Abstract
Membrane fatty acid composition of CaCo-2 cells was modified by incubating the cells for 8 days in medium containing 100 microM 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 micrograms/mg 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. 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.
Highlights
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 results suggested that A:cholesterol acyltransferase (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
Us-To enrich the cell membranes in the respective fatty acid, ing CaCo-2 cells as a model for the small intestinal absorp- the medium was removed and replaced with DMEM contive cell, we studied whether membranes could be modified taining 2.5% FCS and 100 pM of the fatty acid attached by an individual fatty acid and whether this, in turn, could to fatty acid-poor albumin (3:1, molar ratio) 3 days after affect intracellular cholesterol metabolism
Summary
Materials was completely evaporated under nitrogen, the fatty acid salt was dissolved in 1.5 ml of hot distilled water and ad-. HMG-CoA was [3H]Oleic acid (2.6 Ci/mmol), purified by TLC, was purchased from P-L Biochemicals, Inc. This solution was stirred well and M199 with 10 mM HEPES was added to make the final conentration. CaCo-2 cells were incubated with 1 ml of the labeled oleic grown in T-75 plastic flasks CaCo-2 cells were incubated with 1 ml of the labeled acetate solution at 37OC in 95% air-5% COB.At the indicated time, the medium was removed and the cells were rinsed thoroughly with ice-cold PBS and scraped. Acyl coenzyme A:cholesterol acyltransferase activity was measured as previously described [8]; the specific activity of oleoyl-CoA was 19,250dpmhmol. 3-Hydroxy-3-methylglutaryl coenzyme A reductase activity was measured as described [12]; the specific activity of HMG-CoA was 23,800 dpmhmol. Fatty acids were determined as previously described [8]
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