Facilitative glucose transporter 9, a unique hexose and urate transporter

Abstract

GLUT9 is a novel, facilitative glucose transporter isoform that exists as two alternative splice variants encoding two proteins that differ in their NH₂-terminal sequence (GLUT9a and GLUT9b). Both forms of GLUT9 protein and mRNA are expressed in the epithelia of various tissues; however, the two splice variants are expressed differentially within polarized cells, with GLUT9a localized predominantly on the basolateral surfaces and GLUT9b expressed on apical surfaces. Protein expression of GLUT9 drops under conditions of starvation but increases with addition of glucose and under hyperglycemic conditions. The substrate specificity of GLUT9 is unique since, in addition to transporting hexose sugars, it also is a high-capacity uric acid transporter. Several recent large-scale human genetic studies show a correlation between SNPs mapped to GLUT9 and the serum uric acid levels in several different cohorts. The relationship between GLUT9 and uric acid is highly clinically significant. Elevated uric acid levels have been associated with metabolic syndrome, obesity, diabetes, hypertension, and chronic renal failure. Although some believe uric acid is elevated as a result of these diseases, there is now evidence that uric acid may play a role in the pathogenesis of these diseases. It is also known that GLUT9 is expressed in articular cartilage and is a uric acid transporter, and thus it is possible that GLUT9 plays a role in gout, a disease of uric acid deposition in the joints. In addition, some studies have suggested that intake of fructose plays an important role in causing elevated serum uric acid levels, especially in diabetes and obesity. It is possible that GLUT9, which seems to be both a fructose and a uric acid transporter, plays an important role in these conditions associated with hyperuricemia.

the facilitative glucose transporters (SLC2A) belong to the facilitated transporter super gene family known as the glucose transporters (GLUTs). These integral membrane proteins are responsible for the facilitated diffusion of glucose and other hexoses into cells. Fourteen GLUTs have been identified. These transporters share several sequence and structural similarities, such as 12 transmembrane helices and conserved amino acid residues (18). The GLUT family is subdivided into three classes on the basis of comparison of amino acid sequences (35). GLUT1–4 are in class 1, GLUT5, −7, −9, and −11 are in class 2, and the last class consists of GLUT6, −8, −10, and −12 and the H⁺/myoinositol transporter (35). GLUT14 is 95% identical to GLUT3 and thought to be a duplication of this class 1 transporter (37). The various GLUTs differ in their affinity for substrates, tissue distribution, and hormonal regulation. For example, GLUT4 is a glucose transporter found in muscle and adipose tissue that localizes to the plasma membrane in response to insulin, whereas GLUT11 is found in heart and skeletal muscle and appears to have a higher affinity for fructose than for glucose (16).

All members of the GLUT superfamily have a core structure of 12 transmembrane helices clustered in two sets of six, between which there is a central aqueous pore (16). The substrate specificity of this family of GLUTs varies greatly. GLUT1, −3, and −4 can transport glucose and galactose but not fructose, whereas GLUT2 can transport all three, and GLUT5 can recognize only fructose and possibly 2-deoxyglucose. The mechanism of this selective specificity was not initially clear. Early studies supported the existence of a central substrate-binding/transport site, which determines hexose specificity. The recent crystal structures solved for E. coli transporters lactose permease and glycerol 3-phosphate transporter suggest that, during the transport cycle, these two clusters undergo a change in conformation such that their binding site moves from an outward- to an inward-facing orientation (1, 17). In the case of the GLUT proteins, sequence alignments revealed the QLS motif in TM7 as a likely substrate selectivity filter (33). As the number GLUT family members increased and several different classes were identified, scanning mutagenesis studies and computer modeling indicate that an aqueous pore is formed by transmembrane helical domains (TMs) 5, 7, 8, 10, and 11 (2, 28). However, the shape of the outer- or inner-facing vestibules permitting entry of the substrates into the pore and access to the putative substrate-binding site is not entirely clear. Mueckler et al. (27) initially reported that a naturally occurring conservative mutation of valine 197 in TM5 to isoleucine (V197I) in GLUT2 resulted in significantly decreased transport capacity in a Xenopus oocyte system. Valine is a smaller residue than isoleucine, suggesting that, if these residues in TM5 faced into the pore, the larger isoleucine might impair the passage of substrate.

Comparison of the sequence alignments of the full family of the 14 GLUT members showed another possible hydrophobic residue, which might be related to fructose specificity, namely Ile-314, found in TM7 of GLUT7. GLUT2 and −5, which recognize fructose, also express Ile at the equivalent position. In contrast, the non-fructose-transporting proteins, GLUT1, −3, and −4, have a valine at the equivalent position. A recent study by Manolescu et al. (22) demonstrated that a single point mutation of a hydrophobic residue in the class 2 family members GLUT5 and −7 and the corresponding residue in GLUT2 abolished fructose transport without affecting the kinetics of glucose transport. This residue in TM7 faces the aqueous pore and lies close to the opening of the exofacial vestibule in the class 2 transporters. In a subsequent study by the same group, the investigators identified a three-amino acid motif (NXI/NXV) found in class 2 transporters GLUT9 and −11, which appears to be critical in determining whether fructose can access the translocation mechanism. These studies all support key roles for the hydrophobic residues lining the aqueous pore at the opening of the exofacial vestibule and suggest that differences among the GLUTs in these key residues may influence substrate specificity and access to the binding site through steric hindrance (29). It is also possible that similar interactions and residues may enable GLUT9 to transport a negatively charged purine derivative, such as urate, instead of a neutral hexose.

Characterization of GLUT9

The identification of GLUT9 came through an attempt to find the transporter responsible for fluorodeoxyglucose uptake in medullary thyroid carcinomas and pheochromocytomas, tissues that did not express any known facilitative glucose transporters (32). A search of the expressed sequence tag database revealed an expressed sequence tag clone with significant homology to GLUT1 and −5 (32). High levels of expression were found in normal human kidney and liver by Northern blot analysis. Later work by our group showed that the GLUT9 gene encodes for two alternative RNAs, the original GLUT9, now referred to as GLUT9a, and a splice variant that encodes a protein with a shorter NH₂ terminus, GLUT9ΔN (4) or GLUT9b. The human GLUT9 gene maps to chromosome 4p15.3–p16 and consists of 12 exons spanning 195 kb and codes for a 540-amino acid protein, whereas the GLUT9b splice variant consists of 13 exons, spans 215 kb of the GLUT9a gene, and codes for a 512-amino acid protein (4). GLUT9 shares 44% amino acid sequence similarity with another class 2 transporter, GLUT7. GLUT9 shows several motifs characteristic of the class 2 transporters, such as a PESPR/PETK in helices 6 and 12, a GRR/GRL in loops 2 and 8, and a PFI in the last extracellular loop (4, 32). In addition, mouse homologs for this gene have also been characterized. The mouse homolog of human GLUT9a is 539 amino acids in length, and the mouse homolog of GLUT9b is 523 amino acids in length (8). Both homologs share 85% identity with their human counterparts. The mouse gene maps to chromosome 5 and spans 12 and 13 exons for GLUT9a and GLUT9b, respectively (19).

Expression of GLUT9

GLUT9 is expressed in several different tissues, as delineated in Table 1. A few specific tissues will be discussed in more detail. GLUT9a RNA is expressed mainly in liver, kidney, and placenta, whereas the GLUT9b isoform is expressed primarily in kidney and placenta (4, 19). Further studies of GLUT9 expression in the kidney have demonstrated expression of protein in the proximal tubule of human kidney and possibly the distal convoluted tubule or connecting tubules of the mouse kidney (4, 19). Interestingly, the two isoforms appear to be differentially expressed in the kidney. Kidneys from diabetic mice show a significant increase in protein expression of GLUT9b compared with kidneys from control mice. This increase in expression is also seen in the liver of diabetic mice as well (19). Confocal microscopy on a polarized Madin-Darby canine kidney cell line stably overexpressing human GLUT9a or GLUT9b has shown that GLUT9a localizes exclusively to the basolateral membrane, whereas GLUT9b localizes to the apical membrane (4). This differential expression pattern is believed to be associated with vectorial reabsorption or excretion of glucose or other substrates to ensure efficient substrate handling in the kidney.

Table 1.

Expression of GLUT9 in tissue

Tissue	Notes	Ref. No.
Kidney	Found in mouse and human; mGLUT9 protein in outer cortex; hGLUT9 protein in proximal tubule; hGLUT9a and     hGLUT9b distinctly located on different cell membranes in polarized cells	4, 19, 32
Liver	Found in mouse and human; mGLUT9 protein expressed on sinusoidal membrane	4, 32
Embryo	Found in mouse; protein differentially expressed at specific developmental stages	8
Articular cartilage	Found in sheep and human; mRNA identified in human chondrocytes and in embryonic and adult sheep chondrocytes	25, 26
Sperm	Found in mouse; expression of both isoforms but differentially located on the sperm cell membranes	20
Testis	Found in mouse; both isoforms expressed in Leydig cells and seminiferous tubule	20
Placenta	Low-level mRNA for both isoforms detected in adult human tissue	4, 32
Heart	Found in human and sheep by mRNA	26, 32
Lung	Low-level mRNA for GLUT9a isoform detected only in adult human tissue	4, 32
Skeletal muscle	Low-level mRNA detected in adult tissue	32
Leukocytes	Low-level mRNA for GLUT9a isoform detected only in adult human tissue	4, 32
Brain	Low-level mRNA for GLUT9a isoform detected only in adult human tissue	4, 32

GLUT9, glucose transporter 9; mGLUT9, mouse GLUT9; hGLUT9, human GLUT9.

GLUT9 localization has also been studied in the murine early embryo as well as sperm and testis. At the one- and two-cell zygote stage in the mouse, GLUT9 is expressed at the plasma membrane. However, later, at the blastocyst stage, GLUT9 is localized perinuclearly in the trophoectoderm, which becomes the placenta, but not the inner cell mass, which develops into the embryo proper (8). Glucose is a critical substrate for early embryo development, and thus far only GLUT1 expression has been identified at the one-cell zygote stage. However, localization of this transporter is not at the plasma membrane at this early stage of development (31), and thus GLUT9 may function as the only GLUT responsible for glucose delivery at this stage. It is not clear why GLUT9 protein would change cell location at a later stage of development, but it is possible that it functions as an alternative substrate transporter, depending on embryonic stage. Mouse testis and sperm also express GLUT9. Both isoforms were detected in mouse testis. Confocal microscopy shows that GLUT9a and -b localize to the intraseminiferous tubule cells and the Leydig cells. In addition, GLUT9a localized to the midpiece of sperm, whereas GLUT9b localized to the acrosome, midpiece, and principal piece of the sperm (20). Fructose is a key energy substrate for male gametes, and thus it is possible that expression at specific locations within the sperm cell membranes may deliver this hexose to critical sites required for motility and transport of the sperm through the female reproductive tract. Moreover, Leydig cells required glucose as substrates for steroidogenesis, and thus expression of GLUT9 may be important for adequate testosterone production.

GLUT9 expression has been demonstrated in a human articular cartilage cDNA library by PCR (26). In addition, GLUT9 is present in both mature and developing articular cartilage of sheep (25). GLUT9 was also found in the pancreatic β-cells of both mouse and human. Interestingly, expression of GLUT9 was decreased by starvation but returned to normal with addition of glucose, suggesting that expression of GLUT9 in β-cells is regulated by glucose (13).

Functional Characteristics of GLUT9

In addition to the structural characteristics and expression profile, another important aspect of glucose transporters is their functional characteristics. There have been several efforts to characterize GLUT9's transport kinetics and substrate specificity. Because GLUT9 has several sugar transporter signatures, much of this work has focused on examining how this transporter binds and transports hexoses and related molecules. Takanaga et al. (34) examined glucose transport in HepG2 cells using a novel technique called intramolecular fluorescent resonance energy transfer. They found that, although GLUT9 expression is low relative to other GLUTs in HepG2 cells, a small interfering (si)RNA-mediated knockdown of GLUT9 significantly decreased glucose flux into cells. Several other studies have examined glucose transport using a Xenopus laevis oocyte expression system. Expression of human GLUT9 resulted in a two- to threefold increase in deoxyglucose uptake compared with control oocytes. Mouse GLUT9 expressed in oocytes resulted in a three- to fivefold increase in deoxyglucose uptake compared with water-injected oocytes (8). In addition, glucose transport was not inhibited by cytochalasin B, a GLUT1 inhibitor (4). Another study using the Xenopus oocyte system found that human GLUT9 could transport glucose and fructose but was a poor deoxyglucose transporter and did not transport galactose (22). The K_m for glucose was found to be 0.61 mM, whereas for fructose the K_m was 0.42 mM. Compared with other GLUTs using a Xenopus oocyte overexpression system (6, 10, 15), GLUT9 is a high-affinity glucose and fructose transporter. The absence of the QLS motif in GLUT9 might be responsible for the low transport rate for deoxyglucose, similar to its well-characterized relative, GLUT5 (7).

Recent studies have raised the possibility that GLUT9 may be transporting the purine derivative urate. Much of this recent work was motivated by genetic data suggesting that single nucleotide polymorphisms (SNPs) mapped to GLUT9 may correlate to serum urate levels (5, 9, 11, 12, 21, 36). These were initially very surprising results because there were no previous data to suggest a relationship between a glucose transporter and urate, the end product of purine metabolism in humans. Anzai et al. (3) have shown that GLUT9 has an affinity for urate similar to uric acid transporter 1 (URAT1), a urate-anion exchanger. The K_m of GLUT9 for urate was found to be 365 μM, and the maximum velocity (V_max) was 5,521 pmol·h⁻¹·oocyte⁻¹. Anzai et al. (3) have also shown that organic anions and other substrates known to interact with URAT1 do not affect urate uptake by GLUT9. Additionally, elevation of external K⁺ facilitated urate uptake. These data have led Anzai et al. (3) to suggest that urate is the only substrate for GLUT9 and that the mechanism of transport is distinct from URAT1 (3). Other groups have also found that GLUT9 is capable of transporting urate. Vitart et al. (36) found the K_m to be 890 μM and the V_max to be 5.33 pmol·min⁻¹·oocyte⁻¹. However, this group additionally reported low but detectable fructose transport and that urate could inhibit uptake of labeled fructose. These data suggest that GLUT9 is both a fructose and urate transporter but is considerably more active as a urate transporter.

Another recent study also examined the transport kinetics of GLUT9 for urate. Caulfield et al. (9) found that both isoforms of human GLUT9 were found to have similar transport kinetics, a K_m of 981 μM, and a V_max of 304 pmol·oocyte⁻¹·20 min⁻¹. In comparison, the class 1 glucose transporters GLUT1 and GLUT2 did not mediate any detectable urate flux. Furthermore, this group also reported that extracellular glucose could accelerate urate efflux by GLUT9. Fructose could also mediate urate efflux, but to a lesser degree. In addition, this group also reported an increase in radiolabeled urate uptake by a cell line overexpressing human GLUT9 compared with nonoverexpressing controls. This increase in urate uptake could be diminished by knocking down GLUT9 expression with siRNA. From these data, Caulfield et al. (9) concluded that GLUT9 is a high capacity urate transporter that can transport urate or hexoses alone or exchange urate for glucose or fructose.

GLUT9 and Serum Urate

Data from several recent large-scale genetic studies led to research into urate transport by GLUT9. These genetic data show a correlation between SNPs mapped to GLUT9 and the serum urate levels in several different cohorts (See Table 2). In the first of these studies, Li et al. (21) identified two SNPs within GLUT9 that were correlated with lower serum urate levels in two separate Italian populations. Another study by Döring et al. (12) identified four SNPs within GLUT9 that were associated with lower serum urate levels, with a more pronounced effect in women. Additionally, this study also found that these SNPs were associated with a decrease in risk for self-reported gout. Several other studies have now confirmed this association with SNPs in GLUT9 gout and serum urate (9, 11, 36). One of these studies also found three SNPs that were associated with fractional excretion of urate, which, when decreased, is a risk factor for hyperuricemia (36). Another study found that the effect of three of the SNPs on urate levels was magnified by BMI (5). Two studies in Japanese patients show that mutations to GLUT9 can lead to hypouricemia, a benign condition characterized by serum urate <3 mg/dl (3, 23). These studies found three different mutations that result in single amino acid alterations to GLUT9 in hypouricemic patients. A mutation to URAT1 that has previously been linked to hypouricemia was not present in these patients. Both groups expressed the mutant GLUT9 proteins in Xenopus oocytes and found significantly decreased transport of urate, suggesting that these mutations may be responsible for the hypouricemia.

Table 2.

SNPs associated with decreased serum uric acid levels

SNP	Location	Notes	Ref. No.
rs1014290	Intron 3	Associated with decreased risk of gout and increased FEUA	36
rs6449213	Intron 4	Associated with decreased risk of gout and increased FEUA; effect of SNP modified by BMI	12, 36
rs13113918	Exon 4	Associated with decreased serum uric acid and increased FEUA	9
rs7442295	Intron 6	Associated with decreased risk of gout and increased fractional excretion of uric acid	5, 9, 12, 21
rs6855911	Intron 7	Associated with decreased risk of gout	5, 12, 21
rs737267	Intron 7	Associated with decreased risk of gout and increased FEUA	36
rs16890979	Exon 8	Associated with decreased risk of gout in white patients	11

SNP, single nucleotide polymorphism; FEUA, fractional excretion of uric acid; BMI, body mass index.

Physiological Role of GLUT9 and Future Directions

Although recent research has revealed much about GLUT9, many important questions related to the physiological role of this transporter remain to be answered. Several studies have now confirmed that GLUT9 can transport urate. Genetic studies strongly suggest that SNPs and mutations in GLUT9 can affect serum urate levels. Furthermore, GLUT9 is expressed in the proximal tubule, a location known to play an important role in serum urate regulation (24). These data strongly suggest that GLUT9 is a urate transporter that plays a role in the regulation of serum urate levels. However, it is still unknown exactly how GLUT9 functions in the kidney. Urate handling by the kidney is thought to involve four steps: glomerular filtration, presecretory reabsorption, tubular secretion, and finally, postsecretory reabsorption (23). More work is needed to understand what role GLUT9 plays in one or more of these processes. In addition, more research is necessary to understand how the identified SNPs affect urate handling by GLUT9. All of the known SNPs associated with serum urate levels lie within introns, so how these SNPs affect protein expression or function is unclear. In addition, the role of GLUT9 as a glucose transporter needs to be examined further. Transport kinetics data regarding the ability of GLUT9 to transport glucose and fructose are not consistent, although structural analysis of GLUT9 coding sequence and amino acid sequence suggests that GLUT9 is a glucose transporter.

Better understanding of the physiological role of GLUT9 could lead to discoveries with significant clinical implications. Glucose transport plays an important role in cell energy metabolism in virtually all tissues. There are data to support that GLUT9 plays a role in glucose transport in liver, pancreatic β-cells, and the early embryo. These tissues are all adversely affected by diabetes, so understanding energy metabolism in these tissues in the context of hyperglycemia and insulin resistance is clinically significant. The relationship between GLUT9 and urate is also highly clinically significant. We now know that GLUT9 is expressed in articular cartilage and is a urate transporter. Therefore, it is possible that GLUT9 plays a role in gout, a disease of urate deposition in the joints. In addition, elevated urate levels have been associated with metabolic syndrome, obesity, diabetes, hypertension, and chronic renal failure (23). Although some believe urate is elevated as a result of these diseases, there is now evidence that urate may play a role in the pathogenesis of these diseases (14, 30). Some have speculated that intake of fructose plays an important role in causing elevated serum urate levels, especially in diabetes and obesity (24). It is possible that GLUT9, which seems to be both a fructose and a urate transporter, plays an important role in these conditions associated with hyperuricemia. More research into the role of GLUT9 in diabetes and the metabolic syndrome may lead to the development of interventions to help prevent or treat these increasingly common diseases.

GRANTS

This research was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Training Grant T32-DK007130/T32-DK-007120 (M. Doblado), NIDDK Grant DK-70351 (K. H. Moley), and an American Diabetes Association research grant awarded to K. H. Moley.