**4.1. GLUT1 (***Slc2a1***)**

More in-depth phylogenetic analysis of the GLUT sequences implied a different clustering of GLUT6 and 8, GLUT10 and GLUT12, and HMIT as more distant family members implying the existence of 5 subclasses based on sequence identities but in addition functional properties. Based on these analysis, the evolutionary more ancient forms HMIT, GLUT10, 12, 6, and 8 are postulated to mediate substrate transport which is linked to electrochemical gradients as it has

The protein sequences of the 14 isoforms protein sequences are between 14-63% identical and 30-79% conserved. Common to all isoforms of the GLUT protein family are the predicted 12 transmembrane helices that are based on the initial hydropathy plot for GLUT1 (Figure 2B). The 12-helix model has been supported by studies applying glycosylation scanning, the use of epitope-tags placed within the extrafacial loops as well as antibodies directed against the predicted extra-and intracellular loops and the N-and C-terminal parts of the proteins. All family members are highly glycosylated membrane proteins harbouring an N-linked glyco‐ sylation site. For Class I and II family members this is positioned in the first exofacial loop between transmembrane helices 1 and 2, while class III family members contain a shorter

Sequence comparisons between all isoforms identified conserved residues that have been termed sugar transporter signatures [3]. These include conserved glycine residues in helices 1, 2, 4, 5, 7, 8 and 10, indicating a critical role in the structure of these helices. In particular helix

Nonetheless, the primary sequences of the various isoforms do not allow prediction of their substrate specificity or kinetics of transport. However, on the basis of mutational analyses, several residues and motifs have been demonstrated to participate in the substrate recognition by GLUT1 as well as other isoforms. GLUT1, 3, and 4, which transport glucose but not fructose,

GLUT2 and 5, which both transport fructose, have a HVA or MGG in this position. Structure function analysis of GLUT2 and 3 chimeras expressed in *Xenopus laevis* oocytes demonstrated that GLUT3 can be converted to a glucose/fructose transporter with GLUT2 transport kinetics when the amino acid sequence of GLUT2 from the beginning of helix *7* to the COOH terminus is inserted into GLUT3. This demonstrates the impact of helix7 on substrate specificity and kinetics of transport. A specific feature of all Class II transporters is their ability to transport fructose, with GLUT5 representing the *bona fide* fructose transporter. Again, helix7 is important for exofacial substrate recognition, since transport of fructose by class II isoforms has been linked to the presence of a NXV/NXI motif. Mutating the isoleucine position significantly reduced fructose transport, while glucose uptake was unaltered. Interestingly, Class II isoforms were unable to transport 2-deoxy-D-glucose (2-DG) or galactose. A valine to isoleu‐ cine mutation in GLUT2 (V165I) within helix 5 abolished 2-DG transport, and introduction of the same point mutation in GLUT1 also resulted in a comparable reduction of 2-DG transport.

extracellular loop 1 and harbor the glycosylation site within the larger loop 9 [3].

7 appears to be important for substrate binding from the exofacial site.

been described for HMIT and GLUT12 [25].

18 Glucose Homeostasis

have the QLS sequence in helix 7.

**3.3. Structural features & substrate specificity**

GLUT1, also known as the HepG2 or erythrocyte sugar transporter, is highly abundant in erythrocyte membranes making up 3-5% of the total erythrocyte proteins. The high amount of GLUT1 in red blood cells allowed the generation and characterization of an antibody that was used for the molecular cloning of GLUT1 from a hepatoma cDNA expression library in 1985 [26]. Although not present in hepatocytes, however, GLUT1 represents the most ubiquitously expressed isoform. The transporter is already found throughout early mammalian embryo development from the oocyte to the blastocyst and is present at high levels in endothelial and epithelial-like barriers of the brain, the eye, peripheral nerves, the placenta and especially in certain tumor cell lines and tissues, although not in hepatocytes. Also, GLUT1 is highly expressed in most routinely used laboratory cell lines.

When assessed in *Xenopus laevis* oocytes, GLUT1 transports glucose with a Km of ~3 mM. Under equilibrium exchange conditions GLUT1 has a Km of 20-21 mM for 3-O-methylglucose and 5 mM for 2-DG. Other hexoses transported by GLUT1 are galactose, mannose and glucosamine. When expressed in *S. cerevisae* rat GLUT1 showed a Km for D-glucose of 3.4 mM and transport was inhibited by cytochalasin B (IC50=0.44µM), HgCl2 (IC50=3.5µM), phloretin (IC50=49 µM) and phlorizin (IC50=355 µM).

## *4.1.1. GLUT1 physiology*

## *Human Physiology-the GLUT1 deficiency syndrome (OMIM #606777)*

Mutations in the GLUT1 gene are causative for an autosomal-dominant disorder that is characterized by infantile seizures, developmental delay, acquired microcephaly, and ataxia and infantile seizures which is assumed to be caused by the decreased rate of glucose transport from the blood into cerebrospinal fluid. Defective glucose transport across the blood-brain barrier was first described 1991 [27] and linked to GLUT1 deficiency in 1998 [28]. About a 100 cases have been identified worldwide, including a wide spectrum of heterozygous mutations, including nonsense, missense, insertion, deletion and splice-site mutations, and hemizygosity of the GLUT1 gene.

*4.2.1. GLUT2 physiology*

fructose is normal in FBS patients.

*Mouse models of GLUT2 deficiency*

display hyperglycemia and develop diabetes.

*Human GLUT2 deficiency and the FANCONI-BICKEL SYNDROME (OMIM #227810)*

Rare homozygous or compound heterozygous mutations within the GLUT2 gene cause a type of glycogen storage diseases (GSD), termed GSD XI. The first patient was described by Fanconi and Bickel's, therefore the GLUT2 deficiency is referred to as Fanconi-Bickel Syndrome (FBS). Thus far, 112 patients have been reported. Analysis of 63 patients revealed a total of 34 different *GLUT2* mutations with none of them being particularly frequent. The clinical symptoms of FBS are hepatomegaly secondary to glycogen accumulation, glucose and galactose intolerance, fasting hypoglycemia, tubular nephropathy, and severely stunted growth. In contrast to the metabolism of glucose and galactose**,** utilization of orally or intravenously administered

Mammalian Sugar Transporters http://dx.doi.org/10.5772/58325 21

Glucose homeostasis in FBS-patients is heavily disturbed and postprandial hyperglycemia is frequently observed. A few patients have been diagnosed with diabetes mellitus and have been treated with insulin. Hypoglycemia in fasting states is a feature of FBS. Hypoglycemia has very frequently been documented and plasma glucose levels as low as 18 mg/dl have been reported in FBS patients. Compared to other types of hepatic glycogen diseases hepatic

No specific treatment exists for patients with FBS. Symptomatic treatment is directed towards stabilization of glucose homeostasis and compensation for renal losses of various solutes. The amelioration of the consequences of renal tubulopathy includes the replacement of water and electrolytes. In order to control renal glucose loss and hepatic glycogen accumulation in FBS, patients receive a diet with adequate caloric intake with slowly absorbed carbohydrates.

The importance of GLUT2 in the regulation of glucose-stimulated insulin secretion from the

Investigations of GLUT2 mutations that have been linked to the Fanconi-Bickel syndrome [29] or patients with transient or permanent neonatal diabetes mellitus [30] indicated an important

The consequences of GLUT2 deficiency have been analyzed in detail for the various tissues where GLUT2 is involved and/or essential in maintaining whole body glucose homeostasis. Early on, the critical role for GLUT2 in the β-cell for glucose stimulated insulin-secretion *in vivo* became apparent in mice with transgenic overexpression of a GLUT2 antisense RNA specifically in pancreatic β-cell cells. Upon an 80% reduction of GLUT2 in β-cells those animals

Whole body GLUT2 knockout in mice results in offspring that appears completely normal at birth, but neonates develop early symptoms similar to type 2 diabetes and do not survive beyond the age of 3 weeks. Homozygous GLUT2 deficiency in mice results in hyperglycemia and elevated plasma levels of free fatty acids and β-hydroxybutyrate. *In vivo* glucose tolerance is abnormal and, *in vitro*, β-cells display a gradual loss of control of insulin gene expression by

role for this transporter in pancreatic β-cell development and function in humans.

adenomas or malignancies have never been observed in patients with FBS.

β-cell of the islets of Langerhans has been well established for mice.

Since ketone bodies bypass the blood-brain barrier and enter the brain via a monocarboxylic acid transporter (MCT1) they provide an alternative energy source for the brain under conditions of GLUT1 deficiency. Accordingly, a ketogenic diet is effective in controlling the seizures and other symptoms of the GLUT1 deficiency syndrome. However, this treatment is less effective regarding neurobehavioral symptoms. Correlations between genotype and phenotype still remain elusive.

#### *Mouse Models of GLUT1 deficiency*

Although representing the first GLUT isoform that was discovered, and despite being well characterized, mouse models for GLUT1 deficiency were only described recently. Mice that are transgenic for a homozygous GLUT1 anti-sense transgene are lethal during gestation; heterozygosity for the GLUT1 anti-sense cDNA was associated with growth retardation and developmental malformations.

In mice, homozygous knockout of GLUT1 was associated with embryonic lethality around day E10dpc and E13-14dpc, while heterozygous animals were viable and showed no differ‐ ences in body weight development and growth. Decreased brain weights were reported, however, histological abnormalities were not found. While plasma glucose levels were normal in the heterozygous animals, glucose was decreased in the cerebrospinal fluid (CSF). As shown by PET-scan analysis, glucose uptake and metabolism were reduced in brains of heterozygous GLUT1 knockout animals. These animals also showed deficits in motor activity, balance and coordination as well as spontaneous cortical seizures. Overall, heterozygosity for GLUT1 in mice resembles features of humans with the GLUT1 deficiency syndrome.

#### **4.2. GLUT2** *(Slc2a2)*

The second transporter of the GLUT family was cloned in 1988 from human liver and kidney cDNA libraries. The initial characterization detected GLUT2 mainly in the liver, kidney and intestine but the transporter was later demonstrated to be present specifically in the insulin producing β-cells of the pancreas. GLUT2 is a low-affinity, high capacity transporter and with a Km in the range of ~17 mM, it has the highest Km for glucose among the known members of the GLUT family. GLUT2 also transports galactose (~92 mM), D-mannose (~125 mM), and Dfructose (~76 mM). Recently, GLUT2 was shown to transport glucosamine with high affinity (Km=~0.8 mM). Structurally, GLUT2 lacks the QLS motif in helix 7 which is thought to confer substrate specificity of the transporter, and which may explain the high affinity for glucosa‐ mine.

GLUT2 is located in the basolateral membrane of the epithelial cells of the intestine and kidney, where it participates in the release of absorbed (via SGLT1 in the intestine) or reabsorbed (via SGLT1 and 2 in the kidney) glucose into the blood stream.
