1Max-Planck-Institut für molekulare Physiologie, 44227 Dortmund; 2Institut für Pathologie, BG-Kliniken Bergmannsheil, 44789 Bochum; and 3Chirurgische Abteilung, Katholisches Krankenhaus Dortmund-West, 44379 Dortmund, Germany
Submitted 21 April 2004 ; accepted in final form 2 June 2004
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ABSTRACT |
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endosomes; enterocytes
In Caco-2 cells, a model for human enterocytes, the major amount of SGLT1 was located in intracellular compartments (13). The intracellular SGLT1 population did not consist of transporters en route from biosynthesis to the plasma membrane, because elimination of transporters from the biosynthesis pathway with cycloheximide did not alter the size or shape of the intracellular SGLT1 pool. Furthermore, intracellular vesicles containing SGLT1 were associated with microtubules. Microtubules are the "railroad tracks" for intracellular vesicular trafficking. Therefore, the intracellular SGLT1 population is very likely to be highly mobile and part of a regulatory mechanism. Actually, there have been several reports of SGLT1-mediated D-glucose uptake regulation likely being related to SGLT1 trafficking. These include regulation of SGLT1-mediated D-glucose uptake by hormones (5), second messengers (17, 25), protein kinase inhibitors/activators (10, 22, 26), and extracellular D-glucose levels (19).
Intracellular trafficking of SGLT1 also is an important issue in the explanation of some pathophysiological states. The autosomal recessive disorder glucose galactose malabsorption syndrome is caused by missense mutations in the SGLT1 gene. Heterologous expression of these mutant SGLT1 genes in Xenopus laevis oocytes led to a complete loss of sodium-dependent D-glucose uptake into oocytes, in contrast to oocytes expressing wild-type SGLT1. Transport deficiency of the mutants was due to a trafficking defect of the SGLT1 protein, which accumulated in intracellular compartments (14). Therefore, more detailed knowledge of SGLT1 trafficking and regulation in epithelial cells may provide clues for novel therapies for trafficking diseases (1, 2) such as glucose galactose malabsorption syndrome.
In the present study, we demonstrate that intracellular SGLT1 in Caco-2 cells resides in endosomes. These intracellular populations of SGLT1 are also present in absorptive cells of the human jejunum, which highlights the validity of the Caco-2 cell model for the investigation of SGLT1 regulation in human enterocytes. We further explored the role of endosomal SGLT1 in the regulation of D-glucose uptake into cells by comparing SGLT1 distribution with D-glucose uptake into Caco-2 cells after exposure of the cells to various stimuli.
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MATERIALS AND METHODS |
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Tissue sample preparation and immunohistochemistry. Surgically removed samples of normal human jejunum were fixed with 5% formalin and then dehydrated in an ascending ethanol series before being embedded in paraffin. For immunofluorescence light microscopy, labeling was performed on 3- to 5-µm-thick paraffin sections according to a previously described incubation protocol (9).
Endosome labeling, immunocytochemistry, and microscopy. Endosomes of Caco-2 cells, which were grown to confluence on poly-L-lysine-coated coverslips, were labeled using a pulse chase protocol. The cell surface was biotinylated with 0.5 mg/ml EZ-Link sulfo-N-hydroxy-succinimidobiotin (Pierce, Rockford, IL) as described previously (23). The biotinylated cells were then chased for 30 min at 37°C to incorporate biotin-labeled proteins into endosomes by fluid-phase endocytosis. Cells were then fixed and used for immunostaining as described previously (13). Biotin was detected by adding Cy2-streptavidin (Amersham Biosciences, Piscataway, NJ) to the secondary antibody solution. Alternatively, Vybrant CM-DiI cell-labeling solution (Molecular Probes, Eugene, OR) was used to stain the plasma membrane before a chase at 37°C as described above. Fluorescence was observed with a Zeiss Axiophot microscope or with a Noran OZ laser scanning confocal microscopic imaging system connected to a Nikon Eclipse TE200 inverted microscope.
Free-flow electrophoresis and -methyl-D-glucose uptake.
The preparation of a cellular organelle fraction from Caco-2 cells, the separation of the cellular organelle fraction by free-flow electrophoresis, the analysis of the fractions obtained by free-flow electrophoresis, and the determination of organelle markers and SGLT1 antigen using a specific antibody were recently described in detail (13). Sodium-dependent uptake of
-[U-14C]methyl-D-glucose (300 mCi/mmol; NEN, Boston, MA) into Caco-2 cells was performed as described previously (4, 15).
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RESULTS |
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Effect of mastoparan. Mastoparan is a tetradecapeptide and a constituent of wasp venom. It is known to activate heterotrimeric G proteins and to enhance apical endocytosis in Madin-Darby canine kidney cells (8). To test whether mastoparan also enhances apical endocytosis in Caco-2 cells, fluid-phase endocytosis of horseradish peroxidase into Caco-2 cells was measured in the absence and presence of mastoparan (Fig. 3). Mastoparan (50 µM) in the incubation medium significantly (>3-fold) enhanced endocytosis of horseradish peroxidase in Caco-2 cells as well.
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Effect of altered extracellular D-glucose levels.
The D-glucose level in the lumen of the small intestine varies depending on the nutritional state of the individual, and the lower limit is probably 0 mM D-glucose after a fasting period. After a meal, the action of membrane-bound hydrolytic enzymes, located in the microvilli of enterocytes, on sugars such as maltose, sucrose, and -limit dextrins results in a high local concentration of D-glucose. Luminal D-glucose concentration in the upper jejunum is estimated to exceed 50 mM and probably is much higher in the unstirred layer close to the site of terminal carbohydrate digestion (7). Actually, these maximal local concentrations were calculated to be as high as 200300 mM (18). Therefore, extracellular D-glucose concentrations of 0100 mM were investigated in the present study.
Caco-2 cells were incubated with D-glucose-free medium or with medium containing 50 or 100 mM D-glucose for 1 h. The incubation media were adjusted with D-mannitol to maintain osmolarity. The cells were then washed, and the sodium-dependent uptake of [14C]--methyl-D-glucose was measured for 10 min (Fig. 7). In cells preincubated with 50 or 100 mM D-glucose, sodium-dependent uptake of
-methyl-D-glucose was significantly inhibited (by 20 and 45%, respectively) compared with preincubation in D-glucose-free medium. The same experiment was also performed in the presence of 50 µM nocodazole, a drug that depolymerizes microtubules. When microtubules were depolymerized, sodium-dependent
-methyl-D-glucose uptake was lower in all instances, but more interestingly, no inhibitory effect due to prior incubation with high D-glucose medium was observed. These data suggest that the regulation of sodium-dependent
-methyl-D-glucose uptake by extracellular D-glucose levels requires an intact microtubule network.
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DISCUSSION |
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Comparable results were obtained in the present study. Short-term (90 min) exposure of Caco-2 cells to mastoparan, a drug known to enhance apical endocytosis, acutely redistributed SGLT1 from the plasma membrane to intracellular sites paralleled by a significant attenuation of sodium-dependent D-glucose uptake into the cells. The general scheme underlying the action of mastoparan and probably many other agents described in the literature is very likely a drug-initiated general change in the sorting of transporters, which leads to decreased or enhanced endocytosis of SGLT1, thereby increasing or decreasing the number of transporters at the cell surface, which ultimately leads to increased or decreased sodium-dependent D-glucose uptake into the cell. However, regulation of SGLT1-mediated D-glucose uptake by extracellular D-glucose levels differs from this scheme. Low extracellular D-glucose increases and high extracellular D-glucose downregulates SGLT1-mediated D-glucose uptake into Caco-2 cells. This occurs without a change in the cellular steady-state distribution of SGLT1.
It is surprising at first glance that SGLT1-mediated D-glucose uptake decreases when Caco-2 cells are challenged with high extracellular D-glucose. In addition, enhanced sodium-dependent D-glucose uptake into brush-border membrane vesicles prepared from rat small intestine that was exposed to high D-glucose was reported previously (19). These conflicting data may be explained by the different methodological approaches used. Another explanation may be the diverse pathways engaged in intestinal D-glucose absorption. Besides cellular uptake mediated by SGLT1, a paracellular pathway for D-glucose (18) and the involvement of apical GLUT2, a transporter allowing facilitated diffusion of D-glucose, also have been postulated. High luminal D-glucose has been reported to induce recruitment of GLUT2 to the brush-border membrane, and the GLUT2-mediated diffusion of D-glucose across the brush-border membrane in that situation represents the major route of D-glucose uptake into enterocytes (12). To circumvent interference from this transporter, we used -methyl-D-glucose exclusively to measure SGLT1-mediated substrate uptake into Caco-2 cells.
-Methyl-D-glucose is a specific substrate for SGLT1, but not for GLUT2 (3). It is therefore possible that total D-glucose uptake via multiple pathways into Caco-2 cells increases after exposure to high extracellular D-glucose, even if the SGLT1-mediated pathway decreases. A decrease in energy requiring transport may be one physiological reason for the downregulation of SGLT1 in enterocytes under high luminal D-glucose conditions. Another reason is probably the prevention of a multiplication of the osmotic challenge to enterocytes, because via SGLT1, with every D-glucose molecule, two sodium ions are transported into the cell as well.
How can regulation of D-glucose uptake without a change in the cellular SGLT1 steady-state distribution be explained? Toward this end, two observations are of significance. The first important observation is that regulation of SGLT1-mediated D-glucose uptake without cellular redistribution of transporters works at all! This is possible only if SGLT1 molecules themselves are regulated in their transport activity. The second important observation is the association of SGLT1-containing endosomes with microtubules, which we found in an earlier study (13). Microtubules are the railroad tracks for vesicular trafficking, which suggests mobility of the cellular SGLT1 pool, and the induction of SGLT1 trafficking with mastoparan in the present study demonstrates that the cellular SGLT1 pool is indeed mobile. Inhibition of this mobility by microtubule depolymerization with nocodazole inhibited the regulatory response to altered extracellular D-glucose levels. Thus SGLT1 activation/inactivation as well as trafficking of transporters seems to be part of the mechanism that regulates SGLT1-mediated D-glucose uptake in response to extracellular D-glucose levels. Taking these properties into account, we assume a mechanism that includes membrane protein cycling and activation/inactivation steps similar to those involved in the regulation of cell surface receptors such as G protein-coupled receptors (21), cytokine receptors (20), and the epidermal growth factor receptor (24). In this hypothetical scheme, SGLT1 in the apical membrane is inactivated after a certain time with regard to D-glucose transport capability and is then endocytosed. Endosomal SGLT1 in turn is eventually activated and then returned to the plasma membrane. Sodium-dependent D-glucose uptake into the cell could then be regulated without altering the cellular steady-state distribution of SGLT1 by changing the velocity of this SGLT1 activation/inactivation cycle, which ultimately determines the amount of transport-active SGLT1 in the apical membrane.
Our present study suggests that in addition to the obvious mode of SGLT1 regulation by shifting transporters between locations, there is another regulatory mechanism that includes SGLT1 trafficking and activation/inactivation steps of the transporter. A challenge for the future is to actually prove the coexistence of cellular pools of transport-active and -inactive SGLT1 and to discriminate between them. This project is presently being pursued in our laboratory.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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