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)
Ácidos Grasos/farmacología , Ácido alfa-Linolénico/metabolismo , Animales , Células CACO-2 , Bovinos , Colesterol/metabolismo , Células Clonales , Medios de Cultivo , Humanos , Cinética , Microvellosidades/efectos de los fármacos , Microvellosidades/metabolismo , Fosfolípidos/metabolismoRESUMEN
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)
Adenocarcinoma/metabolismo , Neoplasias del Colon/metabolismo , Mucosa Intestinal/metabolismo , Lípidos/biosíntesis , Ácido alfa-Linolénico/metabolismo , Antimetabolitos/farmacología , Células CACO-2 , Radioisótopos de Carbono , División Celular/fisiología , Células Clonales , Desoxiglucosa/farmacología , Inhibidores Enzimáticos/farmacología , Ácidos Grasos/metabolismo , Glucosa/metabolismo , Humanos , Intestinos/citología , Ionóforos/farmacología , Cinética , Monensina/farmacología , Oligomicinas/farmacología , Concentración Osmolar , Serina Endopeptidasas/metabolismo , Especificidad por Sustrato , Temperatura , Factores de Tiempo , Ácido alfa-Linolénico/análisisRESUMEN
Feeding rats a diet containing high levels of protein (as casein) increases the secretion and biosynthesis of pancreatic serine proteases. Cholecystokinin (CCK) presumably plays a role in this process although other GI peptides such as the gastrin-releasing peptide (GRP) may be involved. In this article, we describe the kinetics of pancreatic adaptation to a diet containing 45% protein as soybean and fish. Then we report the effect of treatment with either a cholecystokinin-receptor antagonist (MK-329) or a gastrin-releasing peptide-receptor antagonist ([D-F5 Phe6, D-Ala11]-Bn(6-13)OMe, or BIM 26226) on pancreatic adaptation to this diet. Prior to experiments, adult male Fischer rats received a diet containing 22% protein for 1 week. In the first experiment, 48 rats were fed a diet containing 45% protein; they were killed after 0-7 days. In the second experiment, 53 rats were fed the 22- or 45%-protein diet and received three daily injections of either the vehicle alone, MK-329, or BIM 26226 for 7 days before they were killed. When the protein-rich diet was fed for 0-7 days, amylase, in vitro biosynthesis, and mRNA levels were gradually decreased while serine protease biosynthesis was increased, reflecting the general enhancement of chymotrypsinogen, trypsinogen, and elastase mRNA levels. For all these parameters, adaptation leveled off after a 5-day feeding. When the protein diets were fed for 7 days, MK-329 significantly inhibited the adaptation of trypsin (specific activity and mRNA) and elastase (mRNAs) to the 45%-protein diet. BIM 26226 had no effect on pancreatic adaptation to the protein-rich diet.(ABSTRACT TRUNCATED AT 250 WORDS)