Different modes of sodium-D-glucose cotransporter-mediated D-glucose uptake regulation in Caco-2 cells

Saeed Khoursandi,1 Daniel Scharlau,1 Peter Herter,1 Cornelius Kuhnen,2 Dirk Martin,3 Rolf K. H. Kinne,1 and Helmut Kipp1

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


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We recently reported that a considerable amount of the sodium-D-glucose cotransporter SGLT1 present in Caco-2 cells, a model for human enterocytes, is located in intracellular compartments attached to microtubules (Kipp H, Khoursandi S, Scharlau D, and Kinne RKH. Am J Physiol Cell Physiol 285: C737–C749, 2003). A similar distribution pattern was also observed in enterocytes in thin sections from human jejunum, highlighting the validity of the Caco-2 cell model. Fluorescent surface labeling of live Caco-2 cells revealed that the intracellular compartments containing SGLT1 were accessible by endocytosis. To elucidate the role of endosomal SGLT1 in the regulation of sodium-dependent D-glucose uptake into enterocytes, we compared SGLT1-mediated D-glucose uptake into Caco-2 cells with the subcellular distribution of SGLT1 after challenging the cells with different stimuli. Incubation (90 min) of Caco-2 cells with mastoparan (50 µM), a drug that enhances apical endocytosis, shifted a large amount of SGLT1 from the apical membrane to intracellular sites and significantly reduced sodium-dependent {alpha}-[14C]methyl-D-glucose uptake (–60%). We also investigated the effect of altered extracellular D-glucose levels. Cells preincubated (1 h) with D-glucose-free medium exhibited significantly higher sodium-dependent {alpha}-[14C]methyl-D-glucose uptake (+45%) than did cells preincubated with high D-glucose medium (100 mM, 1 h). Interestingly, regulation of SGLT1-mediated D-glucose uptake into Caco-2 cells by extracellular D-glucose levels occurred without redistribution of cellular SGLT1. These data suggest that, pharmacologically, D-glucose uptake can be regulated by a shift of SGLT1 between the plasma membrane and the endosomal pool; however, regulation by the physiological substrate D-glucose can be explained only by an alternative mechanism.

endosomes; enterocytes


DIETARY D-GLUCOSE IS ABSORBED from the lumen of the small intestine by accumulation of D-glucose into enterocytes. This process is energized by simultaneous "downhill" transport of sodium ions and is mediated by the sodium-D-glucose cotransporter SGLT1. Therefore, the plasma membrane transporter SGLT1 is frequently expected to be located exclusively in the luminal brush-border membrane of enterocytes. However, the distribution of SGLT1 in epithelial cells is more complex. In LLC-PK1 cells, a cell line from the porcine kidney proximal tubule, endogenous SGLT1 was not detected in the brush-border membrane but near the apical plasma membrane (11). In excised loops from rabbit jejunum, SGLT1 was located in the brush-border membrane and was also proposed to be in intracellular sites (6).

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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials and cell culture. All chemicals were of the highest purity available and were purchased from Sigma (Deisenhofen, Germany). Other reagents and antibodies were also obtained from Sigma, unless other sources are indicated. Caco-2 cells were acquired from the Deutsche Sammlung für Mikroorganismen und Zellkulturen (Braunschweig, Germany). Cells were grown in 75-cm2 flasks (Falcon, Heidelberg, Germany) at 37°C and 5% CO2 in minimal essential medium (Life Technologies) supplemented with 10% fetal calf serum, 1% nonessential amino acids, and 1% glutamine. Every 2 days, the culture medium was renewed. For cell analysis using free-flow electrophoresis, Caco-2 cells (passages 15–25) were seeded (2 x 105 cells/dish) onto 5-cm petriPERM hydrophilic dishes (Vivascience, Hanover, Germany) and grown for 16–18 days. For D-glucose uptake experiments, Caco-2 cells were seeded (1 x 105 cells/well) onto six-well plates (Falcon) and grown for 16–18 days. Under these growth conditions, Caco-2 cells exhibited high sodium-dependent D-glucose uptake, which is a property of only the fully polarized Caco-2 cell (4).

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 {alpha}-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 {alpha}-[U-14C]methyl-D-glucose (300 mCi/mmol; NEN, Boston, MA) into Caco-2 cells was performed as described previously (4, 15).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SGLT1 distribution in human jejunum. We recently identified a large intracellular population of SGLT1 in Caco-2 cells (13). Although Caco-2 cells are a very common cell culture model for human enterocytes, Caco-2 cells were originally derived from a colon carcinoma. The regulation and distribution of SGLT1 in a cancer cell might be different from that in healthy tissue. We therefore investigated the distribution of SGLT1 in samples from human jejunum, which were obtained from the pathology department of a local hospital. Immunostaining with SGLT1-specific antibodies (13) of paraffin sections from human jejunum are shown in Fig. 1. The major amount of SGLT1 in human jejunum was detected inside absorptive cells, with a more prominent abundance at the apical pole of the cells. Only faint staining of SGLT1 was observed in the brush-border membrane. These observations resemble the results obtained in Caco-2 cells and highlight the validity of this cell culture model for the investigation of SGLT1 regulation in human enterocytes.



View larger version (113K):
[in this window]
[in a new window]
 
Fig. 1. Sodium-D-glucose cotransporter SGLT1 distribution in human jejunum. Paraffin sections from human jejunum were labeled with anti-SGLT1 antibody QIS30 (red) and 4,6-diamidino-2-phenylindole (DAPI) (nuclei, blue). Incubation with only the secondary antibody revealed no significant fluorescence (data not shown). Magnification, x250; inset, x630.

 
Intracellular SGLT1 is located in endosomes. After separation of Caco-2 cell organelles by free-flow electrophoresis, intracellular SGLT1 comigrated with endosomes (13). However, this does not necessarily prove localization of SGLT1 to endosomes, because intracellular SGLT1 could also be located in organelles with properties comparable to those of endosomes during the separation in an electric field. To test whether intracellular SGLT1 is actually located in endosomes, colocalization of SGLT1 with labeled lipids and labeled membrane proteins internalized from the apical surface of Caco-2 cells was investigated (Fig. 2). Intracellular structures containing SGLT1 match in size and shape with parts of the compartments labeled by internalized fluorescent lipids and membrane proteins, suggesting that the intracellular SGLT1 population is formed by endocytosis. However, not all endosomes labeled by internalized fluorescent lipids and membrane proteins also contain SGLT1, indicating that SGLT1 is found only in a subpopulation of the endosomes present in Caco-2 cells.



View larger version (105K):
[in this window]
[in a new window]
 
Fig. 2. SGLT1 is located in endosomes. 0 min: plasma membrane of Caco-2 cells grown to confluence on poly-L-lysine-coated coverslips was labeled with the fluorescent lipid DiI or biotin (red) at 0°C. After fixation of the cells, SGLT1 (green) was stained with a specific antibody. 30 min: in another preparation, DiI or biotin was allowed to be endocytosed from the plasma membrane by incubation of the cells for 30 min at 37°C. The cells were then fixed, and SGLT1 was stained with a specific antibody. DiI fluorescent lipid label is shown in red, and SGLT1 is shown in green. Experiments with DiI were observed using epiflourescence microscopy, and experiments with biotin were observed using confocal microscopy. Incubation with only the secondary antibodies revealed no significant fluorescence (data not shown). Scale bars, 15 µm.

 
The existence of a large network of endosomes containing SGLT1 raises questions about its physiological role. In an earlier study (13), we reported that intracellular compartments containing SGLT1 are attached to microtubules. We therefore assume that intracellular SGLT1 is highly mobile and part of a mechanism by which SGLT1 abundance at the apical cell surface is regulated in response to an altered physiological demand for sodium-dependent D-glucose uptake into enterocytes. To test this hypothesis, we studied the effect of mastoparan, a drug that enhances apical endocytosis in some cells (8), and the effect of altered extracellular D-glucose levels on sodium-dependent {alpha}-methyl-D-glucose uptake and SGLT1 distribution in Caco-2 cells.

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.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 3. Mastoparan enhances apical endocytosis in Caco-2 cells. Caco-2 cells grown to polarity on petriPERM support were incubated at 37°C with medium containing 50 µM mastoparan for 20 min and then with medium containing 50 µM mastoparan and 2 mg/ml horseradish peroxidase (HRP) for 10 min. Control cells were incubated only with HRP for 10 min. The cells were then washed with PBS and lysed with 1% Tween 20. The lysate was cleared by centrifugation (20 min, 20,000 g), and HRP in the supernatant was determined with a commercial peroxidase assay (Sigma Fast). Values are means ± SD; n = 3.

 
Mastoparan treatment had a significant inhibitory effect on sodium-dependent {alpha}-methyl-D-glucose uptake into Caco-2 cells, which is a measure of transport-active SGLT1 present at the apical cell surface. Incubation of Caco-2 cells with various concentrations of mastoparan for 90 min (Fig. 4) significantly decreased {alpha}-methyl-D-glucose uptake into cells in a concentration-dependent manner. To investigate whether this effect was due to a reduced amount of SGLT1 present at the apical cell surface, we determined the distribution of SGLT1 in control cells and in cells incubated with 50 µM mastoparan for 90 min by free-flow electrophoresis (13) (Fig. 5). Incubation of Caco-2 cells with mastoparan significantly diminished the amount of SGLT1 present in the apical membrane in parallel with an increase in the intracellular amount of SGLT1 (Fig. 5A). Interestingly, after treatment of Caco-2 cells with mastoparan, the amount of SGLT1 in early as well as late endosomal fractions increased, with the latter suggesting that the rate of lysosomal SGLT1 degradation may increase in response to a forced enhanced endocytosis rate. A comparison of the ratios between intracellular and apical SGLT1 (Fig. 5B) revealed that in control experiments, there is about twofold more SGLT1 in intracellular compartments than at the cell surface. This ratio increased in mastoparan-treated cells to a fourfold greater intracellular amount of SGLT1. A shift in the subcellular SGLT1 distribution was also detected using immunostaining of endogenous SGLT1 in Caco-2 cells treated with mastoparan (50 µM, 90 min) (Fig. 6). Compared with the distribution in control cells, in mastoparan-treated cells, the compartments containing SGLT1 were condensed to bigger structures that were arranged around the nucleus.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 4. Mastoparan inhibits sodium-dependent {alpha}-methyl-D-glucose uptake into Caco-2 cells. Caco-2 cells grown to polarity in 6-well plates were incubated with various concentrations of mastoparan for 90 min at 37°C. Sodium-dependent uptake of {alpha}-methyl-D-glucose over 10 min was then determined. Sodium-dependent uptake of {alpha}-methyl-D-glucose was also determined in the presence of 0.5 mM phlorizin, a specific inhibitor of SGLT1. Values are means ± SD; n = 3.

 


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 5. Cellular SGLT1 is redistributed after mastoparan treatment. Caco-2 cells grown to polarity on petriPERM support were incubated for 90 min at 37°C with medium containing 50 µM mastoparan. Control cells were not treated with mastoparan. A cellular organelle fraction was prepared from the cells, which was further separated by free-flow electrophoresis. A: fractions obtained by free-flow electrophoresis were analyzed for SGLT1 by ELISA and were further analyzed for various organelle markers: AP, apical plasma membrane; BL, basolateral plasma membrane; EE, early endosomes; LE, late endosomes. Peak fractions of the organelle markers are marked with arrows. Representative results from 3 independent experiments are shown. To quantitate cellular SGLT1 distribution, the area below the curves for apical and intracellular amounts as indicated at top was measured, and the ratio intracellular/apical SGLT1 was calculated. The quantitative results from 3 independent experiments are shown in B. Values are means ± SD; n = 3. *P < 0.01 vs. control (significantly different); Student's t-test.

 


View larger version (82K):
[in this window]
[in a new window]
 
Fig. 6. Mastoparan changes intracellular SGLT1 distribution in Caco-2 cells. Caco-2 cells were grown to confluence on poly-L-lysine-coated coverslips and incubated for 90 min with medium containing 50 µM mastoparan at 37°C. The cells were then fixed and permeabilized. SGLT1 was labeled with a specific antibody (red). Nuclei were stained with DAPI (blue). Control cells were not treated with mastoparan. The fluorescence of the labels was observed using epifluorescence microscopy. Incubation with only the secondary antibody revealed no significant fluorescence (data not shown). Scale bars, 15 µm.

 
These data demonstrate that the cellular pool of SGLT1 can be shifted from the plasma membrane to intracellular compartments and that this shift alters the cell's capability to conduct sodium-dependent D-glucose uptake. However, the regulation of sodium-dependent D-glucose uptake by mastoparan is pharmacologically induced and therefore is not necessarily relevant to the regulation of SGLT1-mediated D-glucose uptake into cells under physiological conditions. To study the effect of a potential, more physiological regulator, we investigated the effect of varying extracellular D-glucose levels on SGLT1-mediated {alpha}-methyl-D-glucose uptake into Caco-2 cells.

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 {alpha}-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 200–300 mM (18). Therefore, extracellular D-glucose concentrations of 0–100 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]-{alpha}-methyl-D-glucose was measured for 10 min (Fig. 7). In cells preincubated with 50 or 100 mM D-glucose, sodium-dependent uptake of {alpha}-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 {alpha}-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 {alpha}-methyl-D-glucose uptake by extracellular D-glucose levels requires an intact microtubule network.



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 7. Extracellular D-glucose regulates sodium-dependent {alpha}-methyl-D-glucose uptake into Caco-2 cells. Caco-2 cells grown to polarity in 6-well plates were incubated with media containing various concentrations of D-glucose for 1 h at 37°C. D-Mannitol was used to adjust the osmolarity to the same level in all experiments. Sodium-dependent uptake of {alpha}-methyl-D-glucose over 10 min was then determined. Sodium-dependent uptake of {alpha}-methyl-D-glucose was also determined in the presence of 0.5 mM phlorizin, a specific inhibitor of SGLT1. The experiment was repeated with cells that were preincubated with 50 µM nocodazole 30 min before and during incubation at various D-glucose concentrations. Values are means ± SD; n = 3. *P < 0.01 vs. control (significantly different); Student's t-test. **P > 0.05 (not significantly different); Student's t-test.

 
Investigation of the subcellular SGLT1 distribution by free-flow electrophoresis after exposure of Caco-2 cells to D-glucose-free or high D-glucose medium revealed no significant changes (Fig. 8A). In all instances, the ratio between intracellular and apical SGLT1 was ~2:1 (Fig. 8B).



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 8. Extracellular D-glucose does not affect cellular SGLT1 distribution. Caco-2 cells grown to polarity on petriPERM support were incubated for 1 h at 37°C with medium containing various concentrations of D-glucose. D-Mannitol was used to adjust the osmolarity to the same level in all experiments. A cellular organelle fraction was prepared from the cells, which was further separated by free-flow electrophoresis. A: fractions obtained by free-flow electrophoresis were analyzed for SGLT1 by ELISA and were further analyzed for various organelle markers: AP, BL, EE, and LE. The peak fractions of the organelle markers are indicated with arrows in A. A representative result observed in 3 independent experiments is shown. To quantitate cellular SGLT1 distribution, the area below the curves for apical and intracellular amount, indicated at top, was measured, and the ratio intracellular/apical SGLT1 was calculated. The quantitative results from 3 independent experiments are shown in B. Values are means ± SD; n = 3. The values are not significantly different from each other (P > 0.05; Student's t-test).

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Several studies have demonstrated regulation of SGLT1-mediated sodium-dependent D-glucose absorption, which includes transcriptional long-term regulation caused by changes in dietary carbohydrate levels (16). However, there also have been reports of acute short-term effects on sodium-dependent D-glucose uptake into cells. SGLT1-mediated D-glucose uptake into cells was rapidly altered by prior treatment with forskolin (25), cAMP or the cAMP-raising agent cholera toxin (17), the peptide hormone glucagon-like peptide 2 (5), epidermal growth factor (6), and high extracellular D-glucose (19). In several of these examples, a shift of SGLT1 between the plasma membrane and other cellular locations has been postulated or directly demonstrated.

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 {alpha}-methyl-D-glucose exclusively to measure SGLT1-mediated substrate uptake into Caco-2 cells. {alpha}-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.


    ACKNOWLEDGMENTS
 
The skillful and ambitious technical assistance of Jutta Luig and Hendrike Schütz is gratefully acknowledged. We are also indebted to Petra Glitz and Christiane Pfaff for the professional handling and maintenance of the cell culture.


    FOOTNOTES
 

Address for reprint requests and other correspondence: H. Kipp, Max Planck Institute of Molecular Physiology, Otto-Hahn-Str. 11, 44227 Dortmund, Germany (E-mail: helmut.kipp{at}mpi-dortmund.mpg.de)

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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Aridor M and Hannan LA. Traffic jam: a compendium of human diseases that affect intracellular transport processes. Traffic 1: 836–851, 2000.[CrossRef][ISI][Medline]

2. Aridor M and Hannan LA. Traffic jams II: an update of diseases of intracellular transport. Traffic 3: 781–790, 2002.[CrossRef][ISI][Medline]

3. Bissonnette P, Gagne H, Coady MJ, Benabdallah K, Lapointe JY, and Berteloot A. Kinetic separation and characterization of three sugar transport modes in Caco-2 cells. Am J Physiol Gastrointest Liver Physiol 270: G833–G843, 1996.[Abstract/Free Full Text]

4. Blais A, Bissonnette P, and Berteloot A. Common characteristics for Na+-dependent sugar transport in Caco-2 cells and human fetal colon. J Membr Biol 99: 113–125, 1987.[ISI][Medline]

5. Cheeseman CI. Upregulation of SGLT-1 transport activity in rat jejunum induced by GLP-2 infusion in vivo. Am J Physiol Regul Integr Comp Physiol 273: R1965–R1971, 1997.[Abstract/Free Full Text]

6. Chung BM, Wallace LE, Hardin JA, and Gall DG. The effect of epidermal growth factor on the distribution of SGLT-1 in rabbit jejunum. Can J Physiol Pharmacol 80: 872–878, 2002.[CrossRef][ISI][Medline]

7. Debnam ES and Unwin RJ. Hyperglycemia and intestinal and renal glucose transport: implications for diabetic renal injury. Kidney Int 50: 1101–1109, 1996.[ISI][Medline]

8. Eker P, Holm PK, van Deurs B, and Sandvig K. Selective regulation of apical endocytosis in polarized Madin-Darby canine kidney cells by mastoparan and cAMP. J Biol Chem 269: 18607–18615, 1994.[Abstract/Free Full Text]

9. Herter P, Kuhnen C, Müller KM, Wittinghofer A, and Müller O. Intracellular distribution of {beta}-catenin in colorectal adenomas, carcinomas and Peutz-Jeghers polyps. J Cancer Res Clin Oncol 125: 297–304, 1999.[CrossRef][ISI][Medline]

10. Hirsch JR, Loo DD, and Wright EM. Regulation of Na+/glucose cotransporter expression by protein kinases in Xenopus laevis oocytes. J Biol Chem 271: 14740–14746, 1996.[Abstract/Free Full Text]

11. Ikari A, Nakano M, Kawano K, and Suketa Y. Up-regulation of sodium-dependent glucose transporter by interaction with heat shock protein 70. J Biol Chem 277: 33338–33343, 2002.[Abstract/Free Full Text]

12. Kellett GL and Helliwell PA. The diffusive component of intestinal glucose absorption is mediated by the glucose-induced recruitment of GLUT2 to the brush-border membrane. Biochem J 350: 155–162, 2000.[CrossRef][ISI][Medline]

13. Kipp H, Khoursandi S, Scharlau D, and Kinne RKH. More than apical: distribution of SGLT1 in Caco-2 cells. Am J Physiol Cell Physiol 285: C737–C749, 2003.[Abstract/Free Full Text]

14. Martin MG, Lostao MP, Turk E, Lam J, Kreman M, and Wright EM. Compound missense mutations in the sodium/D-glucose cotransporter result in trafficking defects. Gastroenterology 112: 1206–1212, 1997.[ISI][Medline]

15. Matosin-Matekalo M, Mesonero JE, Delezay O, Poiree JC, Ilundain AA, and Brot-Laroche E. Thyroid hormone regulation of the Na+/glucose cotransporter SGLT1 in Caco-2 cells. Biochem J 334: 633–640, 1998.[ISI][Medline]

16. Miyamoto K, Hase K, Takagi T, Fujii T, Taketani Y, Minami H, Oka T, and Nakabou Y. Differential responses of intestinal glucose transporter mRNA transcripts to levels of dietary sugars. Biochem J 295: 211–215, 1993.[ISI][Medline]

17. Nath SK, Rautureau M, Heyman M, Reggio H, L'Helgoualc'h A, and Desjeux JF. Emergence of Na+-glucose cotransport in an epithelial secretory cell line sensitive to cholera toxin. Am J Physiol Gastrointest Liver Physiol 256: G335–G341, 1989.[Abstract/Free Full Text]

18. Pappenheimer JR. On the coupling of membrane digestion with intestinal absorption of sugars and amino acids. Am J Physiol Gastrointest Liver Physiol 265: G409–G417, 1993.[Abstract/Free Full Text]

19. Sharp PA, Debnam ES, and Srai SK. Rapid enhancement of brush border glucose uptake after exposure of rat jejunal mucosa to glucose. Gut 39: 545–550, 1996.[Abstract]

20. Stroud RM and Wells JA. Mechanistic diversity of cytokine receptor signaling across cell membranes. Sci STKE 2004: re7, 2004.

21. Tan CM, Brady AE, Nickols HH, Wang Q, and Limbird LE. Membrane trafficking of G protein-coupled receptors. Annu Rev Pharmacol Toxicol 44: 559–609, 2004.[CrossRef][ISI][Medline]

22. Veyhl M, Wagner CA, Gorboulev V, Schmitt BM, Lang F, and Koepsell H. Downregulation of the Na+-D-glucose cotransporter SGLT1 by protein RS1 (RSC1A1) is dependent on dynamin and protein kinase C. J Membr Biol 196: 71–81, 2003.[CrossRef][ISI][Medline]

23. Webster P. Early intracellular events during internalization of Listeria monocytogenes by J774 cells. J Histochem Cytochem 50: 503–518, 2002.[Abstract/Free Full Text]

24. Wiley HS, Shvartsman SY, and Lauffenburger DA. Computational modeling of the EGF-receptor system: a paradigm for systems biology. Trends Cell Biol 13: 43–50, 2003.[CrossRef][ISI][Medline]

25. Williams M and Sharp P. Regulation of jejunal glucose transporter expression by forskolin. Biochim Biophys Acta 1559: 179–185, 2002.[ISI][Medline]

26. Wright EM, Hirsch JR, Loo DD, and Zampighi GA. Regulation of Na+/glucose cotransporters. J Exp Biol 200: 287–293, 1997.[Abstract/Free Full Text]





This Article
Abstract
Full Text (PDF)
All Versions of this Article:
287/4/C1041    most recent
00197.2004v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (1)
Google Scholar
Articles by Khoursandi, S.
Articles by Kipp, H.
Articles citing this Article
PubMed
PubMed Citation
Articles by Khoursandi, S.
Articles by Kipp, H.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2004 by the American Physiological Society.