Critical Roles of Two Hydrophobic Residues within Human Glucose Transporter 9 (hSLC2A9) in Substrate Selectivity and Urate Transport.

High blood urate levels (hyperuricemia) have been found to be a significant risk factor for cardiovascular diseases and inflammatory arthritis, such as hypertension and gout. Human glucose transporter 9 (hSLC2A9) is an essential protein that mainly regulates urate/hexose homeostasis in human kidney and liver. hSLC2A9 is a high affinity-low capacity hexose transporter and a high capacity urate transporter. Our previous studies identified a single hydrophobic residue in trans-membrane domain 7 of class II glucose transporters as a determinant of fructose transport. A mutation of isoleucine 335 to valine (I355V) in hSLC2A9 can reduce fructose transport while not affecting glucose fluxes. This current study demonstrates that the I335V mutant transports urate similarly to the wild type hSLC2A9; however, Ile-335 is necessary for urate/fructose trans-acceleration exchange to occur. Furthermore, Trp-110 is a critical site for urate transport. Two structural models of the class II glucose transporters, hSLC2A9 and hSLC2A5, based on the crystal structure of hSLC2A1 (GLUT1), reveal that Ile-335 (or the homologous Ile-296 in hSLC2A5) is a key component for protein conformational changes when the protein translocates substrates. The hSLC2A9 model also predicted that Trp-110 is a crucial site that could directly interact with urate during transport. Together, these studies confirm that hSLC2A9 transports both urate and fructose, but it interacts with them in different ways. Therefore, this study advances our understanding of how hSLC2A9 mediates urate and fructose transport, providing further information for developing pharmacological agents to treat hyperuricemia and related diseases, such as gout, hypertension, and diabetes.

Correction: New fluorinated fructose analogs as selective probes of the hexose transporter protein GLUT5.

Correction for 'New fluorinated fructose analogs as selective probes of the hexose transporter protein GLUT5' by Olivier-Mohamad Soueidan, et al., Org. Biomol. Chem., 2015, 13, 6511-6521.

New fluorinated fructose analogs as selective probes of the hexose transporter protein GLUT5.

Facilitated hexose transporters (GLUTs) mediate the transport of hexoses and other substrates across the membranes of numerous cell types, and while some are expressed ubiquitously (e.g., GLUT1), others are more tissue specific (e.g., GLUT5). These properties have been exploited for the imaging of cancer cells by the use of hexose based probes, including fluorinated hexose derivatives for use with positron emission tomography (PET). However, design of new probes has been hampered by a limited understanding of how GLUT transporters interact with their substrates at the molecular level. Two fluorinated fructose surrogates designed for uptake by the GLUT5 transporter are described here: 3-deoxy-3-fluoro-D-fructose (3-FDF) and 1-deoxy-1-fluoro-2,5-anhydromannitol (1-FDAM). Synthesis (both cold and radiolabeled) and in vitro analysis of their transport characteristics in two breast cancer cell lines (EMT-6 and MCF-7) expressing GLUT5 are detailed. Both analogues are readily taken up into both cancer cell lines, with uptake mediated primarily by GLUT5. They also have low IC50 values, indicating a high affinity for the transporter, suggesting that the uptake of these probes would be unaffected by endogenously circulating fructose. Selective uptake by GLUT5 was also demonstrated in Xenopus oocytes. Finally, these results are the first demonstration that a hexose existing predominantly in the pyranose ring structure (3-FDF) is transported by GLUT5, strongly suggesting that this transporter can handle both furanose and pyranose forms of fructose.

Synthesis and characterization of 6-deoxy-6-fluoro-D-fructose as a potential compound for imaging breast cancer with PET.

FDG-based imaging with positron emission tomography (PET) has been widely used in the detection of cancer, but has not reached its full potential. In breast cancer, the glucose/fructose transporter GLUT2 and the fructose transporter GLUT5 are known to be overexpressed in transformed tissues, implicating that a fructose-based analogue would be a useful target for the improved imaging of breast cancer. We have successfully synthesized the fluorinated fructose compound, 6-deoxy-6-fluoro-D-fructose (6FDF) and examined its potential for transport and accumulation in breast cancer cells. Expression analysis of GLUT isoforms was performed on two GLUT5 expressing breast cancer cell lines using western blotting and immunocytochemistry. Uptake and inhibition studies were undertaken using [14C]-labelled hexoses. Transport inhibition studies showed dose dependent inhibition of fructose transport in both cell lines by the newly synthesized 6-deoxy-6-fluoro-D-fructose (6FDF). Also, near linear uptake over time of [14C]-labelled 6FDF was observed in both cell lines. It appears that 6FDF may have great promise for use in in vivo PET imaging of breast cancer. Ongoing work will confirm the efficacy of this compound in imaging in mouse models.

GLUT Characterization Using Frog Xenopus laevis Oocytes.

Xenopus laevis oocytes are a useful heterologous expression system for expressing glucose transporters (GLUTs) and examining their functions. In this chapter, we provide a detailed protocol on oocyte extraction and preparation for GLUT9 protein expression. Furthermore, we describe the determination of GLUT9 overexpression level by biotinylation and Western blotting analysis. Finally, we also describe how GLUT9-expressing oocytes can be used to measure urate kinetics by radioisotopes as well as two-microelectrode voltage clamping techniques.

Mouse glucose transporter 9 splice variants are expressed in adult liver and kidney and are up-regulated in diabetes.

A novel glucose transporter (GLUT), mouse GLUT9 (mGLUT9), was recently cloned from mouse 7-d embryonic cDNA. Several splice variants of mGLUT9 were described, two of which were cloned (mGLUT9a and mGLUT9a Delta 209-316). This study describes the cloning and characterization of another splice variant, mGLUT9b. Cloned from adult liver, mGLUT9b is identical to mGLUT9a except at the amino terminus. Based on analysis of the genomic structure, the different amino termini result from alternative transcriptional/translational start sites. Expression and localization of these two mGLUT9 splice variants were examined in control and diabetic adult mouse tissues and in cell lines. RT-PCR analysis demonstrated expression of mGLUT9a in several tissues whereas mGLUT9b was observed primarily in liver and kidney. Using a mGLUT9-specific antibody, Western blot analysis of total membrane fractions from liver and kidney detected a single, wide band, migrating at approximately 55 kDa. This band shifted to a lower molecular mass when deglycosylated with peptide-N-glycosidase F. Both forms were present in liver and kidney. Immunohistochemical localization demonstrated basolateral distribution of mGLUT9 in liver hepatocytes and the expression of mGLUT9 in specific tubules in the outer cortex of the kidney. To investigate the alternative amino termini, mGLUT9a and mGLUT9b were overexpressed in kidney epithelium cell lines. Subcellular fractions localized both forms to the plasma membrane. Immunofluorescent staining of polarized Madin Darby canine kidney cells overexpressing mGLUT9 depicted a basolateral distribution for both splice variants. Finally, mGLUT9 protein expression was significantly increased in the kidney and liver from streptozotocin-induced diabetic mice compared with nondiabetic animals.

Identification of Key Residues for Urate Specific Transport in Human Glucose Transporter 9 (hSLC2A9).

Human glucose transporter 9 (hSLC2A9) is critical in human urate homeostasis, for which very small deviations can lead to chronic or acute metabolic disorders. Human SLC2A9 is unique in that it transports hexoses as well as the organic anion, urate. This ability is in contrast to other homologous sugar transporters such as glucose transporters 1 and 5 (SLC2A1 &SLC2A5) and the xylose transporter (XylE), despite the fact that these transporters have similar protein structures. Our in silico substrate docking study has revealed that urate and fructose bind within the same binding pocket in hSLC2A9, yet with distinct orientations, and allowed us to identify novel residues for urate binding. Our functional studies confirmed that N429 is a key residue for both urate binding and transport. We have shown that cysteine residues, C181, C301 and C459 in hSLC2A9 are also essential elements for mediating urate transport. Additional data from chimæric protein analysis illustrated that transmembrane helix 7 of hSLC2A9 is necessary for urate transport but not sufficient to allow urate transport to be induced in glucose transporter 5 (hSLC2A5). These data indicate that urate transport in hSLC2A9 involves several structural elements rather than just a unique substrate binding pocket.

Erratum: Automated synthesis and dosimetry of 6-deoxy-6-[(18)F]fluoro-D-fructose (6-[(18)F]FDF): a radiotracer for imaging of GLUT5 in breast cancer.

6-Deoxy-6-[(18)F]fluoro-D-fructose (6-[(18)F]FDF) is a promising PET radiotracer for imaging GLUT5 in breast cancer. The present work describes GMP synthesis of 6-[(18)F]FDF in an automated synthesis unit (ASU) and dosimetry calculations to determine radiation doses in humans. GMP synthesis and dosimetry calculations are important prerequisites for first-in-human clinical studies of 6-[(18)F]FDF. The radiochemical synthesis of 6-[(18)F]FDF was optimized and adapted to an automated synthesis process using a Tracerlab FXFN ASU (GE Healthcare). Starting from 30 GBq of cyclotron-produced n.c.a. [(18)F]fluoride, 2.9 ± 0.1 GBq of 6-[(18)F]FDF could be prepared within 50 min including HPLC purification resulting in an overall decay-corrected radiochemical yield of 14 ± 3% (n = 11). Radiochemical purity exceeded 95%, and the specific activity was greater than 5.1 GBq/µmol. Sprague-Dawley rats were used for biodistribution experiments, and dynamic and static small animal PET experiments. Biodistribution studies served as basis for allometric extrapolation to the standard man anatomic model and normal organ-absorbed dose calculations using OLINDA/EXM software. The calculated human effective dose for 6-[(18)F]FDF was 0.0089 mSv/MBq. Highest organ doses with a dose equivalent of 0.0315 mSv/MBq in a humans were found in bone. Injection of 370 MBq (10 mCi) of 6-[(18)F]FDF results in an effective whole body radiation dose of 3.3 mSv in humans, a value comparable to that of other (18)F-labeled PET radiopharmaceuticals. The optimized automated synthesis under GMP conditions, the good radiochemical yield and the favorable human radiation dosimetry estimates support application of 6-[(18)F]FDF in clinical trials for molecular imaging of GLUT5 in breast cancer patients.[This corrects the article on p. 248 in vol. 4.].

Automated synthesis and dosimetry of 6-deoxy-6-[(18)F]fluoro-D-fructose (6-[(18)F]FDF): a radiotracer for imaging of GLUT5 in breast cancer.

6-Deoxy-6-[(18)F]fluoro-D-fructose (6-[(18)F]FDF) is a promising PET radiotracer for imaging GLUT5 in breast cancer. The present work describes GMP synthesis of 6-[(18)F]FDF in an automated synthesis unit (ASU) and dosimetry calculations to determine radiation doses in humans. GMP synthesis and dosimetry calculations are important prerequisites for first-in-human clinical studies of 6-[(18)F]FDF. The radiochemical synthesis of 6-[(18)F]FDF was optimized and adapted to an automated synthesis process using a Tracerlab FXFN ASU (GE Healthcare). Starting from 30 GBq of cyclotron-produced n.c.a. [(18)F]fluoride, 2.9 ± 0.1 GBq of 6-[(18)F]FDF could be prepared within 50 min including HPLC purification resulting in an overall decay-corrected radiochemical yield of 14 ± 3% (n = 11). Radiochemical purity exceeded 95%, and the specific activity was greater than 5.1 GBq/µmol. Sprague-Dawley rats were used for biodistribution experiments, and dynamic and static small animal PET experiments. Biodistribution studies served as basis for allometric extrapolation to the standard man anatomic model and normal organ-absorbed dose calculations using OLINDA/EXM software. The calculated human effective dose for 6-[(18)F]FDF was 0.0089 mSv/MBq. Highest organ doses with a dose equivalent of 0.0315 mSv/MBq in a humans were found in bone. Injection of 370 MBq (10 mCi) of 6-[(18)F]FDF results in an effective whole body radiation dose of 3.3 mSv in humans, a value comparable to that of other (18)F-labeled PET radiopharmaceuticals. The optimized automated synthesis under GMP conditions, the good radiochemical yield and the favorable human radiation dosimetry estimates support application of 6-[(18)F]FDF in clinical trials for molecular imaging of GLUT5 in breast cancer patients.

Radiopharmacological evaluation of 6-deoxy-6-[18F]fluoro-D-fructose as a radiotracer for PET imaging of GLUT5 in breast cancer.

INTRODUCTION: Several clinical studies have shown low or no expression of GLUT1 in breast cancer patients, which may account for the low clinical specificity and sensitivity of 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) used in positron emission tomography (PET). Therefore, it has been proposed that other tumor characteristics such as the high expression of GLUT2 and GLUT5 in many breast tumors could be used to develop alternative strategies to detect breast cancer. Here we have studied the in vitro and in vivo radiopharmacological profile of 6-deoxy-6-[(18)F]fluoro-D-fructose (6-[(18)F]FDF) as a potential PET radiotracer to image GLUT5 expression in breast cancers. METHODS: Uptake of 6-[(18)F]FDF was studied in murine EMT-6 and human MCF-7 breast cancer cells over 60 min and compared to [(18)F]FDG. Biodistribution of 6-[(18)F]FDF was determined in BALB/c mice. Tumor uptake was studied with dynamic small animal PET in EMT-6 tumor-bearing BALB/c mice and human xenograft MCF-7 tumor-bearing NIH-III mice in comparison to [(18)F]FDG. 6-[(18)F]FDF metabolism was investigated in mouse blood and urine. RESULTS: 6-[(18)F]FDF is taken up by EMT-6 and MCF-7 breast tumor cells independent of extracellular glucose levels but dependent on the extracellular concentration of fructose. After 60 min, 30±4% (n=9) and 12±1% (n=7) ID/mg protein 6-[(18)F]FDF was found in EMT-6 and MCF-7 cells, respectively. 6-deoxy-6-fluoro-d-fructose had a 10-fold higher potency than fructose to inhibit 6-[(18)F]FDF uptake into EMT-6 cells. Biodistribution in normal mice revealed radioactivity uptake in bone and brain. Radioactivity was accumulated in EMT-6 tumors reaching 3.65±0.30% ID/g (n=3) at 5 min post injection and decreasing to 1.75±0.03% ID/g (n=3) at 120 min post injection. Dynamic small animal PET showed significantly lower radioactivity uptake after 15 min post injection in MCF-7 tumors [standard uptake value (SUV)=0.76±0.05; n=3] compared to EMT-6 tumors (SUV=1.23±0.09; n=3). Interestingly, [(18)F]FDG uptake was significantly different in MCF-7 tumors (SUV(15 min) 0.74±0.12 to SUV(120 min) 0.80±0.15; n=3) versus EMT-6 tumors (SUV(15 min) 1.01±0.33 to SUV(120 min) 1.80±0.25; n=3). 6-[(18)F]FDF was shown to be a substrate for recombinant human ketohexokinase, and it was metabolized rapidly in vivo. CONCLUSION: Based on the GLUT5 specific transport and phosphorylation by ketohexokinase, 6-[(18)F]FDF may represent a novel radiotracer for PET imaging of GLUT5 and ketohexokinase-expressing tumors.

Isolation of intestinal brush-border membranes.

This unit provides protocols for isolating intestinal brush-border membranes from rat, pig, and cow. These membranes can be used for immunoblotting or other analytical techniques. They will also spontaneously form closed vesicles which allow for flux assays to be performed using rapid filtration techniques. Overall the isolation procedures take approximately 3.5 hr. The resulting isolated membranes can be stored under liquid nitrogen or at -70 degrees C for a week or more depending upon the species.

Identification of a hydrophobic residue as a key determinant of fructose transport by the facilitative hexose transporter SLC2A7 (GLUT7).

Until recently, the only facilitated hexose transporter GLUT proteins (SLC2A) known to transport fructose were GLUTs 2 and 5. However, the recently cloned GLUT7 can also transport fructose as well as glucose. Comparison of sequence alignments indicated that GLUTs 2, 5, and 7 all had an isoleucine residue at position "314" (GLUT7), whereas the non-fructose-transporting isoforms, GLUTs 1, 3, and 4, had a valine at this position. Mutation of Ile-314 to a valine in GLUT7 resulted in a loss of fructose transport, whereas glucose transport remained completely unaffected. Similar results were obtained with GLUTs 2 and 5. Energy minimization modeling of GLUT7 indicated that Ile-314 projects from transmembrane domain 7 (TM7) into the lumen of the aqueous pore, where it could form a hydrophobic interaction with tryptophan 89 from TM2. A valine residue at 314 appeared to produce a narrowing of the vestibule when compared with the isoleucine. It is proposed that this hydrophobic interaction across the pore forms a selectivity filter restricting the access of some hexoses to the substrate binding site(s) within the aqueous channel. The presence of a selectivity filter in the extracellular vestibule of GLUT proteins would allow for subtle changes in substrate specificity without changing the kinetic parameters of the protein.

Characterization of the human SLC2A11 (GLUT11) gene: alternative promoter usage, function, expression, and subcellular distribution of three isoforms, and lack of mouse orthologue.

GLUT11 (SLC2A11) is a class II sugar transport facilitator which exhibits highest similarity with the fructose transporter GLUT5 (about 42%). Here we demonstrate that separate exons 1 (exon 1A, exon 1B, and exon 1C) of the SLC2A11 gene generate mRNAs of three GLUT11 variants (GLUT11-A, GLUT11-B, and GLUT11-C) that differ in the amino acid sequence of their N-termini. All three 5'-flanking regions of exon 1A, exon 1B and exon 1C exhibited promoter activity when expressed as luciferase fusion constructs in COS-7 cells. 5'-RACE-PCR, quantitative real-time PCR, and Northern blot analysis performed with specific probes for exon 1A, 1B and 1C demonstrated that GLUT11-A is expressed in heart, skeletal muscle, and kidney, GLUT11-B in kidney, adipose tissue, and placenta, and GLUT11-C in adipose tissue, heart, skeletal muscle, and pancreas. Surprisingly, mice and rats lack the SLC2A11 gene. When expressed in Xenopus oocytes, all three GLUT11 isoforms transport glucose and fructose but not galactose. There was no apparent difference in the subcellular distribution of the three isoforms expressed in COS-7 cells. Our data indicate that different promoters and splicing of the human SLC2A11 gene generate three GLUT11 isoforms which are expressed in a tissue specific manner but do not appear to differ in their functional characteristics.

Rapid insertion of GLUT2 into the rat jejunal brush-border membrane promoted by glucagon-like peptide 2.

A possible role for GLUT2 transiently expressed in the rat jejunal brush-border membrane (BBM) under the influence of glucagon-like peptide 2 (GLP-2) was investigated using in vivo perfusion of the intestinal lumen as well as isolation of membrane proteins and immunohistochemistry. A 1 h vascular infusion of GLP-2 in vivo doubled the rate of fructose absorption and this increase could be blocked by luminal phloretin. Immunohistochemistry of frozen sections of rat jejunum showed the expression of GLUT2 in both the basolateral and BBMs of mature enterocytes. Perfusion of the intestinal lumen with 50 mM D-glucose or vascular infusion of 800 pM GLP-2 for 1 h increased the expression of GLUT2 in the BBM. Quantification of these changes using Western blotting of biotinylated surface-exposed protein showed a doubling of the expression of GLUT2 in the BBM, but the effects of glucose and GLP-2 were not additive. These results indicate that vascular GLP-2 can promote the insertion of GLUT2 into the rat jejunal BBM providing a low-affinity/high-capacity route of entry for dietary hexoses.

Cloning and functional characterization of the human GLUT7 isoform SLC2A7 from the small intestine.

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 (K(m) = 0.3 mM) and fructose (IC(50) = 0.060 mM). Galactose, 2-deoxy-d-glucose, and xylose were not transported. Uptake of 100 microM d-glucose was not inhibited by 200 microM phloretin or 100 microM 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.