Chris I. Cheeseman

SLC2A9 Is a High-Capacity Urate Transporter in Humans

Background Serum uric acid levels in humans are influenced by diet, cellular breakdown, and renal elimination, and correlate with blood pressure, metabolic syndrome, diabetes, gout, and cardiovascular disease. Recent genome-wide association scans have found common genetic variants of SLC2A9 to be associated with increased serum urate level and gout. The SLC2A9 gene encodes a facilitative glucose transporter, and it has two splice variants that are highly expressed in the proximal nephron, a key site for urate handling in the kidney. We investigated whether SLC2A9 is a functional urate transporter that contributes to the longstanding association between urate and blood pressure in man. Methods and Findings We expressed both SLC2A9 splice variants in Xenopus laevis oocytes and found both isoforms mediate rapid urate fluxes at concentration ranges similar to physiological serum levels (200–500 μM). Because SLC2A9 is a known facilitative glucose transporter, we also tested whether glucose or fructose influenced urate transport. We found that urate is transported by SLC2A9 at rates 45- to 60-fold faster than glucose, and demonstrated that SLC2A9-mediated urate transport is facilitated by glucose and, to a lesser extent, fructose. In addition, transport is inhibited by the uricosuric benzbromarone in a dose-dependent manner (K i = 27 μM). Furthermore, we found urate uptake was at least 2-fold greater in human embryonic kidney (HEK) cells overexpressing SLC2A9 splice variants than nontransfected kidney cells. To confirm that our findings were due to SLC2A9, and not another urate transporter, we showed that urate transport was diminished by SLC2A9-targeted siRNA in a second mammalian cell line. In a cohort of men we showed that genetic variants of SLC2A9 are associated with reduced urinary urate clearance, which fits with common variation at SLC2A9 leading to increased serum urate. We found no evidence of association with hypertension (odds ratio 0.98, 95% confidence interval [CI] 0.9 to 1.05, p > 0.33) by meta-analysis of an SLC2A9 variant in six case–control studies including 11,897 participants. In a separate meta-analysis of four population studies including 11,629 participants we found no association of SLC2A9 with systolic (effect size −0.12 mm Hg, 95% CI −0.68 to 0.43, p = 0.664) or diastolic blood pressure (effect size −0.03 mm Hg, 95% CI −0.39 to 0.31, p = 0.82). Conclusions This study provides evidence that SLC2A9 splice variants act as high-capacity urate transporters and is one of the first functional characterisations of findings from genome-wide association scans. We did not find an association of the SLC2A9 gene with blood pressure in this study. Our findings suggest potential pathogenic mechanisms that could offer a new drug target for gout.

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.

GLUT2 is the transporter for fructose across the rat intestinal basolateral membrane.

BACKGROUND The exact roles of disaccharidases and GLUT5 in the brush border membrane and GLUT2 in the basolateral membrane in the absorption of fructose across the intestine have not been fully determined. This paper describes characterization of fructose transport across the jejunal basolateral membrane using isolated membrane vesicles. METHODS Transport of fructose was measured using rapid filtration of vesicles. Luminal perfusion in vivo with glucose and fructose before vesicle preparation was used to assess modulation of GLUT2 activity. Western blotting measured the abundance of GLUT2 in the membrane. RESULTS The maximal rate of transport for fructose was 1100 pmol/mg protein/s and the Michaelis constant was 16 mmol/L. Fructose and glucose could completely inhibit the transport of each other. Perfusion of the intestinal lumen with fructose or glucose saline for 4 hours produced a fourfold increase in maximal fructose transport. CONCLUSIONS These data indicate that the one transport protein, GLUT2, is responsible for moving both fructose and glucose out of the enterocyte across the basolateral membrane under basal conditions. The activity of this, or a closely related carrier, is rapidly upregulated by the presence of hexoses in the intestinal lumen, explaining the potentiation of fructose absorption by luminal glucose and obviating any need to involve apical disaccharidases.

Inhibition of glucose absorption in the rat jejunum: A novel action of alpha-D-glucosidase inhibitors

BACKGROUND & AIMS alpha-D-Glucosidase inhibitors act primarily by decreasing disaccharide hydrolysis and thus reduce the amount of free monosaccharides available for absorption. A novel action of alpha-D-glucosidase inhibitors is presented, indicating a direct effect on free glucose absorption by the rat jejunum. METHODS The jejunum was isolated and free hexose was measured using in vivo single-pass luminal perfusion and dual vascular and luminal single-pass in vitro perfusion. Xenopus oocytes were injected with RNA transcript encoding recombinant sodium-glucose cotransporter 1, and uptake of 3H-labeled 3-O-methyl-D-glucopyranose (3-O-MG) was assessed. RESULTS Acarbose (0.1 mg/mL), added to the lumen, decreased D-glucose absorption by 20% in vivo. Addition of 0.1 or 1.0 mg/mL acarbose to the lumen in vitro decreased the appearance of 3-O-MG in the vascular effluent by 28% and 60%, respectively. Accumulation of D-glucose within the enterocytes was decreased significantly by 67% and 79% when acarbose (1 mg/mL) or phloridzin (2 mmol/L), respectively, were present in the luminal perfusate. In contrast, acarbose did not affect the transport rate of free D-fructose and did not inhibit 3-O-MG uptake in oocytes expressing sodium-glucose cotransporter 1. CONCLUSIONS The findings indicate that alpha-D-glucosidase inhibitors act specifically on the entry of free glucose into the enterocyte, an additional means by which they can reduce postprandial hyperglycemia.

A highly conserved hydrophobic motif in the exofacial vestibule of fructose transporting SLC2A proteins acts as a critical determinant of their substrate selectivity

The substrate specificity of the facilitated hexose transporter, GLUT, family, (gene SLC2A) is highly varied. Some appear to be able to translocate both glucose and fructose, while the ability to handle 2-deoxyglucose and galactose does not necessarily correlate with the other two hexoses. It has become generally accepted that a central substrate binding/translocation site determines which hexoses can be transported. However, a recent study showed that a single point mutation of a hydrophobic residue in GLUTs 2, 5 & 7 removed their ability to transport fructose without affecting the kinetics of glucose permeation. This residue is in the 7th transmembrane helix, facing the aqueous pore and lies close to the opening of the exofacial vestibule. This study expands these observations to include the other class II GLUTs (9 & 11) and shows that a three amino acid motif (NXI/NXV) appears to be critical in determining if fructose can access the translocation mechanism. GLUT11 can also transport fructose, but it has the motif DSV at the same position, which appears to function in the same manner as NXI and when all three residues are replaced with NAV fructose transport lost. These results are discussed in relation to possible roles for hydrophobic residues lining the aqueous pore at the opening of the exofacial vestibule. Finally, the possibility that the translocation binding site may not be the sole determinant of substrate specificity for these proteins is examined.

Effect of hyperglycemia on D-glucose transport across the brush-border and basolateral membrane of rat small intestine

Experimental hyperglycemia leads to an increase in the capacity of the rat small intestine to absorb glucose. This effect occurs within hours from the onset of hyperglycemia and is thought to involve an induction of glucose transport in the brush-border and/or basolateral membrane of the intestinal epithelium. We devised a protocol for the simultaneous preparation of brush-border vesicles and basolateral vesicles from rat small intestine to determine the locus for the induction of glucose transporter in hyperglycemic rats. A 6 h period of intravenous infusion with a 30% glucose solution had no effect on the initial rate of glucose uptake across jejunal or ileal brush-border vesicles when measured in the absence of a Na+ gradient, suggesting that enhanced glucose uptake is not dependent on an increase in the number of Na+-dependent secondary active glucose transporters in the brush-border. Hyperglycemia did not effect the rate of glucose uptake across ileal basolateral vesicles but did cause a 78% increase in the initial rate of carrier-mediated D-glucose uptake across jejunal basolateral vesicles. The induction of glucose transport in the jejunal basolateral membrane was characterized by a rapid rate of glucose equilibration across the vesicles (t 1/2 = 46 s sorbitol infused controls, 18 s hyperglycemia) and a 75% increase in the Vmax for carrier-mediated glucose uptake with no significant change in Kt. When the rats were pretreated with cycloheximide prior to intravenous infusion, the initial rate of D-glucose uptake dropped to 13% of that seen in jejunal basolateral vesicles prepared from untreated rats. These results suggest a rapid turnover rate for the Na+-independent glucose transporter in the basolateral membrane of the enterocyte. An increase in the number of functioning glucose transporters in the basolateral membrane may play an important role in the short-term induction of glucose absorption by the jejunum of the hyperglycemic animal.

Cholecystokinin Decreases Intestinal Hexose Absorption by a Parallel Reduction in SGLT1 Abundance in the Brush-Border Membrane

The dual lumenaly and vascularly perfused small intestine was used to determine the mechanism by which cholecystokinin octapeptide (CCK-8) decreases the rate of glucose absorption. With CCK-8 in the vascular perfusate the rate of 3-O-methyl-d-glucose absorption decreased, whereas the rate of d-fructose absorption was unaffected. The substrate pool size within the tissue during steady-state transport, in the presence and absence of CCK-8, was estimated by compartmental analysis of the 3-O-methyl-d-glucose washout into the vascular bed. When CCK-8 was included in the vascular perfusate, the absorptive cell pool size decreased when compared with untreated tissue. Both the steady-state hexose absorption data and the washout studies indicated that the locus of action of CCK-8 was the SGLT1 transporter located in the brush-border membrane. The SGLT1 protein abundance in isolated brush-border membranes, as quantified by Western blotting, showed a decrease that paralleled the decrease in the steady-state transport rate induced by CCK-8. These results indicate that CCK-8 diminishes the rate of intestinal hexose absorption by decreasing SGLT1 protein abundance in the brush-border membrane of the rat jejunum and therefore provides evidence for acute enteric hormonal regulation of the rate of glucose absorption across the small intestine.

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.

Crypt cell production rate, enterocyte turnover time and appearance of transport along the jejunal villus of the rat

Intestinal nutrient absorption is subject to adaptation with, for example, diabetes, diet lipid variations (isocaloric semisynthetic diets enriched with saturated (S) or polyunsaturated (P) fatty acids), ileal resection and abdominal irradiation. These models were used in rats to assess dynamic morphology and distribution of amino acid transporter along the villus. The enterocyte migration rate (EMR) was measured using [3H]thymidine; the vincristine metaphase arrest technique was used to determine the crypt cell production rate (CCPR); quantitative autoradiography was used to assess the time and age of enterocytes when the uptake of 1 and 20 mM [3H]leucine and [3H]lysine was initiated along the villus. The enhanced jejunal uptake of nutrients which occurs after a 50% distal enterectomy was associated with a fall in EMR and CCPR, yet the enhanced nutrient uptake which occurs in diabetes is not associated with any alteration in EMR, CCPR, enterocyte transport pool (ETP), i.e., the length of the enterocyte column along with the villus containing amino acid transporter) or expression of transporter along the villus. The reduced uptake of nutrients in rats fed P as compared with S was associated with increased rather than decreased ETP and age of the enterocytes at the tip of the villus. The reduced nutrient uptake which occurs 3 days after abdominal irradiation was associated with increased EMR and CCPR, and reduced ETP and age of enterocytes of the tip of the villus. However, 14 days after irradiation when nutrient transport remains reduced, these parameters have returned to normal. Thus, alterations in nutrient transport may be associated with changes in the dynamic morphology of the intestine, but the two processes are not necessarily interdependent. We speculate that the changes in the dynamic morphology of the intestine, and the changes of amino acid transport which occurs in these models of intestinal adaptation, are independently controlled.

Na+- and K+-dependent uridine transport in rat renal brush-border membrane vesicles

The transport of uridine into rat renal brush-border membrane vesicles was investigated using an inhibitor-stop filtration method. Uridine was not metabolized under these conditions. The rapid efflux of intravesicular uridine was prevented by adding 1 mM phloridzin to the ice-cold stop solution. In the presence of inwardly directed gradients of either Na+ or K+, zero-trans uridine uptake exhibited a transient overshoot phenomenon indicating active transport. The overshoot was much more pronounced with Na+ than K+ and it was not observed when either Na+ or K+ was at equilibrium across the membrane. The K+-induced overshoot was not due to the presence of a membrane potential alone, as an inwardly directed gradient of choline chloride failed to produce it. The amplitude of the overshoot was increased by raising either the Na+ or K+ concentration outside the membrane or by using more lipophilic anions (reactive order was NO3- greater than SCN- greater than Cl- greater than SO4(2-). Zero-trans efflux studies showed that the uridine transport is bidirectional. Li+ could substitute poorly for Na+ but not at all for K+. Stoichiometries of 1:1 and greater than 1:1 were observed for Na+: uridine and K+: uridine coupling, respectively. A preliminary analysis of the interactions between Na+ and K+ for uridine uptake showed complex interactions which can best be explained by the involvement of two different systems for nucleoside transport in the rat renal brush-border membrane, one requiring Na+ and the other K+ as transport coupler.