CoA:cholesterol acyltransferase activity and caused similar directional changes in the rate of cholesteryl ester synthesis in isolated intestinal cells. These same sterols, however, failed to affect cytosolic cholesterol esterase activity in vitro.-Field, F. J. Intestinal cholesterol esterase: intracellular enzyme or contamination of cytosol by pancreatic enzymes?J. Lipid Res. 1984. 2 5 389-399. Supplementary key words cholesterol esterification small intestine amylase acylcoenzyme A:cholesterol acyltransferase Essentially all exogenous and endogenous cholesterol that is absorbed from the intestinal lumen into the absorptive cell is in the form of the unesterified sterol. When cholesterol is eventually secreted into the mesenteric lymphatic system as part of the core lipids in chylomicrons and very low density lipoproteins, approximately 80% of the cholesterol has been esterified. The presumed site for this esterification reaction is the intestinal absorptive cell (1). Two mechanisms have been described for the esterification of absorbed cholesterol in the intestine. It has previously been postulated that subunits of the pancreatic enzyme, cholesterol esterase, penetrate the brush border membrane of the enterocyte, aggregate to an active enzyme once inside the cell, and catalyze cholesterol esterification through a reversal of the hydrolytic reaction (2). Intestinal cholesterol esterase is located predominantly in the cytosol of the intestinal cell (3). More recently, using an immunocytochemical technique, the reaction product (cholesterol esterase antigenic determinants) was uniformly distributed within the intestinal absorptive cell, lamina propria, and submucosa. No antigens were found in the microvillar membrane (4). Conjugated trihydroxybile salts are an absolute requirement for the catalysis of cholesterol esterification by cholesterol esterase (5,6). It is felt that the bile salts protect the enzyme from proteolytic degradation and act as a cofactor for the enzyme. Cholesterol esterase does not require energy (7) and fatty acids are esterified to cholesterol without first being esterified to their coenzyme A derivatives. The pH optimum for this cytosolic enzyme is 6.2, with less than 5% of the esterifying activity being present at pH 7.4 (5, 6). In contrast to cholesterol esterase, acylcoenzyme A:cholesterol acyltransferase (ACAT), a microsomal enzyme, has been described in the intestine of humans (8), rats (9), guinea pigs (lo), rabbits (1 l), and subhuman primates.’ Intestinal ACAT has been localized to the endoplasmic reticulum, with highest specific activity being observed in the rough membranes (12). The enzyme is inhibited by bile salts (1 3). Energy is necessary for ACAT to catalyze cholesterol esterification (9). It is a requirement that fatty acids first be “activated” to their CoA-derivatives prior to esterification. The pH optimum for ACAT is 7.2 (9). Information is accumulating on the regulation of intestinal ACAT by fats, cholesterol and other sterols, particularly 25-hydroxycholesterol, 8-sitosterol, progesterone, and ethinyl estradiol (1 2-1 4). There is a marked disparity between these two mechanisms of cholesterol esterification in the intestine. Yet, Abbreviations: ACAT, acylcoenzyme A:cholesterol acyltransferase; ’ Armstrong, M. , and F. J. Field. Unpublished data. TLC, thin-layer chromatography Journal of Lipid Research Volume 25, 1984 389 by gest, on N ovem er 6, 2017 w w w .j.org D ow nladed fom it is still not clear which mechanism is primarily responsible for the esterification of cholesterol in the gut. The data presented in this report support the hypothesis that cholesterol esterase activity in intestinal cytosol is a contamination of this subcellular fraction by pancreatic enzymes which are present in the lumen during cell fractionation. Progesterone, ethinyl estradiol, and 25-hydroxycholesterol, sterols which regulate intestinal microsomal ACAT activity but which do not regulate cholesterol esterase activity in vitro, affect the rate of cholesterol esterification in isolated intestinal cells in the same direction that ACAT activity is affected. Thus, it is concluded that ACAT is more likely responsible for catalyzing cholesterol esterification in the intestine than cholesterol esterase. MATERIALS AND METHODS [ l-'4C]Oleoyl coenzyme A, [4-'4C]cholesterol, [9,10'H]oleic acid, and [ 1 ,2-3H(N)]cholesterol were purchased from New England Nuclear. Oleoyl coenzyme A, oleic acid, cholesterol, taurocholic acid, starch substrate, agarose Type VII, progesterone, maltose, 8-NADH, and dithiothreitol were from Sigma. 1,3,5( lO)-Estratrien-l7ethinyl-3,1 7-diol and 5-cholesten-3,25-diol were from Steraloids. Lipid standards were from Nu-Chek, Inc. All other reagents were reagent grade.