RESUMEN

OBJECTIVES: The present study examines the kinetic of plasma triacylglycerol (TAG) after sequential ingestion of lunch and dinner as well as the contribution of dietary fat ingested at lunch to subsequent post-dinner TAG composition. METHOD: Six healthy subjects were included. After standardized breakfast (7: 30AM), 2 mixed meals with fat loads composed of 44 g olive oil (rich in oleic acid) at lunch (12PM) and 44 g sunflower oil (rich in linoleic acid) at dinner (7PM) were ingested. [1-13C] palmitate was added in lunch only. Plasma TAG and chylomicron-TAG (CMTAG) levels were measured sequentially after meals. [1-13C] palmitate enrichment and concentrations of oleic acid and linoleic acid were measured in all lipid fractions. RESULT: Post-dinner plasma TAG peak was delayed as compared to lunch (3 hours vs 1 hour, p=0.002) whereas the magnitude of the postprandial peaks was not significantly different between lunch and dinner (2.4+/-0.3 vs 2.0+/-0.4 mmol/L, p=0.85). [1-13C] palmitate enrichment was maximal 5 hours after lunch in all lipid fractions and decreased slowly thereafter. After dinner ingestion, the rate of decline of [1-13C] palmitate enrichment plateaued during the first 60 minutes. Oleic acid increased slightly and immediately after dinner and remained the predominant fatty acid in all lipid fractions during the first hour after dinner. A delayed peak of plasma and CM-TAG was observed after dinner as compared to lunch without difference in the magnitude of peaks. CONCLUSION: The contribution of dietary fat ingested at lunch to post-dinner lipemia is confirmed despite the relatively long lasting interval between the 2 meals (7 h) and the absence of any early peak of plasma TAG after dinner.

Asunto(s)

Asunto(s)

RESUMEN

We investigated the influence of four different culture media: 20% fetal bovine serum (FBS), 5% FBS, 5% FBS supplemented with 10 mg x L(-1) linoleic acid (18:2(n-6)) or alpha-linolenic acid (18:3(n-3)) on alpha-linolenic acid apical uptake in clone TC7 of human intestinal Caco-2 cell line. Neither cellular viability nor cell monolayer integrity and permeability were altered by the four culture conditions. Our results show that the different culture media led to changes in alpha-linolenic acid maximal rate of uptake (Vmax) but did not alter the apparent transport constant (Km). Reducing FBS concentration from 20% to 5% increased significantly the rate of alpha-linolenic acid uptake, which was further increased by supplementation of the medium with 18:2(n-6) or 18:3(n-3). Supplementation with essential fatty acids led to a marked enrichment of brush-border membrane phospholipids in polyunsaturated fatty acids of the corresponding series and decreased significantly the levels of monounsaturated fatty acids. Saturated fatty acids, unsaturation index, and cholesterol/fatty acid ratios were unchanged. No clear relation could be established between the changes in membrane lipid composition and the alterations of alpha-linolenic acid uptake. These results indicate a weak influence of membrane lipid composition in the modulation of the uptake. Therefore, the increase of uptake following long-term supplementation of TC7 cells with essential fatty acids could be attributed to an increase of the expression of membrane protein(s) involved in the apical uptake of long-chain fatty acids. This remains to be established.

Asunto(s)

RESUMEN

The uptake kinetics of alpha-linolenic acid (18:3(n - 3)), an essential fatty acid, were investigated in the human intestinal cell line Caco-2. Four clones (PD10, PF11, PD7 and TC7) from the heterogeneous parental Caco-2 cells population were used. After a screening step using isolated cells, the TC7 clone was selected for the study of alpha-linolenic acid uptake. [1-(14)C]linolenic acid dissolved in 10 mM taurocholate was presented to the microvillus plasma membrane (apical side) of TC7 differentiated cells, grown on a semi-permeable polycarbonate membrane. The results show that the initial rate of uptake is not a linear function of the 18:3(n- 3) monomer concentration in the incubation medium. In the monomer concentration range studied (0.2 to 36 microM) apical uptake was saturable and followed Michaelis-Menten kinetics (V(max) = 15.4 +/- 0.6 nmol/mg protein per min, K(m) = 14.3 +/- 1.3 microM). In addition, it was temperature- and energy-dependent but was apparently unaffected by the sodium gradient and intracellular metabolic fate of 18:3(n - 3). Excess of unlabeled saturated or unsaturated long chain fatty acids (C16 to C22) led to a 27-68% reduction of [1-(14)C]linolenic acid uptake. Likewise basolateral uptake was saturable (V(max) = 4.9 +/- 0.7 nmol/mg protein per min, K(m) = 8.7 +/- 2.9 microM). These facts argue in favour of the existence in these human intestinal cells of a carrier-mediated transport system for alpha-linolenic acid and probably other long chain fatty acids as well.

Asunto(s)

RESUMEN

The mechanisms by which ET-18-OCH3 (1-O-octadecyl-2-O-methyl-sn-glycero-3 -phosphocholine) and other analogues of alkyl-lysophospholipids exert their antineoplastic effects are not yet fully elucidated. Possible interference with mechanisms involving intracellular pH (pHi) regulation was examined by measuring the effect of ET-18-OCH3 on the activity of the Na+/H+ exchanger in the breast cancer-derived cell line MCF-7. When ET-18-OCH3 was added to culture medium at 10 muM (determined as a noncytotoxic but cytostatic concentration), it led to an intracellular acidification (0.15 pH unit). It also decreased the rate of pHi recovery by Na+/H+ exchange following artificial acidification. Kinetic parameters of the exchange indicated that this was due to a decrease in the affinity of the exchanger for both transported ions, rather than to a decrease in the number of exchanger proteins in the membrane (same maximal efflux rate for treated and untreated cells). These results suggest that Na+/H+ exchanger inhibition and subsequent cytoplasmic acidification participate in the mode of action of ET-18-OCH3, and could be used for modulation of tumor-cell chemosensitivity or their subsequent commitment into programmed cell death.

Asunto(s)

RESUMEN

In a previous study we showed that intestinal uptake of alpha-linolenic acid (18:3n-3) was carrier-mediated and we suggested that a plasma membrane fatty acid protein was involved in the transport of long-chain fatty acids. To further test this hypothesis, the mechanism of linoleic acid (18:2n-6) uptake by isolated intestinal cells was examined using a rapid filtration method and 20 mM sodium taurocholate as solubilizing agent. Under these experimental conditions transport of [1-14C]linoleic acid monomers in the concentration range of 2 to 2220 nM was saturable with a Vm of 5.1 +/- 0.6 nmol/mg protein/min and a Km of 183 +/- 7 nM. Experiments carried out in the presence of metabolic inhibitors, such as 2,4-dinitrophenol and antimycin A, suggested that an active, carrier-mediated mechanism was involved in the intestinal uptake of this essential fatty acid. The addition of excess unlabeled linoleic acid to the incubation medium led to a 89% decrease in the uptake of [1-14C]linoleic acid, while D-glucose did not compete for transport into the cell. Other long-chain polyunsaturated fatty acids added to the incubation mixture inhibited linoleic acid uptake by more than 80%. The presence of alpha-linolenic acid (18:3n-3) in the incubation medium caused the competitive inhibition (Ki = 353 nM) of linoleic acid uptake. The data are compatible with the hypothesis that intestinal uptake of both linoleic, and alpha-linolenic acid is mediated by a membrane carrier common to long-chain fatty acids.

Asunto(s)

RESUMEN

Na(+)-H+ exchange activity was studied in a human breast cancer cell line. At steady state, the intracellular pH (pHi) of the cells was 7.23 +/- 0.01, and intracellular buffering capacity (beta i) was 44 +/- 4 mM/pH unit. pHi was controlled by a Na(+)-H+ antiporter that was reversible, electroneutral, inhibited by 5-(N-methyl-N-isobutyl)amiloride, and dependent on extracellular Na+. The exchanger function depended on internal H+ concentration, according to an allosteric activation mechanism obeying the model of Hill. The exchanger was inactive at pHi > or = 7.22, and its maximal activity was reached at pHi < 6.60. The exchanger was stimulated by osmotic shrinking but was unaffected by growth factors (epidermal growth factor, insulin-like growth factor I) or by serum. When cells were grown in a medium supplemented with linoleic or alpha-linolenic acids, large quantities of the additional fatty acid accumulated in membranes, saturated fatty acids increased, and monounsaturated fatty acids decreased. These changes reduced cell proliferation but had no effect on the steady-state value of pHi, on beta i, or on the kinetic parameters of the Na(+)-H+ exchanger. Therefore, in this system, cell proliferation is not directly related to the activation of the Na(+)-H+ exchanger.

Asunto(s)

RESUMEN

The effect of calcium upon the uptake of oleic acid solubilized with 10 mM taurocholate was investigated using an in vitro model of isolated enterocytes. The addition of Ca2+ to the incubation medium (Hanks' medium) led to a decrease in oleic-acid uptake. This uptake inhibition was dependent on both the amount of Ca2+ and the fatty-acid concentration, since the inhibitory effect was significant for 10 microM but not 100 microM oleic acid. The determination of the monomeric activity of oleic acid indicated that the decrease in fatty-acid uptake was not linked to the formation of insoluble calcium soaps. The replacement of Hank's medium by several mineral waters containing between 0.3 and 11.7 mM Ca2+ significantly reduced the uptake of both 10 and 100 microM oleic-acid. The ionic composition of these waters was correlated with the decrease of initial rate of oleic acid uptake, but Ca2+ and other ions could interfere by synergetic effects with the fatty-acid-absorption mechanism. It is concluded that the ion-induced inhibition of oleic-acid uptake is not due to the formation of insoluble soaps but rather to a direct effect on the fatty-acid transport in membranes. Whether the fatty-acid binding protein in the plasma membrane is involved in the effect of Ca2+ on fatty-acid transport remains to be established.

Asunto(s)

RESUMEN

The intestinal uptake of [1-14C]linolenic acid [18:3(n-3)], an essential fatty acid, was investigated in isolated hamster intestinal cells using a rapid filtration method and 20 mmol/L taurocholate as solubilizing agent. Under these conditions, the initial rate of alpha-linolenic acid uptake was not a linear function of external monomer concentrations in the range of 2 to 2250 nmol/L, but rather the transport system was characterized by saturation kinetics with Vmax = 11.37 nmol.mg protein-1.min-1 and Km = 382 nmol/L. Temperature and metabolic poisons (2,4-dinitrophenol, antimycin A) drastically decreased the initial rate of uptake, as did replacement of Na+. The presence of excess unlabeled alpha-linolenic acid in the incubation medium significantly inhibited the uptake of [1-14C]linolenic acid, whereas L-alanine and D-glucose had no effect. Other long-chain fatty acids (saturated or unsaturated), as well as cholesterol, inhibited the uptake of [1-14C]linolenic acid. We concluded that an active, carrier-mediated mechanism was involved in the intestinal transport of alpha-linolenic acid. Inhibition data are compatible with the hypothesis that intestinal uptake of alpha-linolenic acid is mediated by a carrier common to long-chain fatty acids.

Asunto(s)

RESUMEN

The intracellular pH (pHi) of isolated hamster intestinal cells was determined with three techniques: the null-point method with digitonin, the distribution of the weak acid 5,5-dimethyloxazolidine-2,4-dione, and the trapped fluorescent indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. In physiologic saline, the pHi determined with the fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein was 7.00 +/- 0.03 (n = 20) but was 6.68 +/- 0.04 (n = 25) when there was no Na+ in the medium, indicating an important role of external Na+ in maintaining pHi at neutral level. When Na+ was added to an incubation medium lacking the ion, the pHi increased. The time-course of this alkalinization depended on the Na+ concentration, whereas K+ had only a slight effect. Amiloride (1 mM) completely inhibited the Na+ effect, thereby showing the basic role of the Na+/H+ antiport in the regulation of pHi. The effect of 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and furosemide showed that there also exists an anionic component in the process, probably of the Cl-/OH- (HCO3-) exchange type. These results show that the amiloride-sensitive Na+/H+ antiport plays an important but not exclusive role in maintaining the pHi neutral or slightly alkaline in isolated intestinal cells from the hamster.

Asunto(s)

RESUMEN

The cytoplasmic pH (pHi) was determined in isolated rat intestinal cells with four methods. The pHi of cells in physiological saline buffered with Hepes (pH 7.3) at 37 degrees C was close to 7.0. The most reliable method, using the fluorescent pH indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), furnished a mean value of 7.03 +/- 0.05 (n = 42). The buffering capacity of intestinal cells determined with this fluorescent indicator was 62 +/- 5 mmol.l-1.pH-1. The mechanism governing the control of cytoplasmic pH was also investigated with BCECF, varying the Na+ concentration inside and outside the cells. When intestinal cells were suspended in a sodium-free medium in the presence or absence of ouabain, they became acidified. The process was reversed when Na+ was added to the incubation medium. An identical phenomenon occurred when the cells were artificially acidified with NH4Cl. Additional experiments led to the conclusion that isolated rat intestinal cells have an Na+/H+ exchanger independent of Cl- and inhibited by amiloride. This exchanger plays an important but not exclusive role in the control of pHi. The presence of other exchangers and the high buffering power of the cells explains the high stability of pHi noted in this study.

Asunto(s)

RESUMEN

The uptake of biotin by isolated hamster intestinal cells was investigated in the zone of physiological concentrations. Uptake of the vitamin was not a linear function of the external concentrations and kinetics could be saturated [Km = 1.12 microM, Vmax = 33.9 pmol/(mg protein X min)]. Metabolic inhibitors (antimycin A, 2,4-dinitrophenol) had no effect, so uptake is not energy dependent. Ouabain and N-ethylmaleimide, inhibitors of cation gradients, had no effect on biotin uptake, thus showing the absence of cotransport. The inhibition of uptake by analogs of biotin, that is, biocytin and DL-thioctic acid, indicates that the terminal carboxyl group and the thiophane ring play a role in the recognition of biotin. Counterflow experiments showed competitive inhibition of efflux when excess biotin was present in the cells. These findings are consistent with biotin uptake by isolated hamster enterocytes being a process of facilitated diffusion.

Asunto(s)

RESUMEN

Isolated intestinal mucosa cells of rats were used to investigate the intestinal transport of biotin. This method utilizing a double-label isotope technique showed that uptake could not be saturated, even in a wide range of biotin concentrations (0.01-2 microM). A metabolic inhibitor (antimycin A) did not prevent cell uptake of biotin. The transport mechanism was independent of temperature (Q10 = 1.04). When excess biotin was added to the incubation medium, there was no efflux of the vitamin from intestinal cells. The results also showed that the cells did not concentrate the vitamin, regardless of its concentration in the incubation medium. The mechanism of biotin uptake by rat cells at physiological concentrations is thus a passive diffusion phenomenon.

Asunto(s)

Asunto(s)