Fatty acid uptake and metabolism in a human intestinal cell line (Caco-2): comparison of apical and basolateral incubation

Free fatty acids can enter the enterocyte via the apical or basolateral plasma membrane. We have used the Caco-2 intestinal cell line to examine the polarity of free fatty acid uptake and metabolism in the enterocyte. Differentiated Caco-2 cells form polarized monolayers with tight junctions, and express the small intestine-specific enzymes sucrase and alkaline phosphatase. Cells were grown on permeable polycarbonate Transwell filters, thus allowing separate access to the apical and basolateral compartments. Total uptake of [3H]palmitate bound to bovine serum albumin (palmitate-BSA 4:1) was twofold higher (P less than 0.05 or less) at the apical surface than at the basolateral surface. The relative apical and basolateral membrane surface areas of the Caco-2 cells, as measured by partition of the fluorophore trimethylammonium-diphenylhexatriene TMA-DPH), was found to be 1:3. Thus, apical fatty acid uptake was sixfold higher than basolateral uptake per unit surface area. Analysis of metabolites after incubation with submicellar concentrations of [3H]palmitate showed that the triacylglycerol to phospholipid (TG:PL) ratio was higher for fatty acid added to the apical as compared to the basolateral compartment (20% at 60 min, P less than 0.025). Little fatty acid oxidation was observed. Preincubation with albumin-bound palmitate, alone or with monoolein, increased the incorporation of both apical and basolateral free fatty acids into TG. The results suggest that the net uptake of long-chain free fatty acids across the apical plasma membrane is greater than uptake across the basolateral membrane. In addition, a small increase in the TG:PL ratio for apically, compared to basolaterally, added free fatty acids suggests that polarity of metabolism occurs to a limited extent in Caco-2 enterocytes.     

Proline uptake by monolayers of human intestinal absorptive (Caco-2) cells in vitro.

Monolayers of the Caco-2 human intestinal cell line exhibit active and passive uptake systems for the imino acid L-proline. The active transport component is saturable and it is responsible for about two thirds of the observed flux over the nanomolar concentration range, at 37 degrees C and pH 7.4. In contrast to L-phenylalanine, specific L-proline uptake has a high degree of sodium dependency and the efficiency of the carrier system is significantly reduced when protein synthesis (cycloheximide), Na+/K(+)-ATPase (ouabain) or cellular metabolism (sodium azide) are inhibited. The expression of the L-proline carrier by Caco-2 cells was under some degree of nutritional control. Glucose deficiency, over the time scale of the experiment, had no effect. The temperature-dependence of the specific uptake process followed the Arrhenius model with an apparent activation energy of 93.5 kJ nmol-1. This pathway also displayed Michaelis-Menten concentration-dependence with a Ksdm of 5.28 mM and a maximal transport flux (Jsdmax) of 835 pmol min-1 (10(6) cells)-1. Although the passive component was unchanged, the pH of the donor phase exerted a profound effect on the active carrier component. Within the physiological pH range a local maximum efficiency was found at pH 7.4 but dramatic increases were noted as pH 5.0 was approached. In competition studies, with 100-fold excess of a second amino acid, strong inhibition of uptake was found with alpha-aminoisobutyric acid, L-alanine and L-serine whereas moderate inhibition was observed with glycine, D-proline and gamma-aminoisobutyric acid. Aromatic and branched amino acids showed weak (L-valine) or no interaction (L-phenylalanine, L-leucine) with the carrier system. These data indicate that the carrier system for the uptake of L-proline has many features in common with the A system for amino acid transport.     

Characteristics of lysophosphatidylcholine in its inhibition of taurine uptake by human intestinal Caco-2 cells

The characteristics of lysophosphatidylcholine (LPC) in its inhibition of the taurine uptake by human intestinal Caco-2 cells were investigated. By treating the cells with 200 microM of LPC, the taurine uptake was rapidly decreased by approximately 60%. This decrease was accompanied by an increase in the Km value for the uptake. A rapid uptake of LPC itself by the cells was also observed. The inhibitory activity of LPC was specific to the uptake of taurine and certain amino acids, while the uptake of glucose, glutamic acid and peptide (glycylglutamine) was not affected by LPC. The activity was dependent on the structure of a polar head and the bound fatty acid. The phosphorylcholine residue was likely to have played an important role, and surface active LPC with fatty acids of C14 or longer was highly inhibitory. These results suggest that the interaction of LPC with the taurine transporter in the intestinal cell membrane was the cause of the reduced taurine uptake.     

Characterization of carnosine uptake and its physiological function in human intestinal epithelial Caco-2 cells

Carnosine (beta-Ala-L-His) is known to have the physiological functions of an antioxidant. Although dietary carnosine is thought to be absorbed across intestinal epithelial cells, the mechanism for this absorption is not yet well understood and its function in the intestinal tract is also obscure. The intestinal transport of carnosine was characterized in the present study by using human intestinal Caco-2 cells, and its physiological function in these cells was further examined. The carnosine uptake was proton-dependent, being activated by lowering the apical pH value. Its uptake was significantly inhibited by other dipeptides, whereas it was not inhibited by other amino acids. These characteristics of the carnosine uptake strongly suggest its transport into the cells via peptide transporter 1 (PepT1). Since carnosine has antioxidative activity, we studied its effect on the H2O2-induced secretion of inflammatory cytokines in Caco-2 cells. The H2O2 induced increase in IL-8 secretion was inhibited by a pretreatment with carnosine for 3 h, this inhibition being presented in a dose-dependent manner. These results suggest that carnosine had a protective effect against oxidative stress in intestinal epithelial cells.     

Lactobacillus plantarum extracellular chitin-binding protein and its role in the interaction between chitin, Caco-2 cells, and mucin

In the present work, we describe the adhesion capabilities of a recombinant Lactococcus lactis strain producing an extracellular protein from Lactobacillus plantarum. Our results show that this protein may offer the bacterium a mechanism to bind to N-acetylglucosamine-containing polymers, such as human mucins, present in different environments.     

Pre-treatment of Caco-2 cells with zinc during the differentiation phase alters the kinetics of zinc uptake and transport(2)

The Caco-2 cell model was used to study the efficiency of absorption and endogenous excretion of zinc (Zn) regulated by dietary Zn concentration. Cells were seeded onto high pore-density membranes and maintained in medium supplemented with 10% FBS. After confluence, cells were treated with 5 or 25 μmol Zn/L for 7 d, and Zn uptake and transport were measured in both apical (AP) and basolateral (BL) directions by using ⁶⁵Zn. Similar cells were labeled with ⁶⁵Zn and the release of Zn to the AP and BL sides was measured. The AP uptake of Zn in cells exposed to 25 μmol Zn/L was slower (p < 0.05) than that in cells exposed to 5 μmol Zn/L. The AP to BL transport rate in the 25 μmol Zn/L group was only 40% (p < 0.05) of that in the 5 μM group. In contrast, the rate of BL Zn uptake was 4-fold higher in cells treated with 25 μmol Zn/L than in those treated with 5 μmol Zn/L (p < 0.05). The BL to AP transport rate was 2-fold higher in cells treated with 25 μmol Zn/L than in those treated with 5 μmol Zn/L (p < 0.05). Basolateral uptake was 6 to 25 times greater (p < 0.05) than AP uptake for cells treated with 5 and 25 μmol Zn/L, respectively. The rate of Zn release was enhanced about 4-fold (p < 0.05) by 25 μmol Zn/L treatment. Release to the BL side was 10 times greater than to the AP side. Zn-induced metallothionein (MT), thought to down-regulate AP to BL Zn transport, was 4-fold higher (p < 0.001) in the 25 μmol Zn/L group than in the 5 μM group, but the rate of BL Zn release was higher in cells treated with 25 μmol Zn/L than in those treated with 5 μmol Zn/L (p < 0.05). Induced changes in transport rates by media Zn concentrations could involve the up- and/or down-regulation of Zn influx and efflux proteins such as the ZIP and ZnT families of Zn transporters.     

Mechanism(s) of butyrate transport in Caco-2 cells: role of monocarboxylate transporter 1

The short-chain fatty acid butyrate was readily taken up by Caco-2 cells. Transport exhibited saturation kinetics, was enhanced by low extracellular pH, and was Na(+) independent. Butyrate uptake was unaffected by DIDS; however, alpha-cyano-4-hydroxycinnamate and the butyrate analogs propionate and L-lactate significantly inhibited uptake. These results suggest that butyrate transport by Caco-2 cells is mediated by a transporter belonging to the monocarboxylate transporter family. We identified five isoforms of this transporter, MCT1, MCT3, MCT4, MCT5, and MCT6, in Caco-2 cells by PCR, and MCT1 was found to be the most abundant isoform by RNase protection assay. Transient transfection of MCT1, in the antisense orientation, resulted in significant inhibition of butyrate uptake. The cells fully recovered from this inhibition by 5 days after transfection. In conclusion, our data showed that the MCT1 transporter may play a major role in the transport of butyrate into Caco-2 cells.     

Glutamine regulates the expression of proteins with a potential health-promoting effect in human intestinal Caco-2 cells

Glutamine is an essential amino acid for the enterocytes with respect to maintaining the gut mucosal integrity and function. This study was conducted to explore a molecular basis for the beneficial effects of glutamine on intestinal cells by searching for glutamine-dependent changes in the proteome. Caco-2 cells were exposed to different concentrations of L-glutamine with or without L-methionine sulfoximine, an inhibitor of the glutamine synthetase activity. 2-DE combined with MALDI-TOF-MS was used to identify proteins whose expression is changed by glutamine. To assess the relative protein synthesis rate, incorporation of L-[2H5]glutamine into individual proteins was monitored. The expression levels of 14 proteins changed significantly with the glutamine availability. Examples of differentially expressed proteins with potential health-promoting effects on the intestine are plasma retinol-binding protein, ornithine aminotransferase, apolipoprotein A-I, mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase, and acyl-CoA synthetase 5. Expression of these proteins was not changed by arginine deprivation. The differential change in the expression levels of the proteins was not correlated with their rate of synthesis, excluding an effect of glutamine depletion on general protein synthesis. Together, this study shows a gene-specific effect of glutamine on intestinal cells.     

Sorting of endogenous plasma membrane proteins occurs from two sites in cultured human intestinal epithelial cells (Caco-2)

We studied the postsynthetic sorting of endogenous plasma membrane proteins in a polarized epithelial cell line, Caco-2. Pulse-chase radiolabeling was combined with domain-specific cell surface assays to monitor the arrival of three apical and one basolateral protein at the apical and basolateral cell surface. Apical proteins were inserted simultaneously into both membrane domains. The fraction targeted to the basolateral domain was different for the three apical proteins and was subsequently sorted to the apical domain by transcytosis at different rates. In contrast, a basolateral protein was found in the basolateral membrane only. Thus, sorting of plasma membrane proteins occurred from two sites: the Golgi apparatus and the basolateral membrane. These data explain apparently conflicting results of earlier studies.     

Mechanisms of provitamin A (carotenoid) and vitamin A (retinol) transport into and out of intestinal Caco-2 cells

The purpose of this study was to compare the mechanisms of intestinal retinol (ROL) and carotenoid transport. When differentiated Caco-2 cells were incubated with ROL for varying times, cellular ROL plateaued within 2 h, whereas retinyl ester (RE) formation increased continuously. ROL and RE efflux into basolateral medium (BM) increased linearly with time, ROL in the nonlipoprotein fraction and REs in chylomicrons (CMs). In contrast to carotenoids, ROL uptake was proportional to ROL concentration (0.5-110 microM). ROL efflux into BM occurred via two processes: a) a saturable process at low concentrations (<10 microM) and b) a nonsaturable process at higher concentrations. When ROL-loaded cells were maintained on retinoid-free medium, free ROL, but not REs, was secreted into BM. Glyburide significantly reduced ROL efflux but not ROL uptake. Inhibition of ABCA1 protein expression by small interfering RNAs decreased ROL efflux but not carotenoid efflux. Scavenger receptor class B type I (SR-BI) inhibition did not affect ROL transport but decreased carotenoid uptake. The present data suggest that a) ROL enters intestinal cells by diffusion, b) ROL efflux is partly facilitated, probably by the basolateral transporter ABCA1, and c) newly synthesized REs, but not preformed esters, are incorporated into CM and secreted. In contrast to ROL transport, carotenoid uptake is mediated by the apical transporter SR-BI, and carotenoid efflux occurs exclusively via their secretion in CM.     

Androgen regulation of stage-dependent cyclin D2 expression in Sertoli cells suggests a role in modulating androgen action on spermatogenesis

Regulation of spermatogenesis involves stage-dependent androgen action on Sertoli cells, but the pathways involved are unclear. We assessed if cyclin D2 could play a role. In rats, Sertoli cell nuclear, stage-dependent immunoexpression of cyclin D2 switched on after Day 10 and persisted through Day 35, but disappeared by adulthood. However, ethane dimethane sulfonate (EDS)-induced testosterone withdrawal in adult rats for 6 days induced stage-dependent cyclin D2 immunoexpression in Sertoli cells, with highest expression at stages IX-XII and nondetectable at stages VI-VIII (opposite that for androgen receptor [AR] immunoexpression). In EDS-treated rats, a single injection of testosterone but not of estrogen reversed this change in 4 h, and testosterone administration from the time of EDS treatment prevented expression of cyclin D2 in Sertoli cells. The EDS-induced changes in cyclin D2 immunoexpression were matched by changes in expression of Ccnd2 (cyclin D2) mRNA in isolated stage-dissected tubules. Treatment of adult rats with flutamide induced stage-dependent cyclin D2 immunoexpression in Sertoli cells within 18 h, and confocal microscopy revealed that immunoexpression of AR and cyclin D2 were mutually exclusive within individual seminiferous tubules in these animals. Sertoli cell-selective ablation of the AR in mice using Cre/loxP technology also resulted in stage-dependent Sertoli cell cyclin D2 immunoexpression. Downstream from cyclin D2 action is retinoblastoma 1 (RB1), a tumor suppressor protein, immunoexpression of which paralleled stage-dependent AR expression in Sertoli cells; RB1 stage specificity disappeared after EDS treatment. These results point to a non-cell cycle role for cyclin D2 and RB1 in mature Sertoli cells in the stage-dependent mechanisms regulated by AR expression and androgen action.     

Microtubular organization and its involvement in the biogenetic pathways of plasma membrane proteins in Caco-2 intestinal epithelial cells

We characterized the three-dimensional organization of microtubules in the human intestinal epithelial cell line Caco-2 by laser scanning confocal microscopy. Microtubules formed a dense network approximately 4-microns thick parallel to the cell surface in the apical pole and a loose network 1-micron thick in the basal pole. Between the apical and the basal bundles, microtubules run parallel to the major cell axis, concentrated in the vicinity of the lateral membrane. Colchicine treatment for 4 h depolymerized 99.4% of microtubular tubulin. Metabolic pulse chase, in combination with domain-selective biotinylation, immune and streptavidin precipitation was used to study the role of microtubules in the sorting and targeting of four apical and one basolateral markers. Apical proteins have been recently shown to use both direct and transcytotic (via the basolateral membrane) routes to the apical surface of Caco-2 cells. Colchicine treatment slowed down the transport to the cell surface of apical and basolateral proteins, but the effect on the apical proteins was much more drastic and affected both direct and indirect pathways. The final effect of microtubular disruption on the distribution of apical proteins depended on the degree of steady-state polarization of the individual markers in control cells. Aminopeptidase N (APN) and sucrase-isomaltase (SI), which normally reach a highly polarized distribution (110 and 75 times higher on the apical than on the basolateral side) were still relatively polarized (9 times) after colchicine treatment. The decrease in the polarity of APN and SI was mostly due to an increase in the residual basolateral expression (10% of control total surface expression) since 80% of the newly synthesized APN was still transported, although at a slower rate, to the apical surface in the absence of microtubules. Alkaline phosphatase and dipeptidylpeptidase IV, which normally reach only low levels of apical polarity (four times and six times after 20 h chase, nine times and eight times at steady state) did not polarize at all in the presence of colchicine due to slower delivery to the apical surface and increased residence time in the basolateral surface. Colchicine-treated cells displayed an ectopic localization of microvilli or other apical markers in the basolateral surface and large intracellular vacuoles. Polarized secretion into apical and basolateral media was also affected by microtubular disruption. Thus, an intact microtubular network facilitates apical protein transport to the cell surface of Caco-2 cells via direct and indirect routes; this role appears to be crucial for the final polarity of some apical plasma membrane proteins but only an enhancement factor for others.     

Differentiation of liver epithelial (stem-like) cells into hepatocytes induced by coculture with hepatic stellate cells

The liver is believed to contain stem cells that can differentiate into either hepatocytes or biliary epithelial cells. In the present study, we established a nonhepatocytic epithelial cell line from the normal livers of adult rats. The established cells, designated HSL cells, were immunoreactive against alpha-fetoprotein, but neither albumin nor cytokeratin 19. To demonstrate the differentiation potential of HSL cells in vitro, the cells were cocultured with hepatic stellate cells as a mixture or separately using insert wells. Consequently, although coculture with hepatic stellate cells rendered HSL cells able to produce albumin, the mixed coculture system mimicking the hepatic environment elicited this phenomenon more effectively than the separated coculture system. In conclusion, HSL cells have immature properties and the potential to differentiate into mature cells. Not only the extracellular matrices but also soluble factors, which are produced by hepatic stellate cells, induce this maturation, demonstrating the importance of the hepatic environment for hepatocyte differentiation.     

Nocodazole, a microtubule-active drug, interferes with apical protein delivery in cultured intestinal epithelial cells (Caco-2)

The polarized delivery of membrane proteins to the cell surface and the initial secretion of lysosomal proteins into the culture medium were studied in the polarized human intestinal adenocarcinoma cell line Caco-2 in the presence or absence of the microtubule-active drug nocodazole. The appearance of newly synthesized proteins at the plasma membrane was measured by their sensitivity to proteases added either to the apical or the basolateral surface of cells grown on nitrocellulose filters. Nocodazole was found to reduce the delivery to the cell surface of an apical membrane protein, aminopeptidase N, and to lead to its partial missorting to the basolateral surface, whereas the drug had no influence on the delivery of a basolateral 120-kD membrane protein defined by a monoclonal antibody. Furthermore, nocodazole selectively blocked the apical secretion of two lysosomal proteins, cathepsin D and acid alpha-glucosidase, whereas the drug had no influence on their basolateral secretion. These results suggest that in Caco-2 cells an intact microtubular network is important for the transport of newly synthesized proteins to the apical cell surface.