**4.12. Class III family members**

of uric acid absorption, leading to severe hypouricemia complicated by nephrolithiasis and exercise-induced acute renal failure. Therefore, GLUT9 is essential for renal reabsorption of uric acid-increased expression is associated with hyperuricemia and gout, while loss of

GLUT9 deficiency in mice leads to hyperuricemia, massive hyperuricosuria, and an early-onset nephropathy, which his is in contrast to the condition in humans where dysfunctional GLUT9 is associated with hypouricemia. Hyperuricemia in mice due to GLUT9 deficiency appears to be a result of impaired urid acid uptake by the liver and therefore inability to be degraded to allantoin by uricase. The nephropathy in GLUT9 knockout animals is characterized by obstructive lithiasis, tubulointerstitial inflammation, and progressive inflammatory fibrosis of the cortex. In contrast, liver-specific GLUT9 inactivation in adult mice results in severe hyperuricemia and hyperuricosuria, in the absence of urate nephropathy or any structural abnormality of the kidney. The deficiency of GLUT9 in mice showed that it represents a functional uric acid transporter *in vivo,* allowing GLUT9 to be identified as a major player in urate homeostasis due to its dual role in uric acid handling in the kidney and in the liver. Whether GLUT9 at all plays a role as a (glucose)/fructose transporter that potentially links

fructose uptake by the liver and uric acid metabolism remains to be seen (Figure 4A).

The human GLUT11 was cloned by PCR on the basis of sequence information obtained from ESTs and a genomic sequence. Three variants of GLUT11 (GLUT11-A, GLUT11-B, and GLUT11-C) have been identified that only differ in their N-terminal sequences. Since for each of the variants the corresponding 5' sequences upstream of exon 1 exhibit promoter activity, transcriptional regulation is assumed to occur by alternative promoter usage. The three isoforms are expressed in a tissue specific manner: GLUT11-A is present in heart, skeletal muscle and kidney; GLUT11-B is expressed in placental, adipose, and kidney tissue; while GLUT11-C is found in adipose, heart, skeletal muscle and pancreatic tissue. Glu‐ cose transport activity for GLUT11 was detected in liposomes reconstituted with GLUT11 containing membranes. The transporter shows affinity for glucose with a Km of 0.16 mM when measured in *Xenopus laevis* oocytes. GLUT11 also transports fructose but not galactose and shows a rather low affinity for cytochalasin B. Mutational analysis of the DSV motif in GLUT11 that corresponds to the "fructose" transporter motif "NAI" also showed that also in GLUT11 this particular region of helix 7 determines substrate selectivity of the transporter. Endogenous GLUT11 protein was localized in heart and skeletal muscle tissue with an antibody raised against the C-terminus of GLUT11 that does not distinguish between the different variants. Human GLUT11 is expressed exclusively in slow-twitch muscle fibres and is unaffected by physiological and pathophysiological conditions except in primary myopathy. Surprisingly, the human SLC2A11 gene has not orthologue in the

function leads to severe hypouricemia.

*Mouse Models of GLUT9 deficiency*

28 Glucose Homeostasis

**4.11. GLUT11** *(SLC2A11)*

rat and mouse genome.

Class III isoforms share specific features that are unique to this Class. First, structurally all Class III members carry their N-glycosylation site at the fifth extracellular loop. Common to all the isoforms is the presence of an internalization signal (dileucine or YSRI in case of GLUT10) that retains these transporters at an intracellular localization under steady state conditions. Thus far, a stimulus for translocation of Class III isoforms leading to plasma membrane localization has only been proposed for GLUT13 which has not been confirmed.

#### **4.13. GLUT6** *(SLC2A6)*

GLUT6 (formerly designated GLUT9) was cloned from human leucoyte cDNA by (RACE)- PCR on the basis of murine ESTs and the human genomic sequence. GLUT6 mRNA is expressed predominantly in the brain, spleen and peripheral leucocytes. Hexose transport for GLUT6 was only shown when reconstituted in liposomes, where GLUT6 transport activity was found in the presence of 5 mM but not 1 mM glucose. GLUT6 exhibits a low cytochalasin B binding affinity. Thus far, no other data on kinetics of transport and potential substrates have been published for this isoform.

GLUT6 contains an N-terminal dileucine motif that is responsible for intracellular retention of the protein when overexpressed in primary rat adipocytes. GLUT6 is only detected at the plasma membrane when the dileucine residues are mutated to alanine or when clathrin dependent endocytosis is blocked by overexpression of a dominant-negative dynamin mutant. However, no cell-surface translocation of GLUT6 is observed in response to stimuli such as insulin, phorbol ester or hyperosmolarity. A gene expression profiling study aimed to identify deregulated in chronic lymphocytic leukaemia associated with Trisomy 12 identified Slc2a6 among the seven genes with the strongest correlation. Although a significant deregulation was not confirmed subsequently by real-time PCR analysis, the specific expression of GLUT6 in leucocytes and the spleen might indicate an important role for this transporter in normal physiology for this cell lineage.

The biochemistry, cell biology and physiology of GLUT6 is currently not well understood.

#### **4.14. GLUT8** *(SLC2A8)*

GLUT8 (formerly GLUTX1) was the first isoform of the extended *SLC2A* family to be identified by database mining. The human, rat and mouse cDNA were cloned by 5' and 3' RACE-PCR from testis cDNA samples. The transporter is mainly expressed in the testes, and lower levels are found in the brain (cerebellum), adrenal gland, liver, spleen, brown adipose tissue and lung. Functional characterization of GLUT8 in *Xenopus laevis* oocytes immediately revealed the intracellular retention of the transporter due to the presence of a dileucine based motif in its N-terminus. When the two leucine residues are changed to alanines GLUT8 localizes to the plasma membrane in mammalian cells and *Xenopus laevis* oocytes that allowed the determination of transport kinetics for the protein. GLUT8 shows a high affinity for glucose with a Km of ~2 mM. Glucose transport can be competitively inhibited with fructose, galactose, and cytochalasin B. The intracellular localization of GLUT8 raised the question of whether this isoform would be insulin-responsive and thereby compensate for GLUT4 in the respective knockout mice which are lacking the insulinresponsive glucose transporter yet near normal glucose tolerance. Indeed, in blastocysts of mice, GLUT8 has been found to account for insulin-stimulated glucose uptake. However, various groups performed extensive studies in primary rat adipocytes, 3T3L1 adipocytes, insulin-responsive CHO-cells as well as neuronal cell types such as N2A, PC12, and hippocampal neurons that all failed to identify a stimulus that leads to a plasma mem‐ brane translocation of GLUT8.

**4.15. GLUT10** *(SLC2A10)*

the transporter supports these findings.

Danish, Finns, and Taiwanese populations.

*4.15.1. GLUT10 physiology*

*Mouse models of GLUT10 deficiency*

GLUT10 has been cloned from human liver cDNA by 3' and 5' RACE PCR based on an EST sequence that was identified via a homology search with known GLUT protein sequences. GLUT10 mRNA is present in the human heart, lung, brain, liver, skeletal muscle, pancreas, placenta and kidney. Expression of GLUT10 is also detected in human and mouse white adipose tissue as well as human and mouse adipocyte cell lines SGBS and 3T3L1, respectively. One remarkable structural feature is the absence of the PESPR motif just after helix 6 that is conserved for all the GLUT isoforms. Heterologous expression of GLUT10 in *Xenopus laevis* oocytes demonstrated 2-DG transport with high affinity (Km ~0.3 mM). Uptake of 2-DG can be

Mammalian Sugar Transporters http://dx.doi.org/10.5772/58325 31

Although no localization study has been performed for GLUT10 clearly demonstrating a plasma membrane or intracellular localization as seen for other Class III family members, immunocytochemical studies indicated an intracellular localization for GLUT10 under steadystate conditions. The presence of the potential internalization motif YSRI at the C terminus of

GLUT10 is located at the chromosomal region 20q12-13.1, a susceptibility locus that has been linked to type 2 diabetes in Caucasian Americans. Therefore, there has been particular interest in GLUT10 as a potential candidate or susceptibility gene involved in the disease. However, polymorphisms in GLUT10 were not associated to type 2 diabetes in Caucasian Americans,

Deficiency for GLUT10 in humans has been found to be associated with ATS, a rare autosomal recessive connective tissue disease that is characterized by widespread arterial involvement with elongation, tortuosity, and aneurysms of the arteries. Homozygous mutations (deletion, non-sense, and missense) for GLUT10 were found in six families with ATS. It is currently

Two groups reported the phenotypic analysis of mice with amino acid substitutions G128E or S150F in GLUT10 [34, 35]. Both substitutions are located in exon2 of GLUT10 and are conserved among the rodent and human protein. Based on predictions, the substitutions G128E and S150F were expected to interfere with the helix structure of transmembrane regions 4 and 5, respec‐ tively. The mice strains were generated after screening a mutant mouse library that was based on N-ethyl-N-nitrosurea (ENU) mutagenesis in healthy C3HeB/FeJ males. Callewaert et al. [34] did not report any of the vascular, anatomical, or immunohistological abnormalities as encountered in patients with ATS. Both mutant strains appear normal at birth, gained weight appropriately and survived to adulthood. The animals showed normal heart rhythm, heart structure, and ventricular function. No specific arterial tortuosity, stenosis, dilatation, or aneurysm in cerebral vessel pattern was noted. However, histopathology revealed thickening

competed with galactose and glucose and inhibited with phloretin.

*GLUT10 and Arterial tortuosity syndrome (ATS, OMIM# 208050) in Humans*

unknown how loss of GLUT10 leads to this connective tissue disorder [33].

The intracellular localization of GLUT8 is also observed *in vivo* under steady-state conditions. In the testis immunofluorescence microscopy shows intracellular localization of GLUT8 in a late-endosomal/lysosomal compartment. In the brain, immunogold labeling electron-micro‐ scopy localized GLUT8 in synaptic dense core vesicles of nerve terminals and secretory granules of vasopressin neurons.

The intracellular retention signal of GLUT8 has been characterized and found to contain the consensus sequence [D/E]XXXL[L/I] that represents a late-endosomal/lysosomal sorting motif. The DEXXXLL sorting signal of GLUT8 interacts with the adaptor proteins AP-1 and AP-2 and controls trafficking of the protein. Interestingly, both GLUT8 and GLUT12 contain a [D/ E]XXXL[L/I] sorting signal; however, the exact composition of the XXX residues seems to finetune the routing and trafficking of the two proteins.

#### *4.14.1. GLUT8 physiology*

#### *Mouse Models of GLUT8 deficiency*

GLUT8 knockout mice appear healthy and exhibit normal growth, body weight development and glycemic control, indicating that GLUT8 does not play a significant role in maintenance of whole body glucose homeostasis. Offspring distribution from heterozygous matings indicated a deviation from the expected Mendelian distribution regarding birth of GLUT8 homozygous animals. This observation was attributed to a decreased sperm motility of GLUT8 deficient spermatocytes that is associated with lower ATP levels and a reduced mitochondrial membrane potential, while number and survival rate of spermatozoa is unchanged. The reduced amount of homozygous GLUT8 offspring is not related to impaired preimplantation embryo development – as might have been suggested by antisense studies in embryos-since mating of knockout mice produced viable, normally developing offspring in numbers comparable to those of a wildtype intercross. GLUT8 deficiency is associated with behavioural alterations indicating a significant physiological role for this isoform in the central nervous system. GLUT8 deficient mice have an increased proliferation of hippocampal cells and behavioral tests show increased arousal, a tendency to altered grooming, and a reduced risk assessment in those animals. However, despite the in depth characterization of the cell biology of GLUT8 and its physiology *in vivo* based on knockout mice, the function of the protein remains thus far unknown.
