Cloning and functional characterization of the human GLUT7 isoform SLC2A7 from the small intestine
Qiang Li ,*
Andrei Manolescu,*
Mabel Ritzel,
Sylvia Yao,
Melissa Slugoski,
James D. Young,
Xing-Zhen Chen, and
Chris I. Cheeseman
Membrane Protein Research Group, Department of Physiology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada
Submitted 9 September 2003
; accepted in final form 15 March 2004
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ABSTRACT
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Facilitated glucose transporters (GLUTs) mediate transport of sugars across cell membranes by using the chemical gradient of sugars as the driving force. Improved cloning techniques and database analyses have expanded this family of proteins to a total of 14 putative members. In this work a novel hexose transporter isoform, GLUT7, has been cloned from a human intestinal cDNA library by using a PCR-based strategy (GenBank accession no. AY571960). The encoded protein is comprised of 524 amino acid residues and shares 68% similarity and 53% identity with GLUT5, its most closely related isoform. When GLUT7 was expressed in Xenopus oocytes, it showed high-affinity transport for glucose (Km = 0.3 mM) and fructose (IC50 = 0.060 mM). Galactose, 2-deoxy-D-glucose, and xylose were not transported. Uptake of 100 µM D-glucose was not inhibited by 200 µM phloretin or 100 µM cytochalasin B. Northern blotting indicated that the mRNA for GLUT7 is present in the human small intestine, colon, testis, and prostate. Western blotting and immunohistochemistry of rat tissues with an antibody raised against the predicted COOH-terminal sequence confirmed expression of the protein in the small intestine and indicated that the transporter is predominantly expressed in the enterocytes' brush-border membrane. The unusual substrate specificity and close sequence identity with GLUT5 suggest that GLUT7 represents an intermediate between class II GLUTs and the class I member GLUT2. Comparison between these proteins may provide key information as to the structural determinants for the recognition of fructose as a substrate.
human glucose transporter 7
FACILITATED GLUCOSE TRANSPORTERS mediate transport of sugars across the cell membrane by using the chemical gradient of sugars as the driving force. Early cloning efforts identified five members of this family of proteins (GLUT15, SLC2A15); however, recently improved cloning techniques and database analyses have expanded the family to include up to a total of 14 possible members (7, 10, 19, 20, 25, 27, 28). On the basis of sequence homology each has been assigned to one of three classes (I, II, and III), of which class II has been proposed to be made up of GLUT5, -7, -9, and -11 (14). GLUT5 is a fructose transporter expressed predominantly in the apical membrane of the small intestine (where it serves to absorb this hexose from the diet) and also in spermatozoa, which rely on fructose as an energy source (1, 2, 26). Unlike the class I GLUT isoforms, (GLUT1, -2, -3, and -4), hexose transport mediated by GLUT5 is unaffected by cytochalasin B (CB) and glucose is a very poor substrate (23). GLUT11 expression appears to be limited to heart and skeletal muscle, and it can transport glucose, the uptake of which is inhibited by fructose (32). Thus these two closely related proteins would both appear to be able to transport fructose. Currently, there are no reports on the functional characterization of the putative GLUT9 (SLC2A9) (25) mRNA, which is largely expressed in spleen, peripheral lymphocytes, and brain, as indicated by Northern blots. This protein should not be confused with the class III protein now assigned as GLUT6 (SLC2A6) (10, 19) but which was initially named GLUT9. This leaves the one remaining member of class II, GLUT7, with no known expression pattern or characterization of functional activity. In this paper we report on work showing the isolation and characterization of this fourth member of the class II proteins, GLUT7 (SLC2A7). Xenopus oocyte expression experiments using human GLUT7 (hGLUT7) indicate that this 524-amino acid protein can transport glucose and fructose with high affinity and that its transport activity is unaffected by CB and phloretin. Tissue distribution appeared to be limited to the small intestine, colon, prostate, and testis.
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MATERIALS AND METHODS
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Isolation of a cDNA encoding the hGLUT7 gene.
On the basis of the alignment of human GLUT5 cDNA (GenBank accession no. NM_003039) and human genomic contig AL356306
[GenBank]
(replaced by AL035703
[GenBank]
in the NCBI database presently), three forward and three reverse primers were designed based on conserved regions of both DNA sequences. One specific band of
0.8 kb was amplified by using a human intestinal cDNA library (Clontech, Palo Alto, CA) that was different from the GLUT5 sequence. This new product was then used to search the database for a unique coding region, and, by using automated computational analysis, we found a putative full-length sequence already deposited in the database (GenBank accession no. XM_060424) annotated by the International Human Genome Sequencing Consortium. Therefore, we designed primers based on the unique regions of the putative gene and attempted to amplify the full-length cDNA from a human intestinal cDNA library.
The PCR reaction (30 µl) contained 50 ng of cDNA library, 2.5 units of Taq-Deep Vent DNA polymerase (100:1), and 10 pmol each of the 5'- and 3'-oligonucleotide primers 5'-TACGGGATCCCGATGGAGAACAAAGAGGCG GGAACCCCT-3' (BamHI site underlined) and 5'-CGGAATTCCGCAGGGCCGCTAAAAGGAAGTTTC-3' (EcoRI site underlined). Amplification for 1 cycle at 94°C for 5 min, 57°C for 55 s, 72°C for 100 s and for 32 cycles at 94°C for 55 s, 57°C for 55 s, 72°C for 100 s (Robocycler 40 temperature cycler; Stratagene, La Jolla, CA) generated visible PCR products of the predicted size.
The PCR reaction mixture was resolved on a 1% agarose gel. A band at 1.7 kb was isolated and purified (QIAEX II gel extraction kit; Qiagen). This product was first ligated into pGEM-T (Promega, Madison, WI) and then subcloned into the enhanced Xenopus expression vector pGEM-HE (33) by using BamHI and EcoRI. By providing additional 5'- and 3'-untranslated sequences from a Xenopus
-globin gene, the pGEM-HE construct gave significant functional activity that was used in the subsequent characterization of the human protein. The authenticity of the construct was confirmed by sequencing. The open reading frame of this putative cDNA is 1,575 bp long and could encode a 524-amino acid protein, which we have assigned as GLUT7 (SLC2A7).
Expression of recombinant hGLUT7 in Xenopus oocytes.
Plasmid containing the hGLUT7 gene was linearized with NheI and in vitro transcribed with T7 polymerase mMESSAGE mMACHINE (Ambion). Adult female Xenopus laevis were obtained from Nasco (Fort Atkinson, WI) and housed at 18°C on a 12:12-h h light-dark cycle. Stage V/VI oocytes were harvested from anesthetized frogs and placed in Modified Barth's Medium (MBM) [in mM: 88 NaCl, 1 KCl, 0.33 Ca(NO3)2, 0.41 CaCl2, 0.82 MgSO4, 2.4 NaHCO3, and 2.5 Na pyruvate, with 0.1 mg/ml penicillin, 0.05 mg/ml gentamicin sulfate, and 10 mM HEPES at pH 7.5]. The follicular layer was removed by treatment for 2 h with 0.02 g/ml type I collagenase (Sigma). After selection, hypertonic phosphate treatment was applied to all oocytes subjected to microinjection. Before mRNA microinjection the oocytes were incubated in MBM at 1618°C for 24 h to restore their osmolarity. The oocytes were injected with 1050 nl (
20 ng) GLUT7 synthetic mRNA transcript and incubated for 35 days at 1618°C before functional uptake assays.
Northern blotting.
Northern blot analysis of the possible GLUT7 mRNA expression in spleen, thymus, prostate, testis, ovary, small intestine, colon, and leucocytes as well as heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas were performed using normal tissue mRNA Northern blots (Clontec catalog no. 77591, batch 7090547 and no. 77601, lot 9041180). Each lane was normalized against expression of the
-actin gene. A PCR-based fragment of 813 bp corresponding to the last 271 amino acids of the COOH-terminal end of the wild-type hGLUT7 was generated as a hybridization probe. The forward primer was 5'-CTGAGAGGCCACACGGACAT-3', and the reverse primer was 5'-cggaattccgcagggccgctaaaaggaagtttc-3'. Briefly, the probe was subcloned in a pCR II-TOPO vector (Invitrogen) and linearized with restriction endonucleases SacI and NotI for the subsequent production of the antisense and sense probes, respectively. Digoxigenin (DIG)-labeled antisense and sense riboprobes were synthesized by in vitro transcription with T7 and SP6 RNA polymerase and DIG RNA labeling mix (Roche Diagnostic, Mannheim, Germany) according to the manufacturer's instructions. The DIG labeled probes were hybridized to the blot under conditions of high stringency (85°C for 5 min). After blots were washed, the signal was recorded on Kodak-XAR5 film after 5 min exposure.
Preparation of isolated plasma membranes.
Isolated brush-border membranes from rat jejunum and ileum were made using a standard technique routinely employed in this laboratory (21). Briefly, mucosal scrapings were taken from the jejunum and ileum on ice and the divalent cation precipitation method was used to isolate the brush-border membranes from intracellular and basolateral membranes. Na+-K+-ATPase activity, a marker for basolateral membranes, was reduced to 30% of the initial homogenate levels, and alkaline phosphatase activity was enriched 17-fold.
Western blotting and immunohistochemistry.
Two polyclonal antibodies were raised against the unique 11-amino acid sequence of the COOH-terminal tail of GLUT7 (PTASPAKETSF) by Alpha Diagnostics International (San Antonio, TX). Testing of the affinity-purified product in Western blots indicated that antibody 7440 recognized the expression of GLUT7 protein in plasma membranes of mRNA-injected oocytes but was negative for water-injected oocytes.
Oocytes were embedded in OCT (10% polyvinyl alcohol, 4% polyethylene glycol) embedding medium (Shandon) and flash frozen in liquid nitrogen. Ten-micrometer-thick sections were cut on a cryostat (Leica cryostat, Richmond Hill, ON, Canada), placed on slides, and stored at 20°C. On the day of use, sections were brought to room temperature and fixed with methanol for 90 s. Following a 5-min wash with PBS, the sections were treated with 1% SDS for 5 min to increase antigen exposure and were again thoroughly washed in PBS (3 times for 5 min each). The sections were then treated with 10% goat serum (0.1% Tween 20) to decrease nonspecific binding of the secondary antibody, followed by a rapid rinse with PBS and incubation with primary antibody. The primary anti-GLUT7 antibody was diluted 1:200 in a 25% wt/vol milk solution (25 g powdered milk in 100 ml PBS with 0.05% Tween 20) and placed on slides for 24 h at 4°C, followed by washes with high-salt (3 times normal NaCl) PBS and then PBS. The slides were then incubated with 1 mg/ml biotinylated goat anti-rabbit secondary antibody (Chemicon) diluted 1:200 in PBS for 1h at room temperature. The sections were washed in high-salt PBS and PBS and then treated with streptavidin-conjugated FITC (Amersham) for 30 min in the dark. After three 5-min washes with PBS in the dark, the slides were mounted with Vectashield (Vector, Burlingame, CA) and sealed with nail polish. They were then viewed and photographed under a confocal microscope (Zeiss LSM510).
Radiotracer flux assays.
The influx experiments were performed at 20°C with 510 oocytes for each condition and 14C- or 3H-labeled hexose at a specific activity of 1 µCi/ml. Oocytes were washed with ice-cold MBM to stop the incubation, and then individual oocytes were placed in vials and dissolved in 0.5 ml 5% SDS for 30 min. Finally, scintillation fluid (5 ml) was added to each vial and radio activity was measured with a Beckman LS6500. All experiments were performed 37 times, and the results were compared with the influx values obtained with water-injected oocytes. In experiments in which phloretin was added, ethanol was used as the initial solvent for the stock solution and subsequent dilution resulted in 1% ethanol being present in the uptake solutions. Similarly, DMSO was the solvent employed for CB, again present at a final concentration of 1% in the uptake solutions. Separate experiments showed that at this concentration neither of these solvents had any effect on glucose uptake into water-injected oocytes or those expressing GLUT7.
Kinetic analysis.
D-Glucose uptake was measured over a range of concentrations from 0.05 to 6 mM in 30-min incubations, which had been determined to be within the linear slope of uptake. Uptake was corrected for nonspecific uptake by using water-injected eggs from the same batch of eggs in each experiment. ENZFITTER software was used to determine the transport kinetics for the GLUT7-mediated glucose uptake by nonlinear regression analysis. Conventionally, a nonmetabolized substrate is used for kinetic measurements to avoid the possibility of the kinetic parameters of a rate-limiting intracellular metabolic pathway superseding the kinetic constants of the transporter. In this study 2-deoxy-D-glucose (2DG) was found not to be a substrate, and so we focused on the naturally occurring hexoses. However, it should be noted that when the sodium-dependent glucose transporter (SGLT1) is expressed in oocytes there is no discernible difference in the rates of transport between D-glucose and
-methyl-D-glucoside. SGLT1 is expressed at very high levels in oocytes and achieves a far greater rate of uptake than we observe with GLUT7, so we concluded that hexokinase cannot be rate limiting under our conditions (9, 24).
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RESULTS
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PCR employing the unique primers for the putative GLUT7 sequence with a human intestinal cDNA library generated a 1.7-kb cDNA product that gave codes for a predicted amino acid sequence shown in Fig. 1. This 524-amino acid sequence, when aligned with that for GLUT5, shows 68% similarity and 53% identity between the two proteins. Both the nucleotide and amino acid sequence data have been deposited with GenBank (accession no. AY571960). This clone was then used to produce mRNA for injection into oocytes and functional characterization of the protein.

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Fig. 1. Alignment of human glucose transporter GLUT5 and GLUT7 primary sequences. Sequences were compared using Clustal X software. Identical residues are highlighted.
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GLUT7 functional studies.
The uptake of a panel of hexoses into mRNA- and water-injected oocytes showed that GLUT7 mediated the transport of glucose and fructose. Figure 2A shows a typical experiment. There was no significant GLUT7-mediated flux of D-galactose, 2DG, or D-xylose. The mold metabolite CB, known to inhibit transport activity of many GLUTs, had no significant effect on the uptake of glucose mediated by GLUT7 (Fig. 2B). Similarly, 200 µM phloretin had no effect on glucose uptake (Fig. 2C).

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Fig. 2. A: GLUT7 mediated hexose fluxes in Xenopus oocytes. Oocytes were injected with GLUT7 mRNA or water 5 days before the measurement of uptake of radiolabeled substrates (100 µM). Bars represent uptake measured over 30 min into 810 oocytes injected with mRNA (hatched bars), water (open bars), and net uptake (filled bars). Error bars represent the standard error of the mean. 2DOG, 2-deoxy-D-glucose. B: effect of the addition of 100 µM cytochalasin B (CB) on the uptake of 100 µM D-glucose into oocytes injected with GLUT7 mRNA C: effect of addition of 200 µM phloretin on 100 µM D-glucose uptake. In these experiments, 1820 oocytes were used for each condition.
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The time course of 100 µM glucose uptake into oocytes expressing GLUT7 was linear for at least 40 min (data not shown), and so 30-min incubations were chosen for subsequent kinetic experiments. This length of incubation maximized the uptake signal while remaining on the linear portion of the uptake curve. Uptake of glucose over the concentration range 0.055.0 mM was curvilinear and, when corrected for uptake into water-injected oocytes, exhibited Michaelis-Menten type kinetics with a Km of 0.20.4 mM (Fig. 3). Fructose inhibition of glucose uptake indicated an IC50 of 60.3 ± 25.8 µM, indicating that GLUT7 also has a high affinity for this hexose (Fig. 4).

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Fig. 3. Kinetics of GLUT7-mediated glucose uptake into Xenopus oocytes. Glucose uptake using 30-min incubation periods over a range of concentrations (conc) is shown. The uptake is corrected for the uptake measured into the same batch of oocytes injected with water, and the fitted curve is that solved by nonlinear regression for a single Michaelis-Menten component with a Km of 0.3 mM and a Vmax of 20 pmol·oocyte1·30 min1.
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Fig. 4. Inhibition of GLUT7-mediated glucose uptake by fructose. The uptake of 100 µM D-glucose was measured in the presence of increasing concentrations of cold D-fructose. Incubations lasted 30 min, and the data points represent the mean uptake into 45 individual oocytes corrected for uptake into water-injected oocytes under identical conditions. The curve was fitted by nonlinear regression analysis, which gave an IC50 of 60.3 µM.
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Tissue expression pattern.
Northern blotting of intestinal mRNA with the antisense cDNA DIG-labeled probe corresponding to the COOH-terminal coding sequence for the last 271 amino acids of hGLUT7 gave a band corresponding to a transcript of 1.35 kb. This was detected in the small intestine and colon as well as weaker signals in the testis and prostate. Spleen, thymus, ovary, and leucocytes gave no detectable signal (Fig. 5). A second blot showed no signal in the heart, brain, placenta, lung, liver, skeletal muscle, kidney, or pancreas (data not shown).

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Fig. 5. Tissue distribution of GLUT7 by Northern blotting. The digoxigenin (DIG)-labeled partial GLUT7 cRNA corresponding to amino acids M254M524 was used as a probe. Lanes were loaded with mRNA normalized to the same -actin RNA concentration. After hybridization, the blot was exposed to film for 5 min before developing. Lane 1, spleen; lane 2, thymus; lane 3, prostate; lane 4, testis; lane 5, ovary; lane 6, small intestine; lane 7, colon; lane 8, leucocytes. The bands correspond to a size of 1.35 kb. Marker indicates size in kilobases.
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Protein expression was determined by using a commercially prepared antibody raised against the 11 COOH-terminal amino acids of the hGLUT7 sequence. The specificity of the polyclonal antibody 7440 was clearly demonstrated by comparing the staining pattern of water-injected Xenopus oocytes with that of oocytes 5 days after injection with hGLUT7 mRNA. The mRNA injection clearly led to the expression of novel protein in the plasma membrane, which was detected by the antibody using Western blotting, whereas there was no binding of the antibody to membranes from water-injected eggs (Fig. 6B). Western blotting of pooled oocyte plasma membranes from oocytes injected with GLUT7 mRNA showed a single protein band (Fig. 6B), whereas in membranes from eggs expressing GLUT5 no band was observed, supporting the conclusion that antibody 7440 can specifically detect the expression of the hGLUT7 protein. Conversely, the GLUT5 antibody failed to detect GLUT7 protein (Fig. 6A). Western blotting of brush-border membranes prepared from the jejunum and ileum of Sprague-Dawley rats also reacted with the GLUT7 antibody, giving a band at a position equivalent to an apparent molecular weight of 53 kDa. The expression was low in the jejunum and higher in the ileum (Fig. 6C).

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Fig. 6. A: Western blot of oocyte membranes prepared from oocytes 5 days after injection with human GLUT7 mRNA (shown to be transporting glucose and fructose) or with GLUT5 mRNA (shown to transport fructose) or injected with water and probed with anti-GLUT5 antibody. B: same membranes probed with anti-GLUT7 antibody 7440. C: Western blot of rat jejunal and ileal brush-border membranes probed with the anti-GLUT7 antibody 7440.
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Immunohistochemistry of hGLUT7 mRNA-injected oocytes, using the same GLUT7 antibody, showed significant expression of protein in the plasma membrane, whereas there was no signal detected in water-injected eggs (data not shown). This confirmed that the antibody could also be used with tissue slices as well as for Western blots. Immunohistochemistry of rat frozen intestinal tissue with the 7440 antibody showed expression of an hGLUT7-like protein (Fig. 7) that corresponded to the Western blotting of rat membranes with strong staining in the ileum. However, although Northern blotting for human tissues indicated the presence of GLUT7 mRNA in the colon, no staining was detected in rat tissue for this region of the gastrointestinal tract. Examination of the staining patterns in the rat ileum indicated that the protein was predominantly expressed in the brush-border membrane with only a very weak signal in the lateral or basal membranes. The protein was not found in the underlying muscle layers nor within the villus core, indicating that GLUT7 is not expressed in endothelial or muscle cells. Some bright signals evident in both control and test tissues represent autofluorescence of mast cells in the villus core. The specificity of the binding of antibody 7440 was again confirmed by the inability of the preabsorbed primary antibody to bind to proteins in the ileal brush-border membrane (Fig. 7B).

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Fig. 7. Immunohistochemistry of rat ileum (A) and colon (C) probed with the primary antibody 7440 raised against the putative last 11 amino acids of human GLUT7 and ileum probed with the antibody preabsorbed with the antigenic synthetic peptide (B). Frozen sections (11 µm) of rat intestine were probed with the primary antibody overnight at 4°C. The presence of GLUT7 was visualized with an FITC-tagged secondary antibody. Images were captured with a Zeiss LSM510 confocal microscope.
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DISCUSSION
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The sequence alignment of GLUT5 and -7 shows that the two human proteins are 68% similar and 53% identical (Fig. 1). GLUT7 appears to contain many of the so called "signature sequences" that are believed to be typical of the GLUT family of proteins. The proline-containing motif (PESPR) between transmembrane segments (TMs) 6 and 7 is present in both GLUT5 and -7. There are two QLS motifs within TM 7 of GLUT1, -3, and -4, and the one equivalent to position 279281 of GLUT1 has been suggested to be part of the glucose selectivity filter (29). This first motif is missing from GLUT2 (replaced with HVA), which is a low-affinity glucose and fructose transporter, and from the fructose transporter GLUT5 (replaced with MGG). The second QLS motif present in TM 7 is found at position 283285 in GLUT1, -3, -4, and -5 and is also present in GLUT7. It has previously been proposed that QLS 279281 forms a high-affinity bond with the C1 hydroxyl on glucose and is part of the exofacial recognition site. Additionally, exchange of the QLS from GLUT3 into the equivalent position in GLUT2 largely removed its ability to transport fructose, and the insertion of HVA from GLUT2 into GLUT3 induced low-affinity fructose transport (29). However, the absence of either HVA or QLS at this site in GLUT7, which binds both glucose and fructose with very high affinity (Km < 1.0 mM), would suggest that other residues that line the pore must also be involved in the recognition of substrates.
It is of interest that although GLUT1 contains two tryptophan residues at 388 and 412, both GLUT5 and -7 have only the one tryptophan at the position equivalent to 412. This ditryptophan "motif" has been implicated in the CB binding site, and although GLUT5 has previously been shown to be insensitive to this fungal inhibitor we can now report that GLUT7 is also not inhibited by CB. This additional finding certainly lends support for the role of these two tryptophans in the binding of CB to GLUT proteins. Similarly, just as GLUT5 is insensitive to inhibition by phloretin (5), so is GLUT7, which sets these class II transporters apart from those in class I.
The vast majority of membrane proteins are glycosylated, and GLUT7 appears to be no exception, given that the bands detected on the Western blots are relatively diffuse, typical of glycosylated proteins. The GLUT proteins belonging to classes I and II all appear to be glycosylated on the first extracellular loop found between the TMs 1 and 2. In contrast, preliminary data for class III indicates glycosylation on loop 9 between TMs 9 and 10 (32), although the functional significance of this is not known. Examination of the primary sequence of GLUT7 indicates that within the first putative extracellular loop there are three asparagine residues that could provide a site for N-linked glycosylation. N46 is probably too close to the cell membrane, leaving the two most likely sites as N59 and N69, and of these two N59 appears to be best conserved within the class I and II proteins.
Of considerable interest is the substrate specificity of this GLUT7 isoform, which can transport both glucose and fructose. None of the other hexoses tested appeared to be substrates, including galactose, 2DG, and xylose. This inability to handle galactose and 2DG is surprising given that the other GLUT that can recognize both glucose and fructose is GLUT2, for which galactose and 2DG are also a substrates. This would indicate that comparison between the selectivity filters of these three transporters should lead to some insights regarding the key residues involved in binding to the substrates.
The GLUT7 affinity for glucose is also very high compared with the majority of GLUTs, with a Km of
0.3 mM, which is almost an order of magnitude lower than that of GLUT5 for fructose (3) and two orders of magnitude lower than that of GLUT2 (4, 13, 15). The only other GLUT that has been shown to have such a low Km is GLUT10, using 2DG as the substrate (8). Similarly, the ability for GLUT7 to transport fructose at a comparable rate for the same concentration as glucose (0.1 mM) and the IC50 of 60 µM shows that GLUT7 also has a high affinity for fructose. It is not clear, currently, as to why GLUT7 would have such a high affinity for its substrates, but presumably it must be related to the physiological role this protein plays.
The distribution of GLUT7 appears to be limited to the more distal region of small intestine, the ileum, which contrasts with SGLT1, GLUT5, and GLUT2. These latter hexose transporters are found predominantly in the jejunum with a much lower protein abundance in the ileum (3, 6, 12, 30). The presence of the message for this protein in the testis and prostate is of interest given that the need for fructose as a substrate for sperm is well documented and GLUT7 can transport both glucose and fructose (1). Additional work needs to be done to determine just where in these tissues the transporter is expressed.
Although is not possible at this stage to definitively assign a physiological role for GLUT7 in the intestine, it is possible to draw some inferences from the data currently available. The low Km for this transporter suggests that it deals with low ambient concentrations of its substrates, glucose and fructose. Therefore, although it does not seem likely that GLUT7 plays a key role in taking up glucose from the diet in the initial stages of digestion and absorption, it may be important toward the end of the meal when luminal concentrations of glucose and fructose in the ileum are low. There is a rare genetic disease in humans, glucose/galactose malabsorption, in which point mutations in the SGLT1 lead to failure of the protein to be inserted into the brush-border membrane (18, 22, 31). This results in malabsorption of the hexoses (glucose and galactose) taken up by this transporter. The uptake of fructose in the jejunum, however, which enters via different pathways, appears to be relatively normal in these patients. There are at least two known possible routes of entry for fructose across the brush-border membrane: via GLUT5, which is specific for fructose, and via the newly identified pathway of transiently expressed apical GLUT2. GLUT5 appears to be expressed in a constitutive manner and is not regulated by the presence of luminal hexoses during the course of a meal (11), although its expression can adapt to maintained changes in the diet. In contrast, GLUT2 is inserted into the jejunal brush-border membrane within minutes when glucose is present in the intestinal lumen (16, 17). This effect appears to be mediated by the uptake of glucose via SGLT1 and so will not operate in glucose/galactose malabsorbers in whom SGLT1 is nonfunctional.
Could GLUT7 play a significant role in absorbing fructose and glucose in these patients? This seems unlikely for two reasons. First, GLUT7 appears to be a high-affinity transporter, which usually means a low Vmax and hence low capacity. Thus it is unlikely that GLUT7 would have the required capacity that can already be achieved via GLUT2 and -5, which have much higher Km values, unless GLUT7 is present in high abundance. Second, if GLUT7 were able to handle a significant component of the nutrient load of fructose, then it should also be able to absorb significant amounts of glucose and so, at least partially, compensate for the absence of SGLT1 in patients with glucose/galactose malabsorption. This does not appear to be the case. Clearly, additional work is required to determine the contribution that GLUT7 makes to the absorption of the nutrient carbohydrate load in the small intestine.
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GRANTS
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This work was supported by grants from the Canadian Institutes for Health Research and the Alberta Heritage Foundation for Medical Research. Q. Li is supported by a fellowship from the Kidney Foundation of Canada. A. Manolescu is the recipient of a J. B. Collip studentship for Diabetes Research from the Muttart Diabetes Research & Training Centre.
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ACKNOWLEDGMENTS
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We thank Dr. H. G. Joost for invaluable discussions during the course of this work. This work could not have been completed without the invaluable technical assistance of Debbie O'Neill.
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FOOTNOTES
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Address for reprint requests and other correspondence: C. I. Cheeseman, Dept. of Physiology, Rm. 722, Medical Sciences Bldg., Univ. of Alberta, Edmonton, AB T6G 2H7, Canada (E-mail: chris.cheeseman{at}ualberta.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.
* Q. Li and A. Manolescu contributed equally to this work. 
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