Abstract
Dietary fatty acids (FAs) crossing the apical plasma membrane of small intestinal enterocytes are targeted to different metabolic pathways than serum FAs crossing the basolateral membrane. This apparent compartmentalization of FA metabolism in enterocytes was further investigated using a model human enterocyte-like intestinal cell line. [3H]Oleic acid bound to bovine serum albumin (BSA) was added to the apical or basolateral surfaces of confluent monolayers of Caco-2 cells growing on uncoated polycarbonate filters. In other experiments, [3H]oleic acid incorporated into micelles with taurocholate (+/- 2-monoacylglycerol) was added apically. Caco-2 cells absorbed oleic acid bound to BSA from both the apical and basolateral surfaces at the same rate. Oleic acid in micellar solution was absorbed more efficiently than oleic acid bound to BSA. Regardless of its site or mode of presentation, the majority of the incorporated oleic acid was found in triglycerides. Only a small fraction was subjected to beta-oxidation or esterification into phospholipids. Most of the incorporated oleic acid was still retained intracellularly at 24 h. The polarity of triglyceride secretion was influenced by the experimental conditions. Triglyceride secretion was not significantly polarized when oleic acid-BSA was presented apically. However, the ratio of basolateral to apical secretion at 24 h was 9:1 for oleic acid-BSA presented basolaterally. For oleic acid in taurocholate micelles there was a trend toward polarity of secretion to the apical media (apical to basolateral ratio = 2:1). The inclusion of 2-monoacylglycerol in oleic acid-taurocholate micelles did not augment triglyceride synthesis or secretion. These differences indicate that compartmentation of FA metabolism in Caco-2 cells is influenced by the site of FA presentation. Northern and Western blot hybridization studies indicated that the liver fatty acid-binding protein but not the intestinal fatty acid-binding protein gene is expressed in these cells. The absence of this latter 15 kDa protein indicates that it is not required by Caco-2 cells for the synthesis of triglycerides or for the polarized export of triglyceride. These studies indicate that the Caco-2 cell line will be a useful model system for studying the polarization of FA trafficking/metabolism in enterocytes and defining the role of intracellular fatty acid binding proteins in these processes.
Highlights
Dietary fatty acids (FAs)crossing the apical plasma membrane of small intestinal enterocytes are targeted to different metabolic pathways than serum FAs crossing the basolateral membrane
They support the de novo synthesisof FAs, lipids, and cholesterol and are a major site of production of extracellular lipid transport proteins, apolipoproteins A-I, A-IV, and B-48.The enterocyte is exposed to dietary FAs at its apical surface and to plasmaderived endogenous FAs at its basolateral surface
Caco-2 cells (12 days postconfluent) grown on Costar inserts were incubated with 0.5 m~ ['Hloleic acid bound to bovine serum albumin (BSA) (OA-BSA) added to the apical media or to the basolateral media or with ['Hloleic acid-taurocholate (OA-TC) micelles or with ['H]oleic acid-taurocholatemonoacylglycerol micelles (OA-TGMAG) added to the apical media
Summary
Penicillin, streptomycin, nonessential amino acids, Lglutamine, and Dulbecco’s minimum essential medium (DMEM) were purchased from Mediatech (Herndon, VA). [3H]Oleic acid-taurocholate micelles containing 0.25 mM 2-monopalmitoy1glycerol were prepared by incubating radiolabeled sodium oleate (0.5 mM) and 0.25 mM 2-monopalmitoy1glycerol in serum-free media containing 8 mM taurocholate for 1 h at 37°C. To determine the incorporation of [3H]oleicacid into radioactive lipids, an aliquot (-50,000-100,000 cpm) of media or cell lipid extract, along with lipid standards (arachidonic acid, triolein, diolein, and cholesteryl oleate) was spotted on a thin-layer chromatography plate (20 cm x 20 cm, Silicagel G25, Brinkmann Instruments, Inc., Westbury, NY) which was subsequently developed in hexane-ethyl ether-acetic acid 80:20:1 [14]. Cell lipid extracts (-50,000 cpm) along with phospholipid standards [phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidic acid (PA), lysophosphatidylcholine (LPC), and sphingomyelin (SM)] were spotted on a thin-layer chromatography plate and developed first with chloroform-methanolacetic acid-water 50:25:8:4 (1st dimension) and with the same solvents in a ratio of 50:7.5:8:2 (2nd dimension). The Student's &testfor unpaired observations was used for calculation of Pvalues
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
