Inhibition of acylcoenzyme A: cholesterol acyltransferase activity by PD128O42: effect on cholesterol metabolism and secretion in CaCo-2 cells.

The regulation of cholesterol uptake and secretion by acylcoenzyme A:cholesterol acyltransferase (ACAT) was investigated in the human intestinal cell line, CaCo-2. A new ACAT inhibitor, PD128042 (CI-976), was first characterized. The addition of the fatty acid anilide to membranes prepared from CaCo-2 cells inhibited ACAT activity without altering the activities of HMG-CoA reductase, fatty acid Co-A hydrolase, or triglyceride synthetase. PD128042 was a competitive inhibitor of ACAT with 50% inhibition occurring at a concentration of 0.2 micrograms/mL. When added to the medium of CaCo-2 cells at a concentration of 5 micrograms/mL, PD128042 inhibited oleate incorporation into cholesteryl oleate by 92% and increased oleate incorporation into triglycerides and phospholipids by 51% and 38%, respectively. After incubating CaCo-2 cells with the ACAT inhibitor, the rate of newly synthesized cholesterol decreased by 75% and membranes prepared from these cells contained significantly less HMG-CoA reductase activity. PD128042 significantly decreased the basolateral secretion of newly synthesized cholesteryl esters without affecting the secretion of newly synthesized triglycerides or phospholipids. The inhibitor decreased the esterification of labeled exogenous cholesterol which was taken up by the cell from bile salt micelles. Moreover, after 16 hr of ACAT inhibition, less labeled unesterified micellar cholesterol was associated with the cell. The esterification of cholesterol in CaCo-2 cells plays an integral role in the uptake of cholesterol through the apical membrane and its eventual secretion at the basolateral membrane.