Modification of CaCo2 CaCo2 CaCo2 cell membrane fatty acid composition by eicosapentaenoic acid and palmitic acid : effect on cholesterol metabolism

Membrane fatty acid composition of CaCo-2 cells was modified by incubating the cells for 8 days in medium containing 100 p~ eicosapentaenoic acid or palmitic acid. The effect of membrane fatty acid changes on cholesterol metabolism was then studied. Cells incubated with eicosapentaenoic acid had significant changes in membrane fatty acid composition with an accumulation of 20:5 and 22:5 and a reduction in monoenoic fatty acids compared to cells grown in palmitic acid. Intracellular cholesteryl esters could not be detected in CaCo-2 cells grown in the presence of the n-3 polyunsaturated fatty acid. In contrast, cells incubated with the saturated fatty acid contained 2 pglmg protein of cholesteryl esters. Cells grown in eicosapentaenoic acid, however, accumulated significantly more triglycerides compared to cells modified with palmitic acid. The rate of oleic acid incorporation into triglycerides was significantly increased in cells incubated with eicosapentaenoic acid. CaCo-2 cells modified by eicosapentaenoic acid had lower rates of HMG-CoA reductase and ACAT activities compared to cells modified with palmitic acid. The incorporation of the two fatty acids into cellular lipids also differed. Palmitic acid was predominantly incorporated into cellular triglycerides, whereas eicosapentaenoic acid was preferentially incorporated into phospholipids with 60% of it in the phosphatidylethanolamine fraction. I The data indicate that membrane fatty acid composition is significantly altered by growing CaCo-2 cells in eicosapentaenoic acid. These modifications in membrane fatty acid saturation are accompanied by a decrease in the rates of cholesterol synthesis and cholesterol esterification. Murthy, S. , E. Albright, S. N. Mathur, and F. J. Field. Modification of CaCo-2 cell membrane fatty acid composition by eicosapentaenoic acid and palmitic acid: effect on cholesterol metabolism. J Lipid Res. 1988. 29: 773-780. Supplementary key words teryl esters phosphatidylethanolamine membranes HMG-CoA reductase ACAT cholesCaCo-2 cells are derived from a human colon adenocarcinoma. When fully differentiated these cells have the morphological and biochemical characteristics of small intestinal absorptive cells (1). These cells have been shown to have microvillar brush border membranes; they form tight junctions at confluency; and they maintain their polarity when cultured on plastic or cellulose filters (2). These cells are proving to be useful models for the study of many parameters of enterocyte function such as glucose and ion transport (3), sodium-dependent Pi transport (4), and lipoprotein secretion (5). Recently, we have used these cells to study the regulation of acyl coenzyme A:cholesterol acyltransferase (ACAT) activity by cholesterol which is absorbed into the cell via bile salt micelles (6). The results suggested that ACAT activity was stimulated by the absorbed cholesterol and this regulation was secondary to the expansion of the ACAT substrate pool by the incoming cholesterol. This supported previous data whereby intestinal ACAT activity was’ stimulated in the intestines of animals ingesting a diet supplemented with cholesterol (7). Our laboratory has also been interested in the effects of dietary fat saturation on cholesterol metabolism in the intestine. In an earlier study, intestinal ACAT activity was significantly increased in rabbits fed a diet enriched in polyunsaturated fatty acids compared to the activity in intestines from animals ingesting a diet supplemented with saturated fat (8). In a more recent study, intestinal ACAT activity was increased and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity was decreased in rabbits fed menhaden oil (a fish oil enriched in n-3 polyunsaturated fatty acids) compared to the activities observed in the intestines of animals ingesting a diet supplemented with cocoa butter, a saturated fat (9). In both of these dietary studies, the alterations in the intestinal microsomal membrane fatty acid compositions reflected the composition of the ingested oils. It was postulated that the changes in membrane fatty acid saturation, independently of the membrane cholesterol content, regulated the activities of these two enzymes (8,9). This is not a new concept. Changes in cellular membrane fatty acid saturation have been correlated with the regulation of many membrane-bound enzymes and other cellular functions (10, review). The Abbreviations: HMG, 3-hydroxy-3-methylglutaryl; ACAT, acylcoenzyme A:cholesterol acyltransferase; TLC, thin-layer chromatography; PBS, phosphate-buffered saline. Journal of Lipid Research Volume 29, 1988 773 by gest, on N ovem er 6, 2017 w w w . j.org D ow nladed fom difficulty in interpreting the results from the above dietary studies, however, is that the dietary oils contain a mixture of fatty acids, all of which may have diverse effects on the parameters being studied, Le. , cholesterol metabolism. Using CaCo-2 cells as a model for the small intestinal absorptive cell, we studied whether membranes could be modified by an individual fatty acid and whether this, in turn, could affect intracellular cholesterol metabolism. Since there has been recent enthusiasm for the ingestion of fish oil (an oil rich in eicosapentaenoic acid) for its hypolipidemic effect (ll), we investigated the effect of this fatty acid on membrane fatty acid composition and cholesterol metabolism in CaCo-2 cells and compared this to the effect of a saturated fatty acid, palmitic acid. The results suggest that membrane fatty acid saturation can be significantly modified by growing CaCo-2 cells in the presence of eicosapentaenoic or palmitic acid. These modifications are associated with the regulation of HMGCoA reductase and ACAT activities.