Effect Of EPA And D On Chylomicron Assembly In Caco-2 Cells

The absorption of dietary lipid not only determines energy supply to the body tissues, but also affects the incorporation of dietary fatty acid into animal products. Before its entry into circulatory system, long chain fatty acid derived from dietary fats is supposed to be absorbed into enterocytes and finally secreted as chylomicron, which is a meticulous and multi-step process involved in fatty acid transportation and chylomicron assembly. In order to throw light upon the fatty acid-specificity observed in fatty acid transportation in enterocytes, a vitro model for the assembly of chylomicron in enterocytes was established using Caco-2 cells. The validity was valued by different indexes reflecting the morphology and integrity of cell monolayer, coupled with alteration in relevant enzyme/protein expression. With this proposed model, the influence of n-3 polyunsaturated fatty acids on chylomicron assembly in Caco-2 cells was revealed. In order to explain the fatty acid-specificity discovered in chylomicron assembly, intercellular accumulation as well as extracellular secretion of triglyceride and apolipoprotein B, considered as the indispensable substrates for chylomicron assembly, was detected by the integration of radioisotopes. Given that L-FABP could channel and co-transfer with fatty acid into muclei for regulating gene expression, L-FABP-targeted overexpression vector and interference vectors were constructed and confirmed after the relationship between fatty acid treatment and L-FABP expression being identified, which lays the foundation for further investigation into mechanism.1. Establishment of Caco-2 cell monolayer model. Transepithelial electrical resistance was adopted to value the integrity and permeability of cell monolayer. TEER stably increased during the differentiation of Caco-2 cells in a time-dependent manner, and the average of TEERs at the end of the differentiation rises to 570Ω·cm2. The formation of functional structure was confirmed by transmission electron microscopy. Brush border microvilli and tight junction, both of which are the representative characteristics of enterocytes, were observed in Caco-2 cells grown on transwell for 21 days. As one of the brush border enzymes with the feature of polarized secretion, alkaline phosphatase concentration in separate chambers implies the process of differentiation. According to our results, it took as long as 18 days to distinguish the polarized secretion of alkaline phosphatase. When Caco-2 cells were completely differentiated for 21 days, alkaline phosphatase was almost only present in the apical medium. The activity of alkaline phosphatase measured in apical medium was 10 times than that in basolateral medium. Liver fatty acid binding protein is a critical protein in lipid metabolism and its expression indicates the improvement of physiological function of cells. The expression of L-FABP was enhanced along with the differentiation of Caco-2 cells, resulting in high expression in well-differentiated cells.2. Effect of DHA and EPA on chylomicron assembly in Caco-2 cells. Cell viability after fatty acid treatment was concerned by MTT test. With regard to our results, the cell viability was 95% even the fatty acid concentration increased to 600μmol/L. The addition of [1,2,3-3H]glycerol into apical chamber was designed to unveil the triglyceride synthesis. Compared with the control, the accumulation and secretion of [3H]trialyceride increased during the incubation with all fatty acids in a time-dependent manner(P<0.01). Only when the incubation time prolonged to 36 hours, the triglyceride synthesis stimulated by certain fatty acid showed difference. Compared with OA, cells incubated with 400μmol/L EPA significantly caused 16.5% and 21% reductions in the levels of [3H]trialyceride accumulation and secretion(P<0.01). Meanwhile, the reduction caused by DHA was 23.3% and 28%, respectively(P<0.01). Additionally, there was no difference at any time point between EPA and DHA(P>0.05). The addition of [1-14C] fatty acid into apical chamber was calculated to determine the uptake of fatty acid. The disappearance of labeled fatty acids from apical medium and the cellular uptake were similar during the incubation(P>0.05). During the initial 12h~24h, approximately 50%~80% of the added fatty acid had entered the cells. The contents of acid-precipitable products in basolateral medium were also similar in cells treated with OA, EPA and DHA(P > 0.05). Apolipoprotein synthesis was investigated with the addition of [35S]methionine into apical chamber. Although the three fatty acids all increased the incorporation of label into either cellular or medium apo B48 and apo B100 compared with the control(P<0.01), less labeled apo B accumulation and secretion by cells incubated with DHA and EPA were observed(P<0.01). Secreted lipoprotein was isolated by sequential ultracentrifugation. Fatty acid treatments all increased chylomicron secretion compared with the control(P<0.01). Remarkably, compared with OA, 400μmol/L EPA and DHA resulted in a lower secretion of chylomicron(P<0.01).3. Identification of relationship between fatty acid treatment and L-FABP expression and the construction of L-FABP overexpression/interference vector. Treatments with OA, EPA and DHA for 36 h facilitated the L-FABP expression in Caco-2 cells with no distinction. Four designed sh RNA vectors were respectively co-transfected into 293 T cells with overexpression vector, followed by western blot examining L-FABP expression. It came to the conclusion that overexpression vector translated precisely into L-FABP and sh RNA2 plasmid achieved the highest interference efficiency. L-FABP expression in Caco-2 cells transfected with overexpression vector was higher than that in control treatment as expected. Whereas the expression of L-FABP was successfully inhibited in Caco-2 cells after transfection with the interference plasmid, compared with the control. The possibility of controling L-FABP expression in Caco-2 cells would lay the foundation of explaining whether the influence of fatty acid on chylomicron assembly is dependent on the L-FABP/PPARα signaling pathway or not.It turned out that, under the certain cultural condition, polarized and differentiated Caco-2 cells can be used in subsequent experiment, serving as an in vitro enterocytes-like model for nutrient absorption and metabolism. The exposure of DHA and EPA reduced the secretion of chylomicron compared with OA, not because of the different uptake efficiency, but partly explained by the inhibitory effect on synthesis of triglyceride and apo B. Here we discussed the effects of dietary LCFA on CM assembly in enterocytes and its possible mechanism, providing theoretical basis underlying dietary fatty acid transportion in enterocytes.