Divisions of 1 Medical Genetics and 2 Gastroenterology, Research Center, Hôpital Sainte-Justine, Department of Pediatrics, Université de Montréal, Montreal, Quebec, Canada H3T 1C5
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ABSTRACT |
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L-Carnitine is derived both from
dietary sources and biosynthesis. Dietary carnitine is absorbed in the
small intestine and then distributed to other organs. Previous studies
using Caco-2 cells demonstrated that the transport of
L-carnitine in the intestine involves a carrier-mediated
system. The purpose of this study was to determine whether the uptake
of L-carnitine in Caco-2 cells is mediated by the recently
identified organic cation/carnitine transporter (OCTN2). Kinetics of
L-[3H]carnitine uptake were investigated with
or without specific inhibitors. L-Carnitine uptake in
mature cells was sodium dependent and linear with time.
Km and Vmax values for saturable
uptake were 14.07 ± 1.70 µM and 26.3 ± 0.80 pmol · mg
protein1 · 6 min
1,
respectively. L-carnitine uptake was inhibited
(P < 0.05-0.01) by valproate and other organic
cations. Anti-OCTN2 antibodies recognized a protein in the brush-border
membrane (BBM) of Caco-2 cells with an apparent molecular mass of 60 kDa. The OCTN2 expression was confirmed by double immunostaining. Our
results demonstrate that L-carnitine uptake in
differentiated Caco-2 cells is primarily mediated by OCTN2, located on
the BBM.
intestine; brush-border membrane; organic cation transport; anti-OCTN2 antibodies; valproate.
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INTRODUCTION |
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-HYDROXY-
-TRIMETHYLAMINOBUTYRATE
(L-carnitine) plays an important role in lipid
metabolism by facilitating the transport of long-chain fatty acids
across the mitochondrial inner membrane followed by fatty acid
-oxidation (3, 35). Carnitine, supplied principally in
the diet, is absorbed in the intestine and transported to many other
tissues by putative transporters. Recently, a new family of organic
cation transporters, designated organic cation/carnitine transporter
(OCTN) has been described (43). Some novel members of this
family, namely OCTN1, -2, and -3, are also capable of transporting
carnitine, each with distinct characteristics (18, 41,
43). OCTN2, the most important carnitine transporter, was
recently cloned (42, 55, 56). Mutations in this protein cause the autosomal recessive inborn error, systemic carnitine deficiency (SCD) (4, 17, 30, 50). OCTN2 has 12 putative transmembrane domains, is expressed in several tissues, such as kidney,
and transports carnitine in an Na+-dependent manner, with
high-affinity and pH-dependence (19, 32, 42, 55, 56).
OCTN2 also plays an important pharmacological role, because it mediates
the transport of many drugs, including tetraethylammonium (TEA),
pyrilamine, valproate, and verapamil (55).
Recently, Nakanishi et al. (28) described the
identification of a second energy-coupled carnitine transporter. This
transporter, called amino acid transporter system B0,+
(ATB0,+), is primarily as an amino acid transporter
(29) and plays an important role in their absorption in
the intestine. It is expressed in the intestine, lung, and mammary
gland and has an Na+- and Cl-coupled
transport system for neutral and cationic amino acids (28,
29).
In the intestine, the mechanism of L-carnitine uptake remains unclear. Earlier studies (9) using human intestinal biopsy specimens suggested that intestinal L-carnitine uptake occurred by a carrier-mediated process. However, a subsequent study (21) using rat intestinal brush-border membrane (BBM) vesicles concluded that the uptake process is the result of passive diffusion. Human-derived intestinal epithelial cell lines constitute in vitro models for the study of small intestinal epithelia. Among these, Caco-2 cells readily undergo enterocyte-like differentiation and when grown to confluency, adopt many of the absorptive characteristics of villus absorptive cells (20, 25). In culture, Caco-2 cells progress through distinct states: homogeneously undifferentiated (subconfluence), heterogeneously polarized and differentiated (0-20 days postconfluence), and homogeneously polarized and differentiated (30 days postconfluence) (49). By using these cells in their second state, i.e., with a strong discrepancy in cellular morphology and ultrastructural brush border organization, McCloud et al. (25) have shown that the L-carnitine uptake involves a carrier-mediated system (Km =45 µM) that is temperature, Na+, and energy dependent. However, the exact nature and identity of the carnitine transporter in Caco-2 cells has not been defined.
We hypothesized that carnitine uptake by Caco-2 cells is mediated by OCTN2 on the basis of its functional properties, i.e., Na+-dependence, pH-dependence, and inhibition by valproate (18, 42, 56). The results of our studies indicate that OCTN2 is indeed expressed on the BBM of Caco-2 cells.
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MATERIALS AND METHODS |
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Cell culture. Caco-2 cells were obtained from the American Type Culture Collection (Manassas, VA) at passage 17. Cells were grown in 75-cm2 plastic flasks (Corning Glass Works, Corning, NY) in MEM (GIBCO-BRL, Grand Island, NY) supplemented with decomplemented FCS 10% (GIBCO-BRL), penicillin/streptomycin 1% (GIBCO-BRL), and DMEM nonessential amino acid solution (GIBCO-BRL). The cells were maintained at 37°C in a 95% air-5% CO2 atmosphere. After confluence (70-90%), cells were split by using 0.05% trypsin (0.5 nM) in EDTA (GIBCO-BRL).
Sucrase activity. To assess cellular differentiation, sucrase activity was measured. The Caco-2 cells (between passages 34 and 37) were plated (5 × 106/well) onto 12-well plates (Corning Glass Works) in MEM supplemented with 5% FCS. Undifferentiated (subconfluent), partially differentiated (7-10 days postconfluence) and mature (10-15 postconfluence) Caco-2 cells, were scraped in maleate buffer and sonicated, and sucrase activity was assayed by the method of Messer and Dahlqvist (26) as modified by Lloyd and Whelan (22).
Carnitine uptake.
Carnitine uptake studies were carried out according to McCloud's
method (25), with minor modifications. Caco-2 cells
(between passages 34 and 37) were plated (5 × 106/well) onto 12-well plates (Corning Glass Works) in MEM
supplemented with 5% FCS. The cells were used for uptake studies
10-15 days after confluence, and all the experiments were
conducted at 37°C. The mature cells were incubated in 1 ml of
prewarmed Krebs-Ringer phosphate buffer (in mM: 123 NaCl, 4.93 KCl,
1.23 MgSO4, 0.85 CaCl2, 5 glucose, 5 glutamine,
and 20 NaH2PO4, pH 7.4) containing methyl-L-[3H]carnitine (Amersham Pharmacia
Biotech, Buckinghamshire, UK) at a concentration of 50 nM. After
different incubation periods, the medium was aspirated and cells were
immediately washed with ice-cold phosphate buffer and digested at
70°C with 0.5 ml of 1 N NaOH. The reaction was neutralized after
1 h by adding 0.5 ml of 1 N HCl. The cells were then collected,
scintillation liquid was added, and radioactivity was counted. In
another series of experiments, the Na+, pH,
Cl dependence, and the effect of some ATB0,+
substrates (leucine, alanine, tryptophan) on carnitine uptake were explored.
Assays with inhibitors. For the inhibitor experiments, mature Caco-2 cells (10-15 postconfluence) were preincubated for 30 min with 500 µM pyrilamine, verapamil, TEA, or valproate. All these drugs were purchased from Sigma (St. Louis, MO). Protein determinations were done on parallel wells by the method of Lowry et al. (23).
Western blot analysis. Rabbit polyclonal antibodies were raised against a synthetic polypeptide from Research Genetics (Huntsville, AL), 5'-QWQIQSQTRMQKDGEESPT-3', corresponding to amino acids 532-550 of mouse OCTN2 (17). Undifferentiated (subconfluence), partially differentiated (7-10 days postconfluence) and mature (10-15 days postconfluence) Caco-2 cells (71-90% confluence) were washed three times with saline and collected with a scraper. Thereafter, cells were centrifuged for 5 min at 24°C (850 g). The pellet was suspended in 200 µl of saline and sonicated for 20 s. BBMs and basolateral membranes (BLMs) of mature (10-15 days postconfluence) Caco-2 cells were isolated as described by Schmitz et al. (37), and the degree of their purity was assessed by measuring the enrichment of marker enzymes, alkaline phosphatase for the BBM (15), and Na+,K+-ATPase for the BLM (27). The enrichment factor of alkaline phosphatase in BBM was 10-fold, similar to that reported recently by Hirohashi et al. (14). For the BLM, the relative enrichment of Na+,K+-ATPase over the homogenate was eight. The membranes were dispersed ultrasonically and sample buffer 2 × [4% SDS, 20% glycerol, 200 mM DTT, 120 mM Tris (pH 6.8), and 0.002% bromphenol blue] was then added to Caco-2 cell homogenates or to the isolated membrane preparations. Proteins were denatured in a boiling water bath for 5 min before use for Western blot analysis. Samples were first separated by electrophoresis on a 7.5% SDS-PAGE. The proteins were transferred to polyvinylidene difluoride (PVDF) membrane Immobilon (Millipore, Bedford, MA). The PVDF membrane was incubated in PBS buffer (in mM: 150 NaCl, 2.5 KCl, 5 Na2HPO4, 1.5 KH2PO4) containing 0.02% sodium azide and 12% skim milk. Subsequently, the membranes were incubated overnight with polyclonal anti-peptide antibody (in PBS buffer containing 0.02% sodium azide and 1% BSA), rinsed with the above buffer with 0.1% Tween 20 without skim milk, then incubated with secondary antibody [goat anti-rabbit IgG, horseradish peroxidase-linked whole antibody (Sigma)]. The membranes were washed as before and the ELISA substrate added (Boehringer Mannheim, Laval, QC, Canada). Molecular weights were estimated by using prestained SDS-PAGE broad-range standards (Bio-Rad, Hercules, CA).
Immunofluorescence. Caco-2 cells (between passages 34 and 37) were grown on glass coverslips and were maintained in 95% air-5% CO2 atmosphere at 37°C. They were grown in a complete medium consisting of MEM containing 1% penicillin/streptomycin, 1% DMEM nonessential amino acids, and 5% decomplemented FCS. The cells were employed for experiments 10-15 days after confluence. They were first fixed in 3.8% paraformaldehyde for 30 min at room temperature. After brief washings, the cells were permeabilized for 10 min at room temperature in 0.2% Triton X-100 and blocked with 3% BSA in PBS. The primary antibodies anti-OCTN2 and anti-alkaline phosphatase (Medicorp, Montreal, QC, Canada), diluted 1:800 and 1:80 in PBS, were added and incubated with cells for 90 min at 4°C. After brief washings, fluorescein-conjugated goat anti-rabbit IgG (Biosource International, Montreal, QC, Canada) (1:500) and rhodamine-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA) (1:400) were used as secondary antibodies, at a dilution of 1:500. The cover glass was washed, mounted in Vectashield medium (Vector Laboratories, Burlingame, CA), and examined by fluorescence microscopy by using a Nikon Eclipse E600 microscope.
Statistical analysis. Results are reported as means ± SE of at least triplicate samples. Statistical analyses were performed by using ANOVA with GraphPad InStat (GraphPad Software, San Diego, CA). For the determination of uptake kinetic parameters, the Graph PRISM version 3.0 program was used. Inhibition studies using valproate were analyzed by using linear regression of Lineweaver-Burk plots.
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RESULTS |
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Assessment of the state of differentiation of Caco-2 cells. Sucrase activity, determined before each experiment, was 0.015 ± 0.005 IU/g protein for undifferentiated (subconfluent cells) and 3.2 ± 1.1 IU/g protein for partially differentiated (7-10 postconfluent cells). Sucrase activity was 7.9 ± 1.5 IU/g protein at 10-15 days postconfluence, confirming cellular maturation and differentiation.
L-[3H]carnitine uptake.
We studied carnitine transport in undifferentiated and mature Caco-2
cells using L-[3H]carnitine at a
concentration of 50 nM. Uptake by mature Caco-2 cells was linear
initially and reached an equilibrium at ~8 min at a rate of 0.426 pmol · mg
protein1 · min
1 (Fig.
1). In undifferentiated Caco-2 cells,
uptake values were very low, indicating an absence of carnitine
transport (Fig. 1).
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Effect of cationic drugs on L-carnitine uptake.
Subsequent uptake experiments were performed by using mature Caco-2
cells, in the presence of Na+. Preincubation for 30 min
with 500 µM of verapamil, pyrilamine, valproate, or TEA revealed in
each case, a significant decrease in
L-[3H]carnitine uptake (Table
1). As shown in Table 1, unlabeled carnitine but not proline (at a concentration of 500 µM, 30 min preincubation) inhibited L-[3H]carnitine
uptake (P < 0.01) (Table 1).
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OCTN2 identification and localization.
Western blot analysis revealed a protein with an apparent molecular
weight of 60 kDa labeled by the mouse OCTN2-specific antibody in
partially and mature Caco-2 cell homogenates (Fig.
4A). In contrast,
undifferentiated Caco-2 cells showed no immunoreactivity with this
antibody (Fig. 4A). To localize the OCTN2, BBM and BLM were
isolated from mature Caco-2 cells and probed with the specific antibodies. The OCTN2 antibody recognized a 60-kDa protein in immunoblots of BBM from mature Caco-2 cells (Fig. 4B). No
bands were detected in BLM (Fig. 4B).
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Immunofluorescence.
Immunostaining with anti-OCTN2 polyclonal antibodies revealed strongly
positive fluorescence (Fig.
5A). The distribution of fluorescence with anti-OCTN2 was approximately the same when the cells
were stained by anti-alkaline phosphatase (Fig. 5B), which is specific to the apical membrane. Colocalization of OCTN2 and alkaline phosphatase (Fig. 5C) revealed that OCTN2 was
restricted to the apical membrane of mature Caco-2 cells.
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DISCUSSION |
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The present study demonstrates the presence of a specific transporter for the uptake of L-carnitine in mature Caco-2 cells. To pursue our hypothesis that OCTN2 may be responsible for this observation, we have determined its characteristics in the presence and absence of inhibitors of OCTN2-mediated transport and confirmed the presence of OCTN2 by Western blot analysis and immunofluorescence. We observed that mature Caco-2 cells are able to transport L-carnitine, whereas undifferentiated cells are not. Transport occurs only when the cells attain their ultrastructural brush-border organization, up to 10-15 days postconfluence (Fig. 1). Previous studies on Caco-2 cells done with a variety of soluble molecules have shown that transport occurs only after cell polarization (13). Caco-2 cells have been shown to express the transport systems of nutrients, such as carbohydrates (1, 24), amino acids (11, 31), dipeptides (45), and bile acids (12), only when they acquire other properties of differentiated enterocytes. In our studies (Fig. 1), the carnitine transport in Caco-2 cells appears to be mediated by a membrane transporter, because its uptake was only observed in mature cells (polarized epithelia). Hamilton et al. (9) reported that human small intestine tissue is capable of carnitine uptake, whereas colonic tissue is not. Of note, although undifferentiated Caco-2 cells resemble the colon cancer from which they are derived, mature Caco-2 cells are morphologically and functionally like human enterocytes (13, 20, 34). Our results are therefore consistent with those of Hamilton et al. (9).
Na+ and pH dependence of carnitine uptake. Carnitine transport by mature Caco-2 cells was Na+ dependent (Fig. 2A). At low concentrations or in the absence of Na+, L-carnitine transport was significantly lower. These results support the involvement of an Na+-dependent carrier-mediated system for L-carnitine uptake in Caco-2 cells (25). Studies using everted rings of rat intestine, gut sacs, and perfused intestinal segments in vivo (39), as well as those performed in isolated guinea pig enterocytes (7) and using intestinal biopsies (9) as well as human skin fibroblasts (44), have demonstrated the presence of an Na+-dependent carnitine transporter. This distinguishes it from the majority of organic cation uptake mechanisms so far reported at apical membranes of the intestine, most of which are Na+ independent (16). In guinea pig jejunal cells (7) and in HLF cells (58), L-carnitine uptake was also strongly diminished when Na+ was depleted from the buffer or replaced isosmotically by other monovalent cations, such as Li+, K+, or choline.
We also examined the effect of pH variation on L-carnitine uptake by mature Caco-2 cells. Results revealed a modest pH dependence (Fig. 2B). Raising the pH of the extracellular buffer from 7.4 (control) to 8.5 reduced L-carnitine uptake. In contrast, acidification of the extracellular buffer from 7.4 to 5.5 had no significant effect. Variation in pH also had a significant effect on carnitine uptake in cultured human hepatoma HLF cells (58). Previous reports (33) exhibited a variable and slight pH-dependency for the transport of L-carnitine in human embryonic kidney (HEK)-293 cells transfected by human OCTN2 (hOCTN2).Kinetics and inhibition of carnitine transport.
L-Carnitine uptake in Caco-2 cells is a saturable process
(Fig. 3A) with high affinity (Km,
14.07 ± 1.70 µM) similar to the known properties of OCTN2. In
humans, OCTN2 is expressed in many tissues including intestine
(42). OCTN2 is unique in that it transports carnitine with
a high affinity (Km 4.3 µM) in an Na+-dependent manner, and organic cations in an
Na+-independent manner. Recently, Bleasby et al.
(2) reported that 1-methyl-4-phenylpyridinium absorption
by Caco-2 cells is mediated by an Na+-dependent
transport mechanism. This raises the possibility of OCTN2 in
these cells, because 1-methyl-4-phenylpyridinium can inhibit carnitine
uptake via OCTN2 (55, 56).
Immunochemical identification and localization. To further explore the concept that the uptake of L-carnitine in Caco-2 cells is mediated by OCTN2, a mouse OCTN2-specific antibody was produced and used against undifferentiated, partially differentiated, and mature Caco-2 cells. Western blot experiments in homogenates of partially differentiated and mature Caco-2 cells showed immunoreactivity to the antibodies against OCTN2 (Fig. 4A). The molecular size (60 kDa) of the detected bands is close to the estimated size (63 kDa) of OCTN2. Comparing mature Caco-2 cells (10 to 15 days postconfluence) with the partially differentiated Caco-2 cells (7 to 10 days postconfluence), the amount of the protein increased, suggesting that the OCTN2 expression level depends on the differentiation stage. Previous studies (25), reported the involvement of a carrier-mediated system, that is Na+ and energy-dependent in partially differentiated Caco-2 cells. Our results, as well as those of McCloud et al. (25) done on Caco-2 cells, demonstrate the primary role of OCTN2 in the transport of carnitine.
Absence of reactivity with the anti-OCTN2 antibody in immature Caco-2 cells supports the absence of transport of L-carnitine in undifferentiated cells and also confirms that OCTN2 is expressed only when the cells attain their BBM organization. Indeed, the anti-OCTN2 antibodies recognized a protein of 60 kDa only in immunoblots of BBM preparation from mature Caco-2 cells. Staining was absent in BLM preparations (Fig. 4B). Western blot analysis provided further evidence for the presence of OCTN2 in mature Caco-2 cells BBM. To confirm OCTN2 and its localization, double immunohistochemical staining was undertaken. Staining with anti-OCTN2 polyclonal antibodies (Fig. 5A) revealed the presence of positive punctates on the apical surface. The same fluorescence distribution was observed with the staining with antialkaline phosphatase, which is a specific marker of BBM. When the images were superposed, OCTN2 and alkaline phosphatase, were colocalized (Fig. 5C). These results indicate that OCTN2 is localized on the BBM of mature Caco-2 cells. Besides OCTN2, the OCTN transporter subfamily includes two other members, OCTN1 and OCTN3, each with the ability to transport carnitine with differing characteristics (18, 41). Human OCTN1 is expressed in several tissues including intestine and transports carnitine weakly in an Na+-dependent manner (41, 43, 57). Rat intestinal OCTN1 interacts with carnitine with a very low affinity and in an Na+-independent manner (54). In mice, OCTN1 mediates carnitine uptake in an Na+-dependent mode with low affinity, although its expression in intestine has not been reported (41). OCTN3 has only been cloned in mice (41) and mediates carnitine uptake in an Na+-independent mode. It is expressed predominantly in testis and weakly in kidney and was not detectable in gut. Both human and mouse OCTN1 and OCTN2 transport carnitine in an Na+-dependent manner (41, 43, 57). The carnitine transport activity of OCTN1 is significantly lower than OCTN2 (57). On the basis of this affirmation, OCTN1 is not likely to be responsible for the 50% retained carnitine uptake activity in Na+-deprived conditions seen in Fig. 2A. Tamai et al. (41) have also cloned a new carnitine transporter, OCTN3, from the mouse. When expressed in HEK-293 cells, it exhibits Na+-independent carnitine uptake and shows a higher specificity and affinity for carnitine. Among mouse tissues examined, OCTN3 was strongly expressed in testis and weakly in kidney and not in the intestine. This transporter has been investigated only in mouse and not yet in humans. Before implicating OCTN3 in the retained carnitine uptake activity in Caco-2 cells in Na+-depleted condition, one would have to demonstrate that this transporter exists in humans with specific tissue distribution and functional characteristics. In our studies, the 50% retained carnitine uptake activity in Na+-deprived conditions could be explained by the implication of some other unidentified carnitine transporter, which may be Na+-independent and expressed only when the Caco-2 cells attain differentiation. Nakanishi et al. (28) reported the cloning of a new carnitine transporter, called ATB0,+, from mouse colon and expressed functionally in mammalian cells (HRPE). The process of carnitine transport by ATBo,+ was Na+ and Cl ![]() |
ACKNOWLEDGEMENTS |
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We acknowledge the technical support given by Dr. Idriss Djilali-Saiah and the secretarial assistance of Sylvie Julien.
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FOOTNOTES |
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This work was supported by grants from the Dairy Producers of Canada (to E. Seidman and I. Qureshi) and Canadian Institute of Health Research Grant MT-15448 (to I. Qureshi and G. A. Mitchell).
Address for reprint requests and other correspondence: I. A. Qureshi, Division of Medical Genetics, Hôpital Sainte-Justine, 3175, Cote Sainte-Catherine, Montreal, QC, Canada H3T 1C5 (E-mail: aqureshi{at}justine.umontreal.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 10, 2003;10.1152/ajpgi.00220.2002
Received 7 June 2002; accepted in final form 4 January 2003.
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