Effects of eicosapentaenoic acid and docosahexaenoic acid on chylomicron and VLDL synthesis and secretion in Caco-2 cells

Abstract

The present research was undertaken to determine the effects of EPA (20 : 5 n-3) and DHA (22 : 6 n-3) on chylomicron and VLDL synthesis and secretion by Caco-2 cells. Cells were incubated for 12 to 36 h with 400 μM OA, EPA, and DHA; then 36 h was chosen for further study because EPA and DHA decreased de novo triglycerides synthesis in a longer incubation compared with OA (P < 0.01). Neither the uptake nor oxidation was different in response to the respective fatty acids (P > 0.05). Compared with OA, intercellular and secreted nascent apolipoprotein B48 and B100 were decreased by EPA and DHA (P < 0.01). Both DHA and EPA resulted in a lower secretion of chylomicron and VLDL (P < 0.01). In contrast to OA, EPA and DHA were preferentially incorporated into phospholipids instead of triacylglycerols (P < 0.01). These discoveries demonstrated that exposure of DHA and EPA reduced the secretion of chylomicron and VLDL partly by regulating the synthesis of TG and apoB.

Figure 1

Effect of OA, EPA, and DHA on Caco-2 cell triacyl[³H]glycerol accumulation and secretion. The cells plated on filter membranes and cultured for 21 days were incubated with DMEM containing delipidated FCS in the absence or presence of 400 μM OA, EPA, or EHA, respectively, for the time specified in the figure. The incorporation of [1,2,3-³H]glycerol into triacyl[³H]glycerol accumulated in cells (a) and secreted into basolateral medium (b) was estimated as described in Section 2. OA: oleic acid; EPA: eicosapentaenoic acid; DHA: docosahexaenoic acid. Data are expressed as mean ± SD of 6 different experiments. Means without a common letter differ between fatty acids, P < 0.01.

Figure 2

Percent uptake of EPA, DHA, and OA by Caco-2 cells. Caco-2 cells plated on filter membranes and cultured for 21 days were incubated in serum-free DME medium containing 400 μM [l-¹⁴C]OA, [l-¹⁴C]EPA, or [l-¹⁴C]DHA. After 12, 24, and 36 h of incubation, acid-precipitable products were determined from the apical medium (a), cells (b), and basolateral medium (c). The results are expressed as % of added acid-precipitable radioactivity/mg cell protein. Data are expressed as mean ± SD of 6 different experiments. Means without a common letter differ, P < 0.01.

Figure 3

Oxidation of EPA, DHA, and OA in CaCo-2 cells. The cells plated on filter membranes and cultured for 21 days were incubated in serum-free DME medium containing 400 μm [l-¹⁴C]OA, [l-¹⁴C]EPA, or [l-¹⁴C]DHA for 36 h. (a) CO₂ was collected and acid-soluble radioactivity in the incubation medium was measured as described in Section 2. (b) The sum of CO₂ and acid-soluble radioactivity as % of total added radioactivity is given. Data are expressed as mean ± SD of 6 different experiments. Means without a common letter differ, P < 0.01.

Figure 4

Incorporation of [l-¹⁴C]OA, [l-¹⁴C]EPA, and [l-¹⁴C]DHA into cell-associated and secreted lipids. Caco-2 cells plated on filter membranes and cultured for 21 days were incubated with [l-¹⁴C]OA, [l-¹⁴C]EPA, or [l-¹⁴C]EHA for 36 h. Cells were harvested and basolateral medium was collected for cell-associated (a) and secreted (b) lipids as described in Section 2. OA: oleic acid; EPA: eicosapentaenoic acid; DHA: docosahexaenoic acid; TG: triacylglycerol; PL: phospholipid; FFA: free fatty acids; CE: cholesteryl ester. Data are expressed as mean ± SD of 6 different experiments. Means without a common letter differ, P < 0.01.

Figure 5

Effect of OA, EPA, and DHA on Caco-2 cell nascent apolipoprotein synthesis and secretion. The cells plated on filter membranes and cultured for 21 days were incubated with 400 μM OA, EPA, or DHA for 35 h. After 35 hours, cells were incubated in methionine-free medium with albumin alone or respective fatty acid for 1 h. Labeled [³⁵S] methionine was added to the apical well. Apolipoproteins B-48 and B-100 were analyzed after the incubation with [³⁵S] methionine by SDS gel electrophoresis. The cells were harvested and basolateral medium was collected for de novo cell (a) and medium (b) apolipoprotein synthesis as described in experimental procedures. OA: oleic acid; EPA: eicosapentaenoic acid; DHA: docosahexaenoic acid. Data are expressed as mean ± SD of 6 different experiments. Means without a common letter differ between fatty acids, P < 0.01.

Figure 6

Effect of OA, EPA, and DHA on Caco-2 cell lipoprotein synthesis and secretion. Caco-2 cells plated on filter membranes and cultured for 21 days were incubated with OA, EPA, and DHA for 36 h. Cells were harvested and basolateral medium was collected for lipoprotein isolation as described in Section 2. CM: chylomicrons; VLDL: very low-density lipoprotein; OA: oleic acid; EPA: eicosapentaenoic acid; DHA: docosahexaenoic acid. Data are expressed as mean ± SD of 6 different experiments. Means without a common letter differ between fatty acids, P < 0.01.