Trafficking of exogenous fatty acids within Caco-2 cells.

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.     

Effects of intestinal fatty acid-binding protein overexpression on fatty acid metabolism in Caco-2 cells

Intestinal fatty acid-binding protein (I-FABP) is a cytosolic protein expressed at high levels (up to 2% of cytosolic proteins) in the small intestine epithelium. Despite cell transfection studies, its function is still unclear. Indeed, different effects on fatty acid metabolism depending on the cell type and the amount of I-FABP expressed have been reported. Furthermore, a decrease in fatty acid incorporation has been unexpectedly obtained when I-FABP reached 0. 72% of cytosolic proteins in fibroblasts (Prows et al. 1997. Arch. Biochem. Biophys. 340: 135). In the present study, the effect of a high level of I-FABP similar to amounts present in the small intestine was investigated in the human colon adenocarcinoma cell line, Caco-2. After transfection with human I-FABP cDNA, a clone expressing 1.5% I-FABP and unchanged level of liver FABP was selected. These cells, which had a lower rate of proliferation as compared with mock-transfected cells, developed the typical morphological characteristics of differentiated enterocytes. Incubation of differentiated cells with [(14)C]palmitate showed a 34% reduction (P < 0.01) of fatty acid incorporation, whereas the relative distribution of radiolabel into triglycerides was not affected. A nonsignificant 21% reduction of fatty acid incorporation was observed with another clone expressing 10-fold less I-FABP. In conclusion, a high level of I-FABP expressed in a differentiated enterocyte model inhibited fatty acid incorporation, by a mechanism which remains to be defined.     

Modulation of cholesterol concentration in Caco-2 cells by incubation with different n-6 fatty acids

Incorporation of exogenous cholesterol was compared in human adenocarcinoma colon cells (Caco-2) after incubation with 100 microM of either linoleic acid (LA, 18:2n-6), gamma-linolenic acid (GLA, 18:3n-6), arachidonic acid (AA, 20:4n-6) or adrenic acid (or n-6 docosatetraenoic acid, DTA, 22:4n-6). In both cells 7 days after seeding and 14 days after confluency, incubation with LA significantly raised the proportion of 18:2n-6 but not its long-chain metabolites in cellular phospholipid. Incubation with GLA increased the levels of 18:3n-6, 20:3n-6, and 20:4n-6. Incubation with AA increased the levels of 20:4n-6 and 22:4n-6, and incubation with DTA increased the levels of 22:4n-6 as well as its retro-conversion metabolite, 20:4n-6. A subsequent addition of cholesterol (180 microM) to the medium significantly raised the cellular cholesterol level but less so in the cells 7 days after seeding incubated with GLA. The increase in cellular cholesterol level was generally greater in the cells of 7 days after seeding, particularly those incubated with long-chain highly unsaturated n-6 fatty acids, than in those of 14 days after confluency. These findings suggest that the cell growth and the extent of unsaturation in cell membrane phospholipid fatty acids modulate the incorporation of the exogenous cholesterol into the Caco-2 cells.     

Effects of medium-chain fatty acids on intracellular calcium levels and the cytoskeleton in human intestinal (Caco-2) cell monolayers

The effects of medium-chain fatty acids (MCFA) on intracellular calcium (Ca2+) levels and actin filaments in the Caco-2 monolayer were investigated. A site-dependent increase in intracellular Ca2+ levels caused by decanoic acid (C10) at 13 mM was observed by confocal laser scanning microscopy. The area in which the intracellular Ca2+ levels was increased was measured by image analysis, and increased to 11% of the total area of the monolayer within 1 minute. This was maintained for 5 minutes, and decreased thereafter. The other MCFAs did not significantly increase the intracellular Ca2+ levels. Obvious morphological changes of actin filaments were induced by only C10 among C8-C14. The area in which actin filaments were depleted was also quantified, and the increase in area became significant after 40 minutes. The area of the actin-depleted spot corresponded to the area occupied by 5 to 10 cells as well as that in which the intracellular Ca2+ level was increased. The effectiveness of only C10 suggested that the mechanism of the absorption enhancement by C10 would be different from that by the other MCFAs, or that C10 has some additional physiological functions although the mechanism of the enhancement is the same as for the other MCFAs.     

Effects of growth regulators on fatty acids of soybean suspension cultures

Soybean suspension cultures were grown for 24 weeks in the absence of plant growth regulators and in the presence of 1 ppm levels each of an auxin (indole-3-butyric acid), a cytokinin (kinetin) and a gibberellin (gibberellic acid), individually and in all possible combinations. Cells grown in the presence of the auxin with and without gibberellin contained relatively greater amounts of palmitic and smaller amounts of polyunsaturated acids than did cells grown under other regimens. The combination of cytokinin and gibberellin caused a higher proportion of linoleic and a lower proportion of linolenic acids than in cells of the other groups. Neither of these regulators by itself produced the effect, and addition of auxin to the other two diminished the effect.     

Effect of polyunsaturated fatty acids on the growth of fish cells in culture

• 1.1. The effects on growth of supplementing the medium with (n-3) and (n-6) polyunsaturated fatty acids (PUFA) were investigated in Atlantic salmon (AS) and turbot (TF) cell lines.
• 2.2. Neither cell line grew in the absence of serum, and addition of increasing percentages of serum resulted in graded increases in cell growth in both cell lines.
• 3.3. The growth of AS cells was stimulated by supplementing the medium with both (n-6)PUFA and (n-3)PUFA at 5–25 μM, especially 18:3(n-3) and 20:5(n-3).
• 4.4. Intermediate concentrations (15–20 μM) of 18:2(n-6) and 18:3(n-3) increased cell growth in TF cells, although only after 8 days in culture.
• 5.5. In contrast, both (n-3) and (n-6)PUFA at 25 μM tended to inhibit the growth of TF cells, and in longer incubations caused cell death.
• 6.6. The inhibition of TF cell growth rate and, in particular, the cell death induced by 25 μM PUFA could be abolished by the addition of vitamin E to the medium.

     

Effect of polyunsaturated fatty acid-enriched phosphatidylcholine and phosphatidylserine on butyrate-induced growth inhibition, differentiation and apoptosis in Caco-2 cells

Phospholipids are fascinating in terms of important bio-functional compounds. The present work investigated the effect of polyunsaturated phosphatidylcholine (PC) and phosphatidylserine (PS) on butyrate-induced growth inhibition, differentiation and apoptosis using Caco-2 cells. Growth inhibition of Caco-2 cells became apparent 24 h after addition of PC while it took 48 h with PS. Alkaline phosphatase activity of Caco-2 cells increased with combined PC or PS and sodium butyrate (NaBT) at 72 h, indicating that PC and PS enhanced cell differentiation in the presence of NaBT. An increased enrichment factor was also found when cells were treated with combinations of PC or PS and NaBT. These results suggest that marine PC and PS can be considered to be potentially useful colon cancer chemotherapy agents with high bio-availability.     

Effects of fatty acids on angiogenic activity in the placental extravillious trophoblast cells

Fatty acids regulate angiogenesis although no such information is available in first trimester placental trophoblast cells despite the fact that angiogenesis is a critical step involving these cells in early placentation. We investigated effects of different fatty acids on angiogenesis, their uptake and metabolism and expression of lipid metabolic genes in first trimester placental trophoblast cells using HTR-8/SVneo cell line. Fatty acid uptake by these cells exhibited a saturable kinetics. Uptake of AA was consistently greater compared with that of EPA and DHA throughout the incubation period of 180 min. Use of triacsin C, an inhibitor of acyl-CoA synthetase, significantly inhibited fatty acid uptake as well as fatty acid induced cell proliferation in these cells. Angiogenic effect (as measured by tube formation) of these fatty acids was in the following order DHA>EPA>AA>OA. Angiogenic effect of these fatty acids (AA, EPA, OA) was significantly decreased in ANGPTL4 knocked down cells, indicating ANGPTL4 may be involved at least in part in fatty acid induced angiogenesis. In addition, these fatty acids altered expression of several lipid metabolic genes such as ADRP, FABP4, FABP3, and COX-2 those are involved in angiogenesis. All these data suggest that fatty acids regulate angiogenic processes in these cells via different mechanisms.     

Effects of essential fatty acids on mediators of mast cells in culture

The objective of this study was to investigate the effects of alpha-linolenic acid (18:3n-3) and linoleic acid (18:2n-6) on the fatty acid composition and the activity and release of mast cell mediators in the canine mastocytoma cell line C2. Cells were cultured in Dulbecco's modified Eagle's medium mixed with 50% Ham's F12 (containing linoleic acid 0.14 micro M). The basic medium (DEH) was supplemented with 0.14 micro M alpha-linolenic acid. 14.0 micro M alpha-linolenic acid (DEH-n-3) or 14.0 micro M linoleic acid (DEH-n-6) was added. Eight days after culturing of C2 in DEH-n-3 we measured elevated levels of n-3 fatty acids up to 22:3. The tryptase activity and the stimulated PGE2 production and histamine release were reduced. In contrast, after culturing of C2 in DEH-n-6 we determined elevated levels of n-6 fatty acids up to 20:3, increased tryptase activity and stimulated histamine release. Thus 18:3n-3 has anti-inflammatory effects in cultured canine mastocytoma cells.     

Effects of tributyltin on barrier functions in human intestinal Caco-2 cells

The effect of tributyltin (TBT) on human intestinal epithelial cell functions was investigated by using human intestinal Caco-2 cell monolayers. We paid particular attention to the effect of TBT on two barrier functions: the tight junction as a physical barrier and MDR1/P-glycoprotein as a biological barrier. A loss of monolayer integrity was apparent from the TBT treatment and the paracellular permeability was increased by TBT. On the other hand, the activity of P-glycoprotein, which was examined by measuring the accumulation of Rhodamine-123 and daunomycin, was increased by prolonged TBT treatment in a concentration-dependent manner (1-100 nM). Furthermore, it was clarified by Western and Northern blots that this increase was accompanied by the increased expression of MDR1 mRNA and protein. The activation of a multidrug resistance transporter P-glycoprotein by TBT would cause a disorder of the human intestines by changing the drug pharmacokinetics.     

Effects of long-chain fatty acids on human urothelial cells in organ culture

It has been suggested that tumour-derived cells are differentially sensitive to the anti-proliferative and cytotoxic effects of long chain n-3 and n-6 polyunsaturated fatty acids (PuFAs). We have previously shown that PuFAs are also growth suppressive to highly proliferative normal human urinary bladder uro-epithelial (NHU) cells grown in monolayer culture. To determine if the effects on NHU cells are directly related to the proliferative index, we have studied the effects of long chain fatty acids in a bladder organ culture system, where proliferation and differentiation of the urothelium is under homeostatic control. A 50 microM concentration of fatty acids was chosen as this concentration of PuFA was profoundly growth inhibitory to NHU cells in monolayer culture. In organ culture, 50 microM PuFAs had no detectable effect on the proliferation or on the preservation of urothelial differentiated histioarchitecture, as assessed using a panel of phenotypic markers. These results suggest that the effects of PuFA may be modulated by the tissue microenvironment.     

不同饵料对卤虫生长、总脂含量及脂肪酸组成的影响

为了提高养殖卤虫的饵料营养价值,了解其不同生长阶段营养成分变化情况,采用单因子试验研究了8种饵料（三角褐指藻、小球藻、微绿球藻、酵母液、三角褐指藻＋小球藻＋微绿球藻、三角褐指藻＋酵母液、小球藻＋酵母液和微绿球藻＋酵母液）对卤虫生长、总脂含量及脂肪酸组成的影响,结果表明：不同饵料种类对卤虫生长、总脂含量及脂肪酸组成的影响显著（P＜0.05）,增长率,以三角褐指藻＋酵母液最优;总脂含量、以三角褐指藻最优（19.67%）,除酵母液外,与其它饵料相差不显著（P＞0.05）;脂肪酸组成效果,以微绿球藻组最优（EPA：18.01%,DNA：0.55%,（n-3）HUFA：19.08%）,与三角褐指藻组相差不大（P＞0.05）,显著高于其它各组（P＞0.05）.同时以三角褐指藻为饵料,研究了卤虫不同生长阶段（体长2、4、6、8、10 mm）总脂含量、脂肪酸组成变化,结果表明：卤虫体长2～10 mm总脂含量为14.27%～20.93%,随体长的增长降低;EPA、DHA及（n-3）HUFA的含量,均随体长的增长降低,EPA含量为：10.47%～20.77%,DNA含量为：0～0.70%,（n-3）HUFA含量为：10.85%～22.01%.结论认为,卤虫以三角褐指藻或三角褐指藻＋酵母液为饵料培养营养价值最佳,其体长小于6 mm营养价值较佳.     

Effects of perfluoroalkyl carboxylic acids on the uptake of sulfobromophthalein via organic anion transporting polypeptides in human intestinal Caco-2 cells

We performed a detailed investigation of the uptake of sulfobromophthalein (BSP) from the apical membrane of Caco-2 cells, which is a substrate for organic anion transporting polypeptides (OATPs), and calculated the kinetic parameters of BSP uptake as follows: K_m = 13.9 ± 1.3 μM, V_max = 1.15 ± 0.07 nmol (mg protein)^?1 (5 min)^?1, and k_d = 38.2 ± 0.53 μL (mg protein)^?1 (5 min)^?1. Coincubation with medium-chain (C7–C11) perfluoroalkyl carboxylic acids (PFCAs), such as perfluoroheptanoic acid (PFHpA, C7), perfluorooctanoic acid (PFOA, C8), perfluorononanoic acid (PFNA, C9), perfluorodecanoic acid (PFDA, C10) and perfluoroundecanoic acid (PFUnDA, C11), significantly decreased BSP uptake by 27–55%, while coincubation with short- (C3–C6) and long-chain (C12–C14) PFCAs decreased the uptake only slightly. Dixon plotting suggested that PFOA, PFNA and PFDA competitively inhibited the BSP uptake with inhibition constant (K_i) values of 62.2 ± 1.3 μM, 35.3 ± 0.1 μM and 43.2 ± 0.3 μM, respectively. PFCAs with medium-chains could be substrates for OATPs, probably OATP2B1, which is the most abundantly expressed OATP isoform in Caco-2 cells.     

Common mechanisms of monoacylglycerol and fatty acid uptake by human intestinal Caco-2 cells

Free fatty acids (FFA) andsn-2-monoacylglycerol (sn-2-MG), the twohydrolysis products of dietary triacylglycerol, are absorbed from thelumen into polarized enterocytes that line the small intestine.Intensive studies regarding FFA transport across the brush-bordermembrane of the enterocyte are available; however, little is knownabout sn-2-MG transport. We therefore studied the kineticsof sn-2-MG transport, compared with those of long-chain FFA(LCFA), by human intestinal Caco-2 cells. To mimic postprandial luminaland plasma environments, we examined the uptake of taurocholate-mixed lipids and albumin-bound lipids at the apical (AP) and basolateral (BL)surfaces of Caco-2 cells, respectively. The results demonstrate thatthe uptake of sn-2-monoolein at both the AP and BL membranes appears to be a saturable function of the monomer concentration ofsn-2-monoolein. Furthermore, trypsin preincubation inhibits sn-2-monoolein uptake at both AP and BL poles of cells.These results suggest that sn-2-monoolein uptake may be aprotein-mediated process. Competition studies also support aprotein-mediated mechanism and indicate that LCFA and LCMG may competethrough the same membrane protein(s) at the AP surface of Caco-2 cells.The plasma membrane fatty acid-binding protein (FABP_pm) isknown to be expressed in Caco-2, and here we demonstrate that fattyacid transport protein (FATP) is also expressed. These putative plasmamembrane LCFA transporters may be involved in the uptake ofsn-2-monoolein into Caco-2 cells.

     

Effects of n-3 fatty acids on growth and survival of J774 macrophages

To further understand potential mechanisms underlying the protective effects of eicosapentanoic acid (EPA) against atherosclerosis, J774 macrophages were used to explore cellular responses to growth in the presence of PUFA in vitro. Clonogenic assays indicated that 15 microg/ml of EPA killed over 90% of J774 populations. Docosapentaenoic acid (DPA) was more cytotoxic than either EPA or docosahexaenoic acid (DHA). EPA was shown to be elongated to DPA. Cytotoxicity induced by EPA was not inhibited by the presence of alpha-tocopherol (a-toc) in the medium. Immunological screening for caspase enzymes and microscopic examination indicated that apoptosis was not the major cause of cell death. Proliferation assays demonstrated that total cell numbers of EPA-treated cells were not significantly different to control cells. Increasing does of EPA were correlated with increasing levels of intracellular malondialdehyde (MDA). These observations suggest that EPA may influence the growth parameters of macrophages whilst inducing moderately elevated levels of oxidative stress.     

Effects of copper on the expression of metal transporters in human intestinal Caco-2 cells

The final step in the biosynthesis of nicotinamide-adenine dinucleotide, a major coenzyme in cellular redox reactions and involved in intracellular signaling, is catalyzed by the enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT). The X-ray structure of human NMNAT in complex with nicotinamide mononucleotide was solved by the single-wavelength anomalous dispersion method at a resolution of 2.9 A. Human NMNAT is a symmetric hexamer whose subunit is formed by a large six-stranded parallel beta-sheet with helices on both sides. Human NMNAT displays a different oligomerization compared to the archaeal enzyme. The protein-nicotinamide mononucleotide interaction pattern provides insight into ligand binding in the human enzyme.     

游离脂肪酸对大鼠胰岛细胞胰岛素信号转导蛋白表达的影响

目的 :探讨游离脂肪酸是否对大鼠胰岛细胞某些胰岛素信号转导蛋白的表达产生一定的影响。方法 :分离、培养新生SD大鼠胰岛细胞 ,分别与软脂酸 (0 .2 5mmol/L)或油酸 (0 .12 5mmol/L)孵育 12、2 4、36h ,提取蛋白后用Western印迹法检测胰岛素信号转导蛋白 (cPKCα、Grb2、ERK2 )的水平。结果 :软脂酸和油酸孵育 12h后 ,大鼠胰岛细胞cPKCα、Grb2和ERK2的蛋白水平同对照组相比均未发生显著变化 (P >0 .0 5 ) ;孵育 2 4h后胰岛细胞Grb2的蛋白水平同对照组相比未发生显著变化 (P >0 .0 5 ) ;cPKCα的蛋白水平同孵育 12h后相比显著上调 (P <0 .0 5 ) ;ERK2的蛋白水平同对照组相比明显下降 (P <0 .0 5 )。软脂酸和油酸孵育 36h后大鼠胰岛细胞cPKCα的蛋白水平同对照组及孵育 12h后相比显著上调 (P <0 .0 5 ) ;而Grb2和ERK2的蛋白水平同对照组及孵育 12h后相比均明显下降 (P <0 .0 5 )。结论 :游离脂肪酸可通过上调cPKCα和降低Grb2和ERK2的蛋白水平来阻滞胰岛素的信号转导 ,这可能是游离脂肪酸引起胰岛素抵抗的机制之一